Gerbino, P.G.
ABSTRACT
Small green algae are commonly observed growing only on the
backs of mid-intertidal limpets. A closer examination was made of
this relationship.
Several different green algae were found growing on the shells
of mid-intertidal limpets. Of these, Percursaria dawsonii (Hollenberg
& Abbott, 1968), and two species of Enteromorpha (Link, 1820) are
morphologically very similar.
The most heavily foliated limpets were found living on vertical
landward faces of wave-exposed rocks between +1 and +4 feet of Mean
Lower Low Water. Fronds of one species of Enteromorpha, labelled
E. sp.2, achieved greater length at sites which were more exposed to
waves than where wave action was less severe.
Enteromorpha sp.» did not survive on limpet shells which were
permanently affixed to mid-tidal rocks. In culture, E. sp., continued
to grow on these empty shells. Additionally, E. sp.» grew in culture
on granite, plastic, wood, Pollicipes plates, Mytilus shell, and free¬
floating in the culture medium.
A procedure for statistically measuring and comparing the cells
of these algae is described and the relationship of one alga to its
hosts is discussed.
Gerbino, P.G.
INTRODUCTION
The presence of green algae growing on the shells of
intertidal molluscs has been observed by many investigat-
ors (Setchell and Gardner, 1920; Smith, 1944; Schlumpberger,
1979). Some algae, most notably Percursaria dawsonii (Hollenberg &
Abbott 1968), are described as virtually host-specific to
some of these molluscs. (Hollenberg and Abbott, 1968).
The nature of this epizoic relationship has never
been described. Recently Schlumpberger (1979) surveyed
the microalgae growing on the tiny mollusc, Tricolia
pulloides (Carpenter, 1865), but no work has been done
regarding the green algae of the larger limpets.
This short project was undertaken to identify the
green algae found on the shells of some mid-tidal limpets
and to investigate some of the parameters governing this
relationship.
MATERIALS AND METHODS
Three transects were taken along lines of sight at each
location. Beginning at the uppermost boundary between sand
or rock and terrestrial vegetation, samples of all
Enteromorpha species and other green algae whose identity
was not readily apparent were taken by hand. If algae
was found on a mollusc shell, the animal was taken intact.
All sampling was done at low tide and lower limit
of transects was determined by water level. This ranged
from -1.5 115 May 1980) to +1.3 (24 May 1980) feet of
Mean Lower Low Water.
Transect width varied from a few feet to several feet
Gerbino, P.G.
depending on terrain and quantities of acceptable samples
found. Samples were marked according to location.
Tide level, weather conditions, and time of day were re¬
corded for each transect.
Algae were identified according to standard taxonomic
procedure with the following exceptions. Because differen¬
tiation among three of the algae was very difficult, more
accurate methods of identification were required. Cell
dimensions were recorded accurately and systematically for
20 cells from each alga in question. Cell length was
always recorded along the axis of the frond and width across
the frond. For each alga, mean cell length and width were
computed and statistics utilized to determine significant
differences among the samples.
The most prolific alga found on the limpets was select-
ed for further investigation. This was a species of
Enteromorpha (Link, 1820) which was given the label,
Enteromorpha species (abbreviated E. sp.2).
Determination offood of limpets was by microscopic
analysis of gut contents performed immediately after
eating was observed.
Kenic cultures of algae were obtained by isolating
individual plants in 10 ml petri dishes. To each was
added 5 ml of sea water filtered at 0.45 um and 1 ml of
Provasoli's nutrient solution modified with 2.40 ugei-:of
NaMoo and 3.73 ug'1" of Cuso. In addition, 0.5 ml Geo.
and 1 ml penicillin solution were added to control diatom
and bacteria proliferation. Cultures were maintained at
15° C. Photoperiod was 10 hours/day of 41 microeinsteins
Gerbino,P.G.
per M-2.1-1 fluorescent light.
In all cases where algae growth was monitored on the
limpet shells, the soft parts of the limpets were removed
first.
Where shells were affixed to granite in the intertidal,
Poxy Putty (Permalite Plastics, Newport Beach, CA) was
used as a strong water-proof adhesive.
RESULT:
Several different green algae were observed on mid-
tidal limpets. They were identified as Rhizoclonium
implexum (Kutz, 1845), two species of Ulva (Linnaeus, 1753),
Percursaria dawsonii, Enteromorpha intestinalis ((L.)Link,
1820), and two undescribed species of Enteromorpha
(cf. E. flexuosa ((Roth)J. Agardh, 1883)).
Measurements of cell dimensions from Percursaria
dawsonii, Enteromorpha sp.j, and E. sp.2 are tabulated in
figure 1. Statistical analysis of mean cell lengths using
pair-wise associations and the Student's t-test confirmed
the presence of a significant difference at the .05 level
between E. sp., and both E. sp., and P. dawsonii cell lengths.
Analysis of cell widths failed to confirm a significant
difference between any two algae species.
These data are plotted graphically in figures 2 and 3,
and indicate that cell size should only be one of several
criteria used to discriminate among these algae.
The most abundant algae found growing exclusively on
limpet backs were (in descending order) Enteromorpha sp.
Percursaria dawsonii, and E. sp.. For all these algae
the most heavily foliated limpets were found on the landward
Gerbino, P.G.
side of wave-exposed rocks.
In 11 transects taken at four Monterey Peninsula sites:
Mussel Point, Point Pinos, Pescadero Point, and Stillwater
Cove; E. sp.» was found growing only on the shells of
mid-tidal limpets and occasionally in the furrows between
plates of Pollicipes polymerus (Sowerby, 1833) or among
the ridges of Tetraclita squamosa rubescens (Darwin, 1854)
Enteromorpha sp., was never observed growing on rocks,
other algae, invertebrates other than previously specified,
or free-floating in tide pools.
As a rough estimate of differences among the growing
conditions at each site, maximum frond length of E. sp.
was measured by haphazardly selecting 10 long fronds from
limpets at each site and recording the longest frond in
each group (Figure 4).
Limpets found harboring E. sp.» include Collisella
limatula (Carpenter, 1864); C. scabra (Gould, 1846);
C. digitalis (Rathke, 1833); C. pelta (Rathke, 1833);
Notoacmea scutum (Rathke, 1833); and Lottia gigantia
(Sowerby, 1834). All these limpets were taken from the
mid-intertidal zone extending from +1 to +4 feet of
Mean Lower Low Water.
In culture, cut E. sp., fronds attached and grew on
granite, plastic, wood, and sterilized limpet shell frag-
ments, and grew free-floating in the culture medium.
The spores of E. sp., settled and grew on sterilized
ytilus, Tetraclita, and limpet shell fragments, as well as
granite, and plastic petri dishes (Figure 5). Qualitatively,
Gerbino, P.G.
it was noted that individual plants which settled and
grew on granite, Mytilus shell, wood and plastic were
easily pulled free, whereas those attaching to limpet shell
were more strongly attached.
It was frequently observed that when two or more
foliated limpets, regardless of species, were placed in a
container 12'x16"x6" or smaller, some limpets would crawl
upon the backs of other limpets. Analysis of gut contents
of the climbing limpets confirmed the presence of E. sp..
as a food consumed by limpets.
In field tests, 20 limpets were removed from their
intertidal rocks, the soft body parts removed, and the
shells with attached algae permanently affixed to the same
mid-tidal granite rock from which they had been taken.
All that remained after three days were algal patches of
cells and a few bleached E. sp., bushes.
In tests to determine the necessity of a live limpet
for algal growth, 30 limpet shells with E. sp.» and
Percursaria growth were maintained in culture as previous-
ly and in a sea water table with a continuous unfiltered flow,
they not only survived, but grew larger. The sole excep¬
tion was a series of cultures in which the algae began to
die after the presence of a fungus was noted.
SUMMARY
Several different green algae were found growing on the
shells of mid-intertidal limpets. Some of these are very
similar morphologically.
Gerbino,P.G.
The most heavily foliated limpets were found living
on vertical landward faces of wave-exposed rocks.
Enteromorpha sp., fronds achieved their greatest length
at sites which were most exposed to waves.
Enteromorpha sp., will continue to grow in culture after
the limpet has been removed. In addition, E. sp., grew
in culture on several smooth substrates and free-floating
in the culture medium. In the field, E. sp., did not survive
on limpet shells from which the soft body parts had been
removed.
DISCUSSION
Enteromorpha sp. appears to require a highly special-
ized micro-habitat. Doty and Newhouse (1954) assert that
the three major boundries to marine algae life are
suitable substrate, temperature and salinity. Additionally,
Harlin (1978) notes Enteromorpha intestinalis, in partic-
ular, requires a diet with high levels of fixed nitrogen.
Meeting the need for large amounts of fixed nitrogen could
be done by growing near sewage outfall areas which is well
documented for E. intestinalis (Boalch, 1957) or by growing
in areas which receive a large amount of wash from waves,
This only partially explains the mid-tidal habitat of
E. sp. 2.
The species of molluscs that Enteromorpha sp., occurred
on were found on the landward side of wave-swept rocks.
This seems consistant with the observation that many, if
not most, organisms occurring in the intertidal must protect
Gerbino, P.G.
themselves from the brunt of the crashing waves. Thus,
occurrence of E. sp.» only on parts of the rock not in
direct confrontation with oncoming waves may indicate
that any algae germinating on more exposed surfaces is
rapidly eroded.
The pitted shell tops of the mid-tidal limpets seem
necessary to provide the proper surface for E. sp..
since the smooth granite rock of the Monterey Peninsula
was found to be an unsuitable substrate for firm attachment.
The eroded top layers of limpet shells, termed the intrita¬
calx by D'Attilio and Radwin (1971), would be acting in a
similar fashion to specialized substrates such as various-
ly textured rocks associated with Enteromorpha species in
the Netherlands (Nienhuis, 1969) and at the North Sylt
Wadden Sea, Germany (Gertraud, 1976).
Finally, in wave-protected, but highly washed areas
where limpets are found, E. sp.» most likely occurs nowhere
but on limpet backs because of predation. This conclusion
is in agreement with Southward (1956) who found that
limpets eat virtually all the Enteromorpha around them
except that which is on their backs. Any of thesegalgae
that manages to develop away from the limpets and escape
their predation must still escape predation by foraging
Tegula and many other herbivorous mid-tidal invertebrates.
Several problems were encountered in the course of
this research. Differentiation among the two undescribed
Enteromorpha species and Percursaria dawsonii was a slow
tedious procedure. Because of the similarities in cell size
Gerbino, P.G.
and plant morphology among these species which all occur
on the same limpets, it was not always appareat which was being
observed. Compounding this problem was the lack in the
literature of described species of Enteromorpha which
corresponded to the algae being collected (Setchell and
Gardner, 1920; Smith, 1944; Abbott and Hollenberg, 1976).
The statistical analyses which were performed on the
cell dimensions of these algae aided only partially in
identification within the group. Larger sample sizes of cell
measurements are needed from these species to determine
whether they are, in fact, new or different species of
Enteromorpha.
As with any organism of such small stature, identifica-
tion in the field was difficult. Especially within the
infinitely irregular terrain of the rocky intertidal, any
transect which fails to uncover such a tiny alga must be
viewed with cautious skepticism.
Finally, achieving sporulation in culture was success¬
ful only once. More often, fronds matured, but rather than
releasing their spores, they retained them effecting in situ
germination and gross polymorphism of the fronds. Possible
reasons for this include undetected variations in temper-
ature, amount of light, or fixed nitrogen levels in the
culture environment (Yarich, 1976). Since the incidence
of sporulation in culture and a further observation of
sporulation in the field coincided closely with a new moon
and a full moon, respectively, the possibility of lunar
periodicity as described by Smith (1947) for Ulva exists
and merits further investigation.
Gerbino, P.G.
ACKNOWLEDGMEN
I wish to express my gratitude to Isabella A. Abbott
for the guidance she has given me and the patience she
has had with me. I would also like to thank Kathy French,
Celia Smith, and Bill Magruder for their continued
assistance. Finally, I wish to thank Chris Agel for her
help with putting this paper together.
Gerbino, P.G.
10
LITERATURE CITED
ABBOTT, ISABELLA A. & GEORGE J. HOLLENBERG
1976. Marine Algae of California. Stanford Univer-
sity Press, Stanford, CA 827pp.
BOALCH, G.T.
1957. Marine algal zonation and substratum in Beer
Bay South-East-Devon. Jour. Mar. Biol. Assn. U.K.
36,519-528
D'ATTILIO, ANTHONY & G.E. RODWIN
1971. The intritacalx, an undescribed shell layer in
mollusks. The Veliger 139(4): 344-347
DOTY, MAXWELL S. & JAN NEWHOUSE
1954. The distribution of marine algae into estua-
rine waters. Am. Jour. Botony 41(6): 508-515
GERTRAUD, LUTHER
1976. Fouling studies on natural-stone substrate in
the tidal zone of the North Sylt Wadder Sea. Algar
Helgol. Wiss. Meeresunters. 28(3/4): 318-51
HARLIN, MARYLIN M.
1978. Nitrogen uptake by Enteromorpha spp. (Chloro-
phyta): application to aquaculture systems. Aquacul-
ture 15(4): 373-376
HOLLENBERG, GEORGE" J. & ISABELLA A. ABBOTT
1968. New species of marine algae from California.
Can. Jour. Bot. 46: 1235-1251
NIENHUIS, P.H.
1969. The significance of the substratum for inter-
Gerbino, P.G.
tidal algal growth on the artificial rocky shore of
the Netherlands. Int. Rev. ges. Hydrobiol 54,207-
215
SCHLUMPBERGER, JAY M.
1979. A survey of the microalgae living on the shell
of the small prosobranch mollusc, Tricolia Pulloides
(Carpenter, 1865). Unpublished MS. on file at Hop-
kins Marine Station Library, Pacific Grove, CA 17 pp.
SETCHELL, WILLIAM A. & N.L. GARDNER
1920. The marine algae of the pacific coast.
Uni¬
of California Publications in Botony, San Francisco
8(2): 139-374
SMITH, GILBERT M.
1944. Marine algae of the Monterey Peninsula Calif-
ornia. Stanford University Press, Stanford, CA
122 pp.
SMITH, GILBERT M.
1947. On the reproduction of some pacific coast
species of Ulva. Am. Jour. Botany 31: 80-87
YARISH, C.
1976. Polymorphism of selected marine Chaetophorceae
(Chlorophyta). Br. Phycol. Jour. 11: 29-38
Gerbino, P.G.
12
EXPLANATION OF FIGURES
Table of cell measurements from 20 cells of
Figure 1:
Enteromorpha sp., E. sp.», and Percursaria
dawsonii
Scattergrams illustrating the degree of
Figure 2:
correlation between lengths and widths of cells
measured in Figure 1
Figure 3:
Comparison of mean cell sizes, standard devia¬
tions, and ranges for algal cells tabulated
in Figure 1
Figure 4:
Comparitive lengths of Enteromorpha sp., fronds
obtained at four Monterey Peninsula sites
Table of results of experiments in which E. sp.
Figure 5:
was cultured on various substrates and of
observed, naturally occurring E. sp., habitats
in the field
Gerbino,
CELL SIZES FROM THREE MORPHOLOGICALLY
SIMILAR EPIZOIC GREEN ALGAE
PERCURSARIA DAWSONI!
E. SP.2
CELL
E. SP.
w ()
WIDTH
LENGTH
6
X-8.2u
X=6.7u x=6.7u x=6.9u x=6.5u x=7.4u
5=2.14
s=1.59 s=2.21 s=0.88 s=1.43 s=0.99
t-test, .05 level
E. sp. length significantly different than
E. sp.2 and P. dawsonii.
All other comparisons: no significant differences.
Figure 1
Gerbino,P.G
6
ai
CELL LENGTH VS. CELL WIDTH
FOR THREE EPIZOIC GREEN ALGAE
ee.
*.
CELL WIDTH (U)
Figure 2
10
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Figure 4

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38
Gerbino, P.G.
TABLE OF RESULTS OF CULTURING ON
SUBSTRATES VS. FIELD OBSERVATIONS
SUBSTRATE
E. SP.2 FOUND
SETTLING
IN FIELD
ACHIEVED IN CULTURE
LIMPET SHELL
POLLICIPES
+
SHELL
TEGULA SHELL
MYTILUS SHELL
GRANITE
GLASS
NA
PLASTIC
NA
woop
FREE FLOATING
Figure 5