Marine brachyuran crabs’ osmoregulatory and metabolic responses upon warming and seawater dilution challenges: the non-native Charybdis helleri is more sensitive than the native Menippe nodifrons.

Marine brachyuran crabs’ osmoregulatory and metabolic responses upon warming and seawater dilution challenges: the non-native Charybdis helleri is more sensitive than the native Menippe nodifrons. | 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 Marine brachyuran crabs’ osmoregulatory and metabolic responses upon warming and seawater dilution challenges: the non-native Charybdis helleri is more sensitive than the native Menippe nodifrons. Leonardo de Paula Rios, Carolina A. Freire This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5595652/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Jan, 2026 Read the published version in Biological Invasions → Version 1 posted 5 You are reading this latest preprint version Abstract The presence of non-native organisms challenges ecosystems under the influence of climate change. Comparisons of physiological performance between ecologically-similar native and non-native species contribute to invasion studies. We examined two decapod crustaceans in Estuarine Complex of Paranaguá (ECP), Brazil: the non-native Charybdis hellerii and the native Menippe nodifrons. Crabs were acclimated to control (26 °C) and elevated (30 °C) temperatures for one week in full-strength seawater (35‰), and were then submitted to dilute seawater (30, 25, and 20‰) for 6 hours. Hemolymph was assayed for osmolality, chloride, magnesium, and lactate; muscle samples were evaluated for hydration levels. Dissolved oxygen and ammonia production were assessed in the experimental water. Both species were impacted by low salinity, with an synergistic effect from elevated temperatures. However, C. hellerii was more affected than M. nodifrons, displaying less capacity to keep stable muscle hydration levels upon seawater dilution, a steeper decrease in dissolved oxygen, higher ammonia excretion, and higher lactate, as compared to the native crab. The non-native C. hellerii was physiologically challenged to a much higher degree than the native species. Although C. hellerii has established populations in the ECP, its sensitivity (synergistic deleterious effect) to salinity reductions and rising temperatures may limit its further spread in areas with intense fluctuating abiotic conditions. These data can support modelling efforts of the trends in these species distribution where C. helleri is invasive. This result may also be indicative of the undergoing process of invasion; similar approaches could contribute to invasion science involving other marine/estuarine crabs. Crustacea Decapoda invasion metabolism osmoregulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Climate change and biological invasions constitute especially relevant threats to biodiversity worldwide (Walther et al. 2009; Wallingford et al. 2020; Robinson et al. 2020; King et al. 2021). Despite their significance, their effects are commonly examined in isolation, although there is data in the literature that points to a close relationship between climate change and an increase in the pace and intensity of biological invasions (Dukes and Mooney 1999; Walther et al. 2009; Christensen et al. 2020; Ricciardi et al. 2021; Cuthbert and Briski 2021). Species have experienced population geographical shifts in recent decades, attributed to rising temperatures in their original habitats, and frequently causing invasion of new habitats (Parmesan 2006; Wallingford et al. 2020; Rubenstein et al. 2023). In addition, climate change may alter the likelihood of introduction and adaptive success of non-native species (Walther et al. 2009; Blackburn et al. 2011; Giangrande et al. 2020; Robinson et al. 2020; King et al. 2021; Ricciardi et al. 2021). Heatwaves, in particular, can trigger massive die-offs of native species, creating vacant ecological niches which invasive species can exploit (Occhipinti-Ambrogi 2007). In addition to facilitating the escape, introduction, and expansion of the range of invasive species, climate change and extreme events may alter abiotic conditions, including temperature, salinity, pH, and oxygen availability of local habitats. For example, Sanford et al. (2014) examined how ocean acidification caused by elevated CO 2 in Tomales Bay, California (USA) might increase the vulnerability of native Olympia oysters to predation by invasive snails. Their experiment showed that 48% more oysters were consumed under high CO 2 conditions. Smaller oysters were predated more than larger oysters, highlighting how environmental stress could amplify the impact of invasive predators. These changes in temperature and CO 2 levels can synergistically increase the stress to which aquatic populations are exposed, directly impacting ecosystem health and negatively affecting human activities such as fishing (Occhipinti-Ambrogi 2007; Robinson et al. 2020; Ricciardi et al. 2021; Rubenstein et al. 2023). In southern Brazil, the only non-native decapod crustacean species established along the Estuarine Complex of Paranaguá, Paraná state (ECP) is the portunid crab Charybdis hellerii (A. Milne-Edwards, 1867). It is thought to have been introduced through the dispersal of larvae by the release of ballast water from cargo ships (Mantelatto and Dias 1999; Soares et al. 2022; Cintra et al. 2023). This species originates from the Indo-Pacific region, where it occurs in estuaries, intertidal regions, under rocks or in corals (Watanabe et al. 2014; Sant’Anna et al. 2015; Negri et al. 2018). It is currently distributed along the east coast of America (from the United States to southern Brazil), as well as some regions of the Mediterranean Sea (Negri et al. 2018; Siqueira et al. 2021). The introduction of this species into ecologically sensitive areas like Brazilian estuarine areas can have severe consequences, as C. hellerii competes with native species for habitat and food (Sant’Anna et al. 2012, 2015; Watanabe et al. 2022). In some Brazilian states such as São Paulo and Santa Catarina (regions adjacent to the ECP), C. hellerii has outnumbered some local crab populations (Sant’Anna et al. 2012, 2015; Siqueira et al. 2021). In the ECP, C. hellerii co-occurs with the native species Menippe nodifrons (Menippidae), a species found along the Brazilian coast, and both inhabit rocky shores, reefs, shallow water beaches and mid-coastal regions (Santana et al. 2009; Sant’Anna et al. 2012; Occhi et al. 2019). The distribution of M. nodifrons overlaps with the environments recently colonized by C. hellerii . In non-native areas, C. hellerii has been found inhabiting rocks, corals, mud and sand bottoms, intertidal and subtidal regions (Stanski et al. 2022). In addition to its ecological importance, M. nodifrons serves as a fishing resource in certain regions of the northern hemisphere and in parts of Brazil, including the ECP (Oshiro 1999; Bertini et al. 2007), while C. hellerii is not (yet) a target of artisanal fishing for consumption in this region of the Brazilian coast. Physiological tolerance plays a crucial role in the competition between native and non-native species. Species which are more sensitive to environmental fluctuations, such as in pH, dissolved oxygen, temperature and salinity, may face exclusion or replacement by other, more tolerant species. Multiple environmental stressors can elicit three distinct types of effects on organisms: synergistic, additive, or antagonistic. A synergistic effect arises when the combined influence of two or more stressors produces an impact that exceeds the sum of their individual effects. An additive effect is observed when the cumulative impact of multiple stressors equals the sum of their individual effects, indicating no significant interaction that either amplifies or diminishes their influence. Conversely, an antagonistic effect occurs when the combined impact of two or more stressors is less than the sum of their individual effects, suggesting an interaction that mitigates or partially neutralizes their overall influence (Dinh et al. 2022). Therefore, the comparison of physiological responses between a native and a non-native sympatric species to saline and thermal challenges can contribute to predictions of ecological impact of invasion (Christensen et al. 2020; Ricciardi et al. 2021; Cuthbert and Briski 2021; Boardman et al., 2022). We hypothesize that the non-native species C. helleri is more tolerant to warming and seawater dilution than the native M. nodifrons, mainly due to the invasive potential of C. hellerii , whose populations are already established in estuaries adjacent to the study site. Materials And Methods Specimen collection and maintenance Individuals of Charybdis hellerii and Menippe nodifrons were captured by cylindrical traps in the Bay of Paranaguá, Paraná, Brazil (25°31'44.3"S 48°28'23.1"W) or were obtained through active capture on the Banana Island, also located in the Bay of Paranaguá (25°25'28.3"S 48°24'25.2"W), in the summer months of February and November-December 2020. Animals were placed in plastic containers filled with water from the collection site, maintained under constant aeration, and transported to the Laboratory of Comparative Physiology of Osmoregulation (LFCO) at the Department of Physiology, Federal University of Paraná. At LFCO, the animals remained in a 250-liter stock aquarium filled with seawater for a duration of ~7 days, maintaining a density of ~1 individual for 10 liter. In each field collection, only one species was captured. Thus, both species did not inhabit the stock aquarium at the same time. Aeration was constant, and an external filter ensured good water quality. The filtration system utilizes a three-stage process comprising mechanical, chemical, and biological treatment. Water quality was systematically monitored through both visual assessment of turbidity and total dissolved solids as well as quantitative chemical analysis of ammonia and nitrite concentrations. Regular system maintenance is performed according to manufacturer specifications, including periodic replacement of filter media and implementation of weekly partial water changes (~30% volume). Salinity was of 34-35‰, temperature 26.28±0.64 °C (average and standard deviation), pH at ± 8.3, dissolved oxygen in the water ~8.0 mg/L and light conditions were according to the natural photoperiod (13h Light: 11h Dark). The animals were fed small pieces of fish fillet every two days, and feeding was halted 24 hours before the experiments. Following this initial phase, some individuals were utilized for control (temperature) experiments, maintaining temperature at 26 °C but reducing the salinity, according to the experimental protocol described below. The remaining specimen were acclimated in the same stock aquarium for seven days with a temperature increase of 4 °C above the annual average observed in summer (from 26 °C to 30 °C). These temperature data were added to Table S2 in the supplementary material. Others parameters in the stock aquarium water remained constant and the temperature elevation occurred gradually at a rate of 0.5 °C per hour between 8:00 am and 4:00 pm during the first of seven days of acclimation to the higher temperature. After this process, the final temperature was 30.47±0.57 °C (mean and standard deviation). These temperature data were added to Table S3 in the supplementary material. Experiments For the experiments, animals were transferred from the stock aquarium to individual small aquaria filled with 2 liters of brackish water of salinity 35, 30, 25 or 20‰. Specimens of the control group were retrieved from the stock tank kept at 26 °C, while experimental animals were retrieved from the stock tank after being acclimated to the increased temperature of 30°C. Experimental time of exposure was always for 6 hours. Crabs maintained in the stock aquarium at 26 °C or 30°C were then exposed to identical osmotic challenges: 35, 30, 25 and 20‰, for 6 hours. The entire procedure was replicated six times, with additional batches of crabs maintained at either 26 or 30 °C. Thus, the total N used was 96 individuals (2 temperatures x 4 salinities x 6 replicates = 48 crabs of each species). Following the experimental exposure to the distinct salinities for 6 hours, individuals were covered with crushed ice in a styrofoam box, and were cryoanesthesized for approximately 5 minutes without direct contact with the ice. A hemolymph sample was obtained through puncturing the arthrodial membrane of a pereiopod during anesthesia. Subsequently, they were euthanized by opening the carapace using sharp material, and immediately sectioning the central nervous cord. After euthanasia, samples of muscle tissue were removed from the fifth pair of pereiopods (adapted for swimming) in C. hellerii and the right chelipod in M. nodifrons . Hemolymph and muscle samples were stored in a -20°C freezer for posterior assays of osmolality, chloride, magnesium and lactate concentrations (hemolymph) and determination of muscle water content. Water samples were obtained from the experimental vials to analyze dissolved oxygen concentration and initial and final ammonia levels. Osmo/ion-regulatory assays Hemolymph osmolality was determined in undiluted hemolymph samples using a Vapor Pressure Micro-Osmometer (VAPRO 5520, Wescor, USA). Hemolymph chloride and magnesium levels were assayed in diluted samples (1:5 for chloride and 1:25 for magnesium), using colorimetric kits (#115 for chloride and #50 for magnesium, from Labtest, Lagoa Santa, Brazil) under, respectively, 450 and 505 nm. The decrease in hemolymph osmolality, which is the sum of inorganic ions (such as chloride and magnesium) and organic osmolytes such as amino acids, for example - can lead to systemic imbalances in the acid-base system - chloride is used in the exchange for bicarbonate, besides being the main inorganic ion present in the hemolymph - and magnesium is a cofactor of enzymes, such as Na + /K + -ATPase, and helps in the transport of oxygen, occupying a binding site in the hemocyanin molecule. Thus, changes in these parameters can indicate deviation from a situation of homeostasis or physiological ‘comfort’ (see Freire, 2025). Muscle water content (MWC) was determined by weighing muscle samples (wet weight) in pre-weighed and sealed 2 mL Eppendorf tubes (Bioprecisa FA2104 N, precision 0.1 mg, Brazil). After remaining for 48 hours at 60 °C inside an oven, the samples were weighed again (dry weight), and the weight loss (water) was expressed as a percentage using the formula: MWC (%) = [(wet weight – dry weight) / wet weight] x 100 The increase in muscle water content (water influx from hemolymph osmolality decrease) may indicate that the tissue has been challenged beyond its capacity to maintain hydration levels stable, also being a sign of physiological distress (see Freire, 2025). Metabolic assays All experiments were conducted with constant aeration for 5 hours. Then, aeration was interrupted and saturation dissolved oxygen levels were immediately measured (mg/L) (oximeter YSI 55, YSI, Yellow Springs, USA). At the end of this last one hour, the final dissolved oxygen levels were again measured. The difference between these two values were used to calculate dissolved oxygen concentration per gram of the animal, according to: Magnitude of decrease in dissolved oxygen concentration (µgO 2 /g.weight.h) = (initial O 2 – final O 2 )*vial volume mL*1000/g crab weight High dissolved oxygen concentration indicates an increase in metabolism through the consumption of energy reserves, which leads to the need to search for more resources to maintain optimal internal conditions for survival. Lactate was determined in undiluted hemolymph samples under 550 nm (Labtest kit #138). Lactate analyses were performed on the same day and the storage time in the -20°C freezer was less than 4 hours. An increase in lactate concentration indicates activation of anaerobic metabolism or use of energy reserves to achieve energy demand. Before placing the crab in the experimental vial, a water sample was obtained for initial ammonia concentration values. After the 6 hours of experiment had elapsed, another water sample was taken for final ammonia levels. Ammonia concentration was determined using the Spectro Kit Ammonia Indotest (#2542, Alfakit, Florianópolis, Brazil), with absorbance read at 630 nm (as in Freire et al., 2020). High ammonia concentration in the water indicates an increase in amino acid metabolism, either to meet energy demands or using these amino acids to regulate osmolality. Statistical Analyses We performed a PCoA to detail the correlation between all variables, illustrating the distinct responses of the 2 crabs to the same set of challenges. Then we chose to do a principal component analysis (PCA) as an initial exploratory analysis for both species, considering the following variables: hemolymph osmolality, chloride, magnesium, lactate, muscle water content, dissolved oxygen concentration and excreted ammonia and then, a PERMANOVA. We evaluated two possibilities of inclusion of factors in the model: salinity x temperature and temperature x salinity and their interactions. When the PERMANOVA test revealed statistical differences (p ≤ 0.05), comparisons among conditions were performed, and differences among salinity and temperature conditions were evaluated separately. For this additional analysis, two-way ANOVAs (analysis of variance) were applied to the data, with “temperature” and “salinity” as fixed factors, when data met the requirements of normality and homogeneity of variances. When they did not, the Scheirer-Ray-Hare test was performed. For both tests, the tests of Tukey test were employed as post hoc test, to localize group differences. In order to directly compare the two species, Student’s t-test were used,or Mann-Whitney test, if data were not normally. Before the Scheirer-Ray-Hare test, the data were transformed in several ways: Ranks, Log10, Ln, exponential, square root. However, none of them were effective. We did not use size as a covariate (in an ANCOVA, for example) because we had already performed a t-test to compare the difference between the size of the two species, and the result was that there was no significant difference (P = 0.947). There are some studies that point out how we should not directly compare species, or else, consider “species” as a level of source of variation for any measured variable because they are not independent (Garland and Adolph 1994; Garland et al. 2005). These results are available in Table S1 until S12, in the supplementary material. Results The PCoA analysis conducted for both species demonstrated that the first two axes explain most of the variation found (43.01% and 22.3%, respectively) in relation to the physiological variables analyzed. Furthermore, the points related to Charybdis hellerii exposed to a temperature of 30°C are very distant from the others, evidencing the significant effect that temperature has on the measured variables (Fig. 1). The PCA analysis in Menippe nodifrons showed that the first axis explains 39.1% of the total variance, while the second axis explains 22.7%. Both axes present differentiated grouping between osmoregulation and metabolism variables. However, the osmoregulatory parameters (osmolality, chloride and magnesium) have high negative values on first axis. Conversely, the metabolic parameters (dissolved oxygen concentration, lactate synthesis and excreted ammonia) have high positive values on second axis (Fig. 2). The PCA analysis for C. hellerii was similar: the first axis accounted for 51.8% of the explained variance, while the value for the second axis was 16.6%. As observed for M. nodifrons , the variables associated with osmoregulation showed strong negative correlations on first axis and the metabolic variables showed strong positive correlations on second axis (Fig. 3). In the PERMANOVA analysis conducted for M. nodifrons , a significant effect of salinity (Pseudo-F = 23, p = 0.001) and time (Pseudo-F = 6, p = 0.015) on physiological parameters was observed. No significant interaction was detected between these two factors (Pseudo-F = 0, p = 0.776), as presented in Table 1. Salinity accounted for the majority of the variation among the groups (R² = 60%), whereas temperature explained only 5% of the observed variation. The lack of a significant interaction between salinity and temperature suggests that these factors operate independently in influencing the physiological responses. Table 1. Results for two-way permutational multivariate analysis of variance (PERMANOVA) between the Salinity and Temperature for physiological parameters of Menippe nodifrons d.f. SS MS Pseudo F R 2 P Salinity 3 309474.5691 103158.1897 23 0.599 0.001*** Temperature 1 24493.64932 24493.64932 6 0.047 0.015* Salinity:Temperature 3 6419.431802 2139.810601 0 0.012 0.776 Residuals 40 176175.4293 4404.385732 0.341 Total 47 516563.0795 10990.70382 1.000 d.f.: degrees of freedom. SS: sum of squares. MS: mean squares. Significance codes: *** p<0.001; ** p<0.01; *p< 0.05; . p<0.1 For C. hellerii , a significant interaction between temperature and salinity was observed in explaining the variation in the measured variables (Pseudo-F = 2, p = 0.047). Additionally, significant independent effects of temperature (Pseudo-F = 39, p = 0.001) and salinity (Pseudo-F = 43, p = 0.001) were detected, as presented in Table 2. Salinity accounted for the majority of the variation among the groups (R² = 60%), while time explained only 18% of the variation. The interaction between temperature and salinity contributed to 3% of the total variation observed. Table 2. Results for two-way permutational multivariate analysis of variance (PERMANOVA) between the Salinity and Temperature for physiological parameters of Charybdis hellerii d.f. SS MS Pseudo F R 2 P Salinity 3 391335.2579 130445.086 43 0.600 0.001*** Temperature 1 119292.8907 119292.8907 39 0.183 0.001*** Salinity:Temperature 3 20750.57404 6916.858012 2 0.032 0.047* Residuals 40 121223.8568 3030.596419 0.186 Total 47 652602.5795 13885.16126 1.000 d.f.: degrees of freedom. SS: sum of squares. MS: mean squares. Significance codes: *** p<0.001; ** p<0.01; *p< 0.05; . p<0.1 Both crab species displayed decreased hemolymph osmolality when exposed for 6 hours to decreasing salinities (Fig. 4A). Acclimation to a higher temperature resulted in enhanced decrease in hemolymph osmolality in both species at salinities 25 and 20‰. Comparatively, the non-native C. hellerii displayed even lower hemolymph osmolality than M. nodifrons under both thermal and hypo-saline challenges (Fig. 4A). Compatibly, muscle hydration was less controlled by the non-native C. helleri , which displayed higher levels of tissue hydration when compared to the more stable levels observed in the native M. nodifrons (Fig. 4B), across all experimental salinities (results of the statistical tests are presented in Supplementary Tables S1, S2, S6 and S7). The response of hemolymph chloride to reduced salinities followed the same pattern observed above for osmolality (Fig. 5A), again, in general, with lower values for C. hellerii than those of M. nodifrons . For M. nodifrons , acclimation to a warmer temperature led to increased hemolymph chloride at salinity 30‰ (Fig. 5A and Supplementary Table S1). Decreased salinities also led to reduced levels of hemolymph magnesium (Fig. 5B and Supplementary Table S1). Comparatively, C. hellerii displayed lower hemolymph magnesium concentration than M. nodifrons when warm-acclimated, for all experimental salinities (Fig. 5B). Acclimation to raised water temperature resulted in significant decrease in dissolved oxygen concentration, especially for C. helleri , at all salinities. Dissolved oxygen concentration was markedly lower for the native M. nodifrons . In the native species, the effect of warm acclimation was noted in salinities 30 and 20‰ (Fig. 6A). Dissolved oxygen concentration was higher in C. hellerii than in M. nodifrons , specifically upon acclimation to increased temperature and at the salinities of 30 and 25‰ (Fig. 6A). Warming caused elevated hemolymph lactate; in C. hellerii , at 20‰, but in M. nodifrons at 25 and 20‰. Lactate was higher in the invasive C. helleri than in M. nodifrons at the control temperature, at 25‰ (Fig. 6B). Acclimation to warmer temperature also led to increased ammonia excretion in both species, and essentially for all salinities (Fig. 6C), again with higher levels noted for the non-native C. hellerii than for the native M. nodifrons , in particular in the lower salinities (Fig. 6C). Results of the statistical tests are presented in Supplementary Tables S1, S2, S6 and S7). Discussion Climate change may interfere, either enhancing or attenuating biological invasion processes. Using physiological tools, we assessed whether warming, associated to a common estuarine challenge - seawater dilution - would distinctly affect the invasive species Charybdis helleri , when compared to the native and sympatric Menippe nodifrons . The findings demonstrate that when exposed only to hyposalinity, both species have similar responses. However, when the temperature factor is introduced along with hyposalinity, the non-native species has, surprisingly, a lower physiological performance. This trend is evidenced by the dispersion of points representing C. hellerii in the PCoA graph, in a distinct pattern from the result obtained with the native crab (Fig. 1). Both species experienced hemolymph osmolality dilution as a result of exposure to lower salinities at both temperatures. A recently published article reported that C. helleri can regulate its extracellular fluid when external salinity decreases; when exposed to higher salinities, its extracellular fluid matches the external one, as expected in general for estuarine brachyuran crabs (Occhi et al. 2019). However, C. hellerii has a lower hemolymph osmolality regulatory ability than other portunids that inhabit the same region, such as Callinectes danae and C. ornatus (Freire et al. 2011, 2020; Rios and Freire 2023). A direct comparison with these Callinectes species highlights the evidently lower osmoregulatory capacity of C. hellerii . Under the most challenging condition, warming (30°C) and lowest salinity (20‰, equivalent to ~600 mOsm/kg.H 2 O), these species present the following osmolality values: 788±10 mOsm/kg.H 2 O in C. danae , 766±28 mOsm/kg.H 2 O in C. ornatus , and 701±26 mOsm/kg.H 2 O in C. helleri (personal observation, unpublished data). Thus, the 3 species mentioned can maintain a higher internal osmolality than the external environment, but C. hellerii is the least efficient. While comparing these species is pertinent (all three belong to the Portunidae family and cohabit the same region in southwestern Atlantic), C. helleri is not a “swimming crab” as the Callinectes crabs, belongs to distinct genus, and is ecologically closer to species of semi-terrestrial decapods, such as M. nodifrons (Santana et al. 2009; Negri et al. 2018; Occhi et al. 2019; Stanski et al. 2022) . Muscle hydration remained stable in both species at 26 °C, but muscle tissues of C. helleri displayed increased hydration in the individuals acclimated to the higher temperature. This means that the invasive species was unable to control the influx of water from the external environment into the extracellular environment of the muscle tissue, the hemolymph. Similar results were found in Clibanarius taeniatus and Clibanarius virescens , two hermit crab species from intertidal areas with ecological similarities to C. hellerii and M. nodifrons . Dunbar and Coates (2004), exposed these crabs to rising temperatures and decreasing salinity over 6 hours. Both species showed increased body weight, indicating tissue hydration increase. In addition, it had been shown that C. hellerii shows a similar tendency for water uptake in muscle tissue when exposed to gradual salinity reductions (30‰ to 20‰ and 10‰) over 24 hours, indicating sensitivity to prolonged hypo-saline conditions (Occhi et al. 2019). The ion regulation patterns are compatible with the osmolality results: C. hellerii and M. nodifrons behave similarly and there is hemolymph dilution in chloride and magnesium concomitant with seawater dilution, especially from 35 to 30‰, with levels remaining more stable upon further seawater dilution. However, warming leads to improved chloride stability in M. nodifrons , but further decreased values in C. hellerii , again highlighting the higher regulatory capacity of M. nodifrons . As for hemolymph magnesium, its levels remain stable in the lowest salinities in M. nodifrons , unaffected by warming, while again, warming causes further decreases in C. hellerii . Temperature increase can affect the ionic regulation capacity of crustaceans in different ways, either increasing, decreasing, or not affecting this regulation, with no consistent trend observed in previous studies (Weber and Spaargaren 1970; Diaz et al. 2004; Dobrzycka-Krahel et al. 2014; Borecka et al. 2016). In our study, PERMANOVA analysis indicates that the combination of temperature and salinity affected only the non-native species C. helleri : thus, a synergistic effect of both stressors on the invasive crab. The influence of warming combined with hyposalinity on the ionic regulation of decapods remains relatively unexplored. For example, the hermit crab species C. taeniatus and C. virescens have osmoregulatory patterns similar to M. nodifrons and C. helleri. Upon increased temperature and decreased salinity, the hermit crabs have impaired ionic regulatory capacity, resulting in significant hemolymph dilution and decrease in the concentration of the main ions sodium, potassium, calcium and magnesium (Dunbar and Coates 2004) . All three proxies for metabolic activation, reduction in dissolved oxygen (putatively implying oxygen consumption by the aerobic metabolism of the crab), hemolymph lactate production and ammonia excretion were very distinctively more activated in the combination of warming and seawater dilution, in the non-native C. hellerii , than in the native M. nodifrons, as demonstrated by the PCA plots (Fig. 2 and 3). Salinity did not strongly influence the reduction in water dissolved oxygen pattern in both species; however, a synergistic effect of warming with hyposalinity was observed, resulting in metabolic activation, especially in the non-native C. hellerii . Increase in aerobic metabolism – in our case here, a much more intense decrease in dissolved oxygen upon warming in the experimental vials containing the invasive crab - from this combination of stressors is a general outcome in estuarine decapods - of variable lineages and osmoregulatory capacities. A similar response of increased oxygen consumption when exposed to warming with hyposalinity was observed in the crab Scylla serrata (Chen and Chia 1996), and penaeid shrimps Litopenaeus stylirostris (Diaz et al. 2004), Marsupenaeus japonicus (Chen and Lai 1993; Setiarto et al. 2004), and the palaemonid prawns Palaemon peringueyi (Allan et al. 2006), Palaemon macrodactylus, Palaemon longirostris and Palaemonetes varians (Lejeusne et al. 2014). We must acknowledge that we did not use a closed respirometer, and thus our oxygen reduction data should not be directly ascribed to consumption by the animal, and we prefer not to calculate Q10 values here, for caution. However, given that crabs from both species remained still during the experimental period inside the experimental vials, we can propose that the sharp difference in the pattern of reduction in dissolved oxygen during the hour of measurement between the two species reflects metabolic activation (upon warming) to a much higher degree in the invasive species C. helleri than in the native M. nodifrons" , also supported by ammonia and lactate data . Remarkably elevated Q₁₀ values (reaching 4.9) and other physiological indicators of enhanced metabolic activity were documented in juvenile Scylla paramamosain under thermal stress conditions (20, 25, 30, and 35°C) (Liu et al., 2022). Notably, such pronounced Q₁₀ values may represent a physiological signature of recent invasion events, signifying the initial acclimatization phase of invasive species to novel environmental conditions, reflecting thermal sensitivity in the new environment (Keller & Taylor, 2008; Boardman et al., 2022; Marochi et al. 2024). This recent invasion is further evidenced by the standard deviation values obtained from dissolved oxygen analyses, which were significantly higher in the invasive species compared to the native species. (Supplementary Tables S1), also a sign of high degree of stress. This possibility of more intense activation of metabolism in initial phases of biological invasion should be further examined in the literature. Lactate synthesis can occur due to the absence of available oxygen or function as an auxiliary energy substrate when energy demand is not fully met by aerobic respiration (Sherwood et al. 2013; Spicer 2014; Lee et al. 2023). The increase in lactate concentration in crustaceans occurs in response to physical activity, exposure to hypoxia or anoxia, reduced salinity or increased temperature (Lorenzon et al. 2007; Maciel et al. 2008; Jost et al. 2012; Sherwood et al. 2013; Freire et al. 2017, 2020). Although there are no records of how the combined effect of temperature and salinity change affects lactate production in crustaceans, our study demonstrates that the non-native species C. hellerii activates anaerobic metabolism as well, when challenged with warming and seawater dilution, and to a much higher degree than the native crab. Moreover, the increased lactate production in the invasive crab when compared to the native crab confirms our data on concomitant reduction of dissolved oxygen, thus strengthening the idea of metabolic activation due to stress of warming. Both species demonstrated increased ammonia excretion into water upon warming and seawater dilution challenge. But again, this increase in excretion upon warming combined with seawater dilution was much more pronounced in C. hellerii than in M. nodifrons . Increased water ammonia must come from the crab. Thus, ammonia data confirms oxygen data, allowing us to make the point that warming strongly results in metabolic activation of the invasive crab, when compared to the native species. Reviews on decapod crustaceans suggest that ammonia excretion tends to rise as salinity decreases (Weihrauch 2004; Henry et al. 2012; Romano and Zeng 2013; Leone et al. 2017). Ammonia is the primary nitrogenous compound excreted by decapod crustaceans in freshwater and even in brackish waters, primarily produced through catabolism of amino acids (Larsen et al. 2014). In osmoregulatory species, the free amino acids generated are also used during the osmoregulation process (Strefezza et al. 2019). In relation to non-native crustacean species, the literature offers diverse examples: while amphipod invaders like Crangonyx pseudogracilis and Dikerogammarus villosus appear more sensitive to high ammonia concentrations, the crayfish Procambarus clarkii exhibits mechanisms to counteract ammonia toxicity (Prenter et al. 2004; Gergs et al. 2013; Normant-Saremba et al. 2015; Shen et al. 2021). Estuarine non-native crustacean species, such as Carcinus maenas, Hemigrapsus takanoi, Hemigrapsus sanguineus and Charybdis japonica showcase high ammonia excretion rates, probably due to the increase in metabolism resulting from the experimental treatment (Watanabe et al. 2009; Fowler et al. 2011; Fehsenfeld and Weihrauch 2016; Landeira et al. 2020). It is evident that C. hellerii requires a greater energy supply to cope with the stress caused by the synergistic effect of the combination of warming and decreased seawater salinity. This reduces the scope for activity directed towards other functions, as a trade-off, decreasing invasive power under these conditions (Freire and Sampaio 2021; Rato et al. 2021; Marochi et al. 2024). Although C. hellerii exhibits certain apparent physiological disadvantages compared to M. nodifrons , particularly in light of potential future climate change scenarios, it is important to acknowledge that other ecological factors within the Paranaguá Estuarine Complex also play critical roles. These factors are likely to be key determinants in the persistence or decline of C. hellerii population. Currently, C. hellerii population is considered established in the Paranaguá Estuarine Complex (Metri et al. 2020). However, based on the capture locations of these individuals, this population appears to be restricted to rocky shores and the euhaline zone, where salinity exceeds 30 ‰ (Lana et al. 2001; Marone et al. 2005; Occhi et al. 2019). Interspecific competition may be one factor contributing to this restriction. The preference for rocky shores as the primary habitat for C. hellerii could be a strategy to avoid competition for space and resources with other portunids in the region, such as C. danae and C. ornatus , which have larger populations and are potential predators of C. hellerii (Sant’Anna et al. 2012). The presence of predators can limit the distribution and prevent the establishment of invasive species (Ruesink 2007; Silva et al. 2018; Rato et al. 2021). On the other hand, it is common for predators not to recognize invasive species as potential prey (Llewelyn et al. 2010; Silva et al. 2018). In Brazil, there are few reports of C. hellerii being predated by native species, with the octopus species Octopus vulgaris (Sampaio and Rosa 2006), Octopus insularis (Silva et al. 2018) and the goldspotted snake eel Myrichthys ocellatus (Siqueira et al. 2021) being notable exceptions. Of these species, only Octopus vulgaris and Myrichthys ocellatus are found along the coast of Paraná state (Hackradt and Félix-Hackradt 2009; Amado et al. 2015). Reproductive aspects can also mitigate the impact of restricted physiological performance. Although C. hellerii exhibits lower reproductive potential (producing fewer eggs per spawning event) compared to M. nodifrons and other local portunids, such as C. danae and C. ornatus , its reproductive period remains constant, whereas the aforementioned species experience reproductive peaks during spring and summer (Mantelatto and Garcia 2001; Sant’Anna et al. 2015; Marochi et al. 2021). This continuous reproductive strategy reduces competition for resources and enhances the potential for population expansion. Moreover, in future climate change scenarios, particularly with rising temperatures, M. nodifrons may be more severely impacted. A study by Marochi et al. (2021) concluded that increased temperatures could reduce larval survival in M. nodifrons . Additionally, M. nodifrons larvae exhibit a low capacity for acclimatization to warming waters (Marochi et al. 2024). While similar studies on C. hellerii are lacking, other species of the same genus have demonstrated high larval survival rates under the salinity and temperature conditions used in this study, such as: Charybdis feriatus (Baylon and Suzuki 2007; Soundarapandian et al. 2010) and C. japonica (Fowler et al. 2011). Another notable factor is the potential overlap in the diets of C. hellerii and M. nodifrons , which may lead to competition for food resources. Non-native species often display greater voracity than their native counterparts, giving them a competitive advantage in acquiring food (Sant’Anna et al. 2012, 2015; Siqueira et al. 2021). In this case, both species primarily feed on other invertebrates, particularly low-mobility organisms such as mollusks (Izar et al. 2023). However, in C. hellerii , feeding preferences are influenced by predator density. At moderate densities (30 crabs/m³), the preference is for mollusks, but at higher densities (60 crabs/m³), the preference shifts towards other crabs (Izar et al. 2023).The enhanced ability of the non-native species to acquire food resources may compensate for its lower physiological performance, providing the energy required for osmoregulation and other physiological functions. As a result, this dynamic poses a potential threat to the viability of M. nodifrons populations. Still another feature to be mentioned is that the lower physiological performance of the non-native species may be related to the fact that its invasion is relatively recent. For instance, while the invasive crab Eriocheir sinensis began its invasion of the United Kingdom in the 1970s, its population did not experience significant growth and expansion until the 1990s (Hänfling et al. 2011). Thus, it is plausible that C. hellerii has not yet fully optimized its fitness in an environment that differs from its native habitat (see Lee and Bell 1999, for freshwater invasion). Similarly, the first records of C. hellerii in the São Paulo state (a region just north of the Paranaguá estuarine complex) date back to 1995 (Negri et al. 2018). Less than two decades later, its population had already become the second most abundant among crustaceans inhabiting rocky shores, surpassed only by M. nodifrons (Sant’Anna et al. 2012; Izar et al. 2023). This highlights the importance of continued monitoring of C. hellerii populations in the coming years to better understand potential adaptations and ecological dynamics. Although no published studies have analyzed the population dynamics of C. hellerii in the Paranaguá Estuarine Complex (PEC), other factors beyond poor physiological performance may limit local expansion. One of them is the limitation of the distribution range of this species, which shows that C. hellerii is at the southern limit of its distribution. Tropical invasive species often face challenges in establishing populations in higher-latitude regions with greater species diversity, which can hinder the growth and expansion of C. hellerii populations, as demonstrated by Sant’Anna et al. (2015) at Armação do Itapocoroy, Santa Catarina state (a region just South of the Paranaguá estuarine complex). Conclusion The use of physiological tools within an ecological framework, presents a complementary and mandatory approach for examining the novel interactions between non-native and native species in environments where these species did not co-occur naturally before human action (see Boardman et al., 2022). Biological invasions and current and future climate change will certainly be among the most important drivers accounting for future ecosystems. In this study, we opted for osmoregulatory and metabolic approaches to analyze the physiological performance of two species of decapod crustaceans, one non-native species and one native species, which already co-occur along an estuarine complex in southern Brazil. The findings reveal that, although the two species exhibit similar responses under saline stress alone, the introduction of temperature as a factor alongside salinity yields divergent results due to a synergistic effect. Specifically, the non-native species C. hellerii shows inferior physiological performance compared to the native species M. nodifrons , which is not usually the expected result. However, despite this lower physiological performance, other ecological traits, such as feeding strategies and reproductive patterns, may provide compensatory advantages for C. hellerii . Moreover, the physiological data generated can contribute to comparative studies in areas with analogous ecological characteristics undergoing biological invasion processes. These insights can also be incorporated into future models aimed at predicting the impacts of climate change on local populations and understanding the dynamics of coexistence between non-native and native species in shared environments. Ongoing monitoring of both C. hellerii and M. nodifrons populations is crucial for understanding their long-term viability and the broader ecological consequences of this invasion. Declarations Acknowledgments and Funding Authors gratefully acknowledge the financial support by CAPES (Federal Government of Brazil) through a PhD fellowship to LPR, and CNPq through Research Grants to CAF (# 302829/2015-6 and 307760/2019-7). Retrieval of crabs from nature for this study was authorized by ICMBio/SISBIO (Ministry of Environment, Brazil), number 20030, renewed annually, issued to CAF. Author Contributions Both authors contributed to the conception and design of the study. LPR performed the experiments. Data, statistics, and conclusions were extensively and comprehensively discussed by both authors. The initial draft of the manuscript was written by LPR and underwent thorough edition by CAF. Both authors concur with the final submitted version. Declarations Conflict of interests The authors declare that there are no conflicts of interest. Ethics approval Ethical approval was deemed unnecessary for the nature of this study, but experimental animals were, nonetheless treated with care and respect, and were submitted to cold-anesthesia before euthanasia. References Allan EL, Froneman PW, Hodgson AN (2006) Effects of temperature and salinity on the standard metabolic rate (SMR) of the caridean shrimp Palaemon peringueyi . 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Brooks/Cole, Belmont, USA Silva EJD, Bezerra LEA, Xavier I (2018) The tropical Octopus insularis (Mollusca, Octopodidae): a natural enemy of the exotic invasive swimming crab Charybdis hellerii (Crustacea, Portunidae) Siqueira MA, Vieira MLM, Moraes D, et al (2021) Predation on the invasive swimming crab Charybdis hellerii (Crustacea, Decapoda) by Myrichthys ocellatus (Actinopterygii, Ophichthidae): the first record of consumption by a native fish. Neotropical Biodivers 7:155–159. https://doi.org/10.1080/23766808.2021.1920298 Soares MO, Xavier FRDL, Dias NM, et al (2022) Alien hotspot: Benthic marine species introduced in the Brazilian semiarid coast. Mar Pollut Bull 174:113250. https://doi.org/10.1016/j.marpolbul.2021.113250 Soundarapandian P, Ilavarasan N, Varadharajan D, et al (2010) Effect of Salinity on Growth and Survival of Portunid Crab, Charybdis eriata Larvae. Int. J. Pharm. Biol. Arch. 4(1): 150-156 Spicer JI (2014) What can an ecophysiological approach tell us about the physiological responses of marine invertebrates to hypoxia? J Exp Biol 217:46–56. https://doi.org/10.1242/jeb.090365 Stanski G, Boos H, Amaro Pinheiro MA (2022) Animais marinhos exóticos invasores no Sul do Brasil. Rev CEPSUL - Biodiversidade E Conserv Mar 11:e2022002. https://doi.org/10.37002/revistacepsul.vol11.2336e2022002 Strefezza TF, De Andrade IM, Augusto A (2019) Reduced pH and elevated salinities affect the physiology of intertidal crab Minuca mordax (Crustacea, Decapoda). Mar Freshw Behav Physiol 52:241–254. https://doi.org/10.1080/10236244.2019.1681898 Wallingford PD, Morelli TL, Allen JM, et al (2020) Adjusting the lens of invasion biology to focus on the impacts of climate-driven range shifts. Nat Clim Change 10:398–405. https://doi.org/10.1038/s41558-020-0768-2 Walther G-R, Roques A, Hulme PE, et al (2009) Alien species in a warmer world: risks and opportunities. Trends Ecol Evol 24:686–693. https://doi.org/10.1016/j.tree.2009.06.008 Watanabe S, Wilder MN, Strüssmann CA, Shinji J (2009) Short-Term Responses of the Adults of the Common Japanese Intertidal Crab, Hemigrapsus Takanoi (Decapoda: Brachyura: Grapsoidea) at Different Salinities: Osmoregulation, Oxygen Consumption, and Ammonia Excretion. J Crustac Biol 29:269–272. https://doi.org/10.1651/08-2998R.1 Watanabe TT, López-Greco LS, Zara FJ (2022) Seminal fluid and spermatophore production in a western Atlantic invasive swimming crab, Charybdis hellerii , reveals a different pattern to Portunoidea. Arthropod Struct Dev 66:101137. https://doi.org/10.1016/j.asd.2021.101137 Watanabe TT, Sant’Anna BS, Hattori GY, Zara FJ (2014) Population biology and distribution of the portunid crab Callinectes ornatus (Decapoda: Brachyura) in an estuary-bay complex of southern Brazil. Zool Curitiba 31:329–336. https://doi.org/10.1590/S1984-46702014000400004 Weber RE, Spaargaren DH (1970) On the influence of temperature on the osmoregulation of crangon crangon and its significance under estuarine conditions. Neth J Sea Res 5:108–120. https://doi.org/10.1016/0077-7579(70)90007-4 Weihrauch D (2004) Ammonia excretion in aquatic and terrestrial crabs. J Exp Biol 207:4491–4504. https://doi.org/10.1242/jeb.01308 Supplementary Files SupplementarymaterialBINVD2400674.docx Cite Share Download PDF Status: Published Journal Publication published 09 Jan, 2026 Read the published version in Biological Invasions → Version 1 posted Reviewers agreed at journal 08 Apr, 2025 Reviewers invited by journal 08 Apr, 2025 Editor invited by journal 31 Mar, 2025 Editor assigned by journal 29 Mar, 2025 First submitted to journal 28 Mar, 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5595652","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":440260097,"identity":"e1a494f6-4c38-4fd4-8672-7fe308e9abba","order_by":0,"name":"Leonardo de Paula Rios","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYBACAwYGxgcJBjZyIM6BB0RqYTb4UJBmDNaSQKQWNskZHw4nNoB4xGmR7n0gzWPAnD4/7PBDoC12croNhLTIHDcw5jFgy914O80AqCXZ2OwAIS0SaQzJPAY8uRtnJ4C0HEjcRoyWwzwGEumGs9M/EK2FsXGGgUGCvHQOsbbIHGNm+GCQYLhBOqfgQIIBEX6xn93G/iPhz395+dnpmz98qLCTI6iFQQJmHVilASHlyFrkG4hRPQpGwSgYBSMSAADPi0MkDIfyxwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-9269-0640","institution":"Associação MarBrasil","correspondingAuthor":true,"prefix":"","firstName":"Leonardo","middleName":"de Paula","lastName":"Rios","suffix":""},{"id":440260098,"identity":"7e15035e-f109-4fa7-a2fa-cedf8606290d","order_by":1,"name":"Carolina A. Freire","email":"","orcid":"","institution":"Universidade Federal do Parana","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"A.","lastName":"Freire","suffix":""}],"badges":[],"createdAt":"2024-12-06 18:50:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5595652/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5595652/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10530-025-03747-6","type":"published","date":"2026-01-09T15:59:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80341279,"identity":"7cfb3b91-4768-4685-b1db-8abca87051a2","added_by":"auto","created_at":"2025-04-10 17:54:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114688,"visible":true,"origin":"","legend":"\u003cp\u003eResponses of both crab species to a same set of environmental challenges, demonstrated through a PCoA (Principal Coordinates Analysis). The non-native \u003cem\u003eCharybdis hellerii\u003c/em\u003e and the native \u003cem\u003eMenippe nodifrons\u003c/em\u003e were acclimated to 26 °C (control) and 30 °C, and then exposed for 6 hours to salinities 35‰ (control), 30‰, 25‰ and 20‰.\u003c/p\u003e","description":"","filename":"Fig1PCoA.png","url":"https://assets-eu.researchsquare.com/files/rs-5595652/v1/f7cc77098281b2a06430249a.png"},{"id":80341280,"identity":"f253d2bd-3edf-4f42-8c6e-7958c3c5ea4e","added_by":"auto","created_at":"2025-04-10 17:54:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1302129,"visible":true,"origin":"","legend":"\u003cp\u003ePCA analysis demonstrating the visualization of complex patterns in multivariate data, in relation to the following physiological variables:(Cl, chloride; Lac, lactate; Mg, magnesium; MWC, muscle water content; NH3, excreted ammonia; Oc, dissolved oxygen concentration; Osm, osmolality) throughout salinity and temperature in \u003cem\u003eMenippe nodifrons\u003c/em\u003e. Axis PC1 explained 39.1% of the variation, and axis PC2 explained 22.7% of total data variation.\u003c/p\u003e","description":"","filename":"Fig2pcaMenippe.png","url":"https://assets-eu.researchsquare.com/files/rs-5595652/v1/b3b7711303e8964c06bfd6d9.png"},{"id":80341281,"identity":"dca7cd38-eae2-4f8f-a44e-0eafcf138a86","added_by":"auto","created_at":"2025-04-10 17:54:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1284789,"visible":true,"origin":"","legend":"\u003cp\u003ePCA analysis demonstrating the visualization of complex patterns in multivariate data, in relation to the following physiological variables: (Cl, chloride; Lac, lactate; Mg, magnesium; MWC, muscle water content; NH3, excreted ammonia; Oc, dissolved oxygen concentration; Osm, osmolality) throughout salinity and temperature in \u003cem\u003eCharybdis hellerii\u003c/em\u003e. Axis PC1 explained 51.8% of the variation, and axis PC2 explained 16.6% of total data variation.\u003c/p\u003e","description":"","filename":"Fig3pcaCharydis.png","url":"https://assets-eu.researchsquare.com/files/rs-5595652/v1/312e2b1e6f145d5449bb7645.png"},{"id":80341640,"identity":"c253f5b4-76df-4e60-93d4-f6c8787d5a56","added_by":"auto","created_at":"2025-04-10 18:02:25","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":570098,"visible":true,"origin":"","legend":"\u003cp\u003eMarkers of stability in hemolymph concentration and muscle hydration.\u003cstrong\u003e \u003c/strong\u003eHemolymph osmolality (A) and Muscle water content (B) of the non-native \u003cem\u003eCharybdis hellerii\u003c/em\u003e and the native \u003cem\u003eMenippe nodifrons\u003c/em\u003e acclimated to 26 °C (control) and 30 °C, and then exposed for 6 hours to salinities 35‰ (control), 30‰, 25‰ and 20‰ for 6 hours *: effect of acclimation to a higher temperature (26 x 30 °C) for each species; \u0026amp;: difference between experimental salinities and control (35‰); ≠: difference between species in their respective experimental condition; the grey line indicates isosmotic point; n = 6 (values according to Prosser, 1973 for reference seawater).\u003c/p\u003e","description":"","filename":"Fig4cap3boxrebuttal01.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5595652/v1/087956768aa9dc794d3e3364.jpg"},{"id":80341639,"identity":"22cbf54f-c5df-4104-80b2-d0bcad276aa5","added_by":"auto","created_at":"2025-04-10 18:02:25","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":547040,"visible":true,"origin":"","legend":"\u003cp\u003eMarkers of stability in hemolymph ionic concentrations. Hemolymph chloride (A) and magnesium concentrations (B) of the non-native \u003cem\u003eCharybdis hellerii\u003c/em\u003e and the native \u003cem\u003eMenippe nodifrons\u003c/em\u003e acclimated to 26 °C (control) and 30 °C, and then exposed for 6 hours to salinities 35‰ (control), 30‰, 25‰ and 20‰ for 6 hours *: effect of acclimation to a higher temperature (26 x 30 °C) for each species; \u0026amp;: difference between experimental salinities and control (35‰); ≠: difference between species in their respective experimental condition; the grey line indicates isosmotic point; n = 6 (values according to Prosser, 1973 for reference seawater).\u003c/p\u003e","description":"","filename":"Fig5cap3boxrebuttal01.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5595652/v1/fe0b490ee8cb2e2988410ac7.jpg"},{"id":80341284,"identity":"53e5aa4f-743f-4465-90b4-4a965e39868a","added_by":"auto","created_at":"2025-04-10 17:54:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2858656,"visible":true,"origin":"","legend":"\u003cp\u003eMarkers of metabolic activity.\u003cstrong\u003e \u003c/strong\u003eMagnitude of decrease\u003cstrong\u003e \u003c/strong\u003ein dissolved oxygen concentration\u003cstrong\u003e \u003c/strong\u003e(A), lactate concentration (B), and ammonia excreted into the water (C) for the non-native \u003cem\u003eCharybdis hellerii\u003c/em\u003e and the native \u003cem\u003eMenippe nodifrons\u003c/em\u003e acclimated to 26 °C (control) and 30 °C, and then exposed for 6 hours to salinities 35‰ (control), 30‰, 25‰ and 20‰ for 6 hours.*: effect of acclimation to a higher temperature (26 x 30 °C) \u0026nbsp;for each species; \u0026amp;: difference between experimental salinities and control (35‰); ≠: difference between species in their respective experimental condition; n = 6.\u003c/p\u003e","description":"","filename":"Fig6cap3boxrebuttal01.png","url":"https://assets-eu.researchsquare.com/files/rs-5595652/v1/63af36d468e9052dab701e67.png"},{"id":100069455,"identity":"3eea72d5-e872-4250-91d6-08ba07a1a960","added_by":"auto","created_at":"2026-01-12 16:14:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7337723,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5595652/v1/5255c5da-2bbb-4907-968a-96edb0b1c1c1.pdf"},{"id":80341294,"identity":"14effc28-5b8a-4f69-bde3-1bbbcc0ff748","added_by":"auto","created_at":"2025-04-10 17:54:25","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":170404,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialBINVD2400674.docx","url":"https://assets-eu.researchsquare.com/files/rs-5595652/v1/d69e8e7bdf642848e547c7a2.docx"}],"financialInterests":"","formattedTitle":"Marine brachyuran crabs’ osmoregulatory and metabolic responses upon warming and seawater dilution challenges: the non-native Charybdis helleri is more sensitive than the native Menippe nodifrons.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eClimate change and biological invasions constitute especially relevant threats to biodiversity worldwide (Walther et al. 2009; Wallingford et al. 2020; Robinson et al. 2020; King et al. 2021). Despite their significance, their effects are commonly examined in isolation, although there is data in the literature that points to a close relationship between climate change and an increase in the pace and intensity of biological invasions (Dukes and Mooney 1999; Walther et al. 2009; Christensen et al. 2020; Ricciardi et al. 2021; Cuthbert and Briski 2021). Species have experienced population geographical shifts in recent decades, attributed to rising temperatures in their original habitats, and frequently causing invasion of new habitats (Parmesan 2006; Wallingford et al. 2020; Rubenstein et al. 2023). In addition, climate change may alter the likelihood of introduction and adaptive success of non-native species (Walther et al. 2009; Blackburn et al. 2011; Giangrande et al. 2020; Robinson et al. 2020; King et al. 2021;\u0026nbsp;\u0026nbsp;Ricciardi et al. 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHeatwaves, in particular, can trigger massive die-offs of native species, creating vacant ecological niches which invasive species can exploit (Occhipinti-Ambrogi 2007). In addition to facilitating the escape, introduction, and expansion of the range of invasive species, climate change and extreme events may alter abiotic conditions, including temperature, salinity, pH, and oxygen availability of local habitats. For example, Sanford et al. (2014) examined how ocean acidification caused by elevated CO\u003csub\u003e2\u003c/sub\u003e in Tomales Bay, California (USA) might increase the vulnerability of native Olympia oysters to predation by invasive snails. Their experiment showed that 48% more oysters were consumed under high CO\u003csub\u003e2\u003c/sub\u003e conditions. Smaller oysters were predated more than larger oysters, highlighting how environmental stress could amplify the impact of invasive predators. These changes in \u0026nbsp;temperature and CO\u003csub\u003e2\u003c/sub\u003e levels\u0026nbsp;can synergistically increase the stress to which aquatic populations are exposed, directly impacting ecosystem health and negatively affecting human activities such as fishing (Occhipinti-Ambrogi 2007; Robinson et al. 2020; Ricciardi et al. 2021; Rubenstein et al. 2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn southern Brazil, the only non-native decapod crustacean species established along the Estuarine Complex of Paranaguá, Paraná state (ECP) is the portunid crab \u003cem\u003eCharybdis hellerii\u003c/em\u003e (A. Milne-Edwards, 1867). It is thought to have been introduced through the dispersal of larvae by the release of ballast water from cargo ships (Mantelatto and Dias 1999; Soares et al. 2022; Cintra et al. 2023). This species originates from the Indo-Pacific region, where it occurs in estuaries, intertidal regions, under rocks or in corals \u0026nbsp;(Watanabe et al. 2014; Sant’Anna et al. 2015; Negri et al. 2018). It is currently distributed along the east coast of America (from the United States to southern Brazil), as well as some regions of the Mediterranean Sea\u0026nbsp;(Negri et al. 2018; Siqueira et al. 2021). The introduction of this species into ecologically sensitive areas like Brazilian estuarine areas can have severe consequences, as \u003cem\u003eC. hellerii\u003c/em\u003e competes with native species for habitat and food\u0026nbsp;(Sant’Anna et al. 2012, 2015; Watanabe et al. 2022). In some Brazilian states such as São Paulo and Santa Catarina (regions adjacent to the ECP), \u003cem\u003eC. hellerii\u003c/em\u003e has outnumbered some local crab populations\u0026nbsp;(Sant’Anna et al. 2012, 2015; Siqueira et al. 2021). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the ECP, \u003cem\u003eC. hellerii\u003c/em\u003e co-occurs with the native species \u003cem\u003eMenippe nodifrons\u0026nbsp;\u003c/em\u003e(Menippidae), a species found along the Brazilian coast, and both inhabit rocky shores, reefs, shallow water beaches and mid-coastal regions\u0026nbsp;(Santana et al. 2009; Sant’Anna et al. 2012; Occhi et al. 2019). The distribution of \u003cem\u003eM. nodifrons\u003c/em\u003e overlaps with the environments recently colonized by \u003cem\u003eC. hellerii\u003c/em\u003e. In non-native areas, \u003cem\u003eC. hellerii\u003c/em\u003e has been found inhabiting rocks, corals, mud and sand bottoms, intertidal and subtidal regions (Stanski et al. 2022). In addition to its ecological importance, \u003cem\u003eM. nodifrons\u003c/em\u003e serves as a fishing resource in certain regions of the northern hemisphere and in parts of Brazil, including the ECP \u0026nbsp;(Oshiro 1999; Bertini et al. 2007), while \u003cem\u003eC. hellerii\u003c/em\u003e is not (yet) a target of artisanal fishing for consumption in this region of the Brazilian coast.\u003c/p\u003e\n\u003cp\u003ePhysiological tolerance plays a crucial role in the competition between native and non-native species. Species which are more sensitive to environmental fluctuations, such as in pH, dissolved oxygen, temperature and salinity, may face exclusion or replacement by other, more tolerant species.\u0026nbsp;Multiple environmental stressors can elicit three distinct types of effects on organisms: synergistic, additive, or antagonistic. A synergistic effect arises when the combined influence of two or more stressors produces an impact that exceeds the sum of their individual effects. An additive effect is observed when the cumulative impact of multiple stressors equals the sum of their individual effects, indicating no significant interaction that either amplifies or diminishes their influence. Conversely, an antagonistic effect occurs when the combined impact of two or more stressors is less than the sum of their individual effects, suggesting an interaction that mitigates or partially neutralizes their overall influence\u0026nbsp;(Dinh et al. 2022).\u0026nbsp;Therefore, the comparison of physiological responses between a native and a non-native sympatric species to saline and thermal challenges can contribute to predictions of ecological impact of invasion (Christensen et al. 2020; Ricciardi et al. 2021; Cuthbert and Briski 2021; Boardman et al., 2022). We hypothesize that the non-native species \u003cem\u003eC. helleri\u003c/em\u003e is more tolerant to warming and seawater dilution than the native \u003cem\u003eM. nodifrons,\u0026nbsp;\u003c/em\u003emainly due to the invasive potential of \u003cem\u003eC. hellerii\u003c/em\u003e, whose populations are already established in estuaries adjacent to the study site.\u0026nbsp;\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003eSpecimen collection and maintenance\u003c/p\u003e\n\u003cp\u003eIndividuals of \u003cem\u003eCharybdis hellerii\u003c/em\u003e and \u003cem\u003eMenippe nodifrons\u003c/em\u003e were captured by cylindrical traps in the Bay of Paranaguá, Paraná, Brazil (25°31'44.3\"S 48°28'23.1\"W) or were obtained through active capture on the Banana Island, also located in the Bay of Paranaguá (25°25'28.3\"S 48°24'25.2\"W), in the summer months of February and November-December 2020. Animals were placed in plastic containers filled with water from the collection site, maintained under constant aeration, and transported to the Laboratory of Comparative Physiology of Osmoregulation (LFCO) at the Department of Physiology, Federal University of Paraná. At LFCO, the animals remained in a 250-liter stock aquarium filled with seawater for a duration of ~7 days, maintaining a density of ~1 individual for 10 liter. In each field collection, only one species was captured. Thus, both species did not inhabit the stock aquarium at the same time. Aeration was constant, and an external filter ensured good water quality.\u0026nbsp;The filtration system utilizes a three-stage process comprising mechanical, chemical, and biological treatment. Water quality was systematically monitored through both visual assessment of turbidity and total dissolved solids as well as quantitative chemical analysis of ammonia and nitrite concentrations. Regular system maintenance is performed according to manufacturer specifications, including periodic replacement of filter media and implementation of weekly partial water changes (~30% volume).\u0026nbsp;\u0026nbsp;Salinity was of 34-35‰, temperature 26.28±0.64 °C (average and standard deviation), pH at ± 8.3, dissolved oxygen in the water ~8.0 mg/L and light conditions were according to the natural photoperiod (13h Light: 11h Dark). The animals were fed small pieces of fish fillet every two days, and feeding was halted 24 hours before the experiments. Following this initial phase, some individuals were utilized for control (temperature) experiments, maintaining temperature at 26 °C but reducing the salinity, according to the experimental protocol described below. The remaining specimen were acclimated in the same stock aquarium for seven days with a temperature increase of 4 °C above the annual average observed in summer (from 26 °C to 30 °C).\u0026nbsp;These temperature data were added to Table S2 in the supplementary material.\u0026nbsp;Others parameters in the stock aquarium water remained constant and the temperature elevation occurred gradually at a rate of 0.5 °C per hour between 8:00 am and 4:00 pm during the first of seven days of acclimation to the higher temperature. After this process, the final temperature was 30.47±0.57 °C (mean and standard deviation). These temperature data were added to Table S3 in the supplementary material.\u003c/p\u003e\n\u003cp\u003eExperiments\u003c/p\u003e\n\u003cp\u003eFor the experiments, animals were transferred from the stock aquarium to individual small aquaria filled with 2 liters of brackish water of salinity 35, 30, 25 or 20‰. Specimens of the control group were retrieved from the stock tank kept at 26 °C, while experimental animals were retrieved from the stock tank after being acclimated to the increased temperature of 30°C. Experimental time of exposure was always for 6 hours. Crabs maintained in the stock aquarium at 26 °C or 30°C were then exposed to identical osmotic challenges: 35, 30, 25 and 20‰, for 6 hours. The entire procedure was replicated six times, with additional batches of crabs maintained at either 26 or 30 °C. \u0026nbsp;Thus, the total N used was 96 individuals (2 temperatures x 4 salinities x 6 replicates = 48 crabs of each species).\u0026nbsp;Following the experimental exposure to the distinct salinities for 6 hours, individuals were covered with crushed ice in a styrofoam box, and were cryoanesthesized for approximately 5 minutes without direct contact with the ice. A hemolymph sample was obtained through puncturing the arthrodial membrane of a pereiopod during anesthesia. Subsequently, they were euthanized by opening the carapace using sharp material, and immediately sectioning the central nervous cord. After euthanasia, samples of muscle tissue were removed from the fifth pair of pereiopods (adapted for swimming) in \u003cem\u003eC. hellerii\u003c/em\u003e and the right chelipod in \u003cem\u003eM. nodifrons\u003c/em\u003e. Hemolymph and muscle samples were stored in a -20°C freezer for posterior assays of osmolality, chloride, magnesium and lactate concentrations (hemolymph) and determination of muscle water content. \u0026nbsp;Water samples were obtained from the experimental vials to analyze dissolved oxygen concentration\u0026nbsp;and initial and final ammonia levels.\u003c/p\u003e\n\u003cp\u003eOsmo/ion-regulatory assays\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHemolymph osmolality was determined in undiluted hemolymph samples using a Vapor Pressure Micro-Osmometer (VAPRO 5520, Wescor, USA). Hemolymph chloride and magnesium levels were assayed in diluted samples (1:5 for chloride and 1:25 for magnesium), using colorimetric kits (#115 for chloride and #50 for magnesium, from Labtest, Lagoa Santa, Brazil) under, respectively, 450 and 505 nm. The decrease in hemolymph osmolality, which is the sum of inorganic ions (such as chloride and magnesium) and organic osmolytes such as amino acids, for example - can lead to systemic imbalances in the acid-base system - chloride is used in the exchange for bicarbonate,\u0026nbsp;besides being the main inorganic ion present in the hemolymph - and magnesium is a cofactor of enzymes, such as Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e-ATPase, and helps in the transport of oxygen, occupying a binding site in the hemocyanin molecule. Thus, changes in these parameters can indicate deviation from a situation of homeostasis or physiological ‘comfort’ (see Freire, 2025).\u003c/p\u003e\n\u003cp\u003eMuscle water content (MWC) was determined by weighing muscle samples (wet weight) in pre-weighed and sealed 2 mL Eppendorf tubes (Bioprecisa FA2104 N, precision 0.1 mg, Brazil). After remaining for 48 hours at 60 °C inside an oven, the samples were weighed again (dry weight), and the weight loss (water) was expressed as a percentage using the formula:\u003c/p\u003e\n\u003cp\u003eMWC (%) = [(wet weight – dry weight) / wet weight] x 100\u003c/p\u003e\n\u003cp\u003eThe increase in muscle water content (water influx from hemolymph osmolality decrease) may indicate that the tissue has been challenged beyond its capacity to maintain hydration levels stable, also being a sign of physiological distress (see Freire, 2025).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMetabolic\u0026nbsp;assays\u003c/p\u003e\n\u003cp\u003eAll experiments were conducted with constant aeration for 5 hours. Then, aeration was interrupted and saturation dissolved oxygen levels were immediately measured (mg/L) (oximeter YSI 55, YSI, Yellow Springs, USA). At the end of this last one hour, the final dissolved oxygen levels were again measured. The difference between these two values were used to calculate dissolved oxygen concentration per gram of the animal, according to:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMagnitude of decrease in dissolved oxygen concentration\u0026nbsp;(µgO\u003csub\u003e2\u003c/sub\u003e/g.weight.h) = (initial O\u003csub\u003e2\u003c/sub\u003e – final O\u003csub\u003e2\u003c/sub\u003e)*vial volume mL*1000/g crab weight\u003c/p\u003e\n\u003cp\u003eHigh\u0026nbsp;dissolved oxygen concentration\u0026nbsp;indicates an increase in metabolism through the consumption of energy reserves, which leads to the need to search for more resources to maintain optimal internal conditions for survival. \u0026nbsp;Lactate was determined in undiluted hemolymph samples under 550 nm (Labtest kit #138). Lactate analyses were performed on the same day and the storage time in the -20°C freezer was less than 4 hours.\u0026nbsp;An increase in lactate concentration indicates activation of anaerobic metabolism or use of energy reserves to achieve energy demand.\u003c/p\u003e\n\u003cp\u003eBefore placing the crab in the experimental vial, a water sample was obtained for initial ammonia concentration values. After the 6 hours of experiment had elapsed, another water sample was taken for final ammonia levels. Ammonia concentration was determined using the Spectro Kit Ammonia Indotest (#2542, Alfakit, Florianópolis, Brazil), with absorbance read at 630 nm (as in Freire et al., 2020).\u0026nbsp;High ammonia concentration in the water indicates an increase in amino acid metabolism, either to meet energy demands or using these amino acids to regulate osmolality.\u003c/p\u003e\n\u003cp\u003eStatistical Analyses\u003c/p\u003e\n\u003cp\u003eWe performed a PCoA to detail the correlation between all variables, illustrating the distinct responses of the 2 crabs to the same set of challenges. Then we chose to do a principal component analysis (PCA) as an initial exploratory analysis for both species, considering the following variables: hemolymph osmolality, chloride, magnesium, lactate, muscle water content, dissolved oxygen concentration and excreted ammonia and then, a PERMANOVA. We evaluated two possibilities of inclusion of factors in the model: \u003cem\u003esalinity x temperature\u003c/em\u003e and \u003cem\u003etemperature x salinity\u0026nbsp;\u003c/em\u003eand their interactions. When the PERMANOVA test revealed statistical differences (p ≤ 0.05), comparisons among conditions were performed, and differences among salinity and temperature conditions were evaluated separately. For this additional analysis, two-way ANOVAs (analysis of variance) were applied to the data, with “temperature” and “salinity” as fixed factors, when data met the requirements of normality and homogeneity of variances. When they did not, the Scheirer-Ray-Hare test was performed. For both tests, the tests of Tukey test were employed as \u003cem\u003epost hoc\u0026nbsp;\u003c/em\u003etest, to localize group differences. In order to directly compare the two species, Student’s t-test were used,or Mann-Whitney test, if data were not normally. Before the Scheirer-Ray-Hare test, the data were transformed in several ways: Ranks, Log10, Ln, exponential, square root. However, none of them were effective. We did not use size as a covariate (in an ANCOVA, for example) because we had already performed a t-test to compare the difference between the size of the two species, and the result was that there was no significant difference (P = 0.947). There are some studies that point out how we should not directly compare species, or else, consider “species” as a level of source of variation for any measured variable because they are not independent (Garland and Adolph 1994; Garland et al. 2005). These results are available in Table S1 until S12, in the supplementary material. \u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe PCoA analysis conducted for both species demonstrated that the first two axes explain most of the variation found (43.01% and 22.3%, respectively) in relation to the physiological variables analyzed. Furthermore, the points related to \u003cem\u003eCharybdis hellerii\u003c/em\u003e exposed to a temperature of 30\u0026deg;C are very distant from the others, evidencing the significant effect that temperature has on the measured variables (Fig. 1).\u003c/p\u003e\n\u003cp\u003eThe PCA analysis in \u003cem\u003eMenippe nodifrons\u003c/em\u003e showed that the first axis explains 39.1% of the total variance, while the second axis explains 22.7%. Both axes present differentiated grouping between osmoregulation and metabolism variables. However, the osmoregulatory parameters (osmolality, chloride and magnesium) have high negative values on first axis. Conversely, the metabolic parameters (dissolved oxygen concentration, lactate synthesis and excreted ammonia) have high positive values on second axis (Fig. 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe PCA analysis for \u003cem\u003eC. hellerii\u003c/em\u003e was similar: the first axis accounted for 51.8% of the explained variance, while the value for the second axis was 16.6%. As observed for \u003cem\u003eM. nodifrons\u003c/em\u003e, the variables associated with osmoregulation showed strong negative correlations on first axis and the metabolic variables showed strong positive correlations on second axis (Fig. 3).\u003c/p\u003e\n\u003cp\u003eIn the PERMANOVA analysis conducted for \u003cem\u003eM. nodifrons\u003c/em\u003e, a significant effect of salinity (Pseudo-F = 23, p = 0.001) and time (Pseudo-F = 6, p = 0.015) on physiological parameters was observed. No significant interaction was detected between these two factors (Pseudo-F = 0, p = 0.776), as presented in Table 1. Salinity accounted for the majority of the variation among the groups (R\u0026sup2; = 60%), whereas temperature explained only 5% of the observed variation. The lack of a significant interaction between salinity and temperature suggests that these factors operate independently in influencing the physiological responses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Results for two-way permutational multivariate analysis of variance (PERMANOVA) between the Salinity and Temperature for physiological parameters of \u003cem\u003eMenippe nodifrons\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003ed.f.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003eSS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 92px;\"\u003e\n \u003cp\u003eMS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003ePseudo F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 140px;\"\u003e\n \u003cp\u003eSalinity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e309474.5691\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e103158.1897\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.599\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.001***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 140px;\"\u003e\n \u003cp\u003eTemperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e24493.64932\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e24493.64932\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.047\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.015*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 140px;\"\u003e\n \u003cp\u003eSalinity:Temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e6419.431802\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e2139.810601\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.776\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 140px;\"\u003e\n \u003cp\u003eResiduals\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e176175.4293\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e4404.385732\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.341\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 140px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e516563.0795\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e10990.70382\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ed.f.: degrees of freedom. SS: sum of squares. MS: mean squares. Significance codes: *** p\u0026lt;0.001; ** p\u0026lt;0.01; *p\u0026lt; 0.05; . p\u0026lt;0.1\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eC. hellerii\u003c/em\u003e, a significant interaction between temperature and salinity was observed in explaining the variation in the measured variables (Pseudo-F = 2, p = 0.047). Additionally, significant independent effects of temperature (Pseudo-F = 39, p = 0.001) and salinity (Pseudo-F = 43, p = 0.001) were detected, as presented in Table 2. Salinity accounted for the majority of the variation among the groups (R\u0026sup2; = 60%), while time explained only 18% of the variation. The interaction between temperature and salinity contributed to 3% of the total variation observed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Results for two-way permutational multivariate analysis of variance (PERMANOVA) between the Salinity and Temperature for physiological parameters of \u003cem\u003eCharybdis hellerii\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003ed.f.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003eSS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 92px;\"\u003e\n \u003cp\u003eMS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003ePseudo F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 140px;\"\u003e\n \u003cp\u003eSalinity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e391335.2579\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e130445.086\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.001***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 140px;\"\u003e\n \u003cp\u003eTemperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e119292.8907\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e119292.8907\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.183\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.001***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 140px;\"\u003e\n \u003cp\u003eSalinity:Temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e20750.57404\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e6916.858012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.032\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.047*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 140px;\"\u003e\n \u003cp\u003eResiduals\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e121223.8568\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e3030.596419\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.186\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 140px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e652602.5795\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e13885.16126\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ed.f.: degrees of freedom. SS: sum of squares. MS: mean squares. Significance codes: *** p\u0026lt;0.001; ** p\u0026lt;0.01; *p\u0026lt; 0.05; . p\u0026lt;0.1\u003c/p\u003e\n\u003cp\u003eBoth crab species displayed decreased hemolymph osmolality when exposed for 6 hours to decreasing salinities (Fig. 4A). Acclimation to a higher temperature resulted in enhanced decrease in hemolymph osmolality in both species at salinities 25 and 20\u0026permil;. Comparatively, the non-native \u003cem\u003eC. hellerii\u003c/em\u003e displayed even lower hemolymph osmolality than \u003cem\u003eM. nodifrons\u003c/em\u003e under both thermal and hypo-saline challenges (Fig. 4A). Compatibly, muscle hydration was less controlled by the non-native \u003cem\u003eC. helleri\u003c/em\u003e, which displayed higher levels of tissue hydration when compared to the more stable levels observed in the native \u003cem\u003eM. nodifrons\u003c/em\u003e (Fig. 4B), across all experimental salinities (results of the statistical tests are presented in Supplementary Tables S1, S2, S6 and S7).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe response of hemolymph chloride to reduced salinities followed the same pattern observed above for osmolality (Fig. 5A), again, in general, with lower values for \u003cem\u003eC. hellerii\u003c/em\u003e than those of \u003cem\u003eM. nodifrons\u003c/em\u003e. For \u003cem\u003eM. nodifrons\u003c/em\u003e, acclimation to a warmer temperature led to increased hemolymph chloride at salinity 30\u0026permil; (Fig. 5A and Supplementary Table S1). Decreased salinities also led to reduced levels of hemolymph magnesium (Fig. 5B and Supplementary Table S1). Comparatively, \u003cem\u003eC. hellerii\u003c/em\u003e displayed lower hemolymph magnesium concentration than \u003cem\u003eM. nodifrons\u003c/em\u003e when warm-acclimated, for all experimental salinities (Fig. 5B).\u003c/p\u003e\n\u003cp\u003eAcclimation to raised water temperature resulted in significant decrease in dissolved oxygen concentration, especially for \u003cem\u003eC. helleri\u003c/em\u003e, at all salinities. Dissolved oxygen concentration was markedly lower for the native \u003cem\u003eM. nodifrons\u003c/em\u003e. In the native species, the effect of warm acclimation was noted in salinities 30 and 20\u0026permil; (Fig. 6A). Dissolved oxygen concentration was higher in \u003cem\u003eC. hellerii\u003c/em\u003e than in \u003cem\u003eM. nodifrons\u003c/em\u003e, specifically upon acclimation to increased temperature and at the salinities of 30 and 25\u0026permil; (Fig. 6A). Warming caused elevated hemolymph lactate; in \u003cem\u003eC. hellerii\u003c/em\u003e, at 20\u0026permil;, but in \u003cem\u003eM. nodifrons\u003c/em\u003e at 25 and 20\u0026permil;. Lactate was higher in the invasive \u003cem\u003eC. helleri\u003c/em\u003e than in \u003cem\u003eM. nodifrons\u003c/em\u003e at the control temperature, at 25\u0026permil; (Fig. 6B). Acclimation to warmer temperature also led to increased ammonia excretion in both species, and essentially for all salinities (Fig. 6C), again with higher levels noted for the non-native \u003cem\u003eC. hellerii\u003c/em\u003e than for the native \u003cem\u003eM. nodifrons\u003c/em\u003e, in particular in the lower salinities (Fig. 6C). Results of the statistical tests are presented in Supplementary Tables S1, S2, S6 and S7).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eClimate change may interfere, either enhancing or attenuating biological invasion processes. Using physiological tools, we assessed whether warming, associated to a common estuarine challenge - seawater dilution - would distinctly affect the invasive species \u003cem\u003eCharybdis helleri\u003c/em\u003e, when compared to the native and sympatric \u003cem\u003eMenippe nodifrons\u003c/em\u003e. The findings demonstrate that when exposed only to hyposalinity, both species have similar responses. However, when the temperature factor is introduced along with hyposalinity, the non-native species has, surprisingly, a lower physiological performance.\u0026nbsp;This trend is evidenced by the dispersion of points representing \u003cem\u003eC. hellerii\u003c/em\u003e in the PCoA graph, in a distinct pattern from the result obtained with the native crab (Fig. 1).\u003c/p\u003e\n\u003cp\u003eBoth species experienced hemolymph osmolality dilution as a result of exposure to lower salinities at both temperatures. A recently published article reported that \u003cem\u003eC. helleri\u003c/em\u003e can regulate its extracellular fluid when external salinity decreases; when exposed to higher salinities, its extracellular fluid matches the external one, as expected in general for estuarine brachyuran crabs (Occhi et al. 2019). However, \u003cem\u003eC. hellerii\u003c/em\u003e has a lower hemolymph osmolality regulatory ability than other portunids that inhabit the same region, such as \u003cem\u003eCallinectes danae\u0026nbsp;\u003c/em\u003eand \u003cem\u003eC. ornatus\u003c/em\u003e (Freire et al. 2011, 2020; Rios and Freire 2023). A direct comparison with these \u003cem\u003eCallinectes\u003c/em\u003e species highlights the evidently lower osmoregulatory capacity of \u003cem\u003eC. hellerii\u003c/em\u003e. Under the most challenging condition, warming (30°C) and lowest salinity (20‰, equivalent to ~600 mOsm/kg.H\u003csub\u003e2\u003c/sub\u003eO), these species present the following osmolality values: 788±10 mOsm/kg.H\u003csub\u003e2\u003c/sub\u003eO in \u003cem\u003eC. danae\u003c/em\u003e, 766±28 mOsm/kg.H\u003csub\u003e2\u003c/sub\u003eO in \u003cem\u003eC. ornatus\u003c/em\u003e, and 701±26 mOsm/kg.H\u003csub\u003e2\u003c/sub\u003eO in \u003cem\u003eC. helleri\u0026nbsp;\u003c/em\u003e(personal observation, unpublished data). Thus, the 3 species mentioned can maintain a higher internal osmolality than the external environment, but \u003cem\u003eC. hellerii\u003c/em\u003e is the least efficient. While comparing these species is pertinent (all three belong to the Portunidae family and cohabit the same region in southwestern Atlantic), \u003cem\u003eC. helleri\u003c/em\u003e is not a “swimming crab” as the \u003cem\u003eCallinectes\u003c/em\u003e crabs, belongs to \u0026nbsp;distinct genus, and is ecologically closer to species of semi-terrestrial decapods, such as \u003cem\u003eM. nodifrons\u003c/em\u003e (Santana et al. 2009; Negri et al. 2018; Occhi et al. 2019; Stanski et al. 2022)\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMuscle hydration remained stable in both species at 26 °C, but muscle tissues of \u003cem\u003eC. helleri\u0026nbsp;\u003c/em\u003edisplayed increased hydration in the individuals acclimated to the higher temperature.\u0026nbsp;This means that the invasive species was unable to control the influx of water from the external environment into the extracellular environment of the muscle tissue, the hemolymph. Similar results were found in \u003cem\u003eClibanarius taeniatus\u003c/em\u003e and \u003cem\u003eClibanarius virescens\u003c/em\u003e, two hermit crab species from intertidal areas with ecological similarities to \u003cem\u003eC. hellerii\u003c/em\u003e and \u003cem\u003eM. nodifrons\u003c/em\u003e. Dunbar and Coates (2004), exposed these crabs to rising temperatures and decreasing salinity over 6 hours. Both species showed increased body weight, indicating tissue hydration increase. In addition, it had been shown that \u003cem\u003eC. hellerii\u003c/em\u003e shows a similar tendency for water uptake in muscle tissue when exposed to gradual salinity reductions (30‰ to 20‰ and 10‰) over 24 hours, indicating sensitivity to prolonged hypo-saline conditions (Occhi et al. 2019).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe ion regulation patterns are compatible with the osmolality results: \u003cem\u003eC. hellerii\u003c/em\u003e and \u003cem\u003eM. nodifrons\u003c/em\u003e behave similarly and there is hemolymph dilution in chloride and magnesium concomitant with seawater dilution, especially from 35 to 30‰, with levels remaining more stable upon further seawater dilution. However, warming leads to improved chloride stability in \u003cem\u003eM. nodifrons\u003c/em\u003e, but further decreased values in \u003cem\u003eC. hellerii\u003c/em\u003e, again highlighting the higher regulatory capacity of \u003cem\u003eM. nodifrons\u003c/em\u003e. As for hemolymph magnesium, its levels remain stable in the lowest salinities in \u003cem\u003eM. nodifrons\u003c/em\u003e, unaffected by warming, while again, warming causes further decreases in \u003cem\u003eC. hellerii\u003c/em\u003e. Temperature increase can affect the ionic regulation capacity of crustaceans in different ways, either increasing, decreasing, or not affecting this regulation, with no consistent trend observed in previous studies (Weber and Spaargaren 1970; Diaz et al. 2004; Dobrzycka-Krahel et al. 2014; Borecka et al. 2016). In our study, PERMANOVA analysis indicates that the combination of temperature and salinity affected only the non-native species \u003cem\u003eC. helleri\u003c/em\u003e: thus, a synergistic effect of both stressors on the invasive crab. The influence of warming combined with hyposalinity on the ionic regulation of decapods remains relatively unexplored. For example, the hermit crab species \u003cem\u003eC. taeniatus\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;C. virescens\u003c/em\u003e have osmoregulatory patterns similar to \u003cem\u003eM. nodifrons\u003c/em\u003e and \u003cem\u003eC. helleri.\u0026nbsp;\u003c/em\u003eUpon increased temperature and decreased salinity, the hermit crabs have impaired ionic regulatory capacity, resulting in significant hemolymph dilution and decrease in the concentration of the main ions sodium, potassium, calcium and magnesium\u0026nbsp;(Dunbar and Coates 2004)\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll three proxies for metabolic activation, reduction in dissolved oxygen (putatively implying oxygen consumption by the aerobic metabolism of the crab), hemolymph lactate production and ammonia excretion were very distinctively more activated in the combination of warming and seawater dilution, in the non-native \u003cem\u003eC. hellerii\u003c/em\u003e, than in the native \u003cem\u003eM. nodifrons,\u0026nbsp;\u003c/em\u003eas demonstrated by the PCA plots (Fig. 2 and 3). Salinity did not strongly influence the reduction in water dissolved oxygen pattern in both species; however, a synergistic effect of warming with hyposalinity was observed, resulting in metabolic activation, especially in the non-native \u003cem\u003eC. hellerii\u003c/em\u003e. Increase in aerobic metabolism – in our case here, a much more intense decrease in dissolved oxygen upon warming in the experimental vials containing the invasive crab - from this combination of stressors is a general outcome in estuarine decapods - of variable lineages and osmoregulatory capacities. A similar response of increased oxygen consumption when exposed to warming with hyposalinity was observed in the crab \u003cem\u003eScylla serrata\u003c/em\u003e (Chen and Chia 1996), and penaeid shrimps \u003cem\u003eLitopenaeus stylirostris\u003c/em\u003e (Diaz et al. 2004), \u003cem\u003eMarsupenaeus japonicus\u003c/em\u003e (Chen and Lai 1993; Setiarto et al. 2004), and the palaemonid prawns \u003cem\u003ePalaemon peringueyi\u003c/em\u003e (Allan et al. 2006), \u003cem\u003ePalaemon macrodactylus, Palaemon longirostris and Palaemonetes varians\u003c/em\u003e (Lejeusne et al. 2014). We must acknowledge that we did not use a closed respirometer, and thus our oxygen reduction data should not be directly ascribed to consumption by the animal, and we prefer not to calculate Q10 values here, for caution. However, given that crabs from both species remained still during the experimental period inside the experimental vials, we can propose that the sharp difference in the pattern of reduction in dissolved oxygen during the hour of measurement between the two species reflects metabolic activation (upon warming) to a much higher degree in the invasive species \u003cem\u003eC. helleri\u003c/em\u003e than in the native \u003cem\u003eM. nodifrons\"\u003c/em\u003e, also supported by ammonia and lactate data\u003cem\u003e.\u0026nbsp;\u003c/em\u003eRemarkably elevated Q₁₀\u0026nbsp;values (reaching 4.9) and other physiological indicators of enhanced metabolic activity were documented in juvenile \u003cem\u003eScylla paramamosain\u003c/em\u003e under thermal stress conditions (20, 25, 30, and 35°C) (Liu et al., 2022). Notably, such pronounced Q₁₀\u0026nbsp;values may represent a physiological signature of recent invasion events, signifying the initial acclimatization phase of invasive species to novel environmental conditions, reflecting thermal sensitivity in the new environment (Keller \u0026amp; Taylor, 2008; Boardman et al., 2022; Marochi et al. 2024). This recent invasion is further evidenced by the standard deviation values obtained from dissolved oxygen analyses, which were significantly higher in the invasive species compared to the native species. (Supplementary Tables S1), also a sign of high degree of stress. This possibility of more intense activation of metabolism in initial phases of biological invasion should be further examined in the literature. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLactate synthesis can occur due to the absence of available oxygen or function as an auxiliary energy substrate when energy demand is not fully met by aerobic respiration (Sherwood et al. 2013; Spicer 2014; Lee et al. 2023). The increase in lactate concentration in crustaceans occurs in response to physical activity, exposure to hypoxia or anoxia, reduced salinity or increased temperature (Lorenzon et al. 2007; Maciel et al. 2008; Jost et al. 2012; Sherwood et al. 2013; Freire et al. 2017, 2020). Although there are no records of how the combined effect of temperature and salinity change affects lactate production in crustaceans, our study demonstrates that the non-native species \u003cem\u003eC. hellerii\u003c/em\u003e activates anaerobic metabolism as well, when challenged with warming and seawater dilution, and to a much higher degree than the native crab. Moreover, the increased lactate production in the invasive crab when compared to the native crab confirms our data on concomitant reduction of dissolved oxygen, thus strengthening the idea of metabolic activation due to stress of warming.\u003c/p\u003e\n\u003cp\u003eBoth species demonstrated increased ammonia excretion into water upon warming and seawater dilution challenge. But again, this increase in excretion upon warming combined with seawater dilution was much more pronounced in \u003cem\u003eC. hellerii\u003c/em\u003e than in \u003cem\u003eM. nodifrons\u003c/em\u003e. Increased water ammonia must come from the crab. Thus, ammonia data confirms oxygen data, allowing us to make the point that warming strongly results in metabolic activation of the invasive crab, when compared to the native species. Reviews on decapod crustaceans suggest that ammonia excretion tends to rise as salinity decreases (Weihrauch 2004; Henry et al. 2012; Romano and Zeng 2013; Leone et al. 2017). Ammonia is the primary nitrogenous compound excreted by decapod crustaceans in freshwater and even in brackish waters, primarily produced through catabolism of amino acids (Larsen et al. 2014). In osmoregulatory species, the free amino acids generated are also used during the osmoregulation process (Strefezza et al. 2019). In relation to non-native crustacean species, the literature offers diverse examples: while amphipod invaders like \u003cem\u003eCrangonyx pseudogracilis\u003c/em\u003e and \u003cem\u003eDikerogammarus villosus\u003c/em\u003e appear more sensitive to high ammonia concentrations, the crayfish \u003cem\u003eProcambarus clarkii\u003c/em\u003e exhibits mechanisms to counteract ammonia toxicity (Prenter et al. 2004; Gergs et al. 2013; Normant-Saremba et al. 2015; Shen et al. 2021). Estuarine non-native crustacean species, such as \u003cem\u003eCarcinus maenas, Hemigrapsus takanoi, Hemigrapsus sanguineus\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Charybdis japonica\u003c/em\u003e showcase high ammonia excretion rates, probably due to the increase in metabolism resulting from the experimental treatment (Watanabe et al. 2009; Fowler et al. 2011; Fehsenfeld and Weihrauch 2016; Landeira et al. 2020). It is evident that \u003cem\u003eC. hellerii\u003c/em\u003e requires a greater energy supply to cope with the stress caused by the synergistic effect of the combination of warming and decreased seawater salinity. This reduces the scope for activity directed towards other functions, as a trade-off, decreasing invasive power under these conditions (Freire and Sampaio 2021; Rato et al. 2021; Marochi et al. 2024).\u003c/p\u003e\n\u003cp\u003eAlthough \u003cem\u003eC. hellerii\u003c/em\u003e exhibits certain apparent physiological disadvantages compared to \u003cem\u003eM. nodifrons\u003c/em\u003e, particularly in light of potential future climate change scenarios, it is important to acknowledge that other ecological factors within the Paranaguá Estuarine Complex also play critical roles. These factors are likely to be key determinants in the persistence or decline of \u003cem\u003eC. hellerii\u003c/em\u003e population. Currently, \u003cem\u003eC. hellerii\u003c/em\u003e population is considered established in the Paranaguá Estuarine Complex (Metri et al. 2020).\u0026nbsp;However, based on the capture locations of these individuals, this population appears to be restricted to rocky shores and the euhaline zone, where salinity exceeds 30 ‰\u0026nbsp;(Lana et al. 2001; Marone et al. 2005; Occhi et al. 2019). Interspecific competition may be one factor contributing to this restriction. The preference for rocky shores as the primary habitat for \u003cem\u003eC. hellerii\u003c/em\u003e could be a strategy to avoid competition for space and resources with other portunids in the region, such as \u003cem\u003eC. danae\u003c/em\u003e and \u003cem\u003eC. ornatus\u003c/em\u003e, which have larger populations and are potential predators of \u003cem\u003eC. hellerii\u003c/em\u003e (Sant’Anna et al. 2012). The presence of predators can limit the distribution and prevent the establishment of invasive species\u0026nbsp;(Ruesink 2007; Silva et al. 2018; Rato et al. 2021). On the other hand, it is common for predators not to recognize invasive species as potential prey\u0026nbsp;(Llewelyn et al. 2010; Silva et al. 2018). In Brazil, there are few reports of \u003cem\u003eC. hellerii\u003c/em\u003e being predated by native species, with the octopus species \u003cem\u003eOctopus vulgaris\u003c/em\u003e (Sampaio and Rosa 2006), \u003cem\u003eOctopus insularis\u003c/em\u003e (Silva et al. 2018)\u0026nbsp;and the goldspotted snake eel \u003cem\u003eMyrichthys ocellatus\u003c/em\u003e (Siqueira et al. 2021)\u0026nbsp;being notable exceptions. Of these species, only \u003cem\u003eOctopus vulgaris\u003c/em\u003e and \u003cem\u003eMyrichthys ocellatus\u003c/em\u003e are found along the coast of Paraná state\u0026nbsp;(Hackradt and Félix-Hackradt 2009; Amado et al. 2015).\u003c/p\u003e\n\u003cp\u003eReproductive aspects can also mitigate the impact of restricted physiological performance. Although \u003cem\u003eC. hellerii\u003c/em\u003e exhibits lower reproductive potential (producing fewer eggs per spawning event) compared to \u003cem\u003eM. nodifrons\u003c/em\u003e and other local portunids, such as \u003cem\u003eC. danae\u003c/em\u003e and \u003cem\u003eC. ornatus\u003c/em\u003e, its reproductive period remains constant, whereas the aforementioned species experience reproductive peaks during spring and summer\u0026nbsp;(Mantelatto and Garcia 2001; Sant’Anna et al. 2015; Marochi et al. 2021). This continuous reproductive strategy reduces competition for resources and enhances the potential for population expansion. Moreover, in future climate change scenarios, particularly with rising temperatures, \u003cem\u003eM. nodifrons\u003c/em\u003e may be more severely impacted. A study by Marochi et al. (2021) concluded that increased temperatures could reduce larval survival in \u003cem\u003eM. nodifrons\u003c/em\u003e. Additionally, \u003cem\u003eM. nodifrons\u003c/em\u003e larvae exhibit a low capacity for acclimatization to warming waters (Marochi et al. 2024). While similar studies on \u003cem\u003eC. hellerii\u003c/em\u003e are lacking, other species of the same genus have demonstrated high larval survival rates under the salinity and temperature conditions used in this study, such as: \u003cem\u003eCharybdis feriatus\u003c/em\u003e (Baylon and Suzuki 2007; Soundarapandian et al. 2010) and \u003cem\u003eC. japonica\u003c/em\u003e (Fowler et al. 2011). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnother notable factor is the potential overlap in the diets of \u003cem\u003eC. hellerii\u003c/em\u003e and \u003cem\u003eM. nodifrons\u003c/em\u003e, which may lead to competition for food resources. Non-native species often display greater voracity than their native counterparts, giving them a competitive advantage in acquiring food\u0026nbsp;(Sant’Anna et al. 2012, 2015; Siqueira et al. 2021). \u0026nbsp;In this case, both species primarily feed on other invertebrates, particularly low-mobility organisms such as mollusks (Izar et al. 2023). However, in \u003cem\u003eC. hellerii\u003c/em\u003e, feeding preferences are influenced by predator density. At moderate densities (30 crabs/m³), the preference is for mollusks, but at higher densities (60 crabs/m³), the preference shifts towards other crabs (Izar et al. 2023).The enhanced ability of the non-native species to acquire food resources may compensate for its lower physiological performance, providing the energy required for osmoregulation\u0026nbsp;and other physiological functions. As a result, this dynamic poses a potential threat to the viability of \u003cem\u003eM. nodifrons\u003c/em\u003e populations.\u003c/p\u003e\n\u003cp\u003eStill another feature to be mentioned is that the lower physiological performance of the non-native species may be related to the fact that its invasion is relatively recent. For instance, while the invasive crab \u003cem\u003eEriocheir sinensis\u003c/em\u003e began its invasion of the United Kingdom in the 1970s, its population did not experience significant growth and expansion until the 1990s (Hänfling et al. 2011). Thus, it is plausible that \u003cem\u003eC. hellerii\u003c/em\u003e has not yet fully optimized its fitness in an environment that differs from its native habitat (see Lee and Bell 1999, for freshwater invasion). Similarly, the first records of \u003cem\u003eC. hellerii\u003c/em\u003e in the São Paulo state (a region just north of the Paranaguá estuarine complex) date back to 1995 (Negri et al. 2018). Less than two decades later, its population had already become the second most abundant among crustaceans inhabiting rocky shores, surpassed only by \u003cem\u003eM. nodifrons\u003c/em\u003e (Sant’Anna et al. 2012; Izar et al. 2023). \u0026nbsp;This highlights the importance of continued monitoring of \u003cem\u003eC. hellerii\u003c/em\u003e populations in the coming years to better understand potential adaptations and ecological dynamics.\u003c/p\u003e\n\u003cp\u003eAlthough no published studies have analyzed the population dynamics of \u003cem\u003eC. hellerii\u003c/em\u003e in the Paranaguá Estuarine Complex (PEC), other factors beyond poor physiological performance may limit local expansion. One of them is the limitation of the distribution range of this species, which shows that \u003cem\u003eC. hellerii\u003c/em\u003e is at the southern limit of its distribution. Tropical invasive species often face challenges in establishing populations in higher-latitude regions with greater species diversity, which can hinder the growth and expansion of \u003cem\u003eC. hellerii\u003c/em\u003e populations, as demonstrated by Sant’Anna et al. (2015) at Armação do Itapocoroy, Santa Catarina state (a region just South of the Paranaguá estuarine complex).\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe use of physiological tools within an ecological framework, presents a complementary and mandatory approach for examining the novel interactions between non-native and native species in environments where these species did not co-occur naturally before human action (see Boardman et al., 2022). Biological invasions and current and future climate change will certainly be among the most important drivers accounting for future ecosystems. In this study, we opted for osmoregulatory and metabolic approaches to analyze the physiological performance of two species of decapod crustaceans, one non-native species and one native species, which already co-occur along an estuarine complex in southern Brazil. The findings reveal that, although the two species exhibit similar responses under saline stress alone, the introduction of temperature as a factor alongside salinity yields divergent results due to a synergistic effect. Specifically, the non-native species \u003cem\u003eC. hellerii\u003c/em\u003e shows inferior physiological performance compared to the native species \u003cem\u003eM. nodifrons\u003c/em\u003e, which is not usually the expected result.\u0026nbsp;However, despite this lower physiological performance, other ecological traits, such as feeding strategies and reproductive patterns, may provide compensatory advantages for \u003cem\u003eC. hellerii\u003c/em\u003e. Moreover, the physiological data generated can contribute to comparative studies in areas with analogous ecological characteristics undergoing biological invasion processes. These insights can also be incorporated into future models aimed at predicting the impacts of climate change on local populations and understanding the dynamics of coexistence between non-native and native species in shared environments.\u0026nbsp;Ongoing monitoring of both \u003cem\u003eC. hellerii\u003c/em\u003e and \u003cem\u003eM. nodifrons\u003c/em\u003e populations is crucial for understanding their long-term viability and the broader ecological consequences of this invasion.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments and Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors gratefully acknowledge the financial support by CAPES (Federal Government of Brazil) through a PhD fellowship to LPR, and CNPq through Research Grants to CAF (# 302829/2015-6 and 307760/2019-7). Retrieval of crabs from nature for this study was authorized by ICMBio/SISBIO (Ministry of Environment, Brazil), number 20030, renewed annually, issued to CAF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth authors contributed to the conception and design of the study. LPR performed the experiments. Data, statistics, and conclusions were extensively and comprehensively discussed by both authors. The initial draft of the manuscript was written by LPR and underwent thorough edition by CAF. Both authors concur with the final submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was deemed unnecessary for the nature of this study, but experimental animals were, nonetheless treated with care and respect, and were submitted to cold-anesthesia before euthanasia.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAllan EL, Froneman PW, Hodgson AN (2006) Effects of temperature and salinity on the standard metabolic rate (SMR) of the caridean shrimp \u003cem\u003ePalaemon peringueyi\u003c/em\u003e. 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J Exp Biol 207:4491\u0026ndash;4504. https://doi.org/10.1242/jeb.01308\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biological-invasions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"binv","sideBox":"Learn more about [Biological Invasions](https://www.springer.com/journal/10530)","snPcode":"10530","submissionUrl":"https://submission.nature.com/new-submission/10530/3","title":"Biological Invasions","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Crustacea, Decapoda, invasion, metabolism, osmoregulation","lastPublishedDoi":"10.21203/rs.3.rs-5595652/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5595652/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The presence of non-native organisms challenges ecosystems under the influence of climate change. Comparisons of physiological performance between ecologically-similar native and non-native species contribute to invasion studies. We examined two decapod crustaceans in Estuarine Complex of Paranaguá (ECP), Brazil: the non-native Charybdis hellerii and the native Menippe nodifrons. Crabs were acclimated to control (26 °C) and elevated (30 °C) temperatures for one week in full-strength seawater (35‰), and were then submitted to dilute seawater (30, 25, and 20‰) for 6 hours. Hemolymph was assayed for osmolality, chloride, magnesium, and lactate; muscle samples were evaluated for hydration levels. Dissolved oxygen and ammonia production were assessed in the experimental water. Both species were impacted by low salinity, with an synergistic effect from elevated temperatures. However, C. hellerii was more affected than M. nodifrons, displaying less capacity to keep stable muscle hydration levels upon seawater dilution, a steeper decrease in dissolved oxygen, higher ammonia excretion, and higher lactate, as compared to the native crab. The non-native C. hellerii was physiologically challenged to a much higher degree than the native species. Although C. hellerii has established populations in the ECP, its sensitivity (synergistic deleterious effect) to salinity reductions and rising temperatures may limit its further spread in areas with intense fluctuating abiotic conditions. These data can support modelling efforts of the trends in these species distribution where C. helleri is invasive. This result may also be indicative of the undergoing process of invasion; similar approaches could contribute to invasion science involving other marine/estuarine crabs.","manuscriptTitle":"Marine brachyuran crabs’ osmoregulatory and metabolic responses upon warming and seawater dilution challenges: the non-native Charybdis helleri is more sensitive than the native Menippe nodifrons.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 17:54:20","doi":"10.21203/rs.3.rs-5595652/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-08T23:06:08+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-08T18:45:45+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Biological Invasions","date":"2025-03-31T19:25:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-29T12:08:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biological Invasions","date":"2025-03-28T15:57:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biological-invasions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"binv","sideBox":"Learn more about [Biological Invasions](https://www.springer.com/journal/10530)","snPcode":"10530","submissionUrl":"https://submission.nature.com/new-submission/10530/3","title":"Biological Invasions","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9e6a7065-c932-485a-85f1-13464ae7d3b8","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:06:57+00:00","versionOfRecord":{"articleIdentity":"rs-5595652","link":"https://doi.org/10.1007/s10530-025-03747-6","journal":{"identity":"biological-invasions","isVorOnly":false,"title":"Biological Invasions"},"publishedOn":"2026-01-09 15:59:23","publishedOnDateReadable":"January 9th, 2026"},"versionCreatedAt":"2025-04-10 17:54:20","video":"","vorDoi":"10.1007/s10530-025-03747-6","vorDoiUrl":"https://doi.org/10.1007/s10530-025-03747-6","workflowStages":[]},"version":"v1","identity":"rs-5595652","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5595652","identity":"rs-5595652","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}