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.     

Mitochondrial DNA variation and population genetic structure of the white-clawed crayfish, Austropotamobius pallipes pallipes

Here we report a high level of mtDNA variationfrom RFLP analysis within A. p. pallipes from 21 French and 4British populations. Seventeen haplotypes were recorded among 269individuals. Nucleotide sequence divergence (p) among mtDNAhaplotypes ranged from 0.0019 to 0.0425. Nucleotide diversity ()ranged from 0.01 to 0.006 (mean = 0.0031) and from 0 to 0.001(mean = 0.00037) for populations located in southern and northernFrance, respectively. Graphical representation from principalcoordinate analysis based on Nei's genetic distance values amongpopulations showed two groups, cluster A, including the 16populations sampled in the north-western and north-easternFrance, England and Wales and cluster B containing 9 populationssampled in the central and southern of France. No geneticdifferences were noted among the most northern French and Englishpopulations whereas marked interpopulation genetic diversity wasobserved in southern populations. Such genetic heterogeneityamong populations of A. p. pallipes appears to be related tohabitat fragmentation and subsequent recolonizations fromrefugial areas during the Pleistocene. Implications for theconservation of A. p. pallipes are discussed.     

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.     

Ranging behaviour of a large freshwater invertebrate, the white-clawed crayfish Austropotamobius pallipes

• 1 Radio‐telemetry and mark‐recapture methods were used to study the summer movements of adult and juvenile white‐clawed crayfish, Austropotamobius pallipes from a wild population in a small braided stream, Dalton Beck, North Yorkshire, U.K. Radio‐transmitters were attached to the chelae of 18 large (> 35 mm carapace length) crayfish and individuals were subsequently located to within 0.15 m. Additionally a total of 888 crayfish were marked with carapace brands, and 83 were recaptured.
• 2 Radio‐tracked crayfish exhibited significantly greater local activity at dusk (21.00–00.00) than at dawn (03.00–06.00), or during morning (09.00–12.00) and afternoon (15.00–18.00) monitoring periods.
• 3 The greatest movements of radio‐tracked crayfish occurred within 2 days of release. After this time, periods of residence were interspersed by movements to new locations, interpreted as establishment of ephemeral home areas. It is suggested that the initial large movements were the result of a ‘fright response’ following capture.
• 4 Movements varied widely between individuals, some moving more than 300 m in 10 days, while others showed little movement over an equivalent time period. Mean (±SE) daily movements were 4.6 ± 3.0 m for males and 1.5 ± 1.0 m for females. Although crayfish often used specific home sites for in excess of 7 days, displaced animals did not return to home sites.
• 5 The total distances travelled and the mean distance travelled per day by individual radio‐tagged crayfish did not differ significantly between upstream or downstream directions or between males and females. This was also the case for marked crayfish used in mark‐recapture studies.
• 6 Positive correlations between distance moved per day and size (carapace length) were found for downstream movements by male and female crayfish, but not for upstream movements.
• 7 Some preliminary observations of the response of crayfish to flood events suggested that these could be catastrophic with two out of five tracked crayfish found dead after a high stream‐discharge event.

     

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.     

Histopathological survey of pathogens and commensals of white-clawed crayfish (Austropotamobius pallipes) in England and Wales

A histopathological survey of white-clawed crayfish (Austropotamobius pallipes) from six rivers in England and Wales was conducted between November 2007 and September 2011. A. pallipes bacilliform virus (ApBV) was present in four samples; infected crayfish showed pathological responses ranging from mild hypertrophy of infected nuclei and emargination of chromatin through to loss of architecture of the hepatopancreatic cells. Crayfish were found to be hosts to two different ciliate species, mites, nematodes, digeneans and the microsporidian Thelohania contejeani. The variation in disease presence between populations highlights the importance of conducting appropriate surveys of native crayfish prior to movement of animals to refugia sites for relocation, restocking or breeding programmes.     

Diversity of mitochondrial DNA of the endangered white-clawed crayfish (Austropotamobius italicus) in the Po River catchment

1. The strong contraction of the European populations of white‐clawed crayfish has stimulated research of its population genetics. As part of a conservation project to introduce and improve Italian populations of Austropotamobius italicus, the genetic diversity of 16 populations from 10 tributaries of the Po River drainage was evaluated by sequencing a 494 bp length of cytochrome oxidase subunit I (COI mtDNA). 2. The evolutionary tree topologies and the parsimony network of 26 haplotypes separated into two clusters, on the basis of high average genetic distance (6.51 ± 0.42%). This finding indicated the presence of distinct evolutionary lineages, other than different allopatric sources. 3. An ancestral haplotype (Nav) was identified and 13 haplotypes, found mainly as singletons, were independently connected to Nav by short branches with one mutation site. Unimodal mismatch distribution and the goodness‐of‐fit of the observed data reflected a model of sudden population expansion, after the last glacial bottleneck event. 4. A general moderate level of genetic variability (h = 0.22–0.7 and π = 0.045–0.27%) was highlighted within A. italicus populations inhabiting Alpine and Apennine tributaries of the Po River. Analysis of molecular variance (amova ) revealed significant genetic structuring across all hierarchical levels, indicating that a major proportion of genetic variance is structured among localities within drainages. Multiple adjacent localities, strictly connected by the Po River, shared the two most frequent haplotypes (Nav and Laz), while remote populations, far from the Po River inflow, move towards a mtDNA fixation. 5. The results of this study indicate that distinct populations should be treated as separate entities for management. The key implication of the data is that ad hoc measures for successful programmes of recovery and management strategies for A. italicus conservation, such as the phylogeographic study of haplotype distribution at a local scale and the genetic variability of populations used as stock breeders, should be considered.     

A study of reproductive females of the freshwater crayfish Austropotamobius pallipes

Details of the female annual reproductive cycle in the population from Northumberland are given. Mating occurs in October and is followed by egg laying in early November. Eggs are carried by females until the following July–August when hatching occurs. The smallest berried female caught was 25 mm carapace length (C.L.) and this was chosen as the minimum size for adult females. Only 40–50% of adult females, hand-caught, were berried in the Spring following egg laying; but this percentage was low largely because few females in the size classes below 28 mm C.L. were berried. The reasons for adult females being non-reproductive in a year are discussed.     

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).     

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.     

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.     

Reproduction in an Irish lake population of the crayfish Austropotamobius pallipes (Lerehoullet)

SUMMARY. 1. Mature crayfish were collected in 1984 from an alkaline Irish lake before and after the mating season.
2. There was some correlation between frequency of mating and decrease in vasa deferentia weight in captive males, but in the field vasa deferentia weight losses were highly variable. The data indicate that one third of adult males may not have mated in the wild.
3. All females appear to have spawned, whether or not mated, with an average 85% reduction in ovary weights over the season.
4. Pleopodal egg counts directly after laboratory spawning were on average 81% of ovarian counts; in the field, an estimated 50 days after spawning, they were only 61% of estimated ovarian egg numbers.
5. Average ovarian egg size was positively correlated with female size. Also, larger crayfish had a wider range of egg sizes than smaller crayfish.
6. It is suggested that aggression and competition for shelter among brooding females may result in a progressive loss of eggs.     

Respiration in relation to ion uptake in the crayfish Austropotamobius pallipes (Lereboullet).

1. O2 uptake was determined for periods of 23-46 h in salt-depleted crayfish held in deionized water (DW) or Na-free media at 10 degrees C. These media were replaced by artificial lakewater media (ALW) containing 0-2-0-6 mM Na and O2 uptake was again determined for periods of 24-66 h. 2. During net ion uptake in ALW the metabolic rate was either elevated or depressed. Standard metabolism in ALW altered by amounts equivalent to 0-1 - 15-5% (mean 6-4 (15) +/- 4-4% S. D.) of the metabolic rate measured during salt-depletion. On three occasions the metabolic rate was elevated by 22-0 - 66-7%, but some of this increase may have been due to locomotor activity. 3. The calculated values for thermodynamic work involved in ion transport were 0-056 - 0-268 J/10 g. h at 10 degrees C, or 1-5 - 7-2% of the mean standard metabolic rate. Most of the observed changes in metabolic rate lie within the limits of experimental error (ca. +/- 7%). Hence the energetic cost of ion transport is too small for direct measurement in intact crayfish.     

Population genetic structure of the endangered crayfish Austropotamobius pallipes in France based on microsatellite variation: biogeographical inferences and conservation implications

1. One important goal in conservation biology is to characterise evolutionary lineages within endangered species before management decisions are taken. Here, we assess population differentiation in the freshwater crayfish Austropotamobius pallipes, an endangered species endemic to western Europe and provide valuable information for the conservation of French populations. 2. Analysis of five microsatellite loci in 44 populations revealed very different within population levels of genetic diversity (0.000 < H₀ < 0.564). Two groups, corresponding to northern and southern French populations, showed a high degree of genetic differentiation in both allele frequencies and allele sizes. Comparison of these results with previous studies of A. pallipes strongly suggests that the divergence between northern and southern populations could have occurred during the last glaciation period of the Pleistocene from one Atlantic and one Mediterranean refuge. 3. Evidence for genetic admixture between these two lineages was revealed by correspondence analyses in southern populations, probably as the result of artificial translocations. 4. French populations appeared significantly differentiated among the different river drainages and were highly structured within rivers. The impact of population size, population bottlenecks and founder events on the population genetic differentiation are discussed. 5. Based on these results, we propose the designation of two evolutionarily significant units for A. pallipes in France. Our data also support the maintenance of separate demographic management strategies for crayfish inhabiting different river systems. However, genetic analyses will have to be combined with demographic and ecological data for sustainable conservation programmes.     

Phylogeography of the white-clawed crayfish (Austropotamobius italicus) in Spain: inferences from microsatellite markers

The white-clawed crayfish (Austropotamobius italicus), a cornerstone of Spain’s aquatic ecosystems, was once widely distributed throughout much of the country. Unfortunately, its populations have suffered very strong declines over the last 40 years due to the spread of introduced species (red swamp and signal crayfishes), diseases, habitat loss and other anthropogenic impacts. The present work examines the genetic variation in 23 Spanish and four Italian populations of white-clawed crayfish via the analysis of microsatellite loci. The data show genetic variation in the Spanish populations to be affected by drastic and successive bottlenecks. Notwithstanding, the diversity of these Spanish populations in terms of observed heterozygosity is similar to or even higher than that recorded for other European populations studied using these same markers. North-central Spanish populations are clearly differentiated from the country’s remaining populations; they should be considered distinct management units. Processes occurred in historical and recent times, such as genetic drift and translocations, contribute greatly to this genetic structure. These data provide useful information for conservation of this species, since the preservation of its population structure and genetic variability should be goals for management decisions.     

Breeding in the crayfish, Austropotamobius pallipes: mating patterns, mate choice and intermale competition

1. The breeding behaviour of the white-clawed crayfish, Austropotamobius pallipes, which is in decline throughout Europe, was analysed. 2. Observations and experiments focused on: (i) several aspects of mating behaviour, showing the primary role of the female during courtship, the patterns of mature male receptivity and the possible existence of a mating stimulus produced by either a mating pair or a receptive female; (ii) the potential for mate choice either by males (males were not choosy, mating with the first receptive female they met) or by females (that rejected the smallest males and males with only one cheliped) and (iii) intermale fighting before copulation (larger males copulated more often, with the exception of some ’sneakers‘). 3. Sperm competition occurred when a new male was presented with a female after a previous male‘s spermatophore had been deposited; the new male cleaned the rival male‘s spermatophore by feeding on it before copulation. This behaviour has also been observed in some pseudoscorpions, millipedes and collembolans, but always in cases where the spermatophore is deposited on the substratum. Introduction     

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.