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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: tplatt@saintmarys.edu).

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.

LITERATURE CITED

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Evans, N. A. 1985. The influence of environmentaltemperature upon transmission of the cercariae ofEchinostoma liei (Digenea: Echinostomatidae). Parasi-tology 90:269–275.

Evans, N. A., and D. M. Gordon. 1983. Experimentalstudies on the transmission dynamics of the cercariae ofEchinoparyphium recurvatum (Digenea: Echinostoma-tidae). Parasitology 87:167–174.

Haas, W., B. Beran, and C. Loy. 2008. Selection ofthe host’s habitat by cercariae: from laboratory

experiments to the field. Journal of Parasitology 94:1233–1238.

Keeler, S. P., and J. E. Huffman. 2009. Echinostomes inthe second intermediate host. Pages 60–87 in B. Friedand R. Toledo, eds. The Biology of the Echinostomes.Springer Science + Business Media, New York.

Loy, C., W. Motzel, and W. Haas. 2001. Photo- and geo-orientation by echinostome cercariae results in habitatselection. Journal of Parasitology 87:505–509.

McCarthy, A. M. 1999. Phototactic responses of thecercaria of Echinoparyphium recurvatum during phasesof sub-maximal and maximal infectivity. Journal ofHelminthology 73:63–65.

Meyrowitsch, D., N. Ø. Christensen, and O. Hindsbo.1991. Effects of temperature and host density on thesnail-finding capacity of cercariae of Echinostomacaproni (Digenea: Echinostomatidae). Parasitology102:391–395.

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Pechenik, J. A., and B. Fried. 1995. Effect of temperatureon survival and infectivity of Echinostoma trivolviscercariae: a test of the energy limitation hypothesis.Parasitology 111:373–378.

Platt, T. R., L. Burnside, and E. Bush. 2009. The role oflight and gravity in the experimental transmission ofEchinostoma caproni (Digenea: Echinostomatidae)cercariae to the second intermediate host, Biomphalariaglabrata (Gastropoda: Pulmonata). Journal of Parasi-tology 95:512–516.

Platt, T. R., H. Greenlee, and D. A. Zelmer. 2010. Theinteraction of light and gravity on the transmission ofEchinostoma caproni (Digenea: Echinostomatidae)cercariae to the second intermediate host, Biomphalariaglabrata (Gastropoda: Pulmonata). Journal of Parasi-tology 96:325–328.

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4 COMPARATIVE PARASITOLOGY, 79(1), JANUARY 2012