Author: Kayla Ruff
Peer Reviewer: Parsa Nayyara
Professional Reviewer: Dr. Shea Tuberty
Abstract
The purpose of this investigation was to determine the potential threat posed by the understudied invasive land planarian Bipalium kewense to North Carolinian ecosystems. It was hypothesized that the land planarian B. kewense will significantly impact North Carolina ecosystems because of its reproductive capabilities and its voracious predatory habits. To test the predatory behaviors of B. kewense, 15 specimens were placed in separate 669 mL containers along with one Eisenia fetida earthworm as a potential prey item. The artificial habitats were analyzed every two days to determine whether predation on earthworms was occurring. To test regeneration rates, 10 land planarians from the first part of the experiment were cut into anterior, medial, and posterior fragments of approximately the same size and placed in 3 separate containers. The segments were monitored every two days to determine survival and whether cephalization was occurring. Only 7% of earthworms survived after 10 days. Furthermore, 97% of the B. kewense fragments survived, and 27% of the specimens asexually reproduced without being cut into multiple pieces. To verify the statistical significance of this study result, a two-tailed Student’s t-test was performed. The P-Value was 0.00071. Additionally, survivorship (and total mortality) was plotted over time, a P-Value of 0.038 and R2 value of 0.92 in this ecological study indicate the presence of B. kewense significantly impacted earthworm survival percentages over time. Based on its reproductive potential and predatory habits, it can be concluded that B. kewense is a high-risk invasive species to North Carolina. Future study is needed to determine possible natural or artificial control methods and the usefulness of employing B. kewense as a biocontrol mechanism for invasive earthworms.
Introduction
Bipalium kewense (aka the hammerhead worm) is an invasive and predatory land planarian that has the potential to significantly alter native ecosystems in North Carolina (Choate & Dunn, 1998). Bipalium kewense is native to Southeast Asia and is thought to have arrived in the United States through the horticulture trade (Watermolen, 2008). In a French study regarding invasive land planarians, researchers recorded a total of 36 sightings of Bipalium kewense in mainland France as well as French territories overseas (Justine, et al., 2018). This indicates the global spread of Bipalium kewense and validates the need for research in other areas to determine the population distribution of Bipalium kewense. In North Carolina, there has been very little research on Bipalium kewense regarding its predatory behavior and potential ecological impacts. Bipalium kewense has been present in North Carolina since at least the 1950s (Bertone, et al., 2020), yet its impact on native ecosystems is virtually unknown.
The diet of B. kewense consists almost entirely of detritivorous earthworms which feed on decaying organic material (leaves, plants, and animals) and are essential to the overall productivity of soil. Without earthworms, soil organic matter content and overall soil quality would be greatly reduced (Clements, et al, 1991).
A study in New Zealand showed that hammerhead worms reduced yields of agricultural grass by about six percent (Murchie & Gordon, 2012). Despite the fact there are many invasive earthworms that are negatively impacting some ecosystems in North America, the earthworms found in Alexander County, North Carolina are vital for the health of soil ecosystems (Callaham, et al., 2002). For example, Eisenia fetida, or the red wiggler, is very commonly used in compost bins because of their ability to organically fertilize the soil (Heeter, 2019). It is possible that the presence and expansion of the invasive land planarian B. kewense could deplete earthworm populations and affect the overall health of soil ecosystems. Furthermore, the reproductive potential of B. kewense increases the likelihood that the population of hammerhead worms in North Carolina will continue to increase, making range expansion likely.
Land planarians are hermaphroditic, which means they have both male and female reproductive organs and can reproduce sexually and asexually by fragmentation. Because of their unique reproductive abilities, the planarian’s population is extremely difficult to control (Sluys, 2019). For instance, if someone were to step on a flatworm with the intent of killing it, it would likely break into several pieces with the potential of developing into several individual flatworms. Land planarians in North Carolina are little known and have not been studied extensively. In fact, the 15 specimens of B. kewense collected for this study were collected from Alexander County, North Carolina, which has no records for the presence of this species. Because of the potential of B. kewense to wreak havoc on soil ecosystems in North Carolina, it can be classified as a high-risk species which is a species that is likely to cause negative impacts in ecosystems (Mazzotti, Briggs-Gonzalez, n.d.). The research in this study is crucial in better understanding the predatory habits and reproductive potential of this invasive species. The results can be used to estimate predation rates and predict impacts on soil communities.
Methods:
Experiment 1: Predation
In this study, the predation rates of the land planarian Bipalium kewense were examined. To begin, 15 hammerhead worms were collected from a residential community in Alexander County, North Carolina at a latitude of 35.801310 degrees North and -81.316280 degrees West. The specimens were collected at night in early October by overturning rocks and woody debris and by examining areas of moss, leaves, and soil. The flatworms tended to be more active and easier to collect on warm moist nights. Each hammerhead worm was then placed in a separate well-ventilated 669 mL container with a damp paper towel, moss, and soil. The moss and soil were used to best simulate the natural habitat of the worms, while the purpose of the paper towel was to hold moisture. Then, fifteen Eisenia fetida earthworms were collected and placed in a container together with soil and leaves. The separate containers of terrestrial flatworms and earthworms were then placed in a dark room with a nearly constant temperature of 70 degrees for a 10-day incubation period. During this time, the survival rates of both the land planarians and the earthworms were recorded. Survival rates from this part of the study served as a control for comparison to data collected from the predation containers. Following the 10-day acclimation period, one earthworm was placed in each of the hammerhead worm habitats. The terrariums were checked every two days to determine if predation of earthworms had occurred. The number of surviving earthworms and predation events were recorded over the 10-day period. After 10 days, the predation rates were no longer being monitored and a study of regeneration rates in B. kewense was conducted.
Experiment 2: Regeneration
To test the rates of regeneration for Bipalium kewense, 10 of the hammerhead worm specimens from the first part of the experiment were selected. To begin, each specimen was placed on a dissecting tray and a scalpel was used to cut the flatworms into thirds to produce anterior, medial, and posterior segments. The segments were placed in separate, well-ventilated containers containing damp paper towels. There were three containers: one with the 10 anterior segments, one having 10 medial segments, and one with 10 posterior segments. The containers were monitored every two days to observe whether the fragments remained alive and if cephalization was occurring in the medial and posterior fragments. If cephalization were occurring, a head shape that is visible to the naked eye would be formed. Cephalization would indicate that the fragments were developing into individual flatworms with the capabilities of the original specimens.
Data Analysis and Statistical Methods
All data were recorded in Excel Spreadsheets and graphs were constructed based on the collected data. A two-tailed Student’s t-test was performed to determine the statistical significance of the impact of Bipalium kewense on the survival rate of earthworms during the predation study. Further statistical evidence was obtained by determining the R-squared value and P-Value for the regression line of survival rate of earthworms versus time when living in a terrarium with B. kewense.
Results:
Experiment 1: Predation Study
After the 10-day incubation period, 100 percent of the original 15 earthworms and 15 specimens of Bipalium kewense survived in the control treatment (Fig. 1A). In the experimental treatment, the survival rate of earthworms showed a sharp decline from 100% (15 earthworms) to 33% (5 earthworms) between the start of the experiment and Day 2 of the study. Predation rates slowed after Day 2 and the number of earthworms steadily decreased from 33% (7 earthworms) to 7% (1 earthworm) between Day 2 and Day 10 of the Predation Study (Fig. 1 B). The effect of the presence of B. kewense was statistically significant when compared to earthworm survival rates in the control group without B. kewense. A t-test yielded a P-Value of 0.00071 which is well below the 0.05 threshold. Additionally, a P-Value of 0.038 was calculated and an R2 of 0.9228 was determined from a regression analysis of the graph of survival rate versus time for earthworms in the presence of B. kewense (Fig.1 C). This indicates that the presence of B. kewense is significantly affecting the survival rate of earthworms over time. Furthermore, the hammerhead worms were very active hunters and moved quickly until they located the earthworms in the containers. They would then attack the earthworms and immediately wrap their bodies tightly against the earthworms (Fig. 1D). In many cases, the earthworm prey was noticeably larger than the B. kewense predators. Once the earthworms were secured, the B. kewense specimens would produce a large quantity of a mucus-like secretion that covered the prey (Fig. 1E).
Experiment 2: Regeneration Study
After 13 days, 100 percent of the posterior and medial fragments obtained from the original Bipalium kewense specimens survived. The survival rate of anterior fragments was 100 percent between days 1 through 7 but decreased to 90 percent on day 9 of the study. The survival rate for anterior fragments remained at 90 percent from days 9 through 13 (Fig. 2A). The cumulative percent survival of all fragments was 97 percent (29 out of 30). Observations for cephalization of the medial and posterior B. kewense fragments were made throughout the duration of Experiment 2 (Regeneration Study). Head development was first observed on the posterior fragments after 5 days and after 7 days on the medial fragments (Fig. 2B). All fragments were showing signs of cephalization or were fully cephalized after 12 days of initially being cut (Fig. 2C/D).
Discussion:
Based on the results of the predation and regeneration studies, Bipalium kewense has the potential to significantly alter soil ecosystems in North Carolina. The data collected from this study are extremely important for the future understanding of this invasive species. When earthworms were first placed with B. kewense, predation was almost immediate in some of the containers. Hammerhead worms definitely are not sit and wait predators based on observations in this study. The primitive hammerhead worms stalk, attack, and kill their prey much like lions, tigers, wolves, and other larger and more familiar predators. During predation, Bipalium kewense releases a mucus-like secretion that covers the prey. This tetrodotoxin slowly paralyzes the earthworms and possibly deters predators along with powerful digestive enzymes that liquify earthworm tissues that can be absorbed directly into the body of B. kewense (Ritson-Williams, et al., 2006).
The presence of Bipalium kewense caused a significant decline in the earthworm population in the experimental group when compared to the control group (Fig. 1A and 1B; p<.05). Only one earthworm survived in the experimental group after day 10 of the study; Whereas, 15 earthworms in the control group survived over a 10 day period. Conversely, survival rates of B. kewense specimens were the same in the control and experimental groups. The sharp decline of earthworms during the first 3 days of the predation study was due to predation by B. kewense. There was a strong negative correlation between earthworm survival over time due to the presence of B. kewense (Fig. 1C; P-value=0.038, R2=.9228). These results indicate Bipalium kewense is a voracious predator with potential to decimate earthworm populations.
The earthworms found in Alexander County and used in this study, Eisenia fetida, are crucial to increasing plant productivity (McLaughlin, 2009). According to this study, earthworm populations have the potential to decline significantly in the presence of B. kewense. Earthworm species such as Eisenia fetida improve soil quality by making soil rich in nutrients through their castings or excrement (Pennington, 2020). The nitrogen produced in earthworm castings is readily available for plants to use, thus increasing overall plant productivity (Lines-Kelly, 1993).
Conversely, the presence of invasive earthworm species such as Amynthas agrestis (also known as the Asian jumping worm) causes a significant depletion of soil quality and a decrease in plant productivity. Asian jumping worms diminish the top layer of leaf litter that covers forest floors which leaves soil bare and exposed (Sever, 2020). This changes the chemistry of soil, reduces protection for species that live on the forest floor, prevents the rebuilding of forests, and increases the introduction of invasive plant species (Price-Christensen, 2020). Researchers conducted a study in the Great Smoky Mountains National Park where they found the invasive jumping worms in over half of the sites they sampled (Ivey, 2016). This further provides evidence of the growing expansion of the Asian jumping worm and the need for control methods.
Observations in this study showed Bipalium kewense feeding on its prey above the soil surface which demonstrates that it could potentially be used as a biological control for invasive epigeic species such as Amynthas agrestis (Maddi, 2019). Ultimately, the results of this study indicate that Bipalium kewense is an efficient and voracious predator of earthworms with potential to negatively impact soil productivity in areas with beneficial earthworm species or possibly improve soil productivity in areas dominated by damaging or invasive earthworm species.
Cephalization was very rapid in all fragments (Fig. 2C/D). The 33 percent difference in cephalization rates between medial and posterior fragments could be due to the location of the cut sites that formed the fragments, but more research is needed on this topic. The high survival and development rates of B. kewense fragments demonstrate the remarkable resilience and reproductive capabilities of this invasive species. Results from this study indicate the expansion of B. kewense could be extremely difficult to control. Simply stepping on a flatworm to kill it may cause it to break into many separate fragments each of which could develop into a mature flatworm. In fact, 27 percent of the 15 original flatworms in this study self-fragmented in their individual containers without being cut. The regeneration study demonstrates that B. kewense has the potential to rapidly expand its range because of its reproductive and survival capabilities.
Conclusion:
Because their diet consists primarily of earthworms, Bipalium kewense could deplete earthworm populations and potentially disrupt native soil ecology and nutrient cycling. The reproductive capabilities of B. kewense make it very difficult to control. Potential predators of B. kewense include small snakes, salamanders, frogs, lizards, and birds; but previous studies have shown hammerhead worms to be very distasteful (Stokes et al., 2014). Thus, biological control of this species is unlikely. Future research is needed to fully realize the potential detrimental impacts of B. kewense and possible control methods. B. kewense has not been heavily researched by scientists, and they have attributes that could potentially be vital for future research. Because of their voracious appetite for earthworms, they could potentially be used as a biological control against invasive earthworm species. Their ability to regenerate and undergo cephalization is revolutionary for stem cell research (Gentile, et al., 2011). There have been studies regarding stem cells in aquatic planarians but not in land planarians (Karami, et al., 2015). Aquatic planarians have proteins similar to those found in humans, in addition to having pluripotent adult stem cells. Land planarians, however, have not been studied as heavily, and their contributions to research are unknown.
