Marta Jaskulak

PhD Agnieszka Rorat
email: agnieszkarorat@gmail.com

Ligia Kurianska-Piatek

Sebastian Hofman

Janusz Bigaj

Franck Vandenbulcke

Barbara Plytycz

E Fetida

Species-specific Cd-detoxification mechanisms in lumbricid earthworms Eisenia andrei, Eisenia fetida and their hybrids

Keywords: Eisenia sp., hybrids, cadmium, Gene expression, detoxification, Bioaccumulation

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Cadmium (Cd) contamination of soils is a ubiquitous environmental problem that has resulted from uncontrolled industrialization, unsustainable urbanization, and intensive agricultural practices [START_REF] Khan | Soil contamination with cadmium, consequences and remediation using organic amendments[END_REF]. The dispersion of cadmium in the environment had been increasing in the past few decades all over the world. As an example, currently, Cd contamination can be found as a direct result of most industrial activities such as, mining, smelting, transport, burning of fossil fuels, wastewater irrigation systems, and as a by-product of synthetic phosphate fertilizers [START_REF] Grobelak | Cadmium-induced oxidative stress in plants, cadmium toxicity, and tolerance in plants: from physiology to remediation[END_REF]. Being a toxic element, Cd is a high threat to soil quality, food safety, and human health [START_REF] Shahid | Cadmium Bioavailability, Uptake, Toxicity and Detoxification in Soil-Plant System[END_REF]. To date, numerous studies have investigated the toxic impact of Cd in plants and animals, including humans.

Research focused on human exposure to cadmium shows that Cd induces several types of cancers, neurotoxic effects as well as kidney damage, bone demineralization, and the disruptions of the immune system [START_REF] Buha | Bone mineral health relates to environmental cadmium exposureexperimental and human data[END_REF]. Overall, industrial activities involved in a large scale dispersal of heavy metals to the environment are associated with genotoxic and carcinogenic hazards, which creates a crucial challenge for regulatory authorities [START_REF] Eom | Low-Level Environmental Cadmium Exposure Induces Kidney Tubule Damage in the General Population of Korean Adults[END_REF]. Improvements in the technologies of contaminant detection and quantification in environmental samples are essential [START_REF] Mahajan | Role of Phytoremediation in Reducing Cadmium Toxicity in Soil and Water[END_REF]. Nevertheless, novel approaches that could take into account not only chemical hazards but its biological and ecological effects are currently needed for accurate safety evaluation [START_REF] Khan | Soil contamination with cadmium, consequences and remediation using organic amendments[END_REF].

Earthworms, living in close contact with soil, are constantly confronted with different pollutants, among the others high cadmium concentrations [START_REF] Wang | Integration of earthworms and arbuscular mycorrhizal fungi into phytoremediation of cadmium[END_REF]. Moreover, they are a crucial part of soil fauna, which helps to maintain soil structure and organic matter cycling via soil ingestion and excretion, making them key regulators of soil processes. Hence, earthworms are widely used as model organisms for the evaluation of the hazardous effects of different terrestrial toxicants [START_REF] Yu | Joint toxic effects of cadmium and four pesticides on the earthworm (Eisenia fetida)[END_REF].

Compared to other lumbricid species, composting earthworms from Eisenia genus are very resistant to most pollutants, including heavy metal contamination. Nevertheless, most of the early studies are focused on the Eisenia sp., not taking into account the distinction between E. fetida and E. andrei [START_REF] Rombke | DNA barcoding of earthworms (Eisenia fetida/andrei complex) from 28 ecotoxicological test laboratories[END_REF][START_REF] Suleiman | Determination of the performance of vermicomposting process applied to sewage sludge by monitoring of the compost quality and immune responses in three earthworm species: Eisenia fetida, Eisenia andrei and Dendrobaena veneta[END_REF]. Proper distinction of the red earthworm E. andrei and striped E. fetida on basis of morphological criteria is sometimes unequivocal, thus shall be connected with species delimitation by the species-specific DNA sequences of the mitochondrial and/or nuclear genes. Such molecular markers revealed a clear distinction between Ea and Ef species, and at least two distinct mitochondrial lineages of the latter, described as Ef1 and Ef2 (e.g. [START_REF] Pérez-Losada | Phylogenetic species delimitation of the earthworms Eisenia fetida (Savigny, 1826) and Eisenia andrei Bouché, 1972 (Oligochaeta, Lumbricidae) based on mitochondrial and nuclear DNA sequences[END_REF][START_REF] Otomo | Usefulness of DNA Barcoding in Ecotoxicological Investigations: Resolving Taxonomic Uncertainties Using Eisenia Malm 1877 as an Example[END_REF][START_REF] Rombke | DNA barcoding of earthworms (Eisenia fetida/andrei complex) from 28 ecotoxicological test laboratories[END_REF]. Combined analysis of sequences of the haploid mitochondrial genes (of the maternal origin) and diploid nuclear genes of maternal-paternal origin made possible to find out hybrids between laboratory-mated (Ea+Ef1) or (Ea+Ef1) pairs (Plytycz et al. 2018a,b;2020;[START_REF] Podolak | Annual Reproductive Performance of Eisenia andrei and E. fetida 2 in Intra-and Inter-Specific Pairs and Lack of Reproduction of Isolated Virgin Earthworms[END_REF]).

Among the progeny of hermaphroditic Ea+Ef pairs appeared both specimens of the pure Ea or Ef species (derived by self-fertilization) and hybrids derived from Ea ova fertilized by Ef spermatozoa (called here Ha), but non Hf hybrids from Ef ova fertilized by Ea spermatozoa.

These later appeared only among progeny of Ha hybrids mated with Ef pure species but were very rare and sterile. The Ha hybrids were relatively common and fertile, giving offspring with Ea species consisting off second (and then third) generations of Ha hybrids and pure Ea specimens (Plytycz et al. 2018a,b;[START_REF] Podolak | Annual Reproductive Performance of Eisenia andrei and E. fetida 2 in Intra-and Inter-Specific Pairs and Lack of Reproduction of Isolated Virgin Earthworms[END_REF]), but fertility of hybrids was gradually diminished in the subsequent generations [START_REF] Plytycz | Impairment of reproductive capabilities in three subsequent generations of asymmetric hybrids between Eisenia andrei and E. fetida from French, Hungarian and Polish laboratory colonies[END_REF]. The question arose whether hybrids are more sensitive to stressing factors than specimens of the pure parental species?

Our previous laboratory studies on effects of cadmium were performed on E. fetida [START_REF] Brulle | cDNA cloning and expression analysis of Eisenia fetida (Annelida: Oligochaeta) phytochelatin synthase under cadmium exposure[END_REF], Bernard et al. 2010) and more recently on E. andrei [START_REF] Homa | Dermal exposure of Eisenia andrei earthworms: Effects of heavy metals on metallothionein and phytochelatin synthase gene expressions in coelomocytes[END_REF][START_REF] Takacs | Exposure of Eisenia andrei (Oligochaeta; Lumbricidea) to Cadmium Polluted Soil Inhibits Earthworm Maturation and Reproduction but not Restoration of Experimentally Depleted Coelomocytes or Regeneration of Amputated Segments[END_REF], Rorat et al. 2017). We have documented that exposure of E. andrei earthworms to soil spiked with high concentration of cadmium chloride (500 mg kg -1 air-dried soil), caused massive cadmium bioaccumulation in earthworm bodies without lethal effects; it caused inhibition of maturation of juveniles, inhibition of cocoon production by adults without adverse effects on regenerative processes [START_REF] Takacs | Exposure of Eisenia andrei (Oligochaeta; Lumbricidea) to Cadmium Polluted Soil Inhibits Earthworm Maturation and Reproduction but not Restoration of Experimentally Depleted Coelomocytes or Regeneration of Amputated Segments[END_REF]. Cadmium exposure even enhanced regeneration of amputated posterior segments that corresponded with induction of expression of metallothionein gene of this species [START_REF] Rorat | Protective role of metallothionein during regeneration in Eisenia andrei exposed to cadmium[END_REF].

In our previous studies, the mechanisms of detoxification of Cd were investigated on transcriptomic level and some genes were identified as involved or potentially involved in those processes, namely, metallothionein (mt), glutathione S-transferase (gst), lysenin (lys), superoxide dismutase (sod), phytochelatin synthase (pcs) [START_REF] Brulle | cDNA cloning and expression analysis of Eisenia fetida (Annelida: Oligochaeta) phytochelatin synthase under cadmium exposure[END_REF], Bernard et al. 2010[START_REF] Homa | Dermal exposure of Eisenia andrei earthworms: Effects of heavy metals on metallothionein and phytochelatin synthase gene expressions in coelomocytes[END_REF]. Among the others, it has been shown that the protective mechanisms were mainly based on a strong induction of a gene coding the cadmium metallothionein (mt2) in earthworms [START_REF] Homa | Dermal exposure of Eisenia andrei earthworms: Effects of heavy metals on metallothionein and phytochelatin synthase gene expressions in coelomocytes[END_REF][START_REF] Engelmann | Phenotypic and functional characterization of earthworm coelomocyte subsets: Linking light scatter-based cell typing and imaging of the sorted populations[END_REF]. The implication of the other genes mentioned before is not fully elucidated. Even if the two Eisenia species are very resistant to stressors and show similar responses overall, we noted specificities, in particular at the molecular level, which we decided to investigate in this work.

Therefore, it was reasonable to compare effects of cadmium exposure on E. andrei, two distinct lineages of E. fetida (Ef1 and Ef2) and on hybrids appearing among progeny of Ea+Ef pairs mated in the laboratory settings (Plytycz et al. 2018a[START_REF] Plytycz | Impairment of reproductive capabilities in three subsequent generations of asymmetric hybrids between Eisenia andrei and E. fetida from French, Hungarian and Polish laboratory colonies[END_REF][START_REF] Podolak | Annual Reproductive Performance of Eisenia andrei and E. fetida 2 in Intra-and Inter-Specific Pairs and Lack of Reproduction of Isolated Virgin Earthworms[END_REF]. For this, the accumulation of Cd and the level of expression of genes directly or indirectly involved in the response to Cd were measured in pure species and their hybrids investigated at the same laboratory conditions.

The main goal of the first part of our experiments was to compare the life traits and the capacity of cadmium accumulation in whole bodies of adult earthworms from genetically defined Ea, Ef1, Ef2 and their hybrids (Ha). Earthworms were exposed for four weeks to commercial soil either unpolluted (control) or moderately or higly spiked with cadmium chloride at the same concentrations as that used during our previous studies [START_REF] Takacs | Exposure of Eisenia andrei (Oligochaeta; Lumbricidea) to Cadmium Polluted Soil Inhibits Earthworm Maturation and Reproduction but not Restoration of Experimentally Depleted Coelomocytes or Regeneration of Amputated Segments[END_REF][START_REF] Rorat | Protective role of metallothionein during regeneration in Eisenia andrei exposed to cadmium[END_REF]. The second part aimed for gaining insight into cadmium-related defense mechanisms at transcriptomic level in coelomocytes extruded from a new set of Ea, Ef1, Ef2, and Ha/Hf earthworms exposed for 0 days (control), 2 days or 7 days to moderately Cd-polluted soil in mesocosms. Overall, the upregulation of mt and sod genes and downregulation of lys genes were more pronounced in hybrids than in parental species.

Materials and methods

Earthworms

Adult composting E. andrei (Ea) and E. fetida (Ef) earthworms deriving from laboratory stocks at the University in Lille (France) were cultured for a decade in the laboratory of the Institute of Zoology and Biomedical Research of the Jagiellonian University (Krakow, Poland), and in parallel for last four years in the laboratories of Rzeszow University (Poland).

Earthworms were cultured in boxes with commercial soil at room temperature and fed ad libitum on boiled/dried tea, nettle and dandelion leaves. For several years some of them were used for studies on progeny of laboratory-paired genotyped virgin specimens giving in offspring specimens of E. andrei (Ea), two mitochondrial lineages of E. fetida (Ef1 and Ef2) species, and interspecific hybrids (H) derived either from Ea ova (Ha) or E1/Ef2 ova (Hf), each of them individually delimited by species-specific sequences of the mitochondrial COI gene and diploid nuclear 28S rRNA gene (Plytycz et al. 2018a;2020;[START_REF] Podolak | Annual Reproductive Performance of Eisenia andrei and E. fetida 2 in Intra-and Inter-Specific Pairs and Lack of Reproduction of Isolated Virgin Earthworms[END_REF]. Some of these genetically delimited adult (clitellate) specimens were used for experiments performed in the same laboratory conditions [START_REF] Plytycz | Impairment of reproductive capabilities in three subsequent generations of asymmetric hybrids between Eisenia andrei and E. fetida from French, Hungarian and Polish laboratory colonies[END_REF][START_REF] Podolak | Annual Reproductive Performance of Eisenia andrei and E. fetida 2 in Intra-and Inter-Specific Pairs and Lack of Reproduction of Isolated Virgin Earthworms[END_REF]. One group of earthworms (54 individuals) was used for the measurement of cadmium accumulation in the whole earthworm bodies, the second group (50 individuals) served as a source of coelomocytes for the measurement of the expression of stress-related genes.

Soil preparation

The air-dried commercial soil (PPUH BIOVITA, Tenczynek, Poland organic soil, 51.7% organic matter, pH=6.11) was spiked either with water (control) or with cadmium chloride (CHEMPUR, Piekary Slaskie, Polska), 500 mg kg -1 , as described previously [START_REF] Takacs | Exposure of Eisenia andrei (Oligochaeta; Lumbricidea) to Cadmium Polluted Soil Inhibits Earthworm Maturation and Reproduction but not Restoration of Experimentally Depleted Coelomocytes or Regeneration of Amputated Segments[END_REF][START_REF] Rorat | Protective role of metallothionein during regeneration in Eisenia andrei exposed to cadmium[END_REF]. Control and Cd-polluted soils were kept separately or mixed 1:1, giving samples of unpolluted control soil, and those with high (425 ± 45.66 mg kg -1 ) or very high (835 ± 35.712 mg kg -1 ) cadmium concentration.

Experimental design

Experiment 1 Cd accumulation in earthworms: In total 54 earthworms were exposed either to unpolluted control soil or to soil with high cadmium concentration during 4 weeks in mesocosms. Briefly, four individuals from each group of species: Ea, Ef1, Ef2 and Ha were placed in the plastic boxes (of 9 cm diameter) with perforated lids. At the end of the exposure period, the earthworms were depurated overnight in boxes with wet lignin substrate (Paso-Trading, Polska), and then sacrified by freezing and prepared for the metal accumulation analysis.

Experiment 2: Gene expression in Cd-exposed earthworms: In total 50 individuals were used as a source of coelomocytes, i.e. the immune cells present in earthworm coelomic cavity, noninvasively retrieved from coelomic cavity by a mild elctric shock as described in 2.4.3.1. E.

andrei, E. fetida, and their Ea-derived (Ha) and Ef-derived hybrids (Hf) were exposed for 2 and 7 days to highly polluted soil in mesocosms (boxes with with perforated lids, 9 cm diameter). The level of gene expression of glutathione S-transferase (gst), lysenin (lys), metallothionein (mt), superoxide dismutase (sod) and phytochelatin synthase (pcs) was assessed in extruded coelomocytes, and related to the expression of two stable house-keeping genes -actin (act) and ribo13 (r13).

Analyses

Earthworm genotyping

Supravitally amputated tail tips of numerically coded adult earthworms were used for DNA extraction and genetic analysis using oligonucleotide primers amplifying species-specific variants of mitochondrial COI genes and nuclear 28S rRNA genes, as described earlier (Plytycz et al. 2018a,b;2020;[START_REF] Podolak | Annual Reproductive Performance of Eisenia andrei and E. fetida 2 in Intra-and Inter-Specific Pairs and Lack of Reproduction of Isolated Virgin Earthworms[END_REF].

As previously established, mitochondrial species-specific sequences of the COI gene were called either 'a' or 'f'/'f2' for Ea and Ef1/Ef2, respectively, while the diploid species-specific nuclear markers of the 28S rRNA gene were called either 'A' or 'F' for Ea and Ef, respectively. In a results, the investigated specimens were genotyped either as pure species, Ea (aAA), Ef1 (fFF), Ef2 (f2FF), or inter-specific hybrids, either Ea-derived aAF, Ef-derived fFA with the two first letters (aA-, fF-, f2F) representing ova-derived genes, i.e. maternity, and the last letter (A or F) representing spermatozoa-derived genes, i.e. paternity.

Cadmium content

Cadmium content in soil and in whole earthworm bodies was measured by slightly modified methods used in our previous studies [START_REF] Takacs | Exposure of Eisenia andrei (Oligochaeta; Lumbricidea) to Cadmium Polluted Soil Inhibits Earthworm Maturation and Reproduction but not Restoration of Experimentally Depleted Coelomocytes or Regeneration of Amputated Segments[END_REF][START_REF] Rorat | Protective role of metallothionein during regeneration in Eisenia andrei exposed to cadmium[END_REF]). Soil samples were dried at 105°C for 24 h and then divided into subsamples about 0.5 g of dry soil mass of each. Cadmium concentration in soil was determined after half open wet digestion in a quartz glass with 10 mL of mixture of concentrated HNO3 (69.0-70.0% to trace metal analysis, Baker Instra-Analyzed) and HClO4 (70% a.r, Chem-Lab) (7:1 v/v), using hot plates.

Cadmium concentrations were measured by atomic absorption spectrometry (AAS) with a flame and atomic absorption spectrometry with a graphite furnace nebulizer (Perkin-Elmer) to achieve the desired method sensitivity. Accuracy was verified by analyzing four blanks and three replicates of standard certified material (Loamy Sand 10 CRM033-50G) with the samples.

Prior to the analysis of cadmium concentration in whole earthworm bodies, earthworms were kept for 2 days on wet filter papers to remove their gut content, frozen, and then dried in vacuum drier in 70°C for 24 h to achieve constant dry mass. For determination of cadmium concentration in earthworm bodies, the wet digestion method was applied but to reach the required ratio 1: 20 only 4 mL of concentrated HNO3 (69.0-70.0% to trace metal analysis, Baker Instra-Analyzed) were used. Cadmium concentrations in earthworm bodies were measured by atomic absorption spectrometry methods with four blanks and three replicates of standard certified material (Bovine Liver SRM 1577c) with the sample in digestion.

The cadmium concentrations were calculated on a dry mass of each sample of soil or earthworms and are presented in mg kg -1 (μg g -1 ).

Gene expression analysis

Extrusion of coelomocytes extrusion via electrostimulation

After 24 h depuration on wet lignin (Paso-Trading, Poland), earthworms were placed individually on petri dishes and stimulated for 30 s with a 4.5 V electric current to expel coelomic fluid with suspended coelomocytes through the dorsal pores [START_REF] Homa | Dermal exposure of Eisenia andrei earthworms: Effects of heavy metals on metallothionein and phytochelatin synthase gene expressions in coelomocytes[END_REF][START_REF] Rorat | Protective role of metallothionein during regeneration in Eisenia andrei exposed to cadmium[END_REF]. Pellets of coelomocyte samples were resuspended in 2 mL of TriReagent (Molecular Research Center Inc., USA) for RNA extraction.

Extraction of RNA and reverse transcription

Expression of five target and two house-keeping genes (HKG) was performed on the total RNA extracted from coelomocytes by TRI Reagent® (Molecular Research Center, USA), according to manufacturer's instructions. Overall, coelomocytes' pellets from each sample were suspended in 2 mL of Tri Reagent and 1 mL was later used for each extraction. Samples were then treated with DNase I, RNase-free (1 U/µL) (Thermo Fisher Scientific, USA) according to manufacturer's instructions. The quantity and quality of isolated RNA were analyzed spectrophotometrically using spectrometer with a LVis nano plate (SPECTROstar Nano, BMG LABTECH, USA, LVis Plate, BMG LABTECH, USA). RNA integrity was confirmed on 2% agarose gel stained with ethidium bromide (Ethidium Bromide Solution (10 mg mL -1 ), Thermo Scientific, USA). The amount of isolated RNA allowed us to perform the reverse transcription of 1.5µg cDNA using QIAGEN OMNISCRIPT RT KIT (QIAGEN, Germany), according to manufacturer instructions. cDNA samples were then stored in -20 o C until further analyses.

Real-time qPCR amplification.

The mRNA levels of chosen genes were determined by real-time quantitative PCR after reverse transcription. For qPCR, Agilent Mx3000P QPCR System (Agilent Technologies, USA) with 2X Takyon for SYBR Assay -No ROXqPCR Kits for SYBR Assay (Eurogentec, Belgium) were used, and reactions were performed on 96-well qPCR plates (Eppendorf twin.tec ® PCR Plates, Eppendorf AG, Germany). All qPCR reactions were performed as follows: 95°C 5 min and 40 cycles at 95°C 30 s, 55°C 30 s, and 72°C 60 s. After 40 cycles, the last step of the program consisted of 72°C for 5 min. The Eisenia sp. specific primes were used for actin (act), ribosomal protein S13 (r13), metallothionein (mt), glutathione Stransferase (gst), phytochelatin synthase (pcs), lysenin (lys) and superoxide dismutase (sod) [START_REF] Brulle | cDNA cloning and expression analysis of Eisenia fetida (Annelida: Oligochaeta) phytochelatin synthase under cadmium exposure[END_REF][START_REF] Homa | Dermal exposure of Eisenia andrei earthworms: Effects of heavy metals on metallothionein and phytochelatin synthase gene expressions in coelomocytes[END_REF] after the validation of their compatibility with hybrids.

The selected primers are presented in Table 1. Expression levels were determined as the number of cycles needed for the amplification to reach a threshold fixed in the exponential phase of PCR reaction (Ct). Levels of expression and amplification effectiveness of target genes were compared to the expression of two constitutively expressed and validated housekeeping genes. qPCR efficiency of each target gene was calculated according to the method previously described (Chaâbene et al., 2017). Overall, the relative expression level was calculated according to the following formula:

Data analysis

HKG stability was quantified using two software's: BestKeeper and NormFinder. All results are expressed as means ± standard deviation, n=3-6. Descriptive statistics and statistical analyses were produced using the OriginPro 2015 software and Statistica. Different letters "a", "b" "c", "d" on top of bars indicate statistically significant differences according to the 1 one-way analysis of variance.

Results

Genetic species delimitation

Cytoplasme

Cadmium content in soil and in whole earthworm bodies

The cadmium concentration in the commercial unpolluted soil was very low (L; 0.11 ± 0.,107 mg kg -1 ); in soil spiked with cadmium chloride was very high (H; 835 ± 35.712 mg kg -1 dry weight) and intermediate in 1:1 mixture of these soil samples (M; 425 ± 45.66 mg kg -1 dry weight) (Table 2). Cadmium concentrations in earthworms living in unpolluted soil were low (0.69 -1.12 mg kg -1 ) and did not differ significantly between Ea, Ef1, Ef2, and Ha groups of earthworms.

Cadmium

Gene expression in earthworms

Reference genes

For determination of the stability of chosen housekeeping genes, the NormFinder and BestKeeper software were used. Overall, the expression of both chosen reference genes was stable across all experimental conditions and species (Appendix 1).

Expression of gst and pcs genes

The expression level of pcs and gst was undetectable in coelomocytes of E. andrei in both clean and Cd-contaminated soil. However, a negligible expression of those genes was observed for Ef1, Ef3 and Ha exposed to soil contaminated by cadmium (Figures 2A and2B).

Expression of mt, sod, and lys genes

Only two rare Ef-drived hybrids (Hf*) were used for analysis of mt, sod, and lys expression. In unpolluted soil (i.e. on day 0 of cadmium exposure), the expression of mt and sod was very low while it was relatively in a case of lys. In each instance, it was similar to the expression of these genes in other specimens from unpolluted soil, i.e. on day 0 of cadmium exposure (Fig.

3, right columns of rows A, B, and C).

In all tested organisms, a significant induction of the expression of mt and sod was observed after exposure to cadmium (Figure 4, rows A and B), while expression of lys was drastically diminished (Fig. 3, row C).

Expression of mt

The expression rate of metallothionein was negligible in earthworms exposed to clean, uncontaminated soil. In coelomocytes of Ef1, Ef2 and Ha, the expression of mt was significantly (p < 0.05) induced after already two days of exposure, and in Ef1 and Ha continued to increase further until the 7-day timepoint. However, in coelomocytes of E.

andrei, the induction of mt expression occurred later than for other earthworms, and was significant only after 7 days post exposure (Figure 3, row A, left column). At each time point the interspecies differences were statistically insignificant (Fig. 3, row A, right column).

Expression of sod

Similar results were observed for the expression rate of sod (Figure 3, row B). Once again, the expression rate for all organisms living in clean soil was almost undetectable while the exposure to cadmium caused a significant increase in sod expression. However, two days post-exposure, the expression rate of sod in E. andrei was still almost no detectable, whereas for all other organisms was already significantly (p < 0.05) induced. At day 7 day after cadmium exposure, expression of sod significantly increased versus day 2 in Ea, Ef2, and Ha (Fig. 3, row B, left column). The interspecies differences were insignificant on day 0 and day 2, while on day 7 was significantly higher in Ha than in Ea, Ef1, and Ef2 (Fig. 3, row B, right column).

Expression of lys

The expression of lysenin gene was highly influenced by exposure to different soil conditions across all tested organisms. In all cases, its expression was at its highest in coelomocytes of earthworms exposed to clean soil and the Cd contamination caused a severe and significant (p < 0.05) downregulation of that expression (Fig. 3, row C). Moreover, for Ef2 and Ha, the downregulation was significant for both, 2 and 7 day post exposure, whereas for Ea and Ef1 it was downregulated by 2 days post-exposure, and then the expression rate stabilized and stayed at the same level without further downregulation (Figure 3, row C, left column).

Interspecies comparisons revealed the lack of statistically significant differences in day zero and day 2 after cadmium exposure while on day 7 lys expression differed significantly between species, being the highest in Ef1, and then in Ea, Ef2 and Ha (Fig. 3, row C, right part).

Discussion

Adult specimens of hermaphroditic lumbricid earthworms E. andrei and E. fetida, the latter with two distinct lineages Ef1 and Ef2, and interspecific Ha hybrids developed from Ea ova fertilized by Ef spermatozoa were fully viable during four week exposure to cadmiumpolluted soil (425 ± 45.66 mg kg -1 or 835 ± 35.712 mg kg -1 ) despite a high cadmium accumulation in the whole earthworm bodies. Bioaccumulation was dose-dependent, similar in Ef1/Ef2 lineages of Ef, and lower than that in Ea. In contrast, Ea-derived hybrids reached plateau of Cd-accumulation capacity already at lower cadmium concentration in soil.

Hypothetically, all investigated specimens from genus Eisenia are able to survive in Cdcontaminated environments due to adapting the same or similar detoxification mechanisms to those described previously at transcriptomic level in representatives of E. andrei and E. fetida [START_REF] Homa | Dermal exposure of Eisenia andrei earthworms: Effects of heavy metals on metallothionein and phytochelatin synthase gene expressions in coelomocytes[END_REF], Rorat et al. 2017), while data concerning hybrids were vastly unexplored.

Therefore comparative studies on the expression of genes involved in Cd detoxification were imperative. Second part of previous investigations revealed that the expression of stressrelated genes upon exposure to Cd-polluted soil at 425 ± 45.66 mg kg -1 was either upregulated (mt and sod), down-regulated (lys) or unchanged (gst and psc).

Expression of genes for metallothionein and phytochelatin synthase

In a study by [START_REF] Brulle | cDNA cloning and expression analysis of Eisenia fetida (Annelida: Oligochaeta) phytochelatin synthase under cadmium exposure[END_REF] performed on E. fetida, the induction pattern of genes encoding phytochelatin synthase was significantly increased in earthworms exposed to a low concentration of cadmium (8 mg kg -1 ). However, in higher concentrations, no difference was noticed between Cd-exposed earthworms and earthworms not exposed to any contamination, which suggests that pcs expression is only induced during low exposure to cadmium.

Similarly to our study, expression of metallothionein was highly up-regulated after Cd exposure, and in the mentioned [START_REF] Brulle | cDNA cloning and expression analysis of Eisenia fetida (Annelida: Oligochaeta) phytochelatin synthase under cadmium exposure[END_REF] study, it was also shown to be dose-dependent in high Cd concentrations. Such relation between different responses of the expression of pcs and mt genes is of interest when considering their role in metal detoxification in animals. In low concentrations of Cd, the expression of pcs was shown to be significant and responsible for metal chelation in worm cells (probably via the synthesis of Phytochelatins (PCs) although the presence of PCs has not been demonstrated), whereas such induction was only noticed at a small exposure level. This is on the contrary to the effects of high concentrations of Cd, where similarly to our study, pcs expression patterns did not show any change of their level over control animals, whereas the expression rate of mt was substantially upregulated. In another study, focused on Caenorhabditis elegans, the toxicogenomic analysis after exposure to cadmium also showed no effect on the expression of pcs-1 gene [START_REF] Cui | Toxicogenomic analysis of Caenorhabditis elegans reveals novel genes and pathways involved in the resistance to cadmium toxicity[END_REF]. Moreover, a recent transcriptomic study on the same species: C. elegans, found similar results -after exposure of nematods to cadmium (300µM) and zinc (500µM), the expression change of pcs-1 was determined via qPCR followed by normalization by the invariant ribosomal rla-1. The results showed, that the pcs response to Zn and Cd contamination was at best negligible, and the change in comparison to control animals was not significant (p < 0.05) [START_REF] Essig | Deletion of Phytochelatin Synthase Modulates the Metal Accumulation Pattern of Cadmium Exposed C. elegans[END_REF]. By combining these studies, we can see that phytochelatins and metallothioneins possibly act together to protect the cells against Cd. Phytochelatins have more of an immediate role at lower concentrations and metallothioneins coming later with higher induction. In addition, a comparison between the chosen gene expression profiles in earthworms exposed or unexposed to Cd showed upregulation of two genes for all tested earthworms after Cd exposure -metallothionein and superoxide dismutase as well as down-regulation of lysenin. Such alterations between the expressions of those genes suggest that earthworms can oppose and reduce the toxic effects of Cd through several different pathways. Moreover, in the presented study, we noticed that the mt overexpression due to Cd exposure occurs not only in E. fetida but also in E. andrei and hybrids between those species, which suggest, that this biomarker is suitable for both species.

However, results suggest that E. andrei (aAA) activate its detoxification mechanisms slower than E. fetida (fFF) and their hybrids (aAF). Moreover, fast up regulation of expression of mt genes in hybrids suggests, that they obtained the nuclear allele responsible for a fast reaction from E. fetida, which showed similar tendencies, and not from E. andrei. In addition, such results could indicate that perhaps, Ef earthworms possess another detoxification mechanism or a specific regulation mechanism that is responsible for faster mt expression in comparison to Ea. Thus, by cloning of the genes coding mt in both species and studying of the regulation of the gene is needed to broaden our understanding of such response to metal exposure.

Similar to our study, the induction of overexpression of the mt gene was recently shown in E.

andrei earthworms after exposure to copper, which is a widely used fungicide, especially in organic farming [START_REF] Mincarelli | Evaluation of gene expression of different molecular biomarkers of stress response as an effect of copper exposure on the earthworm EIsenia Andrei[END_REF]. Moreover, an induction of mt expression was also noticed in other earthworms including Dendrobaena octaedra and Lumbricus rubellus exposed to Cu [START_REF] Mustonen | Metallothionein gene expression differs in earthworm populations with different exposure history[END_REF][START_REF] Höckner | Metallothionein gene activation in the earthworm (Lumbricus rubellus)[END_REF]. The population of Dendrobaena octaedra exposed to Cu-Ni contaminated soil was shown to have higher mt expression levels in comparison to earthworms not exposed to metals. What is even more interesting, during artificial exposure experiment, exposure affected the mt expression, but only in the earthworms collected from the clean, unpolluted soil, showing that there is a delay in the mt response of earthworms which were not exposed to metals in the past. On the contrary, earthworms collected from the Cu-Ni smelting site showed not only higher but also constant levels of mt expression [START_REF] Mustonen | Metallothionein gene expression differs in earthworm populations with different exposure history[END_REF]. Such results reflect on the role of the mt expression and the homeostasis of several metals and protection against oxidant damage.

Superoxide dismutase and Glutathione S-transferase 20

Glutathione S-transferase (GST) is an enzyme known to take part in the metabolism of lipophilic organic contaminants and in cellular protection against oxidative stress. A study by [START_REF] Mkhinini | Effect of treated wastewater irrigation in East Central region of Tunisia (Monastir governorate) on the biochemical and transcriptomic response of earthworms Eisenia andrei[END_REF] showed an induction of gst expression after the exposure of E. andrei to treated wastewater (TWW) containing several trace metals. However, such effects were observed in earthworms exposed to TWW for more than 20 days. Our study showed no effect of cadmium exposure to the expression of this gene in a 7-day period, which could suggest that the gst mechanism is induced after prolonged, chronic exposure to heavy metals, but it is not an immediate response to the presence of contamination. In another study, the antioxidant gene expression of E. fetida was explored after worm exposure to various concentrations of hexabromocyclododecane. The results showed a significant up-regulation of the sod gene but no significant differences in the expression rate of gst, which further suggests its involvement in protection against the oxidative damage occurs during a prolonged period of time [START_REF] Shi | Antioxidant gene expression and metabolic responses of earthworms (Eisenia fetida) after exposure to various concentrations of hexabromocyclododecane[END_REF]. Moreover, a recent study of [START_REF] Maity | Oxidative stress responses of two different ecophysiological species of earthworms (Eutyphoeus waltoni and Eisenia fetida) exposed to Cd-contaminated soil[END_REF] focused on the oxidative stress response of E. fetida and Eityphoeus waltoni exposed to Cd, the overexpression of the gst gene increased significantly post-exposure and was dose-dependent. However, in E. fetida, after 2 days post-exposure, the expression of gst remained the same as in control animals, which also suggest its late-onset. In a transcriptomic study by [START_REF] Chai | Transcriptome analysis of genes expressed in the earthworm Eisenia fetida in response to cadmium exposure[END_REF], focused on cadmium toxicity in E. fetida, a significant up-regulation od sod genes was also observed in earthworms exposed to 10, 30, 60 mg/kg of Cd 2+ in dry soil, respectively.

Lysenin

Lysenin is a protein involved in immunity processes produced by coelomocytes and chloragocytes [START_REF] Munguira | Lysenin Toxin Membrane Insertion Is pH-Dependent but Independent of Neighboring Lysenins[END_REF]. The presence of lysenin and lysenin-related proteins was documented in E. andrei and E. fetida and their putative hybrids [START_REF] Swiderska | Data on proteins of lysenin family in coelomocytes of Eisenia andrei and E. fetida obtained by tandem mass spectrometry coupled with liquid chromatography[END_REF]).

In the present study, the expression rate of lys gene was significantly down-regulated post Cd exposure in all tested animals (Ea, Ef, Ha). A similar decrease in lys expression was noticed by Rorat et al. (2017) in E. andrei andBrulle et al. (2011) in E. fetida. However, the mechanisms behind such response are still unknown. In a study by [START_REF] Wang | Analysis of earthworm Eisenia fetida proteomes during cadmium exposure: An ecotoxicoproteomics approach[END_REF] the expression of lys was also shown to diminish after earthworms were exposed to E. coli.

Besides, [START_REF] Wu | Evaluation of phenanthrene toxicity on earthworm (Eisenia fetida): An ecotoxicoproteomics approach[END_REF], discovered that lysenin-related protein was significantly upregulated post-exposure to PAH phenanthrene. Hence, lysenin expression in earthworms can have a complex response to exposure to different contaminants. Overall, the results imply the existence of a complex regulatory network that needs to be further explored in future studies.

Hybrids response to environmental stressors

We have shown previously that Eisenia andrei (Ea) and E. fetida (Ef) of French provenance are capable of hybridization, as viable offspring appeared among progeny of laboratory-mated inter-specific Ea+Ef pairs. Recently we have shown that hybridization is not unique for French Ea/Ef earthworms, but is shared by laboratory-mated earthworms from French, Hungarian, and Polish laboratory cultures [START_REF] Podolak | Annual Reproductive Performance of Eisenia andrei and E. fetida 2 in Intra-and Inter-Specific Pairs and Lack of Reproduction of Isolated Virgin Earthworms[END_REF][START_REF] Plytycz | Impairment of reproductive capabilities in three subsequent generations of asymmetric hybrids between Eisenia andrei and E. fetida from French, Hungarian and Polish laboratory colonies[END_REF]), but fertility of hybrids is gradually diminished in the subsequent generations [START_REF] Plytycz | Impairment of reproductive capabilities in three subsequent generations of asymmetric hybrids between Eisenia andrei and E. fetida from French, Hungarian and Polish laboratory colonies[END_REF].

Inter-specific hybrids were detected in natural populations of Ea and Ef from Scandinavia (Martinsson and Erseus, 2018) thus further data on hybridization in natural environment, even such highly polluted, including compost heaps, are expected.

Both E. andrei and E. fetida earthworms as well as their hybrids are perfect cadmium bioaccumulators and can survive in heavily contaminated soil due to efficient protective mechanisms. Mt and sod upregulation and lys down-regulation are especially pronounced in hybrids. Therefore we shall exclude such a possibility that the improper functioning of investigated stress-related genes is the main factor responsible for gradual impairment of hybrid fertility in the subsequent generations [START_REF] Plytycz | Impairment of reproductive capabilities in three subsequent generations of asymmetric hybrids between Eisenia andrei and E. fetida from French, Hungarian and Polish laboratory colonies[END_REF].

Interestingly, similar to our results, recent research on several species of plants indicated that hybrids can outperform their parental species after exposure do different stressors (both biotic and abiotic). As an example, a couple of types of poplar hybrids had been shown to better adapt to suboptimal temperatures and demonstrate superior growth, particularly under the less favorable, suboptimal temperature conditions [START_REF] Guet | Genetic variation for leaf morphology, leaf structure and leaf carbon isotope discrimination in European populations of black poplar (Populus nigra L.)[END_REF]. Overall, the interspecific hybridization has recently become of-interest to forest geneticists for tree improvement by assessing the possible feasibility of transferring desirable traits such as higher resistance to contamination, droughts, or diseases, among species [START_REF] Juranović-Cindrić | Influence of F 1 hybridization on the metal uptake behaviour of pine trees (Pinus nigra x Pinus thunbergiana ; Pinus thunbergiana x Pinus nigra)[END_REF]. Apart from trees, another study showed a variation between Cd uptake in maize hybrids, which can be used for phytoremediation of Cd-contaminated soil as well as for the selection and breeding of new, Cd-tolerant genotypes of maize [START_REF] Akhtar | Photosynthesis and growth response of maize (Zea mays L.) hybrids exposed to cadmium stress[END_REF][START_REF] Wang | Characterization of Cd translocation and accumulation in 19 maize cultivars grown on Cd-contaminated soil: Implication of maize cultivar selection for minimal risk to human health and for phytoremediation[END_REF].

Similar tendencies had been observed for the accumulation of Cr in sunflower hybrids, which is currently used in the selection and breeding of Cr-tolerant genotypes of sunflowers [START_REF] Farid | Phyto-management of Cr-contaminated soils by sunflower hybrids: physiological and biochemical response and metal extractability under Cr stress[END_REF]. However, the molecular basis of such differential accumulation and the differences in the genetic makeup of both hybrids and parent species are scarcely researched and need to be further investigated in both, plants and earthworms exposed to contamination [START_REF] Wang | Characterization of Cd translocation and accumulation in 19 maize cultivars grown on Cd-contaminated soil: Implication of maize cultivar selection for minimal risk to human health and for phytoremediation[END_REF], Naeem et al., 2016[START_REF] Juranović-Cindrić | Influence of F 1 hybridization on the metal uptake behaviour of pine trees (Pinus nigra x Pinus thunbergiana ; Pinus thunbergiana x Pinus nigra)[END_REF].

Conclusions

The insights gained in the presented study have improved our understanding of metal resistance of E. fetida, E. andrei and their hybrids. The results indicate that all of them are very efficient cadmium bio-accumulators with similar mechanisms of Cd detoxication.

Consistently, upregulation of mt and sod expression was fastest in hybrids, slower in Ef and slowest in Ea. Hypothetically Ef and Ea differ in their mechanisms controlling expression of stress-related genes and immunity-connected genes thus further studies of these genes and their interactions in hybrids are necessary.

  -rich ova with plenty mitochondria are fertilized by spermatozoa with very scarce cytoplasm and a few mitochondria. DNA barcoding based on specific sequences of mitochondrial haploid COI gene shows three distinct branches; 'a' for Ea or 'f'/'f2'for two mitochondrial lineages of Ef, namely Ef1 and Ef2[START_REF] Plytycz | Impairment of reproductive capabilities in three subsequent generations of asymmetric hybrids between Eisenia andrei and E. fetida from French, Hungarian and Polish laboratory colonies[END_REF]. Species-specific maternal-paternal diploid nuclear sequences of 28S rRNA gene, are either 'AA' (for Ea) or 'FF' for Ef or 'AF' for interspecific hybrids. Fertilization of 'aA' ovum by 'A' spermatozoon gives aAA zygote of the pure Ea species, while 'aA' ovum fertilized by 'F' spermatozoon gives Ea-derived aAF hybrid. The 'fF' or 'f2F' ova fertilized by 'A' spermatoza give fFA or f2FA hybrids, respectively (Figure1). All sequences are deposited in GenBank under the following accession numbers: MT646836-MT646899 and MT649303-MT649399. All the earthworms survived the experiment but cadmium exposure inhibited cocoon production almost completely (data not shown).

Figure 1 .

 1 Figure 1. Maximum-likelihood phylogram for the species-specific sequences 'a' or 'f'/'f2' of

Figure 2 .

 2 Figure 2. Relative expression level of gst (A) and pcs (B) in coelomocytes of groups of

Figure 3 .

 3 Figure 3. Relative expression level of mt (row A), sod (row B) and lys (row C) in

Table 1 .

 1 qPCR primer sequences

	Gene Forward primer 5'-3'	Reverse primer 5'-3'	Amplicon	Accession
				size (bp)	number
	r13	5'CGCACGGTTTTAGTTTCT3'	5'CCATGCGAGTCTCGAAG3'	148	MG076964
	act	5'CCATCCATCGTCCACAG 3'	5'GCATGTGTGAGTCCTAATTT3'	147	EY893028.1
	mt	5'AAAGTGAGTGCTTGCC3'	5'ACTGATGACAGAGTTCCG3'	120	KP770991.1
	gst	5'TGGAAGTGGAGGTTTCCTTG3' 5'TGAGGTGGCCAAGTCTTTTC3' 161	EY892760.1
	pcs	5'TCATGGTCCTGAACACG3'	5' CACAAGTTGCCGAAACTC 3'	154	KP770990.1
	lys	5'CGGCAACAAACGTCTAC3'	5'GTGAAATACAGGCAGAAGC 3' 163	MG076962.1
	sod	5'GGTGCTCACTTCAACCCATT3' 5'AAGATCGTCCACCAGCTCAT3' 167	DQ286712.1

Table 2 .

 2 concentrations in whole bodies of earthworms kept in soil with M or H cadmium Concentration of Cd in soil and earthworms' bodies after 4-week exposure in unpolluted (L), intermediate (M) or very high (H) soil. Results shown as means ± SD, n=3 -6.

	pollution were overlapping (113.79 -376.73 and 252.68 -454.18 mg kg -1 , respectively), with
	lowest accumulation capacity in Ef2 earthworms (113.79 and 252.68 mg kg -1 in M and H soil,
	respectively), but these values were not significantly different from those in the second
	lineage of the Ef species, i.e. Ef1 (203.43 and 337.66 respectively). The whole body Cd
	bioaccumulation in Ea reached 316.43 mg kg -1 in the M soil and was significantly higher in

the Ea from the H soil (454.19 mg kg -1 ). Cadmium bioaccumulation was most efficient in the Ha hybrids reaching plateau already in the M soil, as the values at M and H soil were similar (376.73 and 309.246 mg kg -1 , respectively).

Acknowledgements Investigations are financially supported the National Centre of Science (2016/23/B/NZ8/00748) and by the Jagiellonian University (K/ZDS/005405).


Thomas Sordel

Stéphanie Garnier-Raveaud

Fabien Sauter

# , Fabien Sauter § Catherine Pudda

Frédérique Marcel

Michel De Waard

Christophe Arnoult

Michel Vivaudou

François Chatelain

Nathalie Picollet-D'hahan
email: picolletdhahan@dsvsud.cea.fr

Anne Simon

Michel De Waard^

Christophe Arnoult^

Michel Vivaudou°

Hourglass SiO2 coating increases the

Keywords: planar patch-clamp, BK(Ca) channels, IRK1 channels, PECVD coating, AFM, SEM

INTRODUCTION

Ion channels represent a large family of potential drug targets (Venter et al., 2001, Wang andLi, 2003), and have thus become important objects in the drug discovery/screening process. However, the lack of high-throughput assays has hampered their use for screening purposes and still constitutes a bottleneck in lead optimization steps (Xu et al., 2004[START_REF] Wood | [END_REF]. Since 1998, efforts have been made to automate electrophysiology (Xu et al., 2004) and several important technological advances have been made. To increase the throughput of patch-clamp recordings, attempts have been made to replace the traditional pipette with a planar substrate. Glass was the first material used for planar patch-clamp recording because this material has good dielectric properties and interacts well with membrane lipids (Fertig et al., 2003). Nevertheless, microstructuring processes involving glass are complex and require specific equipments. A wide range of other materials, and sometimes a hybrid assembly of different materials, have been also tested : PDMS (Sigworth and Klemic, 2002), SiO 2 coated plastic chips (Wang and Li, 2003), SiO 2 coated silicon chips (Asmild et al., 2003), quartz (Stett et al., 2003b) and encapsulated silicon chips in a plastic matrix (Seo et al., 2004). Nevertheless, stable high-resistance seals (giga-seals) are still elusive. The success rates on the "best" materials remain low, ranging between 30 and 50%, depending on the cell type (Stett et al., 2003a, Owen andSilverthorne, 2002). The optimum choice of suitable material and surface coatings has yet to be defined (Xu et al., 2003) because of the complex nature of the seal. Even under optimal conditions, seal resistance values remained consistently below the minimum that is required for highly resolved currents and single channel analysis. Therefore, highthroughput screening for agonists or antagonists of ion channels using planar substrates is mainly done in surrogate cell systems in which the channel of interest is stably or transiently overexpressed (Chien, 2003).

The optimisation of planar patch-clamp cell sealing involves both biological and technological approaches. Some authors introduced new procedures in order to improve the quality of the cell suspension (Bruggemann et al., 2003) or optimize the identification of cells that are suitable for patchclamping [START_REF] Mc Dowell | [END_REF].

Here, we describe a method that is based on the modification and characterization of the dimension and shape of the pores, surface roughness and chip capacitance in relation to the quality of the seal. We have focused on silicon-based chips coated with a SiO 2 layer, the production of which offers high accuracy and flexibility of microstructuration and surface functionalisation. This approach has led to a model of PECVD SiO 2 coating defining an "hourglass" shape of the hole (Fig. 2). We demonstrate that by decreasing the size of the microhole and modifying shape and roughness, the seal resistance in comparison to flat thermal SiO 2 treatment was enhanced. Secondly, decreasing chip capacitance allowed ion channels of low conductance and endogenous channel activities to be recorded in a whole-cell configuration mode. Two transfected cell lines (CHO and HEK-293) displayed the electrophysiological signatures of the expected BK(Ca) and IRK1 potassium channel activities and those of cationic endogenous channels. (nomenclature officielle: BK(Ca) = K Ca 1.1) Our approach relies on a growing database of experimental correlations allowing the optimization of chip design to potentially shift from whole cell to highly resolved unitary currents recordings and from recombinant to native channel studies. We show here that the planar interface can be adapted to specific cell types by modifying and controlling the features of the chip, thus greatly expanding the application options to a wide range of different cell lines, primary cultures and isolated native cells.

MATERIAL AND METHODS

Device assembly

The patch-on-a-chip device was described previously (Picollet-D'Hahan et al., 2004). The chip consists of 9 independent cell-patch sites, each defined by a couple of o-rings (Inner diameter I.D. = 3 mm, Outer diameter O.D. = 5 mm) which act as walls of individual chambers. The so-defined top and bottom compartments communicate through a hole of 2.5 µm diameter in a 2.12 µm thick silica membrane. The compartments are connected to a network of microfluidic capillaries, allowing solutions to be supplied and exchanged independently. Two printed circuit boards (PCB) with 9 Ag/AgCl planar electrodes were assembled in a "sandwich" manner and connected to a patch-clamp amplifier.

Chip passivation

Two types of chips were studied: chips coated with a SiO 2 layer grown with a thermal oxidation (thermal SiO 2 ) and chips with an additional PECVD SiO 2 layer on the upper side. The fabrication process of the chips was described previously (Picollet-D'Hahan et al., 2004). PECVD was deposited at 300°C with a rate of 1300 Å/min. This resulted in a layer with a thickness of 0.5 µm to 1.5 µm. Three PECVD SiO 2 layer thicknesses were investigated (Table 1) and studied in terms of chip resistance and capacitance values.

Atomic Force Microscopy measurements

Atomic Force Microscopy (AFM) experiments were performed with a Dimension TM 3100 AFM (Digital Instruments, Santa Barbara, CA) and a Nanoscope IV controller (Digital Instruments) equipped with a large-range scanner (maximum XY scan range of 90 µm x 90 µm with vertical Z range of 6 µm).

Measurements were obtained in the AFM contact mode by using silicon nitride cantilevers with a 0.12 N/m nominal spring constant, and a scanning velocity of 30 -180 µm/s. Images were recorded in the constant force mode with minimal external force.

Cell preparation

Human Embryonic Kidney 293 (HEK-293) cells were stably transfected with pcDNA3.1-HA-mBKc (Creacell, La Tronche, France), a plasmid construct carrying the coding sequence of mBKc (mSlo A48206). The cells were cultured in Dulbelco's modified Eagle's medium supplemented with 10% (V/V) foetal calf serum, 1.2 mg/ml geneticin, 1% penicillin-streptomycin and 1% L-glutamine in flasks. Chinese Hamster Ovary (CHO) cells were stably transfected with pcDNA3.1-HA-mIRK1 (Creacell), a plasmid carrying the coding sequence of the IRK1 protein. The cells were cultured in Ham F12 medium supplemented with 10% (V/V) foetal calf serum, 1.2 mg/ml geneticin, 1% penicillin-streptomycin and 1% L-glutamine in flasks.

Cells were pre-washed with Phosphate Buffered Saline (PBS, Invitrogen, Cergy-Pontoise, France). To detach the cells prior to the experiment, Accutase, rather than trypsin/EDTA, proved to be most effective (Tao et al., 2004). Accutase-isolated cells revealed better membrane integrity and optimised morphology.

In addition, this enzyme has been reported to not alter ion channel functionality. The number of aggregates was reduced and the suspension more homogeneous, something that is particularly important in order to exclusively immobilize one individualized cell onto the microhole. After detachment, cells were centrifuged at 360 x g and resuspended in a mixture of PBS/Accumax at a concentration of 2.10 5 cells/ml and kept at 4°C under agitation. Immediately prior to the planar patch-clamp experiments, the cells were concentrated in electrophysiological solution to 5.10 5 cells/ml.

Electrophysiology

Experimental conditions were similar to those used in conventional patch-clamp experiments, where the "bath" solution becomes the upper chamber solution and the intrapipette solution the lower chamber solution. The upper chamber solution contained (in mM): 118 NaCl, 5.6 KCl, 2.4 CaCl 2 , 1.2 MgCl 2 , 10 HEPES, 11 glucose, pH 7.4 (with NaOH), conductivity 1.32 S/m. In inhibition experiments, BaCl 2 and iberiotoxin (Sigma, St Quentin Fallavier, France) were added. The lower chamber contained (in mM): 30 KCl, 110 K-aspartate, 1 MgCl 2 , 0.1 CaCl 2 , 1 EGTA, 10 HEPES, pH 7.3 (with KOH), conductivity 1.17 S/m. Experiments were conducted at room temperature (20°C) and cells were studied in the whole-cell mode. For each experiment, one microhole out of 9 was examined as followed: the bottom chamber of the device was filled with the solution first, followed by the upper chamber. Care was taken to eliminate bubbles in the fluidic circuit. Microhole resistance (Rh) was monitored by applying a square wave of voltage (5 mV amplitude and 10 ms duration) across the microfluidic chambers through the microhole with Ag/AgCl electrodes. The cellular suspension was then injected into the upper chamber (~2,500 cells/chamber) and a negative pressure of -100 mbar to -200 mbar was applied to the lower chamber in order to attract one cell onto the microhole while simultaneously monitoring patch resistance. This phase of the process is successfully completed within 30 seconds to get one cell onto the microhole. A suction was then maintained until a seal was established. Then the negative pressure could be released. Wholecell configuration was obtained by applying a 1 V pulse during 100 µs to snap the isolated membrane patch. Subsequently, voltage control and current recordings from the cell membrane were performed.

Acquisition was performed with the Multiclamp 700A amplifier (Axon Instruments, Union City, USA).

All traces were sampled at 20 kHz and filtered at 2 kHz. The current-voltage (I-V) relationships were obtained from voltage ramps from 0 mV to + 120 mV with 10 mV increments over 400 ms for BK(Ca) channels, and from + 60 mV to -120 mV with 10 mV decrement during 200 ms for IRK1 channels.

RESULTS

High-resistance seals were previously obtained with non-transfected Jurkat cells on 2.5 µm diameter holes structured in a 2 µm thick silica membrane (Picollet-D'Hahan et al., 2004). However, seal resistances depended very much, as was expected, on the type of cells used. In addition, the seals did not prove to be tight enough for whole-cell mode measurements of transfected CHO cells. Thus, we explored novel ways to modify cell patch-site parameters such as microhole size and surface properties. Hydrophilic surfaces were previously shown to promote the attachment of hydrophilic cell membranes (Corey and Stevens, 1983). In order to test whether the seal was sufficiently tight for use on cells expressing ion channels, the chip surface was passivied with SiO 2 PECVD leading to a more hydrophilic surface. In addition, the PECVD coating was expected to reduce the microhole diameter to less than 2.5 µm and so lead to higher hole resistance values and possibly higher seal resistance values. Chips with a thermal SiO 2 surface layer and chips with an additional PECVD SiO 2 layer on the upper side were used to assess the effect of the additional PECVD layer on the performance of the chip in terms of pore dimension, pore shape, surface roughness, chip capacitance and then weighed against seal quality.

PECVD SiO 2 coating alters the physical properties of microholes

Both thermal and PECVD SiO 2 coated chips were observed by electron microscopy in order to determine microhole size and shape. On thermal SiO 2 chips, the nominal microhole diameter of 2.5 µm showed a well-defined round circle (Fig. 1A). A 1.5 µm PECVD layer reduced the microhole diameter to 1.7 µm while introducing roughness around the aperture (Fig. 1B).

The influence of the passivation process on surface topography was analysed by AFM (atomic force microscopy). The thermal SiO2 surface appeared extremely clean and homogeneous (Fig. 1C). In contrast, asperities and irregularities were present on the PECVD SiO 2 surface (Fig. 1D). Some roughness was determined on either slide in a zone of 7 µm diameter around the microhole and divided into 4 areas (Fig. 1C,D). The arithmetic average of the absolute values of the surface height deviations (measured from the mean plane) was calculated for each area, with a value of 2.4 ± 0.1 nm (n=4) on thermal SiO 2 surfaces and 9.0 ± 1.4 nm (n=4) on PECVD SiO 2 surfaces. Flatness in the immediate vicinity of the microhole was quantified and showed a crown measuring 45 nm in height and a width of 800 nm on the PECVD SiO 2 surface (Fig. 1D,F).

Modelling of patch-site resistance and capacitance

Reproducibility and accuracy of the SiO 2 PECVD process were evaluated by measuring the microhole resistance Rh of each chip for the different thicknesses of the PECVD SiO2 layer: 0, 0.5, 1 and 1.5 µm.

Rh, which is an indirect measure of microhole diameter, was determined for a device that contained an intrapipette saline solution but no cells. The results showed a clear correlation between resistance and diameter (Table 1). Typically, the mean experimental resistance for a 2.5 µm diameter aperture was 0.55 ± 0.1 MΩ and increased to 1.15 ± 0.1 MΩ with a 1.5 µm SiO 2 layer. In order to further investigate the influence of PECVD SiO 2 deposition on Rh, two models were developed to study the importance of the microhole shape. System resistance can be modelled by only taking into account the cell-patch site resistance [START_REF] Matthews | IEEE-EMBS International Conference on Neural Engineering[END_REF]. The cell-patch site can be modelled as a conductor with a resistance that matched that of the intrapipette solution in the microhole (Fig. 2A). In model 1, it was assumed that the PECVD deposition led to a cylindrical shape of the aperture (Fig. 2B). Theoretical values reported in Fig. 2D were calculated using Eq. 1 giving the resistance for a cylindrical shape as a function of electrolyte resistance ρ, hole height l and surface S of the aperture :

S l R h ρ =
Eq. 1

Fig. 2D shows that the theoretical microhole resistance values which were calculated on the basis of a cylindrical shape are higher than the experimental values.

In model 2, which is based on Scanning Electron Microscopy (SEM) observations (Fig. 1B), it was assumed that PECVD deposition led to an "hourglass" shaped hole. It was expected that holes would have the axial symmetry depicted in Fig. 2C and an ellipsoidal curve was used to model the shape of holes. The equation allowing the determination of the microhole diameter as a function of the height z is:

( ) ( ) 2 1 2 0 0 0 2 1 ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ + - - - = e e z d d φ φ φ φ
Eq. 2 with the initial microhole diameter Ø 0 =2.5 µm, the measured microhole diameter Ø d after PECVD deposition, the thermal SiO 2 thickness e 0 and the deposit thickness e d . Eq. 1 was discretized (???? Ca veut dire???) on the total height (e d + e 0 ) in order to calculate theoretical resistances. Fig. 2D and Table 1 show good agreement between the average resistance of four different cell-patch-site diameters and the modelled cell-patch site resistance.

In our device (Fig. 2A), the Si-based structure leads to a high capacitance since it functions as a capacitor between the electrophysiological medium of the upper (zone A) and lower (zone D) chambers and the isolating SiO 2 layers (zone B divided in B 1 and B 2 ) on either side of the silicon layer (zone C). The model for the capacitance is provided by Eq. 3 where C PS stands for the total capacitance of the cell-patch site, C AD and C AC for the front and C CD for the back capacitances.

1 1 1 - ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ + + = CD AC AD PS C C C C Eq. 3 d S C r ε ε 0 = Eq. 4
The capacitances reported in Table 1 were calculated using Eq. 3 and 4, with the insulating layer dielectric constant ε r (4.5 for SiO 2 ), the surface S of the surrounding fluid, and the layer thickness d. In our configuration, C AD can be ignored because the surface of the microhole SiO 2 membrane was only 0.004 mm² compared to the 7 mm² taken into account for the C AC and C CD contributions.

As expected, the cell-patch site capacitance, measured with a 263A potentiostat and a 1025 frequency response detector (Princeton Applied Research, Oak Ridge, USA) was reduced after PECVD passivation.

Capacitance measurements performed before and after the positioning of a 1.5 µm PECVD layer showed a good correlation with theoretical results (Table 1).

PECVD SiO 2 coating improves seal frequency and quality

Chips with a thermal SiO 2 layer and chips with a 1.5 µm thick PECVD layer were experimentally characterized by evaluating seal values and cell membrane stability on the substrate whereas the two others intermediate PECVD thicknesses (0.5 and 1 µm) were used to study the evolution of microhole shape during the deposition process. Cell trapping and sealing were performed as described in the Methods section. The influence radius of suction through the microhole in which a cell is attracted was experimentally observed to be around 30-50 µm. Under this condition, cells in suspension within a distance of 40 µm could be reliably positioned on the microhole and remained in this position even when the negative pressure was released. Immediately after trapping the cell, a negative pressure ranging from 0 mbar to -250 mbar was applied and the cell formed a seal on the patch aperture. The results indicate that PECVD SiO 2 passivation increased the seal success rate from nearly 0% (no seal higher than 100 MΩ on n=30 measurements without a PECVD layer) to 17% (15 seals higher than 100 MΩ on n=90 measurements with the PECVD layer). Taking into account all seals, the mean resistance of a cell on microhole varied from 30 MΩ without PECVD layer (n=30) to 155 MΩ (n=90) with PECVD layer. More precisely, with a PECVD layer, 9 seals out of 15 are set in a range of 100 MΩ to 200 MΩ, 2 seals in a range of 200 to 500 MΩ, 2 seals in a range of 500 to 1000 MΩ and 2 seals superior to 1 GΩ (Fig. 3). The highest seal resistance reached 7 GΩ. 73% of the seals that were higher than 100 MΩ were lost within 2 minutes. In order to test the setup with different cell lines and different types of channels, inward rectifier IRK1 channels were analysed using transfected CHO cells. Potassium currents were recorded at voltage sweeps from +60 mV to -120 mV (by -10 mV decrements) for 200 ms. Inward rectifying currents were inactivated in the presence of 1 mM BaCl 2 (Fig. 5A andB) and 10 mM BaCl 2 , respectively (Fig. 5D). The Ba 2+ block of the inward current reversed readily upon washout (Fig. 5D).

Low conductance and endogenous current recordings

Whereas 1 mM BaCl 2 did not affect the outward current at positive potential, 10 mM BaCl 2 led to the activation of cationic endogenous channels (Fig. 5C andD). Upon washout, this cationic current disappeared.

DISCUSSION

Potassium channels, including both Ca 2+ -activated and inwardly rectifying K + channels, are the major class of ion channels involved in the generation of membrane potentials. The ability to control and regulate the resting membrane potential is due to an unusual conduction feature, which is now commonly referred to as inward and outward rectification. Such a definite electrical signature, perfectly characterized by conventional patch-clamp techniques, makes BK(Ca) and IRK channels attractive models for the validation of new planar patch-clamp chips. Large conductance calcium-and voltage-activated K + channels are also referred to as "BK" (déjà dit) or "maxi-K" channels because of their high single channel conductance (250 -300 pS), eliciting a large membrane current. The activation of BK channels leads to a small increase in extracellular K + which shifts the reversal potential for K + towards a more positive value.

This leads to the activation of an IRK1 conductance of 23 to 30 pS, which is weaker than that of BK channels. In addition, both voltage-gated ion channels are targets for known drugs or divalent ions as well as potential targets for new biosensor approaches (Straub et al., 2001). For these reasons, efforts are being concentrated very much on understanding the properties of K + channels as well as identifying new antagonists or activators (Busse et al., 1993, Nilius andDroogmans, 2001).

We have focused on optimising the coupling of the cells to planar chips in order to be able to record planar patch-clamps of resolved BK and IRK1 currents. We recorded ionic currents from BK(Ca) channels expressed in HEK-293 cells, as well as currents from IRK1 channels expressed in CHO cells, with known values of 250 pS and 30 pS, respectively. Current traces and current-voltage curves were similar to those obtained using conventional patch-clamping (data not shown). The capabilities of our device for the detection of drug-induced modifications of ion channel functions were examined using iberiotoxin, a specific blocker of the BKs as well as Ba 2+ , a divalent cation which is known to inhibit IRK1 currents. The onset and offset of the reversible Ba 2+ block demonstrated the capacity of our device to introduce and remove compounds within seconds. In addition, Ba 2+ is not only known to inhibit inward rectifying IRK1 currents but also to permeate through endogenous cationic channels at positive potentials.

However this effect was not observed at a concentration of 1 mM, as was previously described in the literature (Skryma et al., 1994). With our device, Ba 2+ permeation could only be observed at concentrations of 10 mM and higher.

It is generally agreed that the seal resistance value, the substrate surface state and the substrate (chip or pipette) capacitance are critical parameters in patch clamp experiments. We demonstrated that a 150 MΩ resistance seal, a 10 nm surface roughness and a 54 pF chip capacitance allowed ionic currents to be recorded in the whole-cell mode, permitting the pharmacological characterization of BK(Ca) and IRK1 channels.

Improving the frequency and quality of membrane sealing

Our experiments have shown that PECVD SiO 2 passivation leads to 155 MΩ -7 GΩ seal resistances.

Initially, we hypothesized that the reduction of aperture diameters from 2.5 µm to 1.7 µm, which is a value recently reported to be optimal for such applications (Pantoja et al., 2004), favoured the sealing process. However, similar results could not be obtained in experiments involving micro-structured chips using a mask of 1.7 µm diameter apertures (data not shown). This clearly indicates that aperture diameter is not the only factor which determines seal quality. Seal quality also depends on the length of membrane invagination in the microhole and the total contact surface. The optimal range of 1-2 µm holes should be a compromise between the surface offered by the microhole to the cell membrane and the elasticity of the cell membrane during aspiration. We assumed that PECVD SiO 2 deposition leads to larger sidewalls and/or smoother angles of the microhole which provide a better contact area for seal formation (Sigworth and Klemic, 2002). The "hourglass" shape of the aperture, demonstrated by SEM observations, and the microhole resistance value, seem to favour formation and quality of the required seal. Rough edges were previously said to prevent adequate seal formation, so that care should be taken to obtain a clean and smooth surface around the microhole (Sigworth andKlemic, 2002, Corey andStevens, 1983). For many, this apparently favours a good contact between the cell membrane and the substrate, only leaving a gap of a few Å (Corey andStevens, 1983, Sakmann andNeher, 1984). While qualitative considerations have been advanced as "seal rules" or "seal recipe", no scale or roughness values were given in most studies.

However, our experiments showed that, despite the roughness caused by the PECVD SiO 2 layer the seal values were actually higher than those obtained on smooth, thermal SiO 2 . It can therefore be assumed that an average roughness of 10 nm has a positive influence on the resistance value by increasing the contact area between the cell membrane and the substrate or that the sole physico-chemical properties of the SiO2-coated surface improve seal quality. Future investigations will focus on determining the optimal degree of roughness, for example by modifying PECVD deposit parameters such as temperature or pressure, for optimal seal performances.

Device performances

Silicon introduces a capacitance due to the free charge carrier density of silicon (Fertig et al., 2003). This inherent high capacitance is the main drawback of silicon chips since electrical noise increases with capacitance [START_REF] Wonderlin | Neuromethods: Neurophysiological Techniques: Basic Methods and Concepts[END_REF]. The chips under investigation were initially coated with 2.12 µm thermal SiO2 but the capacitance of 75 pF was too high to allow high resolution current recordings. The addition of an insulating layer of 1.5 µm SiO 2 PECVD reduced the chip capacitance from 75 to 54 pF.

This range of capacitance must still be reduced further towards the values of 2 -20 pF obtained with micropipettes. In a near future, we expect to decrease our capacitance to less than 10 pF by reducing the fluid contact surface and/or increasing the insulator layer. It is indeed desirable that the device capacitance be no higher than the cell capacitance (10-100 pF) so that the cell membrane remains the dominant noise factor [START_REF] Matthews | IEEE-EMBS International Conference on Neural Engineering[END_REF].

The ability of a patch-clamp device to allow rapid and robust solution exchange with low sample solution consumption is one of the prerequisites for drug screening and ligand-gated channel analysis. The system under investigation allows the handling of small volumes in the analysis chambers of 4 µl and is therefore Inward rectifying K + currents were elicited by a voltage pulse from +60 to -90 mV ( ) and were inhibited in the presence of 10 mM BaCl 2 ( ). The part of the curve ( ) at positive potentials shows the activation of endogenous Ca 2+ channels by 10 mM BaCl 2 . The I-V curve (x) was plotted after blocker washout and shows the reversible effect of BaCl 2 . P/4 leak subtraction was displayed.

  Potassium currents were recorded without leak subtraction from HEK-293 cells stably expressing BKtype Ca 2+-activated channels. Current traces in Fig. 4A represent results from the execution of voltage command protocols with sweeps from 0 to +120 mV (by 10 mV increments) during 400 ms and demonstrate the effects of iberiotoxin (IBTX, 100 nM), a selective blocker of this channel type. Under these conditions, the outward currents were strongly inhibited by IBTX. The curves describing the current-voltage relationship, after leak current compensation, indicate an 88 % inhibition of BK(Ca) channels at +120 mV (Fig 4.B).

  suitable for drug screening applications. This was demonstrated by the block of BK(Ca) channels with 20 µl of 100 nM iberiotoxin circulating in the microfluidics capillaries; traditional patch-clamp recordings usually require much larger volumes. Further optimizations are underway to decrease the compound volume to below 10 µl.This work constitutes a promising intermediary step showing whole-cell recordings and reversible detection of drug-induced modulation of ion channel activity. Using the multi-parametrical method described in this paper, we aim to produce new chips allowing a higher number of seals and higher seal resistance values. High resolution recordings and reduced reagents consumption open new application fields of the planar patch-clamp technology.

Figure 1 :

 1 Figure 1: Cell-patch site made of thermal SiO 2 (left column) and PECVD SiO 2 (right column).

Figure 2 :

 2 Figure 2: Schematic cross section representation of silicon chip capacitance (not scaled) and

Figure 3 :

 3 Figure 3: Distribution of seal resistances.

Figure 4 :

 4 Figure 4: Whole cell K + currents recorded from BK(Ca) channels with the chip device.

Figure 5 :

 5 Figure 5: Whole cell K + currents recorded from IRK1 channels with the chip device.

Table 1 : Effect of SiO 2 PECVD deposition on typical electrical characteristics of chips.

 1

	Three different PECVD SiO 2 thicknesses (e d ) were investigated: 0.5, 1 and 1.5 µm. Microhole diameter
	(Ø d ) measured with SEM, theoretical (th) and experimental (exp) microhole resistance (R h ) and cell-patch
	site capacitance (C) are reported for each type of PECVD deposit. Resistance was calculated according to
	equations 1 and 2 using model 2 (see text) and capacitance according to Eq. 3 and 4 (see text). A good
	correlation of resistance and capacitance with the thickness of PECVD SiO 2 layers was observed. (*): no
	measurement performed.
	SiO 2 e d	Ø d µhole	Microhole resistance	Capacitance
	thickness	(µm)	(R h , MΩ)	(C, pF)
	(µm)
			R h (th)	R h (exp)	C (th)	C (exp)
	0	2.50	0.46	0.55	70	75
	0.5	2.20	0.62	0.65	62	-(*)
	1.0	1.95	0.87	0.85	58	-(*)
	1.5	1.70	1.24	1.15	51	54

ACKNOWLEDGMENTS

We would like to thank Chris Lingle for providing the BK cDNA.