Steve Smith
email: steve.smith@vetmeduni.ac.atorcid

Franz Hoelzl

Sandrine Zahn

François Criscuolo

Telomerase activity in ecological studies: what are its consequences for individual physiology and is there evidence for effects and trade-offs in wild populations

Keywords: Telomeres, trade-off, immunity, reproductive effort, life history

Increasing evidence at the cell level highlights the balance between investment in growth, reproduction and somatic maintenance in wild populations. Studies of telomere dynamics have informed researchers about the loss and gain of telomere length both on a seasonal scale and across the lifespan of individuals. In addition, telomere length and telomere rate of loss seems to have evolved differently among taxonomic groups, and relate differently to organismal diversity of lifespan.

So far, the mechanisms behind telomere maintenance remain elusive, although many studies have inferred a role for telomerase, an enzyme/RNA complex known to induce telomere elongation from lab studies. Exciting further work is also emerging that suggests telomerase (and/or its individual component parts) has a role in fitness that goes beyond the maintenance of telomere length. Here we review the literature on telomerase biology and examine the evidence from ecological studies for the timing and extent of telomerase activation in relation to life history events associated with telomere maintenance. We suggest that the underlying mechanism is more complicated than originally anticipated, possibly involves several complimentary pathways, and is likely associated with high energetic costs. Potential pathways for future research are numerous and we outline what we see as the most promising prospects to expand our understanding of individual differences in immunity or reproduction efficiency.

Introduction

The burgeoning field of telomere dynamics has added new perspectives and directions to the study of ecology and evolutionary biology. The interest is testament to the fact that telomeres represent an intriguing biomarker that provides the link between individual physiology, life history, and fitness. As the field has developed, so too has the realisation that the maintenance of telomeres is at the centre of this balance. Studies so far, both experimental [START_REF] Herborn | Stress exposure in early post-natal life reduces telomere length: an experimental demonstration in a long-lived seabird[END_REF][START_REF] Nettle | An experimental demonstration that early-life competitive disadvantage accelerates telomere loss[END_REF] and correlational (Hoelzl, Cornils, Smith, Moodley, & Ruf, 2016a;[START_REF] Turbill | Daily torpor is associated with telomere length change over winter in Djungarian hamsters[END_REF], indicate that telomere restoration is costly (reviewed in [START_REF] Young | The role of telomeres in the mechanisms and evolution of life-history trade-offs and ageing[END_REF]. However, the mechanisms underlying telomere maintenance remain elusive and evolutionary biologists have turned to clinical research for answers. The obvious candidate as regulator within the system is the enzyme complex telomerase. Telomerase comprises an RNA component (TERC) and a catalytic protein domain (TERT) essential for the reverse transcriptase activity of the enzyme (see Box 1). Telomerase is known to catalyse the elongation and maintenance of telomeres by adding telomeric repeats to the chromosomal ends and thus counteracting losses that occur during end replication and oxidative stress [START_REF] Harley | Telomeres shorten during ageing of human fibroblasts[END_REF][START_REF] Von Zglinicki | Oxidative stress shortens telomeres[END_REF]. For most organisms, telomerase is not active throughout all life stages and across all tissue types [START_REF] Haussmann | Telomerase Expression Is Differentially Regulated In Birds Of Differing Life Span[END_REF][START_REF] Lingner | Reverse Transcriptase Motifs in the Catalytic Subunit of Telomerase[END_REF][START_REF] Wright | Telomerase activity in human germline and embryonic tissues and cells[END_REF], strongly suggesting tradeoffs between life history stages such as growth, maintenance and reproduction. As a potential regulator of these trade-offs, measurement telomerase is of increasing interest for evolutionary biologists trying to understand the process of senescence (see Box 2).

Box 1

The structure and function of telomerase -Telomerase in its active state is composed of a DNA polymerase enzyme complex (telomerase reverse transcriptase; TERT) that carries its own RNA template (telomerase RNA component; TERC, TER or TR) to add telomeric repeats to the ends of chromosomes.

-In vertebrates, TERT is composed of the essential telomerase N-terminal domain (TEN), the C-terminal extension domain (CTE), the telomerase RNA-binding domain (TRBD) and the reverse transcriptase domain (RT). The TEN domain stabilizes the DNA-RNA duplex in the active site of the enzyme [START_REF] Akiyama | The telomerase essential N-terminal domain promotes DNA synthesis by stabilizing short RNA-DNA hybrids[END_REF], CTE is necessary for stable binding of the enzyme to the DNA [START_REF] Hossain | Functional Analysis of the C-terminal Extension of Telomerase Reverse Transcriptase. A Putative "Thumb" Domain[END_REF], TRBD binds the RNA-template (Lai et al, 2001) and RT is the catalytic domain [START_REF] Dey | Current Perspectives of Telomerase Structure and Function in Eukaryotes with Emerging Views on Telomerase in Human Parasites[END_REF].

-TERC is considered a non-coding RNA that contains the template region for adding telomeric repeats to the ends of chromosomes. Its total size is highly variable between taxa and ranges from 133 nucleotides (nt) in the fish Nothobranchius furzeri to over 2000 nt in the fungus Neurospora crassa (http://telomerase.asu.edu/; [START_REF] Podlevsky | The Telomerase Database[END_REF]. In vertebrates, the template-RNA is flanked by the template boundary element (TBE) at the 5' side. The pseudoknot (PK) is located downstream of the template and is essential for TERT-TERC interaction and function of the holoenzyme. Furthermore, TERC contains several structures that interact with TERT and other proteins (e.g. dyskerin protein complex) to ensure functionality (reviewed in [START_REF] Logeswaran | Monophyletic Origin and Divergent Evolution of Animal Telomerase RNA[END_REF]; http://telomerase.asu.edu/).

-Telomeric repeat variants (e.g. TTAGGG -in vertebrates) differ between taxonomic groups. Correspondingly, both TERT and TERC have a distinct composition of subunits and regions in different taxa. In insects for example, TERT lacks the TEN domain while nematodes lack TEN and large fractions of CTE (reviewed in [START_REF] Mason | Telomerase structure function[END_REF]. In TERC, the arrangement and composition of elements (PK, TBE, ect.) is variable between taxa, but even more pronounced, the sequence and structure within these elements is highly variable between taxonomic groups [START_REF] Logeswaran | Monophyletic Origin and Divergent Evolution of Animal Telomerase RNA[END_REF].

-Trafficking of TERT and TERC between nucleus and cytoplasm is not fully understood. In yeast (Saccharomyces cervicia), the assembly of the telomerase holoenzyme likely takes place in the cytoplasm before it is imported into the nucleus, while in humans, TERT and TERC are separated in the nucleus during most of the cell cycle and are only assembled during S phase. Such separation is likely to be necessary to avoid unregulated telomerase activity (reviewed in [START_REF] Gallardo | Telomerase biogenesis. The long road before getting to the end[END_REF]. Telomerase activity is detectable throughout the cell cycle but only a small fraction of TERC is colocalized with telomeres during S phase (the stage where telomere elongation takes place in vivo), suggesting a low elongation rate per cell cycle (this is true for both, human and yeast; [START_REF] Gallardo | Telomerase biogenesis. The long road before getting to the end[END_REF][START_REF] Teixeira | Telomere Length Homeostasis Is Achieved via a Switch between Telomerase-Extendible and -Nonextendible States[END_REF].

-A study by [START_REF] Schmidt | Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres[END_REF] reveals that telomerase diffuses throughout the nucleoplasm and that the enzyme frequently binds TPP1 (a protein of the shelterin complex) at telomeres. These bindings occur thousands of times per S phase, but only a very limited number of these probing events are sufficiently long to allow telomere elongation.

-After telomerase is guided to the chromosomal end, and forms stable "bonds", elongation is carried out only on the G-rich overhang (either resulting from terminal RNA primer removal on the lagging strand, or by exonuclease dependent resection, reviewed in Procházková [START_REF] Procházková Schrumpfová | Composition and Function of Telomerase-A Polymerase Associated with the Origin of Eukaryotes[END_REF]. Therefore, telomerase binds to the distal nucleotides of these overhangs and adds a new telomeric repeat (in vertebrates: GGTTAG). This elongation step can be repeated multiple times via a translocation mechanism and DNA Pol [alpha]-primase can fill the complementary strand [START_REF] Diede | Telomerase-Mediated Telomere Addition In Vivo Requires DNA Primase and DNA Polymerases α and ẟ[END_REF].

-In multicellular organisms, telomerase activity is usually detected in all early developmental stages. After organogenesis, telomerase activity is restricted to proliferating stem cells, while it is downregulated in somatic cells (reviewed in Procházková Schrumpfová, Fojtová, & Fajkus, 2019). In mammals, the tendency to downregulate somatic telomerase activity is body mass dependent, where species of weight smaller than 1 kg (e.g. mice) keep telomerase activity throughout their life, while larger animals downregulate telomerase in somatic tissues (likely to reduce the risk of cancer; [START_REF] Seluanov | Telomerase activity coevolves with body mass, not lifespan[END_REF].

Senescence is defined in evolutionary biology as the relaxation of natural selection at old-ages, attributed to the reduced fitness contribution of older breeders due to poor reproductive performances and survival rates [START_REF] Gaillard | An integrative view of senescence in nature[END_REF]. Williams' theory of senescence stipulates that it should be found in all living organisms that have, among other prerequisites [START_REF] Gaillard | The Williams' legacy: A critical reappraisal of his nine predictions about the evolution of senescence[END_REF], genes that will be beneficial early in life and detrimental later on (i.e. (the antagonistic pleiotropy of aging, [START_REF] Williams | Pleiotropy, natural selection, and the evolution of senescence[END_REF]. At a more mechanistic level, senescence, which promotes cell division arrest and apoptosis, could be considered as a cell response to aging stress, with both positive and negative impacts on individual fitness. For instance, cell senescence also promotes regular tissue repair, and protects against cell immortalization and cancer, while at the same time triggering proinflammatory or tissue degeneration processes (reviewed in [START_REF] Autexier | The Structure and Function of Telomerase Reverse Transcriptase[END_REF][START_REF] Campisi | Aging, Cellular Senescence, and Cancer[END_REF].

Because telomeres and telomerase stand at the crossroad of these cell mechanisms, we propose here that the study of telomerase will benefit from being considered in the context of its pleiotropic effects on individual fitness and through its modulation of life-history trade-offs. To do so, we highlight that (i) telomerase genes vary in sequences and expression, (ii) telomerase may act via non-telomererelated pathways, (iii) telomerase may have pleiotropic effects due to dynamic changes in expression and thus mediate trade-offs among traits. We then address what could be the costs associated to telomerase activation, and propose a few avenues for studying the putative telomerase -fitness link.

Box 2: Measurement of telomerase activity: previous studies and methods employed

For more than a decade, evolutionary ecologists have focussed on telomeres [START_REF] Haussmann | Telomere length provides a new technique for aging animals[END_REF], and very rapidly to telomerase activity [START_REF] Haussmann | Telomerase activity is maintained throughout the lifespan of long-lived birds[END_REF], in order to better understand individual variability in lifespan [START_REF] Haussmann | Longer telomeres associated with higher survival in birds[END_REF]. However, evaluations of telomerase activity are not a simple prospect for most of the species of ecological interest. Logistical constraints and/or specific expertise requirements are the major challenges. For instance, storage and pre-treatment of samples to preserve telomerase activity is inherently complicated ❶. Thus the development of reliable telomerase activity (TA) assessment methodologies is essential to allow research in evolutionary biology to develop and understand covariation of age-or tissue-related telomerase activity with animal life-histories. This schematic representation shows the most common methods employed to measure TA in human and cancer research. The methods are presented following two main categories: ❷ detection of telomerase products and ❸ amplification of these products (TRAP-based methods). Figure is adapted from [START_REF] Mensà | The telomere world and aging: Analytical challenges and future perspectives[END_REF].

The Ecological Link to Telomerase

Early studies of telomere dynamics in non-model organisms reported individual differences in telomere attrition related to age [START_REF] Aubert | Telomere Dynamics and Aging[END_REF]2020), birth order [START_REF] Noguera | Embryonic and postnatal telomere length decrease with ovulation order within clutches[END_REF], birth timing [START_REF] Eisenberg | Delayed paternal age of reproduction in humans is associated with longer telomeres across two generations of descendants[END_REF], early life stress [START_REF] Cram | Effects of early-life competition and maternal nutrition on telomere lengths in wild meerkats[END_REF][START_REF] Price | Telomeres and Early-Life Stress: An Overview[END_REF], reproductive output [START_REF] Beaulieu | Oxidative status and telomere length in a long-lived bird facing a costly reproductive event[END_REF][START_REF] Monaghan | Do telomere dynamics link lifestyle and lifespan?[END_REF], rewarming during hibernation [START_REF] Hoelzl | Telomeres are elongated in older individuals in a hibernating rodent, the edible dormouse (Glis glis)[END_REF], and habitat suitability [START_REF] Angelier | Telomere length, non-breeding habitat and return rate in male American redstarts[END_REF] among others. After this initial interest in telomeres as a reliable biomarker for stress and biological aging, evidence began to emerge that, in some species at least, telomere length could be actively restored or even elongated [START_REF] Foley | Growing old, yet staying young: The role of telomeres in bats' exceptional longevity[END_REF][START_REF] Haussmann | Telomeres shorten more slowly in long-lived birds and mammals than in short-lived ones[END_REF][START_REF] Hoelzl | Telomeres are elongated in older individuals in a hibernating rodent, the edible dormouse (Glis glis)[END_REF]. Despite initial scepticism, further studies confirmed active telomere maintenance across multiple species. Telomerase activity was considered the most likely mechanism responsible for telomeric restoration and once again ecological researchers borrowed from clinical fields and cancer research to find the best way to measure telomerase activity in an ecological setting (see Box 2). Subsequent work has implicated an association between telomerase activity levels and telomere dynamics in a broad range of taxa. [START_REF] Mu | Season-Specific Changes in Telomere Length and Telomerase Activity in Chinese Pine (Pinus tabulaeformis Carr.)[END_REF] found a link between seasonal leaf growth, telomere length and telomerase activity in the Chinese Pine (Pinus tabulaeformis). Interestingly, telomere length also increased with temperature throughout the year whereas telomerase activity showed a negative association, suggesting a potential lag between stimulus and response. A strong association between telomerase activity and telomere length was observed in multiple strains of zebra fish with a marked drop in both detected in older individuals [START_REF] Anchelin | Behaviour of Telomere and Telomerase during Aging and Regeneration in Zebrafish[END_REF]. In a follow-up experimental study [START_REF] Anchelin | Premature aging in telomerase-deficient zebrafish[END_REF], telomerase-knockdown zebra fish showed premature aging and reduced lifespan in the first generation. Restoration of telomerase activity in the second generation led to rescue of telomere length and survival confirming the causal role of telomerase in the association. Likewise, [START_REF] Hatakeyama | Telomere attrition and restoration in the normal teleost Oryzias latipes are linked to growth rate and telomerase activity at each life stage[END_REF] demonstrated in Japanese medaka (Oryzias latipes) that telomere attrition and restoration are linked to growth and telomerase activity respectively, and that a critical loss of telomere homeostasis is associated with mortality.

Recently, an intriguing link with embryo corticosterone levels was shown in yellow-legged gulls (Noguera et al. 2020). Hatchlings from corticosterone-injected eggs were shown to have both higher levels of telomerase activity and longer telomeres. Telomerase was also shown to induce the synthesis and repair of TTAGG-telomeric repeats in Lepidoptera (Gong et al. 2015) and this association seems to be widespread in most orders of insects (Korandova et al. 2014). Combined, these studies largely demonstrate that telomere length and telomerase is closely coupled as expected based on in vitro studies and tightly regulated by environmental factors.

Crucially though, many studies have not detected the expected link between telomere maintenance and telomerase activity (see citations in Supp. Table 1). Clinical studies in humans and experimental work show that in many conditions, telomere length is decoupled from telomerase activity [START_REF] Xie | Early telomerase inactivation accelerates aging independently of telomere length[END_REF] and that telomerase acts in diverse functions at cellular and physiological levels (de Punder, [START_REF] De Punder | Stress and immunosenescence: The role of telomerase[END_REF][START_REF] Haendeler | Mitochondrial Telomerase Reverse Transcriptase Binds to and Protects Mitochondrial DNA and Function From Damage[END_REF]Sahin & DePinho, 2012;[START_REF] Saretzki | Telomerase, mitochondria and oxidative stress[END_REF].

Work in honeybees (Apis mellifera) also could not find a link between telomere length and telomerase activity, which was found to be regulated in a developmental and caste-specific manner [START_REF] Korandová | Activity of telomerase and telomeric length in Apis mellifera[END_REF]; see also [START_REF] Koubová | Telomerase activity is upregulated in the fat bodies of pre-diapause bumblebee queens (Bombus terrestris)[END_REF]. The inconsistent findings from studies of the telomere -telomerase relationship raise questions about the ubiquity of a single telomere repair mechanism but, perhaps more interestingly, also suggests alternative roles for telomerase and its subunits in life history trade-offs. Resolving the links in these trade-offs promises to open the door to exciting avenues of research in ecology and evolutionary biology.

Linking life-history trade-offs and individual fitness with telomerase?

The essential requirement for a biological variable to play a role in evolution is to show variation among individuals in a population (along with transmission to the next generation and conferring fitness effect). Telomerase is formed via the assemblage of two main subunits coded by highly conserved genes (TERT, TERC), which are also characterized by a recent evolution history [START_REF] Hrdličková | Insights into the evolution of mammalian telomerase: Platypus TERT shares similarities with genes of birds and other reptiles and localizes on sex chromosomes[END_REF]. More importantly, polymorphisms of those genes do exist in humans, their discovery originating from the study of telomere diseases such as congenital dyskeratosis [START_REF] Savage | TINF2, a Component of the Shelterin Telomere Protection Complex, Is Mutated in Dyskeratosis Congenita[END_REF][START_REF] Vulliamy | The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita[END_REF]. In those patients, the RNA component of the telomerase (TERC) and an associated regulating protein (dyskerin, DCK1 gene) are mutated, leading to defective telomere maintenance by telomerase and to premature ageing phenotypes [START_REF] Marrone | Dyskeratosis congenita: molecular insights into telomerase function, ageing and cancer[END_REF]. Human TERT mutations were also found to be associated with different, rather late-life, diseases [START_REF] Calado | Telomeres in disease[END_REF], and TERT single nucleotide polymorphisms (SNPs) have been identified in humans, often associated with an increase in cancer risks (Rafnar et al., 2009, together with shortened telomeres in this study), or bone marrow failure [START_REF] Yamaguchi | Mutations in TERT, the Gene for Telomerase Reverse Transcriptase, in Aplastic Anemia[END_REF]. Enhanced telomerase activity is found in more than 9 out of 10 cancer cells [START_REF] Shay | A Survey of Telomerase Activity in Human Cancer[END_REF]. So how could telomerase defects enhance cancer risks? Causality is thought to relate to the capacity of hematopoietic cells (and then immune cells) to up-regulate telomerase when activated.

Given that the level of TERT expression may be the rate-limiting mechanisms of control of telomerase activity [START_REF] Counter | Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase[END_REF], TERT deleterious mutations may shorten telomere length of leucocytes, and then decrease the ability of the immune system to survey cell immortalization and eliminate cancer cells [START_REF] Calado | Telomeres in disease[END_REF]. Therefore, focusing on the individual variation in TERT expression and telomerase activity in white-blood cells, and characterising how it may modulate immunosenescence [START_REF] De Punder | Stress and immunosenescence: The role of telomerase[END_REF], may provide the first insights into the telomerase -fitness relationship. In addition, telomerase polymorphisms have been previously associated with non-cancer diseases, with significantly modified soma protective effects [START_REF] Snetselaar | Telomerase Reverse Transcriptase Polymorphism rs2736100: A Balancing Act between Cancer and Non-Cancer Disease, a Meta-Analysis[END_REF]. TERC variants were shown to have different longevities in humans [START_REF] Soerensen | Genetic variation in TERT and TERC and human leukocyte telomere length and longevity: a cross-sectional and longitudinal analysis[END_REF], thereby suggesting that some telomerase mutations may carry putative fitness benefits.

Telomerase: a two-headed coin with telomere length and non-telomere length sides

The studies cited above mainly explain their results based on the protective action of telomerase on telomeres, which is of prime interest for biologists seeking for telomerase -fitness relationships but also renders difficult any conclusion on telomerase effect per se. However, noncanonical effects of telomerase do exist (i.e. not mediated by the maintenance of telomere length) and should be taken into account to explore further the potential fitness benefits provided by this enzyme.

Insights for the putative importance of these non-canonical roles in shaping life-histories were recently illustrated in an interspecific comparison study of TERT sequences and expressions [START_REF] Lai | The protein subunit of telomerase displays patterns of dynamic evolution and conservation across different metazoan taxa[END_REF].

Highly variable patterns of alternative splicing of the catalytic subunit TERT was described even in closely related species, suggesting that different functions of telomerase may have evolved in relation to lineage specific life histories [START_REF] Lai | The protein subunit of telomerase displays patterns of dynamic evolution and conservation across different metazoan taxa[END_REF]. However, to date, the nature of those non-canonical roles (and their control by alternative splicing) are almost exclusively studied in humans and seem restricted to a few main impacts of TERT expression: mitochondrial functioning, enhanced cell proliferation (via the activation of growth factor signalisation pathway), control of the gene expression (not only oncogenes, e.g. immune genes) and regulation of telomerase activity during early-life development [START_REF] De Punder | Stress and immunosenescence: The role of telomerase[END_REF][START_REF] Smith | Telomerase modulates expression of growthcontrolling genes and enhances cell proliferation[END_REF][START_REF] Ulaner | Telomerase Activity in Human Development Is Regulated by Human Telomerase Reverse Transcriptase (hTERT) Transcription and by Alternate Splicing of hTERT Transcripts[END_REF][START_REF] Xiang | hTERT Can Function with Rabbit Telomerase RNA: Regulation of Gene Expression and Attenuation of Apoptosis[END_REF]. Even if we need additional aspects of telomerase activity or TERT to be better described (like non-telomeric DNA maintenance, [START_REF] Gorbunova | Expression of Human Telomerase (hTERT) Does Not Prevent Stress-induced Senescence in Normal Human Fibroblasts but Protects the Cells from Stress-induced Apoptosis and Necrosis[END_REF], those observations are of broad interest for evolutionary biologists. They suggests that either due to genetic variance or post-transcriptional regulation, individuals may differently react to environmental factors, for which the physiological response needs the activation of TERT expression and/or telomerase activity. One intriguing observation was made in planarian worms, an animal showing tissue regeneration capacity and asexual or sexual reproduction. In this model, alternative splicing of TERT seems to regulate the mechanisms of telomere elongation either in the somatic cells during fission (asexual reproduction) or in the germ-line cells during sexual reproduction [START_REF] Tan | Telomere maintenance and telomerase activity are differentially regulated in asexual and sexual worms[END_REF]. Together with similar observations in colonial ascidians or urochordate animals, it suggests that variation in telomerase expression has coevolved with specific life-history traits like mode of reproduction [START_REF] Lai | The protein subunit of telomerase displays patterns of dynamic evolution and conservation across different metazoan taxa[END_REF], extreme lifespan or somatic regeneration capacity (Tan et al., 2012 and herein cited references).

Tackling the telomerase -fitness link from its pleiotropic nature

We have briefly introduced so far that the role of telomerase may go beyond its accepted contribution to the maintenance of telomere length (non-canonical impacts of the TERT or TERC subunits), which may be associated with species' traits. However, to tackle the telomerase -fitness hypothesis, we may also reconsider the question through the prism of its main period of high activity (at least in most birds and mammals), early-life development, and of its primary role in cell replication.

Could telomerase have a dual and age-related impact on fitness, via individual health and lifespan components? By favouring developmental processes and cell renewal in early life, do telomerase activity and non-canonical TERT/TERC expression have positive effects on individual reproductive output, while leading to increased cancer risks in later life? When thinking of such pleiotropies of telomerase and fitness relationships, the first objective of future studies should be to evaluate how early and late-life expression of telomerase may modulate individual fitness at young, reproductive and post-reproductive adult stages. For instance, TERT alternative splicing is recognized as being involved in modulating telomerase activity during development in humans, but only its consequence in terms of telomere length has so far been evaluated (reviewed in [START_REF] Shay | Telomeres and telomerase: three decades of progress[END_REF]. This question needs to be re-evaluated in biological models where individual health and fitness can be measured over life. Along the same lines, the pleiotropies of non-canonical effects should also be studied. In fact, TERT ectopic expression in mice fibroblasts triggers cell growth by repressing growth-inhibiting pathways [START_REF] Geserick | Expression of mTert in primary murine cells links the growth-promoting effects of telomerase to transforming growth factor-β signaling[END_REF]. This suggests that individual variation in TERT expression may underline individual variation in growth rates, and thereby contribute to the growth -lifespan trade-off [START_REF] Metcalfe | Growth versus lifespan: perspectives from evolutionary ecology[END_REF].

Tackling the telomerase -fitness link from its dynamic nature

An important second way of studying telomerase in an evolutionary context will be to search for acute telomerase activity or TERT expression in response to stress under natural conditions.

Telomerase is mainly described as active during embryogenesis and early-life in most animals [START_REF] Bekaert | Telomere biology in mammalian germ cells and during development[END_REF][START_REF] Prowse | Developmental and tissue-specific regulation of mouse telomerase and telomere length[END_REF], and then is shutdown likely to avoid cancer-risks [START_REF] Shay | Telomeres and telomerase: three decades of progress[END_REF]. However, this general view has been regularly amended by data collected on several taxa, suggesting that telomerase activity could be maintained at adulthood in certain conditions. For instance, beside the maintained telomerase activity in the soma of insects, lizards or fish (reviewed in [START_REF] Gomes | Telomere biology in Metazoa[END_REF], repression of telomerase activity was not found as an unalterable characteristic in mammals and birds. Telomerase activity actually covaries (negatively) with species body mass in rodents [START_REF] Gorbunova | Coevolution of telomerase activity and body mass in mammals: From mice to beavers[END_REF] and life-long telomerase activity characterizes some long-lived small seabirds [START_REF] Haussmann | Telomerase activity is maintained throughout the lifespan of long-lived birds[END_REF], while its expected positive impact on telomere length was described in hibernating (Hoelzl et al., 2016a;[START_REF] Turbill | Seasonal variation in telomere length of a hibernating rodent[END_REF] as well as in non-hibernating species [START_REF] Criscuolo | Seasonal variation in telomere dynamics in African striped mice[END_REF][START_REF] Fairlie | Lifelong leukocyte telomere dynamics and survival in a free-living mammal[END_REF]. The latter studies highlighted that telomerase may be turned-on at certain life stages, likely in some particular cells (e.g. stem or hematopoietic cells) and raises the question of whether telomerase forms part of the response-repertoire an organism possesses to face stress [START_REF] Beery | Chronic stress elevates telomerase activity in rats[END_REF], or which may constrain adaptive abilities [START_REF] Choi | Reduced telomerase activity in human T lymphocytes exposed to cortisol[END_REF]. Given the ability of immune cells to express high telomerase activity when activated [START_REF] Bodnar | Mechanism of Telomerase Induction during T Cell Activation[END_REF][START_REF] Hiyama | Activation of Telomerase in Human Lymphocytes and Hematopoietic Progenitor Cells[END_REF][START_REF] Yamada | Up-regulation of telomerase activity in human lymphocytes[END_REF], future studies would benefit from a focus on immune cells, and how changes in telomerase activity (i.e. increases) vary at the individual level in response to immune challenge and what are the collateral effects on other somatic tissues. Still, while exploratory, those studies will probably uncover only part of the global picture as they will focus solely on the dynamics of telomerase activity. Adding the non-canonical functions of telomerase via the characterization of TERT / TERC dynamics of expression over age and in response to stress is vitally important to establish how telomerase may affect individual fitness or bestow individuals with specific life-history trait associations.

Telomerase as a mediator of the reproductive trade-off

The different age-related risks of telomerase activity have so far been conceptualized based on our present knowledge of telomerase implication in cell-growth or renewal processes [START_REF] Morrison | Telomerase Activity in Hematopoietic Cells Is Associated with Self-Renewal Potential[END_REF] and in cell immortalization and the associated cancer risk [START_REF] Tian | Evolution of telomere maintenance and tumour suppressor mechanisms across mammals[END_REF]. However, the risk has never been considered in relation to the cost of reproduction in adults, neither for telomerase activity nor at the level of subunit expression and of their non-canonical effects.

When studying the cost of reproduction, defined as decreased future reproductive and survival rates [START_REF] Stearns | The evolution of life histories[END_REF], one may consider telomerase as one of the pleiotropic regulators that may affect adult breeders current success (via the maintenance of telomere length) and their future prospects (via the non-canonical TERT / TERC protective effects, see below). Because the cost of reproduction may be mediated through telomere erosion or other cell defects of the adult soma and of gametes, using telomerase as the response variable will extend our understanding of reproductive costs beyond the "Y model" of energy allocation between reproduction and lifespan (reviewed in [START_REF] Harshman | The cost of reproduction: the devil in the details[END_REF]. Such a possibility has been illustrated in a single cell-organism, yeast, where a role for telomerase in response to DNA replication stress, but also in additional maternal cell ageing, was highlighted [START_REF] Xie | Early telomerase inactivation accelerates aging independently of telomere length[END_REF]. Such a telomerase-based fitness component may also relate to the regulation by telomerase of stem cells capacity to be mobilized and punctually proliferate [START_REF] Calado | Telomerase: not just for the elongation of telomeres[END_REF], thereby modulating cell renewal rate and organ performance in breeders. Again, focusing on the immune system, as it is the somatic tissue where telomerase activity is more likely to be recorded, seems the best alternative if we are to evaluate how telomerase may mediate the ageing outcomes of reproduction [START_REF] Schulenburg | Introduction. Ecological immunology[END_REF].

Evidences for a cost of telomerase activation

The first cost to consider for telomerase activity in an evolutionary biology sense is energetic.

A previous review on the role of telomeres in the evolution of life-history traits underlined the importance (and lack) of obtaining accurate data on how costly it is for a cell, and thereby for an organism, to maintain telomere length (and thus indirectly in activating telomerase, [START_REF] Young | The role of telomeres in the mechanisms and evolution of life-history trade-offs and ageing[END_REF].

Ecophysiological studies have suggested that maintenance of telomere length may be interpreted as energetically costly since, for instance, food supply during bad years allowed better telomere maintenance in free-living edible dormice (Glis glis), (Hoelzl et al., 2016a;[START_REF] Nowack | Always a price to pay: Hibernation at low temperatures comes with a trade-off between energy savings and telomere damage[END_REF]. Still, those studies did not disentangle the specific impact of telomerase activation from telomere lengthening. The current absence of clear data showing increased ATP consumption associated with telomerase activity allows an alternative view that telomerase may modulate in return the efficiency of the cellular energy-generating machinery.

The intriguing telomerase and mitochondrial relationship

Interestingly, among the non-canonical cell functions of telomerase so far described (reviewed in [START_REF] Arndt | New prospects for targeting telomerase beyond the telomere[END_REF], one intriguing finding is the mitochondrial localization of the TERTtelomerase subunit, recurrently reported by independent research groups [START_REF] Ahmed | Telomerase does not counteract telomere shortening but protects mitochondrial function under oxidative stress[END_REF][START_REF] Haendeler | Mitochondrial Telomerase Reverse Transcriptase Binds to and Protects Mitochondrial DNA and Function From Damage[END_REF][START_REF] Moslehi | Telomeres and Mitochondria in the Aging Heart[END_REF]). This appears to be of interest for studying the TERT specific role in the context of life-history trade-offs, since the mitochondria stands at the crossroads of the production of cell energy (i.e. ATP) and of deleterious by-products of cell respiration (i.e. reactive oxygen species (ROS) [START_REF] Criscuolo | Mitochondrial Uncoupling Proteins: New Perspectives for Evolutionary Ecologists[END_REF]Sahin & DePinho, 2012;[START_REF] Speakman | Uncoupled and surviving: individual mice with high metabolism have greater mitochondrial uncoupling and live longer[END_REF]. The first observation came from TERT deficient mice strains that showed compromised mitochondrial biogenesis through decreased mitochondrial content in somatic tissues, such as the heart and liver, as well as through decreased ATP mitochondrial synthesis [START_REF] Sahin | Telomere dysfunction induces metabolic and mitochondrial compromise[END_REF]. The cellular pathways implicated in those negative effects have been clearly established. The lack of TERT expression leads to the suppression of the PGC activation pathway [START_REF] Sahin | Telomere dysfunction induces metabolic and mitochondrial compromise[END_REF] with cascading negative effects on gluconeogenesis, fatty acid oxidation or oxidative balance (Sahin & DePinho, 2012).

Whether those changes are telomere-length dependent or due to non-canonical functions of TERT remains under debate. TERT was found to bind to genes coding for the complex I of the respiration chain, a major site of superoxide production, to improve respiratory chain activity (of complex I and of maximal respiration rate) and to protect mitochondrial DNA from oxidative stress [START_REF] Haendeler | Mitochondrial Telomerase Reverse Transcriptase Binds to and Protects Mitochondrial DNA and Function From Damage[END_REF]. However, at least one study found higher levels of mitochondrial DNA damages due to hydrogen peroxide when TERT was re-localized within the mitochondria, and suggested that the mitochondrial -telomerase relationship was part of the oxidative stress pathway that triggers cell apoptosis [START_REF] Santos | Mitochondrial hTERT exacerbates free-radical-mediated mtDNA damage[END_REF]. In addition, TERC -/-mice but which retain TERT expression, presented the same mitochondrial dysfunction as TERT-deficient mice, strongly suggesting a telomere length deficiency (and not a non-canonical) explanation (Sahin & DePinho, 2012;[START_REF] Strong | Phenotypes in mTERT⁺/⁻ and mTERT⁻/⁻ Mice Are Due to Short Telomeres, Not Telomere-Independent Functions of Telomerase Reverse Transcriptase[END_REF]. In the future, studying the effects of telomerase on mitochondrial functioning and then on the bioenergetics of cells needs to be extended in two ways. First, towards the study on how telomerase, mitochondrial dysfunction and signalization pathways well-known to control the energy trade-offs of organism ageing (e.g. IGF1 and mTOR) are interconnected (reviewed in Sahin & DePinho, 2012). Whether the telomerase regulation of the mitochondrial functioning is direct or indirect is not of primary importance for evolutionary studies, but how telomerase may modulate those signalization pathways will uncover new sides of the individual variability in the growth and ageing trade-off.

Second, we need to extend those studies to a broader range of species, starting with those that maintain telomerase activity after early-life development. As with telomeres, telomerase, mitochondria and IGF1 / mTOR pathways are highly conserved over evolution. Exploring how they differently interact in species that evolved variable growth rates, metabolism and lifespans would be highly valuable. For instance, telomerase activity is not found in dipterian insects [START_REF] Sasaki | Detection and distribution patterns of telomerase activity in insects[END_REF], but whether non-canonical functions of telomerase play a role in individual fitness in those animals remains unexplored.

The best models to start with: the TERT -/-knock-out animals

On longevity trade-off…

Tackling the role of telomerase in regulating the trade-offs among cellular and life-history traits will be of paramount importance in the coming years. While it remains indescribably complex to decipher causality in telomere biology and ageing, one natural starting place is to examine how lifehistory traits like growth, reproduction and lifespan are combined in biological models that experimentally lack telomerase activity, such as the TERT or TERC -/-organisms [START_REF] Anchelin | Premature aging in telomerase-deficient zebrafish[END_REF][START_REF] Blasco | Telomere Shortening and Tumor Formation by Mouse Cells Lacking Telomerase RNA[END_REF]. Those knock-out models have provide us with some clues that need to be explained in a life-history context. Up-to-date, only one study has indirectly addressed the variation of expression of telomerase and of TERT / TERC subunits in relation to lifespan evolution and extrinsic risks of mortality [START_REF] Hartmann | Telomeres shorten while Tert expression increases during ageing of the short-lived fish Nothobranchius furzeri[END_REF]. In short (3 months) and longer-lived (6 months) killifish, Nothobranchius furzeri, TERT and TERC gene expression correlate with telomerase activity and are equally expressed in both strains, but increase with age in some tissues of the long-lived population.

Since telomere ends erode with age in both strains, we may expect some non-canonical roles for telomerase subunits to play a role in lifespan in this animal. Using such populations of the same species, that have naturally evolved life-history trait differences due to ecological constrains (i.e. the short-lived killifish originates from temporary pools with eggs drying in the mud until the next generation, and so we short adult lifespan expectancy), we may extend our understanding of the impact of telomerase and telomerase subunits' expression to divergent evolution of fast pace of life (i.e. fast growth and high reproductive investment).

…reproduction trade-off… TERC -/-mice show progressive telomere shortening over generations, with reduced cell turnover, bone marrow failure, and reproductive impairment only in the sixth generation of homozygous mutants (TERT -/-mice presented similar phenotypes [START_REF] Herrera | Disease states associated with telomerase deficiency appear earlier in mice with short telomeres[END_REF][START_REF] Strong | Phenotypes in mTERT⁺/⁻ and mTERT⁻/⁻ Mice Are Due to Short Telomeres, Not Telomere-Independent Functions of Telomerase Reverse Transcriptase[END_REF] for consistent results in a mouse strain with a different genetic background). Telomerasemutated C. elegans worms (trt-1) can survive the absence of full telomerase activity and show no particular deficiencies, apart from becoming sterile after few generations (Lackner & Karlseder, 2012).

While being rather anecdotal for molecular and cell biologists focusing on lifespan, those studies conducted on very different species may say something about the control of reproduction by age signalling, but may also have missed how impaired telomerase activity may modulate the reproductive output of adults. If accumulation of DNA damage over generations is the main explanation for the late appearance of specific sterile phenotypes in knockout models, we may expect that reproduction, or any other activity either energy consuming or potentially stressful, would also be more detrimental for the future reproductive and survival prospects of TERT -/-or TERC -/-breeders. Unfortunately, the original work on mice said nothing about the reproductive success of those mice [START_REF] Blasco | Telomere Shortening and Tumor Formation by Mouse Cells Lacking Telomerase RNA[END_REF], while mutated worms did show a decrease in brood size [START_REF] Lackner | Organismal propagation in the absence of a functional telomerase pathway in Caenorhabditis elegans[END_REF]. Additional telomerase mutants like TERT -/-zebrafish (in which telomerase is active through life and present in the ovary even in infertile old females) develop normally until adulthood [START_REF] Anchelin | Premature aging in telomerase-deficient zebrafish[END_REF] and start with normal clutch size and viability (i.e. until 1 year-old). However, TERT -/-zebrafishes then present a fast senescence and premature sterility [START_REF] Henriques | Consequences of telomere shortening during lifespan[END_REF]. Interestingly in this study, TERT -/+ and TERT +/+ zebrafish increased their reproductive performances after 1 year, while the female TERT -/-presented a reduced number of eggs per spawn [START_REF] Anchelin | Premature aging in telomerase-deficient zebrafish[END_REF]. Still, there were no detailed data on how, for instance, the heterozygote strain may suffer from a lowered reproductive success than the control one. Such a question remains a key point to evaluate the putative telomerase -fitness links. Again, given that TERT has been found to have non-canonical roles in inducing neutropenia in zebrafish [START_REF] Alcaraz-Pérez | A non-canonical function of telomerase RNA in the regulation of developmental myelopoiesis in zebrafish[END_REF], it is unknown whether this could also modulate reproductive success via gamete viability (see [START_REF] Anchelin | Premature aging in telomerase-deficient zebrafish[END_REF] for deleterious effect on sperm).

…or immune trade-off.

Since cell proliferation is one of the active arms of immunity, a last obvious context where telomerase impact should be evaluated in knock-out models relates to the cost of mounting an immune response. Telomerase seems to be involved in replicative capacity of immune cells [START_REF] Allsopp | Telomerase Activation and Rejuvenation of Telomere Length in Stimulated T Cells Derived from Serially Transplanted Hematopoietic Stem Cells[END_REF] and TERC -/-mice show a reduced spleen size with a reduced rate of proliferation of either B and T lymphocytes following immune challenge [START_REF] Blasco | Immunosenescence phenotypes in the telomerase knockout mouse[END_REF]. In addition, a higher proportion of neutrophils has been recorded [START_REF] Herrera | Disease states associated with telomerase deficiency appear earlier in mice with short telomeres[END_REF] (maybe as a compensatory response to lymphocyte weakness) and macrophages of TERC -/-mice are characterized by an "oldphenotype" with reduced proliferative capacity and higher intracellular ROS concentrations [START_REF] Sebastián | Telomere Shortening and Oxidative Stress in Aged Macrophages Results in Impaired STAT5a Phosphorylation[END_REF]. As neutrophils and macrophages are central in the set-up and control of the inflammatory and innate immune responses [START_REF] Desai | Leukocyte function in the aging immune system[END_REF], evaluating how TERC -/individuals react to inflammatory challenges commonly used in ecophysiological studies (i.e. lipopolysaccharide injection) will be of interest, both in terms of energy expenditure of collateral oxidative damage, and ultimately of future reproductive and survival prospects.

Conclusion: there is a lot to gain in studying telomerase for evolutionary biologists

Our present knowledge of the role of telomerase in modulating cell ageing, bioenergetics or signalization pathways (all mechanisms underlying the individual phenotype and ultimately its lifehistory traits such as immunity, growth or reproductive rates) is largely lacking. Using knock-out models of telomerase genes has provided us with a unique tool to study how the combination of physiological and life history traits is likely to be altered when compared to wild-type strains. Of course, heterozygotes of mutated genes (TERT -/+ or TERC -/+) will be more valuable as they will take us beyond the on/off (i.e. sterile/fertile) phenotypes. In addition, the study of telomerase must be extended to a broader range of species in non-models animals like birds, fish, mammals or insects, where descriptive studies on change in telomerase activity with age or tissue have already paved our way (e.g. [START_REF] Hartmann | Telomeres shorten while Tert expression increases during ageing of the short-lived fish Nothobranchius furzeri[END_REF][START_REF] Jemielity | Short telomeres in short-lived males: what are the molecular and evolutionary causes?[END_REF][START_REF] Koubová | Telomerase activity is upregulated in the fat bodies of pre-diapause bumblebee queens (Bombus terrestris)[END_REF][START_REF] Mcaloney | Polymorphisms within the Telomerase Reverse Transcriptase gene (TERT) in four breeds of dogs selected for difference in lifespan and cancer susceptibility[END_REF][START_REF] O'hare | Telomerase gene expression in the chicken: Telomerase RNA (TR) and reverse transcriptase (TERT) transcript profiles are tissue-specific and correlate with telomerase activity[END_REF][START_REF] Wang | Telomerase activity in the bats Hipposideros armiger and Rousettus leschenaultia[END_REF]. Adding original data on, more particularly, the pleiotropic action of canonical and non-canonical activities of telomerase, and exploring how they may have co-evolved with particular life-history traits form our next scientific objective.

Acknowledgements

Data accessibility. This article has no additional data. Competing interests. We declare we have no competing interests. Funding. We received no funding for this study

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Supplementary Information

Supplementary Table 1: XXXX