We used longitudinal data collected from Seychelles warblers to investigate the association between host age, senescence, and GM characteristics. We found no evidence of senescent declines in the GM; both bacterial alpha diversity and composition remained largely invariable with respect to age in adults and did not differ in an individual’s terminal year. Instead, environmental factors, including season and variation in mean territory quality across the study period, appeared to have the greatest impact on the GM during adulthood. Within individuals, we also found no evidence of increased GM personalisation or instability in older age groups, even in the terminal year. This is despite including some relatively very old individuals in the dataset (the oldest individual was c.a. 17 years of age, Additional file 1: Figure S2).
Seychelles warblers have a median lifespan at fledging of 5.5 years [41, 42] and, at the population level, there is evidence of reproductive and survival senescence from approximately six years of age [43, 44]. In our analysis, we included samples from 66 individuals older than six years of age. Of these, 37 individuals had at least two samples taken longitudinally, including 19 individuals with samples taken before and after 6 years of age. There was a maximum of 4.8 years between longitudinal samples for putatively senescent individuals (mean 1.4 years ± 0.2 SE, Additional file 1: Figure S2). These sample sizes are either comparable to, or greater than, the few studies that have identified statistically significant effects of age on GM structure in other wild systems (e.g. Bennett et al. (2017), N = 14 post-prime individuals sampled cross-sectionally; Trosvik et al. (2018), N = 70 post-prime individuals sampled cross-sectionally; Sadoughi et al. (2022), N = 11 senescent individuals sampled longitudinally over 1.5 years). Furthermore, our study also included 97 individuals (out of a total of 273 adults) that had a sample taken in their terminal year of life allowing us to additionally test for changes in the GM close to death. Thus, this was a robust dataset with which to investigate the relationships between host age, senescence, and the GM. As such, the absence of statistical significance is unlikely to be due to a lack of power to detect effects that are large enough to be biologically meaningful to the host.
A wealth of studies on humans have identified a decline in bacterial diversity and shifts in GM composition in older age groups [e.g. 13, 15, 20]. Only a few studies have been undertaken in the wild however, cross-sectional studies on lemurs (Lemur catta) [28] and geladas (Theropithecus gelada) [29] have also demonstrated shifts in GM composition, although not GM alpha diversity, between reproductively mature and post-prime adult individuals. We found no statistical evidence of an association between age and GM alpha diversity, and only identified extremely limited shifts in GM composition in adult Seychelles warblers. This was the case even after controlling for the possibility of differential rates of damage accumulation by investigating GM differences in the terminal year of life [34]. This is consistent with the findings of several other studies on wild non-human primate populations in which GM diversity and composition remained largely invariable with respect to chronological age during adulthood [31, 32]. However, these analyses were either cross-sectional with small sample sizes for old individuals (N = 12 samples from old individuals in [31]) or, where longitudinal sampling did take place, did not control for the time interval between sampling points when comparing GM similarity [32]. Cross-sectional analyses can suffer from issues such as selective disappearance which mask processes occurring within individuals, and GM turnover can confound comparisons made between samples taken at different time points. A recent study on wild meerkats also demonstrated that GM diurnal cycling remained consistent with chronological age, suggesting that functionality is largely maintained even in old individuals [33]. However, although repeat samples were taken from individuals, this study did not investigate within individual dynamics per se. Furthermore, none of the aforementioned studies controlled for differential damage accumulation by including a measure of biological condition such as time to death. Thus, we provide a more robust test of the association between age, senescence, and the GM and, by doing so, corroborate the results of these previous studies.
The discrepancy between studies on wild animals and those on humans might be explained, in part, by lifestyle and behavioural factors that change with age in human populations but that don’t exist in wild systems. For example, in humans, medication intake and the probability of living in residential care increase with age, whilst physical activity and dietary quality decrease, all of which can directly impact the GM [70, 71]. It is possible that certain factors that impact the GM also vary with age in some wild mammalian species. For example, dental wear and tooth loss increase with age in some non-human primates [72, 73] which can, in turn, influence an individual’s food choices and their ability to extract nutrients from dietary components [74]. However, Seychelles warblers ingest their food whole, feed almost exclusively on insects throughout their lives, and show no decline in foraging efficiency with age [75]. Thus, such effects are unlikely to be universal across wild species. This could potentially explain some of the observed variation in GM-ageing patterns identified amongst different wild taxa.
Our findings are also in contrast to studies on captive animals which have demonstrated shifts in overall GM composition with increasing age [e.g. 14, 16, 76]. However, captive animals are often housed in highly controlled conditions and frequently harbour unrealistically low levels of GM diversity [22, 77]. In wild systems, environmental variation can strongly impact the GM [33, 78, 79] and may override host intrinsic effects observed in the laboratory. Indeed, time of day, seasonal differences, and changes in mean territory quality were strong determinants of GM composition in Seychelles warblers, consistent with previous studies on this system [6, 45, 80]. GM similarity also declined with an increasing number of days between samples suggesting a high level of turn-over within the GM. Thus, although Seychelles warblers demonstrate both reproductive and survival senescence [43, 44], constant environmental uptake of microbes may override any largescale effects of senescence on the GM.
In addition to the lack of change in overall GM composition, only six (out of 54) individual core genera were associated with age in adult Seychelles warblers. One genus that decreased in abundance with increasing host age was in the family Ruminococcaceae. This is one of the most abundant families in the human GM [81]. Members of the Ruminococcaceae are obligate anaerobes, interact directly with the mucosal layer of the gut epithelium and produce important short chain fatty acids such as butyrate [82]. The abundance of this family has also been shown to decrease with age in several long-term human studies [11, 83, 84] and reduced abundances have been associated with increased gut inflammation and chronic intestinal disorders such as Crohn’s disease [85, 86]. Thus, it is possible that certain members of the GM are linked to senescent declines in the Seychelles warbler. However, this Ruminococcaceae genus made up a relatively small fraction of the overall GM (c.a. 1% average relative abundance in adults). Thus, further tests of its functionality, for example through metagenomic sequencing, would be needed to understand its role within the Seychelles warbler GM and whether a decline in its abundance in older age groups is biologically significant to the host.
Aside from Ruminococcaceae, the core genus Gordonia also decreased in abundance with increasing host age, while four other genera (Kineococcus, Pseudonocardia, Quadrisphaera, and a genus in the family Micromonosporaceae) increased with age. These genera are all in the phylum Actinobacteria which is widely distributed in the environment. They are also aerobic, and produce a range of natural products including antimicrobials [87–91]. It’s possible that environmental bacteria may increase in abundance within the GM as host immune function declines with age and/or as other key bacterial genera, such as the Ruminococcaceae, are lost. However, due to their widespread distribution, it is also plausible that these bacteria are constantly acquired from the host’s environment (e.g. via their diet) and may gradually accumulate and persist within the GM by outcompeting other microbes. Each actinobacterial genus that was associated with age was present at < 1% mean relative abundance in adults, suggesting that they may only play a very minor role within the gut ecosystem. Furthermore, despite these few patterns, the vast majority of core genera showed no association with age suggesting senescence was not associated with a large-scale restructuring of the GM.
Many more taxa were associated with variation in environmental factors, including time of sampling, season, and mean territory quality across sampling periods. This is consistent with other studies showing that environmental dynamics can play a significant role in structuring the GM of wild animals [33, 78, 79]. For example, a study on wild meerkats showed that diurnal shifts in GM composition outweighed an association between the GM and host chronological age [33]. Circadian GM dynamics have been identified in human and wild animal studies, and may be driven by differences in foraging regimes and dietary intake throughout the day [33, 92]. In the Seychelles warbler, the genera Lactococcus and Enterococcus showed the largest increases in abundance between samples collected in the morning and afternoon. Both genera are lactic acid producing bacteria and play an important role in dietary carbohydrate fermentation [93]; as such, these genera may gradually increase in response to an influx of nutrients as individuals start to forage [92]. However, both genera are also found in insect microbiomes [94–96] and so it is possible that these taxa accumulate passively as feeding increases throughout the day and are mainly transient colonisers of the GM that don’t carry out a fermentative function.
Aside from time of day, differences in mean territory quality between sampling periods and season were also associated with GM differences. As territory quality (a measure of insect abundance) is likely to be linked to climatic factors, such as rainfall and temperature, which may also influence microbial abundances, it is possible that these GM differences were driven by differential exposure to microbes in the external environment. Seasonal GM differences could also be driven by similar processes. However, variation in the GM could also be linked to physiological change associated with host stress under low quality conditions [97]. Furthermore, since Seychelles warbler predominantly reproduce in the major breeding season (June-September) some of the seasonal differences in adult GM composition could be linked to physiological changes associated with host reproduction [98, 99]. Further work, including dense sampling of the same individual pre- and post-reproductive attempts would be needed to understand if this is the case.
We found no evidence that changes in the GM were more extreme in an individual’s terminal year of life when damage accumulation is expected to be at its greatest due to senescence, and only two genera were more abundant in terminal year samples (Friedmanniella and Microbacterium). These are both environmental microbes that are frequently isolated from insects [100, 101] and neither of them were associated with host age. The Seychelles warbler benefits from very low levels of extrinsic mortality during adulthood [40]. Indeed, an absence of natural predators, lack of human disturbance, and a relatively constant and benign climate, enables many individuals to reach an old age, providing the opportunity to detect and study senescence in this species [2]. Thus, although it is possible that a very small proportion of mortality in our study was stochastic, it is very unlikely that this would be at a level high enough to override any significant association between senescence and the GM. Therefore, the fact that we found little change in GM alpha diversity, composition, or stability in the terminal year, and that this relationship did not depend on age, suggests that the GM is largely unaffected by host senescence in the Seychelles warbler. Our previous work has shown that differential survival is associated with differences in both bacterial and fungal GM composition in the Seychelles warbler, however, in both cases, survival was assessed over much shorter periods post sampling (in most cases less than 3 months post sampling, but in some cases less than 5 months) [45, 80]. In this study we chose to look at changes over a longer period (the terminal year) to try to identify factors linked to senescence rather than the more immediate changes in the GM just prior to death which could be a consequence of a rapid decline in health. We expected senescent changes in the GM to accumulate gradually over longer periods, but we found no evidence that this was the case.
Aside from overall GM alpha diversity and composition, we found no evidence of increased GM personalisation, or reduced within-individual GM stability, with increasing age or in the year leading up to death in adult Seychelles warblers. This also contrasts with long-term studies on humans [15, 46, 102], and a recent longitudinal study on wild macaques (Macaca assamensis) [30], which identified increasing GM heterogeneity amongst individuals in older age groups. These studies were unable to determine the cause of these differences; whilst greater heterogeneity could be driven by reduced GM stability, it may also be driven by other factors such as reduced social contact amongst elderly individuals [11, 30]. Social interaction strength predicts GM similarity in wild mice, Apodemus sylvaticus [69], and reduced social network size has also been associated with lower GM diversity [69, 103]. However, social isolation may not be a hallmark of ageing in all species. Seychelles warblers are cooperative breeders that often live in groups consisting of a breeding pair and subordinate individuals, some of which may help with reproductive attempts [104–106]. The presence of subordinates does not decline as dominant breeding individuals age and, indeed, the recruitment of helpers increases in elderly females [41, 107]. Thus, there is currently no evidence to suggest that warblers become less social with age. Consequently, there may be ample opportunity for microbes to be shared amongst individuals in this population regardless of age.
In conclusion, our study finds little evidence of senescent changes in the GM of the Seychelles warbler. Although the abundance of a very small number of individual genera were associated age, overall GM alpha diversity and composition remained stable throughout adulthood in this species. Instead, environmental factors were the major driver of GM differences amongst adult warblers. Whilst this contrasts with studies on humans and captive animals, our findings add to the growing body of literature reporting mixed effects of age in wild populations. Further work is needed to better understand whether variation in lifestyle and behavioural factors drive the observed variation in senescent changes in the GM of different taxa.