This research project aims to characterise the gut microbiome of the dog and closely related wild-living species, the grey wolf and the red fox to investigate if the composition of the microbiome could be linked to domestication. By Associate Professor Anders Johannes Hansen, Natural History Museum of Denmark, University of Copenhagen What makes particular species susceptible to domestication has been a fundamental scientific question for years and the current consensus is that genetics holds the answer. However, investigations have shown that the microorganisms that live on humans and animals are vital to health (their microbiomes), and behaviour, and that the composition can be altered by diet. Based on this, and the fact that the wild progenitor of many domestic animals first made contact with humans due to scavenging on human waste, we aim to characterise the gut microbiome of the dog and closely related wild-living species, the grey wolf and the red fox to investigate if the composition of the microbiome could be linked to domestication. Background Investigations have shown that the microbiome of humans and animals are of vital relevance to both health, and behaviour, and that the microbiome’s composition can be influenced and altered by external factors like the diet1. Changes in microbiome are significantly related to stress behaviour in humans2, and stress, anxiety, and depression-related behaviour in mice3. Hence, as a diet and behaviour are both linked to the gut microbiome, and as the wild progenitor of many domestic animals first made contact with humans due to scavenging on human waste, changes in the microbiome could play an important role in the domestication of species. Humans, dogs and wolves have been interacting for at least the last 15,000 years since the domestication of the dogs. The relationship has always been of interest to researchers and lately, with return of the wolf in Denmark this relationship has become strongly of interest within the public, as evident from the activity across numerous media and social media platforms. Hence, as this group of animals (Canidae) and the human interaction has scientific interest and has caught the public eye, so we decided to investigate if the domestication of the dog could be facilitated by changes in the microbiome. Microbiomes More than 20 stool samples from dogs (Canis lupus familiaris), wolves (Canis lupus lupus) and the less closely related red foxes (Vulpes vulpes) were collected in Denmark and Greenland and their microbiomes were investigated. Multiple fecal samples were analysed for each species and from each sampling location allowing for the microbial compositional comparison between species and geographical location. The microbiome was generated using 16S metabarcoding and subsequent bioinformatics analysis. The bacterial diversity (as measured by molecular richness, OTU) was similar between the dog and wolf with around a mean of 220 OTUs, but significantly higher in the fox with more than twice that amount (see figure above). The reason for this discrepancy is not clear but it could indicate that foxes have a different and more diverse diet than the two Canis lupus subspecies The composition of the gut microbiome varies (fig bacteria distribution) between the 3 (sub)species, but more between the dogs/wolves and the foxes. Clostridia and Fusobacteria constitutes most of the microbiome of the dog and a great deal of the wolf, this could be because of the genetically closeness of these animals, but could also be a response to a more diet with high content of meat and fat, which has been shown in cats, where a high fat diet increases the amount of clostridia in the microbiome. Lactobacillales is found in a higher proportion in the fox than in the dog, but not in the wolf. Lactobacillus is found in omnivores and herbivores, which is expected given the fox is the most likely to scavenge food, and therefore have the more omnivorous diet when compared to the exclusively carnivorous wolf. The high proportion of flavobacteria, sphingobacteria and proteobacteria in the foxes, and to a lesser degree the wolves, could also be a response to the differences in a more wild diet. The dog microbiome is dominated by the fusobacteria where the different bacteria groups of the wolf and fox microbiome are more equally divided. This could be because most of the scat samples found in Denmark probably are from non-feral dogs, and the ones from Greenland is from kept sled dogs and hence the dogs are probably being fed a less divers diet compared to the wild-living wolves and foxes. Multidimensional scaling (fig below) revealed that the microbiome composition varied both geographically and between species. In particular, the red fox samples were significantly different from the dog and wolf samples, which may reflect their distinct evolutionary history (as they differ in genus to the other two). However, differences between their life histories, such as a more scavenging based life style could also cause the observed pattern. Citizen Science This preliminary study of the Canidea microbiome in Denmark and in Greenland is an excellent example of the synergy that can be generated through collaborations between volunteers and researchers. Most of the samples and relevant metadata utilised in this study have been collected through a citizens network Ulvetracking.dk. They have through the last 4-5 years collected scat and saliva samples from predatory mammals in Jutland and Fyn resulting in more than 350 samples from Denmark alone. The Research Team The most active contributors to the data generation were Cecilie S. Jensen, Alba Rey-Iglesia, Christopher James Barnes and Anders J. Hansen who was PI on the grant from the Carlsberg Foundation who supported this research project. However, a long list of people (see below) has contributed significantly to obtained results either by providing information, samples or participated in the discussion of the results. Anders Johannes Hansen about the Grant from the Carlsberg Foundation With the seed money received from the Carlsbergfondet it was possible to make the first steps towards testing the influence of mirobiomes on canids like foxes, dogs and wolves. Thereby help creating the for my future scientific career trajectory and strengthen my ability to secure funding in the future. The role and influence of canids in modern human society is vividly exemplified by the on-going debate concerning wolves in the Danish nature. Hence, understanding the relevant factors influencing the interaction of canids with humans is of paramount importance if unwanted incidences should be avoided. The result of this study helps unravelling one of the possible key factors influencing the human canid relationship and thus have direct influence on decision making in our society. What Next? The results of the investigation revealed that we need to include more samples of the included taxonomic groups and that we should consider including captive individuals of foxes and wolves. Hence, we are collecting wolf and fox samples from zoological gardens in the UK, Sweden and Denmark to complement our results from the wild living individuals. Whether the microbiome from the captive wolves are more similar to domesticated dogs, or to wild wolves will provide us with the ability to disentangle the effects of genetic and environmental variation in determining host microbiome. Furthermore, these results could also show if the interaction with humans have an influence on the composition of the microbiome in a captive compared to wild-living individual, which is necessary if the microbiome should be one of the dog domestication drivers. Impact The final analysis and finding of this investigation will be disseminated in scientific papers and conferences in the near future. So far, the preliminary results have been presented at an interdepartmental seminar between the Molecular Biology and Genetics, Danish Centre for Environment and Energy both at Aarhus University and the Natural History Museum of Denmark at University of Copenhagen. The results generated in this project has influenced a series of public interviews and discussions and press releases by the grant holder and collaborators which has led to more than 100 appearances in printed and online media. The results have not led to any direct private collaborations yet, but the results generated could be used in connection to improving and developing new canine food products. Additional Contributors Carsten Rahbek, Natural History Museum of Denmark, University of Copenhagen; Charlotte Hansen, Natural History Museum of Denmark, University of Copenhagen; Frederik Seersholm, Department of Environment and Agriculture, Cutin University; Johannes Lang, Institut für Tierökologie und Naturbildung; Kent Olsen, Naturhistorisk Museum Aarhus; Kim Møller, Ulvetracking.dk; Niels Martin Schmidt, Department of Bioscience - Arctic Ecosystem Ecology, Aarhus University; Marlene EM Restrup, Natural History Museum of Denmark, University of Copenhagen; Mike Bunce, Department of Environment and Agriculture, Cutin University; Mikkel Sinding, Natural History Museum of Denmark, University of Copenhagen; Morten Meldgaard, Ilisimatusarfik; Olivier Gilg, F-21440 Francheville, France; Paw S. Rasmussen, Ulvetracking.dk; Pernille S. Olsen, Natural History Museum of Denmark, University of Copenhagen; Peter Sunde, Department of Bioscience, Aarhus University; Sabina Haaning, Ulvetracking.dk; Tatiana Feuerborn, Department of Bioinformatics and Genetics, Swedish Museum of Natural History; Tom Gilbert, Natural History Museum of Denmark, University of Copenhagen; Tina B. Brand, Natural History Museum of Denmark, University of Copenhagen. References 1. Turnbaugh, P. J. et al. The Effect of Diet on the Human Gut Microbiome: A Metagenomic Analysis in Humanized Gnotobiotic Mice. Science Translational Medicine 1, 6ra14–6ra14 (2009). 2. Foster, J. A. & Neufeld, K.-A. M. Gut–brain axis: how the microbiomeinfluences anxiety and depression. Trends in Neurosciences 36, 305–312 (2013). 3. Ezenwa, V. O., Gerardo, N. M., Inouye, D. W., Medina, M. & Xavier, J. B. Animal Behavior and the Microbiome. Science 338, 198–199 (2012).