Evidence for Senescence Among Microorganisms

Certain attributes of the population biology of microorganisms need to be kept in mind when one attempts to interpret whether senescence occurs. First, although vital stains are available for determining the live/dead status of microbes (Ericsson et al. 2000; Nelson et al. 2002) viability, at least for bacteria, is defined operationally as the ability to grow to detectable levels in or on a recovery medium (Postgate and Calcott 1985). Survival is thus equated in practice with the ability to divide repeatedly. By this criterion, all aclonal organisms would be dead! In fact, as judged by other criteria, noncultivable bacterial cells can be alive (e.g., Roszak et al. 1984), though replication potential may be lost at least in a fraction of such populations (Ericsson et al. 2000; see also the bacterial ‘persisters’ phenomenon, Chap. 7).

Second, importantly and as noted earlier, it is the clonal lineage, not the individual cell that is of ecological and evolutionary interest. This is comparable to the genet/ramet distinction in macroorganisms. Strictly speaking, for mortality statistics, the appropriate comparison would be among numerous clones, over time, as opposed to the population dynamics within a single ‘clone’.

The latter, however, is what bacteriologists typically study, as in growth curve experiments. Usually microbe cultures have an age structure and consequently cells will not be physiologically uniform throughout the population at any given time as well as over time (Yanagita 1977), unless precautions have been taken to do the experiments in ‘continuous culture’ (e.g., Dykhuizen 1990). Such procedures maintain a tightly controlled environment with replenishment of nutrients and removal of staling products. Otherwise, potential senescence effects, i.e., those due to some intrinsic organismal dysfunction, are confounded with those arising from externally imposed stress due to a drastically changing environment.

Third, estimating the distribution of clones of microorganisms in nature, other than within an arbitrarily delimited local area such as a field, is of necessity grossly incomplete due to the typically vast dissemination of clonal fragments (typically propagules such as spores, bacterial cells, etc.). This is much more difficult operationally than tracking plant and animal clones discussed above, but for different reasons. The problem in microbial ecology surrounds the unexcelled dispersal powers of bacteria and fungi (as well as some of the protists), not the lack of sophisticated identification or tracking methods. Although it is rarely possible to census an entire clonal population, in some situations it is possible to estimate the extent of emigration (Lindemann et al. 1982) or immigration (Andrews et al. 1987) from a source.

Finally, the occurrence of dormancy or quiescence, frequently under starvation conditions, together with various physiological states of activity and growth rate within a clonal population (Chap. 7 and Kester and Fortune 2014; Palkova et al. 2014), complicate the interpretation of senescence. This is somewhat analogous to modifications in the life cycle of reptiles and amphibians caused by diapause, diet, and temperature.

Bacteria. To explore the phenomenon of senescence among prokaryotes we might begin by asking whether it occurs among the relatively few differentiated species, i.e., those that have more than one morphotype. To do so we return to the example at the outset, Caulobacter crescentus (Fig. 6.1), which lives in aquatic environments. As noted earlier, the organism consists of a flagellated swimming cell called a ‘swarmer’ that eventually ejects its flagellum, settles, and differentiates into an elongated, sessile, ‘stalked cell’ tipped with an adhesive holdfast. Undifferentiated swarmer cells cannot replicate but stalk cells produce successive swarmer cells asexually, operating essentially like stem cells (Curtis and Brun 2010).

Ackermann et al. (2003) used microscopy to follow age-specific output by the stalked cells in three replicated in vitro experiments (actually subpopulations) of the same strain of C. crescentus. Some cells produced up to 130 progeny in the approximately 300 h monitored, but division rate decreased or halted over time in many other cells. From this result they concluded that “senescence can indeed evolve in bacteria if there are systematic differences between the two cells emerging from division”. However, what is supposedly ‘senescing’ here is a particular cell type, effectively a ramet, not the bacterial clone. Assuming this phenomenon is real, i.e., occurs in nature, what the results imply is that the genetic individual will continue indefinitely by the released swarmers, which in turn will differentiate, etc., as ongoing cycles repeat themselves, even though there may be some attenuation in the rate of clonal expansion.

Stewart et al. (2005) examined cell division through time in the well-known bacterium Escherichia coli. The rod-shaped cell divides in the middle forming two seemingly identical daughter cells. However, they find that the fission products are physiologically different: one new end (‘pole’) per cell is produced during division, implying that one of the ends of each progeny cell is preexisting from a previous division (‘old pole’, distal from the axis of division) and one is newly synthesized (‘new pole’, along the division axis). Old poles persist through multiple cell cycles and can be discriminated microscopically from new poles. At each division the cell inheriting the old pole is somewhat slower to divide and over time the effects on this lineage are cumulative.

The older a pole cell is, the slower its growth rate and offspring production; there was some evidence that the older lineage also has an increased incidence of death. This physiological and reproductive asymmetry is associated with the polar localization of cell components, including proteins and peptidoglycan, known to accompany bacterial growth (Saberi and Emberly 2013; Kysela et al. 2013; for related studies on another rod-shaped bacterium and possible medical implications, see Aldridge et al. 2012). However, whether the inequality is a cause or consequence of bacterial cell aging is unknown (Stewart et al. 2005). Wang et al. (2010), using different experimental conditions, reached somewhat different conclusions with respect to the nature of population dynamics in E. coli; Rang et al. (2011) reanalyzed the data from both studies and provided a reconciliation.

The foregoing studies (as do those of microorganisms generally as recounted in other examples below) elucidate the process of cellular 'aging' and are not evidence for clonal senescence. This situation recounted above is analogous to the aging and death rates of individual ramets in an aspen clone. Importantly, the bacterial genetic individual continues indefinitely, as does the aspen genetic individual and fitness may actually improve with time. Thus, for comparisons with senescence in unitary organisms, the closest approximation is among clones, i.e., how different microbial clones behave over time.

The trajectory of a given clone should also be followed, as judged by its replicative potential at various ages (as evidenced by generation time or in competition assays between representatives of younger vs. older populations). For example, the long-term evolutionary dynamics of 12 subpopulations of E. coli cultured from a common ancestor have been studied since 1988 by Lenski and colleagues who find, among other things, that fitness apparently increases ‘without bound’ (Wiser et al. 2013). To date this remarkable experiment has proceeded over some 30 years, >50,000 bacterial generations, and is ongoing. There is no evidence from such work that these bacteria senesce.

Yeasts and filamentous fungi. The classic phenomenon of replicative senescence in some yeasts is, at least superficially, similar to so-called ‘aging’ in bacteria noted above. Some 60 years ago, Mortimer and Johnson (1959) determined that although the budding yeast Saccharomyces cerevisiae continues to divide indefinitely when cultured under favorable conditions, the individual ‘mother’ cells have a finite ‘replicative life span’. Many ensuing studies have embellished this basic point (see Steinkraus et al. 2008; Henderson and Gottschling 2008).

In budding yeasts (as opposed to fission yeasts, below), the result of division is two morphologically different cells, a mother and a daughter that originate from the bud (i.e., replication is clearly asymmetrical, though aberrant division occurs in the oldest mothers resulting in indistinguishable mothers and daughters; Steinkraus et al. 2008). Mortimer and Johnson (1959) followed the replicative history of 36 mother cells by micromanipulation and microscopy. They reported a mean life span of ~24 (±8 S.D.) generations and also noted a progressively longer generation time as mothers aged, with early generations taking 60-100 min and late ones up to 6 h.

When budding ceased there was visible evidence (granularity, lysis) that most mothers died (Subsequent work has shown that such post-replicative cells can remain in a viable and metabolically active state for days and given rise to the term ‘chronological life span’; Fabrizio and Longo 2003; Steinkraus et al. 2008). Interestingly, the replicative age of the mother is not passed to daughters early in her life span though in the latter half of their lives mothers produce daughters with progressively shorter replicative life spans. There are numerous hypotheses to explain such observations and to account for a presumed ‘senescence factor’ (Henderson and Gottschling 2008).

Using a different yeast model wherein the rod-shaped organism divides into two superficially identical daughters much as does E. coli, Coelho et al. (2013) report the absence of aging in the fission yeast Schizosaccharomyces pombe under standard culture conditions. Lineages followed by time-lapse microscopy showed no progressive increase in replication time or mortality rate. A mutant cell line that divided off-center into larger and smaller cells likewise showed no increase in division time.

Cell death was random, not preceded by aging phenomena, and correlated with inheritance of protein aggregates that possibly interrupt cytokinesis or formation of the cell walls in the daughters. Cells exposed to heat or oxidation treatments to simulate environmental stress underwent asymmetry in aggregate segregation whereby the lineage inheriting the large aggregates aged whereas their sisters inheriting few such aggregates did not age. The authors suggest that asymmetrically induced segregation of damage has evolved to partition damage into a cell line that is sacrificed so that the other escapes.

In the filamentous fungi, intraclonal ‘aging’ or localized regression or senescence of ramets analogous to that in degenerating and regenerating benthic invertebrate clones unquestionably occurs (Trinci and Thurston 1976). On balance the evidence is against senescence of an entire clone. In the absence of robust demographic data, inferences must be made from the characteristics in culture or estimated age and size (terrain occupancy) in nature. Extensive and extremely old clones of various fungi have been mapped (see Chaps. 4 and 5 and Dickman and Cook 1989; Smith et al. 1992; Bendel et al. 2006). However, as noted at the outset of this section, increasing evidence shows that length of life and senescence are poorly correlated (Baudisch et al. 2013; Jones et al. 2014).

Furthermore, in many if not most circumstances the vast and occasionally global dispersal of fungal clones, typically by asexual spores, means that clones are discontinuous and their full extent impossible to estimate accurately (Kohli et al. 1992; Goodwin et al. 1994; Anderson and Kohn 1998). It was established long ago from continuous growth experiments in so-called racing tubes (Ryan et al. 1943; Gillie 1968) that clones can grow indefinitely (Fawcett 1925; Perkins and Turner 1988; Gow and Gadd 1995), although not necessarily continuously (Bertrand et al. 1968).

There is evidence from laboratory culture that strains of some fungi senesce (Griffiths 1992; Griffiths and Yang 1993); in Podospora anserina the phenomenon has been attributed to mitochondrial DNA instability (Albert and Sellem 2002) and similarly aberrant lines have been associated with cytoplasmically transmissible factors (Bertrand 2000). Senescence in this coprophilic (dung- inhabiting) fungus has been ascribed possibly to its colonization of an ephemeral resource (Geydan et al. 2012; van Diepeningen et al. 2014).

Diatoms. The clonal dynamics of diatoms resemble bacteria and are very interesting. Unlike bacteria, however, each diatom cell is covered by a rigid silica wall (frustule) formed in two components, the slightly larger ‘lid’ (epitheca) overlapping the smaller diameter ‘bottom’ (hypotheca) somewhat like a petri dish. At division, one progeny cell receives the parental epitheca and regenerates a new hypotheca. Hence this cell is the same size as the parent, as will be one of its descendants at each subsequent cell division.

The other daughter cell receives the smaller hypotheca, which becomes its epitheca, and a new hypotheca is generated. This cell is thus smaller than the parent and will give rise to a lineage of sequentially smaller cells (Yanagita 1977). The consequence of this asexual division protocol is the rapid dilution of the larger daughter lineage by the successively smaller. Eventually a threshold in the lineage of progressively smaller cells is reached that triggers either sexual reproduction, whereupon cell size is restored, or the cells become critically small and die (Chepurnov et al. 2004).

Of course either event marks the end of that clonal progression. Time-lapse imagery of a strain of the centric diatom Ditylum brightwellii showed that the smaller (hypothecal) lineage actually inherited more and possibly ‘better’ parental material at each division than the epithe- cal lineage, and that it divided about 4% faster (Laney et al. 2012). This shortens the average interval between rounds of sexual reproduction and the authors suggest may be a strategy to increase representation of the ancestral genome in a population. The generality (other strains and species) and realism (applicability in nature) of these intriguing findings await further work. Cultural studies suggest that some diatoms exist for exceptionally long periods as asexual populations; however, the population dynamics and rates of mortality over time of such clones await further study. Therefore, there is no evidence for clonal senescence, though such diatom data are embedded in the senescence literature. Even if there were, this form of ‘senescence’ would appear to be unique, unlike senescence as construed in unitary organisms.

Protozoa. The amoebozoa and alveolates such as Paramecium reproduce asexually by dividing in half, as well as sexually. The evidence for senescence in this group is mixed. Some, perhaps most, species display abnormalities that approximate senescence if cultured over long periods of time, though lineages are rejuvenated by sexual reproduction. Other species persist with little if any evidence for declining rates of fission. Woodruff (1926), in what is now a classic experiment, grew a single culture of P aurelia asexually for >11,000 generations. He sampled the culture periodically and found no evidence for a sustained decline, rather (p. 437) “that there are inherent, normal [his emphasis], minor, periodic accelerations and depressions of the fission rate due to some unknown factor in cell phenomena.”

Other ciliates such as Tetrahymena are referred to as ‘immortal’. Bell (1988b) in a definitive assessment of the early work in protozoology concluded that the general trend for species in fission rate was negative but that some cultures can be propagated for thousands of generations without perceptible decline and that “although very general, senescent decline is not universal” (p. 43). He (and others) interpret metazoan senescence as being mechanistically different from protozoan senescence, the former ascribed to an unavoidable consequence of selection for an optimal life history, and the latter a nonadaptive consequence of accumulating deleterious mutations reflecting Muller’s ratchet (Chap. 2).