Corentin Iltis
email: corentin.iltis@gmail.com.

Jérôme Moreau

Guillaume Gamb

Corentin Manière

Céline Boidin-Wichlacz

Aurélie Tasiemski

Denis Thiéry

Philippe Louâpre

Day/night variations of feeding and immune activities in larvae

Keywords: Basal immunity, ecological and evolutionary immunology, nychthemeral variations, haemocyte types, Tortricidae

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

Introduction

The fundamental observation that organisms do not constitutively maintain immunity at its maximum levels has given rise to the field of evolutionary and ecological immunology [START_REF] Schmid-Hempel | Variation in immune defence as a question of evolutionary ecology[END_REF][START_REF] Schulenburg | Introduction. Ecological immunology[END_REF][START_REF] Siva-Jothy | Insect immunity: an evolutionary ecology perspective[END_REF]. Its underlying theory predicts that the expression of immune traits can be adjusted in a dynamic way, depending on environmental factors like population density [START_REF] Barnes | Density-dependent prophylaxis in the mealworm beetle Tenebrio molitor L. (Coleoptera: Tenebrionidae): cuticular melanization is an indicator of investment in immunity[END_REF], food availability and quality [START_REF] Ponton | Integrating nutrition and immunology: a new frontier[END_REF][START_REF] Vogelweith | Host plant variation plastically impacts different traits of the immune system of a phytophagous insect[END_REF], temperature [START_REF] Raffel | Negative effects of changing temperature on amphibian immunity under field conditions[END_REF] or parasitism threat [START_REF] Schmid-Hempel | On the evolutionary ecology of specific immune defence[END_REF][START_REF] Sheldon | Ecological immunology: costly parasite defences and trade-offs in evolutionary ecology[END_REF]. Indeed, resource allocation to immunity is predominantly shaped by the intensity of exposure to pathogens and parasitic faunas (parasites, parasitoids), which is spatially and temporally dynamic [START_REF] Lindström | Immunological investments reflect parasite abundance in island populations of Darwin's finches[END_REF][START_REF] Martinez-Bakker | The influence of biological rhythms on hostparasite interactions[END_REF]. Accordingly, spatial variations in levels of immunity have been found between distinct host populations experiencing geographically heterogeneous pressures exerted by these natural enemies [START_REF] Promislow | Host ecology shapes geographical variation for resistance to bacterial infection in Drosophila melanogaster[END_REF][START_REF] Lindström | Immunological investments reflect parasite abundance in island populations of Darwin's finches[END_REF][START_REF] Vogelweith | Geographical variation in parasitism shapes larval immune function in a phytophagous insect[END_REF]. Temporal fluctuations in immunity may allow an organism to cope with recurrent variations in threat of infection resulting from seasonality (seasonal scale) or natural alternance of day and night conditions (daily scale) [START_REF] Martin | Seasonal changes in vertebrate immune activity: mediation by physiological trade-offs[END_REF][START_REF] Martinez-Bakker | The influence of biological rhythms on hostparasite interactions[END_REF]Westwood et al. 2019).

Virtually all host species are confronted with daily rhythmic, predictable changes in their abiotic (e.g. light, temperature) and biotic environment, including exposure to natural enemies [START_REF] Martinez-Bakker | The influence of biological rhythms on hostparasite interactions[END_REF]Westwood et al. 2019). For instance, most parasitoid species exhibit daily fluctuations in their patterns of foraging and oviposition, such that their hosts are expected to face varying parasitism pressures over the course of a day, culminating during the peak of parasitoid activity [START_REF] Bertram | Temporal shifts in conspicuousness: mate attraction displays of the Texas field cricket, Gryllus texensis[END_REF][START_REF] Fleury | Adaptive significance of a circadian clock: temporal segregation of activities reduces intrinsic competitive inferiority in Drosophila parasitoids[END_REF].

Similarly, some parasites are able to schedule their burst of emergence in the environment at specific times of the day to maximise transmission rates during a restricted time interval that coincides with host activity [START_REF] Lu | Evolution in a multi-host parasite: chronobiological circadian rhythm and population genetics of Schistosoma japonicum cercariae indicates contrasting definitive host reservoirs by habitat[END_REF]. Daily rhythms in host exposure to natural enemies can also arise from host activity profiles, as convincingly illustrated by food-borne infections. Natural food of both animal and vegetal origins is far from being totally aseptic and, instead, harbours various microbial consortia partly constituted by pathogenic bacteria, fungi and viruses [START_REF] De Filippis | Recent past, present, and future of the food microbiome[END_REF][START_REF] Turner | The plant microbiome[END_REF]. Thus, natural food can be considered as a potential source of oral infection for a host through ingestion of intestinal pathogens and parasites [START_REF] Vogelweith | Food-mediated modulation of immunity in a phytophagous insect: an effect of nutrition rather than parasitic[END_REF]. Given that many host species display a daily structure of food intake with defined peak and trough (rather than feeding evenly throughout 24 h), it is assumed that the risk of food-borne infection should predictably fluctuate over the course of a day and peak during the active phase of host feeding (Westwood et al. 2019).

Faced with such fluctuating environmental pressures, insect hosts rely on basal and inducible immunity to defend themselves against infectious enemies [START_REF] Gillespie | Biological mediators of insect immunity[END_REF][START_REF] Kanost | Innate immune responses of a lepidopteran insect, Manduca sexta[END_REF][START_REF] Schmid-Hempel | Evolutionary ecology of insect immune defenses[END_REF]. Basal immunity corresponds to the background, readily available protection displayed by the host to prevent potential infection, that is, levels of immune defences prior to a challenge. This immune maintenance is conferred by the presence of effectors constitutively expressed in the insect haemolymph, such as the number of haemocytes (blood immune cells of arthropods and molluscs) recruited during various cellular processes and the melanisation cascade catalysed by the phenoloxidase enzyme (PO) produced from inactive prophenoloxidase (PPO) [START_REF] Cerenius | The prophenoloxidase-activating system in invertebrates[END_REF][START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF][START_REF] Strand | The insect cellular immune response[END_REF]. Conversely, inducible immunity is mobilised upon an actual infection and refers to the insect ability to mount an appropriate immune response to fend off the multiplying intruder. As part of this immune response, the insect can upregulate the expression of the immune effectors mentioned above, and synthesise de novo specific immune agents (e.g. anti-microbial peptides) following the recognition of a pathogenic signal [START_REF] Brown | Activation of host constitutive immune defence by an intestinal trypanosome parasite of bumble bees[END_REF][START_REF] Bulet | Antimicrobial peptides in insects: structure and function[END_REF][START_REF] Schmid-Hempel | Evolutionary ecology of insect immune defenses[END_REF].

The existing body of evidencegathered so far on dipteran insect models including Drosophila melanogaster (Drosophilidae) and Anopheles stephensi (Culicidae)suggests time-of-day dependent variations in inducible levels of insect immunity: the strength and efficiency of the immune response expressed to combat a deadly microbial intruder [START_REF] Lee | Circadian regulation in the ability of Drosophila to combat pathogenic infections[END_REF][START_REF] Murdock | Complex environmental drivers of immunity and resistance in malaria mosquitoes[END_REF][START_REF] Stone | The circadian clock protein timeless regulates phagocytosis of bacteria in Drosophila[END_REF]). In the diurnal species D. melanogaster for instance, the immune response to an artificial bacterial infection (phagocytic activity, expression of immune-related genes coding for anti-microbial peptides) is more elevated in case of challenge occurring during the resting rather than the active phase [START_REF] Lee | Circadian regulation in the ability of Drosophila to combat pathogenic infections[END_REF][START_REF] Stone | The circadian clock protein timeless regulates phagocytosis of bacteria in Drosophila[END_REF]. This daily enhancement of inducible immunity is accompanied with faster bacterial clearance and improved host survival upon the few hours after the infection [START_REF] Lee | Circadian regulation in the ability of Drosophila to combat pathogenic infections[END_REF][START_REF] Stone | The circadian clock protein timeless regulates phagocytosis of bacteria in Drosophila[END_REF]. Comparatively to this literature on host inducible immunity, little is known about the way daily cycles may shape the insect investment in basal levels of immunity, in anticipation of the risk of infection. Still, a dynamic investment in host basal immunity all along the day could be expected, considering that keeping immune defences at high levels of readiness constitutively should result in severe energetic losses and even collateral damage for the host (immunopathology) [START_REF] Schmid-Hempel | Variation in immune defence as a question of evolutionary ecology[END_REF][START_REF] Schulenburg | Introduction. Ecological immunology[END_REF]. For example, daily rhythms in host basal immunity could have evolved to cope with periodic and highly predictable risk of encounter with enemies, as would be the case for food-borne infections (Westwood et al. 2019). In this context, host exposure to enemies should periodically oscillate between maximum and minimum threats of infection occurring during active and resting phases of feeding, respectively (Westwood et al. 2019).

This study proposes to explore the day/night variations in feeding activity and basal immunity (i.e. using non-experimentally challenged individuals) in an insect host. Laboratory work was conducted on larvae of the grapevine moth Lobesia botrana (Lepidoptera: Tortricidae), which have evolved a defensive strategy relying on a relatively important investment in basal immunity (i.e. a high basal activity of the PO/PPO system) by comparison with closely related tortricid species [START_REF] Vogelweith | Defense strategies used by two sympatric vineyard moth pests[END_REF]. They can therefore be expected to adjust the expression of these core defensive traits to contend with varying threat of infection over the course of a day. On the one hand, larval behaviour was monitored to quantify the day/night amount of time spent feeding as a surrogate for the intensity of exposure to food-derived enemies in natural conditions. Larvae of this species can be expected to display a day/night structure of feeding activity as a way to cope with nychthemeral variations in the nutritional value (e.g. amounts of defensive compounds) of grape berries they feed upon [START_REF] Carbonell-Bejerano | Circadian oscillatory transcriptional programs in grapevine ripening fruits[END_REF]. On the other hand, this work examined the day/night basal levels of several effectors involved in insect cellular and humoral immunity. Focus was placed on systemic immunity (haemolymph) because immune traits in the circulating compartment can be affected in case of oral infection, with demonstrated consequences for the outcomes of the infection process [START_REF] Freitak | The maternal transfer of bacteria can mediate trans-generational immune priming in insects[END_REF]Wu et al. 2016). For cellular immunity, quantitative and qualitative measurements focused on haemocyte load and the relative abundances of the different haemocyte types, respectively.

The haemocyte pool of insects is indeed constituted by several types of immune cells, each fulfilling a specific defensive function (e.g. phagocytosis, encapsulation, PO secretion) as a component of host immunocompetence [START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF][START_REF] Strand | The insect cellular immune response[END_REF]Vogelweith et al. 2016). For humoral immunity, measures encompassed the activity of the PO/PPO system, which controls the production of melanin and intermediate compounds with cytotoxic properties to kill microscopic and macroscopic intruders [START_REF] Cerenius | The prophenoloxidase-activating system in invertebrates[END_REF]. As frequently observed in other Lepidopteran species, it may be expected that L. botrana larvae behave so they restrict their feeding activity to a specific phase, either daytime or nighttime, rather than foraging at constant rates throughout the day [START_REF] Goodspeed | Arabidopsis synchronizes jasmonate-mediated defense with insect circadian behavior[END_REF][START_REF] Niepoth | Comparing behavior and clock gene expression between caterpillars, butterflies, and moths[END_REF][START_REF] Suszczynska | Circadian regulation of caterpillar feeding and growth[END_REF]. Likewise, they are expected to condense their investment in basal immunity to daytime or nighttime, rather than keeping these immune defences at costly, high levels of readiness all along the day.

Material and methods

Insect stock and rearing methods

This study was performed with L. botrana individuals originating from a large laboratory stock, comprised of several thousand of caged moths (INRAe, Villenave d'Ornon, France).

The genetic diversity of the stock was preserved by ensuring a yearly supply with wild individuals picked from nearby vineyards. This strain has been already used in former studies focused on the immunity of L. botrana larvae [START_REF] Iltis | When warmer means weaker: high temperatures reduce behavioural and immune defences of the larvae of a major grapevine pest[END_REF][START_REF] Iltis | Are life-history traits equally affected by global warming? A case study combining a multitrait approach with fine-grain climate modeling[END_REF][START_REF] Vogelweith | Defense strategies used by two sympatric vineyard moth pests[END_REF].

Since its establishment a few years ago and until the start of the experiments, the population has been maintained without diapause on an artificial diet (see composition below) under controlled rearing conditions (temperature: 20 ± 0.5°C, relative humidity: 60 ± 5%, photoperiod: L17:D6 and 1 h of dusk, luminosity: 650 lux during photophase and 100 lux at dusk). Such a diapause-free biological cycle is unlikely to have impacted the basal immunity of this laboratory-reared population over the daily scale examined in this study, since immune variations related to diapause strategies should operate on longer timescales (e.g. seasonal scale). Moreover, a previous work has reported that L. botrana individuals belonging to the laboratory stock used here display similar patterns of investment in basal and inducible immunity by comparison with insects collected in field conditions during summer period [START_REF] Vogelweith | Defense strategies used by two sympatric vineyard moth pests[END_REF]. A few bands of waxed paper (5 x 10 cm) were hung inside the rearing cages as suitable oviposition supports for mated females. Once covered with eggs (after 2-3 days), the papers were renewed and placed in plastic boxes under a moist surface to minimise egg mortality due to desiccation.

Within the 24 h following hatching from the eggs, neonate larvae were collected with a soft paintbrush and deposited individually in microtubes containing 1.5 ml of artificial culture medium [START_REF] Thiéry | Relative performance of European grapevine moth (Lobesia botrana) on grapes and other hosts[END_REF]. In order to focus on basal immunity and avoid immune challenge due to pathogen ingestion, larvae were fed with a rearing medium supplemented with antifungal and antibacterial substances strongly limiting microbial presence in larval food (composition for 1000 ml: 1000 ml permuted water, 15 g agar, 86.6 g maize flour, 41.3 g wheat germ, 45.5 g beer yeast extract, 6 g ascorbic acid, 3.4 g mineral salt, 128 mg pyrimethanil, 2.7 g benzoic acid, 2.8 mg methyl 4-hydroxybenzoate and 5 ml 95% ethanol). Tube caps were pierced for air circulation and covered with a piece of fine mesh fabric to prevent larvae from escaping. Individuals were then transferred to climate chambers (ST 2/2 BASIC, Pol-Eko Aparatura, Wodzisław Śląski, Poland) to complete their development until behavioural and immune measurements. Within these devices, larvae were all raised under a realistic cycle of diurnally changing conditions of light and temperature (temperature ± 0.1°C, relative humidity: 50 ± 10%, photoperiod: L18:D6, luminosity: 650 lux during photophase). Rearing incubators were programmed to run a 'mean summer day', defined according to recent climatic observations collected between 15 July and 15 August in Burgundy (Eastern France), where L. botrana naturally occurs (for more details, see [START_REF] Iltis | Are life-history traits equally affected by global warming? A case study combining a multitrait approach with fine-grain climate modeling[END_REF]. Emphasis was placed on summer period because L. botrana is mainly active over this date range, as evidenced by one peak of adult emergence observed in field conditions [START_REF] Barnay | Survey of natural populations of Trichogramma (Hym., Trichogrammatidae) in the vineyards of Alsace (France)[END_REF][START_REF] Martín-Vertedor | Global warming affects phenology and voltinism of Lobesia botrana in Spain[END_REF]. The daily cycle of temperatures met by larvae throughout their development was constituted by six thermal segments of 4 h each: 17.8, 22.3, 24.9, 22.9, 19.0 and 16.4°C. To reflect a natural alternance of diurnal and nocturnal conditions, lights were switched off during the 19.0 and 16.4°C thermal segments. Abiotic conditions inside the incubators were tracked with independent data loggers (Hobo, Onset Computer Corporation, Bourne, USA) and did not deviate from the regime programmed throughout the experiments. To enable simultaneous collection of daytime and nighttime data during each working day (~ 10 h), larvae were randomly distributed among different incubators with shifted phases (i.e. differing in the onset timing of daytime and nighttime).

Experiments involved individuals reaching their 4 th larval instar (i.e. at a larval age of about 2 weeks after hatching), the penultimate stage of larval development in this species [START_REF] Thiéry | Relative performance of European grapevine moth (Lobesia botrana) on grapes and other hosts[END_REF]. This selection procedure thus enabled to exclude individuals close to metamorphosis (late 5 th instar larvae), which may undergo profound behavioural and physiological modifications as they start to pupate and stop feeding. Larvae were randomly divided among two experimental groups because each group required distinct temporal organisation for measurements (see below). Group 1 was reserved for behavioural surveys (time budget allocated to feeding activity). Group 2 was devoted to immune assays (haemocyte load, activity of the PO/PPO system, relative abundances of different haemocyte types).

Group 1: behavioural measurements of larval feeding activity

Temporal organisation

The feeding behaviour of each tested individual was measured during four specified time intervals of 1 h, termed as blocks, each being associated with a given thermal segment (daytime: 22.3 and 24.9°C, nighttime: 19.0 and 16.4°C) (Fig. 1). Importantly, each block was disposed in the central part of its respective thermal segment of 4 h, such that measurements started 1 h 30 min after the onset and ended 1 h 30 min before the offset of the thermal segment (Fig. 1). This allowed to avoid any excessive abrupt change in thermal conditions that could disturb larval behaviour during the tests (i.e. through a short-term larval behavioural response to rapidly changing thermal conditions). Of note, this temporal organisation also excluded the confounding effects due to sudden light transition between daytime and nighttime (or inversely), which could also yield similar stress-related effects on the expression of larval feeding behaviour.

Behavioural measurements of larval feeding activity

Behavioural trials were conducted on 47 individuals (n = 47 for daytime, n = 47 for nighttime) to quantify the amount of time each spent feeding during daytime and nighttime.

Once they reached their 4 th larval instar, larvae were gently extracted from their rearing tube and individually placed in standardised compartmentalised Petri dishes (100 mm diameter x 15 mm height, 4 compartments of 29.4 cm 3 each) covered with a thin layer of fresh rearing medium (~ 10 ml per compartment). The lid was placed on the Petri dish to restrict larva's movements within the boundaries of its assigned compartment and pierced to allow air circulation in the different compartments. Therefore, each larva was confined to a single compartment with accessible food ad libitum and impossibility of interacting with other individuals in adjacent compartments. Petri dishes were kept within the incubators from which larvae originated throughout the 24 h of survey. After an acclimation period of 15 min, the 24-h sequence started to be recorded with a camera (AD7013MTL, Dino-Lite, Almere, Netherlands) fixed on a tripod to keep vision on the four compartments simultaneously. All the sequences were initiated during the 24.9°C thermal segment (photophase). Non-heating red light lamps were placed inside the incubators to enable nighttime observations without disturbing larval behaviour. Light and temperature conditions were spatially homogeneous inside the climate chambers and all tested larvae were presumably exposed to the same abiotic conditions during the tests. Behavioural surveys were restricted to 24 h in a way to avoid larval moulting between fourth and fifth instar during the tests, which could have deeply disturbed feeding behaviour. Indeed, L. botrana larvae require on average a few days to pass through their fourth instarespecially in males which develop faster than females as larvae [START_REF] Thiéry | Relative performance of European grapevine moth (Lobesia botrana) on grapes and other hosts[END_REF] such that extending behavioural tests to several days of continuous monitoring could have resulted in a large proportion of individuals entering premoulting or moulting stage during the experiments. Furthermore, behavioural tests were not supposed to be stress-inducing as they were conducted within the rearing incubators, under the same abiotic conditions that larvae encountered throughout their development, and without possibility of antagonistic interactions between larvae in adjacent compartments.

Hence, a relatively short acclimation period (15 min) was deemed sufficient by comparison with former studies involving a 24-h adjustment time prior to behavioural measurements of feeding activity as a way to allow larvae to acclimate to a sudden environmental change between rearing and test conditions, which did not occur in the present work [START_REF] Havko | Stimulation of insect herbivory by elevated temperature outweighs protection by the jasmonate pathway[END_REF][START_REF] Herden | Shifting Nicotiana attenuata's diurnal rhythm does not alter its resistance to the specialist herbivore Manduca sexta[END_REF].

Once the 24 h elapsed, the videotapes were collected and further analysed with DinoCapture software (version 2.0, Dino-Lite, Almere, Netherlands). The feeding behaviour of each tested individual was examined during daytime (two blocks) and nighttime (two blocks) (i.e. four blocks throughout the 24-h sequence). During each block, larval behaviour was continuously monitored to record the amount of time spent feeding. Feeding was scored in case of rhythmic head movement in a stationary position, with head lowered on the medium surface, indicating biting and food ingestion [START_REF] Bernays | Foraging in nature: foraging efficiency and attentiveness in caterpillars with different diet breadths[END_REF]). Locomotion is infrequent in L. botrana larvae since they usually complete their development within a silk nest comprised of an aggregate of several floral buds or grape berries. Hence, the simple distinction between feeding and resting (no obvious head movement) remains relevant with regard to the biological features of the species. The larval investment in feeding was calculated as the time budget (in min) allocated to this activity during each block.

Group 2: immunological parameters

Temporal organisation

By contrast with long-lasting behavioural trials, collections of immune data were instantaneous, and thus required a different scheduling (Fig. 1). Immune samplings were dispersed throughout daytime and nighttime to obtain an integrative picture of larval immunity, representative of changes occurring in both light and temperature conditions.

Sampling periods excluded the first 25% of each phase (daytime or nighttime), that is, the first 4 h of daytime (which lasted 16 h) and the first 2 h of nighttime (which extended over 8 h) (Fig. 1). As mentioned in case of behavioural trials (Group 1), this procedure enabled to avoid confounding stress physiological effects possibly arising from steep changes in light conditions during the transition from daytime to nighttime, or inversely. Similarly, samplings were never conducted during the first 20 min of a thermal segment to ensure temperature stabilisation and larval acclimation to the shift in thermal conditions occurring between two successive thermal segments. For haemocyte load and activity of the PO/PPO system, immune samplings were dispersed over three thermal segments of daytime (22.3, 24.9 and 22.9°C) and two thermal segments of nighttime (19.0 and 16.4°C). For the relative abundances of haemocyte types, immune samplings occurred during one thermal segment of daytime (24.9°C) and one thermal segment of nighttime (16.4°C) (Fig. 1).

Haemocyte load and activity of the PO/PPO system Basal levels of haemocyte load and activity of the PO/PPO system were assessed on a total of 211 individuals (n = 102 for daytime, n = 109 for nighttime), following standardised methods formerly developed [START_REF] Vogelweith | Host plant variation plastically impacts different traits of the immune system of a phytophagous insect[END_REF]). Immune measurements were run at room conditions (temperature: 20 ± 0.5°C, relative humidity: 60 ± 5%, luminosity: 650 lux). Prior to the haemolymph samplings, larvae were transferred to sterile microtubes and dipped in an ice-filled box for 15 min to facilitate handling, because L. botrana larvae are particularly active when manipulated. Individuals to be tested during nighttime were maintained in constant darkness until haemolymph collection by covering the box with an opaque lid.

Larvae were placed on a cold surface and wounded in the dorsal part of their abdomen (using a sterile entomological pin) to collect a droplet of haemolymph (~ 1 µl) with a cold sterile glass capillary (Hirschmann Laborgeräte, Eberstadt, Germany). Haemolymph sampling usually requires no more than 10 sec, such that the abrupt shift from darkness to light experienced by some individuals (in case of nighttime measurements) was unlikely to affect their immunity, even on the very short term. The 1 µl volume of haemolymph was mixed with 20 µl of filtered, cold phosphate-buffered saline (hereafter PBS, 10 mM, pH 7.4). Tubes were homogenised before 10 µl of the solution were immediately pipetted into a Neubauer haemocytometer under a phase contrast microscope (Primo Star, Zeiss, Oberkochen,

Germany) at a magnification of 400x for a standardised estimation of the haemocyte load. The remaining 11 µl of diluted haemolymph were supplemented with 10 µl of filtered PBS and instantly snap-frozen in liquid nitrogen, before being stored at -27°C for subsequent measurements of PO/PPO system activity.

The activity of the PO/PPO system was extrapolated spectrophotometrically, distinguishing the activity of the functional PO extract naturally present in the sample (i.e. PO activity) from the potential activity of the whole PO/PPO system (i.e. total PO activity). This latter immune trait can be defined as the activity of both spontaneously functional PO and proenzyme stock, and its estimation required the cleavage of PPO into PO by means of chymotrypsin addition. Frozen haemolymph samples were gently thawed on ice for 15 min and then centrifuged (4000 g, 15 min, 4°C) before two supernatant aliquots of 5 µl each were pipetted into microplate wells. The first aliquot was mixed with 160 µl of diluted PBS solution (35 ml ultrapure water, 5 ml filtered PBS) and was reserved for measurement of PO activity. The second aliquot was supplemented with 160 µl of chymotrypsin solution (35 ml ultrapure water, 5 ml filtered PBS, 2.45 mg trypsin) to quantify total PO activity. The reaction started after the addition of a colorimetric substrate comprised of 20 µl of L-Dopa solution (40 ml ultrapure water, 160 mg L-Dopamine). This enzymatic reaction proceeded for 40 min at 30°C in a microplate reader (Versamax, Molecular Devices, San Jose, USA). Absorbance curves were fitted to regular absorbance recordings (every 15 s) at 490 nm. These curves were then analysed with SOFT-Max Pro software (version 4.0, Versamax, Molecular Devices, San Jose, USA) to extract the maximum enzymatic activity Vmax (the maximum speed of conversion of L-Dopamine into dopachrome by PO), which can be approximated by the maximum slope of the absorbance curve during its starting linear phase. All immune measurements were finally reported to 1 µl of pure haemolymph to enable comparisons between experimental groups.

Description and relative abundances of different haemocyte types

Flow-cytometric analyses were run on 30 individuals (n = 15 for daytime, n = 15 for nighttime) to discriminate the different haemocyte types present in the haemolymph and assess their relative abundances. The method used to collect one droplet of haemolymph was identical to the one described for measurements of haemocyte load, PO and total PO activities (see above). For fixation, the haemolymph samples were flushed into 0.5 ml microtubes containing 9 µl of a 4% paraformaldehyde solution diluted with sterile filtered PBS. The mixture was then gently homogenised and tubes were conserved at 4°C before analytical experiments. Haemolymph samples were processed through a flow cytometer (LSR Fortessa X20, Becton Dickinson, San Jose, USA). Cells were discriminated based on their complexity/granularity (side scatter) and size (forward scatter), with 10,000 threshold events counted per sample. Results were analysed using Kaluza software (version 2.1, Beckman Coulter, Brea, USA). Relative abundances were expressed in proportions of the total cell count assessed in the haemolymph sample.

Statistical analyses

The aim of this study was to investigate the day/night fluctuations of larval feeding activity and basal immunity while accounting for the possible variability related with realistic changes in temperature conditions occurring within daytime and nighttime. Hence, statistical models for almost all traits (feeding activity, haemocyte load, PO and total PO activities) incorporated a fixed-effect factor termed as 'phase' (daytime versus nighttime) alongside with a randomeffect factor termed as 'timepoint' and represented by the timing at which measurements were taken within each of these two phases (thermal segment sampled). Data for the relative abundances of haemocyte types were collected during one single thermal segment for each phase and, therefore, the statistical models only encompassed the fixed effect driven by the phase.

Data for time budget allocated to feeding activity met the assumptions required for parametric tests (normality and homoscedasticity), and were therefore analysed through a univariate analysis of variance (ANOVA) with mixed effects, also integrating the identity of the tested individual as an additional random effect. As overdispersed count data, haemocyte load was processed using a Generalised Linear Mixed Model (GLMM) with a negative binomial error structure. Data for PO activity required a log-transformation to be normalised, while those for total PO activity naturally complied with the assumptions of parametric tests.

Consequently, PO and total PO activities were studied by means of ANOVAs with mixed effects. The relative abundances of the different haemocyte types were compared between daytime and nighttime with Student's t-tests for independent samples. Normality and homoscedasticity were assessed with a Shapiro-Wilk and a Levene test, respectively, conducted on the residuals of the fitted model. All statistical analyses were run using R software (version 4.0.3, R Core Team).

Results

Group 1: behavioural measurements of larval feeding activity The time budget devoted to feeding was significantly impacted by phase (i.e. day/night cycle) (ANOVA with mixed effects, 𝜒 1 2 = 5.61, P = 0.02). Larvae spent a greater amount of time feeding (+16%) during nighttime than daytime (Fig. 2). As a random effect, the timepoint of data collection during the different phases did not affect larval feeding activity (𝜒 1 2 = 3.61, P = 0.06) (Fig. 2).

Group 2: immunological parameters

Haemocyte load and PO activity were not significantly modulated by phase (haemocyte load:

GLMM negative binomial error, 𝜒 1 2 = 2.34, P = 0.12; PO activity: ANOVA with mixed effects, 𝜒 1 2 = 1.23, P = 0.27) (Fig. 3(A), 3(B)). Total PO activity was significantly impacted by phase (ANOVA with mixed effects, 𝜒 1 2 = 7.16, P = 0.007): individuals sampled during nighttime exhibited higher basal levels of total PO activity (+26%) comparatively to those sampled during daytime (Fig. 3(C)). As a random effect, the timepoint of data collection during the different phases had no effect on any of the three immune traits inspected (haemocyte load: 𝜒 1 2 = 0.03, P = 0.87; PO activity: : 𝜒 1 2 = 0, P = 1; total PO activity: 𝜒 1 2 = 0,

P = 1) (Fig. 3(A), 3(B), 3(C)).
Flow-cytometric analyses combined with microscopic observations led to the discrimination of four haemocyte types circulating in the haemolymph of 4 th instar L. botrana larvae: prohaemocytes, oenocytoids, granulocytes and plasmatocytes (Fig. 4). Prohaemocytes are the smallest cells, displaying a round shape, a low cytoplasmic granularity and a high nuclear cytoplasmic ratio (N:C, i.e. ratio of the size of the nucleus to the size of the cytoplasm). Oenocytoids are intermediate in size and granularity, with a centred nucleus and a high N:C (4:1). Granulocytes are relatively large cells with high granularity, easily identified by the inclusion of numerous granules and vesicles in the cytoplasm. Plasmatocytes are among the biggest immune cells with an off-centred nucleus and a low N:C (between 1:2 and 1:1).

Interestingly, three haemocyte types were found to be significantly influenced by phase: prohaemocytes (Student's t-test, t28 = -2.26, P = 0.03), oenocytoids (t28 = 4.76, P < 0.001) and plasmatocytes (t28 = -4.03, P < 0.001). The relative abundances of prohaemocytes and plasmatocytes were higher for individuals sampled during nighttime by comparison with those sampled during daytime (Fig. 5). The inverse pattern was observed for the relative abundance of oenocytoids, which was higher for individuals sampled during daytime relative to those sampled during nighttime (Fig. 5). By contrast with these three haemocyte types, the relative abundance of granulocytes did not significantly differ between daytime and nighttime (t28 = -0.76, P = 0.45) (Fig. 5).

Discussion

This study explored the existence of day/night fluctuations of feeding behaviour (presumably influencing the dynamic of exposure to food-borne enemies in natural conditions) and basal immunity in the larval stage of a non-experimentally challenged insect. In terms of behaviour, results outline that L. botrana larvae spent a greater amount of time feeding at night, which could imply that the highest risk of food-borne infection would occur during this time interval. In terms of immunity, both the humoral and cellular branches were impacted by the day/night cycle. Collectively, data gathered on PO and total PO activities indicate that (i) L.

botrana larvae maintained an amount of active enzyme (PO activity) at constant basal level throughout 24 h, and (ii) they increased the inactive proenzyme stock (PPO) involved in total PO activity during nighttime. Although haemocyte load remained unaffected by the day/night cycle, the balance of the different types constituting the haemocyte pool was highly dynamic all along the day. Out of the four haemocyte types identified through flow-cytometric analyses (prohaemocytes, oenocytoids, granulocytes, plasmatocytes), the relative abundances of three types were found to vary between daytime and nighttime. At night, the relative abundances of prohaemocytes and plasmatocytes increased, whereas the one of oenocytoids decreased. These data provide the first evidence of nychthemeral variations of basal immunity in an insect model, leading to possible implications for the general understanding of the temporal structure of insect immunity.

This work identified four out of the five haemocyte types recovered in Lepidoptera: only spherulocytes were not discriminated owing to their rarity and morphological/cytological similarity with prohaemocytes [START_REF] Ribeiro | Insect haemocytes: what type of cell is that?[END_REF][START_REF] Strand | The insect cellular immune response[END_REF]Vogelweith et al. 2016). However, their relative abundance can be deemed negligible comparatively to prohaemocytes, which were the most abundant haemocyte type in the haemolymph samples.

Such a predominance remains unsurprising for immature larvae used in this study, given that prohaemocyte abundance progressively declines with larval age due to differentiation in other types [START_REF] Ghoneim | Effects of Novaluron and Cyromazine, chitin synthesis inhibitors, on the larval hemogram of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae)[END_REF]. The nighttime increase in the relative abundances of prohaemocytes and plasmatocytes could compensate for the concurrent decrease in the one of oenocytoids. Indeed, the circulating stock of oenocytoids is more likely renewed through differentiation of already circulating haemocytes as progenitor cells (prohaemocytes and plasmatocytes), rather than mitosis or release of oenocytoids from the hematopoietic organs [START_REF] Liu | Hemocytes and hematopoiesis in the silkworm, Bombyx mori[END_REF][START_REF] Nakahara | Two hemocyte lineages exist in silkworm larval hematopoietic organ[END_REF][START_REF] Strand | The insect cellular immune response[END_REF]. The nighttime drop in the relative abundance of oenocytoids could be related with the concomitant increase in total PO activity, since oenocytoids are labile cells functioning as the site of PPO synthesis and releasing this proenzyme in the haemolymph through lysis (González-Santoyo and Córdoba-Aguilar 2012; [START_REF] Jiang | Immunity in Lepidopteran Insects[END_REF]). This study is among the first ones to demonstrate that the composition of the insect haemocyte pool in different cell types can display nychthemeral variations, in the absence of immune challenge [START_REF] Islam | Diurnal rhythm of hemocyte population in an insect, Schizodactylus monstrosus Drury[END_REF]. In that respect, it suggests that the mechanisms causing these variations (e.g. haemocyte differentiation, migration between compartments) can operate rather quickly (on a daily scale).

In an ecological context, this nighttime elevation of several immune parameters could translate into a higher ability of L. botrana larvae to combat a potential infection during nocturnal time. The nighttime production of PPO (increase in total PO activity) might lead to a more efficient melanisation response to an infection. Indeed, this immune process relies on the activation of the available PPO stock rather than an additional synthesis of this proenzyme following challenge [START_REF] Adamo | Estimating disease resistance in insects: phenoloxidase and lysozymelike activity and disease resistance in the cricket Gryllus texensis[END_REF]. For instance, studies conducted on several insect models (mosquitoes, bumblebees) reported no significant upregulation of immune-related genes encoding PPO production as an immediate response to the infection [START_REF] Cui | Molecular characterization of a prophenoloxidase cDNA from the malaria mosquito Anopheles stephensi[END_REF][START_REF] Erler | Dynamics of immune system gene expression upon bacterial challenge and wounding in a social insect (Bombus terrestris)[END_REF]). However, there is no broad empirical support to a positive relationship between basal levels of PO/PPO system activity and successful host defence against pathogens and parasites (Adamo 2004; González-Santoyo and Córdoba-Aguilar 2012; [START_REF] Mucklow | Variation in phenoloxidase activity and its relation to parasite resistance within and between populations of Daphnia magna[END_REF]).

This suggests that host immunocompetence is a multifactorial trait that cannot be assessed through measurements of PO/PPO system activity alone [START_REF] Mucklow | Variation in phenoloxidase activity and its relation to parasite resistance within and between populations of Daphnia magna[END_REF]. Besides this humoral aspect of immunity, the present study appears to indicate an increased ability of L.

botrana larvae to encapsulate an infectious enemy throughout nighttime, because plasmatocytes act as the main capsule-forming agents in Lepidoptera [START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF][START_REF] Strand | The insect cellular immune response[END_REF]. Of note, variations in the abundance of haemocyte types mediating encapsulation response were shown to correlate positively with the degree of host resistance to an infection involving multicellular organisms [START_REF] Fors | Differences in cellular immune competence explain parasitoid resistance for two coleopteran species[END_REF][START_REF] Gerritsma | Natural variation in differentiated hemocytes is related to parasitoid resistance in Drosophila melanogaster[END_REF]). As a next step, it would be worth investigating whether the nighttime upregulation of immune traits highlighted here can translate into an actual elevation of larval resistance (decreased infection rates) or tolerance (improved survival rates) in face of an experimental oral infection, using antibiotic-free food allowing for the development of potentially pathogenic microorganisms. In the same vein, further investigations should also examine the relation between daily patterns of host immunity and both the intensity (e.g. bacterial load present in the larval food) and the nature (infectious enemy involved) of the infection threat [START_REF] Vogelweith | Food-mediated modulation of immunity in a phytophagous insect: an effect of nutrition rather than parasitic[END_REF].

In natural environments, the day/night contrast in feeding activity reported here could reflect the daily dynamic of food-borne infection of L. botrana larvae, likely to ingest microbes (yeasts, bacteria and fungi) developing in consortium on the grape surface [START_REF] Barata | The microbial ecology of wine grape berries[END_REF][START_REF] Vogelweith | Food-mediated modulation of immunity in a phytophagous insect: an effect of nutrition rather than parasitic[END_REF]. Many Lepidopteran larvae (in both moths and butterflies)

have been observed feeding predominantly at night, and this behaviour is thought to have evolved as a strategy of predator and parasitoid avoidance, or a way to take advantage of a nighttime reduction in the amount of toxins contained in plant tissues [START_REF] Goodspeed | Arabidopsis synchronizes jasmonate-mediated defense with insect circadian behavior[END_REF][START_REF] Suszczynska | Circadian regulation of caterpillar feeding and growth[END_REF]. In vineyards, mortality of L. botrana larvae is chiefly driven by parasitoids belonging to the Hymenoptera and Diptera orders, but the daily activity profiles of these natural enemies remain fairly undocumented [START_REF] Thiéry | Biological protection against grape berry moths. A review[END_REF]. Focusing on the plantinsect interaction, some transcripts related with the synthesis of defensive compounds in the flesh of grape berries were found to be upregulated during daytime (e.g. terpenoid biosynthetic genes) [START_REF] Carbonell-Bejerano | Circadian oscillatory transcriptional programs in grapevine ripening fruits[END_REF]. Such nychthemeral variations of plant nutritional value could have possibly driven the evolution of a nocturnal pattern of foraging in carpophagous L. botrana larvae.

This work has implications with regard to the evolutionary dynamic of host-pathogen, host-parasite or host-parasitoid interactions. Indeed, its results imply that the susceptibility of a host species to an infection should fluctuate over the course of a day, and a similar phenomenon has been described regarding the virulence and transmission of the infectious enemy (reviewed by Westwood et al. 2019). It thus suggests that the daily rhythms inherent to host resistance (immunity) and enemy virulence could be a major determinant of the outcomes of the infection and the evolutionary trajectory of the interaction (Martinez-Bakker and Helm 2015; [START_REF] Prior | Periodic parasites and daily host rhythms[END_REF]Westwood et al. 2019). Extrapolating the results and conclusions of this study to natural settings would require accounting for the bottom-up regulation of plant traits (largely influenced by grape variety) on the nychthemeral variations of larval immunity. This is especially true given that the abundance and composition of microbial communities developing on the berry surface is influenced by grape cultivar, and so should be the selective pressures exerted on the day/night structure of larval immunity [START_REF] Barata | The microbial ecology of wine grape berries[END_REF][START_REF] Bokulich | Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate[END_REF].

Additionally, there should be value in uncovering the role (if any) played by light and temperature conditions in shaping the immune response patterns to day/night conditions here highlighted. This work was not designed to quantify specifically the importance of these abiotic variables for the fluctuations of insect immune function. This would require, for instance, comparing different cycles of light and temperature conditions, uncoupling these two environmental parameters, or more thoroughly controlling for the effect of the timing of data acquisition (e.g. by evenly distributing timepoints of data collection and standardising their number and duration for both day and night). From a mechanistic perspective, several processes can be proposed to explain the possible influence of fluctuating light and temperature conditions on the insect immunity. On the one hand, the insect could plastically adjust its immunity to short-term changes in light and/or temperature occurring throughout the day. For instance, temperature is known to be a critical determinant of immune function in L.

botrana larvae, such that cooler conditions during nighttime may explain the concomitant rise in some immune traits like total PO activity, pending that such thermally-induced plasticity in immune traits can occur on a daily scale [START_REF] Iltis | When warmer means weaker: high temperatures reduce behavioural and immune defences of the larvae of a major grapevine pest[END_REF][START_REF] Iltis | Are life-history traits equally affected by global warming? A case study combining a multitrait approach with fine-grain climate modeling[END_REF]. On the other hand, another plausible explanation to these results would lie in the chronobiological route, involving an endogenous biological rhythm (orchestrated by a circadian clock) to account for the nychthemeral variations observed. This cycle could be entrained by daily recurrent fluctuations of light and temperature, since they may both act as environmental cues (i.e.

'Zeitgebers') driving the circadian machinery and regulating its outputs in natura [START_REF] Bell-Pedersen | Circadian rhythms from multiple oscillators: lessons from diverse organisms[END_REF][START_REF] Vanin | Unexpected features of Drosophila circadian behavioural rhythms under natural conditions[END_REF]). Identifying the nature of these mechanisms should help to examine the importance of day/night transitions (i.e. dusk and dawn in natural conditions) in shaping the daily profiles here reported, considering that environmental changes occurring during these transitions could serve as external cues enabling the insect to adaptively anticipate the upcoming environment. In the present investigation, it appears unlikely that transitions between the two phases would have driven the daily pattern responses of immunity observed, provided that data collection was evenly distributed over each of the different timepoints. Therefore, individuals sampled at the very end of the last timepoint for daytime and nighttime (approximating dusk and dawn conditions, respectively) only represented a minor proportion of the overall dataset, but more mechanistic investigations are clearly needed at this point to ascertain this statement.

In conclusion, this study evidences a marked day/night contrast in the basal levels of expression of several immune traits in an insect model. It could be proposed that the increase in basal immunity at night has evolved with a predominantly nocturnal pattern of feeding in L.

botrana larvae (i.e. an endogenous immune cycle). This feeding activity profile has been supposedly shaped by daily dynamics in the strength of bottom-up and top-down forces mediated by plant and natural enemies, respectively, on the behaviour of the phytophagous insect. However, further investigations are required to test the hypothesis postulating that L.

botrana larvae adjust their investment in basal immunity as a specific response pattern to day/night variations in feeding activity and intensity of exposure to food-borne enemies. This work was conducted with almost aseptic and artificial food to investigate day/night variations in insect basal immunity. Hence, as a next step, the functional consequences of these immune fluctuations have to be carefully examined at the level of biotic interactions, in the event of more ecologically relevant risks of oral infection (e.g. larvae feeding upon antibiotic-free food). This procedure would help ascertaining the role of daily variations of immunity in preventing and/or dealing with infection, and estimate the specificity of these processes in face of attacks from a wide range of potential enemies [START_REF] Stone | The circadian clock protein timeless regulates phagocytosis of bacteria in Drosophila[END_REF] Represented values are means (± 95% c.i.). Significant differences are shown by asterisks

Fig. 1 .Fig. 2 .

 12 Fig. 1. Temporal design of the experiments and scheduling of the different behavioural

Fig. 3 .

 3 Fig. 3. Responses of haemocyte load (A), PO activity (B) and total PO activity (C) to the

Fig. 4 .

 4 Fig. 4. Lobesia botrana larvae were fixed in 4% paraformaldehyde solution and then

Fig. 5 .

 5 Fig. 5. Responses of the relative abundances of the four haemocyte types found in

FiguresFig. 1 .

 1 Figures

Fig. 2 .

 2 Fig. 2.Response of the time budget allocated to feeding (during an observation of 1 h) to the

Fig. 3 .

 3 Fig. 3. Responses of haemocyte load (A), PO activity (B) and total PO activity (C) to the

Fig. 5 .

 5 Fig. 5. Responses of the relative abundances of the four haemocyte types found in

  ;Westwood et al. 2019). Besides, other biotic (plant) and abiotic parameters (light, temperature) could have shaped the immune pattern observed, which could also evolve in response to the need of temporally segregating incompatible physiological processes or align immunity with other physiological rhythms(Westwood et al. 2019). Thus, assessing the contribution of such environmental variables to this day/night structure of host immunity would be of high relevance to identify the in situ environmental drivers of rhythms in traits underpinning host resistance and to extrapolate the results of this study to a broader ecological context. Such investigations should hold promise for improving the general understanding of the temporal variations of insect immunity through the lens of evolutionary and ecological immunology. contamination. Journal of InsectPhysiology, 77,[55][56][57][58][59][60][61] https://doi.org/10.1016/j.jinsphys.2015.04.003Vogelweith, F., Moret, Y., Monceau, K., Thiéry, D., & Moreau, J. (2016). The relative abundance of hemocyte types in a polyphagous moth larva depends on diet. Journal of

	Insect Physiology, 88, 33-39. https://doi.org/10.1016/J.JINSPHYS.2016.02.010
	Westwood, M. L., O'Donnell, A. J., de Bekker, C., Lively, C. M., Zuk, M., & Reece, S. E.
	(2019). The evolutionary ecology of circadian rhythms in infection. Nature Ecology and
	Evolution, 3, 552-560. https://doi.org/10.1038/s41559-019-0831-4
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	lepidopteran insects. Developmental and Comparative Immunology, 64, 65-74.

https://doi.org/10.1016/j.dci.2016.02.010

Acknowledgements

We thank Lionel Delbac and Samuel Ribeiro for their technical help in maintaining the insect stock and performing the experiments, respectively. We also want to thank Sébastien Zito and Benjamin Bois for providing the climatic data used in this study. We are grateful to Nathalie Jouy for her contribution to flow-cytometric analyses. This study was financially supported by the Conseil Régional de Bourgogne Franche-Comté through the Plan d'Actions Régional pour l'Innovation (PARI) and two other funding sources (FABER LOUAPRE AGREE-BGS,