STUDY OF CERCARIAL PENETRATION
ABSTRACT
A possible new marine cercaria found in the snail
Batallaria attramentaria at Elkhorn Slough, California
was described. A survey was performed on possible inter-
mediate hosts and the cercaria's penetration behavior in
one of them, Gillichthys mirabilis. As the cercaria aged,
its activity and penetration ability decreased. The
duration of successful cercarial penetration was dependent
upon the level of cercarial activity prior to attempted
penetration.
STUDY OF CERCARIAL PENETRATION
INTRODUCTION
The complicated life cycle of digenetic trematodes
is fascinating. Some have been known to have as many as
five hosts in a typical life cycle (Olsen, 1974). At
Elkhorn Slough a possibly undescribed species of marine
cercaria was discovered in the snail Batallaria attramentaria.
One or many of the common birds in the slough is thought to
be the definitive host. Two of the common fish in the area
are Gillichthys mirabilis, "Longjaw Mudsucker", and
Gasterosteus aculeatus, "Stickleback". The cercaria were
observed to infect both these fish. A preliminary description
of the cercaria was performed as well as studies dealing
with its natural host and penetration behavior in Gillichthys
mirabilis. Also the effect of time and temperature on
the activity and penetration successof the cercaria was
explored.
METHODS AND MATERIALS
The snail Batallaria attramentaria was collected from
Elkhorn Slough and stored in the dark using a tarpaulin
covered aquarium with running ocean water. Gillichthys
mirabilis, "Longjaw Mudsucker", were collected from the
tide pools at Elkhorn Slough with dip nets. Cercaria were
collected by placing three snails in a 45 ml plastic
container positioned 18 cm from a 75 watt incandescent light
source for 1 hour. Cercaria were concentrated by filtering
the water through a 50 micron Nitex filter and gently
STUDY OF CERCARIAL PENETRATION
washing the cercaria into a beaker. Concentrations as high
as 7000 cercaria/ml were collected and then diluted to a
standard concentration of 1000 cercaria/ml.
Descriptions of cercaria were made by observing live
specimens on a phase contrast microscope and some stained
slide preparations of unshed cercaria. The slides were
of the hepato-pancreas area of infected Batallaria
attramentaria. The snails were fixed with 5% Glutaraldehyde
then 9% Formaldehyde and lastly embedded in JB4 plastic.
A 2  section was cut and stained with methylene blue.
A survey of possible intermediate hosts was conducted
by placing a living host in a 8 cm X 10 cm opened glass
container partially filled with sea water at 20°C and under
constant light. Cercaria were added to bring the final
concentration of cercaria to 100 cercaria/ml. If the host
was relatively small, additional hosts were added to the
container. The host was checked every 10 minutes for 30
minutes for the presence of penetrating cercaria. All
experiments began within 2 hours after cercaria collection.
Penetration behavior was studied using a cross section
of Gillichthys mirabilis placed in sea water under a
dissection microscope. Cercaria were added to the petri
dish and their behavior observed.
Penetration tests were conducted by preparing replicate
samples of cercaria (over 500 cercaria/ml) and placing them
at 20°C under natural light. At designated intervals the
cercaria were placed in a 8 cm finger bowl containing a live
STUDY OF CERCARIAL PENETRATION
Gillichthys mirabilis and 100 ml of ocean water. Two hours
later the Gillichthys mirabilis was gently washed and placed
in a new container. Two days later the fish were dissected
and the number of cercaria per 1.4 cm of spine was recorded.
Since cercaria concentrated in the spinal column of infected
Gillichthys mirabilis, that area was used as a spot assay
for cercarial penetrations.
RESULTS
Description of cercaria
The cercaria is biocellate and parapleurolophocercous.
The body is covered with minute spines (1 u long) and bears
at least 18 slender long projections. Body measurements
based on 2 electron microscope photographs are: 80 u length,
60 u width.
The square black eyespots are located approximately
one-third the length of the body from the anterior end. The
anterior one-half of the body is clear while the posterior
one-half contains 14 granular gland cells. There are two
sets of spines in the mouth region. (Fig. 1) The most
posterior of these is a set of 17-18 spines arranged in an
elipse and pointing toward the ventral side of the cercaria.
Anterior to these is a second set of 5-6 spines that may be
pointed forward or ventrally as a unit. Both sets are
thought to act as scraping devices during penetration of
the next host. The exits of the penetration gland ducts
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STUDY OF CERCARIAL PENETRATION
are located just anterior to the oral sucker. (Fig. 2)
They are arranged in a 3-4-4-3 pattern as they pass
dorsally under the oral sucker. The measurements of the
ellipsoid oral sucker are: 30-35 u length, 27-32 u width.
The excretory bladder may take a V, Y or oval shape.
40 flame cells, 20 on a side, are connected via ducts to
the excretory bladder. (Fig. 3) The complete duct system
was never satisfactorily determined, so a flame cell
formula is not presented. The flame cell duct system in
the figure was formed by combining results from several
different specimens.
The tail is approximately 325 u long and 16 u wide
and contains two fins. The anterior lateral fin is
133 u long and 44 u wide. The posterior dorsal-ventral
fin is 211u long and 25 u at its widest point. There is
20 u overlap between the posterior part of the lateral fin
and the anterior dorsal part of the dorsal-ventral fin.
The cercaria swims in a zig-zag or spiral motion. It
generally swims upward and then slowly sinks head first.
RESULT
Penetration behavior
Cercarial penetration behavior began when the cercaria
swam 1-4 mm and made contact with the fish. Upon contact.
the cercaria lost its tail. About one-half of the time the
tail would not separate from the body until it actively
STUDY OF CERCARIAL PENETRATION
jerked. Several cases were observed where the tail would
not separate from the body. In these cases the tail move-
ment repeatly dislodged the cercaria from the host's skin.
Immediately following tail loss, the cercaria
rhythmatically contracted and elongated.
This behavior
presumably aided the flow of penetration gland enzymes to
the fish skin and/or the scraping action of the oral sucker
spines.
Upon penetration the cercaria would migrate within the
fish through a series of peristaltic motions. The cercaria
migrated to an open space and encysted. Preferred areas
for encystment include the spinal column, orbit and lower
buccal cavity. Encystment of over half of the cercaria is
complete within two days. In areas of high cercarial
infestation, the encystment period seemed to be delayed.
RESULTS
Survey of natural hosts
In the survey of natural hosts the response was
positive if the cercaria demonstrated penetration behavior
within thirty minutes after the introduction of the host.
The cercaria was found to penetrate the following
hosts: Gillichthys mirabilis (Longjaw Mudsucker),
Gasterosteus aculeatus (Threespine Stickleback) and to
a much lesser extent Clinocottus analis (Wooly Sclupin).
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STUDY OF CERCARIAL PENETRATION
They seemed to prefer to penetrate the fins rather than the
skin of the fish.
Cercaria did not respond to the following hosts:
Hermissenda crassicornis (Opisthobranchia), Callianass:
californiensis (Callianassidae) and Pachygrapsus crassipes
(Grapsidae).
There was some chance that the respiration of the host
would allow some cercaria to penetrate through the gills or
surrounding structures. However, dissection of all of the
hosts on the latter list revealed no metacercaria three days
after infection.
RESUITS
Cercarial penetration
The success of cercarial penetration is time dependent
with the highest rates during the first few hours. (Fig. 4)
At 20° C, the cercaria began to lose their penetration
ability after approximately 8 hours and by 20 hours it was
down to 4% of its highest value.
Observations were made on cercaria stored at 0° C for
2 days. It took over 24 hours for these cercaria to lose
their penetration ability.
RESULTS
Activity of cercaria
The activity of the cercaria, as assayed by their
frequency of swimming, is both time and temperature
STUDY OF CERCARIAL PENETRATION
dependent. (Fig. 5) The temperature of 70° C is lethal to
the cercaria after 20 minutes. At 45° C the cercaria remain
alive for 10 hours but lost their activity after 6 hours.
At lower temperatures the cercaria will stay alive for
progressively longer periods of time. The longest living
cercaria was stored at 0° C for 72 hours.
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DISCUSSION
The cercaria is very similar to both Parasitictodora
hancocki (Martin 1950) and Stictodora tridactyla (Martin
and Kuntz 1955). One of the major differences in both cases
is the number of flame cells. The flame cell formula for
Parasitictodora hancocki is 2 (313)(3+3)- 24 and the same
for Stictodora tridactyla is 2((3+343).(343-3): 36. The
other major difference is the location of the excretory
bladder exit tube. Both Parasitictodora hancocki and
Stictodora tridactyla have inverted "T" shaped exit tubes
in the tail. An excretory bladder exit tube has never been
observed in the undetermined cercaria.
There was much variability observed in the number of
flame cells and oral spines, and the arrangement of
penetration gland duct exits in the Elkhorn Slough Cercaria.
The experiment to test cercarial penetration was
hindered because some of the Gillichthys mirabilis were
infested with metacercaria when collected. If a large
number of metacercaria were found in or near the spine of
10
STUDY OF CERCARIAL PENETRATION
Gillichthye mirabilis when the fish was dissected, the data
was not scored.
The species of the metacercaria was not
known. It was easily distinguishable from the newly formed
metacercaria by both its color and size.
Host penetration must be an energy consuming process.
Since the cercaria does not feed, it must have a limited
energy supply. Consequently, cercaria must find and
penetrate a host before this energy supply is depleted.
If the activity of the cercaria is increased or decreased,
then its rate of energy consumption will rise or lower
respectively. Itwas expected that the less active cercaria
would be able to infect a host for a longer period of time.
This theory was confirmed when a batch of cercaria which was
stored at 0° C for 24 hours was observed to penetrate a
Gillichthys mirabilis.
Because of the small sample size in Figure 5, it is
difficult to make any specific conclusions. It is known
that the cercaria have a limited energy resource. After a
certain length of time they will run out of energy and die.
What factors influence this time span? Is there a point
where the cercaria are still alive but no longer can
penetrate or encyst? These are a few questions that may
be answered in a more lengthy study.
Final identification of the cercaria and a complete
life cycle for the trematode is also work that this report
left unfinished.
11
STUDY OF CERCARIAL PENETRATION
This report did establish that there may be a previously
undescribed cercaria in Elkhorn Slough. In addition it
showed that cercarial penetration of an intermediate host
seems to be limited by the energy previously stored in the
cercaria.
Two species of fish were infected by the cercaria in
this study. In only one of these fish, Gillichthys mirabilis
did metacercaria develop. In Gasterosteus aculeatus the
cercaria would not or could not encyst. The brain case
and spinal column were checked in several different
Gasterosteus aculeatus with the same results--no metacercaria.
The cercaria must have penetrated and then died. Some loose
cell masses were found inside the Gasterosteus aculeatus
to support this hypothesis. If this observation is valid in
the field then Gasterosteus aculeatus may be introduced to
a marine environment and help control trematodes. The fish
would absorb and essentially destroy many cercaria without
allowing the life cycle to continue.
STUDY OF CERCARIAL PENETRATION
ACKNOWLEDGMEN
I would like to thank the faculty and staff at Hopkins
Marine Station for a very enjoyable and rewarding quarter.
I would especially like to thank Professor Gary E. Wagenbach
for his steady guidance and open office door. Inside I was
able to gain much strength and inspiration. Special thanks
goes to Kathy French and Faylla Chapman for there constant
smiles as I pestered them for equipment and supplies.
And of course to Dan and Janie - for without them the
slough just would not be quite the same.
12
STUDY OF CERCARIAL PENETRATION
LITERATURE CITED
MARTIN, W.E. 1950. Parasitictodora hancocki n.gen, n.sp.
(Trematoda: Heterophyidae), with observations on its
life cycle. J. Parasit. 36: 360-370.
—, W.E., and Kuntz R.E. 1955. Some Egyptian
heterophyid trematodes. J. Parasit. 41: 374-382.
OLSEN, O.W., 1974 Animal Parasites - Their life cycles
and ecology. Third Edition University Park Press,
Baltimore, Maryland 567 pages.
14
STUDY OF CERCARIAL PENETRATION
Explanation of Figures
D-VF Dorsal-ventral fin
E
Eyespot
EB Excretory Bladder
FL
Flame Cells
G Genital Primordium
LF
Lateral fin
OS Oral Sucker
P
Penetration Glands
PD Penetration Gland Ducts
Figure 1. Diagram of oral spines in dorsal and lateral views
Figure 2. Cercaria of unidentified species
Figure 3. Penetration gland ducts
Qualitative study of the activity of cercaria
Figure 4.
at varying temperatures. Activity was measured
by the frequency the cercaria exhibited some
type of swimming behavior. All cercaria were
collected between O and 1 hours of the study
and stored for 1 hour in vials at 20°0.
Two hours after collection, the cercaria were
exposed to the temperature change.
STUDY OF CERCARIAL PENETRATION
Study of the change in successful penetrations
Figure 5.
as the cercaria age. The entire experiment was
performed at 20°C. For each data point one
Gillichthys mirabilis was used. Two days
after infection, the number of cercaria within
1.4 cm of fish spine was recorded and expressed
as a percentage of the two hour assay.
number of cercaria in Nth hour assay
% - 100 X
number of cercaria in 2 hour assay
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