age-related change in phototaxis by cercariae of echinostoma caproni (digenea: echinostomatidae)
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Age-Related Change in Phototaxis by Cercariae of Echinostomacaproni (Digenea: Echinostomatidae)Author(s): Thomas R. Platt and Rose M. DowdSource: Comparative Parasitology, 79(1):1-4. 2012.Published By: The Helminthological Society of WashingtonDOI: http://dx.doi.org/10.1654/4541.1URL: http://www.bioone.org/doi/full/10.1654/4541.1
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Age-Related Change in Phototaxis by Cercariae of Echinostoma caproni(Digenea: Echinostomatidae)
THOMAS R. PLATT1
AND ROSE M. DOWD
Department of Biology, Saint Mary’s College, Notre Dame, Indiana 46556, U.S.A.
ABSTRACT: Cercarial dispersal is the result of fixed action patterns in response to reliable environmental cues. We tested the
effect of age on the preference of Echinostoma caproni cercariae for light or dark. Individual cercariae were isolated within
10 min of release from Biomphalaria glabrata and placed in a CarolinaTM Deep-Well Slide. Half of the slide (top and
bottom) was covered with electrical tape to exclude light. The entire chamber of the slide was observed on low power of a
dissecting microscope so the cercaria was readily visible whenever it was in the lighted portion of the slide. The amount of
time a cercaria spent in the light and the number of times it crossed from light to dark during a 5-min period at 0, 1, 2, and 4 hr
were determined (n 5 20). The mean amount of time cercariae spent in the light declined significantly from immediately
after release (127.7 sec) as compared to 1 hr (68.4 sec), 2 hr (51.6 sec), and 4 hr (10.6 sec) postemergence. The same pattern
was seen in the average number of times cercariae crossed from light to dark in a 5-min period: 10.7, 7.2, 6.95, and 1.5,
respectively. Cercariae showed no preference for light or dark immediately upon release (P 5 0.119), nor was there a
correlation between the amount of time spent in the light and the number of crossings at this time period. Cercariae spent a
significantly greater amount of time in the dark with age (1–4 hr), and the number of crossings at each of these time periods
was highly correlated with the time spent in the light. These findings suggest that light is not an important cue for E. caproni
cercariae immediately upon release; however, they develop a strong preference for darker habitats, or an aversion to light, as
they age.
KEY WORDS: Digenea, Echinostomatidae, Echinostoma caproni, cercariae, cercarial age, phototaxis, behavior.
The responses of trematode cercariae to environ-
mental factors have been described as fixed action
patterns (Sukhdeo, 1990; Sukhdeo and Sukhdeo,
2004). Trigger cues (environmental stimuli) have
been identified as unambiguous signals that provide
reliable information about the environment to which
cercariae respond, through innate locomotor activity,
to locate ecospaces where there is a high probability
of encountering the next host in the life cycle. The
most frequently studied trigger cues are light, gravity,
temperature, and water currents, and cercariae
respond in a predictable manner to these stimuli.
Temporal changes in cercarial behavior to envi-
ronmental stimuli have been reported for several
species of trematode cercariae; however, few have
been investigated closely. McCarthy (1999) found
that young cercariae (,30 min after release) of
Echinoparyphium recurvatum (Echinostomatidae)
were positively phototactic; however, at 2 hr, the
same cercariae demonstrated a negative phototaxis
in a simple choice experiment. McCarthy (1999)
correlated this change in phototaxis to increased
infectivity with cercarial age as demonstrated by
Evans and Gordon (1983).
The purpose of the current study was to assess the
response of Echinostoma caproni cercariae to light
under similar circumstances to determine if an age-
related change in response to light might be a
common attribute of echinostome cercariae.
MATERIALS AND METHODS
Observation chambers were constructed using CarolinaTM
Deep-Well Slides (Carolina Biological, Burlington, NorthCarolina, U.S.A.). A slide consists of a capped plastic well,2.5 cm in diameter, with a volume of ,1 ml. Blackelectrician’s tape was used to cover half of the bottom of theslide and half of the cap, which effectively excluded lightfrom half of the chamber.
Individual cercariae were collected within 10 min ofemergence from infected Biomphalaria glabrata maintainedin well water at room temperature (22uC). A single cercariawas transferred to the well of the slide via pipette.Additional water from the same beaker was added to fillthe well, and the cap was secured, taking care to align theedges of the tape on the cap with the tape on the bottom ofthe slide. The slide was placed on the stage of an OlympusTL2 dissecting microscope equipped with a 6-V, 20-Watttungsten bulb. Observations were initiated immediatelyusing illumination from above and below the slide. Lightintensity was 800 6 30 lux, as determined using an Extechlight meter (Model 401025; Extech Instruments, Waltham,Massachusetts, U.S.A.). The amount of time the cercariaremained in the lighted portion of the chamber during a5-min interval was determined with a stopwatch, and thenumber of times the cercaria crossed from light to dark wasnoted and recorded. Each cercaria was assessed for 5 min at0, 1, 2, and 4 hr after emergence. The experiment consistedof 20 replicates.1 Corresponding author (e-mail: [email protected]).
Comp. Parasitol.79(1), 2012, pp. 1–4
1
A repeated measures analysis of variance (ANOVA) wasperformed to compare the differences in the amount of timespent in the light and the number of times the cercariaecrossed from the light to the dark. A type I error rate of 0.05was used. For multiple comparisons, a Bonferroni test wasused with a type I error rate of 0.00833 (0.5/6). A one-sample t-test of the differences between the number ofseconds spent in the light and dark was used to determine ifthere was a preference at each time interval. The nullhypothesis was m 5 0. A Pearson correlation was used todetermine the relationship between the number of timescercariae crossed from light to dark and the amount of timespent in the light. The ANOVA was done using SYSTAT(ver. 6.0). The remaining tests were conducted usingMinitabH (2006).
RESULTS
The mean number of seconds the cercariae spent in
the light declined over the 4-hr observation period
(Fig. 1). The time spent in the light declined
significantly for all pairs (P , 0.001), except at times
1 and 2. At time 0, immediately after emergence, the
mean was 128 sec spent in the light. There was a
significant decrease between time 0 and time 1
(P 5 0.001) but no significant change between times
1 and 2 (P 5 0.73). At time 4, the mean dropped to
10.55 sec spent in the light. There was no significant
difference in the amount of time spent in the
light or dark for newly emerged cercariae (,10 min;
P 5 0.119); however, cercariae spent significantly
more time in the dark at all other times (Table 1).
The mean number of times the cercariae crossed
between the light and dark halves is shown in
Figure 2. The differences between all pairs were
significant, except between times 1 and 2 (P 5 0.73).
At time 0, the mean number of cercarial crossings
was 9.75. As the cercariae aged, the number of
crossings between the light and dark halves declined
until 4 hr, when the mean was 1.70. There was no
correlation between the number of crossings and time
spent in the light for newly emerged cercariae (r 5
0.34; P 5 0.142); however, there were significant
correlations at 1 hr (r 5 0.871; P , 0.001), 2 hr (r 5
0.724; P , 0.001), and 4 hr (r 5 0.876; P , 0.001).
All cercariae were observed at the end of the trials,
and they were alive and active.
DISCUSSION
In the current study, young E. caproni cercariae
(,10 min old) showed no preference for the light or
dark portions of the test chamber. This, combined
with the absence of a correlation between the time
spent in the light and the number of times cercariae
crossed from light to dark, suggests that light is not an
important factor in the early stages of the search for a
second intermediate host. As E. caproni cercariae
aged (.1 hr old), the amount of time spent in the
dark increased; coupled with the high correlation
between the amount of time spent in the light and the
number of times the cercariae crossed from light to
dark, this strongly suggests a developing preference
for darker microhabitats or, alternatively, an avoid-
ance of lighted areas.
Results of the current study support McCarthy’s
(1999) findings for E. recurvatum; however, the
behavior of E. caproni cercariae appears more
complex. While E. caproni cercariae do tend to
Figures 1, 2. Response of Echinostoma caproni cercar-iae to light/dark chamber tests. 1. Mean number of seconds(6SD) Echinostoma caproni cercariae spent in the light halfof a test chamber during a 5-min trial at 4 different timeperiods (n 5 20). 2. Mean number of times (6SD)Echinostoma caproni cercariae crossed from the light tothe dark half of a test chamber during a 5-min trial at 4different time periods (n 5 20).
2 COMPARATIVE PARASITOLOGY, 79(1), JANUARY 2012
spend more time in the dark with age, the relationship
is more dynamic than could have been determined by
the static design employed by McCarthy (1999).
Results from the current study would predict that
recently emerged cercariae of E. recurvatum would
have been evenly distributed between light and dark,
instead of significantly higher numbers congregating
on the light side of the observation chamber
(McCarthy, 1999). The phototactic response of the
2 species may be quite different. Loy et al. (2001)
noted different responses to light (both direction and
intensity) and gravity in cercariae of 4 species of
echinostomes.
Echinostoma caproni cercariae demonstrate a clear
preference for the top of the water column in both
behavioral (Haas et al., 2008) and transmission (Platt
et al., 2009, 2010) studies. In vertical trials at both
low and high light intensities, Haas et al. (2008)
found E. caproni cercariae in the top 10–15% of the
water column. This pattern was not significantly
altered when light was introduced from the bottom of
trial cuvettes (Haas et al., 2008). Similarly, the
placement of light at the bottom of transmission
chambers did not significantly alter the larger number
of metacercariae found in sentinel snails at the top of
the water column in transmission studies, nor did the
absence of light (Platt et al., 2010). These results all
suggest that a negative geotaxis is the dominant
trigger cue for E. caproni cercariae (Platt et al., 2010)
in nature.
Platt et al. (2009) found that sentinel snails in the
dark half of a simple choice experiment were more
heavily infected with metacercariae of E. caproni in a
horizontal chamber over an 8-hr period, which would
support the preference of E. caproni cercariae for the
dark as they age, when gravity is eliminated as a
confounding variable. Haas et al. (2008) reported that
E. caproni cercariae shift their response to light with
age; they were initially negatively phototactic but
became positively phototactic after 5 hr. These trials
were conducted in a horizontal chamber, eliminating
gravity as a factor and are difficult to reconcile with
the results from the current study.
The differences in the number of times cercariae
crossed from light to dark could be attributed to the
exhaustion of energy stores; however, Evans (1985)
reported that nearly 80% of Echinostoma liei (5E.caproni) cercariae were alive more than 15 hr after
release at 25uC, and were still capable of infecting
snails. Pechenik and Fried (1995) reported similar
values for cercariae of Echinostoma trivolvis, as did
Evans and Gordon (1983) for cercariae of E.recurvatum. Meyrowitsch et al. (1991) demonstrated
a maximum swimming rate of approximately 2.5 mm/
sec for E. caproni cercariae at 25uC at 2 hr
postemergence; the swimming rate declined with
age, but they still averaged ,1.7 mm/sec at 8 hr
postemergence. If energy exhaustion were the only
explanation for the differences in the current study,
we would have expected to find equal numbers of
cercariae on the light and dark sides of the chamber at
the final time period. All cercariae were alive at the
end of the experiment, although we did not assess
swimming speed or infectivity in the current study.
The releaser response of cercariae must result in
actions that will ultimately bring the cercariae into
host space + host time (Combes et al., 1994) in order
to increase the probability of encountering a suitable
intermediate host. This equation must also account
for ‘‘parasite time,’’ as the nonfeeding cercariae must
accomplish this feat while they possess sufficient
energy to complete the search process, contact, and
infect the second intermediate host. Recently
emerged cercariae demonstrated no preference for
light or dark as determined by the amount of time
spent on either half of the test chamber and the
absence of a correlation between the time spent in the
light versus the number of crossings. This suggests
that recently emerged cercariae were actively search-
ing the ‘‘host space’’ with little regard for light
conditions. As cercariae age, they remained on the
dark side of the chamber longer and quickly returned
Table 1. The effect of the age of Echinostoma caproni cercariae on the percentage of time spent in the dark during a5-min trial (n = 20).
Cercarial age Mean Minimum MaximumOne-sample t-test(light vs. dark)*
,10 min 57.3 8.0 94.7 P 5 0.119
1 hr 77.2 23.3 100 P , 0.001
2 hr 82.7 46.7 100 P , 0.001
4 hr 96.5 59.3 100 P , 0.001
* Time in light 2 time in dark. H0: m 5 0.
PLATT AND DOWD—PHOTOTAXIS IN ECHINOSTOMA CAPRONI CERCARIAE 3
to the dark side when they did venture into the light.
Cercariae of E. caproni, like many species of
echinostomes, show broad co-accommodation for
the second intermediate host (Keeler and Huffman,
2009), and this includes both aquatic gastropods and
amphibians (see Platt et al. [2009] for a review of the
life cycle of E. caproni). Therefore, cercarial
behaviors that bring E. caproni to the surface and
permit searching of both lighted and shaded micro-
habitats for a second intermediate host must have
been more successful over evolutionary time (Haas
et al., 2008; Platt et al., 2009).
ACKNOWLEDGMENT
The authors thank Dr. Richard J. Jensen, Depart-
ment of Biology, Saint Mary’s College, for his
advice, statistical expertise, and producing the
figures.
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4 COMPARATIVE PARASITOLOGY, 79(1), JANUARY 2012