R, Lane - The Effect of Ammonium Nitrate on Dionaea muscipula Trap Closure

The Effect of Ammonium Nitrate on Dionaea muscipula Trap Closure

Abstract

The Venus flytrap (Dionaea muscipula) is known for its unique trap closure mechanism, allowing the plant to capture and digest prey as a nutritional supplement in nitrogen-poor environments. However, escalating fertilizer usage in the Venus flytrap’s natural habitat, the subtropical wetlands of North and South Carolina, threatens the species. This study focuses on the impact of NH4NO3, a common component of chemical fertilizers, on the Venus flytrap's trap closure mechanism. While previous work has focused on the effect of chemical fertilizers on Venus flytrap closure kinetics, they have failed to address the direct effect of NH4NO3 on the plant, offering an opportunity to isolate and understand the specific impact of this common fertilizer component on trap closure. This study exposed Venus flytraps to either NH4NO3 (100 mg in 5 mL water) or water (negative control). Their trap closure times were assessed over three days using a cotton string as a mechanical stimulus. This assay was optimized by a replicate study of Volkov et al.’s 2008 work on the effect of ion channel blockers, TEACl and BaCl2, on trap closure. Recordings of trap closure were rewatched to quantify the observable stages of trap closure, and three time values were reported: when the cotton string touched the second trigger hair, when the trap closure mechanism was activated, and when the Venus flytrap closed. The findings indicate that NH4NO3 significantly increases the total time required for trap closure; the mean closure time for Venus flytraps exposed to NH4NO3 was 3.82 seconds, while the negative control plants closed in 0.94 seconds (p < 0.05). These findings suggest that exposure to NH4NO3 may hinder the Venus flytrap’s ability to capture and digest prey efficiently, which may have cascading effects on the larger ecosystem. The underlying mechanism for how NH4NO3 affects trap closure times is a knowledge gap, but the results highlight the potential impact of human-induced nitrogen sources on Venus flytrap physiology.

Abstract

Presentation Recording

Background and Introduction

Methods

Results and Discussion

Supplemental Materials

Lane R. '23

Works Cited

Presentation Recording

Background and Introduction

In nutrient-rich soil, plants can synthesize amino acids from nutrients acquired from the surrounding soil (Okumoto et al., 2011). Typically, plants convert inorganic nitrogen (NO3- and NH4+) from the soil into nitrogen-containing organic molecules, including amino acids (Okumoto et al., 2011). Once synthesized, amino acids are stored in sink organs (e.g., developing roots and leaves, flowers, and seeds) and support the plant's growth, development, and nutrition (Pate et al., 1981; Hildebrandt et al., 2015). However, carnivorous plants, or CPs, are almost exclusively found in nutrient-poor sites, such as swamps and bogs (Fagerberg et al., 1991). As a result, CPs have evolved to acquire nutrients through "reduced," or supplementary nitrogen sources, such as insects (Fagerberg et al., 1991). This capacity gives CPs a competitive advantage in nutrient-poor environments by allowing them to obtain amino acids from their prey (Brewer et al., 2011). Thus all CPs have the same goal to ensure their survival: attracting, capturing, and digesting prey (Juniper et al., 1989).

Despite their shared objective, CPs employ a variety of mechanisms, including both passive and active approaches. For example, the pitcher plant (Nepenthes) utilizes a "pitfall" trap to capture its prey (Mithöfer, 2017). The pitcher plant secretes a sticky fluid that captures the insects that fall into its large basin-like trap, allowing the plant can easily capture and retain its prey (Kang et al., 2021). Meanwhile, the Venus flytrap (Dionaea muscipula) utilizes a "snap trap" to capture its prey (Mithöfer, 2017). The Venus flytrap's excitable capture mechanism has intrigued scientists for decades. Darwin was the first to report the Venus flytrap's ability to imprison prey by rapidly closing its trap when sensory trigger hairs are activated, which has inspired an array of modern-day research projects and publications (1875).

Research on the Venus flytrap is particularly relevant now as the species is at risk of extinction. In 1979, there were approximately 4 million Venus flytraps in North and South Carolina, yet, by 2015, less than 35,000 estimated flytraps remained (Waller et al., 2016). Indigenous Venus flytraps are found exclusively in the subtropical of North and South Carolina, limited to fourteen counties across the two states (U.S. Fish & Wildlife Service, 2022). According to the North Carolina Wildlife Organization, nutrient runoff poses a significant threat to these ecosystems (2015). Additionally, research has shown over a 400% increase in fertilizer usage in the last 60 years (Howard et al., 2012).

High levels of nutrient runoff often cause increased levels of nitrogen, one of the most abundant chemicals in nutrient runoff, in surrounding soil and water (Environmental Protection Agency, 2019). As stated earlier, carnivory in Venus flytraps is an adaptation that responds to the nutrient-deficient soils of the Carolina coastal plains (Brewer et al., 2011). Therefore, Venus flytraps are potentially disadvantaged in nutrient-rich soils (Brewer et al., 2011). Research on these disadvantages is crucial for developing strategies that can aid the Venus flytrap’s survival. Howard et al. studied the effect of soil nitrogen levels on trap closure (2012). They watered Venus flytraps with a fertilizer solution (10 grams of commercial fertilizer in 1 gallon of distilled water), collecting data on the total duration of trap closure, which is the time between the stimulation of the trigger hairs and when the lobes ceased to move (Howard et al., 2012). They found that plants grown in nitrogen-sufficient soil (22.5-30 ppm) closed more slowly (16.12 seconds) than those grown in nitrogen-deficient soil (7.5-15 ppm) (3.58 seconds) (Howard et al., 2012). Howard et al. argue that, as nitrogen levels increase in the coastal plains of North and South Carolina, Venus flytraps may become less responsive to prey (2012). Understanding the physiology of trap closure is necessary for studying the effects of nutrient runoff on the Venus flytrap, as the plant's survival relies on this mechanism (Brewer et al., 2011).

Recently, studies have revisited the original ideas surrounding the physiology of trap closure proposed by Darwin in the 19th century. In 1991, Fagerberg et al. defined three distinct stages of trap closure: capture, appression, and sealing. Capture is the only rapid movement of the trap (appression and sealing happen over 30 minutes), which occurs immediately after the trap is stimulated, lasting 0.3 to 0.5 seconds (Fagerberg et al., 1991; Volkov et al., 2008). The kinetics of capture is most relevant; the faster the Venus flytrap closes, the more likely it will trap an insect and successfully acquire nutrients from its prey (Fagerberg et al., 1991). The mechanics of capture are particularly relevant to trap closure kinetics research, which breaks down the process into three distinct sub-sections: stimulus perception, electrical signal transmission, and induction of mechanical and biochemical responses (Volkov et al. 2008). Stimulus perception, the first substage of capture, is characterized by a mechanically silent period with no observable movement after the trap has been exposed to a physical stimulus (Volkov et al., 2008). The second stage of capture is distinguished by the contraction of the upper and lower lobes of the trap (Volkov et al., 2008). In the third subsection of capture, the lobes tighten, snapping the trap shut (Volkov et al., 2008). In order to fully understand the mechanics of this unique behavior, scientists also investigated the underlying physiological changes that elicit such a complex response.

Contact with prey triggers and action potential (AP) in Venus flytraps, which is a rapid change in voltage across a membrane (Henrich et al., 2018; Sukhov et al., 2011). Because two action potentials are required to trigger trap closing, a stimulus must activate two unique trigger hairs before the plant initiates the closing mechanism (Brown, 1916). To conduct these electrical signals, Venus flytraps have evolved to have a network of nerve cells capable of long-distance electrical signaling (Fromm, 2012; Hedrich et al., 2016). When a stimulus makes contact with a Venus flytrap, an electric current is generated, transmitting signals throughout the phloem to activate trap closure (Hedrich et al., 2016). This stage typically lasts 0.068 seconds (Volkov et al., 2008), and was supported by Hedrich et al.’s multiple measurements of phloem potential specifically in sieve tubes (2016). Therefore, the Venus flytrap's ability to detect and act on prey relies on AP generation facilitated by the phloem network (Hedrich et al., 2016). In plants, the phloem facilitates the transportation of sugar and the flow of ionic solutes, allowing it to be a suitable conduit for electrical currents (Armstrong & Hille, 1998). Thus, for Venus flytraps, the phloem can be compared to a "wired network" that transmits signals similar to how electrical signals are conducted through neurons in animals (Hedrich et al., 2016). However, it’s important to understand the importance of ion channels for generating electrical signals on a molecular level.

In Venus flytraps, a stimulation of trigger hairs activates mechanosensitive ion channels, which are transport proteins that allow ions to diffuse across the phospholipid bilayer of cell membranes (Henrich et al., 2018); (Sibaoka, 1969). Many ion channels in Venus flytraps are gated channels, ion channels that open and close in response to a physical stimulus (Henrich et al., 2018; Sibaoka, 1969). Like most cells, the cellular membranes of Venus flytraps are phospholipid bilayers (Brown, 1916). However, charged molecules, such as those transmitted via AP, cannot diffuse through cell membranes as they are repelled by the hydrophobic tails of the phospholipid bilayer making ion channels integral to the trap closure mechanism (Henrich et al., 2018). In Venus flytraps, Ca2+ and K+ Ion channels are integral to the trap closure mechanism (Hedrich et al., 2018). Therefore, observable changes in trap closure are almost always related to the physiology of ion channels (Hedrich et al., 2018). Volkov and colleagues found that even millimolar solutions of ion channel blockers, like BaCl2 and tetraethylammonium chloride (TEACl), decreased the speed of trap closure (2008). For example, when traps were exposed to 20 μL 10 mM TEACl, the length of the plant’s mechanical response, the third stage of capture, increased from 0.09 seconds to 0.28 seconds. Ultimately, without functional ion channels, Venus flytraps would not be able to close their traps and effectively capture prey (Henrich et al., 2018).

Venus flytraps are a suitable model organism for investigating the mechanical capture of insects in CPs. Capture mechanics can be quantified by analyzing observable changes in Venus flytrap behavior (Volkov et al., 2008). By recording data when (1) the stimulus triggers the second trigger hair, (2) the lobes (the upper and lower leaf) first cave in, and (3) the initial contact between lobes, Volkov et al. could distinguish the subsections of capture (2008). Additionally, Venus flytraps can be artificially stimulated through various chemical and mechanical stimuli (Volkov et al., 2008). Volkov et al. found that using a mechanical stimulus like a cotton thread can be a straightforward and replicable artificial stimulus; they reproduced the same results 109 times on different Venus flytrap plants using a cotton thread while only producing 34 replicates with gelatin, a chemical stimulus (2008).

This year, I will research the effect of soil nitrogen levels on trap closure, partially replicating Howard et al.’s 2012 study. However, I will apply Volkov et al.’s methods to analyze data on the three subsections of capture: stimulus perception, electrical signal transmission, and induction of mechanical and biochemical responses (2008). This study will address knowledge gaps on the effect of nutrient runoff on the subsections of capture. The hypothesis of this work is that Venus flytraps grown in nitrogen-sufficient soil will be less responsive to physical stimuli than those grown in nutrient-poor soil because there is less need to supplement nitrogen through prey (Howard et al., 2012). Last year’s research on ion channel blockers informed my understanding of trap closure kinetics; to best understand how anthropogenic sources, such as nutrient runoff, affect the Venus flytrap, we must study the physiology of trap closure.

Studying trap closure is especially relevant to general Venus flytrap conservancy as the plant relies on prey to survive. Venus flytraps have evolved to survive in a specific microenvironment, living only in the nutrient-poor bogs of North and South Carolina (DuMond, 1973). So if nutrient levels increase in the surrounding soil, carnivory, and therefore trap closure, will no longer provide a selective advantage for the Venus flytrap (Luken 2007). Additionally, as a photosynthetic predator, Venus flytraps play a distinct role in their microenvironment to regulate insect populations. Therefore, if Venus flytraps go extinct, these bogs will lose a vital predator, which will likely disrupt the predator-to-prey balance in these bogs (Howard et al., 2012). Venus flytraps are also one of the first species to regenerate in bogs after forest fires, which is essential to the bog’s resistance to natural disasters and recovery, especially in light of increased forest fires due to climate change (Luken 2007). As the most threatening anthropogenic crisis, climate change, increases the threat and scale of natural disasters this quality will become even more crucial (Lopez et al., 2015). Ultimately, the extinction of the Venus flytrap would be a significant ecological loss that could be avoided through a deeper understanding of trap closure in the presence of excess NH4NO3, a common nitrogen source used in commercial fertilizers.

Methods

Safety and Precautions

Personal protective equipment (including goggles, a lab coat, and gloves) was worn when handling NH4NO3 (MSDS), BaCl2 (MSDS), and tetraethylammonium chloride (TEACl) (MSDS) and while testing Venus flytraps exposed to NH4NO3, BaCl2, and TEACl to prevent skin and serious eye irritation. Additionally, BaCl2 is highly toxic if ingested. PPE was also worn during soil tests to prevent skin and eye irritation from LaMotte Nitrogen CTA Tablets (Product # 1382) (MSDS) and Floc-Ex Tablets (Product # 5504A) (MSDS).

General Overview

Preliminary research in 11th grade was based on Volkov et al.'s 2008 work on the effects of ion and water channel blockers on the kinetics of Dionaea muscipula (Venus flytrap) closure. Two 10 μL drops of 5 mM BaCl2 and 10 mM tetraethylammonium chloride (TEACl) were placed on the midrib of the trap using a P-20 micropipette. In 12th grade, the experiment monitored the effect of adding 100 g of NH4NO3 to soil on the kinetics of trap closure over three consecutive days.

Model Organism and Care

Adult-sized Venus flytraps were purchased from Joel's Carnivorous Plants (Product # B0BBWGGK55). The Venus flytraps were shipped with their rhizomes wrapped in a wet paper towel to preserve moisture. Upon arrival, the plants were transferred to 250 mL plastic nursery pots with a 1:2 perlite-peat moss soil blend (i.e., 14 grams of perlite and 28 grams of peat moss to create 48 total grams of the substrate). This setup facilitated high water retention and proper drainage, and emulated the flytrap's natural, acidic bog habitat (Brittnacher, 2019; Joel's Carnivorous Plants). The soil's mass was determined using a Carolina Electronic Balance Scale (Product # OB2143). Each Venus flytrap was labeled with a letter so the plant could be easily identified throughout the experiment. Groups of three to four potted Venus flytraps were arranged in 10 x 20 inch propagation trays, each filled with 1 L of reverse osmosis (RO) water. RO water was used because Venus flytraps are highly sensitive to the minerals and other impurities found in tap or mineral water (Brittnacher, 2019; Joel's Carnivorous Plants). Each tray was then covered with a humidity dome to maintain high humidity levels essential for the plant's growth (Carolina Biological Supply, 2012). In between trials, the trays were stored on a Johnny's Seeds seedling light cart (Product # 7026), set to a 12:12 hour light-dark photoperiod with four 40 W lights positioned 36 cm above the plants (Brittnacher, 2019) (Image 1).

Image 1: Growing conditions replicate the Venus flytrap's natural environment, subtropical wetland bogs, in a classroom setting.

Due to evaporation, 500 mL of RO water was added to each propagation tray weekly. Additionally, water was replaced entirely every four weeks, or immediately if white mold was identified in the water, to maintain water quality and prevent the growth or spread of mold (Joel's Carnivorous Plants). If mold was identified directly on the plant, any infected traps were pruned using dissection scissors (Joel's Carnivorous Plants). The plant was quarantined in a separate propagation tray to prevent the mold from spreading to healthy plants (Joel's Carnivorous Plants).

Replicate Study

To measure the effects of TEACl and BaCl2 on the rate of trap closure, a 10 mM solution of TEACl and a 5 mM solution of BaCl2 were prepared (Volkov et al., 2008). Using a Flinn Scientific Analytical Balance Scale (Product # OB2160), 828 mg of TEACl (Sigma Aldrich; Product # 3563247) and 610 mg of BaCl2 dihydrate (Flinn Scientific; Product # B0004) were measured. Both compounds were added to volumetric flasks with 500 mL of RO water to create their respective solutions. Both solutions were covered with parafilm and stored between dosings in a dry and well-ventilated location. RO served as the negative control.

To prepare for data collection, two 10 μL drops of either water, 10 mM TEACl, or 5 mM BaCl2 were placed on a Venus flytrap’s midrib, the interior of the trap where the leaf hinges, using a P-20 micropipette (Image 2). Before dosing the Venus flytraps, a photo of each plant was taken. Any traps accidentally triggered by the micropipette were reported, as those particular traps may not have been dosed with the solution. Additionally, photos of each trap before and after (22 - 26 hours later) dosing were captured and stored in a Pre-Dosing vs. Post-Dosing table in case exposure to either TEACl or BaCl2 changed the appearance of the trap.

Image 2: Solution on midrib.

The next day, traps were tested by brushing two trigger hairs with an 8 cm cotton string (Volkov et al., 2008). Only fully open traps of at least 2.5 cm in size were tested. Traps under 2.5 cm are often immature and may not have fully developed trap-closing mechanisms. Therefore, they may not respond to stimuli in the same way as mature traps (Howard et al., 2012). A tripod-mounted iPhone 11 was placed directly in front of a plant to record a focused video of each trap's closure upon mechanical stimulation with a cotton string. The string was held lengthwise above the trap, dropped into it, and then quickly removed by pulling down, and sliding across the trap to activate the trigger hairs (Volkov et al., 2008). After each trial, the camera was repositioned to capture the next trap, and the process was repeated. Each downward-facing trap was propped upward using a 5 cm plastic plant tag placed in the soil beneath it. This allowed a clear angle of the interior of the trap to be easily captured on camera. A Google Drive link to each video was stored in a table titled “Videos of Trap Closing Stimulated by Cotton Thread” with the date, plant, and trap number.

Negative Control (N-Depleted Soil)

To monitor the effects of NH4NO3 on the rate of trap closure, each Venus flytrap was separated into two groups: one treated with 100 mg of NH4NO3 (experimental group) and another treated with only RO water (negative control). The negative control group was placed in a propagation tray labeled "Group 1 - N-Deficient Soil," and the experimental group was placed in a tray labeled "Group 2 - N-Sufficient Soil" to help differentiate between the two groups of plants. 5 mL of RO water was added to a nursery pot containing 45 g of soil substrate and one Venus flytrap. Peat moss and perlite are naturally nitrogen-depleted soil sources, generating N-depleted conditions for the Venus flytraps (Joel's Carnivorous Plants). After being dosed with RO water, the traps were tested, as previously described, to document any changes in the rate of trap closure.

Experimental Arm (N-Sufficient Soil)

The experimental arm investigated the effect of NH4NO3 added to the soil on the rate of trap closure. Howard et al. exposed Venus flytraps to 14-14-14 NPK fertilizer to study the effect of soil nitrogen levels on the rate of trap closure (2012). Although Howard et al. exposed the Venus flytraps to all the nutrients found in 14-14-14 NPK fertilizer, they only monitored the soil’s nitrogen levels due to the plant’s particular sensitivity to the nutrient. By monitoring only soil nitrogen levels, Howard et al. was likely able to observe how these plants react to changes in their environment that might affect their traditional nitrogen acquisition strategies (i.e., catching and digesting insects). NH4NO3 has been known to support plant growth and is commonly found in commercially available fertilizers, including 14-14-14 NPK fertilizer (Errebhi et al., 1990). Thus, substituting NH4NO3 is sufficient as it is the nitrogen source in 14-14-14 NPK fertilizer, eliminating potential confounding effects of other nutrients. After using an analytical balance scale to measure 100 mg of NH4NO3, the chemical was dissolved in 5 mL of RO water. The solution was gradually added to a nursery pot containing 45 g of soil substrate and one Venus flytrap in approximately 1 mL doses to prevent it from draining through the pot. The first round of data collection (i.e., triggering the traps) occurred 22 to 26 hours after the initial dosing of NH4NO3. The traps were rested daily for the next two days to ensure that the Venus flytraps had sufficient time to absorb the NH4NO3. Additionally, this three-day period allowed for a qualitative assessment of the NH4NO3's effect on overall plant health. The goal of the experimental design was to study the effect of NH4NO3 on Venus flytrap closure without subjecting the plants to harm.

Soil Nitrogen Assay

A LaMotte NPK Soil Test (Product # 3-5880) was used to determine the nitrogen concentration in the soil from a scale of N0 to N4 (Howard et al., 2012). Soil tests were completed on the first (i.e., the first day of testing the traps) and third day of data collection (i.e., the last day of testing the traps) to ensure the plant was growing in soil with the appropriate nitrogen level. Following the manufacturer’s instructions, 4 g samples of soil from each pot were measured into a plastic weigh boat using a Flinn Electronic Balance (Product # OB2143). Each sample was labeled with the plant's letter and placed in a Hamilton Beach Food Dehydrator (Product # 32100) at 40 °C for 1 hour. Then, each dried soil sample was added to a 15 mL centrifuge tube with 15 mL of RO water and a LaMotte Floc-Ex Tablet (Product # 5504A) and mixed. Each test tube was labeled with the plant's letter to track each soil sample between steps. The samples were then arranged in a Premiere XC-1000 Bench-Top Centrifuge (4000 RPM) (Product # BZA635853) for two minutes to allow the soil to settle to the bottom of the tube. Using a 3 mL dropper pipette, 10 mL of the supernatant was transferred to a LaMotte Square Test Tube (Product # 0106). The supernatant was treated with one LaMotte Nitrogen CTA Tablets (Product # 1382), mixed, and placed in a protective sleeve (LaMotte; Product # 0106-FP) for 5 minutes. Then, the solution color was compared to the LaMotte Nitrogen Color Chart (Product # 1382). N-0 indicated nitrogen-depleted soil (≈ 0-7.5 ppm); N-1 indicated nitrogen-deficient soil (≈ 7.5-15 ppm); N-2 indicated nitrogen-adequate soil (≈ 15-22.5 ppm); N-3 indicated nitrogen-sufficient soil (≈ 22.5-30 ppm) (LaMotte, n.d.). The soil nitrogen concentration and a photo of the soil test result were recorded and stored in a table titled “Soil Nitrogen Concentration.”

Data Analysis

For more information on the mechanical stimulation and camera setup for data collection, refer to the "Replicate Study" section above. Once testing concluded, all video recordings were rewatched, and the following time values were reported: (1) when the cotton thread touched the second trigger hair, (2) when the trap closure mechanism was activated, and (3) when the Venus flytrap closed (Image 2) (Volkov et al., 2008). The change of the leaf curvature from convex to concave characterized the second value (Volkov et al., 2008). The third value was determined when there was no remaining space between the trap's upper and lower lobes. The times for each data point were recorded and stored in Google Sheets. The second value was subtracted from the first value to find the time required for trap closing to initiate (A), and the third value was subtracted from the first value to find the time required for the trap to fully close (B) (Image 3). The values for the trap activation time (A) and trap closure time (B) were calculated automatically by Google Sheets using the values for stages 1, 2, and 3.

Image 3: The three observable stages of trap closure used for data analysis.

Then, the mean closure times for both N-sufficient and N-depleted conditions were calculated (values A and B) and compared using a two-tailed independent t-test to determine if the results were statistically significant. Additionally, the same trap was compared across multiple days using a correlated two-tailed t-test (i.e., the delay in trap closure and the time required for the trap to close for the same trap across the first, second, and third days of data collection). If a trap fell out of frame, or the video became too blurry to determine the stages of trap closure, the data for that trap was omitted as it was no longer possible to accurately record values for times 1, 2, and 3. Additionally, if no movement was observed after 60 seconds, "no closure" was recorded in the analysis table, and the trap’s data was omitted from the average (Howard et al., 2012). If an individual trap does not close, it may be a sign that the trigger hairs on that particular trap are damaged or not functioning properly (Volkov et al., 2013). If many traps on a Venus flytrap refuse to close, it could indicate poor overall health for the entire plant (Joel's Carnivorous Plants).

Results and Discussion

Preliminary research in 11th grade was based on Volkov et al.'s 2008 work on the effects of ion channel blockers (TEACl and BaCl2) on the kinetics of trap closure. To quantify video recordings of trap closure, each video was rewatched, and the following three values were reported: (1) when the cotton thread touched the second trigger hair, (2) when the trap closure mechanism was activated, and (3) when the Venus flytrap closed (Volkov et al., 2008). For the positive control (10 mM TEACl) and experimental arm (5 mM BaCl2), 2 10 µL drops of solution were placed on a trap's midrib 24 hours before testing (Volkov et al., 2008).

Figure 1A: Average Trap Activation Time (s) 24-Hours Post-Midrib Dosing (10 mM TEACl vs. H2O)

Figure 1A represents the mean activation time (s) after exposure to a mechanical stimulus for Venus flytraps dosed with 2 10 µL drops of either water (blue bar; n = 12) or 10 mM TEACl (green bar; n = 9). Error bars represent the standard deviation for the mean of each data set. This data was analyzed using a two-tailed independent t-test, and no significant difference was observed in trap activation time (p = 0.11; p > 0.05).

Figure 1B: Average Trap Closure Time (s) 24-Hours Post-Midrib Dosing (10 mM TEACl vs. H2O)

Figure 1B represents the mean closure time (s) for Venus flytraps dosed with 2 10 µL drops of either water (blue bar; n = 12) or 10 mM TEACl (green bar; n = 9). Error bars represent the standard deviation for the mean of each data set. This data was analyzed using a two-tailed independent t-test, and a significant difference was observed in trap closure time (p = 0.0012; p < 0.05).

When activated, the Venus flytrap's trigger hairs act as mechanosensors, generating an electric signal that acts as an action potential, ultimately reaching the motor cells responsible for closing the trap (Volkov et al., 2007). TEACl is a known K+ channel blocker in plants; exposure to TEACl interferes with the changes in turgor pressure required for trap closure by inhibiting the efflux of K+ ions from the plant’s cells (Volkov et al., 2000). In this case, TEACl served as a positive control because the mechanism for how the compound impacts trap closure is already well-understood. Volkov et al. found that 2 10 µL drops of 10 mM TEACl placed on the midrib of the trap 24 hours before data collection “dramatically decreased the speed of trap closure” but observed no increase in activation time (2008).

Like Volkov et al., an increase in trap closure time was also observed for traps exposed to TEACl but no increase in trap activation time was observed during the replicate study. The mean activation time was 0.46 seconds and 0.36 seconds for Venus flytraps dosed with water and 10 mM TEACl, respectively (Figure 1A). The means were analyzed using a two-tailed independent t-test, and no significant difference was found (p = 0.11; p > 0.05). Volkov et al. reported a trap activation time of ≈ 0.5 seconds for Venus flytraps exposed to TEACl and the negative control (2008). The mean time trap closure time was 0.86 seconds and 1.42 seconds for Venus flytraps dosed with water and 10 mM TEACl, respectively (Figure 1B). The means were analyzed using a two-tailed independent t-test, and a significant difference was found (p = 0.0012; p < 0.05). Volkov et al. found a mean closure time of approximately 0.5 seconds and 1.3 seconds for Venus flytraps dosed with water and 10 mM TEACL, respectively.

Figure 1C: Average Trap Activation Time (s) 24-Hours Post-Midrib Dosing (5 mM BaCl2 vs. H2O)

Figure 1C represents the mean activation time (s) after exposure to a mechanical stimulus for Venus flytraps dosed with 2 10 µL drops of either water (blue bar; n = 12) or 5 mM BaCl2 (red bar; n = 9). Error bars represent the standard deviation for the mean of each data set. This data was analyzed using a two-tailed independent t-test, and no significant difference was observed in trap activation time (p = 0.76; p > 0.05).

Figure 1D: Average Trap Closure Time (s) 24-Hours Post-Midrib Dosing (5 mM BaCl2 vs. H2O)

Figure 1D represents the mean closure time (s) for Venus flytraps dosed with 2 10 µL drops of either water (blue bar; n = 12) or 5 mM BaCl2 (red bar; n = 9). Error bars represent the standard deviation for the mean of each data set. This data was analyzed using a two-tailed independent t-test, and no significant difference was observed in trap closure time (p = 0.76; p < 0.05)

Like TEACl, BaCl2 is a K+ channel blocker in plant, but BaCl2 exposure can act as a Ca2+ channel inhibitor (Volkov et al., 2007). Ca2+ channel play an important role in the signal transduction from the trigger hairs after exposure to a stimulus to the cells responsible for trap closure (Hodrick and Sievers, 1988). For the trap closure process to commence, mechanically-gated channels at the base of each trigger hairs open, leading to an influx of Ca2+ ions (Volkov et al., 2007). The opening of Ca2+ channels helps propagate the action potential, eventually leading to the opening of voltage-gated K+ channels (Proko et al., 2021). Therefore, by inhibiting Ca2+ channels, exposure to BaCl2 should induce a delay in trap closure (Volkov et al., 2007). Volkov et al. found that 2 10 µL drops of 5 mM BaCl2 placed on the midrib of the trap 24 hours before data collection resulted in a 3-second delay in trap activation, but caused no increase in trap closure time (2008). Although Ca2+ channels are crucial to AP propagation from the trigger hairs to the rest of the trap, BaCl2 was selected as the experimental arm because the effects on trap closure are less well-known as the overall trap closure mechanism is more directly tied to the action of the K+ channels, which is directly impacted by TEACl exposure.

The mean trap activation time was 0.46 seconds and 0.44 seconds for Venus flytraps dosed with water and 5 mM BaCl2, respectively. The means were analyzed using a two-tailed independent t-test, and no significant difference was found (p = 0.76; p > 0.05). The time required for trap closure was 0.86 seconds (water) and 0.89 seconds (BaCl2), showing no significant difference as determined by the same statistical analysis (p = 0.76; p > 0.05). These results demonstrate that BaCl2 had no significant observable effect on any part of Venus flytrap closure during the replicate study, which was highly unexpected.

However, because there were statistically significant results for Venus flytraps dosed with TEACl, there may have been an issue with the BaCl2 itself rather than an underlying issue with the experimental design or set-up. Therefore, the trap closure assay (using a cotton string as a mechanical stimulus) and data analysis methods adapted from Volkov et al.’s 2008 study were verified by the results for TEACl and the negative control during the replicate study. Despite the successful TEACl trials, applying solution directly to the Venus flytrap’s midrib had limitations. In particular, confirming whether the solution had penetrated the plant was impossible. Therefore, the senior year work moved to soil-based dosing, which can be more easily measured.

Figure 2: Diagram of Experimental Set-Up: Negative Control vs. Experimental Arm (NH4NO3 vs. H2O)

This diagram illustrates the experimental design for the senior year research on the effect of NH4NO3 on trap closure. On Day 1, Venus flytraps were exposed to either 100 mg NH4NO3 in 5 mL water or 5 mL water added to the soil. Both groups of Venus flytraps were mechanically stimulated on Days 2, 3, and 4 to measure trap closure times. The bar graph represents the experiment’s expected results: Venus flytraps exposed to NH4NO3 (purple bars) would exhibit longer trap activation and closure times than those exposed to water (blue bars).

With a reliable measure of trap closure, the senior year research focused on the effect of increased fertilizer usage and nutrient runoff on the Venus flytrap by studying the impact of NH4NO3, a common component of chemical fertilizers, on the Venus flytrap's closure mechanism. Figure 2 depicts the experimental set-up of the senior year research on the effect of NH4NO3 on trap closure. The hypothesis was that Venus flytraps exposed to NH4NO3 would exhibit a longer trap activation and closure times time because increased nitrogen in the substrate would decrease the plant’s need for insect capture, possibly resulting in reduced efficiency of trap closure. Although traps were tested daily for three consecutive days, after performing a two-tailed correlated t-test, no statistical significance was found in trap activation time, trap closure time, or total time across the three days (for total time for all combinations, p > 0.05: Day 1 vs. Day 2, p = 0.72; Day 2 vs. Day 3, p = 0.59; Day 1 vs. Day 3, p = 0.79). Because of this, data was pooled from all three days, and those means were compared.

Figure 3: Average Total Time (s) of Trap Closure (s) (NH4NO3 vs. H2O)

Figure 3 represents the mean total closure time (s) for Venus flytraps exposed to either 5 mL of water (blue bar; n = 24) or 100 mg of NH4NO3 dissolved in 5 mL of water (purple bar; n = 25). This data is pooled across three days of data collection. Error bars represent the standard deviation for the mean of each data set. This data was analyzed using a two-tailed independent t-test, and a significant difference was observed in trap activation time (p = 2.7 x 10-20; p < 0.05).

Figure 3 represents the mean total closure time (s) for Venus flytraps exposed to either 5 mL of water or 100 mg of NH4NO3 dissolved in 5 mL of water. This metric was used to gain a full-picture view of the Venus flytrap's physiological response to NH4NO3. The total time represents the interval from exposure to a mechanical stimulus to when the trap stopped moving, or the first value to the third value, as explained previously in the replicate study conducted during junior year. The values were averaged for each condition, and the means were compared using a two-tailed independent t-test. This data reveals a significant increase in the total time required for trap closure for Venus flytraps exposed to NH4NO3 (p = 2.7 x 10-20; p < 0.05). Figure 3 shows that, on average, Venus flytraps exposed to NH4NO3 closed in 4.17 seconds after exposure to a mechanical stimulus, while negative control plants closed in 1.43 seconds.

Figure 4A: Average Trap Activation Time (s) (NH4NO3 vs. H2O)

Figure 4A represents the mean activation time (s) after exposure to a mechanical stimulus for Venus flytraps exposed to either 5 mL of water (blue bar; n = 24) or 100 mg of NH4NO3 dissolved in 5 mL of water (purple bar; n = 25). This data is pooled across three days of data collection. Error bars represent the standard deviation for the mean of each data set. This data was analyzed using a two-tailed independent t-test, and no significant difference was observed in trap activation time (p = 0.12; p > 0.05).

Figure 4B: Average Trap Closure Time (s) (NH4NO3 vs. H2O)

Figure 4D represents the mean closure time (s) for Venus flytraps exposed to either 5 mL of water (blue bar; n = 24) or 100 mg of NH4NO3 dissolved in 5 mL of water (purple bar; n = 25). This data is pooled across three days of data collection. Error bars represent the standard deviation for the mean of each data set. This data was analyzed using a two-tailed independent t-test, and a significant difference was observed in trap closure time (p = 6.3 x 10-22; p < 0.05).

Figures 4A and 4B break down trap closure into the two key stages described earlier, trap activation time and trap closure time, for Venus flytraps exposed to either NH4NO3 or water. Although statistical significance was found in the total time required for trap closure (Figure 3), no significant increase in trap activation time was observed (Figure 4A). The mean delay in trap closure was 0.51 seconds and 0.34 seconds for Venus flytraps exposed to water and NH4NO3, respectively. The means were analyzed using a two-tailed independent t-test, and no significant difference was found in trap activation time (p = 0.12; p > 0.05). However, when comparing the trap closure stage, the effect of NH4NO3 was more evident. The mean closure time was substantially longer for Venus flytraps exposed to NH4NO3 (3.82 seconds) and water (0.94 seconds). The means were analyzed using a two-tailed independent t-test, and a significant increase in trap closure time was observed (p = 6.3 x 10-22; p < 0.05).

These results indicate that the contrast in total trap closure times (Figure 3) can be attributed entirely to changes in the trap closure time, rather than the trap activation time. These results are similar to the TEACl results (Figure 1A and 1B), but no significant difference was observed in the trap activation time, but a considerable difference in trap closure time was observed. However, in this case, the increase in trap closure time is even more pronounced after NH4NO3 exposure. The exact mechanism for how NH4NO3 affects trap closure times is a knowledge gap. However, the observed effects of NH4NO3 on trap closure suggest a similar mechanism to TEACl. Because TEACl is a K+ channel inhibitor, these results suggest the involvement of K+ channel regulation in response to nitrogen-rich soil. K+ channels are crucial to the trap closure movemebt but not trap activation: by interfering with the efflux of K+ from the cells in the trap, these channels control the osmotically-driven water movement necessary for Venus flytrap to snap down on prey (Volkov et al., 2008). However, further research is needed to verify this assertion.

One possible outcome of this experiment was ammonium poisoning, so the plants were assessed for any signs of deterioration for two weeks following the NH4NO3 application. Ammonium poisoning in Venus flytrap is a condition that results from exposure to excess NH4+ levels and could result in wilted and blackend traps, ultimately leading to plant death (Britto et al., 2001). There were no observable signs of damage, suggesting that the changes observed in trap closure were due to the plant’s physiological response to the altered environment rather than potential harm caused by NH4NO3 exposure.

Although the study was conducted with no significant errors, there are still sources of error that future researchers should address. In October 2022, many Venus flytraps were infected with white mold (likely caused by excess humidity) that spread across the replicates, eventually killing all the plants. Because of this outbreak, data collection was delayed for two months, impacting the number of replicates available for testing and the timeline for the experiment. However, even with the dead plants, the negative control and experimental arm were still completed on-time with new, healthy plants.

Another source of error could have been the NH4NO3 concentration in each pot. Although 100 mg NH4NO3/5 mL water was consistent across the experimental arm, plants may have had varying amounts of water after dosing as the plants were watered indirectly through a bottom-watering system. This could have resulted in a variation in each plant's final concentration of NH4NO3 or variations in how quickly the chemical additive leached out of the substrate and into the excess water in the bottom of the tray. However, all plants were sealed under one humidity dome, ensuring a constant humidity level for all Venus flytraps and minimizing water level variation within the pots. Therefore, variations in final NH4NO3 concentrations are less likely to be influenced by differences in evaporation rates or water loss due to humidity fluctuations. LaMotte soil tests were used to determine the soil N concentration on Days 2 and 4 of the experiment, but these results had to be interpreted based on varying shades of red using the provided colormetric scale. Soil test results for the experimental arm were dark red, indicating an N concentration of at least 22.5 ppm in each pot. However, as the colorimetric scale did not have finer differentiations in color to distinguish between small concentration increments, it was difficult to discern if the there were significant differences in the N concentration across the plants that could potentially affect the experimental outcomes. The concentration was definitely ≥ 22.5 ppm N, however, one cannot conclusively know the final concentration of NH4NO3 in each pot using this approach.

Habitat destruction, such as converting land for agricultural use, is one of the greatest threats to the Venus flytrap (Cross et al., 2020). Additionally, research has shown a 400% increase in fertilizer usage in North Carolina during the past 60 years (Howard et al., 2012). Exposure to NH4NO3 can increase trap closure times in Venus flytraps, indicating that this standard component of chemical fertilizer can have unintended consequences on the behavior of this unique carnivorous plant. Because Venus flytraps are highly sensitive to environmental changes, they are considered an indicator species, meaning that their abundance in an environment can be used as a barometer of the health and well-being of its surrounding ecosystem (Jennings and Rohr, 2011).

The Venus flytrap plays a crucial role in the subtropical wetlands of North and South Carolina. This behavioral change could affect other species in the Venus flytrap's local environment; most directly, small insects and spiders, the Venus flytrap’s natural prey (Schultze et al., 2012). This could ultimately lead to an overpopulation of insects and spiders, disrupting the predator-prey balance in the Venus flytrap's native environment, and have a cascading effect on the ecosystem as a whole. Future research should focus on understanding the long-term consequences of environmental changes, such as increased fertilizer usage and other human activities, on the Venus flytrap's carnivorous behavior and its role in the local ecosystem. This information could help inform and develop effective conservation strategies. Ultimately, these bogs present a unique environment that is truly worth preserving; their distinct ecological conditions have provided the specific selective pressures necessary to foster the unique biodiversity seen in the Venus flytrap.

Supplemental Materials

Junior Year (2021 - 2022)

Senior Year (2022 - 2023)

Lane R. '23

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