Search

Any initiative aimed at the management of a threatened species needs a good knowledge of its environmental requirements. Aims of this study were to individuate suitable areas for the reintroduction of crayfish species belonging to the Austropotamobius pallipes complex and to evaluate the causes of the local extinction in Tuscany (Italy) of some populations. Between May 2003 and September 2004, we collected ecological data from 19 streams of 4 catchments, 9 watercourses where crayfish were present (WI) and 10 where they were present in the recent past and absent now (WO), and we compared them. Multivariate analyses were performed using chemico-physical and biotic parameters to examine the relationships between their values and the presence of crayfish. The results did not allow us to find significant differences between the two categories of streams, suggesting their suitability for crayfish reintroduction.

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.

This paper summarizes the various themes discussed in a roundtable session on the uses and usefulness of genetics for conservation of our European crayfish heritage. After reviewing the importance of characterizing the different genetic stocks (ESU: Evolutionary Significant Units) of a species, where morphological criteria used for taxonomy, are utilizable with caution, the present state of knowledge of the phylogeny of European species was determined, in particular for the genus Austropotamobius which occupies most studies. There was a lively debate on the elevation to species rank of A. italicus. The status of knowledge of the taxonomy of Astacus astacus has been presented. Finally, given a major increase in works describing the genetic variability of natural populations of European crayfish, it was evident that an up to date inventory of the different research teams in this field should be developed, for several reasons – (i) to describe these teams and their managers (thus facilitating contact) (ii) to list the species studied and the markers used (iii) to give some recommendations on standardizing the use of certain markers or genes so as to allow comparison between the results collected by the various teams, and to apply them on a broader geographic scale.

In France, the distribution of the white-clawed crayfish, Austropotamobius pallipes (Lereboullet, 1858), is restricted, fragmented and mainly located in headwaters. To preserve this indigenous species, it is necessary to characterize its ecological requirements (water and habitat quality). With this aim in view, a two-year study is being conducted in the Deux-Sèvres department (Western France) since November 2002. Nine brooks from four different catchments are monitored regularly; eight of the nine brooks harbour whiteclawed crayfish populations. Two sampling sites are surveyed per brook, the first being where the crayfish population is located and the second 2 to 3 km downstream. Physicochemical parameters (18) are measured twice monthly and biotic factors are estimated twice yearly. In this study, the I.B.G.N. (Indice Biologique Global Normalisé) protocol based on the determination of macroinvertebrates was used as a biotic index of biological water quality. Results of this preliminary study on two brooks (Thouet and Verdonnière) show that physico-chemical and biological data considered separately do not provide reliable information about A. pallipes ecological requirements. However, the use of multivariate analyses (Principal Component Analysis) to combine abiotic and biotic factors highlights a good correlation between these parameters. Organic matter appears to be a better discriminating factor than mineral matter affecting presence or absence of the whiteclawed crayfish.

In the past, conservation programmes for a given species consisted in restoring the habitat and in translocating individuals without knowledge of their taxonomic status. Even if managers wanted to be informed, the “traditional taxonomy”, based on morphological characters, was discouraging because it could indicate several types of classification. This is the case for Austropotamobius pallipes, considered as a species complex. Today, conservation genetics aims to maintain, on one hand, the genetic specificity of populations (genetic integrity principle) and, on the other hand, the genetic diversity within and between populations (biodiversity principle), these basic principles being considered both at the level of protection measures and management measures. As an endangered species, A. pallipes is subjected to a loss of genetic diversity, a result of deterioration of water quality responsible for habitat fragmentation, with populations being confined to headwaters of the catchments. Consequently a certain degree of genetic variability must be maintained within the species because it governs the adaptation potential: the populations must be capable of responding to new environmental conditions. In A. pallipes, recent studies from several countries attempt to first describe the distribution of the present natural populations and secondly, by studying mitochondrial DNA, to clarify the taxonomy (number and identification of the present species and subspecies by phylogenetic inferences) and to assess the biogeographical history. These two preliminary steps are fundamental before defining conservation units and working at the catchment level, using highly polymorphic nuclear markers. The new approach has provided a good framework for research, with more frequent dialogues between geneticists and managers.

A survey of the macroinvertebrates of three brooks harbouring the white-clawed crayfish was conducted in Haute-Vienne department (France). Its aim was to increase our understanding of these ecosystems to help the conservation of A. pallipes. These brooks run through pastoral areas with well-developed riparian vegetation, which offers an important shade. Water temperature, dissolved oxygen concentration, pH, and conductivity fell within the ranges found for this species. A total of 34, 31, 29 taxa and 1 502, 1 364, 2 707 individuals of macrobenthos were collected in Holme, Besque and Bellecombe streams, respectively. Results showed good to very good water quality with IBGN scores ranging from 15 (Bellecombe) to 17 (Holme and Besque), reflecting a limited impact of the anthropogenic disturbances. Taxa diversity were high for Holme and Besque with Shannon index around 3.2, translating a great heterogeneity of habitat and an equilibrated faunal community. Bellecombe showed a limited diversity with Shannon index of 1.42, resulting from the presence of numberous Chironomidae. This brook suffers probably weak organic pollution which could be related to the low water flow observed during the sampling. The similarity test according to Jaccard index showed high percentage of common taxa among ETP (Ephemeroptera-Trichoptera-Plecoptera) between all sites. The high similarity of benthic macroinvertebrate communities could be an useful criteria to identify brooks for restocking purpose.

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.

The phylogenetic relationships of the genus Austropotamobius were reviewed by the compilation of two recent genetic studies based on the partial nucleotide sequence of the mitochondrial RNA 16S gene. The results showed a well-resolved phylogeny revealing four distinct clades: A1, A2, A3 and B, supported by high bootstrap values. The clades A (including A1, A2, A3) and B are separated by a high genetic divergence (5%). In accordance with morphological and nuclear data, two species could be defined: A. italicus and A. pallipes, respectively. The average of genetic divergence within the major group A and B was 2.1% ± 1.2 and 0.9% ± 0.6 respectively. Three clades A1, A2 and A3 correspond mainly to crayfish sampled from Austria-Switzerland, South of Balkans and Italy-Spain respectively. On the basis of morphological, genetic and distribution data, a new systematic - based on two species A. pallipes and A. italicus with 3 subspecies A. i. carinthiacus, A. i. carsicus and A. i. italicus - could be proposed for the white-clawed crayfish species complex. We rejected the specific status of A. berndhauseri and A. i. lusitanicus given to the Swiss and Spanish crayfish respectively.

A protected population of the white-clawed crayfish, Austropotamobius pallipes pallipes (Lereboullet), has been studied between 1995 and 1996. 1,511 crayfish were collected over the period of study including trap and hand catches. 356 animals exceeded the minimum legal size -90 mm TL (Total Length)-, representing 23.6% of the total catches. Catch Per Unit Effort (C.P.U.E.) ranged from 1.2 in February to 8.6 in October. The sex ratio of total catch was biased in favour of females (1:l.g). Males are only dominant during the cold period where water temperature is less than 10°C. Thelohania seems to be cyclic in this population with high levels of infected crayfish during both summers. No infected crayfish were found between January and March. Estimated densities ranged from 4.4 to 2.2 adults.m2 of brook area. Mating activity started in mid to end November as day-length shortened and water temperature fell below 10°C. Berried females ranged from 58.5 to 100.4 mm in TL. Mean pleopodal egg number declined over the duration of the incubation period (mean = 55 eggs per female in December to 30 eggs per female in May). The maximum number of eggs was 85 for a female (85 mm TL) captured in November. In the beginning of May, 40% of females sampled had no eggs. All the results seem to show an over-density of crayfish in this population and recommendations to regulate it are discussed.

Mitochondrial DNA (mtDNA) variation in the white-clawed crayfish, Austropotamobius pallipes pallipes, was examined by restriction endonuclease analysis of samples obtained from three geographical locations representing two very différent habitats : the Crochatiere and the Magot for brooks and the Pinail for ponds. This study used a new type of molecular marker in crayfish. The method of mtDNA extraction was not based on the clear lysate method or on ultracentrifugation and used no end-labelling détection. It is discussed according to the literature about marine crustaceans. MtDNA was digested with 6 endonucleases. The molécule size of the mtDNA of the white-clawed crayfish was approximately 17750 ± 580 base pairs. Two restriction enzymes produced polymorphic digestion patterns, defining a total of three haplotypes (1-3). There was a shared haplotype 2 between individuals among the two brooks. The haplotype 3 was only found in individuals obtained from ponds of Pinail.

A mass mortality was detected in the downstream section of one of the most extensive French populations of the endangered white-clawed crayfish, Austropotamobius pallipes, on June 26, 2013. This population occupied a 12 km stretch of the La Lucelle brook, with an estimated size of around 150 000 individuals. The presence of the crayfish plague pathogen was quickly diagnosed as the cause of the mortality, and monitoring was carried out to follow the spread of the disease from 15 July 2013 for one year. Results showed that after a fast spread between 15 and 25 July 2013 (upstream progression of mortality for about 4 km), the mortality front was limited to a stretch of a few hundred meters from August until December 2013. During winter, mortality was always observed, confirming that disease remained active. In April 2014, the mortality front was halted by a large dam in the brook (2 m high), 0.56 km from brook source. Two months later, 30 live crayfish were observed above the dam. On the 30 August 2014, no crayfish were found above the dam. Infected individuals analysed for microsatellite markers confirmed the Pacifastacus leniusculus strain of Aphanomyces astaci at the origin of this outbreak. Before the crayfish plague spread upstream of the large dam, a sample of 576 individuals was collected from upstream of the dam and translocated to another stream in the same French department. In July 2014, observations by night confirmed the presence of translocated white-clawed crayfish in the receiving brook.

Several alien crayfish of North American origin have become established in Europe in recent decades, but their identification is often confusing. Our aim was to verify the taxonomic status of their European populations by DNA barcoding. We sequenced the cytochrome c oxidase subunit I (COI) gene fragment of individuals representing all American crayfish known from European waters, and compared the results with reference sequences from North America. Our results confirm the morphological identification of Orconectes juvenilis from a population in eastern France, and of the marbled crayfish (Marmorkrebs), i.e., a parthenogenetic form of Procambarus fallax, from south-western Germany. Sequences of most individuals of presumed Procambarus acutus from the Netherlands were similar to American P. cf. acutus, but one was divergent, closer to a sequence of a reference individual of P. cf. zonangulus. However, divergences among three American P. cf. zonangulus samples were also high, comparable to interspecific variation within cambarid species complexes. The divergence between O. immunis from Europe and America also reached values corresponding to those observed among distinct Orconectes species. Genetic variation in the American range of these crayfish should therefore be further studied. Our study shows that DNA barcoding is useful for the rapid and accurate identification of exotic crayfish in Europe, and also provides insights into overall variation within these taxa.

The redclaw crayfish, Cherax quadricarinatus, was introduced to Martinique Island for aquaculture purposes in 2004, in an attempt to revitalize the freshwater crustacean aquaculture sector. In 2015, three wild populations were discovered during an electrofishing survey on fish diversity. In 2018, a specific crayfish survey was performed at night using spotlighting and baited traps at 34 sites throughout the island. The species was mostly found in the center and northern part of the island, specifically, a total of 105 specimens were captured in eight streams and five closed water bodies. We sequenced a 491 base-pair fragment of the COI gene to understand the invasion history and pathway from the presumed source population at the Mangatal hatchery. Among the eight haplotypes found, three were dominant, of which, two occurred in the Mangatal hatchery. As crayfish are sold alive, there is a high risk of further human-mediated introductions across the island hydrographic basins. Thus, the distribution of this species could rapidly expand throughout Martinique freshwater ecosystems, with ecological impacts on native communities yet to be determined and requiring urgent investigation.

The Eurasian minnows of the genus Phoxinus are small cyprinid species, widely distributed across Europe and Asia. Currently, there are at least 15 species in Phoxinus, with preliminary data suggesting more to be described. Despite the discernible increase in research integrating molecular phylogenetic approaches with traditional taxonomy and systematics, inter- and intraspecific relationships in the genus Phoxinus are still poorly known. The aims of this research were to: (i) compile data on the distribution of Phoxinus species in Croatia and Bosnia and Herzegovina from the sampling conducted by authors from 2006 to 2016 and from literature sources, and (ii) provide a detailed insight on distribution ranges of genetic clades and species of Phoxinus in the western Balkans. Additional 118 localities in Croatia and 8 locations in Bosnia and Herzegovina were added to already known 160 localities from the literature data. Molecular analyses of mitochondrial DNA indicate: (i) the presence of Phoxinus marsilii in Croatia (Drava drainage, the Papuk Mountain), around 260 km south of its known distribution range in Hungary, and (ii) an exceptional genetic variability of P. lumaireul in the Western Balkans.