leucothrix mucor infestation of benthic crustacea, fish eggs, and

LEUCOTHRIX
CRUSTACEA,
MUCOR INFESTATION
OF BENTHIC
FISH EGGS, AND TROPICAL
ALGAE1
Paul W. Johnson,2 John McN. Sieburth,
Department
of Bacteriology
and Biophysics, and Graduate
University
of Rhode Island, Kingston
School of Oceanography,
02881
Akella Sastry,
Graduate
School
of Oceanography,
University
of Rhode
Island,
Kingston
02881
C. R. Amold,
Narragansett
Marinc
Game Fish Laboratory,
Narragansett,
Rhode
Island
02882
and
Maxwell S. Doty
Department
of Botany,
University
of Hawaii,
HonoluIu
96822
ABSTRACT
Appendages and eggs of benthic marine crustacea arc often populated with the characteristic filaments of the bacterium
Leucothrix
mucor. Planktonic
crustacea and fish eggs
fret of L. mz~co~ become infested when held in aquaria in the absence of antibiotics.
Isolates from these organisms are grossly indistinguishable
from isolates from algae. Although
L. mucor is not a pathogen, it may be involved in high mortalities by causing pelagic eggs
to sink below the surface and by interfering
with the filtering
apparatus of larval forms.
Antibiotics
that prevent development
of L. mucor ( and other microorganisms ) reduce the
mortality
in developing
eggs and larvae. Direct microscopic
examination
of 48 seaweeds
from the lagoon at Majuro Atoll in the Marsha11 Islands for L. rntzor indicated its presence
on 81% of the samples. When 18 randomly chosen samples were put in enrichment culture,
detection increased to lOO%, These observations are at variance with a previous report that
L. mucor is either absent or rare in warm waters.
INTRODUCTION
Leucotlarix
~UCOT ( Oersted)
( Harold
and Stanier 1955) has been isolated from
enrichment cultures of decomposing algae
( IIaroId
and Stanier 1955; Pringshcim
1957), from the thalli of fresh algae (Iewin
1959) and from seawater (Anderson and
Heffernan 1965). The habitat of this organism is generally considered to be macroscopic algae, where it grows as an epiphyte
( Brock 1966). Although Leucothrix
filaments have been seen by other observers
on marine animals,3 apparently only An1 This work was supported in part by the Smithsonian Institution
in regard to logistics and by
grants from the Biological
Oceanography
(BO
programs of the
18000 ) and Sea Grant (GH44)
National
Science Foundation
to J.M.S.
2 Present address: Department
of Microbiology,
University
of Massachusetts, Amherst
01002.
s R. A. Lewin has observed Leucothrix-like
filaments on the antennae of the hermit crabs at La
Jolla, California,
and S. W. Watson saw a heavy
LIMNOLOGY
AND
OCEANOGRAPHY
962
derson and Conroy (1968) have reported
Leucothrix-like
bacteria colonizing the eggs
and plcopods of a benthic crustacean; this
organism was not isolated and compared
with L. mucor. Kelly and Brock (1969)
stated that this most obvious and usually
ubiquitous microorganism is rare or absent
on seaweeds from warmer waters of the
Gulf oE Mexico, Atlantic Ocean, and Florida Bay, but they gave no data and advanced no explanation.
IIere WC report field observations and
laboratory isolations showing that L. mucar is widespread on benthic crustacea,
dcvcloping eggs and larvae, and detritus.
It may constitute a nuisance to those who
maintain
and rear such organisms in
aquari a. A 3-week visit to Majuro Atoll
(east cncl, 7” 05’ N, 171” 23’ E) in the Marinfestation
on the gills of horseshoe crabs undergoing a mass mortality
in the tanks at the Marine
Biological Laboratory,
Woods Hole, Massachusetts.
NOVEMBER
1971,
V. X6(6)
LEUCO'L'IIRIX
MUCOR
shall Islands afforded an opportunity
to
determine the relative abundance of L.
mucor in this warm-water lagoon. Grateful
acknowledgment
is made to Alan Campbell and I. E. Wallen who arranged the
field trip; to Yuri I. Sorokin with whom the
fieldwork was conducted; to John Iaman,
the district medical offcer who provided a
small boat and laboratory supplies; to the
many Marshallesc and Americans who rcscued us from a logistic nightmare; and to
T. J. Smayda for the live Sargasso Sea
specimens.
OCCU~UXENCE
ON
ANIMALS
As a problem in undergraduate research
in bacteriology, one of us (P.W.J.) enriched
an d isolated a number of strains of L.
mucor from the red algae Polysiphonin
lanosa and Chondrus crispus. Another of
us (J.M.S. ), while preparing a lecture on
the microbial flora and diseases of marine
animals, recognized that certain Leptothrix-like
and chainlike microbial
forms
(Dannevig
1919; Oppenheimer
1955))
which have been observed to cover the
surface of cod eggs and to cause them to
sink and die, could be Leucothrix.
Before
using an algal isolate of L. mucor in an
attempt to infect eggs removed from a
gravid rock crab ( Cancer irroratus ) , we
examined the eggs microscopically
to determine if the commonly encountered “fungal mycelium” (Anderson and Conroy 1968;
Wolf 1958) was present. Conspicuous fungallike growth between the eggs turned
out, on closer examination, to be L. mucor
(see
Fig, 1A) with its 2-p-wide long filamcnts terminating in gonidia. Although it
has been stated that “fungus infection is
not apt to be confused with any other
condition” (Wolf 1958, p. 1)) it is undcrstandable that zoologists and even microbiologists could interpret the long filaments
of L. mucor as fungal mycelium, especially
at low magnifications.
Lewin (1959) used
direct streaking to show that the organisms
are present in substantial numbers in the
original material. However, only cnrichmcnt culture ( Harold and Starrier 1955)
INFESTATION
963
pcrmittcd the growth of isolated colonies
on OZR agar ( Sieburth 1967).
Both the crab egg and algal isolates
formed typical fingerprint colonies on agar,
which arc seen at 100X as the single filament of a young colony lengthens and coils
upon itself to form a fingerprintlike
swirl.
Isolates from both sources in broth culture
formed filaments with terminal gonidia,
which became motile and aggregated to
form sessilc rosettes, giving rise to filamcnts in small tufts. This indicated that
the crab isolates wcrc L. mucor and that
marine animals as well as algae can serve
as a substrate.
In an attempt to substantiate our suspicion that the filamentous
organism observed on fish eggs (Danncvig 1919; Oppenhcimcr 1955) is L. mucor, we made a
series of observations on eggs from spawning to hatching. Newly spawned eggs of
the cod Gadus morhua and winter flounder
Pseudopleuronectes americanus were free
of bacteria. ‘Within 2 to 3 days of incubation, in either the running seawater of the
spawning tank or in the static water of
aquaria, filaments oE L. mucor became apparent. Heavily infested eggs upon death
bccamc rapidly overgrown with nonfilamentous bacteria. Typical L. mucor isolates were obtained from the P. americanus
eggs. Suspended and scdimentcd detritus
in the aquaria was overgrown with L. mucar filaments.
Net catches of copepods,
predominantly
Acartia clausi, which were
added to the rearing tanks as food for developing larvae, also became populated
with L. mucor filaments (Fig. 1D).
The presence of L. mucor on the copepods in the aquaria, but not on fresh plankton, indicated that the organisms were
being infected by the seawater system,
Water samples from the running seawater
system were enriched by a dialysis sac
(% volume) containing OZR broth (Sieburth 1967) which was aerated. Leucothrix
mucor filaments grew on the external surface of the sac, showing the presence of
this organism in the running seawater system. A possible source of this L. mucor
arc. 1.
specimrns
Phase contrast photomicrographs
showing I.iwolhril~U,COI filaments on natnrally infcctcd
(filament
diametm:
2 g). Sp~cirnrns of the rock <I-ah Cancer irromtus:
t-egg
from egg
mass showing typical filaments; B-the eye of a ya,m~ larva showing atypical short straight nonse~i.
tate filments,
note the bulbous tip on several filaments;
C&typical
filaments on the eye of an older
larva. D-The
copepod Acartia clausi; E-the
setae of the pleopods of a mature grass shrimp Palaemmete~ pugio; F-heavy
infestation of pleopod of a green crab Carcinus maem.
may be the epiphytic
flora from seaweeds
near the intake.
A fresh net tow of zooplankton
dominated
by the third naupliar
stage of the copepod Pseudocalanusminutus was divided into two portions of sterile
seawater, one maintained
as a control and
the other inoculated
with a L. mucm isolate from P. lanosa. After 24 hr of aerated
incubation
at 22C, the controls were still
free of filaments,
but the inoculated
group
had L. mucor filaments
on the periopods,
antcnnac,
and uropods.
LEUCOTHRIX
TADLE
1.
The natural
occurrence
the authors
MUCOR
965
INPESTATLON
of Leucothrix
rnucor
on crustaceans as observed
on variozls dates, from April 1969-May
1970
by one or more
of
Organs
Location
Pngurus longicarpus
Planes minutes
Car&us
maenas
Cancer irroratus
Cancer borealis
Lithodes maia
Ilomarus americanus
Palaemonetes pugio
Antennae
~lcopocls and egg mass
Antennae and plcopods
Egg mass
Egg mass
Egg mass
Egg mass
Plcopods and uropods
Crangon septemspinosa
Unidentified
species
of prawn
Pleopods
Plcopods
Narragansett
Bay
Sargasso Sea
Narragansett
Bay
Narragansett
Bay
Narragansett
Bay
Narragansett
Bay
Pt. Judith, R.I.
Narragansett
Bay
Pettaquamscutt
R.
Narragansett
Bay
Majuro Atoll,
Marshall Is.
Host
In addition to the characteristic
filaments composed of flexible chains of cells
with terminal gonidia (Fig. lC), both algae and crustacea often have short straight
nonseptate filaments (Fig. 1B). One’s first
reaction might be to regard these as a
different organism. However, closer cxamination of young specimens such as shown
in Fig, 1B indicated that these shorter
filaments might also be L. mucor.
The
bulbous cells seen at the tips of several
filaments in Fig. 1B have been observed
on slide cultures (Snellen and Raj 1970).
Isolates from P. americanus eggs also had
a similar appearance as young cultures on
agar; the gonidia germinated to yield filamcnts up to 14 p long without observable
septa. Diluted broth cultures also yielded
this form. It appears that, under conditions of minimal nutrients, gonidia germinate and form short, stiff filaments without
subdivision,
This form has apparently not
been recognized as a part of the life cycle (Harold and Stanier 1955; Pringsheim
1957; Brock 1966; Snellen and Raj 1970)
or as a naturally occurring form of L.
mucor.
A number of freshly caught decapod
crustaceans have been examined for the
presence of L. mucor filaments, So far the
planktonic forms appear free of L. mucor,
although benthic forms are quite heavily
infested. From 4 to 20 specimens of each
species were examined. A list of appreciably infested species of crustacea is given
and egg mass
in Table 1. The marked infestation of some
specimens is illustrated in Fig. IF, showing
the L. mucor filaments on an edge of a
pleopod of C’arcinus maenus. The most examined species so far is Palaemonetes pugio. Every specimen from a number of
locations has had populated pleopods (Fig.
1E) and uropods.
There are two problems in determining
the occurrence of L. mucor on crustaceans.
One is that since filaments appear to break
off from specimens preserved with alcohol
and formaldehyde,
only fresh specimens
can be observed. The second is that only
specimens or body parts thin enough for
phase contrast microscopy can be observed.
The scanning electron microscope, which
is not restricted in its depth of field, should
prove useful in determining the distribution of this microorganism on marine animals. The presence of L. mucor on Planes
minutus maintained in Sargasso Sea water
indicates that this microorganism may bc
present in at least one pelagic habitat,
Leucothrix mucor appears more susccptible to antibiotic control than the natural
bacterial flora of seawater ( Oppenheimer
1955). Six isolates from algae, crustacea,
and seawater were used to determine the
minimal inhibitory
concentrations in OZR
broth after 4 days incubation
at 22C.
These were : penicillin, 0.1 mg/liter; strcptomycin, 5.0 mg/liter; penicillin and streptomycin ( 1: 1 s/s), 0.5-0.7 mg/liter;
and
chloromycetin, 0.7-0.9 mg/liter. There was
966
JOHNSON,
SIEDU1~TI1,
SASTRY,
little strain variation. The USCof penicillin
and streptomycin at 25 mg/liter prevented
L. mucor development on cod eggs and
the usual mortality; levels of streptomycin
as 10~ as 4 mg/liter prevented the mass
mortalities of C. irroratus larvae observed
in the untreated controls,
The possible ecological role of a L. mucar epiflora on marine arthropods, particularly the benthic crustacea, is still not
known. Anderson and Stephens (1969) observed that the apparent uptake of 3% glycine by Artemia, Limnoria, and Tigriopus
was drastically reduced by preincubation
with antibiotics and concluded that the
microorganisms responsible for the uptake
were apparently associated with the exoskeleton and not the gut. They stated
( p, 248) “It is possible to think of the
arthropod and its associated epiflora as an
ecosystem. Amino acids might contribute
to the nutrition
of epiflora which could
then be cropped periodically and serve as
a food source for the arthropod. This is
possible, but we have no evidence that this
is the case.” We have observed that Crangon septemspinosa, a nonselective feeder,
cats bacterial films and L. mucor filaments,
and that P. pugio apparently grooms or
grazes its pleopods which are heavily infested with L. mucor. Studies on the
nutritional
value of L. mucor for grazing
organisms, including the host animal, are
indicated.
L. mucor strains from
Representative
C. irrora,tus ATCC 25906, P. americanus
ATCC 25907, and P. r)u@o ATC,C 25908
have been deposited in the American Tse
Culture Collection, Rockville, Maryland.
Interference with egg and larval development by filamentous bacteria also occurs
natuns, the
in Ereshwater. Sphaerotilus
equivalent of L. mucor in freshwater, has
been incriminated.
The death of shad fry
(Lincoln and Foster 1943) and emerging
walleye larvae ( Smith and Kramer 1963)
have been attributed to S. natans growth
arising from papermill wastes. In polluted
streams, S. nutcms was found to decrease
of gill-breathing
insects
the production
ARNOLD,
AND
DOTY
(Gaufin and Tarzwell 1955), and S. natans
growing in a Danish stream polluted by
silage juice was believed to bc the cause
of trout egg mortality in the anoxic slime
mats and the death of bottom-living
invertebrates entangled in the bacterial filamcnts ( Rasmussen 1955 ) .
OCCURRENCE
ON
TROPICAL
ALGAE
The islands on the southern rim of Majuro Atoll are joined by causeways modifying water circulation but permitting access to the length of the elliptical lagoon.
A microbiological
field laboratory was set
up at a remote inn on Anenelibu Island
which was central to the sampling areas
an d away from the center of population.
All seaweed samples were collected by
snorkeling at four stations representative
of the lagoon. Samples l-10 were collected
on 26 February 1970 at Ririkku; 11-20 on
28 February at Uotjaa; 21-30 on 1 March
at Rotoin; 31-40 on 3 March at Ririkku
again; and 41-50 on 6 March at Kaku
Cape. The algae were teased or sectioned
and wet mounts were sealed by forming a
thin bead of silicon grease on the edges of
the cover slip. A Wild M-11 field microscope with phase contrast condenser and
objectives was used to examine thcsc prcparations: first at 125~ for the presence and
abundance of 2-p-wide filaments, then at
500~ to confirm the presence of terminal
gonidia on the filaments. Eighteen random
samples were used to prepare enrichment
samples ( Harold and Stanier 1955). The
surface film was examined at intervals up
to 9 days for L. mucor filaments. Attempts
wcrc made to isolate L. mucor from these
enrichments as well as by direct streaking
( Lewin 1959). Failure to obtain pure cultures appeared to be due to rapid overgrowth by gliding and flagellated microorganisms, Air and water temperatures
remained near 28C during this period.
The relative abundance of II. mucor on
48 identified samples of algae is shown in
Table 2. A low population indicates that
filaments were present but difficult to find,
a high population indicates numerous fila-
LEUCOTHRIX
TABLE
2.
Relative
abundance
MUCOR
of Leucothrix
mwor
967
INE’.ESTATS.ON
on tropical
algae at Majuro
Atoll,
Marshall
Islands
L. mucor population
saG:lc
Cyanophyceae
Hytlrocoleus
lyngb yaceus
L yugb ya majuscula
Lyngbya
spp.
Schixothrix
spp,
Schixothrix
tenerimum
Chlorophyceae
Boocllea spp.
Caulerpa brachypus
Caulerpa urvilliana
Clarlophora
spp,
Dictyosphaeria
cauernosa
Halimeda
taenicola
Lobophora
variegata
Microdictyon
spp.
Rhixoclonium
spp.
Rhodophyceae
Centroceras
Ceramium
clauulatum
spp.
“Gelidium”
IIypnea
spp.
spp.
Jania spp.
Lauren&a
spp.
Polysiphonia
spp.
Sp yriclia
35
22
11
29
26
30
48
13
20
48
41
45
46
47
14
39
10
36
37
44
5
42
24
27
34
38
43
50
Low
Medium
corallincs
Phacophyceac
Dictyota
spp.
Ectocarpus in&us
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Paclina japonica
Sphacelaria
Totals
tribuloicles
X
X
X
X
3
8
23
46
3
1
33
6
19
9
48
High
X
filamentosa
Unidentified
--
28
31
32
17
14
16
21
25
49
Unclctcctccl
X
X
X
X
X
X
9
14
4
2;
968
JOHNSON,
SIEI3URTH,
SASTRY,
3.
enrichment
TABLE
-_--
Comparison
of direct examination
and
methods for detection of presence of
Leucothrix mncor on tropical algae
--___
Detection
Sai?O?e
3
6
8
9
11
12
13
14
Either
-I-
+
Spyridia filamentosa
Caulerpa brachypus
+
-
+
Unidentified
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
-t
-t
+
+
+
+
+
+
+
+
-I-tc
-t
14
12
18
Dictyota
spp.
Padina japonica
Spyridia filamentosa
Spyridia filamentosa
Sphacelaria tribuloides
Caulerpa urvilliana
Unidentified
Halimeda
Ceramium
green
taenicola
Lyngbw
18
22
23
25
27
29
30
31
33
of L. mtwor
EnrichDirect
ment
c
+
+
-/+
-
+
-
-
+
and
SPP.
mixture
Schixothrix
spp.
Laurencia
spp.
Caulerpa urvilliana
Dictyosphaeria
cavernosa
Hydrocoleus
lyngbyaceus
Ectocarpus in&us
Totals
+
+
+
+
-t
mcnts in many fields, and a medium population falls between. Leucothrix mucor was
present on all four algal classes examined.
Only 9 of the 48 specimens were free of
filaments which could be definitely rccognized as L. mucor. Fourteen ( 29%) were
sparsely populated and 25 (52%) were
moderately to heavily populated, thus 81%
of the samples had detectable L. mucor.
The results of the comparison of direct examination and enrichment for the
detection of L. mucor are given in Table
3. Although 14 were positive on microscopic examination, only 12 yielded positive enrichment
cultures.
Six of those
with microscopically
detectable L. mucor
were negative on enrichment, but all four
of those negative on direct examination
yielded positive enrichment cultures, suggesting that all 18 of the samples contained
L. mucor.
It must be borne in mind that very few
marine algae occur in nature without other
algae as epiphytes.
Most mature algal
ARNOLD,
AND
DOTY
specimens are assemblages with one dominant species. Others are mixtures such as
samples 14, 43, 46, and 48. Sample 15 was
so complex, containing species of Schixothrix, Lohophora, Jania, and Laurencia
among others, that it was discarded from
the results. In enrichment culture, L. mucar could arise from a few cells epiphytic
on any of the algae in the assemblage. For
this reason, no special note should be taken
as to which species support L. mucor on
enrichment. Species of host algae are given
as an indication of the wide spectra of
algae, or their algal epiphytes, that can
support L. mucor.
A strain of L. mucor, isolated during
winter from P. lanosa in Narragansett Bay
was taken to Majuro to see if a cold-water
strain would survive and attach in tropical
water, The culture remained viable at 28C
over a 3-week period and a subculture was
used to inoculate zooplankton
obtained
with a No. 10 net. Filaments attached to
the antennae, pleopods, and periopods
were detectable after 24 hr; zooplankton
in the control aquarium remained free during the several days of observation. Three
small unidentifed
prawns present in the
algal mat on a dead coral at Majuro were
examined. All had a few typical L. mucor
filaments attached to their periopods and
uropods.
Of 12 fresh algal specimens from Waikiki Beach (near the University
of Hawaii Beach Laboratory
and Aquarium)
collected enroute to Majuro, 7 contained
appreciable numbers of L. mucor. An apparent 100% infestation of the algal samples at Majuro, with 52% being moderate
to heavy, indicates that L. mucor is far
from absent or rare in at least this one
warm-water
environment.
The collateral
observations in Hawaii and with the Rhode
Island strain indicate that the abundance
of L. mucor at Majuro Atoll is not an isolated instance, that temperate isolates can
thrive
in the tropics, that this organism
also occurs cpibiotically on tropical benthic
crustaceans and may be more abundant in
warm water than suggested by Kelly and
Brock ( 1969).
LEUCOTIIRIX
MUCOR
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