Nicolas Ferry
email: ferrynicolas@me.com

Marion Cordonnier

Florence D Hulot

Farisayi Dakwa

Lovelater Sebele

Stéphane Dray

Hervé Fritz

Marion Valeix

Heterogeneity of water physico-chemical characteristics in artificially pumped waterholes: do African herbivores drink at the same locations and does it lead to interference competition?

Keywords:

In many semi-arid savanna ecosystems, surface water is scarce and only found in artificially pumped waterholes at the end of the dry season, leading to high large mammal densities and competition. Further, the modification of the physico-chemical characteristics of the drinking water over the dry season (e.g. through faeces accumulation) could enhance competition. Indeed, elephants, considered as key-competitors, and other herbivores by aggregating near the trough where clear water arrives could compete for this resource. We studied the drinking locations of eight herbivore species around pumped waterholes in relation to these water characteristics in Hwange National Park, Zimbabwe. We identified differences of the physico-chemical characteristics of the water in different sections of pumped waterholes at the end of the dry season. Elephants drank the water in or close to the trough, whereas other species drank further in the waterhole, except roan and sable antelopes which were indifferent about where they drank. Interference competition with elephants for the access to water close to the trough was not detected for zebras and kudus. We discuss possible directions for future research to enhance our understanding of waterhole use by herbivores.

Introduction

Species distribution patterns depend on the spatial distribution of resources [START_REF] O'neill | Resource utilization scales and landscape pattern[END_REF][START_REF] Pearson | The spatial extent and relative influence of landscape-level factors on wintering bird populations[END_REF]. Patches of resources represent attractive areas in the landscape, around which animals are likely to aggregate and may temporarily form mixed-species groups [START_REF] Waser | Primate polyspecific associations: do they occur by chance?[END_REF][START_REF] Stensland | Mixed species groups in mammals[END_REF]. The co-occurrence of different species can lead to interspecific competition (exploitative or interference, [START_REF] Schoener | Field experiments on interspecific competition[END_REF], forcing some species to avoid more competitive ones at both spatial [START_REF] Durant | Living with the enemy: avoidance of hyenas and lions by cheetahs in the Serengeti[END_REF][START_REF] Tannerfeldt | Exclusion by interference competition? The relationship between red and arctic foxes[END_REF] and temporal scales [START_REF] Ziv | Interference competition and temporal and habitat partitioning in two gerbil species[END_REF][START_REF] Valeix | Interference competition and temporal niche shifts: elephants and herbivore communities at waterholes[END_REF]. The intensity of these competition processes increases as resource quantity or accessibility decreases. This is typically the case of surface water resources in arid and semi-arid ecosystems, where water resources dry up as the dry season progresses. In many arid and semi-arid savanna ecosystems, surface water during the dry season is provided mainly through artificial pumping of underground water. These pumped waterholes often represent the only source of surface water available to animals at the end of the dry season. This scarcity of water leads to the aggregation of a wide range of large mammal species around pumped waterholes, sometimes at very high levels of abundance [START_REF] Weir | Daily occurrence of african game animals at water water holes during dry weather[END_REF][START_REF] Valeix | Temporal dynamics of dry-season water-hole use by large African herbivores in two years of contrasting rainfall in Hwange National Park, Zimbabwe[END_REF].

While several studies have focused on the use of pumped waterholes by large wild mammal species [START_REF] Western | Water availability and its influence on the structure and dynamics of a savannah large mammal community[END_REF][START_REF] Redfern | Surface-water constraints on herbivore foraging in the Kruger National Park, South Africa[END_REF][START_REF] Hayward | Waterhole use by African fauna[END_REF], our understanding of the underlying processes of co-occurrence patterns of different species is still https://doi.org/10.1016/j.jaridenv.2019.104014 Received 23 May 2018; Received in revised form 8 April 2019; Accepted 26 August 2019 incomplete. Interspecific competition is likely to be a major process taking place between species simultaneously exploiting water resources. For example, [START_REF] Valeix | Interference competition and temporal niche shifts: elephants and herbivore communities at waterholes[END_REF] showed a temporal avoidance of African elephants Loxodonta africana by several other herbivores species. However, a recent study revealed that when zebras Equus quagga, and to a lesser extent kudus Tragelaphus strepsiceros, co-occur with elephants around pumped waterholes at the end of the dry season, they tend to all aggregate in the same specific sections of the waterhole area [START_REF] Ferry | Interspecific interference competition at the resource patch scale: do large herbivores spatially avoid elephants while accessing water?[END_REF]. One hypothesis suggested to explain this unexpected result is that it is not only the presence or the quantity of the resource that matters but its quality. Here, we thus focused on the physico-chemical characteristics of water. The documented patterns of species positions around pumped waterholes may result from a passive aggregation with species being attracted by the same characteristics of the water (e.g. sufficient level of some mineral nutrients, low organic matter content) in particular areas of the waterholes. Numerous studies have reported that animals select patches with food of higher quality (e.g., [START_REF] Langvatn | Feeding-patch choice by red deer in relation to foraging efficiency[END_REF][START_REF] Wilmshurst | Forage quality and patch choice by wapiti (Cervus elaphus)[END_REF][START_REF] Van Der Wal | Trading forage quality for quantity? Plant phenology and patch choice by Svalbard reindeer[END_REF][START_REF] Hochman | Effects of food quality, diet preference and water on patch use by Nubian ibex[END_REF]. However, studies on the influence of the quality of water resources on wild animals drinking behaviour are rare (but see [START_REF] Wanke | Water quality for game in drylands: a case study from the Khaudum National Park, Namibia[END_REF][START_REF] Chamaillé-Jammes | Spatial relationship between elephant and sodium concentration of water disappears as density increases in Hwange National Park, Zimbabwe[END_REF]. At a fine scale, nothing is really known about the role of the spatial heterogeneity of water characteristics in waterholes on drinking choices and surface water use.

Herbivores are active carriers of nutrients when they urinate and defecate in water [START_REF] Naiman | Large animals and system-level characteristics in river corridors[END_REF][START_REF] Masese | Are large herbivores vectors of terrestrial subsidies for riverine food webs?[END_REF][START_REF] Subalusky | The hippopotamus conveyor belt: vectors of carbon and nutrients from terrestrial grasslands to aquatic systems in sub-Saharan Africa[END_REF][START_REF] Hulot | Mega-herbivores subsidies in artificial pounds in Hwange National Park, Zimbabwe[END_REF]. It is therefore possible that the combined effect of evaporation and accumulation of herbivore faeces and urine in waterholes as the dry season progresses leads to a change in the physico-chemical characteristics of the water in most waterholes (e.g. [START_REF] Gereta | Wildlife-water quality interactions in the serengeti national Park, Tanzania[END_REF][START_REF] Strauch | Interactions between soil, rainfall, and wildlife drive surface water quality across a savanna ecosystem[END_REF][START_REF] Msiteli-Shumba | Driving factors of temporary and permanent shallow lakes in and around Hwange National Park, Zimbabwe[END_REF]) and to a spatial gradient of these characteristics in pumped waterholes. In these artificially pumped waterholes, the presence of an area where the clear pumped water arrives could lead herbivores to actively seek for this water with specific physico-chemical characteristics [START_REF] Wanke | Water quality for game in drylands: a case study from the Khaudum National Park, Namibia[END_REF]. Under this water gradient hypothesis, elephants and most other herbivores are expected to end up being aggregated as the dry season progresses just because the water coming from the trough, which represents the area where fresh water arrives in the waterhole, attracts them all.

Here, we studied the physico-chemical characteristics of the water in different areas of pumped waterholes in the semi-arid savanna of Hwange National Park, Zimbabwe, where we also monitored the positioning of different herbivore species when they drink at these waterholes. We tested the hypothesis that the heterogeneity of water physicochemical characteristics in a waterhole explains aggregation patterns at attractive areas of the waterhole, and ultimately may lead to interference competition between elephants and other herbivore species. We predicted (i) the existence of a gradient of water physico-chemical characteristics at the waterhole scale at the end of the dry season, (ii) the attraction of troughs where pumped underground water emerges, represented by a low distance between drinking herbivores and the trough, and (iii) an interference competition between elephants (the largest and a potentially aggressive species [START_REF] Valeix | Interference competition and temporal niche shifts: elephants and herbivore communities at waterholes[END_REF] and other herbivore species for the access to water of better physico-chemical properties, resulting in a higher distance to the trough when drinking in the presence of elephants compared to situations without elephant.

Materials and methods

Study site

The study area (~7 000 km 2 ) is located in the northern region of Hwange National Park which covers ~15 000 km 2 of semi-arid dystrophic (low nutrient soil) savanna in north-western Zimbabwe (19°00′ S, 26°30′ E; Fig. 1). The vegetation is primarily woodland and bushland savanna. The long-term mean annual rainfall is ~600 mm, which falls primarily between November and April, and the dry season stretches from May to October. The surface water available to animals is found in natural waterholes and rivers as well as artificially pumped waterholes. Waterholes are depressions that are fed by rainwater during the rainy season. During the dry season, many waterholes are supplied with pumped underground water. Water is extracted from boreholes using solar panel-powered pumps (so water is extracted during the day). Water is first pumped into an open-top concrete ground-level small drinking trough, and then flows continually from the trough to the depression (see Appendix 1 for pictures). Natural water bodies dry up during the dry season and only artificially pumped waterholes offer drinking water throughout the year and at the end of the dry season. In pumped waterholes, the pumping is continuous during the day when most herbivore species come to drink [START_REF] Valeix | Interference competition and temporal niche shifts: elephants and herbivore communities at waterholes[END_REF] and the waterhole is then continually fed with pumped water. Eight herbivore species were monitored: two browsers (giraffe Giraffa camelopardalis and greater kudu), two mixed-feeders (African elephant and impala Aepyceros melampus) two woodland grazers (roan antelope Hippotragus equinus, and sable antelope Hippotragus niger) and two grassland grazers (warthog Phacochoerus africanus and Burchell's zebra).

Data

Water physico-chemical characteristics

We sampled the physico-chemical characteristics of the water in four monitored pumped waterholes (Fig. 1) in October 2017, i.e. at the end of the dry season. A water sample was collected in three different areas for each waterhole. The first sample was taken in the water near the trough (A), the second at the furthest location from the trough (C), and the third at an intermediate distance (B) (Fig. 1). For the four waterholes, the mean distance between A and C is 73.2 m (ranging from 61 m at Shapi to 87 m at Guvalala) and represents the maximal distance to the trough when animals were drinking. Water samples were taken before 12 p.m. to minimize variability in the time of the day. We measured near the shoreline temperature, dissolved oxygen concentration, pH, turbidity and chlorophyll a concentration with a YSI 6600 VZ Multiparameter water probe. Conductivity was measured with a Hanna HI 98312 portable conductivity meter. Water conductivity measures the concentration in ions dissolved in water (calcium, magnesium, sodium, potassium, chloride, sulphate, nitrate and bicarbonate for the major ions). Water samples for laboratory analyses were collected on site at the deepest point with a bottle fastened to a 3 m pole. Water was stored on ice in 1-L polyethene bottles and was filtered back to the laboratory with GF/F 47 mm filters. Total nitrogen and total organic carbon concentrations and water hardness were determined from unfiltered water samples. Ammonium, nitrite, nitrate and orthophosphate concentrations were determined from filtered water samples. Chemical analyses were realized with a Hach DR 3900 portable data logging spectrophotometer and reagents in accordance with the manufacturer's procedure.

Drinking position

Observations of drinking herbivores were made at the same four monitored pumped waterholes (Fig. 1) between August and November 2016, i.e. at the end of the dry season. Every day, observations were done from 6 a.m. until 6 p.m. at one of the study pumped waterholes. Observers were inside a car parked at approximatively 100m from the waterhole as a compromise between disturbance and quality of the observation. Each time an herbivore or a group of herbivores drank at the waterhole, the position of the closest individual to the trough (focal individual) was recorded. In order to calculate the distance between the drinking focal individual and the trough, we recorded the angle to the north (with a compass) and the distance from the car (with a range finder) of the focal individual and the trough. For all drinking observations of the herbivores, we recorded if elephants were present at the waterhole or not.

Drinking position and water physico-chemical characteristics were not studied the same year, but 2016 and 2017 were characterized by similar annual rainfall (438.3 and 477 mm respectively). Water availability depends on rainfall during the rainy season and evaporation and pumping during the dry season. Morphological characteristics (depth, shape) of the waterholes and trough position did not change between 2016 and 2017. We therefore assume that water had a comparable gradient of physico-chemical characteristics between these two years.

Analyses

Analyses were conducted using R v. 3.3 software (R Development Core Team, 2004).

Water physico-chemical characteristics

In order to assess the existence of a gradient of water physico-chemical characteristics inside waterholes, we performed multivariate analyses. First, a Principal Component Analysis (PCA, package ade4, [START_REF] Dray | The ade4 package: implementing the duality diagram for ecologists[END_REF] was performed on the thirteen variables. We then removed the effect of the heterogeneity between waterholes with a within-class analysis and performed a between-class analysis to estimate the percentage of the remaining variability explained by the differences between the three locations A, B and C.

Drinking position

We first assessed the difference of position where animals drank at the waterhole between each study herbivore species by performing a mixed-model (package lmer, [START_REF] Bates | The Lmer Package for R: Linear Mixed-Effects Models Using S4 Classes[END_REF] on the distance to the trough when drinking. The species was considered as a fixed effect and the waterhole monitored as a random effect. A pairwise post-hoc comparison was then performed on the mixed-model between each pair of species (package lsmeans, [START_REF] Lenth | Least-squares means: the R package lsmeans[END_REF]. The p-values were adjusted with the Tukey method. We then assessed the influence of elephant presence on the drinking position of other herbivore species. We performed a mixed-model with the distance to the trough when drinking as the dependant variable. The interaction between the presence of elephants when drinking and the herbivore species was introduced as an explanatory term in the fixed part of the model. This analysis could be carried out for kudus and zebras only as these two species were the only ones recorded both in the absence of elephants and with elephants at the study waterholes. The waterhole monitored was considered as a random effect.

Results

Water physico-chemical characteristics

After the within-class analysis removed the variability due to differences between waterholes, the between-class analysis revealed that 31% of the remaining variability was explained by differences of water physico-chemical characteristics between the locations A, B and C. As the distance to the trough increases (from A to C), the concentration of total organic carbon, dissolved oxygen, total nitrogen and ammonium, and the conductivity increased whereas the temperature, the turbidity and the nitrate concentration decreased (Fig. 2, Fig. 3).

Drinking position

The mixed model allowed estimating the mean ( ± SE) drinking distance to the trough for the eight study species, which ranged from 15.5 m to 58.5 m (Fig. 4). Post-hoc tests revealed three different groups: (i) elephants, which drank the closest to the trough, (ii) roan and sable antelopes with an intermediate estimated mean distance from the trough, and (iii) all other study herbivore species (zebras, kudus, impalas, giraffes and warthogs), which drank the furthest from the trough (Fig. 4). A high variability was observed between waterholes, especially for roan and sable antelopes, which were observed to drink at highly variable distances from the trough (Fig. 5). No effect of the presence of elephants on the drinking distance from the trough was observed neither for zebras (β ± SE = 0.77 ± 6.56, t = 0.118, p = 0.9), nor for kudus (β ± SE = -1.11 ± 8.23, t = -0.13, p = 0.9).

Discussion

In this study, we identified differences of water physico-chemical characteristics at the scale of the waterhole at the end of the dry season. We then showed that elephants drank the water that flowed from the trough, that all other studied species drank relatively far from the trough except roan and sable antelopes, which were indifferent about where they drank. Finally, we did not detect any spatial patterns indicative of interference competition for the access to freshly pumped water between elephants and zebras or kudus.

Water physico-chemical characteristics

Our results revealed differences of water physico-chemical characteristics in different sections of pumped waterholes at the end of the dry season. Indeed, troughs are set up between the outlet of the pump and the waterhole and provide water of the same characteristics as the underground water to the animals because the trough is continually flushed [START_REF] Wanke | Water quality for game in drylands: a case study from the Khaudum National Park, Namibia[END_REF]. This water is generally less salty and conductive than surface water. Given that there is no input of rainwater in the dry season, evaporation and changes due to fauna activity lead to changes of the physico-chemical characteristics of the water in the waterhole [START_REF] Wanke | Water quality for game in drylands: a case study from the Khaudum National Park, Namibia[END_REF][START_REF] Msiteli-Shumba | Driving factors of temporary and permanent shallow lakes in and around Hwange National Park, Zimbabwe[END_REF], in particular with the concentration of ions which lead to an increase in conductivity and salinity.

Far from the trough, the water is enriched in organic matter (high concentration of total organic carbon, total nitrogen) and degradation product (ammonium). This organic matter may be inherent to the presence of microorganisms in the waterhole [START_REF] Strauch | Interactions between soil, rainfall, and wildlife drive surface water quality across a savanna ecosystem[END_REF], but also to urine and faeces of the numerous animals drinking in the waterhole. This last source of contamination is indeed frequently mentioned in the literature (e.g. [START_REF] Gereta | Wildlife-water quality interactions in the serengeti national Park, Tanzania[END_REF][START_REF] Strauch | Interactions between soil, rainfall, and wildlife drive surface water quality across a savanna ecosystem[END_REF][START_REF] Hulot | Mega-herbivores subsidies in artificial pounds in Hwange National Park, Zimbabwe[END_REF]. These organic matter inputs may stimulate phytoplankton growth, leading to high concentrations of chlorophyll a and dissolved oxygen, and lead to the high turbidity and water eutrophication observed in Hwange National Park [START_REF] Msiteli-Shumba | Driving factors of temporary and permanent shallow lakes in and around Hwange National Park, Zimbabwe[END_REF]) and can intensify heterogeneity of water characteristics between trough and the remaining waterhole. According to the criterions of the Department of Water and Sanitation in South Africa (DWAF, 1996), the water far from the trough is hypertrophic. No guideline exists for wildlife concerning water quality. However, the criterions for domestic water show that the high chlorophyll a concentration and turbidity are symptomatic of degraded water with significant taste and odour problems, secondary bacteria growth and risk of diseases due to infectious agents (DWAF, 1996). (caption on next page)

Drinking position

Several studies have documented how freshwater parameters influence terrestrial animals' drinking behaviour. For example, [START_REF] Auer | Water Availability and Chemical Water Quality as Important Factors for Sustainable Wildlife Management in the Etosha National Park and for Domestic Stock in Farming Areas of Namibia[END_REF] showed that an increase of the water salinity led herbivores to sniff and taste the water extensively, try many positions at the waterhole, and sometimes leave the waterhole without drinking. [START_REF] Stommel | Large mammals in Ruaha National Park, Tanzania, dig for water when water stops flowing and water bacterial load increases[END_REF] studied the response of some African mammals to total aerobic bacterial load and suggested that digging is an adaptation to avoid poor quality water and potentially pathogenic microbes. However, none focused on the heterogeneity at the scale of the waterhole.

Our results showed that elephants drank most of the time the water from the trough, except in Ngweshla (but only three observations observed at that waterhole). This confirms what [START_REF] Ramey | Desert-dwelling African elephants (Loxodonta africana) in Namibia dig wells to purify drinking water[END_REF] observed where elephants damaged borehole infrastructure to access freshly pumped water, with lower coliform counts, even when water was available in the waterhole. The "single-hit" epidemiological model [START_REF] Haas | Estimation of risk due to low doses of microorganisms: a comparison of alternative methodologies[END_REF] which assumes that exposition (here ingestion) to one pathogenic organism only can be sufficient to induce disease could explain these results. Following this model, elephant drinking more water than other herbivores (from 4-fold to 40-fold increase compared to giraffe and impala respectively, Table 1), have a higher probability to ingest pathogens and develop disease than other species. This would lead elephant to drink water from the troughs, which can be more difficult to access and represent smaller areas than the remaining waterhole, but being "safer" in terms of pathogens.

However, this model does not explain patterns observed for other species. Roan and sable antelopes do not show specific patterns and have a high variability in their drinking position. The five other species (zebra, kudu, giraffe, warthog and impala) drank at locations remote from the trough, which was unexpected, especially for giraffes, which drank very far from the trough in spite of being the second species in terms of water consumption (Table 1). [START_REF] Haas | Estimation of risk due to low doses of microorganisms: a comparison of alternative methodologies[END_REF] described another less stringent model, where a minimal effective dose inherent to the organism is needed to observe infection or disease [START_REF] Haas | Estimation of risk due to low doses of microorganisms: a comparison of alternative methodologies[END_REF]. If this threshold increases with body mass, and that the quantity of pathogens ingested increase with the amount of water consumed, we would expect species consuming equivalent amount of water per kilogram to have the same drinking position behaviour, which is not the case (Table 1). This suggests that, if pathogen load may be an important driver of the drinking behaviour of herbivores, it is not sufficient to explain the drinking position patterns. The drinking position could therefore be the result of a compromise between pathogen avoidance and mineral requirements. Future experimental research based on choice of water of different quality and different pathogen load could help understanding the decision making of these species.

Finally, other confounding factors inherent to the waterholes could interact or override water physico-chemical characteristics to explain herbivores' drinking position such as topography (slopes) and vegetation cover near waterholes. Indeed, the surrounding vegetation can influence perceived predation risk and can be beneficial (e.g. escape) or detrimental (e.g. camouflage for ambush predators) for herbivores (e.g. Valeix et al., 2008 and references therein). It could explain the variability between but also within waterholes, with unequal distances to cover upon the location at the waterhole. This is something that will need further investigation in the future.

Interference competition with elephants

A previous study revealed that elephants do not prevent other herbivores from drinking at waterholes [START_REF] Valeix | Do elephants prevent other African herbivores from using waterholes in the dry season?[END_REF]. Here, we showed that zebras and kudus drank far from the trough whether elephants are present or absent, which is not supporting a hypothesis of interference competition with elephants for the access to fresh water. Fig. 3. Physico-chemical characteristics of the three points (A, B and C) respectively close, intermediate and far from the trough in the four waterholes ("G" = Guvalala, "K2" = Kennedy 2, "N" = Ngweshla, "S" = Shapi). The thirteen studied physico-chemical characteristics are presented: dissolved oxygen (DO), total organic carbon (TOC), total nitrogen (N), ammonium (NH4+), nitrite (NO2), nitrate (NO3), orthophosphate (PO4) and chlorophyll a (Chl.a) concentration and conductivity (Conductivity), hardness (Hardness), turbidity (Turbidity) and temperature (Temperature). Regarding DWAF vol. 7 (1996) for aquatic ecosystem monitoring, the target water quality range is 7 mg l -1 for ammonium (NH4+), between 80 and 120% for dissolved oxygen (DO) and the water is considered as hypertrophic if orthophosphate (PO4-) is above 0.25 mg l -1 . Regarding DWAF vol. 1 (1996) on domestic water monitoring, if the chlorophyll a (Chl.a) is above 10 mg l -1 the water "has a distinct murky appearance, becoming increasingly green in colour with significant taste and odour problems and secondary growth bacteria on the distribution system", if the hardness is below 50 the water is considered as soft, if it is between 150 and 200 the water is considered as "moderately hard". Finally, if the turbidity is above 10 the water carries an associated risk of disease due to infectious disease agents and chemicals adsorbed onto particulate matter. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) The water-gradient hypothesis seems therefore unlikely to explain the spatial aggregation patterns observed by [START_REF] Ferry | Interspecific interference competition at the resource patch scale: do large herbivores spatially avoid elephants while accessing water?[END_REF], where zebras and kudus got closer to elephants at waterholes, as the dry season progressed. Another hypothesis that has been advanced to explain the observed aggregation patterns in this previous study is that zebras and kudus get close to elephants in order to decrease their perceived predation risk, as adult elephants are almost invulnerable to predation and may sometimes deter predators. Regarding the other herbivore species, we did not highlight any interference competition for the access of fresh water as they usually drank when elephants were absent and yet they did not drink at the trough. Only sable and roan antelopes are likely to face interference competition with elephants, as they are the only ones drinking sometimes relatively close to the trough. However, sable and roan antelopes almost never drank at waterholes when elephants were present, suggesting either a temporal avoidance of elephants [START_REF] Valeix | Interference competition and temporal niche shifts: elephants and herbivore communities at waterholes[END_REF] or different temporal niches at waterholes driven by other factors (e.g. food requirements, predation pressure).

Conclusion

At the end of the dry season, waterholes are characterized by heterogeneous physico-chemical characteristics of the water, with marked differences between the trough and the rest of the waterhole. In our study, we measured some physico-chemical characteristics of water but it would be interesting to identify relevant nutrient and water organoleptic properties for wildlife. Ultimately, it would help to define what a water of good quality for wildlife is. Water physico-chemical characteristics alone do not explain the observed drinking locations of herbivores, which may result from several factors. . Future experimental research controlling for water characteristics (pathogen load and mineral content) and environmental co-variables (e.g. predation pressure, topography) is required to disentangle the drivers of the drinking behaviour of large mammals at waterholes.

Table 1

Characterization of the species mean drinking distance to the trough and water-dependence defined through the interval between successive waterhole visits and water consumption.

Fig. 1 .

 1 Fig. 1. Map of the four monitored pumped waterholes in Hwange National Park, Zimbabwe. Locations of B and C (in blue, corresponding respectively to locations at intermediate and the longest distance from the trough) and A (in red, corresponding to the trough) for each waterhole. Aerial photos of the waterholes were taken from Google Map and scale bar is the same for all four waterholes. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2 .

 2 Fig. 2. Graphical results of the between-class principal component analysis. Top: position of the four waterholes (black dots) with confidence ellipses around the categories defined by the distance to the trough (A, B and C). Bottom: position of the thirteen variables: dissolved oxygen (DO), total organic carbon (TOC), total nitrogen (N), ammonium (NH4+), nitrite (NO2), nitrate (NO3), orthophosphate (PO4) and chlorophyll a (Chl.a) concentration and conductivity (Conductivity), hardness (Hardness), turbidity (Turbidity) and temperature (Temperature).

Fig. 4 .

 4 Fig. 4. Estimated mean ( ± SE) of the distance to the trough for drinking individuals of the nine study herbivore species that visited the four study pumped waterholes in Hwange National Park, Zimbabwe. The number of observations (n) for each species is provided. Different letters (a, b, c) indicate significant differences obtained by pairwise post-hoc comparison on the mixed-model between each pair of species. Red line indicates maximum distance averaged between the four waterholes. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5 .

 5 Fig. 5. Distance to the trough for drinking individuals of the eight study herbivore species that visited the four study pumped waterholes in Hwange National Park, Zimbabwe. Dotted lines represent the maximum drinking distance at the time of the monitoring for each study waterhole.

  kg) Water consumption per kilogram (mililitter.kg -1 )

Acknowledgements

We thank the Zimbabwe Parks and Wildlife Management Authority for providing authorizations to carry out this research. We sincerely thank Adolf Mawoyo, Tawanda Tarakini and Martin Muzamba for their help with data collection on water physico-chemical characteristics. We are grateful to Wilderness Safari Trust and in particular Arnold Tshipa for data on annual rainfall. We also thank fervently all rangers who accompanied us on the field. We benefited from useful comments from Norman Owen-Smith and Izak Smit that helped improved the quality of this manuscript.

Funding sources

This research was conducted under the Hwange Environmental Research Development (HERD) programme from the French 'Centre National de la Recherche Scientifique' (CNRS) and 'Centre de

Coopération Internationale en Recherche Agronomique pour le Développement' (CIRAD), within the study area of, and with funding support from, the CNRS Zone Atelier Hwange (CNRS-Hwange LTSER). The fieldwork was carried out under the framework of Nicolas Ferry's PhD supported by a grant from the French 'Ministère de la Recherche' through the 'Ecole Doctorale E2M2' of 'Université Claude Bernard Lyon 1'.

Ethics

Experiments were conducted following the ARRIVE guidelines on the use of animals in research, and permits were issued by Zimbabwe Parks and Wildlife Management Authority (ZPWMA).

Authors' contributions

NF, MC, FD, and FH collected the data; NF, SD, HF and MV designed the study, and all authors contributed to writing the manuscript, and approved the final version of the manuscript.

Conflicts of interest

We declare we have no competing interests.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jaridenv.2019.104014.


A Velasco

Aiste Lengvenyte

J Rodriguez-Revuelta

L Jimenez-Treviño

Philippe Courtet

M P Garcia-Portilla

J Bobes

P A Sáiz

Garcia-Portilla Mp

Sáiz Pa

María Paz García-Portilla

Neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, and monocyte-to-lymphocyte ratio in depressed patients with suicidal behavior: A systematic review

Keywords: Neutrophil/lymphocyte ratio, Platelet/lymphocyte ratio, Monocyte/lymphocyte ratio, Suicidal behavior, Depression

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

Introduction

Suicidal behavior (SB) is a serious public health concern. More than 700,000 people die by suicide every year, representing one death every 40 seconds on average [START_REF]Suicide worldwide in 2019: global health estimates[END_REF]. Factors contributing to increased risk of suicidal behaviors are diverse and complex, but epidemiological studies indicate that the vast majority of attempted and completed suicides occur in people with mental disorders with mood disorders being the most frequently associated with SB [START_REF] Bachmann | Epidemiology of Suicide and the Psychiatric Perspective[END_REF][START_REF] Dong | Suicide-related behaviours in schizophrenia in China: a comprehensive meta-analysis[END_REF].

Understanding the pathophysiology of suicide is still a long-term goal. The evidence increasingly indicates a possible role of the immune-inflammatory response in the development and maintenance of depression and SB [START_REF] Velasco | Neutrophil-to-lymphocyte ratio: A potential new peripheral biomarker of suicidal behavior[END_REF]. Inflammation has been associated with increased risk of suicidal behaviors above and beyond the risk associated with depression [START_REF] Bergmans | Inflammation as a unique marker of suicide ideation distinct from depression syndrome among U.S. adults[END_REF]. Neuroinflammatory processes are a pathophysiological mechanism that is essential for understanding SB in depressed patients. To explain the role of the immune system in the pathophysiology of suicide, a comprehensive model has been proposed. In this model, sleep disturbances, stress, childhood abuse, and infections induce dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis that is associated with a chronic low-grade inflammatory state and increased risk of suicidal behaviors [START_REF] Vasupanrajit | Inflammation and nitro-oxidative stress in current suicidal attempts and current suicidal ideation: a systematic review and meta-analysis[END_REF]. It has therefore been suggested that inflammatory biomarkers are potentially useful in predicting and monitoring suicide risk in patients with depression [START_REF] Courtet | Neuroinflammation in suicide: Toward a comprehensive model[END_REF].

Neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and monocyte-tolymphocyte ratio (MLR) indexes are convenient and cost-effective blood indicators of inflammatory status [START_REF] Demirkol | Cambridge University Press Accepted manuscript: Authors' Copy and inflammatory markers[END_REF].

NLR is the most studied hematological parameter [START_REF] Puangsri | Potential usefulness of complete blood count parameters and inflammatory ratios as simple biomarkers of depression and suicide risk in drug-naive, adolescents with major depressive disorder: Simple peripheral markers for inflammation in drug-naive, adolescents with major depressive disorder[END_REF]. Neutrophils are the first defense cells of the innate immune system, representing an active nonspecific inflammatory mediator of https://doi.org/10.1192/j.eurpsy.2023. [START_REF] Mazza | Neutrophil/lymphocyte ratio and platelet/lymphocyte ratio in mood disorders: A metaanalysis[END_REF] Published online by Cambridge University Press phagocytosis and apoptosis functions [START_REF] Rungelrath | Neutrophils in innate immunity and systems biology-level approaches[END_REF], and lymphocytes represent the regulatory or protective component of the immune system [START_REF] Özyurt | Increased neutrophil-lymphocyte ratios in depressive adolescents is correlated with the severity of depression[END_REF][START_REF] Chaplin | Overview of the immune response[END_REF][START_REF] Ivković | Neutrophil-to-lymphocyte ratio predicting suicide risk in euthymic patients with bipolar disorder: Moderatory effect of family history[END_REF]. NLR is the ratio between two different immune pathways reflecting the intensity of chronic stress. It may be more informative and perhaps less changed by unknown factors. PLR index is related to stress. The presence of stress activates the sympathetic nervous system, increases platelets, and induces endothelial permeability. When this permeability occurs, neutrophils and macrophages appear, generating peripheral inflammation [START_REF] Zheng | Elevated levels of monocytelymphocyte ratio and platelet-lymphocyte ratio in adolescents with non-suicidal selfinjury[END_REF]. Some studies suggest that PLR could be a better predictor than NLR for determining the severity of inflammation [START_REF] Kayhan | Cambridge University Press Accepted manuscript: Authors' Copy lymphocyte and platelet-lymphocyte ratios with the severity of major depression[END_REF][START_REF] Turkmen | Platelet-tolymphocyte ratio better predicts inflammation than neutrophil-to-lymphocyte ratio in endstage renal disease patients[END_REF]). An elevated level of MLR is associated with an overexpression of immunological genes that increases the production of cytokines related to monocytes and, as a consequence, activates microglia in the brain, causing neuroinflammation [START_REF] Zheng | Elevated levels of monocytelymphocyte ratio and platelet-lymphocyte ratio in adolescents with non-suicidal selfinjury[END_REF].

These indexes have been suggested as new indicators of low-grade inflammation and have been used as systemic inflammation prognostic scores in diseases such as cancer, coronary heart disease, and pancreatitis [START_REF] Isaac | Elevated neutrophil to lymphocyte ratio predicts mortality in medical inpatients with multiple chronic conditions[END_REF] and are also being investigated in neuropsychiatric disorders such as Alzheimer's disease, schizophrenia, bipolar disorder, and major depressive disorder (MDD) [START_REF] Özyurt | Increased neutrophil-lymphocyte ratios in depressive adolescents is correlated with the severity of depression[END_REF][START_REF] Mazza | Neutrophil/lymphocyte ratio and platelet/lymphocyte ratio in mood disorders: A metaanalysis[END_REF]. Recently, a meta-analysis [START_REF] Mazza | Neutrophil/lymphocyte ratio and platelet/lymphocyte ratio in mood disorders: A metaanalysis[END_REF] reported that inflammatory activation occurs in mood disorders and that NLR and PLR may be useful to detect this activation. NLR has been found to increase in depressed patients compared with healthy controls (HC) [START_REF] Kayhan | Cambridge University Press Accepted manuscript: Authors' Copy lymphocyte and platelet-lymphocyte ratios with the severity of major depression[END_REF][START_REF] Demir | Neutrophillymphocyte ratio in patients with major depressive disorder undergoing no pharmacological therapy[END_REF] and in depressed patients with SB. Moreover, studies have suggested that NLR may be a significant predictor of SB in MDD [START_REF] Velasco | Neutrophil-to-lymphocyte ratio: A potential new peripheral biomarker of suicidal behavior[END_REF][START_REF] Ekinci | The connections among suicidal behavior, lipid profile and low-grade inflammation in patients with major depressive disorder: a specific relationship with the neutrophil-to-lymphocyte ratio[END_REF] and could be more elevated in patients with recent Suicide Attempt (SA) [START_REF] Orum | Mean platelet volume and neutrophil to lymphocyte ratio as parameters to indicate the severity of suicide attempt[END_REF]. In parallel, increased PLR levels have also been associated with diagnosis and severity of depression [START_REF] Kayhan | Cambridge University Press Accepted manuscript: Authors' Copy lymphocyte and platelet-lymphocyte ratios with the severity of major depression[END_REF][START_REF] Sunbul | Increased Neutrophil/Lymphocyte Ratio in Patients with Depression is Correlated with the Severity of Depression and Cardiovascular Risk Factors[END_REF]. Finally, MLR was significantly higher in adolescents with SA than in HC [START_REF] Ucuz | Can suicide behavior and seasonality of suicide be predicted from inflammatory parameters in adolescents?[END_REF].

However, potential mechanisms underlying inflammatory processes in depression and suicidal behavior have yet to be fully elucidated. Biomarkers would provide more https://doi.org/10.1192/j.eurpsy.2023.18 Published online by Cambridge University Press personalized methods for their assessment and treatment and would help to enhance our understanding of suicidal pathophysiology and improve prevention [START_REF] Dickerson | The association between immune markers and recent suicide attempts in patients with serious mental illness: A pilot study[END_REF]. No previous reviews have examined NLR, PLR, and MLR in depressed patients with/without SA and Suicidal Ideation (SI) and HC. Therefore, we aimed to explore if there are significant differences in NLR, PLR, and MLR in i) depressed patients with or without a lifetime history of SA; ii) depressed patients with a lifetime history of SA vs healthy controls; and iii) depressed patients with SI before and after treatment.

Materials and methods

A systematic literature search was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement [START_REF] Page | The PRISMA 2020 statement: an updated guideline for reporting systematic reviews[END_REF]. The protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO) (CRD42022361238).

Search criteria

We systematically searched PubMed, Web of Science, and Scopus databases in September 2022. A single search strategy has been used for each of the databases: Studies of neutrophil-to-lymphocyte ratio were systematically searched using the terms "(NLR OR neutrophil-to-lymphocyte ratio OR neutrophil-to-lymphocyte index OR In the same way, studies of platelet-to-lymphocyte ratio were systematically searched using the terms "(PLR OR platelet-to-lymphocyte ratio OR platelet-to-lymphocyte index OR platelet-to-lymphocyte rate OR platelet to lymphocyte ratio OR platelet to lymphocyte index OR platelet to lymphocyte rate OR platelet lymphocyte ratio OR platelet lymphocyte index OR platelet lymphocyte rate OR platelet/lymphocyte ratio OR platelet/lymphocyte index OR platelet/lymphocyte rate) AND (depressive disorder OR depressive disorders OR depression OR mood disorder OR mood disorders OR major depression) AND (suicide OR suicidal behavior OR suicidal attempt OR suicidal thoughts OR self-mutilation)."

Finally, studies of monocyte-to-lymphocyte ratio were systematically searched using the terms "(MLR OR monocyte-to-lymphocyte ratio OR monocyte-to-lymphocyte index OR monocyte-to-lymphocyte rate OR monocyte to lymphocyte ratio OR monocyte to lymphocyte index OR monocyte to lymphocyte rate OR monocyte lymphocyte ratio OR monocyte lymphocyte index OR monocyte lymphocyte rate OR monocyte/lymphocyte ratio OR monocyte/lymphocyte index OR monocyte/lymphocyte rate) AND (depressive disorder OR depressive disorders OR depression OR mood disorder OR mood disorders OR major depression) AND (suicide OR suicidal behavior OR suicidal attempt OR suicidal thoughts OR self-mutilation)."

We reviewed titles and abstracts to select potentially relevant papers. After this screening process, we reviewed the full texts and checked the references in the included studies, metaanalyses, and systematic reviews to identify additional studies. Some data was extracted from previous meta-analyses and systematic reviews.

Eligibility criteria

Case-control studies and cross-sectional data from longitudinal studies that compared NLR, PLR, and/or MLR indexes among depressed patients with SB, depressed patients without SB, https://doi.org/10.1192/j.eurpsy.2023.18 Published online by Cambridge University Press and healthy controls were included, based on the following criteria: (i) patients with MDD according to standardized diagnostic criteria; (ii) measurement of NLR, PLR, and/or MLR in young people and adults; (iii) patients with current SI and history of SB. Only articles in English published in the last 5 years were included. Conference and meeting abstracts, metaanalyses, reviews, and pilot studies were excluded (see Supplementary Table ).

Data extraction and assessment of methodological quality

Data were extracted by two independent authors (A.V. and P.A.S.) and verified by the other two (J.R. and L.J.). Extracted data included author, year of publication, country, diagnosis, study population, sample size, age, ratios of females, depression and suicide scales, type of outcome (SI/SA), and type and quality of the study. Results were ordered according to indexes (Table 1). The methodological quality of the included studies was assessed using the Oxford criteria [START_REF] Howick | Explanation of the 2011 Oxford Centre for Evidence-Based Medicine (OCEBM) Levels of Evidence (Background Document)[END_REF]. Only medium-and high-quality papers were included in the final review.

Any disagreements between reviewers were resolved by discussion and consensus.

Results

Study selection and characteristics

A total of 86 studies were identified from electronic databases and, after removing duplicates, there were 37 single records to be screened. After reading titles and abstracts, we identified 21 full-text articles to be assessed for eligibility but excluded 10 studies after the full text was read (the inclusion and exclusion process is depicted in Figure 1). Of those, 11 studies met the inclusion and quality criteria and were selected for this review.

A total of 10 papers were rejected for the following reasons: (i) type of article (review, letter, meeting abstract): 2 articles were meeting abstracts and 2 articles were letters to the editor; (ii) 2 articles did not include a study of suicidal behavior, 2 articles did not specify depressed https://doi.org/10.1192/j.eurpsy.2023.18 Published online by Cambridge University Press patients, and 1 study included parameters of peripheral inflammation other than NLR, PLR, and MLR; and (iii)1 article was published in the Turkish language (see Supplementary Table ).

All included studies were published between August 2017 and September 2022. Five studies were conducted in Turkey [START_REF] Demirkol | Cambridge University Press Accepted manuscript: Authors' Copy and inflammatory markers[END_REF][START_REF] Ekinci | The connections among suicidal behavior, lipid profile and low-grade inflammation in patients with major depressive disorder: a specific relationship with the neutrophil-to-lymphocyte ratio[END_REF][START_REF] Meydaneri | Can Neutrophil Lymphocyte Ratio Predict the Likelihood of Suicide in Patients with Major Depression?[END_REF][START_REF] Yagci | Biochemical predictors in presentations to the emergency department after a suicide attemp[END_REF][START_REF] Önen | Blood Count Parameters as Inflammation Indicators in Children and Adolescents Diagnosed with Depressive Disorder[END_REF], one in India [START_REF] Adhikari | Neutrophil-lymphocyte ratio and c-reactive protein level in patients with major depressive disorder before and after pharmacotherapy[END_REF], two in Spain [START_REF] Velasco | Neutrophil-to-lymphocyte ratio: A potential new peripheral biomarker of suicidal behavior[END_REF][START_REF] Martínez-Botía | Sex-dependent grades of haematopoietic modulation in patients with major depressive episodes are associated with suicide attempts[END_REF], one in Sweden [START_REF] Grudet | Vitamin D and inflammation in major depressive disorder[END_REF], one in Israel [START_REF] Amitai | Neutrophil to-lymphocyte and platelet-to-lymphocyte ratios as biomarkers for suicidal behavior in children and adolescents with depression or anxiety treated with selective serotonin reuptake inhibitors[END_REF], and one in Thailand [START_REF] Puangsri | Potential usefulness of complete blood count parameters and inflammatory ratios as simple biomarkers of depression and suicide risk in drug-naive, adolescents with major depressive disorder: Simple peripheral markers for inflammation in drug-naive, adolescents with major depressive disorder[END_REF].

The 11 records included in the review yielded a total sample of 1,701 participants, of which 819 were patients with MDD and suicidal behavior (including current SI and lifetime SA), 494 were control patients (MDD without SB), and 388 were healthy controls. We included data from (i) six studies of NLR, PLR, and/or MLR in depressed patients with or without SB (4,20,27,31-33); (ii) six studies of NLR, PLR, and/or MLR in depressed patients with SB vs HC [START_REF] Puangsri | Potential usefulness of complete blood count parameters and inflammatory ratios as simple biomarkers of depression and suicide risk in drug-naive, adolescents with major depressive disorder: Simple peripheral markers for inflammation in drug-naive, adolescents with major depressive disorder[END_REF][START_REF] Ekinci | The connections among suicidal behavior, lipid profile and low-grade inflammation in patients with major depressive disorder: a specific relationship with the neutrophil-to-lymphocyte ratio[END_REF][START_REF] Yagci | Biochemical predictors in presentations to the emergency department after a suicide attemp[END_REF][START_REF] Önen | Blood Count Parameters as Inflammation Indicators in Children and Adolescents Diagnosed with Depressive Disorder[END_REF][START_REF] Martínez-Botía | Sex-dependent grades of haematopoietic modulation in patients with major depressive episodes are associated with suicide attempts[END_REF][START_REF] Grudet | Vitamin D and inflammation in major depressive disorder[END_REF]; (iii) two studies of NLR in depressed patients with SI before (with or without monotherapy 4 weeks prior) and after pharmacotherapy (2 to 12 weeks after treatment), one of which also explored PLR [START_REF] Demirkol | Cambridge University Press Accepted manuscript: Authors' Copy and inflammatory markers[END_REF][START_REF] Adhikari | Neutrophil-lymphocyte ratio and c-reactive protein level in patients with major depressive disorder before and after pharmacotherapy[END_REF], respectively (Table 1).

Patients with major depressive disorder with or without suicidal behavior

In three studies with patients with moderate to severe depression, NLR was reported to be higher in suicide attempters compared with depressed patients without a history of SA [START_REF] Velasco | Neutrophil-to-lymphocyte ratio: A potential new peripheral biomarker of suicidal behavior[END_REF][START_REF] Ekinci | The connections among suicidal behavior, lipid profile and low-grade inflammation in patients with major depressive disorder: a specific relationship with the neutrophil-to-lymphocyte ratio[END_REF][START_REF] Amitai | Neutrophil to-lymphocyte and platelet-to-lymphocyte ratios as biomarkers for suicidal behavior in children and adolescents with depression or anxiety treated with selective serotonin reuptake inhibitors[END_REF]. NLR could be potentially used as a biomarker to predict recent and past SA (4).

However, these results were not confirmed in other studies. First, in two studies there was a (non-significant) tendency towards an increase in NLR in patients with a history of SA vs without any [START_REF] Meydaneri | Can Neutrophil Lymphocyte Ratio Predict the Likelihood of Suicide in Patients with Major Depression?[END_REF][START_REF] Martínez-Botía | Sex-dependent grades of haematopoietic modulation in patients with major depressive episodes are associated with suicide attempts[END_REF]. One study examining the association between current SI and NLR in patients with MDD found no differences between patients with and without current SI in NLR [START_REF] Grudet | Vitamin D and inflammation in major depressive disorder[END_REF] Regarding the PLR index in patients with MDD, two studies reported PLR to be higher in patients with a history of SA vs those without any [START_REF] Velasco | Neutrophil-to-lymphocyte ratio: A potential new peripheral biomarker of suicidal behavior[END_REF][START_REF] Amitai | Neutrophil to-lymphocyte and platelet-to-lymphocyte ratios as biomarkers for suicidal behavior in children and adolescents with depression or anxiety treated with selective serotonin reuptake inhibitors[END_REF]. Conversely, this difference was not observed in three other studies [START_REF] Ekinci | The connections among suicidal behavior, lipid profile and low-grade inflammation in patients with major depressive disorder: a specific relationship with the neutrophil-to-lymphocyte ratio[END_REF][START_REF] Meydaneri | Can Neutrophil Lymphocyte Ratio Predict the Likelihood of Suicide in Patients with Major Depression?[END_REF][START_REF] Martínez-Botía | Sex-dependent grades of haematopoietic modulation in patients with major depressive episodes are associated with suicide attempts[END_REF] (Table 2).

Finally, only two studies explored the MLR index in relation to SB in MDD, with no reported statistically significant differences between depressed patients with and without SB (4,31) (Table 2)..

Patients with major depressive disorder and suicidal behavior vs healthy controls

In three studies, NLR was reported to be higher in depressed patients with a history of SA vs HC (9,20,28). However, three other studies reported no statistically significant differences (29,31,32) (Table 3).

Regarding the PLR index, two studies reported that PLR was higher in depressed patients (including SA, SI and nSI) vs HC [START_REF] Yagci | Biochemical predictors in presentations to the emergency department after a suicide attemp[END_REF][START_REF] Ucuz | Can suicide behavior and seasonality of suicide be predicted from inflammatory parameters in adolescents?[END_REF]. Conversely, in two studies, this difference was not observed (20,31) (Table 3). MLR was investigated in only two out of six studies, with inconsistent results. MLR was reported to be higher in depressed patients with SA vs HC (9). However, MLR was reported to be decreased in depressed patients with SB vs HC (31) (Table 3).

Depressed patients with suicidal ideation before and after treatment for depression

There were two studies that evaluated NLR before and after antidepressant treatment. None of the studies included MLR.

Discussion

SB is a leading cause of death and disability worldwide [START_REF] Klonsky | Suicide, Suicide Attempts, and Suicidal Ideation[END_REF]. Detecting and identifying potential biomarkers of peripheral inflammation in suicidal behavior has the potential to provide the knowledge needed to understand the pathophysiology of SB, develop personalized therapies, and improve prevention. To date, this is the first review that has examined NLR, PLR, and MLR in depressed patients with and without SA and current SI versus healthy controls. NLR, PLR, and MLR indexes emerge as relatively stable biomarkers of systemic inflammation [START_REF] Mazza | Anxiety and depression in COVID-19 survivors: Role of inflammatory and clinical predictors[END_REF], which, in turn, is cost-effective and easily accessible. Perhaps, for this reason, most of the studies included in this systematic review were conducted in low-income countries, which seem to be interested in this option given the potential value for clinical application.

https://doi.org/10.1192/j.eurpsy.2023.18 Published online by Cambridge University Press According to our review, in depressed patients, NLR was higher in patients with a history of SA, suggesting that, if confirmed in larger studies, it could be a biomarker of suicidal vulnerability in these patients. Although this result was not observed in all studies, in the majority there was a tendency for increased NLR. To the best of our knowledge, only one study that examined the differences in patients with and without SI did not find this association [START_REF] Grudet | Vitamin D and inflammation in major depressive disorder[END_REF]. However, it has been previously suggested that SA and SI are different phenomena with different explanations and predictors [START_REF] Galfalvy | A genome-wide association study of suicidal behavior[END_REF], and it seems that the relationship between the increase in NLR and suicidality occurs only in SA and not in SI [START_REF] Grudet | Vitamin D and inflammation in major depressive disorder[END_REF]. In addition, NLR was higher in depressed patients with SA vs HC. However, these differences were not observed in depressed patients with SI vs HC. Mounting evidence indicates that activation of the immune-inflammatory response is linked to the development and maintenance of depression and SB [START_REF] Velasco | Neutrophil-to-lymphocyte ratio: A potential new peripheral biomarker of suicidal behavior[END_REF][START_REF] Courtet | Neuroinflammation in suicide: Toward a comprehensive model[END_REF][START_REF] Mazza | Neutrophil/lymphocyte ratio and platelet/lymphocyte ratio in mood disorders: A metaanalysis[END_REF]. Some studies suggest that PLR could be a better predictor than NLR for determining the severity of inflammation [START_REF] Kayhan | Cambridge University Press Accepted manuscript: Authors' Copy lymphocyte and platelet-lymphocyte ratios with the severity of major depression[END_REF][START_REF] Turkmen | Platelet-tolymphocyte ratio better predicts inflammation than neutrophil-to-lymphocyte ratio in endstage renal disease patients[END_REF]. Our review shows inconsistent results regarding PLR in MDD patients (including SB). However, when compared with HC, the difference in the PLR index is observed more clearly in depressed patients. This phenomenon might be explained by the fact that platelets are one of the first cells to start an inflammatory cascade (cytokines, chemokines, the serotonin pathway), and patients with depression had a loss of equilibrium in hematopoietic production, resulting in an imbalance or distress in modulation [START_REF] Martínez-Botía | Sex-dependent grades of haematopoietic modulation in patients with major depressive episodes are associated with suicide attempts[END_REF].

The present review found no evidence for the link between MLR and SB in depressed patients, in contrast to some studies than showed MLR was higher in the manic episodes of bipolar disorder compared with euthymic states [START_REF] Özdin | A comparison of the neutrophil-lymphocyte, plateletlymphocyte and monocyte-lymphocyte ratios in schizophrenia and bipolar disorder patients -a retrospective file review[END_REF]. However, despite not being related to suicidal behavior, high MLR in young people appears to be associated with self-harm when compared with young people without this behavior [START_REF] Zheng | Elevated levels of monocytelymphocyte ratio and platelet-lymphocyte ratio in adolescents with non-suicidal selfinjury[END_REF].

https://doi.org/10.1192/j.eurpsy.2023.18 Published online by Cambridge University Press Finally, discrepant, and limited results were found regarding SI changes and inflammatory indexes following antidepressant treatment. These results may indicate that: (i) not all depressed patients show changes in inflammatory response [START_REF] Adhikari | Neutrophil-lymphocyte ratio and c-reactive protein level in patients with major depressive disorder before and after pharmacotherapy[END_REF]; (ii) the mechanisms underlying the relationship between inflammation and suicide are still unclear [START_REF] Russell | Evidence for an association between inflammatory markers and suicide: a cohort study based on 359,849 to 462,747 Taiwanese adults[END_REF]: and (iii) it has not been determined whether inflammation is a causative factor or a consequence of depression [START_REF] Özyurt | Increased neutrophil-lymphocyte ratios in depressive adolescents is correlated with the severity of depression[END_REF]. However, we also need to keep in mind that inflammatory response is influenced by multiple factors, such as body mass index, use of tobacco and other psychoactive substances, duration and severity of illness, resistance to antidepressant treatment, other psychiatric comorbidities, unbalanced diet, lack of exercise, and stress or traumatic life events in childhood or adulthood [START_REF] Adhikari | Neutrophil-lymphocyte ratio and c-reactive protein level in patients with major depressive disorder before and after pharmacotherapy[END_REF][START_REF] Martínez-Botía | Sex-dependent grades of haematopoietic modulation in patients with major depressive episodes are associated with suicide attempts[END_REF][START_REF] Del Giudice | Rethinking IL-6 and CRP: Why they are more than inflammatory biomarkers, and why it matters[END_REF].

Limitations

First, our results are mostly based on cross-sectional studies, and a causal relationship between NLR and SI and SB in patients with MDD cannot be inferred. Second, other inflammatory parameters were not assessed in the review, precluding us from concluding whether increased NLR is an independent biomarker or is related to other immune and inflammatory changes in depressed patients. Third, the studies included in the systematic review are very heterogeneous (i.e., sample characteristics, small sample sizes, recruitment, and assessment of depression and suicidality), and therefore results remain preliminary and cannot be generalized to any specific population. Finally, due to the small number and small scale of studies included, we cannot exclude publication and reporting bias in those studies, possibly biasing the results of the systematic review.

Conclusion

In conclusion, the present review found preliminary evidence for an association between NLR and SB in patients with MDD. Our results reinforce the idea that neuroinflammatory

  First, Demirkol et al. (2019) studied depressed patients (n = 74) with monotherapy four weeks before treatment and found a decrease in NLR during and after treatment with antidepressant therapy (AD) and bright light therapy (BLT), with a greater decrease compared with AD monotherapy [NLR mean (SD) = 2.31(1.05) pretreatment vs 2.25 (0.96) after treatment vs 1.9 (0.9) 2 weeks after treatment; p < 0.001]. In addition, HDRS scores and SI were also https://doi.org/10.1192/j.eurpsy.2023.18 Published online by Cambridge University Press significantly decreased after treatment [HDRS mean (SD) = 20.69 (4.21) pretreatment vs 16.69 (5.96) after treatment vs 15.14 (5.33) 2 weeks after treatment; p < 0.001; and SI median (Q1-Q3): 5 (3-10) pretreatment vs 5 (1-9) after treatment vs 3.5 (0-7) 2 weeks after treatment; p < 0.001] (8). However, in a sample of 50 depressed patients without antidepressant treatment the previous month, Adhikari et al. (2018) found a significant increase in NLR after 12 weeks of antidepressant treatment only in females [NLR mean in females (SD) = 2.55 (0.87) pretreatment vs 2.85 (0.89) 12 weeks after treatment; p < 0.001] (30). Regarding PLR, only one study examined PLR levels in depressed patients with SI before and after treatment and concluded there was no significant change in PLR during AD treatment [PLR mean (SD) = 118.61 (39.01) pretreatment vs 118.16 (40.57) after treatment vs 117.96 (40.82) 2 weeks after treatment; p = 0.985] (8).

  (Table2).

https://doi.org/10.1192/j.eurpsy.2023.18 Published online by Cambridge University Press

  PRISMA 2020 flow diagram for new systematic reviews that included database searches

		Identification of studies via databases
	Id en tifi	Records identified from databases (n = 86)	Records removed before
	ca		screening:
	tio		Duplicate records removed
	n		(n = 49)
	Sc
	re	Records screened
	en	(n = 37)
	in
	g
			Records excluded based on title
			and abstract
			(n = 16)
	Eli
	gi bil	Reports assessed for eligibility	Reports excluded (n = 10):
	ity	(n = 21)	Reason 1. Type of article (n = 4)
			Reason 2. Language (n = 1)
			Reason 3. Another outcome (n =
			5)
	In cl ud	Studies included in review (n = 11)
	ed

https://doi.org/10.1192/j.eurpsy.2023.18 Published online by Cambridge University Press

Figure 1. https://doi.org/10.1192/j.eurpsy.2023.18 Published online by Cambridge University Press

Table 2 . Neutrophil-to-lymphocyte ratio, Platelet-to-lymphocyte ratio, and Monocyte-to-lymphocyte ratio in Major Depressive Disorder with Suicidal Behavior versus non-Suicidal Behavior patients

 2 Major Depressive Disorder; MLR: Monocyte/lymphocyte ratio; NLR: Neutrophil/lymphocyte ratio; PLR: Platelet/lymphocyte ratio; SA: Suicide Attempt; SB: Suicidal Behavior; SI: Suicidal Ideation; SD: Standard Deviation.

	Author, date

https://doi.org/10.1192/j.eurpsy.2023.18 Published online by Cambridge University Press https://doi.org/10.1192/j.eurpsy.2023.18 Published online by Cambridge University Press

Table 3 . Neutrophil-to-lymphocyte ratio, Platelet-to-lymphocyte ratio, and Monocyte-to-lymphocyte ratio in Major Depressive Disorder with Suicidal Behavior versus Healthy Controls

 3

	Author, date

https://doi.org/10.1192/j.eurpsy.2023.18 Published online by Cambridge University Press

Funding source

This research was partially supported by the Government of the Principality of Asturias PCTI-2021-2023 IDI/2021/111, the Fundación para la Investigación e Innovación Biosanitaria del Principado de Asturias (FINBA), and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación. The funders had no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript. https://doi.org/10.1192/j.eurpsy.2023.18 Published online by Cambridge University Press Accepted manuscript: Authors' Copy

Accepted manuscript: Authors' Copy processes may be important in the pathophysiology of suicidal behavior in depressed patients.

NLR could be an attractive, convenient, and cost-effective trait marker of suicidal vulnerability in patients with MDD. Future large-scale replication studies are needed to confirm the observed associations and to examine the apparently understudied role of PLR and MLR in depressed patients in greater depth.

Acknowledgment

The authors wish to thank Sharon Grevet for her English assistance.

Conflict of interest

All authors declare no conflict of interest relative to this study.