Anne Ramat

Martine Simonelig
email: martine.simonelig@igh.cnrs.fr

Functions of PIWI proteins in gene regulation: new arrows added to the piRNA quiver

Keywords: maternal-to-zygotic transition, mRNA decay, mRNA stabilization, piRNAs, PIWI proteins, translation

piRNAs and PIWI proteins play key functions in a wide range of biological and developmental processes through the regulation of cellular mRNAs, in addition to their role in transposable element repression. Evolutionary studies indicate that these PIWI functions in mRNA regulatory programs, occurring in both germ and somatic cells, are ancestral.

Recent advances have widely expanded our understanding of these functions of PIWI proteins, identifying new mechanisms of action and strengthening their importance through their conservation in distant species. In this review, we discuss the latest findings regarding piRNA/PIWI dependent mRNA decay in germ cells and during the maternal-to-zygotic transition in embryos combined with new modes of action of PIWI proteins in mRNA stabilization and translational activation, and piRNA-independent roles of PIWI proteins in cancer.

The piRNA pathway and the range of its functions

Piwi-interacting RNAs (piRNAs; see Glossary) are a class of small non-coding RNAs expressed mainly in animal germlines. They are primarily a defense mechanism for the germline genome against transposable element (TE) mobilization and invasion (reviewed in [START_REF] Czech | piRNA-Guided Genome Defense: From Biogenesis to Silencing[END_REF][START_REF] Huang | piRNA Biogenesis in Drosophila melanogaster[END_REF][START_REF] Ozata | PIWI-interacting RNAs: small RNAs with big functions[END_REF][START_REF] Yamashiro | PIWI-Interacting RNA in Drosophila: Biogenesis, Transposon Regulation, and Beyond[END_REF]). piRNAs are 21 to 32 nucleotides in length and associate with specific ARGONAUTE proteins of the PIWI clade: MIWI, MILI and MIWI2 in the mouse; PRG-1 in Caenorhabditis elegans; Piwi, Argonaute 3 (Ago3) and Aubergine (Aub) in Drosophila. In mice and Drosophila, a large proportion of piRNAs originate from arrays of defective TEs assembled in loci that have originally been called "piRNA clusters". These clusters adapt by insertion of newly invading transposons, producing new piRNAs that repress cognate active TEs [START_REF] Brennecke | An epigenetic role for maternally inherited piRNAs in transposon silencing[END_REF][START_REF] Khurana | Adaptation to P element transposon invasion in Drosophila melanogaster[END_REF].

The biogenesis of piRNAs is tightly intertwined with their role as TE silencer. A unified model of piRNA biogenesis has recently been reported, which explains piRNA production in most species, indicating an evolutionary conservation for about 800 million years [START_REF] Gainetdinov | A Single Mechanism of Biogenesis, Initiated and Directed by PIWI Proteins, Explains piRNA Production in Most Animals[END_REF] (Box 1).

Beside this well documented role in TE repression, functions of PIWI proteins and piRNAs in the regulation of gene expression have also been described early. Cellular mRNA regulation by piRNAs and PIWI proteins contribute to various cellular and developmental processes, such as embryonic patterning in Drosophila and spermatogenesis in the mouse, through the decay of large numbers of mRNAs [START_REF] Barckmann | Aubergine iCLIP Reveals piRNA-Dependent Decay of mRNAs Involved in Germ Cell Development in the Early Embryo[END_REF][START_REF] Gou | Pachytene piRNAs instruct massive mRNA elimination during late spermiogenesis[END_REF][START_REF] Rouget | Maternal mRNA deadenylation and decay by the piRNA pathway in the early Drosophila embryo[END_REF][START_REF] Zhang | MIWI and piRNA-mediated cleavage of messenger RNAs in mouse testes[END_REF]. PIWI proteins were first discovered based on their conserved functions in germline stem cell self-renewal [START_REF] Cox | A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal[END_REF], and these functions were shown more recently to involve cellular mRNA regulation [START_REF] Ma | Aubergine Controls Germline Stem Cell Self-Renewal and Progeny Differentiation via Distinct Mechanisms[END_REF][START_REF] Rojas-Rios | Aubergine and piRNAs promote germline stem cell selfrenewal by repressing the proto-oncogene Cbl[END_REF]. These proteins are found in a variety of stem cells, including somatic stem cells, and required for stem cell stemness in primitive species, such as planaria, which are known to be associated with high regenerative capacity (reviewed in [START_REF] Rojas-Rios | piRNAs and PIWI proteins: regulators of gene expression in development and stem cells[END_REF]). In these species a large proportion of piRNAs are produced from genes (genic piRNAs) suggesting functions of the piRNA pathway in gene regulation. In fact, genic piRNAs are abundant in many species, consistent with conserved functions of PIWI proteins in post-transcriptional regulation of developmental programs, with recent evolutionary studies proposing that these PIWI/piRNA functions could be ancestral [16,[START_REF] Praher | Characterization of the piRNA pathway during development of the sea anemone Nematostella vectensis[END_REF].

In this Review, we focus on recent advances made in uncovering both the biological functions of PIWI proteins, and their mechanisms of action. While PIWI proteins act mostly by mRNA slicing (or cleavage) through their endonuclease activity, upon base-pairing of target mRNA with piRNAs, novel mechanisms of action of PIWI proteins in mRNA stabilization and translational activation have been identified. Recent studies have also revealed the conserved function of the piRNA pathway in maternal mRNA decay during the maternal-to-zygotic transition, and established piRNA-independent roles of PIWI proteins in tumorigenesis and metastasis. These new findings largely expand the breadth of piRNA and PIWI protein functions in healthy development and pathological processes, through a variety of molecular mechanisms.

Degradation of cellular mRNAs by piRNAs and PIWI proteins

Since PIWI proteins cleave TE mRNAs, they can be easily considered as being able to degrade cellular mRNAs. This would require either the insertion of TE-related sequences into mRNA untranslated regions (UTRs), the production of piRNAs from genes with similar sequence (e.g. pseudogenes), or a low complementarity-based targeting of mRNAs with piRNAs produced from TE or repeated sequences. These different modes of cellular mRNA recognition by piRNAs have indeed been described in various contexts and species (reviewed in [START_REF] Rojas-Rios | piRNAs and PIWI proteins: regulators of gene expression in development and stem cells[END_REF]).

One such example has been the cause of experimental troubles for decades with UAS transgenes in the Drosophila germline but has only been identified recently as ensuing from piRNA-mediated regulation (Box 2). Beyond this technical example, piRNA-mediated degradation of cellular mRNAs regulates a whole cohort of genes essential for various developmental programs. Mouse spermatogenesis is one of those. In mouse testes, whereas fetal piRNAs are produced from TE sequences and regulate TEs, pachytene piRNAs that are produced in adults at the pachytene stage of meiosis are unrelated to TEs.

Pachytene piRNAs are mostly loaded into MIWI, a cytoplasmic PIWI protein. CLIP (crosslinking and immunoprecipitation) assays of MIWI revealed its bindings to a large repertoire of cellular mRNAs pointing to a role in post-transcriptional regulation of gene expression [START_REF] Zhang | MIWI and piRNA-mediated cleavage of messenger RNAs in mouse testes[END_REF][START_REF] Vourekas | Mili and Miwi target RNA repertoire reveals piRNA biogenesis and function of Miwi in spermiogenesis[END_REF]. Different modes of MIWI-dependent mRNA decay have been reported. piRNAs produced from ancient TEs target the same TEs inserted into long noncoding RNAs or mRNA 3'UTRs, leading to mRNA cleavage by MIWI slicer activity [START_REF] Watanabe | Retrotransposons and pseudogenes regulate mRNAs and lncRNAs via the piRNA pathway in the germline[END_REF]. piRNAs produced from pseudogenes present in piRNA clusters were also proposed to repress their cognate mRNAs [START_REF] Watanabe | Retrotransposons and pseudogenes regulate mRNAs and lncRNAs via the piRNA pathway in the germline[END_REF]. However, a recent evolutionary study revealed a lack of functional constraint upon pseudogene-derived piRNA targeting, suggesting a minimal contribution of pseudogene piRNAs in mRNA regulation [START_REF] Gebert | Primate piRNA Cluster Evolution Suggests Limited Relevance of Pseudogenes in piRNA-Mediated Gene Regulation[END_REF]. Finally, incomplete base-pairing of mRNAs with pachytene piRNAs was shown to lead to either mRNA slicing by MIWI, generating genic piRNAs by ping-pong, or mRNA decay by deadenylation through the recruitment by MIWI of the CAF1 deadenylase [START_REF] Gou | Pachytene piRNAs instruct massive mRNA elimination during late spermiogenesis[END_REF][START_REF] Zhang | MIWI and piRNA-mediated cleavage of messenger RNAs in mouse testes[END_REF][START_REF] Wu | The evolutionarily conserved piRNA-producing locus pi6 is required for male mouse fertility[END_REF] (Key Figure: Figure 1A). These two outcomes depend on the level of base-paring between piRNAs and their target mRNAs, and on the formation of the MIWI-CAF1 complex taking place specifically in late spermatids. Importantly, both piRNA cluster gain-of-function and lack-of-function approaches showed that defects in mRNA regulation by pachytene piRNAs lead to mouse male sterility, demonstrating the key role of this regulatory mechanism in sperm maturation [START_REF] Wu | The evolutionarily conserved piRNA-producing locus pi6 is required for male mouse fertility[END_REF][START_REF] Goh | piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis[END_REF]. Removal of piRNAs produced from different piRNA clusters does not systematically lead to phenotypic defects, possibly due to redundancy. In addition, a small number of piRNAs within a piRNA cluster are actually responsible for mRNA cleavage and sperm defects, consistent with the high level of base-pairing required for slicing [START_REF] Wu | The evolutionarily conserved piRNA-producing locus pi6 is required for male mouse fertility[END_REF].

A recent study in the planarian Schmidtea mediterranea, revealed that one of the three PIWI proteins also targets a large repertoire of mRNAs leading either to their decay through ping-pong, or to binding without degradation [START_REF] Kim | Planarians recruit piRNAs for mRNA turnover in adult stem cells[END_REF]. Again mRNA slicing by PIWI depends on the level of piRNA complementarity: a strong base-pairing in particular across nucleotides 10-11 leading to mRNA cleavage, whereas uncleaved mRNAs show a lack of base-pairing at nucleotides 10-11 and after nucleotide 16 [START_REF] Kim | Planarians recruit piRNAs for mRNA turnover in adult stem cells[END_REF].

The piRNA pathway is rather different in C. elegans in that mRNA targeting by piRNAs (called 21U-RNAs) induces the production from target mRNAs of secondary antisense small RNAs (called 22G-RNAs), that loaded into other ARGONAUTE proteins (WAGOs or CSR-1) are the actual effectors of mRNA target regulation (reviewed in [START_REF] Ozata | PIWI-interacting RNAs: small RNAs with big functions[END_REF]). In this species, piRNAs target most germline mRNAs through partial complementarity similar to microRNA seed-based interactions [START_REF] Shen | Identification of piRNA Binding Sites Reveals the Argonaute Regulatory Landscape of the C. elegans Germline[END_REF][START_REF] Zhang | The piRNA targeting rules and the resistance to piRNA silencing in endogenous genes[END_REF]. This leads to transcriptome-wide mRNA regulation by piRNAs, whereas their role in regulating TEs remains modest [START_REF] Barucci | Small-RNA-mediated transgenerational silencing of histone genes impairs fertility in piRNA mutants[END_REF][START_REF] Reed | Widespread roles for piRNAs and WAGO-class siRNAs in shaping the germline transcriptome of Caenorhabditis elegans[END_REF].

Strikingly, the silencing of a specific C. elegans mRNA, xol-1 by an X-linked piRNA plays a key role in sex determination and dosage compensation [START_REF] Tang | A Sex Chromosome piRNA Promotes Robust Dosage Compensation and Sex Determination in C. elegans[END_REF]. This piRNA developmental function is remarkable because it appears to be conserved in a distant species of nematode, C. briggsae, and more surprisingly, a similar regulation also takes place in the silk worm Bombyx mori that is separated from nematodes by 700 million years. Indeed in B. mori, a female specific piRNA represses through ping-pong, the Masculinizer mRNA that controls sex determination and dosage compensation in males [START_REF] Kiuchi | A single female-specific piRNA is the primary determiner of sex in the silkworm[END_REF]. This piRNAmediated regulation is essential for female differentiation. Therefore, mRNA degradation by piRNAs has crucial functions in various developmental processes related to sexual reproduction such as germ cell development, sex determination and dosage compensation.

Another important role of piRNA-mediated mRNA decay has been recently reported in reproductive isolation between species. In Drosophila testes, the most abundant piRNAs are unrelated to TEs and originate from repeated sequences: the Suppressor of Stellate (Su(Ste)) and AT-chX loci located on the Y and X chromosomes, respectively [START_REF] Aravin | Dissection of a natural RNA silencing process in the Drosophila melanogaster germ line[END_REF][START_REF] Kotov | piRNA silencing contributes to interspecies hybrid sterility and reproductive isolation in Drosophila melanogaster[END_REF][START_REF] Nagao | Biogenesis pathways of piRNAs loaded onto AGO3 in the Drosophila testis[END_REF][START_REF] Nishida | Gene silencing mechanisms mediated by Aubergine piRNA complexes in Drosophila male gonad[END_REF].

Deletion of the Su(Ste) locus leads to significant increase of Stellate (Ste) mRNA and protein levels, abnormal spermatocyte development and male sterility, thus revealing the biological importance of Ste mRNA repression by Su(Ste) piRNAs [START_REF] Aravin | Dissection of a natural RNA silencing process in the Drosophila melanogaster germ line[END_REF][START_REF] Kotelnikov | Peculiarities of piRNA-mediated post-transcriptional silencing of Stellate repeats in testes of Drosophila melanogaster[END_REF]. However, Ste and Su(Ste) are not conserved in related Drosophila species. On the other hand, AT-chX piRNAs show sequence similarity with vasa mRNA that encodes a key determinant of germ cell specification [START_REF] Nishida | Gene silencing mechanisms mediated by Aubergine piRNA complexes in Drosophila male gonad[END_REF]. vasa is not highly regulated by AT-chX piRNAs though, due to low complementarity. Unexpectedly, AT-chX piRNAs show stronger base-pairing with vasa mRNA from a closely related species, D. mauritiana [START_REF] Kotov | piRNA silencing contributes to interspecies hybrid sterility and reproductive isolation in Drosophila melanogaster[END_REF]. Interspecies crosses between D. melanogaster and D. mauritiana produce males with reduced number of germ cells and meiotic failure, resulting in part from repression of D. mauritiana vasa mRNA by AT-chX piRNAs, and derepression of Ste mRNA due to the lack of Su(Ste) piRNAs [START_REF] Kotov | piRNA silencing contributes to interspecies hybrid sterility and reproductive isolation in Drosophila melanogaster[END_REF]. Therefore, the repression of cellular mRNAs by piRNAs and PIWI proteins is not only involved in germline development and sexual reproduction, but also appear to contribute to reproductive isolation between closely related species. Intriguingly, a recent study revealed the high divergence of pachytene piRNAs between placental mammals and even within the human population, and proposed that piRNA diversity might act in reproductive isolation [START_REF] Ozata | Evolutionarily conserved pachytene piRNA loci are highly divergent among modern humans[END_REF].

A key role of piRNAs and PIWI proteins in the maternal-to-zygotic transition

In most species, the zygotic genome is silent in the early embryo, therefore, maternally deposited mRNAs orchestrate the first steps of development. Maternal mRNAs are then progressively eliminated, and the zygotic genome starts to be expressed taking control of development, in a process called the maternal-to-zygotic transition (MZT). Maternal mRNA decay is accomplished by two pathways that are maternally and zygotically encoded (reviewed in [START_REF] Vastenhouw | The maternal-to-zygotic transition revisited[END_REF]). Recent data have highlighted the conservation of piRNA and PIWI protein function in maternal mRNA decay during the MZT (Figure 2).

The role of piRNAs in maternal mRNA degradation in Drosophila has been the first clear example of a cellular mRNA regulation by piRNAs with a role in development. nanos (nos) mRNA is one of the maternal mRNAs that gets degraded in the early Drosophila embryo. It encodes a key determinant of posterior patterning and germ cell development. nos mRNA regulation involves a complex interplay between degradation, stabilization and translational control allowing the distribution of Nos protein as a gradient emanating from the posterior pole of the embryo. nos mRNA is initially present in the whole embryo, with about 4% being localized and stabilized at the posterior pole, in the so-called germ plasm [START_REF] Bergsten | Role for mRNA localization in translational activation but not spatial restriction of nanos RNA[END_REF][START_REF] Trcek | Drosophila germ granules are structured and contain homotypic mRNA clusters[END_REF].

The rest of nos mRNA in the somatic part of the embryo is degraded by both the maternal and zygotic pathways of mRNA decay [START_REF] Thomsen | Genome-wide analysis of mRNA decay patterns during early Drosophila development[END_REF]. The cytoplasmic PIWI protein Aub loaded with piRNAs is provided maternally to the embryo and contributes to nos mRNA decay; it is therefore a component of the maternal pathway of mRNA decay [START_REF] Rouget | Maternal mRNA deadenylation and decay by the piRNA pathway in the early Drosophila embryo[END_REF] (Figure 2). Aub cooperates with two other components of this pathway, the RNA binding protein Smaug and the CCR4-NOT deadenylation complex. nos mRNA decay by Aub does not involve its endonuclease activity but rather deadenylation by CCR4-NOT. piRNAs produced from two TEs, roo and 412, target nos 3'UTR with partial base-pairing. piRNA target sites in nos sequence, as well as the piRNAs themselves are necessary for nos mRNA decay and embryonic development, revealing an intriguing role of TEs in this developmental process [START_REF] Rouget | Maternal mRNA deadenylation and decay by the piRNA pathway in the early Drosophila embryo[END_REF]. Aub CLIP assays in Drosophila embryos have identified more than 600 mRNAs bound by Aub in a piRNA-dependent manner [START_REF] Barckmann | Aubergine iCLIP Reveals piRNA-Dependent Decay of mRNAs Involved in Germ Cell Development in the Early Embryo[END_REF]. Aub is involved in the decay of germ cell mRNAs that, similarly to nos, are unstable in the soma and accumulate in the germ plasm to participate in germ cell development. Interestingly, some of these mRNAs produce genic piRNAs with ping-pong signatures, indicating cleavage by Aub. Thus, Aub degrades maternal mRNAs by two mechanisms: cleavage and recruitment of the CCR4-NOT complex [START_REF] Barckmann | Aubergine iCLIP Reveals piRNA-Dependent Decay of mRNAs Involved in Germ Cell Development in the Early Embryo[END_REF][START_REF] Rouget | Maternal mRNA deadenylation and decay by the piRNA pathway in the early Drosophila embryo[END_REF].

A recent study revealed a similar piRNA-mediated maternal mRNA decay in the mosquito Aedes Aegypti [START_REF] Halbach | A satellite repeat-derived piRNA controls embryonic development of Aedes[END_REF]. In this system, piRNAs are not produced from TE sequences but from satellite repeats that have been conserved in mosquitos for more than 200 million years. Two piRNAs derived from satellite repeats associate with the PIWI protein Piwi4 and degrade maternal mRNAs in mosquito embryos. Injection of oligonucleotides antisense to piRNAs in early embryos leads to early developmental arrest and increased levels of target mRNAs. These two piRNAs are not maternally deposited but expressed from the zygotic genome. Therefore, in Aedes Aegypti, piRNAs are part of the zygotic pathway of maternal mRNA decay. These piRNAs act similarly to specific microRNAs in Drosophila and zebrafish, which are also expressed zygotically and target a large number of maternal mRNAs to induce their decay during the MZT [START_REF] Bushati | Temporal reciprocity of miRNAs and their targets during the maternal-to-zygotic transition in Drosophila[END_REF][START_REF] Giraldez | Zebrafish MiR-430 Promotes Deadenylation and Clearance of Maternal mRNAs[END_REF]. Analysis of base-pairing requirement of mosquito piRNAs have shown the implication of the seed and additional base-pairing either directly following the seed, or in the 3' part of the piRNA. However, complementarity at the potential cleavage site -positions 10/11 of the piRNA-was dispensable, indicating a cleavage-independent mode of mRNA decay [START_REF] Halbach | A satellite repeat-derived piRNA controls embryonic development of Aedes[END_REF].

Remarkably, in C. elegans a role for 22G-RNAs produced from mRNA targeting by piRNAs has also been reported in maternal mRNA decay during the MZT. 22G-RNAs loaded into the Argonaute protein CSR-1 are provided maternally to the embryo and initiate the degradation of complementary mRNAs through CSR-1 cleavage activity [43].

These studies point to a widespread role amongst animals of the piRNA pathway in the MZT, a key developmental program driving early embryogenesis. How did piRNAs become players of this developmental process? Various mechanisms contribute to mRNA clearance during the MZT [START_REF] Vastenhouw | The maternal-to-zygotic transition revisited[END_REF]. As an efficient system to degrade mRNAs, the piRNA pathway might have been coopted to provide an additional level of mRNA decay during the MZT and increase the robustness of the process.

piRNAs and PIWI proteins in stabilization of cellular mRNAs

Low base-pairing between piRNAs and their target mRNAs does not allow slicing by PIWI proteins, but rather the recruitment of regulators such as the CCR4-NOT complex [START_REF] Gou | Pachytene piRNAs instruct massive mRNA elimination during late spermiogenesis[END_REF][START_REF] Rouget | Maternal mRNA deadenylation and decay by the piRNA pathway in the early Drosophila embryo[END_REF].

Recruitment of different regulators would mediate various outcomes in mRNA regulation, including positive regulation.

In the early Drosophila embryo, Aub and piRNAs are part of the developmental program leading to germ cell mRNA decay in the soma [START_REF] Barckmann | Aubergine iCLIP Reveals piRNA-Dependent Decay of mRNAs Involved in Germ Cell Development in the Early Embryo[END_REF], but Aub is also a core component of germ granules that compose the germ plasm, where these mRNAs are stabilized [START_REF] Trcek | Drosophila germ granules are structured and contain homotypic mRNA clusters[END_REF][START_REF] Little | Independent and coordinate trafficking of single Drosophila germ plasm mRNAs[END_REF]. These conflicting data were analyzed using nos mRNA as a paradigm.

Aub and piRNA target sites in nos 3'UTR were shown to be required for nos mRNA stabilization in the germ plasm [START_REF] Dufourt | piRNAs and Aubergine cooperate with Wispy poly(A) polymerase to stabilize mRNAs in the germ plasm[END_REF]. At the mechanistic level, Aub directly interacts with the germline specific cytoplasmic poly(A) polymerase Wispy that accumulates in the germ plasm, inducing mRNA polyadenylation and stabilization (Key Figure: Figure 1B). In line with this positive role of Aub and piRNAs, Aub was reported to be involved in anchoring germ cell mRNAs to the germ plasm during late oogenesis [START_REF] Vourekas | Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm[END_REF]. The molecular basis of the switch in Aub function between soma and germ plasm remains unknown, although additional factors, in particular specific components of the germ plasm such as Oskar or Tudor, a direct Aub interactor, are likely to be involved.

Germ granules are conserved structures in germ cells of many species. They contain mRNAs required for germline specification and development, as well as PIWI proteins, suggesting that this role of PIWI proteins in mRNA stabilization and storage in germ granules might be conserved. Consistent with this, recent studies in C. elegans have revealed that piRNA/PIWI-mediated mRNA accumulation in germ granules prevent their silencing [START_REF] Dodson | Germ Granules Coordinate RNA-Based Epigenetic Inheritance Pathways[END_REF][START_REF] Ouyang | P Granules Protect RNA Interference Genes from Silencing by piRNAs[END_REF].

Translational activation: a new mode of action of piRNAs and PIWI proteins

Early reports pointed toward a role of PIWI proteins in the regulation of translation. Both MIWI and MILI were found to associate at different steps of mouse spermatogenesis with the mRNA cap-binding complex that is required for translation activation [START_REF] Grivna | MIWI associates with translational machinery and PIWIinteracting RNAs (piRNAs) in regulating spermatogenesis[END_REF][START_REF] Unhavaithaya | MILI, a PIWI-interacting RNA-binding protein, is required for germ line stem cell self-renewal and appears to positively regulate translation[END_REF]. More recently, Aub was shown to regulate protein levels without affecting cognate mRNA poly(A) tail length in germline stem cells of Drosophila ovaries, indicating a potential role in translational regulation [START_REF] Rojas-Rios | Aubergine and piRNAs promote germline stem cell selfrenewal by repressing the proto-oncogene Cbl[END_REF]. A link was also reported between Aub and eIF4 translation initiation factors in Drosophila cultured cells [START_REF] Ma | Aubergine Controls Germline Stem Cell Self-Renewal and Progeny Differentiation via Distinct Mechanisms[END_REF].

Two recent studies have established the role of PIWI proteins in translational activation and revealed the underlying mechanisms, in mouse spermatids and Drosophila embryos [START_REF] Dai | A Translation-Activating Function of MIWI/piRNA during Mouse Spermiogenesis[END_REF][START_REF] Ramat | The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm[END_REF]. While analyzing pachytene piRNA-induced mRNA decay in a spermatocyte-derived cell line, an increase in protein levels without affecting mRNA levels was found instead for specific piRNAs, suggesting a role in translational activation [START_REF] Dai | A Translation-Activating Function of MIWI/piRNA during Mouse Spermiogenesis[END_REF]. This positive effect was shown to require MIWI, and a combination of CLIP assays and ribosome profiling identified a large group of mRNAs potentially regulated by MIWI. AU-rich elements (AREs) in 3'UTRs were found as the common element in these mRNAs, and indeed the insertion of an ARE was sufficient to switch the regulation of an mRNA from decay to translational activation. The HuR protein, known to bind AREs, associated with MIWIactivated mRNAs and contributed to translational activation (Key Figure: Figure 1C, Figure 3). Importantly, many mRNAs subject to MIWI-dependent translational activation are involved in spermatid development, and the regulation of two such mRNAs by MIWI was demonstrated to play a crucial role in acrosome formation [START_REF] Dai | A Translation-Activating Function of MIWI/piRNA during Mouse Spermiogenesis[END_REF].

In the Drosophila embryo, translational regulation of germ cell mRNAs is intimately linked to their stability. These mRNAs are translationally repressed and unstable in the soma, whereas they are stabilized and translated in the germ plasm. Consistent with this, the nos translational repressor complex contains components involved in both translational repression and mRNA decay such as Smaug and the CCR4-NOT deadenylation complex [START_REF] Gotze | Translational repression of the Drosophila nanos mRNA involves the RNA helicase Belle and RNA coating by Me31B and Trailer hitch[END_REF]. Aub being involved in nos mRNA stabilization in the germ plasm [START_REF] Dufourt | piRNAs and Aubergine cooperate with Wispy poly(A) polymerase to stabilize mRNAs in the germ plasm[END_REF], its potential role in translational activation was analyzed. Because Oskar protein drives germ plasm assembly at the posterior pole of embryos, overexpression of Oskar in the whole embryo was used to mimic the germ plasm. In this context, Aub is indispensable for nos mRNA translation [START_REF] Ramat | The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm[END_REF].

piRNAs are also involved in this Aub function as Nos protein levels depends on both piRNA biogenesis and the presence of piRNA target sites in nos 3'UTR. Polysome profiling revealed the role of Aub at the initiation step of translation [START_REF] Ramat | The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm[END_REF].

These two studies identify a new function of PIWI proteins in promoting translation and strongly suggest an evolutionary conservation of this function. Remarkably, several aspects of this PIWI mode of action show striking similarities in mouse and Drosophila. First, both MIWI and Aub activate translation at the level of initiation, by directly recruiting the translation initiation complex eIF3 [START_REF] Dai | A Translation-Activating Function of MIWI/piRNA during Mouse Spermiogenesis[END_REF][START_REF] Ramat | The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm[END_REF] (Figure 3). Recent data have highlighted regulatory functions of eIF3, in addition to its role in basal translation [START_REF] Meyer | 5' UTR m(6)A Promotes Cap-Independent Translation[END_REF][START_REF] Thakor | Cellular mRNA recruits the ribosome via eIF3-PABP bridge to initiate internal translation[END_REF][START_REF] Valasek | Embraced by eIF3: structural and functional insights into the roles of eIF3 across the translation cycle[END_REF]. MIWI and Aub bind eIF3 through the eIF3f and eIF3d subunits, respectively. eIF3d plays an especially important role in the varied modes of eIF3-based regulation, involving both cap-dependent and -independent translation [START_REF] Lee | eIF3 targets cell-proliferation messenger RNAs for translational activation or repression[END_REF][START_REF] Lee | eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation[END_REF][START_REF] Rode | Differential Requirement for Translation Initiation Factor Pathways during Ecdysone-Dependent Neuronal Remodeling in Drosophila[END_REF]. Second, both MIWI and Aub directly associate with the poly(A) binding protein (PABPC1 in the mouse, PABP in Drosophila), another actor in translation initiation [START_REF] Dai | A Translation-Activating Function of MIWI/piRNA during Mouse Spermiogenesis[END_REF][START_REF] Ramat | The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm[END_REF][START_REF] Wu | Domain-functional analyses of PIWIL1 and PABPC1 indicate their synergistic roles in protein translation via 3'-UTRs of meiotic mRNAs[END_REF]. Third, the localization of PIWI protein interaction with eIF3 was also similar in mouse and Drosophila. In both cases, immunostaining revealed eIF3 foci in close proximity to, rather that inside, PIWI-containing germ granules [START_REF] Dai | A Translation-Activating Function of MIWI/piRNA during Mouse Spermiogenesis[END_REF][START_REF] Ramat | The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm[END_REF], suggesting that PIWI-dependent active translation takes place at the edge of germ granules (Figure 3).

These data reveal a high level of conservation of PIWI-dependent translational activation,

adding a new fundamental PIWI mechanism of action in mRNA regulation, which might be conserved in other animal species.

piRNA-independent functions of PIWI proteins

PIWI proteins are mostly specific to the germline, however, PIWI genes are aberrantly expressed in different types of human cancers, and are part of cancer-testis antigens that are testis-restricted genes induced in somatic cancers [START_REF] Litwin | The meaning of PIWI proteins in cancer development[END_REF][START_REF] Muhammad | piRDisease v1.0: a manually curated database for piRNA associated diseases. Database : the journal of biological databases and curation[END_REF]. The mechanisms of action of PIWI proteins in cancers have remained puzzling because their expression is not linked to the presence of large amounts of piRNAs [63]. A recent study identified the function of PIWIL1 (the homologue of mouse MIWI) independent of piRNAs in pancreatic cancer metastasis [START_REF] Li | piRNA-independent function of PIWIL1 as a co-activator for anaphase promoting complex/cyclosome to drive pancreatic cancer metastasis[END_REF]. Unloaded PIWIL1 was shown to interact with the anaphase promoting complex/cyclosome (APC/C) that is involved in protein homeostasis through the ubiquitinproteasome system. This interaction activates APC/C-dependent degradation of the cell adhesion-related protein Pinin, contributing to cancer cell migration and metastasis [START_REF] Li | piRNA-independent function of PIWIL1 as a co-activator for anaphase promoting complex/cyclosome to drive pancreatic cancer metastasis[END_REF] (Key Figure: Figure 1D). A function of MIWI independent of piRNAs in protein regulation has also been described in mouse spermatogenesis. MIWI sequesters in the cytoplasm and inhibits the activity of the E3 ubiquitin ligase RNF8 known to play a role in histone ubiquitylation and degradation. The timely degradation of MIWI in late spermatids leads to RNF8 nuclear translocation and histone degradation and replacement by protamines, a process essential for chromatin compaction and spermatid maturation [START_REF] Gou | Ubiquitination-Deficient Mutations in Human Piwi Cause Male Infertility by Impairing Histone-to-Protamine Exchange during Spermiogenesis[END_REF] (Key Figure: Figure 1D). Although MIWI loading is not required for its interaction with RNF8, MIWI does associate with piRNAs in late spermatids and its loading is necessary for its degradation via APC/C [START_REF] Zhao | piRNA-triggered MIWI ubiquitination and removal by APC/C in late spermatogenesis[END_REF]. This PIWI function is relevant to human male fertility since mutations producing undegradable PIWIL1 were found in patients with azoospermia [START_REF] Gou | Ubiquitination-Deficient Mutations in Human Piwi Cause Male Infertility by Impairing Histone-to-Protamine Exchange during Spermiogenesis[END_REF].

Finally, another piRNA-independent function of PIWIL1 was recently reported in gastric cancer. Although mRNA binding by PIWI proteins without piRNAs has been previously proposed in germ cells [START_REF] Ma | Aubergine Controls Germline Stem Cell Self-Renewal and Progeny Differentiation via Distinct Mechanisms[END_REF][START_REF] Vourekas | Mili and Miwi target RNA repertoire reveals piRNA biogenesis and function of Miwi in spermiogenesis[END_REF], this PIWI protein mode of action has not been formally demonstrated in germ cells and was not confirmed in following studies [START_REF] Gou | Pachytene piRNAs instruct massive mRNA elimination during late spermiogenesis[END_REF][START_REF] Zhang | MIWI and piRNA-mediated cleavage of messenger RNAs in mouse testes[END_REF][START_REF] Rojas-Rios | Aubergine and piRNAs promote germline stem cell selfrenewal by repressing the proto-oncogene Cbl[END_REF]. piRNAs are not present in gastric cancer cells and PIWIL1 was shown to bind mRNAs independently of piRNAs and repress them through the recruitment of nonsense-mediated mRNA decay components, promoting cancer cell proliferation, migration, and metastasis [START_REF] Shi | PIWIL1 Promotes Gastric Cancer via a piRNA-Independent Mechanism[END_REF]. How PIWIL1 directly binds mRNA without piRNAs remains to be analyzed. These studies reveal emerging roles of PIWI proteins independent of piRNAs. More examples of such PIWI mechanisms of action remain to be discovered in the future in particular within pathological contexts, where PIWI proteins are expressed independently of the whole cohort of genes required for piRNA biogenesis.

Concluding remarks

Since their discovery as a germline-specific defense mechanism against TEs, piRNAs and PIWI proteins have been involved in a wide range of regulatory processes through the regulation of non-TE mRNAs. Because they were shaking up a "dogma", these PIWI/piRNA functions unrelated to TEs have been reluctantly accepted. However, it is not surprising that, as an efficient system for mRNA decay, the piRNA pathway has been repurposed to cellular mRNA regulation. It is striking that the same developmental programs are regulated by piRNAs and PIWI proteins in very distant species. Biological functions of piRNA/PIWIdependent mRNA regulation include germline development and specific processes linked to sexual reproduction such as sex determination and dosage compensation [START_REF] Tang | A Sex Chromosome piRNA Promotes Robust Dosage Compensation and Sex Determination in C. elegans[END_REF][START_REF] Kiuchi | A single female-specific piRNA is the primary determiner of sex in the silkworm[END_REF]. Massive maternal mRNA decay during the MZT in early embryos also depends on piRNAs and PIWI proteins in both Drosophila and Aedes whose lineages separated 260 million years ago.

Evolutionary studies are required to understand whether the regulation of these developmental programs by the piRNA pathway is conserved or was repeatedly co-opted for during evolution. Nonetheless, the presence of genic piRNAs across a wide range of species, including primitive bilaterian and non-bilaterian species, such as planarians and cnidarians, indicates that mRNA regulation by piRNAs and PIWI proteins is evolutionary ancient and conserved [START_REF] Barckmann | Aubergine iCLIP Reveals piRNA-Dependent Decay of mRNAs Involved in Germ Cell Development in the Early Embryo[END_REF][START_REF] Zhang | MIWI and piRNA-mediated cleavage of messenger RNAs in mouse testes[END_REF]16,[START_REF] Praher | Characterization of the piRNA pathway during development of the sea anemone Nematostella vectensis[END_REF][START_REF] Gebert | Primate piRNA Cluster Evolution Suggests Limited Relevance of Pseudogenes in piRNA-Mediated Gene Regulation[END_REF][START_REF] Kim | Planarians recruit piRNAs for mRNA turnover in adult stem cells[END_REF][START_REF] Jensen | Conserved Small Nucleotidic Elements at the Origin of Concerted piRNA Biogenesis from Genes and lncRNAs[END_REF]. Many questions remain to be solved and a challenge for future studies is to understand how mRNA regulation by piRNAs and PIWI proteins is conserved, although not at the sequence level (see Outstanding Questions).

Indeed, in contrast to microRNAs whose sequences are conserved through evolution, those of the piRNA are not-even between closely related species. piRNAs are extremely diverse and their pool can evolve rapidly within a species (Box 1). In addition, the low level of basepairing required to target mRNAs indicate that most mRNAs might potentially be regulated by piRNAs. How is selectivity achieved? Cooperation with other regulatory pathways, e.g. RNA binding proteins, mRNA modifications, are likely to contribute strongly. Consistent with this, piRNA-based regulation is often redundant with other regulatory mechanisms and involved in fine-tuning developmental processes [START_REF] Rojas-Rios | piRNAs and PIWI proteins: regulators of gene expression in development and stem cells[END_REF]. Increasing the piRNA repertoire also provides novel opportunities for mRNA regulation that may be fixed (or discarded) by natural selection, leading to new piRNA biological functions. This capacity of piRNAs to evolve rapidly might also participate in speciation through reproductive isolation between related species mediated by both detrimental piRNA-dependent mRNA regulation, and derepression of transposable elements in interspecies crosses.

Positive mechanisms of action of piRNAs and PIWI proteins in mRNA stabilization and translational activation have been discovered. Again, the parallels in the underlying mechanisms seems remarkable with PIWI proteins recruiting the same translation initiation factors across very distant species. In addition, PIWI proteins act independently of piRNAs in cancer progression and metastasis. These studies reveal the high versatility of PIWI proteins in gene regulation and highlight the importance of these PIWI functions in developmental and pathological processes. Future analyses are likely to identify additional biological functions of piRNAs and PIWI proteins, particularly ones linked to germline development and sexual reproduction. They might also reveal new mechanisms of action of PIWI proteins and should allow for a better understanding of their connection with other regulatory mechanisms.

Box 1. piRNA biogenesis and function in transposable element repression

The unified model of piRNA biogenesis proposes that piRNA production involves two interconnected mechanisms: phasing and ping-pong amplification. Interestingly, these two mechanisms are physically separated in the cell, ping-pong taking place in nuage composed of membrane-less ribonucleoprotein granules localized at the periphery of germ cell nuclei, and phasing occurring at the mitochondrial outer membrane [69-73]. First, piRNA cluster genes [START_REF] Rosenkranz | piRNA cluster database: a web resource for piRNA producing loci[END_REF] are transcribed into long noncoding transcripts by RNA polymerase II via specific transcription factors or chromatin regulators [START_REF] Huang | piRNA Biogenesis in Drosophila melanogaster[END_REF][START_REF] Ozata | PIWI-interacting RNAs: small RNAs with big functions[END_REF] (Key Figure: Figure 1). The piRNA precursor transcript is then exported at the periphery of the nucleus in the nuage, with a specific export complex containing the Nuclear export factor 3 (Nxf3) in Drosophila [START_REF] Elmaghraby | A Heterochromatin-Specific RNA Export Pathway Facilitates piRNA Production[END_REF][START_REF] Kneuss | Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export[END_REF].

There, Drosophila Ago3 associated with a maternally-inherited piRNA recognizes and cleaves a complementary piRNA precursor transcript, generating a 5' monophosphorylated RNA that interacts with Aub. New piRNAs can then be produced either by ping-pong or phasing. In the ping-pong cycle, the piRNA precursor is cleaved again further downstream by Ago3 to generate the piRNA 3' end. This new piRNA loaded into Aub targets and drives slicing of complementary TE transcripts, generating piRNAs that are loaded into Ago3. In turn, piRNA-loaded Ago3 binds to and slices piRNA precursor transcripts to generate piRNAs that associate with Aub. This reciprocal cleavage by Aub and Ago3 results in the production of complementary piRNAs with an overlap of ten nucleotides, a signature of the ping-pong amplification cycle (reviewed in [START_REF] Czech | piRNA-Guided Genome Defense: From Biogenesis to Silencing[END_REF]). For piRNA generation by phasing, the cleaved piRNA precursor transcript interacting with Aub travels with the RNA helicase Armitage to the mitochondrial outer membrane [START_REF] Ge | The RNA-Binding ATPase, Armitage, Couples piRNA Amplification in Nuage to Phased piRNA Production on Mitochondria[END_REF]. The endonuclease Zucchini (MitoPLD in the mouse) directed by Aub cleaves the piRNA precursor transcript generating the 3' end of a new piRNA loaded into Aub. The rest of the piRNA precursor transcript is repeatedly bound at its 5' end by Piwi and cleaved by Piwi-guided Zucchini, generating a phased production of Piwi-loaded piRNAs towards the 3' end of the piRNA precursor transcript [START_REF] Han | Noncoding RNA. piRNA-guided transposon cleavage initiates Zucchini-dependent, phased piRNA production[END_REF][START_REF] Homolka | PIWI Slicing and RNA Elements in Precursors Instruct Directional Primary piRNA Biogenesis[END_REF][START_REF] Ishizu | Distinct and Collaborative Functions of Yb and Armitage in Transposon-Targeting piRNA Biogenesis[END_REF][START_REF] Izumi | Zucchini consensus motifs determine the mechanism of pre-piRNA production[END_REF][START_REF] Mohn | Noncoding RNA. piRNA-guided slicing specifies transcripts for Zucchini-dependent, phased piRNA biogenesis[END_REF]. While the phased piRNA production generates de novo piRNAs increasing piRNA diversity, the ping-pong amplification cycle increases the pool of pre-existing piRNAs.

In addition to this post-transcriptional silencing of TEs, piRNAs also repress TEs transcriptionally. piRNAs produced by phasing and loaded into nuclear PIWI proteins (Piwi in Drosophila, MIWI2 in the mouse) travel to the nucleus where they interact with TE nascent transcripts. PIWI proteins then establish repression through a complex network of chromatin remodelers [START_REF] Batki | The nascent RNA binding complex SFiNX licenses piRNA-guided heterochromatin formation[END_REF][START_REF] Fabry | piRNA-guided co-transcriptional silencing coopts nuclear export factors[END_REF][START_REF] Mugat | The Mi-2 nucleosome remodeler and the Rpd3 histone deacetylase are involved in piRNA-guided heterochromatin formation[END_REF][START_REF] Murano | Nuclear RNA export factor variant initiates piRNA-guided cotranscriptional silencing[END_REF][86][START_REF] Zhao | A Pandas complex adapted for piRNA-guided transcriptional silencing and heterochromatin formation[END_REF].

Box 2. Repression of UAS transgenes by Hsp70 piRNAs in Drosophila germ cells

In Drosophila developmental genetics, the UAS/Gal4 inducible expression system set up in the 90's has been unavoidable [START_REF] Brand | Targeted gene expression as a means of altering cell fates and generating dominant phenotypes[END_REF]. This yeast-derived system involves the transcriptional activator Gal4 and its binding sequence UAS upstream of a minimal Hsp70 promoter.

Localized Gal4 expression allows spatial and temporal expression of a cDNA cloned downstream of UAS-Hsp70 promoter. It soon became evident that the system was working in somatic tissues, but not in germ cells [START_REF] Rorth | Gal4 in the Drosophila female germline[END_REF], although the reason was not understood. A recent study investigated this question and showed that the piRNA pathway is responsible for Hsp70-driven repression of UAS transgenes in the germline [START_REF] Deluca | Efficient Expression of Genes in the Drosophila Germline Using a UAS Promoter Free of Interference by Hsp70 piRNAs[END_REF]. Indeed, the Hsp70 gene clusters produce germline specific piRNAs that target the Hsp70 sequence present in UAS expression vectors. Furthermore, the presence in the genome of a Hsp70-containing transgene stimulates cleavage of endogenous Hsp70 transcripts, increasing the amount of Hsp70 piRNAs [START_REF] Olovnikov | De novo piRNA cluster formation in the Drosophila germ line triggered by transgenes containing a transcribed transposon fragment[END_REF]. Reducing Hsp70 regions in new UAS vectors results in high expression in germ cells, clearly demonstrating that piRNA base-pairing with a cellular mRNA can efficiently induce its repression [START_REF] Deluca | Efficient Expression of Genes in the Drosophila Germline Using a UAS Promoter Free of Interference by Hsp70 piRNAs[END_REF]. required for regulation. Additional complementarity in the microRNA 3' region is also generally necessary to achieve regulation.

Slicing or cleavage: ARGONAUTE-mediated cleavage of target mRNAs, occurring between nucleotides 10 and 11 from the 5' end of the complementary piRNA.

Spermiogenesis: complex differentiation program at the end of spermatogenesis where spermatids differentiate and mature into spermatozoa. This program includes chromatin compaction leading to transcription repression, and thus depends, after this step, on regulation of stored mRNAs.

Transposable elements (TEs): DNA sequences that have the ability to move into a genome. They lead to various types of genomic alterations and rearrangements that may be deleterious. However, they also increase genome diversity and thus play a major role in genome dynamics and evolution. polymerase Wispy that directly associates with Aub, might also participate in translational activation through polyadenylation. In both the mouse and Drosophila systems, PIWI interactions with the translation machinery take place at the periphery of germ granules, suggesting actual localized translation at the edge of germ granules.

Figure 1 (

 1 Figure 1 (Key Figure): The different mechanisms of action of PIWI proteins. (Top panel) Transcriptional (left) and post-transcriptional (right) repression of TEs by piRNAs and PIWI proteins. piRNA precursor transcripts are produced by the RNA polymerase II from piRNA cluster genes and exported to the nuage (blue). Here, piRNA-loaded Ago3 slices a complementary piRNA precursor transcript, generating a cleaved RNA with a new 5' end that is bound by Aub. This RNA fragment is cleaved into piRNAs either via phasing, by the Zucchini endonuclease (Zuc) at the mitochondrial outer membrane, or via ping-pong in the nuage. In the second part of the ping-pong cycle, piRNA loaded-Aub targets and cleaves a complementary TE mRNA, generating piRNAs loaded into Ago3. Reciprocal cleavage by Aub/Ago3 contributes to TE post-transcriptional repression and amplification of the piRNA pool. Piwi loaded with piRNAs during phasing translocates to the nucleus, associates with TE nascent transcripts and recruits chromatin remodelers, inducing transcriptional silencing through histone modifications (red dots). (Bottom panel) Molecular mechanisms of PIWI and piRNA functions in the regulation of gene expression. (A) PIWI-dependent cellular mRNA degradation. PIWI proteins promote mRNA decay through either the recruitment of deadenylation factors -the CAF1 deadenylase or the CCR4-NOT complex-or mRNA cleavage, depending on the level of base-pairing between piRNAs and target mRNAs. (B) PIWI-dependent mRNA stabilization. In Drosophila oocytes and early embryos, Aub binds germ cell mRNAs through piRNA base-pairing. Whereas in the soma, Aub binding leads to the decay of these mRNAs (A), in the germ plasm (green) Aub accumulates through direct interaction with Tudor and contributes to localization of the same mRNAs. Aub directly recruits the cytoplasmic poly(A) polymerase Wispy, leading to polyadenylation and stabilization in the germ plasm. Components of germ granules such as Tudor, Vasa and Oskar, are likely to participate in the switch in Aub function from mRNA decay to stabilization between soma and germ plasm. (C) PIWI-dependent translational activation. PIWI proteins directly recruit the eIF3 translation initiation factor and the poly(A) binding protein (PABP), thus contributing to mRNA 5'-3' end interactions and leading to translational activation. (D) piRNA-independent functions of PIWI proteins. PIWI proteins interact with the APC/C complex acting either as an activator (in cancer cells), or a target (in spermatids) of APC/Cmediated protein degradation. (Left panel) In cancer cells, unloaded-PIWI bound to APC/C triggers the ubiquitylation and degradation of the tumor suppressor Pinin, facilitating cancer cell migration and metastasis. (Right panel) In elongated mouse spermatids, timely APC/Cdependent degradation of PIWI allows the release and nuclear translocation of the ubiquitin ligase RNF8, essential for histone exchange with protamines.

Figure 2 :

 2 Figure 2: Function of piRNAs and PIWI proteins during the MZT. (Top panel) Profiles of maternal unstable mRNAs (blue lines) and of zygotic mRNAs (red line) are shown. Both maternal and zygotic factors contribute to maternal mRNA decay leading to a sequential degradation. The dashed line indicates the start of the bulk of zygotic transcription. (Bottom panels) In Drosophila, Aub and TE piRNAs are part of maternal factors required for maternal mRNA decay, together with Smaug and the CCR4-NOT complex. Aub promotes maternal mRNA clearance by deadenylation or cleavage. In Aedes, Piwi4 is provided maternally, whereas satellite repeat piRNAs that target maternal mRNAs are expressed by the zygotic genome. Therefore, Piwi4/piRNAs are part of the zygotic pathway of mRNA decay. Satellite repeat piRNAs target maternal mRNAs with loose base-pairing and contribute to their clearance by an unknown mechanism (indicated by a question mark).

Figure 3 :

 3 Figure 3: Translational activation by piRNAs and PIWI proteins. (Left panel) Temporal mRNA regulation by MIWI/piRNAs during mouse spermiogenesis. MIWI and piRNAs are key factors of mRNA regulation during spermiogenesis. They are involved in mRNA decay by MIWI-dependent cleavage in spermatocytes and round spermatids, and by deadenylation through the recruitment of CAF1 in elongating spermatids. In addition, MIWI and HuR activate translation of ARE-containing mRNAs in round spermatids. MIWI recruits the eIF3 translation initiation factor through direct interaction with eIF3f; it also directly associates with PABPC that coats the mRNA poly(A) tail. On the other hand, HuR bound to the mRNA through ARE motifs recruits eIF4G3 known to bridge the cap-binding protein eIF4E and PABPC, a process facilitating translation initiation. These protein interactions activate capdependent translation. The temporal regulation depends in part on stronger MIWI/eIF3f/HuR interactions in round spermatids, and on stronger association between CAF1 and MIWI in elongating spermatids. (Right panel) Spatial mRNA regulation by Aub/piRNAs in the Drosophila embryo. nos maternal mRNA translation is repressed in the somatic part of the embryo and its targeting by piRNA/Aub contributes to its decay. In contrast, in the germ plasm (green), Aub promotes nos mRNA stabilization and translational activation. Aub recruits PABP by direct interaction, and eIF3 at least via its association with the eIF3d subunit. Aub activates translation initiation, possibly by a cap-independent mechanism, bypassing the repression imposed by the eIF4E-binding protein Cup. The poly(A)

Acknowledgments

Work in the Simonelig lab is supported by UMR9002 CNRS-University of Montpellier, ANR (ANR-17-CE12-0011-01, ANR-19-CE12-0031), FRM, Fondation ARC, the Labex EpiGenMed and AFM Telethon.

Glossary

Deadenylation: the process of poly(A) tail shortening where adenosine nucleosides are removed from the 3' end of mRNAs by a poly(A)-specific 3' exonuclease. The CCR4-NOT complex that is conserved from yeast to human, is the predominant deadenylase. It is composed of eight proteins including two deadenylases CCR4 and CAF1/POP2. Deadenylation triggers full mRNA decay. Seed: the seed sequence has been defined for microRNAs and corresponds to nucleotides 2 to 7 from the microRNA 5' end, where perfect complementarity with the target mRNA is

Conflicts of Interest

The authors declare no conflicts of interests.


David Beaune

Yann Sellier

Stream restorations with meanders increase dragonfly and damselfly diversity and abundance, including an endangered species

Keywords: Dragonflies, Damselflies, Freshwater, Wetland, Insect conservation, meander, 43 Zygoptera, Epiprocta, lotic water, Coenagrion mercuriale 44

Introduction

The Odonata order including damselflies (Zygoptera) and dragonflies (Epiprocta) did not escape the global change and overall biodiversity crises. One tenth of the species are listed as threatened of extinction (assigned as critically endangered, endangered, or vulnerable in the IUCN Red List; [START_REF] Clausnitzer | Odonata enter the biodiversity crisis debate: the first global assessment of an insect group[END_REF]. The group varies from ubiquitous species adapted to degraded water through to pristine water specialists sensitive to ecological change. Most of them are affected by pollution, habitat destruction, and climate change which impact their aquatic ecosystems [START_REF] Clausnitzer | Guardians of the watershed. Global status of dragonflies: critical species, threat and conservation[END_REF]. Hence this group is considered to be a bioindicator for wetlands [START_REF] De Paiva Silva | Adult Odonata abundance and community assemblage measures as indicators of stream ecological integrity: a case study[END_REF][START_REF] Kutcher | Adult Odonata conservatism as an indicator of freshwater wetland condition[END_REF]. Human activities can degrade wetlands and lead to ecosystem simplification and homogenization [START_REF] Lougheed | Wetland degradation leads to homogenization of the biota at local and landscape scales[END_REF] and remaining protected areas are rare and isolated in an ocean of anthropogenic systems [START_REF] Trombulak | Review of Ecological Effects of Roads on Terrestrial and Aquatic Communities[END_REF][START_REF] Cushman | Effects of habitat loss and fragmentation on amphibians: a review and prospectus[END_REF]. That is why wetland conservation involves habitat restoration projects. For Odonata, habitat conservation/restoration projects can be categorized for two types of systems: lotic and lentic.

There are Odonata in lotic (flowing water) and lentic (still water) ecosystems. Examples of a lotic ecosystem include: creeks, streams, runs, rivers, springs, brooks and channels. Ponds, basin marshes, ditches, reservoirs, seeps, lakes, and vernal/ephemeral pools are lentic systems. Globally the lotic species are at greater risk than those in lentic waters [START_REF] Clausnitzer | Odonata enter the biodiversity crisis debate: the first global assessment of an insect group[END_REF] because lotic habitats are less predictable and stable in space and time. Lentic species are more generalized and have a higher dispersal capacity, resulting in larger ranges and wider ecological preferences [START_REF] Korkeamäki | Distribution and habitat specialization of species affect local extinction in dragonfly Odonata populations[END_REF][START_REF] Corbet | Dragonflies: behaviour and ecology of Odonata[END_REF][START_REF] Grewe | Recent range shifts of European dragonflies provide support for an inverse relationship between habitat predictability and dispersal[END_REF].

In protected areas, stakeholders have to conserve habitat diversity in order to keep ecological functioning and biological diversities. This mission implies sometimes, active restoration projects of previous existing habitat such as lotic habitats; artificially degraded, destroyed or plugged. In the West of France, the Pinail National Nature Reserve, is the only National Reserve of the Vienne department. Among the Odonata order (Insecta: Pterygota), 50 from 138 described in Europe were assessed in the reserve (Sellier, unpub. Data).

Restoration projects aiming to recreate running water by meandering were undertaken (during winter of 2010 and 2011) in order to increase ecological niches and thus biodiversity (especially for lotic species). To our knowledge, there is no study to date illustrating the positive ecological impact of stream meandering on Odonata diversity and abundance comparing data before and after restoration. This study aims to illustrate this positive ecological restoration effect.

The study focused on southern damselfly Coenagrion mercuriale (Charpentier, 1840) (Odonata: Zygoptera) return or neocolonization in streams that were blocked and sloweddown by biomass accumulation. C. mercuriale is one of Europe's most threatened damselflies and is listed in the European Habitats directive. C. mercuriale has a south-west European distribution and has declined in countries on the northern and eastern boundaries of its distribution, including Britain. It is highly specialized, being restricted to fragmented biotopes in Europe. These are small lowland heathland streams and ditches on old water meadow ditch systems on chalk streams. Within these biotopes, a requirement for a thermally advantageous microclimate (e.g. use of unshaded, permanently flowing, small watercourses with perennial herbaceous aquatic vegetation) restricts the species further to patches of early successional habitat with lotic water [START_REF] Purse | Dispersal characteristics and management of a rare damselfly[END_REF]. C. mercuriale was occasionally observed within the reserve from 1988 in the Hutte and Rivau streams. However, occurrence decreased over time hence the stream restoration project's primary objective is to restore local populations of C. mercuriale.

The goal of this paper is to assess whether Odonata (dragonflies and damselflies) community abundance and species richness is influenced by restoration of lotic habitats. The study focusses on the endangered species C. mercuriale.

We test the hypothesis that additional species (such as lotic species) would be recruited to the study sites, specifically Coenagrion mercuriale.

Materials and Methods

Study area

The National Nature Reserve of the Pinail (N 46° 42' 2.698"-E 0° 31' 13.378"), (Réserve Naturelle Nationale du Pinail, www.reserve-pinail.org) is a unique concentration of >6000 ponds with 3000 permanent water holes distributed over 142 ha [START_REF] Préau | Amphibian response to the non-native fish, Lepomis gibbosus: the case of the Pinail Nature Reserve, France[END_REF], Figure 1). It is surrounded by 4166 ha of forest mainly classified within Natura 2000 status (and also ZSC: special zone of preservation and ZPS: zone of special protection), ZICO (zone of Europe community interest for birds) (European Nature Protection area network) and ZNIEFF of 1 and 2 types: natural Zone of Ecological Interest Fauna and Flora (French natural inventory of biodiversity) and a RAMSAR site in 2020. The reserve is covered with diversified Erica moors on acid and oligotrophic ground (podzol). This site forms a very rich ecocomplex where 2 500 species (fauna, flora, fungi) are listed [START_REF] Beaune | The use of Chara spp.(Charales: Characeae) as a bioindicator of physico-chemical habitat suitability for an endangered crayfish Austropotamobius pallipes in lentic waters[END_REF][START_REF] Beaune | Freshwater acidification: an example of an endangered crayfish species sensitive to pH[END_REF][START_REF] Beaune | Native top-predator cannot eradicate an invasive fish from small pond ecosystems[END_REF].

Lentic to lotic restoration

Historically, humans have extracted rock from the site for more than 1200 years. This includes the extraction of grindstone (Pierre meulière) resulting in thousands of open ponds.

They also dug channels, probably to transport materials. The channels ran from the Pinail plateau to the lower streams linked to the rivers Vienne and Clain. Human exploitation has stopped since the 20th century and protection of the site officially started in 1980. With phytomass accumulation plus limited water flow, the channels of 'La Hutte', 'Rivau' and 'Reu' were blocked and eventually a succession of long ponds formed. Permanent water flows run underground except during exceptionally dry events. Restoration consisted of the digging of a succession of meanders increasing the length of the stream and the microhabitat heterogeneity, and the reconnection of water bodies to restore water flow and current.

Restoration actions were achieved in the winter of 2011 for 'la Hutte' and 'Rivau', consisting of unblocking channels and recreating meanders (increasing sinuosity) thereby improving instream habitat heterogeneity with some islands and banks of sand or pebbles or rock. See For the Rivau (20 m), the works took place on a single day in October 2010. Work was completed by the Nature Reserve team with a 3.6-ton mini-excavator. In addition to the remodeling of the stream, a pond has been created in parallel with the Rivau.

For the two restorations, no external material (gravel) was added. The sands, gravel and stones came from the site avoiding changes in the water pH and other components.

Research and restoration protocols have been approved by the direction régionale de l'Environnement, de l'Aménagement et du Logement Nouvelle-Aquitaine (DREAL N.A.) and the National Reserve scientific committee.

Odonata inventories

Since 1995, Odonata (diversity and abundance) have been monitored on the site at least one morning in June and July of each year following [START_REF] Langlois | Méthode de suivi des milieux ouverts par les Rhopalocères dans les réserves naturelles de France[END_REF]. The day was selected according to favorable weather conditions: sunny without rain nor wind, which could bias the census. For each transect inventory, one experienced biologist walked the same stream bank at a constant speed of 2 km/h. Each imagines (adults) were recorded in a virtual corridor of 5m wide (5m high from the ground and 2.5m on each side of the biologist) and were exhaustively identified at the species level by direct recognition or by capture with a butterfly net for difficult species to identify (i.e. Lestes spp). The two streams: La Hutte and Rivau were monitored between 10:30-13:00 with a total transect length of 400m and 90m respectively. Censuses are still ongoing and are also performed at other sites of the reserve following the same protocol. The restoration's positive impacts on species richness, species emergence and species abundance were tested for 1/la Hutte stream (over its entire length: 400 m) and 2/ the Rivau stream (area limited to the restored portion: 20 m).

Community analyses

Species richness and abundance (individual number) were calculated for each stream;

Hutte and Rivau with one transect inventory in June and one in July (annual census that failed these conditions were excluded, see Table 3 & 4 for the analyzed years). The actual species richness was not attained with the monitorings. Using R (R Development Core Team, 2020), the extrapolated cumulative diversity richness was estimated by random resampling using several methods: bootstrap, jackknife of degrees 1 and 2, and Chao [START_REF] Palmer | The estimation of species richness by extrapolation[END_REF].

These four estimation methods consider differently the occurrences of rare species and the importance of replication [START_REF] Chao | Estimating the population size for capture-recapture data with unequal catchability[END_REF][START_REF] Colwell | Estimating terrestrial biodiversity through extrapolation[END_REF] and were calculated with the SPECPOOL function of the Vegan-R-package [START_REF] Oksanen | Vegan: community ecology package. R package version 2[END_REF]. Cumulative diversity richness curves provide a good estimate of the representativeness of the sampling effort and were also drawn to estimate the number of species by extrapolation with infinite sampling and compare the species diversity of the streams BEFORE and AFTER the restoration. The first year after restoration, the curves were obtained with the SPECACCUM function Vegan-R package.

Classical species diversity indices were calculated for each available year of census at the two streams: Shannon-Wiener Index (H; H is low under strong dominance of one single species and higher as the number of species increases in an assemblage, especially if they are equally distributed), Simpson's Index (λ ; the index represents the probability that two individuals randomly selected from a sample belong to different species, ranging from 0 to 1) and Pielou evenness (J ; constrained between 0 and 1. The less variation in communities between the species, the higher J is. In other words, an even community is 1).

The compositional dissimilarity among the annual census BEFORE and AFTER restoration was tested using the Bray-Curtis index [START_REF] Bray | An ordination of the upland forest communities of southern Wisconsin[END_REF] and Horn-Morisita overlap index, still using the Vegan-R-package [START_REF] Oksanen | Vegan: community ecology package. R package version 2[END_REF]. The Bray-Curtis dissimilarity is a statistic used to quantify the compositional dissimilarity between different sites (here the same site but BEFORE and AFTER restoration), based on counts at each site. The Horn-Morisita overlap index is less sensitive to hyper-abundant species. Ordinations of the annual census are graphically displayed by non-metric multidimensional scaling (NMDS), in two dimensions; using Bray-Curtis and Horn-Morisita distances separately. NMDS is an indirect gradient analysis approach which produces an ordination based on a distance or dissimilarity matrix. NMDS attempts to represent, as closely as possible, the pairwise dissimilarity between objects (yearly census) in a low-dimensional space (with species as vectors).

Furthermore, the analysis of multivariate homogeneity of group (BEFORE and AFTER restoration) dispersions (variances) is performed using BETADISPERSER function. Non-Euclidean distances between objects and group centroids are handled by reducing the original distances to principal coordinates (PCo); the distances of the group are eventually tested with PERMUTEST for Permutation test of multivariate homogeneity of groups dispersions (variances).

Differences in the species richness, abundance of Odonata, diversity indexes (H, λ, J) BEFORE and AFTER restoration were tested with Wilcoxon rank sum test (non-parametric data) for each stream: Hutte and Rivau. The first year after restoration was considered nonrepresentative as it was highly perturbated and was excluded from analyses (2011 for Rivau and 2012 for Hutte). All statistical tests were considered significant at the 0.05 level.

The southern damselfly

Among species inventoried, the endangered Coenagrion mercuriale (Charpentier, 1840) was a focus for annual abundance. This species was the main target for the restoration projects due to its conservation and patrimonial status. Consequently, the total number of individuals inventoried on June-July annual transects described below were compared BEFORE and AFTER the restoration on both streams.

Results

Odonata inventories

The number of insects (abundance) and species (diversity) significantly increased in both streams after restoration (Table 1, Figure 4,5,6).

Rivau : Before the restoration, an average of 6.8 ±SD 2.6 individual Odonata were counted during the June -July annual census (N= 5 years) with transects, increasing significantly to 52.5 ± 40.0 Odonata after the restoration (N= 8) see Table 1, with W test); from 5.4 ±SD 1.8 species before to a significant increase of 9.9 ± 3.6 species. A total of 13 different species were observed during the annual June-July census (N = 5) before restoration and 25 species after restoration (8). The extrapolated calculated diversities of the Rivau range from 15 to 18 species before restoration and from 29 to 37 species after restoration (Table 2, Figure 4,5).

Hutte : Before the restoration, an average of 64.1 ±SD 21.9 individual Odonata were counted during June and July annual census (N= 10 years), increasing significantly to 303 ± 120 Odonata after the restoration (N= 8; Table 1, with W test) ; from 12.9 ± 3 species before to a significant increase of 15.7 ± 3.4 species. A total of 28 different species were observed during the annual June-July census (N=10) before restoration and 31 species after restoration (7). The extrapolated calculated diversities of the Hutte range from 31 to 38 species before restoration and from 35 to 43 species after restoration (Table 2, Figure 4,6).

Occurrence of some species happened after the restoration. It is not possible to confirm that those species were not present in the streams before the restoration. However ten species were observed in the Hutte stream only after the restoration namely : Calopteryx virgo (Linnaeus, 1758), Chalcolestes viridis (Vander Linden, 1825), Coenagrion scitulum (Rambur, 1842), Crocothemis erythraea (Brullé, 1832), Gomphus graslinii Rambur, 1842, Leucorrhinia pectoralis (Charpentier, 1825), Onychogomphus forcipatus (Linnaeus, 1758), Orthetrum cancellatum (Linnaeus, 1758), Somatochlora metallica (Vander Linden, 1825), Sympetrum meridionale (Selys, 1841). Some of these species inhabit running water systems (lotic; see Table 3). Inversely some species were not observed anymore after 2011 including Aeshna affinis Vander Linden, 1820, Aeshna cyanea (O. F. Müller, 1764), Aeshna isoceles (O. F. Müller, 1767), Aeshna mixta Latreille, 1805, Cordulia aenea (Linnaeus, 1758), Gomphus simillimus Selys, 1840. Some of these species inhabit lentic water systems (see Table 3). All the species absent in the stream after restoration were observed within the reserve ponds.

Similarly on the Rivau : 13 species were observed after restoration, namely : Calopteryx virgo (Linnaeus, 1758), Coenagrion mercuriale (Charpentier, 1840), Coenagrion puella (Linnaeus, 1758), Coenagrion pulchellum (Vander Linden, 1825), Coenagrion tenellum (Villers, 1789), Enallagma cyathigerum (Charpentier, 1840), Ischnura elegans (Vander Linden, 1820), Ischnura pumilio (Charpentier, 1825), Lestes dryas Kirby, 1890, Libellula depressa Linnaeus, 1758, Libellula quadrimaculata Linnaeus, 1758, Platycnemis pennipes (Pallas, 1771), Sympetrum meridionale (Selys, 1841). Some of these species inhabit running water systems (lotic; see Table 4). One lentic water species was not observed after the restoration namely Lestes virens vestalis Rambur, 1842 (see Table 4). All the species absent in the stream after restoration were observed within the reserve ponds. Some species including Ceriagrion tenellum, Orthetrum coerulescens became hyperabundant (Tables 3 &4), affecting the diversity indexes and eveness of the community (see Table 1).

Community analyses

Odonata communities BEFORE and AFTER the restoration seem dissimilar for both streams Hutte and Rivau due to new species observed in both streams (see Table 3 & 4, and below for details about the species, and the yearly transects analyzed). Graphically, the Odonata communities ordinations by non-metric multidimensional scalings (NMDS, in two dimensions display; using Bray-Curtis and Horn-Morisita distances) show no overlap for the small community of the Rivau (20m) but slight overlapping for the larger Hutte 400m's community (Figure 7). For the Rivau: the two centroids or spatial medians drawn draw by 'spider diagram' where each point (annual census) is connected to the group centroid BEFORE and AFTER restoration are distant; accordingly, the ellipsoid hulls, standard error ellipses of both group coordinates are not overlapped (Figure 7a). For the Hutte: a slight overlap is visible (Figure 7b). However, the analyses of multivariate homogeneity of groups BEFORE and AFTER restoration using Permutation tests for homogeneity of multivariate dispersions did not demonstrate significant dissimilarities. Using Bray-Curtis distance: Rivau: F=0.0204 ; pvalue = 0.9 and Hutte : F=2.6992 ; p-value = 0.12 (99 permutations). Using Horn-Morisita distance= Rivau: F=0.9705; p-value = 0.28 and Hutte: F= 2.7004; p-value = 0.12 (99 permutations).

Indeed, several species are still present before and after restoration with 57% common species for Hutte (21/37spp) and 46% for Rivau (12/26 spp). Thus, communities are not significantly distant (i.e. not significantly different) accordingly to this test.

The southern damselfly

The restoration projects targeted the establishment or increase in the Coenagrion mercurial populations. This damselfly was never observed on the Hutte stream and rarely on the Rivau before restoration (see Table 2). Coenagrion mercurial was observed on both Hutte and Rivau the first year after restoration (Figure 8). After the restoration the number of observed insects was significantly higher in the Rivau (W = 39, p-value = 0.0289) and Hutte (W = 71.5, p-value = 0.004501) than before (with great annual variations): the number is from 0 to 4.3 ±SD 4.8 on the Rivau and from 1 ±SD 2.6 to 9.9 ± 7.8 on the Hutte. See (Table 1,2 & Figure 9).

Discussion

Restoration of rectilinear streams aims to broaden the spectrum of relevant habitats to increase stream biodiversity. In particular meanders create micro-current conditions with a variety of current speeds and water acceleration, thus creating various substratum deposits, from sandy banks to gravels. This restoration also alters the riparian flora such as into the the area. Odonata respond to these riparian changes and actively select new habitats [START_REF] Carchini | Species distribution and habitat features in lentic Odonata[END_REF][START_REF] Remsburg | Aquatic and terrestrial drivers of dragonfly (Odonata) assemblages within and among north-temperate lakes[END_REF]. New habitats lead to additional species into the system. Our study demonstrates the rapid increase in both Odonata species diversity and abundance in two restored streams. The number of observed species almost doubled on the Rivau (from 5.4 observed species to 9.9 spp). By extrapolation the total species number on site increased from 15-18 spp to 29-37 spp. The abundance also greatly increased with 770% more individuals on the Rivau. Similarly, on the 400m Hutte stream, the extrapolated diversity increased from 31-38 spp to 35-43 spp; as well as the abundance with 475% more individuals. These restoration projects created new habitats leading to local biodiversity enrichment and conservation success. This was also described with Coenagrion mercurial selecting tussock-forming monocots, Juncus inflexus L., 1753 and Deschampsia cespitosa (L.) P.Beauv., 1812 [START_REF] Rouquette | Roosting site selection in the endangered damselfly, Coenagrion mercuriale, and implications for habitat design[END_REF]. These are used for oviposition, as perching sites and the roots provide the main larval habitat (Rouquette and Thompson 2005). All these microhabitats provide for several distinct and required activities such as foraging, mate-seeking, pairing, oviposition and nocturnal roosting suiting more selective species [START_REF] Corbet | Dragonflies: behaviour and ecology of Odonata[END_REF]. Changes in flora can affect the Odonata communities [START_REF] Rouquette | Roosting site selection in the endangered damselfly, Coenagrion mercuriale, and implications for habitat design[END_REF]. In our study the Odonata quickly responded to new habitat availability and heterogeneity. Species such as Coenagrion mercurial which favor running waters, gained new lotic habitats in both streams after restoration (similarly to Calopteryx virgo, Gomphus graslinii, Onychogomphus forcipatus who arrived after restoration). Additional populations of this endangered species is promising, especially for a rather sedentary species and poor disperser [START_REF] Watts | Molecular and ecological evidence for small-scale isolation by distance in an endangered damselfly, Coenagrion mercuriale[END_REF][START_REF] Purse | Dispersal characteristics and management of a rare damselfly[END_REF]. Movements are more likely within than between patches of suitable habitat over short to medium distances (50-300 m) and the likelihood of C. mercuriale recolonizing sites naturally (with only 1-11% of emigration rate) is within 1-3 km of other populations (particularly within large clusters) [START_REF] Purse | Dispersal characteristics and management of a rare damselfly[END_REF]. For this species it is vital to add or increase existing populations within suitable sites to facilitate stepping-stone dispersal movements.

Another management action to conserve this species within perennial herbaceous aquatic vegetation in small watercourses, is to maintain early successional habitat by rotational cutting or grazing. Long-term regional persistence is likely to depend on metapopulation dynamics and insects occupying a shifting habitat mosaic (Thomas & Hanski 1997). In our study, annual change seems to be present. Is it due to weather factors, drying up of the stream (drought), and possibly the current change in plants? Indeed, severe 2017 and 2019 droughts with negative effects on Odonata abundance are visible in Table 3 and4 andin the C. mercuriale occurrences (Figure 8) and should be investigated. The lowest occurrences post restoration are in 2017 and 2019. Climate change may reduce the water amount input to the reserve in the near future as suggested by recent models [START_REF] Préau | Habitat patches for newts in the face of climate change: local scale assessment combining niche modelling and graph theory[END_REF][START_REF] Préau | Predicting suitable habitats of four range margin amphibians under climate and land-use changes in southwestern France[END_REF] and this may counteract the positive effect of the restoration. Change in species occurrence could be affected by the change in the riparian flora succession and deserve further investigations. Other species may appear and disappear over time. More generally our study demonstrates that meandering stream increases the diversity and abundance of Odonata and is a good restoration strategy for artificially rectilinearized streams. This was demonstrated on two scales: fine scale with 20m streams and longer with 400m. On the finest scale, changes in the community are more detectable with more dissimilar species occupying the modified habitat. This kind of conservation action are beneficial for the biodiversity of the river basin and thus support a good ecological status by improving its biological quality, in agreement with one of the objectives of the EU Water Framework Directive 2000/60/EC (EU Council 2000). Pinail National Nature Reserve, Vienne. France.

Figure

  Figure 2 & 3. Regarding the Hutte stream, work was performed over 9 days and 400 m. Restoration began in September 2011 with vegetation clearing. Work using a 20-ton mechanical excavator began on December 12, 2011 with the unblocking of three landings that blocked the stream flow. This classic public works excavator has proven to be unsuitable for the finer works of meandering. Work therefore resumed from October 1, 2012 with a 6-tonne excavator. A final day of fine work (reconstruction of banks) was done by hand during the winter. The cost of this intervention was € 11,500.

  Rivau and Hutte. Firstly, Characeae appeared (Chara virgata Kütz., 1834, Nitella translucens (Pers.) C.Agardh, 1824, Nitella tenuissima (Desv.) Coss. & Germ.), and various species of Juncus became abundant (Juncus articulatus L., Juncus acutiflorus Ehrh. ex Hoffm., Juncus bulbosus L.). Secondly, Schoenus nigricans L. and Cladium mariscus (L.) Pohl disappeared, and Carex viridula Michx. subsp. oedocarpa (Andersson) B.Schmid, Baldellia ranunculoides (L.) Parl., Carex elata subsp. elata All., 1785 and Eleocharis multicaulis (Sm.) Desv colonized

Figures:Figure 1

 1 Figures:

Figure 2

 2 Figure 2 The Hutte stream (400m) before 12/12/2011 (left) and after 01/01/2012 restoration (meandering). Pinail Nature Reserve, Vienne. France. ©Y. Sellier

Figure 5

 5 Figure 5 Mean species number and abundance of Odonata on the Rivau (20m, June-July), before (5 census) and after (8) restoration of the stream (boxplots displaying median, IC95% =notch, IQR). Pinail National Nature Reserve, Vienne. France.

Figure 6

 6 Figure 6 Mean species number and abundance of Odonata on the Hutte (400m, June-July), before (10 census) and after (7) restoration of the stream (boxplots displaying median, IC95% =notch, IQR). Pinail National Nature Reserve, Vienne. France.

Figure 7a

 7a Figure 7a Odonata community ordinations by non-metric multidimensional scaling (NMDS, in two dimensions display; using Bray-Curtis (left) and Horn-Morisita distances (right), with ellipsoid hull gathering all the group coordinates, 'spider web diagram' connecting all the annual census coordinates to the group centroids with standard error full ellipse displays) of the Rivau 20m stream (no overlap); showing differences in Odonata communities before and after the restoration. (each dot represents an annual census; each red cross represents a factor species). Pinail National Nature Reserve, Vienne. France.

Figure 7b

 7b Figure 7b Odonata community ordinations by non-metric multidimensional scaling (NMDS, in two dimensions display; using Bray-Curtis (left) and Horn-Morisita distances (right), with ellipsoid hull gathering all the group coordinates, 'spider web diagram' connecting all the annual census coordinates to the group centroids with standard error full ellipse displays) of

Figure 8 Figure 9

 89 Figure 8 Southern damselfly Coenagrion mercuriale (Charpentier, 1840) (Odonata: Zygoptera) in 2016 at the Hutte 400m stream, present after restoration. Pinail Nature Reserve, Vienne. France. ©Y. Sellier

Figure 10

 10 Figure 10 The Rivau 20m (left) and Hutte 400m streams after restoration. Pinail Nature Reserve, Vienne. France. ©Y. Sellier

Table list :

 list Table1Mean Odonata species richness, abundance, diversity (H, λ, J indexes) (mean ± SD) in June-July of two streams: Hutte 400m and Rivau 20m after annual census (June-July), before and after restoration. Pinail National Nature Reserve, Vienne. France. Statistical

	significances: *: p-value ≥0.05; **: p-value ≥0.01; ***: p-value ≥0.001.
	Stream		Rivau Rivau Rivau		Hutte Hutte Hutte
	Restoration	Before	After	Wilcoxon	rank	Before	After	Wilcoxon rank sum
			(5)	(8)	sum test		(10)		(7)	test
	Species		5.4 ±	9.9 ±	*		12.9 ±	15.7 ±	*
	diversity		1.8	3.6	W = 34. p-value =	3.0		3.4	W = 55.5, p-value =
					0.02315				0.02365
	Abundance	6.8 ±	52.5	**		64.1 ±	303 ±	***
			2.6	± 40	W = 40. p-value =	21.9		120.5	W = 70, p-value =
					0.002129				0.0003773
	Shannon-		1.59 ±	1.73	NS		2.01 ±	1.77 ±	NS
	Wiener	Index	0.33	±	W = 25. p-value =	0.32		0.21	W = 19, p-value =
	(H)			0.38	0.2618				0.0615
	Simpson's		0.65 ±	0.77	NS		0.80 ±	0.72 ±	*
	Index (λ)		0.32	±	W = 16. p-value =	0.07		0.06	W = 12, p-value =
				0.07	0.7382				0.01250
	Pielou		0.49 ±	0.34	**		0.31±	0.26 ±	***
	evenness (J)	0.07	±	W = 2. p-value =	0.03		0.03	W = 4, p-value =
				0.07	0.003108				0.000617
	N Coenagrion	0 ± 0	3.8 ±	*		1	±	9.9 ±	**
	mercuriale			4.7	W = 39. p-value =	1.5		7.7	W = 71.5, p-value =
					0.0289				0.002250

Table 2

 2 Observed and estimated (4 extrapolation methods: Chao, Jackkniffe degree 1 and 2, Bootstrap) Odonata species richness (mean ± SD) of two streams: Hutte 400m and Rivau 20m after annual census (June-July), before and after restoration. Pinail National Nature Reserve, Vienne. France.

		Species	Chao	Jackknife1 Jackknife2 Bootstrap Nb	of
		observed				annual
						census
	Hutte	28	35.4±7.5	34.3±2.7	37.8	30.9±1.5	10
	(Before)
	Hutte	31	39.8±7.9	39.9±3.9	43.0	34.6±2.1	7
	(After)
	Rivau	13	15.5±3	17±1.8	18.2	15±1.2	5
	(Before)
	Rivau	25	33.9±8	32.9±4.2	37.1	28.6±2.2	8
	(After)

Table 3

 3 Hutte 400m stream monitoring of the Odonata, before and after restoration in winter 2012. Numbers are individual observations per species for one day in June and one in July, with similar weather conditions (without rain and win). Blue bars are relative abundance. Species are sorted in 4 habitat types (following Poitou-Charentes Nature, 2019).

Table 4

 4 Rivau stream 20m monitoring of the Odonata, before and after restoration in winter 2010. Numbers are individual observations per species for one day in June and one in July, with similar weather conditions (without rain and win). Blue bars are relative abundance Species are sorted in 4 habitat types (following Poitou-Charentes Nature, 2019). Pinail National Nature Reserve, Vienne. France.

Acknowledgment

Funding: This work was supported by the direction régionale de l'Environnement, de l'Aménagement et du Logement Nouvelle-Aquitaine (DREAL N.A.), the communauté de

commune de Grand Châtellerault (CAGC), the syndicat mixte Vienne Aval (SMVA), Agence de l'eau Loire-Bretagne. We are grateful to Michel Bramard (OFB) for technical advices and Pascal Dubech, Valentine Dupont for support with field works. Authors have no competing interests to declare.