Of Fish and Men
Which role can fish play in aging research?
A big one, if you ask Dario Valenzano. He does research on killifish, an extraordinarily short-lived vertebrate. His research gives valuable insights into the role of the immune system in the aging process – not only in killifish, but in humans as well.
by Peter Kohl
»Wow, that’s an old fish.« The excitement in Dario Valenzano’s voice is almost tangible when he discovers the old fish in the aquarium tank. »We have to give him a name. Perhaps Methuselah?« he suggests, laughing. But why exactly is the researcher on aging so happy to see a fish that was born in October 2015? For comparison, a fish that is often used in laboratories around the world, the zebrafish Danio rerio, has a lifespan of up to seven years. But Valenzano’s lab performs research on the turquoise killifish Nothobranchius furzeri, and this fish has a lifespan of four to nine months, depending on the strain.
That is why this species is so interesting for a researcher on aging – although this was not always Valenzano’s specialization. Today he is a Principal Investigator at the Max Planck Institute for Biology of Ageing and a member of the Cluster of Excellence on Aging Research CECAD. As a researcher, he started out studying the evolution of facial expression in primates. But in 2002, the aquarium enthusiast Stefano Valdesalici (now the president of the Italian Killifish Association) talked to Professor Alessandro Cellerino, Valenzano’s doctoral advisor. Soon after personally picking up the first batch of short-lived killifish embryos, it was all up with Valenzano. »In my first two years of killifish research I was on my own. I was the caretaker, the technician and the experimenter. After two years, with enough preliminary results, we got our first grant and could hire a technician. But until then it was a lot of hard work with hardly any weekends off.« The effort paid off, though. Today, at the age of 38, Valenzano heads his own group at a Max Planck Institute in Cologne and has gained worldwide renown as an expert on killifish: »Today, one of my tasks is to help other scientists kick-start their own research on killifish. We send out protocols and embryos on a weekly basis and often receive requests for collaboration – it is a terrific thing! The more people there are in the community, the better.«
The intestines are a battleground – or a place of cooperation?
Still, what is it that makes this tiny fish so interesting for an entire community of researchers? Aren’t there already enough model organisms to perform research on? There certainly are, but they all have advantages and disadvantages. For example, a commonly used lab animal is the fruit fly Drosophila, which usually lives about 30 days, or the roundworm Caenorhabditis elegans, which lives for two to three weeks. This makes them interesting for aging research, but there are disadvantages too.
Even though these organisms share many genes with humans, there are also big differences. For example, invertebrates like worms and flies completely lack an adaptive immune system. Another lab animal, the mouse, is quite similar to humans when it comes to biological defense strategies. But with a lifespan of more than two years, researchers would have to wait for quite a while to learn more about the aging process. »That is why the killifish is perfect for our questions regarding the immune system. Similar to us, fish also have immune cells like B- and T-lymphocytes in their body to fight against threats.« Specifically, they look at the intestines, which are very important interfaces between the organism and the outside world. »The intestine can be considered a battlefield between the host and other organisms like bacteria.
But it can also be something different than a battlefield, a place where cooperation or a mutualistic relationship can take place. Bacteria provide nutrients and metabolites to the host. The role of the immune system is to recognize and respond to pathogenic bacteria. We are specifically interested in studying how immune system function declines over time upon aging. « Valenzano is particularly interested in the characterization of the important regions in the genome that are responsible for the antibody diversity of the fish: »We want to see how the antibody repertoire changes with aging and if that affects the aging process of the organism.« One of the striking things about this organism is that for every organ they have looked at so far, they see an aging process. »Either every organ has its own intrinsic aging clock or there is a master regulator of aging that causes everything to fail with age. One hypothesis is that the immune system is fading. I think that this fish provides a great opportunity to study how the immune system plays a role in aging.«
To explain how the immune system interacts with the gut microbiota, Dario Valenzano found an interesting analogy. He sees the immune system as a gardener who takes care of plants and gets rid of unwanted weeds. »When the gardener gets old, the weeds take over and the ones that grow faster will completely overtake the pretty plants. This could be something similar to what is happening in the fish or in us.« But it is possible to help this inner gardener. »Maybe, when the gardener gets older, a niece or a friend will bring some nice flowers. That might trigger him to take better care of the garden again. But if the garden is really full of weeds and completely taken over by stubborn shrubs – in some ways a very resilient environment – it might be difficult to bring it back to a healthy and youth-like state. A very harsh treatment might be necessary: the garden needs to be completely redone to get it back on track.« At this point he leaves the analogy again and returns to his research: »What we are wondering now is: Can the gut microbiome play a role in the aging process? Can we interfere with aging by manipulating the microbial community associated with a given organism? We know that the microbiome importantly contributes to metabolism and physiology; many of our diseases are characterized by changes in the gut microbial community. What if we try to rejuvenate the microbiota? Would that affect the host’s aging process?« Hearing this idea, one can understand Valenzano’s excitement as a treasure-hunter with a special goal: collecting fish poop. »See the poop here?« he says, pointing to an aquarium. »That’s like gold to us!«
As interesting as killifish might be for research, keeping and breeding them requires special training and dedicated personnel. This has to do with the fish’s complex lifestyle and the harsh environment in which it dwells – the African savannah in Zimbabwe and Mozambique. In the wild, killifish live in ponds that usually desiccate during the dry season. This has created a very strong evolutionary pressure for the fish to grow very rapidly, reaching sexual maturation in about three weeks. When you have little time to mate, you should hurry up. Once their work – reproduction – is done, the pressure of natural selection becomes weaker with regard to aging and remaining strong. »That is possibly the reason for the short lifespan and the early onset of illnesses like cancer in this species.« To establish the killifish as an organism for the study of aging in the lab, several milestones were necessary. To mimic the wildlife conditions, the aquarium has a twelve hour day and night light cycle, with a half hour sunrise and sunset. The temperature is kept constant at 28°C. The most interesting part of the life cycle comes six to seven days after the eggs are fertilized. While fish usually need water to survive, this fish needs fresh air. The eggs are collected and plated on a substrate of moist and sterile coconut-fiber. After three weeks, the eggs are ready to see water again and the fish can hatch – or they can be kept for months and sometimes years in a cool incubator! »We can synchronize the hatching by controlling the incubation temperature: for instance, I can decide that they will hatch next week on Wednesday. That makes experimental planning a lot easier for us.«
An ironing board and a WhatsApp group are helpful in the savannah
Even though Dario Valenzano’s team can keep up to 2,000 fish in the institute’s fish facility, from time to time he runs fieldwork in Africa to collect fish tissue samples from wild fish populations. Modern technology and social media are a great help for him – in the form of a WhatsApp group with national park rangers. »Rangers know the favorite spots where I collect fish in the park and I am in contact with them to receive updates about the weather and the water levels in the ponds. If they tell me ›Dario, it’s too dry,‹ then I don’t go. The past year was extremely dry. 2016 was an El Niño year, so we were forced to cancel the expedition. It basically did not rain at all. Hopefully the coming season will be better.« Another helpful tool is a simple ironing board. »In the field you have to improvise. We use an ironing board that we purchased at a local market as a mobile working bench, and it works great. We now have a hand-cranked centrifuge, so we can isolate and bring back to the lab pellets of bacteria that we take from water samples.«
Another thing he and his team have to deal with during their expeditions are the sometimes challenging African conditions: »Last time my student and I got Dengue fever. And then there is wildlife – buffalos, lions, hyenas and elephants, which in this area can be quite aggressive as they are often threatened by intense poaching. It can be tough if you have never been to the bush. Every time I choose someone new to come with me, it is a difficult decision.« But in the face of these objective risks, the researchers take every possible precaution. More than ten years of experience in the field and full support from the national park rangers significantly increase safety. And even if some risks remain – Dario Valenzano is willing to take them to reveal the secrets of aging.