A marbled crayfish posed by a lake. Photo: Ranja Andriantsoa / The Atlantic

By Sarah Zhang
5 February 2018
(The Atlantic) – No one knows exactly when the clones first appeared, but humans only became aware of them in the early 2000s.
It was a German aquarium owner who first brought it to scientists’ attention. In 1995, he had acquired a bag of “Texas crayfish” from an American pet trader, only to find his tank inexplicably filling up with the creatures. They were all, it turns out, clones. Sometime, somewhere, the biological rule that making baby crayfish required a mama crayfish and papa crayfish was no longer inviolate. The eggs of the hobbyist’s all-female crayfish did not need to be fertilized. They simply grew into copies of their “mother”—in a process known as parthenogenesis.Crayfish specialists were astonished. No one had seen anything like it. But the proof was before their eyes and in 2003, scientists dubbed the creatures marbled crayfish, or Marmorkreb in German.Scientists quickly realized the marbled crayfish were not just in German aquariums. The self-replicating creatures were out in the wild, and they were aggressive invaders. “Every single one has the ability to reproduce. Every single one could start a new population,” says Zen Faulkes, a crustacean researcher at the University of Texas at Rio Grande Valley who keeps a map of marbled crayfish invasions. You can easily buy marbled crayfish online (though they are now banned in the European Union and some U.S. states). The species has shown up in the wild in Germany, Italy, Slovakia, Sweden, Japan, and Madagascar. “We’re being invaded by an army of clones,” says Faulkes.For the first time, scientists have now fully sequenced the DNA of the marbled crayfish. In fact, they sequenced not one but 11 crayfish—including those originating from German pet shops as well as wild ones caught in Madagascar. The creatures are indeed clones of each other, all descended from a single crayfish that somehow gained the ability to reproduce on its own. They had remarkably little genetic diversity. At most four letters in their entire DNA sequence differed in a meaningful way.Another intriguing fact, says Frank Lyko, who led the study, is that marbled crayfish are triploid, meaning they have three sets of chromosomes. Most crayfish—and most other animals—have two sets, one inherited from the mother and the other from the father. It’s unclear, however, whether these three sets of chromosomes are the cause or consequence of its self-cloning ability. Despite having the DNA sequence in hand, “the reason and origin of parthenogenesis is still somewhat mysterious,” says Gerhard Scholtz, a zoologist at the Humboldt University of Berlin who first described the marbled crayfish in 2003. [more]

A Pet Crayfish Can Clone Itself, and It’s Spreading Around the Worlda, Representative genotyping results of 24 animals collected on four distinct collection sites. The heatmap indicates sequence similarities with the marbled crayfish reference sequence. P. fallax and P. alleni were included as controls. Cyt b, cytochrome b; SNPs, single-nucleotide polymorphisms. b, Marbled crayfish by-catch in a traditional fishing tool or 'tandroho'. c, Distribution of marbled crayfish on Madagascar (as of March 2017) in major biogeographical zones55, as indicated. Red dots indicate discovery sites where the presence of marbled crayfish was confirmed by DNA sequencing. White dots indicate sites where no marbled crayfish were found. The small central yellow circle indicates the distribution range reported for the year 2007. Graphic: Gutekunst, et al., 2018 / Nature Ecology and Evolution

5 February 2018 (DKFZ) – Some years ago, a freshwater crayfish which reproduced alone in the aquarium puzzled pet owners and scientists. This could only be explained by a phenomenon called parthenogenesis, a natural form of asexual reproduction. Now, genome sequencing and comparative studies of individual animals have shown that in fact all specimen descend from a single mother crayfish. The Marmorkrebs clone is a separate species (Procambarus virginalis), which, as genetic studies have shown, has split from the slough crayfish found in the Everglades (Procambarus fallax) some 30 years ago.In a current publication, Frank Lyko and his team from the German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ) in Heidelberg have provided proof that the all-female offspring of the marbled crayfish is genetically identical. Lyko explains: “We could detect only a few hundred variants in a genome that is larger than the human genome. That is an incredibly small number.” The minute variations can be ascribed to natural mutations. The DKFZ researchers counted 3.5 billion base pairs in the crayfish genome, which makes it about seven percent larger than the human genome.In another part of the study, a scientist in Madagascar additionally examined how well the crayfish is able to spread in the wild via parthenogenesis. Its enormous reproductive success came as a surprise to the scientists. “It was known that the crayfish can establish itself in the wild after releases from the aquarium,” Lyko said. “But the news was that it can spread so rapidly and massively.”Apart from subtropical Madagascar, Marmorkrebs can also be found, for example, in Sweden, Japan, Freiburg, Hanover, or Heidelberg. This is evidence of its remarkable adaptability –without any sexual reproduction. According to textbook knowledge, it is precisely the “invention of sex” and the resulting mix of paternal and maternal genes that enlarge genetic variety, thus facilitating rapid adaptation to adverse environmental conditions.However, although all marbled crayfish are born with the same genes, they are able to adapt to a wide variety of habitats. This is made possible by epigenetic mechanisms. These are regulated by small chemical tags attached to DNA, the genetic substance. Epigenetic mechanisms basically regulate the interpretation of genetic information. They work like switches that turn genes on or off. “Epigenetic variants are often influenced by genetic variants. In Marmorkrebs, however, epigenetic variation is independent, because there is virtually no genetic variation,” Lyko explained.This epigenetic regulation makes the marbled crayfish extremely interesting for tumor researchers. A crayfish for cancer research – what sounds like a play of words in German (“Krebs” is the word for the disease and also for ‘crayfish’), has in fact become reality. Lyko said: “Marmorkrebs is an animal that reproduces clonally and therefore represents a model of a central aspect in tumor development.” A tumor is also capable of adapting to its environment, for example, by developing resistance against anticancer drugs. Scientists have known for a couple of years, partly from studies at the DKFZ, that epigenetic mechanisms also play a key role in these processes. They can influence cancer risk and the disease course.A phenomenon called clonal evolution occurs both in Marmorkrebs and in tumors. Lyko: “Tumor genomes also evolve clonally, because they go back to a single original cell.” A variety of factors play a role in this process, such as chance mutations in genes, genetic drift, selective pressure and epigenetic adaptation to the environment. The team led by Lyko now wants to use marbled crayfish to further investigate their role. According to Julian Gutekunst, who is the first author of the present publication, the key question to be pursued is “which impact environmental factors have on epigenetics and gene regulation.” These findings may enlarge our knowledge about processes in tumors and open prospects for new approaches in tumor treatment.Julian Gutekunst, Ranja Andriantsoa, Cassandra Falckenhayn, Katharina Hanna, Wolfgang Stein, Jeanne Rasamy and Frank Lyko: “Clonal genome evolution and rapid invasive spread of the marbled crayfish”, Nature Ecology & Evolution DOI: 10.1038/s41559-018-0467-910.1038/s41559-018-Frank Lyko: “The marbled crayfish (Decapoda: Cambaridae) represents an independent new species”,
Zootaxa 2017, DOI: /10.11646/zootaxa.4363.4.638/s41559-018-0467-9

A clonal crayfish from nature as a model for tumors

ABSTRACT: The marbled crayfish Procambarus virginalis is a unique freshwater crayfish characterized by very recent speciation and parthenogenetic reproduction. Marbled crayfish also represent an emerging invasive species and have formed wild populations in diverse freshwater habitats. However, our understanding of marbled crayfish biology, evolution and invasive spread has been hampered by the lack of freshwater crayfish genome sequences. We have now established a de novo draft assembly of the marbled crayfish genome. We determined the genome size at approximately 3.5 gigabase pairs and identified >21,000 genes. Further analysis confirmed the close relationship to the genome of the slough crayfish, Procambarusfallax, and also established a triploid AA’B genotype with a high level of heterozygosity. Systematic fieldwork and genotyping demonstrated the rapid expansion of marbled crayfish on Madagascar and established the marbled crayfish as a potent invader of freshwater ecosystems. Furthermore, comparative whole-genome sequencing demonstrated the clonality of the population and their genetic identity with the oldest known stock from the German aquarium trade. Our study closes an important gap in the phylogenetic analysis of animal genomes and uncovers the unique evolutionary history of an emerging invasive species.

Clonal genome evolution and rapid invasive spread of the marbled crayfish