Life History Responses of Four Invasive Crayfish Species Under Prolonged Suboptimal Temperatures

Synopsis

Biological invasions are strongly shaped by temperature, especially in poikilothermic organisms, where thermal regimes influence life-history traits, thereby determining both their competitive potential and geographic distribution. However, comparative evidence on how suboptimal thermal conditions modulate interactions among co-occurring invasive species remains scarce. We experimentally compared the growth, survival, and reproductive performance of the invasive parthenogenetic marbled crayfish Procambarus virginalis with three widespread North American crayfish invaders in Europe: the spiny-cheek crayfish Faxonius limosus, the signal crayfish Pacifastacus leniusculus, and the red swamp crayfish Procambarus clarkii. Experiments were conducted under prolonged suboptimal temperature conditions (∼16°C over 45 weeks), followed by a short-term temperature increase (∼20°C). Across three independent laboratory trials, we assessed species performance in single-species and mixed-species stocks. Despite reduced absolute growth rates at low temperature, marbled crayfish rapidly compensated for their initially smaller size and outperformed spiny-cheek crayfish in growth and survival. In contrast, marbled crayfish were consistently suppressed when co-occurring with the larger and more aggressive red swamp crayfish, whereas interactions with signal crayfish resulted in temporary growth advantages but ultimately size convergence. Survival patterns reflected a combination of size asymmetries, behavioral dominance, and intraspecific aggression, with marbled crayfish exhibiting notably high survival in single-species stocks across all trials. Reproductive development was strongly temperature-constrained. While marbled crayfish readily formed glair glands and ovulated eggs at 16°C, successful hatching occurred only after the temperature was raised. Our results demonstrate that suboptimal thermal conditions do not eliminate competitive asymmetries among invasive crayfish but instead reshape invasion outcomes in species-specific ways. These findings highlight the marbled crayfish’s capacity to persist and interact competitively even in colder environments, with important implications for invasion dynamics under ongoing climate change.

Introduction

The introduction of species beyond their native ranges can result in the establishment of self-sustaining non-native populations that spread and generate ecological, economic, or sociocultural impacts (Haubrock et al. 2025a, b), thereby qualifying them as invasive non-native species (see Soto et al. 2024, for further reference). Freshwater ecosystems are particularly vulnerable to biological invasions (Sayer et al. 2025), and numerous invasive taxa not only impact native communities but also interact competitively with other non-native species (Gherardi et al. 2011; Havel et al. 2015). Among these, freshwater crayfish rank among the most damaging invaders globally (Lodge et al. 2012).

Across taxa, invasion success is shaped by environmental filters that interact with species’ thermal performance and life–history schedules (Blackburn et al. 2014). In poikilotherms in particular, temperature scales core processes, such as metabolism, feeding, growth, aggression, and reproduction (Angilletta Jr 2009), thereby modulating establishment, spread, and the strength and direction of biotic interactions (O’Connor et al. 2011). This framework predicts that both suboptimal and elevated temperatures can re–rank competitive hierarchies among co–occurring invaders when rivals differ in thermal tolerance, plasticity, or behavioral dominance (Kordas et al. 2011; Urban et al. 2016).

Within this broader context, freshwater crayfish provide a well-established model group for studying temperature-sensitive invasion dynamics. Europe is disproportionately affected by non-native crayfish of North American origin, which frequently displace native species through competition and transmission of the crayfish plague pathogen Aphanomyces astaci, to which European crayfish are highly susceptible (Ion et al. 2024; Mojžišová et al. 2024; Oficialdegui et al. 2025). Three long-established invaders (the spiny-cheek crayfish Faxonius limosus, signal crayfish Pacifastacus leniusculus, and red swamp crayfish Procambarus clarkii) are now widespread (Kouba et al. 2014). More recently, the parthenogenetic marbled crayfish, Procambarus virginalis, has expanded rapidly across the continent, largely through repeated pet-trade releases (Patoka et al. 2016; Sánchez et al. 2024). Continued introductions increase the likelihood of co-occurrence among invasive taxa, creating conditions for competitive reordering under varying environmental constraints (Weiperth et al. 2020; Balzani et al. 2025).

This trend towards over-invasion scenarios raises critical questions about species’ competitive performance under varying environmental conditions, which can be optimal for some species but suboptimal for others (Hudina et al. 2011; Kouba et al. 2016; Balzani et al. 2025). Consequently, understanding how environmental factors, such as temperature, mediate establishment success, range expansion, and interspecific interactions is essential for predicting relative abundance patterns and long-term persistence in invaded communities (Carvalho et al. 2022; O’Hea Miller et al. 2024). Species such as the marbled crayfish and red swamp crayfish are typically regarded as warm-water taxa, although both exhibit substantial tolerance to low temperatures, as demonstrated by their persistence in relatively cold climates (Veselý et al. 2015; Haubrock et al. 2019; Hamr 2024; Mengal et al. 2026). However, their introductions have often occurred in thermally influenced habitats, including naturally warm waters or sites affected by thermal pollution in temperate regions (Ercoli et al. 2019; Bláha et al. 2022). Besides crayfish, diverse warm-water taxa, including aquatic plants, molluscs, shrimps, fish, and turtles, can use these habitats as a source for outward spread into surrounding habitats and for gradual acclimation to temperate climate conditions (Lőkkös et al. 2016; Lipták et al. 2018; Šajna et al. 2023; Takács et al. 2025). Nevertheless, releases associated with irresponsible or unaware pet ownership can occur virtually anywhere, including environments with suboptimal thermal conditions, as evidenced by records from Poland, Sweden, and the North American Great Lakes region (Bohman et al. 2013; Maciaszek et al. 2022; Hamr 2024).

To assess whether suboptimal thermal conditions act as a selective filter that reshapes competitive hierarchies among co-occurring non-native crayfish, we conducted controlled comparative experiments focusing on their growth, survival, and reproduction. We examined the performance of marbled crayfish relative to spiny-cheek crayfish, signal crayfish, and red swamp crayfish under suboptimal temperature conditions (∼16°C) across three independent trials. Using invasive crayfish as a model system, these experiments provide mechanistic insight into how temperature constrains invader performance and modulates interspecific competitive outcomes. Because the same thermal and behavioral mechanisms govern many freshwater invaders, our comparative, low–temperature approach offers a transferable framework for anticipating invasion trajectories under suboptimal thermal regimes. This knowledge is essential for improving risk–assessment frameworks and anticipating the dynamics of current and emerging crayfish invasions in temperate freshwater systems under present–day climatic conditions and future scenarios.

Materials and methods

Animal origin and juvenile selection

Egg-bearing females of spiny-cheek crayfish and signal crayfish were collected from established feral populations in the Czech Republic, specifically from the Blanice river in Protivín (49.186°N, 14.221°E) and Křesanovský brook in Vimperk (49.059°N, 13.761°E), respectively. Females were transported to the laboratory several weeks before hatching and were gradually acclimated to the experimental temperature regime (i.e., ∼16°C). The other two species originated from closed laboratory cultures maintained at the Research Institute of Fish Culture and Hydrobiology in Vodňany and were derived from locally sourced pet-trade individuals. The red swamp crayfish has not yet been established in the Czech Republic (Kouba et al. 2014). At the time of the experiments (conducted from 2019 to 2021), existing marbled crayfish populations (Patoka et al. 2016) were insufficient to obtain the required number of egg-bearing females. Although logistically demanding, only juveniles that first reached independence (accompanied by the onset of exogenous feeding) on the same day were selected and matched across species to ensure strict comparability of performance. Signal crayfish juveniles reach independence at the second developmental stage, whereas juveniles of the remaining species were used at the third developmental stage (Kawai and Kouba 2022).

Experimental design

To better understand the performance of juvenile marbled crayfish relative to the abovementioned invasive species under suboptimal temperature conditions, we conducted three independent laboratory experiments comparing growth, survival, and reproduction. Each trial included marbled crayfish and one of the three additional species, and comprised three experimental groups: two monocultures (one per paired species) stocked at an initial density of 16 juveniles per aquarium (hereafter referred to as “single-species” stocks), and one communal stock with the same total density but an equal species ratio (1:1; eight juveniles of each species; hereafter referred to as “mixed-species” stock). We replicated each experimental combination five times, resulting in 15 aquaria per trial and 45 aquaria in total across all trials.

In our previous study (Kouba et al. 2021), conducted under near-optimal warm conditions (∼22°C), marbled crayfish extruded and carried pleopodal eggs for the first time between weeks 14 and 16 of culture (∼16–18 weeks of age, including the early postembryonic development of juveniles; Guo et al. 2019). Based on this work, the present study focused on species performance under suboptimal temperature conditions (∼16°C). Seitz et al. (2005) reported that marbled crayfish cease reproduction at temperatures of 15°C or below. As growth and maturation are strongly temperature-dependent, with lower temperatures slowing growth and delaying maturation, the experimental period under the selected temperature regime was extended to 45 weeks. During this period, marbled crayfish ovulated; however, successful hatching did not occur (see Results for details). Consequently, the temperature was increased to ∼20°C for an additional 9 weeks (until week 54) in the experimental groups involving marbled crayfish and red swamp crayfish, the only species that consistently formed glair glands and ovulated eggs within the initial 45-week period. The spiny-cheek crayfish and signal crayfish stocks were terminated at that point, while their corresponding marbled crayfish stocks continued. The light: dark photoperiod was maintained at 12:12 h.

Although each species has a distinct thermal optimum, 16°C constitutes a biologically suboptimal condition for all four taxa, enabling meaningful performance comparisons without introducing confounding species–specific temperature treatments. It can be more restrictive for the warm–water red swamp crayfish and marbled crayfish than the cold–adapted crayfish species (Westhoff and Rosenberger 2016). Even for the latter group, natural summer temperatures commonly exceed 20°C (Bohman et al. 2016), indicating that 16°C lies below the typical thermal range experienced by all species. We therefore applied a single, uniform temperature regime to ensure direct interspecific comparisons under identical environmental constraints.

Culturing conditions and feeding

Unsexed juveniles were randomly stocked into a laboratory recirculating rearing system consisting of glass aquaria (37 cm width × 55 cm length × 31.5 cm height; usable volume 55 L), which served as culture units. To minimize aggression and cannibalism, shelters were provided in the form of a fired clay brick (6.5 × 28.5 × 13.5 cm) containing 39 cross holes (26 and 13 holes with cross-sectional profiles of 1 × 3 cm and 1 × 1 cm, respectively) placed in each aquarium (Kouba et al. 2011). Consequently, the number of available individual shelters exceeded the initial number of stocked juveniles by more than twofold.

As crayfish grew during the experiment, two blocks of joined polypropylene tubes, each comprising five tubes (length 10 cm, inner diameter 35 mm), were added as larger shelters after 9 weeks of culture. Each block consisted of three longitudinally joined tubes forming the base, with two additional tubes arranged pyramidally in a second layer (Veselý et al. 2017).

In all trials, crayfish were fed ad libitum once daily with defrosted brine shrimp (Artemia sp.) nauplii and chironomid larvae during the first 6 weeks to minimize resource limitation and isolate temperature and interspecific interaction effects from density-dependent food competition. Thereafter, the diet was changed to defrosted chironomid larvae supplemented with commercial pellets (Granugreen, Sera, Heinsberg, Germany). Aquaria were cleaned three times per week (Monday, Wednesday, and Friday), and the system was replenished with drinking tap water as required.

To minimise handling stress, individual weights (after removal of excess surface water using absorbent tissue paper) were recorded every 3 weeks using an analytical balance (Kern and Sohn GmbH, Balingen, Germany) for a total of 45 weeks. As individuals grew and matured, they were also sexed and examined for the presence of glair glands and attached eggs (for details see Das et al. 2025). For marbled crayfish and red swamp crayfish, the only species exhibiting consistent formation of glair glands and egg deposition within the initial 45-week period, reproductive parameters were monitored under increased water temperature of ∼20°C for another 9 weeks.

Water temperature (T,°C), dissolved oxygen (DO, mg L⁻¹; measured using Oxi 315i, WTW GmbH, Weilheim, Germany), and pH (measured using a pH 315i, WTW GmbH, Weilheim, Germany) were recorded daily and remained generally stable throughout the experiment (Table 1).

Table 1.

Basic water quality parameters (temperature,°C; dissolved oxygen, DO, mg L⁻¹; and pH) were recorded in each trial comparing marbled crayfish P. virginalis with comparator species (spiny-cheek crayfish F. limosus, signal crayfish P. leniusculus, or red swamp crayfish P. clarkii) under the respective temperature regimes.

	First 45 weeks						Additional 9 weeks
	T		DO		pH		T		DO		pH
Trial	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
F. limosus	16.1	0.3	8.7	0.7	7.7	0.2	20.2	0.6	7.8	0.6	7.8	0.1
P. leniusculus	16.0	0.3	8.5	0.6	7.7	0.3	20.0	0.9	8.1	0.6	7.8	0.3
P. clarkii	16.1	0.6	9.3	0.8	7.3	0.4	20.2	0.4	7.9	0.5	7.2	0.2

Note: Data are presented as means with their respective SDs.

Statistical analyses

Weight data were evaluated for normality and homogeneity of variances using the Kolmogorov–Smirnov and Levene’s tests, respectively. Because several groups violated test assumptions, comparisons of weights between single- and mixed-stock treatments were conducted using non-parametric Kruskal–Wallis tests, followed by post hoc multiple comparisons of mean ranks. Sex-related weight differences in sexually reproducing species were assessed at week 45 using nonparametric Mann–Whitney U tests for both single- and mixed-stock treatments. Nonparametric survival analyses were performed using the Kaplan–Meier method implemented in the survival package in R (Therneau and Grambsch 2000) to test for significant differences among specific pairwise assemblages. Additionally, survival rates, calculated as % survival from the initial stocking densities, were arc-sine transformed and assessed as weight data. Statistical analyses other than survival analyses were conducted using Statistica 14.0.0.15 for Windows (TIBCO Software Inc). Data are presented as mean and their respective SD unless specified otherwise, and results were considered statistically significant at P < 0.05.

Results

Growth

The initial body mass of juveniles at the onset of exogenous feeding varied substantially among species across all trials. Based on a subsample of individuals not subsequently used in the experiment, spiny-cheek crayfish juveniles were almost twice as heavy as marbled crayfish (mean = 11.0, SD = 1.5 mg vs. mean = 5.7, SD = 0.7 mg; n = 20 per species). A similar pattern was observed in the comparison with red swamp crayfish (mean = 11.8, SD = 1.3 mg vs. mean = 5.1, SD = 0.3 mg; n = 10 for both species). The greatest disparity occurred in the signal crayfish trial, in which juveniles were more than four times heavier than marbled crayfish (mean = 24.2, SD = 2.2 mg vs. mean = 5.3, SD = 0.6 mg; n = 10 for both species).

All species exhibited pronounced but distinct growth trajectories (Fig. 1 and Fig. S1). Despite their initially smaller size, marbled crayfish reached body masses comparable to those of the larger spiny-cheek crayfish within 3 weeks of culture. From the sixth week onwards, marbled crayfish exceeded spiny-cheek crayfish in size in both single-species and mixed-species stocks (Fig. S1). No statistically significant differences in body mass were detected between single- and mixed-species stocks within species. After 45 weeks, only a single-stocked spiny-cheek crayfish exceeded 5 g in body mass, whereas several individuals surpassed 10 g in the marbled crayfish in both single- and mixed-stocks (Fig. S2). Spiny-cheek crayfish males and females were similarly sized in both types of stock after 45 weeks (Fig. S3).

Fig. 1.

Mean body weight (g) over time of marbled crayfish Procambarus virginalis in experimental trials with (A) spiny-cheek crayfish Faxonius limosus, (B) signal crayfish Pacifastacus leniusculus, and (C) red swamp crayfish Procambarus clarkii under single-species and mixed-species stocking conditions. In each panel, darker shades represent single-species stocks and lighter shades represent mixed-species stocks. Points indicate weekly means, and shaded areas represent 95% confidence intervals. For data presented as mean and SD, see Fig. S1; for individual measurements, see Fig. S2.

Signal crayfish juveniles remained significantly larger for at least the first 9 weeks of culture (Fig. 1 and Fig. S1). However, marbled crayfish exhibited greater growth during this period. Body masses of both species converged between weeks 12 and 18, after which marbled crayfish prevailed in size (weeks 21–30). With the onset of reproductive development (see below), growth increments in marbled crayfish declined, and body sizes of both species became largely comparable again, with no significant differences detected at weeks 42 and 45 (Fig. S1). Despite higher growth rates and larger mean body mass of marbled crayfish over most of the experimental period, signal crayfish ultimately achieved greater individual sizes. After 45 weeks, only three marbled crayfish individuals exceeded 10 g (maximum 16.64 g) in single-species stocks, whereas twelve signal crayfish individuals surpassed this threshold (single- and mixed-species stocks combined; maximum 25.32 g; Fig. S2). Signal crayfish males were heavier than females in their single stock after 45 weeks (Fig. S3).

Marbled crayfish remained the smaller species throughout the last trial with the red swamp crayfish (Fig. 1 and Fig. S1). When comparing single- and mixed-species stocks, the smaller marbled crayfish was substantially suppressed when cultured together with the red swamp crayfish, and, vice versa, red swamp crayfish attained on average larger sizes, albeit not statistically significant in the given species (Fig S1). The initial growth rates of both species were substantial, but decelerated around week 24, when signs of maturation were more broadly pronounced in both species (see below). At this time, the first red swamp crayfish individual achieved 30 g (Fig. S2). At 45 weeks, the red swamp crayfish individuals usually ranged between 10 and 20 g, but individuals above this were also common. On the contrary, the largest marbled crayfish attained only 7.96 g even in the single-species culture. Red swamp crayfish males were heavier than females in their single stock after 45 weeks (Fig. S3).

Survival

In the spiny-cheek crayfish trial, marbled crayfish achieved the highest survival when held in mixed stock (65.0% after 45 weeks; Fig. S4), while spiny-cheek crayfish exhibited the lowest survival in mixed stock (27.5%; Fig. 2, Table S1). Survival of marbled crayfish in single-species stock was also high (52.2%) and exceeded that of spiny-cheek crayfish in both single- (35.0%) and mixed-species stocks.

Fig. 2.

Kaplan–Meier survival curves for marbled crayfish Procambarus virginalis in trials with spiny-cheek crayfish Faxonius limosus (A), signal crayfish Pacifastacus leniusculus (B), and red swamp crayfish Procambarus clarkii (C) under single-species and mixed-species stock conditions throughout the experiment. Darker and lighter colours denote single-species and mixed-species stocks, respectively.

In contrast, survival curves were more balanced in the signal crayfish trial (Fig. S4 and Table S1). Signal crayfish were larger throughout the experiment, although marbled crayfish temporarily dominated intermediate growth phases (Fig. 1). The lowest survival occurred in the signal crayfish single-species stock (36.3%; Fig. 2 and Fig. S4). Final survival was similar among marbled crayfish single-species stock (51.3%), marbled crayfish mixed-species stock (42.5%), and signal crayfish mixed-species stock (50.0%).

In the red swamp crayfish trial, survival patterns resembled those of the spiny-cheek trial but under reversed size asymmetry (Fig. 2), as marbled crayfish remained substantially smaller (Fig. 1). Marbled crayfish showed the highest survival in single-species stock (78.8%), but survival declined sharply in mixed stock with red swamp crayfish (42.5%; Fig. S4 and Table S1). Red swamp crayfish also exhibited low survival in single-species stock (42.5%), significantly lower than marbled crayfish single-species survival, while mixed-stock red swamp crayfish showed intermediate survival (60.0%; Fig. S4 and Table S1).

Reproduction

Developed glair glands were first observed in red swamp crayfish at weeks 18 and 21 in mixed- and single-species stocks, respectively (Table 2). Although male sexual development was not systematically monitored, the first reproductively mature red swamp crayfish male (Form I) was detected in the mixed-species stock during this period. In marbled crayfish, females bearing glair glands first appeared between weeks 24 (single-species stocks in the trials with spiny-cheek crayfish and red swamp crayfish) and 27 (mixed-species stock in the trial with spiny-cheek crayfish, and both stocks in the trial with signal crayfish). In these groups, the number of females with glair glands was the highest between weeks 33 and 36. Glair gland prevalence peaked between weeks 33 and 36. Marbled crayfish showed a higher probability of glair gland formation than red swamp crayfish, except in the mixed-species stock with red swamp crayfish, where the first case occurred in week 51. In spiny-cheek crayfish, the first female with glair glands was recorded after 42 weeks (mixed) and 45 weeks (single-species; Table 2).

Table 2.

Absolute numbers of females with developed glair glands and those with attached eggs (+) recorded throughout the experiment.

	Week
Trial	Group	18	21	24	27	30	33	36	39	42	45	48	51	54
A	P. virginalis single	–	–	2 + 0	21 + 0	26 + 0	25 + 0	31 + 1	11 + 17	7 + 6	7 + 8	3 + 7	2 + 1	2 + 4
	F. limosus single	–	–	–	–	–	–	–	–	–	1 + 0	NA	NA	NA
	P. virginalis mixed	–	–	–	13 + 0	16 + 0	17 + 0	16 + 4	5 + 13	6 + 3	2 + 5	2 + 2	1 + 2	–
	F. limosus mixed	–	–	–	–	–	–	–	–	1 + 0	1 + 0	NA	NA	NA
B	P. virginalis single	–	–	–	8 + 0	19 + 0	20 + 0	23 + 1	12 + 8	13 + 8	9 + 7	9 + 5	4 + 4	4 + 2 (1)
	P. leniusculus single	–	–	–	–	–	–	–	–	–	–	NA	NA	NA
	P. virginalis mixed	–	–	–	4 + 0	14 + 0	12 + 0	15 + 1	9 + 4	8 + 2	3 + 2	0 + 2	3 + 0	2 + 0
	P. leniusculus mixed	–	–	–	–	–	–	–	–	–	–	NA	NA	NA
C	P. virginalis single	–	–	4 + 0	12 + 0	20 + 0	29 + 0	21 + 1	21 + 9	16 + 15	0 + 18	12 + 21	3 + 22 (8)	3 + 18 (7)
	P. clarkii single	–	1 + 0	5 + 0	6 + 0	5 + 0	7 + 0	7 + 0	6 + 0	7 + 0	6 + 0	4 + 1	2 + 1	4 + 3
	P. virginalis mixed	–	–	–	–	–	–	–	–	–	–	–	1 + 2	1 + 3
	P. clarkii mixed	1 + 0	3 + 0	6 + 0	9 + 0	9 + 0	10 + 0	10 + 0	8 + 0	8 + 0	9 + 0	9 + 1	9 + 0	9 + 0

Numbers in brackets indicate ovigerous marbled crayfish that successfully hatched juveniles in some stocks at weeks 51 and 54 (highlighted in bold in brackets). Note that absolute numbers of evaluated individuals varied with the number of animals stocked and their survival (single- vs. mixed-species stocks; Fig. S4) and that marbled crayfish comprised females only. In general, we do not expect that marbled crayfish females were capable of completing two full reproductive cycles over the duration of the experiment. Data are shown for marbled crayfish Procambarus virginalis in trials with spiny-cheek crayfish Faxonius limosus (A), signal crayfish Pacifastacus leniusculus (B), and red swamp crayfish Procambarus clarkii (C).

Ovigerous marbled crayfish were first recorded at week 36 in the trials with spiny-cheek crayfish and signal crayfish across stocks (Table 2), with peak frequencies occurring shortly thereafter (week 39 in trials with spiny-cheek and signal crayfish, and later in the red swamp crayfish trial). Neither the spiny-cheek crayfish nor the signal crayfish produced eggs during the experiment. Despite frequent oviposition by marbled crayfish, no clutch hatched during the initial 45 weeks at 16°C, and many eggs subsequently showed mortality (Fig. S5). Red swamp crayfish also failed to ovulate under this temperature regime (Table 1). Following the temperature increase to 20°C, red swamp crayfish ovigerous females appeared from week 48 onward. Marbled crayfish maintained in the mixed stock with the red swamp crayfish also developed glair glands and ovulated eggs in week 51. Although none of these clutches hatched, several marbled crayfish in the single-species stock eventually carried viable hatchlings or developing juveniles in the trial with red swamp crayfish after week 51, and a single marbled crayfish female also finally hatched in the trial with signal crayfish.

Discussion

Water temperature is a dominant driver of invasion success in aquatic ecosystems by shaping population dynamics, species interactions, and ultimately invasion trajectories of freshwater taxa (Rahel and Olden 2008). By experimentally comparing three long-established invasive crayfish species in Europe (the spiny-cheek crayfish, the signal crayfish, and the red swamp crayfish) with the newly emerging marbled crayfish under prolonged suboptimal thermal conditions (Seitz et al. 2005; Westhoff and Rosenberger 2016), this study demonstrates that low temperatures constrain absolute performance but do not necessarily eliminate competitive asymmetries among these invaders, with outcomes depending on additional ecological, biological and environmental factors.

Growth

Despite reduced growth rates under suboptimal temperature conditions (∼16°C) compared to near-optimal environments (at ∼22°C; cf. Kouba et al. 2021), interspecific hierarchies among the tested invasive crayfish species remained consistent. Marbled crayfish rapidly overcame its smaller initial size and exceeded spiny-cheek crayfish within 3 weeks, demonstrating that their growth advantage extends beyond warm conditions (Kouba et al. 2021). This is notable given the higher thermal requirements of this species compared with spiny-cheek crayfish (Simčič et al. 2014; Faiad et al. 2023). In ecological and biogeographical studies, temperature preferences and tolerance limits are typically inferred from the range of environmental temperatures in a species’ native distribution (Zhang et al. 2020). Marbled crayfish presumably originate from Florida (Maiakovska et al. 2021), whereas spiny-cheek crayfish are native to the northeastern USA and southeastern Canada (Taylor et al. 2007), suggesting lower temperature requirements. Although the effect was modest, marbled crayfish in mixed-species stocks with spiny-cheek crayfish attained larger sizes than those in single-species cultures. Based on maximum body size alone, the species are broadly comparable, typically not exceeding 10 cm in total length, with individuals around 12 cm being rare (Hamr 2002; Chybowski 2007; Vogt 2021). Marbled crayfish consistently exhibited faster growth across both temperature regimes (Kouba et al. 2021; this study), thus conferring a competitive advantage.

Signal crayfish are native to western North America, while cryptic diversity within the species suggests a more complex phylogeographic structure than previously assumed (Taylor et al. 2007; Larson et al. 2012). The species exhibits the lowest temperature requirements, closely resembling those of generally cold-water European astacids (Westhoff and Rosenberger 2016; Viana et al. 2023). Nonetheless, marbled crayfish were able to compensate for their initially smaller size even at ∼16°C. This advantage, however, is temporary, as signal crayfish ultimately attain substantially greater size and mass, reaching a maximum of around 16 cm (Kirjavainen and Westman 1999; Buřič et al. 2021).

By contrast, as occurred at 22°C (Kouba et al. 2021), red swamp crayfish maintained a clear size advantage throughout the experiment, most pronounced in mixed-species stocks. Although native to relatively warm regions of northeastern Mexico and the southeastern USA (Taylor et al. 2007), the species exhibits substantial thermal plasticity (Westhoff and Rosenberger 2016), as further evidenced by its quasi-global distribution, including temperate regions (Oficialdegui et al. 2020; Ion et al. 2024). A previous study emphasised the temporal consistency of temperature as a crucial driver of red swamp crayfish distribution (Guareschi et al. 2024). Whereas marbled crayfish in the wild typically do not exceed 20 g, red swamp crayfish commonly reach 30–50 g due to remarkable growth rates (up to 50 g within 3–5 months; Gherardi et al. 2011), and attain total lengths of up to 15 cm (Vogt 2021). This pattern is particularly evident in males, which are generally larger in crayfish due to higher moulting frequencies and greater size increments per moult, both of which are closely linked to lower reproductive investment (Reynolds 2002).

Survival

In accordance with Kouba et al. (2021), marbled crayfish maintained in single-species stocks consistently exhibited high survival rates (above 50% after 45 weeks of culture), suggesting relatively low intraspecific competition. This pattern may partly reflect the surplus of food and shelters provided under experimental conditions, but may also be linked to species-specific traits, including its all-female population structure and the absence of reproductive agonistic interactions, as crayfish males are generally more aggressive (Gherardi 2002). In addition, the frequent occurrence of females bearing glair glands, attached eggs, or early juveniles (Das et al. 2025), together with associated pheromonal cues (Kubec et al. 2019), may modulate aggressive interactions within the population. Finally, the comparatively small and not robust chelae of marbled crayfish (Vogt 2021) may reduce the likelihood of severe injuries during intraspecific encounters.

Nevertheless, reduced intraspecific density conferred certain advantages, as evidenced by the mixed-species stocks in which marbled crayfish were competitively dominant over spiny-cheek crayfish. Beyond size-mediated effects, survival outcomes are closely linked to behavioral interactions. Linzmaier et al. (2018) demonstrated that marbled crayfish exhibit greater aggressiveness than spiny-cheek crayfish during agonistic encounters, even when the latter are larger. Similarly, Kubec et al. (2022) reported dominance of marbled crayfish over similarly sized spiny-cheek and signal crayfish, particularly at higher temperatures (tested temperatures of 16°C, 20°C, and 24°C), from which comparable conclusions can be inferred indirectly. Chucholl et al. (2008) documented the dominance of calico crayfish Faxonius immunis over spiny-cheek crayfish, while Hossain et al. (2020) independently demonstrated the superiority of marbled crayfish over calico crayfish alone. Collectively, these findings suggest that behavioral dominance is a significant contributor to survival in interspecific assemblages of marbled crayfish and spiny-cheek crayfish under variable environmental conditions.

In contrast, marbled crayfish experienced substantial survival costs at sub-optimal and near-optimal temperatures when co-occurring with larger red swamp crayfish (Kouba et al. 2021; this study). Red swamp crayfish are characterised by pronounced aggressiveness, a trait that has contributed to their recognition as a globally successful invader (Souty-Grosset et al. 2016). Although marbled crayfish can be effective opponents in direct interactions, this appears to occur only when individuals are of similar size (Jimenez and Faulkes 2011; Hossain et al. 2019). Given the pronounced size disparity between the species under natural conditions, marbled crayfish are likely to be competitively disadvantaged in syntopy. A similar outcome can be expected in co-occurrence with signal crayfish, despite the marbled crayfish’s temporary early-growth advantage.

Reproduction

From an eco-evolutionary perspective, substantial growth and high survival have limited relevance unless they ultimately translate into successful reproduction. Our experimental conditions lacked seasonal changes in temperature and natural photoperiod shifts, both of which can play specific roles in regulating crayfish reproduction (Harlıoğlu and Farhadi 2017). These cues are arguably more important for temperate species such as the signal crayfish and the spiny-cheek crayfish than for more warm-water counterparts (the marbled crayfish and the red swamp crayfish), as further discussed below. It should also be acknowledged that mimicking diel and/or seasonal temperature and light patterns may provide additional information, thereby better reflecting the situation in the wild. Also, the diet available in natural habitats is typically more diverse and may include nutrients that better support somatic growth and maturation. On the contrary, resources such as food, but also shelter, may become limited in the natural environments. Despite these limitations, the experiment still allows for informative comparisons and meaningful insights into reproductive patterns among the studied crayfish species.

First, despite the considerable duration of the experiment at 16°C and the absence of a seasonal temperature decline typical of temperate climates, maturation of signal crayfish females was not expected. Although the largest individuals reached body sizes comparable to the smallest mature individuals of the species after 45 weeks of culture, signal crayfish males generally first mature in their second year of life, with females typically maturing a year later (Lewis 2002). Importantly, successful reproduction in signal crayfish likely requires additional environmental cues, such as photoperiod shortening and temperature decline, as mating and egg attachment normally occur in autumn, followed by overwinter egg incubation and hatching in late spring or early summer (Kouba 2007; Yazicioglu et al. 2018).

In the present study, only two spiny-cheek crayfish females developed glair glands in both mixed- and single-species stocks after 43 and 45 weeks, respectively. By contrast, under warmer conditions (22°C), glair gland formation occurs substantially earlier, at approximately 13 weeks of culture (Kouba et al. 2021). These observations indicate that the experimental conditions employed were sufficient to induce glair gland development, but likely suboptimal for completing the full reproductive cycle. Under highly favourable conditions, some individuals can reproduce within their first year (Kozák et al. 2007). Spiny-cheek crayfish mate in autumn, while ovulation typically occurs the following spring, after a second peak in mating activity (Van den Brink et al. 1988; Buřič et al. 2009). This temporal pattern suggests that exposure to winter conditions serves as a critical reproductive cue. Juveniles hatch ~40–50 days post-ovulation, most commonly in June (Kozák et al. 2006). Both autumn-only and spring-only mating events are sufficient for successful reproduction, as spermatophores are retained in the female’s seminal receptacle, the annulus ventralis, which is a feature conserved across Cambaridae (Buřič et al. 2013). Remarkably, under experimental isolation from males, females were capable of reproducing via facultative parthenogenesis, a reproductive strategy unique among decapods, though not yet documented in natural populations (Buřič et al. 2011). Although exceedingly rare, ovulation during autumn has also been observed; however, the environmental triggers underlying such atypical timing, as well as the winter survival of early juveniles, remain poorly understood (Weiperth et al. 2020).

The red swamp crayfish is well known for its rapid growth and early sexual maturation (Souty-Grosset et al. 2016). When reared at 22°C, females bearing glair glands appeared after approximately 12 weeks of culture; however, despite frequent copulation, no oviposition occurred before experimental termination at week 15 (Kouba et al. 2021). In the present study, females with glair glands were first detected after 18 and 21 weeks in mixed-species and single-species stocks, respectively. Ovigerous females were observed only at week 45, occurring 3 weeks after water temperature was increased to 20°C. Nevertheless, no hatchlings were recorded before the experiment concluded at week 54. Collectively, these findings indicate that the experimental conditions approached the threshold required by this species to reproduce.

Females of marbled crayfish bearing glair glands were first observed at weeks 24 and 27, with ovigerous females appearing from week 34 onwards. The only exception occurred in the mixed-species treatment with red swamp crayfish, where marbled crayfish were strongly suppressed, and the first females bearing glair glands and eggs were not detected until week 51. These results demonstrate that marbled crayfish are capable of regularly forming glair glands and ovulating at 16°C, closely matching the lower reproductive threshold of 15°C reported by Seitz et al. (2005). Notably, successful hatching was, however, observed only after the temperature was increased to 20°C, occurring at weeks 51 and 54. Eggs ovulated shortly after the temperature increase also failed to develop successfully, suggesting that exposure to elevated temperatures is required during at least part of egg maturation, rather than for ovulation or early embryonic development per se.

Support for this interpretation comes from both laboratory and field observations. Under controlled conditions, marbled crayfish exhibit two reproductive peaks, including one in autumn (Vogt 2015). Similarly, wild populations show evidence of late-season reproduction. Lipták et al. (2017) reported numerous ovigerous females and individuals carrying attached juveniles in late October in Slovakia, while Dobrović et al. (2021) documented females with juveniles in October and November in Croatia, which is in line with observations in the Czech Republic (Buřič et al. 2025). During these periods, water temperatures frequently fall below the aforementioned thermal thresholds, yet the offspring appeared fully viable. What remains unresolved is the extent to which such early juveniles, presumably with limited energetic reserves, can survive overwintering conditions.

Under warmer conditions (22°C), the onset of marbled crayfish reproduction is substantially accelerated, with females bearing glair glands and eggs observed as early as weeks 12 and 14 after the onset of exogenous feeding (Kouba et al. 2021). To this, we should add some 2 weeks, which cover the first two mother-dependent developmental stages in this species (Guo et al. 2019). Still, this timing is earlier than previously reported (Seitz et al. 2005) and may be further shortened under more optimal conditions, such as improved dietary quality (Das et al. 2024a; 2024b). Together, these pronounced reproductive plasticities across a broad thermal range likely contribute to the exceptional invasive potential of marbled crayfish, particularly under ongoing climate warming.

Conclusions

Our comparative experiments show that prolonged exposure to suboptimal temperatures reshapes competitive outcomes through combined effects on growth, survival, and reproductive thresholds. These patterns reflect general temperature–dependent mechanisms common across poikilothermic organisms, including shifts in behavioral dominance, changes in activity and feeding, and limits on successful recruitment. Because such mechanisms influence ecological sorting in many freshwater invaders, the crayfish system used here illustrates a broader principle: environmental filtering rarely acts uniformly and often reorganises competitive hierarchies in ways that may either promote coexistence or strengthen differences. The strongest constraints we observed were on reproduction rather than on growth or survival, which suggests that establishment under new climates is often restricted more by unsuccessful recruitment than by adult performance. As climate regimes become more variable, these processes are likely to produce complex and context–dependent invasion trajectories. Species will benefit not only from broad thermal tolerance but also from the ability to maintain competitive and reproductive function across fluctuating environmental conditions. By identifying these mechanistic pathways, our study provides a framework for predicting which invaders are most likely to persist, interact, or dominate under changing thermal conditions, in crayfish and in other temperature–sensitive freshwater organisms.

Author contributions

Antonín Kouba (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Validation, Writing – original draft, Writing – review & editing), Koushik Das (Investigation, Writing – review & editing), Wei Guo (Investigation, Writing – review & editing), Kateřina Marková (Investigation, Writing – review & editing), Lukáš Veselý (Formal analysis, Investigation, Writing – review & editing), Francisco J. Oficialdegui (Formal analysis, Writing – review & editing), Boris Lipták (Formal analysis, Writing – review & editing), Jan Kubec (Formal analysis, Writing – review & editing), Anna Koubová (Investigation, Writing – review & editing), Martin Bláha (Investigation, Visualization, Writing – review & editing), Hamid Niksirat (Investigation, Writing – review & editing), Jiří Patoka (Formal analysis, Writing – review & editing), András Weiperth (Formal analysis, Writing – review & editing), Phillip J. Haubrock (Data curation, Formal analysis, Validation, Visualization, Writing – review & editing), Miloš Buřič (Conceptualization, Formal analysis, Validation, Writing – review & editing)

Funding

This research was funded by the Czech Science Foundation (No. 19-04431S) and by the Ministry of Education, Youth, and Sports of the Czech Republic, which supported the South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses (project CENAKVA, No. LM2018099).

Data availability

The data underlying this article will be shared on request to the corresponding author.