George A Brusch Iv

Heulin Benoit

Dale F Denardo

Dehydration during egg production alters egg composition and yolk immune function

Keywords: Hydration, Immunocompetence, Innate immunity, Maternal-offspring conflict, Osmotic challenge

Parent-offspring conflicts occur when resources are limited for allocation, and, historically, energy has been the primary currency of focus when examining these trade-offs. Water is a fundamental resource that has received far less consideration for parent-offspring conflicts.

Previous research suggests that, when water is limited, reproductive females are compromised in favor of developing embryos. However, these studies limited their assessments to standard metrics such as clutch size and mass. We tested the hypothesis that the mother-offspring conflict over limited water resources leads to finer scale morphological and physiological impacts on the eggs in Children's pythons (Antaresia childreni). We predicted that water deprivation during gravidity alters female investment into her eggs, impacting egg water content and shell development. Additionally, we predicted that the yolk in these dehydrated eggs would have enhanced immune performance metrics, as has been documented in dehydrated adults. We found that eggs from water-deprived females were dehydrated as indicated by reduced percent water and greater yolk osmolality compared to eggs from females that received ad libitum water. We also found that eggs from dehydrated mothers had thinner shells and higher water loss rates. The impacts were not entirely negative as dehydrated eggs had higher antimicrobial capabilities. Also, thinner and more permeability eggshells might allow for elevated rates of rehydration from nest substrate. Overall, by examining an array of egg traits, we demonstrated that dehydration of gravid females impacts the eggs, not just the females as previously reported. As a result, the mother-offspring conflicts are indeed two-sided.

Introduction

Reproduction requires a considerable amount of resource investment. When resources are limited, this can lead to a resource trade-off between the parent and offspring [START_REF] Trivers | Parent-offspring conflict[END_REF].

Historically, the currency of interest in studies of parent-offspring conflicts has been energy [START_REF] Calow | The cost of reproductiona physiological approach[END_REF][START_REF] Doughty | Detecting life history trade-offs: Measuring energy stores in "capital" breeders reveals costs of reproduction[END_REF][START_REF] Reznick | Big houses, big cars, superfleas and the costs of reproduction[END_REF][START_REF] Kotiaho | Costs of sexual traits: A mismatch between theoretical considerations and empirical evidence[END_REF], and the outcomes determined by examining the post-reproductive female body condition [START_REF] Rutherford | Placental efficiency and intrauterine resource allocation strategies in the common marmoset pregnancy[END_REF]Tadif, 2008, Fowden and[START_REF] Fowden | Maternal-fetal resource allocation: Co-operation and conflict[END_REF] along with the quantity and quality of her resulting young [START_REF] Denardo | Are females maternal manipulators, selfish mothers, or both? Insight from pythons[END_REF][START_REF] Schwarzkopf | Selfish mothers" use "maternal manipulation" to maximize lifetime reproductive success[END_REF][START_REF] Shine | Manipulative mothers and selective forces: The effects of reproduction on thermoregulation in reptiles[END_REF]. However, such approaches ignore other currencies as well as more cryptic developmental metrics that might be influenced by such conflicts. In oviparous species, the egg provides the early developmental environment for the embryo, which can have profound impacts on fitness [START_REF] Lindström | Early development and fitness in birds and mammals[END_REF], Monaghan, 2008). Therefore, consideration of egg metrics can provide a more complete understanding of the repercussions of parent-offspring conflicts.

Water is a fundamental resource that has received far less consideration as a potential currency to mediate maternal-offspring trade-offs. The impacts of maternal hydration have been explored after gestation (Hanson et al., 1994) and as a potential proximate cue for reproductive timing [START_REF] Nelson | Simulated drought influences reproduction in male prairie voles[END_REF], Gesquiere et al., 2008[START_REF] Bukovetzky | Reproductive and metabolic responses of desert adapted common spiny male mice (Acomys cahirinus) to vasopressin treatment[END_REF]. However, there is far less known about the direct consequences of maternal hydration during gestation. Recent studies show that, when water is limited, reproductive female snakes face significant increases in plasma osmolality [START_REF] Brusch | When less means more: Dehydration improves innate immunity in rattlesnakes[END_REF][START_REF] Dupoué | Water restriction causes an intergenerational trade-off and delayed mother-offspring conflict in a viviparous lizard[END_REF] and may catabolize greater amounts of muscle mass relative to fat in order to free up bound water [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF]. Loss of muscle mass not surprisingly has been associated with reduced performance by the postreproductive female [START_REF] Lourdais | Structural and performance costs of reproduction in a pure capital breeder, the Children's Python Antaresia childreni[END_REF]. As a result, it is assumed that the water aspects of

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A N U S C R I P T the parent-offspring conflict are often compromised in favor of the developing embryos [START_REF] Dupoué | Intergenerational trade-off for water may induce a mother-offspring conflict in favour of embryos in a viviparous snake[END_REF]. However, maternal hyperosmolality can also enhance maternal immune performance [START_REF] Brusch | When less means more: Dehydration improves innate immunity in rattlesnakes[END_REF] and reduce egg size [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF], indicating a much more complex relationship between the mother and her offspring. This newly appreciated complexity deserves further study, especially considering that past studies have relied on relatively broad, easily assessable metrics of reproductive output to examine potential conflicts for hydric resources (e.g., egg mass or volume, [START_REF] Dupoué | Intergenerational trade-off for water may induce a mother-offspring conflict in favour of embryos in a viviparous snake[END_REF][START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF]. More detailed examinations of egg quality may reveal relatively cryptic impacts of the parent-offspring conflict over water resources. Accordingly, we examined the impact of maternal dehydration on reproductive output in the Children's python (Antaresia childreni), which is a capital-breeding, oviparous squamate that has proven to be a valuable study system for examining parent-embryo interactions (e.g., Stahlschmidt and DeNardo, 2008, 2009a[START_REF] Stahlschmidt | The role of python eggshell permeability dynamics in a respiration-hydration trade-off[END_REF][START_REF] Lorioux | Maternal influences on early development: Preferred temperature prior to oviposition hastens embryogenesis and enhances offspring traits in the Children's python, Antaresia childreni[END_REF][START_REF] Lourdais | Structural and performance costs of reproduction in a pure capital breeder, the Children's Python Antaresia childreni[END_REF].

Regarding water limitations, female Children's pythons deprived of water during gravidity are hyperosmotic, experience greater muscle catabolism, and produce lighter eggs [START_REF] Brusch | When less means more: Dehydration improves innate immunity in rattlesnakes[END_REF][START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF]. We furthered our understanding of water-based parent-offspring trade-offs by testing the hypothesis that the mother-offspring conflict over limited water resources leads to morphological and physiological impacts on the eggs. We predicted that water deprivation during gravidity alters female investment into her eggs and thus impacts egg water content and shell development, two critical investments into the egg that occur in the oviduct. Furthermore, we predicted that the yolk in these dehydrated eggs would have enhanced immune performance metrics, as has been documented in dehydrated adults [START_REF] Brusch | When less means more: Dehydration improves innate immunity in rattlesnakes[END_REF] and dehydrated latestage embryos [START_REF] Brusch | Egg desiccation leads to dehydration and enhances innate immunity in python embryos[END_REF].

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Material and methods

Study Species

Children's pythons (Antaresia childreni) inhabit the wet-dry tropics of northern Australia, where eggs are laid towards the end of the dry season so that offspring hatch at the start of the wet season [START_REF] Wilson | A Complete Guide to Reptiles of Australia[END_REF]. Thus, reproductive females naturally face dramatic challenges to water balance during the period when egg development occurs [START_REF] Brusch | When less means more: Dehydration improves innate immunity in rattlesnakes[END_REF].

Additionally, female Children's pythons brood their eggs and, in doing so, the eggs have minimal contact with substrate for water absorption but also have reduced evaporative water loss (Stahlschmidt et al., 2008).

Experimental Design

All work was conducted under the oversight of the Arizona State University Institutional Animal Care and Use Committee (protocol # 17-1532R). Snakes used for this study were part of a longterm colony at Arizona State University, AZ, USA, and were housed individually in 91 x 71 x 46 cm cages (Freedom Breeder, Turlock, CA, USA). Snakes were deprived of food from the onset of overwintering until oviposition (~5 months) since they typically will not eat when reproductively active. To stimulate the reproductive cycle, snakes were over-wintered for six weeks (mid-December through January) by providing them a light-dark cycle of 6:18 h. During the dark cycle, room temperature was 15 °C, while during the light cycle room temperature was 20 °C and supplemental sub-surface heat was provided under one end of each cage to allow for thermoregulation. After overwintering, room temperature was increased to a constant 31.5 °C,

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which is the preferred temperature of gravid Children's pythons [START_REF] Lourdais | Thermoregulation during gravidity in the children's python (Antaresia childreni): A test of the preadaptation hypothesis for maternal ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T thermophily in snakes[END_REF], with a 12L:12D light cycle (see [START_REF] Brusch | When less means more: Dehydration improves innate immunity in rattlesnakes[END_REF] for breeding protocol). Males were rotated through the female cages so that all females were with multiple males with each visit separated by 2-3 days. Once females underwent their peri-ovulatory shed (which approximates the time of ovulation; [START_REF] Lourdais | Thermoregulation during gravidity in the children's python (Antaresia childreni): A test of the preadaptation hypothesis for maternal ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T thermophily in snakes[END_REF], each snake was alternately assigned to one of two maternal treatment groups of similar mean body mass: water (number of reproductive females and mean mass ± SEM: n = 9, 542±33 g) or no water (n = 10, 507±27 g) provided for the duration of gravidity (i.e., ovulation to oviposition; 22±1 d). Deprivation of water during this time leads to ecologically relevant levels of dehydration (control females: 297±13 mOsm kg -1 , deprived females: 354±12 mOsm kg -1 ; Brusch et al., 2018).

At oviposition of the fertile eggs, clutch size (number of eggs) and clutch mass were measured, and the clutch was moved to a 1.0 L plastic container filled with ~0.5 L of moistened perlite substrate (1:3 by weight) and stored in an environmental chamber at 31.5 ºC.

Concurrently, an egg from each clutch was placed on a scale at room temperature (~25 ºC) and weighed (±0.01 g) every 10 min for 1 h before being returned to the clutch. A separate egg was selected and weighed, after which a 1 cm 2 eggshell section was removed and stored in 95% ethanol. These cross-sections were air-dried at room temperature and mounted with double-sided transparent tape onto brass stubs. They were then coated with gold-palladium using a sputtering fine coater (JEOL JFC 1000) for structural analyses using scanning electron microscopy ([SEM], JEOL JSM 6301F SEM. Photographs (x600) of cross sections from each eggshell were used to take serial measurements (n = 20 for each eggshell) of the fibrous layer, calcareous crust, and total thickness (fibrous + calcareous). From this same egg, a 1 mL yolk sample was collected (see details below) and the remaining egg contents were moved to a pre-weighed, flat aluminum

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dish, re-weighed, and placed in an isothermal chamber at 60 ºC. The mass of the dish and egg contents were re-weighed every 24 h until mass stabilized. Percent water of the egg contents were calculated as: % water = 100 x [(initial mass of dish with egg contents) -(final mass of dish with egg contents) (initial mass of dish with egg contents) -(mass of dish)]

Yolk Preparation

Using slight modifications to the water dilution methods described in [START_REF] Akita | Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain[END_REF] and [START_REF] Kim | Immunoglobulin separation from egg yolk: a serial filtration system[END_REF], yolk plasma proteins were separated from the granules and lipids. Briefly, 1 ml of egg yolk was diluted with 9 mL Nanopure water (pH of 5, acidified with 0.1 N HCl).

Samples were then kept at 4 ºC for 2 h, after which they were centrifuged at 10,000 g for 2 h at 4 ºC. Aliquots of the fluid (50 µL) were separated into vials and frozen at -80 °C to be used within 30 d to measure osmolality and evaluate immune function. Yolk osmolality was determined using a vapor pressure osmometer (±3 mOsm kg -1 ; model 5600; Wescor Inc., Logan, Utah, USA) with samples run in triplicate as described in [START_REF] Brusch | When less means more: Dehydration improves innate immunity in rattlesnakes[END_REF].

Immune Assays

To examine immunocompetence, we performed several plasma-based innate immune function assays. To evaluate the involvement of complement (C') and natural antibodies (NAbs) in reacting to a novel, eukaryotic antigen, we used sheep red blood cells (sRBC, SBH050, Hemostat Laboratories, Dixon, CA, USA) to quantify agglutination and lysis, which are standard measures of soluble constitutive immunity [START_REF] Matson | A hemolysis-hemagglutination assay for characterizing constitutive innate humoral immunity in wild and domestic birds[END_REF]. Briefly, 20 µL of egg yolk plasma were

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serially diluted with phosphate buffered saline (PBS) along a row of a 96-well plate, after which 20 µL of 1% sRBC were added to each well. Plates were incubated at 31.5 ºC for 90 min and then placed at room temperature (~25 °C) for 20 min after which point they were scanned at 600 dots per inch (Hewlett-Packard Co., ScanJet 3670) for agglutination images. After an additional 70 min, plates were centrifuged for 5 min (500 rpm, Sorvall, Newtown, CT, USA) and the supernatant was dispensed into a clean 96-well plate. Absorbance values were measured (405 nm, Bio-Rad, Hercules, CA, USA) to calculate lysis scores. Hemolytic-complement activity was expressed in CH 50 units mL plasma -1 , where 1 CH 50 unit equals the reciprocal of the dilution of plasma required to lyse 50% of the sRBC.

Bactericidal activity was also assessed to determine the ability of egg yolk plasma proteins to kill a prokaryotic microorganism (French and Neuman-Lee, 2012). We used two species of gram-negative bacteria, Escherichia coli and Salmonella enterica. In brief, we combined 1:1 egg yolk dilution with CO 2 -independent media plus 4 nm L-glutamine, agar broth, and either 10 5 colony-producing units of E. coli (Lot#483-478-1, ATCC 8739, MicroBioLogics, St. Cloud, MN, USA) or 10 4 colony-producing units of S. enterica (Lot#501-13-1, ATCC 51741, MicroBioLogics, St. Cloud, MN, USA) on a 96-well microplate. Absorbance values were measured (300 nm, Bio-Rad, Hercules, CA, USA) immediately and again after 12 h of incubation at 37 ºC. Bactericidal ability percentages were calculated as one minus the mean absorbance for each sample, run in triplicate, divided by the mean absorbance for the positive control, and then multiplied by 100.

Statistical Analysis

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We used linear mixed-effect models to examine the effect of maternal treatment (water or no water) on egg (mass, percent water, and mass loss after an hour) and yolk (osmolality, agglutination and lysis scores, and bacterial killing ability of E. coli and S. enterica) metrics. We used maternal treatment as a fixed effect and maternal plasma osmolality nested within maternal ID as a random effect. Within maternal ID, we included parameters to address potential size effects (using a body condition indexstandardized residuals from a linear regression using mass and SVL). To test the effect of maternal treatment on eggshell thickness measurements (calcareous, fibrous, and total) we used a repeated measures analysis of variance (rmANOVA) with maternal treatment and measurement (to account for variation in thickness measurements) as fixed effects, and individual egg as a random effect. We tested for compound symmetry to ensure linearity of repeated thickness measurements within eggshells and a homogenous relationship between samples. We checked to ensure the data met the assumptions for parametric testing and used transformations where necessary. We performed all statistical analyses in R, version 3.3.2 (R Development Core Team 2016) with the packages lme4 [START_REF] Bates | Fitting linear mixed-effects models using lme4[END_REF] and car [START_REF] Fox | An {R} Companion to Applied Regression[END_REF]. Significance was set at α = 0.05.

Results

When comparing the masses of the eggs randomly selected to be sacrificed for yolk and eggshell samples we found that eggs from water-deprived females were significantly lighter that those from females with water (F 1,17 = 5.35, p = 0.034; Fig. 1A). The eggs from the water-deprived females also had significantly lower percent water (F 1,17 = 4.65, p = 0.041; Fig 1B) and higher yolk osmolality (F 1,17 = 8.59, p = 0.001; Fig. 1C) compared to eggs from the water-provided females. Additionally, eggs from water-deprived females had higher percentages of E. coli (F 1,17

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A N U S C R I P T = 7.77, p = 0.010; Fig. 2A) and S. enterica (F 1,17 = 5.31, p = 0.016; Fig. 2B) killed. However, we did not detect any significant differences between treatment groups for agglutination (F 1,17 = 0.27, p = 0.612; Fig. 2C) and lysis (F 1,17 = 0.01, p = 0.900) values (Fig. 2D). Eggs from water-deprived females had greater mass loss after 1 h in a dehydrating lab environment (F 1,17 = 4.62, p = 0.046; Fig. 3). Additionally, eggshells from water-deprived females had thinner fibrous layers (F 1,17 = 4.88, p = 0.044) and thinner total thickness (F 1,17 = 4.44, p = 0.047; Fig. 4), but there was no significant treatment-based difference in calcareous

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A N U S C R I P T crust thickness (F 1,17 = 0.01, p = 0.909; Table 1). We did not detect any significant interactions between mass loss under dehydrating conditions and eggshell thickness measurements (all p > 0.05).

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Discussion

When considering the relative physiological costs of reproduction, there is frequently an emphasis on energy balance and how limited energetic resources can create maternal-offspring conflicts and life-history trade-offs [START_REF] Stearns | The Evolution of Life Histories[END_REF][START_REF] Walker | The trade-off between number and size of offspring in humans and other primates[END_REF][START_REF] Bleu | Experimental litter size reduction reveals costs of gestation and delayed effects on offspring in a viviparous lizard[END_REF]. In these energetic contexts, limited intake or finite internal reserves results in associated costs to mothers (e.g., reduced self-maintenance, [START_REF] Lochmiller | Trade-offs in evolutionary immunology: Just what is the cost of immunity?[END_REF]Deerenberg, 2000, Martin et al., 2008) or offspring (e.g., reduced size, [START_REF] Stearns | Trade-offs in life-history evolution[END_REF][START_REF] Reznick | Big houses, big cars, superfleas and the costs of reproduction[END_REF]. While energy is clearly an important resource for reproductive investment, our results provide the first evidence that water balance also poses maternal-offspring conflicts in oviparous vertebrates. Additionally, our study emphasizes the importance of including effects that are more cryptic than the number of eggs and egg mass when exploring consequences of mother-offspring conflicts.

We found that, similar to previous findings [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF], maternal dehydration (i.e., hyperosmolality), caused by water restriction during gravidity, led to the production of lighter eggs (Fig. 1A). As the energy content of the eggs was determined during vitellogenesis, which occurred prior to our water manipulation, the lighter mass of the eggs produced by waterdeprived females is likely attributable to reduced water investment during gravidity, a time when females typically invest considerable amounts of water into their eggs (Stahlschmidt et al., 2011a). This presumption is supported by the findings that eggs from water-deprived females had less percent water and higher yolk osmolality compared to eggs from females that received ad libitum water throughout reproduction (Fig. 1B-C).

Previous studies examining trade-offs between maternal and embryonic hydration during water deprivation concluded that maternal hydration is compromised in favor of embryonic hydration [START_REF] Dupoué | Intergenerational trade-off for water may induce a mother-offspring conflict in favour of embryos in a viviparous snake[END_REF][START_REF] Sparkman | Use of field-portable A C C E P T E D M A N U S C R I P T ultrasonography reveals differences in developmental phenology and maternal egg provisioning in two sympatric viviparous snakes[END_REF]. However, these studies relied on egg

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volume and mass as their lone egg metrics. Our study, which used additional, more cryptic egg quality metrics (e.g., percent water and yolk osmolality) suggests that the effects of maternal dehydration during gravidity are also present in the oviposited eggs. These previously undetected effects add to a growing body of knowledge regarding water flux from mothers to their developing offspring throughout gravidity [START_REF] Brown | Beyond size-number trade-offs: Clutch size as a maternal effect[END_REF], Dupoué et al., 2015, Lourdais et al., 2015[START_REF] Bonnet | The evolutionary economics of embryonic-sac fluids in squamate reptiles[END_REF].

While water balance is critical for embryonic survival (Cagle et al., 1993, Warner and[START_REF] Warner | Nest-site selection in relation to temperature and moisture by the lizard Sceloporus undulatus[END_REF], water imbalance is not purely detrimental as dehydration-based immune enhancement has been documented in multiple taxa (insects, Hoang, 2001;lizards, Moeller et al., 2013;snakes, Brusch and DeNardo, 2017) including adult and embryonic Children's pythons [START_REF] Brusch | When less means more: Dehydration improves innate immunity in rattlesnakes[END_REF][START_REF] Brusch | Egg desiccation leads to dehydration and enhances innate immunity in python embryos[END_REF]. This suggests that there is a complex interplay between osmolality and physiological functions essential for survival. Our results expand the newly appreciated relationship between hydric state and immune function to include freshly oviposited eggs as another life-stage that exhibits a similar pattern. We found that dehydrated eggs had greater antimicrobial capabilities compared to hydrated eggs (Fig. 2A-B). Microbial infection of eggs can drastically reduce clutch viability and, once pathogens pass through the membrane and infect the developing embryo, there is typically a low chance of survival [START_REF] Kiesecker | Influences of egg laying behavior on pathogenic infection of amphibian eggs[END_REF][START_REF] Adamo | Evidence for adaptive changes in egg laying in crickets exposed to bacteria and parasites[END_REF][START_REF] Cook | Trans-shell infection by pathogenic micro-organisms reduces the shelf life of non-incubated bird[END_REF][START_REF] Brandl | Composition of bacterial assemblages in different components of reed warbler nests and a possible role of egg incubation in pathogen regulation[END_REF]. A robust antimicrobial defense is therefore fundamental to the health of developing embryos [START_REF] Shawkey | Do birds differentially distribute antimicrobial proteins within clutches of eggs?[END_REF], Horrocks et al., 2014). Python embryos at oviposition are approximately one-third through development [START_REF] Lourdais | Thermoregulation during gravidity in the children's python (Antaresia childreni): A test of the preadaptation hypothesis for maternal ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T thermophily in snakes[END_REF] and have fully functional livers, which is primary source of synthesis for antimicrobial peptides [START_REF] Van Hoek | Antimicrobial peptides in reptiles[END_REF]. Thus, it is uncertain as to whether the peptides responsible for the bacterial killing ability of the egg are products of the et al., 2003[START_REF] Lourdais | Maternal brooding in the children's python (Antaresia childreni) promotes egg water balance[END_REF], this may be offset by increased ability to ward off potentially lethal pathogens. To better understand the benefits of an enhanced immune system when dehydrated, future studies should examine survival rates between dehydrated and hydrated eggs experimentally exposed to microorganisms, as well as the source of the antimicrobial agents.

Additionally, the cellular mechanisms responsible for dehydration-based innate immune enhancement at any life stage remain unknown and are in need of further study.

In contrast to the positive effects of dehydration on egg bacterial killing ability, agglutination and lysis were not impacted by dehydration (Fig. 2C-D). These results are in accordance with previous findings that found embryonic dehydration later in development (just prior to hatching), resulted in increased bactericidal ability but not enhanced agglutination and lysis [START_REF] Brusch | Egg desiccation leads to dehydration and enhances innate immunity in python embryos[END_REF]. After recognition of foreign pathogens, agglutination is often the next step in an immune response, whereby cells and microorganisms are clumped together (typically in the presence of complement and/or antibodies) and destroyed via phagocytosis or direct membrane disruption (i.e., lysis, [START_REF] Medzhitov | Innate immune recognition: Mechanisms and pathways[END_REF]. Adult Children's pythons have elevated agglutination and lytic abilities when dehydrated [START_REF] Brusch | When less means more: Dehydration improves innate immunity in rattlesnakes[END_REF], which suggests that complement and antibodies are neither invested into eggs by the female nor effectively produced by the embryos. As it is often presumed that maternal antibodies are passed to the offspring through vertical transmission to prime the immunological defenses of their naive offspring (Grindstaff et al., 2003, Boulinier and[START_REF] Boulinier | Maternal transfer of antibodies: raising immuno-ecology issues[END_REF], future studies should focus on the ontogeny of humoral immunity to help understand the discrepancies between adult and embryonic immune responses to dehydration.

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A potential explanation for our immune results is that dehydration leads to a concentration of innate immune molecules. Previous work to address this possibility showed that diluting plasma samples from dehydrated squamate reptiles to adjust for the decreased volume, still resulted in higher immune metrics compared to plasma samples from hydrated animals [START_REF] Brusch | When less means more: Dehydration improves innate immunity in rattlesnakes[END_REF][START_REF] Moeller | The effect of hydration state and energy balance on innate immunity of a desert reptile[END_REF]. This suggests that dehydration leads to an upregulation (either in total number or activity) of innate molecules. Although we did not conduct a similar experiment for our study, it is reasonable to suspect a similar upregulation occurs in dehydrated eggs.

We also found that the eggs from dehydrated mothers had higher water loss rates (Fig. 3, which may be a result of their thinner shells, specifically thinner fibrous layers (Fig. 4; Table 1).

Our results are consistent with previous research that found that the calcareous crust of Children's python eggs does not change from oviposition to hatching, while the fibrous layer gradually thins (Stahlschmidt et al., 2010). This change is believed to support the increased oxygen demands of the growing embryos whose metabolism dramatically increases just prior to hatching. This shell thinning, however, comes with the cost of higher water loss rates (Stahlschmidt et al., 2010). For our study, eggs from dehydrated mothers started with less water and had the potential to lose water faster. However, trans-shell water movement has a bidirectional potential. Eggs with thinner shells may lose water faster but they may also be able to absorb environmental water faster as has been previously documented in other oviparous species [START_REF] Tracy | Interrelations among water and energy relations of reptilian eggs, embryos, and hatchlings[END_REF], Ji and Braña, 1999[START_REF] Verdú-Ricoy | Phenotypic responses to incubation conditions in ecologically distinct populations of a lacertid lizard: A tale of two phylogeographic lineages[END_REF]. Nest site selection is extremely important for Children's pythons (Stahlschmidt et al., 2011b) and dehydrated mothers, who cannot allocate enough water to their eggs without jeopardizing their own hydric needs, may select moister nest sites so that their eggs could absorb environmental moisture.

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Alternatively, thinner eggs shells may simply reflect a reduced ability of dehydrated female to mobilize and deposit egg shell components during gravidity.

Our results provide the first evidence of a water-based mother-offspring intergenerational trade-off in an oviparous vertebrate. While it was previously assumed that dehydrated mothers were burdened with the majority of the water imbalance in the form of increased plasma osmolality [START_REF] Dupoué | Intergenerational trade-off for water may induce a mother-offspring conflict in favour of embryos in a viviparous snake[END_REF][START_REF] Dupoué | Water restriction causes an intergenerational trade-off and delayed mother-offspring conflict in a viviparous lizard[END_REF] and increased muscle catabolism [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF], our exploration of cryptic traits found that eggs are also significantly impacted. It is not clear, however, if these impacts are entirely negative, as dehydrated eggs had higher antimicrobial capabilities and thinner shells which might allow for elevated rates of rehydration. We suspect that these results are not limited to squamates, but may be applicable to many other oviparous organisms. By and large, the vast majority of previous research has focused on the importance of energy allocation during reproduction (Gittleman and Thompson, 1988[START_REF] Lambert | Energetic consequences of reproduction in Atlantic cod (Gadus morhua) in relation to spawning level of somatic energy reserves[END_REF][START_REF] Dugas | Both sexes pay a cost of reproduction in a frog with biparental care[END_REF]. Water is an essential resource that has received far less consideration, and we strongly encourage future work to focus on the role hydration plays on both reproductive mothers and their offspring using a variety of metrics that may help elucidate underappreciated trade-offs.

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Fig. 1 .

 1 Fig. 1. Average (A, egg mass; B, percent water; C, yolk osmolality [mOsm kg -1 ]) measured in

Fig. 2 .

 2 Fig. 2. Average yolk immune scores (A, Escherichia coli bacterial-killing; B, Salmonella

Fig. 3 .

 3 Fig. 3. Average egg mass loss after 1 h in desiccating conditions measured in Children's python

  embryo. Regardless, while reduced water content can impact egg survival (Aubret

  . R. Soc. B Biol.Sci. 364, 1097-1106. https://doi.org/10.1098/rstb.2008.0247 Brusch, G.A., Billy, G., Blattman, J.N., Denardo, D.F., 2017. Reproduction alters hydration state but does not impact the positive effects of dehydration on innate immune function in children's pythons (Antaresia childreni). Physiol.Biochem. Zool. 90,[646][647][648][649][650][651][652][653][654]

Figure 2 Figure 3

 23 Figure2

Figure 4

 4 Figure4

Table 1 .

 1 Eggshell component thickness measurements of Children's python (Antaresia childreni)eggs at oviposition from females with or without water throughout gravidity (mean ± SEM).

	Maternal	Calcareous	Fibrous	Total
	treatment	crust (µm)	layer (µm) *	thickness
				(µm) *
	Water	6.8 ± 0.2	94.8 ± 3.3	101.6 ± 3.6
	No water 6.5 ± 0.7	85.2 ± 2.8	91.8 ± 3.5

* Significantly different between treatment groups

Acknowledgments

This work would not have been possible without assistance from members of the DeNardo lab who helped with breeding colony management as well as Department of Animal Care and Technologies staff who assisted with animal care. We also wish to thank Dr. O. Lourdais for his support of the project and Loic Joanny, engineer of CMBA in the University of Rennes, for his help in getting SEM photos.

Funding

This material is based upon work supported by the National Science Foundation Graduate Research Fellowship (Grant # 1311230), the Peabody Family Memorial Award, and the Graduate and Professional Students Association JumpStart grant at Arizona State University for GABIV. The SEM analysis was funded by CNRS UMR 6553.

Data availability

The datasets supporting this article can be accessed at https://doi.org/10.6084/m9.figshare.6846845.v1

Competing interests

No competing interests declared.

Author contributions

GABIV and DFD designed the study and collected the samples. BH performed the SEM analyses. GABIV conducted all assays, performed the statistical analyses, and led the writing of the manuscript. DD and BH contributed to revisions and gave final approval for publication. Hanson, L., Hahn-Zoric, M., Berndes, M., Ashraf, R., Herias, V., Jalil, F., Bhutta, T.I., Laeeq, A., Mattsby-Baltzer, I., 1994. Breast feeding: Overview and breast milk immunology.

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Pediatr. Int. 36, 557-561. https://doi.org/10.1111/j. 1442-200X.1994.tb03246.x Hoang, A., 2001. Immune response to parasitism reduces resistance of Drosophila melanogaster to desiccation and starvation. Evolution (N. Y). 55, 2353-2358. https://doi.org/10.1111/j. 0014-3820.2001.tb00748.x Horrocks, N.P.C., Hine, K., Hegemann, A., Ndithia, H.K., Shobrak, M., Ostrowski, S., Williams, J.B., Matson, K.D., Tieleman, B.I., 2014. Are antimicrobial defences in bird eggs related to climatic conditions associated with risk of trans-shell microbial infection? Front. Zool. 11, 49. https://doi.org/10.1186/1742-9994-11-49 Ji, X., Braña, F., 1999. The influence of thermal and hydric environments on embryonic use of energy and nutrients, and hatchling traits, in the wall lizards (Podarcis muralis). Comp.


Sébastien Bonthoux
email: sébastienbonthoux@insa-cvl.fr

Simon Chollet

Ianis Balat

Nicolas Legay

Lolita Voisin

Improving nature experience in cities: what are people's preferences for vegetated streets ?

In the current context of strong urban sprawl, it becomes urgent to find urban approaches that simultaneously promote ecological functions and relationships between people and nature in cities. Streets are omnipresent urban elements that can deliver ecosystem services and facilitate people daily interactions with nature. Promoting vegetation in streets can take different forms which have to be combined with people's preferences. Based on photomontages, we assessed people's perceptions and valuations for herbaceous vegetation types associated to various managements and designs of pavements. Using a combination of a local field survey and a French national online survey, we collected a total of 3609 responses representing a large diversity of socio-demographic characteristics. The results of the field survey confirmed those of the online survey. Although there was variability among people valuations, we found that lowly managed pavements with spontaneous vegetation were in average higher valued than highly managed pavements without vegetation. Pavements with spontaneous vegetation were perceived as less kept than pavements without vegetation, but more beautiful and less boring. We found a consensus of high valuations towards pavements containing vegetation integrated in small design interventions (flowers seeded in foot of wall, design of a meadow strip along the pavement), suggesting that people generally accept vegetation with visible signs of human actions or managements. Socio-demographic characteristics partly explained variabilities in photo valuations. As expected, people frequently connected with nature had the highest preferences for vegetated pavements, spontaneous or integrated in designs. These results show that vegetated streets can become daily biodiversity-friendly urban greenspaces appreciated by urban dwellers. We provide recommendations for promoting vegetation in streets that will be useful for politics, urban designers and managers.

Introduction

The current strong urban sprawl causes profound changes in ecological habitats and associated biodiversity [START_REF] Grimm | Global change and the ecology of cities[END_REF]. However, it is now recognized that nature experience is required for improving urban dweller health and well-being (Botzat et al., 2016;Cox et al., 2017b) and that it can change people attitudes towards pro-environmental behaviors (Soga and Gaston, 2016). In this context, it is necessary that researchers, designers and managers propose urban approaches that simultaneously promote ecological functions and relationships between people and nature [START_REF] Aronson | Biodiversity in the city: key challenges for urban green space management[END_REF][START_REF] Gaston | REVIEW: Managing urban ecosystems for goods and services[END_REF]Soga and Gaston, 2016).

Nature in cities can be promoted at various scales in multiple public or private spaces [START_REF] Aronson | Biodiversity in the city: key challenges for urban green space management[END_REF][START_REF] Beninde | Biodiversity in cities needs space: A meta -analysis of f actors determining intra -urban biodivers -592[END_REF]. Land sparing and land sharing have been proposed as two spatial approaches located at both opposite ends of a continuum of nature conservation strategies [START_REF] Lin | Sharing or sparing? How should we grow the world's cities?[END_REF]. Land sparing which consists in introducing large green spaces (e.g. parks) within a compact urban matrix has been shown an adapted strategy for hosting some large animals and uncommon plant species [START_REF] Caryl | Functional responses of insectivorous bats to increasing housing density support 'land-sparing'rather than 'landsharing'urban growth strategies[END_REF][START_REF] Kendal | The importance of small urban reserves for plant conservation[END_REF]Villaseñor et al., 2017) and to develop various people uses including walking, resting or jogging [START_REF] Palliwoda | Human-biodiversity interactions in urban parks: The species level matters[END_REF]. However, this approach induces a travel distance between housing and parks which can be a barrier to frequent people use [START_REF] Soga | Reducing the extinction of experience: association between urban form and recreational use of public greenspace[END_REF]Žlender and Thompson, 2017). Moreover, this approach requires strong political and economic choices in urban planning. Another approach is land sharing where a higher fragmentation of green spaces dispersed through the urban matrix under diversified forms is proposed (e.g. pocket parks [START_REF] Ikin | Pocket parks in a compact city: how do birds respond to increasing residential density?[END_REF], vegetated streets Säumel et al., 2016, small urban grasslands [START_REF] Kendal | The importance of small urban reserves for plant conservation[END_REF]. This approach is interesting to promote various biological communities associated to different local environmental and management conditions [START_REF] Kendal | The importance of small urban reserves for plant conservation[END_REF] and to increase daily contact with nature (Soga et al., 2016). In addition, this strategy seems particularly relevant in old cities where high urban densities can limit space-consuming projects.

Streets are linear elements omnipresent in the whole urban matrix which are often only viewed as corridors for pedestrian and vehicle traffics. By introducing vegetation, streets can become multi-functional by delivering numerous ecosystem services [START_REF] Säumel | Toward livable and healthy urban streets: Roadside vegetation provides ecosystem services where people live and move[END_REF] and represent opportunities to facilitate incidental people daily interactions with nature (e.g looking at vegetation while walking in a street, Cox et al., 2017a). Street vegetation can take different forms according to design and management practices which have to be combined with people's perceptions and preferences to make effective decision-making (Bennet, 2016;Ives and Kendall, 2014;Wallace et al., 2016). In the largest streets and with a relatively high planning budget, planting trees can be an interesting strategy for improving people street valuations [START_REF] Ng | Preferences for street configuration and street tree planting in urban Hong Kong[END_REF]Todorova et al., 2004). More simply, streets can also encompass a large variety of herbaceous plants, cultivated or spontaneous, by changing management practices or by making small interventions (Weber et al. 2014).

In this study, we assessed people's perceptions and valuations for herbaceous vegetation types associated to various managements and designs of street pavements. For that purpose, we conducted two surveys (one local in the field and one online at national scale) were people had to grade photomontages reflecting various management and design scenarios. Following previous studies which found that people generally prefer green infrastructures compared to mineral infrastructures (Botzat et al., 2016;[START_REF] Fischer | Beyond green: Broad support for biodiversity in multicultural European cities[END_REF]White and Gatersleben, 2011), we tested the hypothesis that street pavements with some vegetation are more preferred than pavements highly managed with no vegetation. We also examined the relationships between the valuations of our respondents and their socio-demographic characteristics.

Material and methods

Photomontages and valuation measures

We based our questionnaire on a visual method by producing photomontages representing different pavement vegetation types associated to various management practices and interventions (Fig. 1). We first constructed three photomontages to compare people valuations among a highly managed pavement without vegetation ('Asphalt High manag.') and two types of lowly managed pavements harboring spontaneous vegetation ('Asphalt Low manag.' and 'Sand Low manag.'). The represented vegetation on these photos reflects vegetation structure and composition that spontaneously grow on pavements as observed in France (Bonthoux et al. in revision). Represented dominant species are Erigeron sp. for 'Asphalt Low manag.' and Trifolium repens (L.) for 'Sand Low manag.' (Fig. 1). We then added two photomontages to assess people valuations for vegetation included in small designs. We first represented a vegetation strip at the foot of the wall ('Asphalt Flower') with sowed species which are often used in French cities for their colored flowers (e.g. Iberis sempervirens (L.), Eschscholzia californica (Cham.)). This type of intervention is possible in existing old pavements by using cracks or by removing a small asphalt strip along the wall. We also proposed an intervention consisting in the creation of a grassland strip between the pedestrian path and the road ('Sand Grassland'). All these photomontages reflect vegetation structure present in late spring -early summer which is the period in which vegetation differences between pavement situations is the highest and in which people are frequently outside and experience vegetated pavements.

Finally, we assessed whether people's preferences depend on the visual context by incorporating the five pavement photomontages in open and closed neighborhood visual contexts (Fig. 1).

We used two measures to assess valuations and perceptions of each pavement vegetation type.

First, the ten photos were presented individually in a random order to respondents which had to grade them between 1 (do not like at all) and 10 (like a lot). Second, to measure perceptions, respondents had to answer whether they found that the five pavement types (pavement without the neighborhood visual context, Appendix Fig. 1) were associated or not (i.e. yes or no response) to seven criteria: beautiful, boring, kept, useful for nature, natural, wild, valuable for the city image. Finally, we collected information about socio-demographic variables that could influence people valuations for pavement vegetation including age, gender, qualification level, job or studies in the environmental field or not, frequency of outdoor activities, house or apartment housing and practice of gardening (Table 1). We also asked the city name of residence to know whether respondents live in rural or urban areas (by informing the number of city inhabitants) and in which French region (by informing longitude and latitude, Table 1).

Surveys

A combination of field and online surveys including the same photomontages and collected information was realized between April and June 2017. Comparison of these two types of survey was used to check the robustness of our results and limit methodological biases.

Field face-to-face surveys were performed in the agglomeration of Blois (105 000 inhabitants) which is located in central France. Surveys were conducted in the inner Blois city and in several small villages in the agglomeration. This survey approach permitted us to optimize the age gradient range by interviewing teenagers and elderly persons who were only slightly addressed by the online survey. It also allowed to interview people not interested in the subject. The online survey was used to increase our response sample size. This latter approach can show results consistent with traditional sampling approaches and allows to obtain a cheap, fast and large collection of responses [START_REF] Brickman Bhutta | Not by the book: Facebook as a sampling frame[END_REF][START_REF] Gosling | Should we trust web-based studies? A comparative analysis of six preconceptions about internet questionnaires[END_REF][START_REF] Riva | The use of the internet in psychological research: comparison of online and offline questionnaires[END_REF]. To disseminate the online survey and optimize the respondents variability we sent a web link to students, professors and administrative personnel of several universities with various specialties including environmental field (e.g. ecology, landscape architecture) and other disciplinary areas (e.g. mathematics, physics, computer science). We also posted the link on different social networks and employed a snowballing approach by asking to forward the survey to other networks.

Analyses

Grades associated to the ten photomontages were highly correlated between both field and online surveys (Spearman rank correlation Rho = 0.81, n=10, Appendix Table 1). We thus pooled both dataset before analyses in order to improve the robustness of results.

We first compared means of grades associated to the ten photos using t-tests. We also calculated coefficients of variation to investigate the level of grade variability. To analyze the relationships between the five pavement types and people perceptions we performed a multiple component analysis (MCA) on the response (yes-no) * seven criteria matrix, including in the same matrix the responses for the five pavement types.

To investigate the relationships between photo grades and the ten socio-demographic variables (Table 1), we computed linear models for each of the ten photomontages. All sociodemographic variables were lowly correlated together (r < |0.7|, Appendix Table 2, [START_REF] Dormann | Collinearity: a review of methods to deal with it and a simulation study evaluating their performance[END_REF] and were included in models. We standardized (mean = 0, SD = 1) the quantitative explanatory variables to facilitate comparison of parameter estimates [START_REF] Schielzeth | Simple means to improve the interpretability of regression coefficients[END_REF]. We used a model averaging approach to take into account the uncertainty in the model selection process [START_REF] Burnham | Model Selection and Multimodel Inference: A Practical Information-Theoretic Approach[END_REF]. We fitted all possible models nested within the full model and ranked them on the basis of AIC and assigned them Akaike weights (w i ).

We then averaged the estimated parameters of the 95% confidence set of models (sum of w i > 0.95) weighted by w i . We considered variables as significant when confidence intervals did not overlap zero. Finally, we calculated the percentage of explained variance for the most parsimonious model (i.e. with the smallest AIC) of the confidence set. No spatial autocorrelation was found in model residuals given that longitude and latitude of respondents' municipalities were included as explanatory variables in models.

Results

Diversity of respondents

A total of 271 and 3338 respondents answered the field and online surveys respectively (n total = 3609). Respondents represented a large diversity of socio-demographic groups (Table 1, See Appendix Fig. 2 for the distributions of quantitative variables) and their residence locations were relatively evenly distributed across France as across the rural-urban gradient (Fig. 2, Table 1). The age and qualification level gradients were oriented towards young people and high diploma due to our questionnaire diffusion approach towards university people but nevertheless the whole gradients were correctly represented and no important difference between field and online survey was found.

Grades and criteria associated to the photomontages

Average respondents' grades associated to situations in open visual contexts were always higher than those in closed visual contexts (Fig. 1). Despite small differences in grade orders between both survey approaches (Appendix Table 1), 'Asphalt High manag.' average grade was globally significantly lower than 'Asphalt Low manag.' and 'Sand Low manag.' average grades in both visual contexts. For these three photo grades, variations were relatively high (coefficients of variation ranging between 0.47 and 0.68, Appendix Table 1) indicating divergences in respondents' valuations. Average grades were the highest for 'Asphalt Flower' and 'Sand Grassland' in both visual contexts. Grade variations were relatively low (coefficients of variation ranging between 0.24 and 0.36) for both situations indicating convergences in respondents' valuations.

The first two axes of the MCA accounted for 45% and 26% of the total variance respectively (Fig. 3). The first horizontal axis was correlated to the 'Beautiful', 'Boring', 'Useful for nature' and 'Valuable for the city image' criteria and the second vertical axis was correlated to the 'Kept', 'Natural' and 'Wild' criteria (Fig. 3, Appendix Table 3). 'Asphat High manag.' situation had a small convex hull and was judged 'not Beautiful', 'Boring', 'Kept', 'not Useful for nature', 'not Natural', 'not Wild' and a mix across 'Valuable' and 'not Valuable for the city image'. The 'Asphalt Low manag.' and 'Sand Low manag.' situations had large convex hulls indicating divergent responses and were associated to the same criteria: 'not Kept', 'quite Natural' and 'Wild' and a judgement mix for the 'Beautiful', 'Boring', 'Useful for nature' and 'Valuable for the city image' criteria. 'Asphalt Flower' and 'Sand Grassland' situations were relatively associated to the same criteria: 'Beautiful', 'not Boring', 'Kept, 'Useful for nature', 'Valuable for the city image' and a judgement mixing for the 'Natural' and 'Wild' criteria (Fig. 3).

Relationships between grades and socio-demographic variables

Because results for both visual contexts were very similar, only results for the closed visual context were presented in the main text (Table 2, see Appendix Table 4 for all detailed results). There were numerous significant relationships between grades and sociodemographic variables, but the total explained variance was low in each model (R² ≤ 11%; Table 2). The 'Environment' variable had the strongest effect magnitude on respondents' grades. The 'Housing', 'Population', 'Longitude', 'Latitude' variables were the least significant.

The 'Asphalt High manag.' grading was significantly and negatively related to 'Age', 'Gender.woman', 'Qualification', 'Environment.yes', 'Outdoor' and 'Gardening.yes' variables.

On the contrary 'Asphalt Low manag.', 'Sand Low manag.', 'Asphalt Flower' and 'Sand Grassland' were globally positively related to these variables (Table 2).

Discussion

Promoting nature in cities is challenging for scientists, designers and managers, because it requires to find urban designs improving trade-offs and synergies between biodiversity conservation, associated ecosystem services and people acceptance of spontaneous nature (Threlfall and Kendal 2017[START_REF] Kowarik | Urban wilderness: supply, demand, and access. Urban For[END_REF], Botzat 2016). In addition to traditionally acknowledge green spaces, streets, which are ubiquitous at the city scale, represent a good opportunity to simultaneously promote biodiversity and enhance nature daily experience (Bonthoux et al. in revision). Based on a large collection of data, we showed that people have more preferences for vegetated than for mineral street pavements, regardless of the neighborhood visual context. However, the appreciation of vegetation strongly depends on the way it is promoted.

Our study was based on complementary field and online surveys, strengthening the robustness of the results. In fact, we found that results of field survey confirm the nationwide online survey. Field survey is an interesting method to control and maximize the variability of people surveyed. However, this technique is time consuming and generally leads to modest sample sizes which are geographically restricted, with potential impacts on results robustness and transferability. Online survey can be oriented towards certain people (highly qualified people in this study) but has the advantage of covering large areas and obtaining a high response amount. In the questionnaire we used a moderate number of photos to limit survey duration and to avoid people demotivation. We chose to compare preferences for vegetation pavement reflecting late spring-early summer period when vegetation is the most developed and people are most often outside. In late summer and in autumn, vegetation with a drier or a degraded aspect may be less appreciated. This effect could be reduced by mowing at this time, the vegetation structure would be more homogeneous between pavement vegetation types and we can expect a low variability of people valuations at this period.

Although there was a high variability among people preferences, we found that lowly managed pavements with spontaneous vegetation were in average higher valued than highly managed pavements without vegetation. This result is in accordance with other studies showing that people prefer vegetated to mineral urban elements (e.g. pavement tree pits, [START_REF] Fischer | Beyond green: Broad support for biodiversity in multicultural European cities[END_REF]; house facades and roofs, White and Gatersleben 2011). Our criteria analysis showed that pavements with spontaneous vegetation were perceived as less kept than pavements without vegetation, but more beautiful and less boring. This finding goes against conveyed ideas in municipalities and technical services (in France at least) that strong management is important for urban dwellers. This point highlights the importance of conducting studies like this one to avoid a truncated overview of people preferences. Our results indicate a wide acceptance of spontaneous vegetation in cities that occur among inhabitants but also local decision-makers and managers (unpublished interviews with 11 municipality mayors or technical service leaders conducted in the agglomeration of Blois). In response to the increasing regulatory ban of pesticide use (e.g. prohibition since 2017 in public spaces in France), alternative methods of vegetation management used by technical agents (hand weeding or thermal/steam weeding) are often moderately effective (Bonthoux et al. in revision) and physically difficult. This forces municipalities to change their attitudes on the presence of spontaneous vegetation in public spaces.

Our results showed a strong people consensus of appreciations towards pavements containing vegetation integrated in small design interventions. This is supported by the study of Weber et al. (2014) who found a higher approval for maintained roadside vegetation compared to spontaneous vegetation in Berlin, and with the fact that people generally accept vegetation with visible signs of human actions or managements (Nassauer 1995, Van den Berg andvan Winsum-Westra 2010). Including vegetation in designs also lead to homogeneous plant covers which are often visually preferred compared to scattered patches of vegetation, as it has been shown in other urban habitats like wastelands in which pioneer vegetation is less appreciated than grassland vegetation (Brun et al. 2017[START_REF] Mathey | Public perception of spontaneous vegetation on brownfields in urban areas-Results from surveys in Dresden and Leipzig (Germany)[END_REF]. Interestingly, the grassland strip ('Sand Grassland') which can be composed of a mix of native grasses and forbs was as much appreciated than the flower strip ('Asphalt Flower') which is generally composed of exotic and/or horticultural species. This legitimates the fact of designing urban grasslands [START_REF] Klaus | Urban grassland restoration: a neglected opportunity for biodiversity conservation[END_REF]) that can favor people experience with nature but also other ecosystem services such as plant and animal biodiversity conservation and the maintenance of pollination [START_REF] Blackmore | Evaluating the effectiveness of wildflower seed mixes for boosting floral diversity and bumblebee and hoverfly abundance in urban areas[END_REF].

We found that socio-demographic parameters partly explained variability in photo appreciations. As expected, people frequently connected with nature had the highest preferences for vegetated pavements, spontaneous or integrated in designs. This effect predominated for people working or doing studies in the environmental field. These results are in accordance with another study showing that people who have high nature relatedness spend more time in public or private green spaces [START_REF] Lin | How green is your garden?: Urban form and socio-demographic factors influence yard vegetation, visitation, and ecosystem service benefits[END_REF]. As other studies, we found that females, people with a high qualification level and older people, who generally have more leisure times in gardens, had the highest preferences for vegetation (Cox et al. 2017a[START_REF] Fischer | Beyond green: Broad support for biodiversity in multicultural European cities[END_REF]. Nevertheless, despite the various socio-demographic variables integrated in models a high part of grade variability remained not explained, indicating that other determinants such as childhood experience or education would be important to consider [START_REF] Rupprecht | Memories of vacant lots: how and why residents used informal urban green space as children and teenagers in Brisbane, Australia, and Sapporo, Japan[END_REF].

Conclusion

The results of this study suggest that vegetated streets can become daily biodiversity-friendly urban greenspaces appreciated by urban dwellers. They should be taken into account by politics, urban designers and managers in different ways. In already built-up areas, we suggest to globally reduce management pressure allowing the appearance of moderate spontaneous vegetation, what should be beneficial for biodiversity and not rejected by people. In future constructions, we suggest that civil engineers and architects integrate local vegetation on pavements through innovative designs.

Figure 1 Figure 2 Figure 3

 123 Figure 1 Photomontages used in the questionnaire for the five pavement types and both visual contexts. Below are presented the associated distributions of respondents' grades (from 1=lowest valuation to 10=highest valuation, closed and open visual contexts represented in grey and white respectively, for each photo n=3609). Horizontal black bars are average grades. Average grade values are indicated above grade distributions. Different letters indicate that the average grades are significantly different between situations. Abbreviation: management (manag.)

Table 1

 1 Comparison (mean and coefficient of variation in brackets) of respondents' grades 434 between the field survey (n=271), the online survey (n=3338) and the whole dataset (n=3609).

	435
		Asphalt High	Asphalt High	Asphalt Low	Asphalt Low	Sand Low	Sand Low	Asphalt Flower	Asphalt	Sand Grassland	Sand Grassland
		manag. Closed	manag. Open	manag. Closed	manag. Open	manag. Closed	manag. Open	Closed	Flower Open	Closed	Open
	Field survey	4.01 (0.63)	5.25 (0.40)	2.99 (0.62)	3.94 (0.51)	3.29 (0.63)	4.49 (0.47)	5.36 (0.49)	5.97 (0.42)	5.29 (0.46)	6.75 (0.32)
	Internet survey	3.53 (0.68)	4.55 (0.53)	4.33 (0.53)	5.23 (0.48)	4.85 (0.51)	5.05 (0.47)	7.22 (0.32)	7.79 (0.23)	6.78 (0.35)	7.94 (0.23)
	Both surveys	3.56 (0.68)	4.60 (0.52)	4.23 (0.54)	5.13 (0.49)	4.73 (0.53)	5.01 (0.47)	7.08 (0.34)	7.65 (0.25)	6.67 (0.36)	7.85 (0.24)
	436
	437
	438
	439
	440
	441
	442

Table 2

 2 Correlations between socio-demographic characteristics. a) Correlation (ƞ²) between a quantitative and a qualitative variable b) Cramer's V correlation between each pair of qualitative variables and c) Pearson correlation between each pair of quantitative variables.

	Age	Genre Qualification Environment Outdoor Housing Gardening Population Longitude

Table 3

 3 Correlation between the two first axes of the Multiple Component Analysis and the

	seven criteria
		Axis 1	Axis 2
	Beautiful	0.71	0.07
	Boring	0.59	0.00
	Kept	0.16	0.54
	Useful for nature	0.65	0.04
	Natural	0.29	0.42
	Wild	0.10	0.61
	Valuable for the city	0.64	0.14

Acknowledgements

We thank the 3609 surveyed participants for their time. We also thank 'Agglopolys, Blois agglomeration' for its financial support and two anonymous reviewers for their constructive comments.

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