Field warming increases body size of nocturnal slugs by extending their daily activity time in an alpine meadow

Field warming increases body size of nocturnal slugs by extending their daily activity time in an alpine meadow | 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 Field warming increases body size of nocturnal slugs by extending their daily activity time in an alpine meadow Xingyu Zhou, Xiaoli Hu, Ying Feng, Shuang Xiang, Youjun Chen, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8198076/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 Although global warming is known to shift animal phenology, its effects on daily activity remain poorly understood. We used long-term field warming with large open-top chambers (15 × 15 × 2.5 m), which increased mean annual air temperature by 0.42°C from 2017–2023, to test whether warming alters daily activity time and body size of the nocturnal slug Agriolimax agrestis in an alpine meadow on the eastern Tibetan Plateau. Warming increased slug activity time by 37.17 min (+ 24.86%) in 2022 and 49.55 min (+ 29.16%) in 2023, and increased body size by 64.51% and 57.11%, respectively, without changing growth phenology. A complementary short-term experiment using small open-top chambers (2 × 2 × 2 m) elevated temperature by 0.44°C and similarly increased activity time by 180.18% and body size by 35.63% under controlled feeding. Slug abundance increased by ca.500% in warmed plots from 2019 to 2023, likely driven by enhanced body size and survival. Overall, warming substantially extends daily activity time, thereby enlarging slug body size and promoting population growth, highlighting an overlooked mechanism by which climate warming affects animal traits and demographics in cold regions. alpine meadow body size daily activity rhythm invertebrates simulated warming Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction One of the important consequences of global warming (IPCC 2022 ) is the change in the timing and duration of animal activities. Global warming is reported to advance the emergence of frogs (Gibbs and Breisch 2001 ) and the migration and breeding timing of birds in temperate regions (Both et al. 2004 ). Moreover, these changes may further affect species traits, such as animal body size by altering growth and development rates (Atkinson 1994 ; Ohlberger 2013 ). However, current studies predominantly focus on the impact of climate warming on phenology, the seasonal activity patterns of organisms, while empirical evidence regarding the consequences of warming for the daily activities of animals and their associated consequences is notably scarce (Hut et al. 2012 ; Rafiq et al. 2023 ). More importantly, animal activities at night have particularly been rarely investigated in relation to climate change (Gaston 2019 ; Kolioulis et al. 2024 ). Most organisms exhibit daily activity patterns (i.e., the distribution of activity and resting time over a 24-hour cycle), which represent a crucial adaptation to periodic environmental factors such as photoperiod and temperature (Danilevsky et al. 1970 ; Prabhakaran and Sheeba 2014 ; Goto 2022 ). Climate warming may influence the activity patterns of animals, especially for ectotherms, as temperature serves as a zeitgebe r for ectotherms, directly altering the phase of their daily activity pattern (Danilevsky et al. 1970 ; Nekhaeva et al. 2024 ). Studies have mostly used modelling and laboratory approaches to investigate the effect of temperature increase on daily animal activity patterns and growth. For example, using the niche mapper model, Huang et al. ( 2013 ) predicted that high temperatures would increase the activity duration of the high-altitude pit viper ( Trimeresurus gracilis ). Moreover, using biophysical modelling, Kearney et al. ( 2009 ) reported that warming would limit the activity duration of ectotherms in tropical and desert regions, but tend to extend the activity duration of ectotherms in temperate regions, where ectotherms frequently experience torpor to conserve energy (Borkataki et al. 2021 ; Auteri 2022 ). Consistently, laboratory studies show that ectotherms are sensitive to rising temperatures in their daily activity patterns, primarily due to their limited thermoregulatory capacity (Angilletta 2009 ; Paaijmans et al. 2013 ; Verheyen and Stoks 2019 ). For example, Fraser et al. ( 1993 ) observed that juvenile Atlantic salmon ( Salmo salar ) predominantly foraged during the day when temperatures were above 10°C, but switched to nocturnal foraging when temperatures fell below 10°C. Similarly, through laboratory experiments, Vermunt et al. ( 2014 ) demonstrated that tuatara ( Sphenodon punctatus ) exhibited a combined diurnal-nocturnal activity pattern at 20°C and 12°C, but shifted to strictly diurnal activity pattern at a lower temperature of 5°C. Even for endotherm, for example, the rodent Phyllotis xanthopygus was found to significantly reduce its activity time when exposed to high temperatures under controlled laboratory conditions, (Sassi et al. 2015 ). However, there are few empirical field experiments addressing the effects of temperature on animal daily activity patterns and associated ecological consequences (Comeault et al. 2021; Everling et al. 2022). The lack is important, because lab experiments may distort the natural behavior and physiological responses of animal individuals (Calisi and Bentley 2009 ). For instance, birds reared under laboratory conditions no longer exhibit reproductive cycles regulated by photoperiod as being the case for wild populations (Dawson et al. 2001). In contrast, even in semi-natural experiments (e.g., open-top chambers), animals are allowed to be exposed to natural settings, although some key environmental variables are partially controlled (Calisi and Bentley 2009 ). To address this knowledge gap, we utilized an established and well-maintained field warming platform (large open top chambers; Hu et al. 2020 , 2024 ) to examine the effects of simulated warming on the daily activity and associated ecological consequences of nocturnal slug species ( Agriolimax agrestis ) in an alpine meadow on the eastern part of the Tibetan Plateau. We investigated the responses of the slug daily activity time, body size, and species abundance to the experimental warming. We also investigated the responses of slug activity phenology and food resource preferences to the experimental warming in order to assess to what extent the warming effect on slug daily activity time may contribute to changes in body size and species abundance. In addition, we conducted a complementary field warming experiment using small open-top chambers to test whether simulated warming could directly change slug activity time and body size. Given that slug activity predominantly occurs at night and that the climatic conditions are cold and moist in the alpine meadow, we predict that experimental warming will increase slug activity time at night, potentially leading to an increase in body size. Additionally, as reproductive potential and fecundity are often associated with body size (Honěk 1993 ; Kingsolver and Huey 2008 ), we expected an increase in species abundance, unless warming sufficiently reduces the plant resources available to the slugs. Materials and methods Study site This study was conducted in an alpine meadow in Hongyuan County, Sichuan Province, China (32°48’N, 102°33’E), on the eastern edge of the Tibetan Plateau at an elevation of 3,500 m above sea level. Meteorological data from the Hongyuan climatological station from 1970 to 2023 indicated that the mean annual temperature is 1.7°C and the mean annual precipitation is 756 mm (ranging from 450 mm to 900 mm), with the majority falling from May to September (Hu et al. 2024 ). According to the meteorological data, the mean annual temperature has increased by 0.29°C per decade, with the growing season (May to September) and non-growing season (October to April) temperature increases of 0.26°C and 0.36°C per decade, respectively. The alpine meadow has been intensively grazed by livestock, mainly domestic yaks ( Bos grunniens ) and Hequ horses ( Equus caballus ). Our experiments were conducted in the winter pasture, where grazing is permitted from October to April. The total vegetation coverage of the meadow is over 90%, primarily composed of forbs (e.g., Saussurea nigrescens , Polygonum viviparum , and Potentilla anserine ), sedges (e.g., Kobresia setchwanensis and Carex spp.), and grasses (e.g., Elymus nutans and Deschampsia caespitos ) (Hu et al. 2021 ). In addition to the high diversity of plant species (Xiang et al. 2009 ; Hu et al. 2020 ), arthropods (Wu et al. 2011 ; Xi et al. 2013 ; Hu et al. 2019 ) are also very abundant in the meadow. The study species The slug Agriolimax agrestis is a species of gastropod mollusks, considered a pest in pastures (Baur and Baur 1993 ; Sternberg 2000 ). The slugs, similar to other slug species (Barker 1991 ), display predominantly nocturnal activity and remain inactive during the daytime under intense light conditions. The average field density at the study site is 0.3 to 0.5 individuals m − 2 . As generalist feeders, they consume a wide variety of forb species. They are visibly active from mid-June until the end of September. The slugs lay eggs in September during the late growing season, and the eggs overwinter in a quasi-dormant state until temperatures rise in late May or early June. Typically, eggs are deposited in moist and sheltered locations, such as soil crevices or leaf litter, which provide insulation and protection. The large-scale field warming experiment Six 15 m × 15 m × 2.5 m (height) open-top chambers (OTCs) were randomly constructed in a fenced 1.0 ha winter pasture in October 2014. The sides of the OTCs were covered by thin steel screen. Three chambers were additionally covered with transparent tempered glass, and the roofs were discontinuously covered with 1.5 m × 0.3 m (width) transparent glass strips. Steel screen (with a mesh size of 0.6 mm × 0.6 mm) was also placed 1 m into the soil to prevent rodents from entering. Three OTCs with transparent tempered glass are referred to as “warmed chambers” and the other three chambers as “non-warmed chambers” (for more details, see Hu et al. 2020 , 2021 ). The climate measurement data logger indicated that the mean annual temperature was 5.23°C and 4.81°C at 30 cm aboveground in the warmed and non-warmed chambers, respectively, from 2017 to 2023, with a 0.24°C increase during the growing season and a 0.55°C increase in the non-growing season. Moreover, from 2022 to 2023, when the daily activity time was investigated for the slugs, the experimental warming increased nighttime temperatures by 0.94°C (19:00–6:00) and daytime temperatures by 0.01°C (6:00–19:00). Such asymmetric warming was consistent with observations and the model predictions for about two to three decades later in the study site according to the SSP1–1.9 scenarios (IPCC 2022 ). The vapor pressure deficit was 0.4% to 0.8% higher in the warmed chambers compared to the non-warmed chambers. Additionally, the soil moisture at 5 cm was 20.2% v/v on average in the warmed chambers and 22.4% v/v in the non-warmed chambers during the growing season. Body size and growth phenology We used fresh body weight to measure size. During the growing season (June to September), five to ten individuals were randomly selected from each chamber and weighed in situ with an electronic balance (sartorius BS224S, 10 − 4 g) every 10 days for two consecutive years (2022–2023). All individuals were released after weighing. Growth phenology was defined as the day when slug body size reached 5 mg (the “starting” time of growth) and the day when slug body size reached 90% of the maximum body size (the “ending” time of growth). To reduce the variability among individual observations, we calculated the phenological time by fitting a logistic regression of body size versus collection day for each chamber for each year. Logistic regression was performed using the nls function in R as follows (Kaufmann 1981 ), $$\:\text{W}\text{e}\text{i}\text{g}\text{h}\text{t}=\frac{Asym}{1+exp\left[\left(xmid-day\right)/scal\right]},$$ where Asym is the maximum weight, xmid is the time when weight reaches half of the maximum weight (i.e., xmid = Asym/2), and scal is the growth rate. All such regressions had statistically significant fits ( P < 0.001; see Supporting information: Table S1 ). Using the regression equation, we calculated the starting and ending times for slug growth phenology. Daily activity Daily activities were investigated every 10 to 20 days, and five investigations were conducted for each year. Each observed individual was classified as either “active” or “inactive” (including states of chill-coma (ceased movement due to cold), resting (lack of visible activity driven by their activity patterns and/or endogenic circadian rhythms), or torpor (decreased physiological activities and metabolic rate due to due to unfavorable external conditions) based on their behavior (Eban-Rothschild and Bloch 2008 ; MacMillan and Sinclair 2011 ; Borkataki et al. 2021 ). Slugs were categorized as “active” when they extended their bodies and moved slowly and continuously, with tentacles fully extended and oscillating to explore their surroundings, leaving visible mucus trails (South 1992 ). This behavior was often associated with activities such as foraging. In contrast, slugs were classified as “inactive” when they remained motionless in a fixed position, with their bodies shortened or curled into a droplet-like shape and their tentacles retracted or immobile (South 1992 ). Due to the slugs' limited range of movement, their activities were often confined to a single plant for multiple hours. We flagged the plants with red sticks placed beside them and recorded both the number of “active” and “inactive” individuals hourly. Prior investigations showed that the slugs were inactive in high-light daytime but had two activity periods. One ranged between 19:00 and 22:00 (7–10 pm; Beijing Time) at night, and the other between 6:30 and 9:30 in the morning. (am) Thus, we started our number recording at 6:00 in the morning and 18:00 at night on each observation day. The daily activity time was calculated by summing the products of the proportion of active individuals and the observation interval (60 min) for each observation day. Species abundance Slug abundance was investigated from early July to early August (the period of highest abundance in the year) for each year (2017 to 2023) using a quadrat sampling method. Specifically, twenty 50 cm × 50 cm quadrats were randomly selected in each chamber when the slugs were active (7:00–9:00). Then, we inspected the vegetation to record all visible slugs. Slug diet Slug diet was investigated during the growing season in 2022. During the investigation of slug activities, we recorded the species identity of plants that the slugs were feeding on. Slug feeding was observed using magnifiers. If the slugs were found scraping off plant surfaces with the radula, tearing plant tissues into smaller pieces, their feeding was assumed to have occurred. The feeding activity was typically slow and rhythmic, leaving distinct scraping marks (usually as irregular holes) on the leaves. In total, we recorded 156 feeding events, and the diet included 29 plant species. Additionally, the dietary plants were categorized into graminoids and forbs. Plant community composition and biomass Plant community composition and aboveground plant biomass were investigated to assess whether the food resource of slugs had changed in response to warming. Specifically, the plant community in each chamber was examined using a quadrat sampling method. In each year, we randomly established twenty 50 × 50 cm quadrats in each chamber. Each quadrat was further subdivided into 5 × 5 cm grids. We then documented species presence and abundance following the protocols described by Hu et al., ( 2021 ). In mid-August of each year when the majority of plant species had completed their reproductive phase, all aboveground plant parts were clipped to the ground surface and harvested in sixteen 50 × 50 cm quadrats that were randomly deployed in each chamber. The harvests were carefully sorted into two functional types (graminoids, including grasses and sedges, and forbs, including the rest of the species), dried at 75°C for 72 h, and then weighed (Hu et al. 2021 ). The small-scale field warming experiment In order to remove any potential confounding factors (e.g., changes in the vegetation structure and animal (potential predators, for example) community induced by warming), we conducted a complementary experiment to determine whether warming per se could change the daily activity time and whether warming could increase the body size of the slugs. In the early August 2024, when the slugs grew rapidly during their lives, fourteen slug individuals with similar body size (ranging between 107.9 mg and 196.2 mg) were reared in a small open top chamber (2 m × 2 m × 2.5 m) built of the same material as the large OTCs, and fourteen individuals were reared outside the chamber under a glass roof supported by two steel pillars. The daily mean temperature was 15.49°C outside and 15.93°C inside the chamber, with an increase of 0.80°C during nighttime (19:00–6:00) and 0.07°C during the daytime (6:00–19:00) (Supporting information: Fig. S2). Each slug was individually placed in a plastic pot (diameter 14.7 cm and depth 9.8 cm) lined with a 2 cm layer of soil, and the top of each pot was covered with a mesh to prevent slugs from escaping but ensure air circulation. Fresh leaves of Saussurea nigrescens (the major food resource of slugs) were added and replaced daily in the pot, and water was misted at midday to maintain suitable humidity. The experiment lasted for 18 days from August 8th 2024 to August 25th 2024. We recorded daily activity time and body size of the slugs using the same method as in the field warming experiment. Data analysis All analyses were performed in R 4.3.1 (R Core Team 2023). The slug diet was visualized using the plotweb function in the bipartite package (v.2.18) (Dormann et al. 2008 ). After testing for the error distribution of response variables, we used generalized linear mixed models (GLMMs) in the lme4 package (Bates et al. 2015 ) with warming as the fixed factor, and chamber identity and year as random factors to determine the warming effect on slug abundance (negative binomial error structure) and the proportion of active individuals (binomial error distribution). Similarly, generalized linear mixed models (GLMMs) with warming as the fixed factor, and chamber identity, date and year as random factors to determine the warming effect the body size of slugs (gamma error with log link function). Linear mixed models (LMMs) were used with warming as the fixed factor, and chamber identity as random effects to determine the warming effect on slug growth phenology (starting time, ending time and growing period), biomass of forbs and slug body size in the small-scale experiment. In addition, general linear model was employed to determine the relationship between the percentage of active slugs and air temperature. The difference between warmed and non-warmed chambers was determined using Fisher-Pitman permutation test in the R package coin (v.1.4.3) (Hothorn et al. 2006 ). In addition, independent sample t-tests were conducted to assess the warming effects on body size in the small-scale experiment. Results The phenology and daily activity time Warming did not significantly change the starting time ((LMM: estimate = − 4.52, F = 4.27, P = 0.11, Fig. 1 a) and the ending time (LMM: estimate = -0.37, F = 0.01, P = 0.91, Fig. 1 a) of slug growth in 2022. Moreover, the difference in slug growing period was indistinguishable between non-warmed (96.37 ± 3.73 days on average) and warmed chambers (100.52 ± 3.51 days; LMM: estimate = 4.15 F = 0.66, P = 0.46). Likewise, warming did not significantly change the starting time ((LMM: estimate = − 2.29, F = 1.19, P = 0.39, Fig. 1 b) and the ending time (LMM: estimate = 4.00, F = 1.71, P = 0.32, Fig. 1 b) of slug growth in 2023. Again, the difference in slug growing period was indistinguishable between non-warmed (100.53 ± 6.71 days on average) and warmed chambers (106.82 ± 10.43 days; LMM: estimate = 6.29, F = 2.69, P = 0.24). In 2022, warming extended the daily activity duration by an average of 28.68 ± 9.42 min (mean ± SE; same for hereafter) at night (GLMM: estimate = 1.46, χ 2 = 17.99, P < 0.001, Fig. 2 a, 2 b) and 8.49 ± 2.43 min in the morning (GLMM: estimate = 0.95, χ 2 = 6.64, P = 0.009, Fig. 2 a, 2 b), respectively, leading to a significant average increase of 37.17 ± 7.64 min per day (a 24.86% increase) in activity time (GLMM: estimate = 1.18, χ 2 = 21.89, P < 0.001, Fig. 2 a, 2 b). Similarly, in 2023, warming extended the daily activity duration by an average of 31.70 ± 24.81 min at night (GLMM: estimate = 1.22, χ 2 = 11.97, P < 0.001, Fig. 2 c, 2 d) and 17.85 ± 9.42 min in the morning (GLMM: estimate = 1.20, χ 2 = 11.13, P < 0.001, Fig. 2 c, 2 d), respectively, leading to a significant average increase of 49.55 ± 25.80 min per day (a 29.16% increase) in activity time (GLMM: estimate = 1.22, χ 2 = 23.63, P < 0.001, Fig. 2 c, 2 d). In addition, the proportion of “active” individuals was positively associated with temperature during the growing season in both 2022 (N = 50, R² = 0.40, P < 0.001) and 2023 (N = 56, R 2 = 0.33, P < 0.001). Slug body size and abundance Experimental warming significantly affected body size of the slugs (GLMM: estimate = 0.52, χ 2 = 152.09, P < 0.001; Fig. 3 ). Specifically, the body size increased significantly by 64.51% in 2022 ( Z = -1.526, P = 0.03, Fig. 3 a) and by 57.11% in 2023 ( Z = -1.552, P = 0.05, Fig. 3 b) at the last measurement. Warming did not significantly change slug abundance in 2017 and 2018, but substantially increased slug abundance by an average of 497.33% from 2019 to 2023 (GLMM: estimate = 0.99, χ 2 = 133.34, P < 0.001, Fig. 4 ). Additionally, the percentage of active slugs was positively associated with temperature across the chambers in both 2022 (N = 50, R 2 = 0.40, P < 0.001) and 2023 (N = 56, R 2 = 0.33, P < 0.001). The diet of slugs The slugs were observed to feed on 19 forb species and 5 graminoid species in warmed chambers, and 14 forb species and 1 graminoid species in non-warmed chambers (Fig. 5 ; more details see Supporting information: Table S2). On average, forbs accounted for 93.17% and 97.92% of the observed dietary interactions between slugs and plants in the warmed and non-warmed chambers, respectively. The difference in the proportion of forbs in the slug diet was negligible between the non-warmed and warmed chambers ( Z = 0.78, P = 0.43). Change in food resource Warming significantly decreased the biomass of forbs (the major food source of slugs) (LMM: estimate = -0.44, F = 86.07, P < 0.001, Supporting information: Fig. S1 ). Specifically, the biomass of forbs significantly decreased by 23.22% in 2022 ( Z = 1.98, P = 0.04, Supporting information: Fig. S1 ) and 15.39% in 2023 ( Z = 1.82, P = 0.03, Supporting information: Fig. S1 ). The small-scale experiment Experimental warming significantly increased the daily activity duration (GLMM: estimate = 1.80, χ 2 = 114.95, P < 0.001; Fig. 6 a, 6 b) by 90.56 ± 19.84 min during the night time ( Z = 2.06, P = 0.02, Fig. 6 a, 6 b) and 42.50 ± 22.88 min ( Z = -1.76, P = 0.04, Fig. 6 a, 6 b) during the daytime. The total daily activity duration was over 2 h longer (+ 180.18%) in the warmed chambers than in the non-warmed chambers (Fig. 6 b). Moreover, warming had a significant effect on slug body size (LMM: estimate = 39.53, F = 4.88, P = 0.03; Fig. 6 c), with a 35.63% increase at the final measurement (t-test: t = 1.86, P = 0.03; Fig. 6 c). Discussion Here, we show that the field warming did not change the phenology of slug growth, but it significantly increased the daily activity time by > 20% during the growth period. Moreover, experimental warming significantly increased the body size of slugs by more than 70% and the slug abundance by more than three times in the last years of the warming experiment. The increased slug body size and abundance cannot be accounted for by the change in the food resource, because the food resource (primarily forbs) for the slugs was significantly lower in the warmed chamber compared to the non-warmed ones. Importantly, the complementary small-scale experiment explicitly suggests that warming per se may increase slug body size by increasing activity time. Thus, it can be concluded that a warming-induced increase in daily activity time (and associated increased resource intake of slug individuals) could have reasonably increased the body size and abundance of the slugs in the field warming experiment. To the best of our knowledge, this is one of the first studies experimentally demonstrating that global warming may significantly change daily rhythms of animals, which can have consequences for species' body size and abundance. Experimental warming often reduces individual body size across diverse taxa including both endotherms (Tabh et al. 2025 ) and ectotherms (Zhao et al. 2014 ; Xi et al. 2016 ), presumably because increased temperatures increase body development rate more than growth rate. However, there are also many studies reporting an increase in body size under experimental warming (Yom-Tov et al. 2008 ; Ozgul et al. 2010 ), and they often attribute the increase to the extension of the growing season (Menzel and Fabian 1999 ). A pertinent example is the study by Ozgul et al. ( 2010 ), which reported that the earlier emergence from hibernation in the yellow-bellied marmot resulted in increased body masses due to the extended growing season. Similarly, Yom-Tov et al. ( 2008 ) found that American martens ( Martes americana ) significantly increased their body size in the latter half of the 20th century, and they attributed the finding to the climate warming-induced extension in growing season (over 14 days). However, in our case, the growth period was unchanged by the experimental warming. By contrast, the increase in the slug body size may be attributed to the warming-induced increase in slug daily activity duration. In this study, the two activity periods of slugs are apparently interrupted by the state of “inactivity” due to low temperature. At our study site, where the annual mean temperature is below 2°C, with mean temperatures falling below 0°C for over 260 days annually (Hu et al. 2020 ; Hu et al. 2021 ), the daily activities of ectotherms in these regions likely depend on temperature fluctuations. More specifically, during slug growth (from mid-June to late September), the daily variation in temperature ranges from − 8.18°C to 39.84°C, and even on the hottest day of August of 2022, the lowest temperatures went down to 3.68°C. Therefore, it is not surprising that the experimental warming significantly delayed the ending time of slug activity at night and advanced the beginning time of slug activity in the morning, thereby extending the duration of slug activity significantly. Consistently, slug activity time increased with increasing temperatures across the chambers in both study years. Given that the slugs were at the growth stage during our observations and hence they spent little time mating and reproducing, it can be speculated that the foraging duration would increase with increasing activity time, which may further lead to the increase in body size. The warming-induced increase in slug body size can be theoretically attributed to other factors such as increased food availability and quality. For example, Yom-Tov and Yom-Tov ( 2005 ) documented an increase in the body size of masked shrews Sorex cinereus in Alaska during the latter half of the twentieth century, and they associated it with increased food availability due to climate warming. However, our experimental warming has resulted in a significant decrease in the biomass of forbs (the preferred food source for slugs) since 2018, while increasing the biomass of graminoids (See Supporting information: Fig. S1 , Hu et al. 2021 ). This coincided with an elevated frequency of slugs feeding on graminoids under warming conditions. Moreover, leaves of graminoids are lower in water content and higher in C/N compared to forbs (Lan et al. 2021 ), indicating that slug food quality also decreased in the warmed chambers. Additionally, no predation and parasitism (due to infestation/inflection) events on slugs were observed throughout the experimental period in both warmed and non-warmed chambers, primarily because there were no large predators foraging on the ground during their activity and because parasites (pathogens) were not abundant due to low temperature in the study site. Consequently, our data indicates that warming did not indirectly increase slug body size by increasing food resources and reducing predation/parasitism. It is also worth noting that relative humidity is suggested to be important for slug growth (Dainton 1954 ; South 1992 ; Willis et al. 2008 ). Nevertheless, in this study, the relative humidity was high (mostly above 65% during our observations) in both treatments, and moreover, the vapor pressure deficit was only slightly higher (by ca. 3%) in the warmed than in non-warmed chambers; hence, this minor difference is not likely to have significantly affected the growth rates of slugs. Consistently, the complementary small-scale experiment demonstrates that warming but not other factors increased the activity time of slugs and further increased their body size. It is not surprising that the abundance of alpine slugs significantly increased under warming conditions, given that larger slug individuals are likely to have greater fecundity (Montresor et al. 2012 ) and their eggs can have a higher survival rate in winter. Indeed, studies have shown that enhanced fecundity and higher survival rates under warming conditions can lead to an increase in species abundance in the salamander Hynobius tokyoensis (Kingsolver and Huey 2008 ; Okamiya et al. 2021 ). Despite these insights, the exact mechanisms driving the increase in slug population abundance remain unclear and require further experimental exploration for a better understanding. It is worth noting that active slugs were more likely to be captured by eyes than “inactive” slugs, particularly at the early stage of investigations, and thus the activity time of slugs might have been overestimated. Nevertheless, we conducted the investigations with similar methods in both treatments, allowing the comparison of slug activity time to be possible. In summary, our data shows that warming enhances the daily activity duration of slugs, thereby increasing their body size and species abundance. Importantly, our study provides an underexplored mechanism underlying warming-induced changes in animal individual size, an important species trait governing species interactions and ecosystem properties. Thus, future research should extend beyond the impact of temperature on phenology and animal physiological metabolism to consider the daily activities of animals. In addition, this study reveals that the nocturnal slugs increased their body size primarily by increasing their activity time. This suggests the importance of studying animal night ecology under climate change (Kolioulis et al. 2024 ). Declarations Acknowledgments We thank Z. Huang and S. He for field and laboratory assistance, and the Sichuan Zoige Alpine Wetland Ecosystem National Observation and Research Station for platform support. Declarations of potential conflict of interests Funding: This work was supported by the National Science Foundation of China (32301391 and 32071605). N.E. acknowledges funding by the German Research Foundation (DFG‚ FZT- 118, 202548816; Ei 862/29-1). Conflicts of interest: The authors declare no competing interests. Ethics approval: Not applicable. Consent to Participate : Not applicable. Consent for publication : Not applicable. Availability of data and materials : The data that support the findings of this study are openly available in Dryad Digital Repository after final acceptance. Declaration of authorship Author contributions: Xingyu Zhou : investigation, formal analysis, writing - original draft, writing - review and editing. Xiaoli Hu : investigation, formal analysis, writing - original draft, writing - review and editing. Ying Feng : investigation. Shuang Xiang : investigation. Youjun Chen : investigation. Yuxia Zhang : investigation. Nico Eisenhauer : writing - review and editing. Shucun Sun : conceptualization, project administration, supervision, writing - review and editing. 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Ecol. 48: 423-434 doi: 10.1007/s10452-014-9495-y Supplementary Files AppendixS1.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 26 Jan, 2026 Reviewers invited by journal 23 Dec, 2025 Editor assigned by journal 26 Nov, 2025 First submitted to journal 24 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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07:28:12","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146387,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8198076/v1/60d14870d55c4a445e02b59b.html"},{"id":99311868,"identity":"2ba5b296-7338-454d-8e9e-630f1857448b","added_by":"auto","created_at":"2025-12-31 16:17:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":41785,"visible":true,"origin":"","legend":"\u003cp\u003eThe starting time (a) and ending time (b) of the slug growth as affected by experimental warming in 2022 and 2023.Warming effects among different years were determined by Fisher–Pitman permutation tests. \u003cem\u003ens\u003c/em\u003e, non-significant. The statistical parameters (\u003cem\u003eZ\u003c/em\u003eand \u003cem\u003eP\u003c/em\u003e values) are shown in Supporting information: Table S2.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8198076/v1/3ffe4fd2f17a0c496d90ba60.png"},{"id":98982731,"identity":"d1c47f74-843b-452d-abc0-cf6f3b49cc5c","added_by":"auto","created_at":"2025-12-25 07:28:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":78260,"visible":true,"origin":"","legend":"\u003cp\u003eThe daily activity of the slugs as affected by experimental warming in 2022 (a) and 2023 (b). Linear mixed models with warming as a fixed effect and chamber identity as a random effect were used for statistical analysis. The bars represent the standard errors. ns, non-significant; * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. The statistical parameters (\u003cem\u003et\u003c/em\u003e and \u003cem\u003eP\u003c/em\u003e value) are shown in Supporting information: Table S4.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8198076/v1/2f4d5df3cd3d9456ba225c28.png"},{"id":99312944,"identity":"e44060f3-525d-46db-bf04-8b14055dd80c","added_by":"auto","created_at":"2025-12-31 16:19:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":70543,"visible":true,"origin":"","legend":"\u003cp\u003eThe body size of the slugs during experimental period as affected by experimental warming in 2022 (a) and 2023 (b).Generalized linear mixed models with warming as a fixed effect and chamber identity as a random effect were used for statistical analysis. The bars represent the standard errors. * \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05. The statistical parameters (\u003cem\u003eZ\u003c/em\u003eand \u003cem\u003eP\u003c/em\u003e value) are shown in Supporting information: Table S4.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8198076/v1/dad727a41e1b58586d189ace.png"},{"id":99312522,"identity":"cb4edf47-8601-4ca9-82a3-2a06c357c7ce","added_by":"auto","created_at":"2025-12-31 16:19:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":56435,"visible":true,"origin":"","legend":"\u003cp\u003eThe species abundance of the slugs as affected by experimental warming during experimental period from 2017 to 2023. Generalized linear mixed models with warming as a fixed effect and chamber identity as a random effect was used for statistical analysis. The bars represent the standard errors. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. The statistical parameters (\u003cem\u003eZ\u003c/em\u003e and \u003cem\u003eP\u003c/em\u003e value) are shown in Supporting information: Table S4.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8198076/v1/de611adb6b081c740182f6ca.png"},{"id":99311641,"identity":"ff341e1f-17aa-4adf-8b00-e86ee7d915d6","added_by":"auto","created_at":"2025-12-31 16:16:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":165238,"visible":true,"origin":"","legend":"\u003cp\u003eThe dietary composition of the slugs in both non-warmed and warmed chambers. The plant species, represented by Arabic numerals and arranged from forbs to graminoids, are listed in Supporting information: Table S2. The width of the green boxes is proportional to the relative abundance of plant species in the diet of the slugs. The color of the boxes indicates the functional type of plants (dark green represents forbs, and light green represents graminoids).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8198076/v1/4c6545e82e4bb5efc2b2eabd.png"},{"id":98982735,"identity":"a6c32e5e-acbf-4e47-b49e-171be5dae5a0","added_by":"auto","created_at":"2025-12-25 07:28:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":50116,"visible":true,"origin":"","legend":"\u003cp\u003eThe body size (a), daily rhythm (b), and daily activity duration (c) of the slugs as affected by warming during complementary small-scale experiment (from August 8\u003csup\u003eth\u003c/sup\u003e 2024 to August 25\u003csup\u003eth\u003c/sup\u003e 2024). The bars represent the standard errors. ns, non-significant; * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. The statistical parameters (\u003cem\u003et\u003c/em\u003e\u003cstrong\u003e/\u003c/strong\u003e\u003cem\u003eZ\u003c/em\u003e and \u003cem\u003eP\u003c/em\u003e value) are shown in Supporting information: Table S4.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8198076/v1/2b3d3998a82d796e488efa92.png"},{"id":99322879,"identity":"2fd40c45-d6e9-4acf-a3e8-14147ae1739a","added_by":"auto","created_at":"2025-12-31 16:44:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1202053,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8198076/v1/51c09bb1-4ecb-47a6-a7c8-c797d24c4bad.pdf"},{"id":98982743,"identity":"2d85185e-f391-4393-ba65-57bacfea01dd","added_by":"auto","created_at":"2025-12-25 07:28:12","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":407091,"visible":true,"origin":"","legend":"","description":"","filename":"AppendixS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8198076/v1/664ee4638a678fb9e43ed967.docx"}],"financialInterests":"","formattedTitle":"Field warming increases body size of nocturnal slugs by extending their daily activity time in an alpine meadow","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOne of the important consequences of global warming (IPCC \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) is the change in the timing and duration of animal activities. Global warming is reported to advance the emergence of frogs (Gibbs and Breisch \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) and the migration and breeding timing of birds in temperate regions (Both et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Moreover, these changes may further affect species traits, such as animal body size by altering growth and development rates (Atkinson \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Ohlberger \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, current studies predominantly focus on the impact of climate warming on phenology, the seasonal activity patterns of organisms, while empirical evidence regarding the consequences of warming for the daily activities of animals and their associated consequences is notably scarce (Hut et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Rafiq et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). More importantly, animal activities at night have particularly been rarely investigated in relation to climate change (Gaston \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kolioulis et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMost organisms exhibit daily activity patterns (i.e., the distribution of activity and resting time over a 24-hour cycle), which represent a crucial adaptation to periodic environmental factors such as photoperiod and temperature (Danilevsky et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Prabhakaran and Sheeba \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Goto \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Climate warming may influence the activity patterns of animals, especially for ectotherms, as temperature serves as a \u003cem\u003ezeitgebe\u003c/em\u003er for ectotherms, directly altering the phase of their daily activity pattern (Danilevsky et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Nekhaeva et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Studies have mostly used modelling and laboratory approaches to investigate the effect of temperature increase on daily animal activity patterns and growth. For example, using the niche mapper model, Huang et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) predicted that high temperatures would increase the activity duration of the high-altitude pit viper (\u003cem\u003eTrimeresurus gracilis\u003c/em\u003e). Moreover, using biophysical modelling, Kearney et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) reported that warming would limit the activity duration of ectotherms in tropical and desert regions, but tend to extend the activity duration of ectotherms in temperate regions, where ectotherms frequently experience torpor to conserve energy (Borkataki et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Auteri \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsistently, laboratory studies show that ectotherms are sensitive to rising temperatures in their daily activity patterns, primarily due to their limited thermoregulatory capacity (Angilletta \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Paaijmans et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Verheyen and Stoks \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For example, Fraser et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) observed that juvenile Atlantic salmon (\u003cem\u003eSalmo salar\u003c/em\u003e) predominantly foraged during the day when temperatures were above 10\u0026deg;C, but switched to nocturnal foraging when temperatures fell below 10\u0026deg;C. Similarly, through laboratory experiments, Vermunt et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) demonstrated that tuatara (\u003cem\u003eSphenodon punctatus\u003c/em\u003e) exhibited a combined diurnal-nocturnal activity pattern at 20\u0026deg;C and 12\u0026deg;C, but shifted to strictly diurnal activity pattern at a lower temperature of 5\u0026deg;C. Even for endotherm, for example, the rodent \u003cem\u003ePhyllotis xanthopygus\u003c/em\u003e was found to significantly reduce its activity time when exposed to high temperatures under controlled laboratory conditions, (Sassi et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, there are few empirical field experiments addressing the effects of temperature on animal daily activity patterns and associated ecological consequences (Comeault et al. 2021; Everling et al. 2022). The lack is important, because lab experiments may distort the natural behavior and physiological responses of animal individuals (Calisi and Bentley \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). For instance, birds reared under laboratory conditions no longer exhibit reproductive cycles regulated by photoperiod as being the case for wild populations (Dawson et al. 2001). In contrast, even in semi-natural experiments (e.g., open-top chambers), animals are allowed to be exposed to natural settings, although some key environmental variables are partially controlled (Calisi and Bentley \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo address this knowledge gap, we utilized an established and well-maintained field warming platform (large open top chambers; Hu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) to examine the effects of simulated warming on the daily activity and associated ecological consequences of nocturnal slug species (\u003cem\u003eAgriolimax agrestis\u003c/em\u003e) in an alpine meadow on the eastern part of the Tibetan Plateau. We investigated the responses of the slug daily activity time, body size, and species abundance to the experimental warming. We also investigated the responses of slug activity phenology and food resource preferences to the experimental warming in order to assess to what extent the warming effect on slug daily activity time may contribute to changes in body size and species abundance. In addition, we conducted a complementary field warming experiment using small open-top chambers to test whether simulated warming could directly change slug activity time and body size. Given that slug activity predominantly occurs at night and that the climatic conditions are cold and moist in the alpine meadow, we predict that experimental warming will increase slug activity time at night, potentially leading to an increase in body size. Additionally, as reproductive potential and fecundity are often associated with body size (Honěk \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Kingsolver and Huey \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), we expected an increase in species abundance, unless warming sufficiently reduces the plant resources available to the slugs.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy site\u003c/h2\u003e \u003cp\u003eThis study was conducted in an alpine meadow in Hongyuan County, Sichuan Province, China (32\u0026deg;48\u0026rsquo;N, 102\u0026deg;33\u0026rsquo;E), on the eastern edge of the Tibetan Plateau at an elevation of 3,500 m above sea level. Meteorological data from the Hongyuan climatological station from 1970 to 2023 indicated that the mean annual temperature is 1.7\u0026deg;C and the mean annual precipitation is 756 mm (ranging from 450 mm to 900 mm), with the majority falling from May to September (Hu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). According to the meteorological data, the mean annual temperature has increased by 0.29\u0026deg;C per decade, with the growing season (May to September) and non-growing season (October to April) temperature increases of 0.26\u0026deg;C and 0.36\u0026deg;C per decade, respectively.\u003c/p\u003e \u003cp\u003eThe alpine meadow has been intensively grazed by livestock, mainly domestic yaks (\u003cem\u003eBos grunniens\u003c/em\u003e) and Hequ horses (\u003cem\u003eEquus caballus\u003c/em\u003e). Our experiments were conducted in the winter pasture, where grazing is permitted from October to April. The total vegetation coverage of the meadow is over 90%, primarily composed of forbs (e.g., \u003cem\u003eSaussurea nigrescens\u003c/em\u003e, \u003cem\u003ePolygonum viviparum\u003c/em\u003e, and \u003cem\u003ePotentilla anserine\u003c/em\u003e), sedges (e.g., \u003cem\u003eKobresia setchwanensis\u003c/em\u003e and \u003cem\u003eCarex\u003c/em\u003e spp.), and grasses (e.g., \u003cem\u003eElymus nutans\u003c/em\u003e and \u003cem\u003eDeschampsia caespitos\u003c/em\u003e) (Hu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition to the high diversity of plant species (Xiang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), arthropods (Wu et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Xi et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) are also very abundant in the meadow.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe study species\u003c/h3\u003e\n\u003cp\u003eThe slug \u003cem\u003eAgriolimax agrestis\u003c/em\u003e is a species of gastropod mollusks, considered a pest in pastures (Baur and Baur \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Sternberg \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The slugs, similar to other slug species (Barker \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), display predominantly nocturnal activity and remain inactive during the daytime under intense light conditions. The average field density at the study site is 0.3 to 0.5 individuals m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e. As generalist feeders, they consume a wide variety of forb species. They are visibly active from mid-June until the end of September. The slugs lay eggs in September during the late growing season, and the eggs overwinter in a quasi-dormant state until temperatures rise in late May or early June. Typically, eggs are deposited in moist and sheltered locations, such as soil crevices or leaf litter, which provide insulation and protection.\u003c/p\u003e\n\u003ch3\u003eThe large-scale field warming experiment\u003c/h3\u003e\n\u003cp\u003eSix 15 m \u0026times; 15 m \u0026times; 2.5 m (height) open-top chambers (OTCs) were randomly constructed in a fenced 1.0 ha winter pasture in October 2014. The sides of the OTCs were covered by thin steel screen. Three chambers were additionally covered with transparent tempered glass, and the roofs were discontinuously covered with 1.5 m \u0026times; 0.3 m (width) transparent glass strips. Steel screen (with a mesh size of 0.6 mm \u0026times; 0.6 mm) was also placed 1 m into the soil to prevent rodents from entering. Three OTCs with transparent tempered glass are referred to as \u0026ldquo;warmed chambers\u0026rdquo; and the other three chambers as \u0026ldquo;non-warmed chambers\u0026rdquo; (for more details, see Hu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe climate measurement data logger indicated that the mean annual temperature was 5.23\u0026deg;C and 4.81\u0026deg;C at 30 cm aboveground in the warmed and non-warmed chambers, respectively, from 2017 to 2023, with a 0.24\u0026deg;C increase during the growing season and a 0.55\u0026deg;C increase in the non-growing season. Moreover, from 2022 to 2023, when the daily activity time was investigated for the slugs, the experimental warming increased nighttime temperatures by 0.94\u0026deg;C (19:00\u0026ndash;6:00) and daytime temperatures by 0.01\u0026deg;C (6:00\u0026ndash;19:00). Such asymmetric warming was consistent with observations and the model predictions for about two to three decades later in the study site according to the SSP1\u0026ndash;1.9 scenarios (IPCC \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The vapor pressure deficit was 0.4% to 0.8% higher in the warmed chambers compared to the non-warmed chambers. Additionally, the soil moisture at 5 cm was 20.2% v/v on average in the warmed chambers and 22.4% v/v in the non-warmed chambers during the growing season.\u003c/p\u003e\n\u003ch3\u003eBody size and growth phenology\u003c/h3\u003e\n\u003cp\u003eWe used fresh body weight to measure size. During the growing season (June to September), five to ten individuals were randomly selected from each chamber and weighed \u003cem\u003ein situ\u003c/em\u003e with an electronic balance (sartorius BS224S, 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e g) every 10 days for two consecutive years (2022\u0026ndash;2023). All individuals were released after weighing.\u003c/p\u003e \u003cp\u003eGrowth phenology was defined as the day when slug body size reached 5 mg (the \u0026ldquo;starting\u0026rdquo; time of growth) and the day when slug body size reached 90% of the maximum body size (the \u0026ldquo;ending\u0026rdquo; time of growth). To reduce the variability among individual observations, we calculated the phenological time by fitting a logistic regression of body size versus collection day for each chamber for each year. Logistic regression was performed using the \u003cem\u003enls\u003c/em\u003e function in R as follows (Kaufmann \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1981\u003c/span\u003e),\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{W}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t}=\\frac{Asym}{1+exp\\left[\\left(xmid-day\\right)/scal\\right]},$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eAsym\u003c/em\u003e is the maximum weight, \u003cem\u003exmid\u003c/em\u003e is the time when weight reaches half of the maximum weight (i.e., xmid\u0026thinsp;=\u0026thinsp;Asym/2), and \u003cem\u003escal\u003c/em\u003e is the growth rate. All such regressions had statistically significant fits (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; see Supporting information: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Using the regression equation, we calculated the starting and ending times for slug growth phenology.\u003c/p\u003e\n\u003ch3\u003eDaily activity\u003c/h3\u003e\n\u003cp\u003eDaily activities were investigated every 10 to 20 days, and five investigations were conducted for each year. Each observed individual was classified as either \u0026ldquo;active\u0026rdquo; or \u0026ldquo;inactive\u0026rdquo; (including states of chill-coma (ceased movement due to cold), resting (lack of visible activity driven by their activity patterns and/or endogenic circadian rhythms), or torpor (decreased physiological activities and metabolic rate due to due to unfavorable external conditions) based on their behavior (Eban-Rothschild and Bloch \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; MacMillan and Sinclair \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Borkataki et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Slugs were categorized as \u0026ldquo;active\u0026rdquo; when they extended their bodies and moved slowly and continuously, with tentacles fully extended and oscillating to explore their surroundings, leaving visible mucus trails (South \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). This behavior was often associated with activities such as foraging. In contrast, slugs were classified as \u0026ldquo;inactive\u0026rdquo; when they remained motionless in a fixed position, with their bodies shortened or curled into a droplet-like shape and their tentacles retracted or immobile (South \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Due to the slugs' limited range of movement, their activities were often confined to a single plant for multiple hours. We flagged the plants with red sticks placed beside them and recorded both the number of \u0026ldquo;active\u0026rdquo; and \u0026ldquo;inactive\u0026rdquo; individuals hourly. Prior investigations showed that the slugs were inactive in high-light daytime but had two activity periods. One ranged between 19:00 and 22:00 (7\u0026ndash;10 pm; Beijing Time) at night, and the other between 6:30 and 9:30 in the morning. (am) Thus, we started our number recording at 6:00 in the morning and 18:00 at night on each observation day. The daily activity time was calculated by summing the products of the proportion of active individuals and the observation interval (60 min) for each observation day.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSpecies abundance\u003c/h2\u003e \u003cp\u003eSlug abundance was investigated from early July to early August (the period of highest abundance in the year) for each year (2017 to 2023) using a quadrat sampling method. Specifically, twenty 50 cm \u0026times; 50 cm quadrats were randomly selected in each chamber when the slugs were active (7:00\u0026ndash;9:00). Then, we inspected the vegetation to record all visible slugs.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSlug diet\u003c/h3\u003e\n\u003cp\u003eSlug diet was investigated during the growing season in 2022. During the investigation of slug activities, we recorded the species identity of plants that the slugs were feeding on. Slug feeding was observed using magnifiers. If the slugs were found scraping off plant surfaces with the radula, tearing plant tissues into smaller pieces, their feeding was assumed to have occurred. The feeding activity was typically slow and rhythmic, leaving distinct scraping marks (usually as irregular holes) on the leaves. In total, we recorded 156 feeding events, and the diet included 29 plant species. Additionally, the dietary plants were categorized into graminoids and forbs.\u003c/p\u003e\n\u003ch3\u003ePlant community composition and biomass\u003c/h3\u003e\n\u003cp\u003ePlant community composition and aboveground plant biomass were investigated to assess whether the food resource of slugs had changed in response to warming. Specifically, the plant community in each chamber was examined using a quadrat sampling method. In each year, we randomly established twenty 50 \u0026times; 50 cm quadrats in each chamber. Each quadrat was further subdivided into 5 \u0026times; 5 cm grids. We then documented species presence and abundance following the protocols described by Hu et al., (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn mid-August of each year when the majority of plant species had completed their reproductive phase, all aboveground plant parts were clipped to the ground surface and harvested in sixteen 50 \u0026times; 50 cm quadrats that were randomly deployed in each chamber. The harvests were carefully sorted into two functional types (graminoids, including grasses and sedges, and forbs, including the rest of the species), dried at 75\u0026deg;C for 72 h, and then weighed (Hu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eThe small-scale field warming experiment\u003c/h2\u003e \u003cp\u003eIn order to remove any potential confounding factors (e.g., changes in the vegetation structure and animal (potential predators, for example) community induced by warming), we conducted a complementary experiment to determine whether warming \u003cem\u003eper se\u003c/em\u003e could change the daily activity time and whether warming could increase the body size of the slugs. In the early August 2024, when the slugs grew rapidly during their lives, fourteen slug individuals with similar body size (ranging between 107.9 mg and 196.2 mg) were reared in a small open top chamber (2 m \u0026times; 2 m \u0026times; 2.5 m) built of the same material as the large OTCs, and fourteen individuals were reared outside the chamber under a glass roof supported by two steel pillars. The daily mean temperature was 15.49\u0026deg;C outside and 15.93\u0026deg;C inside the chamber, with an increase of 0.80\u0026deg;C during nighttime (19:00\u0026ndash;6:00) and 0.07\u0026deg;C during the daytime (6:00\u0026ndash;19:00) (Supporting information: Fig. S2). Each slug was individually placed in a plastic pot (diameter 14.7 cm and depth 9.8 cm) lined with a 2 cm layer of soil, and the top of each pot was covered with a mesh to prevent slugs from escaping but ensure air circulation. Fresh leaves of \u003cem\u003eSaussurea nigrescens\u003c/em\u003e (the major food resource of slugs) were added and replaced daily in the pot, and water was misted at midday to maintain suitable humidity. The experiment lasted for 18 days from August 8th 2024 to August 25th 2024. We recorded daily activity time and body size of the slugs using the same method as in the field warming experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eAll analyses were performed in R 4.3.1 (R Core Team 2023). The slug diet was visualized using the \u003cem\u003eplotweb\u003c/em\u003e function in the \u003cem\u003ebipartite\u003c/em\u003e package (v.2.18) (Dormann et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). After testing for the error distribution of response variables, we used generalized linear mixed models (GLMMs) in the \u003cem\u003elme4\u003c/em\u003e package (Bates et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) with warming as the fixed factor, and chamber identity and year as random factors to determine the warming effect on slug abundance (negative binomial error structure) and the proportion of active individuals (binomial error distribution). Similarly, generalized linear mixed models (GLMMs) with warming as the fixed factor, and chamber identity, date and year as random factors to determine the warming effect the body size of slugs (gamma error with log link function). Linear mixed models (LMMs) were used with warming as the fixed factor, and chamber identity as random effects to determine the warming effect on slug growth phenology (starting time, ending time and growing period), biomass of forbs and slug body size in the small-scale experiment. In addition, general linear model was employed to determine the relationship between the percentage of active slugs and air temperature.\u003c/p\u003e \u003cp\u003eThe difference between warmed and non-warmed chambers was determined using Fisher-Pitman permutation test in the R package \u003cem\u003ecoin\u003c/em\u003e (v.1.4.3) (Hothorn et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In addition, independent sample t-tests were conducted to assess the warming effects on body size in the small-scale experiment.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eThe phenology and daily activity time\u003c/h2\u003e \u003cp\u003eWarming did not significantly change the starting time ((LMM: estimate = \u0026minus;\u0026thinsp;4.52, \u003cem\u003eF\u0026thinsp;=\u003c/em\u003e\u0026thinsp;4.27, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.11, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and the ending time (LMM: estimate = -0.37, \u003cem\u003eF\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.91, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) of slug growth in 2022. Moreover, the difference in slug growing period was indistinguishable between non-warmed (96.37\u0026thinsp;\u0026plusmn;\u0026thinsp;3.73 days on average) and warmed chambers (100.52\u0026thinsp;\u0026plusmn;\u0026thinsp;3.51 days; LMM: estimate\u0026thinsp;=\u0026thinsp;4.15 \u003cem\u003eF\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.66, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.46). Likewise, warming did not significantly change the starting time ((LMM: estimate = \u0026minus;\u0026thinsp;2.29, \u003cem\u003eF\u0026thinsp;=\u003c/em\u003e\u0026thinsp;1.19, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.39, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) and the ending time (LMM: estimate\u0026thinsp;=\u0026thinsp;4.00, \u003cem\u003eF\u0026thinsp;=\u003c/em\u003e\u0026thinsp;1.71, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.32, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) of slug growth in 2023. Again, the difference in slug growing period was indistinguishable between non-warmed (100.53\u0026thinsp;\u0026plusmn;\u0026thinsp;6.71 days on average) and warmed chambers (106.82\u0026thinsp;\u0026plusmn;\u0026thinsp;10.43 days; LMM: estimate\u0026thinsp;=\u0026thinsp;6.29, \u003cem\u003eF\u0026thinsp;=\u003c/em\u003e\u0026thinsp;2.69, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.24).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn 2022, warming extended the daily activity duration by an average of 28.68\u0026thinsp;\u0026plusmn;\u0026thinsp;9.42 min (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE; same for hereafter) at night (GLMM: estimate\u0026thinsp;=\u0026thinsp;1.46, \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;17.99, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) and 8.49\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43 min in the morning (GLMM: estimate\u0026thinsp;=\u0026thinsp;0.95, \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;6.64, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), respectively, leading to a significant average increase of 37.17\u0026thinsp;\u0026plusmn;\u0026thinsp;7.64 min per day (a 24.86% increase) in activity time (GLMM: estimate\u0026thinsp;=\u0026thinsp;1.18, \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;21.89, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Similarly, in 2023, warming extended the daily activity duration by an average of 31.70\u0026thinsp;\u0026plusmn;\u0026thinsp;24.81 min at night (GLMM: estimate\u0026thinsp;=\u0026thinsp;1.22, \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;11.97, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec,\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) and 17.85\u0026thinsp;\u0026plusmn;\u0026thinsp;9.42 min in the morning (GLMM: estimate\u0026thinsp;=\u0026thinsp;1.20, \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;11.13, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec,\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), respectively, leading to a significant average increase of 49.55\u0026thinsp;\u0026plusmn;\u0026thinsp;25.80 min per day (a 29.16% increase) in activity time (GLMM: estimate\u0026thinsp;=\u0026thinsp;1.22, \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;23.63, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec,\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the proportion of \u0026ldquo;active\u0026rdquo; individuals was positively associated with temperature during the growing season in both 2022 (N\u0026thinsp;=\u0026thinsp;50, \u003cem\u003eR\u0026sup2;\u003c/em\u003e = 0.40, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 2023 (N\u0026thinsp;=\u0026thinsp;56, \u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.33, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSlug body size and abundance\u003c/h2\u003e \u003cp\u003eExperimental warming significantly affected body size of the slugs (GLMM: estimate\u0026thinsp;=\u0026thinsp;0.52, \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;152.09, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Specifically, the body size increased significantly by 64.51% in 2022 (\u003cem\u003eZ\u003c/em\u003e = -1.526, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) and by 57.11% in 2023 (\u003cem\u003eZ\u003c/em\u003e = -1.552, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) at the last measurement. Warming did not significantly change slug abundance in 2017 and 2018, but substantially increased slug abundance by an average of 497.33% from 2019 to 2023 (GLMM: estimate\u0026thinsp;=\u0026thinsp;0.99, \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;133.34, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Additionally, the percentage of active slugs was positively associated with temperature across the chambers in both 2022 (N\u0026thinsp;=\u0026thinsp;50, \u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.40, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 2023 (N\u0026thinsp;=\u0026thinsp;56, \u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.33, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eThe diet of slugs\u003c/h2\u003e \u003cp\u003eThe slugs were observed to feed on 19 forb species and 5 graminoid species in warmed chambers, and 14 forb species and 1 graminoid species in non-warmed chambers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; more details see Supporting information: Table S2). On average, forbs accounted for 93.17% and 97.92% of the observed dietary interactions between slugs and plants in the warmed and non-warmed chambers, respectively. The difference in the proportion of forbs in the slug diet was negligible between the non-warmed and warmed chambers (\u003cem\u003eZ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.78, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.43).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eChange in food resource\u003c/h2\u003e \u003cp\u003eWarming significantly decreased the biomass of forbs (the major food source of slugs) (LMM: estimate = -0.44, \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;86.07, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Supporting information: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Specifically, the biomass of forbs significantly decreased by 23.22% in 2022 (\u003cem\u003eZ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.98, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04, Supporting information: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and 15.39% in 2023 (\u003cem\u003eZ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.82, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03, Supporting information: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe small-scale experiment\u003c/h2\u003e \u003cp\u003eExperimental warming significantly increased the daily activity duration (GLMM: estimate\u0026thinsp;=\u0026thinsp;1.80, \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;114.95, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) by 90.56\u0026thinsp;\u0026plusmn;\u0026thinsp;19.84 min during the night time (\u003cem\u003eZ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.06, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) and 42.50\u0026thinsp;\u0026plusmn;\u0026thinsp;22.88 min (\u003cem\u003eZ\u003c/em\u003e = -1.76, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) during the daytime. The total daily activity duration was over 2 h longer (+\u0026thinsp;180.18%) in the warmed chambers than in the non-warmed chambers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Moreover, warming had a significant effect on slug body size (LMM: estimate\u0026thinsp;=\u0026thinsp;39.53, \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.88, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), with a 35.63% increase at the final measurement (t-test: \u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.86, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we show that the field warming did not change the phenology of slug growth, but it significantly increased the daily activity time by \u0026gt;\u0026thinsp;20% during the growth period. Moreover, experimental warming significantly increased the body size of slugs by more than 70% and the slug abundance by more than three times in the last years of the warming experiment. The increased slug body size and abundance cannot be accounted for by the change in the food resource, because the food resource (primarily forbs) for the slugs was significantly lower in the warmed chamber compared to the non-warmed ones. Importantly, the complementary small-scale experiment explicitly suggests that warming \u003cem\u003eper se\u003c/em\u003e may increase slug body size by increasing activity time. Thus, it can be concluded that a warming-induced increase in daily activity time (and associated increased resource intake of slug individuals) could have reasonably increased the body size and abundance of the slugs in the field warming experiment. To the best of our knowledge, this is one of the first studies experimentally demonstrating that global warming may significantly change daily rhythms of animals, which can have consequences for species' body size and abundance.\u003c/p\u003e \u003cp\u003eExperimental warming often reduces individual body size across diverse taxa including both endotherms (Tabh et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and ectotherms (Zhao et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Xi et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), presumably because increased temperatures increase body development rate more than growth rate. However, there are also many studies reporting an increase in body size under experimental warming (Yom-Tov et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Ozgul et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and they often attribute the increase to the extension of the growing season (Menzel and Fabian \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). A pertinent example is the study by Ozgul et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), which reported that the earlier emergence from hibernation in the yellow-bellied marmot resulted in increased body masses due to the extended growing season. Similarly, Yom-Tov et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) found that American martens (\u003cem\u003eMartes americana\u003c/em\u003e) significantly increased their body size in the latter half of the 20th century, and they attributed the finding to the climate warming-induced extension in growing season (over 14 days). However, in our case, the growth period was unchanged by the experimental warming.\u003c/p\u003e \u003cp\u003eBy contrast, the increase in the slug body size may be attributed to the warming-induced increase in slug daily activity duration. In this study, the two activity periods of slugs are apparently interrupted by the state of \u0026ldquo;inactivity\u0026rdquo; due to low temperature. At our study site, where the annual mean temperature is below 2\u0026deg;C, with mean temperatures falling below 0\u0026deg;C for over 260 days annually (Hu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), the daily activities of ectotherms in these regions likely depend on temperature fluctuations. More specifically, during slug growth (from mid-June to late September), the daily variation in temperature ranges from \u0026minus;\u0026thinsp;8.18\u0026deg;C to 39.84\u0026deg;C, and even on the hottest day of August of 2022, the lowest temperatures went down to 3.68\u0026deg;C. Therefore, it is not surprising that the experimental warming significantly delayed the ending time of slug activity at night and advanced the beginning time of slug activity in the morning, thereby extending the duration of slug activity significantly. Consistently, slug activity time increased with increasing temperatures across the chambers in both study years. Given that the slugs were at the growth stage during our observations and hence they spent little time mating and reproducing, it can be speculated that the foraging duration would increase with increasing activity time, which may further lead to the increase in body size.\u003c/p\u003e \u003cp\u003eThe warming-induced increase in slug body size can be theoretically attributed to other factors such as increased food availability and quality. For example, Yom-Tov and Yom-Tov (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) documented an increase in the body size of masked shrews \u003cem\u003eSorex cinereus\u003c/em\u003e in Alaska during the latter half of the twentieth century, and they associated it with increased food availability due to climate warming. However, our experimental warming has resulted in a significant decrease in the biomass of forbs (the preferred food source for slugs) since 2018, while increasing the biomass of graminoids (See Supporting information: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Hu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This coincided with an elevated frequency of slugs feeding on graminoids under warming conditions. Moreover, leaves of graminoids are lower in water content and higher in C/N compared to forbs (Lan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), indicating that slug food quality also decreased in the warmed chambers. Additionally, no predation and parasitism (due to infestation/inflection) events on slugs were observed throughout the experimental period in both warmed and non-warmed chambers, primarily because there were no large predators foraging on the ground during their activity and because parasites (pathogens) were not abundant due to low temperature in the study site. Consequently, our data indicates that warming did not indirectly increase slug body size by increasing food resources and reducing predation/parasitism.\u003c/p\u003e \u003cp\u003eIt is also worth noting that relative humidity is suggested to be important for slug growth (Dainton \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1954\u003c/span\u003e; South \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Willis et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Nevertheless, in this study, the relative humidity was high (mostly above 65% during our observations) in both treatments, and moreover, the vapor pressure deficit was only slightly higher (by ca. 3%) in the warmed than in non-warmed chambers; hence, this minor difference is not likely to have significantly affected the growth rates of slugs. Consistently, the complementary small-scale experiment demonstrates that warming but not other factors increased the activity time of slugs and further increased their body size.\u003c/p\u003e \u003cp\u003eIt is not surprising that the abundance of alpine slugs significantly increased under warming conditions, given that larger slug individuals are likely to have greater fecundity (Montresor et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and their eggs can have a higher survival rate in winter. Indeed, studies have shown that enhanced fecundity and higher survival rates under warming conditions can lead to an increase in species abundance in the salamander \u003cem\u003eHynobius tokyoensis\u003c/em\u003e (Kingsolver and Huey \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Okamiya et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Despite these insights, the exact mechanisms driving the increase in slug population abundance remain unclear and require further experimental exploration for a better understanding.\u003c/p\u003e \u003cp\u003eIt is worth noting that active slugs were more likely to be captured by eyes than \u0026ldquo;inactive\u0026rdquo; slugs, particularly at the early stage of investigations, and thus the activity time of slugs might have been overestimated. Nevertheless, we conducted the investigations with similar methods in both treatments, allowing the comparison of slug activity time to be possible.\u003c/p\u003e \u003cp\u003eIn summary, our data shows that warming enhances the daily activity duration of slugs, thereby increasing their body size and species abundance. Importantly, our study provides an underexplored mechanism underlying warming-induced changes in animal individual size, an important species trait governing species interactions and ecosystem properties. Thus, future research should extend beyond the impact of temperature on phenology and animal physiological metabolism to consider the daily activities of animals. In addition, this study reveals that the nocturnal slugs increased their body size primarily by increasing their activity time. This suggests the importance of studying animal night ecology under climate change (Kolioulis et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Z. Huang and S. He\u0026nbsp;for field and laboratory assistance, and the Sichuan Zoige Alpine Wetland Ecosystem National Observation and Research Station for platform support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations of potential conflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the National Science Foundation of China (32301391 and 32071605). N.E. acknowledges funding by the German Research Foundation (DFG\u0026sbquo; FZT- 118, 202548816; Ei 862/29-1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e:\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e:\u0026nbsp;The data that support the findings of this study are openly available in Dryad Digital Repository after final acceptance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of authorship\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions: Xingyu Zhou\u003c/strong\u003e: investigation, formal analysis, writing - original draft, writing - review and editing.\u0026nbsp;\u003cstrong\u003eXiaoli Hu\u003c/strong\u003e: investigation, formal analysis, writing - original draft, writing - review and editing.\u0026nbsp;\u003cstrong\u003eYing Feng\u003c/strong\u003e: investigation.\u0026nbsp;\u003cstrong\u003eShuang Xiang\u003c/strong\u003e: investigation.\u0026nbsp;\u003cstrong\u003eYoujun Chen\u003c/strong\u003e: investigation.\u0026nbsp;\u003cstrong\u003eYuxia Zhang\u003c/strong\u003e: investigation.\u0026nbsp;\u003cstrong\u003eNico Eisenhauer\u003c/strong\u003e: writing - review and editing.\u0026nbsp;\u003cstrong\u003eShucun Sun\u003c/strong\u003e: conceptualization, project administration, supervision, writing - review and editing.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAngilletta MJ (2009) Looking for answers to questions about heat stress: researchers are getting warmer. 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Ecol. 48: 423-434 doi: 10.1007/s10452-014-9495-y\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oecologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"oeco","sideBox":"Learn more about [Oecologia](https://www.springer.com/journal/442)","snPcode":"442","submissionUrl":"https://submission.nature.com/new-submission/442/3","title":"Oecologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"alpine meadow, body size, daily activity rhythm, invertebrates, simulated warming","lastPublishedDoi":"10.21203/rs.3.rs-8198076/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8198076/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough global warming is known to shift animal phenology, its effects on daily activity remain poorly understood. We used long-term field warming with large open-top chambers (15 \u0026times; 15 \u0026times; 2.5 m), which increased mean annual air temperature by 0.42\u0026deg;C from 2017\u0026ndash;2023, to test whether warming alters daily activity time and body size of the nocturnal slug \u003cem\u003eAgriolimax agrestis\u003c/em\u003e in an alpine meadow on the eastern Tibetan Plateau. Warming increased slug activity time by 37.17 min (+\u0026thinsp;24.86%) in 2022 and 49.55 min (+\u0026thinsp;29.16%) in 2023, and increased body size by 64.51% and 57.11%, respectively, without changing growth phenology. A complementary short-term experiment using small open-top chambers (2 \u0026times; 2 \u0026times; 2 m) elevated temperature by 0.44\u0026deg;C and similarly increased activity time by 180.18% and body size by 35.63% under controlled feeding. Slug abundance increased by ca.500% in warmed plots from 2019 to 2023, likely driven by enhanced body size and survival. Overall, warming substantially extends daily activity time, thereby enlarging slug body size and promoting population growth, highlighting an overlooked mechanism by which climate warming affects animal traits and demographics in cold regions.\u003c/p\u003e","manuscriptTitle":"Field warming increases body size of nocturnal slugs by extending their daily activity time in an alpine meadow","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-25 07:28:07","doi":"10.21203/rs.3.rs-8198076/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-01-26T14:40:48+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-23T11:48:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-27T04:47:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oecologia","date":"2025-11-24T21:48:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oecologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"oeco","sideBox":"Learn more about [Oecologia](https://www.springer.com/journal/442)","snPcode":"442","submissionUrl":"https://submission.nature.com/new-submission/442/3","title":"Oecologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f038cd80-42c7-4d97-be5a-30a7180f84dd","owner":[],"postedDate":"December 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:16:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-25 07:28:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8198076","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8198076","identity":"rs-8198076","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}