Plastic pollution reshapes snail intermediate host life history: implications for schistosomiasis transmission

Plastic pollution reshapes snail intermediate host life history: implications for schistosomiasis transmission | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Plastic pollution reshapes snail intermediate host life history: implications for schistosomiasis transmission Ao Yu, Kaitlyn R Mitchell, J Trevor Vannatta, Giulio De Leo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9510130/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Plastic debris is increasingly recognized as an ecological driver of mosquito-borne disease by creating freshwater breeding habitat, yet its consequences for snail hosts of human schistosomiasis transmission remain poorly understood. For these snail hosts, including Bulinus truncatus , plastic pollution may alter population dynamics by modifying life-history processes through surface biofilm-derived nutritional subsidies, habitat alteration, and toxicity-induced reproductive trade-offs. Methods We performed two laboratory experiments that investigated the effects of macro- and microplastics, individually and jointly, on B. truncatus’ life-history traits (survival, growth, egg mass production, hatching) by using environmentally realistic sizes, polymers, concentrations, and biofouling status. To assess the overall impact of plastic pollution on snail demography, we derived a measure of reproductive output under each plastic treatment, i.e., the expected number of cumulative viable offspring produced over the experiment. Results We found that macroplastic boosted somatic growth, egg mass production, and hatching, consistently playing the most critical role in the demographic increase driven by biofilm-derived nutritional subsidies. Microplastics reduced hatching success but improved survival and growth, resulting in a marginal population decline due to toxicity-induced maintenance-reproduction trade-offs. Combined exposure elevated survival, growth, and egg mass production while mitigating microplastic-induced hatching costs, generating positive but attenuated gains due to nutrition-buffered toxicological costs. Collectively, these results demonstrate that plastic exposure can shift snail population trajectories by altering life-history processes that scale to net reproductive output, with biofouling emerging as the primary driver of demographic response. Conclusions Our findings show that plastic pollution increases B. truncatus demographic output by altering its key life-history traits, which prior work has linked to increased cercarial production with critical implications for human transmission risk. These findings identify plastic pollution as a previously unrecognized pathway and demonstrate that ecological transformation of plastic debris is the dominant determinant of host demographic response with implications for integrating environmental management into disease control strategies. Plastic pollution Microplastic Macroplastic Bulinus Schistosomiasis Life history Population dynamics Disease ecology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Plastic pollution has emerged as an Anthropocene-driven crisis, resulting from escalating plastic production and waste accumulation with slow degradation. Global plastic production increased from 5 million tons in the 1950s to 300 million tons in 2020( 1 ), and currently, 79% of the plastic waste ends up either in landfills or the natural environment( 2 ). In recent years, a large body of research has found that plastic pollution increases human malaria and dengue transmission, because this debris creates additional breeding habitats that support the freshwater life stages of the mosquito vectors responsible for the disease spread( 3 – 5 ). By contrast, little is asked about whether other medically important freshwater hosts similarly benefit from plastic pollution. Studies on the effects of plastic on snail-borne disease, including schistosomiasis, remain extremely limited. In particular, the effects of pollution on key snail life-history traits (i.e., survival, growth, fecundity, and hatching) are largely unknown, even though these traits govern population persistence and even minor shifts could influence transmission potential. To address this knowledge gap, it is necessary to focus on specific disease-relevant snail hosts where changes in life history could directly alter human health risk. Human schistosomiasis is among the most prevalent neglected tropical diseases worldwide, second only to malaria in global burden( 6 ), affecting approximately 250 million individuals, placing approximately 800 million at risk, and disproportionately impacting impoverished rural communities( 7 , 8 ). Urogenital schistosomiasis (caused by Schistosoma haematobium ) is transmitted by snails in the genus Bulinus , while intestinal schistosomiasis (caused by Schistosoma mansoni ) is transmitted by Biomphalaria snail species. Because urogenital schistosomiasis is often more prevalent, particularly among school-aged children, Bulinus snails become a priority for investigating life-history traits and demography( 9 , 10 ). Because no effective vaccines are available, current control strategies rely on snail control( 11 – 13 ) and mass drug administration of praziquantel( 14 ), which does not prevent reinfection( 14 ), making accurate risk assessment crucial. Within this context, identifying how plastic pollution may drive snail host life history reveals three major mechanisms: (i) macroplastics function as substrates that promote biofilm growth and provide nutritional and habitat subsidies, (ii) microplastics exert direct toxicological effects following ingestion, and (iii) interactions between macroplastic-associated nutrition and microplastic-induced toxicity jointly shape Bulinus life-history traits. Plastic pollution has the potential to influence Bulinus snails’ life history via multiple mechanisms. These mechanisms differ by plastic form, with macroplastics (> 5 mm) and microplastics (from 1 µm) exerting distinct effects( 15 ). Macroplastics may act as substrates for biological enrichment. Macroplastic surfaces can support dense microbial biofilms (also known as biofouling( 16 , 17 ), periphyton( 17 , 18 ), or plastisphere( 19 )) that serve as nutritious food for freshwater grazers like snails( 20 ). These biofilms are rich in long-chain polyunsaturated fatty acids and sterols, which are essential nutrients that increase both food abundance and diversity and could enhance snail growth, survival, and reproduction( 21 ). Moreover, macroplastic may create a novel habitat and refugia that support larger snail populations. By contrast, the dominant mechanism associated with microplastics is physiological toxicity following ingestion. Ingested microplastics can bioaccumulate and induce oxidative stress, histopathological alterations, developmental toxicity, and behavior changes( 22 ) that may reduce snail survival, fecundity, and hatching success. Notably, the nutritional properties of macroplastic-associated biofilms have the potential to mitigate these effects, as polyunsaturated fatty acids are known antioxidants that can support antioxidant defenses and increase total antioxidant capacity( 23 , 24 ). Therefore, co-exposing snails to nutrient- and antioxidant-rich biofouled macroplastic could potentially buffer microplastic-induced oxidative stress and alter key life-history outcomes. Plastics may also exert indirect environmental effects, such as shifting the water microbial communities and water parameters such as dissolved oxygen, pH, conductivity, etc( 25 , 26 ). Snails depend on these specific water parameters and, therefore, could be affected. Existing research typically analyzes macro- and microplastics separately, emphasizes acute exposure to high doses or abundance, or focuses on adult organisms( 22 , 27 ). Here, we address this gap with two sequential randomized controlled experiments exposing Bulinus truncatus to macro- and microplastics, systematically varying sizes, biofouling status, and abundance. We quantified key life-history traits and modeled demographic outcomes to assess the long-term effects of plastic. Our findings provide novel insights into how plastic pollution shapes the population dynamics of Bulinus snails with implications for schistosomiasis risks. Materials and Methods Lab experimental reagent and setup We conducted two sequential rounds of experiments (Fig. 1 ) to investigate the long-term effects of plastic pollution on B. truncatus ’s life-history response, with consideration of space constraints and personnel capacity. In the first round, we assessed the effect of plastic pollution on 4 treatment groups: negative control, virgin microplastic, biofouled macroplastic, and a combined treatment from September to December 2024. In the second round, we focused only on macroplastic and used 5 treatment groups: negative control, virgin macroplastic of high and low abundance, and biofouled macroplastic of high and low abundance from April to June 2025. We used B. truncatus snails from the Biomedical Research Institute, which originated from Egypt. B. truncatus snails were born and raised under controlled laboratory conditions (~ 25°C, the ambient temperature of the laboratory, 12-hour light/12-hour dark, artificial pond water). In the first round, we housed 3 snails per jar, starting with 75 jars per treatment. In the macroplastic-only treatment and the combination treatment, 57 jars in each treatment were eliminated due to metal contamination from staples, which was intended for experimenting with biofouled macroplastic shaping (Text S1 and Table S1 -S2). Therefore, only 18 jars in the macroplastic-only treatment and the combination treatment remained for the analysis. In the second round, we housed 3 snails per jar, 50 jars per treatment for negative control and biofouled macroplastic of high or low abundance, and 29 jars per treatment for virgin macroplastic of high or low abundance due to the limited total number of snails available from the Biomedical Research Institute. Each glass jar was 470 mL in size and contained 250 mL of water. The snails were fed organic romaine lettuce ad libitum based on the number of snails alive in each jar. We covered each jar with mesh that allowed air exchange but limited evaporation. We changed the water in each jar weekly to maintain water quality( 28 ). Macroplastic reagent and periphyton profiling We picked 500 mL single-use plastic bottles made from Polyethylene Terephthalate (PET) from Crystal Geyser®, given that it is one of the most common macroplastic types found in waterways( 29 , 30 ). All bottles were initially processed by removing labels and cutting them vertically in half to generate plastic sheets. To biofoul macroplastic, plastic sheets were submerged in freshwater, allowing surface microbial growth to develop( 19 , 31 ). Field biofouling was conducted at Jasper Ridge Biological Reserve (Searsville Lake) during experiment round one and the latter half of round two. During the first half of round two, biofouling was conducted under laboratory conditions due to unsuitable field conditions (extreme precipitation, high water velocity, and low temperatures). More details on the field and lab macroplastic biofouling protocols are available in Text S2. All plastic sheets (field- or lab-biofouled or virgin) were trimmed into uniform 5 cm x 4 cm pieces. To explicitly test the effect of macroplastic abundance, we manipulated the number of macroplastic pieces per jar, using three pieces to represent high abundance and one piece to represent low abundance. Round one and two included both abundance levels. Biofouled macroplastic pieces were replaced when 80–100% of jars showed visible periphyton depletion during weekly monitoring, and virgin macroplastic pieces were not replaced throughout the experiment. Additionally, we profiled surface periphyton in experiment round two for each batch of biofouled macroplastic replacement, and 4 batches total. We measured the absolute abundance (dry mass; DM), proportion of organic matter (ash-free dry mass/ dry mass; AFDM/DM), nutritional quality (C:N), and autotroph to heterotroph ratio (chlorophyll a.:AFDM), periphyton physiological condition (chlorophyll a.:pheophytin)( 32 , 33 ), and took representative microscopy pictures, which all used standard protocols with more detail available in Text S3. We compared the lab versus field biofouling methodology across these aspects using a Mann-Whitney U test. Microplastic reagent We purchased Fluorescent Yellow Polyethylene (PE) microplastic spheres with a size range from 10 um to 27 um and a density of 1g/cc from Cospheric. We used virgin polyethylene microplastics, as polyethylene is the most common type of microplastic produced and found in natural systems( 34 , 35 ). Plastic is ubiquitous in the freshwater bodies, including where Bulinus snails are prevalent, with large heterogeneity in polymer type, shape, and size( 36 , 37 ). We used a 1000 ug/L microplastic concentration for the laboratory experiments, higher than the limited sampling report in Africa( 38 ) and Western Europe( 39 ), but lower than other microplastic and freshwater snail studies( 40 – 42 ) to capture the effect of higher but ecologically relevant concentrations of microplastic where the Bulinus snails are present. We prepared the virgin microplastic treatment using weighted PE microspheres and artificial pond water, formulated according to Biomedical Research Institute guidelines( 43 ) Lab experimental data collection We recorded weekly snail survival, shell growth, and egg masses laid for 13 weeks in both rounds (Fig. 1 B), while hatching success was assessed for 12 weeks in each round, as eggs enrolled during the final experimental week required a full 3-week evaluation period and could not be fully assessed within the observation window. We assessed snail mortality by whether the snail was motile, responded to a stimulus, or was obviously decaying (empty shell or shell residuals). We placed snails under the microscope to take shell pictures for length measurement in ImageJ, defined by the distance from the snail shell apex to the basal lip. Growth was calculated as the change in average snail length per jar from the previous week to the current week. We performed length measurement cross-validation with detailed data cleaning decisions reported in the Table S3 and Text S4. We then decanted the water from the jar through a metal sieve, counted and scraped off snail egg masses on the sieve surface, the macroplastic, and the sides of the jar. We disinfected the jar with 70% alcohol wipes between water changes. We placed approximately ten viable eggs in a well plate from each jar to test for hatching success with weekly water changes of the wells. We recorded the number of snails hatched at 1 week and 3 weeks after enrollment, and any egg that failed to hatch after 3 weeks of enrollment was taken as unviable. Lab experimental statistical analysis We estimated snail survival via a mixed-effect proportional hazard regression model using the coxme and survival packages in R. We modeled snail growth with a generalized additive model using Gaussian errors (identity link) and an AR( 1 ) correlation structure; predictors included treatment and a smooth of the previous week’s mean shell length. We analyzed snail egg mass production with a negative binomial generalized linear model (log link) with jar level random effect and analyzed hatching success with a zero-inflated binomial model with jar level random effect created in the glmmTMB package. We made all pairwise comparisons using the emmeans R package with the multivariate t distribution and a Bonferroni p-value correction for multiple comparisons. Mathematical model on snail demography We computed the snails’ expected reproductive output as the number of live snail offspring that a snail would be expected to produce from the beginning of the experiment over the entire duration of the experiment, namely: $$\:{R}_{obs}^{*}\:=\:{\sum\:}_{a\:=\:1}^{12}l\left(a\right)\times\:b\left(a\right)\times\:h\left(a\right)$$ where \(\:a\) is snail age [weeks] counting from the beginning of the experiment; \(\:l\left(a\right)\) is age-specific survival probability at the treatment-level, i.e., the probability that a snail at the beginning of the experiment will survive until week a ; \(\:b\left(a\right)\) is age-specific birth rate at the treatment-level, that is, the mean number of eggs laid per snail. \(\:h\left(a\right)\) is the age-specific hatching rate at the treatment-level, i.e., the fraction of eggs produced in week a that hatch. To quantify the intrinsic variability of snail reproductive output within each treatment, we performed a nonparametric bootstrap at the jar level. Specifically, for each treatment, we (i) resampled jars with replacement while preserving the original sample sizes, (ii) computed the corresponding reproductive output \(\:{R}_{obs}^{*}\) for the resulting pseudo-dataset as described above, and (iii) repeated this procedure 10,000 times. This yielded (iv) a bootstrap distribution of ​​jar-level reproductive output, which we then aggregated by treatment to obtain the mean reproductive output per treatment, and we used as a proxy for the underlying variability in reproductive performance. To assess whether expected reproductive output differed significantly between treatments, we performed a permutation (randomization) test. For each pair of treatments i and j , we first computed the observed difference in the mean treatment-level reproductive output: $$\:{D}_{obs}^{}\:=\:{R}_{obs}^{*}\left(i\right)\:-\:{R}_{obs}^{*}\left(j\right)$$ Under the null hypothesis of no treatment effect, the jar labels “ i ” and “ j ” are exchangeable. We therefore pooled the jars from both treatments and repeatedly ( n perm = 10,000 times) reassigned the labels “ i rep ” and “ j rep ” at random (without replacement) while preserving the original sample sizes. For each permutation, we recalculated the expected reproductive outputs \(\:{R}_{rep}^{*}\) (i) and \(\:{R}_{rep}^{*}\) (j) using the precomputed jar-level \(\:{R}_{obs}^{*}\) for the randomized groups and obtained the permuted difference: $$\:{D}_{rep}^{}\:=\:{R}_{rep}^{*}\left(i\right)\:-\:{R}_{rep}^{*}\left(j\right)$$ The collection of \(\:{D}_{rep}^{}\) values formed the null distribution against which \(\:{D}_{obs}^{}\) was compared. Because we made no directional assumption, we used a two-tailed test, computing the p -value as: $$\:p\:=\:\frac{\#\:\{\left|{D}_{rep}^{}\right|\:\ge\:\:\left|{D}_{obs}^{}\right|\}\:+\:1}{{n}_{perm}^{}\:+\:1}$$ When evaluating multiple pairwise comparisons, we adjusted the resulting p -values using the Bonferroni correction to control for family-wise error (6 comparisons in round one, and 10 in round 2). Results Effect of plastic pollution on snail survival Exposure to microplastics or microplastic-associated treatments increased survival probability in B. truncatus snails, while macroplastics with and without biofouling had no effects. A hazard ratio (HR) near 1 indicates no difference in mortality risk compared to controls. In round one, the HR for snails in virgin microplastic relative to control was 0.55 (95% CI HR [0.40, 0.76], p < 0.001; Fig. 2 A), corresponding to approximately a 45% lower risk of mortality compared to control snails. The HR for snails in the joint treatment with respect to control was 0.57 (95% CI HR [0.32, 0.99]), which is significant from the model output (p 0.05). This estimate corresponds to an approximate 43% reduction in mortality risk relative to controls, which is not statistically robust. The HR for snails in biofouled macroplastic with respect to control is 1.09 (95% CI HR [0.69, 1.70], p > 0.05). Similarly, in the second round, survival probabilities did not differ significantly among any macroplastic treatments over the 13-week experiment (p > 0.05; Fig. 2 A). For coefficients and pairwise comparisons, see Supplement Table S4-S9. Effect of plastic pollution on snail shell growth All plastic treatments significantly enhanced snail growth relative to controls, with growth varying based on the combination of treatments and abundance. In both rounds, snails in all treatment groups exhibited significantly greater weekly shell growth than the control. In round one, snails in the biofouled macroplastic produced the largest mean weekly growth compared to the control (93.3 µm, p < 0.001; Fig. 2 B), followed by the joint treatment (51.2 µm, p < 0.005) and the virgin microplastic (40.1 µm, p < 0.001). In round two, both snails exposed to biofouled macroplastic or virgin macroplastic significantly increased weekly growth compared to control snails (33.2 um, p < 0.005; 27.9 µm, p < 0.05; Fig. 2 B). Specifically, treatment groups with varying macroplastic abundance were ranked by growth from highest to lowest compared to the control as the following: high abundance virgin macroplastic (39.0 µm; p < 0.05), high abundance biofouled macroplastic (36.4 µm; p < 0.01), low abundance biofouled macroplastic (29.6 µm; p 0.05). For coefficients and pairwise comparisons, see Supplement Table S10-S15. Effect of plastic pollution on snail egg mass production Exposure to macroplastic or macroplastic-associated treatments generally increased egg mass production in B. truncatus snails, although the significance varies by plastic biofouling and abundance. In experiment round one, the snails in the joint treatment had a significantly 20.8% higher egg mass production than the control (p 0.05). In round two, both snails exposed to biofouled macroplastic or virgin macroplastic exhibited significantly more egg mass production compared to the control (17.4%, p < 0.001; 10.5%, p < 0.005; Fig. 3 A). The variation in macroplastic abundance did not have a significant effect on egg mass production based on the coefficients and pairwise comparisons in Supplement Table S16-S21. Effect of plastic pollution on snail hatching success Microplastic exposure reduced snail hatching success, while macroplastic exposure markedly improved hatching success, although combined treatments had no significant effect. In round one, virgin microplastic significantly reduced offspring hatching success by increasing the probability of structural zeros by approximately 57% (p 0.05; Fig. 3 B). Biofouled macroplastic or joint treatment did not significantly affect either the probability of structural zeros or conditional hatching success (p > 0.05). In round two, biofouled macroplastic significantly reduced the probability of structural zeros by approximately 36% relative to control (p < 0.001) and increased conditional hatching success by 3.2-fold (p < 0.001; Fig. 3 B), indicating concurrent effects on both hatching occurrences and performance. Virgin macroplastic significantly increased conditional offspring hatching success by 9% (p 0.05). Increasing macroplastic abundance enhanced hatching responses in biofouled treatments (p 0.05). For coefficients and pairwise comparisons, see Supplement Table S22-S30. The overall effect of plastic pollution on snail expected reproductive output Plastic pollution of various sizes and biofouling conditions significantly impacted the snail population. Biofouled macroplastic plays the most critical role in driving expected reproductive output increases, with notable variations observed between experiments (Fig. 4 ). In round one, the expected reproductive output of snails exposed to both biofouled macroplastic and virgin microplastic was 25.3% larger than controls (p < 0.001) (Fig. 4 A). Similarly, snails in biofouled macroplastic alone had a 26.2% increase in viable offspring per snail (p < 0.001), while those in virgin microplastic had an 8.3% decrease in viable offspring per snail (p < 0.001). In round two, all treatments differed significantly from each other in expected reproductive output (p < 0.001; Fig. 4 B). Snails in high or low abundance biofouled macroplastic exhibited an increase of 89.2% and 66.4%, respectively, compared to controls (p < 0.001). Snails in high or low abundance virgin macroplastic treatments had increases of 24.0% and 19.5%, respectively, compared to controls (p < 0.001). For coefficients and pairwise comparisons, see Supplement Table S31-S34. A summary table of the estimated direction and significance between plastic treatments and snail life-history results for both rounds is provided in Fig. 5 . Profiling periphyton growth on biofouled macroplastic We profiled the periphyton characteristics on macroplastics, including abundance, representative microorganisms, organic content ratio, community composition, physiological condition, and nutritional quality. The mean quantity of periphyton on macroplastics was 38.4 mg/100cm 2 (95% CI [3.4, 56.7]) in dry mass. Microscopic pictures were shown in Figure S13 at 40 times magnification of the representative microbial community grown on the surface of biofouled macroplastic. The mean AFDM: DM ratio was 0.25 (95% CI [0.20, 0.36]), and the mean autotrophic index was 1309 (95% CI [998, 1626]). The mean Chlorophyll: pheophytin ratio of the algae was 3.2 (95% CI [2.4, 3.7]), and the mean C: N ratio of lettuce was 10.1 (95% CI [8.8, 11.4]), and of algae is 12.8 (95% CI [10.1, 11.4]). For coefficients in box plots, see Text S5 and Figure S14. Discussion In two controlled laboratory experiments, our results demonstrate that plastic pollution alters snail life-history traits and, in turn, has the potential to substantially reshape population dynamics, with effects contingent on plastic size, biofouling, and abundance (Fig. 4 ). Across experiments, biofouled macroplastics consistently produced the largest demographic increases, including nearly doubling expected viable offspring per snail at high abundance in round two. In contrast, virgin materials exerted weaker and more variable effects: virgin macroplastic yielded modest population gains, whereas virgin microplastic led to slight population declines. The joint treatment of virgin microplastic with biofouled macroplastic produced intermediate increases in per-capita viable offspring. Together, these patterns indicate that ecological transformation of plastic in the aquatic environment (surface biofilm growth and accumulation), rather than the mere presence of debris, largely determines snail population responses, with plastic size and abundance modulating the magnitude of effects. The following sections detail the specific life-history responses and mechanisms underlying these population changes for each pollution scenario (Table 1). Biofouled macroplastic underscores the critical role of periphyton as a nutritional and habitat subsidy that elevates snail growth and reproductive performance, with survival and hatching success showing context dependence between rounds. Before deployment, our analysis showed that periphyton on biofouled macroplastic accumulated at low to medium organic loads and supported diverse, heterotroph-dominant communities with healthy autotrophs (Text S5 and Figure S13-S14). The periphyton nutritional content per dry weight was comparable to lettuce, indicating that periphyton, alongside lettuce ad libitum , increased both dietary abundance and diversity. Periphyton provides essential nutrients such as long-chain polyunsaturated fatty acids and sterols that enhance snail growth, survival, and reproduction( 21 , 44 ). Previous studies found that greater food diversity can optimize snail growth and fecundity( 21 , 45 ). In round one, higher snail mortality or lower hatching success coincided with degraded water quality in some biofouled macroplastic jars, potentially due to either periphyton-driven or mortality-caused water quality deterioration. Prior review documented the concern of pathogenic microorganisms’ adhesion to plastics that can destabilize water quality( 31 ). The two experimental rounds showed non-significant and opposing trends in survival and hatching success, which could reflect natural variability in water quality and shifts in microbial community composition rather than a consistent effect of treatment. These insights suggest that newly formed biofilms on nominally virgin macroplastic may provide similar but attenuated benefits, consistent with its weaker yet positive demographic effects. Virgin macroplastic may enhance snail growth, reproduction, and hatching success by developing nutritious surface biofilms after entering aquatic systems. Plastic was virgin at deployment and not replaced during the experiment. Although we did not directly quantify surface colonization, prior work shows that plastic surfaces rapidly accumulate biofilm over days to weeks( 46 ) that serve as a food source to grazers on timescales that vary with environmental conditions, including light availability, temperature, and dissolved oxygen( 18 , 47 ). Our results are consistent with this mechanism, suggesting that even initially virgin macroplastic can influence snail demography over the course of the 12-week experiment. Similar processes may also occur on microplastic, although this remains untested. While biofouling on larger plastic may provide nutritional subsidies, responses to microplastic particles likely reflect different physiological mechanisms beyond nutrition. Snails exposed to virgin microplastic exhibited increased survival and somatic growth but reduced reproductive output, indicating a trade-off consistent with energy reallocation from reproduction to maintenance. This pattern aligns with reported endocrine-disrupting effects of polyethylene microplastic( 48 ), to which snail reproduction is particularly sensitive due to neuroendocrine regulation( 49 ). Microplastic-associated chemicals may exacerbate oxidative stress and reproductive disturbances( 50 ), via an imbalance between reactive oxygen species and antioxidants that damage cells in the ovum. Similar maintenance-fecundity trade-offs have been documented in polyethylene microplastic exposures( 51 ), supporting this interpretation. Growth gains may also reflect biofilm ingestion along with the nominally virgin microplastic or more efficient food assimilation via microplastic-facilitated shredding in the gut( 50 ). We confirmed snail microplastic ingestion and egestion through microscopy (Figure S15). Notably, this trade-off is not observed under joint exposure to biofouled macroplastic and virgin microplastic, consistent with a nutrition-dependent response to endocrine-disruptors. Joint exposure to biofouled macroplastic and virgin microplastic attenuates the reproduction-maintenance trade-off seen with microplastic alone, yielding a concurrent increase in survival, somatic growth, and egg production. We speculate a nutrition-dependent buffering of microplastic-induced endocrine and oxidative stress, whereby periphyton-derived micronutrients like antioxidants may mitigate reproductive impairment while sustaining somatic benefits. Such nutrient-mediated modulation of contaminant toxicity is well documented across biological systems humans( 52 , 53 ), animals( 54 , 55 ), and in vitro( 56 , 57 ). Periphyton communities containing polyunsaturated fatty acids( 58 , 59 ) with antioxidant properties may enhance antioxidant defenses and increase total antioxidant capacity under oxidative stress following microplastic ingestion( 23 , 24 ). These interacting nutritional and physiological effects likely explain the joint-treatment outcomes, which scale beyond individual life-history traits to shape the population-level consequences. Plastic pollution tips the balance toward B. truncatus : improved survival and fecundity, faster maturation, and could potentially provide colonizable habitat, altered predator-prey dynamics, and create positive feedback that collectively inflates snail populations. Under realistic co-occurring microplastic and macroplastic pollution, our analysis indicates that snail populations would expand, driven by increases in expected reproductive output. We also found that the next generations of snails could reach reproductive maturity earlier with increased somatic growth when exposed to pollutants. Moreover, snails have been documented to colonize macroplastic debris as a substrate for settlement( 60 ). The additional novel habitat and surface nutrition provided by biofouled macroplastic may further increase the carrying capacity of the system if all other ecological factors remain the same, reinforcing the positive feedback loop of a surging snail population. Beyond these direct demographic effects, plastic pollution may also influence snail population dynamics through behavioral and community-level pathways. Although such processes were beyond the scope of this study, research has shown that marine snails exposed to microplastics exhibit increased vulnerability to predation due to reduced vigilance and antipredator responses( 61 ). Macroplastic may additionally function as a physical refuge, as artificial plastic structures are widely used to enhance aquatic habitats( 62 , 63 ). However, these mechanisms remain speculative in freshwater snail systems and would benefit from targeted investigation. Field-based studies are therefore needed to validate whether plastic debris consistently serves as habitat and/or resource subsidies for B. truncatus and to determine how these changes translate to population growth and, as a result, schistosomiasis transmission risk. Plastic pollution may elevate urogenital schistosomiasis risk by increasing B. truncatus abundance and per-capita parasite output, but its direct effects on cercarial production, survival, and infectivity remain a key knowledge gap. B. truncatus snails are the obligate intermediate host of human urogenital schistosomiasis. Independent of plastic contamination, growth in the snail population increases the total number of free-swimming cercariae released into water, raising human infection risk with routine water contact( 64 ). Prior work also shows that faster snail growth and higher resource availability, which are observed in our experiment, boost cercarial production per capita once infected( 65 ). These factors imply a higher human infection risk through both more contributing hosts and greater parasite loads per host. This is assuming the schistosome cercarial production, morphology, longevity, and infectivity remain unchanged despite plastic pollutant exposure, which remains untested. However, comparative studies report divergent cercarial outcomes across systems, which may plausibly reflect differences in the snail organs where parasites develop and reproduce. One study found that high nanoplastic diets yielded the greatest cercarial production in freshwater snail Stagnicola elodes infected with trematode Plagiorchis sp., which develops primarily in gonadal tissues( 66 ). These findings are consistent with compensatory feeding that offsets the energy costs of rediae and sporocysts( 67 ) and sustains elevated cercarial emergence( 68 ). By contrast, another study reported that nanoplastic reduced parasite production in a marine snail Zeacumantus subcarinatus on infected with the trematode Maritrema novaezealandensis , which develops primarily in the guts and other soft tissue( 69 ). It is likely because nanoparticle exposure imposes energetic and tissue‑repair costs on the snail that divert resources from intramolluscan parasite development, and/or nanoparticles inflict direct physical or cellular damage on sporocysts, impairing their capacity to asexually produce cercariae. They also found that cercarial morphology and infectivity were unchanged, but survival declined under high concentrations due to oxidative stress, tegumental damage, or impaired osmoregulation. Conversely, high concentrations of microplastics lowered infection prevalence and intensity for tadpole hosts in an echinostome- Helisoma trivolvis - Rana sylvatica model( 70 ). It likely arises because of draining cercarial energy upon microplastic contact and delaying host encounters beyond the brief infectious window. Given these divergent outcomes, the net impact of plastic pollution on S. haematobium transmission remains uncertain. Future studies are needed to quantify how plastic exposure alters snail susceptibility to infection and subsequent cercarial production, morphology, longevity, and infection success. This experiment has several limitations and strengths. The macroplastics were biofouled in a location not inhabited by B. truncatus , but our profiling of periphyton traits provides a basis for comparison and for evaluating potential effects on snail life-history in different ecological contexts. Snails may have ingested trace microplastic scraped from the macroplastic surfaces, but we consider this exposure negligible relative to the orders-of-magnitude higher doses that produced the effects observed in our microplastic-related treatments. Due to laboratory constraints, we did not quantify the number of eggs per egg mass for each treatment on a weekly basis. Instead, we used the mean number of eggs enrolled in the hatching assay for each treatment and week as a proxy (around 10 eggs, which typically corresponds to one egg mass( 71 )). Because egg masses cannot be subdivided without structural damage, this approach provides a reasonable and consistent approximation across treatments and time points. Despite these limitations, our study is the first to quantitatively examine how plastic pollution shapes the key life-history traits of B. truncatus , an intermediate host of human schistosomiasis. Using various plastic size classes, polymers, biofouling status, and abundance, we capture key dimensions of environmental heterogeneity while isolating their biological effects. We measured life-history traits using standardized protocols to ensure comparability. We found that plastic pollution meaningfully altered the B. truncatus population dynamics: biofouled macroplastic strongly enhances reproductive output; virgin microplastic produces a more subtle demographic decrease; the joint exposure yields concurrent improvement in life-history. This highlights macroplastic biofouling as a key ecological driver of snail population growth, with implications for schistosomiasis transmission. Future field studies are needed to confirm whether B. truncatus utilizes plastic debris, alongside laboratory experiments quantifying snail susceptibility and cercarial production under plastic exposure, to determine how plastic pollution influences the disease transmission risk to inform public health interventions. Declarations Ethics approval and consent to participate Not applicable, as this study did not involve human participants or vertebrate animals. Consent for publication Not applicable. Competing Interest The authors have declared that no competing interests exist. Funding This work was funded by the National Science Foundation under grant numbers DEB-2011179 and ICER-2522282. Ao Yu was partially funded by the Stanford King Center on Global Development’s Graduate Student Research Funding program under project number 26W11E and the Stanford Center for Human and Planetary Health Catalyst Award. Kaitlyn R Mitchell was partially funded by the Stanford University Center for Innovation in Global Health under grant number 353068. Author Contribution AY, KRM, JTV, and GDL designed the research; AY performed the research; AY contributed new reagents and analytical tools; AY, KRM analyzed the data; AY, KRM, JTV, and GDL interpreted the data; AY wrote the paper; AY, KRM, JTV, and GDL all edited and revised the manuscript. Acknowledgement We appreciate the support from the Biomedical Research Institute, Margaret Mentink-Kane, Gabrielle Bate, and Sara Li for providing experimental subjects and maintenance advice. We are grateful to Allina Zhang, Nyssa Kansal, Emily Chu, Jennifer Xu, Katy Zheng, Madeline Dulce, and Bohdan Kamets for their assistance with experimental subject maintenance and laboratory data collection. 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Supplementary Files SIOfficialforPV.docx Additional file 1: Text S1. Plastic shaping and metal contamination in experiment round 1. Table S1. Impact of the number of staples and biofouled macroplastic abundance on snail survival. Table S2. Impact of the number of staples and biofouled macroplastic abundance on snail survival, co-exposed to virgin microplastic. Text S2. Macroplastic biofouling protocols. Text S3. Macroplastic surface periphyton profiling protocols. Table S3. Data cleaning decisions in handling snail growth measurement errors and measurement cross-validation counts with error rates reported. Text S4. data cleaning decisions growth measurements Figure S1. Weekly snail survival of experimental round 1. Table S4. Snail survival model output. Table S5. Pairwise comparison of snail survival output. Figure S2. Weekly snail survival of experimental round 2. Table S6. Snail survival model output. Table S7. Pairwise comparison of snail survival output. Figure S3. Weekly snail survival of experimental round 2 with grouped macroplastic treatment of various abundance. Table S8. Snail survival model output. Table S9.Pairwise comparison of snail survival output. Figure S4. Weekly snail shell length growth of experimental round 1. Table S10. Snail shell length growth model output. Table S11. Pairwise comparison of snail shell length growth model output. Figure S5. Weekly snail shell length growth of experimental round 2. Table S12. Snail shell length growth model output. Table S13. Pairwise comparison of snail length growth model output. Figure S6. Weekly snail shell length growth of experimental round 2 with grouped macroplastic treatment of various abundance. Table S14. Snail shell length growth model output. Table S15. Pairwise comparison of snail shell length growth model output. Figure S7. Weekly snail egg mass production per jar of experiment round 1. Table S16.Snail egg mass production model output. Table S17. Pairwise comparison of snail egg mass production model output. Figure S8. Weekly snail egg mass production per jar of experiment round 2. Table S18. Snail egg mass production model output. Table S19. Pairwise comparison of snail egg mass production model output. Figure S9. Weekly snail egg mass production per jar of experiment round 2 with grouped macroplastic treatment of various abundance. Table S20. Snail egg mass production model output. Table S21. Pairwise comparison of snail egg mass production model output. Fig S10. Weekly snail hatching success of experiment round 1. Table S22.Snail egg hatching success conditional model and zero-inflation model. Table S23. Pairwise comparison of snail egg hatching success (conditional) model output. Table S24. Pairwise comparison of snail egg hatching success (zero-inflated) model output. Fig S11. Weekly snail hatching success of experiment round 2. Table S25. Snail egg hatching success conditional model and zero-inflation model output. Table S26. Pairwise comparison of snail egg hatching success (conditional) model output. Table S27.Pairwise comparison of snail egg hatching success (zero-inflated) model output. Figure S12. Weekly snail hatching success of experimental round 2 with grouped macroplastic treatment of various abundance. Table S28. Snail egg hatching success conditional model and zero-inflation model. Table S29.Pairwise comparison of snail egg hatching success (conditional) model output. Table S30. Pairwise comparison of snail egg hatching success (zero-inflated) model output. Table S31. Summary statistics with mean and standard deviation of cumulative viable offspring per snail from population permutation test between treatments in experiment round 1. Table S32. Pairwise comparisons of population permutation test between treatments in experiment round 1. Table S33. Summary statistics with mean and standard deviation of cumulative viable offspring per snail from population permutation test between treatments in experiment round 2. Table S34. Pairwise comparisons of population permutation test between treatments in experiment round 2. Text S5. Periphyton profiling results and interpretation. Figure S13. Microscopic pictures at 40 times magnification of the representative microbial community grown on the surface of biofouled macroplastic from round 2 experiment. Figure S14. Field versus lab biofouling methodology comparison on all aspects of periphyton. Figure S15. Representative microscopic pictures of snail feces under natural light (A) and UV light (B) to confirm microplastic ingestion and egestion in snail feces. 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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-9510130","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":631700415,"identity":"d457bbfc-914d-4fbf-b2df-45be2909a455","order_by":0,"name":"Ao Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYLCCDyCC+QADAw8DA2MDMToYZzAYMDCwJZCghZmHJC387T1m0jZ//sibszEf+/CGwUZ2wwECWiTOnDGTzm0zMNzZxpY8cw5DmjFBLQw3coBaGgwYN9zvMQa68HAiQS3yIC0WfwzsNxzjAWn5T1iLAUgLA5tBIlTLAcJaDM8cK7bsbTNO3nCMLZlxjkGy8UxCWuSON2+88eOPnO2GY8yHGd5U2Mn2EdLCwMBhgOxOgspBgP0BUcpGwSgYBaNgBAMASLNApisUZ7EAAAAASUVORK5CYII=","orcid":"","institution":"Stanford University","correspondingAuthor":true,"prefix":"","firstName":"Ao","middleName":"","lastName":"Yu","suffix":""},{"id":631700416,"identity":"d089a93e-ffae-4309-8747-85098c3848e3","order_by":1,"name":"Kaitlyn R Mitchell","email":"","orcid":"","institution":"Stanford University","correspondingAuthor":false,"prefix":"","firstName":"Kaitlyn","middleName":"R","lastName":"Mitchell","suffix":""},{"id":631700417,"identity":"0858e74c-af98-479c-9a27-f042f6ed797f","order_by":2,"name":"J Trevor Vannatta","email":"","orcid":"","institution":"Minnesota State University, Mankato","correspondingAuthor":false,"prefix":"","firstName":"J","middleName":"Trevor","lastName":"Vannatta","suffix":""},{"id":631700418,"identity":"5bc22492-e179-49e6-9f72-e1c78d53b9d3","order_by":3,"name":"Giulio De Leo","email":"","orcid":"","institution":"Stanford University","correspondingAuthor":false,"prefix":"","firstName":"Giulio","middleName":"","lastName":"De Leo","suffix":""}],"badges":[],"createdAt":"2026-04-23 19:43:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9510130/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9510130/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108225752,"identity":"c03423be-59ba-451f-9685-f77d37f64f72","added_by":"auto","created_at":"2026-04-30 16:23:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":494492,"visible":true,"origin":"","legend":"\u003cp\u003eSetup for two rounds of experiments (A) and weekly data collection workflow diagram (B). In panel A, Experiment round 1 includes negative control (75 jars), virgin microplastic (75 jars), biofouled macroplastic (18 jars), and a combined treatment (18 jars). In panel B, each week, we recorded snail survival, shell growth, and egg masses, and assessed hatching success. We checked snail mortality and took live snail pictures under the microscope for snail shell measurements using ImageJ. Then, we collected and counted all egg masses from the container. We placed approximately ten viable eggs in a well plate from each jar to test for hatching success, where we recorded the number of snails hatched.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9510130/v1/408e45491b1fb597560554d3.png"},{"id":108225753,"identity":"6101c436-c313-46d1-9784-2e6b7c53b86e","added_by":"auto","created_at":"2026-04-30 16:23:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":289338,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of plastic treatments on snail survival (A) and growth (B) across experimental rounds. Letters above or adjacent to bars denote statistically significant differences among treatments (Tukey-adjusted pairwise comparisons). Hazard ratios (HRs) show the effect of each treatment relative to the control with HR = 1. Model coefficients and full pairwise comparison results are provided in Tables S4-S15. Weekly snail survival and shell growth for experimental rounds 1 and 2 are shown in Figure S1-S6.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9510130/v1/64c0c9c51ffdd3734030984e.png"},{"id":108225757,"identity":"03062c65-2b7d-40d6-a008-48a12e84d497","added_by":"auto","created_at":"2026-04-30 16:23:12","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":366662,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of plastic treatments on snail egg mass production per jar per week (A) and hatching success (B) across experimental rounds. Values represent model-based marginal means, averaged over variation in snail size and density. Letters above or adjacent to bars denote statistically significant differences among treatments (Tukey-adjusted pairwise comparisons). Model coefficients and pairwise comparison results are provided in Tables S16-S30. Weekly snail egg mass production and hatching success for experimental rounds 1 and 2 are shown in Figure S7-S12.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9510130/v1/43d8e762692e21a8fc449d4b.jpeg"},{"id":108225759,"identity":"757d83a0-46e0-498d-bc1f-dd324cd12d25","added_by":"auto","created_at":"2026-04-30 16:23:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":411065,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of the per-snail cumulative viable offspring by the end of the experiment. A) Experiment round 1 with control, biofouled macroplastic only, virgin microplastic only, and joint of both biofouled macroplastic and virgin microplastic. B) Experiment round 2 with control, biofouled macroplastic of high or low abundance, and virgin macroplastic with high or low abundance. Model coefficients and pairwise comparison results are provided in Tables S31-S34.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9510130/v1/4b94773afcc95ff496408301.png"},{"id":108225755,"identity":"7f6bdbf9-a3db-46a4-92ff-43c7218f5540","added_by":"auto","created_at":"2026-04-30 16:23:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":176939,"visible":true,"origin":"","legend":"\u003cp\u003eA summary table of the snail life history response under each pollution treatment in both experimental rounds. The dual-color box indicates the significant output of the model estimate is no longer significant under the Tukey-adjusted multiple pairwise comparison (p \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9510130/v1/0fc6a4eabfb8f5fe2b1da5c9.png"},{"id":108804234,"identity":"d81cc4b9-884a-44ab-9694-3f5e9509b70b","added_by":"auto","created_at":"2026-05-08 15:18:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1859703,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9510130/v1/4830862f-e04a-40d3-b151-531064289d69.pdf"},{"id":108225758,"identity":"1aa1b16e-3faa-450b-b328-9aada76fca8c","added_by":"auto","created_at":"2026-04-30 16:23:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17852321,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: Text S1. \u003c/strong\u003ePlastic shaping and metal contamination in experiment round 1. \u003cstrong\u003eTable S1. \u003c/strong\u003eImpact of the number of staples and biofouled macroplastic abundance on snail survival. \u003cstrong\u003eTable S2.\u003c/strong\u003e Impact of the number of staples and biofouled macroplastic abundance on snail survival, co-exposed to virgin microplastic. \u003cstrong\u003eText S2. \u003c/strong\u003eMacroplastic biofouling protocols. \u003cstrong\u003eText S3. \u003c/strong\u003eMacroplastic surface periphyton profiling protocols. \u003cstrong\u003eTable S3.\u003c/strong\u003e Data cleaning decisions in handling snail growth\u003cem\u003e \u003c/em\u003emeasurement errors and measurement cross-validation counts with error rates\u003cem\u003e \u003c/em\u003ereported. \u003cstrong\u003eText S4. \u003c/strong\u003edata cleaning decisions growth measurements \u003cstrong\u003eFigure S1.\u003c/strong\u003e Weekly snail survival of experimental round 1. \u003cstrong\u003eTable S4.\u003c/strong\u003e Snail survival model output. \u003cstrong\u003eTable S5.\u003c/strong\u003e Pairwise comparison of snail survival output. \u003cstrong\u003eFigure S2.\u003c/strong\u003e Weekly snail survival of experimental round 2. \u003cstrong\u003eTable S6.\u003c/strong\u003e Snail survival model output. \u003cstrong\u003eTable S7.\u003c/strong\u003e Pairwise comparison of snail survival output. \u003cstrong\u003eFigure S3.\u003c/strong\u003e Weekly snail survival of experimental round 2 with grouped macroplastic treatment of various abundance. \u003cstrong\u003eTable S8.\u003c/strong\u003e Snail survival model output. \u003cstrong\u003eTable S9.\u003c/strong\u003ePairwise comparison of snail survival output. \u003cstrong\u003eFigure S4.\u003c/strong\u003e Weekly snail shell length growth of experimental round 1. \u003cstrong\u003eTable S10.\u003c/strong\u003e Snail shell length growth model output. \u003cstrong\u003eTable S11.\u003c/strong\u003e Pairwise comparison of snail shell length growth model output. \u003cstrong\u003eFigure S5.\u003c/strong\u003e Weekly snail shell length growth of experimental round 2. \u003cstrong\u003eTable S12.\u003c/strong\u003e Snail shell length growth model output. \u003cstrong\u003eTable S13.\u003c/strong\u003e Pairwise comparison of snail length growth model output. \u003cstrong\u003eFigure S6.\u003c/strong\u003e Weekly snail shell length growth of experimental round 2 with grouped macroplastic treatment of various abundance. \u003cstrong\u003eTable S14.\u003c/strong\u003e Snail shell length growth model output. \u003cstrong\u003eTable S15.\u003c/strong\u003e Pairwise comparison of snail shell length growth model output. \u003cstrong\u003eFigure S7.\u003c/strong\u003e Weekly snail egg mass production per jar of experiment round 1. \u003cstrong\u003eTable S16.\u003c/strong\u003eSnail egg mass production model output. \u003cstrong\u003eTable S17.\u003c/strong\u003e Pairwise comparison of snail egg mass production model output. \u003cstrong\u003eFigure S8.\u003c/strong\u003e Weekly snail egg mass production per jar of experiment round 2.\u003cstrong\u003e Table S18.\u003c/strong\u003e Snail egg mass production model output. \u003cstrong\u003eTable S19.\u003c/strong\u003e Pairwise comparison of snail egg mass production model output. \u003cstrong\u003eFigure S9.\u003c/strong\u003e Weekly snail egg mass production per jar of experiment round 2 with grouped macroplastic treatment of various abundance. \u003cstrong\u003eTable S20.\u003c/strong\u003e Snail egg mass production model output. \u003cstrong\u003eTable S21.\u003c/strong\u003e Pairwise comparison of snail egg mass production model output. \u003cstrong\u003eFig S10.\u003c/strong\u003e Weekly snail hatching success of experiment round 1. \u003cstrong\u003eTable S22.\u003c/strong\u003eSnail egg hatching success conditional model and zero-inflation model. \u003cstrong\u003eTable S23.\u003c/strong\u003e Pairwise comparison of snail egg hatching success (conditional) model output. \u003cstrong\u003eTable S24.\u003c/strong\u003e Pairwise comparison of snail egg hatching success (zero-inflated) model output. \u003cstrong\u003eFig S11.\u003c/strong\u003e Weekly snail hatching success of experiment round 2. \u003cstrong\u003eTable S25.\u003c/strong\u003e Snail egg hatching success conditional model and zero-inflation model output. \u003cstrong\u003eTable S26.\u003c/strong\u003e Pairwise comparison of snail egg hatching success (conditional) model output. \u003cstrong\u003eTable S27.\u003c/strong\u003ePairwise comparison of snail egg hatching success (zero-inflated) model output. \u003cstrong\u003eFigure S12.\u003c/strong\u003e Weekly snail hatching success of experimental round 2 with grouped macroplastic treatment of various abundance. \u003cstrong\u003eTable S28.\u003c/strong\u003e Snail egg hatching success conditional model and zero-inflation model. \u003cstrong\u003eTable S29.\u003c/strong\u003ePairwise comparison of snail egg hatching success (conditional) model output. \u003cstrong\u003eTable S30.\u003c/strong\u003e Pairwise comparison of snail egg hatching success (zero-inflated) model output. \u003cstrong\u003eTable S31.\u003c/strong\u003e Summary statistics with mean and standard deviation of cumulative viable offspring per snail from population permutation test between treatments in experiment round 1. \u003cstrong\u003eTable S32.\u003c/strong\u003e Pairwise comparisons of population permutation test between treatments in experiment round 1. \u003cstrong\u003eTable S33.\u003c/strong\u003e Summary statistics with mean and standard deviation of cumulative viable offspring per snail from population permutation test between treatments in experiment round 2. \u003cstrong\u003eTable S34.\u003c/strong\u003e Pairwise comparisons of population permutation test between treatments in experiment round 2. \u003cstrong\u003eText S5. \u003c/strong\u003ePeriphyton profiling results and interpretation. \u003cstrong\u003eFigure S13.\u003c/strong\u003e Microscopic pictures at 40 times magnification of the representative microbial community grown on the surface of biofouled macroplastic from round 2 experiment. \u003cstrong\u003eFigure S14.\u003c/strong\u003e Field versus lab biofouling methodology comparison on all aspects of periphyton. \u003cstrong\u003eFigure S15.\u003c/strong\u003e Representative microscopic pictures of snail feces under natural light (A) and UV light (B) to confirm microplastic ingestion and egestion in snail feces.\u003c/p\u003e","description":"","filename":"SIOfficialforPV.docx","url":"https://assets-eu.researchsquare.com/files/rs-9510130/v1/bd548a096c74922a0364d86f.docx"},{"id":108491559,"identity":"7057ccf1-2685-4cb6-9ba2-8bc8e2e77c6b","added_by":"auto","created_at":"2026-05-05 09:54:36","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":170931,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalabstractOfficialforPV.png","url":"https://assets-eu.researchsquare.com/files/rs-9510130/v1/0a83b717276d04fc40f4f58f.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Plastic pollution reshapes snail intermediate host life history: implications for schistosomiasis transmission","fulltext":[{"header":"Background","content":"\u003cp\u003ePlastic pollution has emerged as an Anthropocene-driven crisis, resulting from escalating plastic production and waste accumulation with slow degradation. Global plastic production increased from 5\u0026nbsp;million tons in the 1950s to 300\u0026nbsp;million tons in 2020(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), and currently, 79% of the plastic waste ends up either in landfills or the natural environment(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). In recent years, a large body of research has found that plastic pollution increases human malaria and dengue transmission, because this debris creates additional breeding habitats that support the freshwater life stages of the mosquito vectors responsible for the disease spread(\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). By contrast, little is asked about whether other medically important freshwater hosts similarly benefit from plastic pollution. Studies on the effects of plastic on snail-borne disease, including schistosomiasis, remain extremely limited. In particular, the effects of pollution on key snail life-history traits (i.e., survival, growth, fecundity, and hatching) are largely unknown, even though these traits govern population persistence and even minor shifts could influence transmission potential. To address this knowledge gap, it is necessary to focus on specific disease-relevant snail hosts where changes in life history could directly alter human health risk.\u003c/p\u003e \u003cp\u003eHuman schistosomiasis is among the most prevalent neglected tropical diseases worldwide, second only to malaria in global burden(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), affecting approximately 250\u0026nbsp;million individuals, placing approximately 800\u0026nbsp;million at risk, and disproportionately impacting impoverished rural communities(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Urogenital schistosomiasis (caused by \u003cem\u003eSchistosoma haematobium\u003c/em\u003e) is transmitted by snails in the genus \u003cem\u003eBulinus\u003c/em\u003e, while intestinal schistosomiasis (caused by \u003cem\u003eSchistosoma mansoni\u003c/em\u003e) is transmitted by \u003cem\u003eBiomphalaria\u003c/em\u003e snail species. Because urogenital schistosomiasis is often more prevalent, particularly among school-aged children, \u003cem\u003eBulinus\u003c/em\u003e snails become a priority for investigating life-history traits and demography(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Because no effective vaccines are available, current control strategies rely on snail control(\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) and mass drug administration of praziquantel(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), which does not prevent reinfection(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), making accurate risk assessment crucial. Within this context, identifying how plastic pollution may drive snail host life history reveals three major mechanisms: (i) macroplastics function as substrates that promote biofilm growth and provide nutritional and habitat subsidies, (ii) microplastics exert direct toxicological effects following ingestion, and (iii) interactions between macroplastic-associated nutrition and microplastic-induced toxicity jointly shape \u003cem\u003eBulinus\u003c/em\u003e life-history traits.\u003c/p\u003e \u003cp\u003ePlastic pollution has the potential to influence \u003cem\u003eBulinus\u003c/em\u003e snails\u0026rsquo; life history via multiple mechanisms. These mechanisms differ by plastic form, with macroplastics (\u0026gt;\u0026thinsp;5 mm) and microplastics (from \u0026lt;\u0026thinsp;5 mm to \u0026gt;\u0026thinsp;1 \u0026micro;m) exerting distinct effects(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Macroplastics may act as substrates for biological enrichment. Macroplastic surfaces can support dense microbial biofilms (also known as biofouling(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), periphyton(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), or plastisphere(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e)) that serve as nutritious food for freshwater grazers like snails(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). These biofilms are rich in long-chain polyunsaturated fatty acids and sterols, which are essential nutrients that increase both food abundance and diversity and could enhance snail growth, survival, and reproduction(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Moreover, macroplastic may create a novel habitat and refugia that support larger snail populations. By contrast, the dominant mechanism associated with microplastics is physiological toxicity following ingestion. Ingested microplastics can bioaccumulate and induce oxidative stress, histopathological alterations, developmental toxicity, and behavior changes(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) that may reduce snail survival, fecundity, and hatching success. Notably, the nutritional properties of macroplastic-associated biofilms have the potential to mitigate these effects, as polyunsaturated fatty acids are known antioxidants that can support antioxidant defenses and increase total antioxidant capacity(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Therefore, co-exposing snails to nutrient- and antioxidant-rich biofouled macroplastic could potentially buffer microplastic-induced oxidative stress and alter key life-history outcomes. Plastics may also exert indirect environmental effects, such as shifting the water microbial communities and water parameters such as dissolved oxygen, pH, conductivity, etc(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Snails depend on these specific water parameters and, therefore, could be affected. Existing research typically analyzes macro- and microplastics separately, emphasizes acute exposure to high doses or abundance, or focuses on adult organisms(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Here, we address this gap with two sequential randomized controlled experiments exposing \u003cem\u003eBulinus truncatus\u003c/em\u003e to macro- and microplastics, systematically varying sizes, biofouling status, and abundance. We quantified key life-history traits and modeled demographic outcomes to assess the long-term effects of plastic. Our findings provide novel insights into how plastic pollution shapes the population dynamics of \u003cem\u003eBulinus\u003c/em\u003e snails with implications for schistosomiasis risks.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLab experimental reagent and setup\u003c/h2\u003e \u003cp\u003eWe conducted two sequential rounds of experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) to investigate the long-term effects of plastic pollution on \u003cem\u003eB. truncatus\u003c/em\u003e\u0026rsquo;s life-history response, with consideration of space constraints and personnel capacity. In the first round, we assessed the effect of plastic pollution on 4 treatment groups: negative control, virgin microplastic, biofouled macroplastic, and a combined treatment from September to December 2024. In the second round, we focused only on macroplastic and used 5 treatment groups: negative control, virgin macroplastic of high and low abundance, and biofouled macroplastic of high and low abundance from April to June 2025.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe used \u003cem\u003eB. truncatus\u003c/em\u003e snails from the Biomedical Research Institute, which originated from Egypt. \u003cem\u003eB. truncatus\u003c/em\u003e snails were born and raised under controlled laboratory conditions (~\u0026thinsp;25\u0026deg;C, the ambient temperature of the laboratory, 12-hour light/12-hour dark, artificial pond water). In the first round, we housed 3 snails per jar, starting with 75 jars per treatment. In the macroplastic-only treatment and the combination treatment, 57 jars in each treatment were eliminated due to metal contamination from staples, which was intended for experimenting with biofouled macroplastic shaping (Text S1 and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S2). Therefore, only 18 jars in the macroplastic-only treatment and the combination treatment remained for the analysis. In the second round, we housed 3 snails per jar, 50 jars per treatment for negative control and biofouled macroplastic of high or low abundance, and 29 jars per treatment for virgin macroplastic of high or low abundance due to the limited total number of snails available from the Biomedical Research Institute. Each glass jar was 470 mL in size and contained 250 mL of water. The snails were fed organic romaine lettuce \u003cem\u003ead libitum\u003c/em\u003e based on the number of snails alive in each jar. We covered each jar with mesh that allowed air exchange but limited evaporation. We changed the water in each jar weekly to maintain water quality(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMacroplastic reagent and periphyton profiling\u003c/h3\u003e\n\u003cp\u003eWe picked 500 mL single-use plastic bottles made from Polyethylene Terephthalate (PET) from Crystal Geyser\u0026reg;, given that it is one of the most common macroplastic types found in waterways(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). All bottles were initially processed by removing labels and cutting them vertically in half to generate plastic sheets. To biofoul macroplastic, plastic sheets were submerged in freshwater, allowing surface microbial growth to develop(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Field biofouling was conducted at Jasper Ridge Biological Reserve (Searsville Lake) during experiment round one and the latter half of round two. During the first half of round two, biofouling was conducted under laboratory conditions due to unsuitable field conditions (extreme precipitation, high water velocity, and low temperatures). More details on the field and lab macroplastic biofouling protocols are available in Text S2. All plastic sheets (field- or lab-biofouled or virgin) were trimmed into uniform 5 cm x 4 cm pieces. To explicitly test the effect of macroplastic abundance, we manipulated the number of macroplastic pieces per jar, using three pieces to represent high abundance and one piece to represent low abundance. Round one and two included both abundance levels. Biofouled macroplastic pieces were replaced when 80\u0026ndash;100% of jars showed visible periphyton depletion during weekly monitoring, and virgin macroplastic pieces were not replaced throughout the experiment.\u003c/p\u003e \u003cp\u003eAdditionally, we profiled surface periphyton in experiment round two for each batch of biofouled macroplastic replacement, and 4 batches total. We measured the absolute abundance (dry mass; DM), proportion of organic matter (ash-free dry mass/ dry mass; AFDM/DM), nutritional quality (C:N), and autotroph to heterotroph ratio (chlorophyll a.:AFDM), periphyton physiological condition (chlorophyll a.:pheophytin)(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), and took representative microscopy pictures, which all used standard protocols with more detail available in Text S3. We compared the lab versus field biofouling methodology across these aspects using a Mann-Whitney U test.\u003c/p\u003e\n\u003ch3\u003eMicroplastic reagent\u003c/h3\u003e\n\u003cp\u003eWe purchased Fluorescent Yellow Polyethylene (PE) microplastic spheres with a size range from 10 um to 27 um and a density of 1g/cc from Cospheric. We used virgin polyethylene microplastics, as polyethylene is the most common type of microplastic produced and found in natural systems(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Plastic is ubiquitous in the freshwater bodies, including where \u003cem\u003eBulinus\u003c/em\u003e snails are prevalent, with large heterogeneity in polymer type, shape, and size(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). We used a 1000 ug/L microplastic concentration for the laboratory experiments, higher than the limited sampling report in Africa(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) and Western Europe(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), but lower than other microplastic and freshwater snail studies(\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) to capture the effect of higher but ecologically relevant concentrations of microplastic where the \u003cem\u003eBulinus\u003c/em\u003e snails are present. We prepared the virgin microplastic treatment using weighted PE microspheres and artificial pond water, formulated according to Biomedical Research Institute guidelines(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e)\u003c/p\u003e\n\u003ch3\u003eLab experimental data collection\u003c/h3\u003e\n\u003cp\u003eWe recorded weekly snail survival, shell growth, and egg masses laid for 13 weeks in both rounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), while hatching success was assessed for 12 weeks in each round, as eggs enrolled during the final experimental week required a full 3-week evaluation period and could not be fully assessed within the observation window. We assessed snail mortality by whether the snail was motile, responded to a stimulus, or was obviously decaying (empty shell or shell residuals). We placed snails under the microscope to take shell pictures for length measurement in ImageJ, defined by the distance from the snail shell apex to the basal lip. Growth was calculated as the change in average snail length per jar from the previous week to the current week. We performed length measurement cross-validation with detailed data cleaning decisions reported in the Table S3 and Text S4. We then decanted the water from the jar through a metal sieve, counted and scraped off snail egg masses on the sieve surface, the macroplastic, and the sides of the jar. We disinfected the jar with 70% alcohol wipes between water changes. We placed approximately ten viable eggs in a well plate from each jar to test for hatching success with weekly water changes of the wells. We recorded the number of snails hatched at 1 week and 3 weeks after enrollment, and any egg that failed to hatch after 3 weeks of enrollment was taken as unviable.\u003c/p\u003e\n\u003ch3\u003eLab experimental statistical analysis\u003c/h3\u003e\n\u003cp\u003eWe estimated snail survival via a mixed-effect proportional hazard regression model using the \u003cem\u003ecoxme\u003c/em\u003e and \u003cem\u003esurvival\u003c/em\u003e packages in R. We modeled snail growth with a generalized additive model using Gaussian errors (identity link) and an AR(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) correlation structure; predictors included treatment and a smooth of the previous week\u0026rsquo;s mean shell length. We analyzed snail egg mass production with a negative binomial generalized linear model (log link) with jar level random effect and analyzed hatching success with a zero-inflated binomial model with jar level random effect created in the glmmTMB package. We made all pairwise comparisons using the emmeans R package with the multivariate t distribution and a Bonferroni p-value correction for multiple comparisons.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMathematical model on snail demography\u003c/h2\u003e \u003cp\u003eWe computed the snails\u0026rsquo; expected reproductive output as the number of live snail offspring that a snail would be expected to produce from the beginning of the experiment over the entire duration of the experiment, namely:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:{R}_{obs}^{*}\\:=\\:{\\sum\\:}_{a\\:=\\:1}^{12}l\\left(a\\right)\\times\\:b\\left(a\\right)\\times\\:h\\left(a\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:a\\)\u003c/span\u003e\u003c/span\u003e is snail age [weeks] counting from the beginning of the experiment; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:l\\left(a\\right)\\)\u003c/span\u003e\u003c/span\u003e is age-specific survival probability at the treatment-level, i.e., the probability that a snail at the beginning of the experiment will survive until week \u003cem\u003ea\u003c/em\u003e; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:b\\left(a\\right)\\)\u003c/span\u003e\u003c/span\u003e is age-specific birth rate at the treatment-level, that is, the mean number of eggs laid per snail. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:h\\left(a\\right)\\)\u003c/span\u003e\u003c/span\u003e is the age-specific hatching rate at the treatment-level, i.e., the fraction of eggs produced in week \u003cem\u003ea\u003c/em\u003e that hatch.\u003c/p\u003e \u003cp\u003eTo quantify the intrinsic variability of snail reproductive output within each treatment, we performed a nonparametric bootstrap at the jar level. Specifically, for each treatment, we (i) resampled jars with replacement while preserving the original sample sizes, (ii) computed the corresponding reproductive output \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{R}_{obs}^{*}\\)\u003c/span\u003e\u003c/span\u003e for the resulting pseudo-dataset as described above, and (iii) repeated this procedure 10,000 times. This yielded (iv) a bootstrap distribution of ​​jar-level reproductive output, which we then aggregated by treatment to obtain the mean reproductive output per treatment, and we used as a proxy for the underlying variability in reproductive performance.\u003c/p\u003e \u003cp\u003eTo assess whether expected reproductive output differed significantly between treatments, we performed a permutation (randomization) test. For each pair of treatments \u003cem\u003ei\u003c/em\u003e and \u003cem\u003ej\u003c/em\u003e, we first computed the observed difference in the mean treatment-level reproductive output:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:{D}_{obs}^{}\\:=\\:{R}_{obs}^{*}\\left(i\\right)\\:-\\:{R}_{obs}^{*}\\left(j\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eUnder the null hypothesis of no treatment effect, the jar labels \u0026ldquo;\u003cem\u003ei\u003c/em\u003e\u0026rdquo; and \u0026ldquo;\u003cem\u003ej\u003c/em\u003e\u0026rdquo; are exchangeable. We therefore pooled the jars from both treatments and repeatedly (\u003cem\u003en\u003c/em\u003e\u003csub\u003e\u003cem\u003eperm\u003c/em\u003e\u003c/sub\u003e = 10,000 times) reassigned the labels \u0026ldquo;\u003cem\u003ei\u003c/em\u003e\u003csub\u003e\u003cem\u003erep\u003c/em\u003e\u003c/sub\u003e\u0026rdquo; and \u0026ldquo;\u003cem\u003ej\u003c/em\u003e\u003csub\u003e\u003cem\u003erep\u003c/em\u003e\u003c/sub\u003e\u0026rdquo; at random (without replacement) while preserving the original sample sizes. For each permutation, we recalculated the expected reproductive outputs \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{R}_{rep}^{*}\\)\u003c/span\u003e\u003c/span\u003e\u003cem\u003e(i)\u003c/em\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{R}_{rep}^{*}\\)\u003c/span\u003e\u003c/span\u003e\u003cem\u003e(j)\u003c/em\u003e using the precomputed jar-level \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{R}_{obs}^{*}\\)\u003c/span\u003e\u003c/span\u003e for the randomized groups and obtained the permuted difference:\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:{D}_{rep}^{}\\:=\\:{R}_{rep}^{*}\\left(i\\right)\\:-\\:{R}_{rep}^{*}\\left(j\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe collection of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{D}_{rep}^{}\\)\u003c/span\u003e\u003c/span\u003e values formed the null distribution against which \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{D}_{obs}^{}\\)\u003c/span\u003e\u003c/span\u003e was compared. Because we made no directional assumption, we used a two-tailed test, computing the \u003cem\u003ep\u003c/em\u003e-value as:\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:p\\:=\\:\\frac{\\#\\:\\{\\left|{D}_{rep}^{}\\right|\\:\\ge\\:\\:\\left|{D}_{obs}^{}\\right|\\}\\:+\\:1}{{n}_{perm}^{}\\:+\\:1}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhen evaluating multiple pairwise comparisons, we adjusted the resulting \u003cem\u003ep\u003c/em\u003e-values using the Bonferroni correction to control for family-wise error (6 comparisons in round one, and 10 in round 2).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEffect of plastic pollution on snail survival\u003c/h2\u003e \u003cp\u003eExposure to microplastics or microplastic-associated treatments increased survival probability in \u003cem\u003eB. truncatus\u003c/em\u003e snails, while macroplastics with and without biofouling had no effects. A hazard ratio (HR) near 1 indicates no difference in mortality risk compared to controls. In round one, the HR for snails in virgin microplastic relative to control was 0.55 (95% CI HR [0.40, 0.76], p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), corresponding to approximately a 45% lower risk of mortality compared to control snails. The HR for snails in the joint treatment with respect to control was 0.57 (95% CI HR [0.32, 0.99]), which is significant from the model output (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but nonsignificant from Tukey-adjusted pairwise comparison (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). This estimate corresponds to an approximate 43% reduction in mortality risk relative to controls, which is not statistically robust. The HR for snails in biofouled macroplastic with respect to control is 1.09 (95% CI HR [0.69, 1.70], p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Similarly, in the second round, survival probabilities did not differ significantly among any macroplastic treatments over the 13-week experiment (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). For coefficients and pairwise comparisons, see Supplement Table S4-S9.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffect of plastic pollution on snail shell growth\u003c/h2\u003e \u003cp\u003eAll plastic treatments significantly enhanced snail growth relative to controls, with growth varying based on the combination of treatments and abundance. In both rounds, snails in all treatment groups exhibited significantly greater weekly shell growth than the control. In round one, snails in the biofouled macroplastic produced the largest mean weekly growth compared to the control (93.3 \u0026micro;m, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), followed by the joint treatment (51.2 \u0026micro;m, p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) and the virgin microplastic (40.1 \u0026micro;m, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In round two, both snails exposed to biofouled macroplastic or virgin macroplastic significantly increased weekly growth compared to control snails (33.2 um, p\u0026thinsp;\u0026lt;\u0026thinsp;0.005; 27.9 \u0026micro;m, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Specifically, treatment groups with varying macroplastic abundance were ranked by growth from highest to lowest compared to the control as the following: high abundance virgin macroplastic (39.0 \u0026micro;m; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), high abundance biofouled macroplastic (36.4 \u0026micro;m; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), low abundance biofouled macroplastic (29.6 \u0026micro;m; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and low abundance virgin macroplastic (16.2 \u0026micro;m; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). For coefficients and pairwise comparisons, see Supplement Table S10-S15.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffect of plastic pollution on snail egg mass production\u003c/h2\u003e \u003cp\u003eExposure to macroplastic or macroplastic-associated treatments generally increased egg mass production in \u003cem\u003eB. truncatus\u003c/em\u003e snails, although the significance varies by plastic biofouling and abundance. In experiment round one, the snails in the joint treatment had a significantly 20.8% higher egg mass production than the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The snails in virgin microplastic or biofouled macroplastic were not significantly different from the control (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In round two, both snails exposed to biofouled macroplastic or virgin macroplastic exhibited significantly more egg mass production compared to the control (17.4%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 10.5%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.005; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The variation in macroplastic abundance did not have a significant effect on egg mass production based on the coefficients and pairwise comparisons in Supplement Table S16-S21.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffect of plastic pollution on snail hatching success\u003c/h2\u003e \u003cp\u003eMicroplastic exposure reduced snail hatching success, while macroplastic exposure markedly improved hatching success, although combined treatments had no significant effect. In round one, virgin microplastic significantly reduced offspring hatching success by increasing the probability of structural zeros by approximately 57% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) while conditional hatching success among non-zero observations did not differ from controls (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Biofouled macroplastic or joint treatment did not significantly affect either the probability of structural zeros or conditional hatching success (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In round two, biofouled macroplastic significantly reduced the probability of structural zeros by approximately 36% relative to control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and increased conditional hatching success by 3.2-fold (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), indicating concurrent effects on both hatching occurrences and performance. Virgin macroplastic significantly increased conditional offspring hatching success by 9% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but did not affect structural zeros (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Increasing macroplastic abundance enhanced hatching responses in biofouled treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but not in virgin macroplastic treatments (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). For coefficients and pairwise comparisons, see Supplement Table S22-S30.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eThe overall effect of plastic pollution on snail expected reproductive output\u003c/h2\u003e \u003cp\u003ePlastic pollution of various sizes and biofouling conditions significantly impacted the snail population. Biofouled macroplastic plays the most critical role in driving expected reproductive output increases, with notable variations observed between experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In round one, the expected reproductive output of snails exposed to both biofouled macroplastic and virgin microplastic was 25.3% larger than controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Similarly, snails in biofouled macroplastic alone had a 26.2% increase in viable offspring per snail (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while those in virgin microplastic had an 8.3% decrease in viable offspring per snail (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In round two, all treatments differed significantly from each other in expected reproductive output (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Snails in high or low abundance biofouled macroplastic exhibited an increase of 89.2% and 66.4%, respectively, compared to controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Snails in high or low abundance virgin macroplastic treatments had increases of 24.0% and 19.5%, respectively, compared to controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). For coefficients and pairwise comparisons, see Supplement Table S31-S34. A summary table of the estimated direction and significance between plastic treatments and snail life-history results for both rounds is provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eProfiling periphyton growth on biofouled macroplastic\u003c/h2\u003e \u003cp\u003eWe profiled the periphyton characteristics on macroplastics, including abundance, representative microorganisms, organic content ratio, community composition, physiological condition, and nutritional quality. The mean quantity of periphyton on macroplastics was 38.4 mg/100cm\u003csup\u003e2\u003c/sup\u003e (95% CI [3.4, 56.7]) in dry mass. Microscopic pictures were shown in Figure S13 at 40 times magnification of the representative microbial community grown on the surface of biofouled macroplastic. The mean AFDM: DM ratio was 0.25 (95% CI [0.20, 0.36]), and the mean autotrophic index was 1309 (95% CI [998, 1626]). The mean Chlorophyll: pheophytin ratio of the algae was 3.2 (95% CI [2.4, 3.7]), and the mean C: N ratio of lettuce was 10.1 (95% CI [8.8, 11.4]), and of algae is 12.8 (95% CI [10.1, 11.4]). For coefficients in box plots, see Text S5 and Figure S14.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn two controlled laboratory experiments, our results demonstrate that plastic pollution alters snail life-history traits and, in turn, has the potential to substantially reshape population dynamics, with effects contingent on plastic size, biofouling, and abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Across experiments, biofouled macroplastics consistently produced the largest demographic increases, including nearly doubling expected viable offspring per snail at high abundance in round two. In contrast, virgin materials exerted weaker and more variable effects: virgin macroplastic yielded modest population gains, whereas virgin microplastic led to slight population declines. The joint treatment of virgin microplastic with biofouled macroplastic produced intermediate increases in per-capita viable offspring. Together, these patterns indicate that ecological transformation of plastic in the aquatic environment (surface biofilm growth and accumulation), rather than the mere presence of debris, largely determines snail population responses, with plastic size and abundance modulating the magnitude of effects. The following sections detail the specific life-history responses and mechanisms underlying these population changes for each pollution scenario (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eBiofouled macroplastic underscores the critical role of periphyton as a nutritional and habitat subsidy that elevates snail growth and reproductive performance, with survival and hatching success showing context dependence between rounds. Before deployment, our analysis showed that periphyton on biofouled macroplastic accumulated at low to medium organic loads and supported diverse, heterotroph-dominant communities with healthy autotrophs (Text S5 and Figure S13-S14). The periphyton nutritional content per dry weight was comparable to lettuce, indicating that periphyton, alongside lettuce \u003cem\u003ead libitum\u003c/em\u003e, increased both dietary abundance and diversity. Periphyton provides essential nutrients such as long-chain polyunsaturated fatty acids and sterols that enhance snail growth, survival, and reproduction(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Previous studies found that greater food diversity can optimize snail growth and fecundity(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). In round one, higher snail mortality or lower hatching success coincided with degraded water quality in some biofouled macroplastic jars, potentially due to either periphyton-driven or mortality-caused water quality deterioration. Prior review documented the concern of pathogenic microorganisms\u0026rsquo; adhesion to plastics that can destabilize water quality(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). The two experimental rounds showed non-significant and opposing trends in survival and hatching success, which could reflect natural variability in water quality and shifts in microbial community composition rather than a consistent effect of treatment. These insights suggest that newly formed biofilms on nominally virgin macroplastic may provide similar but attenuated benefits, consistent with its weaker yet positive demographic effects.\u003c/p\u003e \u003cp\u003eVirgin macroplastic may enhance snail growth, reproduction, and hatching success by developing nutritious surface biofilms after entering aquatic systems. Plastic was virgin at deployment and not replaced during the experiment. Although we did not directly quantify surface colonization, prior work shows that plastic surfaces rapidly accumulate biofilm over days to weeks(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) that serve as a food source to grazers on timescales that vary with environmental conditions, including light availability, temperature, and dissolved oxygen(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Our results are consistent with this mechanism, suggesting that even initially virgin macroplastic can influence snail demography over the course of the 12-week experiment. Similar processes may also occur on microplastic, although this remains untested. While biofouling on larger plastic may provide nutritional subsidies, responses to microplastic particles likely reflect different physiological mechanisms beyond nutrition.\u003c/p\u003e \u003cp\u003eSnails exposed to virgin microplastic exhibited increased survival and somatic growth but reduced reproductive output, indicating a trade-off consistent with energy reallocation from reproduction to maintenance. This pattern aligns with reported endocrine-disrupting effects of polyethylene microplastic(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), to which snail reproduction is particularly sensitive due to neuroendocrine regulation(\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Microplastic-associated chemicals may exacerbate oxidative stress and reproductive disturbances(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e), via an imbalance between reactive oxygen species and antioxidants that damage cells in the ovum. Similar maintenance-fecundity trade-offs have been documented in polyethylene microplastic exposures(\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e), supporting this interpretation. Growth gains may also reflect biofilm ingestion along with the nominally virgin microplastic or more efficient food assimilation via microplastic-facilitated shredding in the gut(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). We confirmed snail microplastic ingestion and egestion through microscopy (Figure S15). Notably, this trade-off is not observed under joint exposure to biofouled macroplastic and virgin microplastic, consistent with a nutrition-dependent response to endocrine-disruptors.\u003c/p\u003e \u003cp\u003eJoint exposure to biofouled macroplastic and virgin microplastic attenuates the reproduction-maintenance trade-off seen with microplastic alone, yielding a concurrent increase in survival, somatic growth, and egg production. We speculate a nutrition-dependent buffering of microplastic-induced endocrine and oxidative stress, whereby periphyton-derived micronutrients like antioxidants may mitigate reproductive impairment while sustaining somatic benefits. Such nutrient-mediated modulation of contaminant toxicity is well documented across biological systems humans(\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e), animals(\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e), and in vitro(\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Periphyton communities containing polyunsaturated fatty acids(\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e) with antioxidant properties may enhance antioxidant defenses and increase total antioxidant capacity under oxidative stress following microplastic ingestion(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). These interacting nutritional and physiological effects likely explain the joint-treatment outcomes, which scale beyond individual life-history traits to shape the population-level consequences.\u003c/p\u003e \u003cp\u003ePlastic pollution tips the balance toward \u003cem\u003eB. truncatus\u003c/em\u003e: improved survival and fecundity, faster maturation, and could potentially provide colonizable habitat, altered predator-prey dynamics, and create positive feedback that collectively inflates snail populations. Under realistic co-occurring microplastic and macroplastic pollution, our analysis indicates that snail populations would expand, driven by increases in expected reproductive output. We also found that the next generations of snails could reach reproductive maturity earlier with increased somatic growth when exposed to pollutants. Moreover, snails have been documented to colonize macroplastic debris as a substrate for settlement(\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). The additional novel habitat and surface nutrition provided by biofouled macroplastic may further increase the carrying capacity of the system if all other ecological factors remain the same, reinforcing the positive feedback loop of a surging snail population. Beyond these direct demographic effects, plastic pollution may also influence snail population dynamics through behavioral and community-level pathways. Although such processes were beyond the scope of this study, research has shown that marine snails exposed to microplastics exhibit increased vulnerability to predation due to reduced vigilance and antipredator responses(\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). Macroplastic may additionally function as a physical refuge, as artificial plastic structures are widely used to enhance aquatic habitats(\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). However, these mechanisms remain speculative in freshwater snail systems and would benefit from targeted investigation. Field-based studies are therefore needed to validate whether plastic debris consistently serves as habitat and/or resource subsidies for \u003cem\u003eB. truncatus\u003c/em\u003e and to determine how these changes translate to population growth and, as a result, schistosomiasis transmission risk.\u003c/p\u003e \u003cp\u003ePlastic pollution may elevate urogenital schistosomiasis risk by increasing \u003cem\u003eB. truncatus\u003c/em\u003e abundance and per-capita parasite output, but its direct effects on cercarial production, survival, and infectivity remain a key knowledge gap. \u003cem\u003eB. truncatus\u003c/em\u003e snails are the obligate intermediate host of human urogenital schistosomiasis. Independent of plastic contamination, growth in the snail population increases the total number of free-swimming cercariae released into water, raising human infection risk with routine water contact(\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). Prior work also shows that faster snail growth and higher resource availability, which are observed in our experiment, boost cercarial production per capita once infected(\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). These factors imply a higher human infection risk through both more contributing hosts and greater parasite loads per host. This is assuming the schistosome cercarial production, morphology, longevity, and infectivity remain unchanged despite plastic pollutant exposure, which remains untested. However, comparative studies report divergent cercarial outcomes across systems, which may plausibly reflect differences in the snail organs where parasites develop and reproduce. One study found that high nanoplastic diets yielded the greatest cercarial production in freshwater snail \u003cem\u003eStagnicola elodes\u003c/em\u003e infected with trematode \u003cem\u003ePlagiorchis\u003c/em\u003e sp., which develops primarily in gonadal tissues(\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). These findings are consistent with compensatory feeding that offsets the energy costs of rediae and sporocysts(\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e) and sustains elevated cercarial emergence(\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). By contrast, another study reported that nanoplastic reduced parasite production in a marine snail \u003cem\u003eZeacumantus subcarinatus\u003c/em\u003eon infected with the trematode \u003cem\u003eMaritrema novaezealandensis\u003c/em\u003e, which develops primarily in the guts and other soft tissue(\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). It is likely because nanoparticle exposure imposes energetic and tissue‑repair costs on the snail that divert resources from intramolluscan parasite development, and/or nanoparticles inflict direct physical or cellular damage on sporocysts, impairing their capacity to asexually produce cercariae. They also found that cercarial morphology and infectivity were unchanged, but survival declined under high concentrations due to oxidative stress, tegumental damage, or impaired osmoregulation. Conversely, high concentrations of microplastics lowered infection prevalence and intensity for tadpole hosts in an echinostome-\u003cem\u003eHelisoma trivolvis\u003c/em\u003e-\u003cem\u003eRana sylvatica\u003c/em\u003e model(\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). It likely arises because of draining cercarial energy upon microplastic contact and delaying host encounters beyond the brief infectious window. Given these divergent outcomes, the net impact of plastic pollution on \u003cem\u003eS. haematobium\u003c/em\u003e transmission remains uncertain. Future studies are needed to quantify how plastic exposure alters snail susceptibility to infection and subsequent cercarial production, morphology, longevity, and infection success.\u003c/p\u003e \u003cp\u003eThis experiment has several limitations and strengths. The macroplastics were biofouled in a location not inhabited by \u003cem\u003eB. truncatus\u003c/em\u003e, but our profiling of periphyton traits provides a basis for comparison and for evaluating potential effects on snail life-history in different ecological contexts. Snails may have ingested trace microplastic scraped from the macroplastic surfaces, but we consider this exposure negligible relative to the orders-of-magnitude higher doses that produced the effects observed in our microplastic-related treatments. Due to laboratory constraints, we did not quantify the number of eggs per egg mass for each treatment on a weekly basis. Instead, we used the mean number of eggs enrolled in the hatching assay for each treatment and week as a proxy (around 10 eggs, which typically corresponds to one egg mass(\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e)). Because egg masses cannot be subdivided without structural damage, this approach provides a reasonable and consistent approximation across treatments and time points. Despite these limitations, our study is the first to quantitatively examine how plastic pollution shapes the key life-history traits of \u003cem\u003eB. truncatus\u003c/em\u003e, an intermediate host of human schistosomiasis. Using various plastic size classes, polymers, biofouling status, and abundance, we capture key dimensions of environmental heterogeneity while isolating their biological effects. We measured life-history traits using standardized protocols to ensure comparability. We found that plastic pollution meaningfully altered the \u003cem\u003eB. truncatus\u003c/em\u003e population dynamics: biofouled macroplastic strongly enhances reproductive output; virgin microplastic produces a more subtle demographic decrease; the joint exposure yields concurrent improvement in life-history. This highlights macroplastic biofouling as a key ecological driver of snail population growth, with implications for schistosomiasis transmission. Future field studies are needed to confirm whether \u003cem\u003eB. truncatus\u003c/em\u003e utilizes plastic debris, alongside laboratory experiments quantifying snail susceptibility and cercarial production under plastic exposure, to determine how plastic pollution influences the disease transmission risk to inform public health interventions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eNot applicable, as this study did not involve human participants or vertebrate animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interests exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the National Science Foundation under grant numbers DEB-2011179 and ICER-2522282. Ao Yu was partially funded by the Stanford King Center on Global Development\u0026rsquo;s Graduate Student Research Funding program under project number 26W11E and the Stanford Center for Human and Planetary Health Catalyst Award. Kaitlyn R Mitchell was partially funded by the Stanford University Center for Innovation in Global Health under grant number 353068.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eAY, KRM, JTV, and GDL designed the research; AY performed the research; AY contributed new reagents and analytical tools; AY, KRM analyzed the data; AY, KRM, JTV, and GDL interpreted the data; AY wrote the paper; AY, KRM, JTV, and GDL all edited and revised the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe appreciate the support from the Biomedical Research Institute, Margaret Mentink-Kane, Gabrielle Bate, and Sara Li for providing experimental subjects and maintenance advice. We are grateful to Allina Zhang, Nyssa Kansal, Emily Chu, Jennifer Xu, Katy Zheng, Madeline Dulce, and Bohdan Kamets for their assistance with experimental subject maintenance and laboratory data collection. We thank Rodolfo Dirzo\u0026rsquo;s lab group, Kirsten Verster, Kevin Arrigo\u0026rsquo;s lab group, Gert van Dijken, Taylor Broek, Guangchao Li, Carnegie Science, Ted Raab, Jasper Ridge Biological Reserve, Adriana I. Hern\u0026aacute;ndez, and Brooke Weigel for sharing necessary lab space, facilities, equipment, and/or valuable inputs on analysis protocols. We thank Giulio De Leo\u0026rsquo;s group, Erin A. Mordecai\u0026rsquo;s group, and Veronica Felicia Frans for their constructive feedback that strengthened our submission. All Figures were created or arranged using Biorender.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data and analysis code supporting the findings of this study are available in the Zenodo repository at the following URL: https://doi.org/10.5281/zenodo.19685289\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePadha S, Kumar R, Dhar A, Sharma P. Microplastic pollution in mountain terrains and foothills: A review on source, extraction, and distribution of microplastics in remote areas. Environmental Research. 2022;207:112232. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.envres.2021.112232\u003c/span\u003e\u003cspan address=\"10.1016/j.envres.2021.112232\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJung YS, Sampath V, Prunicki M, Aguilera J, Allen H, LaBeaud D, et al. 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Journal of Helminthology. 2021;95:e66. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1017/S0022149X21000651\u003c/span\u003e\u003cspan address=\"10.1017/S0022149X21000651\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Plastic pollution, Microplastic, Macroplastic, Bulinus, Schistosomiasis, Life history, Population dynamics, Disease ecology","lastPublishedDoi":"10.21203/rs.3.rs-9510130/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9510130/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePlastic debris is increasingly recognized as an ecological driver of mosquito-borne disease by creating freshwater breeding habitat, yet its consequences for snail hosts of human schistosomiasis transmission remain poorly understood. For these snail hosts, including \u003cem\u003eBulinus truncatus\u003c/em\u003e, plastic pollution may alter population dynamics by modifying life-history processes through surface biofilm-derived nutritional subsidies, habitat alteration, and toxicity-induced reproductive trade-offs.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe performed two laboratory experiments that investigated the effects of macro- and microplastics, individually and jointly, on \u003cem\u003eB. truncatus\u0026rsquo;\u003c/em\u003e life-history traits (survival, growth, egg mass production, hatching) by using environmentally realistic sizes, polymers, concentrations, and biofouling status. To assess the overall impact of plastic pollution on snail demography, we derived a measure of reproductive output under each plastic treatment, i.e., the expected number of cumulative viable offspring produced over the experiment.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that macroplastic boosted somatic growth, egg mass production, and hatching, consistently playing the most critical role in the demographic increase driven by biofilm-derived nutritional subsidies. Microplastics reduced hatching success but improved survival and growth, resulting in a marginal population decline due to toxicity-induced maintenance-reproduction trade-offs. Combined exposure elevated survival, growth, and egg mass production while mitigating microplastic-induced hatching costs, generating positive but attenuated gains due to nutrition-buffered toxicological costs. Collectively, these results demonstrate that plastic exposure can shift snail population trajectories by altering life-history processes that scale to net reproductive output, with biofouling emerging as the primary driver of demographic response.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur findings show that plastic pollution increases \u003cem\u003eB. truncatus\u003c/em\u003e demographic output by altering its key life-history traits, which prior work has linked to increased cercarial production with critical implications for human transmission risk. These findings identify plastic pollution as a previously unrecognized pathway and demonstrate that ecological transformation of plastic debris is the dominant determinant of host demographic response with implications for integrating environmental management into disease control strategies.\u003c/p\u003e","manuscriptTitle":"Plastic pollution reshapes snail intermediate host life history: implications for schistosomiasis transmission","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-30 16:23:06","doi":"10.21203/rs.3.rs-9510130/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-05-12T16:36:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-29T10:46:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-28T09:16:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2026-04-23T19:26:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"62e4b3e4-e6df-466f-a946-d440574e929a","owner":[],"postedDate":"April 30th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"5","date":"2026-05-12T16:36:53+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T16:39:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-30 16:23:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9510130","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9510130","identity":"rs-9510130","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}