Parthenogenetic Marbled Crayfish Research Articles

Considerations for protein and amino acids in standardized reference diet for parthenogenetic marbled crayfish Procambarus virginalis model organism

The concept of a standardized reference diet (SRD) is used in laboratory model organisms to ensure nutritional control between studies and laboratories. Although models using the genetically identical, all female parthenogenetic marbled crayfish (Procambarus virginalis) are growing in popularity, research into nutrition in this species still has many knowledge gaps. To fast track the development of a SRD in terms of protein and amino acids (SRDprotein) for this species, we first analyzed the composition of its body amino acids to determine the ideal protein concept (IPC) of indispensable amino acids in wild-caught P. virginalis (which had an unusually high preponderance of leucine and arginine). Then, we strategically evaluated three common clusters of types of fish feed: (1) ornamental fish feed (SER) fortified with a naturally occurring alga (Spirulina). This type of feed was protein-high in arginine and leucine (SER + SPI) that fulfils the species’ IPC for iso-protein (~ 40%), iso-phosphorus (~ 0.8%) and near iso-energetic (~ 475 kcal 100 g−1); (2) freeze-dried live feed consisting of chironomid larvae (CHI) fortified with Spirulina (CHI + SPI) that fulfils the IPC for iso-protein (~ 46%), iso-phosphorus (~ 0.7%) and near iso-energetic (~ 405 kcal 100 g−1); and (3) a commercially standardized ‘starter diet’ for carnivorous fish larvae (FISH) and post-larval shrimps (SHRIMP) with iso-protein (~ 56%) and iso-phosphorus (~ 1.6%). A total of six diets, embracing a diverse range of proteinaceous feeds, were used in a 100-day ad libitum feeding and growth trial. The FISH group outperformed all the other groups (p < 0.05) and our exploratory multivariate analysis revealed an ideal demand of > 44% protein (tailored to deliver high arginine 3% and leucine 4%, followed by the usual lysine > 3.5% and methionine 1.2%) but also the lowest carbohydrate level (21%). For SRDprotein, our findings show that the FISH diet is ideal and suggest the possibilities of using a CHI + SPI diet for further optimization (more economic use of protein and phosphorus).

Considerations for fatty acids in standardized reference diet for parthenogenetic marbled crayfish Procambarus virginalis model organism

Fatty acid accumulation was studied in the parthenogenetic all-female marbled crayfish Procambarus virginalis using six arbitrarily designed experimental feeds and related to individuals with glair glands (sexual maturity) after 100 days of ad libitum feeding at 21 °C, including gravid females from the wild as a reference. Fatty acids 16:0 and 18:1n-9 comprised 40% of the total amount of fatty acids and tended to up-concentrate in bodies. Shorter chain 14:0 depleted from feed to body. Across diets, there was a concomitant decrease in precursor fatty acid and increase in product fatty acid, such as reinforcements in monounsaturated fatty acid (18:1n-9), eicosanoid precursors 20:4n-6 (arachidonic acid, ARA) and 20:5n-3 (eicosapentaenoic acid, EPA) in-vivo, but not 22:6n-3 (docosahexaenoic acid, DHA) except when deficient in CHI or CHI + SPI diets. Saturation kinetics modeling (R2 0.7–0.9, p < 0.05) showed that when the ARA share is ~ 1%, the EPA share is ~ 8%, and the DHA share is ~ 2% in the food lipids, the accumulation of fatty acids in body lipids levels off. The lowest DHA in the CHI (0% glair glands) or CHI + SPI (0–3.9% glair glands) diets, and the lowest ARA in SER (0% glair glands) or SER + SPI (0–3% glair glands) diets, were synchronous with negligible sexual maturity despite a wide range of observed specific growth rates (2.77–3.60% per day), body size (0.44–0.84 g), ≤ 5% crude lipid and 40–46% crude protein feed. The FISH and SHRIMP diets (56% protein, 11–14% lipid) with the highest ARA, EPA, and DHA together seem to be the most conducive diets for sexual maturity (up to 20% of individuals with glair glands). We propose a fatty acid profile mimicking the FISH or SHRIMP diets as a starting point for designing the lipid content required in the marbled crayfish standardized reference diet.

Assessing the invasive potential of parthenogenetic marbled crayfish Procambarus virginalis Lyko, 2017 (Decapoda, Astacidea) in the water bolies of the temperate zone of Europe

The experiment examined the effect of temperature on the parameters of reproduction and growth in female marbled crayfish. At temperatures close to 20 °C, females began to reproduce at 10–11 months with a body weight of 0.84–1.20 g, but up to 80 % of their egg clutches were nonviable. The duration of egg embryogenesis with an increase in temperature from 16–17 to 26–27 °C decreased from 66–69 days to 21–24 days with a lower temperature development threshold equal to 13.1 °C. A similar molting value that determined the growth rate of individuals in juveniles weighing up to 0.34 g was 13–14 °C, but in sexually mature females it decreased to 5 °C. In water bodies of the temperate zone with a short growing season, females were able to reach sexual maturity only in the third summer of life and produced no more than two clutches of eggs during their life cycle. Hence, the invasive potential of marbled crayfish in the temperate zone of Europe is very limited.

Temperature and hypoxia trigger developmental phenotypic plasticity of cardiorespiratory physiology and growth in the parthenogenetic marbled crayfish, Procambarus virginalis Lyko, 2017

Attempting to differentiate phenotypic variation caused by environmentally-induced alterations in gene expression from that caused by actual allelic differences can be experimentally difficult. Environmental variables must be carefully controlled and then interindividual genetic differences ruled out as sources of phenotypic variation. We investigated phenotypic variability of cardiorespiratory physiology as well as biometric traits in the parthenogenetically-reproducing marbled crayfish Procambarus virginalis Lyko, 2017, all offspring being genetically identical clones. Populations of P. virginalis were reared from eggs tank-bred at four different temperatures (16, 19, 22 and 25 °C) or two different oxygen levels (9.5 and 20 kPa). Then, at Stage 3 and 4 juvenile stages, physiological (heart rate, oxygen consumption) and morphological (carapace length, body mass) variables were measured. Heart rate and oxygen consumption measured at 23 °C showed only small effects of rearing temperature in Stage 3 juveniles, with larger effects evident in older, Stage 4 juveniles. Additionally, coefficients of variation were calculated to compare our data to previously published data on P. virginalis as well as sexually-reproducing crayfish. Comparison revealed that carapace length, body mass and heart rate (but not oxygen consumption) indeed showed lower, yet notable coefficients of variation in clonal crayfish. Yet, despite being genetically identical, significant variation in their morphology and physiology in response to different rearing conditions nonetheless occurred in marbled crayfish. This suggests that epigenetically induced phenotypic variation might play a significant role in asexual but also sexually reproducing species.

Phenotypic plasticity in the monoclonal marbled crayfish is associated with very low genetic diversity but pronounced epigenetic diversity.

Clonal organisms are particularly useful to investigate the contribution of epigenetics to phenotypic plasticity, because confounding effects of genetic variation are negligible. In the last decade, the apomictic parthenogenetic marbled crayfish, Procambarus virginalis, has been developed as a model to investigate the relationships between phenotypic plasticity and genetic and epigenetic diversity in detail. This crayfish originated about 30 years ago by autotriploidy from a single slough crayfish Procambarus fallax. As the result of human releases and active spreading, marbled crayfish has established numerous populations in very diverse habitats in 22 countries from the tropics to cold temperate regions. Studies in the laboratory and field revealed considerable plasticity in coloration, spination, morphometric parameters, growth, food preference, population structure, trophic position, and niche width. Illumina and PacBio whole-genome sequencing of marbled crayfish from representatives of 19 populations in Europe and Madagascar demonstrated extremely low genetic diversity within and among populations, indicating that the observed phenotypic diversity and ability to live in strikingly different environments are not due to adaptation by selection on genetic variation. In contrast, considerable differences were found between populations in the DNA methylation patterns of hundreds of genes, suggesting that the environmentally induced phenotypic plasticity is mediated by epigenetic mechanisms and corresponding changes in gene expression. Specific DNA methylation fingerprints persisted in local populations over successive years indicating the existence of epigenetic ecotypes, but there is presently no information as to whether these epigenetic signatures are transgenerationally inherited or established anew in each generation and whether the recorded phenotypic plasticity is adaptive or nonadaptive.

Effect of dietary supplementation with fatty acids on growth, survival, and fatty acid patterns in Procambarus clarkii and Procambarus virginalis: the first comparison of two invasive crayfish species

The red swamp crayfish, Procambarus clarkii, and the marbled crayfish, Procambarus virginalis, are invasive species expanding their ranges on a global scale. Their spread is favored by the fact that these crayfish are also aquaculture species of great economic importance (P. clarkii) or promising prospects in this regard (P. virginalis). This study represents the first attempt to assess the impact of diet supplementation with fatty acids on growth, survival, and fatty acid content in these crayfish species, as well as the first record of fatty acid patterns in the obligate parthenogenetic marbled crayfish. Our results showed that supplementation with saturated palmitic acid increased P. clarkii survival, which has not previously been reported for crustaceans. This positive effect on the part of palmitic acid supplementation was not observed for P. virginalis individuals. Marbled crayfish had significantly higher survival rates than red swamp crayfish regardless of the diet used. The growth of both species was not affected by fatty acid supplementation. Our study indicated that young marbled crayfish had a poorer fatty acid profile than juvenile conspecifics and red swamp crayfish. Fatty acid supplementation and type of diet significantly affected the fatty acid composition in the crayfish tissues. There was an increase in unsaturated α-linolenic acid for P. clarkii and palmitic acid for P. virginalis. Palmitic acid quantitatively dominated in both species regardless of the experimental treatment, with the exception of juvenile P. virginalis, which was fed an oligochaete diet rich in EPA.

Studying phenotypic variation and DNA methylation across development, ecology and evolution in the clonal marbled crayfish: a paradigm for investigating epigenotype-phenotype relationships in macro-invertebrates.

Animals can producedifferent phenotypes from the same genome duringdevelopment, environmental adaptation and evolution, which is mediated by epigenetic mechanismsincluding DNA methylation. The obligatory parthenogenetic marbled crayfish, Procambarus virginalis, whose genome and methylome are fully established, proved very suitable to study this issue in detail. Comparison between developmental stages and DNA methylation revealed low expression of Dnmt methylation and Tet demethylation enzymes from the spawned oocyte to the 256 cell embryo and considerably increased expression thereafter. The global 5-methylcytosine level was 2.78% at mid-embryonic development and decreased slightly to 2.41% in 2-year-old adults. Genetically identical clutch-mates raised in the same uniform laboratory setting showed broad variation in morphological, behavioural and life history traits and differences in DNA methylation. The invasion of diverse habitats in tropical to cold-temperate biomes in the last 20years by the marbled crayfishwas associated with the expression of significantlydifferent phenotypic traits and DNA methylation patterns, despite extremely low genetic variationon the whole genome scale, suggesting the establishment of epigenetic ecotypes. The evolution of marbled crayfish from its parent species Procambarus fallax by autotriploidy a few decades ago was accompanied by a significant increase in body size, fertility and life span, a 20% reduction of global DNA methylation and alteration of methylation in hundreds of genes, suggesting that epigenetic mechanisms were involved in speciation and fitness enhancement. The combined analysis of phenotypic traits and DNA methylation across multiple biological contexts in the laboratory and field inmarbled crayfish may serve as a blueprint for uncovering the role of epigenetic mechanisms in shaping of phenotypes in macro-invertebrates.

Insights into the genetic regulatory network underlying neurogenesis in the parthenogenetic marbled crayfish Procambarus virginalis.

Nervous system development has been intensely studied in insects (especially Drosophila melanogaster), providing detailed insights into the genetic regulatory network governing the formation and maintenance of the neural stem cells (neuroblasts) and the differentiation of their progeny. Despite notable advances over the last two decades, neurogenesis in other arthropod groups remains by comparison less well understood, hampering finer resolution of evolutionary cell type transformations and changes in the genetic regulatory network in some branches of the arthropod tree of life. Although the neurogenic cellular machinery in malacostracan crustaceans is well described morphologically, its genetic molecular characterization is pending. To address this, we established an in situ hybridization protocol for the crayfish Procambarus virginalis and studied embryonic expression patterns of a suite of key genes, encompassing three SoxB group transcription factors, two achaete-scute homologs, a Snail family member, the differentiation determinants Prospero and Brain tumor, and the neuron marker Elav. We document cell type expression patterns with notable similarities to insects and branchiopod crustaceans, lending further support to the homology of hexapod-crustacean neuroblasts and their cell lineages. Remarkably, in the crayfish head region, cell emigration from the neuroectoderm coupled with gene expression data points to a neuroblast-independent initial phase of brain neurogenesis. Further, SoxB group expression patterns suggest an involvement of Dichaete in segmentation, in concordance with insects. Our target gene set is a promising starting point for further embryonic studies, as well as for the molecular genetic characterization of subregions and cell types in the neurogenic systems in the adult crayfish brain.

The invasive parthenogenetic marbled crayfish Procambarus virginalis Lyko, 2017 gets foothold in Belgium

The invasive parthenogenetic marbled crayfish Procambarus virginalis Lyko, 2017 gets foothold in Belgium

Noble Crayfish Are More Sensitive to Terbuthylazine than Parthenogenetic Marbled Crayfish

We investigated the sensitivity of two freshwater crayfish species (Astacus astacus and Procambarus virginalis) during embryonic development to chronic exposure to the herbicide terbuthylazine under laboratory conditions. The assessed parameters included time of embryonic development, survival rate, hatching weight and histopathology of hepatopancreas. LC50 (median lethal concentration) and ED50 (median effective concentration) were estimated. We were able to determine effects of terbuthylazine for every investigated parameter. For noble crayfish, the LC50 value after 45 days was 0.11 mg/L, and the histology of the hepatopancreas showed effects starting from 0.025 mg/L. Other parameters revealed effects starting at concentrations of 1.6 mg/L for weight and 6.4 mg /L for embryonic development time and hatching rate. Marbled crayfish only showed effects concerning the hatching rate and survival rate at concentrations without a clear dose-effects curve. As a conclusion, our data shows the risk of terbuthylazine in existing concentrations in freshwater ecosystems to non-target organisms and also the need of toxicological studies on directly affected species in addition to the use of model organisms.

Evaluation of the suitability of the parthenogenetic marbled crayfish for aquaculture: potential benefits versus conservation concerns

The parthenogenetic marbled crayfish, Procambarus virginalis, is currently being discussed as a promising new candidate for aquaculture that could supply people in developing countries with high-quality protein and income. The main advantage of marbled crayfish is parthenogenetic reproduction. Comparison of growth between marbled crayfish and red swamp crayfish, Procambarus clarkii, the leading species in crayfish aquaculture revealed inferior body size and considerably slower growth in marbled crayfish. Only a very small proportion of the production would meet the size requirement of the international market and could serve as a cash crop. Aquaculture for local markets in extensive outdoor systems is probably economically feasible in developing countries, but the highly invasive and competitive marbled crayfish could easily escape from such sites, invade natural ecosystems and impair the autochthonous fauna and flora. Culture in closed indoor systems would be a safe alternative but this cost-intensive approach is economically not profitable. Because of small body size and slow growth, conservation concerns, and economic reasons, the marbled crayfish is considered unsuitable for aquaculture. It should not be spread around the globe for aquaculture and sustainable fisheries as was earlier done with the congeneric Procambarus clarkii, resulting in devastating ecological effects in numerous countries.

Trophic ecology of invasive marbled and spiny-cheek crayfish populations

North American cambarid crayfish have been highly successful in establishing and spreading across Europe and are now over-invading earlier arrivals in many water bodies. Parthenogenetic marbled crayfish (Procambarus virginalis), which originated from aquarium stocks, are relatively recent invaders and have established in lakes previously invaded by spiny-cheek crayfish (Faxonius limosus). However, the feeding ecology of marbled crayfish and consequential impacts on the non-native species’ coexistence are largely unexplored. By combining laboratory experiments with stable isotope analyses of field samples, we were able to (1) determine food preferences of both species under controlled conditions and (2) explore their trophic niches in three lakes where both species co-occur. In the food-choice laboratory experiments, the two species showed similar food preferences and consumption rates. Consistently, the stable isotope analyses (δ13C and δ15N) highlighted the intermediate trophic position of both species. Marbled crayfish and spiny-cheek crayfish occupied a wide range of trophic positions corresponding to a very generalist diet. However, marbled crayfish were more relying on arthropod prey than spiny-cheek crayfish which fed more on mollusks. This is the first work providing evidence for trophic plasticity of marbled crayfish in lake food webs. Our results suggest that the addition of marbled crayfish increases grazing pressure on macrophytes and macrophyte-dependent organisms and the allochthonous detritus decomposition in ecosystems already invaded by spiny-cheek crayfish. Since both species are listed as invasive alien species of EU concern, further assessments of potentially endangered food organisms are needed.

How should we treat autopolyploid and parthenogenetic animals taxonomically?

Asexually reproducing and polyploid animals are unresolved problems of taxonomy and nomenclature. The present paper discusses this issue from a theoretical perspective and outlines how the problem was treated in the past. The autotriploid, obligately parthenogenetic marbled crayfish is used as an example to elaborate the conditions under which autopolyploids and parthenogens should be described as separate uniparental species.

Culturing embryonic cells from the parthenogenetic clonal marble crayfish (Marmorkrebs) Procambarus virginalis Lyko, 2017 (Decapoda: Astacidea: Cambaridae)

AbstractThe parthenogenetic marbled crayfish, or Marmorkrebs (Procambarus virginalis Lyko 2017), is an emerging model organism. We describe a method to isolate cells from early-stage embryos and culture them in vitro. The identity of the cells was confirmed by sequencing the cytochrome c oxidase subunit I (COI) gene. This technique can be applied for use in the manipulation of embryonic parthenogenetic crayfish cells.

Potential of marbled crayfish Procambarus virginalis to supplant invasive Faxonius immunis

Biological invasions are a growing threat to global biodiversity due to negative impacts on native biota and ecosystem functioning. Research has expanded from investigating native and alien species interactions to examining relationships among alien species. Invasive crayfish may have similar life histories, niche preferences, and adaptation strategies, but their mutual interactions are little understood. This study aimed to quantify interaction patterns of size-matched calico crayfish Faxonius immunis, established in the Rhine River catchment, and the parthenogenetic marbled crayfish Procambarus virginalis, currently spreading throughout Europe. During agonistic interactions in the absence of shelter, marbled crayfish won a significant majority of fights against calico crayfish, but in the presence of shelter there was no significant difference. When sex of calico crayfish was considered in the analysis without shelter, marbled crayfish won a significantly higher number of fights with female calico crayfish. In the absence of shelter, marbled crayfish dominated calico crayfish females in 83.3% and males in 60% of pairs. With available shelter, the dominance of marbled crayfish was 100% and 54.5% over female and male calico crayfish, respectively. The results suggested that sex and resource availability influence agonistic behaviour in the studied crayfish. Marbled crayfish are confirmed to be competitive against the calico crayfish, which has been shown to be dominant over another serious invader in the Rhine River catchment, the spiny-cheek crayfish Faxonius limosus. In natural sympatric populations, the situation may be affected by factors such as size, reproductive variables, water temperature, and predation pressure.

Morphometry, size at maturity, and fecundity of marbled crayfish (Procambarus virginalis)

Morphometric ratios, relationships and condition factors provide information regarding growth patterns, population dynamics and well-being of individuals in particular environments. Ecology of any species is then better understandable through life history traits and reproductive potential. The present study is aimed at exploring the morphometric and reproductive patterns of the parthenogenetic marbled crayfish, Procambarus virginalis. Marbled crayfish exhibited positive allometric growth and statistically significant common length–weight relationships. Abdomen width, claw length, width, and height increase significantly faster than the chosen most robust body size parameter, postorbital carapace length (POCL). Conversely, total length, carapace length, carapace width, and abdomen length grow proportionally slower than POCL. The condition factor was higher than the threshold value of ∼1 indicating the well-being of the crayfish used. Marbled crayfish, at their first reproduction, were recorded as long as 42.8 ± 10.8 (31.5–73.5) mm total length with a mean fecundity of 89.72 ± 56.9 (22–349) eggs. The relative fecundity (per 1 mm of POCL) of females reproducing for the first time was significantly lower compared to those reproducing repeatedly. Fecundity and relative fecundity exhibited a significant linear relationship with the carapace length and weight of mothers. As a tool for total fecundity estimation, the number of eggs on the third pair of pleopods was counted and plotted against size of mothers, and total fecundity. The number of eggs attached to the third pair of pleopods very closely correlates with the carapace length, weight and total fecundity. Based on our results, it can be concluded that abdomen width and claw growth is allometric compared to POCL which confirms the importance of these body parts. Fecundity parameters were confirmed to have a tight relationship to marbled crayfish size. Clutch size estimation can be used in future research based on the close correlation of egg counts on third pleopods and total fecundity.

Still waters run deep: marbled crayfish dominates over red swamp crayfish in agonistic interactions

Intra- and interspecific interactions contribute to the successful establishment and consequent spreading of species in the environment, which became particularly apparent in the context of ongoing biological invasions. The parthenogenetic marbled crayfish, Procambarus virginalis, Lyko 2017 is recently recognized as an emerging invader due to its high adaptability, fast growth, early maturation, and high fecundity. The present study explored the interaction patterns of size-matched (including 15 body parts morphometry evaluation) pairs of marbled crayfish and red swamp crayfish Procambarus clarkii, a well-known highly aggressive and widely distributed invader of freshwater ecosystems. Despite this, marbled crayfish won significantly more fights and establish dominancy in more cases in both premature and mature experimental trials. Premature red swamp crayfish pairs were more active in contact and fight initiation than mature. In mature, the dominance over female red swamp crayfish was 100%, in males it reached 60%. Premature marbled crayfish dominated in more than 75% pairs. Agonistic behaviour and intensity of fights significantly dropped after establishment of dominance in particular (size and sex) pairs. Therefore, we confirmed that sex and age (size) have effects on agonistic behaviour in crayfish as well as the dominance of marbled crayfish within similarly sized specimens. Despite described behavioural patterns, we can expect that the situation in the potential sympatric occurrence of both species will not be as clear as found in experimental conditions due to greater maximal size of red swamp crayfish.

Characterization of locomotor response to psychostimulants in the parthenogenetic marbled crayfish (Procambarus fallax forma virginalis): A promising model for studying the neural and molecular mechanisms of drug addiction

Although scientific research using mammalian models has made great strides in uncovering the enigmatic neural and molecular mechanisms orchestrating the state of drug addiction, a complete understanding has thus far eluded researchers. The complexity of the task has led to the use of invertebrate model systems to complement the research of drug-induced reward in mammalian systems. Invertebrates, such as crayfish, offer excellent model systems to help reveal the underlying mechanisms of drug addiction as they retain the ancestral neural reward circuit that is evolutionarily conserved across taxa, and they possess relatively few, large neurons, laid out in an accessible, modularly organized nervous system. Crayfish offer the benefits of delineated developmental life stages, a large body size suitable for a variety of experimental methods, and stereotyped behaviors. Unique among crayfish is the parthenogenetic marbled crayfish (Procambarus fallax forma virginalis), a species of asexually reproducing, genetically identical clones. With the benefits of reduced individual variation, high fecundity, and easy lab husbandry, the marbled crayfish would make a particularly powerful addition to the animal model repertoire. Here we characterize the locomotor response of juvenile P. f. f. virginalis exposed to the psychostimulant, d-amphetamine sulfate. Custom video-tracking software was used to record the movement patterns of juveniles exposed to water infused with varying concentrations of d-amphetamine sulfate. ANOVA demonstrated that crayfish locomotion was significantly impacted by drug concentration. These psychostimulant effects provide the foundation of P. f. f. virginalis as a model for parsing the neural and molecular mechanisms of drug addiction.

Glair glands and spawning in unmated crayfish: a comparison between gonochoristic slough crayfish and parthenogenetic marbled crayfish

Glair glands and spawning in unmated crayfish: a comparison between gonochoristic slough crayfish and parthenogenetic marbled crayfish

Morphological characterization and genotyping of the marbled crayfish and new evidence on its origin.

The obligately parthenogenetic marbled crayfish, Procambarus virginalis, is the first formally described asexual species of the Crustacea Decapoda. It is a triploid descendant of the sexually reproducing slough crayfish, Procambarus fallax. Here we describe the morphology of cultured and wild marbled crayfish of wide size ranges in detail and photodocument all taxonomically relevant characters. Some morphological traits and coloration showed considerable variation within populations despite the monoclonal nature of marbled crayfish. There were also significant differences between wild and laboratory populations with respect to body proportions, coloration and spination. Comparison with Procambarus fallax revealed no qualitative morphological characters that unambiguously identify the marbled crayfish. Analysis of the mitochondrial cytochrome c oxidase subunit I gene (COI) and nuclear microsatellites of marbled crayfish and Procambarus fallax from different sources indicated that the tri-allelic microsatellite PclG-02 is better suitable than COI to identify the marbled crayfish. A respective identification key is provided. The COI and microsatellites of Procambarus fallax from different areas of Florida and southern Georgia suggest that the parents of the first marbled crayfish may have come from northern Union County, northern Florida.