On their way to the north: larval performance of Hemigrapsus sanguineus invasive on the European coast—a comparison with the native European population of Carcinus maenas

The Asian shore crab Hemigrapsus sanguineus has become invasive in North Europe and it co-occurs and competes with the native European shore crab Carcinus maenas. Both species develop through a feeding and dispersive larval phase characterised by several zoeal and a settling megalopa stage. Larvae of marine crabs are vulnerable to food limitation and warming has the potential to exacerbate the negative effects of food limitation on survival and growth. We quantified the combined effects of temperature and food limitation on larval performance (survival and growth) of H. sanguineus and we compared our results with those reported on performance of C. maenas larvae, under the same experimental design and methodology. Larvae from four females of H. sanguineus collected on Helgoland (North Sea) were experimentally reared from hatching to megalopa, at four temperatures (range 15–24 °C) and two food conditions (permanent vs. daily limited access to food). Larval survival of H. sanguineus was low at 15 °C and increased with temperature, in contrast to the high survival reported for C. maenas larvae in the range 15–24 °C. Food limitation reduced survival and body mass of H. sanguineus larvae at all temperatures, but without evidence of the exacerbating effect caused by high temperatures and reported for C. maenas. By contrast, high temperature (24 °C) mitigated the negative effect of food limitation on body mass on H. sanguineus larvae. Advantages of H. sanguineus over C. maenas appear especially under the increased temperatures expected from climate change.


Introduction
Increasing trade and travel in the last decades has led to biological invasions, i.e. the dispersion and introduction of many species to geographical areas located outside their native range (Hulmen and Weser 2011; Bailey et al. 2020). Dispersion of organisms due to anthropogenic activities occurs through shipping (ballast water, fouling), construction of corridors, aquaculture, fisheries, and food trade (Carlton 2002;Hulme et al. 2008;Molnar et al. 2008;Katsanevakis et al. 2013). The introduction of a new species can lead to the decline in native species richness and abundances, loss of genetic variation in the native community, and an increase in the homogeneity of the invaded communities (Rahel 2000;Pyšek et al. 2012;Geburzi et al. 2018). Whenever the "introduced" species causes an impact on the host ecosystem, it is considered "invasive". Biological invasions are one of the most important threats to biodiversity after changes in land and sea use, animal exploitation, climate change, and pollution (Diaz et al. 2019;IPCC 2019). Invasive species affect the dynamics of native communities at different scales (Hulme 2017), by means of different interactions including competition, predation, and introduction of new diseases (Ruiz et al. 2000;Sakai et al. 2001;Jeschke et al. 2012). Biological invasions have been reported for most marine and estuarine habitats in the world (Katsanevakis et al. 2013;Chan and Briski 2017;Pyšek et al. 2020). Many hypotheses have been proposed to explain biological invasions and the success of the invaders (Simberloff and Von Holle 1999;Gurevitch et al. 2011;Kelley 2014), yet there is still little information on the processes involved in dispersion and establishment of alien species in novel habitats (Bailey et al. 2020;Rato et al. 2021).
Global climate change is causing an increase in the number and the impact of biological invasions (Hulme 2017;González-Ortegón et al. 2020;Pyšek et al. 2020), because of the steady increase in temperature (IPCC 2021), and the increased frequency of extreme events (e.g. heatwaves, Meehl and Tebaldi 2004;Smale et al. 2019). These changes combined with other human activities can act together to help the dispersal and establishment of new species. For example, the oyster Crassostrea gigas was first introduced in the North Sea for aquaculture purposes and was able to establish there due to above-average summer temperatures that helped the species to spread (Diederich et al. 2004;Smaal et al. 2009). Global warming has also led to the poleward expansion of many species (Sorte et al. 2010;Poloczanska et al. 2013;Giménez et al. 2020).
Global warming could affect both invasive species and their native competitors in different ways. How different species respond to changes in environmental variables (e.g. temperature) will ultimately determine individual performance and species interactions. Increases in temperature cause rises in metabolic demands, which should be met by food supply (Gillooly et al. 2001;Somero 2002). However, for organisms living in the marine pelagic realm, where the food distribution is patchy (Paffenhöfer et al. 1987;McManus et al. 2003) increased temperature combined with food limitation could impair growth and survival. The combined effect of food limitation and increased temperature may particularly impact life history stages characterised by high growth rates (Foster and Hirst 2012;Otto et al. 2020). Crustacean larvae are an example where high growth rates are sustained by high feeding rates, which in turn demand higher food availability (Anger 2001). In such case, one would expect that increased temperatures combined with limited access to food may cause reductions in growth rate and survival . However, the nature of the effect could vary between native and exotic competitors because of differences in thermal optimum ranges (Griffith et al. 2021). Hence, a critical question to understand current invasion and future population spread is how increased temperature combined with food limitation drive the performance of both native and exotic species.
The Asian shore crab Hemigrapsus sanguineus is native to the east coast of Asia (20-50° N) (Takahashi et al. 1985;Fukui et al. 1989;Hwang et al. 1993). It is one of the most abundant crab species on rocky beaches and occupies the upper and middle intertidal zones (Kikuchi et al. 1981;Fukui 1988). It has successfully invaded the coasts of North America, North Europe, and the Adriatic and Black Seas (Schubart 2003;Micu et al. 2010) via ballast water (Ai-yun andYang 1991;Kraemer et al. 2007;Epifanio 2013). In northern Europe, H. sanguineus was first recorded in France and the Dutch Delta system in 1999 (Breton et al. 2002) and then spread to the North Sea and Scandinavia (Wolff 2005;Kerckhof et al. 2007;Dauvin and Dufossé 2011;Gittenberg et al. 2010;Gothland et al. 2013;Seeley et al. 2015;Karlsson et al. 2019). H. sanguineus was occasionally found in the Mediterranean and Black Sea (Schubart 2003;Ben Souissi et al. 2004;Micu et al 2010;Ounifi-Ben Amor et al. 2015GBIF.org 2023). There are several factors likely to drive or limit the expansion of H. sanguineus, including the presence of congeneric competitors (e.g. US North Pacific, Steinberg andEpifanio 2011, Lord andWilliams 2017) and low summer temperatures (Stephenson et al. 2009).
In the Atlantic coast of North America, the northern distribution limit of H. sanguineus is determined by the larval thermal tolerance to low temperatures (Epifanio et al. 1998;Stephenson et al. 2009). Hence, as a result of warming, the distribution of H. sanguineus is likely to expand further north (Epifanio 2013;Giménez et al. 2020). H. sanguineus co-occurs and competes with the shore crab Carcinus maenas in Europe and in North America. Both species overlap in diet and habitat use, with juvenile/adults of H. sanguineus outcompeting C. maenas in the use of space and resources. H. sanguineus also predates on C. maenas juveniles affecting their recruitment success (Lohrer and Whitlach 2002;Jensen et al. 2002;Geburzi et al. 2018). For example, in Southern New England H. sanguineus has significantly reduced the recruitment of C. maenas due to direct predation, leading to a decline in densities by 40-90% (Lohrer and Whitlatch 2002). In addition, when in sympatry individuals of C. maenas migrate towards the subtidal zone (Geburzi et al. 2018). The above-mentioned factors, help explain the displacement of C. maenas from environments where it was previously more abundant.
We compared the performance of H. sanguineus and C. maenas from the perspective of the larval phase for co-occurring populations of the German Bight (Helgoland, North Sea). We focus on larvae because larval survival and recruitment are critical contributors to the propagule pressure by H. sanguineus (Simberloff 2009) and for the persistence of populations of C. maenas. Propagule pressure, i.e. a group of individuals of a species arriving in a region to which they are not native, drives the establishment and spreading of invasive populations (Simberloff 2009). Marine larvae in particular, tend to be more sensitive to environmental fluctuations than juveniles or adults (Pandori and Sorte 2019) and larval survival is central to the recovery of populations after environmental disturbances (Cowen and Sponaugle 2009;Pineda et al. 2009;Giménez et al. 2020). In particular, differences in larval survival among co-occurring species may affect the balance of competition, either exacerbating or counteracting the outcome. In theory, counteracting effects may occur in cases of trade-offs between competition and dispersal abilities (Seifan et al. 2013). Because H. sanguineus was first reported in the German Bight very recently -Scrosati et al. 2011, 2009-Jungblut et al. 2017, it is not clear yet whether H. sanguineus would be able to outcompete C. maenas at that local habitat. Unlike C. maenas, larvae of H. sanguineus cannot develop at temperatures below 13-15 °C (Epifanio 2013) which characterise the spring and early summers in the German Bight and coastal North Sea . For H. sanguineus, larvae appear to be released when the temperature surpasses 15 °C which is early/ mid-June depending on the year ). In the case of C. maenas, larval release starts in May (Harms et al. 1994), but the full larval season of both species partially overlap. We know that under food limitation, zoea I of H. sanguineus is more tolerant to short thermal fluctuations than C. maenas (Giménez et al. 2021). However, over the entire larval phase and under low temperatures, food limitation may produce a stronger negative effect on survival of H. sanguineus than that observed in C. maenas.
Here we compared the responses of larvae of Hemigrapsus sanguineus and Carcinus maenas to food limitation under increased temperature, reared in comparable experimental conditions. We first quantified the combined effects of temperature and food limitation on survival and performance of larvae of H. sanguineus. For the first time, we documented growth and survival responses in any population of this species to food limitation over a wide range of temperatures. In particular, we quantified the correlated responses of growth and development, driving size, and reserves at metamorphosis, which for benthic invertebrates, are known to drive the performance of the post-metamorphic stages in the benthic habitat (Giménez et al. 2004;Pechenik 2006;Giménez 2010;Torres et al. 2016). Second, we compared our results on H. sanguineus with those obtained by Torres and Giménez (2020) for a co-occurring population of C. maenas.

Materials and methods
Animal husbandry, larval rearing and experimental design Berried females of H. sanguineus (carapace width 15.8-17.2 mm) were collected on the island of Helgoland (North Sea, German Bight, 54° 10′ 40.9′′ N 7° 53′ 32.4′′ E) during their reproductive season (July-September) and transported to the laboratory.
Females were kept individually in 2-L aquaria with UV-treated filtered (0.2 µm) seawater (32.5‰) permanently aerated, in a temperature-controlled room at 18 °C (± 0.5 °C) with a 12:12 h light: dark cycle. Females were fed every 3 days with shrimps (Crangon crangon) and water was changed daily to ensure high water quality at hatching.
The experimental setup comprised a factorial design, in which we exposed larvae obtained from each given female to different combinations of temperatures and access to food (following Torres and Giménez 2020). This experimental procedure was repeated four times, i.e. once for each of the hatches obtained from four different females, in order to assess potential variations in responses driven by maternal influence. Freshly hatched zoeae were distributed in 8 treatments (4 replicate vessels per treatment, see below for more details), combination of 2 levels of daily access to food (6 or 24 h/day, provided ad libitum) and 4 temperatures (15, 18, 21 and 24 °C). The different temperatures were chosen based on its natural variability in the German Bight: 15 and 18 °C represent temperatures recorded during the larval season of H. sanguineus ; this corresponds to summer temperatures around the local population (Wiltshire and Manly 2004). Temperatures > 20 °C are expected as the consequence of steady warming due to climate change (Schrum et al. 2016), and as the consequence of the expected increment in the frequency of warm summers (Christidis et al. 2015).
Experiments were carried out in temperaturecontrolled rooms and using natural UV-treated filtered (0.2 µm) seawater. When hatching occurred, 50 larvae were sorted into each of 500 mL rearing vessels (4 replicate vessels per treatment for each of the four females) in UV-treated filtered seawater at the temperature of hatching. Freshly hatched Artemia sp. nauplii (Great Salt Lake Artemia, Sanders, USA) were provided as food for the larvae in densities of ~ 5 nauplii/mL (Torres et al. 2021). In the treatment of limited access to food, Artemia sp. nauplii were available for 6 h each day (between 9 a.m. and 3 p.m., following Giménez and Anger 2005;Torres and Giménez 2020). By contrast, in the treatment of permanent access to food, Artemia sp. nauplii were available all day. Water in all treatments was changed daily following standard procedures for larval rearing (Torres et al. 2021). During the daily water change, live larvae were staged and recorded, dead ones were also recorded and removed from the experiments; in addition, we checked that remaining food was present in each rearing vessel.
Body mass and elemental composition (carbon and nitrogen) were measured in freshly hatched larvae (3 replicates, 50 zoea each at the start of each experiment) and in freshly moulted megalopae (sampled within 24 h after moulting). Carbon content is used as a proxy for lipid reserves, used by crustacean larvae to sustain periods of food limitation Anger and Harms 1990); nitrogen content is used as a proxy for protein content (Dawirs 1986;Dawirs et al. 1986). The number of individual megalopae sampled in each of the 4-replicate rearing vessel was on average 5 (Table S1). Larvae were pipetted onto a filter, rinsed with distilled water, gently blotted dry with filter paper, and stored in pre-weighed tin cartridges at − 20 °C for later analysis. To determine the dry weight, samples were freeze-dried for 48 h and weighed using a microbalance (Sartorius SC2, precision 1 µg). Carbon and nitrogen content were then determined using an elemental analyser (vario MICRO cube CHNS analyser, Elementar Analysensysteme).

Data analysis
The response variables were survival, duration of development, body mass, elemental composition (carbon and nitrogen), and instantaneous growth at the megalopa. Survival to each zoeal stage was calculated as the percentage of survivors in relation to the number of organisms at the start of each experiment. Duration of development was calculated as the time elapsed from hatching to reach each developmental stage. Growth rates were estimated as G = log (W f /W 0 )/t. In this formula W 0 is the average mass (dry weight, carbon or nitrogen) at hatching, W f is the corresponding mass of each individual megalopa collected in each rearing replicate and t is the corresponding duration of development of each individual megalopa.
Mixed modelling was carried out in R (function lme and gls from package nlme, Pinheiro et al. 2018, R Core Team 2013 to assess the responses to the different combinations of food availability and temperatures on survival and duration of development. The models contained temperature and food availability as fixed factors and female of origin as a random factor. We performed backwards model selection (Zuur et al. 2009) in two steps. In a first step, we tested the random terms using restricted maximum likelihood (REML), we compared the different models through the corrected Akaike information criteria (AICc) and ranked them. The model with the lowest AICc was selected for further analysis. When the difference between two models was ΔAICc < 3 and the most complex model had the lower AICc we applied hypothesis testing (likelihood ratio tests). When the models differed significantly (p < 0.05), we chose the model with the lowest AICc and when the difference was not significant we chose the simpler model (with the lowest number of parameters). In a second step, the fixed terms were analysed through maximum likelihood (ML). For dry mass, elemental composition, and growth rates at the megalopa we did not get sufficient data in the food limited treatment at 15 °C for female 4 (F4). We therefore, analysed the data using two different starting models: (1) considering all females but without 15 °C and (2) considering all treatment combinations but without F4.
Data for survival was analysed in the logistic (Warton and Hui 2011) and logarithmic scale; as a first step, proportions (p) were re-scaled using the formula p′ = [p (50-1) + 0.5]/50, to avoid inconsistencies associated to log (0) values. Logarithmically-transformed proportions were used to test the multiplicative model, whereby temperature and food limitation would act independently on the survival rates (Piggot et al. 2015). Survival responses consistent with a multiplicative model cannot be tested when the proportions are expressed in the logistic scale . Duration of development was analysed in the raw and the logarithmical scale to test if the effects were additive, multiplicative or interactive. Body mass and elemental composition were analysed in the raw scale. Tukey´s honestly significant difference tests (Tukey´s HSD) were performed to test differences among the different treatments.

Comparison performance H. sanguineus and C. maenas
We compared the performance of H. sanguineus and C. maenas at different temperatures and food conditions through two means. First, we compared the integrative response of body mass and developmental time. We calculated the ratios between the body mass under food limitation and those observed under permanent access to food for each temperature; the same calculation was made for duration of development. For comparison, these standardised values were plotted against those of Carcinus maenas. In this plot, the unit corresponds to the values of body mass (and duration of development) under permanent access to food at each temperature.
We also calculated the ratios of survival (S R ) and growth rates (G R ) between species, i.e. as SR = S H / S C and G R = G H /G C , where S H and S C are the survival to the megalopa of H. sanguineus and C. maenas, respectively and G H and G C the respective growth rates (from hatching to megalopa). We calculated average ratios and used simulations (details in: Supplementary material, "Materials and methods" section. Data analysis: details on model simulation) to incorporate the intraspecific variation in survival and growth associated to variations within and among families. First, for each species, we simulated 1000 values of the survival and growth rates. Survival was simulated from the statistical model fitted to the responses of both species to the different combinations of temperature and access to food; i.e. there were 1000 values for each combination of species, response variable, temperature, and food condition. Growth was simulated from an additional statistical model which also considers survival as covariate; this model incorporates correlations between average survival and growth associated to female-to-female variation in larval performance. The data and models used for C. maenas correspond to Torres and Giménez (2020). The model used for survival of H. sanguineus was that of Table S2; for growth we fitted an additional model based on female-to-female averages (Supplementary material, Materials and methods section. Data analysis: details on model simulation). Simulations were performed using the function simulate of the package nlme (R Core Team 2013) applied to the best fitted models for each variable. Second, for each combination of temperature and food condition a pair of values of survival and growth was randomly sampled (function sample, R Core Team 2013) for each female of each species. The ratio of survival (or growth) was calculated between two randomly chosen females (one per species). This procedure was repeated 4000 times in order to obtain distributions of ratios of survival (and growth) for each combination of temperature and food condition. We then calculated the average of four ratios, comparing the performance of four hatches of H. sanguineus and four of C. maenas; this calculation takes into account that larvae obtained from four different females were used for each study. In addition, this procedure to calculate the average maintained the correlation between survival and growth. Using the simulated ratios of survival and growth, we calculated the centroid and the 90% confidence ellipses which were plotted for each combination of temperature and food (see Fig. 5).

Results
We first describe the survival, development and growth rates for Hemigrapsus sanguineus and we compare them to the data from Carcinus maenas already published   (Fig. 1, left panel); while at 15 °C, food limitation reduced survival by 74%, at 24 °C the survival reduction was 35%. Best models retained food availability and temperature operating in additive or interactive ways depending on the stage (Table S3), but with variations among larvae from different females (Table S4). Survival to megalopa under limited access to food ranged from 0 to 74% depending on temperature and female of origin, with 0% survival occurring at 15 °C and 24 °C for female 3 (Fig. S5). The effects of temperature and limited access to food on survival to zoeae II to IV were small, but increased for survival to ZV and especially to the megalopa (Fig. S6). The sensitivity to temperature varied among larvae from different females; survival at 15 °C was consistently low (13.2%) but survival at 24 °C ranged from 19 to 92% (larvae under permanent access to food) depending on the female (Fig. S5).
The combined effects of temperature and food limitation on the overall survival to megalopa (Fig. 1, left panel) were consistent with a multiplicative model (additive model retained in the logarithmic scale: Table S3). The multiplicative model can be illustrated considering the combination of 24 °C and permanent access to food as the optimal condition. In this case, the observed proportion of survivors under the combination of two stressors experienced simultaneously (15 °C and limited access to food: 0.05) is close to that expected Fig. 1 Hemigrapsus sanguineus and Carcinus maenas. Average survival from hatching to megalopa as a response to temperature and food availability. Data shown as average values ± SE for the four females of origin. Permanent access to food: blue symbols and continuous line; limited access to food: green symbols with dashed line. Percentages on top (only when significantly different): percent difference in survival between permanent and limited access to food treatments for each temperature. Already published data for Carcinus maenas is presented in the right panel, grey background  by the product of the proportions observed when the stressors were experienced in isolation (limited access to food: 0.3; 15 °C: 0.2). On a female-byfemale basis, the effects ranged from synergistic with a strong effect of temperature (F1 and F2) to antagonistic with a strong effect of limited access to food (F3 and F4).
The overall response of H. sanguineus was clearly different from that of C. maenas (Fig. 1) Fig. 1. Percentages on top or below (only when significantly different): percent difference in development time, C growth, C content, and C/N between permanent and limited access to food treatments for each temperature with a multiplicative model, also differs from the strong interactive effect shown by C. maenas where high temperatures exacerbated the effect of food limitation on survival (Fig. 1).
Duration of development to megalopa in H. sanguineus increased with decreasing temperatures in a non-linear pattern (Fig. 2a, left panel); the best model retained the interactive effect of temperature and limited access to food (Table S5). At low temperatures, the effects of food limitation were weak, producing a delay in the metamorphosis of fewer than 3 days (representing less than a 5% change between the two food conditions). However, at 21 and 24 °C the delay was longer than 3 days, representing 14-16% change (Fig. 2a, left panel). The effect of food limitation was weak at early stages and then it became stronger in the zoea IV (Fig. S7); best models retained temperature in development to stages ZII and ZIII, temperature and food availability operating in an additive way in development to ZIV, and interactively in development to ZV and megalopa (Table S5). In larvae from all females reared at 15 °C, there was an extra zoeal stage (zoea VI) regardless of food availability.
Carbon growth rates of H. sanguineus increased with increasing temperatures in both food conditions; food limitation caused a reduction in carbon growth rates, and the effect was stronger at higher temperatures (Fig. 2b, left panel); best models retained the interaction of food availability and temperature (Table S6). Similar effects were also found in terms of dry mass and nitrogen content (Fig. S8a, c). Exposure to food limitation resulted in a reduction in body mass, carbon and nitrogen content (Figs. 2c left panel, S8b, d), but the magnitude of the effect depended on temperature and varied among females. Best models retained food availability and temperature operating interactively (Table S6). Consistently for all females, the effect of food limitation on carbon content was strong in the range 15-21 °C as compared with 24 °C (e.g. 16% vs. 8% decrease in carbon content). Similar effects were found for dry mass and nitrogen content (Fig. S8b, d). Food limitation reduced carbon and nitrogen contents in similar proportions among temperatures resulting in comparable C/N ratios (Fig. 2d, left panel); the exception was 15 ºC where the reduction in nitrogen (19%) was higher than that of carbon (16%).
Duration of development and growth of H. sanguineus larvae were more affected by temperature which contrasts to those of C. maenas, that are more sensitive to food limitation (Fig. 2). In terms of duration of development, larvae of H. sanguineus were more sensitive to temperature than C. maenas, especially at low temperatures; at 15 °C H. sanguineus reaches the megalopa in ca 70 days while C. maenas needs ca 30 days (Fig. 2a). By contrast, limited access to food increased developmental time in a lesser extent in H. sanguineus (< 16% change between the two food conditions) than in C. maenas (> 20%). While growth rates of H. sanguineus increased with temperature irrespective of the food condition, those of C. maenas decreased with temperature but remained constantly low in the food limited treatment (Fig. 2b). H. sanguineus megalopa had higher carbon content and lower C/N ratios than C. maenas (Fig. 2d). The weakest effect of food limitation on carbon content found at the highest temperature in H. sanguineus contrasts to the pattern found in C.

Fig. 3 Hemigrapsus sanguineus and Carcinus maenas.
Integrated responses of body mass and duration of development under the different treatments of temperature and food availability. Data shown as average values ± SE for both variables. Symbols: permanent access to food is represented with blue symbols (indicated as '+') and limited access to food with green symbols (indicated as '−'), temperature is indicated in the graph next to the symbols maenas, where the effect was weakest at the lowest temperature (Fig. 2c).
Integrated growth responses to megalopa of H. sanguineus were characterised by a strong decrease in body mass under food limitation rather than a long delay in development. The delay in metamorphosis did not compensate the effects of food limitation on growth rates. At 24 °C and under permanent access to food larvae reached a maximum threshold of body mass (= 260 µg/ind), and differences between food conditions were small (limited access to food = 240 µg/ind). However, lower thresholds were reached at lower temperatures, especially under limited food availability (Fig. 3, upper side of graph). In addition, H. sanguineus larvae did not compensate for the effect of low temperature on body mass even under permanent access to food (Fig. 3, upper side of graph). Similar patterns characterised the integrated responses in terms of carbon and nitrogen content (Fig. S9).
The range of variation in the integrative response of H. sanguineus is much higher than that of C. maenas larvae (Fig. 3). However, when the duration of development and the body mass are standardised, the opposite pattern arises. Figure 4 compares the effect of food limitation on the integrated responses of body mass and developmental time, for each temperature, of both Carcinus maenas and H. sanguineus. Each variable (V: body mass or duration of development) was expressed for each temperature (T) as a ratio, R: where the subindices L and P represent limited and permanent access to food, respectively. In this representation, the condition of permanent access to food is set to one irrespective of the temperature because the formula becomes: Figure 4 shows that as compared with C. maenas, H. sanguineus extended the development in a small fraction in response to limited access to food. In C. maenas, the proportional reduction in body mass and delay in metamorphosis became larger with temperature (Fig. 4, see also Torres and Giménez 2020); (1) by contrast in H. sanguineus both reduction in body mass and delays in metamorphosis do not show any consistent trend. The results of the simulated ratios of survival vs growth rates of both species showed that at most of the temperatures and food conditions (15 °C and 18 °C permanent and limited access to food and 21 °C limited access to food) larvae of C. maenas will be favoured; but at higher temperatures and especially under conditions of high access to food, larvae of H. sanguineus may be favoured (Fig. 5). The ellipses, indicating the importance of intraspecific variation in the performance of both species, show that H. sanguineus might perform better at higher temperatures, but it is not likely to outperform C. maenas at the lower temperatures that characterise the German Bight.

Discussion
There were three main findings out of our experiments. First, Hemigrapsus sanguineus is able to Fig. 4 Hemigrapsus sanguineus and Carcinus maenas. Summary of standardised responses to the different temperatures under food limited conditions (blue triangles and red diamonds, see Eq. 1). For the standardised values of body mass and developmental time, the values for permanent food conditions represent the unit (green star, see Eq. 2). At the lowest temperature tested, C. maenas extended the developmental time in response to food limitation; larvae compensated for the limited access to food, metamorphosing with high body mass. H. sanguineus did not compensate for the limited access to food and metamorphosed with lower body mass. Symbols: C. maenas: blue triangles, H. sanguineus: red diamonds. Data for H. sanguineus this study, data C. maenas from Torres and Giménez 2020 metamorphose to megalopa under limited daily access to food over a wide thermal range (15-24 °C). Second, within the range of temperatures experienced in the local habitat (German Bight, North Sea), low temperature (15 °C) appears to be the primary limiting driver of larval performance, in terms of survival and growth. Third, when the performance is quantified in relation to that of Carcinus maenas, unfavourable scenarios for H. sanguineus are projected for temperature characterising cool summers. In turn, favourable scenarios are projected for summers characterised by long heatwaves and after coastal warming.

Temperature and food limitation
H. sanguineus larvae were able to metamorphose to megalopa under limited access to food over a wide range of temperatures, with some variations among families (i.e. larvae originated from different females). This is consistent with other studies on decapod crustacean larvae and strengthen the general hypothesis that such larvae can reach metamorphosis as long as they access food patches for a short period of time every day (Sulkin et al. 1998;Giménez and Anger 2005;González-Ortegón and Giménez 2014;D´Urban Jackson et al. 2014;Torres and Giménez 2020). Limited daily access to food is expected in habitats characterised by food patchiness (Paffenhöfer et al. 1987;Durham and Stocker 2012;Prairie et al. 2012;Robinson et al. 2021) and in scenarios where larvae perform diel or tidal migrations (Forward et al. 2001;Queiroga and Blanton 2005;dos Santos et al. 2008;Thygesen and Patterson 2019). In temperate latitudes such as the one of the German Bight, access to prey during night time should be limited to a few hours because H. sanguineus larvae develop in summer when the darker period is restricted to few hours. Hence, H. sanguineus, as other species including C. maenas should be able to survive the larval phase as long as they access prey for a limited amount of time every day.
Food limitation had important consequences in terms of body mass and reserves at metamorphosis to the megalopa as well as growth rates and developmental time. In species with complex life cycles, traits at metamorphosis are relevant for post-metamorphic survival and are the basis for trait-mediated effects of the pelagic environment on recruitment in the benthic habitat (Giménez 2004;Pechenik 2006;Marshall et al. 2008;Torres et al. 2016). Larvae of H. sanguineus under food-limited conditions metamorphosed to megalopae with reduced body mass at all temperatures, suggesting that impacts of food limitation are consistent across the thermal range expected for cool and warm summers. It is important to understand the mechanisms driving body size at metamorphosis as they should mediate effects of warming on species responses (Lowe et al. 2021;Song et al. 2021). While there is a good amount of information on mechanisms driving metamorphosis in species with complex life cycles (Werner 1988;Emlet 1988;Hentschel and Emlet 2000), only a handful of studies have investigated how such mechanisms respond to warming (D'Urban et al. 2014;Torres and Giménez 2020;Griffith et al. 2021). Body mass is a plastic trait, driven by variations in larval growth rates and developmental time, but body mass should vary within upper and lower thresholds set by fitness costs (Werner 1988;Gotthard and Nylin 1995;Hentschel and Emlet 2000;Gotthard et al. 2000). Beyond the upper threshold, costs are associated to the high growth rates needed to achieve a large body mass (e.g. predation). Longer developmental time contributes to larger body mass but also determines the period when Squares and circles denote centroids of 95% confidence ellipses in shown colour. Data for permanent access to food (circles) shown with ellipses as continuous lines and for limited access to food (squares) as dashed lines. Blue: 15 °C, green: 18 °C, purple: 21 °C and red: 24 °C larvae are exposed to pelagic mortality risks (Eckert 2003) and the conditions experienced after settlement (Miron et al. 1999;Jarrett 2003). Because post-settlement conditions fluctuate in seasonal environments, the specific conditions experienced by juveniles are indirectly determined by the duration of the larval phase. For instance, in H. sanguineus, late settlement may result in reduced juvenile growth as individuals may miss most of the summer season where growth rates are enhanced by high temperatures in the intertidal zone. During summers, temperatures in the intertidal zone may boost growth because they are much higher than water temperatures given the exposure to sunlight during low tides (Stephenson 1942;Lewis 1964;Somero 2002). In addition, late settlement may expose individuals to cannibalism (Moksnes et al. 1997;Moksnes 2002Moksnes , 2004 or predation by e.g. juvenile C. maenas. According to life history theory, costs associated to trait changes should drive the evolution of plastic responses to environmental variation; because of trade-offs associated to such costs, the less responsive traits should be those with higher associated costs (Gotthard and Nylin 1995;Gotthard 2000Gotthard , 2004. In the case of H. sanguineus, larvae responded to food limitation mainly through reduction of growth rates; the extension of development time, found to partially compensate effects of food limitation in C. maenas  was rather short. By contrast, H. sanguineus larvae showed a strong plasticity to temperature by extending the development from < 20 days at 24 °C to > 60 days at 15 °C. Given that at the local population, H. sanguineus females appear to release larvae in early summer, only after temperatures reach 15 °C , summers with water temperatures in the range of 15-18 °C would result in late settlement if larvae were to extend further the developmental time. Hence, in the range 15-18 °C and under food limitation, further extension of the larval phase may have higher fitness costs than metamorphosing with a smaller size (but profiting from a warm growing period).

Low temperatures
Our study is in line with others (Stepherson et al. 2009;Giménez et al. 2020), showing that low larval survival occurs at low temperatures (range 12-15 °C). Reduced body mass at metamorphosis found at 15 °C is expected because body mass at stage, peaks at temperatures where physiological performance is optimal (Anger 1998(Anger , 2001. Perhaps the body mass of megalopa of H. sanguineus would decrease at temperatures higher than 24 °C, as expected from the temperature-size rule (Atkinson 1994).
Under low temperature and food limitation, larvae hatching from all females showed a stronger reduction in nitrogen as compared to carbon. This is unlike previously observed responses to stressors in other species (Harms et al. 1994;Torres and Giménez 2020;Torres et al. 2021) where the main characteristic is a stronger reduction in carbon than in nitrogen content. In decapod crustaceans, carbon content is considered a proxy for lipid reserves (Anger and Harms 1990); hence, stress responses of that type are interpreted as a reduction in the accumulation of lipid reserves, but not in the proteins needed to sustain activity or the enzymatic machinery. Given that in decapods, nitrogen is a proxy for protein levels, we hypothesise that the reduction observed in H. sanguineus reflects a thermal limitation in the rate of protein synthesis. Protein synthesis accounts for a great proportion of the specific dynamic action (SDA, Brody 1964) in crustaceans, i.e. the energetic costs incurred by physiological processes related to feeding (including e.g. ingestion, digestion, assimilation and synthesis : Jobling 1993;Wieser 1994), which increase after a meal (Houlihan et al. 1990;Robertson et al. 2001). For example, in Carcinus maenas, protein synthesis accounts for 20-37% of the post-meal oxygen rise (Houlihan et al. 1990). Temperature affects rates of protein synthesis in fasted and inactive crustaceans (McMillan and Houlihan 1988;Whiteley et al. 1996;El Haj and Whiteley 1997). Hence, one would expect that rates of protein synthesis are compromised at lower than optimal temperatures, due to limitations in covering the associated costs (Whiteley et al. , 2001. In this study, the lowest temperatures tested are suboptimal for H. sanguineus, but not for species that are native to North European Seas (e.g. D' Urban et al. 2014;Torres and Giménez 2020). Hence, the differences between H. sanguineus and e.g. C. maenas, in how C:N ratios respond to limited access to prey may reflect interspecific differences in the thermal tolerance range.
Performance of H. sanguineus relative to C. maenas We found that larval performance (e.g. survival) of H. sanguineus at low temperatures is low in relation to that of C. maenas also at low temperatures (Fig. 1). This is relevant to understand the outcome of the balance between larval settlement, and competition between C. maenas and H. sanguineus in the benthos. C. maenas larvae can complete larval development at temperatures as low as 12 °C (Dawirs 1985;Nagaraj 1993). The larval season of both species partially overlap, C. maenas larvae are released from May onwards and megalopae settle in the intertidal zone from the end of June until the end of August (Giménez and Dirk, 2007). Interspecific competition occurs because juveniles of both species develop in the intertidal zone during summer (Geburzi et al. 2018) The balance of larval supply and competition of C. maenas and H. sanguineus must be evaluated considering both spring and summer conditions. For example, a large number of C. maenas juveniles would survive competition in years with strong settlement of C. maenas followed by weak settlement of H. sanguineus due to cool summers (temperature ~ 15 °C).
We hypothesise that summers characterised by long heatwaves and high temperatures due to warming would increase performance of H. sanguineus larvae and exacerbate current competitive advantages already exhibited by the juvenile and adult stages in the benthos (Lohrer and Whitlach 2002;Jensen et al. 2002;Geburzi et al. 2018). Marine heatwaves, i.e. periods of time (> 3-5 days) when temperatures are above a predefined threshold (Meehl and Tebaldi 2004;Hobday et al. 2016), can have drastic consequences in structure and functioning of marine ecosystems (Garrabou et al. 2009;Marbà and Duarte 2010;Wernberg et al. 2013;Mills et al. 2013) and those in the German Bight have become more frequent since the 1990's (Giménez et al. 2022). During summer heatwaves such as those occurring in 2018, water temperatures around the local population reached values above 18 °C ; temperatures in waters of the coastal Wadden Sea (where C. maenas and H. sanguineus co-occur) were much higher (BSH 2019).
Another important aspect considered in our analysis concerns the role of intraspecific variation in performance (IVP). IVP is common in invertebrate larvae (e.g. Appelbaum et al. 2014;Spitzner et al. 2019;Torres et al. 2020), and expected from genetic variation (Marshall et al. 2008;Durrant et al. 2013) or parental effects (Pond et al. 1996;Shama et al. 2014). Important IVP was found in this study as variation in survival, developmental time, and growth among larvae from different females (e.g. Fig. S5), which is also expressed in Fig. 5 as wide ellipses. The ellipses are spread across regions where the relative performance switches from being stronger in H. sanguineus to become stronger in C. maenas. The ellipses surround the area where 90% of the 1000 simulated events for each factor combination are located. Hence, Fig. 5 depicts the importance of the intraspecific variation in the performance of the species, as opposed to what means show; the ellipses show that, for example, there is a possibility for H. sanguineus larvae to be able to perform (i.e. survive and grow) better than C. maenas at low temperatures. Likewise for Carcinus maenas, there is room for their larvae to perform better than those of H. sanguineus at high temperatures under food limitation. Species coexistence is one of the important ecological consequences of intraspecific variation (Bolnick et al. 2011;Appelbaum et al. 2014).
In synthesis, larvae from the invasive crab H. sanguineus are able to complete their development under a wide range of temperatures and under daily limited access to food (for a period as short as 6 h), with variations among families; hence, H. sanguineus larvae should be able to survive starvation periods as long as they can access food patches for a brief time every day. H. sanguineus responds to food limitation through lengthening the development to a much smaller degree as compared to C. maenas; this occurs at expenses of metamorphosing to megalopa with lower body mass. It remains to be seen how such responses affect the balance of costs and benefits of metamorphosing with lower body mass but still profiting from the warm season increasing juvenile growth rates. H. sanguineus shows different responses to high temperatures and food limitation from the native C. maenas . Under cool summer conditions, we would expect limited survival of H. sanguineus larvae which could favour C. maenas. By contrast, under a warming scenario, H. sanguineus should benefit through high larval growth and survival rates. Under this scenario, increased rates of survival and growth in the pelagic habitat should enhance propagule pressure of H. sanguineus, magnifying the effect produced by being the dominant competitor in the intertidal zone. Overall, our study emphasises the importance of integrative studies comparing the performance among native and invasive species across their life cycles, and extending our study towards the juvenile-adult phase. Such approach will help us to understand and predict effects of warming on species replacement.
Acknowledgements We are grateful to Julia Haafke (AWI) for performing the elemental analysis and Tobias Billens for help with larval rearing (University of Greifswald). We would also like to thank two anonymous reviewers whose comments and suggestions improved the quality of the work.
Author contributions GT, LG and NE-N conceived the experimental design. NE-N and GT performed the experiments. NE-N and LG analysed the data. NE-N wrote the first draft. All authors contributed to the writing of the manuscript and gave final approval for publication.
Funding Open Access funding enabled and organized by Projekt DEAL. The authors have not disclosed any funding. This study is part of NE-N doctoral dissertation and was supported by the Bundesministerium für Bildung und Forschung (Project MERGE; grant n° FKZ 01DN20002), Germany.
Data availability All data for this paper will be available from PANGAEA ® Data Publisher https:// www. panga ea. de

Conflict of interest
The authors declare that they have no conflicts of interests.

Human or animal rights
The research presented in this paper complies with national (Germany) and international laws (guidelines from the directives 2010/63/EU of the European parliament and of the Council of 22nd September 2010) on the protection of animals used for scientific purposes.
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