Naturwissenschaften

, Volume 94, Issue 10, pp 843–846

The parthenogenetic Marmorkrebs (marbled crayfish) produces genetically uniform offspring

Authors

  • Peer Martin
    • Institut für Biologie/Vergleichende ZoologieHumboldt-Universität zu Berlin
  • Klaus Kohlmann
    • Department 5, Inland FisheriesLeibniz-Institute of Freshwater Ecology and Inland Fisheries
    • Institut für Biologie/Vergleichende ZoologieHumboldt-Universität zu Berlin
Short Communication

DOI: 10.1007/s00114-007-0260-0

Cite this article as:
Martin, P., Kohlmann, K. & Scholtz, G. Naturwissenschaften (2007) 94: 843. doi:10.1007/s00114-007-0260-0

Abstract

Genetically identical animals are very much in demand as laboratory objects because they allow conclusions about environmental and epigenetic effects on development, structures, and behavior. Furthermore, questions about the relative fitness of various genotypes can be addressed. However, genetically identical animals are relatively rare, in particular, organisms that combine a high reproduction rate and a complex organization. Based on its exclusively parthenogenetic reproduction mode, it has been suggested that the Marmorkrebs (Crustacea, Decapoda, Astacida), a recently discovered crayfish, is an excellent candidate for research addressing the aforementioned questions. However, until now, a study using molecular markers that clearly proves the genetic uniformity of the offspring has been lacking. Here, with this first molecular study, we show that this crayfish indeed produces genetically uniform clones. We tested this with 19 related individuals of various generations of a Marmorkrebs population by means of six different microsatellite markers. We found that all examined specimens were identical in their allelic composition. Furthermore, half of the analyzed loci were heterozygous. These results and the absence of meioses in previous histological studies of the ovaries lead us to conclude the Marmorkrebs propagates apomictically. Thus, a genetically uniform organism with complex morphology, development, and behavior is now available for various laboratory studies.

Keywords

MicrosatellitesApomictic reproductionClonal model organism

Introduction

The Marmorkrebs, a cambarid freshwater crayfish with unknown geographic origin and taxonomic identity, is the only parthenogenetic example of the about 10,000 decapod species known to date (Scholtz et al. 2003). Accordingly, after its emergence in the German aquarium trade in the mid-1990s, it has attracted a number of investigations regarding its anatomy, reproduction, phylogeny, ecology, and development (Vogt and Tolley 2004; Vogt et al. 2004; Seitz et al. 2005; Braband et al. 2006; Vilpoux et al. 2006; Alwes and Scholtz 2006). Nevertheless, the Marmorkrebs is still only known from aquarium populations.

In our study, we examined which of the numerous modes of parthenogenesis occurs in the Marmorkrebs, as not all of them lead to genetically identical progeny (Suomalainen et al. 1987). Because the Marmorkrebs population is exclusively composed of females, its reproduction is clearly thelytokous (Scholtz et al. 2003). There are four main types of thelytoky: apomixis, automixis, and the sperm-dependent modes gynogenesis and hybridogenesis (Simon et al. 2003). Based on its reproduction without the presence of males (Scholtz et al. 2003), we could exclude the latter two mechanisms for the Marmorkrebs. The most important difference between automixis and apomixis is the absence of meiosis in the latter. In the automictic mode, four haploid nuclei result from the meiotic divisions, and diploidy is then restored by fusion between two of these nuclei. With a few exceptions, the genetic composition of the progeny is not absolutely identical to their female parent because of recombination by crossing over during the reduction division (Fig. 1). In contrast, in apomictic systems, meiosis is completely suppressed. Thus, all offspring are true genetically identical clones of their mother (White 1973). Therefore, we analyzed whether the genetic composition of a Marmorkrebs population stayed the same in a number of specimens over several generations. For this examination, we chose microsatellite fragment length analysis because the application of this method allows a higher resolution for discriminating between individuals and more information about the allelic composition (genotype) than other DNA fingerprinting techniques such as restriction fragment length polymorphism (RFLP) analysis (Dowling et al. 1996).
https://static-content.springer.com/image/art%3A10.1007%2Fs00114-007-0260-0/MediaObjects/114_2007_260_Fig1_HTML.gif
Fig. 1

Modes of thelytokous parthenogenesis and their genetic consequences (modified from Suomalainen et al. 1987 and Simon et al. 2003). a and b are two different alleles of a gene. AA and BB represent the alleles during the meiotic chromosome pairing during the premeiotic doubling. Percentage values indicate the proportion of the genotypes within the population.

Materials and methods

Animals were obtained from the laboratory population of our department. In this population, all individuals are offspring from one single mother, which we gained in 2001. Unfortunately, this founder animal died before we started this investigation under circumstances that did not allow the extraction of DNA. Therefore, its data were not at our disposal, and so, we examined an arbitrary adult female with a daughter from summer 2003, and a second female matured in autumn 2004, including its direct offspring. The samples from 2003, one cheliped of the mother and the whole young, were stored deep-frozen, while the more recent material, a pereiopod of the adult and 16 complete juveniles were preserved in pure ethanol. Furthermore, we used muscle tissue of a Procambarus clarkii male for testing the applied methods. Genomic DNA was extracted using the E.Z.N.A.® Tissue DNA Kit (PEQLAB Biotechnologie GmbH). For microsatellite DNA amplification, we followed the slightly modified protocol described in Belfiore and May (2000) applying primers designed by the same authors for microsatellite markers of P. clarkiihttp://www.ncbi.nlm.nih.gov/Genbank). Polymerase chain reactions (PCRs) were performed in a final volume of 15-μl with 10 to 100 ng of genomic DNA, 1× (NH4)2SO4 buffer, 2.5 mM MgCl2, 0.1 mM each deoxyribonucleotide triphosphate (dNTP), 0.2 μM of each forward and reverse primer, and 0.6 U Taq DNA polymerase using a Mastercycler gradient (Eppendorf). Amplification commenced with 95°C for 2 min, followed by 35 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 1 min, and finished with a 5-min extension at 72°C. All 13 microsatellite markers of P. clarkii were tested on the Marmorkrebs, and the suitable ones were used for genotyping the crayfish samples in an automated sequencer (CEQ™ 8000 Genetic Analysis System, Beckman-Coulter). For this, we carried out the PCR amplifications with forward primers labeled on the 5′ end within the near-infrared spectral region absorbing WellRED fluorescence dyes, D2-PA, D3-PA, and D4-PA, (Proligo® France SAS).

Results

Previous tests showed that 7 of the 13 applied microsatellite markers of Procambarus clarkii were not suitable for our study. We obtained PCR products of the marbled crayfish only from the loci PclG-02, PclG-04, PclG-08, PclG-32, PclG-37, and PclG-48. The result of sizing the fragment lengths shows that all examined specimens of the Marmorkrebs were identical at all these loci (Table 1). In all, we found three homozygous genotypes in PclG-04, PclG-32, and PclG-48 and three heterozygous ones in PclG-02, PclG-08, and PclG-37.
Table 1

Tested microsatellite markers and detected identical allele associations in the analyzed individuals of the Marmorkrebs

Locus ID

GenBank® accession number

Repeat motif

Observed genotype (bp)

PclG-07

AF290922

(TCTA)8

PclG-09

AF290924

(TCTA)14

PclG-17

AF290929

(TCTA)14

PclG-27

AF290932

(TATC)4CATC(TATC)8

PclG-28

AF290933

(GATA)22(GA)5

PclG-29

AF290934

(TATC)9

PclG-45

AF290940

(CA)3...(GA)6

PclG-02

AF290919

(GATA)3GAGAA(GATA)5

267–271

PclG-04

AF290921

(TCTA)3...(TCTA)2...(TCTA)29...(TCTA)

160–160

PclG-08

AF290923

(GATA)16

190–202

PclG-32

AF290935

(CT)7...(TC)37...(CA)15...(CA)5

204–204

PclG-37

AF290939

(CA)4CG(CA)15CG(CA)13

84–86

PclG-48

AF290941

(CA)12

147–147

Locus ID is the arbitrarily given name of the region where the microsatellite marker is situated; GenBank® accession number is the number of the locus given at the entry into the database; repeat motif is the DNA sequence of the microsatellite within the locus; observed genotype shows the allele association of the locus consisting of allele fragment lengths represented by number of base pairs (bp); the minus signs stand for the absence of PCR products in case of unsuitable markers.

Discussion

Apomixis is the most widespread parthenogenetic mode among invertebrates (e.g., Simon et al. 2003; Archetti 2004), and the Marmorkrebs seems to fit well into this general picture. The occurrence of apomixis in the Marmorkrebs is strongly supported by our molecular data, as all individuals investigated show identical results in the six loci. In particular, the absence of any genetic differences over a number of generations suggests that no kind of recombination occurs during oogenesis.

The fact that 50% of the examined loci were heterozygous is considered as further indication for apomixis because in most automictic systems, homozygosity increases rapidly (Suomalainen et al. 1987; Maynard Smith 1998). Whether the high degree of heterozygosity is caused by the possibility that the Marmorkrebs resulted from hybridization between closely related bisexual ancestral species (Lokki et al. 1975; Reeder et al. 2002; Simon et al. 2003; Barton 2001) is unknown because of the lack of information about its origin so far. However, despite the relatively low number of genotyped loci, a higher degree of homozygosity would be definitely expected if there were automixis in Marmorkrebs reproduction. In addition, preliminary histological studies in the ovary of the Marmorkrebs could not prove traces of meiosis during oogenesis, which suggests further support for apomictic parthenogenesis (Vogt et al. 2004).

All this evidence together makes a strong case for apomixis in the Marmorkrebs, even if one considers potential methodological problems such as the bias towards allelic diversity based on the application of primers in species for which they are not designed (Dowling et al. 1996), or the less common cases of premeiotic doubling, a reproduction mechanism typically found in unisexual lizards, and achiasmatic central fusion in which automixis leads to genetically identical offspring as well (Cuellar 1971; White 1973; Cole 1984; Maynard Smith 1998; Archetti 2003; Lenk et al. 2005). Thus, all specimens of a Marmorkrebs population are genetically uniform, except for a few accidentally occurring mutations or the rare incidences of mitotic crossover (Lushai and Loxdale 2002; Archetti 2003; Gandolfi et al. 2003; Omilian et al. 2006), and so, this crayfish meets researchers’ demand for a genetically uniform model organism. Furthermore, the apomictic reproduction guarantees a high degree of conservation of genetic information, which in addition to its large oocytes, makes the Marmorkrebs very attractive for transgenic experiments (Nam et al. 2000). Crayfish and their relatives among the decapod crustaceans are established laboratory animals, and they show complex morphology, development, and behavior, including elaborate social interactions (e.g. Vilpoux et al. 2006; Alwes and Scholtz 2006; Sandeman and Sandeman 1991; Scholtz 1992; Drummond et al. 2002; Gherardi 2002; Krasne and Edwards 2002; Abzhanov and Kaufman 2000; Lundberg 2004). On those scores and other positive features, such as the robustness and the high reproductive rate, we are sure that the Marmorkrebs is going to become popular as a laboratory model organism in arthropod research.

Acknowledgements

We would like to thank Petra Kersten (Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Berlin) for technical support in genotyping. We are grateful to Cassandra Extavour for critical comments on the manuscript.

Copyright information

© Springer-Verlag 2007