Michal Knapp
email: knapp@fzp.czu.cz

Philippe Vernon

David Renault

Studies on chill coma recovery in the ladybird, Harmonia axyridis: ontogenetic profile, effect of repeated cold exposures, and capacity to predict winter survival

Keywords: Ageing, chill injury, cold tolerance, invasive species, ontogenetic stage, overwintering

The harlequin ladybird, Harmonia axyridis, is one of the most successful invasive insect species worldwide. We investigated whether (i) chill coma recovery time (CCRt) changes during the ontogenetic development of this species, (ii) CCRt varies in response to repeated cold shocks, and (iii) CCRt could be a good predictor of winter survival ability in adults.

CCRt decreased during larval development, the lowest CCRt values were observed in teneral adults (one and four days old), and significantly higher values were observed for older adults (16 and 32 days old). Repeated cold shocks (two hours at -3 °C), interrupted by short (30 minutes) warm periods (22 °C) resulted in decreased CCRt after the second cold shock, probably depicting an acclimation response, but then CCRt increased with additional cold shocks, likely revealing the accumulation of chill injuries. The CCRt of pre-overwintering individuals was not correlated with their winter survival. This indicates that CCRt is not a reliable measure of cold tolerance in H. axyridis. However, this result could be partially affected by the experimental setup -the use of laboratory-reared individuals who experienced standardized conditions and thus the variability in CCRt of tested animals could be much lower than the variability present in nature. The substantial variation observed over the ontogenetic development of H. axyridis poses important methodological implications for future studies, as animals of the same stage/age should be compared with each other. The observed U-shaped response to repeated cold shocks indicates that the expectation of linearity between cold exposure and insect response is an oversimplification of real situations.

Highlights:

-chill coma recovery time (CCRt) varies significantly during ladybird ontogeny -repeated cold shocks result in a U-shaped CCRt response -pre-overwintering CCRt was not related to winter survival in this species -the relevance of CCRt as a measure of cold tolerance needs to be evaluated in other species

Introduction

Because of their ectothermic nature and small body sizes, insects are particularly prone to rapid changes in their body temperature and have had to develop a wealth of behavioural and physiological responses to survive at low temperatures [START_REF] Teets | Physiological mechanisms of seasonal and rapid cold-hardening in insects[END_REF]. As a result, thermal tolerance is a crucial physiological trait that is significantly related to the distribution of insect species on Earth [START_REF] Araujo | Heat freezes niche evolution[END_REF]. A range of techniques are available for assessing the cold tolerance of insects; while some of them directly measure mortality (e.g., estimation of survival capacities, measurements of lower lethal limits), other techniques are only proxies of insect tolerance to the deleterious effects of low temperatures (e.g., supercooling point, chill coma onset and recovery; see [START_REF] Terblanche | Ecologically relevant measures of tolerance to potentially lethal temperatures[END_REF][START_REF] Sinclair | An invitation to measure insect cold tolerance: Methods, approaches, and workflow[END_REF] for a review).

Chill coma onset and recovery have been described as reliable metrics of thermal plasticity and tolerance in insects [START_REF] Sinclair | An invitation to measure insect cold tolerance: Methods, approaches, and workflow[END_REF]. Chill coma is initiated when neuromuscular function is disrupted as a result of the organism's inability to maintain proper ion balance due to depolarization of the resting membrane potential (MacMillan and Sinclair, 2011;[START_REF] Overgaard | The Integrative Physiology of Insect Chill Tolerance[END_REF]. Entering into chill coma has substantial ecological consequences for an individual, as immobile insects are not able to acquire food or escape from predators. While the measurement of the onset of chill coma can be challenging in immature insects, the recovery from chill coma, i.e., the ability of a specimen to exhibit a predetermined behaviour, can be easily measured in both juveniles and adults (see [START_REF] Sinclair | An invitation to measure insect cold tolerance: Methods, approaches, and workflow[END_REF] for a discussion of this point).

Chill coma recovery time (CCRt) is expected to differ greatly among and within insect species and particularly among populations of different geographical origin [START_REF] Andersen | How to assess Drosophila cold tolerance: chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits[END_REF][START_REF] Bowler | Insect thermal tolerance: what is the role of ontogeny, ageing and senescence?[END_REF][START_REF] Chown | Physiological variation in insects: hierarchical levels and implications[END_REF]. Within-population CCRt variations are also frequently observed, as developmental plasticity (i.e., the environmental conditions experienced during preimaginal development partly determine adult stress tolerance) or individual acclimation (i.e., exposure to mild stress enhances individual stress tolerance; [START_REF] Colinet | Comparing phenotypic effects and molecular correlates of developmental, gradual and rapid cold acclimation responses in Drosophila melanogaster[END_REF][START_REF] Terblanche | The relative contributions of developmental plasticity and adult acclimation to physiological variation in the tsetse fly, Glossina pallidipes (Diptera, Glossinidae)[END_REF] can affect this measure.

Moreover, thermal tolerance can substantially vary over the course of an insect's life, but ontogenetic variations in the responses to low temperatures have been poorly examined compared to heat resistance (see [START_REF] Bowler | Insect thermal tolerance: what is the role of ontogeny, ageing and senescence?[END_REF]. The existing studies have focused on preimaginal development or adult ageing [START_REF] Colinet | Rapid decline of cold tolerance at young age is associated with expression of stress genes in Drosophila melanogaster[END_REF][START_REF] David | Cold stress tolerance in Drosophila: Analysis of chill coma recovery in D. melanogaster[END_REF][START_REF] Halle | The consequences of parental age for development, body mass and resistance to stress in the red flour beetle[END_REF][START_REF] Pujol-Lereis | Analysis of survival, gene expression and behavior following chill-coma in the medfly Ceratitis capitata: Effects of population heterogeneity and age[END_REF], while studies that included measures on both preimaginal and adult stages are commonly focused on a few developmental stages and age classes (e.g., [START_REF] Arias | Plasticity of life-cycle, physiological thermal traits and Hsp70 gene expression in an insect along the ontogeny: Effect of temperature variability[END_REF], but see Jensen et al., 2007 on Drosophila melanogaster).

Differences between individuals in their cold tolerance are expected to have consequences for their overwintering success [START_REF] Denlinger | Low temperature biology of insects[END_REF], but this prediction has only rarely been verified experimentally (e.g., [START_REF] Alford | Could Behaviour and Not Physiological Thermal Tolerance Determine Winter Survival of Aphids in Cereal Fields[END_REF][START_REF] Kostal | Physiological and biochemical analysis of overwintering and cold tolerance in two Central European populations of the spruce bark beetle, Ips typographus[END_REF].

The majority of studies investigating insect cold hardiness focus on the effects of a single exposure to low temperatures [START_REF] Marshall | The impacts of repeated cold exposure on insects[END_REF]. However, in the field, insects can be exposed to knockdown temperatures on multiple occasions over days, and these repeated exposures could affect insect performance in the following ways: 1) cold hardiness will progressively increase across successive exposures, depicting thermal acclimation; 2) cold hardiness will progressively decrease across successive exposures, revealing an accumulation of chill injuries; 3) cold hardiness will exhibit a complex pattern of a U-shaped cold hardiness response curve as a result of acclimation of the insect during the first sequence of the exposure to the cold conditions, followed by an accumulation phase of chill injuries [START_REF] Colinet | Uncovering the benefits of fluctuating thermal regimes on cold tolerance of Drosophila flies by combined metabolomic and lipidomic approach[END_REF]Marshall andSinclair, 2012, 2015).

In this study, we present the results of a pilot study on the thermal biology of the harlequin ladybird, Harmonia axyridis (Pallas, 1773). This species is currently considered one of the most invasive insects worldwide [START_REF] Brown | The global spread of Harmonia axyridis (Coleoptera: Coccinellidae): distribution, dispersal and routes of invasion[END_REF][START_REF] Lombaert | Inferring the origin of populations introduced from a genetically structured native range by approximate Bayesian computation: case study of the invasive ladybird Harmonia axyridis[END_REF], with probable specific abilities to colonize cold environments [START_REF] Barahona-Segovia | Testing the hypothesis of greater eurythermality in invasive than in native ladybird species: from physiological performance to lifehistory strategies[END_REF]. H. axyridis overwinters as an adult and creates large winter aggregations, frequently in human settlements [START_REF] Berkvens | Cold tolerance of the harlequin ladybird Harmonia axyridis in Europe[END_REF]. Previous studies measured supercooling points and lower lethal temperatures in adult H. axyridis originating from European and US invasive populations [START_REF] Berkvens | Cold tolerance of the harlequin ladybird Harmonia axyridis in Europe[END_REF][START_REF] Koch | Cold hardiness of the multicolored Asian lady beetle (Coleoptera : Coccinellidae)[END_REF] and from Japanese native populations [START_REF] Watanabe | Cold tolerance and myo-inositol accumulation in overwintering adults of a lady beetle, Harmonia axyridis (Coleoptera : Coccinellidae)[END_REF]. To our knowledge, patterns of CCRt variation have not been investigated in this species thus far. We addressed three different questions using a single metric -CCRt: 1) we examined the influence of ontogeny on CCRt and hypothesized that CCRt would decrease from the 1 st to 4 th larval instar because of the increasing probability of exposure to low temperatures in nature (late larval instars can experience colder autumn weather in Central Europe); 2) we investigated the effect of repeated cold exposures on CCRt in adult ladybirds and expected CCRt prolongation because of the accumulation of chill injuries during exposures to cold and insufficient damage repair during very short warm periods among subsequent cold exposures; 3) finally, we were interested in the predictive power of pre-overwintering CCRt for winter survival and expected that individuals achieving lower pre-overwintering CCRt would suffer from lower winter mortality. The ecophysiological meaning of CCRt clearly differs for summer active (questions 1 and 2) and winter diapausing insects (question 3). For summer active insects we employed CCRt as a measure that allowed us to investigate physiological changes during the ontogenetic / physiological responses to repeated cold shocks. For winter diapausing insects we tested the ecological relevance of CCRt as a proxy of insect cold tolerance.

Materials and Methods

Insect collection and rearing

The parental individuals used in study were collected August 2015 from shrubs and lime trees in the university campus of the Czech University of Life Sciences Prague, Czech Republic (GPS: 50°8′ N, 14°21′ E; 300 m a.s.l.). After transportation to the laboratory, the beetles were sexed, and parental pairs were formed. Each couple was placed in a separate Petri dish (9 cm in diameter) containing crumpled filter paper strips, which provided a suitable substrate for egg laying. New egg clutches were placed into Petri dishes and exposed to a standardized laboratory condition (16L:8D photoperiod, 70 % humidity, 26 °C). A temperature of 26 °C is the optimal rearing temperature for H. axyridis [START_REF] Hodek | Ecology and Behaviour of the Ladybird Beetles (Coccinellidae)[END_REF][START_REF] Knapp | Gender and Timing during Ontogeny Matter: Effects of a Temporary High Temperature on Survival, Body Size and Colouration in Harmonia axyridis[END_REF]. Newly hatched larvae were fed ad libitum with eggs of Ephestia kuehniella (Zeller, 1879) (Lepidoptera: Pyralidae) and provided with water in cotton wool.

Chill coma induction and chill coma recovery time measurements

For all beetles investigated in the following experiments (sections 2.3., 2.4. and 2.5.), chill coma was induced and chill coma recovery time (CCRt) was measured in a standardized way.

Pools of six individuals were placed individually into clean Petri dishes and transferred to -3 °C for two hours. A freezing climatic chamber made on order by AVIKO-PRAHA a.s. company was employed for this purpose. The temperature inside the climatic chamber was monitored with a datalogger Tinytag PLUS2 (Supporting Information file Fig. A.1). After 2 hours at -3 °C, the Petri dishes were returned to room temperature (22 ± 1 °C) in an airconditioned laboratory and the beetles were turned onto their dorsum. The duration necessary for observing the first movement of an appendage (most often a leg or a palpus) was measured. As ladybirds can often "play dead" (remain motionless despite being already able to move), individuals were stimulated by gentle teasing with an entomological pin every five seconds.

Experiment 1: Effects of ontogeny on chill coma recovery time

Three fecund parental pairs were selected at random, and their offspring were used in the laboratory experiment investigating the ontogenetic development of cold resistance in H. axyridis. Egg clutches, hatched larvae and 2 nd generation adults were housed under the same rearing conditions as the parental pairs (16L:8D, 26 °C, 70 % humidity, food and water ad libitum). Following egg laying, egg clutches were randomly assigned to one of the following eight treatments to assess the effects of ontogenetic stages on CCRt: 1 st larval instar, 2 nd larval instar, 3 rd larval instar, 4 th larval instar, newly hatched adult (one-day old), four-day-old adult, 16-day-old adult and 32-day-old adult. CCRt was measured only once for each individual (not repeatedly in the course of the experiment). Ladybirds are long-lived insects, with adult longevity exceeding a year; therefore, senescence is expected to take place far later than at the age of 32 days [START_REF] Hodek | Ecology and Behaviour of the Ladybird Beetles (Coccinellidae)[END_REF]. The first and second instar larvae were reared together (a complete clutch) in one Petri dish. When the larvae moulted to the third instar, they were assigned to new Petri dishes in groups of four to five individuals. The CCRt of six larvae per parental pair was measured per treatment, and the CCRt of five individuals per sex per parental pair per treatment was recorded for adult treatments.

Experiment 2: Effects of repeated cold shock on the chill coma recovery time of adults

Twelve males and twelve females aged 14 to 20 days obtained from the same parental pairs as those individuals from the previous experiment were reared at a constant 26 °C, photoperiod 16L:8D, 70 % humidity and with food and water ad libitum. One day before the CCRt measurements, the beetles were individually transferred to Petri dishes and were provided with water only (this short food deprivation period was used to reduce the possible confusing effects of variable stomach contents across repeated measurements). Repeated exposures to knockdown temperatures were achieved as follows: each ladybird was transferred to -3.0 °C for two hours, then returned to room temperature (22 ± 1 °C) for 30 minutes before being transferred again to -3.0 °C. This cyclic procedure was repeated four times (i.e., four exposures of two hours at -3.0 °C), and the CCRt was repeatedly measured for each individual after each exposure to the knockdown temperature (four CCRt measurements for each individual).

Experiment 3: Chill coma recovery time and winter survival of adults

Five males and five females per parental pair were selected at random from 10 parental pairs (i.e., 100 ladybirds in total). Ladybird offspring were reared at 26 °C (photoperiod 16L:8D, 70 % humidity) and supplied with food (Ephestia kuehniella eggs) and water ad libitum. On October 7 th , when the ladybirds were 15-25-day-old adults, the beetles were transferred to an 18 °C and 12L:12D photoperiod regime to initiate winter diapause. The insects were maintained individually in Petri dishes and supplied with water but without food (as they naturally stop feeding during winter diapause). On October 14 th , the CCRt was measured (as described above) for each individual. One day later, the thermal regime was changed to fluctuating temperatures and a short photoperiod [8L(12 °C):16D(6 °C)] to mimic outdoor conditions in late autumn. Six days later, the overwintering phase started and the beetles, still maintained in the climatic chamber, were exposed to the mean temperature regime experienced in outdoor shelters in Prague (data were computed as the long-term mean temperature 1 cm below the soil surface; hourly means from the meteorological station situated in the Crop Research Institute, Praha-Ruzyně were used). The beetles were maintained in clear Petri dishes containing a small watered piece of cotton wool to limit desiccation and placed into the climatic chamber set to the overwintering regime. The climatic chamber was adjusted each hour with a new temperature value (the temperatures applied are attached as Supporting Information file Table A.1). The mean temperature during the overwintering phase was 0.7 °C, and the minimum temperature reached was -4.1 °C.

In the course of the overwintering experiment, the beetles were checked at monthly intervals (November 16 th , December 15 th , January 14 th , February 17 th and March 17 th, 2016when the overwintering experiment was terminated). During all inspections, the Petri dishes with beetles were moved to outdoor conditions to minimize unwanted warming of the experimental beetles, the survival of each individual was recorded, and a new piece of watered cotton wool was inserted into each Petri dish (the old one was removed). Live diapausing ladybirds are commonly tightly attached to the substrate, whereas dead individuals have dropped off and are lying freely on their dorsum. Petri dishes with dead individuals were not removed from the experiment as there was a little risk of false death records (an animal seeming to be dead but is still alive); however, no false records were revealed.

Statistical analyses

To analyse ontogenetic changes in the CCRt, a linear mixed-effects model (LME) was run using the "lme" function implemented in the "nlme" package [START_REF] Pinheiro | Nlme: linear and nonlinear mixed effects models[END_REF] for R (R Development Core Team, 2016). Parental pair identity was used as a random effect in our analysis. Ontogenetic stage was used as a fixed effect in our model. Tukey's HSD test was employed to test for significant differences between particular ontogenetic stages using the "glht" function as implemented in the "multcomp" package [START_REF] Hothorn | Simultaneous inference in general parametric models[END_REF]. Raw data serving as inputs for the abovementioned analysis are available in the Supporting Information file (Table A.2).

To analyse the effects of repeated cold exposures on the CCRt, a linear mixed-effects model (LME) was conducted using the "lme" function implemented in the "nlme" package [START_REF] Pinheiro | Nlme: linear and nonlinear mixed effects models[END_REF] for R (R Development Core Team, 2016). Beetle identity was used as a random effect in our analysis. The order of cold exposure (from 1 to 4; a continuous variable) was used as a fixed effect in our model, while a quadratic term was also included in the model for the case where the response would not be purely linear. The significance of the independent variables (order, order 2 ) was tested using F-tests ("anova.lme" function). To test for the real necessity of both terms (order, order 2 ) in the final model, the most parsimonious model based on AIC-values was also selected [START_REF] Akaike | New look at statistical-model identification[END_REF]. Raw data are available in the Supporting Information file (Table A.3).

To analyse the effects of CCRt on H. axyridis survival during winter, the Cox proportional-hazards model (Cox-PH) was run using the "coxph" function implemented in the "survival" package [START_REF] Therneau | A Package for Survival Analysis in S. version 2.38[END_REF] for R (R Development Core Team, 2016). Winter survival in months (one to five months) was used as the response variable, and CCRt was used as an independent variable in the model. Beetles that survived until the end of the experiment (March) were excluded in the model, as their future longevity was unknown. Raw data serving as inputs for the above analysis are available in the Supporting Information file

(Table A.4).
The effects of sex on the CCRt were analysed in the preliminary analyses performed for all experiments. As there were no significant effects of sex in any model, this term was not included in the final models.

Results

There were significant changes in chill coma recovery time (CCRt) during the ontogenetic development of the harlequin ladybird, Harmonia axyridis (LME: F = 19.92, P < 0.001). The CCRt decreased by ca. 22 % in the course of early larval development from the 1 st to 2 nd larval instars and reached the lowest value in teneral adults (between one and four days old adults). The CCRt of older adults (16 or 32 days old adults) was increased and did not differ from the CCRt of the 2 nd , 3 rd and 4 th larval instars (Fig. 1).

Repeated two-hour exposures to sub-zero temperatures interrupted by 30-minute recovery periods at 22 °C resulted in increasing the CCRt from the second cold exposure onwards in H. axyridis. However, the CCRt decreased from the first to the second cold exposure; thus, alongside the significant linear term (LME: F = 23.92, P < 0.001), there was also a significant quadratic term in the final statistical model (F = 11.24, P = 0.001). The most parsimonious model included both terms (order and order²) according to the AIC (Table 1), demonstrating that a U-shaped response to repeated cold exposures was measured in H. axyridis (Fig. 2).

No significant relationship was observed between the CCRt of the pre-overwintering individuals and their ability to survive during the winter (Cox-PH: z = -1.50, P = 0.13). The observed trend was even opposite that of the hypothesized one: the four individuals who had the lowest CCRt died during the first months of the winter, whereas the three individuals with the highest CCRt survived until the last month of the winter (two of them even survived the whole winter; Fig. 3).

Discussion

In this work, we used CCRt to examine different aspects of ladybird thermal physiology: we 1) investigated the physiological changes during ontogeny, 2) examined the physiological responses of summer active ladybirds to repeated cold shocks, and 3) investigated the relevancy of the CCRt as a proxy for winter cold hardiness. Significant variations of the CCRt were measured across the ontogeny of Harmonia axyridis, with the lowest CCRt values observed in teneral adults. Repeated cold shocks altered the CCRt of H. axyridis, and a Ushaped response was measured, with reduced CCRt during the first phase, followed by a CCRt increase when the number of cold shocks was increased. Finally, we found no relationship between the CCRt of pre-overwintering individuals and their subsequent ability to survive during the winter months.

Changes in the degree of heat tolerance during ontogeny are widespread in insects, but adjustments to cold resistance during ontogeny have been less examined (e.g., [START_REF] Bowler | Insect thermal tolerance: what is the role of ontogeny, ageing and senescence?[END_REF][START_REF] Klockmann | Heat resistance throughout ontogeny: body size constrains thermal tolerance[END_REF][START_REF] Knapp | Gender and Timing during Ontogeny Matter: Effects of a Temporary High Temperature on Survival, Body Size and Colouration in Harmonia axyridis[END_REF][START_REF] Zhao | Life stages of an aphid living under similar thermal conditions differ in thermal performance[END_REF].

Relevant experimental data that reported ontogenic variations of insect cold resistance exist mostly for insect model species (e.g., [START_REF] Arias | Plasticity of life-cycle, physiological thermal traits and Hsp70 gene expression in an insect along the ontogeny: Effect of temperature variability[END_REF][START_REF] Colinet | Rapid decline of cold tolerance at young age is associated with expression of stress genes in Drosophila melanogaster[END_REF][START_REF] Jensen | The influence of developmental stage on cold shock resistance and ability to cold-harden in Drosophila melanogaster[END_REF][START_REF] Pujol-Lereis | Analysis of survival, gene expression and behavior following chill-coma in the medfly Ceratitis capitata: Effects of population heterogeneity and age[END_REF] for the review of older studies see [START_REF] Bowler | Insect thermal tolerance: what is the role of ontogeny, ageing and senescence?[END_REF]. In univoltine holometabolous species, differences in coping with cold exposures among stages could reflect adaptive responses, as the development stages can experience different thermal conditions, with the overwintering stage being most often the most coldresistant one [START_REF] Denlinger | Low temperature biology of insects[END_REF]. Nevertheless, adjustments of insect cold hardiness are not only dictated by environmental temperatures, as differences have also been reported during the ontogeny of multivoltine species over the course of the growth season (e.g., [START_REF] Jensen | The influence of developmental stage on cold shock resistance and ability to cold-harden in Drosophila melanogaster[END_REF]. One of possible explanations is that levels of heat-shock proteins (HSPs), which are partially responsible for the actual cold resistance and are also involved in various stress responses unrelated to cold challenge, vary substantially in the course of insect ontogeny [START_REF] Colinet | Rapid decline of cold tolerance at young age is associated with expression of stress genes in Drosophila melanogaster[END_REF], including in the harlequin ladybird [START_REF] Wang | Molecular Cloning and Induced Expression of Six Small Heat Shock Proteins Mediating Cold-Hardiness in Harmonia axyridis (Coleoptera: Coccinellidae)[END_REF].

There is no unified ontogenetic profile of cold resistance observed for a wide range of insect species. In our study, CCRt was significantly higher in the 1 st larval instar compared with later larval instars. Interestingly, [START_REF] Jensen | The influence of developmental stage on cold shock resistance and ability to cold-harden in Drosophila melanogaster[END_REF] observed a distinct pattern of lower lethal temperature causing 50 % mortality (LT 50 ) in Drosophila melanogaster, which slightly increased from the 2 nd to the 3 rd larval instar. We measured the lowest CCRt value in teneral ladybird adults, which is consistent with the finding of [START_REF] Jensen | The influence of developmental stage on cold shock resistance and ability to cold-harden in Drosophila melanogaster[END_REF] for D.

melanogaster, but opposite the pattern reported for Tenebrio molitor [START_REF] Arias | Plasticity of life-cycle, physiological thermal traits and Hsp70 gene expression in an insect along the ontogeny: Effect of temperature variability[END_REF].

Differences in the changes of ontogenetic patterns of CCRts across species may result from the distinct life histories of the studied species, particularly in terms of overwintering strategies. Importantly, the contrasting cold resistance patterns among studies can also be caused by the various proxies that are employed, as these measures are not necessarily correlated [START_REF] Andersen | How to assess Drosophila cold tolerance: chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits[END_REF].

The higher cold resistance of teneral adults (few hours/days old) of D. melanogaster reported by [START_REF] Colinet | Rapid decline of cold tolerance at young age is associated with expression of stress genes in Drosophila melanogaster[END_REF] was also found in our study for H. axyridis. The cold resistance of adult ladybirds subsequently decreased with increasing age, a pattern also observed for D. melanogaster and Ceratitis capitata [START_REF] David | Cold stress tolerance in Drosophila: Analysis of chill coma recovery in D. melanogaster[END_REF][START_REF] Pujol-Lereis | Analysis of survival, gene expression and behavior following chill-coma in the medfly Ceratitis capitata: Effects of population heterogeneity and age[END_REF]. Decreased cold hardiness with age in adult insects was thought to be related to senescence-associated processes [START_REF] Bowler | Insect thermal tolerance: what is the role of ontogeny, ageing and senescence?[END_REF][START_REF] Halle | The consequences of parental age for development, body mass and resistance to stress in the red flour beetle[END_REF]. However, this hypothesis is unlikely for our 32-day-old H. axyridis whose longevity can easily reach one year; senescence is thus expected to take place later in their lives [START_REF] Hodek | Ecology and Behaviour of the Ladybird Beetles (Coccinellidae)[END_REF]; note also that the CCRt slightly decreased from the age of 16 days to 32 days in our study).

Moreover, increasing cold resistance in the course of senescence was observed for the sub-Antarctic fly Anatalanta aptera [START_REF] Lalouette | Ageing and thermal performance in the sub-Antarctic wingless fly Anatalanta aptera (Diptera: Sphaeroceridae): older is better[END_REF]. Altogether, our results have important methodological implications for future studies on insect cold hardiness, as the exact age of experimental animals should be precisely stated, and animals of the same stage/age should be compared with each other in studies investigating issues other than the effects of ontogeny.

Low temperatures cause chill injuries to chill-susceptible insects, and the extent of the injuries depends on the severity of the treatment, i.e., the severity of the temperatures experienced and the duration of exposure [START_REF] Marshall | The relative importance of number, duration and intensity of cold stress events in determining survival and energetics of an overwintering insect[END_REF][START_REF] Overgaard | The Integrative Physiology of Insect Chill Tolerance[END_REF]. Despite the fact that thermal fluctuations are ubiquitous in nature, the majority of ecophysiological studies are focused on exposures of insects to a single period at low temperature [START_REF] Marshall | The impacts of repeated cold exposure on insects[END_REF]; see the literature focusing on fluctuating thermal regimes and repeated cold exposures, e.g., [START_REF] Colinet | Uncovering the benefits of fluctuating thermal regimes on cold tolerance of Drosophila flies by combined metabolomic and lipidomic approach[END_REF][START_REF] Marshall | The relative importance of number, duration and intensity of cold stress events in determining survival and energetics of an overwintering insect[END_REF]. In this study, we observed the changes in CCRt values in H. axyridis subjected to repeated cold shocks and found that values decreased in the first stage followed by a continuous increase from the second exposure onwards. The observed U-shaped response curve depicted the interplay between acclimation (rapid cold-hardening response; [START_REF] Lee | A rapid cold-hardening process in insects[END_REF] elicited by the first cold exposure and the accumulation of chill injuries (e.g., the creation of reactive oxygen species; [START_REF] Lalouette | Metabolic rate and oxidative stress in insects exposed to low temperature thermal fluctuations[END_REF] inflicted by each successive cold shock. The short warm periods may not have been sufficient for restoring the aerobic metabolism of the insects and completely repairing the physiological injuries caused by exposures to low temperatures [START_REF] Colinet | Uncovering the benefits of fluctuating thermal regimes on cold tolerance of Drosophila flies by combined metabolomic and lipidomic approach[END_REF]. A few hours may have been needed for a complete recovery of the injuries accumulated by cold-shocked ladybirds. For example, five cold exposures separated by recovery periods of 24 hours lowered CCRt values in the red flour beetle [START_REF] Scharf | Context-dependent effects of cold stress on behavioral, physiological, and life-history traits of the red flour beetle[END_REF]. The physiological responses of insects to repeated cold stress are still not known in detail and it is possible that rapid switching between stress and injury repair states itself poses some physiological costs [START_REF] Marshall | The relative importance of number, duration and intensity of cold stress events in determining survival and energetics of an overwintering insect[END_REF].

We failed to report any significant relationship between the CCRt of preoverwintering specimens of H. axyridis and their subsequent overwintering success. This finding is surprising as between-and within-species variations in CCRt commonly exist in insects [START_REF] Chown | Physiological variation in insects: hierarchical levels and implications[END_REF]. This variation seems to be adaptive as species' CCRt generally correlates to their distribution ranges (winter conditions experienced; [START_REF] Andersen | How to assess Drosophila cold tolerance: chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits[END_REF][START_REF] Chown | Physiological variation in insects: hierarchical levels and implications[END_REF], and overwintering individuals often have a significantly lower CCRt compared with their relatives sampled during the breeding season [START_REF] Chown | Physiological variation in insects: hierarchical levels and implications[END_REF]. Our data even showed slight support for the opposite pattern, i.e., individuals with the lowest CCRt died in early winter while those with the highest CCRt survived. A possible explanation is a higher metabolic rate in animals with lower CCRt [START_REF] Williams | Adaptation to low temperature exposure increases metabolic rates independently of growth rates[END_REF]. Animals with higher energy demands can die during overwintering due to the depletion of their energy reserves.

A possible shortcoming of our study is that overwintering success was investigated for laboratory-reared insects that experienced standardized environmental conditions. Moreover, their genetic variability may have been limited (all were offspring of 10 parental pairs). This experimental setting may have reduced the differences in CCRt values among individuals compared with the variation existing for individuals in the field. As laboratory rearing could affect ladybird overwintering performance [START_REF] Berkvens | Cold tolerance of the harlequin ladybird Harmonia axyridis in Europe[END_REF], we encourage a repetition of this experiment using pre-overwintering individuals collected directly in the field. As winter survival in insects is a result of complex interactions between individual physiological traits and environmental conditions, future research investigating the effects of individual cold resistance (represented by various measures/proxies) on winter survival under various overwintering conditions for various insect species is needed to disentangle the effects of individual traits on winter survival in insects. If additional studies confirm that CCRt is not the most reliable predictor of insect overwintering success, chill coma temperatures or lower lethal temperatures, both good predictors of the latitudinal distribution of Drosophila species, [START_REF] Andersen | How to assess Drosophila cold tolerance: chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits[END_REF] might be used as better indicators of overwintering success.

Conclusions

This study provides the first measurements of chill coma recovery time (CCRt) in the invasive harlequin ladybird, Harmonia axyridis. We showed substantial changes in CCRt in the course of the ontogenetic development of H. axyridis. The significant ontogenetic signal in H. axyridis cold hardiness poses important methodological implications for future studies, as animals of the same stage/age should be compared with each other. We also showed that repeated exposures to cold shocks interrupted by short warm periods caused a U-shaped cold resistance response. Individual CCRt was a bad predictor of H. axyridis overwintering success. Together with the limited evidence in the existing literature, our results indicated that CCRt is not a reliable measure of insect overwinter success. Future studies investigating the relationship between prewintering CCRt and following winter survival for various insect species are needed to extend the generality of this pattern.

FiguresFig. 1 .

 1 Figures

Fig. 2 .

 2 Fig. 2. Effect of repeated cold shocks on chill coma recovery time in adult Harmonia

Fig. 3 .

 3 Fig. 3. Relationships between individual pre-overwintering chill coma recovery time and

Acknowledgements

We are grateful to Michal Řeřicha and Radek Svoboda for their help with the laboratory rearing of ladybirds and realization of the overwintering experiment and to Oldřich Nedvěd for sharing his knowledge of H. axyridis thermal biology. We are also grateful to six reviewers who significantly improved the language quality of the text and provided us with insightful suggestions on a previous version of the manuscript.

Funding: This study was supported by grant no. 42900/1312/3166 awarded by the Internal Grant Agency of the Czech University of Life Sciences Prague.

Appendices:

The following additional supporting information may be found in the online version of this article. Table 1. Effect of repeated cold shocks on chill coma recovery time -model selection.

The most parsimonious linear mixed effect model (the first line) was selected on the basis of the Akaike information criterion (AIC). The term "order" indicates the linear effects of the cold shock order on chill coma recovery time; the term "order 2 " indicates the quadratic (nonlinear) effect of the cold shock order on the chill coma recovery time. A difference in the AIC-value (∆ AIC) > 2 indicates a significant difference between models, i.e., the model with lower AIC-value is significantly more parsimonious.


Aliou Ly

Vincent Auroux

Ramin Khayatzadeh

Napoléon Gutierrez

Arnaud Fernandez

Olivier Llopis

Highly spectrally pure 90 GHz signal synthesis using a coupled optoelectronic oscillator

Keywords: Optoelectronic oscillator, COEO, microwave photonics, harmonic generation, millimeter wave, phase noise

teaching and research institutions in France or abroad, or from public or private research centers.

I. INTRODUCTION

High frequency (millimeter-wave), spectrally pure and high power (milliwatt range) RF signals are of great interest for applications in high speed signal processing [START_REF] Devgan | A review of optoelectronic oscillators for high speed signal processing applications[END_REF], advanced radar systems [START_REF] Maleki | Optoelectronic oscillators for microwave and mm-wave generation[END_REF], wireless communications, fiber-radio systems and so on. Conventional techniques for generating such signals are based on the multiplication of lower frequency RF sources, which generally results in an important increase of the phase noise and particularly the phase noise floor (far from carrier phase noise). Optical approaches are thus an interesting alternative to improve the phase noise or the tunability of such sources. As an example, heterodyne mixing of two laser modes results in a large tunability and a very operation frequency, but also in poor phase noise. Conversely, state of the art phase noise is obtained from coherent optical frequency division of a stabilized frequency comb [START_REF] Fortier | Generation of ultrastable microwaves via optical frequency division[END_REF][START_REF] Xie | Generation of ultrastable microwaves via optical frequency division[END_REF]. However this technique remains bulky and difficult to set up. Compared to these approaches, the optoelectronic oscillator (OEO) is a good alternative featuring relatively small size and complexity and low phase noise. Indeed, a simple OEO loop consists of a light source, an optical modulator, a fiber delay line, a photodiode, an amplifier and a filter (electronic or photonic) [START_REF] Yao | Optoelectronic microwave oscillator[END_REF][START_REF] Yao | Optoelectronic oscillator for photonic systems[END_REF]. Thus, the different components of the loop can be freely chosen to meet the objectives of the application to which the OEO is dedicated. Oscillation at any high frequency in the millimeterwave (mm-wave) range is theoretically possible [START_REF] Yao | Progress in the optoelectronic oscillator -a ten year anniversary review[END_REF] provided that high bandwidth modulators and high speed photodiodes are available.

However, expansion in frequency range somehow results in an increase of cost accordingly since low noise and high bandwidth RF components are nowadays certainly available but expensive. Consequently, the cost effective generation of pure RF signals in the V, E or W bands remains a challenge. As a trade-off to producing signals in the former bands from lower bandwidth RF components, frequency multiplication turned out to be a good alternative. As an example, it is possible to generate 60 GHz to 100 GHz signals by simply exploiting the nonlinearity of a Mach Zehnder modulator (MZM) [START_REF] Kiuchi | High extinction ratio Mach Zehnder Modulator applied to a highly stable optical signal generator[END_REF][START_REF] Llopis | 50-60 GHz local oscillator transmission over fiber using optical frequency multiplication[END_REF]. A tunable (95-115 GHz) low noise mm-wave source has also been demonstrated by combining nonlinearities in a MZM and four waves mixing in an optical amplifier [START_REF] Garcia | Low phase noise and frequency tunable 95-115 GHz photonic LO based upon DSB-SC modulation in an MZM and FWM in an UL-SOA[END_REF]. The mode-locked laser is also highly attractive for optical multiplication since this device is highly nonlinear and generates a frequency comb with a sufficient span. Thus, Fukushima et al. reported a 10-110 GHz mm-wave signals synthesis by optical injection locking of two lines of an optical frequency comb generator [START_REF] Fukushima | Optoelectronic millimeter-wave synthesis using an optical frequency comb generator, optically injection locked lasers, and a unitraveling-carrier photodiode[END_REF]. Fortier et al. generated a 100 GHz signal by using an electro-optic modulator driven at 10 GHz and an optical etalon to filter two 100 GHz spaced sidebands [START_REF] Fortier | Digitalphotonic synthesis of ultra-low noise tunable signals from RF to 100 GHz[END_REF]. By using an ultra-high finesse etalon as a photonic filter in an OEO, Bagnell et al. produced a 6-60 GHz signal which corresponds to the harmonics of the 1.5 GHz etalon free spectral range [START_REF] Bagnell | Millimeter-Wave Generation in an Optoelectronic Oscillator Using an Ultrahigh Finesse Etalon as a Photonic Filter[END_REF]. It turns out, in most of the experiments described above, that the generation of signals in the W band is either efficient in terms of spectral purity but requires large equipment or is simple but not efficient enough in terms of RF output power and spectral purity. To the best of our knowledge, there is to date no reported work on the synthesis of a mm-wave in the W band from a coupled optoelectronic oscillator (COEO). The COEO is a special case of a mode locked laser. It is actually the coupling of an electronic oscillator and an optical oscillator, both sharing the same active resonator. The COEO is thus the ideal candidate for millimeter wave generation with optics, because of this unique interplay between the extremely high Q of the active optical loop and the RF feedback loop. Harmonic generation from such system is particularly interesting since it benefits from the high spectral purity of the fundamental frequency and of the efficient multiplication factor due to the optical comb generation. The aim of the present paper is to demonstrate that a 30 GHz COEO is a good candidate for frequency generation at W band. Firstly, the COEO at 30 GHz is described. Then, its performances in terms of RF power at 30 GHz and 90 GHz as well as the corresponding spectral purity (phase noise) are presented Highly spectrally pure 90 GHz signal synthesis using a coupled optoelectronic oscillator Aliou Ly, Vincent Auroux, Ramin Khayatzadeh, Napoleon Gutierrez, Arnaud Fernandez, Olivier Llopis, Member, IEEE II. DESCRIPTION OF THE 30 GHZ COEO

The 30 GHz COEO architecture is depicted in Fig. 1. It is based on two coupled loops: an optical loop (black) and an optoelectrical loop (black and purple). The optical loop consists of a semiconductor optical amplifier (SOA) acting both as an active medium and a source of light. It features an output saturation power of 16 dBm and a 30 dB small signal gain. Light from the SOA successively travels an optical isolator (OI) to protect the SOA from reflection, a 50:50 optical coupler (OC1) and a 90:10 optical coupler (OC2) which 90 % output port is connected to a 400 m standard single mode fiber spool. This latter acts as a delay line and an energy storage device with a chromatic dispersion of the order of +6.8 ps/nm. Intra-cavity dispersion management is however of paramount importance to control the optical frequency comb coherence and to prevent from RF power fading effects. Thus, a chirped fiber Bragg grating (CFBG) with a chromatic dispersion of -7 ps/nm, used in reflection, was inserted into the loop for the group velocity dispersion compensation [START_REF] Khayatzadeh | COEO Phase locking and performance optimization[END_REF]. The output of the CFBG is next sent to a 40 GHz bandwidth MZM and a polarization controller (PC) is used to control its losses. Last, a second fiber delay (200 m SMF) is used to direct the light from the optical loop to the electrical loop. The electrical loop consists of a 75 GHz bandwidth U2T photodiode which output is immediately connected to a duplexer (a W band waveguide thanks to a V-W transition).

Both RF frequencies (fundamental and third harmonic) are next generated and, a 10 dB RF coupler (EC) directs 90 % of the power at 30 GHz to a RF filter. This latter is a home built dielectric resonator centered at 30.1 GHz, featuring a loaded quality factor Q of 430 (69 MHz at 3 dB bandwidth) and 5.7 dB transmission losses. This filter is placed between two electrical isolators (ECs) because the resonator is not perfectly 50 Ω matched. Finally, the filtered signal at 30 GHz is successively sent to a RF amplifier with a saturation power of 21 dBm and a gain of 30 dB, then to a RF phase shifter and back to the MZM. Let's see now how our system performs in term of RF power at 30 GHz and 90 GHz as well as the corresponding phase noise.

III. COEO PERFORMANCES

A. Optical frequency comb of the 30 GHz COEO Observation of the spectrum of the optical frequency comb generated from the COEO is primary since it gives clear information on the phase matching between all modes that can oscillate. More precisely, a wide span frequency comb with intense lines will result in ultra-short and low jitter optical pulses [START_REF] Matsko | Theory of coupled optoelectronic microwave oscillator I: expectation values[END_REF][START_REF] Matsko | Theory of coupled optoelectronic microwave oscillator II: phase noise[END_REF] and thus in a low phase noise for the corresponding RF signal accordingly. To this aim, the 10% output port of the 90:10 optical coupler (OC3, Fig. 1) is connected to an optical spectrum analyzer (OSA HP86142A).

We set the bias voltage of the MZM (Vπ around 5V) to Vbias= 4.02 V and optimized the COEO to get the spectrum displayed in Fig. 2. The optimization consisted of acting on the PC and on the RF phase shifter so as to get a large spectrum with lines with a good contrast.

B. RF power at 30 GHz and at 90 GHz

The evolution of the RF power at the fundamental frequency and its third harmonic versus the MZM bias voltage is studied in this section. The power was measured using an Agilent E4448A electrical spectrum analyzer (ESA, Fig. 1). For the RF signal at 30 GHz, the 10% output port of a 90:10 electrical coupler (EC) is connected to the ESA thanks to a K cable. We have assumed this cable induces an attenuation of 2 dB, then measurements to the ESA are accordingly corrected. For the third harmonic at 90 GHz, it has to be down converted since the ESA features only a maximum input frequency of 50 GHz. The harmonic mixer, which is driven by an internal source of the ESA, is calibrated by the manufacturer (Agilent) and a correction factor is directly applied to the ESA to get the effective power at 90 GHz at the mixer input.

Fig. 3 displays the RF powers for both signals (fundamental and third harmonic) at the duplexer output. As it can be seen, the RF power at 30 GHz remains nearly constant with an average value of -5.6 dBm. Indeed, losses increase with the bias voltage but they are compensated, to a certain limit by the RF amplifier in the electrical loop.

Fig. 3: RF powers at the level of the duplexer for the fundamental frequency (30 GHz) and its third harmonic (90 GHz) versus the MZM bias voltage. Inset, frequency response of the photodiode measured at the duplexer output.

The RF power of the 90 GHz signal features the same evolution as the fundamental with a mean value of -15.8 dBm. These results have to be compared to the -10 dBm obtained at 110 GHz by Fukushima et al. and to the -37 dBm obtained at 50 GHz by Llopis et al. In the former case an EDFA was placed before the photodiode in order to generate such RF output power. The photodiode response (inset of Fig. 3) has been measured using the beat signal between two identical DFB lasers independtly controlled in temperature. Considering its almost flat response up to 105 GHz, our compact system is able to generate appreciable RF power at mm-wave in the W band.

C. Spectral purity at 30 GHz and at 90 GHz

A last but not least study is the level of phase noise associated to our 90 GHz mm-wave synthesis. The harmonic generation should introduce a degradation of 9.5 dB compared to the phase noise spectrum at 30 GHz due to the frequency multiplication. Thus, the lower the phase noise at 30 GHz, the better the spectral purity of the RF signal at 90 GHz. The phase noise measurements are performed at 30 GHz and 90 GHz using an Agilent E5052B signal source analyzer combined with a microwave down converter E5053A and external millimeter wave mixers. Two identical mixers are used, in order to perform the correlation technique which allows an improvement of the phase noise floor of about 12 dB (correlation factor of 300). These mixers feature a loss of about 46 dB on the 75 GHz/110 GHz frequency band, which is partially compensated by a 30 dB low noise W band amplifier.

Since theses apparatus are placed into a Faraday cage located in another room, a 20 m fiber has been used to direct the optical signal from the 90% output port of the 90:10 optical coupler (OC3, Fig. 1) to the photodiode (the same as that one in Fig. 1).

The bias of the MZM is fixed at 4.02 V, which corresponds to the optical spectrum in Fig. 2. Phase noise measurements at 30 GHz and at 90 GHz are plotted in Fig. 4 for a correlation factor of 300. Phase noise level at 10 Hz offset frequency as low as -45 dBc/Hz and -33.2 dBc/Hz can be clearly seen for RF signals at 30 GHz and 90 GHz respectively. Then, the noise decreases with a close to 1/f 4 slope up to 100 Hz likely due to thermal drifts of the optical resonator and with a close to 1/f 3 slope between 100 Hz and 2 kHz, which corresponds to flicker frequency noise induced by the 1/f phase noise of the amplifiers (SOA and RF) in the oscillating loops. It reaches -114.4 dBc/Hz at 1 kHz offset frequency for the fundamental and -104.0 dBc/Hz at 1 kHz offset frequency for the third harmonic. Above approximately 3 kHz offset frequency, the noise floor of the E5052B measurement system is reached. Finally, the COEO phase noise performance is effectively transferred to 90 GHz with the only additional noise due to frequency multiplication by 3 (9.5 dB). Such a phase noise level is much lower than the phase noise level available from any frequency multiplication technique and a quartz based synthesizer, as shown in the last paragraph and in Figure 5.

We have compared our phase noise results to recent achievements in the field of low phase noise millimeter-waves synthesis. Figure 5 depicts the phase noise performance at 1 kHz offset of various sources between 50 GHz and 110 GHz. Circular dots represent millimeter wave sources referenced on quartz based microwave synthesizers, using frequency multiplication obtained either through optical or electronic devices. The ellipsoid represents the typical performance which can be obtained with the best microwave synthesizers and this approach. Diamond dots feature sources which are based on optical resonators. The square dot is the special case of a source obtained through optical frequency division. The last case represents the best performance, but it is obtained on a large size and complex system.

IV. CONCLUSION

Harmonic generation from a 30 GHz COEO has been performed to get a high quality frequency reference at 90 GHz.

The optical spectrum and the RF power of the fundamental and the third harmonic have been observed for different bias states of the MZM. Available RF powers as high as -5.6 dBm at 30 GHz and -15.8 dBm at 90 GHz have been demonstrated in addition to the low phase noise level obtained. Comparison of our results to those reported in the literature make us to believe that harmonic synthesis using a compact COEO is particularly efficient for ultra-high frequency and high spectral purity mmwave synthesis. To the best of our knowledge, this is the lowest phase noise level demonstrated at W band using optoelectronic oscillator approach. Future work will address above 100 GHz frequency generation using faster photodiodes, the photodiode being the only frequency limiting device in this system.

Fig. 1 :

 1 Fig. 1: Schematic of the experimental setup. MZM: Mach Zehnder Modulator, SOA: Semiconductor Optical Amplifier, PC: Polarization Controller, ESA: Electrical Spectrum Analyzer. OSA: Optical Spectrum Analyzer. E5052B: phase noise analyzer. SMF: Single Mode Fiber. CFBG: Chirped Fiber Bragg Grating. OI: Optical Isolator. EI: Electrical Isolator. OC: Optical Coupler. EC: Electrical Coupler. D: Duplexer. A: RF amplifier.

Fig. 2 :

 2 Fig. 2: Optical frequency comb of the 30 GHz COEO obtained at a 4.0 V bias voltage of the MZM.

Fig. 4 :

 4 Fig. 4: Phase noise measurements at 30 GHz (blue) and 90 GHz (red) with a cross correlation technique (correlation factor of 300) for a bias voltage of 4.02 V. Dark: phase noise spectrum of the signal source analyzer at 30 GHz. Green: phase noise spectrum of the signal source analyzer at 90 GHz.

Fig. 5 :

 5 Fig. 5: State of the art of high spectral purity generation in the 50-110 GHz range. Our system features about 10 dB lower noise than the best quartz referenced sources.

This work is supported by the French MoD, Délégation Générale à l'Armement (DGA), under the contract DIFOOL, and also by the région Occitanie, under the contract FLEKSY.