the habitat of leucothrix mucor, a widespread marine microorganism

NOTES
AND
persal of some plankton ( Gislen 1948))
they would most likely have been introduced into the Great Lakes and other
lakes before now. Now that E. affinis is
established in three lakes, it should eventually move into all of the Great Lakes
and other nearby watersheds, resulting in
a welcome addition to the monotonous
entomostracan fauna of North America’s
freshwater plankton.
DANIEL J.
E. G.
JERMOLAJEV
FABER
(r-&e
J$. G. KOSSIAKINA)
Ontario Department
South Baymouth,
Ontario.
of Lands and Forests,
REFERENCES
1959.
D.
V.,
AND
D. CLAYTON.
Plankton
in Lake
Ontario.
Physics
Res.
Note
No.
1, 1959.
Div.
Res., Ontario
Dept. Lands and Forests, Maple,
Ontario.
( Mimeo. )
DEEVEY,
G. B. 1948. The zooplankton
of TisANDERSON,
THE HABITAT
COMMENT
303
bury Great Pond.
Bull. Bingham Oceanog.
Collection,
12: l-44.
-.
1960. The zooplankton
of the surface
waters of the Delaware
Bay region.
Bull.
Bingham Oceanog. Collection,
17 : 5-53.
ENGEL,
R. A. 1962. Eurytemora
affinis
a calanoid copepod new to Lake Erie.
Ohio J.
Sci., 62: 252.
GISL~N,
T.
1948. Aerial plankton
and its conditions of life.
Biol. Rev. Cambridge
Phil.
Sot., 23: 109L126.
R.
1931.
British
fresh-water
Cope238 p.
poda, v. 1. Ray Society, London.
JEFFRIES,
H. P. 1962. Salinity-snace
distribution of the estuarine copepod genus EUTZJtemora.
Intern.
Rev. ges. Hydrobiol.,
47:
291-300.
RYLOV, W. M.
1930.
The fresh-water
calanoids of the USSR.
Zn Keys to the determination
of fresh-water
organisms
[in RusSian]. Leningrad,
1930.
THIENJZMANN,
A. 1950. Verbreitungsgeschichte
der Siisswassertierwelt
Europas.
Binnengewasser, 18: 809 p.
WILSON,
G. B.
1932.
The copepods
of the
Woods Hole region; Mass. U.S. Natl. Museum, Bull., 158: l-635.
FVILSON,
M. S. 1953. New Alaskan records of
Eurytemora
( Crustacea,
Copepoda ) . Pacific Sci., 7: 504-512.
-.
1959.
Free living
Copepoda:
Calanoida.
Zn Freshwater
biology, 2nd ed. Wiley, New York, N.Y. 1248 p.
GURNEY,
OF LEUCOTHHX MUCOR, A WIDESPREAD
MARINE MICROORGANISM
Leucothrix mucor (Oersted) is a marine
microorganism
that has been studied
mainly in the laboratory
(Harold
and
Starrier 1955; Pringsheim
1957; Lewin
1959; Brock 1964). The present work
points out that this organism is widespread in the marine environment
and
should be taken into account in any study
of the role of heterotrophic
microorganisms.
L. mucor is excellent for autecological
investigation because it is large and has
characteristic morphological
features that
can be recognized in natural collections;
it grows as an epiphyte on marine algae,
and usually its filaments project perpendicularly from the surface of algal fronds,
permitting
easy microscopic
study; it
characteristic
morphogenetic
undergoes
changes that are probably of ecological
significance; and it is widespread in marine environments, and a study of its ecology may be expected to have some relevance to broader problems of marine
microbiology.
It is often the most common marine microorganism when viewed
microscopically,
but it rarely appears on
agar plate cultures unless special precautions are taken.
The microscopic
identification
of an
epiphytic filament like L. mucor, although
fairly certain, is not unequivocal.
In certain cases, the microscopic identification
was checked by cultural isolation, but
because of the difficulty
of its routine
isolation, this could not be done for all
samples. However, after extensive microscopic examination of pure cultures of L.
mucor in many stages of nutritional
adequacy and in many morphological
condi-
304
NOTES
AND
tions, I feel certain that I would not make
a false positive identification
of L. mucor,
although I might not recognize a particularly atypical form as L. mucor. Thus, a
microscopic survey would lead to underestimates of the occurrence of L. mucor,
rather than overestimates.
A variety of macroscopic algae was collected. When it became clear that L.
mucor occurred most extensively on red
and filamentous
green algae, collections
were concentrated on this group, but attempts were made to take samples of all
species growing together in a given area.
In many cases the algae were identified
only to family or genus, but the species of
some of the red algae were kindly identified by Dr. Richard Norris or Dr. J. T.
Conover. Enrichment
cultures were set
up following Harold and Stanier ( 1955).
Pure cultures were obtained following the
procedure of these authors and of Pringsheim ( 1957). Because gram-negative motile bacteria often spread across the agar
plates and crowded out the L. mucor colonies, the plates were examined under
125~ magnification
12-16 hr after the
initial inoculation.
The characteristic
L.
mucor colonies (Harold and Stanier 1955)
could frequently be detected at that time
and were picked up using fine sterile insect pins and transferred to fresh plates.
A synthetic medium was used containing:
NaCl, 11.75 g; MgCl, .6H20,
5.35 g;
Na$Oh, 2.0 g; CaC12 .2H20, 0.75 g; KCl,
0.35 g; Tris-hydroxymethyl
amino methane, 0.5 g; Na2HP04, 0.05 g; monosodium
glutamate, 1.0 g; agar, 15 g; deionized
water, 1,000 ml, pH 7.6. Temperature of
incubation was 25C.
Routine examination of seaweeds at Friday Harbor, Washington,
revealed that
many of these organisms had epiphytes
that greatly resembled L. mucor filaments.
Although earlier workers had isolated their
cultures in association with seaweeds, it
was felt that it was essential to isolate
pure cultures that were known to have
been derived directly from presumptive
Leucothrix filaments seen in natural material. This was accomplished by taking
COMMENT
red algal fronds containing
presumed
Leucothrix and washing them extensively
in sterile synthetic medium (containing
basal salts) to remove any casually associated bacteria. The washed fronds were
then laid directly on the surface of agar
plates containing basal salts with 0.05%
monosodium glutamate and 0.05% sodium
phosphate.
Immediately
after
inoculation,
the
plates were examined microscopically, and
the presumptive
Leucothrix
filaments
could easily be seen attached to the algal
The plates were incubated
at
fronds.
room temperature and examined at intervals. In some cases, motile gram-negative
rod-shaped bacteria, which are quantitatively insignificant
on the algal filaments,
grew and moved along the moisture channel that formed where algal filament and
agar met and crowded out the L. mucot
colonies.
Such motile
organisms have
some selective advantage over Leucothrix
on agar plates because of their motility
and rapid unicellular
growth. A Leucothrix filament, unable to fragment, forms
a slowly growing colony. The low concentration of glutamate used in the medium helped to retard the growth of
motile bacteria.
In some cases where
contamination
was avoided, the Leucothrix filaments grew and formed characteristic whorl-patterned
colonies along the
edges of the algal frond. Such colonies
were transferred to fresh medium and
subsequently maintained in pure culture,
where they resembled in all respects the
L. mucor cultures isolated by other workers. Thus, by microscopic control of the
isolation process, it has been possible to
show unequivocally
that filaments seen in
natural material are L. mucor.
It was felt that an essential part of the
experimental definition
of the L. mucor
habitat was the establishment of its epiphytic growth in laboratory cultures. In a
sense, such an establishment would meet
the requirements of Koch’s postulates. Axenic cultures of four marine algae were
obtained from Dr. Luigi Provasoli. These
were the red algae Antithamnion
sarni-
ense, Rhodoclmrton sp., Rangia fusco-JJ~Upurea, and the brown alga Splucelaria sp.
The A. .sarniense was grown in medium
ASP, at IX,
the B. fusco-purpurea
in
ASl’&JTA
at 15C, and the Sphncelaria
and Rhodlochorton in ASPlrNTA
at 2OC.
These rncdin are described by Provasoli
(1963). All were grown at about 1,000
lux illumination in an alternating cycle of
14 hr light and 10 hr dark. The cultures
were grown in 16 mm screw-capped tubes
containing 1-2 ml of medium, and the
tubrs wcrc slowly rotated on a tissue culture tube rotator.
Media were changed
every week or two. Samples of the algae
wxc inoculated with pure culture
of L.
muc”r which had bcon grown in the medium described above so that a high proportion of gonidia (Harold
and Stanier
1955) were prrscnt. Onr small loopful of
this suspension was then used t” inoculate
l-2 ml of cnltnre medium containing a
few mm11 algal fronds, zmd the t~nbrs wcw
placed back in the light. Controls containing the ASPc and ASPIs media without
algae present were also inoculated.
When axonic cultures of seaweeds were
inoculated
with gonidial suspensions of
L. ~UC”T (about IO” gonidia/ml),
rapid
nttachmcnt to the algae occurred, so that
after two days of incubation extensive epiphytic growth had taken place on A. sarnienxz, B. fusco-pwpurea,and Sphacelaria
sp. Attachment did not occur with Rhorlochorton sp., hut the L. ~UCOT filaments
grew close to the algal filaments and were
seen wrapped around them in profusion.
Conceivably, the surface of Rhodochorton
sp. is not suitable for attachment by L.
1lzuc0r.
Fig. 1 shows a photomicrograph
of L.
nzucor growing epiphytically
on a pure
culture of Sphacclarin sp Fig. 2 shows for
comparison a photomicrogvaph of L. nwco~
growing on a frond of the leafy red alga
CaZZophyZZis hacnoplzylln
taken dircctlp
from nature. There is il close resemblance
in the two photographs.
A purr culture
of L. ~UCIJT was is&&cd directly from a
portion of the algal frond ndjacerrt to that
shown in Fig. 2.
L. mucw does not grow alone in either
of the media in which alga” were cultured. In addition, if sterile, washed cotton fibers are added to these media, L.
~UC”T will not grow attachrd to their
surface, although it will grow attached to
cotton fibers when cultured in the glutnmate-Tris medium described previously.
It can he concluded that the alga not onl>provides a substratum for the attachment
“t the bacterium but also nutrients for the
growth of the bacterium.
The algal cultures do not seem to be harmed in any
way by the attachment and growth of I,.
mucor and have been maintained through
succrssixvz transfrrs
made over several
months. Thus, by “hservation and expcrimcnt, it has been shown that at least one
natornl habitat of L. mucor is the surfaw
of scawccds, where it grows as a firmly
attached epjphyte.
In nature, n wide variety of filamrntous
heavily
covered with I,. mumr filaments.
Pure cultures
of L. mucor grow well in
liquid
medium
only when rapidly
shaken.
and this is consistent with the requirement
of water movement
for good growth in
nature.
It is not clear whether
the requirrment
of water movement is for aeration or for some other purpose.
Geographically,
L. nwcor is widely distributcd
in temperate
waters.
I ha>e iholatcd
pure cultures
from seaweeds callccted in Puget Sound, Washington,
Loug
Island
Sound,
Connecticut,
Karragansrtt
Bay, Rhode Island, and Cape Rcykjanes
and Faxafloi
Fiord, Iceland.
Harold and
Stan&
(1955) ‘isolated it from Cnlitomia
waters,
Molisch
[ 1912) and Plingsheiln
(1957) observed it in Adriatic
waters, and
Lcwin
(1959) isolated clones from the rep
gion of Woods Hole, hlassnchixetts.
Other
regions from which it has hern reported
(Berger
and Bringmann
1953) are the
Arctic
Ocean (Murmansk,
USSR),
Baltic
Sea (Sweden,
Schleswig-llolstein,
Latvia!,
North
Sea (Helgoland),
Meditrrrancan
(Gulf
of Kaples),
and Rlack Sea (Bay
of Sevastopol).
Berger
and Rringmann
(1953)
consider
L. mucm
to he 3 characteristic
organism
of polluted
mariw
environments
and state (p, 328), “Fiir die
Abwasserbiologie
der Mecrc stcllt sic ein
.md Ic:af>~ red algae has been fowd
tu be
diagnostisch
vbllig sich dcckcndes
Gegencolonized
with L. rnm~~. The r~son
that
stiick
zu
SpAaemtilus
ntltans
da.”
red algae are so readily colonized
may he
To date, all isolates
in pure culture
the nature of the algal sarfaco.
Red algae
have been remarkably
similar
in physiodo not produce large amounts of mucus or
logical and morphogenctic
behavior.
The!
slime, and it would be expected that their
all have similar
temperature
optima and
surfaces would
provide
a reasonable
denutritional
requirements,
and six strains
gree of stability.
Because L. rnuc~r will
isolated
from both Pacific
and Atlantic
attach to glass or cutton, it does not seem
waters (including
one strain each isolated
likely that any specific
surface propertics
by 1,ewin 1959 and Harold
and Stania
arc required
tor attachment.
Further,
the
1955) have identical
DNA base composired algx: comprise many filamentous
spctions (ht. Mandel,
personal
communicaties, and filaments
provide
a greater smThus, the species as defined morn
face area for attachment
than would
a tion).
phologically
comprises
a homogeneous
similar volume of leafy material.
group of strains physiologically
and bioWhere the water is still or slow moving,
chemically.
Id. mucw
is rare, hut it occurs at exL. muuw is far more common in marinr
tremely high densities on red algae growenvironments
than
would
he apparent
ing in rapidly moving water.
Thus, rocky
from quantitative
hactcrial
counts on agal
areas with
much
wave
action
or tidal
plates.
This
discrepancy
may
current always provide
senwecds that arc
cxisl
NOTES
AND
because L. mucor (because of its filamentous growth habit) may be at a competitive disadvantage on agar plates; although
because of its ability to attach to surfaces,
it may be at a competitive advantage in
natural environments having much water
flow. Now that the precise microenvironment of L. mucor has been defined, it
is possible to examine its physiological
ecology.
The collaboration
of M. Louise Brock
was a great benefit to this work. The
work at San Juan Island was done during
two summers as a visiting investigator at
the Friday Harbor Laboratories, University of Washington.
The work in Iceland
was supported by the Surtsey-Iceland Research Committee.
THOMAS
D.
BROCK
Department of Bacteriology,
Indiana University,
Bloomington
47405.
307
COMMENT
REFERENCES
H., AND G. BRINGMANN.
1953. Die
Scheidenstruktur
Abwasserbakteriums
des
Sphaerotilus
nutuns und des Eisenbakteriums
Leptothrix
im elektronmikroskopischen
Bilde
und ihre
Bedeutung
fur
die Systematik
dieser Gattungen.
Zentr. Bakteriol.,
Parasitenk., Abt. II, 107: 318-334.
BROCK, T. D.
1964. Knots in Leucothrix
muCOT. Science, 144: 870-872.
HAROLD,
R., AND R. Y. STANIER.
1955. The
genera Leucothtix
and Thiothrix.
Bacterial.
Rev., 19: 49-58.
LEWIN,
R. A. 1959. Leucothrix
mucor.
Biol.
Bull., 117: 418.
MOLISCH,
H.
1912.
Neue farblose
Schwefelbakterien.
Zentr.
Bakteriol.,
Parasitenk . ,
Abt. II, 33: 60-61.
PRINGSHEIM,
E. G.
1957.
Observations
on
Leucothrix
mucor and Leucothrix
cohaerens
nov. sp. Bacterial. Rev., 21: 69-81.
PROVASOLI,
L.
1963.
Growing
marine
seaweeds, p. 9-17.
In Proc. 4th Intern. Seaweed Symp., Biarritz,
France,
Sept. 1961.
Pergamon, New York, N.Y.
BERGER,
RELATIONSHIP BETWEEN CARBON CONTENT, CELL VOLUME,
AND
AREA IN PHYTOPLANKTON~
Because of the difficulty
in assessing
the carbon content of living phytoplankton in the sea, due to the presence of
detritus and the variability
of phytoplankton carbon : chlorophyll
ratios, we have
sought a relationship between cell carbon
and cell volume which could be used to
estimate the phytoplankton
carbon in seawater from preserved phytoplankton
samples. Previous investigators have noted a
proportionality
between organic matter or
ash-free dry weight and cell volume (Riley
1941; Wright 1959; Cushing 1958; Strickland 1960). But Lund ( 1964) noted that
the ratio ash-free dry weight : cell volume
is not constant but varies about fivefold
in magnitude. We observed similar variation in the carbon : cell volume ratio and
l Supported
by U.S. Atomic Energy Commission Contract No. AT( ll-l)-34,
Project 108, and
by a National
Science Foundation
Fellowship
to
M. M. Mullin.
found that it varies predictably with cell
volume.
The data reported were gathered independently by Mullin at Woods Hole and
Sloan and Eppley at La Jolla without
collaboration until after the measurements
were made.
The following organisms were grown in
axenic cultures, unless otherwise
indicated:
tertiolecta Butcher
Chlorophyceae : Dundiella
Coccolithus
huxleyi
( Lohm. )
Chrysophyceae:
Kamptner,
Syrucosphuera
(Hymenomonus)
elonguta Droop
Bacillariophyceae
: Skeletonema
costatum
(Greville ) Cleve,
CycZoteZZa nuruz: Hustedt,
Thalassiosira
rotula Meunier
(unialgal),
T.
fluviatilis
Hustedt,
Striate&
unipunctutu
Agardh,
Rhizosolenia
setigeru Brightwell,
Ditylum
brightwellii
(West)
Grunow,
Coscinodiscus
sp. ( unialgal),
C. concinnus
W. Smith (unialgal)
Dinophyceae : Peridinium
trochoideum
( Stein )
Lemm., Gonyuulax
po2yedra Stein (unialgal).