Key aims of research on aging are to understand the molecular mechanisms behind the phenotypic changes that occur during aging and to identify novel life span enhancing interventions. Using the TK as a naturally short-lived vertebrate model system, we report the characterization of the changes in GM composition occurring during aging and the discovery of a novel life span enhancing intervention achieved by acutely transferring young GM to middle-age individuals after antibiotic treatment. This intervention resulted in the maintenance of an overall healthier physiological status, a highly diverse and young-like gut microbial community at late age and in an enhanced transcriptional signature of defense responses to bacteria.

Our results show that TK are characterized by a complex GM community, more species-rich than worms and flies in laboratory conditions and of the same order of magnitude of mammals, both in abundance and composition. Indeed, the four most abundant bacterial phyla observed in the TK are also the four most abundant phyla found in humans and mice. However, unlike mammals, Proteobacteria is the most abundant gut bacterial phylum found in killifish, similarly to other aquatic species, such as zebrafish (Roeselers et al., 2011). Since it is the shortest-lived vertebrate to date reproduced in captivity, the TK can become an ideal model to dissect the links between GM diversity and host aging.

Intriguingly, we show that while the gut bacterial diversity of captive-raised TK is lower than that of wild-caught populations, captive fish still recapitulate the core microbial diversity of wild TK. Laboratory-raised fish are not represented by distinct gut bacterial communities, but are rather dominated by a few high-abundance bacterial taxa that are already present in wild killifish populations. This supports the conclusion that fish raised in captivity have a GM community that is representative for the species in nature. Additionally, we found that wild populations of TK show large between-population gut bacterial diversity, with the emergence of population-specific high-abundance taxa. This is plausibly caused by locality-specific differences in ecological conditions, including climate, soil composition, parasites and diversity of food sources (Nezhybová et al., 2017; Reichard et al., 2017).

Analyzing the changes in GM composition between young and old fish, we found that young fish are characterized by a large taxonomic bacterial diversity. Old fish are less OTU-rich, similar to what is observed in human cohorts from different age classes (Claesson et al., 2012). Intriguingly, while individual bacterial diversity (alpha diversity) is higher in young individuals, old fish are more dissimilar from one another, that is, while each individual old fish has a more homogeneous GM composition than young fish, any two old individuals have more divergent bacterial communities compared to young individuals. This result raises the possibility that the changes in composition and relative abundance in GM communities from young to old individuals could be a function of (i) initial individual GM composition, (ii) differences in individual immune system composition and function or (iii) a combination of initial individual GM composition and host immune function.

Aging in this experimental model was not only associated with reduced bacterial species richness, but more specifically with loss of bacterial taxonomic units involved in carbohydrate, nucleotide and amino acid metabolism, which in mice are associated with aging (Langille et al., 2014). These same changes have been associated in humans with unhealthy aging (Claesson et al., 2012; Rampelli et al., 2013), as well as with chronic conditions such as obesity, type two diabetes and insulin resistance (Neis et al., 2015). The shift in microbial composition between young and old fish was consistently characterized by a higher prevalence of Proteobacteria in old individuals, while young individuals were significantly more enriched in Firmicutes, Actinobacteria and Bacteroidetes. Additionally, functional metagenome analysis showed that young fish had GM associated with carbohydrate metabolism, replication and repair, as well as DNA repair, indicating the young GM’s ability to protect itself against assault and thus maintain homeostasis. On the other hand, bacterial pathogenicity in the gut is associated with the accumulation of mutations over time, induced by the failed capacity to repair them (Leimbach et al., 2013). Old GM was enriched in potentially pathogenic bacteria, associated with dysbiosis. Additionally, functional metagenome analysis on the bacterial communities present in old fish guts indeed found them to be associated with host disease. Consistently, while young gut transcriptomes were associated with high expression of host genes involved in cell cycle activity, likely associated with proliferation and differentiation, old gut transcriptomes were associated with host immune responses to pathogenic bacteria, reflecting the prevalence of more pathogenic bacterial taxa.

Although GM transfers from young, healthy donors have found applications in the clinic to treat acute gut infections such as those associated with Clostridium difficile (Lee et al., 2016) and have been proposed to treat obesity, metabolic syndrome and even neurodegenerative diseases (Marotz and Zarrinpar, 2016; Xu et al., 2015), the application of this methodology as an anti-aging intervention has not been explored to date. Remarkably, despite single associations of different bacterial diets have shown to significantly affect life span in invertebrate model systems such as Caenorhabditis elegans (Zhao et al., 2013), a functional test of the role of a complex GM community associated with young age as an intervention aimed at modulating the recipient’s life span has not yet been carried out to date. By acutely exposing middle-age individuals to young fish GM content – after antibiotic treatment – we could prolong life span and retard the age-dependent decline in exploratory behavior, which we showed to decline during normal aging. Noteworthy, our results exclude that the effects of the interventions depend on repopulating the intestine with any GM community or that antibiotic treatment alone, delaying dysbiosis, was sufficient to explain the full extent of life span increase achieved via transfer of young GM.

Additionally, life span was not affected in young fish exposed to GM from old, young, and sham control fish after antibiotic treatment. These results are compatible with a scenario where the age-associated decline of immune function might be responsible for the progressively decreased capacity of the host to (i) maintain the healthy portion of the GM community and (ii) counteract the proliferation of potentially pathogenic gut bacteria. Since we observed that young fish receiving 16-week-old gut microbiota content did not live significantly shorter than young, control fish, it is plausible that dysbiosis, in the context of a young gut immune function, does not lead to increased mortality.

Fish treated with young GM after depletion of their own resident GM community not only maintained a more diverse microbial community at old age compared to wild-type, age-matched control fish, but their microbial community remained more similar to that of young fish. This raises the possibility that bacterial consortia associated with young fish can contribute to increased life span and enhanced individual health status. Based on functional metagenomic analysis, young fish and fish treated with young GM were enriched for bacteria associated with carbohydrate metabolism and DNA repair, both importantly associated with host metabolism, health and longevity.

Young fish, as well as fish treated with young GM, had a high number of bacterial taxa that frequently co-occurred with one another, de facto contributing to a young-associated bacterial network. On the other end, old wild-type controls (16wk), as well as old fish treated with same age GM (Omt), had smaller bacterial networks, possibly resulting from the higher inter-individual variation in GM composition associated with these groups. Remarkably, applying our analyses to a published mouse cohort (Langille et al., 2014), we extended this finding to mammals, confirming that networks built on GM OTU abundance are associated with host’s chronological age. Our network analysis enabled us to identify a subset of highly frequent taxa associated with a young-like status and with prolonged life span in fish treated with young GM. These involved the genera Exiguobacterium, Planococcus, Propionigenium and Psychrobacter, which are key bacterial genera responsible for structuring a healthy GM community in TK. Interestingly, species belonging to each of these genera have been associated with energy metabolism and potential health benefits. Specifically, species of Exiguobacterium and Propionigenium are able to metabolize cellulose and ferment carbohydrates to produce short chain fatty acids, which are known anti-inflammatory mediators and can modulate the immune system. Planococcus species can hydrolyze gelatin to produce essential amino acids for use by the host and certain Psychrobacter species are capable of producing omega-fatty acids. Taken together, these key bacterial genera can produce metabolites capable of maintaining immune system health and having anti-inflammatory effects on the host, both of which have been associated with longevity.

While GM transfers significantly affected the GM composition of experimental fish, their overall gut transcriptional profile showed that old fish clustered together regardless of the treatment. This could be a consequence of the down-regulation of the transcriptional programs associated with growth in all 16-week-old fish groups. However, transcripts involved in defense against pathogens, extracellular matrix components and the Tor pathway, are dramatically different among experimental groups receiving young or same-age gut content, suggesting that these key aspects might ultimately be fundamental modulators of organismal life span and health. Generating a correlation matrix between bacterial abundance and gut transcripts, we could isolate host transcripts whose expression was significantly correlated with specific OTUs. In particular, bacterial genera associated with a healthier and longer life span, such as Psychrobacter and Exiguobacterium, had strong co-occurrence with host genes importantly associated with aging modulation.

The lack of a generalized transcriptional gut rejuvenation in long-lived fish treated with young GM, together with the fact that antibiotic-only treated fish live longer than control, untreated groups, suggest that a delayed onset of dysbiosis could benefit the host and explain, at least in part, the effects on survival. However, since fish receiving young GM live longer and have a young-like locomotor performance compared to the antibiotic-only treated group, it is likely that, independent from dysbiosis, young GM could directly benefit host physiology, possibly influencing metabolism and immune function. Therefore, young GM could possibly lead to beneficial effects on host survival and behavior performance both via delayed dysbiosis and direct favorable effects of subsets of young microbes. Future work will help shed light on what specific aspects of the aging process can be affected by resetting a young-like gut microbiota in aged individuals and whether this intervention can be more broadly applied to other organisms, including mammals.

Our results indicate that improving the ecological diversity of the GM in old individuals helps to restore health and prolongs life span. Our approach could provide a key to slowing aging and retarding the onset of age-associated diseases by specifically targeting the GM. Given its large bacterial taxonomic diversity and the shortest life span for a vertebrate species raised in captivity, the TK could become a key experimental species which will help to shed light on the functional connection between GM dynamics and aging in vertebrates.