# Cadmium tolerance is associated with tissue-specific plasticity of metallothionein gene expression in Gammarus fossarum field populations

Auréline Lalouette, Arnaud Chaumot, Louveline Lepeule, Karen Gaget, Nicolas Delorme, Laura Garnero, Federica Calevro, Davide Degli Esposti

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## To cite this version:

Auréline Lalouette, Arnaud Chaumot, Louveline Lepeule, Karen Gaget, Nicolas Delorme, et al. Cadmium tolerance is associated with tissue-specific plasticity of metallothionein gene expression in Gammarus fossarum field populations. Scientific Reports, 2025, 15 (1), pp.13913. 10.1038/s41598-025-98937-6. hal-05068990

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# Cadmium tolerance is associated with tissue-specific plasticity of metallothionein gene expression in *Gammarus fossarum* field populations

Auréline Lalouette<fnref n="1" />, Arnaud Chaumot<fnref n="1" />, Louveline Lepeule<fnref n="1" />, Karen Gaget², Nicolas Delorme<fnref n="1" />, Laura Garnero<fnref n="1" />, Federica Calevro² & Davide Degli Esposti<fnref n="1" />✉

The metallothionein gene family codes for proteins involved in metal homeostasis and acute detoxification of non-essential toxic metal ions across the tree of life. We have previously documented increased cadmium (Cd) tolerance in field populations of the crustacean *Gammarus fossarum* exposed to chronic metallic contamination of geochemical origin. This tolerance is lost during maintenance of organisms in the laboratory, and is transmitted to offspring via parental effects. This study investigated whether the expression of the Cd-responsive metallothionein gene *mt1* could be related to Cd-tolerance plasticity in *G. fossarum*. In eleven populations with different chronic Cd exposure history, we simultaneously assessed Cd-tolerance (mortality tests) and *G. fossarum mt1* expression levels by RT-qPCR in the gills and caeca of adult males and in neonates. *mt1* expression levels in the two organs were correlated to Cd-tolerance in field organisms and a loss of tolerance was observed in parallel with a decreased expression of *mt1* in the caeca after maintenance in uncontaminated water. We also recorded a greater inducibility of *mt1* expression in offspring of tolerant populations in the laboratory when re-exposed to Cd along with the bi-parental transmission of Cd-tolerance. These results suggest that the control of *mt1* expression is involved in the plasticity of Cd-tolerance in gammarid populations with different histories of Cd exposure.

**Keywords** Adaptation, Crustaceans, Ecotoxicology, Phenotypic plasticity, Metallothionein, Gammarids

In aquatic ecosystems, heavy metal contamination is a major concern as they are continuously released into the environment either through anthropogenic or natural processes <fnref n="1" />. In response to variable metal contamination in the environment, organisms can develop increased tolerance to metals (i.e. changes in sensitivity), notably through the development of transgenerational acclimation mechanisms ²,³. Tolerance to metal exposure may involve different metal regulation mechanisms that notably detoxify and eliminate metallic elements that are incorporated into the organism ³. Among these mechanisms, the expression of members of the metallothionein (*mt*) gene superfamily has been documented to confer tolerance to metal exposure ⁴,⁵. MTs are cysteine-rich, low molecular weight proteins involved in essential metal homeostasis, non-essential metal detoxification and protection against oxidative stress ⁴,⁶. These proteins are highly conserved across the animal kingdom and are considered to be the main defence against toxic metals in invertebrates ⁷,⁸. On the other hand, the involvement of MTs in metal tolerance has been suggested in many terrestrial and aquatic species (as shown in reviews <fnref n="1" />,²,⁵,⁹). A higher basal level and/or a stronger induction capacity of MTs have been observed in tolerant populations than in naïve populations of the same species. For example, higher levels of MTs were found in cadmium (Cd) tolerant populations of *Chironomius riparius* compared with susceptible populations of the same species after field sampling ¹⁰. In contrast, in *Orchesella cincta*, similar levels were found between Cd tolerant and sensitive populations in the field, but with a greater capacity to induce MT in tolerant populations when they are re-exposed to the metal of interest ¹¹. Furthermore, the involvement of *mt* genes and their products in the transmission of metal tolerance to offspring in invertebrates has been suggested in laboratory studies conducted in daphnia ¹²,¹³,

<footnote n="1">¹Laboratoire d'écotoxicologie, INRAE, UR RiverLy, Villeurbanne 69625, France. ²INRAE, INSA Lyon, Université de Lyon, BF2i, UMR 203, Villeurbanne 69621, France. ✉email: davide.degli-esposti@inrae.fr</footnote>
collembola<sup>14</sup>, bivalves<sup>15</sup> and fish<sup>16,17</sup>. However, these findings have been little studied in individuals from natural populations.

Species of the genus *Gammarus* have been studied in the context of the acquisition of metal-induced tolerance and of the long-term effects of metal exposure (e.g.<sup>18–20</sup>). Recently, we have shown that Cd tolerance, can be observed in several populations of *G. fossarum* living in rivers naturally contaminated by geochemical sources of Cd<sup>21,22</sup>. In these populations, tolerance appears to be linked to non-genetic acclimation, and more specifically to phenotypic plasticity, since this tolerance can be temporarily lost in populations and by individuals when they are isolated in Cd-free water<sup>21</sup>. Furthermore, Cd tolerance is transmissible to offspring through parental effects, seems to depend on long-term parental exposure, and can be transmitted by both mother and father<sup>21</sup>. However, the molecular mechanisms mediating these observations are not currently known for this species. Otherwise, a recent work on this species highlighted the toxicokinetic of Cd ions in various organs (gills, caeca, intestine, cephalon and other tissue)<sup>23</sup>. Specifically, Gestin et al.<sup>23</sup> showed that intestines are involved in the uptake, distribution and elimination of Cd, that caeca play a central role in accumulation and elimination function, while gills are hyper-accumulative organs with an important role in storage of Cd. Taking advantage of the results on the toxicokinetic of Cd and to the recent identification of a Cd-responsive metallothionein coding transcript (*mt1*) in the *G. fossarum* transcriptome<sup>24</sup>, this study aimed to: (i) determine the association between *G. fossarum* *mt1* expression and Cd tolerance at different life stages and sub-individual (organ) and (ii) assess parental transmission of *G. fossarum* *mt1* expression and its effects on Cd-tolerance in the offspring.

## Materials and methods

### Selection of the eleven *G. fossarum* populations

Based on our previous works<sup>22</sup> we considered eleven populations of *G. fossarum* inhabiting upstream areas of headwater streams in natural or semi-natural context and presenting a contrast in exposure to Cd-contamination and Cd-tolerance. More precisely, we selected three populations living in rivers naturally contaminated by Cd and presenting tolerance to this metal (Ardillats, Marchampt and Vernay populations), three populations living in rivers presenting intermediate Cd-contamination and tolerance levels (Strenbach, Rauental and Morcille populations) and five populations living in uncontaminated rivers and presenting no tolerance to Cd (Seran, Doulonne, Katlen, Reigne and Vancelle populations). The results of the distribution of individual survival times obtained during acute Cd sensitivity tests on adult male gammarids freshly collected in the field during the sampling campaign of the present study are already supplied in Lalouette et al.<sup>22</sup>. Each population has been assigned a letter as it follows: Ardillats (A), Marchampt (B), Vernay (C), Strenbach (D), Rauenthal (E), Morcille (F), Séran (G), Doulonne (H), Katlen (I), Reigne (J) and Vancelle (K). Furthermore, an active biomonitoring approach (caging and bioaccumulation measurement in transplanted reference organisms) were used to assess populations' field exposure to Cd as described in previous studies<sup>20,25,26</sup>. The average level of Cd contamination over the last ten years (already published in Lalouette et al.<sup>22</sup>) and the location details of the eleven study sites are presented in Supplementary Table 1.

### Sampling and maintenance of Gammarids in the lab

Two sampling campaigns were realised for this study: one in June 2022 (eleven populations collected) and another in October 2022 (five populations collected). For each campaign, gammarids were collected by kick sampling and quickly transported to the laboratory in plastic buckets containing ambient freshwater. They were maintained in the water of their own site at 12 °C and they were fed *ad libitum* with conditioned alder leaves (*Alnus glutinosa*) until used for the experiments (max 24 h). The day after field sampling and for each population, 6 males per population were dissected and the gills and the caeca were individually collected for each organism as described in Gestin et al.<sup>27</sup> to measure *mt1* gene expression (see Sect. 2.6). Organs were flash-frozen in liquid nitrogen and kept at – 80 °C until RNA extraction.

Gammarids were maintained in the laboratory in plastic aquariums (2–11 L) placed in water baths thermos-regulated at 16 °C and with a controlled photoperiod (16/8 night/day). Gammarids were fed *ad libitum* with conditioned alder leaves (*Alnus glutinosa*) and a weekly supply of Tubifex subadults.

### Deacclimation of populations in Cd-free water in the laboratory

Gammarid organisms from populations A, B, C, G and H were collected in October 2022 and were kept in separate 11 L plastic aquaria (around 1000 adult individuals). After 2 months, male adults from each population were collected for Cd sensitivity tests (see Sect. 2.5) and to measure *mt1* gene expression in gills and caeca (see Sect. 2.6) as previously described. In parallel, we sorted 100 gammarids in mating-pairs with females in the last stage of their reproductive cycle (stage D2 with hatched neonates in brood pouch) from the five populations collected in December 2022 (A, B, C, G, H). After one reproduction cycle (approximatively 25 days), neonates from each population were collected for Cd sensitivity tests (*n* = 24; see Sect. 2.5) and to measure *mt1* gene expression (*n* = 6 pool of 10 neonates; see Sect. 2.6). In this same batch of neonates, we studied MT inducibility by measuring *mt1* gene expression after re-exposure at sublethal levels to Cd. For this purpose and for each population, 120 neonates were collected and isolated by pool of 10 neonates in falcons containing a mixture of Evian water and demineralised water (50/50, conductivity approximately 300 µs/cm) with or without CdCl₂ (equivalent to 3 µg/L of dissolved Cd²⁺). For each condition, 6 falcons were considered and exposure was conducted over 3 days. At the end of the 3 days, neonates from both conditions were collected, placed in 2 mL eppendorf tubes and frozen at -80 °C.

### Cross-breeding of tolerant and sensitive populations

In order to assess parental effects on *mt1* expression in the offspring, we carried out cross-breeding between genitors from the population A (tolerant) and population G (sensitive) collected during the campaign of June
2022 as described in Lalouette et al.²¹. Briefly, after sampled in the field, gammarids in mating-pairs were sorted visually with females in stage D2, separated and re-mated between tolerant and sensitive population, resulting in four conditions: AA, GG, AG, GA, where the first letters correspond to the initial of the population name of the male and the second to the initial of the population name of the female. For each condition, two 2-L plastic aquariums containing 50 mating-pairs of gammarids were considered. At the end of the moulting-cycle (approximatively 25 days), neonates from the different conditions were collected for Cd sensitivity tests and to measure the expression of *mt1* gene expression. Moreover, the inducibility of *mt1* was studied following re-exposure to Cd (3 µg/L) during 3 days using the same protocol as described previously (see Sect. 2.3).

## Acute toxicity tests for cd tolerance assessment

Tolerance to Cd was assessed in adult males after 2 months in the laboratory. Similarly, tolerance was also measured in the cohort of neonates born in the laboratory from field populations or from crosses between populations. For that, we performed acute toxicity tests as described in Lalouette et al.²¹,²². For each condition of adult gammarids (source population, laboratory maintenance duration), three replicates of 15 males were exposed to a nominal concentration of 80 µg/L of Cd in 500 mL plastic beakers. For progeny tolerance assessment, 24 neonates to each condition were collected one day after their release from maternal marsupium. The acute toxicity tests were then carried out by individually monitoring the survival of neonates exposed to a nominal Cd concentration of 20 µg/L in 50 mL falcons.³ As previously described²¹,²², the different Cd solution was prepared from stock solutions of Cd at 0.1 g/L. Then, beakers and falcons were kept in a thermo-regulated water bath at 12 °C and mortality was monitoring daily during exposure tests.

## Gene expression analysis

Gene expression of *mt1* was measured by RT-qPCR ²⁴. Here it follows a description of the different steps.

### Selection of the studied organs

We focused on *mt1* gene expression in gills and caeca for adults' males gammarids. Indeed, a preliminary experiment was conducted in the gills, caeca, ovaries and testes of adult individuals from the tolerant (Ardillats) and the sensitive (Séran) population. The preliminary results showed differences between *mt1* gene expression in the caeca and gills, as well as in the ovaries, but not in the testes (Supplementary Fig. 1). Since this study focuses only on male (to avoid confounding factors linked to the reproductive cycle of females), we chose to focus on the caeca and the gills, two organs involved in Cd toxicokinetics in *G. fossarum*.

### Sample

For adult males, we considered 6 biological replicates per organ and population. For neonates, *mt1* gene expression was measured in pools of 10 neonates. For each measurement, 6 pools of 10 neonates were considered. Then, the same protocol was used for adult and neonates.

### Total RNA extraction and cDNA synthesis

The RNeasy fibrous tissues kit (Qiagen, Hilden, Germany) was used to extracted RNA from each organ or pool of neonates and quality and quantity were assessed by spectrophotometry using NanodropOne (Thermofisher, Waltham, MA, USA). RNAs (about 100 ng per sample) were retro-transcribed using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Waltham, MA, USA).

### Quantitative real-time polymerase chain reaction (qPCR)

For target genes, specific primers for *mt1* and *elongation factor (ef)* genes were used (Supplementary Table 2). Following the reverse transcription, cDNAs was diluted (1:5) and amplified using the OneGreen sybr green mix (Ozyme, Montigny-le-Bretonneux, France) in a Bio-Rad CFX96 qPCR platform (Hercules, CA, USA) using the following conditions: 95 °C for 3 min followed by 40 cycles at 95 °C for 10 s, 61 °C for 30 s and 72 °C for 30 s. Three technical replicates were performed for each sample. MTs were quantified using the relative quantification method (ΔCt) using the elongation factor (EF) expression to normalize gene expression across samples, as in Degli Esposti et al. ²⁴. The reference gene *ef* is a stable gene on *G. fossarum* ²⁸. In order to obtain a better visualization of the results, these were transformed using the square root function (√ΔCt).

## Statistical analysis

The data were processed with the R software version 4.4.0 (R Core Team, 2024). The data obtained during acute toxicity tests (sensitivity to Cd) were analysed by Kaplan-Meier type survival analyses (log-rank test) using the “survival” R package. For *G. fossarum* *mt1* expression, the results obtained were analysed using the Pairwise Wilcoxon rank sum test (abbreviation used: PW) with correction for multiple testing by the Benjamini-Hochberg method. All the statistical analyses were performed with a statistical significance level of 5%.

## Results

### Basal level of *G. fossarum* *mt1* gene expression in adults sampled in field

The expression of *mt1* was measured in the gills and the caeca of adult males after field sampling in tolerant, intermediate and sensitive populations (Fig. 1, Supplementary Fig. 2). The results show that *mt1* expression is 8-fold higher in the gills of organisms from contaminated populations than in uncontaminated (PW test: adjusted p-value = 3.10⁻⁹) and 5-fold higher than those from intermediate populations (PW test: adjusted p-value = 0.03) (Fig. 1, Supplementary Fig. 2). Similarly, the intermediate populations showed higher levels of *mt1* expression than the uncontaminated populations (PW test: adjusted p-value = 9.10⁻⁹). In the caeca, *mt1* expression levels (Fig. 1) of organisms from contaminated populations were 2-fold higher than those from uncontaminated
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** *G. fossarum* *mt1* gene expression measured by RT-qPCR in the gills and caeca of adult males after sampled in the field in contaminated (pooled data from populations A, B and C), intermediate (pooled data from populations D, E and F) and uncontaminated (pooled data from populations G, H, I, J and K) populations. Gene expression was normalized using the elongation factor (EF) C~t~ value. Data detailed by population are presented in Supplementary Fig. 1. Red, yellow and green colors correspond respectively to Cd contaminated, intermediate and uncontaminated status of populations. The symbols indicate the statistical significance (p-value) as followed: (ns) > 0.05; (*) ≤ 0.05; (**) ≤ 0.01; (***) ≤ 0.001; (****) ≤ 0.0001.</figcaption></figure>

populations (PW test: adjusted p-value = 8.10⁻⁵). Interestingly, the results reveal a positive correlation between the median survival time (LT50) of each population and the median expression of *mt1* in the gills (Spearman-correlation, ρ = 0.8; p-value = 10⁻³; Fig. 2) and caeca (Spearman-correlation, ρ = 0.7; p-value = 10⁻²; Fig. 2) of adult males.

**Plasticity of *G. fossarum* mt1 gene expression is associated to loss of Cd tolerance**

After two months of maintenance in Cd-free water (T2), we observed a decrease in Cd tolerance compared to those observed just after field sampling (T0) in population A (log-rank test: p-value = 6.10⁻³) and population C (log-rank test: p-value = 8.10⁻⁸) (Supplementary Fig. 3). In particular, the decrease in survival time distribution was driven by a loss of the highest levels of individual tolerance, together with a decrease of the lowest levels down to the range of survival times observed in uncontaminated populations (Supplementary Table 3). For example, only 16% and 14% of the A-T0 and C-T0 individuals respectively, had a mean survival time inferior or equal to 6 days compared to 40% of A-T2 and 44% of C-T2 (Supplementary Table 3). For population B, no tolerance to Cd was observed in gammarids after field sampling at the date tested. Thus, no difference in Cd sensitivity was observed between B-T0 and B-T2.

Gene expression levels of *mt1* in the gills and caeca of adult males before and after the maintenance in Cd-free water were also measured (Fig. 3). There was a significant decrease in *mt1* expression in the caeca of
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Correlation plots between the average relative expression of *G. fossarum* *mt1* in gills (A) and caeca (B) with the median (LT50) of adults survival time (days) of the 11 populations. Red, yellow and green colors correspond to Cd contaminated, intermediate and uncontaminated status of populations, respectively (see Supplementary Table 1).</figcaption></figure>

organisms from populations A and B (Pairwise-Wilcoxon (PW) test: adjusted p-values < 0.03). The same trend was observed, but not significantly, for organisms from population C (PW test: adjusted p-value = 0.58). In contrast, there was no modulation of *mt1* expression in the gills of organisms from these same populations after 2 months in clean water (PW test: adjusted p-values > 0.4). For uncontaminated populations, no difference in *mt1* expression in the caeca or gills was observed over time (PW test: adjusted p-value = 0.26 and p-value = 0.77, respectively).

## G. fossarum *mt1* gene expression and Cd tolerance in the offspring

In order to investigate the relationship between *mt1* gene expression and Cd tolerance in the offspring, we focused on the offspring produced in the lab by genitors exposed on the field and kept in Cd-free water. The results of acute Cd sensitivity tests showed that neonates from the contaminated populations were more tolerant compared to those from the uncontaminated populations (log-rank test: p-value = 10⁻¹²) (Fig. 4A, Supplementary Fig. 4). However, no difference in *mt1* expression was observed between the tolerant and sensitive populations of neonates before the acute exposure (PW test: adjusted p-value = 0.72) (Supplementary Fig. 5). Thus, we tested the capacity of neonates to induce MT by exposing them to 3 µg/L Cd for 3 days, followed by measurement of *mt1* expression. Results showed that *mt1* inducibility is higher in tolerant neonates (Fig. 4B, Supplementary Fig. 5). Neonates from tolerant parents were able, on average, to induce *mt1* expression four times (Fold-change = 8) more strongly than uncontaminated populations (Fold-change = 2).

Finally, crossbreeding experiments between parents issued from populations A and G showed that neonates born from a tolerant father (condition AG) and from a tolerant mother (condition GA) present a distribution of survival times close to those born from two tolerant parents (condition AA) (Fig. 5A). Indeed, the distribution of survival times of GA and AG neonates is not significantly different from AA condition (log-rank test: p-value = 0.3) while it was significantly different to GG condition (log-rank test: p-values = 6.10⁻³). The expression levels of *mt1* were measured in control condition and after a challenge at 3 µgCd/L for 3 days (Fig. 5B, Supplementary Fig. 6). The results show that *mt1* inducibility was higher in AA (Fold-change = 9.2) under Cd conditions than in GG neonates (Fold-change = 3.7). The results for AG neonates do not show significant differences with GG neonate, although, an increasing trend is observed (Fold-change = 5, respectively) (Fig. 5B). In particular, we observed an increase in individual variability in neonates AG and GA, with respectively, 67% and 50% of individuals presenting a fold-change greater than 4, against 100% for AA condition and 17% for GG condition (Supplementary Table 4).

## Discussion

The ability of populations to maintain themselves in metal-contaminated environments depends on the effectiveness of mechanisms that protect the organisms from metal uptake and/or contribute to metal detoxification ²⁹. One of these mechanisms involves the expression of genes belonging to the metallothionein (*mt*) superfamily. Several studies have suggested that metal detoxification by metallothioneins is associated with increased tolerance to metals ². The implication of MTs in response to metal exposure and/or in metal tolerance has also been documented in various arthropod species such as amphipods ¹⁸,³⁰⁻³², daphnids ¹²,¹³,³³ or diptera ³⁴,³⁵. In two species of arthropods, *Chironomius riparius* and *Orchesella cincta*, tolerant individuals from metal-contaminated sites show increased expression of *mt* gene compared with a reference population ¹⁰,³⁶,³⁷. The
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** *G. fossarum* *mt1* gene expression measured by RT-qPCR in the gills and caeca of adult males after sampled in the field (T0) and after maintenance in Cd-free water during 2 months in the lab (T2) in five field populations (A, B, C, G, H). Gene expression was normalized using the elongation factor (EF) C~t~ value. Red and green colors correspond respectively to Cd contaminated and uncontaminated status of populations in their habitat of origin. The symbols indicate the statistical significance (p-value) as followed: (ns) > 0.05; (*) ≤ 0.05; (**%) ≤ 0.01.</figcaption></figure>

up-modulation of MTs expression could also participate to the antioxidant pathways by reducing the oxidative potential of free ion metals (like Cd or Pb) and thus reducing their pro-oxidant effects ⁴,⁶. Similarly, in tolerant populations, it has been suggested that increasing baseline levels of MTs would help to maintain homeostasis in these populations³⁸. However, establishing this link is challenging in field populations since the majority of these studies in the literature compare only one tolerant population *versus* one sensitive population ¹¹,³²,³⁹. In fact, such experimental designs are subject to possible false positive conclusions in relation to the potential influence of confounding biotic or abiotic factors unrelated to contaminant exposure. Thus, we investigated the link between the expression of *G. fossarum* *mt1* described as being specifically induced during short-term Cd exposure in *G. fossarum* ²⁴ and the plasticity of Cd tolerance observed in chronically exposed natural populations of this species ²¹. Firstly, we measured *mt1* expression in Cd-tolerant and Cd-sensitive natural populations after field sampling in two organs, the caeca and the gills, known to be involved in Cd toxicokinetics in *G. fossarum* ²³. Our results showed a correlation between *mt1* expression and Cd tolerance, with higher levels of *mt1* expression in the gills and caeca of tolerant populations. The strength of our study is to consider eleven field populations with similar living habitats except for Cd-exposure, thus limiting the impact of other environmental confounding factors ²².

Our results also showed that in gammarids exposed to Cd in natural environment, *mt1* is differentially expressed in populations with different histories of metal exposure. More specifically, we showed that the average expression of *mt1* is approximately 4 and 6 times higher in the gills of intermediate and tolerant populations, respectively, compared with sensitive populations. Similarly, in the caeca, the average expression was 2 times higher in the intermediate and tolerant populations. Similar results were found in the gills of the mussel *Pyganodon grandis*, where MT concentrations (measured as protein fraction) increased along the gradient of metal contamination at the different study sites ⁴⁰. However, a correlation between tolerance and MT expression is not systematically found. For example, Chiodi Boudet et al. ³⁹ showed different detoxification strategies in *Palaemon argentinus*. When this species is exposed to metals, the main detoxification pathway in tolerant
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Tolerance and *mt1* expression levels in neonates born in the lab in Cd free water after one reproduction cycle (approximatively 25 days) from three contaminated populations and three uncontaminated populations. (A) Survival time of offspring exposed to Cd (20 µg/L) individually one day after their release from maternal marsupium and (B) Fold change of *G. fossarum* *mt1* gene expression measured by RT-qPCR in neonates after a re-exposure at 3 µgCd/L during 3 days compared to non-exposed controls. Data detailed by population are presented in Supplementary Figs. 3 and 4. Red and green colors correspond respectively to Cd contaminated and uncontaminated status of populations in their habitat of origin. The symbols indicate the statistical significance (p-value): (****) ≤ 0.0001.</figcaption></figure>

populations is Cd sequestration in lysosomal vesicles, or also-called metal-rich granules (MRG), whereas sensitive populations mainly detoxify Cd by binding to MTs. Similarly, Vijver et al. ⁴¹ suggest that MRG are responsible for tolerance during long-term exposure to Cd, whereas MTs are involved mainly during short-term exposure.

Afterwards we investigated whether Cd tolerance plasticity was mirrored by *mt1* expression changes. For that, three tolerant populations (population A, B, C) and two sensitive populations (population G, H) were maintained in Cd free water in the lab for 2 months. When individuals from Cd-tolerant populations were no longer exposed to this metal for 2 months, there was a parallel decrease in tolerance and *mt1* expression levels in the caeca, but not in the gills. Cd toxicokinetics models in *G. fossarum* showed that gills store the metal, while caeca are likely involved in detoxification due to their accumulation and depuration capacities ²³. Indeed, it was found that Cd concentrations accumulated in the gills during a 7-day exposure remained stable after a 30-day depuration phase, whereas Cd concentrations in the caeca decreased by 66% ²³. As seen previously with the results obtained after field sampling, tolerant gammarids may have acquired, during acclimation processes, a capacity to retain more Cd in the caeca where it can be sequestered by MTs and then eliminated. Our results suggest that Cd tolerance is possibly due to a physiological system in which *mt1* expression in the gills decreases the body-burden of Cd accumulation and the *mt1* expression in the caeca allowed metal detoxification (Fig. 3). Moreover, one of the hypotheses that could explain the overexpression of *mt1* in the gills of deacclimatised gammarids from contaminated sites may be that MTs have a shorter half-life than Cd and, as a result, epithelial cells must maintain high levels of transcriptional activity of the *mt1* gene. For example, it has been shown that in the winkle *Littorina littorea*, the half-life of MTs in the gills is 69 days, whereas the half-life of Cd is 300 days ⁴². The authors of the study therefore suggested a closed metal cycle in the organ, requiring a constant renewal of MTs to sequester the free Cd present in the organ ⁴². The same observation has also been made in the mussel *Mytilus edulis*, where the half-life of MTs is 25 days compared with 300 days for Cd ⁴³. However, during this same experiment, population B did not show tolerance to Cd (Supplementary Fig. 3). This result can be explained by the temporal variability of Cd contamination in this site ²¹. Despite this, in this population, high levels of *mt1* expression were found in the gills and caeca, followed by the same dynamics as in the two tolerant populations (A and C population), with a decrease in *mt1* expression in the caeca. Therefore, these results underlined the difficulty to decoupling the exposure parameter from the tolerance parameter and raises
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Cross-breeding between sensitive and tolerant populations. The figure presents (A) the Cd tolerance (distribution of individual survival times) and (B) the fold-change of *G. fossarum* *mt1* gene expression measured by RT-qPCR after re-exposure at 3 µgCd/L during 3 days in neonates produced by Cd tolerant population (population A), uncontaminated population (population G) and from population cross breeding. For the legend of the different conditions, the first capital letter corresponds to the first letter of the male's population name and the second capital letter corresponds to the first letter of the female's population name (notation ♂/♀: e.g., the condition AG corresponds to the crossing between a male from A population and a female from G population). The symbols indicate the statistical significance (p-value) as followed: (ns) > 0.05; (*) ≤ 0.05; (**) ≤ 0.01; (***) ≤ 0.001.</figcaption></figure>

questions about temporality where the loss of tolerance appears to be observed more quickly than observed at the molecular level.

We tested the hypothesis that *mt1* expression in offspring depends on parental exposure with the aim of improving our understanding of non-genetic intergenerational transmission of tolerance (i.e. parental effect) previously described in studies by Vigneron et al. ⁴⁴ and Lalouette et al. ²¹. For that, Cd tolerance and *mt1* expression were followed in a cohort of neonates born in Cd free water in the laboratory after one reproduction cycle: these neonates resulted from the fertilization of gametes that had formed in the field, but they developed as embryos in the maternal marsupia in Cd-free laboratory conditions. Firstly, Cd tolerance was found in neonates of the three tolerant populations. Interestingly, no difference was observed in the basal level of expression of *mt1* under control conditions between tolerant and sensitive neonates. However, a higher inducibility of *mt1* expression was observed in tolerant neonates after a short re-exposure to Cd, suggesting that is the ability to overexpress *mt1* in contaminated conditions and not constitutive basal levels of *mt1* expression that are responsible for Cd tolerance in *G. fossarum* neonates. In accordance with these observations, Shaw et al. ⁴⁵ showed similar results in daphnia (*Daphnia pulex*) after multigenerational exposure to Cd over 50 generations. In this study, identical levels of basal MT expression were found between tolerant and naive individuals after fifty generations and higher MT expression was observed in tolerant individuals after a short re-exposure to Cd ⁴⁵. Similarly in the worm *Dendrobaena octaedra*, Fisker et al. ⁴⁶ showed that the Cd-tolerant individuals of the second-generation exhibited a faster response capacity in the overexpression of a MT during Cd exposure. However, the potential role of MT in the transfer of metal tolerance from adults to offspring has rarely been highlighted, particularly in arthropods. In the study by Guan and Wang ¹³, multigenerational exposure to Cd in *Daphnia magna* resulted in an increase in tolerance to this metal and a significant induction of MT in exposed daphnia in all generations, with maximum synthesis in the later generations. The authors then hypothesised that the transgenerational effect of Cd exposure on the rapid induction of MT was due to the increase in Cd concentration in the offspring. Thus, daphnia born from exposed mothers would be more inclined to synthesise MTs ¹³. In this same species, another study suggests the role of MTs in the mercury tolerance observed in the offspring since they assume that mRNA for MTs could be transferred through reproduction ¹².

Finally, in the present study, a symmetrical role was found for both sexes in the transmission of Cd tolerance, although the capacity for inducing *mt1* was not significantly correlated. These results suggest while *mt1* gene
expression play a clear role in conferring tolerance to the population chronically exposed to Cd, other factors are likely involved in the transmission of this phenotype to the offspring. These results support the hypothesis of a link between epigenetics and the transgenerational effect of adaptation to stress in populations (here, the plasticity of Cd sensitivity), as already suggested in our last study²¹ and other authors ⁴⁷⁻⁴⁹.

## Conclusion

This study conducted in eleven field populations of *G. fossarum* suggests a correlation between the expression of the metallothionein gene, *G. fossarum mt1*, and Cd tolerance plasticity. We have shown that *mt1* gene is over-expressed in the gills and caeca (two organs involved in Cd regulation in this species) in field-tolerant populations. This covariation was also confirmed by a parallel decrease in tolerance and MT expression in the caeca during maintenance in Cd free water in the laboratory. Our study also showed that *mt1* expression regulation can be modulated by historical exposure in field populations and support adaptive processes involving transgenerational mechanisms. These results suggested that links can be drawn between the plasticity of metal detoxification mechanisms at the molecular level (*mt1* expression) and the adaptive plasticity of organism sensitivity at the population level. We suggest that epigenetic mechanisms should be further investigated to improve our understanding of intergeneration acclimation observed in these populations.

## Data availability

Raw data on gene expression and acute toxicity will be available upon acceptance in the National data depository https://entrepot.recherche.data.gouv.fr/dataverse/riverly. For more information, please contact the corresponding author.

Received: 14 January 2025; Accepted: 15 April 2025

Published online: 22 April 2025

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## Acknowledgements

The authors benefitted from the French GDR "Aquatic Ecotoxicology" framework which aims at fostering stimulating scientific discussions and collaborations for integrative approaches. The authors thank the ANR (Agence Nationale de la Recherche) program Dyn-Microbiome (ANR-20-CE34-0012). The authors thank Clément Colomb for his assistance in the field experiments especially for the organisms sampling, and Maxence Ruby for his assistance in the molecular experiments.

## Author contributions

Auréline Lalouette: Writing – original draft, Writing – review & editing, Validation, Methodology, Investigation, Formal analysis, Visualization, Conceptualization. Arnaud Chaumot: Writing – review & editing, Validation, Methodology, Funding acquisition, Supervision, Conceptualization. Louveline Lepeule: Investigation, Formal analysis. Karen Gaget: Resources. Nicolas Delorme: Investigation. Laura Garnero: Investigation. Federica Calevro: Resources. Davide Degli Esposti: Writing – review & editing, Validation, Methodology, Supervision, Conceptualization.
**Declarations**

**Competing interests**

The authors declare no competing interests.

**Additional information**

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1038/s41598-025-98937-6.

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