Geographical variation of mitochondrial DNA between populations of the white-clawed crayfish Austropotamobius pallipes

1. The white-clawed crayfish Austropotamobius pallipes has a widespread distribution in Europe, but since the last century its distribution has been severely affected by a combination of factors, particularly the disease crayfish plague, introduced by foreign crayfish species. At present A. pallipes is considered as vulnerable and endangered and measures are being taken in a number of countries to conserve it.
2. The application of genetics to conservation is of practical value particularly where restocking is being considered. This study assesses levels of genetic differentiation from an analysis of mitochondrial DNA (mtDNA) variation by restriction fragment length polymorphism of four populations sampled on a European scale. Six different composite haplotypes were detected among sixty-four crayfish representing the four populations. mtDNA nucleon diversity values within species ranged from 0.63% to 12.35%. A close genetic similarity was found between British and French populations of A. pallipes .
3. Results show that mtDNA can be as a suitable diagnostic marker for measuring genetic diversity between crayfish populations and that such information is of use in planning crayfish conservation strategies.     

Geographical variation of mitochondrial DNA between populations of the white-clawed crayfish Austropotamobius pallipes

1. The white-clawed crayfish Austropotamobius pallipes has a widespread distribution in Europe, but since the last century its distribution has been severely affected by a combination of factors, particularly the disease crayfish plague, introduced by foreign crayfish species. At present A. pallipes is considered as vulnerable and endangered and measures are being taken in a number of countries to conserve it.
2. The application of genetics to conservation is of practical value particularly where restocking is being considered. This study assesses levels of genetic differentiation from an analysis of mitochondrial DNA (mtDNA) variation by restriction fragment length polymorphism of four populations sampled on a European scale. Six different composite haplotypes were detected among sixty-four crayfish representing the four populations. mtDNA nucleon diversity values within species ranged from 0.63% to 12.35%. A close genetic similarity was found between British and French populations of A. pallipes .
3. Results show that mtDNA can be as a suitable diagnostic marker for measuring genetic diversity between crayfish populations and that such information is of use in planning crayfish conservation strategies.     

Immunological evidence for progesterone and estradiol receptors in the freshwater crayfish Austropotamobius pallipes

In this article, we present evidence for progesterone and estradiol receptors (PR and ER, respectively) in the female of the crayfish Austropotamobius pallipes. To our knowledge, this is the first report of sex steroid receptors in crustaceans. By using immunohistochemistry and Western blotting approaches and employing three different antibodies against PR (human PR, chicken PR-hinge region, and chicken PR A/B domain) and antibodies against human ER, we showed the presence of PR in the ovary and hepatopancreas and ER in the hepatopancreas of the freshwater crayfish A. pallipes. The immunological characteristics and the tissue localization suggest a relatedness with both PR and ER in vertebrates along with their involvement in the modulation of reproductive functions in this crustaceans.     

Phenotypical characterization of indigenous freshwater crayfish populations

The morphology of an animal is known to reflect both genetic variation and adaptation to the environment. Thus, phenotypic criteria have been used to characterize indigenous crayfish populations. Twenty-one morphometric parameters were measured on crayfish from 25 waterbodies in the Austrian and Italian parts of Tyrol. They were analysed with hierarchical cluster analysis to obtain the population structure based on morphological similarity between and within the freshwater crayfish species Astacus astacus (noble crayfish), Austropotamobius torrentium (stone crayfish) and Austropotamobius pallipes (white-clawed crayfish). Furthermore, a stepwise discriminant analysis was applied to the morphometric data to test their differentiating power between populations. Both analyses resulted in a clear differentiation of species and populations, and reflected geographic separations. Thus, the developed morphometric methods were shown to be applicable to characterize the phenotype in freshwater crayfish and seem appropriate to be used for stock identification and the effective characterization of management units in decapod crustaceans.     

Aggression and competition for shelter between a native and an introduced crayfish in Europe

1. The introduced North American crayfish, Pacifastacus leniusculus Dana, is expanding its range in Europe and locally often replaces two native crayfish species, Astacus astacus L. and Austropotamobius pallipes Lereboullet. Pacifastacus leniusculus is also expected to invade the habitat of a third native crayfish, the endangered Austropotamobius torrentium Schrank. Interspecific aggressive interactions and competition for shelter were experimentally studied in the laboratory to assess the potential impact of P. leniusculus on A. torrentium .
2. Neither species was inherently dominant in aggressive interactions, but dominance was strongly size-dependent, favouring the larger and faster growing species, P. leniusculus .
3. Access to limited shelter was generally determined by aggressive dominance, although species-specific preference also influenced the outcome of competition for shelter. Austropotamobius torrentium had a higher preference for experimental shelters and often defended these even against larger P. leniusculus .
4. In accordance with theoretical models of animal conflicts, agonistic interactions between equally sized contestants were more severe than between animals of different size.
5. The P. leniusculus used in the present experiment were infected with the crayfish plague, Aphanomyces astaci , to which the animals are resistant. The crayfish transmitted the disease to non-resistant A. torrentium which died ≈ 2 weeks after contact with P. leniusculus .     

Mating and spawning of the crayfish Austropotamobius pallipes (Crustacea: Astacidae)

Mating and spawning of the crayfish Austropotamobius pallipes (Lereboullet) is described and compared with the accounts of this behaviour in Orconectes limnosus, Pacifastacus trowbridgii and Homarus amerieanus.     

Origin and colonization history of the white-clawed crayfish,Austropotamobius pallipes,in Ireland

The presence of the white-clawed crayfish Austropotamobius pallipes complex in Ireland is suspected to be a result of human translocations. Two hypotheses have been formulated about the origin of the crayfish: from British populations or from western French populations. In order to resolve this question, nine Irish crayfish populations (a total of 124 individuals) were sampled along a south-north cline and investigated by combining two molecular markers: mtDNA and RAPDs. The mtDNA marker, analysed by RFLP on the entire molecule, showed an absence of polymorphism within and among Irish populations. The RFLP haplotype found in Irish populations was only recorded in western French populations and was different from those found in English populations. This result may be explained by a human introduction of crayfish to Ireland from western French populations. RAPD analysis showed a clinal reduction of genetic variability within Irish populations from south to north, associated with an increase in their genetic differentiation. A stepwise model of translocation from the south to the north of Ireland is proposed and discussed.     

The setae of Austropotamobius pallipes (Crustacea: Astacidae)

The author presents here the results of an introductory study into the setal armature of the British species of crayfish, Austropotamobius pallipes (Lereboullet, 1858). Using an ordinary optical microscope, it is demonstrated that a considerable variety of setae exist on the body and appendages of A. pallipes. These setae are described, and their distribution indicated. The setae fall into two main groups:
(1) Setae with relatively thick walls and inconspicuous ampulla, and having no basal septum.
(2) Those setae with relatively thin walls, a well-developed ampulla and basal septum. Further subdivisions of these two groups are made on the bases of outgrowths and outlines of the setal wall. Finally, the significance of the distribution and the variation, is discussed in relation to crayfish behaviour and function, linking the observed morphological facts with the results of some current electro-physiological experiments performed on related decapods.     

Branchiobdellidan infestation on endangered white-clawed crayfish (Austropotamobius pallipes) in the UK

Branchiobdellidans or crayfish worms are clitellate annelids and ectosymbionts of freshwater crayfish. An investigation of branchiobdellidan infestation was undertaken in a population of endangered white-clawed crayfish (Austropotamobius pallipes) in the river Aire, UK. Thirty two percent of animals were infested either by the adult parasite or their cocoons (n=107). Parasite burden increased with host size, but did not differ with sex. Observations of crayfish gill tissue revealed a strong positive relationship between melanization of filaments and parasite prevalence and burden. Taxonomic identification revealed that 1 species of branchiobdellidan was present, Branchiobdella astaci. The first sequences were generated for this species and phylogenetically analysed alongside published sequences for 5 other branchiobdellidan species in Europe. The position of B. astaci within the genus Branchiobdella was confirmed, and it was found to cluster as a sister group to B. parasita.     

The distribution of the crayfish, Austropotamobius pallipes, in British waters

SUMMARY. The distribution of Austropotamobius pallipes , the only freshwater crayfish indigenous to the British Isles, is examined using over 300 records collected since 1970. The majority of records are for England where crayfish are found in areas associated with base-rich easily weathered substrata. Waters containing crayfish tend to be in the pH range 7–9, with calcium levels above 5 mg l⁻¹. Crayfish are found in a wide variety of habitats ranging from deep water-filled quarries with a soft substratum to shallow fast-flowing streams.     

SEM observations on the cuticles of some decapod crustaceans

The cuticles of the crayfish Austropotamobius pallipes (Lereboullet), and the crabs Cancer pagurus L. and Carcinus maenas (L.), have been examined with the scanning electron microscope. Laminae are composed of sheets of horizontal fibres which subsequently are into the inter-laminae. The inter-laminae contain sheets of fibres which are at a slight angle from the horizontal between sucessive laminae. The disposition of the pore canals is clearly related to fibre pattern. The sinuous macrofibres described by Dennell (1973) are discrete from the pore canals.     

Fine-scale genetic structure, phylogeny and systematics of threatened crayfish species complex

Systematic uncertainties in the crayfish Austropotamobius pallipes are well grounded by the number of species and subspecies described using different approaches, causing scientists to define this taxon as "complex". However, a key task that conservation programmes are facing regarding the recent and drastic decline of European populations, is the coherent systematic classification of this threatened species. Here we present results obtained by coupling mtDNA and genome analysis suggestive of a novel evolutionary framework to explain the relationships among phylogenetic lineages of A. pallipes. The direct sequencing of mtDNA COI gene fragment revealed a strong geographic structure with four distinct haplogroups separated by a range of 5-25 mutations. However, mitochondrial data were not supported by genomic fingerprinting based on 535 AFLP polymorphisms. Nuclear markers showed an unexpected moderate level of genetic differentiation and the absence of any geographic structure. Consequently, this study proposes that the taxonomic hypothesis of a single species of A. pallipes settling the Italian continental waters, is affected by complex evolutionary events. To solve the paradox, we hypothesized an evolutive scenario in which the separation of ancient mtDNA lineages likely occurred before the latest glacial periods. However, the speciation process remained incomplete due to secondary intensive postglacial contacts that forced the mingling of the genomes, and confounds the phylogeographic signature still detectable within mtDNA. Postglacial dispersion and the following demographic events, such as founder effects, drift and bottlenecks, abruptly depleted the local mtDNA variation, and shaped the current genetic population structure of white-clawed crayfish.     

Growth in the crayfish Austropotamobius pallipes (Crustacea: Astacidae)

SUMMARY. The growth of Austropotamobius pallipes was studied in the River Ouse during 1976–78. Growth of mature crayfish (>2.5 cm carapace length) was followed by determining the relationship between the growth increment at moult and premoult carapace length, together with the frequency of moulting of different categories of crayfish. These data are supplemented by the recapture of marked individuals and the measurement of crayfish held in corves in the river. Growth was limited to the period May–October when water temperatures exceeded 10°C. Growth rates of male and female crayfish are similar until maturity is reached, thereafter males moult twice per year and the majority of females moult once. No crayfish in excess of 3.7 cm carapace length has been observed to moult more than once per year. Growth of juveniles (<2.5 cm carapace length) was estimated from size frequency distributions constructed from regularly taken samples. Growth rates of juveniles showed great variation both between and within year classes. In the hot dry summer of 1976, juveniles exhibited faster growth rates (instantaneous growth rates (G) for 0+ and 1 + crayfish were 0.029 and 0.013 mg mg⁻¹ day⁻¹. respectively) than in other years. Laboratory experiments on the effect of temperature on the growth rate of 0 + crayfish were undertaken; for crayfish at 15°C. G = 0.0138 (0–53 days) and at 10°C, G = 0.0003 (0–90 days). Crayfish held at 10°C failed to undergo a single successful moult. At 20°C crayfish exhibited exponential growth over the first 40 days, with G = 0.0189. declining thereafter to G = 0.012 (40–90 days).     

Phylogeography of the threatened crayfish (genus Austropotamobius) in Italy: implications for its taxonomy and conservation

A nucleotide sequence analysis of a portion of the mitochondrial large ribosomal subunit was performed to define the phylogeography of the threatened crayfish Austropotamobius (Decapoda; Astacidae) in Italy. We collected 61 specimens from 31 localities across the Italian peninsula. For the phylogenetic inference, we combined the 61 Austropotamobius spp sequences obtained from this study with 18 sequences deposited in GenBank and corresponding to Italian, French, Irish, Swiss, and Slovenian locations. Among the analysed sequences, 34 distinct haplotypes were detected. Our results confirmed the presence of both A. pallipes and A. italicus in the Italian peninsula and the existence within the latter species of a strong intraspecific genetic variation, due to the occurrence of four subspecies with a well-defined geographic distribution. From a conservation viewpoint, Italy, with its high haplotype variability, may be considered a 'hot spot' for the genetic diversity of the European native crayfish Austropotamobius. We suggest that re-introduction programs should be conducted with extreme caution in Italy, since not only the two Austropotamobius species but also the four A. italicus subspecies are genetically and taxonomically separate units and require independent conservation plans.     

The ultrastructure and metal-containing inclusions of mature cell types in the hepatopancreas of a crayfish

The ultrastructure of the hepatopancreas of the crayfish Austropotamobius pallipes (Lereboullet) is described in relation to its function in digestion. X-ray microprobe analyses of the cells of this tissue have been obtained after using a variety of fixatives and plasma oxygen etching. The distribution of metals in the digestive cells is considered in relation to the polarization of the cells and their ability to absorb materials across the apical and basal cell membranes.     

Metal-protein binding patterns in the hepatopancreas of the crayfish (Austropotamobius pallipes) during short term cadmium stress

Cd-binding patterns in the hepatopancreas of the crayfish (Austropotamobius pallipes) have been analysed in response to the administration of a single dose of Cd ions. Four main components, with apparent molecular weights of 3-4, 12, 20 and greater than 70 K daltons, have been isolated from soluble cell fractions. The variation in molar metal/protein ratios of these components is discussed in terms of the uptake, transfer and detoxification of Cd in the hepatopancreas. A model is proposed which describes Cd flux at the subcellular level and indicates that metallothionein-like proteins may mediate in this process.     

Phylogenetic and phylogeographic relationships in the crayfish genus Austropotamobius inferred from mitochondrial COI gene sequences

The present study explores the utility of mitochondrial COI gene sequences to reveal phylogenetic and phylogeographic relationships for the entire European freshwater crayfish genus Austropotamobius. The two traditional taxa, Austropotamobius pallipes and Austropotamobius torrentium, were monophyletic, showing similar genetic diversity, with 28 and 25 haplotypes, respectively, and an uncorrected average pairwise divergence of 0.059 and 0.041. A third distinct haplotype clade, in sister relation to A. torrentium, was discovered at the Upper Kolpa drainage in the northern Dinaric area. All populations north and west of the Alps are genetically impoverished (nucleotide diversity (pi)=0.000-0.001), while southern populations are more diverse (pi=0.001-0.034). A. pallipes reaches the highest diversity in the region of Istra, probably its primary center of radiation. The genetic diversity center for A. torrentium is the southern Balkan peninsula. Other potential glacial refugia were identified in Southern France, Northwestern Italy, the Apennine Peninsula, and in the northern Dinaric area. The Iberian Peninsula has been stocked artificially from Northern Italy. Three main periods of radiation were tentatively identified: late Miocene/early Pliocene for the divergence of species and main lineages, the Pleistocene for the divergence within populations south from Alps, and a postPleistocene expansion north and west from Alps.     

Phylogeny and species composition of five European species of Branchiobdella (Annelida: Clitellata: Branchiobdellida) reflect the biogeographic history of three endangered crayfish species

Branchiobdellid annelids are usually found as commensal symbionts associated with crayfish populations, but knowledge of their dispersion and ecology in Europe is generally scarce. We hypothesized that their geographic extension of species and populations may mirror the distribution history of their hosts. We analysed potential host specificities and the geographic distribution of species from the Italian and Austrian Tyrol and Carinthia by characterizing the morphological and genetic features. On the three indigenous crayfish species Astacus astacus, Austropotamobius pallipes and Austropotamobius torrentium , we identified four branchiobdellid species based on morphological characteristics: Branchiobdella hexodonta, Branchiobdella pentodonta, Branchiobdella balcanica and Branchiobdella parasita . In contrast to the morphological classification, phylogenetic analysis using mitochondrial cytochrome c oxidase I (CO-I) sequences identified five main lineages: B. balcanica, B. hexodonta, Branchiobdella italica, B. parasita and B. pentodonta . The arrangement of branchiobdellid species corresponded generally to the geographical distribution of their crayfish hosts' locations but also confirmed previous assumptions of crayfish translocations. Our study provides the first application of ideas on the association of freshwater crayfish and their ectosymbionts to be used for discussing the biogeography of crayfish populations. The phenotypical and genotypical analysis also demonstrated so far ignored effects of human activities at both macro-ecological and micro-ecological levels.     

A Comparison of Trace Metal Accumulation in Indigenous and Alien Freshwater Macro-Decapods

Concentrations of Cd, Cu, Fe, Mg, Mn, Ni, Pb and Zn were determined in the exoskeleton and hepatopancreas of three freshwater macro-decapods widely distributed in Tuscany, Italy. The species were the indigenous Austropotamobius pallipes (the white-clawed crayfish) and Potamon fluviatile (the river crab) and the alien Procambarus clarkii (the red swamp crayfish, in Italy aquacultured since 1977 and invasive since 1989). Water and sediments from the three environments in which they occur were analysed for trace metals to evaluate the degree of contamination of the habitats. Procambarus clarkii showed a significantly higher accumulation of most of the metals analysed, including the non-essential cadmium, nickel and lead. Interspecific differences were not related to exposure to higher concentrations of metals, but indigenous and alien macro-decapods possibly differ in their physiological requirements for essential trace metals and/or in their rates of essential and non-essential metal uptake, accumulation, detoxification and excretion.     

A Comparison of Trace Metal Accumulation in Indigenous and Alien Freshwater Macro-Decapods

Concentrations of Cd, Cu, Fe, Mg, Mn, Ni, Pb and Zn were determined in the exoskeleton and hepatopancreas of three freshwater macro-decapods widely distributed in Tuscany, Italy. The species were the indigenous Austropotamobius pallipes (the white-clawed crayfish) and Potamon fluviatile (the river crab) and the alien Procambarus clarkii (the red swamp crayfish, in Italy aquacultured since 1977 and invasive since 1989). Water and sediments from the three environments in which they occur were analysed for trace metals to evaluate the degree of contamination of the habitats. Procambarus clarkii showed a significantly higher accumulation of most of the metals analysed, including the non-essential cadmium, nickel and lead. Interspecific differences were not related to exposure to higher concentrations of metals, but indigenous and alien macro-decapods possibly differ in their physiological requirements for essential trace metals and/or in their rates of essential and non-essential metal uptake, accumulation, detoxification and excretion.