Benjamin Herran

Nicolas Cerveau

Camille Houdelet

Clémentine Bernier

Catherine Debenest

Carine Delaunay

Maryline Raimond

Joanne Bertaux
email: joanne.bertaux@univ-poitiers.fr

Pierre Greve
email: pierre.greve@univ-poitiers.fr

Pierre Grève

Grève Pierre Or Bertaux

IGFBP-rP1, a strongly conserved member of the androgenic hormone signalling pathway in Isopoda

Keywords: Armadillidium vulgare Insulin-like Growth Factor Binding Protein-related Protein (IGFBP-rP) Insulin/IGF signalling pathway Isopoda Sexual differentiation Wolbachia AGH, androgenic gland hormone, ILP, insulin-like peptide, IAG, insulin-like androgenic gland hormone, IGFBP, insulin-like growth factor binding protein, IGFBP

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

Introduction

In malacostracan crustaceans (e.g. amphipods, decapods and isopods), sexual differentiation relies on a unique proteinaceous hormone, called the Androgenic Gland Hormone (AGH). The latter, produced by the so-called androgenic glands, is necessary and sufficient to induce male differentiation of the malacostracan juveniles. This hormone has been studied for several decades, revealing it belongs to the Insulin-Like Peptide (ILP) Signalling Pathway (ISP), which includes Insulin-like Growth Factors (IGFs) and insulin-related ILPs like the AGH, also called IAG for Insulin-like Androgenic Gland hormone [START_REF] Ventura | From the discovery of the crustacean androgenic gland to the insulin-like hormone in six decades[END_REF]. Yet, the binding peptides of AGH remained unknown until a first discovery in a decapod cDNA library in 2013 [START_REF] Rosen | A Crayfish Insulin-likebinding Protein: another piece in the androgenic gland insulin-like hormone puzzle is revealed[END_REF]. This first AGH partner was called Cq-IGFBP (Insulin-like Growth Factor Binding Protein) due to its belonging to the IGFBP superfamily. In the canonical ISP, these proteins regulate the distribution and bioavailability of their ligand by sequestering it [START_REF] Baxter | Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities[END_REF][START_REF] Kelley | Comparative endocrinology of the insulin-like growth factor-binding protein[END_REF][START_REF] Bach | IGF-binding proteins -the pieces are falling into place[END_REF]. This way, they might either inhibit the action of the ligand or promote it by carrying and releasing it to its target receptors (as a result of various post-translational modifications or upon interactions with cell surface or matrix) [START_REF] Forbes | Insulin-like growth factor binding proteins: a structural perspective[END_REF]. Members of the IGFBP are found widely in metazoans and are particularly well known in vertebrates, in which they bind IGFs and not insulin [START_REF] Hwa | The insulin-like growth factor-binding protein (IGFBP) superfamily[END_REF]. However, using a recombinant Cq-IGFBP, [START_REF] Rosen | A Crayfish Insulin-likebinding Protein: another piece in the androgenic gland insulin-like hormone puzzle is revealed[END_REF] proved the specific binding of the AGH (i.e. Cq-IAG) by Cq-IGFBP, confirming the unusual interaction between this IGFBP and an insulin-related ILP. Moreover, this first description enabled the subsequent identification of IGFBPs in a variety of other decapod species [START_REF] Chandler | Discovery of a novel insulin-like peptide and insulin binding proteins in the Eastern rock lobster Sagmariasus verreauxi[END_REF][START_REF] Huang | Insights into insulin-like peptide system in invertebrates from studies on IGF binding domain-containing proteins in the female mud crab, Scylla paramamosain[END_REF][START_REF] Huang | Does a blue crab putative insulin-like peptide binding protein (ILPBP) play a role in a virus infection?[END_REF][START_REF] Li | Molecular characterization of insulin-like androgenic gland hormone-binding protein gene from the oriental river prawn Macrobrachium nipponense and investigation of its transcriptional relationship with the insulin-like androgenic gland hormone gene[END_REF][START_REF] Ventura-López | Transcriptomic information from Pacific white shrimp (Litopenaeus vannamei) ovary and eyestalk, and expression patterns for genes putatively involved in the reproductive process[END_REF][START_REF] Song | Insulin-like androgenic gland hormone gene in the freshwater chinese mitten crab Eriocheir sinensis: cDNA cloning, expression pattern, and interaction with EsIGFBP7[END_REF].

Interestingly, [START_REF] Li | Molecular characterization of insulin-like androgenic gland hormone-binding protein gene from the oriental river prawn Macrobrachium nipponense and investigation of its transcriptional relationship with the insulin-like androgenic gland hormone gene[END_REF] and [START_REF] Huang | Does a blue crab putative insulin-like peptide binding protein (ILPBP) play a role in a virus infection?[END_REF] chose to call this protein IAGBP and ILPBP, respectively, referring to its specific function as a binding protein of the IAG hormone (belonging to the ILP family, and not IGF). Furthermore, [START_REF] Song | Insulin-like androgenic gland hormone gene in the freshwater chinese mitten crab Eriocheir sinensis: cDNA cloning, expression pattern, and interaction with EsIGFBP7[END_REF] referred to this gene as IGFBP7 whereas [START_REF] Huang | Insights into insulin-like peptide system in invertebrates from studies on IGF binding domain-containing proteins in the female mud crab, Scylla paramamosain[END_REF] preferred to call this protein an IGFBP-rP (IGFBP-related protein). Indeed, structural and phylogenetic studies have brought out the existence of two distinct families within the IGFBP superfamily [START_REF] Hwa | The insulin-like growth factor-binding protein (IGFBP) superfamily[END_REF][START_REF] Rodgers | Phylogenetic analysis of the insulin-like growth factor binding protein (IGFBP) and IGFBP-related protein gene families[END_REF]. The sensu stricto IGFBP (IGFBP1-6) have a high affinity for IGF peptides only (and a low one for insulin) and are found only in vertebrates. They all display two conserved domains: an insulin binding domain (IB), followed by a thyroglobulin domain. On the other hand, a variety of proteins (including IGFBP7) have been described in both vertebrates and invertebrates and belong to a family of proteins with a low affinity for IGF peptides [START_REF] Oh | Synthesis and characterization of Insulin-like Growth Factor-Binding Protein (IGFBP)-7 recombinant human mac25 protein specifically binds IGF-I and-II[END_REF]Radulović et al., 2015) and a high affinity for insulin [START_REF] Yamanaka | Inhibition of insulin receptor activation by insulin-like growth factor binding proteins[END_REF]Radulović et al., 2015). Contrary to IGFBP1-6, their modular architecture can display various structural domains [START_REF] Hwa | The insulin-like growth factor-binding protein (IGFBP) superfamily[END_REF]. Considering the structural, functional and phylogenetic evidence, members of this IGFBP family have been consensually renamed IGFBP-rP since 1998 [START_REF] Baxter | Recommendations for nomenclature of the insulin-like growth factor binding protein superfamily[END_REF]. Thus, as pointed out by [START_REF] Huang | Insights into insulin-like peptide system in invertebrates from studies on IGF binding domain-containing proteins in the female mud crab, Scylla paramamosain[END_REF], given its structure [START_REF] Rosen | A Crayfish Insulin-likebinding Protein: another piece in the androgenic gland insulin-like hormone puzzle is revealed[END_REF] and phylogenetic position [START_REF] Chandler | Discovery of a novel insulin-like peptide and insulin binding proteins in the Eastern rock lobster Sagmariasus verreauxi[END_REF], Cq-IGFBP is actually a homologue of IGFBP-rP1 (also previously known as IGFBP7), just like all the crustaceans IGFBPs identified so far. The IGFBP-rP1 is characterized by an IB domain, followed by a Kazal domain (protease inhibitor) and an immunoglobulin domain (Ig), all domains also found in the described decapod IGFBPs. Its belonging to the IGFBP-rP family explains why Cq-IGFBP displays such an affinity for an insulin-related ILP (Cq-IAG). More recently, the molecular interaction between the AGH and IGFBP-rP1 was confirmed using a yeast-two hybrid assay [START_REF] Song | Insulin-like androgenic gland hormone gene in the freshwater chinese mitten crab Eriocheir sinensis: cDNA cloning, expression pattern, and interaction with EsIGFBP7[END_REF]. In addition to the evidence at the molecular level, the interaction between decapod IGFBP-rP1 and the AGH was also demonstrated at the transcriptional level, using RNAi [START_REF] Li | Molecular characterization of insulin-like androgenic gland hormone-binding protein gene from the oriental river prawn Macrobrachium nipponense and investigation of its transcriptional relationship with the insulin-like androgenic gland hormone gene[END_REF] and modelled in silico [START_REF] Chandler | Understanding insulin endocrinology in decapod Crustacea: molecular modelling characterization of an insulin-binding protein and insulin-like peptides in the eastern spiny lobster, Sagmariasus verreauxi[END_REF]. Interestingly, [START_REF] Chandler | Understanding insulin endocrinology in decapod Crustacea: molecular modelling characterization of an insulin-binding protein and insulin-like peptides in the eastern spiny lobster, Sagmariasus verreauxi[END_REF] predicted that Sv-IGFBP could bind not only the AGH but also two other ILPs (ILP1 and ILP2), which remain undiscovered to date in other malacostracan orders. This characteristic is consistent with the fact that IGFBP-rPs seem to be involved in varied biological processes in invertebrate species with no AGH (metabolism, immunity, growth…) (N. Li et al., 2012;[START_REF] Mulenga | Silencing of three Amblyomma americanum (L.) insulin-like growth factor binding protein-related proteins prevents ticks from feeding to repletion[END_REF][START_REF] Wang | IGFBP7 promotes hemocyte proliferation in small abalone Haliotis diversicolor, proved by dsRNA and cap mRNA exposure[END_REF][START_REF] Wang | IGFBP7 is involved in abalone metamorphosis[END_REF].

The crustacean IGFBP-rPs, due to their additional interaction with AGH, are however the first ones potentially involved in sexual differentiation. Yet, the exact function of IGFBP-rP1 is still difficult to assess.

In crustacean isopods, using mostly the common woodlouse Armadillidium vulgare as a model species, the sexual development has been extensively studied since the late 1950's, revealing the precise anatomical sequence of male gonad differentiation that begins after the third moult following the release of the larvae from the female ventral pouch (stage 4) [START_REF] Suzuki | Morphological studies on sexual differentiation in Armadillidium vulgare[END_REF]. Moreover, male differentiation seems to be especially sensitive and is often altered in isopods, leading to numerous pathological phenotypes such as various intersex individuals [START_REF] Legrand | Rôle des bactéries symbiotiques dans l'intersexualité, la monogénie et la spéciation chez des crustacés oniscoïdes[END_REF][START_REF] Rigaud | Sterile intersexuality in an isopod induced by the interaction between a bacterium ( Wolbachia ) and the environment[END_REF]. Notably, some strains of the well-known bacteria Wolbachia, a common endosymbiont of woodlice, can alter the sexual differentiation of their hosts [START_REF] Bouchon | Evidence for widespread Wolbachia infection in isopod crustaceans: molecular identification and host feminization[END_REF]. These strains, known as feminizing Wolbachia, have drawn attention as they induce the differentiation of genetic male larvae into functional females and occasionally into partially feminized males, called male intersexes. In the functional females induced by the presence of Wolbachia, the androgenic glands do not differentiate so that the AGH is not expressed at all. Besides, these infected females are refractory to the action of the AGH, as androgenic gland grafts do not masculinize them. In contrast, the male intersexes become refractory in the course of male differentiation, which results in mixed sexual characters, in particular the coexistence of hypertrophied androgenic glands and female genital apertures [START_REF] Legrand | Rôle des bactéries symbiotiques dans l'intersexualité, la monogénie et la spéciation chez des crustacés oniscoïdes[END_REF][START_REF] Rigaud | Sterile intersexuality in an isopod induced by the interaction between a bacterium ( Wolbachia ) and the environment[END_REF]. In this context, all the molecular partners of the AGH are putative targets of the feminizing Wolbachia [START_REF] Juchault | Contribution à l'étude du mécanisme de l'état réfractaire à l'hormone androgène chez les Armadillidium vulgare Latr, (crustacé, isopode, oniscoïde) hébergeant une bactérie féminisante[END_REF]. This includes IGFBP-rP1, as well as the transmembrane tyrosine kinase receptors recently described in decapods and now identified in A. vulgare (Herran et al., 2018). Interestingly, some interaction between Wolbachia and the ISP has been described in arthropods [START_REF] Ikeya | The endosymbiont Wolbachia increases insulin/IGF-like signalling in Drosophila[END_REF][START_REF] Grönke | Molecular evolution and functional characterization of Drosophila Insulin-Like Peptides[END_REF]. To date, the molecular investigations of the isopod sexual differentiation are however limited to the AGH, so that subsequent potential alterations in this pathway are not known on the molecular scale either. In this context, the discovery of IGFBP-rP1 in decapods thus raises several questions relating to its existence and function in isopods.

To tackle these issues, we performed the first characterisation of an isopod IGFBP-rP1 (Av-IGFBP-rP1). Its affiliation to the IGFBP-rP family was first established by structural analyses. We then investigated the expression pattern of this gene among tissues of adult males and females and looked for differential expression during development, in both Wolbachia-infected and uninfected lineages of A. vulgare. Av-IGFBP-rP1 displayed a broad and constitutive expression, suggesting a major functional importance. Silencing experiments of the AGH and of the IGFBP-rP1 confirmed their implication in the physiology of the androgenic glands, as some got hypertrophied following the inhibition of these genes. The evolutionary conservation of IGFBP-rP1 was thus investigated in the Isopoda order. Using a transcriptomic approach, as well as RT-PCR and direct Sanger sequencing, we identified 68 IGFBP-rP1 sequences, one for each of the studied species. Homology to IGFBP-rP1 was confirmed by phylogenetic reconstructions. Negative selection was further demonstrated with dN/dS analyses. Altogether, our results showed that this gene is a key element of the ISP in Isopoda, although not responsible on its own for the different developmental process between male and female isopods and probably not the target of Wolbachia.

Material and methods

Animals

Tissue and developmental expression studies were performed on two lineages of the common woodlouse (A. vulgare) coming from our laboratory rearing: the lineage harvested in Celles/Belle (France) in 1991, which is infected by the wVulC Wolbachia strain and the Wolbachiafree lineage, harvested in Nice (France) in 1967. Tissues from intersexes were collected from individuals of the Wolbachia-infected lineage or intersexes generated from males of the uninfected lineage that were previously experimentally infected with Wolbachia [START_REF] Juchault | Contribution à l'étude du mécanisme de l'état réfractaire à l'hormone androgène chez les Armadillidium vulgare Latr, (crustacé, isopode, oniscoïde) hébergeant une bactérie féminisante[END_REF][START_REF] Rigaud | Feminizing endocytobiosis in the terrestrial crustacean Armadillidium vulgare Latr.(Isopoda): recent acquisitions[END_REF]. The silencing experiment was performed with the uninfected lineage only. For the phylogenetic analysis, we included isopod species for which RNAseq data were already available and species either sampled from our laboratory rearing or collected from various field sites in 2015 and 2016. These species represent a wide diversity of ecology (soil-dwelling, terrestrial, coastal, freshwater and marine species) and mostly a diversity in taxonomic ranks (Table S1).

RNA extractions

To study the spatial expression of Av-IGFBP-rP1, six to eight A. vulgare adults for each sex (male, female, intersex) and each lineage were dissected in Ringer's solution (394 mM NaCl, 2 mM KCl, 2 mM CaCl2/2H2O, 2 mM NaHCO3). The following tissues were gradually pooled and frozen in liquid nitrogen: hemolymph (HE), brain (BR), nerve cord (NC), emptied digestive tract (DT), digestive caeca (CK), muscle/fat (MF), legs (LE) and the different parts of the gonads (ovaries (OV) in females; androgenic glands (AG), utricles (UC), seminal vesicles (SV), vasa deferens (VD) and sperm (SP) in males). All the dissections were repeated three times independently (four times for males of the uninfected lineage), requiring around a hundred animals in total. For all the other analyses (RT-PCR as well as RNAseq and RNAi experiments), whole animals were used. To study Av-IGFBP-rP1 expression during development, we sampled pools of larvae just after birth (i.e. when they were released from the ventral pouch, week 0) and one, two, three weeks later. From the fourth week on, juveniles were sampled individually and measured to infer their developmental stage (4-8) according to [START_REF] Suzuki | Morphological studies on sexual differentiation in Armadillidium vulgare[END_REF]. Both dissected tissues or whole animals were homogenized using a Vibra Cell 75185 sonicator (amplitude of 35%). Total RNA was then extracted using the RNeasy kit (Qiagen) or TRIzol® reagent and treated with RNase-free DNase I (Qiagen), according to the manufacturer's protocols. After quantification with NanoDrop™ technology, the RNAs were stored at -80°C.

RT-PCR and RT-qPCR

Reverse transcriptions were achieved on 500 ng of the previously extracted total RNA using the SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific) following the supplier's instructions. Degenerated primers (Sigma-Aldrich) were manually designed using the three sequences of the IGFBP-rP1 of A. vulgare, A. nasatum and Porcellio dilatatus (Table S2), identified by TBLASTX in the corresponding public cDNA libraries, using Cq-IGFBP as query. RT-PCR were carried out using Phusion® High-Fidelity DNA Polymerase and annealing temperatures varying between 45°C and 60°C according to the characteristics of the primers (Table S2), with an elongation time of 60 s. The amplification products were separated on 1.5% agarose gels and visualised with ethidium bromide and UV.

RT-qPCR were performed using Applied Biosystems™ SYBR™ Green master mixes (5 µL Sybergreen 5X, 0.5 µL of each primer, 2.5 µL cDNA and sterile water to 10 µL). Primer sequences are given in Table S2. The reactions were performed on a LightCycler® 480 System (Roche), using the following program: 95°C for 10 min followed by 45 cycles of (95°C for 10 s, 60°C for 10 s, 72°C for 20 s). Melting curves were established (65°C-97°C) to check the specificity of the PCR products. The Av-IGFBP-rP1 expression levels were analysed relatively to the ribosomal protein RbL8 expression [START_REF] Chevalier | Feminizing Wolbachia: a transcriptomics approach with insights on the immune response genes in Armadillidium vulgare[END_REF], using LightCycler® 480 Software. Statistics on the tissue and development RT-qPCR data were calculated using the Kruskal-Wallis test, complemented by Dunn's post-hoc tests implemented in the R package PMCMR [START_REF] Pohlert | The Pairwise Multiple Comparison of Mean Ranks Package (PMCMR)[END_REF]. The resulting pvalues were adjusted using Holm's correction in order to take multiple comparisons into account.

The Wilcoxon-Mann-Whitney test was used to assess the efficiency of the gene silencing in the RNAi experiment.

Sequencing

The 26 IGFBP-rP1 sequences amplified by RT-PCR were treated with 2 µL of EXOAnP solution (0.05 µL of exonuclease I (1 u), 0.1 µL of AnP (0.5 u) and 1.85 µL of sterile water) at 37°C for 1 h. Enzymes deactivation was achieved at 80°C for 20 minutes. New PCR using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) were then performed (3 µL of PCR product, 0.5 µL of BDT, 0.32 µL of primer, 3 µL of 5X buffer and sterile water to 15 µL). The products of the PCR were precipitated with a mix of 50 µL ethanol (100%), 2.2 µL NaOAc (3M, pH 5.2) and 10 µL water. Pellets were washed with 70% ethanol and solubilized in deionized formamide. Finally, DNAs were sequenced on both strands using Sanger method. The final 68 IGFBP-rP1 sequences were deposited in GenBank under accession numbers [pending submission].

RNA silencing

Av-AGH and Av-IGFBP-rP1 cDNAs were first generated by RT-PCR as described above, with specific primers (Table S2). Amplicons were purified using the QIAquick PCR Purification Kit (Qiagen) and then cloned using the pGEM®-T Easy Vector system, according to the supplier's instructions. Inserts were checked with Sanger sequencing as previously described. Single-stranded RNAs were generated in both directions, overnight, at 37°C the using MEGAscript T7 Kit (Ambion), treated with TURBO DNase (15 minutes at 37°C), precipitated with LiCl, washed with 70% ethanol and dissolved in nuclease-free water, according to the manufacturer's instructions.

Hybridisation of forward and reverse RNAs was performed first for 15 minutes at 70°C followed by 10 minutes at room temperature. Double-stranded RNAs (dsRNAs) were quantified and diluted to 1 µg/µL. Adult males were injected with 1 µL of dsRNA (Av-AGH or Av-IGFBP-rP1) or 1 µL of nuclease free water (vehicle group), to account for the trauma response, aging and environmental rearing conditions. These injections were performed using a Hamilton syringe through a hole drilled on the lateral side of the fifth segment of the pereion. Another control group was not injected at all. At one, four, 12 and 28 weeks post-injection, five animals of each group were frozen individually in liquid nitrogen for RNA extraction and another two were dissected to investigate the morphology of the androgenic glands (six per males) and the presence of female genital apertures.

Bioinformatics

The assembly of both newly generated and already published reads was achieved on our servers (Table S1). The raw reads were first filtered with Trimmomatic (leading and trailing quality of 5, minimum length of 36 bp, Illumina adapters removed) [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF]. The trimmed data were then assembled with IDBA-tran assembler (maximum k-mer size of 100 bp) [START_REF] Peng | IDBA-tran: a more robust de novo de Bruijn graph assembler for transcriptomes with uneven expression levels[END_REF]. The IGFBP-rP1 sequences were searched locally with TBLASTX, using first Cq-IGFBP and then Av-IGFBP-rP1 as queries.

The post-translational modifications were predicted using CBS prediction servers (SignalP 4.1 [START_REF] Petersen | SignalP 4.0: discriminating signal peptides from transmembrane regions[END_REF], NetPhos 3.1 [START_REF] Blom | Sequence and structure-based prediction of eukaryotic protein phosphorylation sites[END_REF], ProP 1.0 [START_REF] Duckert | Prediction of proprotein convertase cleavage sites[END_REF]). The conserved domains of the IGFBP-rP1 sequences were obtained using the Conserved Domain Search service (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [START_REF] Marchler-Bauer | CDD: NCBI's conserved domain database[END_REF]. Coding sequences were retrieved with ORF Finder from the Sequence Manipulation Suite (http://www.bioinformatics.org/sms2/orf_find.html). Sequences logos were drawn using the WebLogo toolsite (http://weblogo.berkeley.edu/logo.cgi) [START_REF] Crooks | WebLogo: a sequence logo generator[END_REF].

Phylogeny reconstructions were carried out using the programs implemented in Seaview [START_REF] Gouy | SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building[END_REF]. Nucleotidic coding sequences were aligned using Muscle [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF]. The final set of sites was determined with Gblocks [START_REF] Castresana | Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis[END_REF][START_REF] Talavera | Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments[END_REF].

The reconstructions themselves were performed using PhyML (maximum likelihood method [START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF]), using GTR+I+G model of evolution as determined using jModelTest 2.1.9 [START_REF] Darriba | jModelTest 2: more models, new heuristics and parallel computing[END_REF][START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF]. Branch support was estimated with both the non parametric Shimodaira-Hasegawa-like approximate Likelihood-Ratio Test (SHlike aLRT) [START_REF] Anisimova | Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative[END_REF][START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF] and the bootstrap proportions (1000 repetitions) [START_REF] Felsenstein | Confidence limits on phylogenies: an approach using the bootstrap[END_REF]. Branch supports were further checked with a bayesian method using BEAST 1.8.4 [START_REF] Drummond | Bayesian Phylogenetics with BEAUti and the BEAST 1.7[END_REF], using the same model of evolution, an uncorrelated relaxed clock (log normal) and a run of 50,000,000 generations (log sampling every 1000 generations). Posterior probabilities (PP) were retrieved using TreeAnnotator.

Evidence for natural selection in our IGFBP-rP1 dataset was investigated using PAML [START_REF] Yang | PAML: a program package for phylogenetic analysis by maximum likelihood[END_REF][START_REF] Yang | PAML 4: phylogenetic analysis by maximum likelihood[END_REF]). An overall dN/dS value was calculated with codeml (seqtype=1, model=0). A dN/dS value was calculated for each site with the tools available on the Datamonkey server (SLAC, FEL, REL [START_REF] Pond | Not so different after all: a comparison of methods for detecting amino acid sites under selection[END_REF][START_REF] Murrell | Detecting individual sites subject to episodic diversifying selection[END_REF]) (http://datamonkey.org) (Pond et al., 2005;Pond and Frost, 2005).

Results

Av-IGFBP-rP1 sequence and structure

With a transcriptomic approach using publicly available reads, we identified in A. vulgare a unique transcript coding for the decapod homologue of IGFBP-rP1, named Av-IGFBP-rP1. Its sequence was confirmed using the Sanger method, revealing a coding sequence of 744 bp (247 amino acids). A signal peptide was predicted, with a cleavage site between positions 22 and 23.

Two sites of phosphorylation (Ser145, Ser232) but no R/K cleavage site were predicted.

In keeping with descriptions in Decapoda, three conserved domains were predicted in Av-IGFBP-rP1. It included the IB domain itself, to which the ligand binds, followed by the Kazal domain (a serine protease inhibitor) and finally an Immunoglobulin I-set domain (Fig. 1).

Expression analysis of Av-IGFBP-rP1

The spatial expression of the Av-IGFBP-rP1 mRNA was investigated by RT-PCR (Fig. S1) and RT-qPCR (Fig. 2) in adult A. vulgare. Av-IGFBP-rP1 was expressed in all the individuals, regardless of their sex (male, female or male intersex) or their Wolbachia-infection status. In all cases, we found a broad expression of Av-IGFBP-rP1 in the analysed tissues. The RT-qPCR analyses in the uninfected lineage showed that this expression was especially strong in the nerve cord (NC), the muscles and fat (MF), the digestive tract (DT) and the ovaries (OV) (Fig. 2A). In contrast, this expression was especially weaker in the digestive caeca (CK). The same expression pattern was observed in the Wolbachia-infected lineage (Fig. 2B) and in the intersexes generated experimentally by injecting Wolbachia in uninfected males (Fig. 2A).

The expression of the Av-IGFBP-rP1 mRNA was also investigated in whole individuals at each developmental stage by RT-qPCR (Fig. 3). In the uninfected lineage, Av-IGFBP-rP1 gene expression was elevated after birth (week 0), decreased after the first and second weeks and remained stable until adulthood (stages 4-9) (Fig. 3A). A similar expression pattern was observed during the development in the Wolbachia-infected lineage (Fig. 3B). Overall, this gene was found continuously expressed throughout development.

Silencing of Av-IGFBP-rP1 and Av-AGH

To confirm the implication of Av-IGFBP-rP1 in the ISP, its cross-talk with Av-AGH was investigated using a RNA silencing approach (Fig. 4). One week post-injection (wpi) of the Av-AGH dsRNA, the expression of Av-AGH was successfully inhibited by 98% compared to the vehicle group (p=0.001). The silencing efficiency was still of 95% (p=0.001), 89% (p=0.002) and 64% (p=0.032) at four, 12 and 28 wpi, respectively (Fig. 4A). In the same way, the expression of Av-IGFBP-rP1 in the IGFBP-rP1 silencing experiment was reduced by 88% (p=0.001), 86% (p=0.001), 94% (p=0.002) and 92% (p=0.008) at one, four, 12 and 28 wpi, respectively (Fig. 4B).

Whereas the inhibition of the Av-AGH gene did not impact the expression of the Av-IGFBP-rP1 gene (Fig. 4C), the silencing of Av-IGFBP-rP1 gene induced an increase in the Av-AGH mRNA level, which significantly reached a two-fold increase at four (p=0.019) and 12 wpi (p=0.002), and up to a seven-fold increase at 28 wpi (p=0.016) (Fig. 4D).

The impact of these two silencing experiments on the gonad phenotypes was also investigated (Fig. 5). No altered phenotype could be observed one week after the silencing of both genes. Four weeks after AGH dsRNA injection, half of the investigated androgenic glands appeared hypertrophied (bigger and more nodular) (5/10) (Fig. 5B). Four weeks after IGFBP-rP1 dsRNA injection, hypertrophied androgenic glands also began to appear (2/10) (Fig. 5E). At 12 and 28 wpi, we observed hypertrophy for all of the androgenic glands in the AGH silencing experiment (12/12 and 12/12) (Figs. 5C and5D), as well as the appearance of female genital apertures in half cases (Fig. 5H). In contrast, at 12 and 28 wpi in the IGFBP-rP1 silencing experiment, the androgenic glands seemed normal (hypertrophy: 0/12 and 0/9) in the dissected individuals (Figs. 5F and5G), which did not display female genital apertures either (Fig. 5I).

IGFBP-rP1 in Isopoda: sequences, structure and evolution

Considering the constitutive expression of the IGFBP-rP1 gene in the different tissues and during the development of A. vulgare, as well as the consequence of the silencing experiment, we suspected an essential role of Av-IGFBP-rP1 in the ISP and thus investigated its global conservation in isopods. IGFBP-rP1 sequences were obtained using two complementary approaches: by RT-PCR with degenerated primers and by using both public and newly generated cDNA libraries. After the sequencing of the PCR fragments and assembly of the RNAseq data, a total of 68 IGFBP-rP1 sequences was obtained from isopod species belonging to 18 different families: five consisting of aquatic species and 13 comprising terrestrial species (Table S1).

All of the sequences shared the canonical domains of this protein: the IB domain, the Kazal domain and an Immunoglobulin domain (Ig, Ig_3) or Immunoglobulin I-set domain (Fig. 6). The open reading frame length varied between 720 bp (239 amino acids) for Bragasellus peltatus and Proasellus ibericus and 756 bp (251 amino acids) for Armadillo officinalis. About 68% (26/38) of the terrestrial isopods (Oniscidea) displayed an IGFBP-rP1 coding sequence of 744 bp (247 amino acids) (Fig. 6). Decapod IGFBP-rP1 sequences have a length within the same range, from 747 bp (248 amino acids) for Cq-IGFBP but up to 771 bp (256 amino acids) for Sagmariasus verreauxi. In isopods, 74 amino acids were found completely conserved in all the sequences (Fig. 6). They constituted ~30% of the total sequence and were especially represented in the Ig domain (47/101).

Among them, ~25% were cysteines (18 residues), mainly distributed in the IB domain (10/60) and the Kazal domain (4/39) but rare in the Ig domain (2/101) or out of the conserved domains (2).

Several conserved motifs are described as being implicated in ligand binding: (R/L)xLxxLL in humans [START_REF] Imai | Substitutions for hydrophobic amino acids in the N-terminal domains of IGFBP-3 and -5 markedly reduce IGF-I binding and alter their biologic actions[END_REF][START_REF] Hong | Insulin-like Growth Factor (IGF)-Binding Protein-3 mutants that do not bind IGF-I or IGF-II stimulate apoptosis in human prostate cancer cells[END_REF], RxLxxL in decapods [START_REF] Rosen | A Crayfish Insulin-likebinding Protein: another piece in the androgenic gland insulin-like hormone puzzle is revealed[END_REF] and CGCCxxC in vertebrates [START_REF] Hwa | The insulin-like growth factor-binding protein (IGFBP) superfamily[END_REF][START_REF] Daza | Evolution of the insulin-like growth factor binding protein (IGFBP) family[END_REF]. We found in isopod IGFBP-rP1 the same CGCCxxC determinant as the one identified in vertebrates (Fig. 6, framed in green). In contrast, the homologue region of the decapod motif RxLxxL (Fig. 6, framed in blue) did not correspond to the same sequence. However, a close and overlapping region was found to be similar to the described motif (Fig. 6, framed in orange), even if not fully conserved: (I/V)xLxxL (or (I/V)xxLxL in Proasellus sp.). In this conserved determinant, the second conserved leucine in Isopoda was found homologous to the first conserved leucine of Decapoda.

A phylogeny of all the IGFBP-rP1 sequences in isopods was reconstructed using the whole coding sequence (Fig. 7). The phylogenetic tree of the gene was globally congruent with the known phylogeny of isopod species. In particular, the IGFBP-rP1 of terrestrial isopods clustered together, which is consistent with the monophyly of Oniscidea. More extensively, the monophyly of every isopod family was respected with two exceptions. Indeed, our two representatives of Platyarthridae (Fig. 7, in red) as well as the four species of Philoscidae (Fig. 7, in purple) did not cluster together.

The monophyly of species classified in Philoscidae is however deemed unlikely [START_REF] Schmidt | Phylogeny of the terrestrial Isopoda (Oniscidea): a review[END_REF].

Because of the modularity of the IGFBP superfamily members, we also constructed phylogenies of the different IGFBP-rP1 conserved domains separately, to check whether they evolved similarly or not. However, considering the very high conservation of these domains (e.g. half of the amino acids in the Ig domain strictly conserved in Isopoda), the resolution of these trees was rather poor (data not shown).

To confirm the apparent good conservation of the isopod IGFBP-rP1, we calculated the dN/dS ratios for the whole sequence set and then for each site of the coding sequence. The overall dN/dS ratio of the isopod IGFBP-rP1 alignment was between 0.12 (codeml, model 0) and 0.17 (SLAC and REL), implying a strong purifying selection on this sequence. This was further confirmed by SLAC, FEL and IFEL methods that predicted 177 (73%), 191 (79%) and 173 (72%) negatively selected codons, respectively, 166 (69%) of these codons being found under purifying selection by these three methods.

Discussion

In this study, we identified and described the first molecular partner of the AGH in Isopoda, namely the malacostracan order in which this unique sex hormone was initially identified and characterized [START_REF] Hasegawa | Isolation and properties of androgenic gland hormone from the terrestrial isopod, Armadillidium vulgare[END_REF][START_REF] Martin | Purification and characterization of androgenic hormone from the terrestrial isopod Armadillidium vulgare Latr. (Crustacea, Oniscidea)[END_REF]). In the model A. vulgare, we found Av-IGFBP-rP1 expressed constitutively, without significant difference between males, females or intersex individuals. The lack of clear differential expression between males and females in isopods is consistent with the fact that Wolbachia-uninfected females are still responsive to the AGH, as shown by the grafts of androgenic glands or the AGH injections [START_REF] De Lattin | Die Beeinflussbarkeit sekundärer Geschlechtsmerkmale von Oniscus asellus durch die Gonaden[END_REF][START_REF] Legrand | Induction des caractères sexuels secondaires mâles chez les femelles des Crustacés Isopodes terrestres par implantation testiculaire[END_REF][START_REF] Katakura | Masculinization through implanting testes into the female Armadillidium vulgare, an isopod crustacean[END_REF][START_REF] Katakura | Masculinization of females of the isopod crustacean, Armadillidium vulgare, following injections of an active extract of the androgenic gland[END_REF]). Yet, the expression of IGFBP-rP1 in females could be surprising considering its putative role in male differentiation and that they do not express the AGH. Actually, it is likely that IGFBP-rP1 ensures several functions simultaneously within the organism, including ligand-independent functions [START_REF] Hwa | The insulin-like growth factor-binding protein (IGFBP) superfamily[END_REF] and functions associated with additional ligands. Indeed, the AGH is not the only known member of the ILP family in Crustacea and thus, not the only candidate ligand for IGFBP-rP1. For instance, four ILPs had already been discovered in 2011 in the branchiopod crustacean Daphnia pulex [START_REF] Colbourne | The ecoresponsive genome of Daphnia pulex[END_REF][START_REF] Dircksen | Genomics, transcriptomics, and peptidomics of Daphnia pulex neuropeptides and protein hormones[END_REF] and two others were described in the decapod Sagmariasus verreauxi in 2015 (Sv-ILP1, a possible homologue of DILP7) [START_REF] Chandler | Discovery of a novel insulin-like peptide and insulin binding proteins in the Eastern rock lobster Sagmariasus verreauxi[END_REF] and in 2017 (Sv-ILP2, a possible homologue of DILP8) [START_REF] Chandler | Understanding insulin endocrinology in decapod Crustacea: molecular modelling characterization of an insulin-binding protein and insulin-like peptides in the eastern spiny lobster, Sagmariasus verreauxi[END_REF]. To this date, the function of these ligands cannot be investigated in Isopoda as no homologue of ILP1 or ILP2 was described in any isopod species. Lastly, the exact interactions between the mobile IGFBP-rP1 and its target tissues are not described yet in crustaceans, so that we cannot exclude variations in its function according to the targets, such as feeding activity in the midgut [START_REF] Huang | Does a blue crab putative insulin-like peptide binding protein (ILPBP) play a role in a virus infection?[END_REF] or ovarian development [START_REF] Huang | Insights into insulin-like peptide system in invertebrates from studies on IGF binding domain-containing proteins in the female mud crab, Scylla paramamosain[END_REF][START_REF] Huang | The presence of an insulin-like androgenic gland factor (IAG) and insulin-like peptide binding protein (ILPBP) in the ovary of the blue crab, Callinectes sapidus and their roles in ovarian development[END_REF] for instance.

The analysis of Av-IGFBP-rP1 expression in the different tissues revealed that its spatial expression in adults was very broad, despite the variation in intensity. This is consistent with previous descriptions in decapod models in which IGFBP-rP1 is also expressed in all of the investigated tissues but hemocytes in Scylla paramamosain [START_REF] Rosen | A Crayfish Insulin-likebinding Protein: another piece in the androgenic gland insulin-like hormone puzzle is revealed[END_REF][START_REF] Chandler | Discovery of a novel insulin-like peptide and insulin binding proteins in the Eastern rock lobster Sagmariasus verreauxi[END_REF][START_REF] Huang | Insights into insulin-like peptide system in invertebrates from studies on IGF binding domain-containing proteins in the female mud crab, Scylla paramamosain[END_REF][START_REF] Huang | Does a blue crab putative insulin-like peptide binding protein (ILPBP) play a role in a virus infection?[END_REF][START_REF] Li | Molecular characterization of insulin-like androgenic gland hormone-binding protein gene from the oriental river prawn Macrobrachium nipponense and investigation of its transcriptional relationship with the insulin-like androgenic gland hormone gene[END_REF][START_REF] Song | Insulin-like androgenic gland hormone gene in the freshwater chinese mitten crab Eriocheir sinensis: cDNA cloning, expression pattern, and interaction with EsIGFBP7[END_REF]. More precisely, we found the highest Av-IGFBP-rP1 expression in the nerve cord, muscles and digestive tracts, and the lowest expression in the hemocytes, testis and most notably in the digestive caeca. A similar expression level of IGFBP-rP1 is reported in the hepatopancreas (i.e. digestive caeca) of Callinectes sapidus and Eriocher sinensis [START_REF] Huang | Does a blue crab putative insulin-like peptide binding protein (ILPBP) play a role in a virus infection?[END_REF][START_REF] Song | Insulin-like androgenic gland hormone gene in the freshwater chinese mitten crab Eriocheir sinensis: cDNA cloning, expression pattern, and interaction with EsIGFBP7[END_REF], whereas the other decapod IGFBP-rP1s display a high gene expression in this tissue. Contrary to Av-IGFBP-rP1, all of them also show an elevated expression in the testis. It seems however difficult to conclude on functional differences between these homologues on the basis of their gene expression patterns only. Considering both its broad expression and its role as a binding protein, circulating freely in the hemolymph, it seems plausible that IGFBP-rP1 protein is ubiquitous in the organism despite local transcriptional variations. Besides, this broad spatial expression pattern is consistent with the fact that the AGH action is not restricted to the male gonads, as this hormone indeed acts on several secondary sex characters (copulatory organs [START_REF] Suzuki | Sex-reversal of male Armadillidium vulgare (Isopoda, Malacostraca, Crustacea) following andrectomy and partial gonadectomy[END_REF][START_REF] Takewaki | The effects of gonadectomy on the sex characters of Armadillidium vulgare, an isopod crustacean[END_REF], brushes on the pereiopods, behaviour [START_REF] Barki | Intersexuality and behavior in crayfish: The demasculinization effects of androgenic gland ablation[END_REF][START_REF] Barki | Male-like behavioral patterns and physiological alterations induced by androgenic gland implantation in female crayfish[END_REF][START_REF] Karplus | The influence of androgenic gland implantation on the agonistic behavior of female crayfish (Cherax quadricarinatus) in interactions with males[END_REF]) and is expressed, in some decapod species, by several organs besides the androgenic glands (ovaries, hepatopancreas, nerve cord) (S. Li et al., 2012;[START_REF] Chung | An insulin-like growth factor found in hepatopancreas implicates carbohydrate metabolism of the blue crab Callinectes sapidus[END_REF][START_REF] Huang | An insulin-like androgenic gland hormone gene in the mud crab, Scylla paramamosain, extensively expressed and involved in the processes of growth and female reproduction[END_REF][START_REF] Li | Molecular characterization of insulin-like androgenic gland hormone-binding protein gene from the oriental river prawn Macrobrachium nipponense and investigation of its transcriptional relationship with the insulin-like androgenic gland hormone gene[END_REF].

Likewise, the broad occurrence in the organism is in line with IGFPB-rP1 binding several ILPs with different spatial expression profiles (e.g. Sv-ILP1, Sv-ILP2, Sv-IAG; [START_REF] Chandler | Discovery of a novel insulin-like peptide and insulin binding proteins in the Eastern rock lobster Sagmariasus verreauxi[END_REF]2017).

We also investigated the differential gene expression of Av-IGFBP-rP1 during A. vulgare development. In crustaceans, a similar analysis was performed on Macrobrachium nipponense only, in which Mn-IAGBP transcription tends to the increase, particularly at the beginning of sexual differentiation [START_REF] Li | Molecular characterization of insulin-like androgenic gland hormone-binding protein gene from the oriental river prawn Macrobrachium nipponense and investigation of its transcriptional relationship with the insulin-like androgenic gland hormone gene[END_REF]. By contrast, we observed a significant decrease of Av-IGFBP-rP1 expression after birth, followed by a relatively constant expression. Moreover, the Av-IGFBP-rP1 expression patterns between males and females and between infected and uninfected lineages were similar. This suggests that Av-IGFBP-rP1 is required throughout the life of the animal but that other factors may account for the different developmental paths taken from stage four on by the males and the females or feminized genetic males. Despite the absence of differential gene expression patterns, there are other ways to modulate IGFBP-rP1 activity besides variations of transcription intensity. First, the amount of proteins could be regulated at the translation level, which would require quantifying the IGFBP-rP1 protein. Then, the responsiveness of the different tissues, in particular the gonads, may also fluctuate: undifferentiated gonads may not interact in the same way with IGFBP-rP1 during development or according to their Wolbachia-infection status. Further studies should also investigate the affinity of isopod IGFBP-rP1 for its ligand in different contexts.

IGFBP-rP1 pull-down combined with AGH antibodies may reveal affinity variations between sexes or between infected and uninfected lineages.

In that respect, a role of IGFBP-rP1 in the sex differentiation cannot be excluded, particularly since its implication in the AGH pathway is now well established in decapods (molecular interaction, modelling, silencing...). The isopod model also appeared particularly wellsuited for the gene silencing approach, as only one injection of Av-AGH or Av-IGFBP-rP1 dsRNA could successfully inhibit the expression of the target genes for at least six months. This way, the implication of Av-IGFBP-rP1 in the AGH pathway was also confirmed in Isopoda with RNAi experiments. Indeed, both Av-AGH and Av-IGFBP-rP1 knock-down could induce the hypertrophy of the androgenic glands, which is also observed in naturally Wolbachia-induced intersexes.

However, only the silencing of the Av-AGH induced the appearance of female genital apertures like in intersexes, leading to a more feminized phenotype. Moreover, the silencing of the Av-AGH did not affect the transcriptional level of Av-IGFBP-rP1, whereas the silencing of the latter induced the overexpression of the Av-AGH gene. Interestingly, the genetic interactions between Av-AGH and Av-IGFBP-rP1 are not consistent with the observations on M. nipponense, in which Mn-IAGBP silencing reduces the transcriptional level of Mn-IAG in the androgenic glands and conversely [START_REF] Li | Molecular characterization of insulin-like androgenic gland hormone-binding protein gene from the oriental river prawn Macrobrachium nipponense and investigation of its transcriptional relationship with the insulin-like androgenic gland hormone gene[END_REF]. To better understand the divergent results, it would be interesting to know whether the authors also observed altered phenotypes of the androgenic glands during the Mn-IAGBP silencing experiment.

The importance of IGFBP-rP1 in isopod biology was finally considered on the evolutionary scale. In the whole Isopoda order, each of the 68 studied species, both aquatic and terrestrial, displayed a single sequence of IGFBP-rP1. All of them featured the three same conserved domains already described in decapods [START_REF] Rosen | A Crayfish Insulin-likebinding Protein: another piece in the androgenic gland insulin-like hormone puzzle is revealed[END_REF][START_REF] Chandler | Discovery of a novel insulin-like peptide and insulin binding proteins in the Eastern rock lobster Sagmariasus verreauxi[END_REF][START_REF] Huang | Insights into insulin-like peptide system in invertebrates from studies on IGF binding domain-containing proteins in the female mud crab, Scylla paramamosain[END_REF][START_REF] Huang | Does a blue crab putative insulin-like peptide binding protein (ILPBP) play a role in a virus infection?[END_REF][START_REF] Li | Molecular characterization of insulin-like androgenic gland hormone-binding protein gene from the oriental river prawn Macrobrachium nipponense and investigation of its transcriptional relationship with the insulin-like androgenic gland hormone gene[END_REF][START_REF] Song | Insulin-like androgenic gland hormone gene in the freshwater chinese mitten crab Eriocheir sinensis: cDNA cloning, expression pattern, and interaction with EsIGFBP7[END_REF]. This description, associated with the monophyly of this gene in all Metazoa [START_REF] Huang | Insights into insulin-like peptide system in invertebrates from studies on IGF binding domain-containing proteins in the female mud crab, Scylla paramamosain[END_REF], definitely proves that, despite the varying nomenclature, all homologue genes identified in crustaceans derived from only one ancestral sequence. Furthermore, the various sequences quite grouped according to their taxonomic origin in the phylogenies, both on the isopod (present study) or metazoan scales [START_REF] Chandler | Discovery of a novel insulin-like peptide and insulin binding proteins in the Eastern rock lobster Sagmariasus verreauxi[END_REF][START_REF] Huang | Insights into insulin-like peptide system in invertebrates from studies on IGF binding domain-containing proteins in the female mud crab, Scylla paramamosain[END_REF][START_REF] Huang | Does a blue crab putative insulin-like peptide binding protein (ILPBP) play a role in a virus infection?[END_REF]. This is inconsistent with the observation of [START_REF] Li | Molecular characterization of insulin-like androgenic gland hormone-binding protein gene from the oriental river prawn Macrobrachium nipponense and investigation of its transcriptional relationship with the insulin-like androgenic gland hormone gene[END_REF] regarding a clade gathering vertebrate and arachnid sequences, which can be explained by the absence of tree root in their analysis. Our study, in addition with the purifying selection brought out by the dN/dS analyses, strongly suggests that the function of the IGFBP-rP1 has been well conserved during malacostracan evolution. This is also supported by the conservation in the isopod sequences of most of the negatively charged amino acids in the N-terminal region of IGFBP-rP1 identified in Decapoda to be involved in electrostatic interaction with the ligands [START_REF] Chandler | Understanding insulin endocrinology in decapod Crustacea: molecular modelling characterization of an insulin-binding protein and insulin-like peptides in the eastern spiny lobster, Sagmariasus verreauxi[END_REF].

Studies on the evolution of IGFBP1-6 in vertebrates already revealed a complicated scenario although they are all composed of the same two conserved domains and form a clear monophyletic group [START_REF] Daza | Evolution of the insulin-like growth factor binding protein (IGFBP) family[END_REF]. By contrast, the evolutionary story of the IGFBP superfamily in invertebrates appears even trickier to decipher, all the more so because various proteins sharing partial similarity have now been identified in this superfamily. In Hexapoda for instance, the gene called IGFBP7 but also known as ImpL2 (Ecdysone-inducible gene L2) displays only the Ig domain, so that only this part of the sequence is homologue to the other members of the IGFBP-rP1 family [START_REF] Bader | The IGFBP7 homolog Imp-L2 promotes insulin signaling in distinct neurons of the Drosophila brain[END_REF]. This receptor is however able to bind ILPs [START_REF] Bader | The IGFBP7 homolog Imp-L2 promotes insulin signaling in distinct neurons of the Drosophila brain[END_REF][START_REF] Honegger | Imp-L2, a putative homolog of vertebrate IGF-binding protein 7, counteracts insulin signaling in Drosophila and is essential for starvation resistance[END_REF]. The Insulin-related peptide-Binding Proteins (IBPs) display only Ig domains as well, and are also able to bind several ILPs, including vertebrate ones [START_REF] Gao | Novel protein of IBP from silkworm, Bombyx mori, involved in cytoplasmic polyhedrosis virus infection[END_REF][START_REF] Sloth Andersen | A new secreted insect protein belonging to the immunoglobulin superfamily binds insulin and related peptides and inhibits their activities[END_REF]. [START_REF] Rosen | A Crayfish Insulin-likebinding Protein: another piece in the androgenic gland insulin-like hormone puzzle is revealed[END_REF] reported ImpL2 and IBPs to be, together with Cq-IGFBP, three IGFBP homologues but their structural domains clearly differ, explaining why they were excluded in broader analyses such as in [START_REF] Huang | Insights into insulin-like peptide system in invertebrates from studies on IGF binding domain-containing proteins in the female mud crab, Scylla paramamosain[END_REF]. In the same way, Single Insulin Binding Domain proteins (SIBDs), as their name implies, only display the IB domain, like neuroparsins (NP), which are also invertebrate peptides with only the IB domain [START_REF] Badisco | Neuroparsins, a family of conserved arthropod neuropeptides[END_REF]. All these proteins (IGFBP, IGFBP-rP, IBP, ImpL2, NP, SIBD) are visibly related, yet distantly [START_REF] Rodgers | Phylogenetic analysis of the insulin-like growth factor binding protein (IGFBP) and IGFBP-related protein gene families[END_REF].

But considering the divergence between their sequences and given that none of the conserved domain is shared by all of them, establishing global phylogenies of this superfamily by classical means is impracticable, all the more so when exon shuffling events are likely to be involved [START_REF] Rodgers | Phylogenetic analysis of the insulin-like growth factor binding protein (IGFBP) and IGFBP-related protein gene families[END_REF].

The high conservation of the IGFBP-rP1 sequences in malacostracans, as reflected by the same transcript structure and the close homology, suggests that Av-IGFBP-rP1 is a key element of the ISP, which plays the same role previously described with the decapod IGFBP-rP1, that is the binding of the AGH, among other ILPs. However, the different IGFBP-rP1 expression patterns during development and the different consequences of the IGFBP-rP1 silencing experiments also suggest that its exact function is not that well conserved between the decapod and isopod models.

Further studies on the sexual differentiation in Isopoda will likely focus on a more direct evidence of the interaction between the IGFBP-rP1 and the AGH, as well as a more global integration of these two interacting molecules in the ISP. Indeed, several other promising binding or receptor proteins of ILPs have now been characterized in crustaceans. Firstly, SIBDs have been described in several decapod species, in which they were thought to be involved in the immune response [START_REF] Castellanos | Single IB domain (SIBD) protein from Litopenaeus vannamei, a novel member for the IGFBP family[END_REF][START_REF] Gai | A putative endocrine factor SIBD (single insulin binding domain protein) involved in immune response of Chinese mitten crab Eriocheir sinensis[END_REF] but likely ensure several functions [START_REF] Chandler | Discovery of a novel insulin-like peptide and insulin binding proteins in the Eastern rock lobster Sagmariasus verreauxi[END_REF].

Lastly, new transmembrane receptors of the AGH were recently described in three decapod species: the Tyrosine Kinase Insulin-like Receptors (TK-IR), also called IAG receptors (IAGR) [START_REF] Aizen | Production of recombinant insulin-like androgenic gland hormones from three decapod species: In vitro testicular phosphorylation and activation of a newly identified tyrosine kinase receptor from the Eastern spiny lobster, Sagmariasus verreauxi[END_REF][START_REF] Sharabi | Identification and characterization of an insulin-like receptor involved in crustacean reproduction[END_REF][START_REF] Guo | A putative insulin-like androgenic gland hormone receptor gene specifically expressed in male Chinese shrimp[END_REF]. Two of these receptors have just been identified in the isopod species P. beticus and A. vulgare (Herran et al., 2018), whereas the AGH remains the only ILP identified in this order so far.

Fig. 1 .

 1 Fig. 1. Comparison of the conserved domains in the published decapod IGFBP-rP1s and the newly characterized isopod IGFBP-rP1, the sequence of A. vulgare being shown for illustration. The conserved domains were mapped using CDsearch (Marchler-Bauer et al., 2015).

Fig. 2 .

 2 Fig. 2. Tissue expression of the Av-IGFBP-rP1 mRNA by RT-qPCR. The following tissues were dissected from A. vulgare adults of uninfected (A) and Wolbachia-infected lineages (B): HE: hemocytes, LE: legs, MF: muscle and fat, NC: nerve cord, BR: brain, SG: salivary glands, DT: digestive tract, CK: digestive caeca, OV: ovaries, SP: sperm cells, VD: vasa deferens, SV: seminal vesicle, UT: utricles, AG: androgenic glands.

Fig. 3 .

 3 Fig. 3. RT-qPCR expression profiles of the Av-IGFBP-rP1 mRNA during development in uninfected (A) and Wolbachia-infected lineages (B). Larvae were sampled after birth (0w), one (1w), two (2w) and three weeks (3w) after birth. Then, each step represents a developmental stage (4-8), characterized by a new moult, until adulthood (AM for adult males, AF for adult females).External sex characters start to appear at stage 5, allowing gender-specific sampling.

Fig. 4 .

 4 Fig. 4. RT-qPCR expression profiles of the Av-AGH mRNA in the Av-AGH silencing experiment (A), the Av-IGFBP-rP1 mRNA in the Av-IGFBP-rP1 silencing experiment (B), the Av-IGFBP-rP1 mRNA in the Av-AGH silencing experiment (C) and the Av-AGH mRNA in the Av-IGFBP-rP1 silencing experiment (D). Two kinds of controls were included: animals which were not injected

Fig. 5 .

 5 Fig. 5. Phenotypes of the AG in the silencing experiment. Normal AG in the water-injected group (A). Hypertrophied AG in the AGH silencing group at four (B), 12 (C) and 28 (D) weeks postinjection of the dsRNA (wpi). Hypertrophied AG in the IGFBP-rP1 silencing group at four wpi (E), then non-hypertrophied AG at 12 (F) and 28 (G) wpi. Female genital aperture observed during AGH silencing, 12 wpi (H) but absent during IGFBP-rP1 silencing (I). The dashed lines show the plane of bilateral symmetry whereas the dashed circle shows the female genital aperture.

Fig. 6 .

 6 Fig. 6. Analyses of IGFBP-rP1 conservation in isopod crustaceans. An analysis of the amino acid conservation was carried out with Multiple Align Show on the alignment of all the isopod IGFBP-rP1 sequences (A). Fully conserved amino acids are highlighted in black, substitutions leading to a functional conservation of the amino acid are indicated in grey. The three conserved domains are framed in red. Sequence logos of the three conserved domains of the IGFBP-rP1 (IB, Kazal, Ig) were generated by WebLogo (B). Positively charged amino acids (KRH) are depicted in red, negatively charged amino acids (DE) in blue, polar amino acids (STNQ) in green, apolar amino acids (AVLIWFM) in purple and the others (YGPC) in black. The conserved ligand-binding motifs are framed in green (CGCCxxC) and orange ((I/V)xLxxL). The region homologous to the decapod motif is framed in blue.

Fig. 7 .

 7 Fig. 7. Isopod IGFBP-rP1 phylogeny of the whole cDNA. Sequences aligned with Muscle were trimmed with Gblocks (723 nucleic sites left). A maximum likelihood tree was constructed using PhyML, with GTR+I+G model of evolution and 1000 bootstrap repetitions. Branch supports were confirmed with SH-aLRT and bayesian approaches. Family names are indicated in black except for

Acknowledgements

We are thankful to Alexandra Lafitte, who is responsible for the laboratory husbandries and to Daniel Guyonnet, who is in charge of the Sanger sequencing platform. This study was supported by the French National Centre for Scientific Research (CNRS), the University of Poitiers (France), the 2015-2020 State-Region Planning Contract and the European Regional Development Fund. The work of B.H. is funded by a PhD fellowship from the French Research Ministry.

families of aquatic species (in blue) and families in which the IGFBP-rP1 sequences did not cluster together (in red and purple). S1. Species for which an IGFBP-rP1 sequence was obtained using RT-PCR or RNAseq.

GenBank accession numbers are given for the cDNA libraries assembled using publicly available reads and for all IGFBP-rP1 sequences identified in this study.

Table S2. Primers used to amplify the IGFBP-rP1 sequences by RT-PCR, to amplify the Av-IGFBP-rP1 and RbL8 sequences by RT-qPCR and to amplify the Av-AGH and Av-IGFBP-rP1 sequences for gene silencing. The IGFBP-rP1 primers were designed to amplify only the cDNA and do not amplify genomic DNA.


Material selection and prototype design of a double-fluid circulation PCM storage for SHIP applications

SolarPACES 2021

Pierre GARCIA, Grégory LARGILLER CEA Grenoble, France Corresponding author: Pierre. Garcia@cea.fr  Thermal storage need  To ease the integration of SHIP in an existing process  A unique component with the following functions:

 heat storage from solar field & from industrial process  heat release to the industrial process  direct heat transfer between solar and process loops using different HTFs



  et al., 2019, The Journal of Chemical Thermodynamics, 2019 Proportions mixed and heated 4h @ 250°C, then characterized T onset =152°C, H f =70 J/g, H =130 J/g Density Solid @ 22°C: 2 151 kg/m 3 Liquid @ 185°C: 2 074 kg/m 3 Ca(NO 3 ) 2 not available as anhydrous salt Only Ca(NO 3 ) 2 -4H 2 O in standard conditions Ca(NO 3 ) 2 -4H 2 O: -Start dehydration below 40°C -Fully dehydrated above 160°C -Total mass loss: 30%wtEffect of humidity on binary mixture and impact on the storage unit?1°K/min, 66mg• 50g bulk anhydrous binary mixture left 3 weeks at 17°C/47%Hr in an open containerWater molecules modify the binary nitrate and make it unusable for heat storage Is it possible to regenerate it? 25cm Cross section External view Melting/solidification temperature increases as humidity drops binary nitrate « regenerates » PCM storage prototypes for CSP application  Shell and tube design  A unique HTF PCM tubes & inserts Heat transfer enhancement by aluminum inserts around the vertical finned tubes Tubes Aluminum fins PCM storage design: state of the art 8 Source: Garcia et al., 2020 SolarPACES Conference PCM storage test section  Prototype tested on two facilities Low cost and non corrosive PCM selected and characterized  Not a lot of choice in this temperature range! Very few data in literature  Ongoing work on humidity management and filling procedures  A wide range of prototype designs considered  CFD and 1D system model to assess thermal performances  Ongoing engineering study with promising designs for the 3 options  With cost estimates from potential manufacturers  Perspectives  CHS storage adapted to numerous storage applications: district heating, Carnot batteries, waste heat recovery,…  Concentric designs show high heat transfer rates and can be used as heat exchangers 13 CFD calculation Temperature field in an elementary PCM+fins+inserts volume

Eutectic Mixture NaNO 3 -Ca(NO 3 ) 2 -KNO 3 Very

	Eutectic Mixture	Ca(NO 3 ) 2 -KNO 3	70	152 Affordable, highly hygroscopic

 Thermal storage solution: Combined Heat Storage (CHS) PCM review, selection and testing 3 Type Name ΔH sol/liq (J/g) T°f (°C) Comments low 180 Affordable, highly hygroscopic

Sugar and sugar alcohol d-Mannitol 294-341 165-168 High supercooling and stability issues Eutectic mixture LiNO 3 -NaNO 2 233 156 High Cost, highly hygroscopic Eutectic mixture LiNO 3 -KCl 272 160 High Cost, highly hygroscopic, corrosion issues Eutectic mixture HCOONa-HCOOK 175 176 Scarce existing data, corrosion issues Inorganic salt Potassium thiocyanate 112-114 157-177 Low cycling stability, corrosion issues, toxicity Fatty acid Adipic acid 239-252 152 Flammable at temperatures >170°C, corrosion issues

	Amide	Benzanilide	129-139	161	Produce CMR and flammable gas upon decomposition
	Phenolic acid	Salicylic acid	199	157

-159 Low cycling stability, highly flammable Aromatic hydrocarbon Hydroquinone 179-235 160-173 Low cycling stability, highly flammable, CMR? Sugar and sugar alcohol Dulcitol 246-257 167-185 Low cycling stability, supercooling

Thanks for your attention!

This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 884213, project FRIENDSHIP.

FRIENDSHIP WP5 team: Pierre Garcia, Gilles « Walter » Matringe, Grégory Largiller, Célian Jean