Osmoregulatory Capacity and Non-Specific Food Preferences as Strengths Contributing to the Invasive Success of the Signal Crayfish Pacifastacus leniusculus: Management Implications

Abstract

Various biological traits support the invasive success of different organisms. The osmoregulatory capacity and food preferences of the signal crayfish Pacifastacus leniusculus were experimentally tested to determine if they contribute to its invasive success. The osmotic concentrations of haemolymph were determined after acclimation of the crustaceans to seven salinities from 0 to 20 PSU. Food preferences were tested using Canadian pondweed Elodea canadensis, and rainbow trout Oncorhynchus mykiss. The results showed that the signal crayfish exhibits a hyper-hypoosmotic regulation pattern in the salinity range from 0 to 20 PSU, enabling them to inhabit both freshwater and brackish environments. Furthermore, the study found signal crayfish to have non-specific food preferences, although fish muscle tissue is more beneficial as a source of energy. Both features, osmoregulatory ability and food preferences, can increase the invasive success of this species as it expands into new areas. The ability to survive in higher salinities compared to the coastal waters of the Baltic Sea along the Polish coastline should be considered in targeted management strategies to control the spread of this invasive species.

1. Introduction

The establishment of invasive alien species (IASs) is increasingly becoming a global environmental and human health problem, as evidenced by the transmission of yellow fever and chikungunya fever by the Asian tiger mosquito Aedes albopictus (Skuse, 1894) [1,2]. Invasive success may be associated with climate/habitat match, a history of invasion success, the number of arriving/released individuals, and physiological capacity [3,4]. Understanding the conditions for invasive success is an urgent priority for improving the prediction of their spread and developing efficient management strategies [4,5,6].

The North American signal crayfish Pacifastacus leniusculus (Dana, 1852) is one of the most globally invasive crayfish species, and the most widespread non-native crayfish in Europe [7,8]. Individuals of this species have characteristics that make them desirable for aquaculture, such as large size, high fecundity [9,10,11] and high growth rate [12]. This led to their large-scale introduction into many countries outside of the native range. If they escape from farms into the wild, they could have a negative impact on native crayfish by replacing them and by spreading the crayfish plague Aphanomyces astaci (Schikora, 1906), which decimates native species [13]. The signal crayfish can significantly alter invertebrate biodiversity and displace native fish species from streams, where it competes for food and shelter [14,15,16,17]. However, not all deliberate introductions of signal crayfish resulted in a self-sustaining population [18].

Animal physiology, including the osmoregulation and feeding preferences studied here, is crucial for predicting the spread of organisms by influencing the ability of species to establish and thrive in new environments [19,20,21,22]. One of the main abiotic factors affecting the physiology of aquatic organisms is salinity, which limits the distribution of many IASs [23,24]. Crayfish are thought to be stenohaline organisms requiring low salinity, since they are generally found in fresh waters.

In contrast, an important biotic factor in terms of invasive success is food preference, which allows a species to utilize different food items, and can vary widely depending on the species and the environment they invade. However, both generalist and specialist species can be successful invaders [25].

An established population of non-native signal crayfish inhabits the River Wieprza, a tributary to the Baltic Sea [26]. By experimentally testing the osmoregulatory capacities and food preferences of specimens originating from this population, this study aims to assess the potential for invasive success in expanding their distribution through the brackish Baltic coastal waters and invading neighbouring coastal rivers.

The specific objectives were:

(1)

To determine the osmoregulatory pattern and osmoregulatory capacities of the signal crayfish in a range of salinities from 0 to 20 PSU.

(2)

To determine the food preferences of signal crayfish using two food items:

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aquatic macrophyte, the Canadian pondweed Elodea canadensis (Michaux, 1803);

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fish, the rainbow trout Oncorhynchus mykiss (Walbaum, 1792).

A concise review of crayfish eradication methods is also presented, and the need to expand the area of management is highlighted in reference to the experimental results obtained.

2. Materials and Methods

2.1. Collecting of the Material

A total of 35 specimens of the signal crayfish were collected from the River Wieprza at Darłowo (Poland) using traps and hand nets between May and July 2014. Two types of traps were used, including ‘Pirate’ (manufactured by Bock-Ås, Parainen, Finland) and ‘Evo’. Three key water parameters were measured during collection of the material:

−

temperature ranged between 11.6 and 15.6 °C;

−

salinity was equal to 0.1 PSU;

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oxygenation reached 80.7%.

2.2. Acclimatization to the Laboratory Conditions

Transported animals should be allowed an adequate period of acclimatization to the laboratory conditions before being used in experiments, as transport causes stress associated with physiological changes that may confound subsequent research [27]. The crayfish were transported to the laboratory in plastic boxes and then placed in a tank containing tap water at a temperature of 10 ± 1 °C, which was kept constant before and during the experiments. Each crayfish tank was continually aerated and had PVC pipes on the bottom to provide shelter for the animals. The upper part was covered with a plastic net to prevent the crayfish from escaping. During the acclimatization period (>7 days), the crustaceans were fed with fish flesh or dry granulated food twice a week. Observations regarding the behaviour of the captive animals were recorded throughout the experiments.

2.3. Laboratory Experiments on Osmoregulation

Acclimation experiments were carried out in a range of salinities similar to those present in the Baltic Sea at different areas and depths (ranging from 0 to 20 PSU). Five individuals were kept in seven salinity gradients: 0; 2; 4; 8; 12; 16 and 20 PSU. Typically, at least 3 individuals per gradient are sufficient for osmolality measurements [28,29].

After acclimatization, individuals were stepwise-acclimated to an increased salinity (4 PSU per 48 h) [30,31]. In the case of acclimation to 2 PSU, crayfish were acclimated to water salinity of 2 PSU over 48 h. Salinity variations were prepared using artificial ‘Aqua Medic Reef Salt’ (Aqua Medic, Bissendorf, Germany, https://www.aqua-medic.de (accessed on 30 April 2024)) dissolved in tap water.

After 48 h of acclimation to experimental salinity, haemolymph was extracted from the heart using a needle and syringe [30,31]. Its osmotic concentrations were determined using WESCOR Vapro 5520 Osmometer (Wescor, Logan, UT, USA, http://water.wescor.com (accessed on 30 April 2024)) with two replicates for each individual.

2.4. Laboratory Experiments on Food Preferences

Two years after the osmoregulatory experiments, a behavioural study was carried out on the same animals. Separate tanks were prepared for each individual crayfish, with tap water at a temperature of 10 ± 1 °C. To stabilize the oxygenation and temperature of the water, tanks were kept for 24 h in the laboratory, before starting the experiments. Twenty-eight crayfish were weighed with 0.001 g accuracy, and placed in tanks for a week-long starvation period [32]. The food preference and consumption rate were assessed using fish muscle tissue and aquatic macrophyte. The amount of each type of food (7% of each individual’s mass) was calculated and provided to the respective individuals [32]. After 24 h, food residues were removed, weighed, and dried at 55 °C until a constant dry mass was achieved [32].

The consumption rate was calculated using Klekowski and Fischer’s formula [33] and expressed as means and standard deviations:

$$C={\displaystyle \frac{\left(M_f-M_{fn }\right)\times E_{vf}}{T_f\times N}}=[\mathrm{J}{\mathrm{ind}}^{-1}{\mathrm{h}}^{-1}]$$

where

C—consumption,

M_f—dry mass of food [mg],

M_fn—dry mass of uneaten food [mg],

E_vf—energy value of food [J mg⁻¹],

T_f—time of food exposition in the tank [h],

N—number of crayfish.

2.5. Statistical Analysis

Due to the small number of examined animals, a non-parametric Kruskal–Wallis ANOVA and post-hoc multiple comparisons of the mean ranks test were carried out to test for differences in the osmoregulatory capacity between individuals acclimated to various salinity levels, at the significance level of p = 0.05. A non-parametric Mann–Whitney U test was carried out to test for differences in the choice of food and in the consumption rate, at the significance level of p = 0.05. These analyses were carried out using the STATISTICA 12.0 PL software (StatSoft, Kraków, Poland).

3. Results

3.1. Osmoregulation

3.1.1. Behaviour

No mortality of the signal crayfish was observed at any of the salinities tested. Most of the individuals remained in their shelters at salinities from 0 to 8 PSU. At salinity 12 PSU, an increase in the activity of crayfish was observed, manifesting in a greater number of individuals outside their shelters. Only at the highest salinities of 16 and 20 PSU were crayfish observed outside of their shelters; displaying signs of stress and a desire to escape.

3.1.2. Osmotic Concentrations and Osmoregulatory Capacity

The osmoregulation pattern observed in signal crayfish individuals was hyper-hypoosmotic (Figure 1), with an isoosmotic point at approximately 19 PSU. As salinity increased, there was a corresponding increase in the osmotic concentrations of haemolymph (Figure 1). Simultaneously, there was a decrease in osmoregulatory capacities (Figure 2). The study found that the highest osmoregulatory capacity was observed at a salinity of 0 PSU, while the lowest was observed at a salinity of 20 PSU (Figure 2). The isoosmotic line, prepared using solutions of known salinities and their osmotic concentrations (Figure 1), was used to estimate the osmoregulatory capacity of signal crayfish individuals.

The osmoregulatory capacities of signal crayfish showed differences in various salinities (p = 0.0041, χ² = 30.19608, df = 6) (Kruskal–Wallis ANOVA).

3.2. Food Preferences of the Signal Crayfish

During the experiments, signal crayfish individuals were given the same amount of fish and macrophyte (7% of each individual’s mass): 5123.97 mg and 5127.76 mg wet mass, respectively. The daily consumed food mass was about 1% of their mass (Figure 3). The differences in food choice were not statistically significant (Mann–Whitney U test; the significance level obtained was p = 0.35).

The consumption rate of fish muscle tissue (522.32 ± 431.87 J ind⁻¹h⁻¹) was statistically greater than the consumption rate of aquatic macrophyte (5.13 ± 5.13 J ind⁻¹h⁻¹) (Mann–Whitney U test, p = 0.000027).

4. Discussion

Traditionally, to determine the freezing point of small volumes of haemolymph, the sample is frozen, and then its melting point is observed. The apparatus and procedure are based on the Ramsay method [34]. This involves freezing the sample and then slowly warming it to observe the disappearance of the last ice crystal, with the temperature at which this occurs being the freezing point. It is suggested to observe the ice crystals with a microscope in the inner vessel containing alcohol and to keep the sample under liquid paraffin in a glass tube. In the present study, a new technique with the Vapro osmometer was used. According to the manufacturer’s user manual, the device measures the dew point temperature depression of a specimen with a resolution of 0.00031 °C. The osmolality of the solution is then calculated by taking advantage of its colligative properties, namely the vapour pressure and freezing point. For osmoregulation studies in small animals, the Ramsay method should be used due to the limited volume of haemolymph. However, for larger animals, such as crayfish, a vapour osmometer can be employed.

4.1. Osmoregulatory Capacity and Food Preferences

Invasive success is partly related to the ability of an aquatic organism to function in a wide spectrum of environmental conditions. This study confirms that signal crayfish from the River Wieprza are euryhaline possessing a hyper-hypoosmotic osmoregulation pattern similar to individuals of this species from other populations [30,31], and were capable of osmoregulating within a salinity range of 0 to 20 PSU. Although an increase in salinity was linked to a reduction in osmoregulatory capacities, the individuals in this study displayed the broad salinity tolerance necessary to colonize and thrive in diverse habitats. Taking into account the relatively low salinity levels across the Baltic Sea, ranging from 2 to 3 PSU in the Bothnian Bay to about 20 PSU in the Kattegat, including 6–8 PSU in the Baltic Proper and 7 PSU in Polish coastal waters [35,36]; considering the high osmoregulatory capacities of signal crayfish, it can be concluded that the brackish Baltic Sea cannot limit the dispersal of this species. This conclusion is somewhat corroborated by the capture of a vagrant signal crayfish approximately 15 km from the mouth of the River Wieprza (Figure 4). Previous records from the Baltic Sea indeed validate such possibilities and inform us of their presence in introduced areas, such as the Danish Straits and the Bothnian Sea [37]. The species’ occurrence in brackish waters along the Pacific Coast (its native habitat) is also documented, with salinities reaching as high as 20 PSU [38].

Research focused on the osmoregulatory capacity of other North American crayfish species found in Poland, including the red swamp crayfish Procambarus clarkii (Girard, 1852), marbled crayfish Procambarus virginalis Lyko, 2017, and spiny-cheek crayfish Faxonius limosus (Rafinesque, 1817), also suggests that brackish waters (such as the Baltic Sea) are unlikely to inhibit crayfish dispersal and colonization of coastal rivers by sea [39,40,41,42]. For instance, there are records of the red swamp crayfish in the brackish and marine waters of the Mediterranean Sea [39,40], and moderate mortality rates were observed in individuals exposed to a high salinity concentration of 35.3 PSU [41]. Additionally, red swamp crayfish were found to both grow and mate at salinities of up to 25 PSU [39]. Therefore, there is risk of further dispersal and colonization of lagoons by the red swamp crayfish to rivers throughout the coastal Mediterranean marine environment [39,40,41]. Given the lower salinity in the Baltic Sea, we can expect similar dispersion. The spiny-cheek crayfish, which was introduced more than a century ago, has already been observed along the Polish Baltic coastal waters and nearby rivers, and inhabits the Vistula and Szczecin Lagoons [43]. Experimentally, it was proved that the spiny-cheek crayfish is able to reproduce at both 3 and 7 PSU [44]. To date, a water body in Szczecin is the closest site to the Polish Baltic coast where the marbled crayfish has been noted in the wild [45]. However, if this species is introduced to the Szczecin Lagoon and establishes a population there, predicting its potential further dispersal to areas with higher salinity (>7 PSU) is difficult due to low survival rates, reduced growth, and inhibited reproduction in saline environments [46]. Under laboratory conditions, the marbled crayfish can survive for more than 80 days at 18 PSU [46]. Theoretically, this suggests it could gradually disperse in the wild, using the brackish waters, in this case, the coastal waters of the Baltic Sea, as a biological corridor linking more suitable habitats.

Like the signal crayfish, the red swamp crayfish, when exposed to salinities up to 20 PSU, hyper-regulate their haemolymph osmotic pressure, with their haemolymph becoming isoosmotic at around 20 PSU [47]. At a much lower salinity level (approx. 13 PSU), the haemolymph of the spiny-cheek crayfish becomes isosmotic [43], allowing this species to be a hyper-regulator in freshwaters and at low salinities (up to 13 PSU), and a hypo-regulator at higher salinities between 14 and 35 PSU.

Besides salinity, water temperature is an important metabolic determinant that may limit the further spread of crayfish. While thermal conditions do not limit the expansion of the spiny-cheek crayfish, as evidenced by its widespread occurrence in Poland’s rivers and lakes [48], it also should not be lethal for the red swamp crayfish, which can withstand a temperature of around 2.5 °C in temperate zones ([49], with the cited literature therein). In contrast, a low temperature causes the marbled crayfish numerous mortalities, although it can withstand temperatures below 2 °C for at least one week [50]. Considering the increasing temperature in Europe caused by climate change, the environmental conditions of the rivers flowing into the Baltic Sea will become increasingly suitable for these non-native American crayfish.

Numerous studies have shown a link between organisms’ osmoregulatory capacity and their metabolic rate [51,52]. In freshwater environments, where maintaining high osmotic pressure is crucial, organisms must allocate more energy to life processes. Hence, minimizing energy expenditure is advantageous in brackish waters. However, the inhabitants of such environments must be adaptable to salinity fluctuations, often caused by freshwater inflows from rivers. Thriving in brackish waters demands specialized physiological adaptations to cope with their dynamic conditions.

The signal crayfish is an omnivorous species [53,54,55], and this study confirmed its non-specific food preferences. This feature increases invasive success, because in the case of a lack of preferred food in the environment, the species can consume alternative food sources. Their ability to exploit non-specific food resources also increases their likelihood of survival in new environments, where they may encounter unfamiliar prey or competitors. These results correspond to that of spiny-cheek crayfish, another common non-native crayfish species in Polish waters, which also have non-specific food preferences [32] and, depending on the availability of a given food in the environment, may feed on animal and/or plant matter [53]. It should be mentioned that there is no evidence of a significant shift in diet during the ontogeny of the signal crayfish [54,55].

The signal crayfish had a higher consumption rate of the fish (rainbow trout) muscle tissue at 10 °C = 522.32 [J ind⁻¹ h⁻¹] than aquatic macrophyte, 5.13 [J ind⁻¹ h⁻¹], indicating that it is more beneficial to consume a food source with a large amount of energy, 107 (for rainbow trout) and 4.2 (for macrophyte) (J mg⁻¹ DW) [56,57], respectively. However, the dietary choices made by this species in its native lotic environment are most likely affected by factors other than the nutritional value of food items, which is contrary to the expectations that the diets of omnivores should be based on this factor alone [55]. A lower consumption rate was observed in the spiny-cheek crayfish consuming Atlantic cod Gadus morhua Linnaeus, 1758: 19.59 [J ind⁻¹ h⁻¹] at 12 °C and 15.02 [J ind⁻¹ h⁻¹] at 18 °C [32]. However, there was a much higher consumption rate of a green macroalgae Enteromorpha spp. 43.77 [J ind⁻¹ h⁻¹] at 12 °C and 50.88 [J ind⁻¹ h⁻¹] at 18 °C by the spiny-cheek crayfish compared to the consumption rate of Canadian pondweed by signal crayfish, indicating that the spiny-cheek crayfish prefers plant-based food.

The ability to exploit different environments and food resources allows invasive species to establish populations in various salinity regimes and with a wide array of food resources, further enhancing their invasive success [24,25]. These adaptabilities ensure that the signal crayfish can function in various habitats and utilize available food.

4.2. Implication to Management of the Signal Crayfish

A better understanding of the characteristics of signal crayfish, such as their osmoregulatory capacity and omnivory, will lead to more effective management decisions and actions. To date, no standard methodology for the control of invasive crayfish has been established [58,59,60,61]. Traditional techniques for the control of invasive crayfish are based on manual methods, including trapping and electrofishing; and physical methods, such as structural barriers and drainage interventions. However, more recent solutions apply biological and chemical approaches [58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76].

The oldest method used by humans for capturing crayfish involves manually collecting them by hand for consumption. This method is often used in shallow water bodies or areas where crayfish densities are low [59]. Trapping is a common method used to capture crayfish in infested areas [59,60,61]. Various types of traps have been deployed. They are made of plastic or fish nets, with walls forming a larger chamber with generally narrow entrances that prevent larger fish or mammals, such as the Eurasian otter Lutra lutra (Linnaeus, 1758), from entering. Recently, a new type of trap consisting of narrow plastic funnels, known as artificial refuge traps, has been applied [62,63]. Electrofishing is a more advanced technique, typically used in small clear water streams or shallow ponds, but requiring specialized equipment and trained personnel [60]. Electrofishing involves using an electric current to temporarily immobilize crayfish, making them easier to capture. However, the capture of those in cover or burrows during the operation is less likely [60]. A different strategy is to use a high-intensity electric shock that causes the death of crustaceans in streams [64].

Structural barriers, which help capture the crayfish or prevent their dispersal, include physical barriers, such as nets, fences and weirs; as well as natural barriers like waterfalls or steep terrain [65,66,67]. Drainage and dewatering can be used to temporarily reduce water levels in infested areas, exposing crayfish to desiccation or allowing for manual removal. This technique is effective in small ponds, but may not be feasible in larger or flowing water systems. It is also used in combination with other methods to increase eradication or precision in estimation of the number of crayfish in streams [68,69].

Chemical control methods, such as the application of pesticides or biocides, can be used to eradicate crayfish populations [70,71,72]. However, these methods are often non-selective and can harm non-target organisms.

Biological control methods involve introducing natural predators like eels [73], parasites, or pathogens that specifically target crayfish species ([60], with the literature therein). The use of pheromones in the capture or release of sterilized males to reduce recruitment is also worth mentioning [74]. While biological control can be effective in reducing crayfish numbers, it requires careful consideration to avoid unintended impacts (of fungus, bacteria, viruses) on native species and ecosystems.

When developing management plans for invasive crayfish species, population modelling [75,76] should be included, integrating all relevant species-specific characteristics to predict the results of the different management strategies. Population models can assess the invasion risk posed by a particular species by simulating its potential spread and establishment in new habitats. Models are useful for controlling the spread of invasive crayfish at the early stage [61]. Understanding the interplay between the key mechanisms regulating invasive crayfish populations and different population control approaches is particularly important in this method.

Osmoregulatory adaptability and non-specific food preferences may increase the invasive success of signal crayfish in areas not previously inhabited by this species. Since osmoregulatory ability possesses advantages in the expansion of new environments by IASs [77], the euryhalinity and osmoregulatory ability of the signal crayfish should be considered in management actions. A predictive model should be implemented to create invasion scenarios to support effective management plans. Given that the impact of signal crayfish is evident in the reduction or disappearance of native species, bioengineering habitat modifications [78], and associated economic losses [79], innovative methods of targeted management may mitigate the negative consequences of invasion by this species. It is important to note that the elimination of invasive crayfish species often requires long-term commitment, multi-stakeholder cooperation and adaptive management approaches [58]. Our results, and bycatch record, suggest that the management of signal crayfish should also include the mouths of the Wieprza River and neighbouring rivers, since brackish environments (7–8 PSU) are not lethal for this species.

5. Conclusions

Understanding the interplay between the biological traits of aquatic species (e.g., osmoregulatory capability, food preferences) and habitat characteristics is essential for predicting biological invasion in aquatic ecosystems. Osmoregulatory capacity is a key factor governing the success of signal crayfish in colonizing diverse aquatic environments. This study augments the knowledge that this species displays a high tolerance to a wide range of salinity levels, allowing it to thrive in both freshwater and brackish ecosystems. This increases its potential geographic range, providing a competitive advantage over the native species that may be limited to specific environmental conditions.

The study also shows that signal crayfish have non-specific food preferences, which may play an important role in its invasiveness by having a broad ecological niche. This adaptability ensures that they can find and utilize available food more effectively; and can outcompete and displace native organisms that may be more specialized in their feeding habits.

Awareness of the osmoregulatory capacity of the signal crayfish is crucial for designing effective management strategies. A targeted approach needs to be implemented to limit its potential to colonize neighbouring rivers, not only via freshwater ways but also through the brackish environment of the Baltic Sea.