Study of the reproduction diversity of Enteromorpha prolifera J. Agardh
Lin Apeng1, Shen Songdong1, Wang Jianwei1, Yan Binlun2
(1. Life Sciences School, Soochow University, Suzhou City, Jiangsu Province, 215123; 2. Marine
Biotechnology Key Construction Laboratory, Huaihai Institute of Technology, Lianyungang City,
Jiangsu Province, 226001)
Abstract: Enteromorpha prolifera (Muell.) J. Agardh (Chlorophyta, Ulvophyceae), which distributes
worldly in the inter-tidal zone of sea, is one of the most common fouling green algae. However, the
present understandings of the life history of E. prolifera have been insufficient to explain their
seasonal abundances. Thus it’s essential to investigate how many reproductive strategies were likely
been employed by and contribute to the successful colonization and flourish of the green alga. In the
present work reproduction diversity of E. prolifera was observed and studied systematically by
culturing chopped tissues. Our results showed that there are totally seven pathways of reproduction for
E. prolifera including sexual, asexual and vegetative reproduction. It was indicated that the variety of
the reproductive ways and the large quantity of reproductive cells produced and released during
reproductive season are the two key factors that facilitate the colonization of E. prolifera; and the
reproduction of the alga E. prolifera mainly depends on the asexual ways. The present results
contribute to increase the understanding about how the opportunistic macroalgae successfully
maintain its colonization and its excessive growth.
Key words: colonization; life history; macroalgal blooms; propagation; tissue culture.
Correspondence author: [email protected]
Introduction
Enteromorpha prolifera (Muell.) J. Agardh (Chlorophyta, Ulvophyceae) is one of the dominant
seaweeds in the littoral zone of China. It distributes in a wide variety of coastal water, such as
brackish-waters of inner bays and estuaries and so on and greatly affects the carbon cycle and
recovery of the contaminant water body to border on the sea and often contributes to the formation of
the so-called ‘green tide’, which causes ecological and indirect economic damages (Hiraoka et al.
2004).
The life cycle of Enteromorpha consist of haploid and diploid phases, both of which are
morphologically similar and reproductive haploid or diploid asexual zoospores formed by mitosis of
vegetative cells (van den Hoek and Mann 1996). Sexual reproduction involves fusion of opposite
mating types of haploid gametes, which can also develop into adult thalli parthenogenetically.
Previous studies also have revealed that the reproduction of Enteromorpha is mainly due to the
asexual zoospores that derived from distal end of thalli (Callow 1996; Eriksson 2005). Different types
of life cycle have been reported in Europe (Bliding 1963; Koeman and van den Hoek 1982; Burrows
1991), Korea (Kim et al. 1991), Japan (Masanori et al. 2003) and North America (Kapraun 1970).
Recently, there have been an increasing number of worldwide reports on the formation of huge
mass of benthic algae due to eutrophication, which are termed ‘green tides’ (Fletcher 1996). These
excessive growths largely consisted of various species of green benthic algae and Enteromorpha spp.
is one of them (Hiraoka et al. 2004). As a result in some areas it greatly jeopardizes the production of
the aquatic crops and often affects the scenery of the beaches. So far there are few studies about
Enteromorpha species in china, but abroad, they have been studied intensively. However many of the
previous researches have been focused on the influence of the outward conditions such as the high
nutrient fluxes and currents or wave actions (Hiraoka et al. 2004; McAvoy and Klug 2005); some
others focused on spore bioadhision (Callow et al. 1997; Stanley et al. 1999; Callow et al. 2000; Patel
et al. 2003; Callow et al. 2003). Thus, even though there has been extensive research to determine
the causes of their development, the whole picture of the reproduction of E. prolifera still
unfathomed yet. The regeneration from segments of Enteromorpha species has been investigated
frequently (Eaton et al. 1966, Moss et al. 1976, Lee et al. 1996, Dan et al. 1997; Dan et al. 2002); there
are also some studies of other green alga focused on the regeneration of protoplasts induced by the
cultivation of chopped tissues (Ye et al. 2005; Wang and Tseng 2006; Ye et al. 2006). It was indicated
that cultivating chopped tissues could induce the formation of reproductive cells of the alga (Dan et al.
1997; Dan et al. 2002). The aim of this study was to look into the inward reasons: the reproductive
strategies of the alga, which contribute to its omnipresence, by this relatively simple method.
Materials and methods
In November 2005, thalli of E. prolifera were collected from the culture nets of Porphyra
yezoensis, a common aquatic crop in coastal region, in intertidal zone of Yancheng city, China. The
thalli, in dark green, were chosen and washed with sterile seawater, transported in seawater to the
laboratory of the Life Sciences School, Soochow University and stored in seawater, at 10 Celsius
degree for further use. The healthy thalli were checked carefully under microscope to make sure that
there was no spore formation yet and then washed 3 times in filtered and sterile seawater with writing
brush and washed 3 times (every time 30s) using ultrasonic cleaner (Branson) in 0.7% KI (3.5g KI
dissolved in 500ml distill water) to remove epiphytes. Then the thalli were chopped into small pieces
(under 0.05 cm2) and cultured in nature seawater with additional nitrogen (500 µmol/L), and phosphor
(50 µmol/L) with a photoperiod of 12:12 L: D and a photon flux density of 20 to 75 m mol m-2.s-1 at
20 Celsius degree. The medium was changed daily.
The gametes were released after several days of cultivation, and zygotes were obtained on slides
by combining drops of water containing gametes from sexually different plants.
To study asexual reproduction of E. prolifera, a single gametophyte per dish was cultured for
releasing monosexual gametes (female or male). The following culture of monosexual gametes was
similar to that of zygotes described above.
A reverse microscope (IX71, Olympus optical Co. Ltd, Tokyo, Japan) equipped with C5050
zoom cameras was used to observe and record the growth progress.
Results
It is well known that vegetative cells from the apex of Enteromorpha thalli can transform directly
into zoosporangia or gametangia (Dan et al., 2002), and according to the researches of Dan et al.
reproductive cells could be induced by cultivation of chopped tissues of Enteromorpha (Dan et al.
1997; Dan et al. 2002). In our experiment, after chopped into small pieces (under 0.25 cm2) and under
cultivation, most parts of most segments of E. prolifera were developed into gametangia or
zoosporangia. Totally seven pathways of reproduction were found, including both sexual and asexual
ways.
Sexual reproduction
During cultivation, some cells in segments gradually increased in size and became sphere-shaped,
thus the surface of the segments became irregular. After several days of culture, brown speckles of
gametangia were formed (Fig1). After maturation, numerous gametes were released from gametangia.
There were two kinds of gametes: ‘male’ and ‘female’ gametes, both of which had a red eyespot and
were elliptic in shape and the female ones were slightly bigger than the male. Both male and female
gametes had two flagella attaching to one top of their elliptic bodies (Fig2), which propelling them
around swiftly before and during mating.
Anisogamy. After released, gametes were so active that they run into each other from time to
time. When two oheterosexual gametes met, they circled around each other rapidly and keep in touch
simultaneously; after a while, if everything went well, the process of gametogamy started: the two
gametes firstly bonded and mingled from the top that without flagella (Fig3); later, from lateral side
and formed a triangle (Fig4). Finally, a spherical zygote was formed (Fig5, Fig6) which could later
develop into an offspring. During the whole process the gametes keep rotating and translating at the
same time and they spun together or rotated around each other. Planozygotes could be clearly
distinguished from sexually unfused gametes.
Thalli cultured from the Planozygotes released quadriflagellate meiospores and thalli cultured
from these meiospores could release biflagellate male or female gametes and the cycle restarts again.
During cultivation we also found the phenomenon of conjunction of several (more than 2)
conjoint gametes. The conjunction of gametes some times might get a little complicated: more than
two gametes could mingle together, thus bigger zygotes formed, or a zygote could conjoint with
another zygote (Fig7) and formed a much larger zygote (Fig8). This flexible conjunction sometimes
could cause “miscarriage”: some of the big zygotes disintegrated shortly after the conjunction (Fig9).
This phenomenon was quite common in our observations.
Asexual reproduction
Cultivation of chopped tissues could also cause the formation and release of spores, which also
are capable of movement.
Reproduction from biflagellate spores. The formation of biflagellate zooids was much similar to
that of the gametes, and sometimes in the same segments. During cultivation, in some of the cells of
segments, biflagellate asexual spores formed, which were fewer but larger than gametes, together with
a much bigger spherical aplanospore formed in the same sporangium (Fig10).
Reproduction from quadriflagellate spores. After several days of cultivation, green speckles of
sporangium formed. After maturation motile spores, which were 5-7µm in length and without cell wall
and quadriflagellate pear-shaped cells, were released (Fig11). Spores swam around searching for
suitable substrata to reside and then started development, which were the same as the zygote in
principle.
Reproduction from single gametes. If released gametes fail to conjoin with contrasexual gametes
in certain period of time, they will also settle down. But they develop in a totally different way from
the zygotes and spores. Shortly after settle-down, the gametes become sphere-shaped, smaller than the
zygotes. After several hours, they started to bud from the eyespots (Fig12), thus slender budding pipes
formed, with red eyespot in one side of the pipes. Latter, the pipes became longer and wider,
meanwhile, the plasma of the gametes gradually moves into the pipe, leaving the round cell wall back
there. It seems that they were not capable of developing thalli and they only grow into rhizoid and
later in cultivation, they often died. Those which didn’t die underwent “parthenogenesis”: after a
short–term development, motile quadriflagellate spores were released (Fig13); after attached to
suitable substrata, these spores germinated exactly like spores.
Vegetative propagation
As opportunistic seaweed, E. prolifera is also capable of vegetative propagation, which increases
its reproductive diversity enormously, and enables it to colonize new territory more quickly and more
efficiently. In our experiments, four ways of vegetative propagation of the alga were found.
Regeneration from segments. Under cultivation, cells within segments may develop in 3 different
pathways: 1. The cells from upper ends often developed into new thalli while the cells from lower
ends into rhizoid, thus the whole segment became a new plant (Fig14); sometimes the top cells
developed into new thalli or new branches as we can see, while the lower end cells didn’t develop into
rhizoid. As a result, a new individual without rhizoid formed from the segment (Fig 15).
Regeneration from protoplasts and cells. Shortly after cultivation some protoplasts from the cells
within the segments might be released into the medium and attached to the bottom of the culture plate.
Several days after cultivation they developed new cell wall and then regenerated into new plants or
became sporangia. Some cells, which often weren’t in the ends, might divide into clusters of cells and
developed into new young plants later (Fig16).
Vegetable growth of germ cells. Under certain circumstances, the germ cells, such as biflagellate
spores and quadriflagellate spores etc, might keep undifferentiated and keep dividing after settlement,
which could cause the formation of clones and even rafts of germ cells.
Vegetable growth of filaceous microphyte. After settled down, the germ cells may start
differentiation: one initial germ cell divides into two cells with different differentiation tendency: one
base cell, which usually developes into rhizoid; one top cell, which usually develops into thalli. The
basal cell seems to possess more differentiation potency, which could remain undifferentiated and
developed into strains of loosely connected spherical cells. These cells are relatively bigger and
loosely jointed to each other, which could then develop into clusters of new thallus or new individuals
(Fig17, Fig18). Sometimes the basal cells proliferated without the obvious development of the top
cells, thus filaceous microphytes formed.
Discussion
E. prolifera, as a short-lived filamentous alga with high nutrition value, is the preferred food of
many herbivorous animals, such as fish, snails and wading birds. Due to the feeding of those animals
and as well as certain human activities, e.g. the voyage of large ships, the formation of fragments of
thallus is inevitable. Using chopped tissues, we could simulate and study the development of
fragments formed under nature conditions. The breakage of the alga causes formation and massive
release of moveable germ cells from fragments (Dan et al. 1997; Dan et al. 2002). By producing those
germ cells, such as gametes, spores, protoplast, or both form regeneration directly and indirectly, those
small pieces could attribute to the generation of new individuals greatly.
According to our observation, the spores and gametes often settle in extremely proximity to each
other, which, we assumed, could be caused by the relatively higher suitability of the substrate taken by
pioneer spores (Callow et al. 1997; Callow et al. 2000). In this way, rafts of propagules may be formed.
But to find out the real mechanism that employed by the spores, further studies are needed. Under
different circumstances, the development of the settled spores and zygotes of E. prolifera, which are
fully totipotent, could be different. They can either develop into new individuals or just reproduce
themselves and form clones, which could also cause the formation of rafts of propagules. This ability
may be the key promoting factor that contributes to the quick start and enormously growth of E.
prolifera in spring. Thus it could dominates suitable substrates before higher plant even begin their
growth (Back et al. 2000), which may actually promote and accelerate its colonization (Santelices et al.
1989).
Besides forming germ cells, the cultivation of the segments could also produce other propagules
such as regenerated segments: develop into intact individuals with new branches forming from cells of
upper ends and rhizoid from lower ends, in a way much like Bryoposis hypnoides, a green alga which
could develop into intact individuals from segments (Wang and Tseng 2006), in a relatively shorter
time. In other cases, the cells of the segment firstly divide into clusters of cells and later these clusters
of cells as a whole develop into new young plants, which indicate a process of degeneration and
redifferentiation of the cells. Due to the fact that it can grow without any attachment to the substratum,
and capable of proliferating when supplied with nutrient-enriched seawater such as estuaries that
associated with sewage effluent, therefore huge mass of the alga may formed in coastal areas where
nutrient levels were higher than normal (Worm and Sommer 2001; Hiraoka et al. 2004; McAvoy and
Klug, 2005).
Rhizoid of E. prolifera develops from basal cells through their elongation and division, which
happens cyclically during the formation of rhizoid. At the first stage, the synthesis of the cell wall
leads to the growth of basal cells in length and the stretch of cells into tubiform from lower end.
Secondly, the cell mass, including nuclear core, transfer into the tube, which is much similar to the
formation of root hair of higher plant. Eventually, the cell divides, and then the whole process starts all
over again, until holdfast is formed in the tip of the rhizoid. If the cell masses and nucleuses don’t
move into the tube, the rhizoid cell won’t perform cell division. As the rhizoid can’t help the plant to
obtain nutrients from environment, we assume that the fission deviation and elongation of the rhizoid
could help the plant to get a suitable site for attaching and helps to hold the thalli more firmly. And if
the stretch part missing, the basal cell could form a string of loosely connected spherical cells, which
are multipotent and are capable of forming new plants. We also found that when there is no suitable
site for attaching, for instance, when cultivated on top of seawater-prepared agar, the formation of the
multipotent spherical cells could be stimulated.
The basal cells of the thallus of the alga possess more differentiation potency though out the
whole growth period, during which they could develop into new branches or even new individuals,
especially during the early development of the alga. Some basal cells of the microphyte became big
and spherical and their affiliations with other cells were loose and may break into small pieces
automatically or under the disturbances caused by wave or marine animals. Some cells of them might
even die and caused the breakdown of the filament, through which clusters of cells formed. These
fragments could later develop into new plants. The development and the proliferate of the microphyte
depends on the age of the thalli: the younger the thallus is, the greater the basal cells’ ability developed
into filaceous microphytes with loosely connected spherical “stem cells”; It also depends on the
density of the settled germ cells and the environment: the more remote the settled germ cells from
each other and the more favorable the environment is, e. g. influx of nutrient-enriched seawater, the
greater filaceous microphytes proliferate themselves. In a word, it is a very efficient way for the alga
to reproduce itself in which the alga could quickly occupy suitable substrate nearby and thus paves the
way to its later omnipresence.
Although through contribute to ‘green tide’ Enteromorpha species may cause great damage to
aquacultrue and even ecosystem, some species of Enteromorpha are important aquaculture crops and
have been used as seafood in several countries, particularly in East Asia, or as the basis of the food
chain in many aquaculture operations and as the feeding supplement for domestic animals, due to its
high nutrient value (Brand 1991; Rusig and Cosson 2001; Dan et al. 2002). For example, E. prolifera
is important edible seaweed, in Japan, it is cultivated in a large scale and been consumed in various
ways (Dan et al. 2002). Enteromorpha species could also been used as compositions of a seaweed
biofilter to maintain water quality, to reduce the flow-though rate of seawater and the nutrient load in
effluents (Ellner et al. 1996; Hernandez et al. 2002). Therefore, it’s important both to control the
proliferation of E. prolifera. Those swarmers and other induced propagules have been used in artificial
seeding (Dan et al. 1997), which leaded to great simplification of the procedures; And as in laboratory,
using this method, reproductive cells of E. prolifera can be easily obtained for further experiment.
Thus we could be free from the restriction of the season, because in this way we could induce germ
cells of the alga any time we want, under laboratory conditions, as long as we posses well preserved
thalli of the alga.
The conjugation of gametes of opposite gametes in vast sea under natural conditions, where
merely to found each other isn’t an easy task to fulfill, which could causes the formation of large
numbers of unconjugated gametes. And the unconjugated gametes seem to posses only the potency to
develop into rhizoid but not individuals. This defect often causes the death of some of them. While the
ones left could perform “parthenogenesis” and reproduce form single gametes. This strategy may
greatly enhance the ability of the alga to survive and reproduce under extremely harsh conditions
when syngamy is impossible.
Due to the difficulty of the gametes to meet each other, the fusion of more than two gametes
might not be a normal phenomenon in nature, which we assume could be caused by the extremely
high density of gametes under experimental conditions. Under cultivation several gametangia may
release gametes simultaneously after maturation, especially under certain stimulus, e.g. been moved
into newly changed seawater, and motile gametes run into each other. In such a crowed condition, the
chances of several gametes meeting each other are especially high, thus more than normally two
contrasexual gametes may fuse together. As the mechanism of the phenomenon still isn’t clear, we
hypothesize that, as a lower plant, E. prolifera hasn’t evolved enough to possess the mechanism to
prevent it. However, to confirm whether it is the case, further investigations are needed.
Acknowledgments
We thank Liran Zhou for her critical editorial review of this paper. And this study was supported
by the fund (No. 2005HS006) of Marine Biotechnology Key Structure Laboratory, Huaihai Institute of
Technology, Lianyungang, China.
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Figure illuminations:
Fig. 1 speckles of gametangia
Fig. 2 gametes with two flagellate
Fig. 3 two gametes band together both from the tops
Fig. 4 two gametes band together from lateral sides 1
Fig. 5 two gametes band together from lateral sides 2
Fig. 6 a zygote formed from gametes
Fig. 7 two zygotes conjoining with each other
Fig. 8 a much bigger zygote formed from two zygotes
Fig. 9 a big zygote disintegrating from inside
Fig. 10 a distinguishable sporangium with biflagellate zoids formed among gametangia
Fig. 11 quadriflagellate zoid
Fig. 12 settled gamete budding from their eyespots
Fig. 13 “parthenogenesis”
Fig. 14 a new plant with rhizoid developed from a segment
Fig. 15 a new plant without rhizoid developed from a segment
Fig. 16 cells divide into clusters of cells within segment
Fig. 17 the breakage of a filament caused the formation of new individuals
Fig. 18 new individuals developed from cells of a filament