Fanny Vogelweith
email: fannyvogelweith@gmail.com

Yannick Moret

Denis Thiéry

Lionel Delbac

Jérôme Moreau

Denis Thiery

No evidence of an immune adjustment in response to a parasitoid threat in Lobesia botrana larvae

Keywords: Campoplex capitator, insect immunity, grapevine moth, Phytomiptera nigrina, plasticity, prophylaxis

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

Introduction

Animals live in dynamic environments and face variation in resource availability, climate and risk of infection over their whole lifespan. Thus, to survive and reproduce successfully, these organisms must allocate resources among competing physiological systems, such as immunity and growth (van der [START_REF] Van Der Most | Trade-off between growth and immune function: A meta-analysis of selection experiments[END_REF], to maximize fitness in changing environments. Immunity is one of the major physiological mechanisms regulating host survival [START_REF] Lochmiller | Trade-offs in evolutionary immunology: just what is the cost of immunity?[END_REF]. In insects, an important part of this defense relies on nonspecific and constitutive mechanisms that involve the coordinated action of hemocytes and the phenoloxidase (PO) system [START_REF] Siva-Jothy | Insect Immunity : An Evolutionary Ecology Perspective[END_REF]. Hemocytes are immune cells circulating in the hemolymph involved in the recognition and encapsulation of pathogens [START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF]. Conversely, PO mostly mediates the melanization of foreign objects and operates through the activation of the prophenoloxidase (PPO) cascade, its inactive precursor typically stored in the hemolymph and the hemocytes [START_REF] Cerenius | The prophenoloxidase-activating system in invertebrates[END_REF].

Insect immunity is associated with inherent costs because it requires energy to build up, maintain and use [START_REF] Armitage | Examining costs of induced and constitutive immune investment in Tenebrio molitor[END_REF], and thus reduces an individual's ability to invest into other physiological systems. Moreover, the activation of insect immunity produces chemical substances that are harmful for the producer and induce cumulative damage in its body [START_REF] González-Santoyo | Phenoloxidase : a key component of the insect immune system[END_REF][START_REF] Nappi | Cytotoxicity and cytotoxie molecules in invertebrates[END_REF]. For this reason, individuals are expected to flexibly adjust their investment into the immune system to find an optimized balance between their ability to fight off pathogens on the one hand, and saving energy as well as limiting the accumulation of toxic immune components in their body on the other hand. Organisms can indeed be exposed to different threats such as parasites and parasitoids, which can vary among and between populations, as reported in Drosophila melanogaster [START_REF] Kraaijeveld | Geographical variation in encapsulation ability of Drosophila melanogaster larvae and evidence for parasitoid-specific components[END_REF][START_REF] Tinsley | Genetic variation in Drosophila melanogaster pathogen susceptibility[END_REF][START_REF] Promislow | Host ecology shapes geographical variation for resistance to bacterial infection in Drosophila melanogaster[END_REF], Gammarus pulex [START_REF] Cornet | Variation in immune defence among populations of Gammarus pulex (Crustacea: Amphipoda)[END_REF] and Lobesia botrana [START_REF] Moreau | Species diversity of larval parasitoids of the European grapevine moth (Lobesia botrana, Lepidoptera: Tortricidae): The influence of region and cultivar[END_REF]Vogelweith et al., 2013a). These different threats induce substantial variation in the investment in immunity within and among wild populations [START_REF] Promislow | Host ecology shapes geographical variation for resistance to bacterial infection in Drosophila melanogaster[END_REF][START_REF] Cornet | Variation in immune defence among populations of Gammarus pulex (Crustacea: Amphipoda)[END_REF]Vogelweith et al., 2013a). For example, recent investigations among natural populations of the grapevine moth (L. botrana) revealed that levels of immune defenses in larvae were positively correlated to parasitoid infection prevalence (Vogelweith et al., 2013a). At a geographical scale, populations with high levels of immune defenses in nonparasitized larvae (high hemocyte concentrations, PPO enzyme activities and antimicrobial peptides) were also those exhibiting the highest rates of infection by parasitoids (Vogelweith et al., 2013a).

Although this confirms that parasitoid pressure strongly affects host immune investment [START_REF] Bryan-Walker | Local adaptation of immunity against a trematode parasite in marine amphipod populations[END_REF][START_REF] Promislow | Host ecology shapes geographical variation for resistance to bacterial infection in Drosophila melanogaster[END_REF], the exact cause of such a positive relationship remains unknown. Two non-mutually exclusive selective pressures could shape and drive such a variation in immune traits. First, parasites can mediate a local selection response in favor of (constitutively expressed) high levels of immune defense among hosts, which would result in positive covariation between parasite prevalence and immune defense [START_REF] Lindström | Immunological investments reflect parasite abundance in island populations of Darwin's finches[END_REF][START_REF] Kalbe | Local differences in immunocompetence reflect resistance of sticklebacks against the eye fluke Diplostomum pseudospathaceum[END_REF][START_REF] Tschirren | Parasites shape the optimal investment in immunity[END_REF]. Alternatively, such a positive correlation could arise if hosts plastically adjust their immune response to the (locally varying) risk of infection, since the ability to sense the current risk of parasitism through visual, chemical, or mechanical cues has been reported in some insect species [START_REF] Peacor | Phenotypic modifications to conspecific density arising from predation risk assessment[END_REF][START_REF] Fievet | Predation risk cues associated with killed conspecifics affect the behavior and reproduction of prey animals[END_REF]. For example, when the risk of parasitism increases, insects show prophylactic investment in their immune defense even in the absence of parasites [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][START_REF] Wilson | Density-dependent prophylaxis: evidence from Lepidopterabaculovirus interactions?[END_REF]. The velvetbean caterpillar (Anticarsia gemmatalis) increased investment in primary defense barriers against parasites, such as the midgut, in response to increased conspecific density and an increased risk of infection [START_REF] Silva | Density-dependent prophylaxis in primary antiparasite barriers in the velvetbean caterpillar[END_REF]. Such a plastic modulation of immune defenses can be rapid as reported in adult bumble-bee workers (Bombus terrestris) [START_REF] Ruiz-González | Rapid induction of immune densitydependent prophylaxis in adult social insects[END_REF]. A plastic investment in immunity is then likely to result in positive covariation between parasite prevalence and immune defense. To understand how parasitoids can modulate insect immunity, it is important to know which of the two above detailed selective pressures shapes investment into insect immunity.

To our knowledge, no study experimentally investigated how insect can plastically modulate their immune defenses in response to parasitoid pressure. Here, we test the hypothesis whether the presence of parasitoids in the local environment induces a plastic increase of the investment in immune defense. To this end, we exposed L. botrana larvae to either parasitoids (excluded physical contacts), or no parasitoid for a period of six days and measured immune parameters (hemocyte concentration and PPO activities system) on the last day of exposure. If L. botrana larvae indeed flexibly adjust their immunity to the parasitoid threat, we expected an increase of all immune parameters in the presence of parasitoids. In this scenario, the larvae of L. botrana would assess cues related to the presence of parasitoids and adjust their investment in immunity to match any increased threat of infection. L. botrana appears to be a good candidate because (1) a variation of larval immune parameters with the parasitoid pressure has already been reported among wild populations (Vogelweith et al., 2013a) and (2) the ability of L. botrana larvae to sense the current in the risk of parasitism has been previously demonstrated (Vogelweith et al., 2013b).

Material and methods

Model insect

The European grapevine moth L. botrana (Lepidoptera: Tortricidae) is currently the major grape pest in Europe and has also spread to American viticulture [START_REF] Gilligan | Discovery of Lobesia botrana ([Denis & Schiffermüller]) in California: an invasive species new to North America (Lepidoptera: Tortricidae)[END_REF]. This grapevine moth is a polyphagous insect that completes 2-5 generations a year (depending on latitude) on different grape cultivars, where the larvae can do considerable damage [START_REF] Thiéry | Les ravageurs de la vigne[END_REF]. The larvae used for this experiment come from inbred stock maintained diapause-free at the French National Institute for Agricultural Research (INRA) (Villenave d'Ornon, France) for more than 10 years. This strain is based on a great number of caged adults (several thousand per week) to which wild adults are periodically added. This laboratory strain has conserved some plasticity because considerable variation is found in immune parameters between larvae [START_REF] Muller | Immune benefits from alternative host plants could maintain polyphagy in a phytophagous insect[END_REF]. In addition, a very similar pattern in terms of basal immunity, response to an immune challenge and parasitoid escape behavior have been found between inbred stock and wild populations sampled in French vineyards [START_REF] Vogelweith | Defense strategies used by two sympatric vineyard moth pests[END_REF]. We used larvae from the laboratory to ensure that they were not previously exposed to parasitoids because this could have affected their immunity. Larvae were fed with an ad libitum amount of a semiartificial diet [START_REF] Vogelweith | Food-mediated modulation of immunity in a phytophagous insect: An effect of nutrition rather than parasitic contamination[END_REF], and maintained in boxes (18 × 11.5 × 7 cm) under standard laboratory conditions (22 ± 1°C, 70 ± 10% r.h., and a L16:D8 photoperiod) at a density of approximately 100 individuals per 300 ml of diet.

Parasitoid populations

To mimic imminent parasitoid attack on the larvae, we used the two most common parasitoids of L. botrana larvae: Campolex capitator (Hymenoptera: Ichneumonidae) and Phytomyptera nigrina (Diptera: Tachinidae) [START_REF] Moreau | Species diversity of larval parasitoids of the European grapevine moth (Lobesia botrana, Lepidoptera: Tortricidae): The influence of region and cultivar[END_REF]. These parasitoids are known to be larvalparasitoid only and parasitize larvae of L. botrana from the 2 nd to the 4 th instar in vineyards in the south of France where they occur in sympatry [START_REF] Thiéry | Les ravageurs de la vigne[END_REF]. The parasitism rate of these two parasitoids strongly depends on the year and the vineyard considered [START_REF] Moreau | Species diversity of larval parasitoids of the European grapevine moth (Lobesia botrana, Lepidoptera: Tortricidae): The influence of region and cultivar[END_REF]Vogelweith et al., 2013a). For instance, in 2012, we observed a parasitism rate of 10% by C. capitator and 5% by P. nigra while in 2011, we found a parasitism rate of 90% by C. capitator in the same vineyard (Vogelweith, unpublish data). Both parasitoid species were obtained from parasitized L. botrana larvae collected on Grenache in a vineyard close to Perpignan (southern France) in May 2012 using the procedure by Vogelweith et al. (2013a). In brief, the larvae were collected at the end of their development in grapes and kept in large polyethylene boxes (60 × 40 × 21.4 cm) in the laboratory where they were checked daily for pupation. Pupae were placed individually in glass tubes (70 × 9 mm diameter) closed with cotton plugs and stored under standard laboratory conditions (22 ± 1°C, 70 ± 10% r.h., and L16:D8 photoperiod). The tubes were checked daily for the emergence of either the moth or the parasitoid. All the parasitoids were of the same age and were used two days after their emergence in the experiment.

Experimental design

Fourth instar larvae from the laboratory culture (see above) were exposed in groups of five to one female parasitoid (either C. capitator or P. nigrina) to assess the effect of parasitoid presence on larval immune parameters. In a previous experiment using then same protocol (Vogelweith et al., 2013b), we did not find any difference between control groups either exposed to a non-parasitoid fly or nothing. For this reason, we used additional groups of unexposed L. botrana larvae (empty cup) as sole control. The groups of larvae were placed in plastic boxes (98 × 98 × 49 mm) containing 50 ml of semi-artificial diet [START_REF] Vogelweith | Food-mediated modulation of immunity in a phytophagous insect: An effect of nutrition rather than parasitic contamination[END_REF] (Fig. 1). A plastic cup (30 mm diameter and 30 mm height) containing one female of C. capitator or P. nigrina (n = 6 parasitoids and n = 30 larvae, respectively), or no parasitoid (n = 5 and n = 20 larvae) was positioned in the middle of each box. The central cup was transparent and pierced with small holes, which allowed for visual, olfactory and vibratory stimulation of L. botrana larvae by the parasitoid. The parasitoids were offered a drop of honey as food for the duration of the experiment. The experimental boxes were maintained in standard laboratory conditions (22 ± 1°C, 70 ± 10% r.h., and L16:D8 photoperiod). Six days later, a sample of hemolymph was collected from each larva for measurements of the hemocyte concentration and the activity of the PO-PPO system as described in [START_REF] Vogelweith | Host plant variation plastically impacts different traits of the immune system of a phytophagous insect[END_REF]. The larvae that reached metamorphosis (5%) did not provide hemolymph and were equally distributed among treatments. Note that we did not directly expose the larvae to the parasitoid because of the very aggressive behavior of C. capitator; since this parasitoid is able to perform more than 10 stings in less than a minute (Vogelweith, unpublished data) inducing consequent injuries to the larvae, possibly affecting immune measurements.

Hemolymph collection and measurement of the three immune parameters

We measured three key immune parameters on 4 th instar larvae well known to reflect parasitoid resistance [START_REF] Prévost | Hemocyte load and immune resistance to Asobara tabida are correlated in species of the Drosophila melanogaster subgroup[END_REF][START_REF] Carton | Insect immune resistance to parasitoids[END_REF]: the concentration of circulating hemocytes, as well as the PO and total-PO activity, assays established to be sensitive enough to detect rapid changes in immunity [START_REF] Ruiz-González | Rapid induction of immune densitydependent prophylaxis in adult social insects[END_REF]Vogelweith et al., 2013c).

Each larva was chilled on ice for 20 min, and 1 µl of hemolymph was first extracted with a sterile glass capillary (Hirschmann Laborgeräte, Eberstadt, Germany) from a wound inflicted to the posterior part of the ventral side of the abdomen. This extract was immediately diluted in 25 µl of cold sodium cacodylate/CaCl2 buffer (0.01 M sodium cacodylate, 0.005 M CaCl2; pH 6.5) to measure the two immune parameters. A 10 µl sample of this solution was immediately removed for the counting of hemocytes; the remainder was stored at -27°C for measurement of the enzymatic activity of the PPO system.

The concentration of hemocytes was measured immediately after hemolymph extraction. This measurement was done using a Neubauer Improved Haemocytometer under phase contrast microscopy (magnification x 400), as described in [START_REF] Vogelweith | Host plant variation plastically impacts different traits of the immune system of a phytophagous insect[END_REF]. The activity of the PPO system was estimated by measuring the enzymatic activity of naturally activated PO enzymes (PO activity), and the activity of the proenzyme together with that of the activated PO (total-PO activity). These measurements were based on a spectrophotometric assay described by [START_REF] Vogelweith | Host plant variation plastically impacts different traits of the immune system of a phytophagous insect[END_REF]Vogelweith et al. ( , 2013a[START_REF] Vogelweith | Defense strategies used by two sympatric vineyard moth pests[END_REF].

Body size of larvae

After the collection of hemolymph, larval body size was estimated by measuring the distance between the most distant lateral sides of the head capsule margins (HC width) [START_REF] Delbac | Larval instars determination for the European Grapevine Moth (Lepidoptera: Tortricidae) based on the frequency distribution of headcapsule widths[END_REF]Vogelweith et al., 2013c) using a Nikon SMZ-10A stereoscopic microscope and a VTO 232 video analysis system (Linkam Scientific Instruments).

Statistical analyses

All statistical analyses were conducted using the software R v3.1.2 loaded with the packages car, lme4 and MASS. The hypothesis of a plastic adjustment of immune parameters (hemocyte concentration, PO and total-PO activities) to the parasitoid presence was analyzed using three linear mixed models (LMMs), in which the parasitoid presence, parasitoid species and the body size of the larvae were entered as explanatory variables, whereas the ID of experimental box was used as a random effect. Note that to fulfill homoscedasticity and Gaussian distribution, these models were computed using log+1-transformed hemocyte concentration, PO and total-PO activities. We used Pearson's rank correlation coefficient with a confidence interval of 95 % (C.I.) for the relationship between immune parameters and larval body size (Extract Model Fitted Values). When the C.I. included 0, the correlation was not significant.

Results

The presence of parasitoid and parasitoid species had no effect on hemocyte concentration (F2 = 0.48; p = 0.786), PO (F2 = 0.85; p = 0.651) and total-PO activities (F2 = 2.90; p = 0.235) (Fig. 2). However, all immune parameters decreased with larval body size (Hemocyte concentration:

Discussion

This study aimed to explain the positive relationship between immune parameters and parasitoid prevalence previously observed in populations of L. botrana larvae inhabiting different French vineyards (Vogelweith et al., 2013a). In particular, we tested the hypothesis that the presence of parasitoids in the local environment can induce a plastic increase of the investment of L. botrana larvae into their immune defense. Our results show that the presence/absence of a parasitoid or the species of the parasitoid present in the environment did not affect immunity of L. botrana larvae, although our assays previously allowed us to measure reliable changes in immunity due to environmental variation and our protocol maximized the parasitoid perception by the larvae (Vogelweith et al., 2013b) compared to what presumably occurs under natural conditions. However, our results confirm the negative relationships between larval body size and immunity previously found and discussed in this species (Vogelweith et al., 2013a). To our knowledge, our study is the first to test variation of investment into the larval immune system in response to the presence of parasitoids.

Many animals are able to anticipate potential exposure to parasitoids and/or predators by sensing cues such as kairomones, inducing the alteration of several of their traits (e.g., avoidance behavior, morphological changes, and changes in oviposition site) to reduce the cost of infection/predation [START_REF] Dicke | Chemical Detection of Natural Enemies by Arthropods : An Ecological Perspective[END_REF][START_REF] Sloggett | Parasitoids induce production of the dispersal morph of the pea aphid, Acyrthosiphon pisum[END_REF]. For example, Sloggett and Wisser (2002) have shown that the pea aphid (Acyrthosiphon pisum) produces winged offspring that leave the host plant to flee colonies infected by parasitoids. Our experimental setting prevented any contact of the moth larvae with the parasitoids, and therefore excluded any direct effect of parasitoid infection, but allowed the larvae to assess different (acoustics, chemicals and visual) cues. We are confident that our experimental setup allowed the detection of the parasitoids by the larvae because, using the same protocol, we previously found that larvae of L. botrana accelerated their development in the presence of these parasitoids (Vogelweith et al., 2013b). Such a plastic alteration of the larval growth rate leading to earlier metamorphosis is believed to be adaptive, as it reduces the temporal exposure of the larvae to the parasitoid. Altogether, these results suggest that developmental acceleration appears to be the better option to escape parasitoid infection as compare to enhancing immunity.

Overall, our results allow us to reject the hypothesis of a plastic change in immunity to explain the positive relationship between larval immunity and parasitism pressure in L. botrana. This suggests that the immunity of L. botrana larvae might be shaped byand adapted tolocal conditions in response to the local selective pressure imposed by parasites, as demonstrated in others species [START_REF] Bryan-Walker | Local adaptation of immunity against a trematode parasite in marine amphipod populations[END_REF][START_REF] Kortet | Boldness in anti-predator behaviour and immune defence in field crickets[END_REF]. Because immunity is costly to express [START_REF] Kraaijeveld | Trade-off between parasitoid resistance and larval competitive ability in Drosophila melanogaster[END_REF], natural selection should favor optimized investments into immune defenses matching the local prevalence and severity of parasitic infections [START_REF] Kalbe | Local differences in immunocompetence reflect resistance of sticklebacks against the eye fluke Diplostomum pseudospathaceum[END_REF]. Consequently, on a micro-evolutionary scale, natural selection should favor reduced levels of immune defense in populations subjected to low parasite pressure [START_REF] Kalbe | Local differences in immunocompetence reflect resistance of sticklebacks against the eye fluke Diplostomum pseudospathaceum[END_REF][START_REF] Lindström | Immunological investments reflect parasite abundance in island populations of Darwin's finches[END_REF], whereas on macroevolutionary scale, immunity should protect species from parasitism has already been found in 16 species of Lepidoptera [START_REF] Smilanich | The insect immune response and other putative defenses as effective predictors of parasitism[END_REF]. Further studies should investigate whether local parasitism pressure leads to population-specific micro-evolutionary changes in the immune defense of L. botrana larvae. The monitoring and measurements of both larval immunity and parasitism in different vineyards over several years could help to investigate this second hypothesis.

F1 = 7

 7 .08, p = 0.008, and rho = -0.96, C.I. 95% = [-0.98; -0.95]; PO activity: F1 = 8.59, p = 0.003, and rho = -0.91, C.I. 95% = [-0.94; -0.86]; Total-PO activity: F1 = 5.27, p = 0.021, and rho = -0.73, C.I. 95% = [-0.82; -0.61]).

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Immune response of L. botrana larvae to the presence of a parasitoid. (a) Hemocyte

  Figure 1

Acknowledgments

We thank Maximilian Körner and Jos Kramer for proof-readings. This study was supported by the Centre National de la Recherche Scientifique (CNRS) and by grants from the Agence Nationale de la Recherche (ANR) (Y.M., ANR-07-JCJC-0134, ANR-08-JCJC-0006); the Conseil Regional of Bourgogne (J.M.); and the Alexander von Humboldt Foundation (F.V.).

Conflict of interest

The authors declare no competing financial interests.


Corentin Iltis
email: corentin.iltis@u-bourgogne.fr.

Jérôme Moreau

Karolina Pecharová

Denis Thiéry

Philippe Louapre

Philippe Louâpre

Reproductive performance of the European grapevine moth Lobesia botrana (Tortricidae) is adversely affected by warming scenario

Keywords: Fluctuating thermal regimes, global warming, grapevine pest, Lobesia botrana, population dynamics, sperm quality

teaching and research institutions in France or abroad, or from public or private research centers.   Reproductive performance of the European grapevine moth Lobesia botrana (Tortricidae) is adversely affected by warming scenario.

Introduction

Viticulture is facing new threats and challenges worldwide, owing chiefly to anthropogenic disturbances [START_REF] Fraga | An overview of climate change impacts on European viticulture[END_REF]). In the current climatic context, rising temperatures are predicted to impact grapevine cultivation by modulating the risks of pest outbreaks and emergence of insect-borne diseases [START_REF] Fraga | An overview of climate change impacts on European viticulture[END_REF][START_REF] Reineke | Grapevine insect pests and their natural enemies in the age of global warming[END_REF]. Indeed, grapevine is targeted by numerous insect pests exerting detrimental effects on grape production through feeding on plant tissues and/or by acting as vectors of plant diseases [START_REF] Reineke | Grapevine insect pests and their natural enemies in the age of global warming[END_REF][START_REF] Iltis | When warmer means weaker: high temperatures reduce behavioural and immune defences of the larvae of a major grapevine pest[END_REF]. As ectothermic animals, phytophagous insects are very sensitive to environmental temperature, which regulates their physiology, individual performance and population dynamics in space (distribution range) and time (life cycle duration, phenology) [START_REF] Bale | Herbivory in global climate change research: direct effects of rising temperature on insect herbivores[END_REF][START_REF] Reineke | Grapevine insect pests and their natural enemies in the age of global warming[END_REF]. In view of the many threats global warming poses to the long-term viability of vineyards, to predict how the projected increase in global mean temperature might affect the population dynamics of major grapevine pests and anticipate the possible repercussions on grapevine cultivation has emerged as one major challenge in recent viticultural research [START_REF] Reineke | Grapevine insect pests and their natural enemies in the age of global warming[END_REF][START_REF] Castex | Pest management under climate change: the importance of understanding tritrophic relations[END_REF][START_REF] Iltis | When warmer means weaker: high temperatures reduce behavioural and immune defences of the larvae of a major grapevine pest[END_REF].

The European grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae), is one of the most harmful viticultural pests in the Palearctic region [START_REF] Iltis | When warmer means weaker: high temperatures reduce behavioural and immune defences of the larvae of a major grapevine pest[END_REF]. Quantitative and qualitative damages are triggered by caterpillars while attacking vine fructiferous organs (floral buds, ripening and mature berries). Larval feeding on berries induces severe yield losses because of direct consumption of agricultural production. Moreover, it allows the intrusion of several fungal agents responsible for bunch rot development (Botrytis cinerea, Aspergilus carbonarius, A. niger) and increases the plant susceptibility to other grape berry boring pests like fruit flies [START_REF] Cozzi | Effect of Lobesia botrana damages on black aspergilli rot and ochratoxin A content in grapes[END_REF][START_REF] Reineke | Grapevine insect pests and their natural enemies in the age of global warming[END_REF]. The economic impacts of L. botrana have been rated as 'high', provided that one caterpillar might infest between 2 and 10 berries throughout its development, and that density might reach up to 20-30 larvae per bunch in heavily infested vineyards [START_REF] Delbac | Damage to grape flowers and berries by Lobesia botrana larvae (Denis & Schiffernüller) (Lepidoptera: Tortricidae), and relation to larval age[END_REF]. High levels of economic injury due to L. botrana activity are also suggested by considerable yield losses like those recorded in Greece, amounting from 13.3% to 27% of the total grape production over a 4-year period [START_REF] Moschos | Yield loss quantification and economic injury level estimation for the carpophagous generations of the European grapevine moth Lobesia botrana Den. et Schiff. (Lepidoptera: Tortricidae)[END_REF]).

Predictions about L. botrana population dynamics under global warming scenario are usually derived from physiologically-based demographic models (PBDMs) [START_REF] Gutierrez | Prospective analysis of the invasive potential of the European grapevine moth Lobesia botrana (Den. & Schiff.) in California[END_REF][START_REF] Gutierrez | Climate warming effects on grape and grapevine moth (Lobesia botrana) in the Palearctic region[END_REF]. These mechanistic models rely on equations describing the effects of temperature on physiological processes enabling to infer population growth, above all developmental rates, survival and per capita fecundity (total number of eggs laid by one female) [START_REF] Gutierrez | Prospective analysis of the invasive potential of the European grapevine moth Lobesia botrana (Den. & Schiff.) in California[END_REF][START_REF] Gutierrez | Climate warming effects on grape and grapevine moth (Lobesia botrana) in the Palearctic region[END_REF][START_REF] Gilioli | A modelling framework for pest population dynamics and management: an application to the grape berry moth[END_REF]. PBDMs can be coupled with in situ weather data and climate change scenarios to project the current and future geographical distribution of L. botrana across a lattice of spatial cells. This way, PBDMs allow to anticipate the local dynamics of L. botrana populations in terms of abundance and phenology [START_REF] Gutierrez | Prospective analysis of the invasive potential of the European grapevine moth Lobesia botrana (Den. & Schiff.) in California[END_REF][START_REF] Gutierrez | Climate warming effects on grape and grapevine moth (Lobesia botrana) in the Palearctic region[END_REF]. These models predict that, concurrently with grape yield, the abundance of L. botrana populations should increase northward and in higher elevations of the Euro-Mediterranean region, while the opposite trend should be observed for southern areas where summer conditions will adversely impact the moth vital functions and reduce grape yields [START_REF] Gutierrez | Climate warming effects on grape and grapevine moth (Lobesia botrana) in the Palearctic region[END_REF].

Notwithstanding their ecological realism (i.e. the possibility to include the bottom-up effects of grapevine phenological stage on L. botrana physiology), PBDMs suffer from shortcomings that could limit their ability to accurately forecast the future of L. botrana populations in a rapidly changing climatic context. First, their equations are based on data obtained under constant temperatures [START_REF] Gutierrez | Prospective analysis of the invasive potential of the European grapevine moth Lobesia botrana (Den. & Schiff.) in California[END_REF]. For that reason, their predictions might differ from biological conclusions drawn by comparing the performance of L. botrana under daily fluctuating thermal regimes, which reflect more realistically the day/night variations of temperatures experienced by insects in their natural environment [START_REF] Colinet | Insects in fluctuating thermal environments[END_REF]. Second, by expressing fecundity as a function of temperature and female's age, PBDMs implicitly rule out the importance of male's reproductive performance on female's egg production. Nonetheless, in L. botrana as in many other lepidopteran species, the spermatophore delivered during copulation does not only include sperm but also accessory gland secretions containing nutrients absorbed by female to boost her egg production [START_REF] South | The influence of male ejaculate quantity on female fitness: a meta-analysis[END_REF]. Since L. botrana is a capital breeding species (i.e. with a non-feeding adult stage), the quality of the nutritive ejaculate delivered by male during copulation importantly mediates the resources allocated to egg production and the reproductive output of the pair (number of eggs laid, hatching success) [START_REF] Muller | Male larval nutrition affects adult reproductive success in wild European grapevine moth (Lobesia botrana)[END_REF](Muller et al. , 2016a)). Provided that ejaculate quality is usually temperature-dependent in arthropod species (Zeh et al. 2012;Vasudeva et al. 2014), addressing the effects of warming on male's ejaculatory traits is required for a better understanding of the consequences of climate change for L. botrana reproduction and population dynamics.

In view of the concerns raised above, we performed laboratory experiments to investigate the consequences of local warming scenario for the performance of L. botrana adult females and males. More specifically, our study aimed at investigating the consequences of temperatures experienced by L. botrana individuals during their whole life on the expression of reproductive traits. For this purpose, we compared the performance of each sex separately between two daily fluctuating thermal regimes designed to reflect current or simulate future thermal conditions in our study area (Burgundy, Eastern France). We recorded several surrogates for overall performance in both sexes, including body amounts of four major energetic components available upon moth emergence (proteins, lipids, glycogen, soluble carbohydrates), and adult lifespan. Additionally, we assessed the effects of temperature on the reproductive traits of females (mating success, number of eggs laid, hatching success, oviposition dynamics) and males (mating success, spermatophore volume, sperm quality).

Previous works focused on the consequences of rising temperatures for L. botrana larval performance reported that caterpillars of this species respond to warmer conditions by speeding up their larval development, which implies a reduced time allowed for feeding [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 multi-trait approach with fine-grain climate modeling[END_REF]. Importantly, such findings have been observed when comparing the two fluctuating regimes used in this study [START_REF] Iltis | Are life-history traits equally affected by global warming? A case study combining a multi-trait approach with fine-grain climate modeling[END_REF]. We therefore expect that this plastic reduction in the duration of the feeding stage should negatively adult performance, resulting in lower stocks of energetic components available upon moth emergence, shortened adult lifespan and decreased reproductive performance of both sexual partners, which crucially hinges on resources acquired through the alimentation of larval stages [START_REF] Moreau | Grape variety affects larval performance and also female reproductive performance of the European grapevine moth Lobesia botrana (Lepidoptera: Tortricidae)[END_REF][START_REF] Muller | Male larval nutrition affects adult reproductive success in wild European grapevine moth (Lobesia botrana)[END_REF].

Materials and methods

Thermal regimes

This study involved two daily fluctuating thermal regimes designed to reflect current and expected future thermal conditions in Burgundy, Eastern France (Longvic-Dijon weather station, 47,27°N; 5,09°E; altitude = 219 m) (www.meteofrance.com). Both these two regimes aimed at emulating an average day during summer period (15 th July -15 th August), because L. botrana exhibits high levels of activity (i.e. one peak of adult emergence) over this date range [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 regime used to simulate summer conditions currently found in Burgundy, called the Current Fluctuating Regime (CFR), was built based on a 20-year recording of in situ hourly temperatures over a recent period (1995)(1996)(1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014). The CFR was composed of six segments of temperatures, each lasting 4h and calculated as the mean segment over the 30 days (15 th July -15 th August) and the 20 years studied.

Alongside the CFR, we designed a scenario termed Future Fluctuating Regime (FFR), aimed at simulating one possible trajectory for local climate in Burgundy, and based on summer thermal conditions likely to occur in this region for a period covering the years 2081 to 2100. The FFR was generated according to the projections provided by six different climate simulations (spatial resolution: 12.5 km), each involving a combination of one General Circulation Model and one Regional Climate Model nested within [START_REF] Taylor | An overview of CMIP5 and the experiment design[END_REF][START_REF] Jacob | EURO-CORDEX: new high-resolution climate change projections for European impact research[END_REF]. All these simulations were run with the most pessimistic of the Representative Concentration Pathway (RCP) scenarios, termed RCP 8.5, which predicts the highest positive radiative forcing (i.e. high absorbance of solar and infrared radiation) as a consequence of massive and growing future emissions of greenhouse gas [START_REF] Moss | The next generation of scenarios for climate change research and assessment[END_REF]. The FFR included six segments of temperatures, each lasting 4h and corresponding to the mean segment over the six simulations performed. All meteorological data used to design the thermal regimes are freely available on the Drias portal (www.drias-climat.fr). The comparison of CFR and FFR indicates that global warming is likely to increase both the mean temperature (+ 5.3°C) and the daily thermal range (+ 0.6°C) in our focal area (Table 1).

Moth rearing and larvae collection

An inbred strain of L. botrana provided the individuals used in this study (French National Institute for Agricultural Research, Villenave-d'Ornon, France). Insect stock arose from large numbers of caged adults (several thousand each week), kept without diapause under standard laboratory conditions (20 ± 0.5°C, 60 ± 5% relative humidity, photoperiod of L17: D6 with 1h of dusk). During photophase, luminosity varied between 600 lx (daytime) and 100 lx (dusk).

Bands of waxed paper (5 x 10 cm) were hung inside rearing cages for eggs laying. Once these oviposition supports received a sufficient number of eggs (after 2-3 days), they were renewed and placed in plastic boxes covered with a daily humidified piece of paper towel to protect eggs from desiccation until hatching.

Adults were obtained by initially collecting 2043 newly hatched larvae (age < 24h) from the rearing stock with a fine paintbrush. To avoid any experimental bias attributable to larval competition and subsequent food deprivation (Thiéry et al. 2014), larvae were individually reared in Eppendorf tubes filled with 1.5 ml of artificial culture medium (composition for 1000 ml: 1000 ml water, 15 g agar, 86.6 g maize flour, 41.3 g wheat germ, 45.5 g beer yeast, 6 g ascorbic acid, 3.4 g mineral salt, 128 mg pyrimethanil, 2.7 g benzoic acid, 2.8 g methyl 4-hydroxybenzoate and 5 ml 95% ethanol). Tube lids were pierced for air circulation and covered with a piece of fine mesh fabric to prevent larvae from escaping.

Freshly hatched larvae were then randomly allocated to one thermal regime (CFR or FFR) programmed with climate chambers (ST 2/2 BASIC, Pol-Eko Aparatura, Wodzisław Śląski, Poland). Abiotic conditions inside these incubators were controlled (temperature ± 0.1°C, 50 ± 10% relative humidity, L18: D6, 650 lx). According to a previous study, daily monitoring of pupation started only 2 weeks (if caterpillars were reared in the FFR) or 4 weeks (if they were reared in the CFR) after initial collection to minimise disturbance [START_REF] Iltis | Are life-history traits equally affected by global warming? A case study combining a multi-trait approach with fine-grain climate modeling[END_REF].

Immediately following pupation, chrysalises were delicately extracted from their cocoon and weighted to the nearest 0.1 mg with a balance (Pioneer PA214C, OHAUS, Greifensee, Switzerland). They were then individually transferred into glass tubes (70 x 9 mm diameter) closed with cotton and were returned to their rearing thermal regime to end their development. Tubes were inspected once a day to monitor adult emergence. Freshly emerged moths were sexed and supplied with water ad libitum through daily soaked cotton plugs. They were then randomly distributed among three groups all along the experiments, because the array of measurements included in the study could not have been performed on the same individuals. In Group 1, adults were frozen to subsequently quantify their body amounts of four major energetic components upon adult emergence. In Group 2, adults were held in their rearing thermal regime until death to record adult lifespan. In Group 3, adults were mated with standard individuals (originating from the laboratory insect stock) to assess the reproductive performance of each sex separately.

Group 1: Body amounts of four major energetic components upon emergence Energetic assessments were run on 75 females (n = 37 for CFR, n = 38 for FFR) and 80 males (n = 36 for CFR, n = 44 for FFR). Within the 12h following their emergence, moths were individually placed in Eppendorf tubes and immediately frozen in liquid nitrogen. They were kept at -80°C for later quantification of their energetic states. Wings were cut before moths were crushed in lysis buffer, as they are covered with scales which might disturb the biochemical assays based on absorbance measurements. Four major energetic components involved in energy production in insects were quantified through spectrophotometry methods: proteins, lipids, glycogen and soluble carbohydrates. The total amounts of soluble proteins available was quantified using a DC Protein Assay kit involving Bradford reagent (Bio-Rad, Hercules, USA). The assessments of other energetic components (lipids, glycogen, soluble carbohydrates) were performed with the method described by [START_REF] Foray | A handbook for uncovering the complete energetic budget in insects: the van Handel's method (1985) revisited[END_REF]. The repeatability of the measurements was assessed by calculating the coefficient of variation over two replicates for protein and lipid body amounts, and excluding the individuals for which this coefficient exceeded 30% (see [START_REF] Iltis | Are life-history traits equally affected by global warming? A case study combining a multi-trait approach with fine-grain climate modeling[END_REF]. Repeatability was relatively high, as indicated by the low percentages of individuals removed from the protein dataset (3.2%) and the lipid dataset (0.6%). More specifically, this selection procedure led to the exclusion of 3 females (n = 2 for CFR, n = 1 for FFR) and 2 males (n = 2 for FFR) from the protein dataset, and 1 female (n = 1 for FFR) from the lipid dataset.

Group 2: Adult lifespan

This group involved 76 females (n = 28 for CFR, n = 48 for FFR) and 73 males (n = 24 for CFR, n = 49 for FFR). Immediately following their emergence, freshly emerged moths were individually kept in clean glass tubes (100 x 15 mm diameter) within their rearing thermal regime. They were supplied daily with water ad libitum through soaked cotton plug, and monitored once a day (during water provisioning) until death. We then recorded the adult lifespan (in days), expressed as the time elapsed between imaginal emergence and death.

Group 3: Reproductive performance of females and males

A total of 99 females (n = 42 for CFR, n = 57 for FFR) and 105 males (n = 53 for CFR, n = 52 for FFR) were allowed to mate in order to assess their reproductive performance. All these individuals were virgin and 24h-old. At dusk, one randomly selected 24h-old virgin individual (female or male) reared in either the CFR or the FFR was confined in a glass mating tube ( 100x 15 mm diameter) with one 24h-old virgin standard individual of the opposite sex. These standardised individuals were obtained from the inbred stock population and were weighted as pupae (precision: ± 0.1 mg) with a balance (Pioneer PA214C, OHAUS, Greifensee, Switzerland). They were considered equivalent in terms of quality as sexual partners given the relatively low genetic variation between them and the same standard environmental conditions (see above) they met during development [START_REF] Moreau | Grape variety affects larval performance and also female reproductive performance of the European grapevine moth Lobesia botrana (Lepidoptera: Tortricidae)[END_REF]). Using standardised individuals as sexual partners for individuals assigned to the two thermal treatments enabled to specifically assess the effects of rearing temperature on the reproductive performance of each sex separately (see [START_REF] Moreau | Grape variety affects larval performance and also female reproductive performance of the European grapevine moth Lobesia botrana (Lepidoptera: Tortricidae)[END_REF][START_REF] Muller | Male larval nutrition affects adult reproductive success in wild European grapevine moth (Lobesia botrana)[END_REF]. Mating trials lasted 3h under controlled conditions (23.1 ± 0.5°C, 60 ± 5% relative humidity, 100 lx). This duration was selected because the latency to mate in this species does not exceed 3h in cases of no-choice mating trials (Muller et al. 2016b). Temperature was set at 23.1°C (intermediate temperature between the mean temperature of the CFR and the FFR) during mating experiments, irrespectively of the thermal regime from which tested individuals originated. Luminosity during mating trials was set at 100 lx to reflect dusk conditions, as the reproduction of this species occurs at dusk in natural settings. Mating tubes were examined every 15 min to check if genital coupling was initiated. In this species, the complete transfer of the spermatophore during copulation requires approximatively 1h [START_REF] Muller | Male larval nutrition affects adult reproductive success in wild European grapevine moth (Lobesia botrana)[END_REF](Muller et al. , 2016a, b), b). Hence, a time lapse of 15 min between two successive inspections is short enough to ensure that every effective mating could have been detected. Individuals for which no copulation was observed within 3h were considered unmated and were discarded from the rest of the experiments.

In cases where a female reared in either the CFR or FFR was mated with a standard male, the male was immediately removed after the pair separation and no longer used in the experiments. The female was left in her mating tube and her rearing thermal regime for oviposition. Water was daily provided ad libitum through soaked cotton plug. Each tube was checked every morning (during water supply) through visual inspection to count the number of eggs deposited by female until death. Ten days after the end of laying period (which extended over approximately 6 days within the CFR and 4 days within the FFR), egg tubes were examined with a stereomicroscope at 8x magnification to calculate the proportion of the eggs that successfully hatched (fertile eggs). An incubation period of at least 10 days ensured that all viable larvae had hatched from the fertile eggs (Torres-Vila et al. 1999;[START_REF] Moreau | Grape variety affects larval performance and also female reproductive performance of the European grapevine moth Lobesia botrana (Lepidoptera: Tortricidae)[END_REF][START_REF] Muller | Male larval nutrition affects adult reproductive success in wild European grapevine moth (Lobesia botrana)[END_REF]. Mating was considered successful when the tested female laid at least one fertile egg, thus indicating an effective delivering of the spermatophore during copulation. Based on these observations, we recorded four metrics indicative of female's reproductive performance: (i) mating success (proportion of the females that laid at least one fertile egg), (ii) fecundity (total number of eggs deposited by each mated female), (iii) fertility (proportion of the eggs that successfully hatched) and (iv) laying rate (in eggs.day -1 , expressed as the ratio between the total number of eggs deposited by each mated female and the number of days elapsed between the appearance of the first and the last eggs laid) [START_REF] Moreau | Grape variety affects larval performance and also female reproductive performance of the European grapevine moth Lobesia botrana (Lepidoptera: Tortricidae)[END_REF].

In cases where a male reared in either the CFR or FFR was mated with a standard female, the female was removed at the end of copulation and frozen at -80°C, while the male was no longer used in the experiments. The standard female was further dissected on a glass side to extract the bursa copulatrix containing male's spermatophore. The dissection first enabled to check for the presence of male's spermatophore inside female's reproductive tract (thus indicating successful mating), provided that spermatophore transfer might sometimes fail despite genital coupling. Once the spermatophore was collected, we extrapolated its size (volume), which provides information about the quantity of sperm and energetic substances transferred to female during copulation (Torres-Vila et al. 1999;[START_REF] Muller | Male larval nutrition affects adult reproductive success in wild European grapevine moth (Lobesia botrana)[END_REF]. For this purpose, we measured spermatophore dimensionsincluding length l, width w and thickness tusing a stereomicroscope (Stemi 508, Zeiss, Göttingen, Germany) at 50x magnification (precision ± 1 µm). Considering the spermatophore as an ellipsoid balloon, we then assessed its volume V using the mathematical expression V = π / 6 (l x w x t). Analyses of these photographic data were performed with the ZEN imaging software (version 2.3, Zeiss, Göttingen, Germany). Alongside spermatophore volume, we also evaluated sperm quality following the standard procedure developed by [START_REF] Muller | Male larval nutrition affects adult reproductive success in wild European grapevine moth (Lobesia botrana)[END_REF]. This involved rupturing the spermatophore in a drop of distilled water, delicately stirring the sperm mass to distribute the sperm evenly throughout the water and to isolate encysted eupyrene bundles. Each bundle contains 256 nucleated sperms involved in egg fertilisation. For this reason, the number of eupyrene bundles in the spermatophore can be interpreted as a surrogate for sperm quality, with demonstrated consequences for female's reproductive output [START_REF] Muller | Male larval nutrition affects adult reproductive success in wild European grapevine moth (Lobesia botrana)[END_REF](Muller et al. , 2016a)).

In summary, we included three metrics related to male's reproductive performance: (i) mating success (expressed as the proportion of males that successfully delivered a spermatophore during copulation), (ii) spermatophore volume and (iii) number of eupyrene bundles (sperm quality).

Statistical analyses

For the body amounts of four energetic components, statistical models incorporated thermal regime, sex, their interaction and pupal mass as a single covariate to control for allometric effect on these energetic stocks. The data for body amounts of proteins, lipids and glycogen satisfied both normality and homoscedasticity assumptions. Hence, the variation in the amounts of these energetic components was decomposed by means of Analyses of Covariance (ANCOVAs). The distribution of soluble carbohydrates data was skewed towards zero and thus required a Generalised Linear Model (GLM)-negative binomial error to be analysed.

Adult lifespan was treated as a function of thermal regime, sex and their interaction within a Cox proportional hazards regression model. For all reproductive traits measured in females (mating success, fecundity, fertility, laying rate) and males (mating success, spermatophore volume, number of eupyrene bundles), statistical models included thermal regime as an explanatory variable and the pupal mass of tested individuals along with the one of their standard partners as two covariates. Mating success of both females and males were compared among thermal regimes with GLM-binomial error. Data for fecundity and spermatophore volume met the criteria of normality and homoscedasticity, while for laying rate a square root transformation was needed to reach normality. Consequently, all these data were studied with ANCOVAs. Fertility was expressed in proportions and therefore tested using a GLM-quasi binomial error with a logit link function. As discrete count data, the number of eupyrene bundles was analysed with a GLM-negative binomial error. Normality and homoscedasticity were evaluated with a Shapiro-Wilk and a Levene test, respectively. All statistical analyses were carried out using R 3.6.2 software.

Results

Group 1: Body amounts of four major energetic components upon emergence

The four energetic components were impacted by sex in the same way, but did not respond to thermal regime or to the interaction between thermal regime and sex (Table 2). Females stocked higher amounts of proteins, lipids, glycogen and soluble carbohydrates than males (Table 2). Furthermore, the amounts of the four energetic components were positively influenced by pupal mass: heaviest individuals possessed higher amounts than lightest ones (proteins: F1,145 = 81,2, P < 0.001, slope = 80.1; lipids: F1,149 = 11.1, P < 0.001, slope = 45.1; glycogen: F1,150 = 60.7, P < 0.001, slope = 13.0; soluble carbohydrates: χ 1 2 = 12.7, P < 0.001, slope = 0.21).

Group 2: Adult lifespan

Adult lifespan was impacted by thermal regime (Cox proportional hazards regression model, χ 1 2 = 55.3, P < 0.001), but not by sex (χ 1 2 = 3.06, P = 0.08) or by the interaction between these two factors (χ 1 2 = 1.19, P = 0.28). Individuals of both sexes reared and kept in warm conditions died faster than those reared and held in cool conditions (Fig. 1).

Group 3: Reproductive performance of females and males

Females reared in warm conditions had a lower mating success compared with females reared in cool conditions (GLM-binomial error: χ 1 2 = 7.28, P = 0.01) (Fig. 2A). By contrast with mating success, fecundity (ANCOVA: F1,59 = 0.22, P = 0.64), fertility (GLM-quasi binomial error: χ 1 2 = 0.88, P = 0.06) and laying rate (ANCOVA: F1,59 = 2.70, P = 0.10) did not respond to temperature (Fig. 2B,C,D). Among female's reproductive traits, only fecundity was affected (positively) by female's pupal mass (F1,59 = 4.71, P = 0.03, slope = 10.5) and the pupal mass of the standard male (F1,59 = 4.06, P = 0.04, slope = 11.1): the heavier the female and the standard male mated with, the higher the number of eggs produced.

Among male's reproductive traits, mating success (GLM-binomial error: χ 1 2 = 0.77, P = 0.38) and spermatophore volume (ANCOVA: F1,77 = 0.14, P = 0.71) remained unaffected by thermal regime (Fig. 3A,B). However, spermatophores from males reared in warm conditions contained fewer eupyrene bundles than those produced by males reared in cold conditions (GLM-negative binomial error: χ 1 2 = 18.3, P < 0.001) (Fig. 3C). None of the male's reproductive traits was impacted by either male's pupal mass or the pupal mass of the standard female mated with.

Discussion

Using realistic fluctuating regimes, this study sought to investigate the impacts of global warming on the reproduction of a major grapevine pest, the European grapevine moth (L.

botrana). We found clear evidence revealing that moth physiology and reproduction are influenced by temperatures experienced throughout ontogeny. When reared in warmer conditions complying to a scenario of local warming (FFR), individuals of both sexes incurred a reduced adult lifespan but their body amounts of four major energetic components remained unchanged compared with individuals reared in thermal conditions similar to those currently found in Burgundy (CFR). Focusing on reproductive traits, females that developed in warm conditions underwent reduced mating success (-26%), while males reared in warm conditions suffered decreased sperm quality, that is, a lower number of eupyrene bundles contained in their spermatophore (-50%).

The absence of temperature-related differences in the body amounts of four energetic components suggests that caterpillars are able to compensate for a shortened time allowed for feeding (accelerated larval development) in warm conditions. These findings match the results of a previous study conducted on L. botrana, which outlined an increase in daily gain of body mass (i.e. growth rate) with mean temperature [START_REF] Iltis | When warmer means weaker: high temperatures reduce behavioural and immune defences of the larvae of a major grapevine pest[END_REF]). In agreement with these conclusions, several phytophagous specieswith examples found in the Lepidoptera orderare known to elevate their food intake rate as temperature increases, probably to fuel higher basal metabolic rates induced by warming [START_REF] Lee | Temperature-by-nutrient interactions affecting growth rate in an insect ectotherm[END_REF][START_REF] Bauerfeind | Increased temperature reduces herbivore host-plant quality[END_REF][START_REF] Pandey | Development and food consumption of some lepidopteran pest under increased temperature conditions[END_REF]. We also reported here a reduced adult lifespan for individuals reared and held in warm conditions, presumably because, as capital breeders, L. botrana adults cannot feed to compensate for the higher energetic losses and reserves consumption occurring with warming (for similar results on capital breeding species, see [START_REF] Laughton | Responses to a warming world: integrating life history, immune investment, and pathogen resistance in a model insect species[END_REF]. Such a reduction in adult lifespan should have important repercussions for the reproduction of L.

botrana. In males, it should imply a shorter time allowed for mate sampling in natural and complex habitats, thereby possibly impairing the encounter between sexual partners. In females, it can entail a reduced time available for oviposition and egg dispersion in space, potentially leading to complex consequences for larval density, levels of intraspecific competition and offspring survival on infested bunches (Thiéry et al. 2014).

In females, the 26% reduction in mating success observed when comparing the two thermal regimes can be explained by the effects of temperature on the precopulatory phase of reproduction, that is, the pheromonal communication between females willing to mate and males. In L. botrana, females signal their sexual receptivity by emitting sex pheromones through calling behaviour [START_REF] Muller | Male larval nutrition affects adult reproductive success in wild European grapevine moth (Lobesia botrana)[END_REF](Muller et al. , 2016a)). In our study, rearing temperatures experienced by females before mating tests are likely to have influenced calling behaviour by altering pheromone production in quantitative (titres, duration of calling behaviour) and/or qualitative ways (blends of chemical compounds constituting the olfactory signal for mating) [START_REF] Ono | Effect of temperature on biosynthesis of sex pheromone components in potato tuberworm moth, Phthorimaea operculella (Lepidoptera: Gelechiidae)[END_REF][START_REF] Konopka | Day-night temperature differential, rather than mean temperature, determines age of sexual maturation in Straicosta albicosta (Noctuidae)[END_REF]. This thermal modulation of pheromone emission should further affect the outcomes of the behavioural interaction between sexual partners (e.g. specific recognition, mating success). With respect to this hypothesis, we expect the negative effects of warming on female's mating success to be exacerbated in natural, highly heterogeneous environments, where pheromonal signal plays a key role in facilitating the encounter between sexual partners. Despite its negative effects on mating success, warming did not influence fecundity and fertility, in accordance with the absence of differences regarding female's body amounts of energetic components observed between thermal treatments.

In males, thermal conditions did not influence mating success, suggesting no differences between males reared in the CFR and the FFR in terms of access to reproduction and ability to transfer the sperm material during copulation. Temperature did not either modify spermatophore volume as a surrogate for the quantity of sperm and accessory gland secretions delivered to female during mating. As spermatophore was included in our energetic assays, these results are congruent with the absence of differences in body amounts of energetic components found when comparing males reared in the two thermal regimes.

However, our study showed that male's sperm qualityapproximated by the number of eupyrene bundles contained in the ejaculatewas severely reduced (-50%) in warm conditions. The negative impacts of warming on male's fertility traits are becoming more and more evident in arthropod species, including sperm number (Zeh et al. 2012;[START_REF] Sales | Experimental heatwaves compromise sperm function and cause transgenerational damage in a model insect[END_REF], viability [START_REF] Stürup | When every sperm counts: factors affecting male fertility in the honeybee Apis mellifera[END_REF][START_REF] Sales | Experimental heatwaves compromise sperm function and cause transgenerational damage in a model insect[END_REF]), size (Vasudeva et al. 2014;[START_REF] Iossa | Temperature-induced developmental plasticity in Plodia interpunctella : reproductive behaviour and sperm length[END_REF], and ability to migrate through the female's genital apparatus [START_REF] Sales | Experimental heatwaves compromise sperm function and cause transgenerational damage in a model insect[END_REF]). Our study brings new evidence showing that eupyrene spermatogenesis in butterfly species is very sensitive to prevailing environmental conditions, particularly temperature [START_REF] Hiroyoshi | Effects of environmental conditions and aging on eupyrene sperm movement in male adults of Polygonia c-aureum (Lepidoptera: Nymphalidae)[END_REF][START_REF] Iossa | Temperature-induced developmental plasticity in Plodia interpunctella : reproductive behaviour and sperm length[END_REF]. Considering that the number of eupyrene bundles included in male's spermatophore primarily mediates female's egg production in L. botrana [START_REF] Muller | Male larval nutrition affects adult reproductive success in wild European grapevine moth (Lobesia botrana)[END_REF](Muller et al. , 2016a)), further experimental investigations are needed to determine to what extent the decline in male's fertility observed for individuals reared in warm conditions will lower the reproductive success of the pair. In this regard, it is reasonable to think that the 50% reduction in sperm quality we highlighted will perceptibly reduce female's egg production after mating. For example, a less important decline in the amount of eupyrene sperm (approximately -33%) due to larval alimentation had been shown to negatively impact both female's fecundity (-30%) and fertility (-10%) in a previous study [START_REF] Muller | Male larval nutrition affects adult reproductive success in wild European grapevine moth (Lobesia botrana)[END_REF].

In terms of pest management, our study has two main implications for PBDMs design, which could contribute to improve the ecological realism of these models and the accuracy of their local and global predictions about the future of L. botrana populations. First, our results

show that, using realistic fluctuating thermal regimes, climate change would not impact female's fecundity. By contrast with this absence of difference in female's fecundity observed when comparing fluctuating regimes, equations constituting PBDMsbased on constant temperaturespredict that per capita egg production will likely increase over the range of temperatures here examined (considering an increase in mean temperature from 20.5°C to 25.8°C) [START_REF] Gutierrez | Prospective analysis of the invasive potential of the European grapevine moth Lobesia botrana (Den. & Schiff.) in California[END_REF][START_REF] Gutierrez | Climate warming effects on grape and grapevine moth (Lobesia botrana) in the Palearctic region[END_REF]. We argue that differences between constant and fluctuating regimes probably arise from the negative effects of thermal fluctuations on egg production, which would likely expose the organism to stressful thermal conditions during the hottest hours of the cycle. Such a phenomenon is likely to become more and more frequent if mean temperature and the range of daily fluctuations increase concomitantly as two facets of global warming, as predicted in [START_REF] Colinet | Insects in fluctuating thermal environments[END_REF]. Second, by demonstrating that male's fertility was halved with respect to warming scenario, our study highlights the need for PBDMs to consider (i) how male's reproductive performance is expected to vary according to temperature and (ii) how this variation in male's traits modulates the offspring production of the pair. In light of our findings, we are firmly convinced that the implementation of realistic patterns of fluctuations together with male's reproductive traits as an integral component of L.

botrana reproductive success within PBDMs should substantially improve their reliability as tools to develop adequate pest management practices and policies.

In conclusion, we expect climate change to reduce the reproductive success of L.

botrana and the abundance of this pest over generations in Eastern France due to the negative effects of the local warming scenario observed for adult lifespan and the reproductive performance of both sexual partners. At this point, two considerations should be examined before inferring future pest population dynamics based on the results of this study. First, this work explored the plastic response of one single L. botrana generation that experienced an abrupt shift in mean temperature between rearing conditionswhose mean temperature basically corresponded to the one of the CFRand FFR. Hence, our findings do not account for the insect ability to adapt to a gradual warming experienced over several successive generations, as expected in natural conditions [START_REF] Bale | Herbivory in global climate change research: direct effects of rising temperature on insect herbivores[END_REF][START_REF] Hoffmann | Rapid adaptation of invertebrate pests to climatic stress?[END_REF]. It could therefore be hypothesised that L. botrana populations will be able to cope with an incremental increase in mean temperature through evolutionary response, such that the negative effects of warming on the reproduction of this pest species should be less important within natural populations than suggested in this study. Second, the abundance of L. botrana populations is not solely be determined by the reproductive success of this pest, but also by the mortality of immature stages (mostly eggs and larvae) induced by parasitoids in agroecosystems, which

Figure captions Fig. 1

 captions1 Figure captions

Fig. 2

 2 Fig. 2 Effect of thermal regime (CFR = Current Fluctuating Regime, FFR = Future

Fig. 3

 3 Fig. 3 Effect of thermal regime (CFR = Current Fluctuating Regime, FFR = Future

Acknowledgements

We are grateful to Lionel Delbac who managed the insect stock, Benjamin Bois and Sébastien Zito who designed the thermal regimes, and Samuel Ribeiro for his practical support all along the experiments. We also want to thank the two anonymous reviewers for their insightful comments and relevant suggestions on the manuscript.

Funding: This study was funded 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 and VALEACLIM-BOIS 20184-07116), and the European Union through the PO FEDER-FSE Bourgogne 2014/2020 programs.

future L. botrana abundances relevant in the context of tritrophic interactions (for an exemple with another pest species, see [START_REF] Gutierrez | Geographic distribution and relative abundance of the invasive glassy-winged sharpshooter: effects of temperature and egg parasitoids[END_REF].

Compliance with ethical standards

Conflict of interest: The authors declare they have no conflict of interest.

Ethical approval: All applicable institutional and/or national guidelines for the care and use of animals were followed.

Table 1

Thermal conditions (°C) for the two daily fluctuating thermal regimes involved in this study (CFR and FFR), that were divided into 6 periods of 4h. Daily thermal range corresponds to the difference between daily maximum and minimum temperatures. Table 2 Mean values and their 95% confidence intervals of the body amounts of four energetic components for the two thermal regimes and sexes. For each energetic component, significant differences (P < 0.05) between regimes or sexes are shown by superscript letters. Data for proteins, lipids and glycogen were compared among thermal regimes and sexes with Analyses of Covariance while for soluble carbohydrates, a

Hour

Generalised Linear Model-negative binomial error was performed. All these statistical models accounted for pupal mass as a single covariate.

There was no significant interaction between thermal regime and sex for any of the four energetic components (proteins: F1,145 = 0.88, P = 0.35; lipids: F1,149 = 0.16, P = 0.68; glycogen: F1,150 = 0.93, P = 0.34; soluble carbohydrates: χ 1 2 = 0.24, P = 0.63).

Proteins


On open cavity flow control in the age of artificial intelligence

Session: Applications

The flow dynamics, resulting from the interaction between a boundary layer and a wall cavity open to the main flow, can be adjusted to obtain more or less pronounced nonlinear interactions between the instability modes of the resonant mixing layer and intracavity spanwise centrifugal instabilities [START_REF] Basley | On the modulating effect of three-dimensional instabilities in open cavity flows[END_REF]. This configuration provides a particularly challenging benchmark for testing the effectiveness of control procedures in fluid systems. With the aim of suppressing cavity resonance, we propose a closed-loop control experiment whose controller results from an automatic learning procedure based on a genetic programming algorithm enhanced with a gradient method (gMLC) (gMLC) [START_REF] Cornejo Maceda | Stabilization of the fluidic pinball with gradient-enriched machine learning control[END_REF]. In this experimental study, two open cavity flows are controlled: a narrow-bandwidth regime with a single dominant frequency and a mode-switching regime where two frequencies compete [START_REF] Lusseyran | Dynamical analysis of an intermittency in an open cavity flow[END_REF]. The flow is forced by a dielectric barrier discharge actuator located at the leading edge and is monitored by a downstream hot-wire sensor over the trailing edge. The feedback law is optimized with respect to the monitored fluctuation level. As reference, the self-oscillations of the mixing layer are mitigated with steady actuation. Then, a feedback controller is optimized with gMLC. After a day-long experiment, gMLC learns a feedback control law stabilizing the open cavity flow up to 1 % of its original fluctuation level with approximately 1% of the actuation energy required for similarly effective steady forcing. Intriguingly, optimized laws learned for one regime perform well for the other untested regime as well. The proposed control strategy can be expected to be applicable for many other shear flow experiments. [START_REF] Maceda | Stabilization of a multifrequency open cavity flow with gradient-enriched machine learning control[END_REF].

It should be noted that, in this study, stabilization of the natural self-sustaining oscillations of the cavity is not the result of flow forcing. With control established, the electrode's action is shown to be virtually nil, both in terms of mean and intensity of fluctuations, while the persistence of the feedback loop is shown to be essential to the controller's robustness. The organization of the spatio-temporal structure of the flow under control, and the transient towards the controlled state, have not yet been studied, but these questions will be addressed at the end of the presentation.

François Lusseyran 1 , 3 1

 13 Guy Y. Cornejo Maceda 2,1 , Eliott Varon 1 , & Bernd R. Noack 2,1,Laboratoire Interdisciplinaire des Sciences du Numérique, Université Paris-Saclay, CNRS, 91400 Orsay, France 2 School of Mechanical Engineering and Automation, Harbin Institute of Technology (Shenzhen), University Town, Xili, Shenzhen 518055, PR China 3 Hermann-Föttinger-Institut, Technische Universität Berlin, Müller-Breslau-Straße 8, D-10623 Berlin, Germany francois.lusseyran@lisn.upsaclay.fr

Fig. 1 .

 1 Fig. 1. Diagram of the machine learning control process. The evaluation of one individual constitutes the fast evaluation loop, operating at 250 Hz. The slow learning loop updates the control laws following the gMLC algorithm; it operates at ∼ O(10-2 Hz).

Fig. 2 .

 2 Fig. 2. Characteristics of the best control law controlling the narrow-bandwidth regime (left-hand column) and the mode-switching regime (right-hand column). (a & c) Power spectra of the velocity measured downstream. Black is for the unforced flow, red for the flow controlled and blue for the flow controlled with the recorded actuation employed as an open-loop control. The spectra for the unforced flow (black) and the closed-loop controlled flow (red) are averaged over 20 realizations. The horizontal dashed lines denote the maximum of the spectra in the observation window (yellow background). The vertical axis is in log 10 scale. (b & d) Time series of the velocity measured downstream and voltage level at the terminals of the DBD actuator.A short window is enlarged to display the behavior close to Amin (the proportions are not kept).

Acknowledgements:This work is supported by the National Natural Science Foundation of China under grants 12302293, 12172109, and 12172111, by the Guangdong Basic and Applied Basic Research Foundation under grant 2022A1515011492, and by the Shenzhen Science and Technology Program under grant JCYJ20220531095605012 and intinially supported by by the French National Research Agency (ANR) via FLOwCON project ANR-17-ASTR-0022.


Michele Esposito

Giancarlo Valente

Yenisel Plasencia-Calaña

Michel Dumontier

Bruno L Giordano

Elia Formisano

SEMANTICALLY-INFORMED DEEP NEURAL NETWORKS FOR SOUND RECOGNITION

Keywords: natural sound recognition, deep neural networks, auditory semantics, semantic embeddings, acoustic-to-semantic transformation

Deep neural networks (DNNs) for sound recognition learn to categorize a barking sound as a "dog" and a meowing sound as a "cat" but do not exploit information inherent to the semantic relations between classes (e.g., both are animal vocalisations). Cognitive neuroscience research, however, suggests that human listeners automatically exploit higher-level semantic information on the sources besides acoustic information. Inspired by this notion, we introduce here a DNN that learns to recognize sounds and simultaneously learns the semantic relation between the sources (semDNN). Comparison of semDNN with a homologous network trained with categorical labels (catDNN) revealed that semDNN produces semantically more accurate labelling than catDNN in sound recognition tasks and that semDNN-embeddings preserve higherlevel semantic relations between sound sources. Importantly, through a model-based analysis of human dissimilarity ratings of natural sounds, we show that semDNN approximates the behaviour of human listeners better than catDNN and several other DNN and NLP comparison models.

INTRODUCTION

Human sound recognition involves the transformation of acoustic waveforms into meaningful representations of the sound-producing source or event. Whereas this ability is automatic and effortless in humans, engineering artificial systems that reproduce human recognition performance has proven challenging. In machine learning (ML), sound recognition has been typically formulated as a classification problem, where sounds are assigned to predefined classes based on the analysis of various features extracted from the input Fundings/support: Data Science Research Infrastructure (DSRI; Maastricht University); Dutch Research Council (NWO 406.20.GO.030 to EF); French National Research Agency (ANR-21-CE37-0027-01 to BLG). acoustic signal. Different ML approaches have been proposed, showing promising results in several applications [START_REF] Bansal | Environmental sound classification: A descriptive review of the literature[END_REF]. Recently, deep neural networks (DNNs) have been shown to outperform other conventional ML algorithms. Mimicking similar research on visual object recognition [START_REF] Fukushima | Neocognitron: A selforganizing neural network model for a mechanism of visual pattern recognition[END_REF], sound-toevent DNNs have been used for sound classification tasks [START_REF] Huang | Aclnet: efficient end-to-end audio classification cnn[END_REF], [START_REF] Hershey | CNN architectures for large-scale audio classification[END_REF], [START_REF] Kong | PANNs: Large-Scale Pretrained Audio Neural Networks for Audio Pattern Recognition[END_REF]. Trained on a large-scale dataset of human-labelled sounds (Audioset, [START_REF] Gemmeke | Audio set: An ontology and human-labeled dataset for audio events[END_REF]), Google's VGGish and Yamnet have provided remarkable performances. These networks receive spectrogram representations as input and can classify sounds in up to 527 and 521 classes, for VGGish and Yamnet, respectively. Although a taxonomic organization of labels has been proposed (Audioset, [START_REF] Gemmeke | Audio set: An ontology and human-labeled dataset for audio events[END_REF]), in most cases the information on the (hierarchical) relation between labels is not used explicitly to train the networks (but see Jimenez et. al [START_REF] Jimenez | Sound event classification using ontology-based neural networks[END_REF]). Typically, labels are encoded as binary categorical variables, using one-hot or multi-hot (in case of multiple simultaneous labels) encoding; Fig. 1 (a,b).

Interestingly, recent cognitive neuroscience research has shown that sound-to-event DNNs (including VGGish and Yamnet) provide a good approximation of human listeners' behaviour in several real-world auditory perception tasks [START_REF] Kell | A task-optimized neural network replicates human auditory behavior, predicts brain responses, and reveals a cortical processing hierarchy[END_REF][START_REF] Giordano | Intermediate acoustic-to-semantic representations link behavioural and neural responses to natural sounds[END_REF]. Giordano et al. [START_REF] Giordano | Intermediate acoustic-to-semantic representations link behavioural and neural responses to natural sounds[END_REF] considered behavioural data consisting of perceived sound (dis)similarities, estimated with a hierarchical sorting task [START_REF] Giordano | Hearing living symbols and nonliving icons: Categoryspecificities in the cognitive processing of environmental sounds[END_REF] and examined to what extent sound-to-event DNNs, and other acoustic, auditory perception and semantic (natural language processing, NLP) models could explain these behavioural data. Results not only showed that sound-to-event DNNs outperformed all other models in predicting human sound dissimilarity judgements but also that NLP models, namely word2vec [START_REF] Mikolov | Efficient estimation of word representations in vector space[END_REF] and GloVe [START_REF] Pennington | Glove: Global vectors for word representation[END_REF], predicted variance of behavioural data that could not be accounted for by sound-to-event DNNs trained using categorical labels. These findings suggest that, when listening to (and comparing) real-world sounds, human listeners automatically exploit higher-level semantic information on the sources besides acoustic information.

Inspired by these results, we sought to develop DNNs that -mimicking human perception [START_REF] Heller | Synergy between human and machine approaches to sound/scene recognition and processing: An overview of icassp special session[END_REF] -learn to recognize To this aim, we formulated sound recognition as a deep-learning regression problem of mapping spectrograms onto a continuous, multidimensional space, quantitatively capturing the semantic relations between sound sources and events. In the present study, we obtained this multidimensional space from the word2vec embeddings of linguistic sound descriptions, Fig. 1 (a,c). To evaluate the effect of semantics on sound recognition DNNs, we trained two networks (semDNN and catDNN) with identical architecture (except for the output layer, see below) using, in one case, 300-dimensional word2vec embeddings of the linguistic sound description (semDNN; [START_REF] Mikolov | Efficient estimation of word representations in vector space[END_REF]), and categorical, one-hot encoded single words in the other case (catDNN).

To avoid biases in the comparison, our DNNs were trained from scratch, as all available pre-trained networks have been trained using categorical coding. Furthermore, for training, we curated a dataset of 388,211 sounds (2,584 hours), covering a broad range of real-world sounds (Super Hard Drive Combo [START_REF]SuperHardDriveCombo[END_REF]), characterized by the rich semantic description that we derived from the database metadata (see below).

Based on [START_REF] Giordano | Intermediate acoustic-to-semantic representations link behavioural and neural responses to natural sounds[END_REF], we expected that semDNNs would better approximate human behaviour in auditory cognitive tasks than catDNNs, as the word2vec embeddings preserve the semantic relation between sound sources, which are instead lost with one-hot encoding, Fig. 1 (b,c).

Similar to our approach, other recent studies proposed to combine sound-to-event DNNs with language embeddings. Xie et al. [START_REF] Xie | Zero-shot audio classification with factored linear and nonlinear acoustic-semantic projections[END_REF] combined audio feature embeddings from VG-Gish and semantic class label embeddings from word2vec at the output stage using a bilinear model. However, the audio network was not explicitly trained to learn semantic embeddings in this case. Recently, Elizalde et al. [START_REF] Elizalde | Clap: Learning audio concepts from natural language supervision[END_REF] employed contrastive learning to combine sentence embeddings (BERT, [START_REF] Devlin | Bert: Pre-training of deep bidirectional transformers for language understanding[END_REF]) with an audio encoder into a joint multimodal space. Our present work, however, focuses on evaluating the effects of semantic representation type (continuous vs categorical) on sound-to-event DNNs and assessing networks' ability to predict human perceptions.

METHODS AND MATERIAL

Network Architecture

We developed two different networks: semDNN and catDNN (Fig. 2). Both networks resemble VGGish (four main convolutional blocks; 64, 128, 256, and 512 filters), including the sound preprocessing and log-mel spectrogram input to the network. Compared to VGGish, we added a dropout layer (rate = 0.2; [START_REF] Srivastava | Dropout: A simple way to prevent neural networks from overfitting[END_REF]) and a batch normalization layer [START_REF] Ioffe | Batch normalization: Accelerating deep network training by reducing internal covariate shift[END_REF] after each downsampling operation, and after the fully connected layers. We also applied global average pooling after the last convolutional block to summarize the feature maps into a fixed-length vector. The two networks differed only at the output layer, where semDNN has a 300-units (N dimensions of semantic embedding = 300) layer with linear activation, and catDNN has a 9,960-units (N dictionary words = 9,960) dense layer with sigmoid activation. We additionally trained a convolutional autoencoder (CAE) to assess the network behaviour with acoustic inputs only (no category/semantic label task; same architecture as in Fig. 2 for the encoder and reverted architecture for the decoder; see below for additional control networks). The loss function was adapted to the network task. We used binary cross entropy for catDNN (multi-classification task), an angular distance for semDNN (regression task), and mean square error for the CAE.

Semantically balanced dataset for training, validation, internal and external evaluation

Networks were trained using sounds and labels from Super-Hard Drive Combo [START_REF]SuperHardDriveCombo[END_REF], a collection of 388,211 variablelength sounds (2,584 hrs) covering a wide range of sound sources and events. We then used a natural language processing pipeline to extract a dictionary of 9,960 sound-descriptive words from the database metadata (median nb. words/sound= 3, range = 1-15). The distribution of the word descriptors in the database was unbalanced (Fig. 3). Thus, we created a semantically balanced dataset based on a hierarchical clustering analysis of the word2vec representations of the dictionary (input = pairwise cosine distance; ward-linkage). We considered a clustering solution with K = 300 semantic word clusters (Fig. 3). The resulting balanced dataset included 273,940 sounds (training set = 90% = 246,546 sounds; 1,366,848 frames; validation set 5%; internal evaluation = 5%).

Without further training, the networks were validated on four publicly-available external datasets: FSD50k [START_REF] Fonseca | FSD50K: An Open Dataset of Human-Labeled Sound Events[END_REF], ESC-50 [START_REF] Piczak | Esc: Dataset for environmental sound classification[END_REF], Urban Sound 8K [START_REF] Salamon | A dataset and taxonomy for urban sound research[END_REF] and MSOS [START_REF]MSOS-dataset[END_REF].

Comparative networks evaluation

We compared semDNN and catDNN relative to two predictionaccuracy metrics, one requiring the conversion of semDNN word2vec embedding predictions onto word predictions (Ranking score), and the other requiring the computation of word2vec embeddings of catDNN word predictions (average maximum cosine similarity, see below). To obtain single-word predictions for semDNN, we projected the pre-dicted semantic embeddings, potentially reflecting a mixture of words, onto the single-word embeddings in the dictionary using non-negative least squares (NNLS) regression [START_REF] Lawson | Solving least squares problems[END_REF].

Ranking Score We first sorted the NNLS β-values (semDNN) and the output probabilities (catDNN) in ascending order to compare prediction accuracy. The ranking score was defined as:

m = 1 - rank -1 N -1 ( 1
)
where N is the dictionary length and rank is the position of β/probability corresponding to the "true" label. When labels included multiple words, we averaged the ranking score obtained for every single word.

Average maximum cosine similarity score (AMCSS) For each sound, we computed the cosine similarity between the topN (5 ≤ topN ≤ 15) predicted words and a word in the true label and then considered its maximum value. This operation was repeated for each word in the label. The AMCSS was obtained as the average of these values (e.g. if the true label is 3 words long, 3 max values are obtained and then averaged).

Prediction of human behavioural data

We evaluated to what extent layer-by-layer embeddings of semDNN and catDNN, and of several control networks, including the CAE, and of additional control models, approximated perceived dissimilarity judgements obtained with humans ([10], Exp. 2, hierarchical sorting task; N sounds = 80). We adopted a cross-validated representational similarity analysis (RSA, [START_REF] Kriegeskorte | Representational similarity analysis -connecting the branches of systems neuroscience[END_REF][START_REF] Giordano | Intermediate acoustic-to-semantic representations link behavioural and neural responses to natural sounds[END_REF]), implying the comparison of behavioural data and model representations in the distance domain (model distance = cosine between-sound distances). We considered as additional comparison models. First, two NLP embeddings (word2vec and GloVe [START_REF] Mikolov | Efficient estimation of word representations in vector space[END_REF][START_REF] Pennington | Glove: Global vectors for word representation[END_REF]) to compare our audio-based learning of semantic relations in semDNN with text-based learning. Second, we considered three prepublished categorical sound-to-event DNNs (Yamnet, VG-Gish and Kell [START_REF] Hershey | CNN architectures for large-scale audio classification[END_REF][START_REF] Gemmeke | Audio set: An ontology and human-labeled dataset for audio events[END_REF][START_REF] Kell | A task-optimized neural network replicates human auditory behavior, predicts brain responses, and reveals a cortical processing hierarchy[END_REF]), and three variants of the semDNN network (semDNN unbal , trained with a randomly selected semantically unbalanced dataset, semDNN GloVe , trained to learn GloVe embeddings, and semDNN notrain , the random initialization of an untrained semDNN network).

RESULTS

Internal and external network-prediction accuracy

Fig. 4 shows the ranking score and AMCSS for semDNN vs catDNN, averaged over all evaluation sounds, for the internal (SuperHardDrive) and the four external datasets. In all cases, the ranking score was higher for semDNN, which indicates that training using semantic embeddings produces more accurate predictions than categorical labels. AMCSS was also higher for semDNN in all cases, indicating that Top-N ranked words for semDNN predictions are more semantically related to the true labels than catDNN. This advantage increases when a larger number of words is considered (Fig.

4, arrows).

A relevant hypothesis was that semDNN embeddings would predict higher-order semantic relations between sounds better than catDNN. We tested the MSOS dataset (see 2.2), for which sounds are organized in five macro-classes (effects, human, music, nature, urban). For both semDNN and catDNN, we computed the pairwise cosine distance between sound embeddings in the last intermediate layer (Fig. 2, arrow). Fig. 5 shows that the semDNN embedding (middle panel) reflects better the macro-class organization (left panel) than the catDNN embedding (right panel; correlation with True categorical model = 0.330 and 0.193 for semDNN and catDNN, respectively).

Comparisons human behavioural data

CONCLUSIONS

We systematically investigated the effects of training DNNs for sound recognition with continuous semantic embeddings (word2vec) vs categorical labels (one-hot encoding). We showed that training with continuous embeddings is beneficial, as it produces semantically more accurate labelling of sounds. Importantly, using human behavioural data, we showed that DNNs trained with continuous semantic embeddings approximate human behaviour better than categorical DNNs. Here, we considered word2vec embeddings of the linguistic sound descriptions to retain information on the (linguistic) semantic relations between the sound sources. In the future, the same approach could be extended to different types of semantic embeddings, for example, derived from natural sound ontologies [START_REF] Giordano | What do we mean with sound semantics, exactly? a survey of taxonomies and ontologies of everyday sounds[END_REF].

Fig. 1 :

 1 Fig. 1: Categorical vs Semantic label encoding (a) Labelencoding step for the two approaches. (b,c) Example visualization of one-hot vs word2vec spaces

Fig. 2 :

 2 Fig. 2: Pre-processing and catDNN/semDNN's architecture. Note how the architectures differ only at the last dense layer.

Fig. 3 :

 3 Fig. 3: t-SNE visualization of 25 clusters of the semantic space. Circle size indicates the number of occurrences of word.

Fig. 4 :

 4 Fig. 4: Networks comparison, Sounds-averaged ranking scores (left) and AMCSS (right) for semDNN and catDNN. Arrows indicate increasing top-N from 5 to 15.

Fig. 6 Fig. 5 :

 65 Fig.6shows the results of the RSA of human dissimilarity ratings of natural sounds for SemDNN, catDNN and other

Fig. 6 :

 6 Fig. 6: Behaviour prediction. SemDNN outperforms all other models at predicting perceived sound dissimilarity.

  ). CV = 0.265), and semDNN GloVe (semDNN trained to learn GloVe embeddings, R 2

	compared models. SemDNN (R 2 CV =0.371) outperformed
	catDNN (R 2 CV = 0.199) and other DNNs trained using cate-
	gorical labels (VGGish, R 2 CV = 0.226; Yamnet, R 2 CV = 0.302;
	Kell, R 2 CV = 0.179. Also, embeddings from SemDNN ex-
	plained human behavioural data better than embeddings de-
	rived by applying NLP models (word2vec (R 2 CV = 0.211;
	GloVe, R 2 CV = 0.156) to sound descriptors. These results con-
	firm our hypothesis that a network combining acoustic and
	semantic information approximates human behaviour in au-
	ditory cognitive tasks better than models considering acoustic
	(categorical sound-to-event DNNs) or semantic (NLP) in-
	formation alone. Finally, SemDNN outperformed several
	other DNNs with the same architecture that we trained in
	different configurations to evaluate the effects of individ-
	ual factors: CAE (convolutional autoencoder, R 2 CV = 0.090),
	semDNN notrain (random initialization for untrained semDNN, R 2 CV = 0.034), semDNN unbal (semDNN trained on unbalanced dataset, R 2

CV = 0.191).

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