Clémence Poirotte
email: c.poirotte@gmail.com

Cécile Sarabian

Barthélémy Ngoubangoye

Andrew J J Macintosh

Marie Charpentier

Clemence Poirotte

Cecile Sarabian
email: sarabiancecile@gmail.com

Barthelemy Ngoubangoye

Faecal avoidance differs between the sexes but not with nematode infection risk in mandrills

Keywords: anti-parasitic behaviour, faecal avoidance, gastro-intestinal parasites, Mandrillus sphinx

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INTRODUCTION

The 'behavioural immune system' [START_REF] Schaller | Parasites, behavioral defenses, and the social psychological mechanisms through which cultures are evoked[END_REF] consists of a suite of mechanisms providing a first line of defence against parasites and pathogens before the intervention of the physiological immune system. It allows individuals to detect cues in the environment indicating the presence of contaminants which may trigger emotional responses (i.e. disgust).

This process facilitates functional behavioural reactions (i.e. avoidance) that decrease parasite infection risk, such as avoiding contagious conspecifics or contaminated substrates [START_REF] Hart | Behavioral adaptations to pathogens and parasites: five strategies[END_REF][START_REF] Curtis | Infection-avoidance behaviour in humans and other animals[END_REF]. Avoiding faeces is a common strategy that several animal species may use to decrease exposure to gastro-intestinal parasites, which are present in faeces and are typically transmitted via ingestion or skin penetration [START_REF] Anderson | Nematode parasites of vertebrates: Their development and transmission[END_REF][START_REF] Goater | Parasitism: The diversity and ecology of animal parasites[END_REF]. Faecal avoidance has mainly been demonstrated in foraging contexts in grazing ungulates (for a review see: [START_REF] Coulson | Parasite insight: assessing fitness costs, infection risks and foraging benefits relating to gastrointestinal nematodes in wild mammalian herbivores[END_REF] but similar studies are less common for other taxa, with a few notable exceptions [START_REF] Garnick | Foraging efficiency and parasite risk in eastern grey kangaroos (Macropus giganteus)[END_REF][START_REF] Sarabian | Hygienic tendencies correlate with low geohelminth infection in free-ranging macaques[END_REF].

Despite the obvious benefits, faecal avoidance also entails costs. Indeed, avoiding areas contaminated with faecal material may decrease the amount of available food resources [START_REF] Hutchings | The herbivores' dilemma: trade-offs between nutrition and parasitism in foraging decisions[END_REF]. We therefore expect faecal avoidance to be influenced by food availability and its nutritive value. Accordingly, several species of ruminants were found to avoid feeding in contaminated areas when faced with the choice of a faecally-contaminated versus uncontaminated sward of identical nutritional value [START_REF] Hutchings | The herbivores' dilemma: trade-offs between nutrition and parasitism in foraging decisions[END_REF][START_REF] Fleurance | Importance of nutritional and anti-parasite strategies in the foraging decisions of horses: an experimental test[END_REF]. These same animals, however, selected the faecally-contaminated sward if enriched in azote or proteins. In addition, we also expect animals to modulate their feeding behaviour with the quantity of infectious agents present in faeces, although the importance of this trade-off in mediating behaviour has rarely been investigated.

Nematodes are ubiquitous gastro-intestinal parasites. They may impact the survival and reproduction of a large range of host species, sometimes causing dramatic declines in wild animal populations [START_REF] Gulland | The impact of infectious diseases on wild animal populations -a review[END_REF][START_REF] Albon | The role of parasites in the dynamics of a reindeer population[END_REF][START_REF] Hanssen | Costs of parasites in common eiders: effects of antiparasite treatment[END_REF][START_REF] Pedersen | Cross-species pathogen transmission and disease emergence in primates[END_REF][START_REF] Hillegass | Parasite removal increases reproductive success in a social African ground squirrel[END_REF]. Gastrointestinal nematodes may therefore represent a major selection pressure favouring the evolution of mechanisms to detect and avoid infection. However, the risk of getting infected with nematodes varies according to the age of faeces because nematode eggs and larvae typically require time to develop before reaching their respective infective stages, e.g. infective third stage (L 3 ) larvae or embryonated eggs [START_REF] Anderson | Nematode parasites of vertebrates: Their development and transmission[END_REF]. This developmental process varies with environmental conditions (temperature and humidity) and may last from a few days to a few weeks depending on the species [START_REF] Neveu-Lemaire | Précis de parasitologie vétérinaire[END_REF][START_REF] Stromberg | Environmental factors influencing transmission[END_REF].

Consequently, we expect nematode infection risk, and therefore faecal avoidance, to be high when faecal material has been in the environment for a certain period of time following excretion by infected hosts.

In this study, we aimed to determine whether mandrills (Mandrillus sphinx) exhibit faecal avoidance in both non-feeding and feeding contexts, and whether the presence of nematodes at different developmental stages impacts their behaviour. In both contexts, we predicted that subjects would avoid faecally contaminated areas (non-feeding context) or food (feeding context), with stronger avoidance responses elicited in a feeding context since the latter involves ingestion, a major pathway to infection (e.g. oro-faecal route; [START_REF] Antonovics | The evolution of transmission mode[END_REF]. Furthermore, if mandrills can detect the presence of nematodes, we predicted that subjects would avoid faeces from parasitized individuals more strongly than those from nonparasitized individuals. Finally, we predicted stronger avoidance responses towards aged faeces compared to fresh faeces if mandrills can assess and respond specifically to nematode infection risk.

Decisions about whether or not to exploit a contaminated food resource may also depend on the forager's attributes. For example, in some primates (olive baboons Papio anubis: Müller-Graf, Collins, Packer, & Woolhouse, 1997; Japanese macaques Macaca fuscata: [START_REF] Sarabian | Hygienic tendencies correlate with low geohelminth infection in free-ranging macaques[END_REF] females avoid faecally-contaminated substrates to a greater degree than males. Similarly, women exhibit greater disgust than men towards animals associated with the spread of infectious diseases, such as cockroaches [START_REF] Olatunji | Disgust sensitivity as a mediator of the sex differences in contamination fears[END_REF][START_REF] Prokop | The association between disgust, danger and fear of macroparasites and human behaviour[END_REF]. However, in other primate species (chimpanzees Pan troglodytes: Sarabian, Ngoubangoye, & MacIntosh, 2017; bonobos Pan paniscus: [START_REF] Sarabian | Feeding decisions under contamination risk in bonobos[END_REF], males and females exhibit similar responses to contaminated food. Here, we predicted that male mandrills would be more risk-prone regarding their feeding decisions, i.e. tolerate a higher risk of infection than females to acquire food, as observed in other Papionini species [START_REF] Müller-Graf | Schistosoma mansoni infection in a natural population of olive baboons (Papio cynocephalus anubis) in Gombe Stream National Park, Tanzania[END_REF][START_REF] Sarabian | Hygienic tendencies correlate with low geohelminth infection in free-ranging macaques[END_REF].

METHODS

Study groups

We studied two semi-free-ranging groups of mandrills housed in forested enclosures of 6.5ha (89 individuals) and 3.5ha (57 individuals) at the 'Centre International de Recherches Médicales de Franceville' (CIRMF) in Southern Gabon. These animals forage freely but are supplemented twice a day with fruits, vegetables and monkey chow, which are deposited in an enclosed 'provisioning area'. This area is used to isolate animals for protocols such as health checks and behavioural tests. This provisioning area is coupled with a 'testing area' where we performed all behavioural tests analysed in this study.

Experimental design

Experiment 1: non-feeding context

To study whether mandrills avoid faeces in the absence of food and whether the presence of nematodes influences this behaviour, we compared subjects' responses to three different bamboo sticks, rubbed with 'nematode-positive' faecal samples (i.e. nematode eggs are present), 'nematode-negative' faecal samples (i.e. nematode eggs are absent) and with a common plant used as control.

Faecal sample collection. We used faecal samples routinely collected from another social group of free-ranging mandrills inhabiting the Lékédi park, a private park in Southern Gabon (Peignot et al., 2008, Mandrillus Project: www.projetmandrillus.com). Since 2012, faecal samples have been opportunistically collected for qualitative identification of gastrointestinal parasites (see [START_REF] Poirotte | Environmental and individual determinants of parasite richness across seasons in a free-ranging population of Mandrills (Mandrillus sphinx)[END_REF]. In addition, one gram of these faecal samples was stored at -20 °C for future use. Frozen faecal samples collected from the same donor no more than 3.5 months apart were paired when one sample contained at least one nematode taxon (nematode-positive) and one sample did not contain any nematode taxon (nematodenegative). In total, we obtained 20 pairs of faecal samples collected between February and September 2014 (mean time spent frozen ± standard deviation = 3.3 ± 0.4 months) from 14 donors (nine females aged 13.5 ± 3.8 years and five males aged 8.3 ± 4.1 years). Note that we did not use faecal samples collected from sexually-receptive females to minimize bias due to potential chemosignals of their reproductive status.

Nematode-positive faecal samples may have contained as many as four nematode taxa [START_REF] Poirotte | Environmental and individual determinants of parasite richness across seasons in a free-ranging population of Mandrills (Mandrillus sphinx)[END_REF]: Strongyloides spp., Trichostrongylus spp. and Necator americanus/Oesophagostomum spp. complex (indistinguishable at the egg stage using standard microscopy). Nematode-negative samples did not contain any observable nematode eggs, but this does not necessarily mean that donors were not parasitized because eggs may be excreted intermittently. However, the presence of parasites within the faeces presented to our test subjects, and not the parasite status of the donor, was the variable of interest here.

Behavioural tests. In September 2014, we conducted 33 behavioural tests on 16 subjects from CIRMF (four females aged 13.2 ± 3.5 years; 12 males aged 11.2 ± 5.7 years; mean number of tests per subject ± s.d. = 2 ± 0.7; Appendix Table A1). Faecal samples of one pair (~one gram of nematode-positive and one gram of nematode-negative faecal samples) were rubbed on two bamboo sticks (~30 cm) attached along the fence of the testing area, four metres apart from each other. These samples were thawed 20 minutes before use. In addition, a central control stick, placed at the middle of the two 'faecal sticks', was rubbed with a common herbaceous plant (one species) that did not belong to the mandrill's diet (see Appendix Materials A1). We checked that mandrills did not prefer certain zones of the testing area by presenting three sticks in a similar setting, all rubbed with the control plant, during preliminary tests (Appendix Materials A1).

Each tested subject was first isolated in the provisioning area and was able to see the experimental setup. The test started once the subject entered the testing area and lasted for 10 minutes. During this period, we recorded the time spent in proximity (less than one metre) to each stick. At the end of the test, the subject was released into the provisioning area. The testing area was cleaned before the next test, performed with another pair of faeces rubbed onto new sticks. Each subject did not perform more than three tests in a row. Faecal samples were stored in an icebox between tests and we alternated their position on the sticks between each test. To avoid multiple freeze-thaw cycles of the samples, each faeces pair was thawed only once and used one to three time(s) in the same half-day period.

Experiment 2: feeding context

To study whether mandrills avoid faeces in a feeding context and whether the presence of nematodes influences this behaviour, we compared subjects' responses to three different food items, faecally-contaminated with 'nematode-positive' faeces (i.e. eggs and/or larvae present), 'nematode-negative' faeces (no eggs, no larvae), and non-contaminated. In addition, to investigate whether the presence of different developmental stages of nematodes influenced behavioural responses, we presented subjects with the same faeces at three different times: on the first day (D1), after one week (D7) and after two weeks (D12) following defecation (see Fig. 1).

Faecal sample collection.

On a single day in November and another day in December 2015, we dewormed two individuals from the two social groups with albendazole (15mg/kg) mixed with milk powder and water. One week later, we isolated each dewormed individual along with an untreated individual from the same group. Individuals were separated from each other (in the testing and provisioning areas) and held for approximately 12 hours. We then collected and mixed all faeces excreted by each donor. Faeces collected from a dewormed donor were paired with faeces collected from the untreated donor from the same group. We thus obtained four faeces pairs (two per month, one from each group) in total, collected from six donors (one female aged 11.3 years; five males aged 10.9 ± 2.3 years -two donors were collected twice; Appendix Table A2). We partitioned the mixture of faeces obtained from each donor into two parts, which were either used for behavioural tests or for the coproscopic analyses that were conducted during the two weeks after collection. We maintained faecal samples in plastic boxes at ambient temperature during the testing period (two weeks) to allow nematode eggs to develop into infective stages. The bottom of the boxes was covered with washed, fresh leaves to minimize drying of faeces.

Coproscopic analyses. Following faecal collection, we first counted nematode eggs using the McMaster technique with a saturated sugar flotation solution (1900 grams per liter; [START_REF] Sloss | Veterinary clinical parasitology[END_REF]. We then searched for nematode larvae using the Baermann method [START_REF] Sloss | Veterinary clinical parasitology[END_REF]. At D1, we confirmed that faecal samples collected from dewormed donors contained no or very few nematode eggs per gram (≤ 50; Appendix Table A2) and no larvae. By contrast, faecal samples collected from untreated donors contained numerous eggs per gram (> 1000; Appendix Table A2), although no larvae were present. We repeated these analyses at D7 and D12. At these dates, while dewormed faecal samples were still free of nematodes, faecal samples collected from untreated donors contained transmissible larvae from one to three nematode taxa (Strongyloides spp.: transmissible larvae observed at D7; Necator americanus and Oesophagostomum spp.: transmissible larvae observed at D12; Appendix Table A2).

Behavioural tests. Following faeces collection, we performed 38 'series of tests' with 22 subjects (seven females aged 12.6 ± 3.5 years and 15 males aged 11.7 ± 5.7 years; Appendix Table A3; Fig. 1a). Each series comprised three behavioural tests performed with the same subject and the same faeces pair at D1, D7 and D12 (Fig. 1a,b). Because of time and field constraints across tests, D1 actually spanned between the first and the second days following defecation and faeces were therefore aged 1-2 day(s). Similarly, at D7 and D12, faecal samples were respectively aged 6-8 days and 11-13 days. In detail, we performed in November 20 series of tests involving 20 subjects (one series per subjects, i.e. three test days per subject). In December, we performed 18 series of tests involving 18 subjects, using 16 subjects already tested in November and two individuals that had not yet participated in November (Appendix Table A3). Six series of tests were incomplete because we were unable to isolate the subject at each time stage (for five series: tests only occurred at D1 and D7; for one series: one test was performed at D1). In total, we performed 107 tests. Tested subjects and donors always came from different social groups to avoid possible confounding effects of familiarity.

Before each test, we aligned three large leaves of Marantacea sp. (~20*10 cm) two metres away from each other in the testing area. This plant species does not belong to the mandrill's diet, although it is commonly found in its habitat. We positioned either a nematode-positive or a nematode-negative faecal sample of one pair at the centre of each peripheral 'faecal' leaf (hereafter, 'nematode-positive leaf' and 'nematode-negative leaf').

Faecal samples were of similar size and resembling a natural faecal bolus (~5 cm in diameter).

We left the middle leaf uncontaminated as a control (hereafter, 'control leaf'). We then positioned two identical food items (banana pieces) on each of the three leaves (six food items in total), approximately 7-8 centimetres from each other. On the nematode-positive and nematode-negative leaves, we positioned one food item atop the faecal sample ('centre' position), and one food item five centimetres away from the faecal sample ('side' position).

All food items on faecal leaves were considered to be 'faecally-contaminated food items', but the central and side positions corresponded to high and low contamination levels, respectively.

Note that these two levels of contamination referred to faecal contamination and not to nematode contamination. We also positioned food items at the centre and on the side of the control leaf (hereafter, 'non-contaminated food items') to serve as controls for high and low food contamination, respectively (Fig. 1b).

As before, each subject was first isolated in the provisioning area and was able to see the experimental setup. The test started once the subject entered the testing area and lasted for three minutes, because subjects generally fed (or not) at the very beginning of the tests. We recorded two behavioural responses: (1) whether or not subjects consumed each of the six food items ('feeding decision'; binary variable); and if they did (2) the latency to feed ('feeding latency'; continuous variable in seconds). At the end of the test, the subject was released into the enclosure. The testing area was cleaned before the next test, performed with another subject using new food items deposited on the same faecal samples. We tested whether subjects spent equal time in proximity to each bamboo stick using a non-parametric, one-way ANOVA following rank transformation of the data (time spent near each stick).

Experiment 2: feeding context

In a first set of analyses, we investigated whether male and female subjects avoided faecally-contaminated food items, irrespective of the level of faecal contamination and irrespective of whether the faeces contained nematodes or not. Using Generalized Linear Mixed Models (GLMMs), we first analysed subjects' feeding decisions towards each of the six food items (model 1 with a binomial distribution, with a logit link function; N = 642, corresponding to the number of food items presented throughout the 107 tests) as a function of faecal contamination on food items, subjects' sex and their interaction. We compared the relative fits of the model with and without this interaction using likelihood-ratio tests (LRT).

We then studied the feeding latency towards each food item consumed in males (model 2 with a negative binomial distribution for over-dispersed count data and a log link function; N = 214, corresponding to the number of food items eaten by males throughout the 107 tests) as a function of faecal contamination on food items. Here, we included only males because females almost never fed on faecally-contaminated food items (see Results). In models 1 and 2, we included three random effects: the identity of the faeces pair and the identity of the test (to account for the non-independence of feeding responses towards the six food items within a test) nested within subject identity.

In a second set of GLMMs, we investigated whether males modulated faecal avoidance behaviour according to the level of faecal contamination and the presence of infective or non-infective stages of nematodes within faeces. As previously, we examined male feeding decisions (model 3, with a binomial distribution and logit link function; N = 498, corresponding to the number of food items presented to males throughout the 107 tests) and feeding latency (model 4, with a negative binomial distribution for over-dispersed count data and a log link function; N = 214, corresponding to the number of food items eaten by males throughout the 107 tests) as a function of the type of leaf (three categories: nematode-positive, nematode-negative, and control, i.e. no faeces), the age of faeces (continuous variable in days), the position of food items (two categories: centre versus side of the leaf), and an interaction between type of leaf and age of faeces as well as between type of leaf and position of food items. As above, the significance of these interactions was assessed using likelihood-ratio tests (LRT). We kept interactions in final models only when they outperformed models without the interaction term. We considered the same random effects as above.

All data were analysed using R v. 3.3.3 (R Core Team, 2016). GLMMs were fit using the package lme4 (for models 1 and 3; [START_REF] Bates | Linear mixed-effects models using[END_REF] and glmmADMB (for models 2 and 4; Skaug, Fournier, Magnusson, & Nielsen, 2016), using maximum likelihood estimations.

We used the package lmtest [START_REF] Hothorn | Package 'lmtest': testing linear regression models[END_REF] to conduct all LRT. All raw data used in these analyses and the video of our experimental design are provided in the Supplementary Material.

RESULTS

Experiment 1: non-feeding context

Mandrills did not spend equal time in proximity to each bamboo stick (F 2,30 = 7.01, P < 0.01). While they spent similar amounts of time near sticks rubbed with nematode-positive and nematode-negative faecal samples (P = 0.66), they spent significantly more time near each of them than near the control stick (P < 0.01 in both cases). 0.10; Table 2). Finally, males modulated their avoidance with the level of faecal contamination: they fed less or later on food items with high contamination compared to low contamination (for both behaviours and both types of faecal leaves: P < 0.01). Indeed, males consumed 45.2% of high contamination level food versus 23.2% of low contamination level food, compared to 62.7% and 23.2% for the central and side control positions, respectively).

DISCUSSION

We showed that mandrills exhibited faecal avoidance in a feeding context but, contrary to our prediction, did not avoid faeces in a non-feeding context. In addition, and again contrary to our predictions, mandrills did not differentiate between faeces containing nematode eggs and those without, nor did they discriminate between fresh and old faeces containing different nematode developmental stages. As we did predict, however, females exhibited greater avoidance towards faecally-contaminated food items than did males.

In the non-feeding context, subjects did not avoid faecal material and did not discriminate between faeces with or without nematodes. They even spent more time near each faecal sample compared to the control sample. Nematode infection risk was, however, minimal because this experiment did not involve ingestion behaviour and faeces contained only non-infective nematode eggs. Subjects therefore may have had little incentive to exhibit avoidance behaviour. By contrast, mandrills appeared attracted to faeces in this experiment, possibly because they were collected from unknown conspecifics and may have conveyed important olfactory cues about the donor's social and/or sexual status. A similar experiment recently showed that mandrills selectively avoid faeces containing gastro-intestinal protozoa [START_REF] Poirotte | Mandrills use olfaction to socially avoid parasitized conspecifics[END_REF]. In contrast to nematodes, protozoa are readily infectious when emitted in faeces, which might explain why the behavioural responses observed differed in relation to these different parasites. A specific olfactory detection mechanism may have evolved regarding protozoa and their avoidance, facilitating the reported social avoidance of contagious individuals [START_REF] Poirotte | Mandrills use olfaction to socially avoid parasitized conspecifics[END_REF].

In a feeding context, on the other hand, we found that mandrills fed less or later on faecally-contaminated food items, as expected. However, subjects displayed similar levels of avoidance towards fresh and old faeces irrespective of whether it came from parasitized individuals (containing infective or non-infective stages of nematodes) or from dewormed, unparasitized individuals. Avoiding all types of faeces during foraging may allow mandrills to limit infection with all kinds of pathogens (e.g. protozoa, bacteria and viruses; [START_REF] Poirotte | Environmental and individual determinants of parasite richness across seasons in a free-ranging population of Mandrills (Mandrillus sphinx)[END_REF][START_REF] Setchell | Parasite prevalence, abundance, and diversity in a semi-free-ranging colony of Mandrillus sphinx[END_REF][START_REF] Zwick | Paratuberculosis in a Mandrill (Papio Sphinx)[END_REF], increasing the benefits of non-specific faecal avoidance. Moreover, infective mobile larvae might migrate out of the faeces and contaminate the surrounding area. Consequently, avoiding contaminated areas rather than particular faeces should represent a better strategy to reduce the risk of encountering infective stages of nematodes. Accordingly, mandrills use large home ranges, travel several kilometres a day [START_REF] Brockmeyer | Social organization and space use of a wild mandrill (Mandrillus sphinx) group[END_REF], tend not to stay for several consecutive days in the same area and avoid reusing areas, including sleeping sites, that have been previously contaminated with nematodes [START_REF] Poirotte | Gastrointestinal parasitism and recursive movements in free-ranging mandrills[END_REF]. In line with our results, ungulates do not discriminate between faeces that do or do not contain nematodes [START_REF] Hutchings | Behavioural strategies used by parasitised and non-parasitised sheep to avoid ingestion of gastrointestinal nematodes[END_REF][START_REF] Cooper | Strategies for the avoidance of faeces by grazing sheep[END_REF][START_REF] Brambilla | Don't spit in the soup: faecal avoidance in foraging wild Alpine ibex, Capra ibex[END_REF]. At the same time, however, the presence of infective stages in old faeces increase faecal avoidance while foraging in white-footed mice Peromyscus leucopus [START_REF] Logiudice | Latrine foraging strategies of two small mammals: implications for the transmission of Baylisascaris procyonis[END_REF] and while conspecific coprophagy is a common behaviour in canids, a recent study reported that dogs eat almost exclusively faeces freshly emitted [START_REF] Hart | The paradox of canine conspecific coprophagy[END_REF]. These divergent results may stem from the different costs and benefits of selective foraging strategies, which certainly vary across different host-parasite systems. Such balances may depend on the pathogenicity and the prevalence of the parasites, for example, or the ecology of the host.

Finally, we observed that females exhibited more avoidance than males: while females almost never ate faecally-contaminated food items (5.2%), males sometimes did (34.3%), especially for food items with a low level of contamination (45.2%), probably resulting in higher infection risk. The fact that males generally maximize fitness by increasing mating opportunities, whereas females invest in longevity (Bateman's principle), could explain this sex bias: females invest more in immunity, including behavioural immunity, to maximize fitness gains [START_REF] Rolff | Bateman's principle and immunity[END_REF]. Alternatively, or in concert, the extreme sexual size dimorphism in mandrills [START_REF] Setchell | Growth and ontogeny of sexual size dimorphism in the mandrill (Mandrillus sphinx)[END_REF] might also influence caloric needs and force males to take more risks in their feeding decisions. This sexual bias in sensitivity to infection risk could impact disease epidemiology and dynamics, although in free-ranging mandrills males do not exhibit greater nematode species richness than females [START_REF] Poirotte | Environmental and individual determinants of parasite richness across seasons in a free-ranging population of Mandrills (Mandrillus sphinx)[END_REF]). Yet, in Japanese macaques, sex differences in avoidance behaviours result in higher nematode intensity in males than females [START_REF] Sarabian | Hygienic tendencies correlate with low geohelminth infection in free-ranging macaques[END_REF]. More generally, such differences may contribute to the widely observed male-biased parasitism in vertebrates [START_REF] Goble | Sex as a factor in infectious disease[END_REF][START_REF] Bundy | Sexual effects on parasite infection[END_REF][START_REF] Gregory | Helminth parasite richness among vertebrates[END_REF][START_REF] Poulin | Helminth growth in vertebrate hosts: does host sex matter[END_REF].

Further investigations are now needed to evaluate and understand the causes of sex differences in the expression of hygienic behaviours, and the importance of these behaviours in mediating parasite distributions within host populations. The general parasite avoidance behaviour reported here, along with the specific strategy to avoid contagious parasites found in this study system [START_REF] Poirotte | Mandrills use olfaction to socially avoid parasitized conspecifics[END_REF], suggest that contrasted parasite life history traits influence the costs and benefits of anti-parasite strategies, and may have driven the evolution of different detection mechanisms and diverse behavioural counterstrategies.

<0.001

Model 1 includes data obtained from both sexes, while model 2 includes only data obtained from males. Equation and sample size are given for each model. The reference level is indicated for each categorical level in brackets (sex: male / female, faecal contamination: faecallycontaminated 'C' / non-contaminated 'NC'). For model 1, we ran the model three times, varying the reference level of only one variable at each run to display the parameter estimates for all combinations of sex and faecal contamination. We present the result of comparisons between faecally-contaminated and non-contaminated food items within both sexes, and between sexes within faecally-contaminated and noncontaminated food items. Significant test statistics are highlighted in bold (P < 0.05). Equation and sample size are given for each model. The reference level is indicated for each categorical level in brackets (type of leaf: nematodenegative 'NN'/nematode-positive 'NP'/control; position of food: centre/side). We ran the model five times, varying the reference level of only one variable at each run to display the parameter estimates for all combinations of type of leaf and position of food. We present the result of comparisons between types of faeces within both positions of food, and between positions of food within both types of faeces. Significant test statistics are highlighted in bold (P < 0.05). bars indicate significant differences (P < 0.05) in feeding decision between types of faeces or between positions of food, according to the results of the models presented in Table 2.

  Oeso., Str.Sex (f/m: female/male), age (in years) at the time of the experiment (Oct. 2015), and group of donors are provided. Each faecal sample was analysed at three different times: D1, D7, and D12, which refer to the number of days following faecal emission. Two coproscopic analyses were performed each time: "MM" refers to the quantitative McMaster technique, and the number below indicates the amount of eggs per gram of faeces; "B" refers to the qualitative Baermann technique to retrieve larvae, "0" indicates that no larvae was observed, while "Nec.", "Str." and "Oeso." refer to the presence of Necator Americanus, Oesophagostomum spp. and Strongyloides spp, respectively.

Figure 1 .

 1 Figure 1. Schedule of Experiment 2 (a) and experimental protocol for one series of

Figure 2 .

 2 Figure 2. Feeding decisions of males and females according to faecal contamination (N =

Figure 3 .

 3 Figure 3. Feeding decisions of males according to faecal contamination and nematode

Table 1 .

 1 Summary of the models investigating faecal avoidance.

	Statistical model

Table 2 .

 2 Summary of the models investigating nematode avoidance in males.

	Statistical model	Predictors (reference level)	Estimate s.e.m. z	P (> z)
	Model 3, N = 498	Type of leaf * Position of food
	Feeding decision	NN * side (control, centre)	1.56	0.60	2.57	0.010
	~ Type of leaf * Position of food + Age of faeces	NP * side (control,centre)	0.82	0.59	1.40	0.16
	+ (Test ID|Subject ID)	Type of leaf
	+ (1|Faeces pair ID)	NP (control, centre)	-2.20	0.44	-5.01	<0.001
		NN (control, centre)	-2.68	0.46	-5.83	<0.001
		NP (control, side)	-1.38	0.42	-3.25	0.001
		NN (control, side)	-1.13	0.42	-2.69	0.007
		NP (NN, centre)	0.49	0.44	1.11	0.27
		NP (NN,side)	-0.25	0.40	-0.62	0.54
		Position of food
		centre (side, control)	-0.36	0.42	-0.87	0.39
		centre (side, NN)	-1.91	0.44	-4.35	<0.001
		centre (side, NP)	-1.19	0.42	-2.84	0.004
		Age of faeces	-0.03	0.03	-0.76	0.45
	Model 4, N = 214	Type of leaf * Position of food
	Feeding latency	NN * side (control, centre)	-0.92	0.31	-2.93	0.003
	~ Type of leaf * Position of food + Age of faeces	NP * side (control, centre)	-0.75	0.29	-2.56	0.010
	+ (Test ID|Subejct ID)	Type of leaf
	+ (1|Faeces pair ID)	NP (control, centre)	1.28	0.23	5.58	<0.001
		NN (control, centre)	1.60	0.26	6.26	<0.001
		NP (control, side)	0.52	0.19	2.70	0.007
		NN (control, side)	0.68	0.19	3.53	<0.001
		NP (NN, centre)	-0.32	0.29	-1.12	0.26

Table A1 .

 A1 Details of subjects (N = 16) used during Experiment 1.

		Subjects Sex Age Group Number of tests
		PC	f 11.0	1	2
		30	m 12.9	1	3
		2D11	m 10.8	1	2
		5I1	m 12.1	1	2
		17F	f 20.1	2	3
		6H	f 12.9	2	3
		12M	f 18.0	2	2
		5I3	m 7.4	2	1
		17A6	m 16.1	2	2
		17A10 m 10.7	2	3
		16B	m 25.9	2	2
		17B11 m 10.8	2	1
		17D2A2 m 4.9	2	2
		17F3	m 13.0	2	2
		16I	m 16.0	2	1
		PM	m 4.8	2	2
	567
	568	Sex (f/m: female/male), and age (in years) at the time of the experiment (Sept. 2014) are
	569	provided. Each subject performed one to three tests, involving one to three faeces pairs. The
	570	number of tests performed with each subject is indicated.

Table A2 .

 A2 Details of donors of faecal samples (N = 6) for each faeces pair and results of

	572	coproscopic analyses during Experiment 2.
						Type / Time of analyses
		Faeces	Donor	Donor status D1/	D1/	D7/	D7/	D12/	D12/
		pair	(sex, age, group)	MM	B	MM	B	MM	B
		Pair #1	33 (f, 11.

Table A5 .

 A5 Feeding decisions according to subject's sex, level of faecal contamination and D12 refer to the number of days following faecal emission. 'Low' level of faecal contamination refers to food items positioned five centimetres away from the faecal samples, while 'high' level of faecal contamination refers to food items on faecal samples. Ratios indicate the number of tests during which subjects ate the food item on the total number of tests.

	596	type of leaf.
					Time of the test
		Sex Faecal contamination	Type of leaf	D1	D7 D12
		f	low	Nematode-negative 0/9	0/9	1/6
		f	low	Nematode-positive 2/9	0/9	1/6
		f	none (side of the leaf)	Control	4/9	4/9	4/6
		m	low	Nematode-negative 16/28 10/28 13/27
		m	low	Nematode-positive 13/28 12/28 11/27
		m none (side of the leaf)	Control	19/28 16/28 17/27
		f	high	Nematode-negative 0/9	0/9	0/6
		f	high	Nematode-positive 0/9	0/9	1/6
		f none (centre of the leaf)	Control	4/9	4/9	3/6
		m	high	Nematode-negative 6/28 6/28 5/27
		m	high	Nematode-positive 8/28 8/28 6/27
		m none (centre of the leaf)	Control	16/28 15/28 17/27
		D1, D7,

Acknowledgements

We thank past and present field assistants of the Mandrillus Project and of the Centre International de Recherches Médicales de Franceville (CIRMF) for their help in data collection. This study was supported by the "Station d'Etudes en Ecologie Globale" (INEE-CNRS) and the "Laboratoire International Associé" (CIRMF and INEE-CNRS) to MJEC and by the Leading Graduate Program in Primatology and Wildlife Science of Kyoto University as well as the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and the Japanese Society for the Promotion of Science (JSPS) to CS. Some data used in this manuscript were collected within the framework of the Mandrillus Project, funded by the "Deutsche Forschungsgemeinschaft" (DFG, KA 1082-20-1; 2012-2015) and the "Agence Nationale de la Recherche" (ANR SLEEP 17-CE02-0002, 2017-2020 to MJEC; ANR ERS, 2018-2021 to Elise Huchard). Experimental procedures were approved by the CENAREST Institute (permit number: AR0042/17/MESRS/CENAREST/CG/CST/CSAR) and the Animal Welfare and Animal Care Committee of the Kyoto University Primate Research Institute (#2016-138). Research permissions were granted by the CIRMF. This is a Mandrillus Project publication number # and ISEM 2018-###-SUD.

2: feeding context 282 283 Experiment

The interaction between the presence or absence of faecal contamination of food items and the subjects' sex significantly influenced observed feeding decisions (model 1, LRT, ∆LogLik = 9.01, P < 0.001; Appendix Table A4). Both sexes fed less on faecallycontaminated compared to non-contaminated food items (model 1, P < 0.01, Table 1, Fig. 2, Appendix Table A5). Faecal avoidance was, however, significantly higher in females than in males (model 1, P = 0.01, Table 1, Fig. 2, Appendix Table A5): females and males consumed 5.2% and 34.3% of faecally-contaminated food items versus 47.9% and 60.2% of noncontaminated food items, respectively. When males consumed food items, feeding latency was significantly higher for faecally-contaminated compared to non-contaminated food items (model 2, P < 0.001, Table 1; Appendix Table A5).

The interaction between type of leaf (nematode-positive, nematode-negative and control) and age of faeces did not significantly influence male feeding decisions (model 3, LRT, ∆LogLik = 1.64, P = 0.51; Appendix Table A4) or male feeding latency (model 4, LRT, ∆LogLik = 0.64, P = 0.52; Appendix Table A4), and was therefore excluded from both models. In addition, male responses towards food items did not vary across time (for both behaviours: P > 0.10; Table 2). By contrast, the interaction between type of leaf and position of food items significantly influenced both male feeding decisions (model 3, LRT, ∆LogLik = 2.56, P < 0.01; Appendix Table A4) and male feeding latencies (model 4, LRT, ∆LogLik = 5.60, P < 0.01; Appendix Table A4). We found that males fed less or later on faecallycontaminated compared to non-contaminated food items, for both levels of faecal contamination (all P < 0.01, Table 2). However, male responses towards faecallycontaminated food items did not differ between food items located on 'nematode-positive' and 'nematode-negative' leaves (for both behaviours and both levels of contamination: P > AUTHORSHIP CP, CS, AM and MJEC designed the study, analysed the data, and wrote the article. CP and CS collected the data. BN helped with data collection.

Appendix.

Materials A1. We conducted six preliminary tests during which we presented subjects with three bamboo sticks rubbed with a common herbaceous plant located in similar areas as those used in Experiment 1. We tested whether subjects spent equal time in proximity to each stick using a non-parametric, one-way ANOVA following rank transformation of the data. We found that subjects spent the same amount of time near each stick (F = 0.42, P = 0.67).

Video A1. Experiment 2: Feeding decision towards food items. The adult male feeds on both food items on the control leaf, and one food item the side of a faecal leaf. for the analyses and sample sizes are given for each model. The interaction terms tested are indicated for each model. To assess the significance 593 of an interaction, we compared the full model including the interaction with the model without the interaction. Bold text denotes cases for which we kept the interaction in the model (when P < 0.10). Significant statistical tests are highlighted in bold (P < 0.05).


Simon Pouil
email: simon.pouil1@gmail.com

Reza Samsudin

Jacques Slembrouck

Ahmad Sihabuddin

Gusnia Sundari

Khazaidan Khazaidan

Hari Anang

Brata Kristanto

Domenico Pantjara

Caruso

Hari Kristanto

Brata Pantjara

Domenico Caruso

Nutrient budgets in a small-scale freshwater fish pond system in Indonesia

Keywords: Small-scale fish farming, N and P budgets, giant gourami, environment, Indonesia

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

Introduction

Producing 3 million tons of farmed fish in 2015, Indonesia is the third largest freshwater fish aquaculture producer country after China and India (FAO, 2017). Indonesian aquaculture production has increased six-fold since 2005 (FAO, 2017), after the government made aquaculture development a national priority [START_REF] Philips | Exploring Indonesian aquaculture futures[END_REF][START_REF] Rimmer | A review and SWOT analysis of aquaculture development in Indonesia[END_REF]. This sector plays an increasingly significant role in Indonesian economy, ensuring food availability, household food security and the improvement of living standards for rural communities [START_REF] Rimmer | A review and SWOT analysis of aquaculture development in Indonesia[END_REF]. Indonesian aquaculture is characterized by a high number of fish farmers (3.34 million in 2016;FAO, 2018) and relatively low annual productivity (1.48 t/farmer) compared with other Asian countries (1.72 t/farmer in Asian countries excluding Indonesia and China;FAO, 2018). This is especially true for freshwater fish aquaculture, which is mainly carried out in, micro-and small-scale farms, which represent an estimated > 90% of all fish farms [START_REF] Maskur | Small-scale freshwater aquaculture extension development in Indonesia[END_REF].

Small-scale fish farmers usually practice aquaculture using low-input and low-level technology. This is especially true for aquaculture production in earthen ponds, where traditional practices favor primary productivity and limit inputs of exogenous food, especially commercial pellets. These traditional production systems reportedly use resources efficiently, particularly through integrated aquaculture-agriculture (IAA) practices [START_REF] Nhan | Water and nutrient budgets of ponds in integrated agriculture-aquaculture systems in the Mekong Delta, Vietnam[END_REF].

Nevertheless, the number of studies providing quantitative evidence of such claims remains limited (e.g. [START_REF] Nhan | Water and nutrient budgets of ponds in integrated agriculture-aquaculture systems in the Mekong Delta, Vietnam[END_REF]. In addition, the development of freshwater aquaculture in recent years has resulted in a shift from low-intensity traditional culture methods to monoculture production systems, with increasing intensification accompanied by the use of commercial pellets [START_REF] Edwards | Changes in traditional inland aquaculture in West Java, Indonesia[END_REF][START_REF] Rimmer | A review and SWOT analysis of aquaculture development in Indonesia[END_REF]. Such changes raise many issues about the real effectiveness of current production systems in the use of nutrient resources and the associated environmental impacts.

Intensification of freshwater aquaculture with the increase in fish density and the use of commercial feed may result in the release of significant amounts of nutrients into water. This enrichment in nutrients such as nitrogen (N) and phosphorus (P) intended for aquaculture may contribute to the deterioration in water quality and to eutrophication (Boyd et al., 1998).

Among the fish produced in Indonesian pond aquaculture, the giant gourami Osphronemus goramy Lacepède ( 1801) is one of the main freshwater commodities of economic importance.

This species has been produced in small-scale farms for decades, usually in semi-intensive systems depending largely on natural food, supplemented with fertilizers and plants and commercial pellets in highly variable proportions.

Commercial pellets are one of the main sources of N and P inputs in semi-intensive and intensive aquaculture production systems (e.g. [START_REF] Briggs | A nutrient budget of some intensive marine shrimp ponds in Thailand[END_REF][START_REF] Nhan | Water and nutrient budgets of ponds in integrated agriculture-aquaculture systems in the Mekong Delta, Vietnam[END_REF][START_REF] Phong | Modeling the soil nutrient balance of integrated agriculture-aquaculture systems in the Mekong Delta, Vietnam[END_REF]. Thus, nutrient assimilation by aquaculture organisms has important implications for water quality and the profitability of ponds. In most cases, no more than 20% to 50% of the N and P introduced into ponds through fertilizers and feed are recovered in farmed organisms at harvest, with the rest being diluted into the water column or accumulated in the sediment (e.g. [START_REF] Boyd | Pond aquaculture water quality management[END_REF][START_REF] Hargreaves | Nitrogen biogeochemistry of aquaculture ponds1[END_REF][START_REF] Ketola | Influence of phosphorus in rainbow trout diets on phosphorus discharges in effluent water[END_REF].

Earthen ponds have a demonstrated ability to trap nutrients such as nitrogen and phosphorus, and pond waters usually have high concentrations of nutrients, suspended solids, and oxygen demand than the water bodies into which they discharge [START_REF] Schwartz | Channel catfish pond effluents[END_REF]. Thus, pond effluents are potential sources of pollution in receiving waters [START_REF] Boyd | Guidelines for aquaculture effluent management at the farm-level[END_REF]. In this context, the lack of information regarding the environmental effects of aquaculture pond systems in Indonesia is of particular concern [START_REF] Aubin | Implementing ecological intensification in fish farming: Definition and principles from contrasting experiences[END_REF].

This study aims to quantify the nutrient (N and P) balances in earthen ponds used for the production of giant gourami in Java (Fig. 1). Here, intensified aquaculture production was considered using organic fertilization and commercial pellets as the unique sources of fish 81 feed.

6

Materials and methods

Ponds systems and management

The experiment was conducted for 5 months (152 d from mid-November 2017 to mid-April 2018) in a small-scale fish farm located in the village of Babakan (-6°28'S; 106°42'E; alt: 125 m), Bogor district, West Java, Indonesia (Fig. 1). The investigated aquaculture pond system consisted of four small semi-intensive earthen ponds, whose size varied between 353 and 482 m 2 with depths ranging from 0.46 to 0.55 m (Table 1). Each pond was equipped with PVC pipes for inlet and outlet water; an anti-predator nets held by bamboo poles were placed at a distance of 1 m from the banks. Juveniles of giant gourami (28.6 ± 6.0 g wet weight, 12.2 ± 1.0 cm) were stocked at a density of 6.2 fish m -2 in each pond (Table 1). Gourami were fed three times a day, seven days a week with the same commercial pellets (proteins: 33%, lipids: 5%, humidity 8%, Table 1) throughout the experiment. The daily ration was 3 to 5% of the estimated wet biomass according to fish growth.

Before starting the experiment, the ponds were dried, the bottom were flattened, the banks consolidated with sediment accumulated during the previous production cycle, unwanted species were removed, and the water was limed and fertilized using organic fertilizer (typical composition: poultry manure mixed with rice bran at a 0.5:1 ratio on a dry weight basis, [START_REF] Purnomo | Slow release fertiliser production from poultry manure[END_REF]. The quantity of organic fertilizer was determined after consulting with local fish farmers (i.e. based on empirical knowledge). Fertilizer was added four times during the first three months of the experiment (equiv. 2.1-2.7 kg m -2 kg, see Table 1). Ponds were filled by gravity with water from a reservoir located at <1 km from the farm. The experiment was carried out in open flow water (average water flow 3.7-10.7% d -1 , total volume exchanged: 1328-2730 m 3 ).

Environmental conditions monitoring

Environmental conditions were recorded continuously throughout the experiment using a data logger (Onset HOBO) placed in each pond at 10 cm depth for water temperature or at 50 cm above the water surface in the center of pond B for air temperature and light intensity (Fig. 1).

A rain gauge placed on the production site recorded the daily rainfall. During the entire experiment, the ambient temperature was 28.7±5.0°C (range: 20.4-43.5°C), the daily diurnal (06:00 AM-06:00 PM) light intensity was on average 23,000 lux with maximum values reaching 176,000 lux, rainfall range was 7.8±14.6 mm day -1 (range: 0-75 mm day -1 ) and the water temperature was 30.2±1.9°C (range: 25.7-38.6°C) with no significant differences between the four ponds.

Sampling

Water

Water was sampled in the inlet channel and at the outlet of each pond eight times during the experiment (every two weeks). Prior to each water sampling, on-site measurements were taken (08:00-10:00 AM) to determine pH, dissolved oxygen (DO, mg L -1 ), temperature (T°C), total dissolved solid matter (TDS, mg L -1 ) using a multi-parameter probe (HI 9829 Hanna).

Water samples were transported to the laboratory in a cooler at 4-5°C, then analyzed less than 6 h after sampling. In addition to N and P (see procedures in Section 2. (1999). Data are summarized in Table 2.

Sediment

In each pond, the depth of accumulated substances during the experiment was determined from measurements of the height from the bottom of the pond of the two bamboo poles installed to hold the anti-predator net at the beginning and at the end of the 152-d experiment.

The substances were composed of uneaten fish feed, fish feces, dead plankton, other organic matter and mineral particles, and are hereafter referred to as accumulated sediment [START_REF] Nhan | Water and nutrient budgets of ponds in integrated agriculture-aquaculture systems in the Mekong Delta, Vietnam[END_REF]. Differences in bottom levels recorded between stocking and harvesting were assumed to be due to sediment accumulated during the fish culture period. Accumulated sediments were sampled at harvesting in five different locations in each pond using a 6.35 cm diameter PVC tube. For each pond, sediments taken from the different locations were thoroughly mixed into a composite sample for further analysis. The composite samples were stored at -18°C and then analyzed for N and P (see Section 2.5). In addition, for each pond, a 10-30 g wet sample was dried at 105°C for 24 hours to determine dry weight. The organic matter content was determined with 1 h combustion of the dried sediment subsamples at 500°C.

Commercial feed and fertilizer

The types and quantities of fertilizers and commercial feed (i.e. manufactured pellets) inputs were recorded daily. Prior to use, batches of fertilizer (poultry manure mixed with rice bran)

were analyzed to determine N and P following the procedures described in Section 2.5.

Regular supplies of commercial feed bags were brought to the farm throughout the experiment. For each new bag, samples of pellets were taken and mixed, and then N and P contents of this mixture were analyzed each month using the procedures described in Section 2.5. Pond nutrient inputs, for each type, were calculated by multiplying recorded quantities by measured nutrient concentrations.

Fish and other species

Before storage in the experimental ponds, 200 giant gourami juveniles were individually sampled to determine initial average body weight and biomass. Every month, 50 fish from each pond were sampled and individually weighed to determine growth and adjust the feed ration. The number of dead fish collected on the water surface was recorded daily for each pond throughout the experiment. At harvest, remaining fish were counted to assess survival and the total biomass of produced fish was determined for each pond.

Dry matter (DM), N and P contents in the fish were determined at the beginning (i.e. fish stocked, n=5) and at the end of the experiment (i.e. fish harvested, n=5 per pond) as described in Section 2.5.

Biomass of colonizing invertebrate species such as freshwater snails (mainly Pomacea canaliculata and, to a lesser extent, Filopaludina javanica) and bivalves (Pilsbryoconcha exilis) were recorded and weighed for each pond at the end of the experiment. N and P contents were determined following the procedure indicated in Section 2.5.

Net fish production was determined as the difference between initial and harvested biomass and was calculated on a kg ha -1 year -1 basis.

Nutrient budgets

Pond nutrient budgets were established for N and P on a kg ha -1 year -1 basis. In each pond, nutrient balance was estimated according to the following equation:

∑inputs = ∑outputs ± unaccounted.
The nutrient inputs included (1) commercial feed, (2) fertilizer, (3) fish stocked and (4) inflow water. The nutrient outputs included (1) accumulated sediments, (2) harvested bivalves and snails (ancillary production), (3) harvested fish, and (4) outflow water. The nutrient input and output quantities were calculated by multiplying total weights or volumes by nutrient concentrations measured at the nearest date.

Following [START_REF] Nhan | Water and nutrient budgets of ponds in integrated agriculture-aquaculture systems in the Mekong Delta, Vietnam[END_REF], nitrogen fixation, N volatilization, photosynthesis and absorption were not measured and phosphorus inputs from rainwater and losses of the nutrients through evaporation were assumed negligible. Nutrient dynamics in phyto-and zooplankton were not considered in this study because these compartments represented very little biomass in our production ponds. Nutrient uptake by other colonizing species, mainly shrimp (Atyidae) and fish (Barbonymus gonionotus, Oreochromis niloticus, and Trichogaster sp.) was considered negligible (<20 kg).

Sample analysis

Total Kjeldahl nitrogen (TKN) in water was measured following the Indonesian National For the other samples (bivalves, feed, fertilizer, fish, sediment and snails), total N was estimated based on the crude protein (CP) content measured according to AOAC 2001.11.

Briefly, each sample was weighed and transferred to a sample tube. Digestion was performed using concentrated sulfuric acid at 400°C for 1 h and, after cooling, measurements were read using an automated Kjeldahl analyzer. The conversion factors used for total N estimation were 5.58 for fish, 5.6 for bivalves and snails and 6.25 for the remaining samples. For total P determination, samples were weighed and digested in nitric acid for 20 min at 190°C.

Measurements were then read using an inductively coupled plasma optical emission spectrometer (ICP-OES).

Data analysis

Statistical comparisons were done for the different water parameters measured in the inlet channel and the four earthen ponds. When normality (Shapiro's test) and homogeneity of variance (Levene's test) were confirmed (BOD, chlorophyll a, DO, TDS, temperature, total N), one-way ANOVA was used to determine significant differences among sampling locations. When significant differences were detected, Tukey's test was performed to compare means. In the case of non-normality and/or heterogenous variances (COD, NO 2 -, NO 3 -, pH, PO₄³⁻, total P), data were analyzed using non-parametric Kruskal-Wallis and Siegel and Castellan tests.

Linear regression analysis was used to test for effects of nutrient inputs on fish yields and on nutrient accumulation in sediment.

The level of significance for statistical analyses was set at α = 0.05. All statistics were calculated using R freeware version 3.3 (R Development Core Team, 2016) and figures were plotted using Microsoft Excel 2016.

Results

Nutrient budgets

Overall, results show that commercial feed and organic fertilizer were the major sources of P and N, and that accumulated sediments were the major sinks of these nutrients (Fig. 2 and3, Table 3). Commercial feed inputs accounted for 56.8 ± 4.7% of the total N and 60.4 ± 3.7% of P inputs. Organic fertilizer (poultry manure mixed with rice bran) was the second most important source of nutrients. Interestingly, water inflow accounted only for 5.8 ± 3.3% of N and 0.1 ± 0.1% of P inputs. Harvested fish accounted for 2.9 ± 0.3% of the total N and 0.7 ± 0.1% P inputs (Fig. 2 and 3, Table 3).

A large proportion of nutrients were trapped in the accumulated sediment: 60.6 ± 3.9% of the total N and 77.0 ± 3.5% of P inputs were found in the accumulated sediments at the end of the experiment. The remaining recorded fractions of the nutrients were found in harvested fish (14.9 ± 6.8% for N and 3.1 ± 1.7% for P) and flushed out with the outflow water. The fractions of nutrients found in ancillary productions were very low (<1% for N and P, Fig. 2 and 3, Table 4). Based on our analysis, we found unbalanced budgets for N and P: 19.9 ± 2.1% of N and 19.3 ± 2.6% of P inputs were not recovered in the outputs (Fig. 2 and3, Table 3).

Fish yields and nutrient inputs

After a 5-month growing period in the four ponds (A, B, C and D), fish yields ranged from 2057 to 10,203 kg ha -1 . A pathological event occurred in pond D and the final yield was dramatically reduced compared with the other ponds (2057 kg ha -1 year -1 for pond D and 9772-10,203 kg ha -1 for the three other ponds). For all ponds, monitoring of the observed mortality made it possible to re-adjust the quantities of food distributed according to the estimated biomass determined after each monthly sampling. This method proved to be fairly accurate for ponds A, B and C with relatively close estimated (98 ± 1%) and observed (92 ± 6%) survival rates at the end of the experiment. Nevertheless, for pond D, the survival rate was overestimated (65% vs. 22% measured at the end of the experiment). Thus, the quantity of pellets distributed in this pond were probably higher than daily rationing initially planned.

The quantities of N and P found in the accumulated sediments increased with increasing total nutrient inputs through commercial feed, fertilizer, fish stocked and inflow water (Fig. 4). The total nutrient inputs explained 98% and 87% of the variability of the N and P found in the sediment, respectively.

Discussion

Management and nutrient budgets of giant gourami earthen pond aquaculture

Aquaculture of giant gourami O. goramy in Indonesia is a very old practice [START_REF] Cuvier | Histoire naturelle des poisons : Tome septième[END_REF]. Many small-scale Asian fish farmers produce giant gourami in fertilized ponds to limit the use of high-priced commercial pellets [START_REF] Pengseng | Assessment of fertilizer application intervals for giant gourami (Osphronemus goramy Lacepede) in ponds[END_REF]. In such systems, giant gourami is traditionally fed a combination of local plants such as giant taro (Alocasia macrorrhizos), supplemented with commercial pellets in varying proportions [START_REF] Aubin | Implementing ecological intensification in fish farming: Definition and principles from contrasting experiences[END_REF].

In the present study, the culture system can be classified as upper-level semi-intensive with commercial pellets as a single source of feed (rationing: 4% on average), in line with intensification trends observed over the past years in Indonesian freshwater aquaculture for other fish species [START_REF] Udo | Intensification of smallholder livestock production, is it sustainable? in: Community Empowerment and Tropical Animal Industry[END_REF]. Not surprisingly, commercial pellets were the main source of nutrients in ponds, contributing 50-61% of the total N and 56-65% of the total P inputs. These results are close to those obtained in shrimp aquaculture conducted in semiintensive ponds (71-76% and 53-83% of the total N and total P inputs, respectively; [START_REF] Casillas-Hernández | Nutrient mass balances in semi-intensive shrimp ponds from Sonora, Mexico using two feeding strategies: Trays and mechanical dispersal[END_REF][START_REF] Páez-Osuna | Fluxes and mass balances of nutrients in a semi-intensive shrimp farm in north-western Mexico[END_REF][START_REF] Páez-Osuna | Environmental load of nitrogen and phosphorus from extensive, semi-intensive, and intensive shrimp farms in the Gulf of California ecoregion[END_REF].

Nevertheless, the nutrient budgets established in our production system revealed that organic fertilizer is also an important source of N and P inputs.

One of main organic fertilizers used in Javanese pond aquaculture is poultry manure (from local intensive broiler farms) mixed with rice bran. In our system, the addition of organic fertilizer during the preparation of the ponds and the fish growing period represented 31-37% of inputs in N and 35-43% of inputs in P. The applied fertilizer doses were determined by the local fish farmers involved in the experiment (i.e. 97-124 g m -2 week -1 ) to promote high primary productivity of the ponds. This is a key feature of giant gourami production in ponds.

This species can breathe atmospheric air and can be reared in heavily fertilized ponds [START_REF] Pengseng | Assessment of fertilizer application intervals for giant gourami (Osphronemus goramy Lacepede) in ponds[END_REF], rich in phytoplankton and for which the dramatic variation in dissolved oxygen can be harmful for other fish species produced locally in earthen ponds, such as the Nile tilapia Oreochromis niloticus or the common carp Cyprinus carpio [START_REF] Philips | Exploring Indonesian aquaculture futures[END_REF]. Considering chlorophyll a as a proxy for the level of fertilization in ponds, although there are significant variations during the production cycle, the mean values between 169 and 446 µg L -1 of chlorophyll a in our ponds are well below those (up to 85.8 mg L -1 ) obtained in giant gourami production ponds in Thailand [START_REF] Pengseng | Assessment of fertilizer application intervals for giant gourami (Osphronemus goramy Lacepede) in ponds[END_REF] despite fertilization rates about 10 times lower (maximum of 60 kg N ha -1 and 30 kg P ha -1 applied during a 120 d production cycle using inorganic fertilizers) than those used in our experiment.

In our system, we used organic fertilizer instead of chemicals and water renewal was about 4-11% d -1 during the fish production cycle. These differences likely explain the lower primary productivity observed in our ponds compared with Thai ponds [START_REF] Pengseng | Assessment of fertilizer application intervals for giant gourami (Osphronemus goramy Lacepede) in ponds[END_REF].

Furthermore, nutrient inputs furnished by massive initial fertilization may be trapped in sediment and no longer be available to promote primary production in pond. These results call into question the bioavailability of nutrients supplied by organic fertilizers in our production system and the improvement of fertilization practices should be considered (see Section 4.2).

In traditional Indonesian earthen pond fish farming, accumulated sediments are not regularly removed. Between each production cycle, the bottom of the pond is flattened, the banks restored using part of the accumulated sediment. These ponds are therefore characterized by sediment that is several tens of centimeters thick. In our experiment, the accumulation rate for sediments was on average, 0.91 cm month -1 . Our results showed that accumulated sediments play a major role in nutrient budgets because a large fraction of the N and P inputs settle directly on the sediment, which is also enriched mainly by organic matter: plankton, dead organisms, and wastes produced by living organisms such as fish, snails, and bivalves. Such findings are in accordance with literature, with reports that nutrient trapping in sediments play a key role in the nutrient balance of fish ponds [START_REF] Nhan | Water and nutrient budgets of ponds in integrated agriculture-aquaculture systems in the Mekong Delta, Vietnam[END_REF], see Table 4). Thus, sediments can account for 61% of total N inputs and 77% of total P inputs, an element known to be highly adsorbed on sediment [START_REF] Boyd | Chemical budgets for channel catfish ponds[END_REF][START_REF] Shrestha | Phosphorus fertilization strategy in fish ponds based on sediment phosphorus saturation level[END_REF]). In our system, less N accumulated in the sediment than P likely due to higher recovery of N inputs in harvested fish. We assume that the absence of sludge removal between each production cycle can increase nutrient trapping in sediment. More regular cleaning of the ponds, and the use of the nutrient-rich sludge for other applications (e.g. crop fertilization) could improve the efficiency and the utility of the nutrients inputs of the ponds.

The percentages of N input recovered in fish (5-19%) were similar to that observed in other semi-intensive and intensive fish farming systems, but we found lower values for P (1-5%). In semi-intensive ponds, proportions of N and P in harvested fish are highly variable and can account for 4-53% of N, and 3-24% of total N and P inputs respectively (e.g. [START_REF] Hlaváč | Effects of common carp (Cyprinus carpio Linnaeus, 1758) supplementary feeding with modified cereals on pond water quality and nutrient budget[END_REF][START_REF] Green | Chemical budgets for organically fertilized fish ponds in the dry tropics[END_REF][START_REF] Nhan | Water and nutrient budgets of ponds in integrated agriculture-aquaculture systems in the Mekong Delta, Vietnam[END_REF], see Table 4), whereas 11-27% of N and 18-32% of P total inputs are recovered in harvested fish in intensive systems (e.g. [START_REF] Boyd | Chemical budgets for channel catfish ponds[END_REF][START_REF] Krom | Nutrient budget of a marine fish pond in Eilat, Israel[END_REF][START_REF] Siddiqui | Nutrient budgets in tanks with different stocking densities of hybrid tilapia[END_REF].

Nutrient use efficiency and environmental impacts

The food efficiency of the production system studied was relatively low (0.107 ± 0.002 and 0.030 ± 0.001 kg kg -1 of fish produced for N and P respectively excluding pond D, Table 4).

Such values are lower than other Asian semi-intensive fish aquaculture systems in which commercial feed is not used (approx. 0.240 and 0.120 kg kg -1 of fish produced for N and P respectively; [START_REF] Nhan | Water and nutrient budgets of ponds in integrated agriculture-aquaculture systems in the Mekong Delta, Vietnam[END_REF], but higher than Brazilian semi-intensive pellet-fed tilapia systems (approx. 0.081 and 0.017 kg kg -1 of fish produced for N and P respectively, [START_REF] Osti | Nitrogen and phosphorus flux from the production of Nile tilapia through the application of environmental indicators[END_REF]. This comparison reveals that, in our system, the commercial food is not efficiently assimilated suggesting that conventional intensification is not the best model for the sustainable production of giant gourami. One explanation for this low efficiency involves a mismatch between the feeding strategy and the feeding behavior of this species. In fish species with small mouths, uneaten feed can reach 30% of the feed supplied [START_REF] Meer | Effect of feeding level on feed losses and feed utilization of soya and fish meal diets in Colossoma macropomum (Cuvier)[END_REF][START_REF] Verdegem | Nutrient discharge from aquaculture operations in function of system design and production environment[END_REF]. In contrast, for large-mouthed species, such as European eel or the African catfish Clarias gariepinus, which can swallow pellets whole, uneaten feed is limited to 1-10% [START_REF] Heinsbroek | The effect of feeding level on the apparent digestibilities of nutrients and energy of a reference diet for the European eel, Anguilla anguilla L., and the African catfish, Clarias gariepinus (Burchell, 1822)[END_REF][START_REF] Verdegem | Nutrient discharge from aquaculture operations in function of system design and production environment[END_REF]. Because the giant gourami is a shy and small-mouthed species, uneaten feed likely represents a significant proportion of the nutrients accumulated in the sediment. Nevertheless, characterization of giant gourami diet in aquaculture ponds, using approach such as stable isotope analysis, is needed to confirm the assumption.

Mechanisms regulating the primary production in pond are multifactorial and complex. The relatively low primary production in ponds generously fertilized with organic manure such as in gourami ponds, suggests that farmers' fertilization practices should be investigated and optimized (as suggested in Section 4.1). In opposite to the inorganic forms of nutrients in pond sediments, are immediately available for primary production, organic forms become available through heterotrophic microbial activity, which requires long periods of time [START_REF] Muendo | Sediment accumulation in fish ponds; its potential for agricultural use[END_REF]. The C:N ratio of feed and fertilizer can greatly affect heterotrophic microbial activity [START_REF] Das | Pond Fertilization Regimen[END_REF]. Thus, adjusting the C:N ratio by adding carbohydrates [START_REF] Magondu | Enhancing productivity of Labeo victorianus ponds using maize flour as a carbohydrate source[END_REF] to organic fertilizers or by the use of low protein-toenergy ratio (P:E) fish diets [START_REF] Kabir | Effect of dietary protein to energy ratio on performance of Nile tilapia and food web enhancement in semi-intensive pond aquaculture[END_REF] may promote the growth of heterotrophic bacteria and thus enhance the availability of organic nutrients. Furthermore, little bioturbation was observed in our experiment; ponds showed homogeneous and relatively compact sediment. The presence of burrowing organisms such as bottom-feeding fish (e.g. common carp) may help increase the bioavailability of nutrients trapped in sediments [START_REF] Ritvo | The effects of common carp bioturbation on fishpond bottom soil[END_REF]. However, more research is needed to understand the potential complex interactions between common carp and giant gourami in pond systems.

Despite the low feed efficiency of the system, a large proportion of nutrients was trapped in the sediments and the discharge in inflow water was low (0.0056 ± 0.0012 and 0.0001 ± 0.0001 kg kg -1 of fish produced for N and P, respectively, excluding pond D, Table 4). These values are significantly lower than the discharges observed in the production of giant gourami (0.0459 ± 0.0042 and 0.0226 ± 0.0008 kg kg -1 of fish produced for N and P, respectively) and three other species (C. gariepinus, C. carpio and O. niloticus; 0.0204-0.0499 and 0.0139-0.0226 kg kg -1 of fish produced for N and P, respectively) reared in cages in Indonesia [START_REF] Syandri | Nitrogen and phosphorus waste production from different fish species cultured at floating net cages in lake Maninjau, Indonesia[END_REF]. Thus, the risks of water quality alteration associated with intensification remain low in earthen pond production systems highlighting the ecosystem services rendered by aquaculture ponds through the trapping of nutrients in sediments [START_REF] Brugere | Ecosystem services in freshwater fish production systems and aquatic ecosystems: Recognizing, demonstrating and capturing their value in food production and water management decisions[END_REF]. Such results suggest that environmentally friendly intensification is possible within these production systems without compromising water quality and, for these reasons, further investigations should be done on the ecological intensification of Indonesian earthen pond aquaculture [START_REF] Slembrouck | Choosing floating macrophytes for ecological intensification of small-scale fish farming in tropical areas: A methodological approach[END_REF]. Nevertheless, the limitations of the present study (see Section 4.3) should be considered when interpreting the results.

Study limitations

Nevertheless, the interpretation of some results presented in this study has to be done with caution. Our experiment was performed on four ponds using a unique zootechnical management method within a single fish farm. Thus, some trends, such as the linear increase of nutrient accumulated in sediments relative to the total nutrient inputs, need to be confirmed in further investigations carried out in a larger scale.

Only the major sources and sinks of N and P where considered in the present study. However, for the four ponds studied and for N and P, fewer nutrients were measured in the outputs than in the inputs. Indeed, about 20% of the total N and total P were not recovered in the measured outputs (Fig. 2 and3).

Several processes were not measured in the present study due to technical constraints: N volatilization, denitrification, photosynthesis and respiration all influence the nutrient budget in ponds [START_REF] Nhan | Water and nutrient budgets of ponds in integrated agriculture-aquaculture systems in the Mekong Delta, Vietnam[END_REF]. N volatilization accounts for a non-negligible part of the N budget in fish ponds. N losses by volatilization can reach 15% of the N inputs in intensive channel catfish Ictalurus punctatus ponds [START_REF] Gross | Ammonia volatilization from freshwater fish ponds[END_REF]. Nevertheless, N volatilization depends strongly on pH and to a lesser extent temperature [START_REF] Hargreaves | Nitrogen biogeochemistry of aquaculture ponds1[END_REF] and occurs in alkaline conditions because only unionized ammonia (NH 3 ) can be lost to the atmosphere [START_REF] Hargreaves | Nitrogen biogeochemistry of aquaculture ponds1[END_REF]. In our production system, morning pH was close to 7.0, but the photosynthetic activity occurring during the diurnal period increased the pH throughout the day, which associated with high temperatures [START_REF] Boyd | Pond aquaculture water quality management[END_REF], may enhance N volatilization.

In addition, we assumed that some uncertainties occurred in the estimation of nutrient concentration in the outflow water. Indeed, water flows out of the ponds through overflow PVC pipes, meaning that only the surface water was evacuated. Thus, the sampled water collected in the PVC pipe maybe not fully representative of the low water-exchange ponds. In such ponds, stratification of nutrients and micro-organisms can occur in the water column [START_REF] Danos | Stratification of microorganisms and nutrients in the surface microlayer of small freshwater ponds[END_REF], even if the depth of the ponds did not exceed 0.55 m. Aeration systems (e.g. paddle wheel) ensure better water mixing but are technically difficult to set up due to the absence of electricity at the majority of the production sites.

Conclusion

The present study is one of the first to investigate nutrient use in small-scale freshwater aquaculture in Indonesia. Despite some limitations, it highlights the need for paying special attention to the monitoring of nutrient budgets in such production systems in which intensification has increased over the past years. In the studied semi-intensive production system, commercial feed and, to a lesser extent, organic fertilizers were the most important nutrient sources. Only a small fraction of nutrient inputs was taken up by harvested fish, and the largest fraction accumulated in the sediment with no nutrient enrichment observed in the outflow water. Nevertheless, further investigations at a larger scale are needed to determine the environmental impacts, especially on water quality, of intensification of freshwater aquaculture in Indonesia.

  ). Samples were also taken to determine chlorophyll-a concentration (APHA 2005), biological oxygen demand (BOD), and chemical oxygen demand (COD), according to AOAC

  Standard (SNI) 06-6989.52-2005 using macro Kjeldahl coupled with titration. Total N was then determined as the sum of TKN, NO₃-N and NO2-N. Phosphorus (P) in water was determined based on the procedures described in the SNI 06-6989.31-2005 adapted from APHA 4500-PE.

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure2. Budget of nitrogen (N, expressed as % of the total inputs and kg ha -1 ) in semi-

Figure 3 .

 3 Figure3. Budget of phosphorus (P, expressed as % of the total inputs and kg ha -1 ) in semi-

Figure 4 .

 4 Figure 4. Relationship between total nutrient inputs (through fertilizer, stocked fish, commercial feed and inflow water) and nutrient accumulation in sediments (kg ha -1 year)

  carp (Cyprinus carpio), B: Tilapia (Oreochromis sp.), C: Several fish species, including silver barb (Barbodes gonionotus), carps (Cirrhina mrigala, Cyprinus carpio and Hypophthalmichthys molitrix), tilapia (Oreochromis niloticus), gouramis (Helostoma temminckii, Osphronemus goramy) and catfishs (Clarias macrocephalus x Clarias gariepnus, Pangasius hypophthalmus), D: Hlaváč et al. (2015), E: Green and Boyd (1995) F: Nhan et al. (2008).

Table 1 .

 1 Production parameters of the giant gourami culture system in earthen ponds.

	Figure 4

Table 2 .

 2 Summary of the physical and chemical parameters measured in the four ponds 566 throughout the five-month experiment. Data are expressed as Means ± SD. Measurements and 567 sampling were done between 08:00 and 10:00 AM.

		Parameters	Inlet	Pond A	Pond B	Pond C	Pond D
	On-site measurements
	Temperature (°C)	28.9±1.3 a	28.9±1.5 a	28.9±1.5 a	29.1±1.6 a	29.2±1.6 a
		pH	6.4±1.0 a	6.7±0.6 a	6.8±0.6 a	6.9±0.5 a	6.9±0.6 a
		DO (mg L -1 )	4.7±1.1 a	4.4±2.2 a	5.1±1.9 a	5.5±2.8 a	4.8±2.6 a
	TDS (mg L -1 )	37.6±7.1 a	45.6±7.3 a	47.0±11.6 a	43.8±5.7 a	46.1±4.9 a
	Laboratory measurements
	Chlorophyll a (µg L -1 )	19±9 a	343±129 b	468±300 b	327±152 b	238±260 a
	BOD (mg L -1 )	2.3±0.7 a	3.9±1.8 a	4.2±1.4 a	4.0±1.7 a	3.8±1.5 a
	COD (mg L -1 )	26.6±17.6 a	26.7±16.4 a	28.1±11.8 a	23.9±19.6 a	28.4±20.0 a
		N (mg L -1 )	2.0±0.4 a	1.5±0.4 ab	1.5±0.4 ab	1.3±0.6 b	1.6±0.4 ab
		P (mg L -1 )	0.012±0.019 a	0.025±0.030 a	0.055±0.050 a	0.028±0.035 a	0.029±0.045 a
	NO₃-N (mg L -1 )	1.59±0.29 a	1.21±0.32 ab	1.08±0.30 ab	1.04±0.45 b	1.17±0.39 a
	NO₂-N (mg L -1 )	0.06±0.06 a	0.02±0.01 a	0.02±0.02 a	0.03±0.04 a	0.06±0.11 a
	PO₄³⁻ (mg L -1 )	3.6±3.2 a	2.9±2.4 a	4.3±3.6 a	2.9±2.3 a	3.0±2.0 a
	568	DO: dissolved oxygen, BOD: biological oxygen demand, COD: chemical oxygen demand.

Table 3 .

 3 Budgets (kg ha -1 ) and use efficiency (kg kg -1 fresh fish) of nitrogen (N) and 570 phosphorus (P) in semi-intensive monoculture of giant gourami. Discharge (kg kg -1 of fish produced) = total nutrient quantity flushed out with outflow 575 water/total fish yields. Table 4. Nutrient (N and P) budgets in semi-intensive aquaculture ponds from selected 578 scientific literature. The nutrient budgets were not always balanced see the cited literature for 579 details.

	Inputs/Outputs	Pond A	Pond B	Pond C	Pond D	% of the total inputs (Mean ± SD)
	(a) N -Inputs
	Commercial feed	1066.3	1050.0	1079.6	748.1	56.8 ± 4.7
	Fertilizer	691.7	599.8	546.0	538.4	34.4 ± 2.8
	Stocked fish	50.3	50.0	50.6	50.4	2.9 ± 0.3
	Inflow water	64.5	60.1	109.3	154.8	5.8 ± 3.3
	Total inputs	1899.4	1760.0	1808.7	1491.7	100
	N -Outputs
	Accumulated sediments	1075.5	1056.2	1049.7	986.7	60.6 ± 3.9
	Harvested bivalves	1.2	0.9	0.6	2.3	0.08 ± 0.06
	Harvested fish	322.7	332.8	331.7	69.9	14.9 ± 6.8
	Outflow water	53.0	44.2	69.8	107.8	4.1 ± 2.2
	Harvested snails	10.5	8.1	9.9	1.4	0.4 ± 0.2
	Total outputs	1463.0	1442.2	1461.7	1168.1	80.6 ± 2.2
	Unaccounted	409.8	317.8	323.9	323.6	19.9 ± 2.1
	N -Efficiencies
	Feed efficiency*	0.109	0.105	0.106	0.369	0.172 ± 0.131
	Discharges**	0.005	0.004	0.007	0.053	0.027 ± 0.024

Acknowledgments

This publication is a production of the COFASP ERA-NET project "IMTA-EFFECT" funded by the European Union and French National Research Agency. The authors acknowledge Bang Macho and the local fish farmers in Babakan village for sharing their experiences and for their assistance throughout the duration of the study. Authors also thank laboratory staff of the IPB (Department of Aquaculture, Bogor) and SIG (Saraswanti Indo Genetech, Bogor) laboratories who carried out nutrient and chlorophyll analyses. This is publication ISEM 2019-018 SUD.

560

Figure 3 Commercial feed:


Audrey Beillard

Ethan Golliard

Valentin Gillet

Xavier Bantreil
email: xavier.bantreil@umontpellier.frtxmetro@umontpellier.frfrederic.lamaty@umontpellier.fr

Thomas-Xavier Métro

Dr Jean Martinez

Frédéric Lamaty

+ E Golliard

Dr + V Gillet

Dr X B Antreil

Prof. Dr T .-X Møtro

Expedient Mechanosynthesis of N,N-Dialkyl Imidazoliums and Silver(I)-Carbene Complexes in a Ball-Mill

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

& Mechanochemistry

Expedient Mechanosynthesis of N,N-DialkylI midazoliums and Silver(I)-CarbeneC omplexesi naBall-Mill AudreyB eillard, Ethan Golliard, Valentin Gillet, Xavier Bantreil,* Thomas-Xavier MØtro,* Jean Martinez, and FrØdØric Lamaty* [a] Dedicatedtothe memory of Dr.Guy Lavigne Abstract: The absence of solvent, associated with intensive mechanical agitation, allowed the first mechanosynthesis of high-value silver(I)-carbenec omplexes and the corresponding N,N-dialkylimidazolium precursors. This procedure gave outstanding results in terms of yield and reactiont ime, when compared to solution-based conditions previously described in literature, and was generalized to unprecedented compounds. Silver(I)-carbene complexes could either be obtainedf rom N,N-dialkylimidazolium salts or directly from imidazole and alkyl halides in ao ne-pot two-step procedure withouti solating the imidazolium intermediate. Additionally,a ne fficient one-pot three-step sequence, including imidazole alkylation, silver metalation, andtransmetalationi sr eported.

In addition to their remarkable anticancer and antimicrobial properties, [1] silver(I)-carbenec omplexes ([Ag(NHC)X]) are recognized as au niversal platform for the generation of organometallicc omplexes. [2] This can be explainedb oth by the great diversity of N-heterocyclic carbenes (NHCs) and aw ide range of metalst hat could be used. Besides, [Ag(NHC)X] complexes bearing N,N-dialkyl-substituted NHCs have proved to have interesting biological activities [1a] and electroluminescent properties. [3] However,[ Ag(NHC)X] complexes suffer from chemo-and photosensitivity,a nd their production requires long reaction times and/or excess reagents. In the literature, their syntheses are mostly performed in CH 2 Cl 2 ,ah ighly volatile organic solvent suspected of causing cancera si ndicatedb yt he GHS classification.T he corresponding imidazolium halide precursors were mostly obtained through alkylation of imidazoles, with extended reactiont imes at refluxing temperatures. Ac onvenient way to increase the efficiency and to reducet he environ-mental impact of such reactions is to use solvent-free mechanosynthesis. [4] While the benefits of mechanochemistry have already been demonstrated for the generation of organometallic complexes, [5] the mechanosynthesis of N,N-dialkylimidazolium halides and their corresponding [Ag(NHC)X] complexes has neverbeen reported.A sanextension of our studies on mechanochemistry for organic synthesis [6] and generation of Pd nanoparticles, [7] we detail herein the advantages of using am echanochemical approach for the production of widely used N,N-dialkylimidazolium salts and valuable [Ag(NHC)X]c omplexes (Scheme 1).

Initially,w ef ocusedo nt he synthesis of N,N-dialkylimidazolium salts, which were obtained by nucleophilic substitution of alkyl halides with N-alkyl imidazoles.U nless noted otherwise, all reactions werer ealized without solvent in a1 0mLs tainless steel reactor containing a1 0mmd iameter stainlesss teel ball that was placed undera gitation at 25 Hz in avibratory ball-mill (vbm). The reactionm ixtures were recovered and treated with preferable solvents, such as EtOH, iPrOH, or EtOAc/water. [8] Subsequentf iltration allowed elimination of any metallic particles and isolation of pure products. [6f] For comparison, the best results reported in the literature for experiments performed in solution have been included (Table 1).

The reactiono fb enzylbromide with N-methyl-and N-isopropyl-imidazole yielded imidazoliums 1a•HBr and 1b•HBr,r espectively,i nm ore than 98 %w ithin only 1h(Table 1, entries 1a nd 2). Interestingly,t he synthesis of 1a•HBr was performed with the same efficiency on a4gs cale, using a2 50 mL reactor agitated in aplanetary ball-mill (pbm). Bidentateligand precursors 2a•HBr and 2b•HBr,f eaturingap yridine and an imidazolium moiety,r espectively,w eresynthesizedi n1hand isolated in excellenty ields (> 97 %, Ta ble 1, entries4and 5). In this case, the use of NaHCO 3 (1.5 equiv) was necessary to deprotonate2 -(bromomethyl)pyridine hydrobromide and obtain satisfactory results. Less reactive alkyl chlorides were also mechanically convertedi nto imidazoliums 1a•HCl and 2a•HCl,w ith slightly longer reaction times (< 3.5 h). When 1,2-bis(bromomethyl)benzene and 2,6-bis(bromomethyl)-pyridinew ere used as the startingr eagents, unreported bis-imidazoliums 3a•2HBr, 3b•2HBr, 4a•2HBr,a nd 4b•2HBr,w ereo btainedi navery efficient manner.T he advantage of mechanochemistry was especially highlighted with 4b•2HBr,a so nly 2.2 ho fm illing was necessary to isolate this product in 94 %y ield, while 7d ays in refluxing THF were required for al iterature procedure (isolated in 58 %y ield). [9] The reactiono fi midazole/benzimidazole with benzylb romide or 2-(bromomethyl)pyridine hydrobromide in the presence of NaHCO 3 proved that this ball-milling procedure could also be extended to symmetrical imidazolium salts. Thus, compounds 5a•HBr, 5b•HBr,a nd 6•HBr were isolated with improved yields compared with the previous methods (entries1 1-13).

Notably,i midazoliums 1a•HBr and 5a•HBr were produced in similar yields when synthesized in av bm or in ap bm, showing that the type of agitationh as no influence on the efficiency of the approach( Ta ble 1, entry 1a nd 11). While as olvent-based synthesis of 1a-6•xHX salts previously described in the literature required adaptation of the conditions for each compound, such as long reaction times (up to 7d ays), in some cases refluxing solvents, the use of ball-mill and solvent-free conditions provides ag eneral,u ser-, and eco-friendly method to obtain imidazoliums with improved yields and dramatically reduced reaction times. [START_REF]Calculation of various green metrics support lower environmental impact of this strategy compared to as olvent-based approach[END_REF] Thes uperiority of solvent-free mechanosynthesis over classical solution-based approaches can be explained by the higher reagent concentrations, which, in combination witha ne fficient mixing that avoidsm ass transfer limitations, resultsi nh igher reaction rates.I na ddition, the majority of the N,N-dialkylimidazolium mechanosynthesesp resented in this study required at least one liquid reagent. The capacity of al iquid to present ag reater reactive surface area to other reagents and to potentially serve as as olvent [START_REF] Mikhailenko | [END_REF] appears to us as ag ood rationale, in this case, to explain the higher efficiency of this approach. This methodology provedg eneral,a sn umerous mono-, di-, and tridentate ligand precursors were produced.

The synthesis of the corresponding silver(I)-carbene complexes was next envisaged by the reactiono fi midazoliums 1a-6•xHX with silver(I) oxide in av ibratory ball-mill under solventfree conditions. Thus, metalation of 1a•HBr,w hich was reported to last 24 hi ns olution,w as completed within only ten minutes under milling, furnishing [Ag(1a)Br] in 93 %y ield (Table 2, entry 1). In this case, the higher efficiency of mechanochemical synthesis over as olution-based approach could be attributed to the higherr ate of creationo fn ew surfaces on Ag 2 Op articles caused by impactsa nd shear stress induced by ball-milling. [START_REF] Mikhailenko | [END_REF]21] With this efficient method in hand, various complexes,k nown or unprecedented, were synthesized. After milling, the reaction mixtures were recovered with CH 2 Cl 2 ,f iltered over celite andc oncentrated under vacuum. 1 HNMR 98) [b] 82 %, THF,rt, 16 h [10] 2 iPr 1b•HBr 198 unprecedented 3Me 1a•HCl 294 78 %, solvent-free, 5h,7 0-110 8C [11] 4 Me 2a•HBr 19 7 [b,c] 87 %, CH 3 CN, 48 h, rt [12] 5 iPr 2b•HBr 199 [c] 60 %, dioxane, 12 h, 101 8C [13] [9] 11 Y = CH 5a•HBr 19 4( 99) [b] 87 %, toluene, 72 h, 70 8C [16] 12 Y = N 5b•HBr 2.5 79 72 %, EtOH, 48 h, 79 8C [17] 13 6•HBr 2.5 93 76 %, CH 3 CN, 48 h, 80 8C [18] [a] To tal mass of reactants = 182 mg;v bm = vibratory ball-mill. [b] Realized in ap lanetaryb all-mill( pbm), with at otal mass of reactants = 3977 mg.

[c] Product was recovered by using iPrOH.

analysisoft he crude mixtures revealed complete conversion of the substrates and high purity of the products in all cases.

Except for complexes [Ag( 2a)Cl] and [Ag 2 (4b)Br 2 ]( Ta ble 2, entries 6a nd 10), all previously described silver(I)-carbene complexes were obtained in similaro ri mproved yields and reduced reactiont imes (entries 3, 4, 11,a nd 12). This particular activation methoda lso allowed the synthesis of five unprecedented silver(I)-carbene complexes (entries 2, 5, 8, 9, and 13). Imidazoliums 2a•HX, 2b•HX,a nd 5b•HBr,w hich contain at least one pyridine ring, requiredl ongerm illing times (up to 2.5 h) than their phenyl-containing congeners 1a•HX, 1b•HX,a nd 5a•HBr (< 1h,e ntries 4-6 and 12 versus1 -3 and 11). The reaction of 3a•2HBr with Ag 2 Od id not yield the expected [Ag 2 (3a)Br 2 ]b ut led to the formation of an insoluble solid, which was postulated to be an organometallic polymer (entry 7). Similari ssues were also observed for [Ag 2 (4a)Br 2 ], which was obtained in irreproducible yields (entry 9). However, the presence of am ore hindered iPr substitution inhibited side reactions, thus [Ag 2 (3b)Br 2 ]a nd [Ag 2 (4b)Br 2 ]w ere isolatedi n8 3 and 77 %y ield, respectively (entries 8a nd 10). It is worth noting that this approach provedt ob ep articularly robust as high yields were obtained independently from various parameters knownt oh ave criticali nfluence in solvent-free or solventless mechanochemicalr eactions, such as relative humidity, type and intensity of milling, physical aspects of the reagents, and reactionmixture. [22] To confirmt he structures of the [Ag(NHC)X] complexes obtained by this originalm echanochemicala pproach, several compounds werer ecrystallized in aC H 2 Cl 2 /pentanem ixture and single crystals were submitted to X-ray diffraction analysis. Thus, unprecedented [Ag(1b)Br] was confirmed to be ab is-NHC silver cationic complex [{Ag(1b) 2 } + . AgBr 2 À ] 2 in which the counter anion AgBr 2 À forms adimer (Figure 1). [28] We next envisioned to further demonstrate the efficiency of this novel approachb yp erforming the synthesis of [Ag(NHC)X] complexes in ao ne-pot sequential alkylation/metalation procedure starting from imidazoles. This technique was very efficient for some [Ag(NHC)X] complexes,s uch as [Ag( 1a)Br] and [Ag(1b)Br] (Table 3, entries 1a nd 2). Interestingly,c omplexes [Ag 2 (3a)Br 2 ]a nd [Ag 2 (4b)Br 2 ]w ere obtained with improved yields when synthesized by this one-pot procedure compared to the conditions reported in Table 2( entry 4a nd 6). However, the isolation of [Ag 2 (4a)Br 2 ]w as still difficult (vide supra; entry 5).

The usefulness of mechanochemistryi no rganometallic synthesis was further demonstrated by reacting[ Ag(1a)Br] with [AuCl(SMe 2 )] (Scheme 2). The transmetalation occurred within 30 min, yieldingc omplex [Au(1a)Cl] in 85 %, thus completing [23] 2[ Ag(1b)Br] 0.5 10 min 85 unprecedented 3[ Ag(1a)Cl] 0.55 10 min 76 yield n.a., [a] CH 2 Cl 2 ,r eflux, 24 h, [24] 4[ Ag(2a)Br] 0.5 1h 78 71 %, CH 2 Cl 2 ,r t, 12 h [12] (4a)Br 2 ]1 .1 2h 30-75 [b] unprecedented 10

[Ag 2 (4b)Br 2 ]1 .5 2.5 h7 78 8%,C H 2 Cl 2 ,r t, 24 h [9] 11

[Ag( 5a)Br] 0.5 20 min 80 75 %, CH 2 Cl 2 ,r t, 2h [26] 12

[Ag(5b)Br] 0.6 1h 77 79 %, CH 3 CN, reflux, 16 h [27] 13

[Ag( 6)Br] 0.5 1h 71 unprecedented

[a] n.a. = not available. [b] Reactionw as not reproducible. as eries of three consecutivec hemical steps performed in ab all-mill with high efficiency.M oreover,[ Au(1a)Cl] could also be obtained directly from N-methylimidazole in ao ne-pot three-step procedure, thus avoiding the use of solvents for the isolation of intermediates. In only 1h and 40 min, [Au(1a)Cl] was isolated with a4 7% overall yield (78 %a verage yield for each step).

In conclusion, we have demonstrated that ball-milling represents the method of choice for an expedient synthesis of N,Ndialkyl-substituted NHC precursors and their corresponding [Ag(NHC)X] complexes.T ime-saving one-pot/two-step and one-pot/three-stepp rocedures allowed the direct production of silver(I)-andg old(I)-carbene complexes from the imidazole and alkyl halides withoutt he isolation of intermediates, hence, paving the way for further one-pot multi-step mechanosyntheses of organometallic complexes.

Figure 1 .

 1 Figure 1. Graphical representation of [{Ag(1b) 2 } + •AgBr 2 À ] 2 .Hatoms are omitted for clarity.

Table 1 .

 1 Mechanosynthesisofi midazolium halides.[a]

	Entry	Products	R 1	Product•HX	Time [h]	Yield [%]	Lit. conditions
	1		Me	1a•HBr	19 8(

Table 2 .

 2 Mechanosynthesisof[ Ag(NHC)X] complexes.

	Entry ProductA g 2 O	Time	Yield	Conditions
	[equiv]		[%]
	1[ Ag(1a)Br] 0.55	10 min 93	91 %, CH 2 Cl 2 ,r t, 24 h

Chem. Eur.J.2015, 21,17614 -17617 www.chemeurj.org 2 015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim

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Acknowledgements

UniversitØ Montpellier and CNRS are acknowledged for funding. We thank Prof. J.-M. Campagne and the AM2N team for generously providing [AuCl(SMe 2 )],a nd Dr.S .R icheter and the IMNO team for [AuCl(THT)].F .M Øtro is warmly acknowledged for the production of the graphical abstract.