Behavioral Disruptive Effects of Spathiphyllum Leaf Extract: Insights from Larval Assays in Drosophila melanogaster

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S. Pruthvi, T. M. Aishwarya, K. B. Vijendra Kumar, Kavitha Raj Varadaraju, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6758746/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 13 You are reading this latest preprint version Abstract This study investigates the effects of phytotoxins derived from Spathiphyllum walsii (peace lily) on the behavioral plasticity of Drosophila melanogaster larvae, focusing on their implications for sensory perception and motor function. Phytotoxins, known for their diverse bioactive properties, serve as both a defense mechanism for plants and potential therapeutic agents in medicine. The research involved the extraction of phytochemicals from Spathyiphyllum leaves, followed by a series of behavioral assays to assess larval responses to food stimuli, olfactory cues, and environmental conditions. The results demonstrated that larvae treated with SL extract exhibited significant delays in reaching food sources compared to control groups, indicating impaired sensory and motor functions. Notably, higher concentrations of the extract produced effects similar to those of colchicine, a well-known microtubule inhibitor. Behavioral assays revealed that both treatments disrupted normal larval behavior, suggesting that phytotoxins may influence neural circuits and microtubule dynamics. These results underscore the potential of plant-derived compounds as modulators of neural function and behavior, highlighting both their therapeutic prospects and risks associated with their use. This study highlights the potential of plant-derived compounds in understanding neurobiological processes and offers insights into their therapeutic applications. Further research is warranted to elucidate the mechanisms underlying these effects and to explore the broader implications of phytotoxins in pharmacotherapy and toxicology. Biological sciences/Biochemistry Health sciences/Neurology Spathiphyllum walsii Phytotoxin Drosophila melanogaster Colchicine Photo-taxis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Plant toxins, commonly referred to as phytotoxins, are a diverse array of bioactive compounds synthesized by plants as a defensive strategy against herbivores, pathogens, and various environmental stressors 1 . These compounds play a critical role in plant survival and adaptation, deterring potential threats through their toxic effects 2 . While many phytotoxins can be harmful or even lethal to living organisms, an increasing body of research highlights their potential therapeutic applications in medicine, particularly in the fields of oncology, neurology, and inflammation management 3 . The classification of plant toxins is primarily based on their chemical structure and biological activity 4 . It is evident from previous studies that the efficacy of a range of readily available plant-based extracts and chemicals that may improve brain function and possess other therapeutic properties 5,6 . Among these, alkaloids stand out as nitrogen-containing compounds that frequently exhibit profound pharmacological effects 7 . Well-known examples include morphine, derived from Papaver somniferum , and nicotine from Nicotiana tabacum 8 . Additionally, cardiac glycosides, such as digoxin from Digitalis purpurea 9 , are vital in the treatment of heart conditions due to their ability to enhance cardiac function 10 . Other categories of phytotoxins include toxic proteins like ricin from Ricinus communis 11 and various lectins, which inhibit protein synthesis or induce apoptosis in cancer cells 12 . The biological activities of these compounds are vast, encompassing anti-inflammatory and antimicrobial properties, which are beneficial in a therapeutic context 13 . For instance, flavonoids and tannins are recognized for their antioxidant capabilities, contributing to the prevention of oxidative stress-related diseases 14 . The mechanisms by which plant toxins exert their effects include the inhibition of protein synthesis 15 , modulation of cellular signaling pathways 16 , and the induction of apoptosis 17 . Ribosome-inactivating proteins (RIPs) like ricin disrupt protein synthesis by targeting ribosomes, ultimately leading to cell death—a property being explored for targeted cancer therapies 18 . Despite their therapeutic potential, the use of plant toxins in medicine poses significant challenges. The fine line between effective and toxic doses complicates their clinical application, requiring meticulous research to optimize dosage and delivery methods 19 20 . For example, colchicine, derived from Colchicum autumnale , has a long history of use for gout due to its anti-inflammatory properties and ability to inhibit microtubule polymerization 21 . Its applications extend to treating familial Mediterranean fever and certain cancers. However, careful management of its dosage is essential to avoid toxic effects 22 . In recent years, innovative strategies have emerged to enhance the delivery of plant-derived compounds 23 . Advances in fields such as genomics, proteomics, and nanotechnology are paving the way for novel drug development approaches that can harness the beneficial properties of phytotoxins while minimizing associated risks 24 . For instance, ricin’s selective cytotoxicity towards cancer cells, when coupled with targeted delivery systems, holds promise for developing new cancer therapies 25 . In addition to these considerations, research has begun to explore the effects of plant extract on model organisms, such as Drosophila melanogaster , a holometabolic insect widely used in genetic and developmental studies 26 . The use of Drosophila allows for detailed investigations into the effects of phytotoxins on behavior and physiology, particularly about muscle development and neural function 27 . This research aims to elucidate how both colchicine and extract from Spathiphyllum leaf (SL) , commonly known as the peace lily, impact larval behavior through their effects on neural circuits and microtubule dynamics in Drosophila melanogaster . SL is noted not only for its aesthetic and air-purifying qualities but also for its calcium oxalate crystals, which can induce irritation if ingested 28 , highlighting the need for a comprehensive understanding of its toxicity at cellular levels. Overall, the exploration of plant toxins as a rich source of bioactive compounds presents a promising avenue for the development of innovative treatments across various medical fields, particularly in oncology and inflammatory diseases. Continued research into their mechanisms of action and improved delivery methods could lead to significant advancements in pharmacotherapy, ultimately enhancing the therapeutic landscape derived from nature’s complex biochemical arsenal. Results 2.1. Plant extraction and phytochemical screening The leaf powder of Spathiphyllum walsii was subjected to extraction in water. A yield of 18% was obtained from the SL extract, which was utilized in the subsequent experiment. The phytochemical analysis conducted on the leaf extract of SL identified several active compounds, including alkaloids, phenols, flavonoids, tannins, carbohydrates, and terpenoids, some of which are recognized for their cytotoxic properties (supplementary table 1 ). 2.2. Effect of Colchicine and SL extract on larval behavioral plasticity The study of larval behavioral plasticity in Drosophila using colchicine and SL extract reveals significant insights into the effects of these substances on larval behavior and microtubule dynamics. The results indicate that larvae treated with SL extract exhibited a longer time (160 sec) delay in reaching yeast compared to the control group. In contrast, the colchicine-treated group did not reach the yeast at all. This suggests that both treatments significantly impair the ability of larvae to respond to food stimuli, indicating a potential disruption in motor function or sensory perception. It was noted that higher concentrations (10mg/mL) of the plant extract elicited effects comparable to those observed with colchicine (Figs. 1 & 2 ). This suggests a dose-dependent relationship where increased concentrations of the plant extract lead to more pronounced behavioral impairments, reinforcing the hypothesis that this extract may act through similar pathways as colchicine (Supplementary Fig. 6). 2.3. Effect of colchicine and SL extract on larval olfactory response The effects of colchicine and SL extract on olfactory responses have been studied, revealing significant insights into how these substances influence sensory perception in larvae. The responsive index (RI) for the control group was normal and higher compared to both the colchicine (CC) and plant extract (SLE) groups. This indicates that the control group exhibited typical olfactory responses. The group treated with SL extract showed a response like the colchicine group, suggesting that both treatments may impair olfactory sensitivity or discrimination abilities. Specifically, as the concentration of SL extract increased, the RI decreased, with 10 mg/ml showing (Figs. 3 & 4 ) a notably lower RI than both 5 mg/ml and 2.5 mg/ml concentrations. (Supplementary Fig. 1) The negative response observed in both colchicine and SL extract-treated groups suggests that these treatments lead to altered olfactory behaviors. 2.4. Effect of Colchicine and SL extract on contact chemosensory response Chemosensory assays are critical for assessing an organism's ability to detect and respond to chemical stimuli in its environment. The specific study discussed evaluates the impact of colchicine and SL extract on the chemosensory response in larvae, focusing on their effects on the responsive index (RI). The control group (CC) exhibited a normal and higher RI compared to the groups treated with colchicine and SL extract, indicating intact chemosensory function in the absence of these treatment. The data suggest that SL extract significantly disrupts chemosensitivity more than colchicine. Specifically, the RI was notably lower in the group treated with 10 mg/ml of SL, indicating a strong negative effect on chemosensory behavior. Treatment with colchicine resulted in a decrease in RI, with complete absence observed in larvae treated with 10 mg/ml of SL extract (Figs. 5 & 6 ). This suggests that both treatments lead to a significant reduction in chemosensory behavior, likely due to microtubule disruption within sensory neurons. The study highlighted that at a concentration of 10 mg/ml, SL extract caused a more pronounced disruption in RI when compared to lower concentrations (Supplementary Fig. 1), reinforcing its potential as a potent disruptor of chemosensory responses. 2.5. Effect of Colchicine and SL extract on larval phototaxis behavior. The effects of colchicine and SL extract on photo-taxis responses reveal significant insights into how these substances influence larval behavior in response to light. The control group exhibited a normal, positive, and higher RI compared to both the colchicine and SL extract groups. The colchicine-treated group demonstrated a highly disrupted RI, indicating a negative response to light stimuli. In contrast, the SL extract group showed an RI that was closer to the control group, suggesting less disruption in photo taxis behavior than the colchicine group (Figs. 7 & 8 ). The 10 mg/ml concentration of SL extract resulted in a more pronounced disruption of the RI compared to the lower concentrations of 5 mg/ml and 2.5 mg/ml (Supplementary Fig. 1). This suggests that lower concentrations may have a milder effect on larval phototaxis. 2.6. Effect of Colchicine and SL extract on larval motor function The larval motor function assay in Drosophila is a critical experimental setup used to assess the impact of various treatments on the movement and muscle function of larvae. This assay typically involves measuring the time taken for larvae to reach a designated endpoint under different conditions, such as exposure to colchicine and SL extract. The results indicate that larvae treated with colchicine (CC group) exhibit a significantly higher time delay in reaching the endpoint compared to those treated with SL extract. The SL Extract group shows a similar delay to the CC group, suggesting both treatments impair motor function. In contrast, the control group (CN) demonstrates a shorter delay, indicating normal motor function under untreated conditions (Figs. 9 & 10 ). The assay results also highlight that a concentration of 10 mg/ml leads to a greater time delay compared to lower concentrations of 5 mg/ml and 2.5 mg/ml (Supplementary Fig. 1). This suggests a dose-dependent effect where higher concentrations exacerbate motor function impairment. 2.7. Effect of Colchicine and SL extract on life cycle. The study of Drosophila melanogaster larvae treated with colchicine and extract from SL aims to investigate the effects of these treatments on various developmental stages, including larvae, pupae, and adult flies. The findings highlight significant disruptions in the life cycle due to both colchicine and plant extract treatments. Both colchicine (CC) and plant extract (SLE) groups exhibited a notable delay in larval development. This resulted in an increased duration of the larval stage compared to the control group (CN). Larvae exposed to these treatments showed abnormal morphology, such as altered body shape and size, indicating that colchicine disrupts microtubule dynamics essential for normal development. The percentage of larvae that transitioned to pupae was significantly lower in the CC and SLE groups. Higher concentrations of SLE reduced pupation rates by approximately 10% compared to the control. Pupae from treated groups displayed disrupted development, leading to abnormal pupal cases and delayed metamorphosis. The adult emergence rates were reduced in both CC and SLE groups at a concentration of 10 mg/ml, with an extended duration for the emergence process noted. Adults that did emerge from colchicine-treated larvae exhibited abnormal wing shapes and sizes, further emphasizing the impact of these treatments on adult morphology (Table 1). Observations during reproductive stages revealed that both colchicine and SLE treatments significantly affected reproductive success, leading to reduced fertility and egg viability. Table-1: Life cycle monitor Groups Percentage of Larvae to pupae conversion Time interval between Pupae to fly conversion Percentage of Pupae to Fly conversion CN 100 6 days 100 CC 70 10 days 10 PE [10mg/ml] 90 10 days 90 Discussion Plant bioactive compounds have been extensively studied for their cytotoxic effects 29 . One significant area of interest is how these compounds interact with microtubules, which are critical components of the cell's cytoskeleton and play a vital role in cell division and intracellular transport 30 . Our study highlights the effects of Spathiphyllum and colchicine on Drosophila melanogaster larvae and provides significant insights into how these substances impact larval behavior, sensory perception, motor function, and overall development. The findings highlight the potential cytotoxic properties of Spathiphyllum and its implications for understanding microtubule dynamics in biological systems. In our study, the extraction process involved soaking leaf powder of Spathiphyllum in water for three days, yielding an extract with an 18% yield. Phytochemical screening revealed the presence of various bioactive compounds such as alkaloids, phenols, flavonoids, tannins, carbohydrates, and terpenoids. These compounds are known for their diverse biological activities, including cytotoxic effects that may influence larval behavior and physiology 31 . Plant-derived toxins can lead to neurological impairments in Drosophila larvae, and Drosophila models are emerging models in toxicity research. For instance, exposure to essential oils has been linked to the presence of vacuoles in brain tissue, indicating potential neurotoxic effects 32 . Moreover, specific studies have noted that certain toxins can induce excitatory junctional potential leading to paralysis, demonstrating their profound impact on neuromuscular function 33 . These findings emphasize the role of sensory neurons in mediating responses to toxic substances. The olfactory response assays in our study demonstrated that both treatments significantly affected sensory perception. Control larvae exhibited normal olfactory responses, while those treated with SL showed reduced responsiveness like the colchicine group. The responsive index (RI) decreased with increasing concentrations of SL extract, indicating impaired olfactory sensitivity. This suggests that both treatments adversely affect the larvae's ability to detect and respond to chemical stimuli in their environment. The sensory systems of Drosophila are crucial for detecting environmental cues, including the presence of toxins. Research has identified various chemoreceptors involved in sensing toxic compounds, which are essential for behavioral avoidance strategies 34 . The impairment of these sensory mechanisms due to toxin exposure can lead to increased vulnerability and decreased survival rates. Chemosensory assays further confirmed the negative impact of both treatments on chemosensory behavior. Control larvae displayed intact chemosensory function, while those treated with either colchicine or SL extract exhibited significantly lower RI values. Notably, SL extract at 10 mg/mL caused a more pronounced disruption than colchicine, highlighting its potential as a potent disruptor of chemosensory responses. The disruption in phototaxis can be attributed to the effects of toxins on the sensory pathways involved in light detection. Drosophila larvae utilize specific photoreceptors to sense light, and toxins may interfere with the functioning of these receptors or the neural circuits that process light information 35 . For instance, genetic studies indicate that specific photoreceptor subtypes are essential for light avoidance, and their impairment due to toxin exposure can lead to a complete loss of phototactic behavior 36 . Phototaxis assays in our study revealed that control larvae responded positively to light stimuli, while those treated with colchicine showed significant disruption in their phototactic behavior. The SL extract group demonstrated less disruption compared to colchicine but still exhibited altered responses at higher concentrations. This suggests that while SL extract may impair phototaxis, it does so to a lesser extent than colchicine. Exposure to various plant extracts and environmental toxins can lead to marked reductions in the climbing abilities of Drosophila . For instance, studies have shown that exposure to paraquat (PQ), a herbicide, significantly impairs mobility, mimicking symptoms of neurodegenerative diseases 37 . In experiments, Drosophila that were pre-fed with neuroprotective compounds like Gardenin A showed restored climbing abilities post-exposure to PQ, indicating that certain phytochemicals can mitigate toxin-induced mobility defects 38 . Motor function assessments indicated that both treatments significantly delayed larval movement towards a designated endpoint. The results showed that colchicine-treated larvae had a greater delay than those treated with SL extract. This reinforces the notion that both substances impair motor function but suggests that SL extract may have a milder effect at lower concentrations. Exposure to plant-derived toxins can lead to developmental delays and morphological abnormalities in Drosophila larvae. For instance, studies involving extracts from various plants have indicated that certain toxins can reduce larval growth rates and alter developmental timelines. The presence of these toxins often results in increased mortality rates during the larval stage, particularly at higher concentrations. For example, Euphorbia prostrata showed a mortality rate of 51.64% at a 30% concentration after 72 hours of exposure, indicating significant toxicity that affects overall development 39 . Toxins can also impact the reproductive capabilities of Drosophila . Research indicates that females exposed to certain toxic compounds have reduced fecundity, meaning they produce fewer offspring. In studies involving macro fungi, it was found that female flies were more sensitive to toxins compared to males and exhibited shorter survival times, with some treatments resulting in no offspring production at all 40 . However, excessive exposure to toxic substances typically results in decreased lifespan due to increased mortality 41 . The study also investigated how these treatments affected the overall life cycle of Drosophila . Both colchicine and SL extract resulted in delayed larval development and abnormal morphology. Higher concentrations of plant extract notably reduced pupation rates and led to abnormal adult morphology upon emergence. Furthermore, reproductive success was compromised in both treatment groups, indicating that these substances disrupt not only immediate behaviors but also long-term developmental processes. Conclusion The findings from this study underscore the significant effects of SL extract and colchicine on Drosophila larvae across various behavioral and physiological parameters. The presence of bioactive compounds in SL extract suggests potential applications in pharmacology and toxicology, particularly concerning microtubule dynamics and SSSsensory processing. Future research could explore the specific mechanisms by which these extracts exert their effects and their potential therapeutic applications or ecological impacts. This comprehensive analysis highlights the importance of understanding plant extract biochemical properties and their implications for model organisms like Drosophila , serving as a foundation for further investigations into their biological activities and potential uses in various fields. Materials and Methods 4.1. Collection of plant material Spathyphyllum plant leaves were collected from a local horticulture nursery, Mysuru, Karnataka, India in March 2024. The plant leaves are subjected to shade dry for 15 days, and then the dried leaves are powdered and soaked in water for 3 days on a magnetic stirrer, then the supernatant is collected and transferred into petri plates for drying. Dried aqueous extract was scraped off and weighed to calculate the yield percentage and used for further studies. Extract was screened for the presence of phytochemicals. 4.2. Fly selection and third instar larvae collection Flies stocks used for the present study were Drosophila melanogaster ( Oregon K strain). These flies were obtained from the Drosophila Stock Centre, Department of Zoology, University of Mysore, Mysore, Karnataka. The fly strains were kept in glass vials with a normal Drosophila medium at room temperature (25±2 °C). Fresh egg plates with a small amount of yeast paste in the middle were used to collect eggs from 1–10 day-old adult flies. After 24-hour incubation intervals, the plates were swapped out and left at room temperature, allowing the hatched larvae to develop. The trials investigated larvae from these plates that were in their early third instar (72–78 hours) 42 . 4.3. Collection and Washing of Third-Instar Larvae Wandering third instar larvae was taken from the growth medium. Immediately, larvae were subjected to careful washing using 15% sucrose initially, followed by washing with double-distilled water twice using a small, moistened paintbrush until the larvae became clean of yeast. Gently stir the water containing larvae with the brush to aid in washing, and the water was drained completely using a 1ml Pipette. After the washing procedure, larvae were placed in a small amount of sucrose water and then distilled water until the assays were performed 43 . 4.4. Pre-incubation and treatment of larvae The collected larvae were subjected to pre-incubation prior to the beginning of the experiment. A period of starvation was maintained initially by subjecting 15 larvae simultaneously to double-distilled water for 20 minutes under normal light exposure. After 20 minutes of starvation, the larvae were subjected to a treatment (feeding) period. For this to be carried out, together control and colchicine, and plant extract with different concentrations treated groups were maintained separately. Each group had 3 larvae. In the control group selected larvae followed the incubation period of 10 minutes using double-distilled water under bright light. In colchicine treated group, larvae were administered with 1000µM colchicine (colchicine prepared in 0.1% DMSO) for 10 minutes under bright sunlight, remaining three groups’ larvae are treated with SL extract in the concentration of 10mg/ml, 5mg/ml and 2.5mg/ml for 10 minutes under bright light. All the control, colchicine, and plant treatment was carried out simultaneously, ensuring the feeding of larvae after a brief period of starvation. After the period of pre-treatment of larvae, an assay was carried out immediately. 4.5. Behavioral plasticity Assay Larvae treated with colchicine and SL extract underwent simultaneous and independent assays. A plate was separately kept untreated as a control. In the experimental design, the assay plate was made up of a glass petri plate (100mm x 15 mm) consisting of four quadrants. 20 ml of 10% agar was carefully poured into the petri plate uniformly and placed aside for solidification. At the beginning of the assay, the larvae were placed such that they would spread out 5 mm from the edge of the plate. There was no fixed period followed during the assay; we rather assessed the recognition and crawling speed of control and treated larvae towards yeast at their respective time intervals. The larvae were allowed to reach the yeast, and time was noted down for the time interval of each larva reaching the yeast in both the control and treated groups. The assays were continued until the entire 3 larvae reached the yeast, and the final time was noted down for evaluation. The assays were carried out in singlets for all the groups 42 . 4.6. Olfactory assay In the experimental design, the assay plate was made up of a glass petri plate (100mm x 15 mm) consisting of four quadrants. 20 ml of 10% agar was carefully poured into the petri plate uniformly and placed aside for solidification. Two filter discs (made from Whattman No. 1 filter paper) were placed at opposite edges of the plate in such a fashion that they face diametrically opposite each other. A fruity odorant (an extract from papaver fruit) was added to one disc and diluent (distilled water) added to the other. The larvae were carefully placed in the center of the plate with a smooth pointed brush, and the larvae were allowed to travel for 1 minute. After this duration of one minute, the number of larvae present on each half of the plate was counted (supplementary figure 2). A response index (RI) was calculated by subtracting the number of animals on the control half of the plate (C) from the number on the stimulus half (S) and dividing by the total: RI = (S - C)/(S + C). For consideration, often a small percentage of larvae remain in the center of the plate and are neglected 44 . 4.7. Contact Chemosensory Assay In the experimental design, the assay plate was made up of a glass petri plate (100mm x 15 mm) consisting of four quadrants. 20 ml of 10% agar was carefully poured into the petri plate uniformly and placed aside for solidification. As a chemosensory source, a defined concentration of 0.1M NaCl was used. After solidification, (0.1M) NaCl was carefully spread over the surface of two diametrically opposite quadrants. The other two opposite quadrants were kept as a plain agar surface. The plates were used 10 minutes later just to ensure the uniform diffusion of 0.1M NaCl through the thin superficial layer of 10% agar on the surface of the plate. The pre-incubated and treated larvae were used in the assay. Larvae from each treatment group were carefully placed at the center of the agar plate with the help of a smooth brush and were allowed to migrate for 6 min (supplementary figure 3). The assay was carried out in triplicate. The ultimate number of larvae on each quadrant was taken to counted after six minutes, and responsiveness was determined by considering the average number of larvae moving towards 0.1M NaCl and plain agar 45 . 4.8. Larval Photo-taxis Assay The photo-taxis assay was similar to the contact chemosensory assay with slight modifications 45 . The assay plate was prepared by adding 20 ml of 10% agar uniformly on a glass petri plate and was allowed to solidify. Each of two opposed quadrants was painted (black) to ensure the dark background is created at the bottom of the plate. The other two opposite quadrants were kept transparent (uncolored) for light to enter. The preincubated and treated larvae were used in the assay. Larvae were carefully placed at the center of the plate using a smooth brush and assayed for 8 minutes. The number of larvae migrating towards either side of the background after 8 minutes was noted. The responsive index (RI) was calculated as mentioned earlier in the olfactory assay (supplementary figure 4). 4.9. Larval motor function assay In the experimental design, a 100 mm x 10 mm Petri dish having 10% Agar, 5 parallel tracks of 1cm wide grids were created. The track was moistened with water to ease the crawling. Before the crawling ability test, larvae were carefully placed on an identical dummy track and allowed to acclimatize for 5 minutes before being transferred to the test track. The preincubated and treated larvae were used in the assay. The time consumed to crawl each 1cm track to the ‘finish line’ was recorded (supplementary figure 5). During the course of the test, if the larva turned backwards or returned, the timer was stopped, and it was then again placed back to the starting position, and then timing was resumed 46 . 4.10. Study on the Life cycle of larvae In the study, 50 healthy and active third instar larvae were collected from culture bottles. The larvae were grouped into Control, Colchicine-treated treated and plant extract-treated groups (n=10). The control group was kept untreated, and the Colchicine group was treated with colchicine medium, and the plant extract groups with plant extract medium (2.5, 5, and 10mg/mL). Every day, the size, shape, and instar stages of the larvae were noted and observed. The rate, duration, and morphology of the larvae's pupation (creation of pupal cases) were tracked, and the size, shape, and color of the pupils were documented. Monitoring was done on adult emergence, emergence rate, time, and morphology. It was determined what proportion of each group's larvae developed into pupae. It was noticed how long it took for the larvae to develop into pupae and then turn into flies. A microscope was used for every observation. 4.11. Statistical analysis All data were analyzed using GraphPad Prism (version 10.4.2). Experiments were conducted in triplicate, and results are presented as mean ± standard error of the mean (SEM). Statistical comparisons between groups were performed using one-way ANOVA. A p-value of less than 0.001 (p < 0.001) was considered statistically significant. Declarations Acknowledgement The authors acknowledge Bioscience CLIx LLP for providing the laboratory facilities and scientific guidance essential to the successful completion of this work. Artificial intelligence tools, ChatGPT by OpenAI, and Perplexity were used solely to assist with language editing and improving the clarity. The scientific content was conceived, written, and verified entirely by the authors. Author contribution statement Authors H. S. Pruthvi, T M Aishwarya, K. B. Vijendra Kumar, Kavitha Raj Varadaraju, Karthik N Awathade, K. S. Bhargava Shreevatsa, Chandan Shivamallu and Chandan Dharmashekharwere involved in the conception, design, and analysis, interpretation of the data and drafting of the paper. Kavitha Raj Varadaraju and Karthik N Awathade were also involved in revising it critically for intellectual content and the final approval of the version to be published. All authors agree to be accountable for all aspects of the work. Data availability statement Phytochemical screening of SL extracts, Comparison of activity at different concentrations of plant extract is available in the supplementary material. The videos of the experimental datasets recorded during the current study are available from the corresponding author on reasonable request . Disclosure statement The authors declare no competing interests. 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Precise Delivery of Ricin A-Chain and Photosensitizer by Aptamer-Functionalized Liposome for Targeted Chemo-Photodynamic Synergistic Therapy. ACS Mater. Lett. 6 , 2050–2058 (2024). Baenas, N. & Wagner, A. E. Drosophila melanogaster as an alternative model organism in nutrigenomics. Genes Nutr. 14 , 14 (2019). Lopez-Ortiz, C. et al. Drosophila melanogaster as a Translational Model System to Explore the Impact of Phytochemicals on Human Health. Int. J. Mol. Sci. 24 , 13365 (2023). Wismer, T. Feline Toxins. in August’s Consultations in Feline Internal Medicine, Volume 7 791–798 (Elsevier, 2016). doi:10.1016/B978-0-323-22652-3.00079-7. Zasheva, D. et al. Cytotoxic Effects of Plant Secondary Metabolites and Naturally Occurring Bioactive Peptides on Breast Cancer Model Systems: Molecular Mechanisms. Molecules 29 , 5275 (2024). Weaver, B. A. How Taxol/paclitaxel kills cancer cells. Mol. Biol. Cell 25 , 2677–2681 (2014). Zasheva, D. et al. Cytotoxic Effects of Plant Secondary Metabolites and Naturally Occurring Bioactive Peptides on Breast Cancer Model Systems: Molecular Mechanisms. Molecules 29 , 5275 (2024). Bernardes, L. M. M. et al. Drosophila melanogaster as a model for studies related to the toxicity of lavender, ginger and copaiba essential oils. PLOS ONE 18 , e0291242 (2023). Zhou, K., Luo, W., Liu, T., Ni, Y. & Qin, Z. Neurotoxins Acting at Synaptic Sites: A Brief Review on Mechanisms and Clinical Applications. Toxins 15 , 18 (2022). Depetris-Chauvin, A., Galagovsky, D. & Grosjean, Y. Chemicals and chemoreceptors: ecologically relevant signals driving behavior in Drosophila. Front. Ecol. Evol. 3 , (2015). Asirim, E. Z., Humberg, T.-H., Maier, G. L. & Sprecher, S. G. Circadian and Genetic Modulation of Visually-Guided Navigation in Drosophila Larvae. Sci. Rep. 10 , 2752 (2020). Keene, A. C. et al. Distinct Visual Pathways Mediate Drosophila Larval Light Avoidance and Circadian Clock Entrainment. J. Neurosci. 31 , 6527–6534 (2011). Zhang, X., Thompson, M. & Xu, Y. Multifactorial theory applied to the neurotoxicity of paraquat and paraquat-induced mechanisms of developing Parkinson’s disease. Lab. Invest. 96 , 496–507 (2016). Maitra, U., Harding, T., Liang, Q. & Ciesla, L. GardeninA confers neuroprotection against environmental toxin in a Drosophila model of Parkinson’s disease. Commun. Biol. 4 , 162 (2021). Riaz, B. et al. Toxicity, Phytochemical Composition, and Enzyme Inhibitory Activities of Some Indigenous Weed Plant Extracts in Fruit Fly, Drosophila melanogaster . Evid. Based Complement. Alternat. Med. 2018 , 2325659 (2018). Li, J., Huang, Y., Wang, D., Zhu, N. & Qiao, X. Comparison of toxic effects of 5 macrofungi against Drosophila melanogaster . J. Insect Sci. 23 , 20 (2023). Arsac, J.-N. et al. Chronic Exposure to Paraquat Induces Alpha-Synuclein Pathogenic Modifications in Drosophila. Int. J. Mol. Sci. 22 , 11613 (2021). Min, V. A. & Condron, B. G. An assay of behavioral plasticity in Drosophila larvae. J. Neurosci. Methods 145 , 63–72 (2005). Fye, S., Dolma, K., Jung Kang, M. & Gunawardena, S. Visualization of Larval Segmental Nerves in 3 rd Instar Drosophila Larval Preparations. J. Vis. Exp. 2128 (2010) doi:10.3791/2128. Khurana, S. & Siddiqi, O. Olfactory Responses of Drosophila Larvae. Chem. Senses 38 , 315–323 (2013). Lilly, M. & Carlson, J. smellblind: a gene required for Drosophila olfaction. Genetics 124 , 293–302 (1990). Nichols, C. D., Becnel, J. & Pandey, U. B. Methods to Assay Drosophila Behavior. J. Vis. Exp. 3795 (2012) doi:10.3791/3795. Supplementary Figures Supplementary Figures 2-6 are not available with this version. Additional Declarations No competing interests reported. Supplementary Files larvapaperSupplementarymaterial.docx Supplementary material Phytochemical screening of SL extracts, Comparison of activity at different concentrations of plant extract is available in the supplementary material. The videos of the experimental datasets recorded during the current study are available from the corresponding author on reasonable request. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 14 Oct, 2025 Reviews received at journal 08 Oct, 2025 Reviews received at journal 08 Oct, 2025 Reviews received at journal 28 Sep, 2025 Reviewers agreed at journal 19 Sep, 2025 Reviewers agreed at journal 18 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviewers agreed at journal 10 Sep, 2025 Reviewers invited by journal 09 Sep, 2025 Editor assigned by journal 17 Jul, 2025 Editor invited by journal 17 Jul, 2025 Submission checks completed at journal 14 Jul, 2025 First submitted to journal 14 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6758746","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":515571827,"identity":"caa408a6-24b4-41d1-bb61-5006016a8b6f","order_by":0,"name":"H. S. Pruthvi","email":"","orcid":"","institution":"JSS Academy of Higher Education \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"H.","middleName":"S.","lastName":"Pruthvi","suffix":""},{"id":515571828,"identity":"d883ee76-a231-41a6-9d2b-7fbc0d853001","order_by":1,"name":"T. M. Aishwarya","email":"","orcid":"","institution":"Maharani's Science College for Women, Mysuru","correspondingAuthor":false,"prefix":"","firstName":"T.","middleName":"M.","lastName":"Aishwarya","suffix":""},{"id":515571829,"identity":"f089b025-eae6-4654-bd12-6b45d111aa1d","order_by":2,"name":"K. B. Vijendra Kumar","email":"","orcid":"","institution":"Bangalore Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"K.","middleName":"B. Vijendra","lastName":"Kumar","suffix":""},{"id":515571832,"identity":"2f8da7dc-0fc2-4d4c-8ccf-a9209ab4b824","order_by":3,"name":"Kavitha Raj Varadaraju","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYBACA2YwCWYzHkhgsAHRjQeI1cIA1JIG0tKAXwsyB6jyMIyBG5izMx/+8KPAJo+/vfnBgYdt5+3Wth8G2lJjE41Li2UzW5pkj0FascSZYwYHEs7cTt52JhGo5VhabgMuhx3mMWMGkokbJHKAfqm4nWx2AKiFseEwPi3GnxkM/idukH8D1GJwLtns/EOCWgykGQwOAG3hAdlywM7sBgFboH5JTpxxJg3kl+QEsxtAWxLw+MWc/zAwxP7YJfa3H3748Gebnb3Z+fSHDz7U2ODUggESwSoTiFUOAvakKB4Fo2AUjIKRAQDzb2X4nBHWkQAAAABJRU5ErkJggg==","orcid":"","institution":"Bioscience CLIx LLP","correspondingAuthor":true,"prefix":"","firstName":"Kavitha","middleName":"Raj","lastName":"Varadaraju","suffix":""},{"id":515571834,"identity":"e1bef2dc-97c2-43db-a099-4fd7ec991b52","order_by":4,"name":"Karthik N Awathade","email":"","orcid":"","institution":"Bioscience CLIx LLP","correspondingAuthor":false,"prefix":"","firstName":"Karthik","middleName":"N","lastName":"Awathade","suffix":""},{"id":515571835,"identity":"fcc4984a-17d7-4cf2-9c14-4cc5c3eff8bc","order_by":5,"name":"K. S. Bhargav Shreevatsa","email":"","orcid":"","institution":"JSS Academy of Higher Education \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"K.","middleName":"S. Bhargav","lastName":"Shreevatsa","suffix":""},{"id":515571836,"identity":"86a0a8b2-0c43-4969-9dee-2671437e4069","order_by":6,"name":"Chandan Shivamallu","email":"","orcid":"","institution":"JSS Academy of Higher Education \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Chandan","middleName":"","lastName":"Shivamallu","suffix":""},{"id":515571837,"identity":"f6e56fb2-9fdf-42dd-a3ea-da820f07ad6e","order_by":7,"name":"Chandan Dharmashekar","email":"","orcid":"","institution":"JSS Academy of Higher Education \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Chandan","middleName":"","lastName":"Dharmashekar","suffix":""}],"badges":[],"createdAt":"2025-05-27 11:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6758746/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6758746/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91649416,"identity":"67497245-965e-47b7-9777-b315e5327677","added_by":"auto","created_at":"2025-09-18 16:39:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":46040,"visible":true,"origin":"","legend":"\u003cp\u003eBehavioral plasticity assay of \u003cem\u003eDrosophila melanogaster\u003c/em\u003e larvae. Larvae treated with 10 mg/mL SL extract exhibited delayed responses to food stimuli, while colchicine-treated larvae failed to reach the yeast source. Data indicate significant impairment of larval sensory and motor function.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6758746/v1/d5180405923b91ec37b0b66e.png"},{"id":91649834,"identity":"852ebc1b-ad4c-438c-8331-617bcc36ee97","added_by":"auto","created_at":"2025-09-18 16:47:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":280196,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of behavioral plasticity assay plates. (A) Control group showing normal crawling toward yeast. (B) Colchicine-treated group exhibiting impaired movement. (C) SL extract-treated group (10 mg/mL) with delayed movement.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6758746/v1/45e47f5675505e18dc7312ea.png"},{"id":91649836,"identity":"8d3ed0aa-310a-48d8-8b29-0ee723e0fea1","added_by":"auto","created_at":"2025-09-18 16:47:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75782,"visible":true,"origin":"","legend":"\u003cp\u003eOlfactory response index of larvae across treatment groups. Control larvae showed a positive response (RI = 0.1), while both colchicine and SL extract (10 mg/mL) groups exhibited negative olfactory responses (RI = -0.1), indicating impaired olfactory perception.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6758746/v1/9b0ca4dfff0ea470560837e5.png"},{"id":91650611,"identity":"3c199039-4b8e-4305-8348-c2b8b70f457a","added_by":"auto","created_at":"2025-09-18 17:03:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":385525,"visible":true,"origin":"","legend":"\u003cp\u003eImages of olfactory assay plates. Comparison of larval distribution in (A) control, (B) colchicine, and (C) SL extract (10 mg/mL) treated groups after exposure to odorant stimuli.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6758746/v1/57412c3cbc0b7426a8cda9ed.png"},{"id":91649421,"identity":"de214320-8e0c-4490-9b99-425edc934ec6","added_by":"auto","created_at":"2025-09-18 16:39:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":68949,"visible":true,"origin":"","legend":"\u003cp\u003eChemosensory assay showing RI differences across groups. Larvae treated with SL extract (10 mg/mL) showed a complete absence of response to NaCl stimulus, indicating severe disruption of chemosensory behavior.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6758746/v1/78e67f675901c3771c336b78.png"},{"id":91650613,"identity":"f80f0ed1-c794-4437-9a92-31b3fd7d6fbb","added_by":"auto","created_at":"2025-09-18 17:03:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":508053,"visible":true,"origin":"","legend":"\u003cp\u003eChemosensory assay plate images. Control larvae showed preference for untreated agar regions, while colchicine and SL extract-treated larvae displayed diminished or no preference.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6758746/v1/355b68b43d666f5f791117a6.png"},{"id":91649839,"identity":"65193876-ca31-4769-a6ae-337239ce99c4","added_by":"auto","created_at":"2025-09-18 16:47:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":85866,"visible":true,"origin":"","legend":"\u003cp\u003ePhototaxis response assay of larvae. The control group showed a positive RI (0.1), indicating normal light preference. Colchicine-treated group displayed strong negative response (RI = -0.1), while SL extract (10 mg/mL) showed mild disruption (RI = -0.025).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6758746/v1/e7d2036f23065d21ee24fb09.png"},{"id":91649843,"identity":"03e9751b-2704-44cc-b327-dc987de6fef3","added_by":"auto","created_at":"2025-09-18 16:47:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":595540,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative phototaxis assay plates. (A) Control larvae migrated towards light quadrants. (B) Colchicine group avoided light. (C) SL extract group (10 mg/mL) displayed mild aversion.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6758746/v1/16f437efc58863cebc7b5ec3.png"},{"id":91649433,"identity":"bcde64c7-6d54-490c-a4d1-f36f1ba93ff3","added_by":"auto","created_at":"2025-09-18 16:39:02","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":101842,"visible":true,"origin":"","legend":"\u003cp\u003eLarval motor function assay. Larvae treated with colchicine and 10 mg/mL SL extract took significantly longer to reach the endpoint compared to control, suggesting impaired motor coordination.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6758746/v1/d1130afdecf5bb4f0c7e6ceb.png"},{"id":91650612,"identity":"c3d1dbfd-ab05-403d-8b39-b96e8229ee3b","added_by":"auto","created_at":"2025-09-18 17:03:02","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":295080,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative motor function assay plates. (A) Control larvae showing normal movement. (B) Colchicine and (C) SL extract-treated groups demonstrating reduced locomotion and delayed endpoint reach.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6758746/v1/c1a05a8f115b8136742ba2e7.png"},{"id":91817464,"identity":"3dd260d8-30ea-40e0-82c7-23ccda54fd10","added_by":"auto","created_at":"2025-09-22 06:56:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3975235,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6758746/v1/09d6aba0-e1b3-4604-964f-67769eeae2ac.pdf"},{"id":91650402,"identity":"a16cab02-3d1b-4526-aa40-ed3cbce785b4","added_by":"auto","created_at":"2025-09-18 16:55:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":37451,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhytochemical screening of SL extracts, Comparison of activity at different concentrations of plant extract is available in the supplementary material. The videos of the experimental datasets recorded during the current study are available from the corresponding author on reasonable request\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"larvapaperSupplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6758746/v1/fb8afe47b52f8fc450d392ea.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eBehavioral Disruptive Effects of Spathiphyllum Leaf Extract: Insights from Larval Assays in Drosophila melanogaster\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant toxins, commonly referred to as phytotoxins, are a diverse array of bioactive compounds synthesized by plants as a defensive strategy against herbivores, pathogens, and various environmental stressors \u003csup\u003e1\u003c/sup\u003e. These compounds play a critical role in plant survival and adaptation, deterring potential threats through their toxic effects \u003csup\u003e2\u003c/sup\u003e. While many phytotoxins can be harmful or even lethal to living organisms, an increasing body of research highlights their potential therapeutic applications in medicine, particularly in the fields of oncology, neurology, and inflammation management \u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe classification of plant toxins is primarily based on their chemical structure and biological activity \u003csup\u003e4\u003c/sup\u003e. It is evident from previous studies that the efficacy of a range of readily available plant-based extracts and chemicals that may improve brain function and possess other therapeutic properties \u003csup\u003e5,6\u003c/sup\u003e. Among these, alkaloids stand out as nitrogen-containing compounds that frequently exhibit profound pharmacological effects \u003csup\u003e7\u003c/sup\u003e. Well-known examples include morphine, derived from \u003cem\u003ePapaver somniferum\u003c/em\u003e, and nicotine from \u003cem\u003eNicotiana tabacum\u003c/em\u003e \u003csup\u003e8\u003c/sup\u003e. Additionally, cardiac glycosides, such as digoxin from \u003cem\u003eDigitalis purpurea\u003c/em\u003e \u003csup\u003e9\u003c/sup\u003e, are vital in the treatment of heart conditions due to their ability to enhance cardiac function \u003csup\u003e10\u003c/sup\u003e. Other categories of phytotoxins include toxic proteins like ricin from \u003cem\u003eRicinus communis\u003c/em\u003e \u003csup\u003e11\u003c/sup\u003e and various lectins, which inhibit protein synthesis or induce apoptosis in cancer cells \u003csup\u003e12\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe biological activities of these compounds are vast, encompassing anti-inflammatory and antimicrobial properties, which are beneficial in a therapeutic context \u003csup\u003e13\u003c/sup\u003e. For instance, flavonoids and tannins are recognized for their antioxidant capabilities, contributing to the prevention of oxidative stress-related diseases \u003csup\u003e14\u003c/sup\u003e. The mechanisms by which plant toxins exert their effects include the inhibition of protein synthesis \u003csup\u003e15\u003c/sup\u003e, modulation of cellular signaling pathways \u003csup\u003e16\u003c/sup\u003e, and the induction of apoptosis \u003csup\u003e17\u003c/sup\u003e. Ribosome-inactivating proteins (RIPs) like ricin disrupt protein synthesis by targeting ribosomes, ultimately leading to cell death\u0026mdash;a property being explored for targeted cancer therapies \u003csup\u003e18\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDespite their therapeutic potential, the use of plant toxins in medicine poses significant challenges. The fine line between effective and toxic doses complicates their clinical application, requiring meticulous research to optimize dosage and delivery methods \u003csup\u003e19 20\u003c/sup\u003e. For example, colchicine, derived from \u003cem\u003eColchicum autumnale\u003c/em\u003e, has a long history of use for gout due to its anti-inflammatory properties and ability to inhibit microtubule polymerization \u003csup\u003e21\u003c/sup\u003e. Its applications extend to treating familial Mediterranean fever and certain cancers. However, careful management of its dosage is essential to avoid toxic effects \u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn recent years, innovative strategies have emerged to enhance the delivery of plant-derived compounds \u003csup\u003e23\u003c/sup\u003e. Advances in fields such as genomics, proteomics, and nanotechnology are paving the way for novel drug development approaches that can harness the beneficial properties of phytotoxins while minimizing associated risks \u003csup\u003e24\u003c/sup\u003e. For instance, ricin\u0026rsquo;s selective cytotoxicity towards cancer cells, when coupled with targeted delivery systems, holds promise for developing new cancer therapies \u003csup\u003e25\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn addition to these considerations, research has begun to explore the effects of plant extract on model organisms, such as \u003cem\u003eDrosophila melanogaster\u003c/em\u003e, a holometabolic insect widely used in genetic and developmental studies \u003csup\u003e26\u003c/sup\u003e. The use of \u003cem\u003eDrosophila\u003c/em\u003e allows for detailed investigations into the effects of phytotoxins on behavior and physiology, particularly about muscle development and neural function \u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThis research aims to elucidate how both colchicine and extract from \u003cem\u003eSpathiphyllum leaf (SL)\u003c/em\u003e, commonly known as the peace lily, impact larval behavior through their effects on neural circuits and microtubule dynamics in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e. \u003cem\u003eSL\u003c/em\u003e is noted not only for its aesthetic and air-purifying qualities but also for its calcium oxalate crystals, which can induce irritation if ingested \u003csup\u003e28\u003c/sup\u003e, highlighting the need for a comprehensive understanding of its toxicity at cellular levels.\u003c/p\u003e\u003cp\u003eOverall, the exploration of plant toxins as a rich source of bioactive compounds presents a promising avenue for the development of innovative treatments across various medical fields, particularly in oncology and inflammatory diseases. Continued research into their mechanisms of action and improved delivery methods could lead to significant advancements in pharmacotherapy, ultimately enhancing the therapeutic landscape derived from nature\u0026rsquo;s complex biochemical arsenal.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Plant extraction and phytochemical screening\u003c/h2\u003e\u003cp\u003eThe leaf powder of \u003cem\u003eSpathiphyllum walsii\u003c/em\u003e was subjected to extraction in water. A yield of 18% was obtained from the SL extract, which was utilized in the subsequent experiment. The phytochemical analysis conducted on the leaf extract of SL identified several active compounds, including alkaloids, phenols, flavonoids, tannins, carbohydrates, and terpenoids, some of which are recognized for their cytotoxic properties (supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Effect of Colchicine and SL extract on larval behavioral plasticity\u003c/h2\u003e\u003cp\u003eThe study of larval behavioral plasticity in Drosophila using colchicine and SL extract reveals significant insights into the effects of these substances on larval behavior and microtubule dynamics. The results indicate that larvae treated with SL extract exhibited a longer time (160 sec) delay in reaching yeast compared to the control group. In contrast, the colchicine-treated group did not reach the yeast at all. This suggests that both treatments significantly impair the ability of larvae to respond to food stimuli, indicating a potential disruption in motor function or sensory perception.\u003c/p\u003e\u003cp\u003eIt was noted that higher concentrations (10mg/mL) of the plant extract elicited effects comparable to those observed with colchicine (Figs.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This suggests a dose-dependent relationship where increased concentrations of the plant extract lead to more pronounced behavioral impairments, reinforcing the hypothesis that this extract may act through similar pathways as colchicine (Supplementary Fig.\u0026nbsp;6).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Effect of colchicine and SL extract on larval olfactory response\u003c/h2\u003e\u003cp\u003eThe effects of colchicine and SL extract on olfactory responses have been studied, revealing significant insights into how these substances influence sensory perception in larvae. The responsive index (RI) for the control group was normal and higher compared to both the colchicine (CC) and plant extract (SLE) groups. This indicates that the control group exhibited typical olfactory responses. The group treated with SL extract showed a response like the colchicine group, suggesting that both treatments may impair olfactory sensitivity or discrimination abilities. Specifically, as the concentration of SL extract increased, the RI decreased, with 10 mg/ml showing (Figs.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e3\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e4\u003c/span\u003e) a notably lower RI than both 5 mg/ml and 2.5 mg/ml concentrations. (Supplementary Fig.\u0026nbsp;1) The negative response observed in both colchicine and SL extract-treated groups suggests that these treatments lead to altered olfactory behaviors.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Effect of Colchicine and SL extract on contact chemosensory response\u003c/h2\u003e\u003cp\u003eChemosensory assays are critical for assessing an organism's ability to detect and respond to chemical stimuli in its environment. The specific study discussed evaluates the impact of colchicine and SL extract on the chemosensory response in larvae, focusing on their effects on the responsive index (RI). The control group (CC) exhibited a normal and higher RI compared to the groups treated with colchicine and SL extract, indicating intact chemosensory function in the absence of these treatment. The data suggest that SL extract significantly disrupts chemosensitivity more than colchicine. Specifically, the RI was notably lower in the group treated with 10 mg/ml of SL, indicating a strong negative effect on chemosensory behavior. Treatment with colchicine resulted in a decrease in RI, with complete absence observed in larvae treated with 10 mg/ml of SL extract (Figs.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig23\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This suggests that both treatments lead to a significant reduction in chemosensory behavior, likely due to microtubule disruption within sensory neurons. The study highlighted that at a concentration of 10 mg/ml, SL extract caused a more pronounced disruption in RI when compared to lower concentrations (Supplementary Fig.\u0026nbsp;1), reinforcing its potential as a potent disruptor of chemosensory responses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Effect of Colchicine and SL extract on larval phototaxis behavior.\u003c/h2\u003e\u003cp\u003eThe effects of colchicine and SL extract on photo-taxis responses reveal significant insights into how these substances influence larval behavior in response to light. The control group exhibited a normal, positive, and higher RI compared to both the colchicine and SL extract groups. The colchicine-treated group demonstrated a highly disrupted RI, indicating a negative response to light stimuli. In contrast, the SL extract group showed an RI that was closer to the control group, suggesting less disruption in photo taxis behavior than the colchicine group (Figs.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e7\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig24\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The 10 mg/ml concentration of SL extract resulted in a more pronounced disruption of the RI compared to the lower concentrations of 5 mg/ml and 2.5 mg/ml (Supplementary Fig.\u0026nbsp;1). This suggests that lower concentrations may have a milder effect on larval phototaxis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Effect of Colchicine and SL extract on larval motor function\u003c/h2\u003e\u003cp\u003eThe larval motor function assay in Drosophila is a critical experimental setup used to assess the impact of various treatments on the movement and muscle function of larvae. This assay typically involves measuring the time taken for larvae to reach a designated endpoint under different conditions, such as exposure to colchicine and SL extract.\u003c/p\u003e\u003cp\u003eThe results indicate that larvae treated with colchicine (CC group) exhibit a significantly higher time delay in reaching the endpoint compared to those treated with SL extract. The SL Extract group shows a similar delay to the CC group, suggesting both treatments impair motor function. In contrast, the control group (CN) demonstrates a shorter delay, indicating normal motor function under untreated conditions (Figs.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e9\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig25\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The assay results also highlight that a concentration of 10 mg/ml leads to a greater time delay compared to lower concentrations of 5 mg/ml and 2.5 mg/ml (Supplementary Fig.\u0026nbsp;1). This suggests a dose-dependent effect where higher concentrations exacerbate motor function impairment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Effect of Colchicine and SL extract on life cycle.\u003c/h2\u003e\u003cp\u003eThe study of Drosophila melanogaster larvae treated with colchicine and extract from SL aims to investigate the effects of these treatments on various developmental stages, including larvae, pupae, and adult flies. The findings highlight significant disruptions in the life cycle due to both colchicine and plant extract treatments. Both colchicine (CC) and plant extract (SLE) groups exhibited a notable delay in larval development. This resulted in an increased duration of the larval stage compared to the control group (CN). Larvae exposed to these treatments showed abnormal morphology, such as altered body shape and size, indicating that colchicine disrupts microtubule dynamics essential for normal development. The percentage of larvae that transitioned to pupae was significantly lower in the CC and SLE groups. Higher concentrations of SLE reduced pupation rates by approximately 10% compared to the control. Pupae from treated groups displayed disrupted development, leading to abnormal pupal cases and delayed metamorphosis. The adult emergence rates were reduced in both CC and SLE groups at a concentration of 10 mg/ml, with an extended duration for the emergence process noted. Adults that did emerge from colchicine-treated larvae exhibited abnormal wing shapes and sizes, further emphasizing the impact of these treatments on adult morphology (Table\u0026nbsp;1). Observations during reproductive stages revealed that both colchicine and SLE treatments significantly affected reproductive success, leading to reduced fertility and egg viability.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable-1: Life cycle monitor\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePercentage of Larvae to pupae conversion\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTime interval between Pupae to fly conversion\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePercentage of Pupae to Fly conversion\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6 days\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 days\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePE\u003c/p\u003e\u003cp\u003e[10mg/ml]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 days\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePlant bioactive compounds have been extensively studied for their cytotoxic effects \u003csup\u003e29\u003c/sup\u003e. One significant area of interest is how these compounds interact with microtubules, which are critical components of the cell's cytoskeleton and play a vital role in cell division and intracellular transport \u003csup\u003e30\u003c/sup\u003e. Our study highlights the effects of \u003cem\u003eSpathiphyllum\u003c/em\u003e and colchicine on \u003cem\u003eDrosophila melanogaster\u003c/em\u003e larvae and provides significant insights into how these substances impact larval behavior, sensory perception, motor function, and overall development. The findings highlight the potential cytotoxic properties of \u003cem\u003eSpathiphyllum\u003c/em\u003e and its implications for understanding microtubule dynamics in biological systems.\u003c/p\u003e\u003cp\u003eIn our study, the extraction process involved soaking leaf powder of \u003cem\u003eSpathiphyllum\u003c/em\u003e in water for three days, yielding an extract with an 18% yield. Phytochemical screening revealed the presence of various bioactive compounds such as alkaloids, phenols, flavonoids, tannins, carbohydrates, and terpenoids. These compounds are known for their diverse biological activities, including cytotoxic effects that may influence larval behavior and physiology \u003csup\u003e31\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePlant-derived toxins can lead to neurological impairments in \u003cem\u003eDrosophila\u003c/em\u003e larvae, and Drosophila models are emerging models in toxicity research. For instance, exposure to essential oils has been linked to the presence of vacuoles in brain tissue, indicating potential neurotoxic effects\u003csup\u003e32\u003c/sup\u003e. Moreover, specific studies have noted that certain toxins can induce excitatory junctional potential leading to paralysis, demonstrating their profound impact on neuromuscular function\u003csup\u003e33\u003c/sup\u003e. These findings emphasize the role of sensory neurons in mediating responses to toxic substances.\u003c/p\u003e\u003cp\u003eThe olfactory response assays in our study demonstrated that both treatments significantly affected sensory perception. Control larvae exhibited normal olfactory responses, while those treated with SL showed reduced responsiveness like the colchicine group. The responsive index (RI) decreased with increasing concentrations of SL extract, indicating impaired olfactory sensitivity. This suggests that both treatments adversely affect the larvae's ability to detect and respond to chemical stimuli in their environment.\u003c/p\u003e\u003cp\u003eThe sensory systems of \u003cem\u003eDrosophila\u003c/em\u003e are crucial for detecting environmental cues, including the presence of toxins. Research has identified various chemoreceptors involved in sensing toxic compounds, which are essential for behavioral avoidance strategies \u003csup\u003e34\u003c/sup\u003e. The impairment of these sensory mechanisms due to toxin exposure can lead to increased vulnerability and decreased survival rates.\u003c/p\u003e\u003cp\u003eChemosensory assays further confirmed the negative impact of both treatments on chemosensory behavior. Control larvae displayed intact chemosensory function, while those treated with either colchicine or SL extract exhibited significantly lower RI values. Notably, SL extract at 10 mg/mL caused a more pronounced disruption than colchicine, highlighting its potential as a potent disruptor of chemosensory responses.\u003c/p\u003e\u003cp\u003eThe disruption in phototaxis can be attributed to the effects of toxins on the sensory pathways involved in light detection. \u003cem\u003eDrosophila\u003c/em\u003e larvae utilize specific photoreceptors to sense light, and toxins may interfere with the functioning of these receptors or the neural circuits that process light information \u003csup\u003e35\u003c/sup\u003e. For instance, genetic studies indicate that specific photoreceptor subtypes are essential for light avoidance, and their impairment due to toxin exposure can lead to a complete loss of phototactic behavior \u003csup\u003e36\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePhototaxis assays in our study revealed that control larvae responded positively to light stimuli, while those treated with colchicine showed significant disruption in their phototactic behavior. The SL extract group demonstrated less disruption compared to colchicine but still exhibited altered responses at higher concentrations. This suggests that while SL extract may impair phototaxis, it does so to a lesser extent than colchicine.\u003c/p\u003e\u003cp\u003eExposure to various plant extracts and environmental toxins can lead to marked reductions in the climbing abilities of \u003cem\u003eDrosophila\u003c/em\u003e. For instance, studies have shown that exposure to paraquat (PQ), a herbicide, significantly impairs mobility, mimicking symptoms of neurodegenerative diseases \u003csup\u003e37\u003c/sup\u003e. In experiments, \u003cem\u003eDrosophila\u003c/em\u003e that were pre-fed with neuroprotective compounds like Gardenin A showed restored climbing abilities post-exposure to PQ, indicating that certain phytochemicals can mitigate toxin-induced mobility defects \u003csup\u003e38\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMotor function assessments indicated that both treatments significantly delayed larval movement towards a designated endpoint. The results showed that colchicine-treated larvae had a greater delay than those treated with SL extract. This reinforces the notion that both substances impair motor function but suggests that SL extract may have a milder effect at lower concentrations.\u003c/p\u003e\u003cp\u003eExposure to plant-derived toxins can lead to developmental delays and morphological abnormalities in \u003cem\u003eDrosophila\u003c/em\u003e larvae. For instance, studies involving extracts from various plants have indicated that certain toxins can reduce larval growth rates and alter developmental timelines. The presence of these toxins often results in increased mortality rates during the larval stage, particularly at higher concentrations. For example, \u003cem\u003eEuphorbia prostrata\u003c/em\u003e showed a mortality rate of 51.64% at a 30% concentration after 72 hours of exposure, indicating significant toxicity that affects overall development \u003csup\u003e39\u003c/sup\u003e. Toxins can also impact the reproductive capabilities of \u003cem\u003eDrosophila\u003c/em\u003e. Research indicates that females exposed to certain toxic compounds have reduced fecundity, meaning they produce fewer offspring. In studies involving macro fungi, it was found that female flies were more sensitive to toxins compared to males and exhibited shorter survival times, with some treatments resulting in no offspring production at all \u003csup\u003e40\u003c/sup\u003e. However, excessive exposure to toxic substances typically results in decreased lifespan due to increased mortality \u003csup\u003e41\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe study also investigated how these treatments affected the overall life cycle of \u003cem\u003eDrosophila\u003c/em\u003e. Both colchicine and SL extract resulted in delayed larval development and abnormal morphology. Higher concentrations of plant extract notably reduced pupation rates and led to abnormal adult morphology upon emergence. Furthermore, reproductive success was compromised in both treatment groups, indicating that these substances disrupt not only immediate behaviors but also long-term developmental processes.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe findings from this study underscore the significant effects of SL extract and colchicine on \u003cem\u003eDrosophila\u003c/em\u003e larvae across various behavioral and physiological parameters. The presence of bioactive compounds in SL extract suggests potential applications in pharmacology and toxicology, particularly concerning microtubule dynamics and SSSsensory processing. Future research could explore the specific mechanisms by which these extracts exert their effects and their potential therapeutic applications or ecological impacts. This comprehensive analysis highlights the importance of understanding plant extract biochemical properties and their implications for model organisms like \u003cem\u003eDrosophila\u003c/em\u003e, serving as a foundation for further investigations into their biological activities and potential uses in various fields.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4.1.\u0026nbsp; \u0026nbsp;Collection of plant material\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSpathyphyllum plant\u0026nbsp;\u003c/em\u003eleaves were collected from a local horticulture nursery, Mysuru, Karnataka, India in March 2024. The plant leaves are subjected to shade dry for 15 days, and then the dried leaves are powdered and soaked in water for 3 days on a magnetic stirrer, then the supernatant is collected and transferred into petri plates for drying. \u0026nbsp;Dried aqueous extract was scraped off and weighed to calculate the yield percentage and used for further studies. Extract was screened for the presence of phytochemicals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4.2.\u0026nbsp; \u0026nbsp;Fly selection and third instar larvae collection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlies stocks used for the present study were \u003cem\u003eDrosophila melanogaster\u003c/em\u003e (\u003cem\u003eOregon K\u003c/em\u003e strain). These flies were obtained from the Drosophila Stock Centre, Department of Zoology, University of Mysore, Mysore, Karnataka. The fly strains were kept in glass vials with a normal Drosophila medium at room temperature (25±2 °C). Fresh egg plates with a small amount of yeast paste in the middle were used to collect eggs from 1–10 day-old adult flies. After 24-hour incubation intervals, the plates were swapped out and left at room temperature, allowing the hatched larvae to develop. The trials investigated larvae from these plates that were in their early third instar (72–78 hours) \u003csup\u003e42\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4.3.\u0026nbsp; \u0026nbsp;Collection and Washing of Third-Instar Larvae\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWandering third instar larvae was taken from the growth medium. Immediately, larvae were subjected to careful washing using 15% sucrose initially, followed by washing with double-distilled water twice using a small, moistened paintbrush until the larvae became clean of yeast. Gently stir the water containing larvae with the brush to aid in washing, and the water was drained completely using a 1ml Pipette. After the washing procedure, larvae were placed in a small amount of sucrose water and then distilled water until the assays were performed \u0026nbsp;\u003csup\u003e43\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4.4.\u0026nbsp; \u0026nbsp;Pre-incubation and treatment of larvae\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collected larvae were subjected to pre-incubation prior to the beginning of the experiment. A period of starvation was maintained initially by subjecting 15 larvae simultaneously to double-distilled water for 20 minutes under normal light exposure. After 20 minutes of starvation, the larvae were subjected to a treatment (feeding) period. For this to be carried out, together control and colchicine, and plant extract with different concentrations treated groups were maintained separately. Each group had 3 larvae. In the control group selected larvae followed the incubation period of 10 minutes using double-distilled water under bright light. In colchicine treated group, larvae were administered with 1000µM colchicine (colchicine prepared in 0.1% DMSO) for 10 minutes under bright sunlight, remaining three groups’ larvae are treated with SL extract in the concentration of 10mg/ml, 5mg/ml and 2.5mg/ml for 10 minutes under bright light. All the control, colchicine, and plant treatment was carried out simultaneously, ensuring the feeding of larvae after a brief period of starvation. After the period of pre-treatment of larvae, an assay was carried out immediately.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4.5.\u0026nbsp; \u0026nbsp;Behavioral plasticity Assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLarvae treated with colchicine and SL extract underwent simultaneous and independent assays. A plate was separately kept untreated as a control. In the experimental design, the assay plate was made up of a glass petri plate (100mm x 15 mm) consisting of four quadrants. 20 ml of 10% agar was carefully poured into the petri plate uniformly and placed aside for solidification. At the beginning of the assay, the larvae were placed such that they would spread out 5 mm from the edge of the plate. There was no fixed period followed during the assay; we rather assessed the recognition and crawling speed of control and treated larvae towards yeast at their respective time intervals. The larvae were allowed to reach the yeast, and time was noted down for the time interval of each larva reaching the yeast in both the control and treated groups. The assays were continued until the entire 3 larvae reached the yeast, and the final time was noted down for evaluation. The assays were carried out in singlets for all the groups \u003csup\u003e42\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4.6.\u0026nbsp; \u0026nbsp;Olfactory assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the experimental design, the assay plate was made up of a glass petri plate (100mm x 15 mm) consisting of four quadrants. 20 ml of 10% agar was carefully poured into the petri plate uniformly and placed aside for solidification. Two filter discs (made from Whattman No. 1 filter paper) were placed at opposite edges of the plate in such a fashion that they face diametrically opposite each other. A fruity odorant (an extract from papaver fruit) was added to one disc and diluent (distilled water) added to the other. The larvae were carefully placed in the center of the plate with a smooth pointed brush, and the larvae were allowed to travel for 1 minute. After this duration of one minute, the number of larvae present on each half of the plate was counted (supplementary figure 2). A response index (RI) was calculated by subtracting the number of animals on the control half of the plate (C) from the number on the stimulus half (S) and dividing by the total: RI = (S - C)/(S + C). For consideration, often a small percentage of larvae remain in the center of the plate and are neglected \u003csup\u003e44\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4.7.\u0026nbsp; \u0026nbsp;Contact Chemosensory Assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the experimental design, the assay plate was made up of a glass petri plate (100mm x 15 mm) consisting of four quadrants. 20 ml of 10% agar was carefully poured into the petri plate uniformly and placed aside for solidification. As a chemosensory source, a defined concentration of 0.1M NaCl was used. After solidification, (0.1M) NaCl was carefully spread over the surface of two diametrically opposite quadrants. The other two opposite quadrants were kept as a plain agar surface. The plates were used 10 minutes later just to ensure the uniform diffusion of 0.1M NaCl through the thin superficial layer of 10% agar on the surface of the plate.\u003c/p\u003e\n\u003cp\u003eThe pre-incubated and treated larvae were used in the assay. Larvae from each treatment group were carefully placed at the center of the agar plate with the help of a smooth brush and were allowed to migrate for 6 min (supplementary figure 3). The assay was carried out in triplicate. The ultimate number of larvae on each quadrant was taken to counted after six minutes, and responsiveness was determined by considering the average number of larvae moving towards 0.1M NaCl and plain agar \u003csup\u003e45\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4.8.\u0026nbsp; \u0026nbsp;Larval Photo-taxis Assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe photo-taxis assay was similar to the contact chemosensory assay with slight modifications \u003csup\u003e45\u003c/sup\u003e. The assay plate was prepared by adding 20 ml of 10% agar uniformly on a glass petri plate and was allowed to solidify. Each of two opposed quadrants was painted (black) to ensure the dark background is created at the bottom of the plate. The other two opposite quadrants were kept transparent (uncolored) for light to enter.\u003c/p\u003e\n\u003cp\u003eThe preincubated and treated larvae were used in the assay. Larvae were carefully placed at the center of the plate using a smooth brush and assayed for 8 minutes. The number of larvae migrating towards either side of the background after 8 minutes was noted. The responsive index (RI) was calculated as mentioned earlier in the olfactory assay (supplementary figure 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4.9.\u0026nbsp; \u0026nbsp;Larval motor function assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the experimental design, a 100 mm x 10 mm Petri dish having 10% Agar, 5 parallel tracks of 1cm wide grids were created. The track was moistened with water to ease the crawling. Before the crawling ability test, larvae were carefully placed on an identical dummy track and allowed to acclimatize for 5 minutes before being transferred to the test track. The preincubated and treated larvae were used in the assay. The time consumed to crawl each 1cm track to the ‘finish line’ was recorded (supplementary figure 5). During the course of the test, if the larva turned backwards or returned, the timer was stopped, and it was then again placed back to the starting position, and then timing was resumed \u003csup\u003e46\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4.10.\u0026nbsp;Study on the Life cycle of larvae\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the study, 50 healthy and active third instar larvae were collected from culture bottles. The larvae were grouped into Control, Colchicine-treated treated and plant extract-treated groups (n=10). The control group was kept untreated, and the Colchicine group was treated with colchicine medium, and the plant extract groups with plant extract medium (2.5, 5, and 10mg/mL).\u003c/p\u003e\n\u003cp\u003eEvery day, the size, shape, and instar stages of the larvae were noted and observed. \u0026nbsp;The rate, duration, and morphology of the larvae's pupation (creation of pupal cases) were tracked, and the size, shape, and color of the pupils were documented. Monitoring was done on adult emergence, emergence rate, time, and morphology. \u0026nbsp;It was determined what proportion of each group's larvae developed into pupae. \u0026nbsp;It was noticed how long it took for the larvae to develop into pupae and then turn into flies. A microscope was used for every observation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4.11.\u0026nbsp;Statistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were analyzed using GraphPad Prism (version 10.4.2). Experiments were conducted in triplicate, and results are presented as mean ± standard error of the mean (SEM). Statistical comparisons between groups were performed using one-way ANOVA. A p-value of less than 0.001 (p \u0026lt; 0.001) was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge Bioscience CLIx LLP for providing the laboratory facilities and scientific guidance essential to the successful completion of this work. Artificial intelligence tools, ChatGPT by OpenAI, and Perplexity were used solely to assist with language editing and improving the clarity. The scientific content was conceived, written, and verified entirely by the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors H. S. Pruthvi, T M Aishwarya, \u0026nbsp;K. B. Vijendra Kumar, Kavitha Raj Varadaraju, Karthik N Awathade, K. S. Bhargava Shreevatsa, Chandan Shivamallu and Chandan Dharmashekharwere involved in the conception, design, and analysis, interpretation of the data and drafting of the paper. Kavitha Raj Varadaraju and Karthik N Awathade were also involved in revising it critically for intellectual content and the final approval of the version to be published. All authors agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhytochemical screening of SL extracts, Comparison of activity at different concentrations of plant extract is available in the supplementary material. The videos of the experimental datasets recorded during the current study are available from the corresponding author on reasonable request\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMith\u0026ouml;fer, A. \u0026amp; Boland, W. Plant Defense Against Herbivores: Chemical Aspects. \u003cem\u003eAnnu. Rev. Plant Biol.\u003c/em\u003e \u003cstrong\u003e63\u003c/strong\u003e, 431\u0026ndash;450 (2012).\u003c/li\u003e\n\u003cli\u003eLannoo, N. \u0026amp; Van Damme, E. J. M. Lectin domains at the frontiers of plant defense. \u003cem\u003eFront. 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Methods\u003c/em\u003e \u003cstrong\u003e145\u003c/strong\u003e, 63\u0026ndash;72 (2005).\u003c/li\u003e\n\u003cli\u003eFye, S., Dolma, K., Jung Kang, M. \u0026amp; Gunawardena, S. Visualization of Larval Segmental Nerves in 3\u003csup\u003erd\u003c/sup\u003e Instar Drosophila Larval Preparations. \u003cem\u003eJ. Vis. Exp.\u003c/em\u003e 2128 (2010) doi:10.3791/2128.\u003c/li\u003e\n\u003cli\u003eKhurana, S. \u0026amp; Siddiqi, O. Olfactory Responses of Drosophila Larvae. \u003cem\u003eChem. Senses\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 315\u0026ndash;323 (2013).\u003c/li\u003e\n\u003cli\u003eLilly, M. \u0026amp; Carlson, J. smellblind: a gene required for Drosophila olfaction. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e124\u003c/strong\u003e, 293\u0026ndash;302 (1990).\u003c/li\u003e\n\u003cli\u003eNichols, C. D., Becnel, J. \u0026amp; Pandey, U. B. Methods to Assay Drosophila Behavior. \u003cem\u003eJ. Vis. Exp.\u003c/em\u003e 3795 (2012) doi:10.3791/3795.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Figures","content":"\u003cp\u003eSupplementary Figures 2-6 are not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Spathiphyllum walsii, Phytotoxin, Drosophila melanogaster, Colchicine, Photo-taxis","lastPublishedDoi":"10.21203/rs.3.rs-6758746/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6758746/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigates the effects of phytotoxins derived from \u003cem\u003eSpathiphyllum walsii \u003c/em\u003e\u0026nbsp;(peace lily) on the behavioral plasticity of \u003cem\u003eDrosophila melanogaster\u003c/em\u003e larvae, focusing on their implications for sensory perception and motor function. Phytotoxins, known for their diverse bioactive properties, serve as both a defense mechanism for plants and potential therapeutic agents in medicine.\u003c/p\u003e\n\u003cp\u003eThe research involved the extraction of phytochemicals from \u003cem\u003eSpathyiphyllum \u003c/em\u003eleaves, followed by a series of behavioral assays to assess larval responses to food stimuli, olfactory cues, and environmental conditions. The results demonstrated that larvae treated with \u003cem\u003eSL \u003c/em\u003eextract exhibited significant delays in reaching food sources compared to control groups, indicating impaired sensory and motor functions. Notably, higher concentrations of the extract produced effects similar to those of colchicine, a well-known microtubule inhibitor. Behavioral assays revealed that both treatments disrupted normal larval behavior, suggesting that phytotoxins may influence neural circuits and microtubule dynamics.\u003c/p\u003e\n\u003cp\u003eThese results underscore the potential of plant-derived compounds as modulators of neural function and behavior, highlighting both their therapeutic prospects and risks associated with their use. This study highlights the potential of plant-derived compounds in understanding neurobiological processes and offers insights into their therapeutic applications. Further research is warranted to elucidate the mechanisms underlying these effects and to explore the broader implications of phytotoxins in pharmacotherapy and toxicology.\u003c/p\u003e","manuscriptTitle":"Behavioral Disruptive Effects of Spathiphyllum Leaf Extract: Insights from Larval Assays in Drosophila melanogaster","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-18 16:38:57","doi":"10.21203/rs.3.rs-6758746/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-14T06:42:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T13:22:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T12:50:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-28T08:03:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330429308880727206288547890896750891439","date":"2025-09-19T04:19:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"318968870374457310575285634260080564111","date":"2025-09-19T02:46:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"204762435463842672567190768246055031798","date":"2025-09-15T17:39:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"63818102483734181218543845039088461083","date":"2025-09-10T08:24:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-09T23:44:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-17T05:06:51+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-17T04:57:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-15T03:15:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-15T03:06:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0647e16d-13fa-4697-b64b-50d1be637b90","owner":[],"postedDate":"September 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":54843343,"name":"Biological sciences/Biochemistry"},{"id":54843344,"name":"Health sciences/Neurology"}],"tags":[],"updatedAt":"2026-05-15T05:55:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-18 16:38:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6758746","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6758746","identity":"rs-6758746","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}