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Aquatic invasive species are a major threat to native freshwater biodiversity. The North American signal crayfish Pacifastacus leniusculus was introduced to Great Britain during the 1970s and is now widely distributed throughout England, Wales and Scotland. First recorded in Scotland in 1995, P. leniusculus is now established at more than twenty sites. The only other introduced crayfish species present in Scotland is the white-clawed crayfish Austropotamobius pallipes. A. pallipes is restricted to only two locations in Scotland, Loch Croispol and Whitemoss Reservoir. P. leniusculus negatively impacts macrophytes, invertebrates and fish though ecological and physical processes. Additionally, P. leniusculus has displaced A. pallipes throughout much of its native range within Great Britain due to competition and disease. Consequently, the two A. pallipes populations in Scotland have a high conservation value. This PhD study aimed to improve understanding of P. leniusculus invasion success by examining trophic dynamics and to develop methodologies that could improve the detection and control of P. leniusculus populations in Scotland. Stable isotope analysis was used to determine the diet composition, trophic position and whether an ontogenetic dietary shift occurs in the Loch Ken population of P. leniusculus. Bayesian mixing models indicated that P. leniusculus in Loch Ken do exhibit an ontogenetic dietary shift. Additionally, individuals of all sizes occupied the trophic position of a predator in Loch Ken suggesting that invertebrates and fish constitute an important component of P. leniusculus diet. Stable isotope analysis was used once again to compare the isotopic niche width and diet composition of P. leniusculus populations from Loch Ken and A. pallipes populations from Loch Croispol and Whitemoss Reservoir. At the species level, A. pallipes exhibited a larger niche width than that of P. leniusculus. At the population level, the isotopic signatures of the A. pallipes populations were considerably different from each other suggesting an overestimation of A. pallipes’ niche width at species level. Results showed no dietary overlap between species and Bayesian mixing models suggested P. leniusculus and A. pallipes were consuming different resources, indicating there would be no direct competition for food resources if they were to co-occur. A plus-maze study was used to determine if P. leniusculus exhibited a preference for one of four food attractants (Oncorhynchus mykiss, P. leniusculus, beef or vegetation), which could be used to improve trapping efficiency. In the maze system, P. leniusculus exhibited no preference for any food attractant presented. This would suggest that either the maze was not a good model or food attractants would not improve trapping efficiency of P. leniusculus. Additionally, a comparative investigation into the use of gill nets as a method to control P. leniusculus was conducted. Results showed that the net type and the presence of fish entangled in the net influenced the number of P. leniusculus caught. Finally, environmental DNA (eDNA) was used and evaluated for detection of P. leniusculus. A robust quantitative Polymerase Chain Reaction (qPCR) assay and DNA extraction protocol were developed. Using the developed qPCR assay, P. leniusculus eDNA was detected in controlled aquaria conditions but not in environmental water samples collected from the field. Furthermore, the quantities of P. leniusculus eDNA declined in aquaria conditions while individuals were still present suggesting the mechanisms for eDNA release by P. leniusculus are complex. Stable isotope analysis indicates that P. leniusculus exhibit an ontogenetic dietary shift, and in each life stage, P. leniusculus function as an omnivore but occupy the trophic position of a predator. Niche width analysis revealed that the diet of P. leniusculus was less general than that observed in A. pallipes and thus diet of P. leniusculus may not be responsible for invasive success. Food attractants will not enhance trapping efficiency but nets may present a potential new method to control P. leniusculus. Similarly, eDNA presents a promising new method for rapid detection of P. leniusculus. It will not be possible to eradicate P. leniusculus in Scotland but the findings of this PhD may help prevent establishment of new populations. These results should be incorporated into future management strategies for P. leniusculus populations in Scotland and may have broader applications in Great Britain and Europe.

- ABSTRACT - The crayfish plague pathogen, Aphanomyces astaci, is responsible for substantial declines and local extinctions of native European crayfish populations. As a consequence, the pathogen is now listed among 100 world's worst invasive alien species. The spread of A. astaci is greatly facilitated by its natural hosts, North American crayfish, that thanks to a long co-evolutionary history with the crayfish plague pathogen evolved efficient defence mechanisms. In contrast, European, Australian and Asian crayfish species are highly susceptible to this disease agent. However, progress of A. astaci infection in native European crayfish was observed to differ between distinct pathogen strains, indicating variability in their virulence. Indeed, we demonstrated a relationship between patterns in crayfish immune response and A. astaci virulence in an experimental infection involving the European noble crayfish and three differently virulent crayfish plague strains. The European continent is currently inhabited by at least eight North American crayfish species. The carrier status was confirmed in six of them, including also Orconectes cf. virilis occurring in the Netherlands and the UK. In this country, we detected Aphanomyces astaci presence in some populations of the non-indigenous crayfish species as well...

This dissertation focuses on increasing the understanding of the evolution processes that have contributed to the diversification of freshwater crayfish. Chapter one estimates the divergence time of the three crayfish families and tests the hypothesis that diversification is tied to the break-up of Pangaea, Gondwanna, and Laurasia. I find that the families of crayfish diverged prior to or in association with the break-up of the three super continents. Chapter two addresses the evolutionary history of the genus Cambarus, using molecular data to test hypotheses of relationships based on chela and carapace morphology. The results provide evidence that the morphology used to determine Cambarus relationships do not reflect evolutionary history and that convergent evolution of morphological traits is common in crayfish. Chapter three addresses evolution at the population level and tests for differences in the genetic population structure of two crayfish with different physiological needs. I find that physiological requirements of these crayfish have influenced their population genetic structure. The last chapter addresses a molecular based hypothesis that rates of mitochondrial evolution are reduced in cave crayfish that have increased longevity, reduced metabolism, and restricted diets compared to surface crayfish. I find that cave crayfish rates of mitochondrial evolution do not significantly differ from surface crayfish. Therefore, increased longevity, reduced metabolism, and restricted diets do not slow the rate of mitochondrial evolution as predicted in this group of cave crayfish.

<p>The crayfish plague refractory crayfish, <i>Pacifastacus leniusculus</i>, which can harbour the fungal parasite within melanotic sheath, are found to constitutively express the gene encoding for prophenoloxidase (proPO) after mimicking parasite attack. In contrast, the susceptible crayfish, <i>Astacus astacus</i>, responds to the parasite by increased levels of proPO transcript, particularly in the semigranular haemocytes. The upregulation of proPO could confer a temporary resistance towards the fungal infection, suggesting that additional factors are involved in maintaining the balance between host and parasite. The resistant crayfish may have adapted to the parasite by increasing the transcript level of immune genes. The parasite can be considered as a symbiont since it does not harm the host rather than it activates the immune gene and possibly preventing other pathogens to become established.</p><p>Two serine proteinase genes encoding a subtilisin-like (<i>AaSP1</i>) and a trypsin (<i>AaSP2</i>) enzyme were isolated from the crayfish plague fungus, <i>Aphanomyces astaci</i>. These proteinases are prepropeptides and generate mature proteins of 39 kDa and 29 kDa, respectively. Characterisation of <i>AaSP1</i> suggests that the enzyme may be involved in intracellular control mechanisms rather than playing a role in pathogenesis. The <i>AaSP2 </i>transcript was not controlled by catabolic repression, but was induced by crayfish plasma, implying a role in pathogenesis toward the crayfish host. </p><p>Physiology and genetics of five <i>Aphanomyces</i> strains, which were isolated from moribund crayfish, were characterised with regard to their pathogen diversity. These strains are not virulent against crayfish. Some physiological properties of these strains differed from <i>A. astaci</i>, such as growth rate, germination and production of chitinase. Genetic analysis clearly indicated that they are not related to <i>A. astaci</i> and their name are proposed to be <i>Aphanomyces repetans</i>.</p><p>The crayfish <i>P. leniusculus </i>was found to be susceptible to white spot syndrome virus infection. The virus has a significant effect to the population of crayfish haemocyte. The number and proportion of granular cell from virus-infected crayfish were higher than in controls, indicating granular cells are more resistant to and may interact by some means with the virus.</p><p>Two morphotypes of the crayfish parasite <i>Psorospermium haeckeli</i> obtained from different crayfish hosts of different geographical origin were analysed for ribosomal ITS DNA in order to compare their genetic diversity. The sequence difference between them was found largely in ITS 1 and ITS 2 regions, which was variable in length and showed 66% and 58% sequence similarity. Thus, different morphotypes of <i>P. haeckeli</i> are genetically diverse.</p>

The crayfish plague refractory crayfish, Pacifastacus leniusculus, which can harbour the fungal parasite within melanotic sheath, are found to constitutively express the gene encoding for prophenoloxidase (proPO) after mimicking parasite attack. In contrast, the susceptible crayfish, Astacus astacus, responds to the parasite by increased levels of proPO transcript, particularly in the semigranular haemocytes. The upregulation of proPO could confer a temporary resistance towards the fungal infection, suggesting that additional factors are involved in maintaining the balance between host and parasite. The resistant crayfish may have adapted to the parasite by increasing the transcript level of immune genes. The parasite can be considered as a symbiont since it does not harm the host rather than it activates the immune gene and possibly preventing other pathogens to become established. Two serine proteinase genes encoding a subtilisin-like (AaSP1) and a trypsin (AaSP2) enzyme were isolated from the crayfish plague fungus, Aphanomyces astaci. These proteinases are prepropeptides and generate mature proteins of 39 kDa and 29 kDa, respectively. Characterisation of AaSP1 suggests that the enzyme may be involved in intracellular control mechanisms rather than playing a role in pathogenesis. The AaSP2 transcript was not controlled by catabolic repression, but was induced by crayfish plasma, implying a role in pathogenesis toward the crayfish host. Physiology and genetics of five Aphanomyces strains, which were isolated from moribund crayfish, were characterised with regard to their pathogen diversity. These strains are not virulent against crayfish. Some physiological properties of these strains differed from A. astaci, such as growth rate, germination and production of chitinase. Genetic analysis clearly indicated that they are not related to A. astaci and their name are proposed to be Aphanomyces repetans. The crayfish P. leniusculus was found to be susceptible to white spot syndrome virus infection. The virus has a significant effect to the population of crayfish haemocyte. The number and proportion of granular cell from virus-infected crayfish were higher than in controls, indicating granular cells are more resistant to and may interact by some means with the virus. Two morphotypes of the crayfish parasite Psorospermium haeckeli obtained from different crayfish hosts of different geographical origin were analysed for ribosomal ITS DNA in order to compare their genetic diversity. The sequence difference between them was found largely in ITS 1 and ITS 2 regions, which was variable in length and showed 66% and 58% sequence similarity. Thus, different morphotypes of P. haeckeli are genetically diverse.

For decades scientists believed that herbivory had minimal impact on freshwater ecosystems. We now know that herbivory in freshwater systems equals or exceeds herbivory in terrestrial and marine systems. In extreme cases, herbivores can change clear, macrophyte dominated ecosystems into turbid plankton dominated ecosystems. Even though research on plant-herbivore interactions in freshwater systems has increased, there is still much that is unknown. This thesis is comprised of four studies investigating freshwater plant-herbivore interactions across multiple spatial scales. The first study investigated how induced chemical defenses in Cabomba caroliniana suppress herbivore consumption and growth as well as how this herbivore-generated change in plant chemistry affects the growth of plant associated microbes. At the spatial scale of individual ponds or lakes, consumers that induce their host plants may also be indirectly affecting other consumers and microbial pathogens via changes in this shared resource. The second study moves to an ecosystem scale and investigates how exotic versus native apple snails may impact Everglades' habitats. We investigated plant preference, consumption, growth and conversion efficiencies in the singly native apple snail to occur in the U.S. (Pomacea paludosa) versus four introduced species (P. canaliculata, P. insularum, P. haustrum and P. diffusa). We found that even though plant preferences are similar, invasive snails tend to eat more, grow more rapidly, and sometimes more efficiently than natives. This suggests that invasive species could have a large impact on the environment, especially the abundance of submerged plants. The third study investigated how palatability of freshwater plants varies with latitude (i.e. geographic scale). Increased herbivory at lower latitudes is hypothesized to select for increased plant defenses, which has been shown to be true for tropical forests, salt marshes, and seaweeds. When we contrasted eight confamilial plants collected in Indiana versus Southern Florida, three of four herbivores significantly preferred northern plants. When we evaluated a second set of plants collected from Indiana versus Central Florida, only one of three herbivores preferred the northern plants. Overall, our results suggest a preference for northern plants, but the strength of this relationship was variable. We hypothesize that this variability may be driven by 1) local variance in herbivore pressure that creates variance in plant defenses, and/or 2) the effect of winter length on the survival and feeding rate of herbivores. The final study expanded to a world scale, and investigated herbivore preference for native vs exotic plants. We found that both N. American crayfish and S. American snails preferred exotic plants over confamilial natives, despite responding to different plant characteristics. The single species of apple snail that occurs in N. American showed no preference for native or exotic plants from a N. American perspective, but instead exhibited preferences that correlated with its history of evolution in S. America. As the N. American species is a sister species of the S. American snails, feeding by the N. American snail appears more affected by its S. American lineage than its recent history in N. America. This suggests that phylogenetic legacy will affect choices of the herbivore as well as resistance or susceptibility of plants.

Chemically mediated interactions have important ecological and evolutionary effects on populations and communities. Despite recognition that herbivory can significantly affect the biomass and composition of freshwater macrophyte communities, there are few investigations of chemical defenses among freshwater vascular plants and mosses and none of freshwater red algae. This study compares the palatability of five species of freshwater red algae (Batrachospermum helminthosum, Boldia erythrosiphon, Kumanoa sp., Paralemanea annulata, and Tuomeya americana) that occur in the southeastern United States relative to two co-occurring macrophytes (the chemically defended aquatic moss Fontinalis novae-angliae and the broadly palatable green alga Cladophora glomerata). We assessed the potential role of structural, nutritional, and chemical traits in reducing macrophyte susceptibility to generalist crayfish grazers. Both native and non-native crayfish significantly preferred the green alga C. glomerata over four of the five species of red algae. B. erythrosiphon was palatable, while the cartilaginous structure of P. annulata reduced its susceptibility to grazing, and chemical defenses of B. helminthosum, Kumanoa sp., and T. americana rendered these species as unpalatable as the moss F. novae-angliae. Extracts from these latter species reduced feeding by ~30-60% relative to solvent controls if tissues were crushed (simulating herbivore damage) prior to extraction in organic solvents. However, if algae were first soaked in organic solvents that inhibit enzymatic activity and then crushed, crude extracts stimulated or had no effect on herbivory. B. helminthosum, Kumanoa sp., and T. americana all exhibited "activated" chemical defenses in which anti-herbivore compounds are produced rapidly upon herbivore attack via enzymatic processes. In an additional accept/reject behavioral assay, B. helminthosum extracts reduced the number of crayfish willing to feed by >90%. Given that three of the five red algal taxa examined in this study yielded deterrent crude extracts, selection for defensive chemistry in freshwater rhodophytes appears to be substantial. Activated chemical defenses are thought to be an adaptation to reduce the resource allocation and ecological costs of defense. As such, activated chemical defenses may be favored in freshwater red algae, whose short-lived gametophytes must grow and reproduce rapidly. Roughly 20% of the known chemical defenses produced by marine algae are activated; further examination is needed to determine whether the frequency of activated chemistry is higher in freshwater red algae compared to their marine counterparts. Continued investigation of chemical defenses in freshwater red algae will contribute to among-system comparisons, providing new insights in the generality of plant-herbivore interactions and their evolution.