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# Influence of EE2 exposure, age and sex on telomere length in European long-snouted seahorse (Hippocampus guttulatus)

Nathalie Prévot d'Alvise, Eliette Ascensio, Simone Richard

► To cite this version:

Nathalie Prévot d'Alvise, Eliette Ascensio, Simone Richard. Influence of EE2 exposure, age and sex on telomere length in European long-snouted seahorse (Hippocampus guttulatus). General and Comparative Endocrinology, 2024, 346, pp.114419. 10.1016/j.ygcen.2023.114419. hal-05155841

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Submitted on 10 Jul 2025

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1 Influence of EE2 exposure, age and sex on telomere length in European long-snouted seahorse

2 (*Hippocampus guttulatus*)

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4 Nathalie Prévot D'Alvise*¹, Eliette Ascensio¹ and Simone Richard¹

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6 1. Université de Toulon, Aix Marseille Univ., CNRS, IRD, MIO, Toulon, France

7 e-mail: nathalie.prevot@univ-tln.fr

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9 * Corresponding author :

10 Nathalie Prévot D'Alvise

11 e-mail: nathalie.prevot@univ-tln.fr

12 ORCID of the author: https://orcid.org/0000-0002-1253-9834

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Abstract

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Keywords

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Seahorse; telomere length; 17α-ethinylestradiol; age; sex; telomerase.

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# 1. Introduction

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The state of the Mediterranean environment has been strongly impacted in recent years by both climate change and chronic anthropogenic pollution (Vincent et al., 2011; Foster et al., 2016; Otero-Ferrer et al., 2017; Prévot D'Alvise et al., 2011). These disturbances have a significant impact on ecosystems, including populations of long-snouted seahorses, *Hippocampus guttulatus* (family Syngnathidae). Currently this species is listed as “Data Deficient” and is threatened in the Mediterranean Sea (CITES convention, Appendix II - all species of seahorses - Pollom, 2017). *H. guttulatus* is a sedentary benthic teleost with a relatively short development cycle (less than a year) and a wild lifespan around 1-4 years or more in captivity (5-6 years). Seahorses are unique marine ectotherm fish with a specific life-history traits including male pregnancy, paternal care and monogamy (Jones and Avise 2001; Foster and Vincent, 2004). In this species, the male incubation is a specific form of reproduction similar to other forms of viviparity in mammals and requires stronger morphological and physiological modifications of paternal tissues akin to those of viviparous females (Wilson et al., 2001; Stölting and Wilson, 2007; Stölting, 2010; Albergotti and Guillette Jr, 2011; Whittington et al., 2015). Fertilized eggs, previously deposited by the female in the male brood-pouch, are incubated and embryogenesis takes place. Embryonic and larval developmental stages continue in the brood-pouch for closely to 3 weeks, before free-swimming juvenile releasing from male's pouch (Sommer et al., 2012). Important juvenile developmental changes occur during the first month and sexual dimorphism begins around 3-4 months. Up to this age, it is almost impossible to differentiate the sex, but females can become mature at this period (Ofelio et al., 2018). At 5 months, it is possible to distinguish the future brood-pouch in male and the onset of mating period can begin in 1-1.5 year-old seahorses (Faleiro et al., 2016). The length of the snout, head, pectoral fin or the absence or presence of dermal filaments on the top of the head are also morphometric characteristics used by some authors to identify sexual maturation. (Goffredo et al., 2004; Curtis et al., 2006; Gurkan, 2020). It is also generally accepted that *H. guttulatus* individuals with an average height of over 11 cm are considered sexually mature.

The reasons for *H. guttulatus* increasing scarcity in the Mediterranean sea are multiple: modification or even
disappearance of their natural habitat (seagrass meadows and coral reefs), abiotic changes (pH, salinity, temperature) of the environment, and above all marked anthropization around the coast (Gristina et al., 2015; Pierri et al., 2021; Vivas et al., 2023). Among the pollutants identified and continuously present on Mediterranean coasts, is the synthetic hormone, 17α-ethinyloestradiol (EE2), referenced in the first Watch List (09/14/2014) of the Water Framework Directive. This synthetic hormone, is known to alter the endocrine system and is responsible for more or less dramatic consequences in individuals, and in the longer term on populations. In general, the EE2 concentrations measured in the marine environment varies from < 5 to 29 ng/l and can reach significantly higher values (42-50 ng/l) in effluents leaving wastewater treatment plants (Leet et al., 2011; Wise et al., 2010). Along the Var coast (France), between 3 and 30 meters depth where seahorses live, there are average concentrations of 18-25 ng/l EE2 (internal data). EE2 effects are well documented in teleost fish, but significant differences in EE2-sensitivity were shown due to age, sex and species (Rehberger et al., 2020). In fish, sex differentiation and maturation are controled by hypothalamus-pituitary-gonadal (HPG) axis which regulated gonadotropin releasing. Among these ones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) directly acted on gonads to regulate the steroid hormone synthesis (oestradiol, E2, and testosterone, T). Plasma steroid concentrations can ultimately act on HPG axis by feedback inhibition (Leet et al., 2011). So, estrogen exposure such as EE2, which is a strong E2-agonist, can disturb the signaling pathway of HPG axis and the normal expression of key genes involved in sex differentiation (Qin et al., 2019) and growth (Bhatta et al., 2012). For example, in sub-adult female coho salmon, EE2 alters positively LH and negatively FSH gene expressions which results in a delay in sex differentiation (Harding et al., 2013). Indeed, FSH normally stimulates the aromatase synthesis, enzyme which is responsible for converting androgens to estrogens. In EE2 exposure, the FSH down-expression slows its synthesis disrupting endogene steroid concentrations. A variety of EE2 effects on fish at different levels (molecular, tissue or physiological) have been documented: down-synthesis of testosterone and 11-ketotestosterone in male fathead minnow (Feswick et al., 2016), sex ratio shifted toward more females and body length and weight significantly reduced in male bony fishes (Shved et al., 2008), decreased male secondary sex characteristics in seahorses (Prévot D'Alvise et al., 2020), reduction of the spawning success in adult zebrafish (Cosme et al., 2015),... In general, EE2 exposure result in a decrease in the quality of
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sperm and/or eggs, gestational disturbance, developmental alterations in newborns, delays in sexual differentiation and maturation, etc. Scarce studies reported the deleterious effect of this synthetic hormone on seahorses (Nenciu et al., 2014; Prévot D'Alvise et al., 2020), via the use of very effective and relevant biochemical and/or molecular (gene expression) analysis. Here, we completed our previous study in seahorses by using another tool: Telomere Length (TL).

In recent years, telomere length has proved to be a molecular tool of increasing interest within the scientific community in the fields of ecology and evolution (Young, 2017; Monaghan et al., 2022; Vaiserman and Krasnienkov, 2021; Tobler et al., 2022). This new research area is innovative and promising. However, it should be underlined the difficulty to use TL as a biomarker since it varies considerably across species (ecto or endothermic taxon - Olsson et al., 2018), lifespan, sex, tissues and life-history trade-offs (disease, abiotic stresses). All these parameters related to telomere dynamics, led to a number of sometimes contradictory conclusions. Tobler et al. (2022) are attempted to clarify this multitude of approaches in giving an overview of the hypotheses and classifying them. Among the many current hypotheses, one of these, was to use TL not only for determining the age and/or sex of vertebrates but also for reporting exposure to past and/or chronic (chemical or physical) stress (Angelier et al., 2018; Louzon et al., 2019; Salmón et Burraco, 2022). If we accept this hypothesis, what do we know about telomere dynamics in seahorses? Does telomere length vary (or not) according to age, sex or exposure to a pollutant? Telomeres are repeated DNA sequences located at the end of eukaryotic chromosomes. Recently, telomere sequences in mammals have been found to code two repeating dipeptide proteins: repeating valine-arginine (VR)ₙ and repeating glycine-leucine (GL)ₙ (Al-Turki et Griffith, 2023). Telomere sequences in fish are highly conserved, represented by a hexameric motif (TTAGGG)ₙ and vary between 2-15 Kb (Ocalewicz, 2013). TL can decrease at a critical age in ectotherm fish such as the short-lived medaka, *Oryzias latipes* (Hatakeyama et al., 2008), *Oryzias melastigma* (Au et al., 2009), killifish, *Nothobranchius furzeri* (Hartmann et al., 2009), male Atlantic cod, *Gadus morhua* (Almroth et al., 2012). However, telomere dynamics is variable in other fish. In female rainbow trout, *Oncorhynchus mykiss*, TL depends on the developmental stage: it shortens and then lengthens (Panasiak et al., 2020) whereas it is inverse in zebrafish, *Danio rerio* (Anchelin et al., 2011; Carneiro et al., 2016). In
European sea bass, *Dicentrarchus labrax* (Horn et al., 2008) or the long-lived bigmouth buffalo, *Ictiobus cyprinellus* (Sauer et al., 2021), there is no significant telomeric attrition with age. The relationship between TL and sex depends on the species and lifespan. In vertebrates, females were longer-lived than males and had longer telomeres (Viña et al., 2005; Barrett & Richardson, 2011). Many studies in teleosts showed that sexually mature male killifish (*Nothobranchius sp.*) or medaka (*Oryzias latipes* or *melastigma*) had shorter telomeres and lifespan than females (Reichard et al., 2022; Gopalakrishnan et al., 2013; Yip et al., 2017). In turn, Atlantic cod males (*Gadus morhua*) possessed longer telomeres than females (Almroth et al., 2012). In nine-spined sticklebacks (*Pungilius pungilius*), the variation of TL was sex- and maturation time-dependent: male TL was constant whereas female TL decreased with increasing maturation age (Noreikiene et al., 2017).

The hypothesis of a positive correlation between seahorse age/sex and telomere dynamics should be admissible. It is recognised that telomeres play a fundamental role in the structural integrity and stability of chromosomes but also in cell cycles (DNA replication). This last particularity is interesting because the natural attrition of telomeres in somatic cells occurs during ageing of individuals. Indeed, in endotherm vertebrates TL maintenance in germinal or stem cells is possible due to some enzymes such as telomerase reverse transcriptase (TERT), whose activity is mainly detectable in the early stages of development (Brunori et al., 2005). In teleosts, it has been shown that telomerase activity (or expression) was constitutive at all ages and in all somatic and reproductive tissues (Kappler et al., 1998; Reichert et al., 2013; Panasiak et al., 2023). TERT can naturally decrease in old fish (after sexual maturity), which is why we observe telomere shortening. This case was observed in marine medaka, *Oryzias melastigma*, or in rainbow trout, *Oncorhynchus mykiss*, where male telomerase expression decreased significantly whereas it was constant in females (Peterson et al., 2015; Klapper et al., 1998). However in many ectotherms, telomerase increased during ageing irrespective of sex and tissues while telomeres shorten (Klapper et al., 1998; Hatakeyama et al., 2008; Hartmann et al., 2009; de Abechuco et al., 2014). E.g., male hakes, *Merluccius merluccius*, exhibited higher TERT expression levels in the brain, testis and liver than females. In zebrafish and medaka, studies have revealed that telomerase plays an important role in regeneration and tissue renewal in these species (Anchelin et al., 2011; Henriques et al., 2013; 2017). Despite this observation, telomerase activity and ultimately the variation in TL, is specific to each aquatic species. So, telomerase expression varies
considerably across tissue and taxa, but both TL and telomerase also vary between populations within a species, between sexes, between individuals within a population and between tissues within an individual.

Irrespective of age/sex/tissue/species, telomerase efficacy to counteract telomere shortening can be altered by abiotics parameters. Recent studies report that physical (UV, temperature) and chemical (oxidative stress, pollutants) factors can alter TL (Royle, 2006; Harari et al., 2013; Simide et al., 2016; Coupé et al., 2019; Louzon et al., 2019).

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This review offers the first discoveries made in *Hippocampus guttulatus*, a marine ectotherm fish and a non-model system, on telomere dynamics and expression of telomerase related to age and sex. To perform this study, 110 seahorses were monitored for 6 months in rearing conditions free of pollutants and optimal for their development. 12 adult seahorses (6 males and 6 females) in captivity were also added to this study. We used genomic DNA present from non destructive dermal filament tissue samples or from destructive tail tissue samples at various ages. Since natural attrition of telomeres could be accelerated under the effect of chemical stress, we tested the hypothesis that endocrine disruptors, such as 17α-ethinyloestradiol (EE2), alter the telomere dynamics in young long-snouted seahorses. In this context, 70 juveniles, aged 2 months and still not sexually differentiated, were exposed for a period of 30 days to an environmental concentration of 21 ng/l of EE2. This experimental group was compared to a control group mainly composed to non-EE2 exposed pollutant seahorses. Here, our hypothesis is to show a link between EE2 exposure, telomere length, telomerase expression, and growth in European seahorse.

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## 2. Material and Methods

### 2.1. Biological material

About 200 seahorses, *H. guttulatus*, were obtained from two spawners present at the "Institut Océanographique Paul Ricard" (IOPR, les Embiez - France). The juveniles were raised under optimal conditions (23.5°C ±1; pH 8.02; 39 psu) from birth until 2 months and were fed daily with brine shrimp. 12
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spawners of *H. guttulatus* (6♀ et 6 ♂) aged 19 ± 1 months, present at the IOPR, were also included in this study. All these spawners are F1 generations from seahorses already been collected on several sites (Cavalaire, Le Brusc, Thau) or accidental fishery.

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After 2 months (2M), 70 juveniles were transferred to two distinct 200 L-aquaria under constant conditions (20°C ±0.5; pH 8.0; 39 psu): 70 2-month-old seahorses in a control aquarium (2M-control) and 70 2-month-old seahorses in an aquarium continuously fed with 17α-ethinylestradiol, EE2 (Sigma Aldrich - 296.4 g/mol) (2M-EE2). EE2 concentration in this aquarium remained constant at 21 ng/l EE2 from a buffer tank at 21 ng/l EE2 (flow rate was 15 l/h). Aquaria were supplied with natural seawater. Lighting was provided by a fluorescent tube linked to a timer in order to regulate the photoperiod. Temperature kept constant during the spawning period (from may to the end of september) and during experimental assays, by a 300W-immersion heater. Artificial devices were placed in aquariums so that seahorses could hide and attach themselves with their prehensile tail.

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During the study, whatever the aquarium the seahorses were fed daily and the natural mortality rate was less than 10% among control seahorses. Nevertheless, significant mortality (27%) in the EE2 contaminated aquarium was noted. However, the remaining seahorses showed no behavioural changes (diet, aggressiveness, etc.) or any apparent external malformations and they were kept by the IOPR institute in a special aquarium.

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## 2.2. Sampling

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Sampling started with 2-month-old seahorses (2M), as at a younger age the individuals were at a delicate growth phase (Fig1A). Nevertheless a sampling of six 15 days-old seahorses (0.5M) had carried in order to estimate telomere length in younger seahorses. Thus at 2 months (2M), *H. guttulatus*, had an average length of 6.6 cm (± 0.7) and an average mass of 743 mg (± 240) (Fig1B). From this point onwards, every month (and during 6 months), individuals were randomly sampled from control aquarium (2M, n=10; 3M, n=23; 4M, n=12; 5M, n=14; 6M, n=17). To EE2 contaminated aquarium, twenty individuals were randomly sampled after one month: the experiment was only conducted during 1 month due to a significant mortality. After being anesthetized with MS222 (Tricaine methanesulfonate) according to the procedure outlined in the
« AVMA Guidelines for euthanasia of Animals; 2013 », a piece of dermal filament (5 mm) from older individuals (Fig1C) was removed and a 10% iso-betadine solution was then applied to promote healing and prevent local infection before the return of seahorses in their aquarium. Concerning younger individuals (2 – 6 months), after a lethal MS222 anesthesia, a tail tip (2-3 cm) was removed. All samples were stored in absolute ethanol or RNAlater (Sigma-Aldrich) and placed at -80°C until analysis. At the time of sample collection, the individuals were weighed, measured, and photographed.

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** (A) Schematic whole experiment timeline. (B) *Hippocampus guttulatus*, 2 months old. Average length and mass of 5.21 cm (± 0.15) and 390 mg (± 30) respectively. (C) More or less abundant and long dermal filaments on the head and trunk in older individuals.</figcaption></figure>

## 2.3. DNA Extraction

The extraction of the genomic DNA was then carried out according to the protocol of the DNeasy Blood & Tissue Kit (Qiagen). 25 mg of tail or dermal filaments (tissue present on the surface of the exoskeleton) were crushed in 180 ml of buffer ATL and 20 µl proteinase K in a lysing matrix D® (MP Biomedicals), and vortexed for 40s (x2) with FastPrep-24® (MPBio). The DNA concentration for each individual was determined by the ThermoScientific NanoDrop Spectrophotometer. The purity and quality of each sample
was verified with the ratio A<sub>260</sub>/A<sub>280</sub> [only samples with a ratio ranged from 1.7 to 2.2 (supplementary material) were used in qPCR] as well as by electrophoretic migration on 2% agarose gel. The obtained DNA was diluted with elution buffer (DNeasy Blood & Tissue Kit, Qiagen) to a final concentration of 10 ng/µl and stored at -80°C until the qPCR assays were conducted.

## 2.4. RNA extraction

Extraction of total RNA was then performed with the RNeasy Plus Universal Kit (Qiagen). 50 mg of tissue was ground in 900 µl of Qiazol Lysing reagent (Qiagen) with "matrix D" beads using FastPrep-24® (MPBio). An equivalent volume of 70% ethanol was added and the mix was transferred into the RNeasy mini-spin column placed in a 2 ml collection tube. After centrifugation step (15s, 8000 x g), the column was placed in a new collection tube. 500 µl of buffer RPE were added into the column and centrifuged (15s, 8000 x g). Finally, column was placed in a new 1.5 ml collection tube and 50 µl of RNase-free water was added and centrifuged (1 min, 6000 x g). Eluate was the RNA sample. The RNA concentration for each individual was determined by spectrophotometry (NanoDrop 8000 Spectrophotometer - ThermoScientific, France). The purity and quality of each sample was verified with the ratio A<sub>260</sub>/A<sub>280</sub> as well as by electrophoretic migration on 1.5% agarose gel, and only samples with an RNA integrity ratio ranged from [1.88-2.14] were used to RT-qPCR (supplementary material).

## 2.5. qPCR analysis – Relative length of telomeres

Primer pair designed should amplify hexameric repeats (TTAGGG) presents in DNA sequence. DNA sequences are those published by Cawthon (2002) under the name tel 1 and tel 2. Equivalent seahorse primers (forward telomere, Telo-fwd 5'-GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3' and reverse telomere, Telo-rev 5'-TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTATCCCTA-3') are designed to allow DNA polymerase to extend from its 3'-end when it is hybridized to telomere hexamer repeats. Primer pair intended to amplify the reference gene, actin (Actin-fwd 5'-AGAGGGGTTACTTCTTGACC-3' and Actin-rev 5'-CCGATGTTCTTTCACTAAGG-3'), were designed from *H. guttulatus* contig number 7454 (internal data) using PrimerPlus software
261 gene studied (telomerase, Tert-fwd: 5'-TTTGCCCAAACATTGTCGGC-3'; Tert-rev: 5'-TAGTGCTGCACAAAAAGCCC-3' and actin, Actin-fwd: 5'-TCACTGGCATGGCTTGTTAG-3'; Actin-262 rev: 5'-CAGATGCAACTGCAGAAACC-3'), was determined with *PrimerPlus* software from the contigs 263 generated from the transcriptome made and donated by N. Bierne (*Laboratoire Génome, Populations, 264 Interactions, Adaptation, University of Montpellier-France*). A pool of cDNA from all seahorse tails was 265 used to construct two calibration plots (telomerase and actin genes) with serial ½ dilutions with initial 266 concentration at 150 ng/µl. qPCR efficiencies standard curves were 1.89 and 1.97 respectively. 267

268 The amplification was performed in a volume of 10 µl with 2 µl of cDNA, 5µl of GoTaq qPCR Master Mix 269 (Promega) and 1µl of each primer with a concentration of 10 pmol/µl. Amplifications were carried out with 270 the following conditions: a first denaturation step at 95°C for 10 minutes, then 45 cycles of denaturation and 271 annealing at 95°C for 10s and 58°C for 20s, and a final extension at 72°C for 35s. These steps were followed 272 by a pre-melting step of 95°C for 1s and a melting curve at 58°C for 10s to confirm the presence of single 273 amplicons and the absence of primer-dimer artifacts. 274

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275 **2.7. Statistical analysis**

276 Data are presented graphically as mean ± standard error of the mean (SEM). Before each statistical analysis, 277 all data was subjected to a Normality test via a Shapiro-Wilk test. Unpaired Mann-Whitney tests were used 278 to test the null hypothesis that EE2 exposure causes significant changes in TL and telomerase expression in 279 experimental groups (3M-EE2) and their corresponding controls (3M). To compare telomere dynamics and 280 telomerase expression between each age classes, a Student's *t* test with Bonferroni adjustment p-value 281 method was performed. Complement linear regression analysis were also used to assess correlation between 282 TL (or telomerase expression) and age classes (Pearson correlation). To study the effect of age and sex on 283 telomere length and telomerase, we used two-way ANOVA followed by a *post-hoc* comparisons to know 284 which groups were different. The level of statistical significance was set at p < 0.05. Statistical analysis was 285 performed using the computing environment R (R Core Team version 3.6.3, 2020). 286

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## 3. Results

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### 3.1. Effects of seahorse age on telomere length (TL) and telomerase expression

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The T/S ratios representative of relative telomere length of 18-day old seahorses (noted 0.5M) and of 2, 3, 4, 5, 6M (M: months) and those over 18 months old, reveal the presence of a significant variation of TL between all age classes studied (ANOVA, F<sub>6,88</sub> = 2.882, p-value = 0.013). “Inter age class” analysis shows a "bell-shaped" evolution of telomere length in *H. guttulatus* with a maximum length at 4 months. Thus, we observe significant differences between individuals aged 18 days (0.5M) and those of 4 months (Tukey HSD, p-value = 0.031) and between those over 18 months and 4 months (Tukey HSD, p-value = 0.042) (Fig. 2A). Results show here is both telomere lengthening and telomere shortening in long-snouted seahorses. This telomeric attrition only becomes significant in sexually mature individuals (> 1 year). TL is also positively correlated with body length (Pearson correlation R = 0.3141, p-value = 0.023), irrespective of seahorse age (Fig. 2B). In turn, only juvenile and adult developmental stages are significantly correlated with body length (Pearson correlations: R<sub>juvenile</sub> = 0.4358, p-value = 0.026 and R<sub>adult</sub> = 0.7379, p-value = 0.0061).

<figure><img src="image_5.png" /></figure>

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<caption>**Fig. 2.** (A) The relationship between telomere length and different age classes of seahorses, *Hippocampus*</caption>
302 *guttulatus* (n= 95) (TL expressed as relative T/S ratio) (ANOVA, F<sub>6,38</sub> = 2.882, p-value = 0.013). (B)

303 Regression analysis between telomere length and body length in all age classes (Pearson correlation, R =
304 0.3141, p-value < 0.05) (TL expressed as Log relative T/S ratio). Data are expressed as mean ± Standard
305 Error of Mean. Asterisk represents significant difference (p-value < 0.05).

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307 Seahorse has significantly telomerase expression in somatic tissues from juvenile to adult stages (ANOVA,
308 F<sub>6,52</sub> = 15.37, p-value = 4.89 10<sup>-10</sup>) (Fig. 3A). Irrespective of age, telomerase is also positively correlated with
309 body length (Pearson correlation R = 0.5174, p-value = 8.55 10<sup>-5</sup>). Telomerase expressions are relatively low
310 up to 5 months with a mean of 0.546 ± 0.041, then there is a significant increase by a factor of 4.6 in
311 individuals over 6 months (2.509 ± 0.017) (t test, p-value <0.001). For young and undifferentiated
312 individuals (juvenile developmental stage), our study shows a 2.5-fold increase in TERT mRNA expression
313 between 18-day (0.5M) and 3-month-old seahorses (Fig. 3B). This is positively correlated with their body
314 length, which increased by the same factor (2.8) (Pearson correlation R = 0.7102, p-value = 4.8 10<sup>-5</sup>).
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** (A) Age-dependent telomerase mRNA expression of seahorses, *Hippocampus guttulatus* (n=59) (ANOVA, F<sub>6,52</sub> = 15.37, p-value = 4.89 10<sup>-10</sup>). AU (Arbitrary Unit). (B) Regression analysis between telomerase expression and body length in all age classes (Pearson correlation, p-value <0.001) (TERT expressed as Log tert expression). Data are expressed as mean ± Standard Error of Mean. Different letters represent significant differences over the time period studied: *a* (p<0.001), *b* (p<0.01), *c* (p<0.05).</figcaption></figure>

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## 3.2. Effects of seahorse sex on telomere length and telomerase mRNA expression

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Telomere dynamics are only studied at sexual differentiation and maturation stages. Measurements of the relative T/S ratios of sub-adult and adult seahorses (Fig. 4A), irrespective of their sex, are respectively, on
average 1.10 ± 0.69 and 0.69 ± 0.3. These results show no significant difference in TL between individuals of opposite sexes or between ages (p>0.05). Here, the variation of TL is not sex- and maturation time-dependent. The level of telomerase mRNA expression differs depending on the individual's gender and age (Fig. 4B). Thus, female sub-adults less than 5 months old have a TERT expression level below 1.05 ± 0.07, while the equivalent in males does not exceed 0.60 ± 0.03. Likewise in male and female individuals over 6 months of age, the level of telomerase mRNA expression is 1.89 ± 0.27 and 3.23 ± 0.38, respectively. The strongest pattern in our study shows that female seahorses have always higher telomerase expression than males (ANOVA, F<sub>7,33</sub> = 29.33, p-value = 2.15 10<sup>-12</sup>). The difference between each sex for the same age group is significant each time: 4M (*t* test, p-value < 0.01), 5M (*t* test, p-value < 0.05), 6M (*t* test, p-value < 0.05) and adults (*t* test, p-value < 0.01). Our data show that telomerase expression is sex- and maturation time-dependent from sub-adult stage.
<figure><img src="image_7.png" /></figure>

**Fig. 4. (A) Telomere length (expressed as relative T/S ratio) and (B) level of telomerase mRNA expression in sub-adult and adult seahorses, *Hippocampus guttulatus*. Seahorses identified visually as males (blue) and females (orange). T: testosterone concentration (Prévot D'Alvise et al., 2020). *Data are expressed as mean ± Standard Error of Mean. Asterisks represent significant difference between seahorses with the same age but different sex: * (p<0.05); ** (p<0.01).*

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### 3.3. Effects of EE2 on telomere length and telomerase mRNA expression

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Measurement of the T/S ratios of 3-month-old control seahorses average 1.00 ± 0.088. Seahorses of the same age but exposed for one month to 21 ng/l of EE2 have an average T/S ratio of 1.11 ± 0.125 (Fig.5). These values suggest that the TL of the two groups is the same except for relative uncertainties (Mann-Whitney test, p-value = 0.4431). At the same time, we observe significant difference (t test, p-value = 0.0171) to the expression of telomerase mRNA between 3-month-old control (0.88± 0.11 AU) and EE2-exposed seahorses (1.12± 0.21 AU).

<figure><img src="image_8.png" /></figure>

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**Fig. 5.** Telomere length (expressed as relative T/S ratio) (gray) and level of telomerase mRNA expression (AU) (black) between seahorses (n = 20), *Hippocampus guttulatus*, aged 3 months and previously exposed for one month to 21 ng EE2/l and healthy individuals of the same age (n = 23). AU: Arbitrary Unit, [Average ± SEM]. Asterisk represents significant difference between seahorses with the same age but different conditions (*p*<0.05).

356

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The 3-month-old controls and 3-month-old seahorses exposed to EE2 have an average mass of 0.652 ± 0.139g and 0.486 ± 0.111g respectively and their length is 6.4 ± 0.15 cm and 5.9 ± 0.14 cm (Fig. 6). Our data
359

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show that body parameters (length and mass) of EE2-exposed seahorses are significantly lower than control-seahorse parameters: mass (t test, t = -3.47, p-value = 0.0012) and length (t test, t = -2.50, p-value = 0.0126).
Between the second and third months, in healthy seahorses, body length and mass increase to 27% and 90% respectively. In turn, in EE2-exposed seahorses, growth rates decrease: 17% and 44% respectively to length and mass. Moreover, both body length and mass of the EE2-exposed group are significantly correlated (Pearson correlation, R = 0.893, p-value = 8.60 10⁻¹⁶).

<figure><img src="image_9.png" /></figure>

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368

**Fig. 6.** Mean growth in healthy seahorses, *Hippocampus guttulatus*, and that of 3 month-old seahorses previously exposed for one month to 21 ng EE2/l. (until 3 months: juveniles; 4-6 months: sub-adults; >1 year: adults)

369

370

**4. Discussion**

371

It has been widely shown that TL and telomerase are involved in ageing of terrestrial and marine ectotherms,
but each species has its own pattern that depend on various parameters, such as growth rate, life-history traits, stress, for example (Dantzer and Fletcher, 2015; Axelsson et al., 2020; Burraco and al., 2020a; Panasiak et al., 2022). In this study, TL pattern, irrespective of age, is similar to zebrafish, Atlantic salmon, squamate reptile and amphibians (Anchelin et al., 2011; McLennan et al., 2018; Ujvari et al., 2017; Burraco et al., 2020b). We observed a progressive telomere lengthening from young juveniles to young sub-adults then a gradually shortening until adult stage. Decline in TL from sub-adults may be a consequence of a fast growth, specific to juvenile developmental stage in this species (Blanco and Planas, 2015). Developmental stages between 15 days and 1.5 months are important transitional changes in early development of *H. guttulatus*. Most vital organs continue developing, whereas gut and exosketelon occur during the first month (Whittington et al. 2015; Ofelio et al., 2018). During somatic growth, both high cell turn-over and energy expenditure increase and lead to reactive oxygen species (ROS) production. Oxidative stress accelerates telomere shortening; but in teleosts, telomere attrition during a rapid growth that occurs with every cell division (Ziegler et al., 2017) can be counteracted by telomerase activity (Hatakeyama et al., 2016; Monaghan and Ozanne, 2018; Panasiak et al., 2022). When an organism can no longer maintain a constant telomere length (here from 4 months in seahorses) the shortening process accelerates until it reaches a critical telomeric length. This is an irreversible issue which confirms the end of cell division and ultimately senescence. In order to better understand the change of telomere length in *H. guttulatus*, we looked at both seahorse growth and also the variation in telomerase (TERT) gene expression. Our discovered patterns may be interpreted in the context of specific *H. guttulatus* ontogeny. Surprisingly, in juvenile period with high tissue renewal, that is to say, significant cell proliferation, is accompanied by a high telomere lengthening and a low transcription of the telomerase encoding gene, which is closely linked to growth (Greider and Blackburn, 1989), and more specifically during early developmental stages (de Abechuco et al., 2014). This trend occurs until the onset of sub-adult stage (4 months) and then reverses. Here, energetic costs allocated to the development of organs are more important from birth to 3 weeks (Novelli et al., 2015). During this period, associated with the planktonic phase, the growth of young juvenile seahorses generate a high ROS level which is not compensated by a constant telomerase expression. After this critical period, the same telomerase level and the mature anti-oxidant system counteracts more efficacely ROS and telomere lengthen.
In order to complete and explain whether the TL increase during juvenile stage, telomere length frequency should be performed in further studies. It is possible that during juvenile stages, the shortest telomeres lengthen until a critical age, then these longer telomeres gradually decrease during adult life when cellular division becomes steady (Hatakeyama et al., 2016). Another peculiarity in this species, between 3 and 5 months, expression of telomerase is slightly greater than at an earlier stage (< 2M). This increase could be associated with the onset of sexual differentiation (Yip et al., 2017). During this period, estrogen concentration (here 17β-estradiol) is on average 960.1 µg E2/l ± 36.3 (Prévot D'Alvise et al., 2020). The presence of these estrogenic hormones will thus stimulate the production of telomerase which is estrogen dependent (Gopalakrishnan et al., 2013). The average E2 concentration is especially high in sexually mature seahorses and is accompanied by an increase in the expression of TERT mRNA: it is accentuated significantly when the sexual maturation phase begins around 6 months and is maintained in mature individuals. These results are contrary to those observed in other fish species such as Atlantic cod (*Gadus morhua*), where a significant decrease in TERT expression appears in individuals aged between 4 and 11 months (Abechuco et al., 2014). Although an increase in expression of the gene that codes for TERT is observed throughout the growth of the seahorses, this appears to be insufficient to maintain constant TL beyond 18 months. This phenomenon has also been observed in other fish species (*Oryzias latipes* - Hatakeyama et al., 2008 and 2016; *Oncorhynchus mykiss* - Klapper et al., 1998; *Nothobranchius furzeri* - Hartmann et al., 2009; *Danio rerio* - Henriques et al., 2013), where the level of telomerase expression remains persistent and increasing throughout their life. This current study showed that telomerase is present in somatic tissues from juveniles to adults in European long-snouted seahorses. Another hypothesis could be that independently of TERT activity - which ensures maintenance of TL - the intensity of telomeric shortening is linked to the longevity of individuals, in vertebrates. Thus, it has been demonstrated that a high rate of telomeric attrition (number of bp "lost" / year) is correlated to reduced longevity in individuals (Unryne et al., 2005; Gomes et al., 2011; Dantzer et Fletcher, 2015). Since the lifespan of a seahorse in captivity is 5 to 6 years, this could explain why telomere shortening is premature. Sexual differentiation in seahorses begins around 3 months and in this study it was possible to differentiate male and female seahorses morphologically at 4 months. In fact, male individuals have an elongated black spot on their lower abdomen
which will lead to the formation of the future brood pouch for the fertilized eggs. In addition, the lower end of their abdomen is more rounded than in females. The relative length of telomeres does not vary significantly according to sex for individuals of the same age and between the age groups studied (from 4 months to over 18 months). These results differ from those observed by Gopalakrishnan et al. (2013) in the medaka where the TL is higher in females than in males. However, in seahorses, a tendency towards telomeric shortening emerges with increasing age, notably in 4-month-old females (and males) and those over 18 months old. For the same period and the same individuals, the expression of telomerase mRNA is significantly different between males and females of the same age. It is always higher in females than in males regardless of the age group studied. This tendency is found in other teleost fish species such as *Oryzias latipes*, *Oryzias melastigma* (Gopalakrishnan et al., 2013; Yip et al., 2017). A significant increase in telomerase is also observed in both males and females between the two stages of sexual differentiation and maturation. These results were also observed in European hake, *Merluccius merluccius* (de Abechuco et al., 2014) where significantly higher telomerase transcription levels were found with increasing age / size. Finally, after the sexual maturation stage (individuals over one year old), the level of telomerase transcription is constant in females while it tends to decrease in males. This trend has also been observed in mature rainbow trout, *Oncorhynchus mykiss* (Klapper et al., 1998; Au et al., 2009). The results observed in the long-snouted seahorse can be explained in part by the levels of estrogen (E2) and testosterone (T) measured both in individuals during the sexual differentiation phase (4-5M) as well as in sexually mature individuals (> 18M) (Prévot D'Alvise et al., 2020). E2 concentration is higher in female seahorses of this species than males in both juvenile and adult stages. Our results agree with other studies since it is known that telomerase is estrogen-dependent and that there is not a perfect correlation between telomerase activity and the level of expression of the corresponding mRNAs (Hartmann et al., 2009; Peterson et al., 2015). Likewise, androgens, including testosterone, have been shown to negatively affect telomerase activity (Ravindranath et al., 2001). When these two hormones co-exist, their effect on telomerase expression will depend heavily on their concentration. In this study, irrespective of the age group studied, female individuals will have an E2 content at least 2.5 times greater than that of T (Prévot D'Alvise et al., 2020). This hormonal situation will therefore significantly and positively contribute to the expression of telomerase.
Surprisingly, exposure to 17α-ethinylestradiol for one month in young two-month-old seahorses does not appear to interfere with telomere biogenesis. However, it has been shown that certain organic pollutants (organochlorine and pyrethroid-type pesticides, phthalates) (Molbert et al., 2020) and inorganic pollutants (Coupé et al., 2019) negatively impact TL in certain aquatic organisms, including *Paracentrotus lividus*. It has been shown that telomeric attrition caused by these types of pollutants is mainly due to oxidative stress. This is because the ends of the telomeres have a sequence rich in G, which is very sensitive to this type of stress. Anthropogenic pollutants or the products of their metabolism by the contaminated host, initiate the formation of reactive oxygen species but also induce an imbalance in the pro and antioxidant systems, leading to telomere shortening (Von Zglinicki, 2002). Thus, in juvenile catfish, *Pelteobagrus fulvidraco* (Mo et al., 2019) or female black perch, *Micropterus salmoides* (Colli-Dula et al., 2014), the female contraceptive pill (EE2) generates significant oxidative stress either via over-expression of the *glutathione S transferase* encoding gene and under-expression of the *glutathione peroxidase* gene, or by decreasing the activity of *superoxide dismutase* and *catalase*. The presence of ROS should then promote and/or accelerate the natural shortening of telomeres. However, some studies have revealed that, conversely, certain pollutants such as PAHs, PCBs or PBDEs (PolyBromatided Diphenyl Ethers) do not contribute to telomeric attrition but are significantly involved in the appearance of metabolic and physiological disorders. The results of our study allow us to show for the first time that exposure to EE2 at an environmental concentration for a period of one month, does not induce significant telomeric shortening in juvenile seahorses. Indeed, even if the TL was not affected by this chemical stress, our study showed that EE2 had a significant negative effect on the growth of individuals. This growth retardation of -10% for length and approximately -45% for mass compared to healthy seahorses was also observed in juvenile wild trout, *Salmo trutta*, with renal inflammation, which led to a significant increase of ROS production (Stauffer et al., 2017). While no difference was observed for TL, there is a significant negative effect on seahorse mass and body length. Although short TL and increased telomeric attrition have been observed in Atlantic salmon juvenile, *Salmo salar* (McLennan et al., 2016), in amphibian larvae, *Pelobates cultripes* (Burraco et al., 2017), in young birds (great tit, *Parus major*) (Nettle et al., 2015) and in the sea urchin, *Paracentrotus lividus* (Coupé et al., 2019) when subjected to environmental stress, these two "characteristics" are not always directly related to a delay in development. This is the case
with developing juvenile seahorses exposed to chronic chemical stress, which in this present study was brought about with EE2. A few studies reported the impact of global climate change in terrestrial and marine ectotherms (Sokolova and Lannig, 2008; Kingsolver et al., 2013). Among parameters involved in climate change, global warming severely affects physiology, fitness and ageing dynamics of ectotherms. Warmer temperatures cause an acceleration of growth rates, induce thermal stress and decrease the onset of maturation stage of parents which directly affects their mating period (Buccaro et al., 2020a). In ectotherms, faster growth can alter the anti-oxidant status and increase ROS production which can accelerate proliferative cell rate (so ageing of cells) and *in fine* whole individual. However, when temperature gradually increases, a thermal acclimatization response occurs in ectotherms, but in case of heatwaves, ectotherms lack time for acclimatization (Rodgers, 2021). Recently, Chatelain et al. (2020) showed a link between oxidative stress and telomere shortening. In the same context, environmental marine pollution causes deleterious effects on aquatic species, for example, long-term exposure to nitrate in ectotherms exert a decrease of growth and survival (Gomez et al., 2020). Since somatic growth is regulated by hormones and the release of endocrine disruptors into marine environment increase, early developmental stage of ectotherms is strongly affected (Molbert et al., 2021, Marrone et al., 2021; Wu et al., 2022). If chemical pollution causes a telomere attrition of ectotherms at the early developmental stage, this could lead to shorter telomeres in future offspring. In both short and long term, global climate change has impacts on individual- and intergenerational-level effects of ageing rates in ectotherms. This ability to counter a reduction in telomeric length induced by exogenous stress could therefore not only be linked to the age of the individual but could also depend on the energy allocation made at that moment. Here, healthy 3 month-old juveniles invest energy in growth (length and mass) since all vital organs are developed. The ROS production dependent to growth is supported by the mature antioxidant system, and the constant level of telomerase maintains TL. EE2 exposure alters growth leading to lower ROS production, nevertheless. Telomerase level increased, since its gene expression is estrogen-dependent, whereas TL is constant. During a stress response, glucocorticoids are released. These hormones inhibit various physiological systems, as growth, in order to conserve energy. They also modulate telomere dynamics and oxidative stress (Haussmann et Marchetto, 2010). E.g. Glucocorticoids improve the antioxidant defense and the oxidative damage repair. They also impact TL by decreasing cell
division or telomere maintenance (telomerase and others mechanisms) but their effect is dose dependent. Choi et al. (2008) show that cortisol induces a down-regulation of telomerase activity caused by a reduction in its transcription. Moreover EE2-exposure alters the steroidogenesis. Here, we worked with environmental EE2.
We can suppose that all glucocorticoid effects are minimized. In *H. guttulatus*, therefore it is possible that telomere maintenance in presence of oxidative damage causes others mechanisms than telomerase (Burraco et al., 2020b). Moreover, there are also several points from this study that are worthy of discussion. (1) We don't measure the oxidative stress in juveniles, (2) EE2 exposure was carried out only in 3 month-old juveniles, and (3) TL distribution should be performed via Q-FISH to complete these results. Another hypothesis to explain the lack of a positive correlation between prolonged exposure to EE2, an unaltered TL and an telomerase increase in young juvenile seahorses, could be the involvement of estrogen. Indeed, estrogen is known to initiate the transcription of the telomerase encoding gene or simply to stimulate the catalytic activity of this enzyme. Several studies carried out in fish have thus shown that the activity of telomerase is estrogen-dependent and that as a consequence it is possible to maintain a constant TL (Gopalakrishnan et al., 2013; Ujvari et Madsen, 2009). Knowing that EE2 has an estrogenic power greater than that of E2, it is legitimate to suppose that seahorses exposed for a month to 21 ng EE2/l will exhibit telomerase expression greater than that of healthy individuals. This EE2 induced enzymatic activity (or here over-expression of mRNA TERT – correlation previously showed by Peterson et al., 2015) could therefore minimize the natural telomeric attrition observed in individuals subjected to environmental stress.

525

## 5. Conclusion

Our results showed that in European long-snouted seahorses, like other ectotherms, telomere lengthens until juvenile stage then shortens with age despite concomitant expression of telomerase encoding gene. Here, the variation of TL is not sex- and maturation time-dependent. The only method by which males seahorses can be differentiated from females is via the significant expression of the telomerase as well as the levels of steroid hormones. The strongest pattern in our study shows that female seahorses have consistently higher
telomerase expression than males. We also showed that telomerase is present in somatic tissues from juveniles to adults in *H. guttulatus*. Finally, even though certain pollutants including ethinyloestradiol are known to negatively affect TL in juvenile fish, this is not the case in undifferentiated and developing seahorses. Our results show that pollution at environmentally relevant concentrations by EE2 had negative consequences for juvenile seahorse growth, and were positively correlated with an increase in telomerase gene expression, suggesting this was up-regulated to compensate for any negative effects on telomere length.

Therefore, it is essential to couple the use of TL with other biomarkers in order to attain an overview of the disruption to metabolic and/or physiological pathways.

540

541 Conflict of interest

The authors declare that they have no conflict of interest.

543 Author contributions

All authors contributed to the study conception. DNA sampling preparation and analysis were performed by Ascensio E. RNA and hormonal sampling preparations, then analysis were performed by Prévot D'Alvise N.

All statistical analysis were performed by Richard S. The first draft of the manuscript was written by Prévot D'Alvise N. and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Smith Aden, a native speaker and a professional translator, checked the quality of the English of the complete paper. Katie Dunning checked the quality of the final version.

551 Data transparency

All authors declare that all data and materials support comply with field standards.

553 Funding

This study was funded by the "Comité d'Aide à la Recherche et aux Tranferts Technologiques" de l'IUT de Toulon and "Appel à Projets 2020" (Telomer2020) - University of Toulon (France).
556 Ethical Approval

557 This study was conducted in respecting the welfare of fishes and in according to the procedure outlined in
558 the « *AVMA Guidelines for euthanasia of Animals; 2013* ». Animal research formation approved by
559 Ministère de l'Agriculture et de la Pêche, sous-direction de la santé et de la protection animale N°I-
560 45CNRSCDTA-F1-13.

561

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