Threadworms are versatile research objects and are excellent models for investigating fundamental evolutionary principles. Researchers at the Max Planck Institute (MPI) for Developmental Biology use the Caenorhabditis and Pristionchus threadworm genera to study the molecular mechanisms of biodiversity.
Prof. Dr. Ralf Sommer, director of the MPI in Tübingen, was the first to describe the worm Pristionchus pacificus during his postdoctoral studies at Caltech, the Californian Institute of Technology, in 1996. The worm proved to be a lucky find for research, in particular for the genetic and molecular analysis of the evolution of developmental processes. The classic threadworm (nematode) used for laboratory examinations is the Caenorhabditis elegans. This nematode has become so popular as it only consists of 959 cells, of which the developmental fate is largely unvaried between individuals and can easily be investigated under the microscope. “It turned out that the development of P. pacificus differs considerably from that of C. elegans. This makes the two species excellent candidates for comparative analyses,” said Sommer.
Moreover, P. pacificus can be handled in the laboratory like C. elegans. P. pacificus has a relatively short life cycle of three days and can be cultivated at 20 °C in Petri dishes, using E. coli as food. However, according to Sommer this is not what makes the nematode a popular laboratory organism. “Many nematodes can be cultivated easily, but in the case of Pristionchus, the advantage is that the cultures yield high amounts of proteins and nucleic acids for use in biochemistry and genomics. Up until now, only a handful of other threadworms are known to have this advantage,” said Sommer.
Comparative investigations as the key to understanding
C. elegans and P. pacifus had the last common predecessor approximately 200 to 300 million years ago. Looking into the evolution of the nematodes and investigating the mechanism of their differentiation is a thrilling experience. Sommer commenced his comparative analyses on the nematodes’ egg-laying apparatus (vulva). He found homologous structures in the two species, but also found that they differed at the level of the molecular mechanisms underlying the development of the vulva. In C. elegans, vulva formation is induced by signals from a single specialised cell (anchor cells), while in P. pacificus other cells and signalling processes are involved in the induction of vulva formation.
In an early stage of its development, C. elegans has six cells that are capable of forming a vulva. However, only three cells are needed to do so. Following the signal from the anchor cell, these cells start to divide and differentiate into vulva tissue. The other three vulva precursor cells act as reserves and step in if the first three become nonfunctional, i.e. have been removed during an experiment. P. pacificus does not possess the three extra cells. Programmed cell death leads to a reduction in the number of vulva-forming cells early during the animal’s development. Sommer and his team decided to look for genes that induced apoptosis and in the process they found the gene Ppa-hairy.
The egg-laying apparatus as model organ
“It had not occurred to anybody to consider this gene since nobody knew that P. pacificus possessed genes that C. elegans did not have. Today we know that this is the case,” said Sommer. The comparative analyses eventually showed that the two species had homologous genes at the same time as having genes that were completely different from each other or still others that had overlapping functions. The scientists assumed that the comparison of expression patterns, which reflect the total quantity of gene products, would provide them with two completely different pictures. However, the contrary was true. The researchers found that the expression patterns of the two nematodes were very similar.
Sommer says that whilst this is difficult to explain he already has an idea as to why this should be so. “The key to all this is redundancy. In the two species, the pathways – i.e. molecular signalling chains – are full of redundancies. It has long been speculated that redundancies are the drivers of evolution. “The trick is the following: On the one hand, different genes or signalling chains can have redundant functions, i.e. lead to the same result. On the other hand, the same signalling chains can have different, or even opposite, effects. For example, a signalling chain in C. elegans might drive vulva formation, whereas the same chain impedes vulva formation in P. pacificus. Sommer is now hoping to clarify whether the signalling chains are highly conserved. And if it should turn out that they are highly conserved the next question to be answered relates to the development of biological form versatility. What governs biodiversity? Selection mechanisms or pure chance?
Evolutionary processes are associated with redundancies
With his work on these key evolutionary questions, Sommer is dealing with a highly explosive field in scientific terms, in which two groups are fighting for recognition. The one group consists of selectionists, whilst the other group is made up of neutralists who support the principle of chance. Sommer is extremely reticent to take part in this controversy and does not want to make an either/or decision. “It will most likely take another 20 years before a decision can be reached. I think it is a mistake to think in terms of stereotypes. It is clear that there is selection, but it is also clear that there are many processes that develop without selection,” said Sommer.
The habitat of Pristionchus also raises interesting questions on evolutionary adaptation. In the process of their search the scientists initially found wild-type P. pacificus strains and new Pristionchus species in soil samples. Then they discovered a more abundant source: decaying dead beetles. The researchers found that threadworms survive in the living beetle as inactive permanent forms. Following the death of the beetle, the nematodes become fairly active, feeding and proliferating on the bacteria that grow when the beetle decays. When choosing a mate, most Pristionchus species are very selective; some of them only live on one particular beetle species.
A specific nematode for specific beetles
But how do the nematodes find “their” beetle. Certainly not by using the classical sensory organs, because nematodes have neither ears, nor hearing or common olfactory organs. Chemotactic experiments provide an answer. “We have investigated how beetles find their partners using P. maupasi, a nematode species specialised in May beetles. We washed the beetles with dichloromethane and offered them this solution on a Petri dish that was placed nearby. The beetles moved towards the washing solution, but did not react to pure dichloromethane. We identified phenol as an active component, i.e. sexual attractant of May beetles,” said Sommer who has in the meantime identified the specific chemical attractants of several Pristionchus species.
Sommer is now hoping to elucidate the molecular mechanisms that control the nematode-beetle relationship and why and how they developed during evolution. It still remains to be clarified whether the relationship is parasitic, mutualistic or symbiotic. “We also need to have a closer look at beetle larvae. It is possible that the more effective immune system of adult beetles keeps the nematode population low or inactive,” speculates Sommer, who plans to further investigate the development and evolution of nematodes using genetic screening.