First evidence of the presence of Multixenobiotic Resistance Mechanism activity in freshwater invasive species, signal crayfish Pacifastacus leniusculus (Dana, 1852)

Background and Purpose: The signal crayfish Pacifastacus leniusculus (Dana, 1852) is one of the most successful invasive species of crayfish in European freshwaters, an extremely diverse though endangered group of ecosystems. The main goal of this study was to functionally characterize multixenobiotic resistance (MXR) mechanism defense activity in P. leniusculus tissues for the first time. MXR mechanism protects the cell from a wide variety of toxic compounds, and it is mediated by the transport activity of ATP-binding cassette (ABC) proteins. Materials and Methods: MXR transporter activity dye assay was performed by using fluorescent model substrate rhodamine B (RB) in combination with inhibitors of MXR efflux pumps: MK571 and Verapamil, known to inhibit multidrug resistance-associated proteins (MRP) and P-glycoprotein (P-gp), respectively. In this assay, the increase in intracellular fluorescence of the substrate dye, indicates inhibition of MXR efflux protein pumps. The assay was performed in three different tissues (gills, hepatopancreas, tail muscle). Additionally, tissues were exposed to selected heavy metals – mercury (HgCl2) and zinc (ZnCl2), known to occur in open freshwaters as pollutants. Results: Optimal time for RB accumulation in gills and hepatopancreas was determined to be 30 minutes. RB efflux in gills was inhibited by MK571 and in hepatopancreas by Verapamil, suggesting that multidrug resistanceassociated proteins are dominant in gills of P. leniusculus, and P-glycoprotein in hepatopancreas. Finally, inhibitory effect of mercury (HgCl2: 10 and 20 μM) and zinc (ZnCl2: 5–20 μM) on multixenobiotic resistance mechanism activity in gills, and only mercury in hepatopancreas, was detected. Conclusions: The results for the first time demonstrate the presence of multixenobiotic resistance mechanism efflux activity as an important tissue specific defense mechanism in P. leniusculus and provide the basis for future molecular and toxicological studies of this invasive and adaptable species

Physiological performance of native and invasive crayfish species in a changing environment: insights from Dynamic Energy Budget models

Crayfish are keystone species important for maintaining healthy freshwater ecosystems. Crayfish species native to Europe, such as Astacus astacus and Austropotamobius torrentium, are facing decline and are increasingly endangered by changing climate and invasions of non-native crayfish, such as Pacifastacus leniusculus and Procambarus virginalis. The success of these invasions largely depends on differences in ontogeny between the native species and the invaders and how changes in the environment will affect the ontogeny. Dynamic Energy Budget (DEB) models can be used to investigate such differences because the models capture dependence of metabolism, and therefore ontogeny, on environmental conditions. We develop DEB models for all four species and investigate key elements of ontogeny and metabolism affecting interspecific competition. We then use the DEB models to predict individual growth and reproduction in current and new conditions that are expected to arise from climate change. Although observations suggest that P. leniusculus poses the major threat to native species, our analysis identifies P. virginalis, in spite of its smaller size, as the superior competitor by a large margin—at least when considering metabolism and ontogeny. Our simulations show that climate change is set to increase the competitive edge of P. virginalis even further. Given the prospects of P. virginalis dominance, especially when considering that it is able to withstand and spread at least some crayfish plague strains that severely affect native species, additional research into P. virginalis is necessary

Behavioural determinants of agonistic success in invasive crayfish

Ecosystems today increasingly suffer invasions by multiple invasive species, some of which may share similar advantageous life history traits and ecological niche. In such cases, direct competition can influence invasion success of both species, and provide insights into competition without co-evolution in species equally novel to the environment. We used two widespread crayfish invaders of freshwater ecosystems of Europe, signal crayfish (Pacifastacus leniusculus) and spiny cheek crayfish (Orconectes limosus), to investigate how behavioural decisions in agonistic encounters contribute to competitive advantages in the absence of adaptation to either opponents or an environment. In direct competition against novel but comparable opponents, the key factor for establishing clear dominance of P. leniusculus in interspecific bouts was its greater tendency towards continued engagement in high-intensity fights. With O. limosus individuals consistently retreating from staged bouts as fights became more intense, P. leniusculus individuals did not need to adapt their strategy to be successful, suggesting that their agonistic behaviour intrinsically predisposed them to win. While both species are detrimental to invaded ecosystems, our results indicate that aggressive behaviour of P. leniusculus against unfamiliar opponents could allow it to more easily outcompete other comparable species and consequently present a potentially greater threat for native ecosystems

Evaluating the Efficacy of Approaches to Control Invasive Populations: A Conceptual Model Development for the Signal Crayfish

Invasive crayfish are among the major threats to freshwater ecosystems, with the signal crayfish, Pacifastacus leniusculus, being one of the most successful crayfish invaders in Europe. Approaches to invasive crayfish control range from manual and physical to biological and biocidal control methods. However, all of these approaches have their drawbacks and have limited efficacy. Among traditional approaches with minimal impacts on environment and non-target species, manual control via trapping is the most frequently applied. More innovative approaches comprise, among others, usage of sterile male release technique, whose efficacy in the field is yet to be fully tested, especially how it combines with more traditional approaches. A good alternative to costly and logistically challenging field comparisons of these approaches and their combinations is population modeling. Population models can integrate all relevant species-specific biological and ecological information and can be applied to identify management scenarios of highest impact on invasive crayfish abundances. In this study, we developed a conceptual population model of the invasive P. leniusculus following the Pop-GUIDE framework. In addition, we expanded on the framework to increase its applicability to other fields beyond environmental risk assessment. Finally, we discuss potential application of the model and its future use as a management tool

Changes in population characteristics and structure of the signal crayfish at the edge of its invasive range in a European river

The ability of rapid range expansion is one of the key determinants of invasive species success. In order to investigate potential drivers behind the rapid spread of invasive species, we explored changes in population characteristics and structure along the invasion pathway of a successful invader in European freshwaters, the signal crayfish (Pacifastacus leniusculus). Diverse population parameters such as relative population abundance, size and sex structure, differences in morphometry and frequency of injuries were compared between signal crayfish population samples at three uniformly distributed segments (approximately 40 km apart) in the lower section of the Mura River, which differed in time since invasion. Examined signal crayfish populations exhibited notable differences, with more recently established populations toward invasion front characterized by lower abundance and male-biased sex ratios, which highlighted males as initial dispersers. We also recorded significant increase in the relative claw size, a competitively advantageous and allometric trait for males, in more recently established populations away from source population. The recorded differences in population structure and male morphometry along the invasion pathway could lead to important clues about dynamics of range expansion and population establishment, highlighting the traits that promote dispersal and better response to local conditions in new habitats. Established differences can also provide insights into the development of targeted management responses aimed at invasive species control

Evaluating the Efficacy of Approaches to Control Invasive Populations: A Conceptual Model Development for the Signal Crayfish

Invasive crayfish are among the major threats to freshwater ecosystems, with the signal crayfish, Pacifastacus leniusculus, being one of the most successful crayfish invaders in Europe. Approaches to invasive crayfish control range from manual and physical to biological and biocidal control methods. However, all of these approaches have their drawbacks and have limited efficacy. Among traditional approaches with minimal impacts on environment and non-target species, manual control via trapping is the most frequently applied. More innovative approaches comprise, among others, usage of sterile male release technique, whose efficacy in the field is yet to be fully tested, especially how it combines with more traditional approaches. A good alternative to costly and logistically challenging field comparisons of these approaches and their combinations is population modeling. Population models can integrate all relevant species-specific biological and ecological information and can be applied to identify management scenarios of highest impact on invasive crayfish abundances. In this study, we developed a conceptual population model of the invasive P. leniusculus following the Pop-GUIDE framework. In addition, we expanded on the framework to increase its applicability to other fields beyond environmental risk assessment. Finally, we discuss potential application of the model and its future use as a management tool

Virome Analysis of Signal Crayfish (<i>Pacifastacus leniusculus</i>) along Its Invasion Range Reveals Diverse and Divergent RNA Viruses

Crayfish are a keystone species of freshwater ecosystems and a successful invasive species. However, their pathogens, including viruses, remain understudied. The aim of this study was to analyze the virome of the invasive signal crayfish (Pacifastacus leniusculus) and to elucidate the potential differences in viral composition and abundance along its invasion range in the Korana River, Croatia. By the high-throughput sequencing of ribosomal RNA, depleted total RNA isolated from the crayfish hepatopancreas, and subsequent sequence data analysis, we identified novel and divergent RNA viruses, including signal crayfish-associated reo-like, hepe-like, toti-like, and picorna-like viruses, phylogenetically related to viruses previously associated with crustacean hosts. The patterns of reads abundance and calculated nucleotide diversities of the detected viral sequences varied along the invasion range. This could indicate the possible influence of different factors and processes on signal crayfish virome composition: e.g., the differences in signal crayfish population density, the non-random dispersal of host individuals from the core to the invasion fronts, and the transfer of viruses from the native co-occurring and phylogenetically related crayfish species. The study reveals a high, previously undiscovered diversity of divergent RNA viruses associated with signal crayfish, and sets foundations for understanding the potential risk of virus transmissions as a result of this invader’s dispersal