A partially sex-reversed giant kelp sheds light into the mechanisms of sexual differentiation in a UV sexual system.

In UV sexual systems, sex is determined during the haploid phase of the life cycle and males have a V chromosome whereas females have a U chromosome. Previous work in the brown alga Ectocarpus revealed that the V chromosome has a dominant role in male sex determination and suggested that the female developmental programme may occur by 'default'. Here, we describe the identification of a genetically male giant kelp strain presenting phenotypic features typical of a female, despite lacking the U-specific region. The conversion to the female developmental programme is however incomplete, because gametes of this feminized male are unable to produce the sperm-attracting pheromone lamoxirene. We identify the transcriptomic patterns underlying the male and female specific developmental programmes, and show that the phenotypic feminization is associated with both feminization and de-masculinization of gene expression patterns. Importantly, the feminization phenotype was associated with dramatic downregulation of two V-specific genes including a candidate male-determining gene. Our results reveal the transcriptional changes associated with sexual differentiation in a UV system, and contribute to disentangling the role of sex-linked and autosomal gene expression in the initiation of sex-specific developmental programmes. Overall, the data presented here imply that the U-specific region is not required to initiate the female developmental programme, but is critical to produce fully functional eggs, arguing against the idea that female is the 'default' sex in this species.

Host and pathogen autophagy are central to the inducible local defences and systemic response of the giant kelp Macrocystis pyrifera against the oomycete pathogen Anisolpidium ectocarpii.

Kelps are key primary producers of cold and temperate marine coastal ecosystems and exhibit systemic defences against pathogens. Yet, the cellular mechanisms underpinning their immunity remain to be elucidated. We investigated the time course of infection of the kelp Macrocystis pyrifera by the oomycete Anisolpidium ectocarpii using TEM, in vivo autophagy markers and autophagy inhibitors. Over several infection cycles, A. ectocarpii undergoes sequential physiological shifts sensitive to autophagy inhibitors. Initially lipid-rich, pathogen thalli become increasingly lipid-depleted; they subsequently tend to become entirely abortive, irrespective of their lipid content. Moreover, infected algal cells mount local defences and can directly eliminate the pathogen by xenophagy. Finally, autophagy-dependent plastid recycling is induced in uninfected host cells. We demonstrate the existence of local, inducible autophagic processes both in the pathogen and infected host cells, which result in the restriction of pathogen propagation. We also show the existence of a systemic algal response mediated by autophagy. We propose a working model accounting for all our observations, whereby the outcome of the algal-pathogen interaction (i.e. completion or not of the pathogen life cycle) is dictated by the induction, and possibly the mutual hijacking, of the host and pathogen autophagy machineries.

Giant kelp vegetative propagation: Adventitious holdfast elements rejuvenate senescent individuals of the Macrocystis pyrifera "integrifolia" ecomorph.

Recent findings on holdfast development in the giant kelp highlighted its key importance for Macrocystis vegetative propagation. We report here for the first time the development of adventitious holdfasts from Macrocystis stipes. Swellings emerge spontaneously from different areas of the stipes, especially in senescent or creeping individuals. After being manually fastened to solid substrata, these swellings elongated into haptera, which became strongly attached after 1 month. Within 4 months, new thalli increased in size and vitality, and developed reproductive fronds. Our results suggest the usage of these structures for auxiliary attachment techniques. These could act as a backup, when primary holdfasts are weak, and thus improve the survival rate of the giant kelp in natural beds.

Nuclear DNA level and life cycle of kelps: Evidence for sex-specific polyteny in Macrocystis (Laminariales, Phaeophyceae).

Giant kelp, Macrocystis pyrifera (Linnaeus) C. Agardh, is the subject of intense breeding studies for marine biomass production and conservation of natural resources. In this context, six gametophyte pairs and a sporophyte offspring of Macrocystis from South America were analyzed by flow cytometry. Minimum relative DNA content per cell (1C) was found in five males. Unexpectedly, nuclei of all female gametophytes contained approximately double the DNA content (2C) of males; the male gametophyte from one locality also contained 2C, likely a spontaneous natural diploid variant. The results illustrate a sex-specific difference in nuclear DNA content among Macrocystis gametophytes, with the chromosomes of the females in a polytenic condition. This correlates with significantly larger cell sizes in female gametophytes compared to males and resource allocation in oogamous reproduction. The results provide key information for the interpretation of DNA measurements in kelp life cycle stages and prompt further research on the regulation of the cell cycle, metabolic activity, sex determination, and sporophyte development.

Comparative transcriptome analysis of Discosporangium mesarthrocarpum (Phaeophyceae), Schizocladia ischiensis (Schizocladiophyceae), and Phaeothamnion confervicola (Phaeothamniophyceae), with special reference to cell wall-related genes.

Within the stramenopile lineage, only brown algae (Phaeophyceae) have evolved complex multicellularity, although some other members of the lineage (e.g., Schizocladia in Schizocladiophyceae; Phaeothamnion in Phaeothamniophyceae) also develop simple multicellular thalli. The development of an adherent extracellular matrix (ECM) is considered to be one of the key steps in the evolution of multicellularity, because ECM is involved in adhesion of cells to each other and in cell-cell communication essential for developmental, reproductive, and sophisticated defense systems. Because there are no unicellular organisms within brown algae, we considered that comparison of other stramenopile taxa closely related to brown algae and having multicellular thalli could yield clues to elucidate the evolution of multicellularity in brown algae. In this study, we investigated transcriptomes involved in cell wall polysaccharide metabolism of three stramenopile species, Discosporangium mesarthrocarpum, which is suggested to be one of the most basal taxa within Phaeophyceae, S. ischiensis, and P. confervicola. We employed 454-FLX high-throughput pyrosequencing to generate expressed sequence tag (EST) databases for these species, and performed comparative analyses between these databases and the genome sequence of the brown alga Ectocarpus siliculosus. Results indicate that cell wall polysaccharide metabolism pathways of D. mesarthrocarpum are similar to E. siliculosus, whereas those of S. ischiensis and P. confervicola are significantly different from E. siliculosus, suggesting that the components of the cell wall in S. ischiensis and P. confervicola are likely to be different from those of E. siliculosus.

Intracellular eukaryotic pathogens in brown macroalgae in the Eastern Mediterranean, including LSU rRNA data for the oomycete Eurychasma dicksonii .

For the Mediterranean Sea, and indeed most of the world's oceans, the biodiversity and biogeography of eukaryotic pathogens infecting marine macroalgae remains poorly known, yet their ecological impact is probably significant. Based on 2 sampling campaigns on the Greek island of Lesvos in 2009 and 1 in northern Greece in 2012, this study provides first records of 3 intracellular eukaryotic pathogens infecting filamentous brown algae at these locations: Eurychasma dicksonii, Anisolpidium sphacellarum, and A. ectocarpii. Field and microscopic observations of the 3 pathogens are complemented by the first E. dicksonii large subunit ribosomal RNA (LSU rRNA) gene sequence analyses of isolates from Lesvos and other parts of the world. The latter highlights the monophyly of E. dicksonii worldwide and confirms the basal position of this pathogen within the oomycete lineage (Peronosporomycotina). The results of this study strongly support the notion that the geographic distribution of the relatively few eukaryotic seaweed pathogens is probably much larger than previously thought and that many of the world's marine bioregions remain seriously undersampled and understudied in this respect.

Secondary zoospores in the algal endoparasite Maullinia ectocarpii (Plasmodiophoromycota).

The present paper deals with the ultrastructure of zoospores produced by the plasmodiophorid Maullinia ectocarpii, living in the marine algal host Ectocarpus siliculosus. The zoospores described here are very similar to secondary zoospores of Polymyxa graminis and Phagomyxa sp. (the latter an algal endoparasite, also). Our results indicate that M. ectocarpii produces two types of plasmodia, and suggest that is a species with a complete life cycle, as it is known for all the Plasmodiophormycota that have been studied. Sporogenic and sporangial plasmodia produce, respectively, primary zoospores with parallel flagella within thick walled resting sporangia, and secondary zoospores with opposite flagella within thin walled sporangia.

Publisher Correction: Morphological, genotypic and metabolomic signatures confirm interfamilial hybridization between the ubiquitous kelps Macrocystis (Arthrothamnaceae) and Lessonia (Lessoniaceae).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Morphological, genotypic and metabolomic signatures confirm interfamilial hybridization between the ubiquitous kelps Macrocystis (Arthrothamnaceae) and Lessonia (Lessoniaceae).

Macrocystis pyrifera and Lessonia spicata are economically and ecologically relevant brown seaweeds that recently have been classified as members of two separated families within Laminariales (kelps). Here we describe for the first time the Macrocystis pyrifera x Lessonia spicata hybridization in the wild (Chiloe Island, Southeastern Pacific), where populations of the two parents exist sympatrically. Externally, this hybrid exhibited typical features of its parents M. pyrifera (cylindrical and flexible distal stipes, serrate frond margins and presence of sporophylls) and L. spicata (rigid and flat main stipe and first bifurcation), as well as intermediate features between them (thick unfused haptera in the holdfast). Histological sections revealed the prevalence of mucilage ducts within stipes and fronds (absent in Lessonia) and fully developed unilocular sporangia in the sporophylls. Molecular analyses confirmed the presence of the two parental genotypes for ITS1 nrDNA and the M. pyrifera genotype for two predominantly maternally inherited cytoplasmic markers (COI and rbcLS spacer) in the tissue of the hybrid. A metabolome-wide approach revealed that this hybrid is more chemically reminiscent to M. pyrifera. Nevertheless, several hits were identified as Lessonia exclusive or more remarkably, not present in any of the parent. Meiospores developed into apparently fertile gametophytes, which gave rise to F1 sporophytes that reached several millimeters before suddenly dying. In-vitro reciprocal crossing of Mar Brava gametophytes from both species revealed that although it is rare, interfamilial hybridization between the two species is possible but mostly overcome by pseudogamy of female gametophytes.

Free Fatty Acids and Methyl Jasmonate Trigger Defense Reactions in Laminaria digitata.

Arachidonic acid, linolenic acid and methyl jasmonate (MeJA) were found to be strong triggers of an oxidative burst in the kelp Laminaria digitata. These findings constitute the first report of an oxidative burst in an algal system induced by free fatty acids. The source of reactive oxygen species can be at least partially inhibited by diphenylene iodonium (DPI). Treatment with arachidonic acid increases the levels of a number of free fatty acids [including myristic (C14:0), linoleic (C18:2), linolenic (C18:3) and eicosapentaeneoic (C20:5) acids] and hydroxylated derivatives [such as 15-hydroxyeicosatetraenoic acid (15-HETE), 13-hydroxyoctadecatrienoic acid (13-HOTE) and 15-hydroxyeicosapentaenoic acid (15-HEPE)]. Similar to a previous report of the function of an alginate oligosaccharide-triggered oxidative burst in the establishment of resistance in L. digitata against infection by its brown algal endophyte Laminariocolax tomentosoides, C20:4- and MeJA-induced oxidative bursts seem to be involved in establishing the same protection in L. digitata. Altogether, this study supports the notion that lipid oxidation signaling plays a key role in defense induction in marine brown algae.

Detection of differential host susceptibility to the marine oomycete pathogen Eurychasma dicksonii by real-time PCR: not all algae are equal.

In the marine environment, a growing body of evidence points to parasites as key players in the control of population dynamics and overall ecosystem structure. However, their prevalence and impact on marine macroalgal communities remain virtually unknown. Indeed, infectious diseases of seaweeds are largely underdocumented, partly because of the expertise required to diagnose them with a microscope. Over the last few years, however, real-time quantitative PCR (qPCR) has emerged as a rapid and reliable alternative to visual symptom scoring for monitoring pathogens. Thus, we present here a qPCR assay suitable for the detection and quantification of the intracellular oomycete pathogen Eurychasma dicksonii in its ectocarpalean and laminarialean brown algal hosts. qPCR and microscopic observations made of laboratory-controlled cultures revealed that clonal brown algal strains exhibit different levels of resistance against Eurychasma, ranging from high susceptibility to complete absence of symptoms. This observation strongly argues for the existence of a genetic determinism for disease resistance in brown algae, which would have broad implications for the dynamics and genetic structure of natural populations. We also used qPCR for the rapid detection of Eurychasma in filamentous brown algae collected in Northern Europe and South America and found that the assay is specific, robust, and widely applicable to field samples. Hence, this study opens the perspective of combining large-scale disease monitoring in the field with laboratory-controlled experiments on the genome model seaweed Ectocarpus siliculosus to improve our understanding of brown algal diseases.

The development, ultrastructural cytology, and molecular phylogeny of the basal oomycete Eurychasma dicksonii, infecting the filamentous phaeophyte algae Ectocarpus siliculosus and Pylaiella littoralis.

The morphological development, ultrastructural cytology, and molecular phylogeny of Eurychasma dicksonii, a holocarpic oomycete endoparasite of phaeophyte algae, were investigated in laboratory cultures. Infection of the host algae by E. dicksonii is initiated by an adhesorium-like infection apparatus. First non-walled, the parasite cell developed a cell wall and numerous large vacuoles once it had almost completely filled the infected host cell (foamy stage). Large-scale cytoplasmic changes led to the differentiation of a sporangium with peripheral primary cysts. Secondary zoospores appeared to be liberated from the primary cysts in the internal space left after the peripheral spores differentiated. These zoospores contained two phases of peripheral vesicles, most likely homologous to the dorsal encystment vesicles and K-bodies observed in other oomycetes. Following zoospore liberation the walls of the empty cyst were left behind, forming the so-called net sporangium, a distinctive morphological feature of this genus. The morphological and ultrastructural features of Eurychasma were discussed in relation to similarities with other oomycetes. Both SSU rRNA and COII trees pointed to a basal position of Eurychasma among the Oomycetes. The cox2 sequences also revealed that the UGA codon encoded tryptophan, constituting the first report of stop codon reassignment in an oomycete mitochondrion.

Ultrastructural analysis of flagellar development in plurilocular sporangia of Ectocarpus siliculosus (Phaeophyceae).

Flagellar development in the plurilocular zoidangia of sporophytes of the brown alga Ectocarpus siliculosus was analyzed in detail using transmission electron microscopy and electron tomography. A series of cell divisions in the plurilocular zoidangia produced the spore-mother cells. In these cells, the centrioles differentiated into flagellar basal bodies with basal plates at their distal ends and attached to the plasma membrane. The plasma membrane formed a depression (flagellar pocket) into where the flagella elongated and in which variously sized vesicles and cytoplasmic fragments accumulated. The anterior and posterior flagella started elongating simultaneously, and the vesicles and cytoplasmic fragments in the flagellar pocket fused to the flagellar membranes. The two flagella (anterior and posterior) could be clearly distinguished from each other at the initial stage of their development by differences in length, diameter and the appendage flagellar rootlets. Flagella continued to elongate in the flagellar pocket and maintained their mutually parallel arrangement as the flagellar pocket gradually changed position. In mature zoids, the basal part of the posterior flagellum (paraflagellar body) characteristically became swollen and faced the eyespot region. Electron dense materials accumulated between the axoneme and the flagellar membrane, and crystallized materials could also be observed in the swollen region. Before liberation of the zoospores from the plurilocular zoidangia, mastigoneme attachment was restricted to the distal region of the anterior flagellum. Structures just below the flagellar membrane that connected to the mastigonemes were clearly visible by electron tomography.

Recurrent invasion of mitochondrial group II introns in specimens of Pylaiella littoralis (brown alga), collected worldwide.

The mitochondrial genome of a filamentous brown alga Pylaiella littoralis (strain CCMP 1907) has been reported to contain four group IIB introns in the LSU rRNA gene and three group IIA introns in the cox1 gene. We found extreme variability in the number of group II introns for these two genes by analyzing eight P. littoralis specimens collected at worldwide habitats. The first intron of the LSU rRNA gene from a specimen collected in France and the fourth intron from a specimen harvested in Japan exhibited an exceptionally long evolutionary distance when compared with the cognate introns found in P. littoralis specimens. Moreover, these introns harbored an intact or nearly intact tripartite ORF, suggesting they are the result of a recent invasion of cognate introns. Based on the fact that many of the target sites were intronless, we propose that opportunity of intron infection is the bottleneck step of the group II intron cycle which consists of invasion, degeneration, and complete loss from the target site.

Secondary zoospores in the algal endoparasite Maullinia ectocarpii (Plasmodiophoromycota)

The present paper deals with the ultrastructure of zoospores produced by the plasmodiophorid Maullinia ectocarpii, living in the marine algal host Ectocarpus siliculosus. The zoospores described here are very similar to secondary zoospores of Polymyxa graminis and Phagomyxa sp. (the latter an algal endoparasite, also). Our results indicate that M. ectocarpii produces two types of plasmodia, and suggest that is a species with a complete life cycle, as it is known for all the Plasmodiophormycota that have been studied. Sporogenic and sporangial plasmodia produce, respectively, primary zoospores with parallel flagella within thick walled resting sporangia, and secondary zoospores with opposite flagella within thin walled sporangia.

Oligoalginate recognition and oxidative burst play a key role in natural and induced resistance of sporophytes of laminariales.

Forty-five species of brown algae (Phaeophyceae) were surveyed for their capacity to respond by an oxidative burst to challenges with alginate oligosaccharides. Intertidal frondose brown algae (Fucales) constitutively released high quantities of peroxide. The capacity to recognize oligoguluronates and to react with an oxidative burst was confined to alginate-rich taxa with complex thallus morphology, epitomized by the sporophytes of Laminariales. When kelp sporophytes were impaired in their capacity to perform an oxidative burst by the NAD(P)H oxidase inhibitor diphenylene iodonium, they were readily degraded by their bacterial epiflora. Thus, in these algae, the oxidative response is an essential element of natural resistance. We also report on the establishment of a well-defined experimental system for investigations on kelp immunity, with Laminaria digitata as the host and its phaeophycean endophyte, Laminariocolax tomentosoides, as the pathogen. We found that an alginate-triggered oxidative burst significantly induces resistance in Laminaria digitata against infection. From these findings we infer that oligoalginate signals are important cues in the interaction between laminarialean kelps and potential pathogens.