Abstract
Bacteria were observed on the embryos of Pachygrapsus
crassipes, Pagurus samuelis, and Hopkinsia rosacea. Strains that grew
in culture were isolated, and were identified as Pseudomonadaceae
and Enterobacteriaceae. There were eighteen isolates: eight from
Pachygrapsus crassipes, six from Pagurus samuelis, and four from
Hopkinsia rosacea. None of the strains showed anti-fungal activity
against Lagenidium callinectes. The role of the bacteria isolated from
Pachygrapsus crassipes embryos were studied in greater detail.
Embryos treated with penicillin G, which was effective against seven
of the eight strains of bacteria, had a higher mortality rate, and a
greater likelihood of being infested with other microbes. This finding
suggests a defense role for the bacteria.
Introduction
Bacteria have been cited as a pathogen of eggs and embryos of aquatic
organisms, especially in commercial hatcheries (Fisher, 83, and Harper
and Talbot, 1984). They have been noted to restrict oxygen and foul
crustacean eggs considerably, causing the mother to drop them.
However, studies on shrimp and lobsters have shown
that bacteria found on embryonic surfaces can also perform a useful
service to the developing animal, by keeping off parasites that would
otherwise block gas exchange of the embryo with its surroundings,
and compete for oxygen. The bacteria keep the embryos clear of
Lagenidium callinectes, for example, by producing anti-fungal agents
(Gil-Turnes, 1989, 1992).
The intent of this study was to describe the kinds of
bacteria present on embryos of marine invertebrate species with long
incubation periods, long enough that colonization by bacteria and
fungi may become a potential problem. Bacteria on Pachygrapsus
crassipes, Pagurus samuelis, and Hokinsia rosacea were characterized.
Pachygrapsus crassipes embryos were studied in greater detail.
The female Pachygrapsus crassipes produces large yolky
eggs that are internally fertilized, and then extruded and attached to
the setae on the abdominal pleopods. The female tends this brood
mass, containing some 50,000 eggs, by cleaning and ventilating it for
an average period of 29 days, whereupon the larvae hatch
(Strathmann, 1987, and Hiatt, 1948). Despite her careful preening,
numerous bacteria are present on the surface of the embryos.
In some Brachyura species, gram-negative bacteria,
including known potential pathogens, such as Vibrio, Pseudomonas
and Aeromonas species, are seen in healthy specimens (Brock and
Lightner, 1990). It is interesting to ask whether bacteria covering the
surface of the Pachygrapsus crassipes embryo, too, are a normal, or
even beneficial factor in the crab's development.
This study will compare success of embryos with and
without the bacterial factor. The effect of inhibiting bacterial growth
on the embryos was found to be harmful to the embryo. The embryo
suffered greater infestation by fungi, protozoa and diatoms when
bacterial growth was inhibited.
Materials and Methods
Organisms
Ovigerous crabs (Pachygrapsus crassipes and Pagurus
samuelis), and nudibranchs (Hopkinsia rosacea) were collected from
the rocky shore at Hopkins Marine Station, Pacific Grove, CA. The
specimens were stored in running sea water aquaria at 15 degrees
Celcius. Äfter several days, the nudibranchs produced an egg ribbon.
Visualization of Bacteria
Use of the DNA specific stain, DAPI (4', 6-diamidino-2-
phenyl-indole), allowed visualization of bacteria on the invertebrate
embryos. DAPI (1 mg/ml stock solution) was diluted 1 to 1,000 in
ultraviolet filtered sea water (UVSW). The embryos were soaked for
10 minutes in the stain, were rinsed three times in UVSW, and
observed under the fluorescence microscope under oil immersion.
Isolation of Bacteria
Embryos were collected from each species, and bacteria
were isolated. To isolate surface strains from Pachygrapsus crassipes
and Pagurus samuelis, embryos were held with tweezers and dragged
along the surface of "ÖZR" agar (15g DIFCO 2216 marine agar, 1g
tryptone, 1 g yeast extract, 5 mg ferric citrate, 1 L sea water). A second
procedure was used to isolate other strains that might be within the
egg sheath beneath the surface. Embryos were homogenized in sterile
sea water and diluted ten-fold. This second procedure was used for
the H. rosacea eggs, additionally.
In the attempt to isolate strains of each morphological
type, each colony of distinct morphology that appeared on the agar was
chosen for isolation, in the attempt t. Eighteen strains were selected in
total: eight strains were chosen from the Pachygrapsus crassipes
embryos, strains 1, 2, 4, 5, 7, 8, 19, and 20, six from the Pagurus
samuelis, strains 9, 10, 11, 12, 13, and 14, and four from H. rosacea,
strains 15, 16, 17, and 18. Pure cultures were obtained after two to
three streakings.
Bacteria Identification
Colony characteristics on ÖZR agar were noted with
respect to color, edge continuity, and general texture. Gram staining
was performed on each isolate. Live cells were examined for motility.
The strains were defined as strict aerobes or facultative
anaerobes based upon their growth being strictly on the agar surface or
additionally, beneath the agar at a depth of greater than 2 cm.
Fermentation of glucose was ascertained by a pH change. The strains
were grown up in a medium containing a pH indicator; acid
production caused a color change.
Strains were tested for the presence of catalase, an
enzyme that catalyzes the breakdown of peroxides formed in the cell
during respiratory metabolism, and for the presence of cytochrome C
(oxidase test). A result of the cell's breakdown of tryptophan is indole
accumulation. This property was recorded, also.
The methyl red test was done to detect the compound
acetoin, indicative of a distinct fermentation pathway leading to the
product 2,3-butanediol, in which acetoin is the immediate precursor to
butanediol. Cells were examined for H»S production, and nitrate
reduction, additionally. The results of all these tests are presented in
Table 1.
Testing for Anti-Fungal Properties Among Isolates
A competition experiment was established to ascertain
whether Lagenidium callinectes growth was inhibited by the various
bacterial species, L. callinectes was grown on ÖZR agar, and a plug
removed and transferred to a fresh plate of ÖZR agar. Hyphae grew
out from the plug after several days, at which point, each of the
eighteen strains were streaked 5 mm away from the hyphae in order
to determine whether any of the isolated strains inhibited the growth
of L. callinectes. It was concluded that if the hyphae growth continued
to extend to the bacterial streak, then there was no inhibition (Figure
Bacterial Strain Response to Antibiotics
Each bacterial strain was used to inoculate 3 ml of agar.
The liquefied agar was immediately poured over solidified agar. Discs
containing antibiotics were placed on the overlay. The commercially
manufactured ampicillin discs contained 10 mcg of ampicillin. The
penicillin G discs were prepared by soaking filter paper discs in a 1 g/L
solution of penicillin G for one hour. Growth was checked at 48
hours. In the case that penicillin G inhibited bacterial growth, a
circular zone of inhibition was observed surrounding the disc (Figure
2). Bacterial growth that covered the plate, including the area of the
plate immediately surrounding the discs, marked a lack of inhibition,
a negative result in Table 3.
Antibiotic and Fungal Treatments
Eggs from five specimens of ovigerous Pachygrapsus
crassipes were examined microscopically, and those from different
mothers were found to be in various stages of development. The
female with embryos at the earliest stage of development was chosen
to be used experimentally.
These embryos had completed
gastrulation; the blastomeres were numerous, and there was a slight
withdrawal of blastomeres from one section of the capsule, revealing
perivitelline space. They lacked early appendages, heartbeats, or eye
spots. It was determined that they were approximately 5 days into
development (Hiatt, 1947)
Sterile tweezers were used to remove a cluster of
approximately 200-300 eggs from the female. Altogether, four clusters
were removed. Each cluster was placed in a separate vial within
separate beakers, filled with 900 ml of UVSW. The sides of the vials
were made of Nytex mesh, which allowed water to flow through, but
prevented the embryos from escaping. Each beaker was aerated by a
tube connecting to an air filter system. The first cluster received no
treatment. The second cluster received 90mg penicillin G (0.1g/L) for
6 hours on the first day of experimentation, and 6 hours the next. The
third cluster was treated in the same way as the second, and in
addition, was infected with L. callinectes. The fungus had been grown
up on agar, and agar plugs approximately 0.5cm on a side, thick with
fungal hyphae, were placed in the vial containing the embryos. The
beaker holding the fourth cluster was infected with L. callinectes, only.
Results
Observation of DAPI stained embryos
Colony forming rods of several shapes and sizes were
observed by fluorescence microscopy, using oil immersion. The rods
formed long strings or patches. They covered the surface of the egg
itself, and had also colonized the fibers binding the eggs together. The
bacterial cover was not uniform, but patchy. The rod-shaped bacteria
formed small colonies of ten to fifteen cells, and larger colonies of
approximately fifty cells. Rods also formed colonies in long strings.
Identification of Bacterial Isolates
The bacteria that grew on ÖZR agar were identified to
family. The tests performed on the strains are presented in Table 1.
Gram-negative, aerobic strains were tentatively placed in
the family Pseudomonadaceae (Table 2). Furthermore, if the colonies
were pigmented yellow or orange, the strain was classified as being in
the flavobacterium or cytophaga genera. If the strain was aerobic and
gram negative, but lacked pigment (white or tan) it was classified as
being in the alteromonas or xanthomonas genera (Hayes, et.al, 1979).
Gram negative facultative anaerobes were tentatively placed in the
Enterobacteriacea family.
Four strains in the Pseudomonadacea family, and one in
the Enterobacteriacea family were found on the Pachygrapsus crassipes
embryos. Six strains from the Pseudomonadacea family were isolated
from the Pagurus samuelis embryos; three strains from the
Pseudomonadacea family, and one from the Enterobacteriacea family
were isolated from the Hopkinsia rosacea embryos.
Anti-fungal Properties
None of the 18 isolates inhibited hyphae growth. The
hyphae growth proceeded right up until the edge of the bacterial streak
in each case.
A problem with the L. callinectes culture obtained from
the ATCC was an association of bacteria with the culture. Growth of
the bacterial contaminant was apparently stimulated with any
perturbation of the fungal culture, i.e., when agar plugs were cut from
it, and transferred to new plates. It is difficult to ascertain whether the
contaminant may have affected the results.
Response to Antibiotic Treatment
Ten of thirteen of the strains showed sensitivity to
ampicillin, and thirteen of eighteen strains to penicillin G (table 3).
Some strains showed greater sensitivity than others, but any
noticeable inhibition is recorded as positive.
Effects of Removing Bacteria with Antibiotic
While seven of the eight strains isolated from
Pachygrapsus crassipes may have been sensitive to penicillin in the
antibiotic disc assay, penicillin did not reduce the bacterial density on
the Pachygrapsus crassipes egg surface. Using DAPI to stain the
penicillin G-soaked embryos, and comparing them to stained control
embryos, showed little difference in the density of bacteria on the
surface. As on the control embryos, the bacteria grew in patchy and
string-like colonies. This observation prompted a quantitative
assessment. Äfter a ten-fold dilution, the density of colonies from the
penicillin G-soaked embryos was 7.1x10° colony forming units
(cfu)/ug, compared to 3.8x104 cfu/ug for the control (Table 3). The
penicillin+L. callinectes treatment resulted in 4.6x10° cfu/ug, and the
L. callinectes treatment showed 1.2x10° cfu/ug (Table 4).
Embryos were examined for development, mortality, and
number of epibionts 10, 13, and 18 days after the experimental setup
(corresponding to days 15, 18, and 23 of embryo development, since
embryos were taken from their mother approximately 5 days into
development). In the assessments on days 10 and 13, only 10-20
embryos per treatment comprised the sample. On day 18, all
remaining embryos were examined (100-300 embryos per treatment).
Epibionts
While fungal hyphae were not observed on the
penicillin G or penicillin G+L.callinectes-treated embryos 18 days after
the initial penicillin soaking, many other epibionts appeared heavily
on the penicillin G-treated egg surfaces. By 10 and 13 days into the
treatment, diatom and protozoan activity was high on the eggs
receiving penicillin G. Eighteen days after the start of the treatments,
the eggs were thickly covered with masses of fibers, made up of
smaller, round bodies. This could be an egg mass or a fungus
(apparently not Lagenidium callinectes).
Several transparent worms crawled the surface of the
embryos receiving the L.callinectes -treatment. Besides this change,
the surfaces of eggs in the control treatment and the L.callinectes
-treatment remained as clean at 18 days into the treatment as they had
been at the start of the experiment. They hosted only a few protozoa.
Mortality
Discoloration indicated a dead embryo. In dead embryos,
either the cells were orange, or the extracellular fluid was pink. In the
first assessment, 10 days after the start of the experiment, 50% of the
sample of embryos taken from the control group were dead. None of
the penicillin G- treated were dead. 29% of the
penicillin+L.callinectes-treated were dead. None of the L.callinectes-
treated were dead. Development was not retarded in any case, in
those that remained alive (figures 3 - 6).
On day 13, 25% of the control embryos were dead, 42.5%
of the penicillin-soaked were dead, 45% of the penicillin+L.callinectes¬
treated were dead, 17.5% of the L.callinectes-treated were dead.
Development appeared retarded in the penicillin+L.callinectes treated
embryos.
By day 18, 30.5% of the controls, and 73.3% of the
penicillin- treated were dead, and essentially all of the
penicillin+L.callinectes, and L.callinectes -treated were dead (98.7%
and 99.5%, respectively).
Discussion
The embryos of the three invertebrates under
investigation in this study were shown to host a number of Gram
negative, rod-shaped bacteria. The bacteria were seen in dense
coverage over as much as 50% of the surface of Pachygrapsus crassipes
embryos. The bacteria were identified as Pseudomonadacea and
Enterobacteriacea. They did not inhibit growth of Lagenidium
callinectes on agar. Seven of the eight Pachygrapsus crassipes strains
were sensitive to penicillin-G. After treatment with penicillin G,
Pachygrapsus crassipes embryos suffered from colonization by more
protozoa, diatoms, and worms than control embryos receiving no
treatment. Their mortality rate increased dramatically.
The question under scrutiny in this investigation was
whether the bacterial guests of the embryo protect it from unwanted
parasites, or if these bacteria are only parasites themselves. Microbial
protection of embryos has been reported for the shrimp, Palaemon
macrodactylus, which, like Pachygrapsus crassipes, broods its eggs
externally. In that study, however, it was shown that the bacteria
produced an anti-fungal compound, 2,3-indolinedione (Gil-Turnes et.
al, 1989). Similarly, the bacteria found on Homarus americanus
embryos produced an anti-fungal compound, tyrosol (Gil-Turnes and
Fencial, 1992).
The bacteria isolated in this study showed no anti-fungal
activity, yet the possibility exists that not all bacteria inhabiting the
embryo surface were isolated. Some strains may not have been able to
grow on ÖZR agar, and some strains may have been overlooked
during the initial screening in which bacteria were chosen for further
study from the first platings.
The penicillin G treatments provide an insight into
another possible role of the bacteria. It was found that treatment with
penicillin G resulted in more bacteria, as noted. Because some strains
were resistant to the antibiotic, it may be that after the sensitive strains
were eliminated, the population of the remaining resistant strains
may have exploded.
In a study of the epibiotic flora associated with embryos of
Homarus americanus, the embryos of laboratory spawned females
hosted different types of epibionts from the wild spawned controls,
notably fewer colonial rods. While the wild spawned embryos seemed
unaffected by bacterial associations, a relationship was found between
increase in bacteria and decline in egg clutch size for the lab spawned
embryos. The authors suggest that colonial rods could be protecting
eggs from an unchecked population explosion of other kinds of
bacteria (Harper and Talbot, 1984).
In a study of the bacterial strains on pelagic fish eggs,
some antibiotic treatments stimulated bacterial growth. The author
suggested that the antibiotics in these particular cases either provided
a growth-promoting substance, or merely repressed one or more
strains of bacteria, allowing the other strains better growth conditions
(Oppenheimer, 1955).
In another study, epibionts on the embryos of Cancer
magister were compared before and after treatment with penicillin G
and streptomycin sulfate. The antibiotics prevented non-filamentous
fouling, but algal filaments were resistant to the antibiotic, and
increased in number. In this case, mortalities were reduced.
Antibiotics inhibited harmful epibionts (Fisher, 1976).
For this study, the antibiotic-sensitive strains of bacteria
may have been preventing epibiont infestation. Their removal
prompted the explosion of various protozoa and diatoms. The fibrous
masses covering the penicillin G-Lagenidium callinectes-treated
embryos could be a type of fungus, or the egg masses of a predatory
worm. Indeed, evidence of a parasitic nemertean, Carcinonemertes
epialti, has been reported on Pachygrapsus crassipes embryos (Roe,
1979). However, Carcinonemertes epialti is pigmented, unlike the
observed in this study. Its egg masses are as thick in diameter as the
worm itself, also inconsistent with these findings.
It can be hypothesized that symbiont strains were those
sensitive to penicillin G, and their elimination meant an opportunity
for pathogenic strains to exploit the embryo. The fact that penicillin
treated embryos have higher mortalities and retarded development
supports this hypothesis.
In conclusion, while it may be true that the bacteria
themselves stress the developing Pachygrapsus crassipes embryo by
their own oxygen and nutrient requirements, it is likely that they
protect the embryos from more demanding parasites, capable of
harming the embryo to a greater extent.
Acknowledgements
My thanks go to David Epel, Robin Hayes, and Paul Sund for their
help and expertise.
Literature Cited
Brock, J. A. and Lightner, D.V. (1990) "Diseases Caused by
Microorganisms. Disease of Marine Animals, Vol.III, ed. O. Kinne.
Hamburg, Germany: Biologische Anstalt Helgoland.
Fisher, W.S. (1983) Eggs of Palaemon macrodactylus: II. Association
with Aquatic Bacteria. Biol. Bull., 164: 201-213.
Fisher, W.S., and D.E. Wickham. (1976) "Mortalities and Epibiotic
Fouling of Eggs from Wild Populations of the Dungenous crab, Cancer
magister. U.S. Fish Widl. Serv. Fish Bull.,74. 201-207.
Fisher, W.S. (1976) Relationships of Epibiotic Fouling and Mortalities
of Eggs of the Dungenous Crab. J. Fish. Res. Board. Can.,33: 2849-2853.
Gil-Turnes, M. S. and W. Fencial. (1992) Embryos of Homarus
americanus are Protected by Epibiotic Bacteria. Biol. Bull., 182: 105-108.
Gil-Turnes, M.S. Mark E. Hay, and W. Fencial. (1989) Symbiotic
Marine Bacteria Chemically Defend Crustacean Embryos from a
Pathogenic Fungus. Science, 246: 116-118.
Harper, R.E. and P. Talbot. (1984) Analysis of the Epibiotic Bacteria of
Lobster (Homarus) Eggs and their Influence on the Loss of Eggs from
the Pleopods. Aquaculture, 36: 9-26.
Hayes, R.R., T.A. McMeekin, and J.M. Shewan. (1979) "The
Identification of Gram Negative, Yellow Pigmented Rods.'
Identification Methods for Microbiologists, second edition, ed. F.A.
Skinner, and D.W. Lovelock. New York: Academic Press. 177-187.
Hiatt, R.W. (1948) The Biology of the Lined Shore Crab, Pachygrapsus
crassipes Randall. Pacific Science, Vol. II, No. 3: 135-213.
Oppenheimer, Carl H. (1955) The Effect of Marine Bacteria on the
Development and Hatching of Pelagic Fish Eggs, and the Control of
Such Bacteria by Antibiotics. Copeia, No. 1. 43-49.
Roe, P. (1979) Aspects of the Development and Occurrence of
Carcinonemertes epialti (Nemertea) from Shore Crabs in Monterey
Bay, California. Biol. Bull., 156: 130-140.
Strathmann, M. (1987) Reproduction and Development of Marine
Invertebrates of the Northern Pacific Coast. Seattle: University of
Washington Press. 451-460.
Table 1
02
Tex
MR
8
8
gb
14
gf
20
P
g
1-

E
Key:
St = strain
Co = color, y = yellow, w = white, t = tan, o = orange
Ed = edge, c = continuous, s = rough, d = diffuse
Tex = texture, g = glossy, b = blurry, f = flowery, c = clumpy, t =
transparent
G = Gram stain
M = Motility
O2 = oxygen requirement (+ indicates strict aerobe, - indicates
facultative anaerobe)
F = glucose fermentation
C = catalase
= oxidase (cytochrome C)
H2s
I = indole
MR = methyl red
VP = Voges Proskauer
28 = hydrogen sulfide production
N = nitrate reduction
Table 2
Strain f, organism
1 P. crassipes
2 P. crassipes
4 P. crassipes
5 P. crassipes
7 P. crassipes
8 P. crassipes
9 P. samuelis
10 P. samuelis
11 P. samuelis
12 P. samuelis
13 P. samuelis
14 P. samuelis
15 H. rosacea
16 H. rosacea
17 H. rosacea
18 H. rosacea
19 P. crassipes
Family
Pseudomonadacea
unidentified
Enterobacteriacea
Pseudomonadacea
unidentified
Pseudomonadacea
Pseudomonadacea
Pseudomonadacea
Pseudomonadacea
Pseudomonadacea
Pseudomonadacea
Pseudomonadacea
Pseudomonadacea
Pseudomonadacea
Pseudomonadacea
Enterobacteriacea
Pseudomonadacea
Genera
flavobacterium or
cytophaga
xanthomonas or
alteromonas
flavobacterium or
cytophaga
xanthomonas or
alteromonas
flavobacterium or
cytophaga
flavobacterium or
cytophaga
xanthomonas or
alteromonas
flavobacterium or
cytophaga
flavobacterium or
cytophaga
xanthomonas or
alteromonas
xanthomonas or
alteromonas
flavobacterium or
cytophaga
xanthomonas or
alteromonas
20 P. cri
sipes
Pseudomonadacea
xanthomonas
alteromonas
Table 3
Strain
13
17
18
19
20
Ampicillin sensitive
+
+/-
+-
Penicillin sensitive

+-
+/-
Table 4
Treatment
control
penicillin
penicillin + L.callinectes
L. callinectes
cfu/ug, day 13 of treatment
3.8X104
7.1x 105
46X 105
12x106
Figure Legends
Figure 1: Bacterial strains do not inhibit L. callinectes growth from the
transferred plug (pictured in center).
Figure 2: Strain #20 is sensitive to the antibiotics penicillin G and
ampicillin.
Figure 3: Control group mortalities on day 10, 13 and 18 of artificial
incubation.
Figure 4: Penicillin G-treated embryo mortalities on day 10, 13, and 18
of artificial incubation.
Figure 5: Penicillin G+-L.callinectes-treated embryo mortalities on day
10, 13, and 18 of artificial incubation.
Figure 6: L.callinectes-inoculated embryo mortalities on day 10, 13, and
18 of artificial incubation.
Figure 1
Figure 2
igure
20 -
Figure 4
80
60
Da
Figure 5
0

Day
Figure 6
00
Day