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1. In France, the distribution of the native white-clawed crayfish (Austropotamobius pallipes) is restricted, fragmented and mainly located in headwater streams. To conserve this indigenous species, it is necessary to characterize its habitat preferences. 2. Seven brooks in the Deux-Sevres Departement (western France) containing wild populations of A. pallipes were studied to determine its ranges of tolerance to 19 physical and chemical water parameters. On two brooks, the Sevre Nantaise and the Verdonniere, sites with and without A. pallipes were compared. Each site was sampled twice monthly from November 2002 to November 2004. 3. It was found that the white-clawed crayfish was able to tolerate wide ranges of values of some of the measured parameters. The Magot site harboured the largest A. pallipes population (17.5 crayfish m−2) and had dissolved oxygen concentrations as low as 4.93 mg L−1, while water temperature rose above 20°C for several consecutive days during summer and nitrate concentrations were always found to be above 30 mg L−1. These unusual findings could call into question the status of A. pallipes as a bioindicator of good water quality. 4. Principal component analyses (PCA) suggested that an increase of organic matter was a discriminant factor for the presence or absence of A. pallipes. In addition, sites with and without crayfish on the Sevre Nantaise brook showed showed significant differences (p

Since Charles Elton, ecologists have struggled to account for the distribution and spread of non-indigenous species (NIS). They now realize that the key factor is ‘‘opportunity’’. The more frequently and persistently a foreign plant or animal is exposed to a new environment, the better its odds of invading. Ecologists call this ‘‘propagule pressure’’ (also termed introduction effort), a composite measure expressing the number of individuals released into a region to which they are non-native. It incorporates estimates of the absolute number of individuals involved in any one release event (propagule size) and the number of discrete release events (propagule number) (Lockwood et al. 2005). Propagule pressure has rarely been documented for freshwater organisms except in fish. Duggan et al. (2006), for example, using data on aquaria in stores and historical records of fish introduced into Canadian and US waters, showed a clear relationship between their occurrence in shops and the likelihood of their introduction and eventual establishment.

Summary 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

The distribution of the prawn species belonging to the genus Palaemonetes is not well defined in the Mediterranean, especially in the south of France where controversy exists concerning taxonomic identification at the species level. A molecular diagnostic technique allowed us to determine without any doubt the presence of Palaemonetes varians in the Camargue. These basic results allowed us to undertake the ecological study of the species, which took place in the context of a massive 100-year flood during the winter 1993/94, leading to a rapid and major fall in salinity that remained low until 1997, before increasing. The impacts of low salinity on the catch and life history traits of Palaemonetes varians in the lagoon were investigated from 1993 to 1999. There was a positive correlation between catches and salinity. Although we observed a major decline in recruitment between the years 1993 and 1994, reproduction parameters and growth did not seem to be affected by low salinity. A comparative study of various life history traits between Mediterranean and northern environments populations was also conducted.

Species that can act as indicators of ecosystem health offer a valuable tool in the management of natural resources. Crayfish have been suggested as bioindicators of water quality in Europe and at least one species (Austropotamobius pallipes) has been studied to determine its tolerance to pollution and its potential as a bioindicator. The genus Austropotamobius includes three crayfish species native to western Europe: A. pallipes, A. italicus and A. torrentium. It was hypothesised that because of their geographical and habitat distribution, the three Austropotamobius species might vary in their value as a bioindicator of water quality. Crayfish of species A. pallipes and A. italicus were subjected to three different treatments: hypoxia (treatment 3, approx 3 mg 1(-1) O-2), light hypoxia (treatment 2, approx 5.5 mg 1(-1) O-2) and normoxia (treatment 1, control, approx 8.5 mg 1(-1) O-2). A. torrentium crayfish were only subjected to treatment 1 (control) and 3. Variations in haemolymph sodium, calcium and chloride were used as a biomarker and concentrations were measured before and after treatment to evaluate hypoxia-induced stress. Significant differences in the concentrations of sodium between the control groups (treatment 1, normoxia) and the experimental groups (treatment 3, 3 mg 1(-1) O-2) were found in the species A. pallipes and A. torrentium. Groups of A. italicus did not show any significant difference between treatments in sodium concentrations but in chloride concentrations. Crayfish of all three species demonstrated a disruption in the ion exchange process in hypoxia, but all tolerated very low oxygen concentration for an extended period of time.

Subsequent surveys carried out from 1978 to 2003 demonstrated a high disappearance of Austropotamobius pallipes populations from the region and the alarming spread of non-indigenous species like Procambarus clarkii and Pacifastacus leniusculus. Represented by 137 populations in 1978, A. pallipes is now almost completely restricted to only one department, with 45 populations recorded, representing a decrease of 68% of the population number in 25 years, with a loss of 40% of populations in the last 6 years. Most of the disappearances of crayfish are unexplained. For the 22 others, the main reasons of the disappearance of indigenous crayfish populations were due to (1) habitat destruction by channelisation, (2) decrease of water quality by use of agricultural chemicals, (3) the construction of ponds or lakes changing the physical and chemical parameters of brooks inhabited by indigenous crayfish, (4) the introduction of non-indigenous species, mainly P. leniusculus and recently (5) crayfish plague implicated and characterized in the disappearance of two populations, two additional ones being suspected. Introduced in 1978 in Vienne department, P. leniusculus has now been found in 28 locations. First record of P. clarkii was in 1988 in Charente-Maritime department; since it has spread in most of the hydrographic basin of this department.

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.

International audience; The loss or diminution of European crayfish populations because of both habitat deterioration and competition with alien crayfish - also responsible for the dissemination of the crayfish plague - would reduce the biodiversity at the species level. The topic "What is meant by biodiversity?" in the context of native freshwater crayfish in Europe was discussed during the Kilkenny CRAYNET meeting in order to make the point about the varied meanings of biodiversity from genes and individuals to population levels.

The photoperiodic control of sexual rest in Armadillidium vulgare was investigated using various experimental protocols. When reared in conditions of a Nanda-Hamner (i.e. resonance) protocol from their first parturial moult to their post experimental moult, females showed a weak resonance effect in sexual rest incidence. The transfer from a long day cycle to a symmetrical skeleton photoperiod--consisting of two equal light pulses per 24 h of continuous darkness--revealed the involvement of a circadian oscillatory system in the photoperiodic clock of this species. The data, obtained in the whole experiments, suggested that both oscillator and hourglass features are involved in the photoperiodic response controlling the sexual rest in Armadillidium vulgare. Moreover, when non-24-h light-dark cycles (with a long photophase) were applied, a mechanism responsible of arrest of reproduction also implied a photoperiodic counter which accumulated and added up the photoperiodic information within a sensitive period during post parturial intermoult.

Random amplified polymorphic DNA (RAPD) analysis was performed to characterize the genetic diversity of Austropotamobius pallipes, a threatened freshwater crayfish native to Europe. Four decamer primers which generated six unambiguous polymorphic bands were used to analyse crayfish from 21 populations sampled in the major part of its range. Genetic diversity within populations of A. pallipes, estimated by Shannon's diversity index, ranged from 0 to 0.446 with a mean of 0.159. A UPGMA dendrogram constructed from pairwise PhiST values between populations, revealed three clusters corresponding to populations sampled in the southern, northwestern and eastern part of its range. AMOVA analysis revealed a high genetic structure of A. pallipes populations PhiST=0.814, with 73.11% of the genetic variation distributed between these clusters. It suggests a historical geographical separation of these groups into three refugial areas, probably in the Rhine, Mediterranean and Atlantic basins during recent glaciations. The close genetic relationships between English and western French populations are in accordance with a natural postglacial origin of English populations from individuals having survived in an Atlantic refugium. However, the present results suggest that the Irish stock originated from a human translocation of individuals from an Atlantic refugium.

Variation in mitochondrial DNA (mtDNA) was surveyed, using restriction endonucleases, in the white-clawed crayfish, Austropotamobius pallipes lusitanicus, from 14 populations sampled in Spain. Four additional samples from France (1), Slovenia (1) and Italy (2) were also analysed. Among the 11 haplotypes listed, only one was detected from the 154 animals sampled from Spanish populations. This haplotype was also recorded in the Fosso di Ferfereta population (Italy). Estimates of nucleotide sequence divergence among haplotypes ranged from 0.45% to 17.4%. Interpopulational genetic relationships showed that Spanish populations were closely related to those of Fosso di Ferfereta with a small genetic distance (0.0003) found between them. AMOVA revealed that most of the genetic variance (71.97%) was attributed to variation between European regions. These results are in accordance with a drastic bottleneck event during the history of the Spanish populations. Four suggestions, based on human introduction, selection and recent or ancient historical events are discussed in relation to the lack of genetic variation in the Spanish crayfish stock.

International audience

Several morphological races and subspecies have been described and later included within the terrestrial isopod species Porcellionides pruinosus. During our study of this species, we have worked on specimens from France, Greece, Tunisia and Reunion island. Laboratory crosses have revealed two separate groups of populations: French populations (four localities) in one group, and those from Tunisia, Reunion island and Greece in the other. French individuals were reproductively isolated from those of the other populations. We have undertaken a survey of mitochondrial DNA (mtDNA) polymorphism in these seven populations. We observed two groups of mitotypes corresponding to the two groups of populations. Interfertility experiments between populations and the mitochondrial genetic distances between mitotypes both suggest the presence of two different species, one in France and one in Greece, Tunisia and Reunion island. The two species harbour, respectively, two different Wolbachia lines. Another feature of the molecular genetic analysis was the apparent mitochondrial monomorphism in the French populations and the low variability in the other three populations. The result can be related to the possibility of Wolbachia-induced genetic hitchhiking in these populations.

Sex determination in Armadillidium vulgare may be under the control of two parasitic sex factors that reverse genetic males into functional neo-females. The first feminizing factor (F) is a Wolbachia and the other (f) is probably a sequence of the F bacterial DNA unstably integrated into the host genome. Both of these feminizing factors are mainly maternally transmitted. Here we investigate the mitochondrial DNA polymorphism of wild iso-female lineages harbouring either F or f. Among the four haplotypes present in the population, two were the f-harbouring lineages, while two were common to the F- and f-harbouring lineages. This result suggests that there has been an introgression of the f factor into lineages infected by F Wolbachia. Based on previous data, we propose two different ways to account for such introgression. Given the particular dynamics of feminizing factors (f-harbouring lineages increase in populations at the expense of F-harbouring lineages), such an introgression should prevent the replacement of F-linked mitochondrial types by f-linked mitochondrial types in wild populations.

Non-indigenous crayfish species (NICS) in Europe now outnumber indigenous crayfish species (ICS) 2:1, and it has been predicted that they may dominate completely in the next few decades unless something is done to protect them. Of the ten NICS introduced at least nine have become established in areas occupied by four of the five ICS. A decline in stocks of ICS has been recorded in many countries in the face of increasing populations of NICS. Most European countries retain at least one ICS but all are under threat from habitat loss, deteriorating water quality, overfishing, climate change, and most importantly from NICS and crayfish plague. The threat to ICS is so great in some countries that “ark”sanctuary sites are being established.The three most widely-spread NICS are the North American species: Pacifastacus leniusculus , Orconectes limosus and Procambarus clarkii . These can be considered as “Old NICS”, which were introduced before 1975, compared with the “New NICS”, which were introduced after 1980, such as the North American species: Orconectes immunis , Orconectes juvenilis , Orconectes virilis , Procambarus sp. and Procambarus acutus ; and the Australian species: Cherax destructor and Cherax quadricarinatus , all of which have much narrower ranges in Europe. The North American species are potentially capable of acting as vectors of crayfish plague. Outbreaks of this disease occur regularly where there are high concentrations of vectors.In addition to the NICS currently established in the wild, a further threat exists through the aquarium trade, where many American and Australian species are available via the internet and in aquarist centres. Owners of such species may discard them into the freshwater environment when they grow too big as with some Cherax spp. and Orconectes spp., or multiply too frequently as with Procambarus sp. (a parthenogenetic species). A conceptual model is presented as a possible way forward for protecting the future survival of ICS in Europe.

The present paper deals with reintroductions or restocking as a management strategy for the indigenous crayfish species (ICS) in Europe. The suitability of the target habitat, the stocking material and the stocking procedure itself are paramount during any reintroduction measure: apart from general water quality and structural parameters, a suitable habitat is ideally geographically isolated from other surface waters and human activities such as intensive fishing pressure and agricultural practices. Genetics of stocking material must be considered. However, it is first essential to make sure that the target habitat is free of crayfish plague. Analyses of experiences gathered in various European countries indicate how difficult it is to get the best information as a basis for successful restocking and consequently the discussions recently conducted among European researchers and managers were aimed at achieving consensus and common strategies.

In some natural populations of Armadillidium vulgare, intersex animals are genetic males which are feminized by maternally transmitted symbiotic bacteria. In these intersex males (iM) the fat body synthesizes vitellogenin, although their gonads are testes with hypertrophied—but nonfunctional—androgenic glands. Vitellogenin is present in the hemolymph of males changed experimentally into iM 90 days after inoculataion of the feminizing bacteria. During the molting cycle, vitellogenin synthesis in iM varies as in ovariectomized females or in vitellogenic females, with a peak at the stage D1″. In A. vulgare, vitellogenin synthesis is a neutral character since it can be observed in a genetic male or in an ovareectomized female; however, it is inhibited by the androgenic hormone. In intersex males, vitellogenin synthesis is the result of their refractoriness to androgenic hormone.

Summary Seasonal reproduction in terrestrial Isopods is regulated by a neurohormone (VIH) synthesized in neurosecretory cells located in the protocerebrum median part. VIH restrains vitellogenin synthesis by the fat body. This inhibitory system is driven by the photoperiodic variations. Long days introduce a decrease in synthesis and—or—VIH release, allowing the oocytes to carry out the last vitellogenic phase. Other signals (mating, eggs in brood-pouch) tune the activity of the inhibitory system. Various aspects of photoperiodic and neurohormonal controls of reproduction are discussed: geographical variability and genetic determinism of the photoperiodic response; chemical nature, specificity and VIH mode of action; male physiology and VE synthesis, modalities of VIH synthesis and release.

The genetic structure of a large population of the white-clawed crayfish Austropotamobius pallipes was assessed with five microsatellite markers by sampling six sites along its extent. The level of genetic polymorphism was relatively high with a mean heterozygosity value of 0.394 for the whole population. No genetic differentiation was observed between the different sampled sites, indicating that this population was panmictic. The possibility of extensive gene flow along this brook, downstream as well as upstream, is discussed.

Two maternally inherited sex ratio distorters (SRD) impose female-biased sex ratios on the wood louse Armadillidium vulgare by feminizing putative males. These SRD are (i) an intracytoplasmic bacterium of the genus Wolbachia, and (ii) another non-Mendelian element of unknown nature: the f element. Mitochondrial DNA variation was investigated in A. vulgare field populations to trace the evolution of host-SRD relationships and to investigate the effect of SRD on host cytoplasmic polymorphism. The Wolbachia endosymbionts showed no polymorphism in their ITS2 sequence and were associated with two closely related mitochondrial types. This situation probably reflects a single infection event followed by a slight differentiation of mitochondria. There was no association between the f element and a given mitochondrial type, which may confirm the fact that this element can be partially paternally transmitted. The spreading of a maternally inherited SRD in a population should reduce the mitochondrial diversity by a hitchhiking process. In A. vulgare, however, a within-population mtDNA polymorphism was often found, because of the deficient spread of Wolbachia and the partial paternal inheritance of the f element. The analysis of molecular variance indicated that A. vulgare populations are genetically structured, but without isolation by distance.

The status of Austropotamobius berndhauseri (Crustacea, Decapoda) was re-examined by the combination of two recent genetic studies based on the analysis of the mitochondrial 16S rRNA gene obtained from sampling across the entire range of the white-clawed crayfish species complex. From the phylogenetic tree obtained, the results clearly demonstrate two clusters (A and B) separated by a strong genetic divergence (5.0 ± 1.0%) corresponding to A. italicus and A. pallipes species. Within A. italicus, three mtDNA lineages were found corresponding to A. i. italicus, A. i. carinthiacus, and A. i. carsicus. Austropotamobius berndhauseri specimens are not genetically distinct from A. i. carinthiacus distributed in Austria. This group is more closely related to A. i. italicus (1.4 ± 0.5%) than A. i. carsicus (3.6 ± 0.7%) which is concordant with allozyme data. These data support the redefinition of A. berndhauseri as being A. italicus carsicus.

The mitochondrial DNA (mtDNA) in animals is generally a circular molecule of ∼15 kb, but there are many exceptions such as linear molecules and larger ones. RFLP studies indicated that the mtDNA in the terrestrial isopod Armadillidium vulgare varied from 20 to 42 kb. This variation depended on the restriction enzyme used, and on the restriction profile generated by a given enzyme. The DNA fragments had characteristic electrophoretic behaviors. Digestions with two endonucleases always generated fewer fragments than expected; denaturation of restriction profiles reduced the size of two bands by half; densitometry indicated that a number of small fragments were present in stoichiometry, which has approximately twice the expected concentration. Finally, hybridization to a 550-bp 16S rDNA probe often revealed two copies of this gene. These results cannot be due to the genetic rearrangements generally invoked to explain large mtDNA. We propose that the large A. vulgare mtDNA is produced by the tripling of a 14-kb monomer with a singular rearrangement: one monomer is linear and the other two form a circular dimer. Densitometry suggested that these two molecular structures were present in different proportions within a single individual. The absence of mutations within the dimers also suggests that replication occurs during the monomer phase.

Morphological criteria and mitochondrial DNA (mtDNA) were analyzed to examine the taxonomic and systematic relationships of the endangered crayfish species Austropotamobius pallipes. Morphological data confirmed the difficulty of differentiating the 3 subspecies: A. p. pallipes, A. p. italicus, and A. p. lusitanicus. MtDNA analysis by RFLP using 6 endonucleases revealed 5 haplotypes and allowed unequivocal identification of the subspecies. The nucleotide sequence divergence among haplotypes characteristic of each subspecies ranged from 10.93-13.6% and may be explained by an ancient geographical separation of each subspecies. This distribution of genetic variation agrees with the presumption of 3 refugial regions during the glaciation periods, possibly the Iberian Peninsula, southern France, and the Balkans. UPGMA analysis from nucleotide distances placed A. p. italicus and A. p. lusitanicus in a cluster separate from A. p. pallipes, suggesting that

In animals, mitochondrial DNA (mtDNA) is generally composed of ~16 kb circular monomer molecules. However, two species of terrestrial Crustaceans Armadillidium vulgare and Porcellionides pruinosus (Isopoda: Oniscidea) are exceptions. Their mtDNA is composed of ~14 kb linear monomers associated to ~28 kb circular head-to-head dimers. In order to describe its structure, the complete mtDNA sequence of A. vulgare has been obtained. It does contain the 13 protein coding genes and the 2 ribosomal sub-units generally found in metazoan mtDNA, but not all of the 22 expected transfer RNA (tRNAs). Besides, a surprising heteroplasmy that generates a dual tRNA alloacceptor for both amino acids Alanine and Valine (tRNAAla/Val) has been discovered. This heteroplasmy by the presence of two different genes on a single mitochondrial locus is an unique example in eukaryotes. Interestingly, this heteroplasmy has been observed in a wide range of Oniscidea species carrying an atypical mtDNA. The appearance of the atypical mitochondrial genome in isopods may have permit the appearance of the tRNAAla/Val, and evolutionary forces that allow the maintenance of these two genes essential for mitochondrial translation might conserve the atypical structure of mtDNA., L'ADN mitochondrial (ADNmt) des animaux est généralement constitué de molécules circulaires monomériques de ~16 kb. Cependant, parmi les rares exceptions qui ont été décrites, deux espèces d'Oniscidea Armadillidium vulgare et Porcellionides pruinosus (Crustacés Isopodes terrestres) présentent un ADNmt atypique composé de molécules monomériques linéaires de ~14 kb associées à des dimères circulaires et palindromiques de ~28 kb. Afin de connaître plus en détail sa structure, l'ADNmt atypique d'A. vulgare a été séquencé. Il contient bien les 13 gènes codants pour des protéines et les deux sous unités ribosomales généralement présents dans l'ADNmt des Métazoaires, mais en revanche il ne présente pas l'ensemble des 22 ARN de transferts (ARNt) attendus. De plus, une étonnante hétéroplasmie générant un ARNt alloaccepteur pour les acides aminés Alanine et Valine (ARNtAla/Val) a été découverte. Cette hétéroplasmie est un exemple unique chez les Eucaryotes par la présence de deux gènes différents sur le même locus mitochondrial. De façon surprenante, cette hétéroplasmie a également été observée chez de nombreuses autres espèces d'Oniscidea qui possèdent aussi un génome mitochondrial atypique. Il semble donc que l'apparition de cet ADNmt atypique chez les Isopodes ait permis l'apparition de l'ARNtAla/Val, et que les forces évolutives permettant le maintien de ces deux gènes essentiels à la traduction mitochondriale soient impliquées dans la conservation de cette structure atypique.