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More Than Meets the Eye: Do Himasthla Sp. B Cercariae Use Chemo-orientation?

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Introduction 
Beneath my feet rages a relentless war between predators and prey, parasites and hosts. I start out prostrated in mud, my nose mere inches away from the ground. My knees are sunk almost a foot beneath the surface, making odd squelching noises each time I attempt to move. Equipped with small mesh bags, I am searching for the most elusive organism in Santa Barbara's Carpinteria salt marsh: the sea slug Acteocina. With small, semi-translucent shells, these denizens of the salt marsh lurk beneath mats of algae, avoiding my prying fingers and searching eyes. Ah, the joys of scientific exploration! With mud in every crevice of my body, I continue my search for the tiny, four-millimeter-long slug. It takes me more than four hours to collect 100 of the slugs; at the end of the day I leave the salt marsh exhausted but triumphant.

Acteocina are just one part of the incredibly intricate salt marsh ecosystem. They play host to a variety of digenetic trematodes, which are parasitic flatworms, barely visible to the naked eye. More than 14 different species of aquatic digenetic trematodes live in the Carpinteria salt marsh; they exploit a variety of hosts during their life cycle (Lafferty, 316). Digenetic trematodes have complicated life cycles and use both mollusks and vertebrates as hosts. I wondered how these tiny parasites are able to find their specific hosts in each part of their life cycle. I admit that I once considered parasites to be the scourges of nature. However, after doing research and reading mounds of articles, I realized that parasites are incredible organisms that have evolved to exist in specific niches. I wondered how the digenetic trematode Himasthla sp. B finds its host amid the variety of distractions in this murky Santa Barbara salt marsh. Does the parasite navigate the muddy waters and infect the correct host through trial and error, or does it utilize more complicated methods to find its host?

Background Information 

Juvenile  Cerithidea californica .

Juvenile Cerithidea californica .


There are thousands of different species of digenetic trematodes, many of which employ extraordinary mechanisms in order to attract or find their next host. The life cycle of digenetic trematodes is complicated and includes two larval stages: miracidia and cercariae. Adult digenetic trematodes are found inside vertebrates like birds, dogs, frogs, and humans. Inside this vertebrate host, adult trematodes slowly release their eggs through the host's feces. The eggs hatch into the first larval stage, miracidia, which then encyst in the first intermediate host, usually a mollusk. Inside the first intermediate host, the miracidia release swimming clones, called cercariae, which bore out of the host and use miniscule tails to propel themselves through the water. The cercariae then infect the second intermediate host, almost exclusively a mollusk. The life cycle begins again when a vertebrate host consumes the parasite (Lafferty, 317).

Himasthla Sp. B  cercariae under a light microscope

Himasthla Sp. B cercariae under a light microscope


Himasthla sp. B  trematodes are digenetic trematodes that inhabit California salt marshes. Him. sp. B miracidia will infect only one species for its first intermediate host, the California horn snail, Cerithidea californica (Lafferty, 316).  C. californica  litter the surface of salt marshes and are anywhere from 15 mm to 30 mm in length and have opaque gray shells. Inside C. californica,  the miracidia rapidly produce clones that bore their way out of the snail and into the surrounding water. Using their undulating tails, the cercariae find and infect a suitable second intermediate host (Lafferty, 317).  Him. sp. B 's favored secondary intermediate host is believed to be the sea slug Acteocina. However, cercariae have also been found encysted in C. californica (the first intermediate host) and Spionid polychaetes (roundworms with small spines) .  Because Him. sp. B miracidia will only infect one species while the cercariae have been found encysted in at least three different species, it is believed that the cercariae are not as host-specific as the miracidia (Caullery, 15). Since the cercariae are not host-specific, most researchers believe that they find their hosts by trial and error, bumbling about their environment until they find a host by chance.

Hypothesis 

Identifying cercariae

Identifying cercariae


After trudging through the muddy salt marsh, I couldn't believe that Him. sp. B cercariae found their appropriate hosts by chance. Often aquatic organisms that live in murky water environments where their sense of smell and acuity of vision are compromised use chemical signals to aid in navigation and finding food (Bronmark, 103). In these environments, many organisms use chemo-orientation, the ability to respond to the chemicals released by other organisms, in order to compensate for their weakened senses. To the naked eye, Him. sp. B 's only sensory organs are the two black eyespots on its pearly white head, which distinguish light from dark. After observing Him. sp. B swimming in a dish of saltwater, I noticed that they displayed behaviors that are different from other species of trematodes. The cercariae swim coiled in a sphere, with only a tiny undulating tail to propel them. Occasionally, the cercariae will pause, uncurl, and then resume their swimming. After observing the behaviors of other digenetic trematodes, I noticed that these pauses—or "stops," as I call them—are characteristic of Him. sp. B alone.  I decided to start testing the behavioral changes in Him. sp. B cercariae while in the presence of a potential second intermediate host,  Acteocina,  in an attempt to determine whether the cercariae use chemo-orientation to find potential hosts.

 

Procedure 

Shedding  C. californica .

Shedding C. californica .


To test my hypothesis, I used  Him. sp. B  cercariae,  C. californica, and Acteocina, all collected from the Carpinteria salt marsh. In the salt marsh, all of these organisms inhabit the same environment; however, Acteocina is the preferred host for the cercariae. In the salt marsh, cercariae naturally bore their way out of their first hosts in response to increased heat. In the lab, cercariae have to be harvested artificially by mimicking the conditions that favor their release. To obtain Him. sp. B cercariae in the lab, I positioned C. californica in compartment boxes filled with seawater and heated them under fluorescent lamps for three hours. After three hours, I checked the compartments for Him. sp. B cercariae. Unfortunately, there are over 14 different species of digenetic trematodes that use C. californica as a host. Therefore, to obtain the necessary amount of cercariae for my experiments, I had to shed at least 80 snails at one time. The shedding process does not harm the snails, and most times the snails shed multiple times.

Test 1: Cercaria Stops in the Presence of  Acteocina  and in the Presence of  Acteocina -Treated Water 
For my first test, I decided to compare cercariae behavior while in the presence of Acteocina and in the presence of  Acteocina-treated water to their behavior in a control situation. For Test 1, I used three different tests: a control, a test with an  Acteocina slug, and a test with Acteocina-treated water. I positioned three 6-by-4-inch plastic well plates horizontally. Fifteen wells were used to perform the control tests, while another 15 were used to conduct the tests of water with  Acteocina, and 15 more wells were used to conduct the tests with Acteocina-treated water. Each set of 15 trials were performed three different times, yielding 45 results for each trial.

To complete my control tests, I filled 15 wells three-quarters full of seawater. I used a micropipette and extracted one  Him. sp. B cercaria and placed it into the first well of the control wells. Then I recorded the number of stops the cercaria made in two minutes. I repeated this test for the remaining 14 control wells.

I filled another set of 15 wells three-quarters full of seawater for the tests of water with  Acteocina. I placed one Acteocina into the water and then one cercaria. I recorded the number of times the cercaria stopped within two minutes. I repeated this test another 14 times in the remaining wells. Each set of 15 trials was performed three times, yielding 45 results for each round of experiments. I hypothesized that cercariae behavior would change (in comparison to the control results) in the presence of  Acteocina,  since the slug is their most favored host. If cercariae behavior changed while in the presence of its host, then it would suggest that the presence of Acteocina prompts the change.

For the Acteocina-treated water, I filled 15 wells three-quarters full with seawater. The tests with treated water that I conducted in my lab assessed cercariae behavior in water that had formerly held Acteocina. These tests were designed to test whether Acteocina was releasing a substance into the water that affected cercariae behavior. In earlier observations I had noticed that Acteocina,  like other slugs, release mucus into the water surrounding them. I decided to test whether cercariae behavior would change in the presence of slug mucus. I placed one Acteocina into each well and removed each slug after 30 minutes. I then placed one cercaria into each of the wells and recorded the number of stops made in two minutes. I repeated this test in the next 14 wells, and then each set of 15 trials was performed three times, yielding 45 results.

Test 2: Cercaria Stops in Presence of  Acteocina -Treated Water Without Mucus 

Acteocina sea slugs.

Acteocina sea slugs.


I performed two tests: a control test and an Acteocina-treated water test without slug mucus. I used the same protocol as in Test 1 for the control tests.

After my first series of trials with Acteocina-treated water,  I noticed that the cercariae would occasionally get stuck in the mucus left behind by the Acteocina.  This worried me because the cercariae would uncoil, but they were most likely uncoiling in response to being stuck in the mucus rather than in response to some chemical released into the water. To account for the mucus, I decided to assess the change in cercariae behavior in treated water that lacked the mucus. I filled 15 wells three-quarters full of seawater, and I placed one Acteocina into each of the wells for 30 minutes, after which I removed them. Using the micropipette, I removed the  top  1,000 ml of the water from the  Acteocina-treated well, being careful to not remove any of the mucus. The water was deposited in a new, unused well. I placed one cercaria in each  Acteocina-treated water well without mucus, and then recorded the number of stops made in two minutes. I then repeated each set of 15 trials three times to produce enough reliable data.

Analysis 
After running tests, I determined that the age of the cercariae was negligible in reference to my results. A one-way ANOVA test determined that time did not affect any of my results.
Disregarding the age differences in the cercariae, the mean number of stops made by the cercariae in the control water was 1.3 stops per two minutes. This number was consistent throughout the experiments. Simply by visual observation, I was able to detect a noticeable behavior change in the cercariae. The cercariae appeared to stop more frequently in the  Acteocina-treated water than the control test. The cercariae stopped an average of 7.5 times in the  Acteocina-treated water, and 11.2 times in the water with Acteocina.

Average number of Cercariae stops.

Average number of Cercariae stops.


I used a Kruskal-Wallis test (Chi square= 27.0, DF= 2, P<.0001) on the control results and the water with Acteocina. I determined that the results for the water with Acteocina were highly significant. According to these results, cercariae behavior changed significantly when they were in the presence of a slug. Since cercariae behavior changed significantly, I looked at my data for the Acteocina-treated water. The treated water contained  Acteocina mucus but no slug. A T-test (T- ratio = -1.93, DF = 28, P = 0.06) of the Acteocina-treated water and the water with Acteocina slugs revealed that the cercariae had a tendency to stop more in the water with  Acteocina. Apparently, the cercariae displayed a slight tendency to stop more often in the water with  Acteocina; however, any change in cercariae behavior in the water with Acteocina and in the  Acteocina-treated water was not statistically significant.

After my basic observations, I was worried that the large number of the stops the cercariae made in the Acteocina -treated water and in the water with  Acteocina  were not the result of chemo-orientation but of the cercariae getting stuck in the slug mucus. My second test, of treated water without mucus, accounted for this variable. Statistical analysis revealed that cercariae behavior in the treated water without mucus was almost the same as in the control test. Therefore, it appeared that the mucus was required for cercariae behavior to change.

Conclusion 
In previous experiments involving miracidia and chemo-orientation, the number of "turn-back swimming displays" was used as a standard to identify chemo-orientation in the miracidia of certain species (Haberl, 1999). Turn-back swimming displays were defined as the miracidia making complete, 180-degree turns while in the presence of certain stimuli. The "stops" that I used to assess changes in cercariae behavior appear to be unique to this species of trematode. As with any behavioral experiment, it is hard to single out variables and find a completely accurate method for measuring behavioral change. However, I felt that stops were easier to assess and possibly more accurate than counting turn-back swimming displays.

These experiments confirmed part of my hypothesis in that they proved that cercariae behavior changes while in the presence of an  Acteocina  slug .  Cercariae behavior also changes significantly in the presence of slug mucus; however, cercariae behavior does not change significantly when in  Acteocina- treated water without mucus. Therefore, the mucus is necessary for cercariae behavior to change. From my results, it appears that the behavior change in the cercariae is dependent on the presence of  Acteocina  mucus. Whether the change is dependent upon a chemical in the mucus, or is a result of the cercariae touching the mucus, is difficult to state.

In retrospect, I gained an incredible amount of experience from this experiment. I learned how to run my own tests and control variables, and I truly enjoyed working with the cercariae. I was able to experience research from start to finish, from collecting field samples to formulating a plan to drawing conclusions. I would like to thank my mentor, Brian Fredensborg, and the people of the Kuris Lab of the University of California at Santa Barbara for their unfailing help and insight.

Bibliography 
Bronmark, Christer, and Lars-Anders Hansson. "Chemical Communication in Aquatic Systems: An Introduction."  OIKOS  27 September 1999: 103-109.
Caullery, Maurice.  Parasitism and Symbiosis . London: Sidgwick and Jackson Limited, 1952.
Haberl, B., et al. "Host-finding in Echnostoma caproni: miracidia and cercariae use different signals to identify the same snail species."  Parasitology  (1999): 479-486.
Lafferty, K.D. "The ecology of parasites in a salt marsh ecosystem."  Parasites and Pathogens: Effects on Host Hormones and Behavior . N.E. Beckage and M. Zuk, eds. New York: Springer, 1997.

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