Antipredator behaviour of native freshwater snails (Sulcospira hainanensis) against the critically endangered big-headed turtle (Platysternon megacephalum)

Antipredator behaviour of native freshwater snails (Sulcospira hainanensis) against the critically endangered big-headed turtle (Platysternon megacephalum) | 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 Antipredator behaviour of native freshwater snails (Sulcospira hainanensis) against the critically endangered big-headed turtle (Platysternon megacephalum) Amy Wing Lam FOK, Jia Huan LIEW, Yik-Hei SUNG This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7478776/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Predators can trigger antipredator behaviour in their prey, leading to a cascading effect on community composition and nutrient cycling. The big-headed turtle ( Platysternon megacephalum ) frequently predates on freshwater snails ( Sulcospira hainanensis ) in Hong Kong hill streams. This study aims to detect and measure the antipredator behaviour of S. hainanensis in response to chemical (e.g., olfactory) signals from P. megacephalum and dead conspecifics. The snails were subject to four treatments: 1) no chemical signals (control), 2) chemical cues from dead conspecifics, 3) chemical cues from predators (turtle), and 4) combination of chemical cues from dead conspecifics and predators. Chemical cues from predators were the only treatment triggered a significant prey response in which they hid under a refuge. The wild population of P. megacephalum has declined because of poaching. Our findings suggest that this may lead to cascading effect on the stream ecosystem (e.g., level of primary productivity) via a weakened prey response in snails. Molluscs stream trophic cascades Asian turtles pheromones Figures Figure 1 Figure 2 Figure 3 Introduction Turtles and tortoises perform critical ecological roles, functioning as consumers, seed dispersers, bioturbators, and nutrient cyclers [1]. Existing literature about the ecological functions of turtles and tortoises have mostly focused on some regions of the world, such as North America [2], while information from other regions remain sparse. This is despite the fact that they are nearly universally threatened [3]. Asian freshwater turtle populations have been impacted by habitat loss and degradation, as well as over-exploitation for food, medicinal and pet markets [3], with about 80% of freshwater turtles and tortoises classified as critically endangered, endangered or vulnerable on the IUCN Red List [4]. While there is an urgent need to investigate the ecology of Asian freshwater turtles to better manage them, the scarcity of wild turtle populations can hinder the execution of systematic ecological studies [5]. One major way in which turtles shape their environment is via predator-prey interactions. In some ecosystems, carnivorous and omnivorous freshwater turtles constitute comparable biomass to predatory fish [6], yet turtles might exert a larger influence on prey species because they are larger and can access a wider range of prey items. For example, the common snapping turtle ( Chelydra serpentina ) have larger gape sizes than the bluegill ( Lepomis macrochirus ), so they can predate on larger prey items such as Rana sphenocephala tadpoles [7]. The presence of the turtle was correlated with a higher mortality of the tadpoles and an increase in the mean mass of the surviving tadpoles. Besides larger prey, turtles can consume a large number of hard-shelled snails that may be inaccessible to other vertebrate predators [8]. Turtles can also alter prey behaviour. In snails, chemoreception is highly developed and serves as the primary sensory system [9]. Snails exposed to predator cues alone or together with injured conspecific cues often triggers antipredator behaviours, such as escaping from a predator cue and refuge-seeking above the water surface, within substrates, or under cover [10], [11]. These behaviours may change the snail’s foraging pattern [12], [13] and reduce their ability to reproduce [14], [15], thereby decreasing activity levels and their population, which could ultimately trigger a top-down trophic cascade affecting the base of the food web—algae and macrophytes. Overall, turtles may indirectly influence ecosystem-level processes (e.g. primary productivity and nutrient dynamics) and community structure (e.g. species composition and total species richness) [16], [17], by suppressing primary consumers, such as snails and tadpoles. Research on predator-prey dynamics among Asian freshwater turtles are mostly limited to interactions with the invasive apple snail ( Pomacea canaliculata ) [14], [18], [19]. Researchers found that the Chinese softshell turtle ( Pelodiscus sinensis ) and Reeve’s terrapin ( Mauremys reevesii ) can be effective biological control agents of apple snails, given the large number of snails they consume [19], [20]. The effects of turtles on native prey species in Asia, however, remain largely unknown. For example, in Hong Kong’s mountain streams, a native freshwater snail ( Sulcospira hainanensis ) [21] is the primary prey of the big-headed turtle ( Platysternon megacephalum ) [22], yet we know very little about the dynamics of their predator-prey interaction, and its broader consequences on the ecosystem. Nevertheless, reduced S. hainanensis populations have been shown to result in sharp increases in algal biomass, total species richness and insect biomass and density [23]. This means that changes in predator-prey dynamics between the snail and its major predator would likely have major cascading effects [17], [24], warranting closer investigation. In this study, we assess if P. megacephalum elicit measurable antipredator responses in their primary prey, S. hainanensis . We addressed the following questions: (1) Do the snails hide when exposed to the chemical cues mimicking turtle presence? (2) Do the snails’ response intensify when both the chemical cues of injured conspecifics as well as turtles are present, indicating that turtle predation is more significant than their mere presence? The results of this research will help fill the vast knowledge gaps regarding the ecological roles of freshwater turtles in Asia. Addressing these knowledge gaps is vital for informing conservation efforts and raising public awareness about the importance of freshwater turtle conservation in Asia. Materials and Methods Study Animals Between September and October 2022, we collected two batches of freshwater snails ( S. hainanensis ) in two separate trips (240 in each batch, a total of 480 snails) from a river in the New Territories of Hong Kong, where the big-headed turtle ( P. megacephalum ) occurs in the higher reaches of the river. The snails [mean (± SD) snail width = 12.6 (± 5.4) mm] were acclimatised for 2–3 days before the experiment and released to the collection site after experiment, within one week of collection. They were allotted randomly to two large holding tanks (40 cm × 40 cm × 61 cm) in densities that mirror the wild, i.e., between 100 and 200 individuals per m 2 [25]. The tanks were filled with de-chlorinated, aerated aged tap water (24°C) with a water filter (filtering capacity: 200 L hr -1 ). The snails were provided shelters constructed out of rocks that were collected from the river, and fed algae wafers three times a week. Four wild P. megacephalum were temporarily held in the laboratory and kept individually in separate tanks with a hide, a basking lamp and a water filter. All procedures involving the protected turtles in this study were approved by the Agriculture, Fisheries and Conservation Department, Hong Kong SAR Government (AF GR CON 09/51 Pt 6). Experimental design We placed 10 snails into each experimental glass tank (12.5 cm × 7 cm × 17 cm), which contained 500 mL of aged tap water and a 10 cm x 10 cm tile for refuge. We tested four experimental treatments, mimicking: 1) status quo without additional chemical cues as a control; 2) chemical cues from dead/injured conspecifics; 3) chemical cues from a turtle predator; 4) chemical cues from dead/injured conspecifics and a turtle predator. For each experimental treatment, we added various combinations of mixtures containing chemical cues after the snails have been acclimatised in the experimental tanks for 30 minutes. In treatment 1, which was our experimental control, we added 150 mL of aged tap water. In treatment 2, we added 150 mL of mixture mimicking dead/injured conspecifics. In treatment 3, we added 150 mL of mixtures mimicking the presence of a turtle predator. For treatments 4, we combined 75 mL of mixtures mimicking dead/injured conspecifics with 75 mL of mixtures mimicking the presence of a turtle predator. Mixtures containing chemical cues that mimic dead/injured conspecifics were produced by adding 3.1–4.7 g of homogenised snail tissue per 150 ml of aged tap water, debris (e.g., shells and snail tissues) were filtered with a sieve (aperture: 212µm). Mixtures that contain chemical cues mimicking the presence of a turtle were produced by placing a P. megacephalum (mean head width: 29.8 ± 9.7 mm; mean plastron length: 78.7 ± 19.5 mm) in 300 ml of water for two hours. We conducted a pilot experiment with 120 snails and observed snail behaviour for two hours after exposing to the control and water with turtle cue, we observed five types of behaviour: (1) hiding under refuge (UR)— stationed under a refuge and could not be seen from above; (2) emerged from water (EW)—completely left the water; (3) retreated in shell (IS)— retreated into its shell, aperture was sealed by the operculum; (4) scanning the environment (SE)— scanning behaviour reflected in sweeping movement of antennae while remaining on the same spot, as if alert to danger; and (5) travelling around (TA)— travelled around in the water. Also, in the pilot experiment, we found that most snail behaviour ceased to change after 40 minutes, which is within the range of other similar studies [26], [27]. Therefore, we observed the snails for 40 minutes and recorded the number of snails exhibiting the five types of behaviours at 10 th , 20 th , 30 th and 40 th minute. Statistical analysis We used generalised linear mixed models (GLMMs) with negative binomial distribution to analyse the effects of treatment on snail behaviour [28]. The numbers of snails exhibiting a behaviour in all time slots were added up for each behaviour in each experiment. These accumulative number of behaviours were then used as response variable, while treatment was used as a fixed factor, and the snail group was treated as a random effect. We carried out a GLMM test for each type of behaviour. All analyses were tested using software R v 4.1.3 software [29]; GLMM was performed with the lme4 package, and residuals and overdispersion were checked using the DHARMa package [30]. Results Among the five behaviours, hiding under refuge (UR) was most frequently observed (mean accumulative number of snails per tank = 22.0), followed by travelling around (TA; 14.2), retreated in shell (IS; 6.6), and scanning the environment (SE; 6.2) (Figure 1). Few snails emerged from the water (EW; 0.6) and thus this behaviour was excluded from subsequent analysis. For hiding under refuge (UR), we found that the predator treatment significantly increased UR behaviour compared to the control (Figure 2a; Table 1; p < 0.01). Both treatments with dead/injured snails (i.e., Injured snail, Predator + Injured snails) induced similar number of snails exhibiting UR behaviour as the control. We found that the number of snails that travelled around (TA) was significantly lower in predator treatment (Figure 2b; p < 0.01), while it was similar between the control and other treatments. The number of snails retreated in shell (IS) and scanning the environment (SE) did not differ across treatments (Figure 2c, 2d). Table 1 Estimated effects of four treatments on the number of snails (Sulcospira hainanensis) exhibiting different behaviours in response to the chemical cue of big-headed turtles (Platysternon megacephalum) and injured conspecifics analysed using generalised linear mixed models. The control treatment was used as the reference level. Bold rows indicate significant effects. Treatments Estimate SE z p Hiding under refuge (UR) Injured snail (vs control) 0.13 0.19 0.70 0.49 Predator (vs control) 0.55 0.18 2.97 < 0.0 1 Predator + Injured snails (vs control) 0.10 0.19 0.52 0.60 Travelling around (TA) Injured snail (vs control) -0.02 0.30 -0.07 0.94 Predator (vs control) -0.92 0.31 -2.96 < 0.0 1 Predator + Injured snails (vs control) -0.24 0.30 -0.79 0.43 Scanning the environment (SE) Injured snail (vs control) 0.37 0.38 0.99 0.32 Predator (vs control) -0.08 0.39 -0.21 0.83 Predator + Injured snails (vs control) -0.25 0.39 -0.65 0.52 Retreated in shell (IS) Injured snail (vs control) -0.58 0.53 -1.09 0.28 Predator (vs control) 0.01 0.52 0.01 0.99 Predator + Injured snails (vs control) 0.16 0.51 0.31 0.76 Discussion In this study, we found that the freshwater snail, S. hainanensis , exhibited antipredator behaviour when exposed to the chemical cues of the big-headed turtle ( P. megacephalum ), primarily by moving under refuge, although the snail’s behaviours did not vary substantially in the presence of dead/injured conspecifics. We discuss these findings and implications for conservation below. As a common prey to various kinds of predators — including freshwater turtles [22], [31], [32] — snails adopt a range of antipredator behaviours, with hiding in substratum and escaping above the water’s surface being the more frequently observed behaviours [33], [34], [35]. The expression of these behaviours can be influenced by different factors. For example, predator type affects whether the snails hide near the water’s surface or in the benthic substrate [27], [35]. Previous studies on the freshwater snails Planorbella campanulata , Pomacea paludosa and Po. canaliculata showed that the snails tend to hide in the benthic substrate when exposed to chemical cues from turtles [27], [36], [37], potentially because burying into the substrate is an efficient method for avoiding turtles which hunt visually [15], [37]. In this experiment, we used S. hainanensis which was frequently preyed upon by P. megacephalum [22]. Like other freshwater snails, S. hainanensis adopted a similar antipredator behaviour by hiding under refuge (UR) in the presence of turtle predators (Figure 2a). Snails also adapt their hiding behaviour according to the availability of benthic refuge. When refuge such as sand or vegetation is present, alarmed snails hide by burying themselves or hide among the vegetation [27], [36], [38]. However, when benthic refuge is not available, snails will instead move out of water [39] or into an area that is inaccessible by the predator [15]. In this experiment, a tile was provided for S. hainanensis to hide at the bottom of the tank and the snails of the predator treatment were found to use this benthic refuge more frequently (Figure 2a). This behaviour is beneficial to the snail’s survival in Hong Kong hill streams, where rock crevices on stream bottom provide many avenues for escape because cobbles and boulders are abundant and persistently available year-round (~ 60% in wet season and ~ 70% in dry season) [40]. This may also explain why wild S. hainanensis detach from rock surfaces to drop into the benthic rock crevices when subject to strong taps, which they may perceive as a predatory risk. Similar drop-and-escape behaviour was observed by DeWitt et al. [36] when snails were disturbed by turtles. Previous studies have showed that, beside the chemical cues of predators, snails also respond to the chemical cues of injured conspecifics [26], [27], [34], [41]. However, we found that S. hainanensis behaves similarly when comparing the control and injured snail treatments (Figure 2a-d). This suggests that S. hainanensis do not respond to the chemical cues of injured snails. Alternatively, our protocol for mimicking the presence of dead or injured conspecifics may not be sufficiently stimulating. For the preparation of injured snail cues, Hayashi and Sugiura [26] and Turner et al. [41] crushed live snails to obtain injured snail cues, while McCarthy and Fisher [34] and Ueshima and Yusa [27] did not specify whether live snails were used. If the snails were alive during cue preparation, they would likely have released alarm pheromones, which could trigger antipredator behaviour in conspecifics [42]. In our study, however, S. hainanensis were frozen intact and stored at -20 °C until they were needed for experiment. Park and Sung [43] highlighted that the olfactory cues of predators (i.e., ventral gland excretion) should be used within a specific timeframe: stored at 4 °C within four days or at -20 °C for up to eight days. In this study, the snails were stored at -20 °C but not all were used within eight days. As our results are inconclusive, we propose that future studies should incorporate the use of live snails to trigger the release of alarm pheromones, and that experimental subjects should be exposed to extracted chemical cues as quickly as possible after preparation. Under undisturbed condition, the high density of P. megacephalum likely regulates the population density of S. hainanensis . However, conducting empirical studies to test this in the field is challenging, because populations of Asian freshwater turtles are scarce [44]. Taking a different approach, we documented the influence of P. megacephalum on the behaviour of S. hainanensis , the dominant grazer in these streams. Our results show that S. hainanensis exhibit an antipredator response to the chemical cues of P. megacephalum in the form of seeking refuge. This behaviour may affect how snails utilise resources in hill stream ecosystems. For instance, upon detecting P. megacephalum , the snails are more likely to occupy covered microhabitats, such as rock crevices, and therefore consume more resources in that area [12], [45]. Further, a reduction of resource consumption by snails in open, uncovered area may free up more resources for other grazing macroinvertebrates [23]. This warrants further studies on the cascading impacts of P. megacephalum on ecological processes and community structure in freshwater streams, if robust populations of P. megacephalum can be identified [7], [16], [46], [47]. Our results highlight the ecological consequences of the population extirpation of Asian freshwater turtles. Where Asian freshwater turtle populations persist, there is an urgent need for more studies on their ecological interactions to deepen our understanding of their ecological roles to inform efforts in environmental education and turtle conservation. Declarations We confirm that this manuscript has not been published elsewhere and is not under consideration by another journal. All authors have approved the manuscript and agree with its submission to Discover Animals. Funding Declaration This study was funded by Lingnan University (Faculty Research Grant; project code:102194). Ethics Declaration The methods used in this study were approved by the Department of Health [Permit number: (20-51) in DH/HT&A/8/2/8 Pt.1] and the Agriculture, Fisheries and Conservation Department [Permit number: (95) in AF GR CON 09/51 Pt. 8] of the Hong Kong Special Administrative Region Government, China. Consent to Participate Declaration : not applicable Consent to Publish Declaration : not applicable Competing interests The authors declare that they have no conflicts of interest that could have appeared to influence the work reported in this paper. Author Contribution A.W.L.F. and Y.H.S. acquired funding and wrote the main manuscript text. A.W.L.F. implemented the experiment and collected the data. A.W.L.F., J.H.L., and Y.H.S. assessed the accuracy of the data analysis methods, validated the methodology, and reviewed the manuscript. Acknowledgement We are grateful to the Agriculture, Fisheries and Conservation Department, Hong Kong SAR Government for granting the research permit, and Lingnan University (Faculty Research Grant; project code:102194) for providing research grants. Special thanks are due to Dr. LAU Michael Wai Ning and Dr. FONG Jonathon for their insightful guidance, which significantly improved the quality of this manuscript. Furthermore, we express our gratitude to the supporting team members, CHAN Man Ho Henry, LAM Pui Yin Ivan, LAM Yu Ki Billy, LEE Wing-Him Henry, LEUNG Julia, LEUNG Ka Wah Franco, LIU Hua Bin, SO Halbert, TAN Kai Teck Desmond, YIP Hon Tim, YIP Wing Yan Gena and YUEN Hiu Ching Hilda who contributed to various capacities, including field assistance, animal care and data collection. 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Bernot, “Predator identity and consumer behavior: differential effects of fish and crayfish on the habitat use of a freshwater snail,” Oecologia , vol. 118, pp. 242–247, 1999, doi: https://doi.org/10.1007/s004420050724. B. D. Wisenden, “Olfactory assessment of predation risk in the aquatic environment,” Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences , vol. 355, no. 1401, pp. 1205–1208, Sept. 2000, doi: 10.1098/rstb.2000.0668. D. Park and H. Sung, “Male Hynobius leechii (amphibia: Hynobiidae) discriminate female reproductive states based on chemical cues,” Integrative Biosciences , vol. 10, no. 3, pp. 137–143, 2006, doi: 10.1080/17386357.2006.9647295. T. D. Richardson and K. M. Brown, “Predation risk and feeding in an intertidal predatory snail,” Journal of Experimental Marine Biology and Ecology , vol. 163, no. 2, pp. 169–182, Nov. 1992, doi: 10.1016/0022-0981(92)90047-E. A. M. Turner, R. J. Bernot, and C. M. Boes, “Chemical cues modify species interactions: the ecological consequences of predator avoidance by freshwater snails,” Oikos , vol. 88, no. 1, pp. 148–158, 2000, doi: https://doi.org/10.1034/j.1600-0706.2000.880117.x. L. Buria, R. Albariño, V. D. Villanueva, B. Modenutti, and E. Balseiro, “Impact of exotic rainbow trout on the benthic macroinvertebrate community from Andean-Patagonian headwater streams,” Fundamental and Applied Limnology , vol. 168, no. 2, pp. 145–154, Feb. 2007, doi: 10.1127/1863-9135/2007/0168-0145. M. J. Vanni, “Nutrient cycling by animals in freshwater ecosystems,” Annual Review of Ecology and Systematics , vol. 33, pp. 341–370, 2002, doi: https://doi.org/10.1146/annurev.ecolsys.33.010802.150519. Additional Declarations No competing interests reported. Supplementary Files DataforsnailantipredatorstudyAWLFJHLYHS.csv Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 26 Oct, 2025 Reviewers agreed at journal 20 Oct, 2025 Reviews received at journal 14 Oct, 2025 Reviews received at journal 07 Oct, 2025 Reviewers agreed at journal 07 Oct, 2025 Reviewers agreed at journal 26 Sep, 2025 Reviewers invited by journal 15 Sep, 2025 Editor invited by journal 09 Sep, 2025 Editor assigned by journal 09 Sep, 2025 Submission checks completed at journal 09 Sep, 2025 First submitted to journal 28 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":259812,"visible":true,"origin":"","legend":"\u003cp\u003eStudy animals in this experiment – the freshwater snail, Sulcospira hainanensis (left), and the big-headed turtle, Platysternon megacephalum (right).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7478776/v1/1ec008461f2c6a6a7fc3e930.jpg"},{"id":92064380,"identity":"386aa7b2-fc09-4e68-9594-99e4ea3e5610","added_by":"auto","created_at":"2025-09-24 08:39:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":35312,"visible":true,"origin":"","legend":"\u003cp\u003eMean accumulative number of snails per tank exhibiting different behaviours, arranged in descending order. UR = hiding under refuge, TA = travelling around, IS = retreated in shell, SE = scanning the environment, EW = emerged from water.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7478776/v1/e01d1c4def422f44c6f3278a.jpg"},{"id":92064238,"identity":"48e04fd1-c563-402d-8314-8b3387986f1d","added_by":"auto","created_at":"2025-09-24 08:39:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":120819,"visible":true,"origin":"","legend":"\u003cp\u003eThe number of snails (Sulcospira hainanensis) displaying different behaviours in response to the chemical cue of big-headed turtles (Platysternon megacephalum) and injured conspecifics: (\u003cstrong\u003ea\u003c/strong\u003e): hiding under refuge (UR); (\u003cstrong\u003eb\u003c/strong\u003e): travelling around (TA); (\u003cstrong\u003ec\u003c/strong\u003e): retreated into shell (IS); and (\u003cstrong\u003ed\u003c/strong\u003e): scanning the environment (SE). p \u0026lt; 0.01 represents a significant difference from control.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7478776/v1/6f2f038bfd0fdcd9ed030b71.jpg"},{"id":92064968,"identity":"f949382d-e386-430a-96a1-fc27ae9a3f61","added_by":"auto","created_at":"2025-09-24 08:47:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":979286,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7478776/v1/e89cbac3-f357-4a7c-bf13-06edbd9de983.pdf"},{"id":92064337,"identity":"b027edc3-9368-482a-a1a6-748d7a76af04","added_by":"auto","created_at":"2025-09-24 08:39:19","extension":"csv","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":936,"visible":true,"origin":"","legend":"","description":"","filename":"DataforsnailantipredatorstudyAWLFJHLYHS.csv","url":"https://assets-eu.researchsquare.com/files/rs-7478776/v1/049cf291ce2854fa73b1fea7.csv"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antipredator behaviour of native freshwater snails (Sulcospira hainanensis) against the critically endangered big-headed turtle (Platysternon megacephalum)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTurtles and tortoises perform critical ecological roles, functioning as consumers, seed dispersers, bioturbators, and nutrient cyclers [1]. Existing literature about the ecological functions of turtles and tortoises have mostly focused on some regions of the world, such as North America [2], while information from other regions remain sparse. This is despite the fact that they are nearly universally threatened [3]. Asian freshwater turtle populations have been impacted by habitat loss and degradation, as well as over-exploitation for food, medicinal and pet markets [3], with about 80% of freshwater turtles and tortoises classified as critically endangered, endangered or vulnerable on the IUCN Red List [4]. While there is an urgent need to investigate the ecology of Asian freshwater turtles to better manage them, the scarcity of wild turtle populations can hinder the execution of systematic ecological studies [5].\u003c/p\u003e\n\u003cp\u003eOne major way in which turtles shape their environment is via predator-prey interactions. In some ecosystems, carnivorous and omnivorous freshwater turtles constitute comparable biomass to predatory fish [6], yet turtles might exert a larger influence on prey species because they are larger and can access a wider range of prey items. For example, the common snapping turtle (\u003cem\u003eChelydra serpentina\u003c/em\u003e) have larger gape sizes than the bluegill (\u003cem\u003eLepomis macrochirus\u003c/em\u003e), so they can predate on larger prey items such as \u003cem\u003eRana sphenocephala\u003c/em\u003e tadpoles [7]. The presence of the turtle was correlated with a higher mortality of the tadpoles and an increase in the mean mass of the surviving tadpoles.\u0026nbsp;\u0026nbsp;Besides larger prey, turtles can consume a large number of hard-shelled snails that may be inaccessible to other vertebrate predators\u0026nbsp;[8]. Turtles can also alter prey behaviour. In snails,\u0026nbsp;chemoreception is highly developed and serves as the primary sensory system\u0026nbsp;[9]. Snails\u0026nbsp;exposed to predator cues alone or together with injured conspecific cues often triggers antipredator behaviours, such as\u0026nbsp;escaping from a predator cue\u0026nbsp;and refuge-seeking above the water surface, within substrates, or under cover\u0026nbsp;[10], [11]. These behaviours may change the snail\u0026rsquo;s foraging pattern\u0026nbsp;[12], [13]\u0026nbsp;and reduce their ability to reproduce\u0026nbsp;[14], [15], thereby decreasing activity levels and their population, which could ultimately trigger a top-down trophic cascade affecting the base of the food web\u0026mdash;algae and macrophytes. Overall, \u0026nbsp;turtles may indirectly influence ecosystem-level processes (e.g. primary productivity and nutrient dynamics) and community structure (e.g. species composition and total species richness)\u0026nbsp;[16], [17], by suppressing primary consumers, such as snails and tadpoles.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResearch on predator-prey dynamics among Asian freshwater turtles are mostly limited to interactions with the invasive apple snail (\u003cem\u003ePomacea canaliculata\u003c/em\u003e) [14], [18], [19]. Researchers found that the Chinese softshell turtle (\u003cem\u003ePelodiscus sinensis\u003c/em\u003e) and Reeve\u0026rsquo;s terrapin (\u003cem\u003eMauremys reevesii\u003c/em\u003e) can be effective biological control agents of apple snails, given the large number of snails they consume [19], [20]. The effects of turtles on native prey species in Asia, however, remain largely unknown. For example, in Hong Kong\u0026rsquo;s mountain streams, a native freshwater snail (\u003cem\u003eSulcospira hainanensis\u003c/em\u003e) [21] is the primary prey of the big-headed turtle (\u003cem\u003ePlatysternon megacephalum\u003c/em\u003e) [22], yet we know very little about the dynamics of their predator-prey interaction, and its broader consequences on the ecosystem. Nevertheless, reduced \u003cem\u003eS. hainanensis\u003c/em\u003e populations have been shown to result in sharp increases in algal biomass, total species richness and insect biomass and density [23]. This means that changes in predator-prey dynamics between the snail and its major predator would likely have major cascading effects [17], [24], warranting closer investigation.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In this study, we assess if \u003cem\u003eP. megacephalum\u003c/em\u003e elicit measurable antipredator responses in their primary prey, \u003cem\u003eS. hainanensis\u003c/em\u003e. We addressed the following questions: (1) Do the snails hide when exposed to the chemical cues mimicking turtle presence? (2) Do the snails\u0026rsquo; response intensify when both the chemical cues of injured conspecifics as well as turtles are present, indicating that turtle predation is more significant than their mere presence? The results of this research will help fill the vast knowledge gaps regarding the ecological roles of freshwater turtles in Asia. Addressing these knowledge gaps is vital for informing conservation efforts and raising public awareness about the importance of freshwater turtle conservation in Asia.\u0026nbsp;\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch2\u003e\u003cem\u003eStudy Animals\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eBetween September and October 2022, we collected two batches of freshwater snails (\u003cem\u003eS.\u003c/em\u003e\u003cem\u003ehainanensis\u003c/em\u003e) in two separate trips (240 in each batch, a total of 480 snails) from a river in the New Territories of Hong Kong, where the big-headed turtle (\u003cem\u003eP. megacephalum\u003c/em\u003e) occurs in the higher reaches of the river. The snails [mean (\u0026plusmn; SD) snail width = 12.6 (\u0026plusmn; 5.4) mm] were acclimatised for 2\u0026ndash;3 days before the experiment and released to the collection site after experiment, within one week of collection. They were allotted randomly to two large holding tanks (40 cm \u0026times; 40 cm \u0026times; 61 cm) in densities that mirror the wild, i.e., between 100 and 200 individuals per m\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e[25]. The tanks were filled with de-chlorinated, aerated aged tap water (24\u0026deg;C) with a water filter (filtering capacity: 200 L hr\u003csup\u003e-1\u003c/sup\u003e). The snails were provided shelters constructed out of rocks that were collected from the river, and fed algae wafers three times a week. Four wild \u003cem\u003eP. megacephalum\u003c/em\u003e were temporarily held in the laboratory and kept individually in separate tanks with a hide, a basking lamp and a water filter. All procedures involving the protected turtles in this study were approved by the Agriculture, Fisheries and Conservation Department, Hong Kong SAR Government (AF GR CON 09/51 Pt 6).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eExperimental design\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eWe placed 10 snails into each experimental glass tank (12.5 cm \u0026times; 7 cm \u0026times; 17 cm), which contained 500 mL of aged tap water and a 10 cm x 10 cm tile for refuge. We tested four experimental treatments, mimicking: 1) status quo without additional chemical cues as a control; 2) chemical cues from dead/injured conspecifics; 3) chemical cues from a turtle predator; 4) chemical cues from dead/injured conspecifics and a turtle predator. For each experimental treatment, we added various combinations of mixtures containing chemical cues after the snails have been acclimatised in the experimental tanks for 30 minutes. In treatment 1, which was our experimental control, we added 150 mL of aged tap water. In treatment 2, we added 150 mL of mixture mimicking dead/injured conspecifics. In treatment 3, we added 150 mL of mixtures mimicking the presence of a turtle predator. For treatments 4, we combined 75 mL of mixtures mimicking dead/injured conspecifics with 75 mL of mixtures mimicking the presence of a turtle predator.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMixtures containing chemical cues that mimic dead/injured conspecifics were produced by adding 3.1\u0026ndash;4.7 g of homogenised snail tissue per 150 ml of aged tap water, debris (e.g., shells and snail tissues) were filtered with a sieve (aperture: 212\u0026micro;m). Mixtures that contain chemical cues mimicking the presence of a turtle were produced by placing a \u003cem\u003eP. megacephalum\u003c/em\u003e (mean head width: 29.8 \u0026plusmn; 9.7 mm; mean plastron length: 78.7 \u0026plusmn; 19.5 mm) in 300 ml of water for two hours.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe conducted a pilot experiment with 120 snails and observed snail behaviour for two hours after exposing to the control and water with turtle cue, we observed five types of behaviour: (1) hiding under refuge (UR)\u0026mdash; stationed under a refuge and could not be seen from above; (2) emerged from water (EW)\u0026mdash;completely left the water; (3) retreated in shell (IS)\u0026mdash; retreated into its shell, aperture was sealed by the operculum; (4) scanning the environment (SE)\u0026mdash; scanning behaviour reflected in sweeping movement of antennae while remaining on the same spot, as if alert to danger; and (5) travelling around (TA)\u0026mdash; travelled around in the water. Also, in the pilot experiment, we found that most snail behaviour ceased to change after 40 minutes, which is within the range of other similar studies\u0026nbsp;[26], [27]. Therefore, we observed the snails for 40 minutes and recorded the number of snails exhibiting the five types of behaviours at 10\u003csup\u003eth\u003c/sup\u003e, 20\u003csup\u003eth\u003c/sup\u003e, 30\u003csup\u003eth\u003c/sup\u003e and 40\u003csup\u003eth\u003c/sup\u003e minute.\u0026nbsp;\u003c/p\u003e\n\u003ch2 id=\"_Toc150441679\"\u003eStatistical analysis\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eWe used generalised linear mixed models (GLMMs) with negative binomial distribution to analyse the effects of treatment on snail behaviour [28]. The numbers of snails exhibiting a behaviour in all time slots were added up for each behaviour in each experiment. These accumulative number of behaviours were then used as response variable, while treatment was used as a fixed factor, and the snail group was treated as a random effect. We carried out a GLMM test for each type of behaviour. All analyses were tested using software R v 4.1.3 software [29]; GLMM was performed with the lme4 package, and residuals and overdispersion were checked using the DHARMa package [30].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAmong the five behaviours, hiding under refuge (UR) was most frequently observed (mean accumulative number of snails per tank = 22.0), followed by travelling around (TA; 14.2), retreated in shell (IS; 6.6), and scanning the environment (SE; 6.2) (Figure 1). Few snails emerged from the water (EW; 0.6) and thus this behaviour was excluded from subsequent analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor hiding under refuge (UR), we found that the predator treatment significantly increased UR behaviour compared to the control (Figure 2a; Table 1; p \u0026lt; 0.01). \u0026nbsp;Both treatments with dead/injured snails (i.e., Injured snail, Predator + Injured snails) induced similar number of snails exhibiting UR behaviour as the control. We found that the number of snails that travelled around (TA) was significantly lower in predator treatment (Figure 2b; p \u0026lt; 0.01), while it was similar between the control and other treatments. The number of snails retreated in shell (IS) and scanning the environment (SE) did not differ across treatments (Figure 2c, 2d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e Estimated effects of four treatments on the number of snails (Sulcospira hainanensis) exhibiting different behaviours in response to the chemical cue of big-headed turtles (Platysternon megacephalum) and injured conspecifics analysed using generalised linear mixed models. The control treatment was used as the reference level. Bold rows indicate significant effects.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"596\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 302px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Treatments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003eEstimate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eSE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003ez\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003ep\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 596px;\"\u003e\n \u003cp\u003e\u003cem\u003eHiding under refuge (UR)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 302px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Injured snail (vs control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 302px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;\u003cstrong\u003ePredator (vs control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.55\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.97\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e0.0\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 302px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Predator + Injured snails (vs control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 596px;\"\u003e\n \u003cp\u003e\u003cem\u003eTravelling around (TA)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 302px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Injured snail (vs control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e-0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e-0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 302px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;Predator (vs control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.92\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.31\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-2.96\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e0.0\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 302px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Predator + Injured snails (vs control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e-0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e-0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 596px;\"\u003e\n \u003cp\u003e\u003cem\u003eScanning the environment (SE)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 302px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Injured snail (vs control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 302px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Predator (vs control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e-0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e-0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 302px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Predator + Injured snails (vs control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e-0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e-0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 596px;\"\u003e\n \u003cp\u003e\u003cem\u003eRetreated in shell (IS)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 302px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Injured snail (vs control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e-0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e-1.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 302px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Predator (vs control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 302px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Predator + Injured snails (vs control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we found that the freshwater snail, \u003cem\u003eS.\u003c/em\u003e\u003cem\u003e\u0026nbsp;hainanensis\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003eexhibited antipredator behaviour when exposed to the chemical cues of the big-headed turtle (\u003cem\u003eP. megacephalum\u003c/em\u003e), primarily by moving under refuge, although the snail\u0026rsquo;s behaviours did not vary substantially in the presence of dead/injured conspecifics. We discuss these findings and implications for conservation below.\u003c/p\u003e\n\u003cp\u003eAs a common prey to various kinds of predators \u0026mdash; including freshwater turtles [22], [31], [32] \u0026mdash; snails adopt a range of antipredator behaviours, with hiding in substratum and escaping above the water\u0026rsquo;s surface being the more frequently observed behaviours [33], [34], [35]. The expression of these behaviours can be influenced by different factors. For example, predator type affects whether the snails hide near the water\u0026rsquo;s surface or in the benthic substrate [27], [35]. Previous studies on the freshwater snails \u003cem\u003ePlanorbella campanulata\u003c/em\u003e, \u003cem\u003ePomacea paludosa\u003c/em\u003e and \u003cem\u003ePo. canaliculata\u0026nbsp;\u003c/em\u003eshowed that the snails tend to hide in the benthic substrate when exposed to chemical cues from turtles [27], [36], [37], potentially because burying into the substrate is an efficient method for avoiding turtles which hunt visually [15], [37]. In this experiment, we used \u003cem\u003eS. hainanensis\u003c/em\u003e which was frequently preyed upon by \u003cem\u003eP. megacephalum\u003c/em\u003e [22]. Like other freshwater snails, \u003cem\u003eS. hainanensis\u003c/em\u003e adopted a similar antipredator behaviour by hiding under refuge (UR) in the presence of turtle predators (Figure 2a).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSnails also adapt their hiding behaviour according to the availability of benthic refuge. When refuge such as sand or vegetation is present, alarmed snails hide by burying themselves or hide among the vegetation [27], [36], [38]. However, when benthic refuge is not available, snails will instead move out of water [39] or into an area that is inaccessible by the predator [15]. In this experiment, a tile was provided for \u003cem\u003eS. hainanensis\u003c/em\u003e to hide at the bottom of the tank and the snails of the predator treatment were found to use this benthic refuge more frequently (Figure 2a). This behaviour is beneficial to the snail\u0026rsquo;s survival in Hong Kong hill streams, where rock crevices on stream bottom provide many avenues for escape because cobbles and boulders are abundant and persistently available year-round (~ 60% in wet season and ~ 70% in dry season) [40]. This may also explain why wild \u003cem\u003eS. hainanensis\u0026nbsp;\u003c/em\u003edetach from rock surfaces to drop into the\u0026nbsp;benthic rock crevices when subject to strong taps, which they may perceive as a predatory risk. Similar drop-and-escape behaviour was observed by DeWitt et al. [36] when snails were disturbed by turtles.\u003c/p\u003e\n\u003cp\u003ePrevious studies have showed that, beside the chemical cues of predators, snails also respond to the chemical cues of injured conspecifics [26], [27], [34], [41]. However, we found that \u003cem\u003eS. hainanensis\u003c/em\u003e behaves similarly when comparing the control and injured snail treatments (Figure 2a-d). This suggests that \u003cem\u003eS. hainanensis\u003c/em\u003e do not respond to the chemical cues of injured snails. Alternatively, our protocol for mimicking the presence of dead or injured conspecifics may not be sufficiently stimulating. For the preparation of injured snail cues, Hayashi and Sugiura [26] and Turner et al. [41] crushed live snails to obtain injured snail cues, while McCarthy and Fisher [34] and Ueshima and Yusa [27] did not specify whether live snails were used. If the snails were alive during cue preparation, they would likely have released alarm pheromones, which could trigger antipredator behaviour in conspecifics [42]. In our study, however, \u003cem\u003eS. hainanensis\u0026nbsp;\u003c/em\u003ewere frozen intact and stored at -20 \u0026deg;C until they were needed for experiment. Park and Sung [43] highlighted that the olfactory cues of predators (i.e., ventral gland excretion) should be used within a specific timeframe: stored at 4 \u0026deg;C within four days or at -20 \u0026deg;C for up to eight days. In this study, the snails were stored at -20 \u0026deg;C but not all were used within eight days. As our results are inconclusive, we propose that future studies should incorporate the use of live snails to trigger the release of alarm pheromones, and that experimental subjects should be exposed to extracted chemical cues as quickly as possible after preparation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUnder undisturbed condition, the high density of \u003cem\u003eP. megacephalum\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003elikely regulates the population density of \u003cem\u003eS. hainanensis\u003c/em\u003e. However, conducting empirical studies to test this in the field is challenging, because populations of Asian freshwater turtles are scarce [44]. Taking a different approach, we documented the influence of \u003cem\u003eP. megacephalum\u003c/em\u003e on the behaviour of \u003cem\u003eS. hainanensis\u003c/em\u003e, the dominant grazer in these streams. Our results show that \u003cem\u003eS. hainanensis\u003c/em\u003e exhibit an antipredator response to the chemical cues of \u003cem\u003eP. megacephalum\u003c/em\u003e in the form of seeking refuge. This behaviour may affect how snails utilise resources in hill stream ecosystems. For instance, upon detecting \u003cem\u003eP. megacephalum\u003c/em\u003e, the snails are more likely to occupy covered microhabitats, such as rock crevices, and therefore consume more resources in that area [12], [45]. Further, a reduction of resource consumption by snails in open, uncovered area may free up more resources for other grazing macroinvertebrates [23]. This warrants further studies on the cascading impacts of \u003cem\u003eP. megacephalum\u003c/em\u003e on ecological processes and community structure in freshwater streams, if robust populations of \u003cem\u003eP. megacephalum\u003c/em\u003e can be identified [7], [16], [46], [47].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur results highlight the ecological consequences of the population extirpation of Asian freshwater turtles. Where Asian freshwater turtle populations persist, there is an urgent need for more studies on their ecological interactions to deepen our understanding of their ecological roles to inform efforts in environmental education and turtle conservation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eWe confirm that this manuscript has not been published elsewhere and is not under consideration by another journal. All authors have approved the manuscript and agree with its submission to Discover Animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDeclaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Lingnan University (Faculty Research Grant; project code:102194).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe methods used in this study were approved by the Department of Health [Permit number: (20-51) in DH/HT\u0026amp;A/8/2/8 Pt.1] and the Agriculture, Fisheries and Conservation Department [Permit number: (95) in AF GR CON 09/51 Pt. 8] of the Hong Kong Special Administrative Region Government, China.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate Declaration\u003c/strong\u003e: not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish Declaration\u003c/strong\u003e: not applicable\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest that could have appeared to influence the work reported in this paper. \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.W.L.F. and Y.H.S. acquired funding and wrote the main manuscript text. A.W.L.F. implemented the experiment and collected the data. A.W.L.F., J.H.L., and Y.H.S. assessed the accuracy of the data analysis methods, validated the methodology, and reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe are grateful to the Agriculture, Fisheries and Conservation Department, Hong Kong SAR Government for granting the research permit, and Lingnan University (Faculty Research Grant; project code:102194) for providing research grants. Special thanks are due to Dr. LAU Michael Wai Ning and Dr. FONG Jonathon for their insightful guidance, which significantly improved the quality of this manuscript. Furthermore, we express our gratitude to the supporting team members, CHAN Man Ho Henry, LAM Pui Yin Ivan, LAM Yu Ki Billy, LEE Wing-Him Henry, LEUNG Julia, LEUNG Ka Wah Franco, LIU Hua Bin, SO Halbert, TAN Kai Teck Desmond, YIP Hon Tim, YIP Wing Yan Gena and YUEN Hiu Ching Hilda who contributed to various capacities, including field assistance, animal care and data collection.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the supplementary information file.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJ. E. Lovich, J. R. Ennen, M. Agha, and J. W. Gibbons, \u0026ldquo;Where Have All the Turtles Gone, and Why Does It Matter?,\u0026rdquo; \u003cem\u003eBioScience\u003c/em\u003e, vol. 68, pp. 771\u0026ndash;778, Sept. 2018, doi: https://doi.org/10.1093/biosci/biy095.\u003c/li\u003e\n\u003cli\u003eJ. W. Gibbons and J. E. 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Sung, \u0026ldquo;Male \u003cem\u003eHynobius leechii\u003c/em\u003e (amphibia: Hynobiidae) discriminate female reproductive states based on chemical cues,\u0026rdquo; \u003cem\u003eIntegrative Biosciences\u003c/em\u003e, vol. 10, no. 3, pp. 137\u0026ndash;143, 2006, doi: 10.1080/17386357.2006.9647295.\u003c/li\u003e\n\u003cli\u003eT. D. Richardson and K. M. Brown, \u0026ldquo;Predation risk and feeding in an intertidal predatory snail,\u0026rdquo; \u003cem\u003eJournal of Experimental Marine Biology and Ecology\u003c/em\u003e, vol. 163, no. 2, pp. 169\u0026ndash;182, Nov. 1992, doi: 10.1016/0022-0981(92)90047-E.\u003c/li\u003e\n\u003cli\u003eA. M. Turner, R. J. Bernot, and C. M. 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Vanni, \u0026ldquo;Nutrient cycling by animals in freshwater ecosystems,\u0026rdquo; \u003cem\u003eAnnual Review of Ecology and Systematics\u003c/em\u003e, vol. 33, pp. 341\u0026ndash;370, 2002, doi: https://doi.org/10.1146/annurev.ecolsys.33.010802.150519.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-animals","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Animals](https://link.springer.com/journal/44338)","snPcode":"44338","submissionUrl":"https://submission.springernature.com/new-submission/44338/3","title":"Discover Animals","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Molluscs, stream, trophic cascades, Asian turtles, pheromones","lastPublishedDoi":"10.21203/rs.3.rs-7478776/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7478776/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePredators can trigger antipredator behaviour in their prey, leading to a cascading effect on community composition and nutrient cycling. The big-headed turtle (\u003cem\u003ePlatysternon megacephalum\u003c/em\u003e) frequently predates on freshwater snails (\u003cem\u003eSulcospira hainanensis\u003c/em\u003e) in Hong Kong hill streams. This study aims to detect and measure the antipredator behaviour of \u003cem\u003eS. hainanensis\u003c/em\u003e in response to chemical (e.g., olfactory) signals from \u003cem\u003eP. megacephalum\u003c/em\u003e and dead conspecifics. The snails were subject to four treatments: 1) no chemical signals (control), 2) chemical cues from dead conspecifics, 3) chemical cues from predators (turtle), and 4) combination of chemical cues from dead conspecifics and predators. Chemical cues from predators were the only treatment triggered a significant prey response in which they hid under a refuge. The wild population of \u003cem\u003eP. megacephalum\u003c/em\u003e has declined because of poaching. Our findings suggest that this may lead to cascading effect on the stream ecosystem (e.g., level of primary productivity) via a weakened prey response in snails.\u003c/p\u003e","manuscriptTitle":"Antipredator behaviour of native freshwater snails (Sulcospira hainanensis) against the critically endangered big-headed turtle (Platysternon megacephalum)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-24 08:37:31","doi":"10.21203/rs.3.rs-7478776/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-26T13:30:46+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"314280413340103042411315906139639565300","date":"2025-10-20T12:32:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-14T21:48:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-07T14:14:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55708200991357111252273345257120501781","date":"2025-10-07T11:23:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60922178302891385645399969841572872403","date":"2025-09-26T13:49:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-16T00:22:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-09T16:17:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-09T08:47:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-09T08:46:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Animals","date":"2025-08-28T09:34:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-animals","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Animals](https://link.springer.com/journal/44338)","snPcode":"44338","submissionUrl":"https://submission.springernature.com/new-submission/44338/3","title":"Discover Animals","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"030b8d29-df1d-488a-bff3-3a2509aca7b9","owner":[],"postedDate":"September 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-25T08:38:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-24 08:37:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7478776","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7478776","identity":"rs-7478776","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}