Attempting to Solve the Life Cycle of Distoichometra bufonis Dickey 1921, (Cestoda: Nematotaeniidae) while Teaching a Field Course!
By Matthew Bolek, Associate Professor of Integrative Biology, Oklahoma State University
I just defended my PhD, and I was driving to my favorite place in the world, Cedar Point Biological Station (CPBS) in Western Nebraska, to teach a three week course on the Natural History of the Invertebrates. Field courses at CPBS are rather demanding, and attempt to expose students to as much biological diversity as humanly possible during a three-week time frame. In a typical CPBS field course, students attend class from 8:00 am to 9:00 pm or later, five days a week. In addition to the field work and class work, students work in groups, and develop an independent project on a specific topic of their choice. At the end of the three-week session, each group of students presents a 15 min scientific presentation and turns in a manuscript on their specific project formatted to be submitted to a journal.
During the five-hour drive from Lincoln Nebraska to CPBS, I remembered some words of advice from John Janovy Jr., my PhD adviser, indicating that the most educationally rewarding class projects are ones that are extremely challenging, and difficult for students to solve. As I brainstormed for a challenging project to engage the seven students, that were registered for my course, on the wonders of invertebrate zoology and field biology, I thought we should attempt to solve the life cycle of one of the most notorious tapeworms at CPBS and that was Distoichometra bufonis!
Distoichometra bufonis is a nematotaeniid cestode, and currently no life cycles are known for any member of this group of tapeworms. Nematotaenid cestodes infect frogs, toads, salamanders and lizards across the world, and D. bufonis is one of the most common parasites of Woodhouse’s toads at CPBS. Although the life cycle of D. bufonis is not known, previous work by one of John Janovy’s graduate students, Lee Hardin suggested that D. bufonis has a terrestrial life cycle and newly metamorphosed toads become infected when toads ingest some terrestrial invertebrate that is small enough for a 15 mm toad to eat! It seemed like the perfect invertebrate zoology project. My idea was, that over the three-week period, we would examine (1) the development of the tapeworm in toads, (2) the behavior of gravid proglottids, (3) evaluate the stomach content of toads for potential intermediate hosts, and (4) observe what terrestrial invertebrates visited toad feces containing gravid proglottids of D. bufonis!
That summer, we made some remarkable observations on the biology of D. bufonis. First, we discovered that as the proglottids of D. bufonis matured, they develop dense areas on the lateral sides and below the tegument, which appeared as dark spots under a microscope. More importantly, those dense areas were more pronounced in gravid proglottids but absent in immature proglottids (Fig 1). Additionally, mature and gravid proglottids contained paruterine capsules, located medially but with no opening for the eggs to be released. However, under cover slip pressure, the tegument above the dense areas of gravid proglottids disintegrated (Fig. 1), suggesting a mechanism for how the eggs of D. bufonis were released.
(Field Note continues below Fig. 1)
The second intriguing observation we made, was on the behavior of gravid proglottids. In petri dishes of water or sand, gravid proglottids actively crawled around but appeared to make no progress, commonly circling back to where they started (Fig. 2). However, when we observed the behavior of gravid proglottids released in toad feces, another biologically relevant observation was made. In each case, gravid proglottids actively crawl to the surface of toad feces, quickly compressed, became inactive and eventually dried (Fig. 2). Observations under a microscope, revealed that as the proglottids compressed the paruterin capsules containing the eggs were displaced laterally, and in the process eggs were released through the disintegrated tegument above the dark dense areas of each proglottid (Fig. 2)!
(Field Note continues below Fig. 2)
Finally, our field observations indicated that the only invertebrates visiting fresh toad feces were ants. Ants were observed removing insect remains out of toad feces and taking them back to their colonies. Importantly, our toad stomach analyses indicated that the same species of ants comprised up to 50% of Woodhouse’s toad’s diets! As you can imagine, we all wondered if ants could be the intermediate host for D. bufonis? Previous work by Lee Hardin indicated that young of the year toads recruited one to two tapeworms per week and in the process, toads consumed over 4000 invertebrates. So to find out we needed to dissect thousands of ants and find an unknown larval stage of D. bufonis. However, we had two problems, first, student projects were due in two days, and second, dissecting 3-5 mm ants proved to be extremely difficult. As a result, we decided to do one more experiment, and that was to find out if ants actually have an interest in D. bufonis proglottids. We collected freshly released gravid proglottids from infected toads, and carefully place them on coverslips, allowing the proglottids to compress and dry. We then marked the location of each proglottid on coverslips, placed the coverslips next to ant colonies and watched. And to our amazement, ants removed each proglottid one by one and took them back to their colony, suggesting ants might be ingesting the eggs of D. bufonis.
As a class, we never solved the life cycle of D. bufonis, but that summer was one of the most rewarding and educational teaching experiences in my academic career. A few years later, I attempted another try at solving the life cycle of D. bufonis at CPBS. I collected 45 newly metamorphosed toads from a location where I thought D. bufonis did not occur. I dissected 15 of those toads and all were negative for tapeworms. I then divided the remaining 30 toads into two equal groups of experimental and time T control toads and placed them into two separate tanks. Everyday for three weeks, I aspirated hundreds of ants from a location where I knew toads were infected with D. bufonis and fed those ants to the experimental toad group; whereas the time T control toads were fed hundreds of aspirated midges. At the end of the three-week session, I anxiously dissected my ant fed toads. Of the 15 toads I dissected, the last toad had a 5 mm D. bufonis in his small intestine! I was ecstatic!!!! However, my excitement quickly disappeared, when I discovered that one of my time T control toads contained one mature D. bufonis. And once again the life cycle of the notorious tapeworms at CPBS eluded me. That was my last summer teaching at CPBS. After all I was now an Assistant Professor at OSU, I needed to get tenure, and there was little time for solving the life cycle of a notorious tapeworm that resided in the small intestine of toads out in western Nebraska. If you are interested in more details about this project, you can visit the following website (http://www.matthewbolek.com/Teaching/NHI%202006d/index.html).