By Bjarne Røsjø
Oslo, Norway.- A tiny carp that can only be found in the waters in peat swamp forests in Indonesia and Malaysia, is a peculiar creature. The world’s smallest fish has a very small and strange genome, and it lives like a larva throughout its whole life. In addition, this microfish lives in water that is as acidic as Cola or Pepsi, with the same colour as tea.
The tiny carp Paedocypris progenetica was quickly identified as the world’s smallest vertebrate when it was discovered in 2006: The maximum size of an adult fish is only 8 millimetres. The species retained this “world’s smallest” status until American researchers in 2014 discovered a frog that was even smaller – only 7 mm. But P. progenetica is still the world’s smallest fish, and certainly one of the strangest.
Researchers at the University of Oslo (UiO) and cooperating institutions now have investigated the genes of two closely related species, living in the same watery environment in Southeast Asia’s peat swamp forests. The two carp species P. carbunculus and P. micromegethes are perhaps a few millimetres larger than the minor in the genus, but they are equally strange.
“Before we started our research, we knew that these fish never grow up to be adults in the usual sense. As biologists, we say that their bodies are truncated. Instead of evolving into adults, their development stops in a way that makes them look like larvae throughout their whole lives”, says researcher Martin Malmstrøm.
“We have discovered that these fish are not strange only in their appearance, but they also have a very strange genome”, adds postdoctoral fellow Ole Kristian Tørresen at the UiO’s Department of Biosciences. The scientific paper summarizing their new findings was presented in April on the front page of the journal Genome Biology and Evolution.
A fishy Peter Pan
The small carp in the genus Paedocypris – the Latin name translates roughly into “baby carp” – makes you think about Peter Pan: The boy who refuses to grow up. Petrolheads might instead draw comparisons between a normal car and a go-cart: the latter performs perfectly fine in it environment, even if it has no roof, doors, windows, bonnet, luggage compartment etc.
The baby carp are similarly lightened versions of their larger relatives, like the common carp. Both their genes and their body functions are reduced to a minimum of what is necessary to survive. The two Paedocypris-species in question are tiny, remain at the larval stage for a lifetime, have a skull consisting of cartilage instead of bone tissue, and about 40 of their skeletal bones are never properly developed.
Acidic like Coke, coloured like tea
The baby carp have never been found outside the peat swamp forests that are indigenous to parts of Indonesia and Malaysia. The climate in these forests is so moist that the soil is constantly saturated with water.
“In these forests, the ground is covered with up to ten meters thick layers of flooded twigs and branches, which decompose very slowly and eventually develop into thick layers of acidic peat. The water contains only small amounts of oxygen, lime and nutrition. In addition, the water is as acidic as Coke or Pepsi – with a pH around 2.5 – and the water has the same colour as tea. You wouldn’t think that this is an environment suitable for fish”, says Malmstrøm.
That’s probably why the famous American evolutionary biologist Daniel H. Janzen, after a visit in 1974, said that it was a waste of time to look for wildlife in these waters. The statement has later turned out to be one of his few mistakes, because we now know that there are between 200 and 300 species of fish there. Approximately one hundred of these species are only found in the acidic waters.
Their immune system is normal, but that’s it
Martin Malmstrøm became interested in the baby carp after discovering, together with Professor Kjetill S. Jakobsen and other researchers at the UiOs Department of Biosciences, that the immune system of the Atlantic cod (Gadus morhua) lacks the part that attacks intrusive viruses and bacteria with antibodies. Humans without the same vital part, which is called MHC II, would run the risk of dying from a tiny scratch. But the cod is doing just fine.
“Our first idea was that Paedocypris lives in water that is too acidic for bacteria to survive there, so that probably meant that the fish could make do with a simplified immune system, like the cod. But no! When we took a closer look, we found that the baby carp’s immune system is almost embarrassingly normal”, Malmstrøm explains.
Luckily, Malmstrøm, Tørresen and their partners found a lot of other strange things, when they studied the tiny carp’s DNA. It turned out that these fish have very small genomes, without important structures that are found in other fish and vertebrates.
“The most important finding is that these carp have lost a lot – approximately 20 percent – of what is called Hox genes. This is a group of genes that have an overall control function in the embryo”, explains Malmstrøm.
The Hox genes are spread along the DNA strands in a defined order, and this order governs the order in which the different body parts are developed in the embryo.
“Attempts have been made to manipulate the order of the Hox genes in fruit flies. This made it possible to breed fruit files with legs where their antennas should have been. If the Hox gene that controls the development of the thorax is repeated, the result is a fruit fly with two thoraxes”, explains Malmstrøm.
Imagine a Hox gene that controls the development of a body segment with two legs in a small invertebrate: Duplication of this gene would cause two body segments with legs to arise. Repeat this duplication a lot of times, and maybe you would end up with a millipede.
A list of simplifications
The researchers have also discovered that the baby carp’s genome is very simplified in another area. The so-called introns – DNA sequences found in DNA, but not in mature RNA – can be quite bulky in most vertebrates. But in Paedocypris, the introns are much reduced. The result is that the exons (DNA sequences that are also found in RNA) lie much closer to each other.
“A gene consists of both introns and exons, and the exons contain the actual recipes for the proteins that are going to be produced. The introns are placed between the exons and can cause different groups of exons to produce different proteins. In this way, one gene can encode more than one protein. The length of the introns doesn’t matter too much, but the role of the introns is that they enable the cell to produce more proteins than the number of genes that are contained in the genome”, explains Ole Kristian Tørresen.
This is the most surprising discovery in the genome of the baby carp: That the intron areas in their DNA are much reduced, so that they are shorter than in related species. In addition, the baby carp have lost much of what is called transposons, that is, “jumping genes” or bits of DNA that can change its position in the genome of a cell.
“You mentioned that these tiny carp live in an acidic environment with little oxygen, little lime and little nutrition. Are their genetic simplifications and life-long larval stage an adaptation to a hostile environment, in order to conserve energy?”
“It is tempting to think like that”, Tørresen answers, “but we should be careful in concluding about a causal connection. You see, there are also other tiny fish species living in the same waters, but they have completely normal genomes and normal bodies. Besides, we know that some small fish species have large genomes, and some large fish species have small genomes. As if that’s not enough, researchers have also found paedomorphic or “childlike” fish species that have a full set of Hox genes”.
A threatened environment
Malmstrøm and Tørresen are concerned about the future of the Southeast Asian peat swamp forests, which are disappearing at an alarming rate. These forests grow on top of extensive reservoirs of fossil coal, and the result is that large areas are being chopped down, drained and excavated each year. This has made Indonesia the world’s fourth largest coal producers.
“I sincerely hope that the sequencing of these unique genomes can draw more attention to the peat swamp forests, so that they can be managed and protected in a better way in the future. We are only beginning to explore the life in these strange forests, and what we found in Paedocypris may indicate that the forests are a genetic treasure trove”, sums up Malmstrøm.
Modelling the model
The two researchers also imagine that the species in the genus Paedocypris can become more interesting to other researchers. The adult individuals are namely very similar to the larvae of zebrafish (Danio rerio), which have for many years been an important model organism for researchers.
“These baby carp have already lost, by nature’s own hand, many important genes that researchers often knock out in zebrafish in order to study them. So, here we have a fish that is simplified by nature instead of by humans. We believe that scientists that are planning to test a hypothesis by knocking out genes with an adult zebrafish, can start with testing in Paedocypris instead, where some of the relevant genes are already silenced. Thus, Paedocypris has the potential to become a model of the model fish”, says Tørresen.
Martin Malmstrøm was working as a post doctor at the Department of Biosciences at the UiO when he wrote the scientific article that was published in April 2018. Today, he is a project manager in the Norwegian Scientific Committee for Food and Environment, an independent and interdisciplinary committee that gives scientific risk assessments on issues concerning healthy foods and environmental issues.
Martin Malmstrøm, Ralf Britz, Michael Matschiner, Ole K. Tørresen, Renny Kurnia Hadiaty, Norsham Yaakob, Heok Hui Tan, Kjetill Sigurd Jakobsen, Walter Salzburger & Lukas Rüber: The most developmentally truncated fishes show extensive Hox gene loss and miniaturized genomes. Genome Biology and Evolution, 19. april 2018. https://academic.oup.com/gbe/article/10/4/1088/4938689
Martin Malmstrøm et al.: Evolution of the immune system influences speciation rates in teleost fishes. Nature genetics, 22. august 2016. https://www.nature.com/articles/ng.3645