Derya Deveci

Francisco A Martin

Pierre Léopold

Nuria M Romero

Pierre Leopold
email: pierre.leopold@curie.frnromero@univ-cotedazur.fr

AstA Signaling Functions as an Evolutionary Conserved Mechanism Timing Juvenile to Adult Transition

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

Introduction

In vertebrates, the onset of puberty is marked by an increased production of steroid hormones as a consequence of the activation of the hypothalamic-pituitary-gonadal (HPG) axis [START_REF] Ellison | Puberty as a life history transition[END_REF][START_REF] Plant | Neuroendocrine control of the onset of puberty[END_REF]. In the hypothalamus, this transition coincides with the pulsatile secretion of gonadotropin-releasing hormone (GnRH) from GnRH neurons (GnRHn). GnRHn project their axons onto the pituitary gland where FSH and LH intermediate hormones are produced, which in turn stimulate the production of steroids at the gonads. GnRH pulsatile secretion is the result of several excitatory and inhibitory inputs from afferent neural circuitries [START_REF] Ojeda | Puberty in the Rat[END_REF].

Among them, Kisspeptin (KISS) produced by hypothalamus neurons plays a leading role [START_REF] Navarro | Neuroendocrine control by kisspeptins: Role in metabolic regulation of fertility[END_REF][START_REF] Pinilla | Kisspeptins and Reproduction: Physiological Roles and Regulatory Mechanisms[END_REF][START_REF] Roa | New frontiers in kisspeptin/GPR54 physiology as fundamental gatekeepers of reproductive function[END_REF][START_REF] Tena-Sempere | Timeline: The role of kisspeptins in reproductive biology[END_REF]. Indeed, knock-down of the KISS receptor GPR54 in GnRHn induces hypogonadotropic hypogonadism and impaired onset of puberty [START_REF] Herbison | Control of puberty onset and fertility by gonadotropin-releasing hormone neurons[END_REF]. Therefore, Kiss neuron activity and the regulation of GnRH secretion are crucial for steroid hormone production and puberty initiation. Despite its importance, the timed regulation of KISS and GnRH secretion relies on multifaceted mechanisms whose nature is still poorly understood [START_REF] Lomniczi | Epigenetic control of female puberty[END_REF][START_REF] Sangiao-Alvarellos | Changes in hypothalamic expression of the Lin28/let-7 system and related MicroRNAs during postnatal maturation and after experimental manipulations of puberty[END_REF]. In many invertebrates, the control of sexual maturation also requires the production of steroid hormones. In Drosophila, the transition from juvenile to adult (i.e. metamorphosis) is marked by an increase in the production of ecdysone by the prothoracic gland (PG). Pioneering work in Bombyx and Manduca identified the Prothoracicotropic Hormone (PTTH) as a brain neuropeptide controlling ecdysone production. In Drosophila larvae, PTTH is produced by a pair of bilateral neurons (PTTHn), which project their axons on the PG [START_REF] Kopeć | Studies on the necessity of the brain for the inception of insect metamorphosis[END_REF][START_REF] Wigglesworth | The Physiology of Ecdysis in Rhodnius prolixus (Hemiptera). II. Factors controlling Moulting and "Metamorphosis[END_REF][START_REF] Mcbrayer | Prothoracicotropic Hormone Regulates Developmental Timing and Body Size in Drosophila[END_REF]. ptth gene transcription significantly increases just before the juvenile/maturation transition (JMT) and ablation of PTTHn delays the time to metamorphosis by 4-5 days, highlighting the importance of this neural circuit in controlling JMT.

The onset of sexual maturation also coincides with the end of the growth period.

Therefore, a coordination of growth and sexual maturation is needed to ensure proper adult size and fitness. Larval growth relies on the production and release of insulin-like peptides (Dilps) by specialized nerosecretory cells called insulin-producing cells (IPCs). As in other models, insulin-IGF signaling (IIS) relays nutritional information to organ and tissue growth, and the release of Dilps by the IPCs, relies on the presence of amino acids in the food [START_REF] Koyama | Growth-Blocking Peptides As Nutrition-Sensitive Signals for Insulin Secretion and Body Size Regulation[END_REF][START_REF] Delanoue | Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor[END_REF][START_REF] Géminard | Control of metabolism and growth through insulin-like peptides in Drosophila[END_REF][START_REF] Slaidina | A Drosophila Insulin-like Peptide Promotes Growth during Nonfeeding States[END_REF]. Under limiting food, animals grow slowly and present delayed JMT. However, the coupling mechanism between growth and maturation is not fully understood.

Recent work has highlighted the role of a hormone called Dilp8 in another coupling between organ growth and maturation. In response to tissue damage, Dilp8 activates a neural circuit that interferes with PTTHn, ensuring extra time for tissue repair before the JMT takes place [START_REF] Colombani | Drosophila Lgr3 Couples Organ Growth with Maturation and Ensures Developmental Stability[END_REF][START_REF] Vallejo | A brain circuit that synchronizes growth and maturation revealed through Dilp8 binding to Lgr3[END_REF][START_REF] Garelli | Imaginal discs secrete insulin-like peptide 8 to mediate plasticity of growth and maturation[END_REF]. However, removal of Dilp8 during development does not induce premature pupariation. This suggests that in the absence of this checkpoint signal, inducer signals are still required for the timed activation of PTTHn.

In this study we performed a genetic screen aimed at identifying regulators of PTTHn.

We found that Allatostatin-A (AstA) and its receptor Allatostatin A Receptor 1 (AstAR1) control PTTH secretion, thereby determining the time of metamorphosis. Interestingly, AstA/AstAR1 also controls animal's growth rate by acting on Dilp2 secretion from the IPCs.

We find that AstA/AstAR1 is developmentally regulated with a peak of activation at the onset of metamorphosis. AstAR1 is homologous to the mammalian Kisspeptin receptor, GPR54.

Therefore we propose that AstA neurons (AstAn) provide an evolutionary conserved developmental signal that controls juvenile growth and maturation, ensuring proper adult size and fitness.

Results

AstAR1 induces growth and maturation

To explore the mechanism timing the onset of JMT, we conducted an RNAi-based screen for the modulation of PTTHn activity. For this, we used the NP423-Gal4 (423>) and the ptth-Gal4 (ptth>) drivers, both targeting the PTTHn. When combined with UAS-dicer2 and UAS-ptth-RNAi, these driver lines induce a delay at the larva-to-pupa transition by 24 and 12 hours, respectively (Figure 1A and Figure S1B). In addition, the NP423-Gal4 driver was recombined with the TshG80 transgene to silence Gal4 expression in ventral cord neurons.

We screened a collection of 1300 RNAi constructs targeting genes encoding membraneassociated proteins, nuclear receptors, and synaptic components, for their ability to delay the onset of metamorphosis when targeted to PTTHn (Figure S1A). One of our best hit identified Allatostatin-A receptor 1 (AstAR1), a rhodopsin-like 7 transmembrane G-coupled protein receptor, as a regulator of PTTHn function. As shown in Figure Figure 1A, Figure . S1B and C, reducing AstAR1 expression in PTTHn by using two independent RNAi lines (TshG80-423>AstAR1-RNAi.dcr2 or ptth>AstAR1-RNAi.dcr2) induces a pupariation delay similar to that observed upon ptth silencing (TshG80, 423>ptth-RNAi.dcr2, TshG80, 423>ptth-RNAi2.dcr2 or ptth>ptth-RNAi.dcr2). As a consequence of the delayed development, larvae grow for a longer period, resulting in pupae that are approximately 10% larger than controls (Figure 1B and Figure S1D andE).

To determine AstAR1 expression, we used an AstAR1-Gal4 knock-in line to drive GFP expression [START_REF] Yamagata | Suppression of Dopamine Neurons Mediates Reward[END_REF]. No signal was observed in larval tissues including the gut, fat body, PG and mitotic tissues, suggesting that AstAR1 is mainly expressed in the brain, as previously reported by high-throughput anatomy RNA-Seq data [START_REF] Brown | Diversity and dynamics of the Drosophila transcriptome[END_REF]. Indeed, we observed a signal within the brain lobes in PTTH cell bodies and PTTH axons that extend to the PG (Figure 1C). We validated AstAR1 expression in PTTHn with two other driver lines expressing Gal4 under the control of fragments of the AstAR1 regulatory region [START_REF] Tirian | The VT GAL4, LexA, and split-GAL4 driver line collections for targeted expression in the Drosophila nervous system[END_REF] (Figure S1F and data not shown). Interestingly, we also detected a specific GFP signal in the IPCs, raising the possibility that AstAR1 controls the activity of these cells (Figure 1C and Figure S1F). Indeed, silencing AstAR1 in the IPCs using Dilp2-Gal4 (Dilp2>) and two independent RNAi lines induces an important reduction in pupal size (-14%) due to a significant reduction in larval growth rate (Figure 1E and F and Figure S1H), but does not affect the timing of the developmental transition (Figure 1D and Figure S1G).

Altogether, these data provide evidence that AstAR1 acts both in the IPCs and the PTTHn, exerting a dual function on the animal growth rate and the time of maturation.

AstAR1 controls Dilp2 and PTTH secretion

To understand the function of AstAR1 signaling in systemic growth, we analyzed Dilp2/5 expression levels in the IPCs by both immunostaining and qRT-PCR on Dilp2/5 trancripts. Upon AstAR1 silencing (Dilp2>AstAR1-RNAi), we observed an increase in Dilp2 and Dilp5 staining in the IPCs (Figure 2A and Figure S2A) despite normal Dilp2 and Dilp5 transcript levels (Figure S2B andC). This profile has previously been found in starved conditions to correspond to impaired Dilps secretion [START_REF] Géminard | Control of metabolism and growth through insulin-like peptides in Drosophila[END_REF][START_REF] Slaidina | A Drosophila Insulin-like Peptide Promotes Growth during Nonfeeding States[END_REF]. Indeed, Dilp2>AstAR1-RNAi larvae show significantly reduced levels of Dilp2 in the hemolymph, as observed in starved animals (Figure 2B).

A similar approach was used to evaluated AstAR1 function in PTTHn. Since the ptth gene is transcriptionally controlled during development [START_REF] Mcbrayer | Prothoracicotropic Hormone Regulates Developmental Timing and Body Size in Drosophila[END_REF], we first sought to analyze ptth transcription but did not observe any change in TshG80-423>AstAR1-RNAi.dcr2 animals compared to controls (Figure S2D). Moreover, PTTH immunoreactivity remains unaffected in the PTTHn cell bodies in TshG80-423>AstAR1-RNAi.dcr2 larvae (Figure S2E). However, PTTHn present extensive remodeling of their axonal terminals at the surface of PG cells with enlarged PTTH-containing boutons, reduced bouton number and a significant decrease in the total number of axonal crosses (Figure 2D-H). Since depolarization of the membrane is necessary for neuropeptide secretion [START_REF] Van Den Pol | Neuropeptide Transmission in Brain Circuits[END_REF], we analyzed PTTHn terminal branches after hyperpolarization of PTTHn using the inward rectifier channel Kir2.1 [START_REF] Baines | Altered electrical properties in Drosophila neurons developing without synaptic transmission[END_REF][START_REF] Paradis | Homeostatic control of presynaptic release is triggered by postsynaptic membrane depolarization[END_REF]. Surprisingly, TshG80,423>Kir2.1 larvae shows a defect in PTTH axonal terminals similar to the one observed upon AstAR1 silencing (Figure 2E-H), suggesting that AstAR1 may controls PTTH secretion from PTTHn boutons located on the PG. To check this hypothesis, we performed ELISA tests for PTTH on larval hemolymph, a technique detecting rather strong variations in circulating PTTH levels [START_REF] Yamanaka | Neuroendocrine Control of Drosophila Larval Light Preference[END_REF]. Indeed, we found lower levels of circulating PTTH in the hemolymph of TshG80-423>AstAR1-RNAi.dcr2 larvae compared to controls (Figure 2C).

We therefore conclude from these experiments that AstAR1 directly regulates neuropeptide secretion in both IPCs and PTTHn.

AstAR1 functions are mediated by Allatostatin A-expressing neurons (AstAn)

The small neuropeptide Allatostatin A (AstA) is the only known ligand for AstAR1 receptor [START_REF] Birgül | Reverse physiology in Drosophila: identification of a novel allatostatin-like neuropeptide and its cognate receptor structurally related to the mammalian somatostatin/galanin/opioid receptor family[END_REF][START_REF] Chen | Allatostatin A Signalling in Drosophila Regulates Feeding and Sleep and Is Modulated by PDF[END_REF]. Antibody stainings against AstA reveal two pairs of bilateral AstA-positive neurons (AstAn) in the larval brain [START_REF] Yoon | Immunocytochemical localization of Diploptera punctata allatostatin-like peptide in Drosophila melanogaster[END_REF][START_REF] Zoephel | Peptidomics of the Agriculturally Damaging Larval Stage of the Cabbage Root Fly Delia radicum (Diptera: Anthomyiidae)[END_REF]. In order to characterize these neurons, we used an AstA-Gal4 line from the GAL4 Fly line collection (Janelia Farm), which presents an expression pattern similar but stronger than the previously described AstA-Gal4 [START_REF] Hergarden | Allatostatin-A neurons inhibit feeding behavior in adult Drosophila[END_REF]. Driving RFP expression with this AstA-Gal4, reveals two neural cell bodies in the basolateral protocerebrum, with dense arborizations in both dorsolateral and dorsomedia protocerebrum (Figure 3A and Video S1). Interestingly, PTTHn and IPCs are located in these arborization areas, suggesting that AstAn could contact both populations of neurons (Figure 3B). In order to test direct connexions between AstAn and PTTHn or IPCs, we used the GFP Reconstitution Across Synaptic Partners (GRASP), which generates a GFP signal when two neuronal circuits are in molecular proximity [START_REF] Gordon | Motor Control in a Drosophila Taste Circuit[END_REF]. When pairing AstA-LexA>GFP11 with 423-Gal4>GFP1-10, a cloud of GFP signal appears in the area of the PTTHn (Figure 3C) Additionally, we observed a robust GFP signal surrounding the IPCs when AstA-Gal4>GFP1-10 is paired with Dilp2-LexA>GFP11 (Figure 3D). These data suggest that AstAn make direct contact with both the PTTHn and the IPCs. By using DenMark (which marks dendritic arborisations) and anti-AstA inmunostainining, we observed some AstA neuropeptide in the vicinity of both PTTHn and IPCs dendrites, suggesting that AstA could physically interacts with its receptor in these neurons (Figure 3E andF).

To investigate the role of AstA in the control of PTTH neurons, we first sought to study the phenotype of AstA sk1 null mutant [START_REF] Hentze | The Neuropeptide Allatostatin A Regulates Metabolism and Feeding Decisions in Drosophila[END_REF]. Ast sk1 mutant larvae shows a delay at the onset of metamorphosis (Figure 4A), similar to the one observed after silencing AstAR1 in the PTTHn (see Figure 1A). Despite having an extended growth period, AstA sk1 null animals are slightly reduced in size (Figure 4B). This could be the result of an effect of AstA on the IPCs, as observed in Dilp2>AstAR1-RNAi animals (see Figure 1E). To confirm that AstA controls both larval growth and the time of metamorphosis as AstAR1 does, we silenced AstA ubiquitously using AstA-Gal4 (AstA>). AstA>AstA-RNAi.dcr2 animals show delayed pupariation timing although no significant growth reduction (Figure 4C-D and Figure S3A for control of RNAi effect). The developmental delay in AstA>AstA-RNAi.dcr2 animals correlates with a reduced kinetic of ecdysone production and is rescued by ectopic administration of 20-hydroxyecdysone (Figure S3B and Figure 4C). Lastly, silencing AstA only in the larval brain (syb>AstA-RNAi.dcr2) causes a similar delay in metamorphosis (Figure 4E) and no overgrowth (Figure 4F). This indicates that AstA expressed in the brain controls growth and maturation by activating AstAR1 in the PTTHn and IPCs. To strengthen this data, we simultaniously silenced AstAR1 in PTTHn and IPCs by using Dilp2,NP423>AstAR1-RNAi.dcr2. Consistently, these larvae were delayed at metamorphosis without modification of their pupal size (Figure 4G andH), a phenotype similar to AstA lossof-function. Thus, we conclude that the brain AstA neuropeptide controls growth and maturation through its receptor AstAR1 in IPCs and PTTHn, respectively.

AstA/AstAR1 differentially controls growth and maturation through AstA-N1 and AstA-N2 neurons

Our previous results suggest that AstAn form a neural circuit with the IPCs and the PTTHn to control growth and sexual maturation, but it is not clear whether this is achieved commonly by both AstA neurons or if each neuron has a distinct role. The Coin-Flp technique, which allows mosaic visualization of patterns [START_REF] Bosch | CoinFLP: a system for efficient mosaic screening and for visualizing clonal boundaries in Drosophila[END_REF], revealed the morphological diversity of AstA neurons. The main posterior AstA neuron, referred to as AstA-N1, projects anteriorly towards the PTTH projections, and to some IPC projections (Figure S3C andD).

The second AstA neuron, referred to as AstA-N2, projects towards the dorsomedium protocerebrum where IPCs cell bodies and PTTH projections are located (Figure 3A and Figure S3C-E). These morphological structures suggest that AstA-N1 and AstA-N2 could signal to both PTTHn and IPCs.

In order to dissect the diffferential functions of AstA-N1 and AstA-N2, we use an intersection of Gal4 and Gal80 expression to silence individual AstA neurons. When both AstA neurons are inactivated using Kir2.1 (AstA>Kir2.1) pupariation is delayed (Figure 5A) without affecting pupa size (Figure 5B), analogous to silencing AstA using AstA-Gal4 (see Figure 4C andD). Combining AstA-Gal4 with UAS-Kir2.1 and Tsh-G80, we could selectively silence AstA-N1 neurons, while maintaining AstA-N2 neurons active (as revealed by GFP, see Figure S4A). In these conditions, we observe a similar developmental delay as in AstA>Kir2.1 animals, together with the remodeling of PTTH axon termini (Figure 4A and Figure S4B-D). Once again, final pupal size is not modified, suggesting that larvae grow at a slower rate, as indicated by a slight retention of Dilp2 in IPCs (Figure 4B and Figure S4E).

The effect of AstAN1 on developmental timing and pupal size was confirmed by silencing AstA exclusively in AstAN1 (TshG80, AstA>AstA-RNAi.dcr2) (Figure S4F andG).

Conversely, the forced activation of AstAN1 (TshG80, AstA>TRPA1) slightly advanced the onset of metamorphosis (Figure 5C). In our hands, this an indirect but highly sensitive method to monitor PTTH signaling. Indeed, in these conditions, no obvious change in circulating PTTH levels could be observed (not shown). This indicates that AstA-N1 neurons are necessary and sufficient to control the timing of pupal transition. In addition, since pupae are of normal size despite a prolonged or shorted larval period (Figure S4G and Figure 5D), our data indicate that AstA-N1 also affect the growth rate of the larvae. Altogether, these experiments indicate that AstA-N1 control both larval growth and the timing of maturation while AstA-N2 control larval growth only.

AstA/AstAR1 achieve maximal signaling activity in PTTHn prior to maturation

Growth takes place during larval development and the cessation of growth occurs concomitant with the onset of maturation when PTTH levels raise [START_REF] Mcbrayer | Prothoracicotropic Hormone Regulates Developmental Timing and Body Size in Drosophila[END_REF]. Thus, we test the temporal relevance of AstA/AstAR1 signaling by evaluating AstAR1 expression both in IPCs and PTTHn during early and late larval development. When driven with AstAR1-Gal4, GFP accumulates in the IPCs at constant levels during larval development, suggesting that AstAR1 expression is not controlled by developmental cues in these cells (Figure 6A). By contrast, we detected progressively increasing GFP signal in the PTTHn during larval development (Figure 6B). Indeed, an up-regulation of AstAR1 transcript levels is observed by RT-qPCR on whole larvae towards the end of larval development (Figure S5A). We did not observe any obvious remodeling of AstAn, as shown using an AstA>GFP reporter (Figure S5B). However, AstA immunoreactivity within AstA-N1 dramatically increases during larval development (Figure 5C). This is concomitant with the observed increase in PTTH as previously reported [START_REF] Yamanaka | Neuroendocrine Control of Drosophila Larval Light Preference[END_REF] . We could not quantify AstA peptide levels specifically in AstA-N2 as they are below detection threshold (Figure S4C). Since neuropeptides are often actively secreted by an increase in intracellular calcium [START_REF] Sudhof | Calcium control of neurotransmitter release[END_REF], we used the CaLexA calcium reporter [START_REF] Masuyama | Mapping Neural Circuits with Activity-Dependent Nuclear Import of a Transcription Factor[END_REF] as a marker of neurosecretory activity in these neurons. Indeed, we found increasing levels of CalexA-induced GFP in AstA-N1, suggestive of an increasing neurosecretory activity towards the end of larval development (Figure 6D). In summary, our data indicates that AstA signals in the IPCs promotes growth throughout larval development and in a temporally-regulated fashion in the PTTHn to induce maturation at the end of larval period (Figure 6E).

Discussion

Coordination of growth and maturation by AstA signaling

During juvenile development, both the rate and the duration of growth affect final adult size. It is therefore important to clarify the mechanisms that coordinate growth rate and growth duration. Studies in Drosophila have revealed that PTTH plays a role in such coordination. After larvae reach a so-called critical weight, ptth transcripts rise, subsequently inducing a rise in ecdysone biosynthesis. In addition to this transcriptional control, we now identify AstA/AstAR1 as a signal controlling PTTH secretion. One question remains: how is the control of PTTH production coordinated with the growing status? A possible mechanism could use Dilp8/LGR3 signaling, which delays the onset of metamorphosis in response to tissue damage. Whether this signal interferes with PTTH function during normal development still needs to be clarified [START_REF] Colombani | Drosophila Lgr3 Couples Organ Growth with Maturation and Ensures Developmental Stability[END_REF][START_REF] Garelli | Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing[END_REF]. In this study, we demonstrate that AstA signalling not only times the onset of maturation by regulating PTTH secretion, but also induces larval growth by promoting Dilp secretion. Interestingly, blocking AstA signaling in the IPCs reduces larval growth rate, but this is compensated for by extending the growth period through PTTHn, allowing animals to reach normal body weight. Interestingly, another receptor for AstA, AstAR2 has been recently reported to link AstA signaling with the metabolic status in the adult IPCs [START_REF] Hentze | The Neuropeptide Allatostatin A Regulates Metabolism and Feeding Decisions in Drosophila[END_REF]. Thus, AstAn, PTTHn and IPCs likely define a homeostatic neural circuit that coordinates growth/metabolism and maturation timing.

AstA and KISS: a conserved neural circuitry controling maturation

AstA was initially described as an arthropod-specific hormone inhibiting food intake and juvenile hormone (JH) secretion [START_REF] Lechner | A Genetic Method for Selective and Quickly Reversible Silencing of Mammalian Neurons[END_REF]. However, a comprehensive evolutionary study of the AstA receptor 1 gene revealed that it shares an evolutionary ancestor gene with the mammalian GPR54 receptor gene [START_REF] Felix | Unravelling the Evolution of the Allatostatin-Type A, KISS and Galanin Peptide-Receptor Gene Families in Bilaterians: Insights from Anopheles Mosquitoes[END_REF]. Moreover, the Drosophila AstA peptide and the human KISS peptide share a conserved FGL motif, suggesting that AstA and KISS could originate from a common ancestor [START_REF] Felix | Unravelling the Evolution of the Allatostatin-Type A, KISS and Galanin Peptide-Receptor Gene Families in Bilaterians: Insights from Anopheles Mosquitoes[END_REF]. As previously mentioned, the KISS/GPR54 pathway promotes pulsatile secretion of GnRH, a necessary event for steroid production and sexual maturation in vertebrates [START_REF] Navarro | Advanced vaginal opening and precocious activation of the reproductive axis by KiSS-1 peptide, the endogenous ligand of GPR54[END_REF]. In this study, we identify the AstA/AstAR1 pathway as part of the timer for PTTH secretion, ecdysone production and the onset of sexual maturation. Our findings present unexpected functional parallels with the role of the KISS/GPR54 pathway at the onset of puberty: (i) KISS expression rises during pre-pubertal stages to induce maximum secretion of GnRH [START_REF] Navarro | Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor, GPR54, in rat hypothalamus and potent luteinizing hormone-releasing activity of KiSS-1 peptide[END_REF]; (ii) hypothalamic levels of GPR54 mRNA increases dramatically at pre-puberty stage [START_REF] Han | Activation of Gonadotropin-Releasing Hormone Neurons by Kisspeptin as a Neuroendocrine Switch for the Onset of Puberty[END_REF]; (iii) an increase in KISS-GPR54 signaling occurs during puberty, due to an absence of desensitization to KISS stimulus [START_REF] Roa | Desensitization of gonadotropin responses to kisspeptin in the female rat: analyses of LH and FSH secretion at different developmental and metabolic states[END_REF]. In parallel with this, we find that Drosophila AstA peptide levels rise at the end of development, anticipating the rise in PTTH levels and the onset of metamorphosis. Concomitantly, expression of AstAR1 in PTTHn increases suggesting that these neurons become more sensitive to the AstA signal just before metamorphosis. Further experiments should bring more insight in how AstA protein levels and AstAR1 transcript rise anticipating the onset of metamorophosis. Another feature observed in mammals is the distribution of two KISS neuron subpopulations in the hypothalamus: one in the arcuate nucleus (ARC), and one in the rostral periventricular area of the 3rd ventricle (RP3V). Different roles for the ARC and RP3V KISS neurons have been allocated in either initiation or progression of puberty [START_REF] Mayer | Timing and completion of puberty in female mice depend on estrogen receptor alpha-signaling in kisspeptin neurons[END_REF], but the regulation of these differential actions is poorly understood [START_REF] Pinilla | Kisspeptins and Reproduction: Physiological Roles and Regulatory Mechanisms[END_REF]. Similar to this, we describe here two separate

METHOD DETAILS

RNAi screen in PTTHn

We performed a biased genetic screen to identify to identify positive regulators of PTTHn. We used the NP423-Gal4 line to screen a set of RNAi lines from the Vienna Drosophila RNAi Center (VDRC) for selected GO (Gene Ontology) terms present in the brain: membrane targeted proteins, nuclear receptors, vesicular mediated neurotransmitter components. 1300 lines were tested on their ability to delay metamorphosis. We tested 300 lines per week by crossing 16 virgins containing the NP423-Gal4 driver with 10 males of each RNAi lines. Three replicates were collected from each cross with collections of 4 h. 270 candidates significantly affected developmental timing. In a secondary screen, positive RNAi lines were combined with ptth> ptth>dcr2 tester line. Only one hit presented a strong and robust developmental delay of metamorphosis, AstAR1.

Measuring developmental timing

All developmental timing experiments were conducted at 25°C on standard cornmeal food without added sugar. Embryos were collected after 4 hours of egglaying carried out on agar dishes containing 3% sugar and were incubated at 25°C for 21 hours after which the L1 larvae were transferred into tubes containing standard cornmeal without added sugar. Pupae number from 4 tubes per genotype were scored 3 times a day and ordered by progressive pupariation time and plotted in GraphPad or Microsoft Excel using non-linear regression curve fit.

AstA neuron activation by TrpA1

AstA neurons were activated in a temporal manner by expressing TrpA1. TshG80-AstA>TrpA1 animals were synchronized at the L2 to L3 transition. Larvae were then kept at 25°C and transferred to 29°C after 12 hours to activate the TrpA1-expressing neurons.

Pupariation was then monitored by counting pupae over time.

Pupal volume and growth rate measurements

Larval growth rate was measured by sorting larvae at the transition of L2 into L3 and transferred to standard cornmeal food. At 80, 97 and 100 hours AEL, images of the larvae were captured under bright light by using the Leica M205 FA. Similar, to obtain pupal volume, pupae were aligned in a petridish and captured under bright light. Pupal or larval length (L) and width (W) were obtained with ImageJ by measuring the medial line between anterior-posterior, and by measuring along the axial line, respectively. Pupal or larval volume was calculated in Microsoft Excel by using the formula for prolate spheroid: (π/6)W 2 *L.

Values were normalized to one control condition to give the "∆ pupal volume". Data obtained was plotted in GraphPad to obtain bar graphs.

Ecdysone titers

Biological replicates of 4-15 larvae were homogenized twice in methanol and cleared by centrifugation to give a final combined volume of ∼450 µl. Duplicate samples were dried and resuspended in 50 µl EIA buffer (spi bio 20-HE ELISA kit -A05120). Spi Bio Kit for 20-HE ELISA were used as per manufacturer's recommendation. The standard curve was determined using GraphPad Prism software, non-linear regression curve fit, asymmetric sigmoidal, 5PL, X is log (conc). Per animal, E titers were adjusted for the volume used in the assay.

Rescue by 20-hydroxyecdysone feeding

Forty freshly ecdysed AstA>dcr2 or AstA> AstA-RNAi.dcr2 L3 larvae, grown at 25°C were transferred to a new vial. After 20 h, larvae were washed and transferred to a vial supplemented with either 20-hydroxyecdysone (Sigma; dissolved in 95% ethanol, final concentration 0.2 mg/ml) or 95% ethanol (same volume as 20-HE). Once seeded with L3 larvae, the vials were returned to 25°C and monitored for pupariation.

Immunostaining

Dissected brains were fixed in 4% formaldehyde (Sigma) for 25 min at RT, washed in PBT (0,1% Triton X-100 in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4, [pH 8])) and blocked in PBT containing 10% FBS for 40 min. Brains were incubated in PBT containing the primary antibody overnight at 4°C, washed three times in PBT, incubated with the secondary antibody for 2 hours at 4°C, washed and mounted in Vectashield mounting media with DAPI (Vector laboratories). The following primary antibodies were used: α-PTTH at (1:400, guinea pig), α-dilp2 (1:400, rat), α-AstA (1:20, mouse anti Ast7 cockroach, DSHB #5F10), α-AstA (1:100, rabbit against cockroach A-AST, Jena Bioscience #ABD-062) α-GFP (1:10.000, chicken), α-GFP specifically recognizing reconstituted GRASP (1:100, mouse, Sigma). Secondary antibodies that were used: Alexa Fluor 488 α-chicken (1:400, Thermo Fisher Scientific), Alexa Fluor 488 α-mouse (1:400), Alexa Fluor 546 α-rat (1;400), Alexa Fluor 648 α-guinea pig (1:400), Alexa Fluor 648 αrabbit (1:400). Fluorescent images were acquired with Leica TSC SP5 using the 20x or 40x objective and analyzed using ImageJ.

Quantitative RT-PCR

Larvae were washed in PBS and frozen in liquid nitrogen. Total RNA was extracted by using RNeasy lipid tissue Minikit (QIAGEN) according to the manufacturer's protocol. RNA samples were treated with DNase and then reserve transcribed to cDNA using the SuperScript II reverse transcriptase (Invitrogen). cDNA samples were then used for RT-qPCR (StepOne Plus, Applied Biosystems) upon using PowerSYBRGreen PCR mastermix (Applied Biosystems). dilp2 and ptth transcript levels were obtained by ΔΔCT method using rpl49 as an internal control. In Microsoft Excel, unpaired Student's T tests were used to compare two sets of data and to obtain statistical values. Primers were designed by using the NCBI/Primer-BLAST.

Dilp2 retention in IPCs

Dilp2 levels in the IPCs were quantified upon staining larval brains at 90 hr AED with α-dilp2

(1:400, rat) as described above. Confocal Z stack images were acquired at 12 bit using 0,5 μm step size with identical laser power and scanning settings. Using ImageJ, sum-intensity of Dilp2 in the IPCs was measured to obtain the area and mean intensity value.

Circulating PTTH and Dilp2 in the hemolymph

Total circulating levels of PTTH and Dilp2 in the hemolymph were quantified by sandwich ELISA. F8 Maxisorp loose Nunc-Immuno modules (Thermo Scientific #469949) were coated and incubated overnight at 4°C with α-FLAG (2.5 μg/ml, Sigma-Aldrich F1804) (for Dilp2) or α-PTTH (1:10) in coating buffer (0,2M sodium carbonate/bicarbonate buffer, pH 9,4; BuPH Carbonate-Bicarbonate buffer packs, ThermoFisher #29892). Plates were washed with PBTw0,2% (0,2% Tween-20 in PBS) and blocked with filter sterilized PBS containing 2%

  AstA neurons with different functions. The sole inactivation of AstA-N1 is sufficient to induce a delay, indicating that it has clear timing function. The role of AstA-N2 in timing maturation is not yet established. Future research will be needed to better understand the differential function of AstAn and incoming regulatory signals for AstA-N1 and AstA-N2. AstA/AstAR1 signaling increases with larval volume and could respond to a size threshold. Our experiments suggest a concommitent action of AstA/AstAR1 signaling on growth and maturation. Interstingly, several studies point to the role of Kisspeptin on growth hormone (GH) secretion from the anterior pituitary gland (Luque RM, 2011, Kadokawa H, 2008., Chang JP, 2012., Gutiérrez-Pascual E, 2007).Given the remarkable molecular and functional conservation between KISS and AstA signaling for the control of maturation and growth, the regulation of Drosophila steroid production by AstA signaling should provide further mechanical insights on this major developmental transition.

Figure titles and legendsFigure 1 .

 1 Figure titles and legends

Figure 2 .

 2 Figure 2. AstA-R1 signaling controls the secretion of Dilp2 and PTTH.

Acknowledgements

We thank Gisele Jarretou, Thomas Phil and Alessandra Mauri for technical assistance. We thank all members of the laboratory and the fly community for insightful discussions and comments on the manuscript. We thank the Vienna Drosophila RNAi Center, the Drosophila Genetics Resource Center, the Bloomington stock Center and Kyoto Stock Center for providing Drosophila lines. This work was supported by the CNRS, INSERM (Junior Researcher grant 2015/6 to N.R.), European Research Council (Advanced grant no. 268813 to P.L.), ARC (grant n° PGA120150202355 to P.L.), and the Labex Signalife program (grant ANR-11-LABX-0028-01 to P.L.). F.A.M. is a recipient of a RyC-2014-14961 contract and supported by BFU2014-54346-JIN.

Declaration of interests

The authors declare no competing financial interests. via one-way ANOVA by Graphpad Prism (*p<0,05; **p<0,01; ***p<0,001, ****p<0,0001).

Error bars are represented as mean ± SEM. See also Figure S2.

STAR★Methods

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Nuria M Romero (Nuria.ROMERO@univ-cotedazur.fr)

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Drosophila melanogaster strains were raised at 25°C on standard cornmeal food containing, per liter, 17 g inactivated yeast powder, 83 g corn flour, 10 g agar, 60 g white sugar, and 4.6 g Nipagin. Male and female larvae and pupas were used in all experiments. The following transgenic and mutant flies were used: ptth-Gal4 [START_REF] Mcbrayer | Prothoracicotropic Hormone Regulates Developmental Timing and Body Size in Drosophila[END_REF], Dilp2-Gal4 [START_REF] Brogiolo | An evolutionarily conserved function of the drosophila insulin receptor and insulin-like peptides in growth control[END_REF][START_REF] Rulifson | Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes[END_REF], Tsh-Gal80 [START_REF] Berni | Autonomous Circuitry for Substrate Exploration in Freely Moving Drosophila Larvae[END_REF],

ptth-HA genomic [START_REF] Shimell | Prothoracicotropic hormone modulates environmental adaptive plasticity through the control of developmental timing[END_REF], Ilp2HF [START_REF] Park | A Genetic Strategy to Measure Circulating Drosophila Insulin Reveals Genes Regulating Insulin Production and Secretion[END_REF], Dilp2-LexA [START_REF] Li | Cold-sensing regulates Drosophila growth through insulin-producing cells[END_REF], AstAR1-Gal4 [START_REF] Yamagata | Suppression of Dopamine Neurons Mediates Reward[END_REF], UAS-TRPA1 [START_REF] Hamada | An internal thermal sensor controlling temperature preference in Drosophila[END_REF] From Vienna Drosophila RNAi Center we obtained the following lines: AstA-RNAi (#14398), AstAR1-RNAi (#39222), ptth-RNAi (#102043), UAS-dicer2 (#60008) or (#60009). NP423-Gal4 (#103614, Drosophila Genetic Resource Center, Kyoto). The following lines were obtained from Bloomington Drosophila Stock Center: AstA-Gal4 (#39351), op-LexA-GFP (#58754), UAS-RFP (#58754), CoinFLP-LexA::GAD (#58754), UAS-Kir2.1 (#6595), UAS-BSA overnight at 4°C. The next day, plates were washed with PBTw0,2% before adding hemolymph.

Hemolymph was obtained by bleeding washed larvae on ice, and collected in 50 μL ice cold PBS. Tubes containing the hemolymph were centrifuged at 1,000 x g for one minute and supernatant was collected. The samples were then mixed with α-HA-Peroxidase, High Affinity (3F10) (Roche #12013819001) at a dilution of 1:500 in PBTw0,2% and incubated in the blocked Nunc-Immuno modules overnight at 4°C. The next day, samples were aspirated and the plates were extensively washed with PBTw0,2%. Next, 1-Step Ultra TMB-ELISA Substrate (Thermo Scientific #34029) was added into the wells and incubated for 30 min at RT. The reaction was stopped by adding 2M sulfuric acid and absorbance was measured immediately at 450nm.

Quantification PTTH bouton in PG

PTTH content per bouton was quantified upon staining wandering larvae with α-PTTH as described above. Confocal Z stack images were acquired at 12 bit using a 0,5 μm step size with identical laser power and scanning settings. Using ImageJ, sum-intensity of the PTTH boutons was measured to obtain pixel intensity and area. PTTH per bouton was obtained by pixel intensity * area. The total number of boutons were counted also in ImageJ. PTTH axonal crosses were obtained by using an adaptation of the Sholl's method, by counting the number of each PTTH projection that crossed the centric ring in the hemicircle, starting from the PG [START_REF] Sholl | Dendritic organization in the neurons of the visual and motor cortices of the cat[END_REF].

QUANTIFICATION AND STATISTICAL ANALYSIS

All


Wahiba Ghezali Ramdani

Ayman Karam

Karine De Oliveira Vigier

Sébastien Rio

Anne Ponchel
email: anne.ponchel@univ-artois.fr

François Jerome
email: francois.jerome@univ-poitiers.fr

François Jérôme

Catalytic glycosylation of glucose with alkyl alcohols over sulfonated mesoporous carbons

Keywords: Biomass, Catalysis, Carbohydrates, Glycosylation, Mesoporous carbon

Introduction

With the exponential growth of the world population, and our concerns about climate changing, the chemical industry needs to produce always more and better from resources that are declining or becoming less and less accessible. In this context, carbohydrates represent a huge reservoir of renewable carbon from which a myriad of chemicals can be theoretically produced. [1][2][3][4][5][6][7][8][9][10][11] The implementation of sugar-based processes on a large scale is far from being an easy task and it requires scientists to overcome scientific and technological bottlenecks. For instance, carbohydrates tend to form tar-like materials, also commonly named humins, which not only decreases the overall selectivity of the catalytic process but also leads to a severe deactivation of solid catalysts. [12][13] As a consequence, the space time yield of sugar-based processes are often too low to be competitive with petrochemical processes, for which chemistry has developed very efficient technologies over more than 70 years.

The catalytic Fischer glycosylation of glucose with fatty alcohols is one of the important sugarbased reactions deployed at an industrial scale. [14][START_REF] Balzer | Nonionic surfactants: alkyl polyglucosides[END_REF][START_REF] Rybinski | [END_REF][17][18][19][20][21] This reaction is catalyzed by sulfuric acid and yields amphiphilic alkyl glycosides (AAG) that are valuable biosurfactants, with current applications in the cosmetic and detergence industries. The price of AAG is about 1.5-2 €/kg, which is still too high to compete with fossil derived surfactants exhibiting similar performances.

Optimization of the catalytic Fisher glycosylation reaction is one of the options to decrease the production cost of AAG. In particular, the replacement of H2SO4 by robust, active and recyclable solid acid catalysts is a possible strategy. Beside the intrinsic catalytic performances of solid acids, the absence of neutralization step at the end of the reaction and the easiness in recovery/recycling of solid catalysts represent additional advantages over H2SO4.

From the view point of catalysis, the direct glycosylation of unprotected carbohydrates with fatty alcohols is a difficult reaction. In particular, improving the catalyst activity is essential in order to assure that the glycosylation rate of carbohydrates is higher than their thermal degradation rate to humins, as above mentioned these tar-like materials leading to a rapid deactivation of catalysts. In the field of heterogeneous catalysis, this reaction is even more complex to kinetically control because carbohydrates and fatty alcohols are bulky substrates, which restrict to some extent, their accessibility to the grafted or anchored catalytic sites, thus reducing the catalyst activity.

With these criteria in mind, microporous catalysts and gel-type resins were previously reported. Two catalytic strategies can be distinguished in the current literature. The first option consists in synthesizing AAG by the direct catalytic Fischer glycosylation of glucose with fatty alcohols under biphasic conditions. In this case, zeolite H-BEA (Si/Al = 25), [22] ZnFe2O4 supported on ZrO2, [23] H2SO4/SiO2, [24,25] polyvinyl bound tri-and disulfonate ethylamine chloride [26] and Aquivion PFSA [27] have been previously investigated and good to excellent yields were claimed. Cellulose, and even lignocellulosic biomass, were also successfully employed as a source of glucose in the synthesis of AAGs through glycosylation reaction.

[28] In order to overcome the low miscibility of carbohydrates with fatty alcohols, a second strategy was explored in the literature. It consists in the transglycosylation of pre-synthesized methyl-or butylglycosides with fatty alcohols. Catalysts of the type Amberlyst-15, Nafion SAC-13, zeolite ITQ-2, sulfonated carbons, sulfonated graphene oxides and heteropolyacids were typically used in such approach [28d, 29]. Even though the catalytic transglycosylation of methyl/butylglycosides with fatty alcohols generally led to catalytic reactions with higher space time yields than the direct glycosylation of glucose with fatty alcohols, it has however the main drawback of requiring an extra synthetic step. One should however mention that, in some cases, the glycosylation and transglycosylation reactions were advantageously combined in a successive way in a single reactor, thus avoiding intermediate purification. [28d] In all these examples, the catalyst activity is depending on (i) the strength of acid sites (Bronsted or Lewis) and (ii) the accessibility of acid sites, which is often improved by the presence of micropores or by the ability of catalysts to swell into the reaction media. The release of water as a co-product and the formation of tar-like materials impact, to a more or less extent, the long term stability of catalyst (catalyst coking, surface hydrolysis, etc.). Whatever the catalytic routes (glycosylation vs transglycosylation), good results were generally claimed in terms of yield and selectivity but the stability, the recyclability and the space time yields of these solid catalysts needs to be further improved to be more competitive with H2SO4, the reference catalyst used at the industrial scale for such reaction. [14] In the field of heterogeneous catalysis applied to biomass, carbon-based catalysts are attracting growing interest. [30][31][32][33][34][35][36][37][38][39] The improved stability of carbon materials in the presence of water (common co-product or solvent in carbohydrate chemistry), their ease of surface functionalization, the possibility to tune their surface polarity and prepare them at a nanoscale represent important features which were previously taken as advantages in the catalytic conversion of carbohydrates [40][41]. From all these data, it occurred to us that sulfonated mesoporous carbons could be promising catalysts for the direct catalytic glycosylation of glucose with fatty alcohols, the presence of mesopores being expected to dramatically facilitate the diffusion of carbohydrates within the catalyst backbone while the carbonaceous skeleton should confer to the catalyst a greater stability in the presence of water than metal oxides for instance.

As recently reviewed by Doustkhah et al., sulfonated carbons have been used as solid acid catalysts in a wide range of reactions such as acetalization, C-C bond couplings, Fries and Beckman rearrangement, etc [42]. To the best of our knowledge, the catalytic activity of sulfonated mesoporous carbons has never been reported in the direct Fisher glycosylation of carbohydrates with fatty alcohols, although this family of catalyst gathers the main criteria to be efficient in such reaction. Mesoporous carbons are typically prepared by the so-called hardtemplating synthesis. The mesoporous carbons are thus the negative replica of an inorganic hard template used as mold. Typically, cubically mesoporous MCM-48 aluminosilicate or mesoporous silica such as 3D cubic cage-like SBA-1 and 2D hexagonal SBA-15 were used as an inorganic template and afford mesoporous carbons referred as CMK-1, CMK-2 and CMK-3, respectively [43,44]. Then, these mesoporous carbons can be sulfonated according to different strategies, including sultone, sulfuric acid, etc [42].

In this communication, we thus investigate the catalytic performances of various sulfonated mesoporous carbons in the direct glycosylation of glucose with n-dodecanol. Sulfonated mesoporous carbons were then benchmarked to previously reported solid acid catalysts, and also from various carbohydrates and alkyl alcohols, to assess their potential but also their limitations.

Experimental section

In a typical procedure, glucose (1.0 g, 5.5 mmol) was mixed with n-dodecanol (10 g, 53 mmol) in a 50 mL round-bottom flask, equipped with a magnetic stirring bar. Then, an acid catalyst (1.8 mol % of -SO3H) was added and the solution was heated in an oil bath at 393K under vacuum (15 mmHg) for the desired reaction time. The reaction was monitored by gas chromatography on a Varian 3900 instrument equipped with a flame ionization detector and an HT5 column (30 m x 0.32 mm x 0.25 μm). Before analysis, AAGs were silylated as described in the supporting information. After complete conversion of glucose, the catalyst was filtered off and the excess ndodecanol was removed under vacuum. For recycling experiments, the solid acid catalyst was recovered by filtration at the end of the reaction, washed with ethanol, dried in an oven at 60°C and then re-used as is without any further purification. 1 H and 13 C NMR spectra of the asobtained AAGs are provided in Figures S13 andS14.

Results and discussion

Sulfonated mesoporous carbons were prepared through a nanocasting method adapted from the procedures of Ryoo [43] and Wu [44]. Briefly, the experimental protocol involves (i) the preparation of a mesoporous SBA-15 mold, (ii) the impregnation/infiltration of the sacrificial SBA-15 with an aqueous solution of sucrose containing sulfuric acid, (iii) a thermopolymerization reaction at 373 K and 433 K followed by a pyrolysis under N2 at a controlled temperature and (iv) the removal of the SBA-15 mold by treatment with 1M NaOH in a mixture H2O/ethanol (1/1). According to the temperature of pyrolysis, different CMK-3 samples were obtained and referred as CMK-3-T, with T being the pyrolysis temperature (723, 823, 923, 1073 and 1173 K). The as-obtained CMK-3-T mesoporous carbons was then sulfonated by reaction with ClSO3H/H2SO4 at 353 K for 20 h affording the CMK-3-T-SO3H catalyst. To assess the utility of mesopores in the catalytic experiments, an amorphous carbon, named AC-723-SO3H, was prepared in the same way as CMK-3-723-SO3H, but without using SBA-15 as hardtemplate. Detailed experimental procedures and full characterization have been placed in the supporting information (Table S1 and figure S1-S10). The textural properties determined by N2 adsorption-desorption and proton exchange capacities determined by treatment with a KCl solution (H3O + /K + exchange and titration of released HCl) of all prepared sulfonated carbons are summarized in Table 1. BET [b] (m 2 /g) Pore diameter

(nm) [c] Pore volume (cm 3 /g) [d] Mesopore volume (cm 3 /g) [c] CMK- [a] the -SO3H loading was determined by suspending sulfonated carbons in a KCl solution (exchange H3O + /K + ) followed by a titration of released HCl with NaOH (see SI for more details); [b] Specific surface area calculated from the Brunauer-Emmet-Teller equation in the P/P 0 range of 0.025-0.20; [c] calculated by the Barrett-Joyner-Halenda method; [d] Total pore volume estimated at P/P 0 = 0.95.

Although high pyrolysis temperatures (>823 K) generated CMK-3-T materials with a welldefined mesoporous structure, it was however not beneficial for the subsequent introduction of sulfonic acid groups in a sufficient amount for catalytic applications (Table 1). As a general trend, the higher the pyrolysis temperature, the lower the -SO3H loading, and the higher the mesopority. This result is consistent with previous reports and with our FT-IR, TEM, XRD and N2 adsorption investigations presented in the supporting information. This is actually attributed to a progressive disappearance of sites favorable for the subsequent functionalization of the CMK-3 surface with -SO3H groups at high pyrolysis temperature due to a more complete carbonization of the precursor forming large carbon sheets which generated, in return, a higher mesoporosity [45]. Hence, for a catalytic application, a compromise has to be made between the amount of -SO3H groups and the mesoporosity of the carbon support. Well-ordered carbons are indeed attractive but they bear a negligible amount of -SO3H which means that high amount of catalyst should be used in this case, which is, in our views, not realistic for the present application. In this context, only two CMK-3-T-SO3H materials (with T=723 K and 823 K) have been selected for catalytic experiments.

In catalytic trials, 1 g of glucose was mixed with 10 g of n-dodecanol and heated at 120°C under vacuum (15 mmHg) in the presence of 1.8 mol% of -SO3H sites grafted either on CMK-3-T (T = 723 and 823) or on AC-723. The reaction was monitored by gas chromatography, allowing the conversion, yields and selectivity to be determined with an experimental error of ± 5%. Results are summarized in Table 2. A typical kinetic profile of the reaction is provided in the Figure S11.

Table 2. Optimal results in the catalytic glycosylation of glucose with n-dodecanol at 120°C [a] Entry Catalyst Time (min)

Conv. (%)

TOF (h -1 ) [b] TON [c] Yield (%) [d] Space Time Yield (kg.m - 3 .h -1 ) [e] 1 CMK-3-723 [e] 180 86 nd [f] --- [a] 120°C, 1.8 mol % of -SO3H, n-dodecanol/glucose mass ratio = 10; [b] determined at a conversion of glucose lower than 20%; [c] determined at the maximum yield in AAGs; [d] other product are dodecyclpolyglycosides and polydextrose; [e] it corresponds to the mass of product converted per volume of solution and per unit of time (determined from the AAG yield); [f] not determined, only tar-like materials were formed; [g] 15 wt% (Si/Al = 25).

Because non-sulfonated CMK-3-T materials (with T=723 K and 823 K) bear (only) weak acid sites (-COOH groups), these materials were tested first as control catalysts. Over CMK-3-723, in the above described experimental conditions, 86% of glucose was converted after 3 h of reaction, but no formation of AAGs was observed. Only tar-like materials were produced in this case (Table 2, entry 1). Reversely, over CMK-3-723-SO3H, glucose was completely converted after 3 h of reaction but AAGs were obtained with 85% yield, confirming that strong acid sites are required for the glycosylation of glucose with n-dodecanol (Table 2, entry 2). Other products formed were dodecylpolyglucosides resulting from the slight oligomerization of glucose on ndodecanol. As determined by gas chromatography, produced AAGs were formed as a mixture of dodecyl glucopyranoside and dodecyl glucofuranoside in a ratio pyranoside/furanoside of 20.

Both forms existed also as two anomers (α and β). The α/β ratio was 2 for the major dodecyl glucopyranoside form.

The turn over frequency (TOF) and the turn over number (TON) of CMK-3-723-SO3H were 110 h -1 and 50, respectively (Table 2, entry 2). Under these conditions, the space time yield reached 48 kg.m -3 .h -1 , which is in line with industrial values of the field. Extrapolation of these results to a reactor of 10 m 3 , running 8 000 h per year (typical size of reactor in this field of chemistry), suggests a theoretical production capacity of 3 840 tons per year and per reactor. It is worth noting that CMK-3-823-SO3H afforded similar results, although it was necessary to use a larger amount of catalyst to keep the -SO3H mol% identical in both trials (Table 2, entry 3).

To benchmark CMK-3-723-SO3H, the catalytic performances were further compared under the same conditions to a series of solid acid catalysts previously published in the literature. SBA-SO3H did not yield AAGs due to its rapid deactivation caused by the stoichiometric release of water (surface hydrolysis and/or -SO3H solvation with water, as previously observed [46]), leading to the major thermal degradation of glucose to humins. (Table 2, entry 5). Amberlyst-15, often used in acid-catalyzed reactions, was unfortunately not stable at 120°C and it was also quickly degraded in our experimental conditions, leading again to the major formation of tar-like materials (10% AAG yield at 60% of conversion of glucose, Table 2, entry 6). In contrast, H-BEA zeolite afforded AAGs in 59% yield but with a very low space time yield of 2 kg.m -3 .h -1 (Table 2, entry 7), which may be rationalized by the presence of acid sites with a lower acid strength than -SO3H groups. Interestingly, in terms of catalytic properties, CMK-3-723-SO3H was found as performant as Aquivion PFSA PW 98, one of the best solid catalysts reported so far for this reaction (Table 2, entry 4). [27,28] However, at the end of the reaction, the CMK-3-723-SO3H has a much lower settling time than Aquivion PFSA, this later being swelled in a very large extent in alkyl alcohol. As a consequence, the recycling procedure of CMK-3-723-SO3H was technically much simpler than that of Aquivion PFSA. Recyclability of CMK-3-723-SO3H is discussed later in the manuscript. Interestingly, the CMK-3-723-SO3H catalyst has a TOF in a similar range to that of H2SO4 (Table 1, entry 8). Although the space time yield was lower than the one obtained with H2SO4, it is noteworthy that the maximum yield of AAG was higher with CMK-3-723-SO3H (85%) than with H2SO4 (74%) (Table 1, entries 2 and 8). CMK-3-723-SO3H was next compared to AC-723-SO3H in order to highlight the importance of mesopores in the catalyst performances. Under identical conditions, AC-723-SO3H was nearly not active achieving a very low yield in AAGs of 8% after 3 h of reaction (Table 1, entry 9). Analysis of the used AC-723-SO3H revealed a drastic decrease in the BET surface from 110 to only 3 m 2 /g, suggesting the deposition of tar-like materials on the surface of AC-723-SO3H. In contrast, CMK-3-723-SO3H was successfully recycled at least 8 times with only a slight decrease of the AAGs yield from 85% to 80% (i.e. within the experimental error) which unambiguously demonstrated the usefulness of mesopores as regards accessibility of -SO3H to the reactants and resistance to deactivation (Figure 2). The used CMK-3-723-SO3H (after 1 catalytic cycle) was analyzed by FT-IR and the presence -SO3H sites was still clearly visible with typical bands between 1000 and 1400 cm -1 , as shown in Figure S12. The determination of the H3O + exchange capacity showed a slight decrease of the -SO3H loading from 0.64 to 0.52 mmol/g which may be explained by (1) a -SO3H leaching or (2) a deposition of tar-like materials on the CMK-3-723-SO3H surface (i.e. dilution of -SO3H). By

AAG yield (%)

Catalytic run means of ICP analysis, no trace of sulfur was found into the solution suggesting that the slight decrease in the amount of -SO3H sites was presumably due to the partial deposition of tar-like material on the CMK-3-723-SO3H surface. Note that the total inhibition of tar-like materials is nearly impossible to achieve in such reaction and a deactivation of the catalyst will certainly occur in the long term. For instance, the BET surface dropped from 618 to 130 m 2 /g after 8 cycles. However, in contrast to non-mesoporous catalysts, the presence of mesopores seems limiting the deposition of tar-like materials.

To further confirm the absence of -SO3H leaching, a hot filtration test was performed. To this end, the CMK-3-723-SO3H catalyst was filtered after 20% conversion and the solution was kept heating at 120°C. No formation of AAGs was observed after removal of the CMK-3-723-SO3H catalyst, thus confirming that, in our case, the catalytic reaction was truly of heterogeneous nature.

The scope of CMK-3-723-SO3H was next generalized to other alkyl alcohol and carbohydrate derivatives (Table 3). Replacing n-dodecanol by n-hexadecanol had no effect on the catalytic performances of CMK-3-723-SO3H and AAGs were obtained in 89% yield after 180 min of reaction (Table 3, entry 1). This is an important result since it has been shown that increasing the chain length of AAGs improved their foaming properties and lowered their toxicity, in particular irritability for eyes. [47] From short chain alkyl alcohols such as methanol for instance, the reaction still proceeded well, achieving an AAG yield of 70% (Table 3, entry 2). Regarding carbohydrates, the substitution of glucose by mannose or galactose in the catalytic glycosylation reaction with n-dodecanol was found feasible over CMK-3-723-SO3H, but afforded AAGs with a lower yield of 50% and 30%, respectively, indicating that the reaction conditions initially defined for glucose are not the optimized ones for mannose and galactose (Table 3, entries 3, 4).

These carbohydrates are indeed known to be more sensitive to the reaction temperature (thermal degradation) than glucose. In this context, the same reaction was conducted from mannose at 100°C instead of 120°C, affording the corresponding AAGs with 95% yield (Table 3, entry 5). 5 [c] Mannose n-dodecanol 120 100 95

[a] 120°C, 1.8 mol % of -SO3H (CMK-3-723-SO3H), n-alkyl alcohol/glucose mass ratio = 10; [b] reaction was performed at reflux of methanol; [c] 100°C.

Conclusion

Here, we investigated the catalytic performances, in terms of yield, selectivity, space time yield and recyclability, of CMK-3-723-SO3H and CMK-3-823-SO3H in the Fischer glycosylation of carbohydrates with alkyl alcohols. We were pleased to see that CMK-3-723-SO3H exhibits catalytic performances superior to the commonly used solid acid catalysts. The presence of mesoporous channels resulted in a high catalyst activity. As a consequence, the catalytic glycosylation rate of carbohydrates with alkyl alcohols was greatly improved, to the detriment of the thermal degradation of carbohydrate which remained drastically limited in this case, thus preventing the CMK-3-723-SO3H catalyst from deactivation and allowing its recycling for at least 8 consecutive catalytic cycles. In contrast to Aquivion PFSA, one of the best solid catalyst reported so far for this reaction, CMK-3-723-SO3H has the advantage of being much more easily recovered at the end of the reaction, the recovery of Aquivion PFSA being hampered by its very important swelling leading to a rapid filter clogging. With CMK-3-723-SO3H, AAGs were obtained with 85-95% yield while the space time yields of the glycosylation reaction remained in line with those expected at an industrial scale in this field of research. Being able to prepare mesoporous carbons with a high mesoporosity and high -SO3H loading represents, in our views, an important perspective to this work to prepare even more performant catalysts. ASSOCIATED CONTENT Supporting Information. Materials, Catalysts preparation, FT-IR characterization, Determination of the H3O + exchange capacity, N2 adsorption experiments, XRD analysis, TEM analysis, 1 H and 13 C NMR spectra and kinetic profile of the reaction.

Figure 2 .

 2 Figure 2. Catalytic recycling of CMK-3-723-SO3H (120°C, 1.8 mol % of -SO3H, n-dodecanol/glucose mass ratio = 10; between each cycle, CMK-3-723-SO3H was washed with ethanol)

Table 1 .

 1 Characterization of carbon-based materials

	Catalyst	-SO3H
		(mmol/g )
		[a]

Table 3 .

 3 Transposition of the catalytic process to different alkyl alcohols and sugars[a] .

	Entry	Sugar	Alkyl alcohol	Time (min)	Conv. (%) AAG yield (%)
	1	Glucose	n-hexadecanol	180	100	89
	2 [b]	Glucose	methanol	180	100	70
	3	Mannose	n-dodecanol	60	100	50
	4	Galactose	n-dodecanol	60	66	30

Authors are grateful to the CNRS, the University of Poitiers and the region Nouvelle Aquitaine for the financial supports. The FR CNRS INCREASE 3707 and the chaire TECHNOGREEN are also strongly acknowledged. ABBREVIATIONS AAG, Amphiphilic alkyl glycosides, PFSA, Perfluorinated sulfonic acid, TOF, Turn over frequency, TON, Turn over number.

Author Contributions

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


Lynda Sahraoui

Myriam Thomas

Aurélie Chevillot

Mohamed Mammeri

Bruno Polack

Isabelle Vallée

Jérôme Follet

Hacina Ain-Baaziz

Karim Tarik Adjou
email: karim.adjou@vet-alfort.fr

Molecular characterization of zoonotic Cryptosporidium spp and Giardia duodenalis pathogens in Algerian sheep

Keywords: Cryptosporidium, Giardia, sheep, Algeria, genotyping, zoonosis

Little is known about the presence of Cryptosporidium spp. and Giardia duodenalis in Algerian sheep, nor their potential role as zoonotic reservoirs. This study aimed to investigate the occurrence and distribution of these two protists in lambs. A total of 83 fecal samples were collected from lambs (< 40 days old) from 14 different farms. Samples were screened for Cryptosporidium spp. and Giardia duodenalis presence using immunofluorescent techniques (IF). Nested PCR of the small subunit ribosomal RNA (rRNA) gene, followed by restriction fragment length polymorphism (PCR-RFLP) and sequence analyses were used to identify Cryptosporidium species. C. parvum was further subtyped by sequencing the highly polymorphic 60 kDa glycoprotein (gp60) gene. For G. duodenalis, nested PCR of the glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes was performed and then PCR-RFLP was used to identify G. duodenalis assemblages. Cryptosporidium oocysts and Giardia cysts were detected in 36/83 (43%) and 23/83 (28%) of fecal samples, respectively. Of the 21/36 (58%) Cryptosporidium samples that were positive with IF, 16/21 (76%) were identified as C. parvum, and 5/21 (24%) as C. ubiquitum. From 15 C. parvum isolates, 2 subtypes were identified within the IIa subtype family, including IIaA21G2R1

(3/15) and IIaA13G2R1 (1/15), while IIdA16G1 (11/15) was the only subtype identified from the IId subtype family. Of the 16/23 (69%) G. duodenalis IF-positive samples, the most frequent assemblage was ruminant-specific assemblage E (10/16), followed by assemblage D (4/16), and A + E mixed assemblages (2/16). This study is the first to identify and genotype both Cryptosporidium spp. and Giardia duodenalis in Algerian lambs, and is also the first to describe G. duodenalis assemblage D in small ruminants. The presence of zoonotic C. parvum subtype families (IIa, IId), C. ubiquitum, as well as G. duodenalis assemblage A+E, indicates that sheep could play an important role as a potential reservoir for protists.

Introduction

Cryptosporidium spp. and Giardia duodenalis are common zoonotic enteric protists causing clinical and subclinical infections in farm animals worldwide, and also pose a significant threat to public health [START_REF] De Graaf | A review of the importance of cryptosporidiosis in farm animals[END_REF].

Clinical symptoms of Cryptosporidium infection in small ruminants (lambs and kids) include diarrhea and weight loss, which can be fatal. This not only severely impacts small ruminant farming economies, but also creates a significant transmission risk to humans [START_REF] De Graaf | A review of the importance of cryptosporidiosis in farm animals[END_REF]. Thus far, seven Cryptosporidium species have been isolated from sheep feces, including C. parvum, C. ubiquitum, C. xiaoi, C. hominis, C. andersoni, C. fayeri, and C. suis (Paraud and Chartier, 2012).

In Algeria, sheep and goat populations are currently estimated at 28 and 4.9 million head respectively, while the cattle population is estimated at only 1.9 million head (Ministry of Agriculture and the rural development, 2016). However very little is known about which specific Cryptosporidium species/subtypes infect small ruminants in Algeria. Thus far, only a few studies have characterized Cryptosporidium at a molecular level in calves [START_REF] Baroudi | Molecular characterization of zoonotic pathogens Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in calves in Algeria[END_REF][START_REF] Benhouda | First molecular investigation of Cryptosporidium spp. in young calves in Algeria[END_REF][START_REF] Ouakli | Cryptosporidium-associated diarrhoea in neonatal calves in Algeria[END_REF], and only one recent molecular Cryptosporidium study on sheep isolates exists [START_REF] Baroudi | Zoonotic Cryptosporidium species and subtypes in lambs and goat kids in Algeria[END_REF]. On the other hand, Giardia duodenalis is known to infect numerous mammalian species and consists of at least eight distinct genetic groups or assemblages (A to H), often with different host specificities: assemblage A and B in humans, primates and other mammals; assemblage C and D in dogs and other canids; assemblage E in hoofed livestock; assemblage F in cats; assemblage G in rodents; and assemblage H has been reported in seals and a gull [START_REF] Ballweber | Giardiasis in dogs and cats: update on epidemiology and public health significance[END_REF][START_REF] Ryan | Zoonotic potential of Giardia[END_REF]. In Algeria, G. duodenalis was recently identified in calves, including ruminant-specific assemblage E and zoonotic assemblage A [START_REF] Baroudi | Molecular characterization of zoonotic pathogens Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in calves in Algeria[END_REF], but no data exist for small ruminants.

The farming of small ruminants is one of the main sources of meat production in Algeria and plays a vital role in food security. As stated before, there are more than 32.9 million small ruminants in Algeria; thus farming these animals can improve the living standards of farmers and households, as well as increase the general availability of animal protein for consumption, thus helping to alleviate poverty.

Little is known about the presence of Cryptosporidium spp. and Giardia duodenalis in sheep, nor the role that these animals may play as reservoirs for these parasites. Therefore, the present work aimed to identify Cryptosporidium and Giardia at a molecular level in lambs from different northern Algerian regions.

Materials and Methods

Specimen collection

Between November 2015 to March 2017, 83 randomly selected lamb rectal fecal samples were collected from 14 farms across four northern Algerian provinces located in the North-Center (Djelfa and Msila), North-West (Sidi Bel Abbès), and North-East (Souk Ahras) (Fig. 1).

The Algerian sheep population is estimated at approximately 28 million head. Sheep farming mainly occurs in northern Algeria as the southern regions (Sahara) are too arid. The sheep populations from the northern provinces included in this study represent approximately one quarter (23%) of the total national sheep population. The farms included in this study were arbitrarily designated F1 to F14, and were predominantly extensive herds (free-ranging) (9/14) where animals graze freely during the day and are housed in sheds at night. Whereas in intensive herds (5/14), animals were housed in farm buildings with zero access to grazing (Table 1).

Sampled lambs were less than 40 days old, presenting with or without diarrhea. Fecal samples were individually collected from lambs in plastic boxes, and were then preserved by diluting 1:1 in 5% (wt/vol) potassium dichromate as previously described [START_REF] Bornay-Llinares | Identification of Cryptosporidium felis in a cow by morphologic and molecular methods[END_REF], and conserved at 4°C until use.

Sample processing

All samples were concentrated from 1 g of original fecal matter as previously described [START_REF] Castro-Hermida | Prevalence of Giardia duodenalis and Cryptosporidium parvum in goat kids in western France[END_REF], then screened for the presence of Giardia cysts and/or Cryptosporidium oocysts by direct immunofluorescence assays (IFA) (MeriFluor ® Cryptosporidium/Giardia, Meridian Bioscience Europe, Milano, Italy). Briefly, oocysts and cysts were resuspended in 500 µL of PBS (phosphate buffered saline), then IFA was performed in duplicate using 20 µL of this solution. Entire slides were examined under a fluorescent microscope at 400× magnification. Samples were considered to be positive when at least one Cryptosporidium oocyst or Giardia cyst was observed per slide.

DNA extraction and PCR amplification

Samples with positive IFA results for either parasite then underwent genomic DNA extraction using the QIAamp Mini Kit (Qiagen), according to manufacturer's instructions. To disrupt (oo)cyst walls, an initial step of six freeze-thaw cycles (freezing in liquid nitrogen for 5 min and thawing at 95 °C for 5 min) was incorporated into the protocol.

To detect Cryptosporidium spp. in Cryptosporidium IFA-positive samples, nested PCR was used to amplify an 830 bp fragment of the 18S SSU-rRNA gene as previously described [START_REF] Xiao | Genetic Diversity within Cryptosporidium parvum and Related Cryptosporidium Species[END_REF]. PCR products were analyzed in 2% agarose gel stained with ethidium bromide (0.5 µg/mL).

To confirm the presence of G. duodenalis in Giardia IFA-positive samples, semi-nested PCR was used to amplify the glutamate dehydrogenase (gdh) gene and triose phosphate isomerase (tpi) gene. [START_REF] Read | Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP[END_REF]. Amplification of a 530 bp tpi gene fragment was performed as previously described [START_REF] Sulaiman | Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis[END_REF]. Reactions were then visualized on ethidium bromide-stained (0.5 µg/mL) 2% agarose gels.

PCR-RFLP

In order to identify Cryptosporidium species, positive 18S SSU-rRNA products were subjected to PCR-RFLP analysis using two endonucleases; SspI and MboII (New England BioLabs, France) as previously described [START_REF] Feng | Wide geographic distribution of Cryptosporidium bovis and the deer-like genotype in bovines[END_REF]. Digestion products were separated on 3% MetaPhor agarose (Ozyme, France). The different Cryptosporidium species were identified according to previously described restriction patterns [START_REF] Feng | Wide geographic distribution of Cryptosporidium bovis and the deer-like genotype in bovines[END_REF].

For G. duodenalis, positive gdh PCR products were digested with NlaIV (New England Biolabs), and tpi PCR products with DdeI (New England Biolabs). RFLP analysis to determine the assemblage was directly carried out on PCR products in a 20 µL reaction volume including 10 µL of unpurified PCR product, 7.6 µL sterile water, 0.4 µL restriction enzyme, and 2 µL 10X restriction enzyme buffer. Digestions were incubated at 37°C for 3 or 4 h, for gdh and tpi respectively. Restricted fragments were separated and visualized by electrophoresis on 2% high-resolution grade agarose gel (MetaPhor) stained with ethidium bromide (0.5 µg/mL). A 50 bp DNA ladder (GeneRuler TM , Thermo Scientific TM ) was used as a size marker. The genetic assemblages were differentiated according to previously described restriction patterns [START_REF] Read | Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP[END_REF][START_REF] Sulaiman | Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis[END_REF]. gp60 gene analysis for C. parvum subtyping C. parvum samples were subtyped by nested PCR-sequence analysis of the 60 kDa glycoprotein locus (gp60), and all positive isolates were sequenced as previously described [START_REF] Gatei | Development of a multilocus sequence typing tool for Cryptosporidium hominis[END_REF]. Briefly, the PCR products were sent to Genoscreen (Lille, France), and sequenced in both directions. Consensus sequences were obtained using BioEdit software (version 5.0.6). The C. parvum subtypes were named using the recommended nomenclature system [START_REF] Sulaiman | Unique Endemicity of Cryptosporidiosis in Children in Kuwait[END_REF][START_REF] Xiao | Molecular epidemiology of cryptosporidiosis: An update[END_REF]. The nucleotide sequences obtained from 15 isolates were deposed to GeneBank database under access number from : MK453405 to MK453419.

Results

Results from this study are summarized in Table 1. IIdA16G1 (n = 11), IIaA13G2R1 (n = 1), and IIaA21G2R1 (n = 3) (Table 1).

Giardia duodenalis assemblage occurrence according to age and diarrhea status

G. duodenalis were detected by IFA in 28% (23/83) of fecal samples, of which 69% (16/23) were positive via semi-nested PCR. Three G. duodenalis assemblages were then identified: the ruminant-specific assemblage E (10/16); assemblage A which is infectious for humans and a number of other mammals (livestock, dogs, cats…) (2/16); and assemblage D which has been reported to infect dogs and other canids (4/16). Mixed assemblage A and E infections were identified in two lambs (2/16). The majority of gdh or tpi PCR products identified mono-infections with ruminant-specific assemblage E. These three assemblages were mainly from non-diarrheic lamb samples (9/16), and were found at a higher rate in older lambs (>21 days) than in younger lambs (<21 days).

Discussion

Cryptosporidium and Giardia species are well-known pathogens of both domesticated farm and companion animals and are thus a significant threat to public health. There is considerable genetic diversity within both Cryptosporidium and Giardia duodenalis, as 14 Cryptosporidium species with several different subtypes, and 6 Giardia species with at least 8 G. duodenalis assemblages have been described [START_REF] Cacciò | Unravelling Cryptosporidium and Giardia epidemiology[END_REF]. However, little is known about Cryptosporidium and Giardia occurrence rates in small ruminants in Algeria. This is the first study to identify and perform molecular characterization of Cryptosporidium spp. and G. duodenalis in Algerian lambs, and our analysis revealed a high diversity of Cryptosporidium species and G. duodenlis assemblages within these farm animals.

In this study, IFA was used to screen for the presence of Cryptosporidium oocysts and Giardia cysts prior to performing PCR. In our study, false negative PCRs occurred, indicating that PCR sensitivity was potentially reduced, which could be due to naturally-occuring PCR inhibitors in fecal samples [START_REF] Yu | Comparative Sensitivity of PCR Primer Sets for Detection of Cryptosporidium parvum[END_REF].

In this study, we report that 27 samples from diarrheal animals were positive for Cryptosporidium, and that 10 diarrheal animal samples were positive for Giardia by immunofluorescence. Most of the cryptosporidiosis-positive samples were collected from young diarrheic lambs, while the majority of Giardia-positive samples were from asymptomatic older animals [START_REF] Robertson | Giardia and Cryptosporidium infections in sheep and goats: a review of the potential for transmission to humans via environmental contamination[END_REF]. This could be due to the fact that Cryptosporidium is a neonatal diarrhea agent, whereas Giardia often infects older animals with subclinical symptoms. It must be noted that neonatal diarrhea is not necessarily due to Cryptosporidium presence, as other diarrhea-causing pathogens (salmonella, viruses, coccidia...) were not investigated in this study.

The two Cryptosporidium species (C. parvum and C. ubiquitum) identified in the present study, have previously been reported in small ruminants from Algeria [START_REF] Baroudi | Zoonotic Cryptosporidium species and subtypes in lambs and goat kids in Algeria[END_REF] and from other countries [START_REF] Paraud | parasitism in goats and sheep: interactions with nutrition and control strategies[END_REF]. In this work, C. parvum was the dominant species (in 16/21 animals, compared to 5/21 with C. ubiquitum), comparable to previous small ruminant data [START_REF] Drumo | Evidence of Host-Associated Populations of Cryptosporidium parvum in Italy[END_REF][START_REF] Goma | The prevalence and molecular characterisation of Cryptosporidium spp. in small ruminants in Zambia[END_REF][START_REF] Maurya | Prevalence and risk factors associated with Cryptosporidium spp. infection in young domestic livestock in India[END_REF][START_REF] Mueller-Doblies | Distribution of Cryptosporidium species in sheep in the UK[END_REF][START_REF] Quilez | Cryptosporidium Genotypes and Subtypes in Lambs and Goat Kids in Spain[END_REF][START_REF] Tzanidakis | Occurrence and molecular characterization of Giardia duodenalis and Cryptosporidium spp. in sheep and goats reared under dairy husbandry systems in Greece[END_REF]. The C. xiaoi species was not identified in lambs in the current study, even though it was recently reported to be frequent in small ruminants from Algeria [START_REF] Baroudi | Zoonotic Cryptosporidium species and subtypes in lambs and goat kids in Algeria[END_REF] and other locations (African countries, Asian countries, and some European countries such as Norway and Poland) [START_REF] Kaupke | Diversity of Cryptosporidium species occurring in sheep and goat breeds reared in Poland[END_REF][START_REF] Parsons | Epidemiology and molecular characterization of Cryptosporidium spp. in humans, wild primates, and domesticated animals in the Greater Gombe Ecosystem, Tanzania[END_REF][START_REF] Peng | Infection rate of Giardia duodenalis, Cryptosporidium spp. and Enterocytozoon bieneusi in cashmere, dairy and meat goats in China[END_REF][START_REF] Robertson | Giardia and Cryptosporidium infections in sheep and goats: a review of the potential for transmission to humans via environmental contamination[END_REF].

In this study, the dominant C. parvum isolate subtype present in the lambs was IIdA16G1 (n = 11/16), while subtypes IIaA21G2R1 (n = 3/16) and IIaA13G2R1 (n =1/16) were reported at lower rates. Our results are consistent with multiple other studies, where the C. parvum IId subtype family is dominant in countries such as Spain, Romania, and Australia [START_REF] Díaz | Cryptosporidium species and subtype analysis in diarrhoeic pre-weaned lambs and goat kids from north-western Spain[END_REF][START_REF] Imre | Zoonotic Cryptosporidium parvum in Romanian newborn lambs (Ovis aries)[END_REF][START_REF] Quilez | Cryptosporidium Genotypes and Subtypes in Lambs and Goat Kids in Spain[END_REF][START_REF] Yang | Longitudinal prevalence, oocyst shedding and molecular characterisation of Cryptosporidium species in sheep across four states in Australia[END_REF]. However in other countries (the UK, Poland, New Guinea) IIa subtype families are dominant [START_REF] Connelly | Genetic diversity of Cryptosporidium spp. within a remote population of Soay Sheep on St. Kilda Islands, Scotland[END_REF][START_REF] Kaupke | Diversity of Cryptosporidium species occurring in sheep and goat breeds reared in Poland[END_REF][START_REF] Koinari | Cryptosporidium species in sheep and goats from Papua New Guinea[END_REF]. The identified subtypes pose a real risk to public health, as the IIdA16G1 C. parvum subtype was recently identified in calves and human children from rural regions of northern Tunisia near Algerian borders (Rahmouni et al., 2014), and human Cryptosporidium infections-including subtype IIaA13G2R1-have been reported in immunosuppressed individuals from Malaysia and Ethiopia [START_REF] Adamu | Distribution and clinical manifestations of Cryptosporidium species and subtypes in HIV/AIDS patients in Ethiopia[END_REF][START_REF] Iqbal | High diversity of Cryptosporidium subgenotypes identified in Malaysian HIV/AIDS individuals targeting gp60 gene[END_REF].

The G. duodenalis assemblages identified in this study indicated that lambs mainly carried G. duodenalis assemblage E mono-infections (10/16), which is usually found in hoofed animals, including cattle and small ruminants [START_REF] Geurden | Prevalence and molecular characterisation of Cryptosporidium and Giardia in lambs and goat kids in Belgium[END_REF][START_REF] Paz E Silva | High occurrence of Cryptosporidium ubiquitum and Giardia duodenalis genotype E in sheep from Brazil[END_REF][START_REF] Tzanidakis | Occurrence and molecular characterization of Giardia duodenalis and Cryptosporidium spp. in sheep and goats reared under dairy husbandry systems in Greece[END_REF]. In addition, mixed assemblage A + E infections were identified in two lambs (2/16), similar to reports of zoonotic A + B assemblages in the USA and Australia [START_REF] Santín | Prevalence and molecular characterization of Cryptosporidium and Giardia species and genotypes in sheep in Maryland[END_REF][START_REF] Yang | Longitudinal prevalence, oocyst shedding and molecular characterisation of Cryptosporidium species in sheep across four states in Australia[END_REF].

Interestingly, and to the best of our knowledge, this is the first description of assemblage D in lambs, which has previously only been identified in dog or canids [START_REF] Thompson | The zoonotic significance and molecular epidemiology of Giardia and giardiasis[END_REF]. This assemblage was isolated from a single extensively-farmed herd where working dogs, stray dogs, and other wild canids are numerous. These animals could contaminate the environment (water and pasture) with Giardia cysts, and thus indirectly contaminate the lambs. Of note, it has been reported that intensive contact between dogs and other animals (pigs and other wild animals) could lead to assemblage D transmission [START_REF] Ryan | Zoonotic potential of Giardia[END_REF][START_REF] Sprong | Identification of zoonotic genotypes of Giardia duodenalis[END_REF]. Other large-scale studies are needed to better understand Giardia circulation, particularly of those assemblages not well adapted to ruminants. Zoonotic pathogenic C. parvum and G. duodenalis subtypes are reported to be common in dairy farm calves from the Algiers region [START_REF] Baroudi | Molecular characterization of zoonotic pathogens Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in calves in Algeria[END_REF]. Our results suggest that lambs may thus be an important reservoir for G. duodenalis in Algeria. Further investigations are required to determine whether this observation holds true in other parts of the country, preferably with larger sample sizes to better understand the epidemiology of cryptosporidiosis and giardiosis in lambs.

In conclusion, the present work shows that C. parvum, C. ubiquitum, and Giardia duodenalis commonly occur in Algerian lambs, and that these data strongly suggest that sheep may be an important reservoir of zoonotic C. parvum and Giardia duodenalis in Algeria. However, further research is necessary to characterize the prevalence of various Cryptosporidium species and Giardia duodenalis assemblages in young lambs, goat kids, and calves, and also in other hosts such as humans, to fully understand the transmission dynamics of these protists in Algeria. This is the first report of both Cryptosporidium spp. and G. duodenalis infections in Algerian lambs, and could serve as baseline data for further investigations to better understand cryptosporidiosis and giardiosis epidemiology in Algeria. Tables Table 1. Occurrence of Cryptosporidium species and Giardia assemblages in lambs from Djelfa, Msila, Sidi Bel Abbès and Souk Ahras provinces, Algeria.

Farm locations

No

3. 1 .

 1 Abbes province only). Three C. parvum subtypes were identified with gp60 gene analysis:

Figure 1 .

 1 Figure 1. Map of Algeria indicating the location, geographical distribution, and number

. of farms Extensive farms Intensive farms No. of samples tested Cryptosporidium spp. Giardia duodenalis

						No. of	No. of IFA-	No. of	Cryptosporidium	C. parvum	No. of	No. of IFA-	No. of	Giardia
						Cryptosporidium	positive	Cryptosporidium-	species	subtypes	Giardia	positive	Giardia-	assemblages
						positive samples	samples	positive samples		(No. /Total)	positive	samples from	positive	(No./Total)
						via IF (Farms	from	via PCR			samples via	diarrheic	samples via
						indicated)	diarrheic				IF (Farms	lambs	PCR
							lambs				indicated)
	Djelfa	3	*F9	F10, F11	24	7 (F9,F10,F11)	3	3	C. parvum (3)	IIaA13G2R1(1/3)	7 (F9, F11)	0	4	E (2/4)
														D (1/4)
										IIdA16G1 (2/3)				A+E (1/4)
	Msila	3	F12	F13, F14	17	4 (F12,F14)	4	4	C. parvum (4)	IIdA16G1 (3/4)	2 (F12, F13) 0	2	E (2/2)
	Sidi Bel Abbès	7	F3, F4,	F2	37	22	17	11	C. parvum (6)	IIdA16G1 (6/6)	14 (F2,	10	10	E (6/10)
			F5, F6,			(F2,F4,F5,F6,F7,F8					F3,F4, F5,			D (3/10)
			F7, F8			)			C. ubiquitum (5)	-	F7, F8)			A+E (1/10)
	Souk Ahras	1	F1	-	5	3 (F1)	3	3	C. parvum (3)	IIaA21G2R1(3/3)	0	0	0	-
	Total	14	9	5	83	36	27	21	2 species	3 subtypes	23	10	16	3 assemblages

*F: Farms included in this study were arbitrarily designated from F1 to F14.

Acknowledgements

The authors would like to thank all the private veterinarians in rural Algerian areas for their participation in sampling.

Funding

Not applicable.

Conflict of interest

The authors declare that they have no conflicts of interest.