Kohatsu
ABSTRACT
Carpospores and tetraspores of Erythrocystis
adhered preferentially to the trichoblasts of Laurencia.
Infection of apical pits appeared to result from
fortuitous attachment of spores to the proximal end
of trichoblasts and extension of a unicellular rhizoid
into the pit. No spore motility was observed. Infection
rates in culture were approximately 30% for Laurencia
collected at Stillwater Cove and Hopkins Marine Station,
both areas in Monterey County, California. It is
notable that the Laurencia at Hopkins is not infected
by Erythrocystis in the field.
In a survey of Laurencia pacifica from Stillwater
Cove, it was found that up to 90% of the pits were
infected by Erythrocystis germlings. Male, female
and tetrasporophyte Erythrocystis occurred in the ratio
of 1:1:3. Erythrocystis thalli ()imm) predominated
in the apical pits. The mean lengths of thalli from
four locations on the Laurencia were significantly
different (P(.05): 1*)2*)2*4)3*.
Kohatsu
(2)
INTRODUCTION
Erythrocystis saccata (J.Ag.) Silva is a red alga
which occurs specifically in the terminal pits of
Laurencia pacifica Kyl. and a few related Laurencia
species (Abbott and Hollenberg, 1976). The thalli
are ovate and contain mucilage. Red-pigmented, unicellular
rhizoids penetrate through the pit into the medulla
of Laurencia. At the tip of every branch of Laurencia,
there is a distinct pit filled by densely packed,
short cells. Distally, these cells elongate and become
filamentous trichoblasts.
By the criteria of Setchell (1918), Erythrocystis
is a parasite: it fulfills the two critical characteristics
of host penetration and reduced thallus. The third
criterion of reduced pigmentation is not apparent.
Detailed external (Kylin, 1928) and ultrastructural
(Kugrens, 1971) descriptions have been made on the
form and development of the vegetative and reproductive
structures. There is little known about the infective
mechanism of Erythrocystis. In other red algal parasites,
the process has been described. Feldmann and Feldmann
(1958) described how spores of Janczewskia verrucaeformis
Solms stuck to the host, Laurencia obtusa (Huds.)
Lamour, and formed rhizoids which penetrated the
intercortical cell walls. Kugrens and West (1973)
noted that Choreocolax polysiphoniae Reinsch infects
Polysiphonia lanosa (L.) Tandy by rhizoidal penetration
Kohatsu
through the cortex or germination within a wound. Goff and
Cole (1976) worked on Harveyella mirabilis (Reinsch)
Schmitz and Reinke. Their experiments showed that
the parasite probably infected Holmsella pachyderma
(Reinsch) Sturch through wounds made by algal grazers.
Spores attached themselves to the wound by a rhizoid,
Subsequently, a primary filament extended into the
medulla where it branched and spread. The parasite
emerged in pustules of host and parasite cells.
The present study sought to elaborate various
aspects of the life history of Erythrocystis: how it
infects a host, the nature of the symbiosis, and its
distribution upon the host.
MATERIALS AND METHODS
Erythrocystis saccata and Laurencia pacifica
were collected at Stillwater Cove, Pebble Beach,
California, between -1.0 to +1.0 ft. tide levels.
Collections of uninfected Laurencia pacifica were made
near West Beach at Hopkins Marine Station, Pacific
Grove, California.
The tetraspores of Erythrocystis were used in all
experiments. They were indistinguishable from carpospores
using light microscopy. It was assumed that both
types of spores used a similar method of host infection.
Spores were obtained by: severing the base of
fertile thalli, drawing out the excess mucilage with
paper toweling, quartering the thallus longitudinally,
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and immersing the pieces in filtered seawater. The
settling chambers were swirled approximately every
ten minutes to prevent the spores from clumping.
The hypothesized infection mechanism was that
the spores moved to the pits by creeping and directed
rhizoids into the pits.
Experiment 1
Spores were settled upon a slide in petri dishes.
Laurencia cuttings were aligned perpendicular to the
slide with the apical tips facing the edge of the
slide. Spores were observed after eight hours to
note the direction of rhizoid growth. The dishes
were kept in a cold room at 15 C. The cultures were
illuminated by fluorescent light for 10 hours/day,
at an intensity of 19 uE/mesec.
Experiment 2
Laurencia cuttings were swirled in seawater containing
tatraspores. Eight cuttings were maintained in a
gallon jar with flowing water. Eight were kept in
petri dishes with filtered seawater. Eight were
kept in dry petri dishes to simulate a low tide.
The cuttings in filtered seawater were observed hourly
for approximately nine hours in order to detect spore
movement. The other cuttings were observed after
the same time period to note the infection rate.
Experiments 3,4,5
These were designed to determine the extent to
Kohatsu
(5.
which Erythrocystis relies on Laurencia.
Experiment 3
Spores obtained with swirling were kept in filtered
seawater. Upon germination, 2 ml Provasoli's nutrient
mixture, 1 ml penicillin-streptomycin and 1 ml germanium
dioxide were added to the petri dish.
Experiment 5
Erythrocystis thalli of sizes 5-10 mm were carefully
teased from their pits. Four were placed in petri
dishes with seawater which had Laurencia soaking in
it. Three were maintained in filtered seawater.
Four 20 cm X 20 cm quadrat samples were collected
haphazardly from Laurencia patchds at Stillwater Cove.
The Erythrocystis present were sexed (male, female,
tetrasporophyte, or juvenile), their length was measured
in millimeters, and their location on the host plant
was noted. The convention of 1*, 2*, and 3* branches
was used. In addition, thalli which were located on
a 2* branch, but not at the apex were scored as 2*A
(see fig. 1).
OBSERVATIONS AND RESULTS
Experiment 1
The tetraspores treated in this experiment showed
patches where spores extended their rhizoids in the
same direction. The majority grew away from the
Laurencia tips.
Experiment 2
Kohatsu
(6)
Tetraspores stuck to trichoblasts with some selectivity
to Laurencia from Stillwater Cove and Hopkins Marine
Station. Their attachment ranged from a weak bond
that could be broken by agitating the cutting to
fairly strong adhesion that was only broken by physically
pulling off the spore. Spores stuck anywhere along the
length of the trichoblasts. Approximately 30% of the
spores landed near the pit and germinated a rhizoid
into the pit. The majority of the spores were lost in
the aquarium. The cuttings in the dry petri dishes
appeared to have the same infection rate as the wet
dishes.
Spores did not show creeping activity. After
24 hours in culture, the Laurencia showed signs of
trichoblast deterioration. The spores attached distally
were cast off with the trichoblast. Trichoblast
deterioration occurred in control dishes without
spores, too.
Sampling confirmed that Erythrocystis thalli
greater than 1 mm in length were found primarily in
the apical pits. Further, a test for the analysis
stis
of variance showed that the mean length of Erythroc
in the 1*, 2*, 3*, and 2*A pits were significantly
different (P(.05). See fig. 2
Experiments 3,4,5
The spores collected with swirling degenerated
with only 5% of the spores germinated. Those kept
Kohatsu
(7)
in filtered seawater broke down within a week. The
spores maintained with Laurencia cuttings lasted
twice as long. Spores innoculated into the medulla of
Laurencia cuttings also degenerated before they ever
germinated.
The spores which were allowed to settle undisturbed
fell in concentrated patches. These were surrounded
by a sticky matrix which bound the spores together
and to the dish. These germinated within 24 hours.
The spores did not develop normally. Many formed
trichoblasts alone. Rhizoidal and trichoblast growth
appeared random. The thalli never developed beyond
the cell initial.
Erythrocystis thalli teased from Laurencia and
maintained in filtered seawater bleached within four
days. Those kept in normal seawater with the "essence"
of Laurencia lasted two weeks before bleaching.
Erythrocystis is not overtly detrimental to its
host nor does it appear dependent for photosynthate.
Local cell death is not evident. Laurencia produces
reproductive structures as normal, including spermatangia
in infected pits. As Kugrens (1971) noted, there are
no intimate cellular connections between Erythrocystis
and Laurencia. The present study demonstrated that
Erythrocystis can live for a time without its host.
C is not translocated into the Erythrocystis thalli
(L. Goff, personal communication).
Kohatsu (8)
Observations:
1. Within a pit, either a male, female, or tetrasporophyte
plant occurred, not any combination of these. This
was observed in over 100 pits.
2. Six or more germlings were commonly found in
infected pits. Germlings were common in both the
pit of ultimate branches and in the apical pit of i*
2*, and 3* branches.
3. Thalli (ie.)1 mm in length) were found almost
exclusively in the apical pits. Dissection of several
pits infected by thalli indicated that only one or two
branched plants were present.
4. An Erythrocystis thallus in an apical pit may
arrest apical growth. Proliferous growth of lower
branches or accelerated growth of a single lower
branch follows. In the latter case, the branch grows
in the direction which resumes growth along the line
of the central axis.
SION
This study indicates that Erythroc
tis infection
can occur through the fortuitous settling and sticking
of spores to the proximal portion of Laurencia pacifica
trichoblasts. It is not conclusive that this is the
sole mechanism of infection. The concentration of
spores in the experimental culture dishes was far
reater than would be encountered in the field.
Although spore creeping was not observed, artifacts
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of culturing may have prevented it. Excessive mucus
secretion of spores and early trichoblast deterioration
may have been causes of negative results. The trichoblasts
are normally deciduous in maturing Laurencia, however
their uniform breakdown in culture suggests induced
deterioration.
The relationship between Erythrocystis and Laurencia
is a low level parasitism. Erythrocystis is not damaging
to its host and probably photosynthesizes normally.
Yet, it is clear that Erythrocystis is very dependent
on the Laurencia pacifica pits for normal spore germination
and development. Kugrens (1971) pointed out that
spores germinate and develop with polarity, while there
is no ultrastructural polarity in the tetraspore.
The aberrant growth of tetraspores in culture suggests
that the Laurencia pits may correct for a deficient
polarity system in Erythrocystis spores.
The size distribution (fig. 2) of Erythrocystis
on its host may be interpreted in two ways. Thallus
length may be assumed to correlate with age. Thus,
the primary Erythrocystis, which were the longest, were
the oldest. This opens the possibility that the
Laurencia pits become receptive to infection with
age: the first-formed pit is the first to be infected.
The other possibility is that there is apical
dominance due to hormones from the Erythrocystis
and/or the Laurencia. The enhanced growth observed
Kohatsu
(10)
in branches below some infected pits is reminiscent of
vascular plants in which the primary bud is removed.
Such growth resulted in most of the 2*A pits. The
original 2* apical pit was replaced by the apical
pit of a proliferous branch. In support of this
explanation, the X length of Erythrocystis in the
2* pits was significantly greater than that of the
2*A pits.
More work is needed on the factors which select
for complete development of only one or two Erythrocystis
plants, of one type, within a pit. A form of intra¬
specific competition is suggested. Just as pollen
grains "race" to extend their pollen tubes to the
ovule, Erythrocystis germlings may compete in getting
their rhizoids through the pit space, and into the
medulla. It would be worthwhile to carefully examine
the region of rhizoidal penetration through the
Laurencia cortical layer. It may be that only one
rhizoid gets through.
Among the parasitic red algae, Erythrocystis
holds a rather unique place. Unlike many other red
algal parasites, Erythrocystis has a distinct, uniform
plant body in which all reproductive stages are known.
It has a well-developed cortex and subcortical layer
of cells distinct from the host. Many other parasites
are irregular cushions containing a mixture of host and
parasite cells (eg. Harveyella mirabilis, Janczewskia sp.).
Kohatsu (11)
The lack of tissue differentiation in some has promoted
ideas that some of these parasites (eg. Lobocolax,
Gonimophyllum and Gardneriella species) are actually
bacterial or viral galls.
In comparing the two different parasites of
Laurencia, we find two modes of infection, morphology,
and cell type, however it would interesting to
study the gradation between the two. Although Janzweskia
predominates on Laurencia spectabilis and Erythrocystis
predominates on L. pacifica, both are found on L. subopposita
and L. masonii. From L. spectabilis to L. pacifica, there
is a gradation from cartilaginous to pliant. Goff and
Cole (1976) suggested there may be enzymes used by
the parasites to dissolve host cell walls. The possibility
of a gradation of enzyme specificity is suggested here.
The potential of such work would give support for
the theory of an independent plant evolved into a
parasite and provide insight into the phylogenetic
relationships among the Laurencia species.
Kohatsu (12)
FIGURES
Fig. 1 Shows convention of naming location of Erythrocystis
on Laurencia. 1*, 2*, and 3* signify apical
pits. 2*A signifies a pit location other than
apical.
Fig. 2 Shows mean length of Erythrocystis found in
*, 2*, 2*A and 3* pits.
Fig.1 ERYTHROCYSTIS LOCATION
)274
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Br3
Cultimate branch
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LOCATION ON LAURENCIA
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ACKNOWLEDGMENTS
I am grateful to Dr. Isabella Abbott for her invaluable
ideas and expertise, William Magruder for his generosity,
and many helpful words of encouragement, and Dr. Robin
Burnett who made things significant.
BIBLIOGRAPHY
Abbott, I.A. and Hollenberg, G.J. (1976) Marine Algae
of California. Stanford Univ. Press, stanford,
CA, pp /38-7
Feldmann, J. and Feldmann, G. (1958) Recherches sur
quelques Floridees parasites. Rev.Gen.Bot.
65: 49-124.
Goff, L.J. and Cole, K. (1976) The biology of Harveyella
mirabilis (Cryptonemiales, Rhodophyceae). II1.
Spore germination and subsequent development
within the host Odonthalia floccosa (Ceramiales,
Rhodophyceae). Can.J.Bot. 57: 268-280.
Kugrens, P. (1971). Comparative ultrastructure of
vegetative and reproductive structures in parasitic
red algae. Ph.D. Thesis, University of California,
Berkeley.
Kugrens, P. and West, J.A. (1973). The ultrastructure
of an alloparasitic red alga Choreocolax polysiphoniae.
Phycologia, 12: 175-186.
Kylin, H. (1928) In Entwicklungsgeschichtliche Florideenstudien
Lunds Univ. Arsskr., N.F., 20: 94-102.
Setchell, W.A. (1918). Parasitism Among the Red Algae
Proc.Am.Phil.Soc. 57: 155-172.