Jessica B Von Pein

Claudia J Stocks

Mark A Schembri

| Ronan Kapetanovic

Matthew J Sweet
email: m.sweet@imb.uq.edu.au

Ronan Kapetanovic

An alloy of zinc and innate immunity: Galvanising host defence against infection

Keywords: antimicrobial, bacterial pathogens, host, innate immunity, pathogen, zinc toxicity, zinc transporters

Innate immune cells such as macrophages and neutrophils initiate protective inflammatory responses and engage antimicrobial responses to provide frontline defence against invading pathogens. These cells can both restrict the availability of certain transition metals that are essential for microbial growth and direct toxic concentrations of metals towards pathogens as antimicrobial responses. Zinc is important for the structure and function of many proteins, however excess zinc can be cytotoxic.

In recent years, several studies have revealed that innate immune cells can deliver toxic concentrations of zinc to intracellular pathogens. In this review, we discuss the importance of zinc status during infectious disease and the evidence for zinc intoxication as an innate immune antimicrobial response. Evidence for pathogen subversion of this response is also examined. The likely mechanisms, including the involvement of specific zinc transporters that facilitate delivery of zinc by innate immune cells for metal ion poisoning of pathogens are also considered. Precise mechanisms by which excess levels of zinc can be toxic to microorganisms are then discussed, particularly in the context of synergy with other antimicrobial responses. Finally, we highlight key unanswered questions in this emerging field, which may offer new opportunities for exploiting innate immune responses for anti-infective development.

| INTRODUCTION

The innate immune system provides the first line of defence against infection. Innate immune cells, including monocytes, macrophages, dendritic cells and neutrophils, initiate both inflammatory and antimicrobial responses to clear pathogens. During infection, pathogens require essential nutrients, including transition metals, for growth and survival. Innate immune cells thus restrict the availability of specific transition metals to limit microbial growth, in a process termed nutritional immunity [START_REF] Hood | Nutritional immunity: Transition metals at the pathogen-host interface[END_REF]. Conversely, phagocytes can also traffic toxic concentrations of metal ions as an antimicrobial response against intracellular pathogens [START_REF] Sheldon | Metals as phagocyte antimicrobial effectors[END_REF].

Zinc is a key component of enzymes across all domains of life. This metal is essential for the structure and/or function of many proteins; for example, the catalytic activity of numerous enzymes [START_REF] Andreini | A bioinformatics view of zinc enzymes[END_REF]. Zinc also has roles in several signal transduction pathways, including in inflammatory and immune signalling [START_REF] Haase | Signal transduction in monocytes: The role of zinc ions[END_REF][START_REF] Pyle | Zinc modulates endotoxin-induced human macrophage inflammation through ZIP8 induction and C/EBPβ inhibition[END_REF][START_REF] Yamasaki | Zinc is a novel intracellular second messenger[END_REF]. Despite these essential roles, excessive zinc can be cytotoxic and can cause Abbreviations: CDF, cation diffusion facilitator; IL, interleukin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MT/Mt, metallothionein (HUMAN/murine); NO, nitric oxide; ROS, reactive oxygen species; SLC/Slc, solute carrier (HUMAN/Murine); SPI-1, Salmonella pathogenicity island-1; S. Typhimurium, Salmonella enterica serovar Typhimurium; UPEC, uropathogenic E. coli. tissue damage, organ failure and death [START_REF] Bennett | Zinc toxicity following massive coin ingestion[END_REF][START_REF] Lee | Roles of zinc and metallothionein-3 in oxidative stress-induced lysosomal dysfunction, cell death, and autophagy in neurons and astrocytes[END_REF]. Therefore, zinc homeostasis needs to be tightly regulated [START_REF] Blencowe | Zn(II) metabolism in prokaryotes[END_REF][START_REF] Eide | Zinc transporters and the cellular trafficking of zinc[END_REF]. Innate immune cells can mobilise zinc during infection to achieve either zinc sequestration or to deliver toxic concentrations of zinc to intracellular pathogens [START_REF] Lonergan | Nutrient zinc at the host-pathogen interface[END_REF][START_REF] Sheldon | Metals as phagocyte antimicrobial effectors[END_REF]. Zinc sequestration has previously been extensively reviewed [START_REF] Haase | An element of life: Competition for zinc in hostpathogen interaction[END_REF][START_REF] Hood | Nutritional immunity: Transition metals at the pathogen-host interface[END_REF][START_REF] Jordan | Multimetal nutrient restriction and crosstalk in metallostasis systems in microbial pathogens[END_REF][START_REF] Lonergan | Nutrient zinc at the host-pathogen interface[END_REF][START_REF] Sheldon | Metals as phagocyte antimicrobial effectors[END_REF]. This review will discuss and highlight key unanswered questions about the emerging role of zinc toxicity as an antimicrobial response deployed by the innate immune system during infection.

| ZINC DEFICIENCY AND SUPPLEMENTATION IN INFECTIOUS DISEASE

While zinc toxicity in humans is very rare, approximately 20% of the world population is estimated to be at risk of inadequate zinc intake [START_REF] Wuehler | Use of national food balance data to estimate the adequacy of zinc in national food supplies: Methodology and regional estimates[END_REF]. Zinc deficiency is of particular concern for susceptibility to infectious diseases, due to the established role of zinc in immune functions [START_REF] Gammoh | Zinc in infection and inflammation[END_REF]. Indeed, sepsis severity is associated with low plasma zinc concentrations in adults [START_REF] Besecker | A comparison of zinc metabolism, inflammation, and disease severity in critically ill infected and noninfected adults early after intensive care unit admission[END_REF][START_REF] Hoeger | Persistent low serum zinc is associated with recurrent sepsis in critically ill patients -A pilot study[END_REF]. Low plasma zinc levels are also linked to immunosenescence, an age-related decline in immune function accompanied by recurring infections and chronic inflammation [START_REF] Maywald | Zinc homeostasis and immunosenescence[END_REF][START_REF] Rink | Altered cytokine production in the elderly[END_REF]. Consistent with this, long-term zinc supplementation reduced infection incidence in healthy elderly subjects [START_REF] Prasad | Zinc supplementation decreases incidence of infections in the elderly: Effect of zinc on generation of cytokines and oxidative stress[END_REF]. Furthermore, zinc supplementation in humans improves outcomes for multiple types of infectious disease, such as respiratory tract infections [START_REF] Brooks | Effect of weekly zinc supplements on incidence of pneumonia and diarrhoea in children younger than 2 years in an urban, low-income population in Bangladesh: Randomised controlled trial[END_REF][START_REF] Sazawal | Zinc supplementation reduces the incidence of acute lower respiratory infections in infants and preschool children: A doubleblind, controlled trial[END_REF], pneumonia [START_REF] Bhutta | Prevention of diarrhea and pneumonia by zinc supplementation in children in developing countries: Pooled analysis of randomized controlled trials[END_REF][START_REF] Valavi | The efficacy of zinc supplementation on outcome of children with severe pneumonia. A randomized double-blind placebocontrolled clinical trial[END_REF], diarrhoeal disease [START_REF] Ahmadipour | Treating viral diarrhea in children by probiotic and zinc supplements[END_REF][START_REF] Al Tarique | Zinc influences innate immune responses in children with enterotoxigenic Escherichia coli-induced diarrhea[END_REF][START_REF] Bhutta | Therapeutic effects of oral zinc in acute and persistent diarrhea in children in developing countries: Pooled analysis of randomized controlled trials[END_REF][START_REF] Castillo-Duran | Trace mineral balance during acute diarrhea in infants[END_REF][START_REF] Sazawal | Zinc supplementation reduces the incidence of acute lower respiratory infections in infants and preschool children: A doubleblind, controlled trial[END_REF] and viral infections [START_REF] Matsumura | Zinc supplementation therapy improves the outcome of patients with chronic hepatitis C[END_REF][START_REF] Varadinova | Mode of action of Zn-complexes on herpes simplex virus type 1 infection in vitro[END_REF].

Of particular note, the WHO and UNICEF recommend zinc supplementation for the clinical management of acute diarrhoea in children, as a consequence of multiple studies demonstrating its efficacy in reducing both disease severity and duration [START_REF] Penny | Zinc supplementation in public health[END_REF].

In keeping with the clinical evidence above, the importance of zinc in host defence has been confirmed by modulating dietary intake of zinc in animal models of infection (summarised in Table S1).

For example, zinc deficiency enhanced organ damage and mortality in a murine model of polymicrobial sepsis, with these effects being rescued by zinc supplementation [START_REF] Knoell | Zinc deficiency increases organ damage and mortality in a murine model of polymicrobial sepsis[END_REF]. Furthermore, zinc restriction in mice enhanced Streptococcus pneumoniae infection, likely due to impaired pathogen clearance by phagocytes [START_REF] Eijkelkamp | Dietary zinc and the control of Streptococcus pneumoniae infection[END_REF]. Such studies are consistent with zinc having important roles in facilitating innate immune cell functions during infection.

| EVIDENCE FOR ZINC TOXICITY AS AN INNATE IMMUNE ANTIMICROBIAL RESPONSE

| Mobilisation of zinc by innate immune cells during infection

Zinc is mobilised and concentrated in specific tissues during inflammation and infection. For example, infection of mice with S. pneumoniae resulted in increased zinc concentrations in the lungs, brain, nasopharynx and blood [START_REF] Mcdevitt | A molecular mechanism for bacterial susceptibility to zinc[END_REF], with this pathogen subsequently shown to co-localise with zinc-enriched regions in murine lungs [START_REF] Eijkelkamp | Dietary zinc and the control of Streptococcus pneumoniae infection[END_REF]. Several studies, utilising either fluorescent zinc-binding dyes or direct elemental analysis, have also documented fluctuations in zinc concentrations within immune cells in response to infection or microbial products (Table 1). Zinc accumulation in phagosomes during infection has been observed in multiple cellular systems. Macrophages mobilise and traffic zinc to phagosomes and intracellular compartments that co-localise with various bacterial pathogens, including Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium and Escherichia coli (Table 1).

Subsequent studies have confirmed that zinc vesicles co-localised with an E. coli strain that specifically reports zinc stress [START_REF] Stocks | Uropathogenic Escherichia coli employs both evasion and resistance to subvert innate immunemediated zinc toxicity for dissemination[END_REF]. Zinc accumulation in lysosomes and azurophilic granules has also been observed in neutrophils, implicating zinc delivery to phagocytosed Streptococcus pyogenes [START_REF] Ong | New insights into the role of zinc acquisition and zinc tolerance in Group A Streptococcal infection[END_REF]. Interestingly, in the phagocytic soil amoeba Dictyostelium discoideum that employs phagocytosis to feed on bacteria, zinc was also delivered towards Mycobacterium smegmatis-containing vacuoles [START_REF] Barisch | Localization of all four ZnT zinc transporters in Dictyostelium and impact of ZntA and ZntB knockout on bacteria killing[END_REF]. Combined with evidence below linking such effects to zinc stress, these studies suggest innate immune cells can deliver zinc to poison intracellular bacteria.

| Pathogens resist zinc toxicity by detoxification during infection

Much of the evidence for innate immune zinc toxicity is based on the necessity of bacterial zinc export systems for survival within innate immune cells. As for mammals, bacteria maintain zinc homeostasis by expression of zinc importers and exporters. Zinc export is achieved via P-type ATPases including ZntA [START_REF] Beard | Zinc(II) tolerance in Escherichia coli K-12: Evidence that the zntA gene (o732) encodes a cation transport ATPase[END_REF][START_REF] Rensing | The zntA gene of Escherichia coli encodes a Zn(II)-translocating P-type ATPase[END_REF], as well as the putative zinc exporters CtpC and CtpG [START_REF] Botella | Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human macrophages[END_REF] (Figure 1). In addition, cation diffusion facilitators (CDF) including ZitB [START_REF] Grass | ZitB (YbgR), a member of the cation diffusion facilitator family, is an additional zinc transporter in Escherichia coli[END_REF][START_REF] Wang | ZntR-mediated transcription of zntA responds to nanomolar intracellular free zinc[END_REF] and heavy metal efflux systems such as CzcCBA promote zinc efflux in bacteria [START_REF] Blencowe | Zn(II) metabolism in prokaryotes[END_REF]; Figure 1). Another CDF, CzcD, also mediates zinc export in both Gram-negative bacteria such as Acinetobacter baumannii [START_REF] Alquethamy | The role of zinc efflux during Acinetobacter baumannii infection[END_REF] and Gram-positive bacteria such as S. pyogenes [START_REF] Ong | An antimicrobial role for zinc in innate immune defense against Group A Streptococcus[END_REF]. CzcD performs this function via an antiport mechanism [START_REF] Guffanti | An antiport mechanism for a member of the cation diffusion facilitator family: Divalent cations efflux in exchange for K + and H +[END_REF]. When zinc levels are elevated, the cytosolic regulators ZntR [START_REF] Brocklehurst | ZntR is a Zn(II)-responsive MerRlike transcriptional regulator of zntA in Escherichia coli[END_REF] and GczA/SczA [START_REF] Kloosterman | The novel transcriptional regulator SczA mediates protection against Zn 2+ stress by activation of the Zn 2+ -resistance gene czcD in Streptococcus pneumoniae[END_REF][START_REF] Martin | The zinc efflux activator SczA protects Streptococcus pneumoniae serotype 2 D39 from intracellular zinc toxicity[END_REF] induce zntA and czcD expression, respectively (Figure 1).

Several zinc exporters and detoxification systems are induced in various pathogens during infection of innate immune cells and/or in in vivo models of infection (Table 2). For example, zinc export was upregulated by S. pneumoniae following infection of human neutrophils, the macrophage-like cell line THP-1, and within an intranasal murine infection model [START_REF] Eijkelkamp | Dietary zinc and the control of Streptococcus pneumoniae infection[END_REF]. In addition, zinc efflux systems were upregulated by M. tuberculosis [START_REF] Botella | Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human macrophages[END_REF], S. Typhimurium [START_REF] Kapetanovic | Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages[END_REF]) and E. coli [START_REF] Stocks | Uropathogenic Escherichia coli employs both evasion and resistance to subvert innate immunemediated zinc toxicity for dissemination[END_REF] upon infection of macrophages. Several studies have also highlighted the necessity of pathogen zinc export systems for their survival during infection (Table 2). Human macrophages were able to more efficiently clear M. tuberculosis and E. coli that lack zinc export systems by comparison to isogenic wild-type strains [START_REF] Botella | Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human macrophages[END_REF][START_REF] Stocks | Uropathogenic Escherichia coli employs both evasion and resistance to subvert innate immunemediated zinc toxicity for dissemination[END_REF]. Additionally, zinc-sensitive mutants of S. pyogenes [START_REF] Ong | An antimicrobial role for zinc in innate immune defense against Group A Streptococcus[END_REF][START_REF] Ong | New insights into the role of zinc acquisition and zinc tolerance in Group A Streptococcal infection[END_REF], S. pneumoniae [START_REF] Eijkelkamp | Dietary zinc and the control of Streptococcus pneumoniae infection[END_REF]) and E. coli [START_REF] Kapetanovic | Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages[END_REF] were more susceptible to neutrophil killing. Similarly, S. pneumoniae, S. pyogenes and E. coli which lack regulators required for zinc exporter expression exhibited reduced intracellular survival in THP-1 cells [START_REF] Martin | The zinc efflux activator SczA protects Streptococcus pneumoniae serotype 2 D39 from intracellular zinc toxicity[END_REF], neutrophils [START_REF] Ong | An antimicrobial role for zinc in innate immune defense against Group A Streptococcus[END_REF] and macrophages [START_REF] Stocks | Uropathogenic Escherichia coli employs both evasion and resistance to subvert innate immunemediated zinc toxicity for dissemination[END_REF], respectively. Recent evidence suggests that the innate immune zinc toxicity response is not just limited to bacterial infection. In murine macrophages, the fungal pathogen Candida glabrata required the zinc transporter ZRC1 for survival [START_REF] Riedelberger | Type I interferons ameliorate zinc intoxication of Candida glabrata by macrophages and promote fungal immune evasion[END_REF]. This study implicated a role for ZRC1 in the sequestration of excess zinc into intracellular vacuoles during infection. Together, this literature implies that a variety of pathogens are subjected to zinc toxicity during infection of innate immune cells and employ zinc detoxification systems to resist this response.

| Pathogen evasion of zinc toxicity responses during infection

Pathogens require strategies to resist or evade host innate immune responses to enable survival during infection. In addition to resistance by zinc detoxification systems (described above), some pathogens can also evade macrophage zinc-toxicity responses. Moreover, Salmonella actually employs both resistance and evasion to subvert zinc toxicity responses deployed by macrophages. While wild-type S. Typhimurium did not co-localise with zinc vesicles in primary human macrophages, a Salmonella pathogenicity island-1 (ΔSPI-1) mutant strain co-localised with zinc vesicles, upregulated zntA and was compromised for intramacrophage survival [START_REF] Kapetanovic | Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages[END_REF]. However, a S.

Typhimurium ΔSPI-1/ΔzntA mutant was no more sensitive to killing by human macrophages than an isogenic ΔSPI-1 mutant strain, suggesting that Salmonella also has SPI-1-and ZntA-independent mechanisms to resist macrophage-mediated zinc toxicity (Kapetanovic et al., 2016). Other bacterial pathogens such as uropathogenic E. coli (UPEC) also evade the macrophage zinc toxicity response. In contrast to non-pathogenic E. coli, the majority of intramacrophage UPEC did not report zinc stress, as indicated by a novel zinc-stress reporter system based on zinc-inducible zntA expression [START_REF] Stocks | Uropathogenic Escherichia coli employs both evasion and resistance to subvert innate immunemediated zinc toxicity for dissemination[END_REF].

Consistent with this, intramacrophage survival of zinc-sensitive zntAand zntR-deficient UPEC mutants was not reduced, in contrast to observations with corresponding mutants of a non-pathogenic E. coli strain [START_REF] Stocks | Uropathogenic Escherichia coli employs both evasion and resistance to subvert innate immunemediated zinc toxicity for dissemination[END_REF]. This suggests that UPEC effectively evades macrophage-mediated zinc toxicity. What remains to be determined are the specific zinc toxicity evasion strategies employed by S. Typhimurium and UPEC.

| HOST CELL MECHANISMS UNDERPINNING ANTIMICROBIAL ZINC DELIVERY

Innate immune delivery of zinc for microbial intoxication is likely to be driven by multiple, interconnected mechanisms, including zinc import, zinc export and interactions with metallothioneins (MTs; Figure 2).

Mammalian cells utilise 14 highly conserved solute carrier (SLC) 39A

family members [also referred to as Zrt-, Irt-like proteins (ZIP)] for the import of zinc and/or other metal ions from the extracellular space and the lumen of organelles into the cytoplasm [START_REF] Eide | Zinc transporters and the cellular trafficking of zinc[END_REF][START_REF] Hara | Physiological roles of zinc transporters: Molecular and genetic importance in zinc homeostasis[END_REF]. In addition, mammals possess 10 CDF zinc exporters of the SLC30A family (also referred to as ZnT) that can export zinc and/or other metal ions from the cytoplasm into luminal compartments or the extracellular space [START_REF] Eide | Zinc transporters and the cellular trafficking of zinc[END_REF][START_REF] Huang | The SLC30 family of zinc transporters -A review of current understanding of their biological and pathophysiological roles[END_REF]. It is likely that both SLC39A and SLC30A zinc transporters are involved in the coordinated shuttling of zinc pools towards engulfed bacteria. In this manner, zinc importers may take up zinc from the extracellular environment and/or cellular compartments to maintain adequate cytosol zinc concentrations, while zinc exporters may concentrate zinc ions into vesicles or bacteria-containing compartments.

Various innate immune stimuli induce zinc mobilisation in macrophages. For example, agonists of several Toll-like receptors (TLRs) increased available zinc as determined by intracellular staining with Fluozin-3 in RAW 264.7 cells and human monocytes [START_REF] Brieger | Differential regulation of TLRdependent MyD88 and TRIF signaling pathways by free zinc ions[END_REF]. In addition, TLR 3, 4 and 7 stimulation promoted vesicular zinc accumulation in primary human macrophages and THP-1

F I G U R E 1
Bacterial zinc efflux systems. Bacteria express various exporters which transport zinc from the cytosol to maintain homeostasis in conditions of high zinc availability. Expression of some exporters are regulated by zinc-sensing transcription factors, thus enabling their selective induction under conditions of zinc stress cells that had been differentiated into macrophage-like cells [START_REF] Kapetanovic | Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages[END_REF]. As TLRs initiate late-stage antimicrobial responses [START_REF] Stocks | For when bacterial infections persist: Toll-like receptor-inducible direct antimicrobial pathways in macrophages[END_REF], zinc toxicity may be engaged by innate immune cells to defend against persistent intracellular pathogens. It is therefore likely that these receptors, and potentially other receptors that sense infections and/or danger, regulate the expression and/or functions of SLC39A, SLC30A and/or MT family members to initiate and/or sustain the zinc toxicity response (Figure 2).

Although it is currently unknown if specific importers are involved in zinc-mediated antimicrobial responses, roles for some SLC39A transporters appear likely. For example, SLC39A8 is a manganese and zinc importer [START_REF] Begum | Mycobacterium bovis BCG cell wall and lipopolysaccharide induce a novel gene, BIGM103, encoding a 7-TM protein: Identification of a new protein family having Zn-transporter and Zn-metalloprotease signatures[END_REF][START_REF] Lin | Hepatic metal ion transporter ZIP8 regulates manganese homeostasis and manganese-dependent enzyme activity[END_REF] [START_REF] Girijashanker | Slc39a14 gene encodes ZIP14, a metal/bicarbonate symporter: Similarities to the ZIP8 transporter[END_REF], is also LPS-inducible and regulates cytokine production in human macrophages [START_REF] Sayadi | Zip14 expression induced by lipopolysaccharides in macrophages attenuates inflammatory response[END_REF]. As SLC39A8 and SLC39A14 are both induced by LPS in macrophages, these transporters may be involved in not only inflammatory responses, but also LPS-induced zinc mobilisation for metal ion poisoning. This possibility remains to be investigated.

With respect to SLC30A family members, SLC30A1 expression is induced by LPS in primary human macrophages, with its ectopic expression in monocytic THP-1 cells being sufficient to drive the formation of zinc vesicles and to subject intracellular E. coli to zinc stress [START_REF] Stocks | Frontline Science: LPS-inducible SLC30A1 drives human macrophage-mediated zinc toxicity against intracellular Escherichia coli[END_REF]. Moreover, this study demonstrated that SLC30A1 localised to compartments containing zinc-stressed E. coli.

However, silencing of SLC30A1 in primary human macrophages did not affect bacterial clearance, suggesting that other zinc exporters may also contribute to this response. One candidate that may play a role in the absence of SLC30A1 is SLC30A4. Interleukin (IL)-4 treatment upregulated Slc30a4 in murine macrophages, resulting in an increase in labile zinc within the phagolysosome [START_REF] Subramanian Vignesh | IL-4 induces metallothionein 3-and SLC30A4-dependent increase in intracellular Zn 2+ that promotes pathogen persistence in macrophages[END_REF]. Slc30a4, along with Slc30a7, was also implicated in granulocyte macrophage colony stimulating factor-mediated zinc sequestration in the Golgi in murine macrophages [START_REF] Subramanian Vignesh | Granulocyte macrophagecolony stimulating factor induced Zn sequestration enhances macrophage superoxide and limits intracellular pathogen survival[END_REF]. This sequestration was associated with a zinc starvation response against an intramacrophage fungal pathogen, but it is possible that these transporters may additionally be involved in the delivery of zinc toxicity to different pathogens and/or in different microenvironments.

Consistent with a role for one or more SLC30A family members in the zinc toxicity response in human and mouse macrophages, zinc was rapidly delivered towards M. smegmatis-containing vacuoles via SLC30A homologues in D. discoideum [START_REF] Barisch | Localization of all four ZnT zinc transporters in Dictyostelium and impact of ZntA and ZntB knockout on bacteria killing[END_REF]. The predicted mechanism of action of SLC30A family zinc transporters has been modelled using the E. coli CDF proteins ZitB [START_REF] Chao | Kinetic Study of the Antiport Mechanism of an Escherichia coli Zinc Transporter, ZitB[END_REF] and YiiP [START_REF] Lu | Structure of the zinc transporter YiiP[END_REF], which export zinc via a H + antiport mechanism [START_REF] Chao | Kinetic Study of the Antiport Mechanism of an Escherichia coli Zinc Transporter, ZitB[END_REF][START_REF] Grass | FieF (YiiP) from Escherichia coli mediates decreased cellular accumulation of iron and relieves iron stress[END_REF]. Indeed, SLC30A1 homodimers utilise a H + electrochemical gradient to drive Zn 2+ efflux in exchange for H + in a pH-driven, sodium-independent and calcium-sensitive manner [START_REF] Shusterman | ZnT-1 extrudes zinc from mammalian cells functioning as a Zn 2+ /H + exchanger[END_REF]. Thus, the acidic environment of phagolysosomes might enable the delivery of zinc to pathogens by one or more SLC30A transporters.

Another important factor influencing zinc transport in mammalian cells is control by the MT family. MTs are a family of monomeric metal-binding proteins that regulate cytosolic zinc homeostasis under both steady and non-steady state conditions [START_REF] Colvin | Cytosolic zinc buffering and muffling: Their role in intracellular zinc homeostasis[END_REF]. The MTs are encoded by 10-12 genes (chromosome 16) and 4 genes (chromosome 8) in humans and mice, respectively [START_REF] Cox | The metallothionein-I gene maps to mouse chromosome 8: Implications for human Menkes' disease[END_REF][START_REF] Vašák | Chemistry and biology of mammalian metallothioneins[END_REF]. Some studies have demonstrated that MTs are regulated in innate immune cells during infection [START_REF] Botella | Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human macrophages[END_REF][START_REF] Subramanian Vignesh | Granulocyte macrophagecolony stimulating factor induced Zn sequestration enhances macrophage superoxide and limits intracellular pathogen survival[END_REF]. Moreover, silencing of the transcription factor metalregulatory factor-1, which controls cellular responses to metals, disrupted MT expression, prevented intracellular zinc accumulation and impaired bacterial clearance in human macrophages chronically stimulated with the microbial product muramyl dipeptide [START_REF] Lahiri | Activation of pattern recognition receptors up-regulates metallothioneins, thereby increasing intracellular accumulation of zinc, autophagy, and bacterial clearance by macrophages[END_REF], thus implicating MTs in antimicrobial responses.

More recently, Mt-1 and Mt-2 were shown to be required for zinc intoxication against phagocytosed C. glabrata in murine macrophages [START_REF] Riedelberger | Type I interferons ameliorate zinc intoxication of Candida glabrata by macrophages and promote fungal immune evasion[END_REF]. This study demonstrated that Mts did not co-localise with the pathogen during infection, suggesting that Mts are involved in zinc mobilisation rather than direct delivery of zinc to intracellular pathogens. It is likely that MTs cooperate with zinc transporters, providing a sophisticated network of effector proteins and zinc chaperones to control the distribution of intracellular metal ions for effective inflammatory and antimicrobial responses. Innate immune cells traffic zinc towards pathogen-containing intracellular compartments, likely through the coordinated actions of zinc importers, zinc exporters and metallothioneins (MTs). SLC39A family members import zinc from extracellular environments into the cytoplasm, while members of the SLC30A family export zinc from the cytoplasm for its delivery to pathogens and/or to maintain zinc homeostasis. MTs regulate cytosolic zinc homeostasis and may control the delivery of zinc to zinc transporters in response to infection. Innate immune signalling systems, including Toll-like receptors, induce the expression of various zinc transporters and MTs, as well as zinc vesicle accumulation in immune cells Imlay, 2012). For example, zinc irreversibly binds to manganeseacquisition proteins in S. pneumoniae [START_REF] Couñago | Imperfect coordination chemistry facilitates metal ion release in the Psa permease[END_REF][START_REF] Mcdevitt | A molecular mechanism for bacterial susceptibility to zinc[END_REF], Bacillus anthracis [START_REF] Vigonsky | Metal binding spectrum and model structure of the Bacillus anthracis virulence determinant MntA[END_REF] and Staphylococcus pseudintermedius [START_REF] Abate | Apo, Zn 2+ -bound and Mn 2+ -bound structures reveal ligandbinding properties of SitA from the pathogen Staphylococcus pseudintermedius[END_REF]. Thus, zinc excess can induce manganese deficiency in bacteria, leading to increased sensitivity to oxidative stress [START_REF] Eijkelkamp | Extracellular zinc competitively inhibits manganese uptake and compromises oxidative stress management in Streptococcus pneumoniae[END_REF][START_REF] Mcdevitt | A molecular mechanism for bacterial susceptibility to zinc[END_REF]. This concept has been recently confirmed in a study whereby ionophores that induced zinc accumulation in Streptococcus uberis disabled oxidative stress protection through inhibition of manganese-dependent superoxide dismutase [START_REF] Harbison-Price | Multiple bactericidal mechanisms of the zinc ionophore PBT2[END_REF]. Zinc excess can also dysregulate homeostasis of other trace metal ions. Excessive zinc can induce copper depletion, which may be driven by dysregulation of copper transporters and detoxification systems [START_REF] Hassan | Zinc stress induces copper depletion in Acinetobacter baumannii[END_REF][START_REF] Xu | Zinc excess increases cellular demand for iron and decreases tolerance to copper in Escherichia coli[END_REF]. In addition, zinc excess increases the concentration of iron in E. coli by transiently inducing expression of iron-uptake genes and downregulating iron storage genes [START_REF] Xu | Zinc excess increases cellular demand for iron and decreases tolerance to copper in Escherichia coli[END_REF]. Zinc may also have direct impacts on key cellular processes required for microbial survival. For example, excessive intracellular zinc can disrupt iron-sulfur (4Fe-S) complex biogenesis in E. coli, thereby arresting key metabolic and growth pathways [START_REF] Li | Zinc toxicity and iron-sulfur cluster biogenesis in Escherichia coli[END_REF][START_REF] Xu | Silver(I), mercury(II), cadmium(II), and zinc(II) target exposed enzymic iron-sulfur clusters when they toxify Escherichia coli[END_REF]. In addition, excess zinc impaired capsule synthesis and perturbed glycolysis through inhibition of glycolytic enzymes in S. pyogenes [START_REF] Ong | Zinc disrupts central carbon metabolism and capsule biosynthesis in Streptococcus pyogenes[END_REF]. Thus, high concentrations of zinc can dysregulate many facets of homeostasis in microorganisms to exert its antimicrobial effects.

| Cooperation between zinc and other innate immune antimicrobial pathways

In addition to direct toxicity, zinc may also promote bacterial clearance through indirect mechanisms, particularly through enhancement of, or synergism with, other innate immune antimicrobial pathways. For example, basal and inducible autophagy is dependent on zinc in various cell types [START_REF] Hung | Dopamine-and zincinduced autophagosome formation facilitates PC12 cell survival[END_REF][START_REF] Hwang | Zinc(II) ion mediates tamoxifen-induced autophagy and cell death in MCF-7 breast cancer cell line[END_REF][START_REF] Lee | Roles of zinc and metallothionein-3 in oxidative stress-induced lysosomal dysfunction, cell death, and autophagy in neurons and astrocytes[END_REF][START_REF] Liuzzi | Role of zinc in the regulation of autophagy during ethanol exposure in human hepatoma cells[END_REF]. In human macrophages, signalling via the pattern recognition receptor NOD2 increased intracellular zinc levels to promote autophagy and intracellular clearance of S. Typhimurium, S. aureus and adherent invasive E. coli [START_REF] Lahiri | Activation of pattern recognition receptors up-regulates metallothioneins, thereby increasing intracellular accumulation of zinc, autophagy, and bacterial clearance by macrophages[END_REF]. Therefore, regulated zinc trafficking in innate immune cells may contribute to autophagymediated control of intracellular pathogens.

Innate immune cells use reactive oxygen species (ROS), generated either through the NADPH oxidase system or through mitochondrial activity, as an antimicrobial weapon. Some evidence suggests that zinc may act cooperatively with ROS to exert antimicrobial effects. The growth of zinc-sensitive E. coli mutants was synergistically inhibited when bacteria were cultured in the presence of both zinc and the superoxide anion generator paraquat [START_REF] Stocks | Uropathogenic Escherichia coli employs both evasion and resistance to subvert innate immunemediated zinc toxicity for dissemination[END_REF]. In contrast, zinc chelation by MTs was required for robust ROS generation upon challenge with C. glabrata [START_REF] Riedelberger | Type I interferons ameliorate zinc intoxication of Candida glabrata by macrophages and promote fungal immune evasion[END_REF], S. Typhimurium [START_REF] Wu | Salmonella utilizes zinc to subvert antimicrobial host defense of macrophages via modulation of NF-κB signaling[END_REF] and Histoplasma capsulatum [START_REF] Subramanian Vignesh | Granulocyte macrophagecolony stimulating factor induced Zn sequestration enhances macrophage superoxide and limits intracellular pathogen survival[END_REF] in murine or human macrophages. Therefore, the precise interplay between zinc and ROS during antimicrobial responses is likely to be context dependent.

In addition to ROS, innate immune cells also generate nitric oxide (NO) for free radical attack of intracellular pathogens. There is some evidence that zinc may influence NO production and/or that NO itself can initiate zinc toxicity. Zinc chelation abrogated TLR4-and TLR3-inducible nitric oxide synthase (iNOS/Nos2) expression that is required for inducible NO production in murine macrophages [START_REF] Brieger | Differential regulation of TLRdependent MyD88 and TRIF signaling pathways by free zinc ions[END_REF]. In bacteria, nitrosative stress may also interfere with zinc homeostasis. In Salmonella, NO triggers zinc release from multiple metalloproteins, including those involved in nitrosative stress sensing [START_REF] Henard | The 4-cysteine zinc-finger motif of the RNA polymerase regulator DksA serves as a thiol switch for sensing oxidative and nitrosative stress[END_REF], cell metabolism, protein synthesis [START_REF] Frawley | Nitric oxide disrupts zinc homeostasis in Salmonella enterica; serovar Typhimurium[END_REF] and DNA binding [START_REF] Schapiro | Inhibition of bacterial DNA replication by zinc mobilization during nitrosative stress[END_REF]. In addition, pharmacological inhibition of Nos2 in an intraperitoneal murine infection model restored bacterial loads of a S. Typhimurium

ΔzntAΔzitB mutant to those of wild-type levels in murine spleen and liver, suggesting that NO may induce zinc toxicity in Salmonella [START_REF] Frawley | Nitric oxide disrupts zinc homeostasis in Salmonella enterica; serovar Typhimurium[END_REF]. This suggests that, under nitrosative stress, effective zinc efflux is required to maintain homeostasis and survival of Salmonella during murine infection [START_REF] Frawley | Nitric oxide disrupts zinc homeostasis in Salmonella enterica; serovar Typhimurium[END_REF]. Of note, however, TLR signalling induces the zinc toxicity response [START_REF] Kapetanovic | Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages[END_REF], but not NOS2 expression [START_REF] Gross | Epigenetic silencing of the human NOS2 gene: Rethinking the role of nitric oxide in human macrophage inflammatory responses[END_REF], in human macrophages. Thus, it seems unlikely that NO production contributes to the susceptibility of zinc-sensitive bacterial mutants in these cells.

Zinc toxicity may also be influenced by other metal ions during antimicrobial responses. Copper is utilised by the innate immune system as an antimicrobial weapon against various pathogens [START_REF] Sheldon | Metals as phagocyte antimicrobial effectors[END_REF]. Non-toxic copper concentrations enhanced the cytotoxic effects of zinc on both S. Typhimurium and E. coli ΔzntA mutants, with these effects being rescued by zntA complementation [START_REF] Kapetanovic | Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages[END_REF]. Similarly, co-treatment of A. baumannii with zinc and copper resulted in altered membrane composition and increased susceptibility to ROS [START_REF] Hassan | Zinc stress induces copper depletion in Acinetobacter baumannii[END_REF]. Together, these studies suggest that copper and zinc may act cooperatively to exert an antimicrobial effect. Indeed, the concentrations of both zinc and copper were increased in Mycobacterium-containing phagosomes of murine macrophages under certain conditions [START_REF] Wagner | Elemental analysis of Mycobacterium avium, Mycobacterium tuberculosis, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system[END_REF].

In keeping with this concept, in LPS-stimulated human macrophages, vesicular zinc and copper partially co-localised [START_REF] Kapetanovic | Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages[END_REF]. This raises the possibility of spatial and/or timedependent cooperativity of these two metal ions, supporting the 'brass dagger' model of host defence [START_REF] German | Bacterial killing in macrophages and amoeba: Do they all use a brass dagger?[END_REF]. However, co-supplementation of THP-1 cells with zinc and copper did not affect clearance of A. baumannii [START_REF] Hassan | Zinc stress induces copper depletion in Acinetobacter baumannii[END_REF], suggesting that other exogenous factors and/or cell types may be important for combinatorial antimicrobial effects of these two metal ions.

| FINAL REMARKS AND FUTURE DIRECTIONS

In conclusion, zinc toxicity is emerging as an antimicrobial mechanism employed by innate immune cells to combat many von PEIN ET AL.

microorganisms, particularly bacterial and fungal pathogens. Significant advances have been made in our understanding of immune cell types that utilise zinc toxicity as an antimicrobial response, the pathogens which are subjected to zinc stress during infection, and the innate stimuli that drive this antimicrobial response. However, as highlighted throughout this review, key questions still remain. Specifically, the molecular mechanisms by which zinc is mobilised towards engulfed pathogens, the precise evasion mechanisms employed by pathogens, and the contributions of other antimicrobial pathways to zinc toxicity require further elucidation. Intriguing recent evidence also points to intertwining effects of the seemingly oppositional antimicrobial pathways of zinc sequestration and zinc toxicity. In a murine infection model, A. baumannii was subjected to zinc starvation in the respiratory tract, but experienced zinc intoxication in the spleen [START_REF] Alquethamy | The role of zinc efflux during Acinetobacter baumannii infection[END_REF]. Similarly, another study showed that while neutrophils employ zinc sequestration against extracellular S. pyogenes, phagocytosed bacteria were subjected to zinc toxicity [START_REF] Ong | New insights into the role of zinc acquisition and zinc tolerance in Group A Streptococcal infection[END_REF]. One possibility is that the capacity of concentrated zinc to be cytotoxic to intracellular pathogens is enhanced by initial zinc starvation encountered in extracellular environments and/or in different tissues. In this way, innate immune-mediated zinc starvation may predispose microorganisms to subsequent zinc poisoning.

As increasing rates of antimicrobial resistance threaten global health worldwide, there is an urgent need for the development of novel therapies for infectious diseases. One approach under investigation is harnessing of innate immune antimicrobial responses through (Huntington Study Group Reach2HD Investigators, 2015;[START_REF] Lannfelt | Safety, efficacy, and biomarker findings of PBT2 in targeting Aβ as a modifying therapy for Alzheimer's disease: A phase IIa, double-blind, randomised, placebo-controlled trial[END_REF][START_REF] Villemagne | A randomized, exploratory molecular imaging study targeting amyloid β with a novel 8-OH quinoline in Alzheimer's disease: The PBT2-204 IMAGINE study[END_REF]. These zinc ionophores also have antibacterial activity against a variety of Gram-positive bacterial pathogens and can act synergistically with antibiotics [START_REF] Bohlmann | Chemical synergy between ionophore PBT2 and zinc reverses antibiotic resistance[END_REF][START_REF] Harbison-Price | Multiple bactericidal mechanisms of the zinc ionophore PBT2[END_REF]. Ionophores have also been effective in reversing antibiotic resistance in Gram-negative bacteria [START_REF] Jen | Neisseria gonorrhoeae becomes susceptible to Polymyxin B and Colistin in the presence of PBT2[END_REF]. Therefore, it may also be possible to mimic innate immune-mediated zinc toxicity through application of zinc ionophores. Therapeutics that target zinc homeostasis, either through host or pathogen manipulation, may thus provide new opportunities for combating antibiotic-resistant bacterial infections. Moreover, knowledge of how zinc toxicity responses are engaged by the host and subverted by pathogens will likely deliver fascinating new insights into the host-pathogen dynamic.

|

  Direct antimicrobial impacts of zinc High concentrations of zinc are likely toxic to pathogens through multiple mechanisms. Zinc is predicted to preferentially bind to proteins in place of other first-row transition metal ions, resulting in mismetallation of essential proteins (Braymer & Giedroc, 2014; Stafford et al., 2013; Xu & F I G U R E 2 Innate immune cells mobilise zinc during infection.

'

  host-directed therapies'. Enhancing our understanding of the molecular mechanisms by which zinc is delivered to engulfed pathogens for metal ion poisoning, as well as the subversion mechanisms employed by pathogens to resist and/or evade zinc toxicity, could ultimately guide the development of new anti-infectives. Of note, zinc ionophores induce intracellular zinc accumulation, with one such ionophore recently passing second phase human clinical trials for the treatment of Alzheimer's disease and Huntington's disease

  T A B L E 1 Innate immune cells mobilise zinc in response to specific stimuli and/or infection

	von PEIN ET AL.
	Species	Cell type	Stimulation	Evidence	References
	Human	Monocyte-derived	Chronic NOD2 stimulation	" Intracellular Zn 2+ . Upregulated MT	Lahiri and
		macrophages		expression.	Abraham, (2014)
			M. tuberculosis infection	" Zn 2+ . Lysosomal/endosomal Zn 2+	Botella et al. (2011)
				accumulation.
			E. coli infection	" Intracellular Zn 2+ . Zn 2+ co-localised with	Botella et al. (2011)
				phagolysosomal E. coli.
				Zn 2+ co-localised with Zn 2+ -stressed E. coli.	Stocks et al. (2019)
				Zn 2+ vesicles co-localised with E. coli.	Kapetanovic et al. (2016)
			LPS	Vesicular Zn 2+ accumulation.	Kapetanovic et al. (2016)
			S. Typhimurium infection	Infection induced Zn 2+ vesicle formation. Zn 2+	Kapetanovic et al. (2016)
				co-localised with ΔSPI-1 mutant strain.
		THP-1	LPS	Vesicular Zn 2+ accumulation.	Kapetanovic et al. (2016)
	Mouse	Peritoneal macrophages	TNF (pre/post infection)	" Zn 2+ in M. avium-containing vacuoles.	Wagner et al. (2005)
			IFNγ (post-infection)	" Zn 2+ in M. avium-containing vacuoles.	Wagner et al. (2005)
			M. tuberculosis/M.	" Phagosomal Zn 2+ .	Wagner et al. (2005)
			smegmatis infection
		Bone marrow-derived	IL-4	Mt-3 and Slc30a4-dependent " of labile Zn 2+	Subramanian Vignesh
		macrophages		stores.	et al. (2016))
			E. coli infection	Intramacrophage E. coli reported Zn 2+ stress.	Stocks et al. (2019))
			C. glabrata infection	Zn 2+ accumulation in fungal vacuole.	Riedelberger et al. (2020)
		RAW 264.7	S. Typhimurium infection	" Intracellular Zn 2+ .	Wu et al. (2017)
			C. glabrata infection	Zn 2+ accumulation in fungal vacuole.	Riedelberger et al. (2020)

  Evidence for innate immune-mediated zinc toxicity against intracellular pathogens

	von PEIN ET AL.
	Pathogen	Infection model	Phenotype	References
	Zinc transporters
	M. tuberculosis	Human macrophage	" ctpC, ctpG, ctpV expression. Growth defect in ΔctpC	Botella et al. (2011)
			mutant.
	S. Typhimurium	Mouse infection model	ΔzntAΔzitB strain out-competed by wild-type strain	Frawley et al. (2018)
		(intraperitoneal)	in liver and spleen.
		Human macrophage	" zntA expression and " susceptibility to killing in	Kapetanovic et al. (2016)
			ΔSPI-1 mutant strain. SPI-1 prevents Zn 2+ vesicle
			co-localisation with bacteria.
	E. coli	Human neutrophil	ΔzntA mutant strain more susceptible to killing.	Kapetanovic et al. (2016)
		Human macrophage	" zntA expression.	Kapetanovic et al. (2016)
			# Survival ΔzntA mutant strain.	Botella et al. (2011)
			" zntA expression. # survival ΔzntA mutant strain.	Stocks et al. (2019)
			Intramacrophage E. coli reported Zn 2+ stress
			(zntA-driven fluorescence).
		Murine macrophage	Intramacrophage E. coli reported Zn 2+ stress	Stocks et al. (2019)
			(zntA-driven fluorescence).
		Mouse infection model	# Dissemination of ΔzntA mutant strain in liver	Stocks et al. (2019)
		(intraperitoneal)	and spleen.
	S. pneumoniae	Human neutrophil	" czcD and # phtE (Zn 2+ importer) expression.	Eijkelkamp et al. (2019)
			Zn 2+ supplementation # wild-type and ΔczcD mutant
			survival.
		Mouse infection model (intranasal)	Zn Eijkelkamp et al. (2019)
	S. pyogenes	Human neutrophil	ΔczcD mutant more susceptible to killing.	Ong et al. (2014); Ong
				et al. (2018)
		Mouse infection model	ΔczcD mutants # mortality of mice. ΔczcD mutants	Ong et al. (2014)
		(invasive	had # survival relative to wild-type during
		subcutaneous)	competitive infection.
		Mouse infection model	ΔczcD mutants # initial lesion size, dissemination	Ong et al. (2018)
		(invasive	in blood and mortality of mice.
		subcutaneous)
	C. glabrata	Murine macrophage	Survival defect in ΔZRC1 mutants.	Riedelberger et al. (2020)
	Pseudomonas	THP-1	" cadA and czcC expression during infection.	Ducret, Gonzalez, Leoni, Valentini,
	aeruginosa			and Perron (2020)
	Regulators of zinc exporters
	E. coli	Human macrophage	# Survival ΔzntR mutant strain.	Stocks et al. (2019)
	S. pneumoniae	THP-1	# Survival of ΔsczA mutant.	Martin et al. (2017)
	S. pyogenes	Human neutrophil	ΔgczA mutant more susceptible to killing.	Ong et al. (2014)
		Mouse infection model	# Virulence and # survival of ΔgczA mutants relative	Ong et al. (2014)
		(subcutaneous)	to wild-type strains.

that is induced by LPS in monocytes

[START_REF] Pyle | Zinc modulates endotoxin-induced human macrophage inflammation through ZIP8 induction and C/EBPβ inhibition[END_REF]

and macrophages (Liu T A B L E 2 2+ co-localised with bacteria in lung cavity. " czcD and # phtE expression. # ΔczcD mutant abundance in lungs. Eijkelkamp et al. (2019) THP-1 " czcD expression. Zn 2+ supplementation # wild-type and ΔczcD mutant survival. et al., 2013), with defined roles in regulating inflammatory responses

[START_REF] Hall | Novel role of zinc homeostasis in IL-23 regulation and host defense following bacterial infection[END_REF][START_REF] Liu | ZIP8 regulates host defense through zinc-mediated inhibition of NF-κB[END_REF][START_REF] Pyle | Zinc modulates endotoxin-induced human macrophage inflammation through ZIP8 induction and C/EBPβ inhibition[END_REF]

. SLC39A14, which is closely related to SLC39A8

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14625822, 2021, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cmi.13268 by Inrae -Dipso, Wiley Online Library on [07/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

of 11 von PEIN ET AL. 14625822, 2021, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cmi.13268 by Inrae -Dipso, Wiley Online Library on [07/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

of 11 von PEIN ET AL. 14625822, 2021, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cmi.13268 by Inrae -Dipso, Wiley Online Library on [07/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

ACKNOWLEDGEMENTS

This work was supported by a National Health and Medical Research Council of Australia (NHMRC) project grant (APP1144967). M.J.S. and M.A.S. are supported by NHMRC Senior Research Fellowships (APP1107914 and APP1106930, respectively).

DATA AVAILABILITY STATEMENT

Data sharing not applicable to this article as no datasets were generated or analysed in this review. This review was composed entirely from previously published data.

CONFLICT OF INTEREST

The authors declare no conflict of interest.


Arnaud Dufils
email: arnaud.dufils@inrae.fr

Raphaël Paut
email: raphael.paut@inrae.fr

Grazed orchards in France: different forms of livestock integration and their implications for fruit growers' practices EURAF 2020 Agroforestry for the transition towards sustainability and bioeconomy

Keywords: Orchard, sheep, livestock, integration, agro-silvopastoral systems

), while at the same time saving on phytosanitary treatments and fertilizers.

We carried out semi-structured interviews with 20 farmers in several regions of France (Provence and North-West). These interviews aimed to analyse: (i) farms structure (different productions and their respective area); (ii) the motivations of the farmers to integrate livestock and fruit trees; (iii) the technical adaptations that sylvopastoralism generated; and finally (iv) the services and disservices provided by animals in orchards.

The place of the animal in orchards is highly dependent on the fruit grower's expectations with regard to the provided services, his interest in livestock farming, but it is also closely linked to his ability to make his production system evolve and acquire new skills. We identified three possible organizations depending on the fruit grower degree of involvement into livestock farming:

(i)
Type 1: Farmers cooperation (n=10). A fruit grower may use a flock from another livestock farmer. Thus, shepherds graze their large livestock from the end of the fruit harvest until budburst the following spring on dense orchard areas. This mainly informal arrangement benefits both parties: access to a grass resource for the herd and weed management for the orchard, thus eliminating at least one mechanical or chemical weeding. (ii)

Type 2: Orchard-Livestock punctual integration (n=6). When a producer aims to optimize the prophylactic action of the sheep against specific pests and diseases such as vole (Microtus duodecimcostatus), codling moth (Cydia pomonella), scab (Venturia inaequalis), he acquires a herd, often of limited size. The herd then grazes as quickly as possible after harvest in order to consume fruits leftovers and leaves, potential sources of inoculum. It then leaves the orchards as budbreak begins. In this configuration, the fruit grower has "fallback" plots: grasslands, mountain pastures or wooded plots, for grazing his herd during spring and summer. (iii)

Type 3: Orchard-Livestock quasi-permanent integration (n=4). Some fruit growers have opted to maximize the presence of their herd in orchards. A quasi-permanent pasture except during harvest and lambing periods provides more room for manoeuvre to increase the impact of sheep on the main pests and diseases in the orchard. In particular, by adjusting the frequency and the duration of sheep presence on a given plot. O3.3_14_301

Changes in the orchard and the fruit grower profession

Besides the collaboration with a professional shepherd, when a fruit farmer owns his herd, investments (fences, shelters, veterinary care...) and orchard improvements have to be considered, although they are partially compensated by livestock sales. In quasi-permanent integration (type 3), it may be necessary to raise the fruiting area to at least 1.10 m above ground, raising tree height.

Moreover, the presence of herds becomes an additional factor to be considered in relation to the nature of the normal management: pruning, treatments, harvesting. Because of copper toxicity towards sheep, it is essential to use an alternative to this active ingredient or to exclude the animals from the plot for 20 to 30 days after copper application. A large part of the extra work related to sheep farming is the management of fodder resource, by organising rotational grazing systems between tree rows, using mobile fences in order to move sheep regularly. Observing sheep's behaviour is also a crucial activity to detect any situation of insufficient grass resources, in order to prevent the risk of damaging tree bark.

Finally, sheep farming also involves more on-call duty, especially during the lambing period. New regulatory and sanitary constraints must be respected. It is also necessary to open up to new professional networks (veterinarians, shearers, slaughterhouses, etc.).

A flexible system for more sustainability

The main advantage of the livestock-orchard mixed system is its flexibility as it is compatible with all production methods (conventional, organic, biodynamic) and paves the way to numerous combinations between orchard and livestock [START_REF] De Lacroix | Alternative management of weed control in orchards using animals in Martinique[END_REF]) depending on the objectives and socio-economic contexts of fruit growers (e.g. geese in peach orchards, hens in olive grove, pigs in apple orchards...). The example of sheep and apple orchard integration described here shows that there are many ways in which animals can be integrated, and that this degree of integration has a significant impact on farmers' management practices.

Moreover, through the reduction of the use of inputs, it has a positive environmental impact, contributing to practice changes and a shit of societal vision of fruit growers. Getting beyond the single model of the specialized and intensified orchard is not simple but can be done gradually. New orchard models will have to be designed, site-specific or 'tailor-made' in accordance with the objectives, needs, skills and constraints of each fruit grower.

Figure 1 .

 1 Figure 1. Example of cherry orchard in Provence region corresponding to our Type 2 (credits: Compagnone M.).

0 0 0 0 0 0 0 0 0 0 461 O3.3_14_301


Raphael Paut
email: raphael.paut@inrae.fr

Rodolphe Sabatier
email: rodolphe.sabatier@inrae.fr

Marc Tchamitchian
email: marc.tchamitchian@inrae.fr

Linking scientific and empirical knowledge: an interactive web app to design agroforestry market gardening systems EURAF 2020 Agroforestry for the transition towards sustainability and bioeconomy

Keywords: agroforestry, intercropping, vegetables, fruit trees, decision-support tool

Introduction

Among agroforestry systems, a particular form of intercropping has developed in recent years in Europe: the agroforestry market gardening system [START_REF] Warlop | The smart project: a focus on fruit trees and vegetables agroforestry systems in France[END_REF]. This particular form of professional agroforestry, where vegetables are grown in intercropping with fruit trees or berries, is found mainly in organic agriculture, on small areas (Fig. 1). Farmers combine a wide variety of crops and rely mainly on short food supply chains [START_REF] Léger | Agroforestry market gardening: a strategic choice to improve sustainability in agroecological transition[END_REF]. Due to their highly agro-ecological nature, these systems seem able of addressing many of the specific challenges of developed European regions.

However, the great diversity of crops in terms of phenology (tubers, leaf, root or fruit vegetables, pome, stone or nuts fruits, berries, etc.), the number of combinations, the various biological interactions to take into account and the nature of these interactions make it very difficult to acquire references on these systems. While research is underway to understand the interactions between fruit trees and vegetables [START_REF] Imbert | Pests, but not predators, increase in mixed fruit treevegetable plots compared to control vegetable plots in a Mediterranean climate[END_REF], the nearly unlimited number of crop combinations makes this work very tedious.

In parallel, there is a substantial "grey literature" on this subject. This literature includes popular books, gardening books, websites and specialized magazines. It mainly compiles empirical knowledge resulting from the capitalization of site-specific experiments. The objective of the present work is therefore to combine scientific and grey literature in order to provide a decision support tool for farmers or new agroforestry practitioners.

Material & Methods

We carried out a review on horticultural intercropping systems in the scientific and grey literature. With regard to scientific literature, studies were identified from a search in the Institute for Scientific Information Web of Knowledge and Google Scholar databases. We requested the following terms: (inter-crop* OR agroforestry OR mixed crop*) AND (fruit* OR orchard OR vegetable* OR market gardening). 74 papers were analysed, resulting in 534 experiments (an experiment was defined by a unique crop combination in site and year). With regard to grey literature, books and documents were identified by search engines. 28 documents were analysed, resulting in 6525 combinations. A combination was defined as a unique data on an intercropping, either qualified as a "positive" or "negative" interaction.

Results

All the data from our literature review were combined in a web-based interactive platform. The user can choose one or more crops. Several forms of graphic representations are then available. When data is available in the scientific literature, Land Equivalent Ratio values are provided for the selected crop as well as all the other crops with which it can be intercropped. When it is available in the grey literature, the data is compiled and weighted. The platform also makes it possible to select several crops and generates a network representation (see Fig.

Figure 1 .

 1 Figure 1. An example of market gardening agroforestry system (pear -potatoes -peach intercropping)

Figure 2 .

 2 Figure 2. Screenshots of the web-based application

Contact the corresponding author if the platform is no longer available at the address provided.

0 0 0 0 0 0 0 0 0 0 482 O4.1_2_64


domain: sde math phys

text:
Bou-Laouz Moujahid

Vadaine Rodolphe

Hajduch Guillaume

Fablet Ronan

Deep Learning for Ship Classication on Medium Resolution SAR Imagery

I. INTRODUCTION

The maritime trafc illustrates multiple types of activities ranging from economical to leasure activities. This trafc induces the need of surveillance by local authorities to address the challenges of maritime safety, maritime security, law enforcement and sheries control [START_REF] Emsa | Emsa outlook 2023[END_REF]. Far off the coast, satellite observations allow collecting valuable information on the ongoing activities using a wide range of sensors, including SAR sensors and AIS data collection. A large litterature is available on the use of a single source of data for the surveillance of maritime trafc for instance using SAR imagery [START_REF] Hajduch | Vigisat ground receiving station and emsa cleanseanet services[END_REF][3] or AIS information [START_REF] Nguyen | A multitask deep learning architecture for maritime surveillance using ais data streams[END_REF] alone. The synergetic use of multiple source of data is however rapidely progressing [START_REF] Fablet | Next step for big data infrastructure and analytics for the surveillance of the maritime trafc from ais\& sentinel satellite data streams[END_REF]. Merging multiple type of data allows either to derive better processing for each data type separately [START_REF] Dechesne | Ship identication and characterization in sentinel-1 sar images with multitask deep learning[END_REF] or a real merging of data information to improve the overal martime domain awareness [START_REF] Paladin | Maritime information sharing environment deployment using the advanced multilayered data lake capabilities: Effector project case study[END_REF].

Commercial SAR satellites deliver very high-resolution imagery [START_REF] Airbus | Terrasar-x image product guide: Basic and enhanced radar satellite imagery[END_REF][9] that can be aquired on specic regions of interest for instance for vessel detection. High-resolution observations make the classication of vessel types straightforward as illustrated by a large litterature on vessel classication for high-resolution satellite imagery [START_REF] Lu | Ship classication in high-resolution sar images via transfer learning with small training dataset[END_REF]. However, few studies have addresse medium-resolution SAR imagery like the one of the Copernicus Sentinel-1 SAR constellation [START_REF] Esa | Sentinel-1 what is it ?[END_REF]. Sentinel-1 allows regular access to large dataset on European waters and large areas worldwide. Delivering automated vessel clas-sication tools on such imagery would benet to the analysis the maritime trafc and activities at sea. This paper specically adresses the vessel classication on medium resolution SAR images from the Sentinel-1 SAR mission and investigates the possibility to jointly exploit such satellite imagery datasets with AIS data streams to develop a learning-based classication framework.

In section II we present the data considered for the study. In section III we present the overall methodology of classication. In section IV we present the evaluation of performances. In section V we present the conclusion and proposition of applications of the results.

II. PRESENTATION OF DATA

The obsevation plan of the Sentinel-1 SAR constellation allows to collect over ocean mainly medium resolution imagery using the Interferometric Wide Swath (IW) acquisition mode. This data can be made available as detected images on two avours being GRDM (medium) and GRDH (high) resolution. While the GRDH products are nominally processed by the ESA ground segment, the Collaborative ground stations can generate as well the GRDM avour products [START_REF] Hajduch | Vigisat ground receiving station and emsa cleanseanet services[END_REF].

In this study, we make use of the GRDH products, which has the following characteristics : a swath (rg x az [km]) of 250 x 170, a resolution (rg x az [m]) of 20 x 22 and a pixel spacing (rg x az [m]) of 10 x 10.

CLS made available series of AIS data collected from ground and space receivers. The AIS data collect the location of vessels reporting their positions and information on their speed, route, and type [START_REF]Automatic identication system[END_REF]. While the data broadcasted by AIS can be switched off or altered, it is broadly used by the Earth observation community as a proxy of vessel location and type [START_REF] Vachon | Validation of radarsat-1 vessel signatures with aislive data[END_REF].

With a view to implementing deep learning strategies, we created reference datasets using the synergy between AIS data and Sentinel-1 SAR data. Thanks to AIS data, we estimate the precise location of the ships in the SAR image and extract related information contained in AIS messages metadata (in our case, length and type). Based on this position and the positions of ships detected automatically on SAR satellite images (algorithm described in [START_REF] Hajduch | Ship detection: From processing to instrument characterization[END_REF]), we extract the image of the ship by cropping the SAR image in the area where the ship is located.

III. CONSIDERED LERANING-BASED METHOLOGY

In this section, we detail our deep learning scheme for ship classication method and the associated rationale.

A. Vessel classication case-study

We focused on the categories "Tanker", "Cargo", and "Others". The "Others" category encompasses every other type of vessels. The motivations of this choice relate to the following facts:

• Neural networks are known to require large amounts of data in order to be effective. As a result, we selected the types of vessels that make up the majority of the data.

Table I shows the most represented categories.

Therefore, the choice of a three-class problem offers a good starting testbed, as well as a perspective for more complex problems.

B. Training and test datasets

The performance and accuracy of deep learning networks depend heavily on the quality of the data used for training and testing. The quality of the labeling issued from AIS data poses a signicant challenge for our dataset. To reduce the impact of this quality shortage, we removed all the products that have the type of vessel "Unitised". These products contain a large amount of cargo, in addition to other types of vessels. Additionally, we removed the products that have a vessel size of zero or greater than 400 meters, as this data is likely to be falsied. The training is carried out on 80% of the data, while the test is done on 20% of the data. The data augmentation methods used includes oversampling and horizontal and vertical ips. Using other methods such as Gaussian noise or rotation proved to be disruptive for the models used.

C. Data preprocessing and choice of input

Due to nature of the SAR data, especially the range of observed pixel values and the occurence of outliers, we applied a logarithmic transformation to the data. This technique helps to minimize the effect of outliers and leads to faster convergence of the loss function. In terms of the input for our model, we chose to use a two-band image, which includes backscatter intensity and incidence angle. This selection was made because the incidence angle has a signicant impact on the backscatter intensity and then on the contrast between vessel and sea surface. As a result, we did not use any pre-trained network, as we believed that they would not be able to handle these features relevantly.

D. Neural network architectures

To address the classication problem, we developed ensemble convolutional neural networks (CNNs). The overall architectures of the CNNs used in this work are illustrated in Table III. We used the most well-known concepts in the stateof-the-art in our models. In Model A, we used a Convolutional Block Attention Module [START_REF] Woo | Cbam: Convolutional block attention module[END_REF], in Model D, we used residual layers [START_REF] He | Deep residual learning for image recognition[END_REF], and in Model E, we used the inception module [START_REF] Szegedy | Going deeper with convolutions[END_REF]. One can see that all models resemble more or less Model A. In fact, Model A is our main model. After many experiments, we believe that this is the best model compared to our data and problem. We used the same parameters in all models. We exploited a categorical cross-entropy loss as a training loss function.

We applied an end-to-end training using Adam Optimizer. We used batches of size 64 and a learning rate of 1e-4. This parametrization has shown good results for our classication task. In our nal model, we combined the output of the ensemble of CNN models using an "Averaging" merging. This conguration proved to be the best not only in terms of precision but also in terms of model calibration. It is worth mentioning that Model A is perfectly calibrated, while our nal model is well calibrated, especially for large condence values (> 0.8).

IV. RESULTS

The experiments were conducted to evaluate the performance models A, B, C, D, and E, on the classication task. Table IV presents the total accuracy scores for each model, as well as our nal model that was constructed by taking the average of the outputs from all ve models. The results of the overall test dataset indicate that the total accuracy of our nal model was 79%. In this study, our objective is to attain an optimal balance between recall and precision for all classes, as there is no preference for classifying one class over another. However, some parts of our data pose a challenge in clearly distinguishing between cargo and tanker ships, causing a minor bias towards either the cargo or tanker class. To address this, we have chosen to lean slightly towards the cargo class as it is the most commonly encountered class in practical applications. The table V presents the confusion matrix and detailed metrics for each class.

V. CONCLUSION

This paper has studied the classication of vessels on medium resolution SAR images. To the best of our knowledge, this is the rst time a Deep learning network has been trained to make this task on a relatively large dataset, and which provides a relatively good results. A pratical use case for our network is the identication of discrepancies between a vessel's declared information and the network's predictions when those predictions are superior to Conv" denotes a convolutional layer, "CBAM" denotes a convolutional block attention module using the same parameters cited in reference [START_REF] Woo | Cbam: Convolutional block attention module[END_REF], "FC" denotes a fully-connected layer, "k-s" denotes kernel size, and "Concatenate" denotes a concatenation operation. Unless otherwise stated, we use in the convolution operations a kernel size of size 3, strides = 1, and padding = "same", we use in the pooling operations a pool size = 2, stride =1, and padding ="valid". We use a dropout rate of 0. 0.9, which can trigger an alert for potentially-false AIS data. A large attention is placed in the eld of maritime safety on dark vessels not reporting their location through AIS, while we believe that other alterations of AIS data are possible and under considered, including eroneous declaration of vessel's type (we could call them bright vessels). This paper paves the way for future work to automatic vessel classication on medium-resolution SAR imagery without the need for AIS data. To our knowledge, the SAR imagery is more difcult to alter than the AIS information. This breakthrough could be achieved through the collection of more data and the development of deep learning methods.

Fig. 1 :

 1 Fig. 1: A comparison of the effect of logarithmic transformation on a vessel image which has a very retro-diffusing point is shown. On the left is the original image, and on the right is the image with the logarithmic transformation applied

TABLE I :

 I Description of Data. The category "Others" includes every other type of vessel

  Table II represents the training and test sets.

	Classes	Tanker	Cargo	Others
	Number of images	22474	37173	20805
	Training set	29739	29739	29739
	Test set	4494	7435	4161

TABLE II :

 II Description

of training/Test data. The data augmentation is carried out on the categories "Tanker" and "Others"

TABLE III :

 III Structures of the models. In this table, "

  [START_REF] Crisp | The state-of-the-art in ship detection in synthetic aperture radar imagery[END_REF]. All convolution and FC layers are followed by a RELU activation. Note that Block 8 is the same for all models

	Models	A	B	C	D	E	Averaging
	Total Accuracy 77.8%	77%	76% 77.4%	77.5%	79%

TABLE IV :

 IV Total Accuracy of Models A, B, C, D, E

TABLE V :

 V Confusion matrix and evaluation metrics

• The classication of vessels on medium-resolution radar images using deep learning techniques is a relatively new and under-explored eld of research. For instance, the results presented in[START_REF] Dechesne | Ship identication and characterization in sentinel-1 sar images with multitask deep learning[END_REF] are difcult to reproduce.In[START_REF] He | Ship classication in medium-resolution sar images via densely connected triplet cnns integrating sher discrimination regularized metric learning[END_REF], the training was not done on a large dataset.

AKNOWLEDGMENT

This work was performed under a research contract between CLS and IMT Atlantique. Part of the work was founded supported by "France Relance". We used Sentinel-1 data acquired between 2017 and 2022 as part of the Copernicus Sentinel programme.


Claire Mayer-Laigle

Karl Moving

Rob Whitton

Marie-Joo Le Guen

Yi Chen

Sustainable powders for laser-assisted additive manufacturing

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

Sustainable materials are being used more commonly with additive manufacturing (AM) technique to enable the manufacture of functional products in a sustainable and economically efficient way [START_REF] Ji | 3D printing of biomass-derived composites: application and characterization approaches[END_REF]. Selective laser sintering (SLS), is an AM technique that shows potential in fusing and solidifying biomass powders into 3D porous materials, paving the way in technical applications [START_REF] Xu | 3D printing for polymer/particle-based processing: A review[END_REF][START_REF] Yu | Impact of Particle Size on Performance of Selective Laser Sintering Walnut Shell/Co-PES Powder[END_REF]. However, because of the low thermal stability of biomass under laser sintering, petroleum thermoplastic polymers, such as nylon, are often mixed with biomass powders to increase their processability for SLS applications [START_REF] Ajdary | Selective Laser Sintering of Lignin-Based Composites[END_REF]. In this latter situation, the interfacial interactions between biomass powder and thermoplastic remain one of the most important challenges that need to be addressed [START_REF] Singamneni | Selective laser sintering responses of keratin-based bio-polymer composites[END_REF]. It is therefore important to develop a method that enables SLS printing of biomass powders directly.

In this work, we have developed an AM process for biomass powders using a laser sintering process with a commercial CO2 laser. The laser system is equipped with a chamber that has nitrogen flowing through it to prevent combustion of the powder. By using a layer-by-layer deposition technique, followed by laser sintering, a 3D structure can be directly printed from vegetal waxes and lignocellulosic composite powders. The morphological, chemical, and mechanical properties of the prepared 3D structures have been characterised by scanning electron microscopy (SEM), Fourier transform infrared spectrometer (FTIR) and dynamic mechanical thermal analysis (DMTA). The processability of wax powders with different particle sizes has been studied. This method is simple and requires no additional chemical or physical process, and it is potentially scalable for industrial applications.

  Sustainable powders for laser-assisted additive manufacturing Dr Claire Mayer-Laigle 1,2 , Karl Moving 1 , Rob Whitton 1 , Dr Mari Joo Le Guen 1 , Dr Yi Chen 1 1 Scion, 49 Sala Street, Rotorua , New Zealand, 2 1IATE, Université de Montpellier, INRAE, Institut Agro-Montpellier SupAgro, Montpellier, France Welcome Function and Poster Session 1, Grand Hall, February 7, 2023, 5:35 PM -7:30 PM


est destinée au dépôt

A

  functional ecology approach to define a conceptual method for species mixing design: a case study on nitrogen recycling Malick S Ouattara, Raphaël Paut, Muriel Valantin-Morison, Safia Médiène To cite this version: Malick S Ouattara, Raphaël Paut, Muriel Valantin-Morison, Safia Médiène. A functional ecology approach to define a conceptual method for species mixing design: a case study on nitrogen recycling. XVII. Congress of the European Society for Agronomy, Aug 2022, Potsdam (Germany), Germany. 2023. hal-04329399

A functional ecology approach to define a conceptual method for species mixing design: a case study on nitrogen recycling

Conclusion

The functional tree thus built will be used afterwards to build a hierarchical model which will lead to a tool to assist in the design of species mixtures. This tool will allow to choose the adequate species for nitrogen recycling according to the local conditions.


Raphael Lebeuf

Laura Dubrulle

Véronique Rataj


Raphael Lebeuf

Véronique Rataj

Oxo-functionalization of

come

Overall sequence :

Depending of the reaction conditions, either the enones or the allylic alcohols are obtained in good selectivity with the same catalyst, an organosoluble ammonium salt of molybdate anion without any additional ligand. The catalyst can be easily separated after the reaction by aqueous washes.

Proposed mechanism of the molybdate catalyzed reactions:

84% (+5% enones) 73% (+17% alcohols) (2 isomers) Photooxidation step :

Performed in dichloromethane (1.5 g of methyl oleate / 10 mL) using meso-tetra -phenylporphyrine (TPP, 2 mg) as photosensitizer under irradiation of high pressure sodium lamp and dioxygen bubbling. Full conversion in 8 hours.

Choice of DCM as solvent because :  non flammable  good solubility of the FAMEs  easily removable due to its low boiling point, avoid to heat the peroxides  rather long lifetime of 1 O 2 in this solvent (t 1/2 = 99 µs).

Note: the reactivity rate constant of methyl oleate is twice higher than its quenching rate constant with singlet dioxygen.

(k r = 2.4 10 4 M -1 .s -1 vs k q = 1.2 10 4 M -1 .s -1 in CHCl 3 at 20 o C)

Substrates scope :

Notes:

 triethylamine-N-oxide quantitatively observed in 1 H NMR  "reduction" much slower with the more nucleophilic but less basic pyridine instead of triethylamine  "reduction" much faster and more selective with dodecanthiol, but more expensive material and more difficult to separate than the volatile triethylamine

  access to keto-or hydroxy derivatives of unsaturated fatty Acids TEAM van Gorkum, R.; Bouwman, E. The oxidative drying of alkyd paint catalysed by metal complexes. Coord.Chem. Rev. 2005, 249, 1709-1728.

Figure 1 .

 1 Figure 1. 1 H NMR in Toluene-d 8 of methyl oleate hydroperoxides alone (blue) and in presence of stoichiometric amount of [C 1 C 1 C 8 C 8 N] 2 MoO 4 , first after 15 minutes at room temperature (red), then after 1 hour at 80°C (black).

Figure 1 .

 1 Figure 1. 1 H NMR in Toluene-d 8 of methyl oleate hydroperoxides alone (blue) and in presence of stoichiometric amount of [C 1 C 1 C 8 C 8 N] 2 MoO 4 , first after 15 minutes at room temperature (red), then after 1 hour at 80°C (black).

Figure 1 .

 1 Figure 1. 1 H NMR in Toluene-d 8 of methyl oleate hydroperoxides alone (blue) and in presence of stoichiometric amount of [C 1 C 1 C 8 C 8 N] 2 MoO 4 , first after 15 minutes at room temperature (red), then after 1 hour at 80°C (black).

  Tanielian, C.; Mechin, R. Reaction and quenching of singlet molecular oxygen with esters of polyunsaturated fatty acids. Photochem.Photobiol. 1994, 59, 263-268. proof of an adduct between the hydroperoxide and the molybdate anion :Dual behavior of the catalyst depending of the conditions :

  NMR yields of dehydration into enone or reduction into allylic alcohol of allylic hydroperoxides (0.25 mmol) from several alkenes using [C 1 C 1 C 8 C 8 N] 2 MoO 4 (5 mol%) catalyst. Conditions A = toluene (V T = 0.5 mL), 80 °C, 4h. Conditions B = triethylamine (0.75 mmol), 40 o C, 4h. Conversions = 100%. Dual Catalytic Role of Molybdate Ions for Direct Conversion of Photooxidized into Keto or Hydroxy Derivatives, L. Lebeuf; V. Nardello-Rataj ACS Sustainable Chem. Eng. 2019, on print, DOI: 10.1021/acssuschemeng.8b04643. ,β)-(γ,δ)-dienone isomers are the main isolated ones


Keywords: co-design, conservation agriculture, on-farm experimentation, systemic approach, participatory development

come

Co-design and on-farm experimentation of practices combining conservation agriculture and agroforestry

Co-design and on-farm experimentation of practices combining conservation agriculture and agroforestry

Sieffert A. [START_REF] Ndoli | Farming with trees : A balancing act in the shade[END_REF] (andre.sieffert@adaf26.org), Morinay A. Agroforestry (AF) studies tend not to take much into consideration the different options available for the management of crops per se, generally taking into account classical techniques. However, in these single farming systems, reduced or no-tillage methods using mulch or cover-crops defined as Direct seed Mulch based Cropping systems (DMC) are gaining momentum. According to recent studies, DMC systems result under specific conditions in an enhancement of biological activity of soils and optimization of soil-plant-microorganisms interactions which in turn can enhance the resistance of crops towards plant pathogens. DMC systems have however been rarely combined with AF systems. A straightforward explanation lies in the high level of expertise needed to follow each of the two approaches, which makes their combination highly complex. But more fundamental issues such as the difficulties to manage root competition between trees and vegetables without ploughing are also at stake. The objectives of our work is (1) the co-design and co-implementation with farmers of DMC and AF systems, (2) the study on how these systems and their combination affect the farming system in its whole and (3) the continuous diffusion of the results of this study. We focus on a co-design which is a result from a thorough collaboration between the farmers and our partners which are AF structures and experts in different fields. We started our work in 2016 and are currently collaborating with a network of 34 farms (arable crops, breeding and horticulture) in the South-East of France which are using or developing DMC and/or AF systems (e.g. vegetable-orchards, agroforestry in arable crops) since 2007 and up. Ndoli (2018) [1] has already shown that under specific conditions the combination of DMC and AF systems can lead to a reduced productivity as in the case of maize grown under trees. Hence, one of our aims is to identify technical and design options optimizing the combined management of crops and trees in DMC systems and allowing the success of the farming system in the long run. We direct this identification into an iterative process with the co-design of the farms. The co-design of the DMC and AF systems is implemented based on the diagnosis of the farming system at various levels associated to the farmer's objectives. On-farm experiments are then processed by the farmers in order to test the viability of the design. The ADAF carries out with the farmer and in relation with the technical partners the monitoring and evaluation of various parameters which assess the agronomic, environmental and socio-economic performances of the farm as well as the soil and crops conditions. Focus is also made, through a systemic approach, on the specific linkage between crop culture and tree management (e.g. workforce repartition and adaptation in vegetable-orchards).

  Sieffert André, Antoine Morinay, Tarik Zniber, Alain Canet, Séverin Lavoyer, Pascale Moity-Maïzi, Rodolphe Sabatier, Raphaël Paut, Marc Tchamitchian To cite this version: Sieffert André, Antoine Morinay, Tarik Zniber, Alain Canet, Séverin Lavoyer, et al.. Co-design and on-farm experimentation of practices combining conservation agriculture and agroforestry. 4th World Congress on Agroforestry Strengthening links between science, society and policy, May 2019, Montpellier, France. 2019. hal-04329480 Agroforestry 2019 -Poster L10 AF in practice 4 th World Congress on Agroforestry Strengthening links between science, society and policy

  [START_REF] Ndoli | Farming with trees : A balancing act in the shade[END_REF] , Zniber T. 1 , Canet A. 2 , Lavoyer S. 3 , Maïzi-Moity P. 4 , Sabatier R. 5 , Paut R. 5 , Tchamitchian M.5

1 Association Drômoise d'Agroforesterie, 26160 Pont-de-Barret, France; 2 Arbre et Paysage 32, 32000 Auch, France; 3 French Agroforestry Association, 32 000 Auch, France; 4 Institut des Régions Chaudes, Montpellier SupAgro, 34000 Montpellier, France; 5 Unité Ecodéveloppement, INRA, 84140 Montfavet, France