organisms have become tolerant to the metal pollution, which surrounds ports and docks. They

Transcription

1 TEXT Introduction The genus Ectocarpus shows many of the simplest features in the Phaeophyceae and is one of the best known brown algal genus. It is often seen as a hairy growth in pools mostly attached to rocks and other algae. The plant has branched filamentous, caespitose thallus, green, brown or yellow in colour, measuring from few to about 30 cm in length, and forming generally a bushy habit (Figs.1&2). The Fig.2: Vegetative morphology of Ectocarpus. prostrate parts are rhizoid-like, and often penetrate the substratum. The alga is attached to the substratum by a stipe. It forms a dense mat of tangled filaments and branching pseudodicótoma. The plants are dioecious, because male and female games are formed in gametangia borne on different thalli. Common in the colder seas of the Northern Hemisphere, these Fig.1: Vegetative morphology of Ectocarpus. organisms have become tolerant to the metal pollution, which surrounds ports and docks. They

2 resemble the green algae in their cell wall structure, with an inner wall of cellulose and an outer wall of pectin, but differ in wall composition, cellular contents, and structure of motile cells. Research on Ectocarpus began in the 19th century with descriptions of species and taxonomy, followed by studies aimed at unravelling reproduction and life history ultra structure and photosynthetic activities. Other major aspects that have been studied include the sexual pheromones and infection of Ectocarpus by viruses. A proposition to adopt Ectocarpus as a general model organism for the brown algae was made in 2004 (Peters et al., 2004a). This proposition was based on the facts like high fertility, rapid growth (the life cycle can be completed in three months) and the ease with which genetic crosses can be carried out and relatively small size of genome (200 Mbp), make Ectocarpus an interesting model for genetic and genomic approaches (Bénédicte Charrier et al. 2007) Diversity and taxonomy Dillwyn (1809) published the first valid description of Ectocarpus (using the name Conferva siliculosa) based on material collected by W. J. Hooker on rocks in the sea at Cromer and Hastings. Type material, collected by Hooker in 1807, is housed at BM (BM and BM ) under the name C. confervoides. Lyngbye (1819) described the genus Ectocarpus based on material from Denmark and cited C. siliculosa Dillwyn as basionym. The correct nomenclature, therefore, is E. siliculosus (Dillwyn) Lyngbye (see Silva et al., 1996 for further details). Ectocarpus siliculosus is the type species of the order Ectocarpales, which includes most of the smaller

3 brown algae. There are 410 species (and infra-specific) names in the algal database at present, of which 100 have been flagged as currently accepted taxonomically ( About 16 species of this genus have been reported in India. These occur mostly along east and west coasts in supralittoral zone. Identification of different species of Ectocarpus based on morphology is difficult because of the plasticity of the commonly examined features Systematics position Division: Heterocontophyta Class: Phaeophyceae Order: Ectocarpales Family: Ectocarpaceae Genus: Ectocarpus Fig.4: Plant body of Ectocarpus.

4 Distribution This marine alga is worldwide in distribution and contains many species. A few of them (at least 6) have also been reported to occur in fresh water (Chapman, 1973). The marine species are cosmopolitan in distribution but are more abundant in colder seas of temperate and polar regions. The genus is common along the Atlantic coast, but is scarce along pacific coast. The algae occur as lithophytes along coasts in littoral and sublittoral zones (Fig.3). Some species occur in shallow water on the sides of the tidal pool. Some species grow as epiphytes on the other algal genera, especially on the members of Fucales and Laminariales (e.g. E. tomentosus). Species like E. fasciculatus have been found growing on fins of certain fishes (Epizoic). Some species are endophytic (E. dermonematis) or endozoic (E. fasciculatus) (wholly or partly). Several species of Ectocarpus have their basal filaments endophytic in (and to some extent parasitic upon) the tissues of diverse large algae. The

5 endophytic threads penetrate in all directions within the gelatinous walls of the substratum, often causing some distortion. Some endophytic species are known to produce galls (E. deformans in Laminaria ) and (E. valiantei in Cystoseria). The endophytic threads, however, retain their chromatophores and do not in general enter the cells, although this feature has been observed by Sauvageau in E. minimus. It is noteworthy that in many of the reduced species of Ectocarpus only plurilocular sporangia are known. Morphology The plant body is filamentous and heterotrichous. The thallus is sparsely to profusely branched, with the cells joined end to end in a single series as monosiphonous (uniseriate), differentiated into irregular prostrate attached system and a branched erect system (Fig.4). However, the lower part in some species may become polysiphonous due to longitudinal division. The prostrate portion is found attached to the substratum and remains creeping on it. The branches of the erect system develop from the prostrate system into a tuft. The branches are always lateral and arise just beneath the septa. The branches end either in a point or tapper into a series of narrow, elongated hyaline, vacuolated cells with few or no chromatophores forming a colourless hair. In some species, the prostrate system is not well-defined. In certain other species, the older portions of major branches are ensheathed (corticated) by a layer of descending rhizoidal branches. The fascicles of branches give the species a characteristic appearance. The attachment is commonly assisted by the outgrowth of rhizoids from the lower cells of the main

6 axis. In several species the erect threads tend to coil around one another (e.g. E. fasciculatus and E. siliculosus) or to become mated together (e.g. E. breviarticulatus and E. tomentosus (Fig.5). The prostrate system serves the function of anchorage, with the substratum or on other plants and the erect system is photosynthetic and bears reproductive organs. In the some dwarf (small) species of Ectocarpus with short erect threads, the Fig.5: Some small/dwarf species of Ectocarpus. sporangia may arise terminally upon them as in E. faeroensis, but in such other species they are borne directly on the prostrate base, which is usually well developed and may branch so profusely as to become pseudo-parenchymatous. Apart from the fertile branches, it may bear ordinary photosynthetic threads, which may exhibit a trichothallic meristem and terminate in long hairs. This pseudoparenchymatous disc then bears only plurilocular sporangia with unicellular stalks. There is evidently considerable variation even in one and the same species; thus although the erect threads are often unbranched, they may occasionally show some ramification and bear the sporangia laterally (Fritsch, 1945).

7 Cell structure The cells of Ectocarpus are small, rectangular or cylindrical, uninucleate and contain ribbon-like or band-shaped chloroplast (chromatophores) with pyrenoid.the pyrenoid is stalked, pear-shaped. The cell wall is thick, mucilaginous, consisting of three pectic cellulose layers. The characteristic gelatinous substance present in the cell wall constitute alginic acid and fucoiden. Inner to the cell wall, a cell membrane is present which enriches the protoplast. The protoplast contains one central nucleus and cytoplasm with many chromatophores. The number and shape of chromatophores varies with species (Fig. 6). The photosynthetic pigments are Chlorophyll a, Chlorophyll c, β-carotene and Fucoxanthin. The fucoxanthin masks the chlorophyll and gives the characteristic brown colouration. The cytoplasm contains many vacuoles called as physodes, which

8 contain polyphenols, probably functioning as lysosomes. The cells of haploid filaments are comparatively shorter in length than diploid filaments. The end branches bear long, tapering hyaline and vacuolated cells without chromatophores.the cells are totipotent. The chloroplast genome of Ectocarpus is circular, 139,954 base pairs and has two inverted repeat regions (IR). Gene content of the plastid consists of protein-coding genes, trna genes, and 3 ribosomal RNA genes. The trna-leu gene of E. siliculosus lacks an intron. In Ectocarpus, the cpdna IRs contain two ribosomal operons encoding 16S, 23S and 5S rrna. E. siliculosus plastid genomes are predicted to encode a total of 139 and 144 proteincoding genes, and 26 and 27 trna genes, respectively (Le Corguillé 2009) Plurilocular sporangia of some mature plants of Ectocarpus fasciculatus have been found to contain polyhedral virus-like particles. The particles are ca. 170 nm in diameter, possessing both a shell and core, and are confined to the nuclear regions of the developing spores. A possible cylindrical form of this presumed virus is found in some nuclei. The infection could be systemic within the plant, and lysis of the zoospores occurs if large numbers of the virus-like particles are present (S.B. Clitheroe and L.V. Evans 2004). Reproduction

9 Ectocarpus reproduce both asexually and sexually Asexual reproduction Asexual reproduction takes place by means of biflagellate zoospores produced in two kinds of sporangia, namely multicellular or plurilocular sporangia and one-celled or unilocular sporangia, borne on the sporophyte (diploid) plant. The unilocular sporangia develop haploid meiozoospores or gonozoospores, but the plurilocular sporangia develop diploid zoospores. Plurilocular sporangia are elongated, multilocular, and are sometimes known as neutral sporangia, while unilocular sporangia are oval, and unilocular.the zoospores from the plurilocular sporangia germinate directly into other asexual plants, whereas the meiozoospores from the unilocular sporangia germinate into haploid sexual (gametophyte) plants. All the motile cells have two flagella in the 11- and 5- o'clock positions Development of unilocular sporangia The unilocular sporangia develop from the apical (terminal) cell of the lateral branches which increase considerably in size to take on a globose or ellipsoid

10 shape and functions as a sporangial initial. The number of chromatophores also increases within the developing sporangium. The diploid nucleus of the initial cell first undergoes meiosis, followed by several mitotic divisions to produce 32 to 64 haploid daughter nuclei. There is then cleavage of the cytoplasm to form as many uninucleate daughter protoplast units, with single chromatophores and haploid nuclei. Each unit then metamorphosis into a pyriform biflagellate haploid swarmer, called a meiozoospore. The flagella are unequal and inserted laterally. The posterior flagellum is short and whiplash type (acronematic) and the anterior one is larger and tinsel type (pantonematic). During the liberation of meiozoospores, the apex of the sporangium wall gets dissolved and the haploid meiozoospores are liberated in a gelatinous mass. After seconds, they get free from this mass and swim freely in water. They remain motile for about 30 minutes. Ultimately the meiozoospores come to rest on substratum and lose their flagella. Germination of meiozoospores After swimming for a while, each meiozoospore on contact with suitable substratum withdraw its flagella, becomes rounded and secretes a membrane around it. Within short time, a germ tube is formed, which divides many times and forms a prostrate filament. Some cells of the prostrate filament become active and form erect

11 filaments. The plants developed on the germination of these haploid meiozoospores are gametophytes or sexual plants in the life cycle. In E. siliculosus, the sexual plants which produce meiospores have sixteen chromosomes. The meiospores thus contain the haploid number, eight. Development of plurilocular sporangia These are elongated, conical, multicellular bodies, developed singly on diploid (2n) or asexual (sporophytic) plants. They develop initially like unilocular sporangia at the tip of short lateral branches. The apical cell enlarges and functions as sporangial initial (mother cell). This sporangial initial becomes enlarged and contains numerous chromatophores. It then undergoes repeated mitotic divisions, forming 6-12 cells in a vertical row. The cells than undergo several vertical divisions, as a result of which a multicellular cone-like structure, consisting of several hundred small cubical cells, arranged in transverse tiers, is produced forming a plurilocular sporangium (Fig. 9.). Protoplast of each compartment metamorphoses into a single

12 biflagellate zoospore. There is no reduction division during the formation of zoospore in plurilocular sporangium. So each small cubical cells of plurilocular sporangium contains a single diploid nucleus. Each unit then metamorphoses into a single, uninucleate (2n) and biflagellate zoospore. The zoospores formed from plurilocular sporangia are similar to meiozoospores developed in unilocular sporangia, but are diploid. The zoospores from a sporangium liberate through an apical or lateral aperture, and on germination each produces a sporophytic (2n) plant. Sexual reproduction The sexual reproduction is of both isogamous and anisogamous type; however, the latter is very common. Oogamy is absent. Anisogamy may be of two types: morphological anisogamy (E. secundus), and physiological anisogamy (E. siliculosus). The gametes are produced inside the plurilocular gametangia borne on haploid plants (gametophytes). Development of plurilocular gametangia They are large, elongated, sessile or shortstalked multicellular structures. Morphologically plurilocular gametangia are similar in structure and development to plurilocular sporangia, but are produced by haploid or gametophytic plants. These gametophytic plants in certain species are physiologically of two types, but morphologically similar. The plurilocular gametangia produce haploid gametes. During their development, the terminal cell of the lateral branchlet gets inflated, followed by repeated transverse divisions to produce a vertical row of flat cells. This is followed by longitudinal and

13 transverse divisions, resulting in the formation of several hundred small cubical cells, arranged in transverse tiers. Each cell gives rise to one, sometimes two, biflagellate pyriform gametes, which in structure are similar to zoospores but slightly smaller in size than them. The gametes are liberated from the gametangia, following the same procedure as in the zoospore liberation from the plurilocular sporangia. Fertilization Fertilisation is external in brown algae, involving fusion of naked gametes that have been released into the surrounding sea water. This feature has been exploited widely to study m any aspects of early development (Berger et al., 1994). During fertilization many male gametes encircle the female gamete and get entangled to it by the anterior large flagellum. This stage is called clump formation. Out of the many, only one male gamete

14 fuses with the female gamete, and the remaining gametes go astray and gradually get destroyed. The uniting gametes then form the zygote through plasmogamy and karyogamy (Fig. 10). Majority of species show physiological anisogamy (Fritsch, 1945), but morphological anisogamy is observed in E. secundus. In physiological anisogamy, both the uniting gametes are morphologically similar, but in morphological anisogamy the female gamete is larger than the male gamete. Isogamy is reported in in E. globifer. Here the fusing gametes are similar in every respect looking alike and behaving alike. Fusion occurs between isogametes coming from the same plant or even the same gametangium. Physiological anisogamy occurs in E. siliculosus, which is a dioecious species. Fusion in this species occurs between gametes from different plants. The fusing gametes are morphologically identical, but differ in their sexual behavior. One is less active and often called the female gamete. It becomes passive and motion-less after a short time. The more active male gametes cluster around and fix themselves to the body of the female gamete by forwardly directed flagella. Soon the anchoring flagellum of one of these gametes contract and its body is brought in contact with female gamete. Finally the two bodies fuse nucleus to nucleus and cytoplasm to cytoplasm to form the zygote. Role of pheromones in fertilization Soon after release, the originally motile female gametes begin to settle on a surface and start to secrete a chemical signal - a pheromone. The biological function of this pheromone is the

15 improvement of mating efficiency by attraction of the flagellated, motile male gametes. The chemical structure of the chemical signal has been established as 6-(1Z)-(butenyl) cyclohepta-1,4-diene, popularly called as Ectocarpene). The compound was subsequently proved to be the progenitor of a series of C 11 hydrocarbons (Wilhelm Boland 1995). Ectocarpene was the first isolated algal pheromone. It was isolated from Ectocarpus spp. by Müller et al. in 1971 Structure of Gametes As shown in the figure, the gamete shows the flagellum (FS) with the eukaryotic arrangement of microtubules (i.e. nine pairs surrounding two pairs {9+2}) inside the flagellum. The chromatophore (Ch) is shown with the stacked thylakoids and dense fucoxanthin granules indicated by two arrows. The nucleus (Nu) is centrally located. There are a large number of mitochondria (M) in the cell for respiration. There are several unmarked vacuoles. The cell wall is very thin and the cell membrane is tightly appressed to it (Fig.11). Fig.11. ultrastructure of Gamete in Ectocarpus

16 What is interesting about these gametes is that the "female" gamete is positively thigmotactic. This means if it touches down on a surface, it settles down and attaches to the surface. It then secretes ectocarpene that diffuses into the surrounding water. The "male" gamete has positive chemotaxis: it swims toward the source of ectocarpene. Should water current or other movement start to move the cell away from the source, the male gamete show inverse negative chemotaxis: it changes swimming direction when the decreasing concentration of ectocarpene is detected. Germination of zygote The zygote undergoes germination without any reduction division and rest. On germination, it develops into a sporophytic (2n) plant, which is morphologically similar to the haploid or gametophytic plant. Diploid sporophyte in some species of Ectocarpus (E. siliculosus) bears both unilocular and plurilocular sporangia on Fig.12. the same plant whereas other species (E. reptans and E. confervoides) produce either kind of them. The sexual generation also propagates itself by the parthenogenetic development of about 5 per cent of the unfused gametes of either sex.

17 Life cycle A haploid-diploid life cycle of Ectocarpus is of particular interest, because it implies the existence of genetic control mechanisms that regulate the setting out of the two alternative, independent developmental programmes, influencing development at the level of the whole organism. In the life cycle of Ectocarpus siliculosus the sporophyte and gametophyte are slightly heteromorphic, the gametophyte is dioecious and sex determination is genotypic. Alternation of generations The life history of Ectocarpus is of considerable scientific interest. There occur two distinct generations in the life cycle- the diploid sporophyteic and the haploid gametophytic generations. The sporophytic plants bear both unilocular and plurilocular sporangia. The unilocular sporangia by meiosis produce haploid meiozoospores, and the plurilocular sporangia by Fig.13.Stage in life cycle of Ectocarpus-A,gametophyte (haploid filament); B, diploid filament with sporangia and zoospores(x) after meiosis;c, sporophyte with asexual sporangia forming zoospores (2x) mitosis produces diploid zoospores (Fig.12). The meiozoospores, on germination, give rise to the alternate haploid plants -the gametophytes with 8

18 chromosomes. The diploid zoospores, on the other hand, develop into other sporophytes. The gametophyte plants bear multicellular reproductive structures called plurilocular gametangia. Each cell of these plurilocular gametangia produces a single pyriform gamete. The gametes from two different Ectocarpus plants fuse to form a zygote, which on germination give rise to diploid sporophyte plant. The function of the diploid plant is the production of zoospores and meiospores. It is, therefore, called sporophyte. The function of the haploid plant is the formation of gamete. This gamete-bearing plant is called the gametophyte, and this stage in the life cycle is called the gametophytic phase or sexual generation. These two kinds of plants with different function and different genetic constitution regularly alternate in the life cycle. This is called alternation of generations. Since the two alternating generations comprise plants that are morphologically similar, this type of alternation of generations is called isomorphic. Ectocarpus, thus, exhibits a primitive type of alternation of generations (Fig.13).

19 In E. siliculosus both the sporophyte and gametophyte are morphologically similar in branching pattern, but they are quite different in their mode of development. Unlike the sporophyte, which is formed by mediate differentiation, following bipolar germination and symmetric division of the initial cell (Peters et al., 2004b), the gametophyte exhibits an asymmetric initial cell division and immediate differentiation of an erect thallus. Therefore, the alternation of generations in E. siliculosus involves an alternation between two fundamentally different patterns of initial cell division: symmetric and asymmetric. The sporophyte plant has 16 chromosomes, whereas the gametophyte has 8 chromosomes. Investigations have shown that in Ectocarpus haploid filaments producing gamentangia and gametes are formed during warmer parts of the year while during colder parts diploid filaments bearing unilocular and plurilocular sporangia are formed. Development and morphogenesis The growth in Ectocarpus varies with the region of plant body and also with species. Growth of the prostrate portion is apical while that the erect system

20 shows intercalary, diffuse or trichothalic growth. Definite intercalary meristems are found only in a limited number of species in which the entire growth of the individual branches is restricted to certain zones, each situated at the base of a hair. The cells of the meristem cuts off segments both above and below, adding to the length of the terminal hair. Genetically the thalli are of two kinds, haploid and diploid. However they are alike morphologically. Sexuality and genetics in Ectocarpus As a group, brown algae exhibit very diverse patterns of mating systems, ranging from isogamy (identical gametes) (Fig.14) to oogamy (motile male gametes and non-motile female egg cells). The mating system of the Ectocarpus siliculosus is particularly interesting because it represents a very primitive state of sexuality. Male and female gametes have identical morphologies (isogamy) and differ only in their physiology and behavior (Physiological anisogamy). The motile female gamete settles early and produces a pheromone, whereas the male gamete swims for longer, homing in on the pheromone produced by the female gamete. Sex is determined genetically by a single, Mendelian locus in Ectocarpus. Scientists aim at identifying and characterizing the sex locus in this organism (J. Bothwell et al. 2006). Due to its small size, short lifecycle, high fertility rate, relatively small genome and other such features Ectocarpus, has become a genetic and genomic model organism for the brown algae. The 214 million base pair genome of Ectocarpus has been sequenced by Genoscope and analyzed by an international consortium coordinated by the Algal Genetics Group (Cock et al., 2010). Analysis of the genome sequence provided important insights into how the brown algae have adapted to the intertidal

21 environment, and identified a number of genes that were linked with the evolution of multicellularity in this lineage (Peters et al. 2004) Economic importance Two important phycocolloids, algin and fucoidin are important secondary metabolites, and are used in the manufacture of beer, toothpaste, ice cream, paint, shaving cream, medicine, and soaps, etc.