Gabriel Machado Matos
email: gabrielmatos92@gmail.com

Paulina Schmitt
email: paulina.schmitt@pucv.cl

Cairé Barreto
email: cairebarreto@gmail.com

Natanael Dantas Farias

Guilherme Toledo-Silva
email: guilherme.toledo@ufsc.br

Fanny Guzmán
email: fanny.guzman@pucv.cl

Delphine Destoumieux-Garzón
email: delphine.destoumieux.garzon@ifremer.fr

Luciane Maria Perazzolo
email: l.m.perazzolo@ufsc.br

Rafael Diego Rosa
email: rafael.d.rosa@ufsc.br

marine drugs

Keywords: host defense peptide, antimicrobial peptide, anti-LPS factor, host-microbe relationship, functional diversity, invertebrate immunity, crustacean, antimicrobial activity

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides with a central β-hairpin structure able to bind to microbial components. Mining sequence databases for ALFs allowed us to show the remarkable diversity of ALF sequences in shrimp. We found at least seven members of the ALF family (Groups A to G), including two novel Groups (F and G), all of which are encoded by different loci with conserved gene organization. Phylogenetic analyses revealed that gene expansion and subsequent diversification of the ALF family occurred in crustaceans before shrimp speciation occurred. The transcriptional profile of ALFs was compared in terms of tissue distribution, response to two pathogens and during shrimp development in Litopenaeus vannamei, the most cultivated species. ALFs were found to be constitutively expressed in hemocytes and to respond differently to tissue damage. While synthetic β-hairpins of Groups E and G displayed both antibacterial and antifungal activities, no activity was recorded for Group F β-hairpins. Altogether, our results showed that ALFs form a family of shrimp AMPs that has been the subject of intense diversification. The different genes differ in terms of tissue expression, regulation and function. These data strongly suggest that multiple selection pressures have led to functional diversification of ALFs in shrimp.

Introduction

Anti-lipopolysaccharide factors (ALFs) are multifunctional antimicrobial host defense peptides (AMPs) with the ability to bind to microbial surface molecules. They were initially characterized as potent inhibitors of lipopolysaccharide (LPS)-induced clotting in marine chelicerates, the horseshoe crabs Tachypleus tridentatus and Limulus polyphemus [START_REF] Tanaka | Limulus anti-LPS factor: an anticoagulant which inhibits the endotoxin-mediated activation of Limulus coagulation system[END_REF]. In addition to their LPS-binding properties, they were also shown to be highly active against Gram-negative bacteria [START_REF] Morita | Isolation and biological activities of limulus anticoagulant (anti-LPS factor) which interacts with lipopolysaccharide (LPS)[END_REF]. In the early 2000s, ALF homologues were identified in hemocyte transcriptomes from two penaeid shrimp, Litopenaeus setiferus and Penaeus monodon [START_REF] Gross | Immune gene discovery by expressed sequence tag analysis of hemocytes and hepatopancreas in the Pacific White Shrimp, Litopenaeus vannamei, and the Atlantic White Shrimp, L. setiferus[END_REF][START_REF] Supungul | Identification of immune-related genes in hemocytes of black tiger shrimp (Penaeus monodon)[END_REF]. Although ALF sequences have been extensively identified in many species, these AMPs appear to be exclusive of marine chelicerates and crustaceans.

ALFs are genetically encoded as precursor molecules composed of a leader sequence followed by a mature peptide containing two conserved cysteine residues [START_REF] Tassanakajon | Shrimp humoral responses against pathogens: antimicrobial peptides and melanization[END_REF]. The three-dimensional structure of both horseshoe crab and shrimp ALFs consists of three α-helices packed against a four-stranded β-sheet [START_REF] Yang | NMR structure of rALF-Pm3, an anti-lipopolysaccharide factor from shrimp: Model of the possible lipid A-binding site[END_REF]. In this structure, the two cysteines flank a central β-hairpin of 20 residues stabilized by a single disulfide bond. This central β-hairpin (also known as "LPS-binding domain" or LPS-BD) is the functional domain of ALFs and holds key charged amino acids involved in the recognition and binding of microbial cell wall components, such as LPS from Gram-negative bacteria, lipoteichoic acid from Gram-positive bacteria and β-glucans from fungi [START_REF] Schmitt | An intimate link between antimicrobial peptide sequence diversity and binding to essential components of bacterial membranes[END_REF]. Indeed, the mechanism of action of ALFs is intimately associated with their ability to bind to those microbial moieties. Notably, ALFs are known to be highly active against a broad range of bacteria, fungi and some enveloped viruses [START_REF] Destoumieux-Garzón | Antimicrobial peptides in marine invertebrate health and disease[END_REF].

Different from horseshoe crabs, ALFs form a diverse and multigenic family of AMPs in penaeid shrimp. Shrimp ALFs are composed of five members (Groups A to E), which differ in terms of primary structure and biochemical characteristics [START_REF] Tassanakajon | Shrimp humoral responses against pathogens: antimicrobial peptides and melanization[END_REF]. While ALFs from Groups A and D possess anionic properties, Groups B and C are exclusively composed of cationic peptides. Interestingly, while cationic ALFs exhibit potent antimicrobial activities against a broad range of bacterial and fungal strains [START_REF] Somboonwiwat | Recombinant expression and anti-microbial activity of anti-lipopolysaccharide factor (ALF) from the black tiger shrimp Penaeus monodon[END_REF], anionic ALFs from Group D have impaired antimicrobial properties [START_REF] Rosa | Functional divergence in shrimp anti-lipopolysaccharide factors (ALFs): from recognition of cell wall components to antimicrobial activity[END_REF]. The limited antibacterial activity of Group D ALFs is likely due to the lack of most residues involved in LPS binding of cationic ALFs from Group B [START_REF] Rosa | Functional divergence in shrimp anti-lipopolysaccharide factors (ALFs): from recognition of cell wall components to antimicrobial activity[END_REF]. Group E ALFs were only described in the kuruma prawn Marsupenaeus japonicus as cationic (MjALF-E1) and anionic (MjALF-E2) peptides, with antimicrobial activity restricted to Gram-negative bacteria [START_REF] Jiang | A new group of anti-lipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response[END_REF].

At present, little is known about the evolutionary forces that may have shaped the diversification of ALF sequences in shrimp. To address this question and explore the biological implications of such sequence diversity, we combined a series of molecular, phylogenetic, transcriptional and functional analyses. By using an in silico mapping method, we have identified novel ALF members in different penaeid species. Bayesian phylogenetic reconstructions revealed the existence of seven ALF groups in shrimp: the previously described Groups A to E, and the novel Groups F and G evidenced here. Each ALF group is encoded by a different locus in the shrimp genome. Through a quantitative PCR-based approach, we have assessed the expression of the seven ALF genes in terms of tissue distribution and transcriptional response to two pathogens (Vibrio harveyi and WSSV), but also during different shrimp development stages (from fertilized eggs to larval and post-larval stages) in the shrimp L. vannamei. Finally, we evaluated the antimicrobial activity of synthetic peptides based on the central β-hairpin of the three novel ALFs identified in L. vannamei (Groups E to G) and presented evidence that the sequence diversity of shrimp ALFs can be reflected in their biological properties. Altogether, the tissue distribution, regulation and biological functions of ALF genes reveal that various evolutionary pressures have led to functional diversification of the ALF family in penaeid shrimp.

Results

ALFs from Penaeid Shrimp Comprise a Diverse Family Composed of Seven Members

By using an exhaustive in silico screening approach, we recovered 47 unique ALF sequences (complete CDS) from both publicly available annotated and non-annotated databases for 10 penaeid shrimp species (Decapoda: Penaeidae) (Table S1). With all predicted amino acid sequences in hand, we performed multiple alignments in order to classify the obtained sequences into the five previously described ALF groups (Groups A to E). Surprisingly, from our sequence analysis, shrimp described ALF groups (Groups A to E). Surprisingly, from our sequence analysis, shrimp ALFs clustered into seven distinct groups with specific amino acid sequence signatures (Figure 1A). In addition to already documented ALFs from Groups A to E, we identified here two novel groups that were conveniently named Group F and G (Figure 1A). Across shrimp species, ALFs corresponded to full-length transcripts that encode for precursors composed of a signal peptide (22 to 28 residues), followed by a mature peptide (10.74 to 12.23 kDa) containing two conserved cysteine residues (Figure 1A). Besides their differences in size and molecular weight, shrimp ALFs also displayed contrasting electrostatic characteristics. Notably, while ALFs from Groups B, C and F showed cationic properties, Groups A, D, E and G were composed of anionic ALFs (Figure 1B). However, independently of their differences in primary structure and biochemical properties, the seven ALFs shared a similar three-dimensional architecture: three α-helices packed against a four-stranded β-sheet (Figure 1C). Members of the seven groups were identified in at least four different shrimp species (Farfantepenaeus aztecus, L. vannamei, M. japonicus and P. monodon) (Table S1). Remarkably, Group G ALFs were the only members that were not identified in the genus Fenneropenaeus (F chinensis, F. indicus and F. penicillatus). On the other hand, two different members from Group C were identified in F. chinensis (FcALF2: JX853775 and FcALF3: JX853776), M. japonicus (MjALF-C1: AB210110 and MjALF-C2: KU160498) and P. monodon (ALFPm6: JN562340 and ALFPm7: KX431031).

Our knowledge of ALF intraspecific sequence diversity was also enriched by the discovery of novel sequences in F. aztecus (Groups A to G), F. penicillatus (Groups A to F), L. vannamei (Groups E to G) and P. monodon (Groups D to G). Besides, according to our in silico analyses, some ALFs from M. japonicus were classified in a different Group to that previously categorized by Jiang and colleagues [START_REF] Jiang | A new group of anti-lipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response[END_REF]. For instance, the sequence MjALF-A2 [START_REF] Jiang | A new group of anti-lipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response[END_REF] is actually a member of Group G and not an ALF from Group A, whereas the cationic MjALF-D1 (GenBank: KU160499) belongs to Group F and not to Group D (which gathers anionic sequences only). More surprisingly, the sequence MjALF-E1 (GenBank: KY627760), previously classified as a cationic member of Group E, did not fit in any ALF Group. Indeed, its mature sequence contains an additional cysteine residue (apart of the two cysteines holding the central β-hairpin structure) that is not found in ALFs from either marine chelicerates or crustaceans. Interestingly, coding sequences related to MjALF-E1 were also found in P. monodon and L. vannamei. Unlike the three-cysteine-containing sequences from M. japonicus and P. monodon, the sequences identified in L. vannamei contain four cysteines (Figure S1).

ALF Sequence Diversity Is Gene-Encoded

To gain insights into the origin of the molecular diversity of the shrimp ALF family, we searched for ALF gene sequences in both annotated (GenBank Nucleotide) and non-annotated (Whole-Genome Shotgun Contigs) databases. From our in silico mining analysis, seven unique genomic sequences were identified in P. monodon: ALFPm2 from Group A (GenBank: EF523561), ALFPm3 from Group B (GenBank: EF523562), ALFPm6 from Group C (GenBank: JN562340), ALFPm8 from Group D (GenBank: NIUS010164210), ALFPm9 from Group E (GenBank: NIUS010076396), ALFPm10 from Group F (GenBank: NIUS011801312) and ALFPm11 from Group G (GenBank: NIUS010749450). Each genomic sequence corresponded to a specific ALF member and we found no evidences that ALFs from different Groups could be encoded by a same genomic sequence.

Despite their differences in terms of sequence signatures, all genes shared a similar structural organization: three exons interrupted by two introns (Figure 1D). Every sequence presents a second exon that encodes the four stranded β-sheets, with the two cysteines delimiting the central β-hairpin. This structure holds the seven charged residues involved in LPS binding and is considered as the functional domain of ALFs. As shown in Figure 1D, not all P. monodon ALFs contain those conserved residues found in ALFPm3 from Group B [START_REF] Yang | NMR structure of rALF-Pm3, an anti-lipopolysaccharide factor from shrimp: Model of the possible lipid A-binding site[END_REF]. Regarding the other gene regions, the first exon covers the 5 -untranslated region (UTR), the leader sequence and the hydrophobic N-terminal portion of the mature peptide (the first α-helice), and the third exon encodes the two C-terminal α-helices of the mature peptide and the 3 -UTR.

ALFs Evolved from Gene Duplication Events before Shrimp Speciation

In order to unravel the phylogenetic relationships of the shrimp ALFs, phylogenetic reconstructions were performed with ALF sequences from 38 species of decapod crustaceans (suborders Dendrobranchiata and Pleocyemata) and three species of marine chelicerates (the horseshoe crabs Carcinoscorpius rotundicauda, L. polyphemus and T. tridentatus) (Table S1). Additionally, we analyzed the ALF-related sequences containing three and four cysteine residues and scygonadins (anionic AMPs from crabs that contain two cysteines flanking 17 amino acid residues [START_REF] Rosa | Antimicrobial peptides in crustaceans[END_REF]). Our Bayesian phylogenetic analysis revealed that ALFs comprise a large and diverse gene family in decapod crustaceans. The first striking piece of information is that the three/four-cysteine-containing peptides (including MjALF-E1), as well as scygonadins, are not authentic members of the ALF family since they form a separate and distant clade from all other sequences (Figure 2A). Indeed, the ALF clade gathered sequences from both crustaceans and marine chelicerates. Regarding the crustacean group, ALFs were split into two main clades (Figure 2A). The first clade included ALFs from Group A, while the second clade gathered ALFs from six additional groups (B to G). Interestingly, sequences from non-penaeid species (Pleocyemata) were found in all shrimp ALF groups, but they also formed exclusive groups distinct from those found in penaeids (Figure 2A). Cationic and anionic ALF groups from penaeid shrimp are displayed in blue and red, respectively. Posterior probabilities (Bayesian) and bootstrap values (neighbor-joining) higher than 50% are shown in the nodes. The list of the ALF sequences included in analyses (annotations, sequences and GenBank accession numbers) is provided in Table S1.

Then, an additional phylogenetic tree was constructed to determine the phylogenetic relationships among the seven shrimp ALF groups (A to G) (Figure 2B). In this tree, shrimp ALFs clustered into two main clades: a first clade containing ALFs from Group A and a second clade divided into three branches. Within the second clade, all cationic shrimp ALFs (Groups B, C and F) clustered into one branch, and anionic ALFs from Groups E and G clustered into a second branch (Figure 2B). Group D ALFs clustered in a third branch (Figure 2B). Altogether, our results suggest that the sequence diversity found in the ALF family was likely driven by gene duplication events before the divergence of decapod crustaceans (Dendrobranchiata and Pleocyemata). Notably, the gene expansion and subsequent diversification of the ALF family seems to have occurred in crustaceans and not in marine chelicerates. Actually, in marine chelicerates, only one ALF type was identified (Table S1).

ALFs Are All Expressed in Individual Shrimps and Differentially Modulated in Response to Tissue Damage

We further focused on the transcriptional profiles of shrimp ALFs in terms of tissue distribution. For gene expression analyses, we considered the seven ALFs from the Pacific white shrimp L. vannamei (Litvan ALF-A to -G). First, the gene expression distribution of Litvan ALFs was assessed in eight different tissues of healthy juveniles by semiquantitative RT-PCR analysis. Overall, Litvan ALFs were mainly detected in circulating hemocytes and gills (Figure 3A). Transcripts of Litvan ALF-A and Litvan ALF-B were detected in foregut, midgut, hemocytes, gills and nerve cord, while the expression of Litvan ALF-C was observed in midgut, hemocytes, and gills (Figure 3A). Besides, while the expression of Litvan ALF-E and Litvan ALF-G was exclusively detected in hemocytes and gills, Litvan ALF-F was mainly expressed in the foregut (Figure 3A). Unlike the other ALF groups, the expression of Litvan ALF-D was only detected in hemocytes (Figure 3A). For all genes, no signals were observed in hepatopancreas, hindgut and muscle (Figure 3A).

ALF gene expression was then studied in response to infections and wounding. We first asked whether ALF genes were all transcribed in a single animal or whether their diversity reflected inter-individual sequence variability. Transcripts of the seven ALF genes were detected in the circulating hemocytes of every individual shrimp, as determined by RT-qPCR (Figure 3B). However, important variation was observed in the basal transcription of each gene among individuals (Figure 3B). While the basal gene expression of Litvan ALF-A to Litvan ALF-F varied from 2-to 6-fold among individuals, variations up to 11.3-fold were found for Litvan ALF-G gene expression (Figure 3B).

Next, we analyzed the gene expression profile of Litvan ALFs in response to microbial challenge and injury. Two unrelated shrimp pathogens were chosen: the Gram-negative V. harveyi and the White spot syndrome virus (WSSV). The transcriptional response of Litvan ALFs was quantified by RT-qPCR in shrimp hemocytes 48 h after infections. This time point was chosen on the basis of previous studies from our group [START_REF] Pilotto | Exploring the Impact of the Biofloc Rearing System and an Oral WSSV Challenge on the Intestinal Bacteriome of Litopenaeus vannamei[END_REF][START_REF] Goncalves | Molecular signatures at imminent death: hemocyte gene expression profiling of shrimp succumbing to viral and fungal infections[END_REF][START_REF] Silveira | An immune-related gene expression atlas of the shrimp digestive system in response to two major pathogens brings insights into the involvement of hemocytes in gut immunity[END_REF]. Anionic ALFs from Groups A, D and E did not respond to pathogens nor to injury (Figure 3C). Conversely, cationic ALFs (Groups B, C and F) and the anionic Group G ALF showed significant changes in expression only in response to tissue injury. Indeed, the expression of Litvan ALF-B (2.6-fold), Litvan ALF-C (18.7-fold), Litvan ALF-F (3.6-fold) and Litvan ALF-G (8.3-fold) was significantly induced in circulating hemocytes after the injection of a tissue homogenate prepared from shrimp muscle (Figure 3C). Similarly, the expression of Litvan ALF-B (2.7-fold) and Litvan ALF-F (4.1-fold) also increased after the injection of sterile seawater. The pathogens (V. harveyi and WSSV) did not modulate further ALF expression. Notably, independently of the experimental condition, a high variability in gene expression was observed for all ALFs (Figure 3C).

Some ALF Genes Are Transcribed Early in Shrimp Development, while Others Are Mainly Expressed in Juveniles

Finally, we studied the expression of the three new L. vannamei ALFs (Groups E, F and G) at different stages of shrimp development: fertilized eggs, nauplii, protozoeae, mysis, postlarvae and juveniles. The expression profile of Litvan ALFs from Groups A to D was previously reported [START_REF] Quispe | Transcriptional profiling of immune-related genes in Pacific white shrimp (Litopenaeus vannamei) during ontogenesis[END_REF]. Three distinct patterns of expression were observed for ALF groups E to G over L. vannamei development.

ALFs from Groups E and F were detected at all developmental stages, but Group F expression could only be quantified from nauplius stages (Figure 4). Group E expression did not vary significantly over the entire shrimp development, from larvae to juveniles. In contrast, Group F expression was maximum in protozoea III (ZIII) and then decreased significantly in juveniles (PL17) (Figure 4). Finally, Group G ALF was only expressed from protozoea III (ZIII), and its expression increased significantly up to juvenile stages (PL17) (Figure 4).

Sequence Diversity of Shrimp ALFs Results in Distinct Antimicrobial Properties

The functional domain (central β-hairpin) of the three novel ALF members identified in L. vannamei (Groups E, F and G) was generated by chemical synthesis to evaluate their antimicrobial properties. Indeed, this functional domain is considered a good proxy of the full-length ALF antimicrobial properties [START_REF] Somboonwiwat | Recombinant expression and anti-microbial activity of anti-lipopolysaccharide factor (ALF) from the black tiger shrimp Penaeus monodon[END_REF][START_REF] Rosa | Functional divergence in shrimp anti-lipopolysaccharide factors (ALFs): from recognition of cell wall components to antimicrobial activity[END_REF][START_REF] Li | Functional Diversity of Anti-Lipopolysaccharide Factor Isoforms in Shrimp and Their Characters Related to Antiviral Activity[END_REF]. Minimal inhibitory concentration assays were performed against Gram-positive and Gram-negative bacteria and fungi (yeast and filamentous) (Figure 5). From the three synthetic peptides, Litvan ALF-G 34-55 displayed the broadest range of antimicrobial activity, being effective against all tested Gram-positive bacteria, the Gram-negative V. nigripulchritudo and the filamentous fungus F. oxysporum (Figure 5). This peptide could also affect the growth of the Gram-negative bacteria E. coli and V. harveyi at 40 µM (data not shown), but total inhibition was only observed against V. nigripulchritudo. Additionally, Litvan ALF-G 34-55 exhibited bactericidal activity against the Gram-positive bacteria B. cereus, B. stationis and M. maritypicum. On the other hand, synthetic β-hairpins of Litvan ALF-E 32-53 could inhibit only the growth of marine Gram-positive bacteria (B. stationis and M. maritypicum) and F. oxysporum (Figure 5). Notably, no antimicrobial activity was observed for Litvan ALF-F 30-51 β-hairpin even at 40 µM. None of the synthetic peptides was able to inhibit the growth of the Gram-negative bacteria A. salmonicida, P. aeruginosa, V. alginolyticus and V. anguillarum, and of the yeast C. albicans. Thus, according to their synthetic β-hairpin, Group G and to a lower extent Group E ALFs show a broad spectrum of antimicrobial activities, whereas Group F is devoid of antifungal and antibacterial activity. However, in agreement with a very poor conservation of residues involved in LPS binding (Figure 1D), ALFs from Groups E-G were almost inactive against Gram-negative bacteria (Figure 5C).

Mar. Drugs 2018, 16, x 9 of 17 three synthetic peptides, Litvan ALF-G34-55 displayed the broadest range of antimicrobial activity, being effective against all tested Gram-positive bacteria, the Gram-negative V. nigripulchritudo and the filamentous fungus F. oxysporum (Figure 5). This peptide could also affect the growth of the Gramnegative bacteria E. coli and V. harveyi at 40 µM (data not shown), but total inhibition was only observed against V. nigripulchritudo. Additionally, Litvan ALF-G34-55 exhibited bactericidal activity against the Gram-positive bacteria B. cereus, B. stationis and M. maritypicum. On the other hand, synthetic β-hairpins of Litvan ALF-E32-53 could inhibit only the growth of marine Gram-positive bacteria (B. stationis and M. maritypicum) and F. oxysporum (Figure 5). Notably, no antimicrobial activity was observed for Litvan ALF-F30-51 β-hairpin even at 40 µM. None of the synthetic peptides was able to inhibit the growth of the Gram-negative bacteria A. salmonicida, P. aeruginosa, V. alginolyticus and V. anguillarum, and of the yeast C. albicans. Thus, according to their synthetic βhairpin, Group G and to a lower extent Group E ALFs show a broad spectrum of antimicrobial activities, whereas Group F is devoid of antifungal and antibacterial activity. However, in agreement with a very poor conservation of residues involved in LPS binding (Figure 1D), ALFs from Groups E-G were almost inactive against Gram-negative bacteria (Figure 5C).

Discussion

We showed here that shrimp ALFs are composed of seven distinct members (Groups A to G) with contrasting biochemical properties, activities and expression patterns. Particularly, ALF sequences were found to vary from cationic to anionic with important consequences on their antimicrobial activities. Overall, ALFs comprise the most diverse AMPs found in penaeid shrimp. Indeed, such diversity has not been observed in any other gene-encoded AMP families from shrimp, which are exclusively composed of cationic (penaeidins and crustins) or anionic (stylicins) members [START_REF] Destoumieux-Garzón | Antimicrobial peptides in marine invertebrate health and disease[END_REF][START_REF] Rosa | Antimicrobial peptides in crustaceans[END_REF]. ALF diversity is encoded by at least seven genes that arose from successive duplications and subsequent mutations (nucleotide substitutions and insertion/deletion events) before decapod crustacean speciation occurred. This indicates that strong evolutionary pressures have driven the functional diversification of ALF genes, giving rise to neo-or sub-functionalization and retention in the shrimp genome.

We found that shrimp ALFs are paralogous genes that evolved before the speciation of the suborder Dendrobranchiata (penaeid shrimp). Indeed, ALF diversity, which is the subject of the present study, goes beyond penaeid shrimp and extends to other decapod species from the suborder Pleocyemata (including crayfish, crabs, lobsters, freshwater prawns, etc.). Some ALF members from non-penaeid decapods fall into the seven groups characterized here for penaeid shrimp. However, the ALF diversity found in the suborder Pleocyemata is different from penaeid shrimp (Dendrobranchiata). It is likely that the remarkable gene expansion and diversification of ALF sequences through gene duplication and subsequent mutation have fueled adaptation to different lifestyles and environments (and their associated pathogens) among crustaceans. Here, we have focused our study on shrimp ALFs, as a good sub-representative of ALF diversity. In order to support our hypothesis, we showed that the biological activities and expression patterns of ALF genes have diverged. In particular, we found that some ALFs are antimicrobial, whereas others are not. Some are expressed early during shrimp development, whereas others are expressed in late developmental stages. Finally, ALFs differ in their tissue distribution and responses to tissue damage. However, much more biological data are still needed on the expression and functions of the different ALF members to understand ALF evolution. This ambitious objective will require the development and use of emerging gene-silencing technologies (such as CRISPR-Cas9 and RNA interference) to achieve specific invalidation of closely-related genes in crustaceans and further phenotyping. Similarly, molecular tools such as in situ hybridization could reveal the tissue specificity of ALFs and thus, they would help in uncovering other biological functions. Finally, a classification of all crustacean ALFs (from both decapod and non-decapod species) based on robust phylogenetic reconstructions may avoid misleading classifications and lead to consensus among researchers.

With the identification of two novel ALF groups, we found that ALFs from Groups E and G share a common ancestor gene. Interestingly, Group G is lacking in species of the genus Fenneropenaeus whereas it is found in species of the genera Farfantepenaeus, Litopenaeus, Marsupenaeus and Penaeus. Although it cannot be ruled out that data are missing from publicly accessible databases, the absence of Group G in Fenneropenaeus could result from a gene loss event within this genus. Alternatively, the duplication event that originated these two genes may not have occurred in the genus Fenneropenaeus. Indeed, the evolutionary history of each group traced a particular trajectory in each shrimp species. For instance, while Group C ALFs from F. chinensis (FcALF2 and FcALF3 [START_REF] Li | Functional Diversity of Anti-Lipopolysaccharide Factor Isoforms in Shrimp and Their Characters Related to Antiviral Activity[END_REF]), M. japonicus (MjALF-C1 and MjALF-C2 [START_REF] Jiang | A new group of anti-lipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response[END_REF]) and P. monodon (ALFPm6 and ALFPm7 [START_REF] Ponprateep | Gene silencing reveals a crucial role for anti-lipopolysaccharide factors from Penaeus monodon in the protection against microbial infections[END_REF][START_REF] Soonthornchai | Differentially expressed transcripts in stomach of Penaeus monodon in response to AHPND infection[END_REF]) are composed of two members, in other penaeids this group appears to be composed by a single gene. However, we do not favor this last hypothesis as Group G is found in a diversity of penaeid species. Interestingly, in L. vannamei, Group G ALF was shown here to (i) have broader and more potent antimicrobial activity than Group E ALF, according to their β-hairpin activity, and (ii) to be expressed at late developmental stages whereas Group E expression tends to decrease over ontogenesis. Therefore, it is tempting to speculate that Group E confers antimicrobial protection at larval stages when Group G is still not expressed, whereas Group G provides a selective advantage to the Litopenaeus genus in facing infections at juvenile and adult stages when they are more exposed to different environmental challenges.

We showed that the expression of the seven ALF genes is simultaneous in the circulating hemocytes of a single shrimp. This result is particularly interesting because it suggests that the different ALF members may act synergistically to improve their antimicrobial properties. However, it is still unknown whether they are produced by the same hemocyte populations. Comparatively, the different penaeidin members of L. vannamei (Litvan PEN1/2, -3 and -4) are constitutively expressed by the granular cell populations [START_REF] Destoumieux | antimicrobial peptides with chitin-binding activity, are produced and stored in shrimp granulocytes and released after microbial challenge[END_REF]. Although the expression of ALFs has been detected in hemocytes from juveniles, some members (Groups C, E and F) appeared to be transcribed in larval stages of shrimp development that precede the emergence of these immune cells [START_REF] Muñoz | Expression of penaeidin antimicrobial peptides in early larval stages of the shrimp Penaeus vannamei[END_REF]. Instead, the expression of ALFs from Groups A, B, D and G was quite similar to that observed for other shrimp AMPs that are exclusively produced by hemocytes [START_REF] Quispe | Transcriptional profiling of immune-related genes in Pacific white shrimp (Litopenaeus vannamei) during ontogenesis[END_REF]. On the one hand, the expression of ALFs early in development could be the result of maternal transmission [START_REF] Quispe | Transcriptional profiling of immune-related genes in Pacific white shrimp (Litopenaeus vannamei) during ontogenesis[END_REF][START_REF] Barreto | Specific Molecular Signatures for Type II Crustins in Penaeid Shrimp Uncovered by the Identification of Crustin-Like Antimicrobial Peptides in Litopenaeus vannamei[END_REF] but, on the other hand, those transcripts might originate from other shrimp tissues. Interestingly, in different species, including L. vannamei, the expression of ALFs from Groups C and F was mainly detected in other tissues (digestive system, gills, eyestalk) than in the circulating hemocytes [START_REF] Li | Functional Diversity of Anti-Lipopolysaccharide Factor Isoforms in Shrimp and Their Characters Related to Antiviral Activity[END_REF][START_REF] Ponprateep | Gene silencing reveals a crucial role for anti-lipopolysaccharide factors from Penaeus monodon in the protection against microbial infections[END_REF][START_REF] Soonthornchai | Differentially expressed transcripts in stomach of Penaeus monodon in response to AHPND infection[END_REF]. However, only the expression of ALFs from Group B (ALFPm3 from P. monodon) was studied by immune staining [START_REF] Somboonwiwat | Localization of antilipopolysaccharide factor (ALFPm3) in tissues of the black tiger shrimp, Penaeus monodon, and characterization of its binding properties[END_REF]. More knowledge about the precise sites of ALF production will contribute to understand the involvement of these AMPs in shrimp epithelial defenses, especially those occurring in gills and intestines [START_REF] Silveira | An immune-related gene expression atlas of the shrimp digestive system in response to two major pathogens brings insights into the involvement of hemocytes in gut immunity[END_REF].

One important finding from this study concerns the differential gene expression pattern of shrimp ALFs in response to various challenges. Indeed, the different ALF genes found across penaeids showed to be responsive to various shrimp pathogens, from viruses to bacteria and filamentous fungi [START_REF] Jiang | A new group of anti-lipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response[END_REF][START_REF] Goncalves | Molecular signatures at imminent death: hemocyte gene expression profiling of shrimp succumbing to viral and fungal infections[END_REF][START_REF] Silveira | An immune-related gene expression atlas of the shrimp digestive system in response to two major pathogens brings insights into the involvement of hemocytes in gut immunity[END_REF][START_REF] Ponprateep | Gene silencing reveals a crucial role for anti-lipopolysaccharide factors from Penaeus monodon in the protection against microbial infections[END_REF]. Moreover, RNA interference (RNAi)-mediated gene silencing assays have confirmed that ALFs are directly involved in shrimp survival to infectious diseases [START_REF] Ponprateep | Gene silencing reveals a crucial role for anti-lipopolysaccharide factors from Penaeus monodon in the protection against microbial infections[END_REF][START_REF] De La Vega | Anti-lipopolysaccharide factor in Litopenaeus vannamei (LvALF): A broad spectrum antimicrobial peptide essential for shrimp immunity against bacterial and fungal infection[END_REF][START_REF] Wang | A shrimp C-type lectin inhibits proliferation of the hemolymph microbiota by maintaining the expression of antimicrobial peptides[END_REF]. Additionally, our results provided new evidences for the role of ALFs in other biological processes. Interestingly, while ALFs from Groups A, D and E were not regulated, the expression of the other ALF genes was induced in response to tissue damage. Particularly, ALFs from Groups C and G were shown to be responsive to a tissue homogenate prepared from shrimp muscle (injury control for the WSSV infection), suggesting that they can be modulated by danger/damage-associated molecular patterns (DAMPs). This nonspecific transcriptional response could be associated with additional biological roles involving the promotion of wound healing and the rapid regeneration of tissues [START_REF] Destoumieux | antimicrobial peptides with chitin-binding activity, are produced and stored in shrimp granulocytes and released after microbial challenge[END_REF]. Additionally, we showed that some ALFs are modulated in the shrimp gut in response to infections, suggesting that ALFs can act as a first line of defense in tissues continuously exposed to microbe-rich environments [START_REF] Silveira | An immune-related gene expression atlas of the shrimp digestive system in response to two major pathogens brings insights into the involvement of hemocytes in gut immunity[END_REF][START_REF] Soonthornchai | Differentially expressed transcripts in stomach of Penaeus monodon in response to AHPND infection[END_REF]. Therefore, it is possible that those ALF variants have evolved novel functions associated with the control of the intestinal microbiota. The shrimp intestinal microbiota is a complex and dynamic community that is directly influenced by both biotic and abiotic factors [START_REF] Pilotto | Exploring the Impact of the Biofloc Rearing System and an Oral WSSV Challenge on the Intestinal Bacteriome of Litopenaeus vannamei[END_REF], but probably it is also by the constitutive expression of immune-related genes. In fact, RNAi experiments revealed that ALFs from Groups B [START_REF] Ponprateep | Gene silencing reveals a crucial role for anti-lipopolysaccharide factors from Penaeus monodon in the protection against microbial infections[END_REF] and C [START_REF] Wang | A shrimp C-type lectin inhibits proliferation of the hemolymph microbiota by maintaining the expression of antimicrobial peptides[END_REF] play an essential role in the control of the bacterial communities residing in the hemolymph. However, more functional genomic studies are required to understand the role of ALF in shrimp intestinal defenses.

Another relevant conclusion taken is that the antimicrobial activity of the functional domain of ALFs (central β-hairpin) is associated with its primary sequence rather than to its charge. Despite their differences in primary structure and biochemical features, the seven ALF groups shared a similar tertiary structure. However, the residues involved in LPS binding are not conserved among the seven groups, confirming the neo-functionalization hypothesis proposed by Rosa and colleagues [START_REF] Rosa | Functional divergence in shrimp anti-lipopolysaccharide factors (ALFs): from recognition of cell wall components to antimicrobial activity[END_REF]. Indeed, LPS binding has been demonstrated for the limulus ALF sequence, which shares a common ancestor with all shrimp ALFs. Taking into account previous studies [START_REF] Somboonwiwat | Recombinant expression and anti-microbial activity of anti-lipopolysaccharide factor (ALF) from the black tiger shrimp Penaeus monodon[END_REF][START_REF] Rosa | Functional divergence in shrimp anti-lipopolysaccharide factors (ALFs): from recognition of cell wall components to antimicrobial activity[END_REF][START_REF] Jiang | A new group of anti-lipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response[END_REF][START_REF] Li | Functional Diversity of Anti-Lipopolysaccharide Factor Isoforms in Shrimp and Their Characters Related to Antiviral Activity[END_REF] and the present results, shrimp ALFs have proved to display a diverse spectrum of antimicrobial activity. While some members exhibited a broad range of antimicrobial activity (Groups B and G), some others displayed limited (Groups A, C and E) or very weak action (Groups D and F). One possible explanation is that the effectiveness of the antimicrobial activity of each ALF group is directly proportional to the amount of positively charged amino acids in its central β-hairpin structure [START_REF] Tassanakajon | Sequence diversity and evolution of antimicrobial peptides in invertebrates[END_REF]. However, we showed that the highly cationic central β-hairpin structure of Litvan ALF-F 19-54 (pI = 9.24) was not active against the microorganisms tested in this study. Likewise, synthetic β-hairpins of the FcALF1 (Group F) from F. chinensis was also poorly active against both Gram-positive and Gram-negative bacteria [START_REF] Li | Functional Diversity of Anti-Lipopolysaccharide Factor Isoforms in Shrimp and Their Characters Related to Antiviral Activity[END_REF]. Thus, besides their overall net charge, other features may interfere directly on their biological activities. Given these results, the determination of the amino acid residues involved in the interaction with other microbial surface molecules (peptidoglycan, lipoteichoic acid, β-glucans, etc.) may provide valuable information of the mechanism of action of ALFs against other microorganisms beyond Gram-negative bacteria [START_REF] Schmitt | An intimate link between antimicrobial peptide sequence diversity and binding to essential components of bacterial membranes[END_REF].

Materials and Methods

Database Searches and Phylogenetic Reconstructions

ALF sequences were methodically collected from publicly accessible databases and used for the search of homologous sequences in both annotated and non-annotated databases. Only fulllength coding sequences were considered. Homology searches were performed using tBLASTx at NCBI. Exon-intron boundaries were defined by alignment of the cDNA and genomic sequences. All nucleotide sequences were manually inspected and analyzed using open-access bioinformatics tools. Three-dimensional models for L. vannamei ALFs were built with SWISS-MODEL (https: //swissmodel.expasy.org/) using ALFPm3 NMR resolution (PDB: 2JOB1) as a template. Deduced amino acid sequences were aligned using MAFFT multiple alignment program (https://mafft.cbrc. jp/alignment/server/). Bayesian phylogenetic analysis was conducted in MrBayes 3.1.2 (http: //mrbayes.sourceforge.net/), using WAG + G as substitution model, with two runs of 10 7 generations, sample rate of 1000 and burn-in of 25%. Neighbor-joining analysis was conducted in MEGA X [START_REF] Kumar | Molecular Evolutionary Genetics Analysis across Computing Platforms[END_REF]. Bootstrap sampling was reiterated 1000 times using a 50% bootstrap cutoff. Trees were drawn using FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).

Animals and Tissue Collection

Litopenaeus vannamei juveniles (10 ± 2 g) and at different development stages were obtained from the Laboratory of Marine Shrimp (Federal University of Santa Catarina, Florianópolis, Brazil). Each developmental stage was identified microscopically and collected as previously described [START_REF] Quispe | Transcriptional profiling of immune-related genes in Pacific white shrimp (Litopenaeus vannamei) during ontogenesis[END_REF] while juveniles were acclimated in controlled conditions for at least one week before any experimentation. Hemolymph was collected from the ventral sinus into a precooled modified Alsever solution (27 mM sodium citrate, 336 mM NaCl, 115 mM glucose, 9 mM EDTA, pH 7.0) and hemocytes were isolated by centrifugation. After hemolymph collection, the following tissues were harvested by dissection: foregut, hepatopancreas, midgut, hindgut, muscle, gills and nerve cord. Tissues were rinsed in Tris-saline solution (10 mM Tris, 330 mM NaCl, pH 7.4), homogenized in TRIzol reagent (Thermo Scientific, Asheville, NC, USA) and processed for semiquantitative RT-PCR analysis.

Experimental Infections

Two unrelated shrimp pathogens were chosen for experimental infections, the Gram-negative Vibrio harveyi and the White spot syndrome virus (WSSV). For the bacterial infection, 6 × 10 7 CFU/animal of V. harveyi ATCC 14126 under 100 µL sterile seawater (SSW) or 100 µL SSW (injury control) were injected. For the viral infection, shrimp were injected with 100 µL of a WSSV inoculum containing 3 × 10 2 viral particles. The WSSV inoculum was prepared from muscle tissues of WSSV-infected shrimp as previously described [START_REF] Goncalves | Molecular signatures at imminent death: hemocyte gene expression profiling of shrimp succumbing to viral and fungal infections[END_REF]. Animals injected with 100 µL of a tissue homogenate prepared from WSSV-free shrimp were used as injury control for the viral infection. At 48 h post-infections, hemocytes were collected, pooled (three pools of five animals per condition) and processed for gene expression analysis. Naïve (non-stimulated) animals were used as a control for all experimental conditions.

Semiquantitative RT-PCR Analysis for Tissue Distribution of Gene Expression

Total RNA was extracted using TRIzol reagent (Thermo Scientific, Asheville, NC, USA) according to the manufacturer's protocol. RNA samples were treated with DNase I (Thermo Scientific) at 37 • C for 15 min and precipitated with 0.3 M sodium acetate (pH 5.2) and isopropanol (1:1; v:v). RNA amount and quality were assessed by spectrophotometric analysis and the integrity of total RNA was analyzed by 0.8% agarose gel electrophoresis. First strand cDNA was synthesized from 1 µg of total RNA using the RevertAid Reverse Transcription kit (Thermo Scientific, Asheville, NC, USA) and oligo(dT) 12-18 primers. PCR reactions were carried out in a 15-µL reaction volume containing 1 µL cDNA, 2 mM MgCl 2 , 0.4 mM dNTP Mix, 0.4 µM of each primer (Table 1) and 1 U Taq DNA Polymerase (Sinapse, São Paulo, SP, Brazil). PCR conditions were as follows: 1 cycle of denaturation at 95 • C for 10 min followed by 30-35 cycles of 95 • C for 30 s, 60 • C for 30 s and 72 • C for 30 s. PCR products were analyzed by electrophoresis (1.5% agarose gel) and stained by ethidium bromide. The expression of the LvActin gene was used as endogenous control. RT-qPCR amplifications were performed in a final volume of 15 µL containing 0.3 µM of each primer (Table 1), 7.5 µL of reaction mix (Maxima SYBR Green/ROX qPCR Master Mix 2×; Thermo Scientific, Asheville, NC, USA) and 1 µL of cDNA. The RT-qPCR program was 95 • C for 10 min, followed by 40 cycles of 95 • C for 15 s and 60 • C for 1 min. Melt curve analysis was performed to evaluate primer specificity. The eukaryotic translation elongation factor 1-alpha (LvEF1α) and the ribosomal protein LvL40 were used as reference genes of expression data in hemocytes. Relative transcript levels were determined by the comparative standard curve method using a standard curve derived from 2-fold dilution series of a cDNA pool of all samples. Differences we considered significant at p < 0.05 (one-way ANOVA and Tukey's multiple comparison test). Gene expression of ALFs during shrimp development was assessed in twelve developmental stages as previously described [START_REF] Quispe | Transcriptional profiling of immune-related genes in Pacific white shrimp (Litopenaeus vannamei) during ontogenesis[END_REF].

Peptide Synthesis, Oxidation and Characterization

Synthetic peptides based on the central β-hairpin of ALF-E (Litvan ALF-E 32-53 : GCYVNRSPYLKKFEVHYRADVKCG), ALF-F (Litvan ALF-F 30-51 : GCTYFVTPKVKSFELYFKGRMTCG) and ALF-G (Litvan ALF-G 34-55 : GCSYSTRPYFLRWRLKFKSKVWCG) were obtained in a Liberty Blue automated microwave peptide synthesizer (CEM Corp, Matthews, NC, USA) using Fmoc-protected amino acids (Iris Biotech GmBH (Marktredwitz, Germany) and Rink Amide AM resin (loading: 0.6 meq/g). Fmoc deprotection was carried out with 20% v/v piperidine in DMF, couplings were performed with DIC/OxymaPure activation (1/1 eq) and additional couplings with TBTU/DIEA/OxymaPure activation (1/2/1 eq). Peptides were cleaved with TFA/TIS/DOT/H 2 0 (92.5/2.5/2.5/2.5) [trifluoroacetic acid/triisopropylsilane/2,2-(ethylenedioxy)-diethanethiol/ultrapure water] and purified by RP-HPLC (JASCO Corp., Tokyo, Japan) on a XBridge™ BEH C18 column (100 × 4.6 mm, 3.5 µm) (Waters Corp., Milford, MA, USA) with a 0-70% acetonitrile-water mixture gradient over 30 min at a flow rate of 1 mL/min. Peptides were further lyophilized and analyzed by MALDI-TOF mass spectrometry in a LCMS-2020 ESI-MS (Shimadzu Corp., Kyoto, Japan) to confirm their molecular masses.

Then, peptides were oxidized as previously reported [28]. In brief, 5 mg of the crude peptide were first reduced with 10% β-mercaptoethanol (95 • C for 5 min) then dissolved in 50% (v/v) AcOH/H 2 O and later diluted in 32 mL of oxidation buffer (2 mM guanidinium chloride, 10% isopropyl alcohol and 10% dimethyl sulfoxide). The pH was adjusted to 5.8 with ammonium hydroxide. The peptide solution was subjected to air oxidation at room temperature for 18 h. The peptide solution was then acidified to pH 2.5 and purified using a SPE C18 (Waters Corp., Milford, MA, USA). The peptides were eluted with 5%, 20%, 40%, 60% and 80% acetonitrile in 0.05% TFA ultrapure water at a flow rate of 1 mL/min. The fractions were collected, and the acetonitrile was evaporated on a Savant SPD 1010 SpeedVac Concentrator (Thermo Scientific, Asheville, NC, USA). The fractions were analyzed by MALDI-TOF mass spectrometry.

Antibacterial and Antifungal Assays

The antimicrobial activity of synthetic peptides was assayed against the Gram-positive bacteria Bacillus cereus ATCC 11778, Bacillus subtilis ATCC 6633, Brevibacterium stationis CIP 101282, Microbacterium maritypicum CIP 105733, Micrococcus luteus CIP 5345 and Staphylococcus aureus ATCC 25932, the Gram-negative bacteria Aeromonas salmonicida ATCC 33658, Escherichia coli SBS363, Pseudomonas aeruginosa ATCC 9027, Vibrio alginolyticus ATCC 17749, Vibrio anguillarum ATCC 19264, Vibrio harveyi ATCC 14126 and Vibrio nigripulchritudo CIP103195, the yeast Candida albicans MDM8 and the filamentous fungus Fusarium oxysporum.

Minimum inhibitory concentrations (MICs) were determined in duplicate by the liquid growth inhibition assay, as previously described [29]. MIC values are expressed as the lowest concentration tested that causes 100% growth inhibition. Poor Broth (PB: 1% peptone, 1% NaCl, pH 7.2) was used for standard bacteria, while PB supplemented with 0.5 M NaCl (PB-NaCl) was used as a culture medium for Vibrio strains. For B. stationis and M. maritypicum cultures, PB-NaCl medium was supplemented with 20 mM KCl, 5 mM MgSO 4 and 1.5 mM CaCl 2 . Potato dextrose broth (Kasvi, São José dos Pinhais, PR, Brazil) at half strength was used for cultures of F. oxysporum while Sabouraud medium (1% peptone, 4% glucose, pH 5.6) was used for yeast cultures. The growth of bacteria and yeast was monitored spectrophotometrically (λ = 595 nm), while F. oxysporum hyphae formation was observed in an inverted microscope. After MIC determination, bacterial cultures were plated in nutrient agar for 24-48 h for the determination of the bactericidal activity of the synthetic peptides.

Conclusions

In conclusion, the combination of our molecular, transcriptional and functional data revealed that ALFs comprise the most diverse AMP family found in penaeid shrimp. We showed that they are composed of seven members encoded by different genes that follow a diverse pattern of expression. Our results also strongly suggest that the expansion and diversification of shrimp ALFs have shaped novel functions for this AMP family beyond their primary antibacterial properties. Thus, ALFs represent an attractive model to explore the impacts of the molecular diversity of immune-related genes on host-microbe interactions. Finally, ALFs possess the broadest spectrum of antimicrobial activity when compared to other shrimp AMPs. These bioactive peptides undoubtedly show biotechnological potential for the development of novel antibiotics derived from AMPs, as well as for the development of selective breeding programs.

Figure 1 .

 1 Figure 1. The seven members of the shrimp ALF family. (A) Multiple alignments of the consensus amino acid signature of each ALF member (Groups A to G) found in penaeid shrimp. Identical residues are highlighted in black while "X" indicates any amino acid. Positively and negatively charged residues are displayed in blue and red, respectively. The conserved cysteine bond is indicated by the arrows. The position of α-helices (red helices) and β-sheets (yellow arrows) is based on the three-dimensional (3D) structure of ALFPm3 (PDB: 2JOB). Intragroup amino acid identity values are indicated on the right. (B) Biochemical properties of shrimp ALFs. MW: molecular weight; pI: theoretical isoelectric point; aa: amino acid residues. (C) Predicted 3D structure of L. vannamei ALFs. The structural models were built based on the NMR (nuclear magnetic resonance) structure of ALFPm3. (D) Not-to-scale schematic representation of ALF genes from P. monodon: Group A (ALFPm2: EF523561), Group B (ALFPm3: EF523562), Group C (ALFPm6: JN562340), Group D (ALFPm8: NIUS010164210), Group E (ALFPm9: NIUS010076396), Group F (ALFPm10: NIUS011801312) and Group G (ALFPm11: NIUS010749450). Boxes represent the exons, and lines represent the introns. Multiple alignments of the amino acid sequences encoded by the exon 2 (white box). Triangles (▼) indicate the two conserved cysteines. Residues involved in LPS binding of ALFPm3 are highlighted in black.

Figure 1 .

 1 Figure 1. The seven members of the shrimp ALF family. (A) Multiple alignments of the consensus amino acid signature of each ALF member (Groups A to G) found in penaeid shrimp. Identical residues are highlighted in black while "X" indicates any amino acid. Positively and negatively charged residues are displayed in blue and red, respectively. The conserved cysteine bond is indicated by the arrows. The position of α-helices (red helices) and β-sheets (yellow arrows) is based on the three-dimensional (3D) structure of ALFPm3 (PDB: 2JOB). Intragroup amino acid identity values are indicated on the right. (B) Biochemical properties of shrimp ALFs. MW: molecular weight; pI: theoretical isoelectric point; aa: amino acid residues. (C) Predicted 3D structure of L. vannamei ALFs. The structural models were built based on the NMR (nuclear magnetic resonance) structure of ALFPm3. (D) Not-to-scale schematic representation of ALF genes from P. monodon: Group A (ALFPm2: EF523561), Group B (ALFPm3: EF523562), Group C (ALFPm6: JN562340), Group D (ALFPm8: NIUS010164210), Group E (ALFPm9: NIUS010076396), Group F (ALFPm10: NIUS011801312) and Group G (ALFPm11: NIUS010749450). Boxes represent the exons, and lines represent the introns. Multiple alignments of the amino acid sequences encoded by the exon 2 (white box). Triangles ( ) indicate the two conserved cysteines. Residues involved in LPS binding of ALFPm3 are highlighted in black.

Figure 2 .

 2 Figure 2. ALFs form a diverse antimicrobial peptide family in decapod crustaceans. (A) Bayesian and (B) neighbor-joining trees of ALFs from decapod crustaceans and marine chelicerates (horseshoe crabs).Cationic and anionic ALF groups from penaeid shrimp are displayed in blue and red, respectively. Posterior probabilities (Bayesian) and bootstrap values (neighbor-joining) higher than 50% are shown in the nodes. The list of the ALF sequences included in analyses (annotations, sequences and GenBank accession numbers) is provided in TableS1.

Figure 3 .

 3 Figure 3. Tissue expression distribution of shrimp ALFS and gene modulation in hemocytes in response to pathogen challenge and tissue damage. (A) Semiquantitative RT-PCR analysis of L. vannamei ALFs in shrimp tissues: foregut (FG), hepatopancreas (HP), midgut (MG), hindgut (HG), hemocytes (HE), muscle (ML), gills (GL) and nerve cord (NC). The expression of the LvActin gene was used as endogenous control. The anatomic location of the tissues is indicated in the shrimp image. The Venn diagram summarizes the main sites of expression of L. vannamei ALFs. (B) mRNA basal levels of L. vannamei ALFs in the circulating hemocytes from five individual shrimp. (C) Gene expression profile of ALFs in the hemocytes of shrimp at 48 h after experimental infections with V. harveyi (grey bars) or WSSV (black bars). Results are presented as mean ± standard deviation of relative expressions (three biological replicates) and statistical differences are indicated by asterisks (*) (one-way ANOVA/Tukey, p < 0.05). N: naïve (non-stimulated) shrimp (white bars), S: sterile seawater injury control, V: V. harveyi ATCC 14126 (6 × 10 7 CFU/animal), W-: tissue homogenate inoculum prepared from WSSV-free shrimp, W+: WSSV (3 × 10 2 viral particles/animal).

Figure 4 .

 4 Figure 4. Expression of ALFs during shrimp development. (A) Gene expression profile of Litvan ALF-E, and -G in twelve developmental stages: fertilized eggs at 0-4 h post-spawning fertilized eggs at 7-11 h post-spawning (EII), nauplius I (NI), nauplius V (NV), protozoea I (ZI), protozoea III (ZIII), mysis I (MI), mysis III (MIII), postlarva 2 (PL2), postlarva 9 (PL9), postlarva 17 (PL17). Representative images of the developmental stages are indicated at the bottom of the graph. Results are present as mean ± standard deviation. The red dotted line indicates the expression in hemocytes from juveniles while the solid blue underline highlights the stages at which the expression was detected (valid dissociation curve) but not quantified (Cq values higher than the limit of quantification). Different letters indicate significant differences among the developmental stages and asterisks (*) shows significant differences between each developmental stage and hemocytes from juveniles (one-way ANOVA/Tukey, p < 0.05). (B) Results of principal component analysis showing the relationship among the expression profile of L. vannamei ALFs during shrimp development. (C) The life cycle of the Pacific white shrimp L. vannamei.

Figure 5 .

 5 Figure 5. The antimicrobial spectrum of the novel shrimp ALFs. (A) Predicted three-dimensional structure of the central β-hairpin of Litvan ALF-E, -F and -G. The structural models were built based on the NMR structure of ALFPm3 (PDB: 2JOB). Conserved residues involved in LPS binding of ALFPm3 are displayed in blue. (B) Representative images of the effects of ALFs against F. oxysporum: (B1) fugal spore inhibition (antifungal effect); (B2) fungal spore germination (no antifungal activity). Bars = 20 µm. (C) Spectrum of antibacterial and antifungal activities of synthetic peptides based on the central β-hairpin of Litvan ALF-E, -F and -G. Minimum inhibitory (MIC) and Minimum bactericidal (MBC) concentrations are expressed in µM. nd: non-determined.

Figure 5 .

 5 Figure 5. The antimicrobial spectrum of the novel shrimp ALFs. (A) Predicted three-dimensional structure of the central β-hairpin of Litvan ALF-E, -F and -G. The structural models were built based on the NMR structure of ALFPm3 (PDB: 2JOB). Conserved residues involved in LPS binding of ALFPm3 are displayed in blue. (B) Representative images of the effects of ALFs against F. oxysporum: (B1) fugal spore inhibition (antifungal effect); (B2) fungal spore germination (no antifungal activity). Bars = 20 µm. (C) Spectrum of antibacterial and antifungal activities of synthetic peptides based on the central β-hairpin of Litvan ALF-E, -F and -G. Minimum inhibitory (MIC) and Minimum bactericidal (MBC) concentrations are expressed in µM. nd: non-determined.

4. 5 .

 5 Fluorescence-Based Reverse Transcription Real-Time Quantitative PCR (RT-qPCR)

Table 1 .

 1 Nucleotide sequences of primers used in this study.

	Gene	Forward Primer (5 -3 )	Reverse Primer (5 -3 )	Amplicon
	LvActin 1	TAATCCACATCTGCTGGAAGGTGG	TCACCAACTGGGATGACATGG	846 bp
	LvActin 2	CCACGAGACCACCTACAAC	AGCGAGGGCAGTGATTTC	142 bp
	LvEF1α 2	TGGCTGTGAACAAGATGGACA	TTGTAGCCCACCTTCTTGACG	103 bp
	LvL40 2	GAGAATGTGAAGGCCAAGATC	TCAGAGAGAGTGCGACCATC	104 bp
	LvRpS6 2	AGCAGATACCCTTGGTGAAG	GATGCAACCACGGACTGAC	193 bp
	Litvan ALF-A 1,2	CTGATTGCTCTTGTGCCACG	TGACCCATGAACTCCACCTC	113 bp
	Litvan ALF-B 1,2	GTGTCTCCGTGTTGACAAGC	ACAGCCCAACGATCTTGCTG	123 bp
	Litvan ALF-C 1,2	ATGCGAGTGTCTGTCCTCAG	TGAGTTTGTTCGCGATGGCC	115 bp
	Litvan ALF-D 1,2	TGTGTTGGTTGTGGCACTGG	CAACGAGGTCAATGTCACCG	131 bp
	Litvan ALF-E 1,2	TGCTACGTGAATCGCAGTCC	CGCTTCCTCTTCCGACAATG	100 bp
	Litvan ALF-F 1,2	AAGCTCTCATTCCTGGTCGG	GGGTGTAACGAAGTACGTGC	180 bp
	Litvan ALF-G 1,2	CCGCTGCATGTCAAGTATCC	TCAGCAGTAGCAGTGTCAGC	140 bp

1

Primers for semiquantitative analysis of gene expression (RT-PCR);

2

Primers for quantitative analysis of gene expression (RT-qPCR).

Acknowledgments:

The authors are grateful to the Laboratory of Marine Shrimp (Federal University of Santa Catarina, Florianópolis, Brazil) for providing the shrimp used in this study and to Pedro Ismael da Silva Jr. (Butantan Institute, São Paulo, Brazil) for the culture of Candida albicans MDM8. We are also in debt to Juan Eberhard for English correction and editing.

Funding: This research was supported by the Brazilian funding agencies CNPq (MEC/MCTI/CAPES/CNPq/ FAPs PVE 401191/2014-1 and MCTI/CNPq Universal 406530/2016-5) and CAPES (CIMAR 1974/2014). P.S. was funded by FONDECYT grant 11150009. G.M.M. and N.D.F. were supported by scholarships provided by FAPESC and C.B. received a scholarship provided by CAPES.

Supplementary Materials:

The following are available online at http://www.mdpi.com/1660-3397/16/10/381/ s1. Table S1: Sequences and biochemical properties of ALFs from decapod crustaceans and marine chelicerates. Figure S1: Amino acid sequence alignments of ALF-related sequences containing three and four cysteine residues. The predicted signal peptides are in bold and underlined. Asterisks (*) mark the identical amino acid residues while the cysteines are highlighted with a black background. GenBank accession numbers are indicated in brackets.

Conflicts of Interest:

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.


Erwan Nicol

Taco Nicolaï

Jingwen Zhao

Tetsuharu Narita

Taco Nicolai

Photo-Cross

Keywords:

HAL is

mixing two types of triblock copolymers that formed independent interpenetrated networks [START_REF] Klymenko | Multiresponsive Hydrogels Formed by Interpenetrated Self-Assembled Polymer Networks[END_REF] . The transient crosslinks of one network were subsequently rendered covalent by in-situ UV irradiation. Even though the networks were topologically independent, physical interaction between both networks led to a shift of the percolation concentration (Cp) and to a large increase of the elastic modulus compared to simple additivity. However, no significant enhancement of the gel toughness was observed, which we attribute to the fact that the networks were independent.

Here, self-assembly of triblock copolymers is exploited in a different manner. Two types of copolymers are mixed with the same central PEO block and different relatively short hydrophobic endblocks. Crosslinks formed by one type of endblock can be rendered covalent by in-situ UV irradiation, whereas the other remains dynamic. It will be shown that the two types of endblocks are randomly mixed in hybrid micellar cores. In this manner we were able to synthesize for the first time homogeneous selfassembled hydrogels containing both dynamic and permanent crosslinks with well-defined chain lengths between the crosslinks.

The dynamic network was formed by association of poly(isobutyryloxyethyl acrylate)-b-poly(ethylene oxide)-bpoly(isobutyryloxyethyl acrylate) (PIEA7-b-PEO270-b-PIEA7 called IEI12K). The photo-cross-linkable network was formed by poly(methacryloyloxyethyl acrylate)-b-poly(ethylene oxide)-bpoly(methacryloyloxyethyl acrylate) (PMEA7-b-PEO270-b-PMEA7 and PMEA9-b-PEO800-b-PMEA7 called tPEO12K and tPEO35k, respectively) These copolymers were synthesized following methods published elsewhere [START_REF] Klymenko | Multiresponsive Hydrogels Formed by Interpenetrated Self-Assembled Polymer Networks[END_REF][START_REF] Kadam | Synthesis of Flower-Like Poly(Ethylene Oxide) Based Macromolecular Architectures by Photo-Cross-Linking of Block Copolymers Self-Assemblies[END_REF][START_REF] Nicol | Synthesis of double hydrophilic poly(ethylene oxide)-b-poly(2-hydroxyethyl acrylate) by single-electron transfer-living radical polymerization[END_REF][START_REF] Nicol | Supplemental Activator and Reducing Agent Atom Transfer Radical Polymerization of 2-Hydroxyethyl Acrylate from High Molar Mass Poly(ethylene oxide) Macroinitiator in Dilute Solution[END_REF] (see Supporting Information) according to the route shown in Scheme 1. In aqueous solution IEI12K and tPEO self-assemble into flower-like micelles that can bridge leading to a network above Cp. The apparent molar masses (Ma) and hydrodynamic radii (Ra) of the micelles obtained from light scattering were close to the averages calculated from values obtained with individual systems (see Supporting Information). It follows that either the aggregation number (Nagg) of the hybrid micelles was the same as the calculated average or that hybridization had not occurred. Formation of transient networks by the copolymers caused a sharp increase of the relative viscosity (ηr) with increasing concentration above Cp: Cp ≈ 15 g/L for tPEO12k and IEI12K and Cp ≈ 8 g/L for tPEO35k (Figure S5). The mechanical relaxation of transient gels formed by self-assembly of tPEO/IEI mixtures above Cp was determined by oscillatory shear measurements. The frequency dependence of the storage (G') and loss (G") shear moduli showed a single relaxation mode that shifted to lower frequencies with increasing weight fraction of tPEO (x) in the mixtures (Figure S6). Master curves could be obtained by superimposition of results obtained at different concentrations, different chain lengths and different weight fraction using horizontal (ax) and vertical (bx) shift factors, see Figure 1a. This implies that the relaxation process of all systems was similar with a characteristic relaxation time that depended on the composition.

The relaxation time (τ) was defined here as the inverse of the radial frequency (1/2πν) at which G' crossed G". It was intermediate between that of the individual gels and increased exponentially with x, see Figure 1b. These observations are best explained if we assume that the network was formed by bridged hybridized micelles so that the escape of PMEA was facilitated by the presence of PIEA and the escape of PIEA was slowed down by the presence of PMEA leading to single intermediate relaxation time. The self-assembled transient hybrid gels were subsequently irradiated with UV-light in the presence of a photoinitiator, which caused covalent cross-linking of the PMEA endblocks of tPEO within the micellar cores. In-situ measurements during photocross-linking showed a very rapid increase of G' upon irradiation, see Figure 2a and Figure S7. The appearance of a frequency independent storage modulus higher than G" at low frequencies confirmed the formation of a covalent hydrogel, see Figure 2b and Figure S8. Mixtures with relatively high concentrations of IEI12K still showed significant relaxation of G' at high frequencies. We suggest that this was caused by escape of PIEA blocks implying that permanently cross-linking PMEA by UV-irradiation did not trap the PIEA blocks that were present in the same hybrid cores (scheme 2). At low frequencies the dynamic IEI12K network has completely relaxed and G' at 0.01 Hz is equal to the elastic modulus (G0) of the covalent network. Assuming rubber elasticity and affine deformation, the ideal elastic modulus is obtained if all tPEO chains are elastically active: G0 = CRT/Mn. The calculated moduli are 0.58, 1.15 and 2.31 kPa for tPEO35k hydrogels, at C = 10, 20 and 40 g/L, respectively. As shown in Figure 3, close to Cp individual tPEO hydrogels formed imperfect networks in which the fraction of elastically active chains is small. Increasing the concentration increased the fraction of elastically active chains leading to a fully formed network at C > 40 g/L for tPEO35k. For tPEO concentrations above, but close to Cp, addition of IEI12K triblock copolymer led to an increase of G0, see Figure 3 and Figure S9. Considering that at low frequencies IEI12K chains no longer contributed to the shear modulus, the increase of G0 was caused by an increasing number of elastically active tPEO chains in the network. In the case of highly imperfect tPEO networks close Cp, the increase caused by adding IEI12K could be as much as two orders of magnitude (Figure S9). This means that adding IEI12K improves the connectivity of the tPEO network, though G0 did not reach the values calculated for ideal tPEO networks.

Interestingly, similar improvement of the mechanical properties could be obtained by addition of the diblock copolymer poly(isobutyryloxyethyl acrylate)-b-poly(ethylene oxide)5k (IE). IE is equivalent to the IEI12K triblock copolymer cut in halve and cannot bridge micelles (Figure S10). This means that the effect of IEI12K on the elastic modulus of the tPEO network is not caused by transient bridging of IEI12K, but by the presence of dynamic hydrophobic blocks in the hybrid micellar cores.

As was mentioned above, light scattering measurements showed that Nagg of the hybrid micelles was close to the average of the individual systems. The implication is that added PIEA blocks of either IE or IEI12K substituted PMEA blocks of tPEO in the micellar cores. This caused an increase of the concentration of micelles containing tPEO, which in turn favored the formation of bridges instead of loops by tPEO and therefore an increase of the fraction of elastically active tPEO chains. G0 started to decrease again at relatively high IEI12K concentrations, when the molar fraction of IEI12K in the hybrid micelles (defined as 2nIEI12k/(2nIEI12k+2ntPEO35k) assuming that the PIEA and PMEA blocks are homogeneously distributed in the cores of hybrid micelles) became larger than about 70 -85 mol%, see Figure 3. This can be explained by the fact that at high IEI12K concentrations an increasing fraction of micelles no longer contain sufficient number of tPEO chains to participate in the permanent tPEO network. The non-linear rheological properties of the cross-linked hydrogels were assessed using two kinds of measurements. First, G' and G" were measured for tPEO12k/IEI12K mixtures as a function of the applied strain after in situ photo-cross-linking at 1 Hz. Notice that the dynamic crosslinks of these mixtures were fully relaxed at this frequency. As expected, at constant copolymer concentration, substituting covalent links by dynamic ones led to a decrease of G' and an increase of the fracture strain (Figure S11). Figure 4a shows that if the amount of IEI12K was increased while keeping the tPEO12k concentration constant, not only G0 increased as discussed above, but also the fracture strain in-Time (s) creased. In common covalent hydrogels, increasing the number of elastically active chains increases G0, but decreases the fracture strain [START_REF] Kadam | Structure and Rheology of Self-Assembled Telechelic Associative Polymers in Aqueous Solution before and after Photo-Cross-Linking[END_REF] . Kean et al. [START_REF] Kean | Increasing the Maximum Achievable Strain of a Covalent Polymer Gel Through the Addition of Mechanically Invisible Cross-Links[END_REF] showed that adding very dynamic crosslinks in a covalent organogel improved the toughness but did not enhance G0. Here, a synergetic effect was observed not only for the stiffness (increase of elastic modulus) but also for the nonlinear properties of the hydrogels (increase of the toughness).

Uniaxial stretching experiments were used to assess the evolution of the tensile stress with increasing strain (Figure 4b-c). As expected, gels obtained from tPEO35k exhibited larger extensibility than those obtained from tPEO12k with the same elastic modulus, because the elastically active chains are longer and thus more extensible. The effect of adding IEI12K was to substantially increase the strain at breakage (up to 350%) without reducing the Young modulus thus corroborating the large oscillation amplitude measurements. The better resistance of hybrid hydrogels to fracture can be explained by a higher capacity to dissipate energy when mechanical strain is applied, because the dynamic network can rearrange and thereby prevent stress concentration at the vicinity of the broken permanent chains. A cyclic loadingunloading experiment (Figure S12) showed small hysteresis implying that dynamic rearrangement and the stress reduction was quite fast compared to the macroscopic loading rate [START_REF] Kean | Increasing the Maximum Achievable Strain of a Covalent Polymer Gel Through the Addition of Mechanically Invisible Cross-Links[END_REF] . To conclude, we present here a novel type of hydrogel formed by self-assembly of two types of POE-based triblock copolymers of which one is photo-cross-linkable. The novelty is that the same junction zones contain both dynamic and covalent crosslinks. Formation of hybridized micellar cores before irradiation was confirmed by the evolution of the characteristic relaxation time of a single relaxation mode with the composition of the copolymer mixtures. Photo-cross-linking of the copolymer mixtures at thermodynamic equilibrium very rapidly turned the physical (and potentially injectable) hydrogel into a self-standing covalent hydrogel formed by one of the copolymers with the other remaining dynamic. The coexistence of both dynamic and covalent crosslinks in the same junctions led to synergetic effects that enhanced the elastic modulus and the resistance of the gels to fracture. These block copolymer based hydrogels are noncytotoxic [START_REF] Klymenko | Formation of porous hydrogels by self-assembly of photo-crosslinkable triblock copolymers in the presence of homopolymers[END_REF] and could be interesting candidates for utilization as injectable and in situ photo-cross-linkable scaffolds for tissue engineering.

ASSOCIATED CONTENT Supporting Information

Materials and methods. Structure of tPEO/IEI12K mixtures. Rheological properties of tPEO/IEI12K mixtures before photo-crosslinking. Photo-cross-linked hybrid covalent/dynamic hydrogels.

The Supporting Information is available free of charge on the ACS Publications website. brief description (PDF).

AUTHOR INFORMATION Corresponding Author

Correspondence should be addressed to Erwan Nicol. Erwan.nicol@univ-lemans.fr. Phone: ++33.2.43.83.33.62

Materials and Methods

Block copolymers synthesis

Amphiphilic triblock copolymers were synthesized according to already published procedures [1][2][3][4] . Briefly, a triple hydrophilic poly(2-hydroxyethyl acrylate)-b-poly(ethylene oxide)b-poly(2-hydroxyethyl acrylate), PHEA-b-PEO-b-PHEA, was synthesized by growing two PHEA blocks from a dibrominated PEO (Br-PEO-Br) macroinitiator using SARA-ATRP. In the case of PEO12k of Mn=12 000 g/mol, the synthesis was performed in DMSO 3 Prior to use the copper surface was activated with sulfuric acid. A degree of polymerization of 7 for the PHEA blocks was obtained at 90% conversion. In the case of PEO35k of Mn=35 000 g/mol, the synthesis was performed in water 4 under air with an intensity 0.17 W.cm -1 during 60 s. The measurements were done in the linear response regime unless otherwise specified.

Static and dynamic light scattering measurements were done using a commercial static and dynamic light scattering equipment (ALV-Langen, Germany and LS-Instruments, Switzerland)

operating with a vertically polarized laser with wavelength λ=632 nm. The measured scattered light intensity was corrected for that of the solvent and normalized by that of toluene. The relative excess scattering intensity (Ir) was multiplied with the Rayleigh factor of toluene and expressed in units of cm-1. The measurements were done at 20 °C using a thermostated bath over a range of scattering wave vectors (q=4.π.n.sin(θ/2)/λ), with θ the angle of observation and n the refractive index of the solvent). The electric field autocorrelation function (g1(t)) was calculated from the normalized intensity autocorrelation functions (g2(t)): g2(t)=1+g1(t) 2 and was analyzed in terms of a relaxation time distribution:

( ) ∫ - = τ τ τ log / exp ) (log ) ( 1 d t A t g (1)
At low polymer concentration a single relaxation mode was observed, but at higher concentrations a second slower and broader relaxation mode was observed. All correlation functions could be well-described using a log-normal distribution for the fast mode and a generalized exponential for the slow mode:

( ) [ ] s p k A * - = τ τ τ τ / exp ) (log (2)
The average relaxation rates (Γ=<τ -1 >) of the single mode at low concentrations and of the fast mode at high concentrations were found to be q 2 -dependent and the cooperative diffusion coefficient was calculated as

Dc= Γ.q -2
The large deformation behavior of the gels was studied by uniaxial tensile and loadingunloading tests on an Instron 5565 tensile tester with a 10 N load cell. Samples were rectangular in shape with 5mm width, 3 mm thickness, and 20 mm length (length between clamps). We kept the samples in paraffin oil during all the tests to prevent them from drying.

Structure of tPEO/IEI mixtures

The structure of self-assemblies formed by PMEA-b-PEO-b-PMEA (tPEO) and PIEA-b-PEO-b-PIEA (IEI) triblock copolymers were studied individually and in mixtures. The apparent molar mass (Ma) and hydrodynamic radius (Rha) of individual tPEO12k and IEI aggregates were obtained by static and dynamic light scattering as described in the materials and methods section and are shown as a function of the polymer concentration in Figure S2. As was already reported observed for similar systems in the literature 2,6 Ma and Rha increased with increasing concentration due to the bridging of micelles leading to formation of larger aggregates. At high concentrations, Ma decreased because repulsive interactions dominated 6 . The number of hydrophobic blocks per micellar core (Nagg) could be estimated from Ma measured at 1 g/L, because at this concentration, repulsive interactions between micelles were negligible and micelles were mainly in the form of single flowers 1 . Nagg ≈ 60 was found for tPEO12k micelles and Nagg≈30 for IEI12k micelles. The large difference between tPEO and IEI is probably due to the lower hydrophobicity of the PIEA blocks which contained about 15% residual hydrophilic units whereas PMEA blocks did not. Nagg of tPEO35k was also approximately 30 (Figure S3).

The calculated values are close to the experimental data at low polymer concentrations where the effect of interaction is small. This suggests that either hybridization did not occur or that Nagg of the hybrid micelles was the same as the calculated average.

Rheological properties of tPEO/IEI mixtures before photo-cross-linking

The viscosity of individual systems and mixtures was measured as function of the shear rate.

All solutions exhibited Newtonian behavior over large range of shear rates, but the most viscous solutions showed shear thinning at shear rates above 10 s -1 (see Fig S4a andS4b). Interestingly, for a given C, ηr had a logarithmic dependence on x:

log�𝜂𝜂 𝑟𝑟(ℎ𝑦𝑦𝑦𝑦) � = 𝑚𝑚 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 . log�𝜂𝜂 𝑟𝑟(𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡) � + 𝑚𝑚 𝐼𝐼𝑡𝑡𝐼𝐼 . log�𝜂𝜂 𝑟𝑟(𝐼𝐼𝑡𝑡𝐼𝐼) � (5)
This behavior noticeably differs from that exhibited by mixtures of other types of self-assembled triblock copolymers 7,8 . In the case of hydrophobically end-capped PEO, Annable et al. 7 reported that chains with different end-cap alkyl lengths relaxed independently. This implies that the viscosity of the mixtures was governed by the polymers with the longest relaxation time, i.e. the highest viscosity. In the case of mixtures of pH-sensitive amphiphilic triblock copolyelectrolytes the viscosity was also governed by that of the most viscous copolymer as soon as it formed a percolated network 8 . In this latter case, although the evolution of the viscosity was not strictly proportional to the evolution of the mean relaxation time τ (lifetime of a hydrophobic block in a micellar core), it followed the same tendency. The frequency dependence of the shear moduli was determined as a function of x. It showed a single relaxation mode that shifted to lower frequencies with increasing x, see Fig. S6.

Scheme 1 .

 1 Scheme 1. Synthesis of PMEA-b-PEO-b-PMEA (tPEO) and PIEA-b-PEO-b-PIEA (IEI) triblock copolymers.

Figure 1 .Figure 2 .

 12 Figure 1. (a) Master curve obtained by superimposition the relaxation spectra of pure tPEO solutions and tPEO/IEI12K mixtures (reference curve: tPEO35k at C = 50 g/L and T = 20 °C). (b) Evolution of the relaxation time with the weight fraction of tPEO35k in tPEO35k/IEI12K mixtures at Ctot = 50 g/L and T = 20 °C.

Figure 3 .

 3 Figure 3. Evolution of the low frequency storage modulus (measured at 0.01 Hz) as a function of the molar percentage of PIEA blocks in the micelle cores for tPEO35k/IEI12k hybrid hydrogels. The total concentration of block copolymer is indicated in the Figure. Dotted lines are guide to the eye. Arrows show the maximum moduli calculated assuming rubber elasticity and affine deformation.

Figure 4 .

 4 Figure 4. (a) Strain dependence of the elastic modulus (G') measured at f = 1 Hz for tPEO12k/IEI12K hybrid photo-cross-linked hydrogels at constant tPEO concentration (C = 30 g/L) and increasing concentration of IEI12K as indicated in the Figure. Arrows indicate the breakage of the sample. (b) Tensile tests of tPEO based hydrogels after photo-cross-linking (stretch rate v = 0.03 s -1 ). (c) Tensile test of tPEO35k C = 40 g/L -IEI12K C = 80 g/L at the beginning (left) and end (right) of the test.

  g/L tPEO 35k C=40 g/L tPEO 35k C=40 g/L -IEI C=40 g/L tPEO 35k C=40 g/L -IEI C=80 g/L

  with a molar ratio [Br-PEO-Br]/[HEA]/[Me6TREN]/[CuBr2] : 1/8/0.2/0.05. Copper wire was used as a catalyst.

  with a molar ratio [Br-PEO-Br]/[HEA]/[Me6TREN]/[CuBr]/[CuBr2] : 1/10/0.5/0.3/0.2. A degree of polymerization of 9 for the PHEA blocks was obtained at 90% conversion. Polymerizable methacrylate functions were then grafted onto the PHEA-blocks leading to the UV-crosslinkable hydrophobic poly(methacryloyloxyethyl acrylate) (PMEA) blocks. 1 H NMR analysis of both final PMEA-b-PEO-b-PMEA (tPEO) triblock copolymers indicated a quantitative functionalization of the HEA units into methacryloyloxyethyl acrylate ones. The number (Mn) and weight (Mw) average molar mass and dispersity (Đ=Mw/Mn) were determined by Size Exclusion Chromatography (SEC) in THF using light scattering and refractometric detection. Mn=1.42×10 4 g/mol, Mw=1.59×10 4 g/mol, Đ=1.1 were measured for tPEO12k. Mn= 4.21×10 4 g/mol, Mw= 5.53×10 4 g/mol, Đ=1.3 were measured for tPEO35k. Poly(isobutyryloxyethyl acrylate)-b-poly(ethylene oxide)-b-poly(isobutyryloxyethyl acrylate) (PIEA-b-PEO-b-PIEA: IEI) was synthesized by esterification of PHEA-b-PEO12k-b-PHEA with isobutyryl chloride in the presence of triethylamine and DMPA using the same procedure as that used for synthetizing PMEA-b-PEO-b-PMEA 1,5 . 1 H NMR analysis of the resulting IEI triblock copolymer indicated 85% of functionalization of the HEA units into isobutyryloxyethyl acrylate ones (Figure S1). The hydrophobic block was thus a statistical copolymer poly((isobutyryloxyethyl acrylate)0.85-co-(2-hydroxyethyl acrylate)0.15). Mn=1.42×10 4 g/mol, Mw=1.59×10 4 g/mol, Đ=1.1 were measured by multi-detection SEC.

Figure S1. 1 H

 1 Figure S1. 1 H NMR of PIEA-b-PEO-b-PIEA (IEI) copolymer in DMSO-d6 (ν0=400 MHz)

Figure S2 .

 S2 Figure S2. Concentration dependences of (a) the apparent molar mass (Ma) and (b) the apparent hydrodynamic radius (Rha) for the IEI aggregates (open circles), tPEO12k (open squares) and tPEO12k/IEI 50/50 (%v/v) mixtures (filled triangles). Blue dashed lines represent the theoretical evolution of non-hybridizing aggregates mixture (equation (3) and (4)).

Figure S3 . 3 )

 S33 Figure S3. Concentration dependence of the apparent molar mass and apparent hydrodynamic radius of tPEO35k aggregates in water.

Figure S4 .

 S4 Figure S4. Shear rate dependence of the relative viscosity of (a) tPEO12k/IEI hybrid hydrogels at C=50 g.L -1 and (b) tPEO35k/IEI hybrid hydrogels at C=50 g.L -1

Figure S5 .

 S5 Figure S5. (a) Concentration dependence of the relative viscosity of IEI12K, tPEO12k and tPEO35k at T=20 °C. (b) Evolution of the relative viscosity with the weight fraction of tPEO in tPEO/IEI mixtures at various concentrations. Dashed lines are fits to equation (5).

Figure S6 .Figure S7 .Figure S8 .Figure S9 .

 S6S7S8S9 Figure S6. Frequency dependences of the storage (G') and loss (G") moduli of tPEO35k/IEI mixtures at Ctot=50 g/L and T=20 °C.

Figure S10 .Figure S11 .

 S10S11 Figure S10. Evolution of the low frequency storage modulus (measured at 0.01 Hz) as a function of the molar percentage of PIEA blocks in the micelle cores for tPEO12k (C=20 g/L) mixed with IEI triblock copolymer (circles) or IE diblock copolymer (triangle)

Figure S12 .

 S12 Figure S12. Evolution of the tensile stress with strain during cyclic loading-unloading experiments.

ACKNOWLEDGMENT

The authors acknowledge Maëva Robin and Arlette Tachou for their help during the block copolymers synthesis, Boris Jacquette for the SEC characterizations and Corentin Jacquemmoz for the NMR experiments. This work was partially supported by the Région des Pays de la Loire -Bioregate Cluster (project Hydrophobiq).

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. All authors contributed equally.

Notes

The authors declare no competing financial interests.

Supporting Information

Photo-Cross-Linked Self-Assembled Poly(Ethylene