Julien Dhinaut
email: julien.dhinaut@u-bourgogne.fr

Manon Chogne

Yannick Moret

Trans-generational immune priming in the mealworm beetle protects eggs through pathogen-dependent mechanisms imposing no immediate fitness cost for the offspring

Keywords: Host-pathogen interaction, Ecological immunology, Invertebrates, Maternal effects, Fitness costs

Trans-generational immune priming in the mealworm

beetle protects eggs through pathogen-dependent mechanisms imposing no immediate fitness cost for the offspring.

Introduction

In many animals, the immunological experience of mothers can be transferred to otherwise naïve offspring and can protect them against infection. This plastic modulation of offspring immunity is likely adaptive by preparing them for the prevailing parasite environment.

Maternal transfer of immunity has been well studied in vertebrates, in which antibodies produced by infected females can be transferred to the foetus through the placenta and in milk during lactation in mammals, or via the egg yolk in birds, reptiles and fish [START_REF] Hasselquist | Maternal transfer of antibodies in vertebrates: transgenerational effects on offspring immunity[END_REF]. Since newborn vertebrates have limited abilities to produce their own antibodies, this maternal help protects them pending the development of their own immune defence.

Invertebrates lack the antibodies that vertebrate mothers pass to their offspring. However, such a trans-generational effect, also called trans-generational immunity (TGIP), has been observed in many invertebrates too [START_REF] Boots | Maternal effects in disease resistance: poor maternal environment increases offspring resistance to an insect virus[END_REF][START_REF] Freitak | Dietary-dependent trans-generational immune priming in an insect herbivore[END_REF][START_REF] Hernández López | Transgenerational immune priming in honeybees[END_REF][START_REF] Huang | Maternal transmission of immunity to white spot syndrome associated virus (WSSV) in shrimp (Penaeus monodon)[END_REF][START_REF] Little | Maternal Transfer of Strain-Specific Immunity in an Invertebrate[END_REF][START_REF] Moret | Trans-generational immune priming: specific enhancement of the antimicrobial immune response in the mealworm beetle, Tenebrio molitor[END_REF][START_REF] Norouzitallab | Multigenerational immune priming in an invertebrate parthenogenetic Artemia to a pathogenic Vibrio campbellii[END_REF][START_REF] Roth | Paternally derived immune priming for offspring in the red flour beetle, Tribolium castaneum[END_REF][START_REF] Sadd | Transgenerational immune priming in a social insect[END_REF][START_REF] Sadd | Facultative but persistent trans-generational immunity via the mother's eggs in bumblebees[END_REF][START_REF] Tidbury | Within and transgenerational immune priming in an insect to a DNA virus[END_REF][START_REF] Trauer | Parental Legacy in Insects: Variation of Transgenerational Immune Priming during Offspring Development[END_REF][START_REF] Yue | Maternal transfer of immunity in scallop Chlamys farreri and its trans-generational immune protection to offspring against bacterial challenge[END_REF]. TGIP effects were revealed through enhanced immune activity and/or increased survival to infection in primed eggs [START_REF] Dubuffet | Transgenerational immune priming protects the eggs only against gram-positive bacteria in the mealworm beetle[END_REF][START_REF] Moreau | Trans-generational immune priming is constrained by the maternal immune response in an insect[END_REF][START_REF] Sadd | Facultative but persistent trans-generational immunity via the mother's eggs in bumblebees[END_REF][START_REF] Zanchi | Relationship between maternal transfer of immunity and mother fecundity in an insect[END_REF], juveniles [START_REF] Freitak | Dietary-dependent trans-generational immune priming in an insect herbivore[END_REF][START_REF] Moret | Trans-generational immune priming: specific enhancement of the antimicrobial immune response in the mealworm beetle, Tenebrio molitor[END_REF][START_REF] Rahman | Induction and transmission of Bacillus thuringiensis tolerance in the flour moth Ephestia kuehniella[END_REF][START_REF] Rosengaus | Pathogen-induced maternal effects result in enhanced immune responsiveness across generations[END_REF][START_REF] Tidbury | Within and transgenerational immune priming in an insect to a DNA virus[END_REF] and adult offspring [START_REF] Castro-Vargas | Methylation on RNA: A Potential Mechanism Related to Immune Priming within But Not across Generations[END_REF][START_REF] Roth | Paternally derived immune priming for offspring in the red flour beetle, Tribolium castaneum[END_REF][START_REF] Sadd | A distinct infection cost associated with transgenerational priming of antibacterial immunity in bumble-bees[END_REF][START_REF] Zanchi | Differential expression and costs between maternally and paternally derived immune priming for offspring in an insect[END_REF].

While beneficial against repeated parasite attacks across generations, TGIP is also associated to fitness costs. Indeed, investment in immunity is costly on multiple levels [START_REF] Schmid-Hempel | Variation in immune defence as a question of evolutionary ecology[END_REF], and its enhanced activity, due to TGIP, was found negatively impacting primed offspring growth [START_REF] Lorenz | Maternal environment shapes the life history and susceptibility to malaria of Anopheles gambiae mosquitoes[END_REF][START_REF] Roth | Paternally derived immune priming for offspring in the red flour beetle, Tribolium castaneum[END_REF] and reproduction [START_REF] Trauer | Parental Legacy in Insects: Variation of Transgenerational Immune Priming during Offspring Development[END_REF]. However, the magnitude of the cost paid by the offspring to TGIP could be dependent on the bacterial pathogens that immune challenged the mothers [START_REF] Dhinaut | Immune priming specificity within and across generations reveals the range of pathogens affecting evolution of immunity in an insect[END_REF]. For instance, the offspring of the mealworm beetle, Tenebrio molitor, paid a lower cost to TGIP in terms of growth when their mothers were immune challenged by Gram-positive bacteria, which are believed to have being an important part of the selection behind the evolution of TGIP in this insect species [START_REF] Dubuffet | Transgenerational immune priming protects the eggs only against gram-positive bacteria in the mealworm beetle[END_REF][START_REF] Dhinaut | Immune priming specificity within and across generations reveals the range of pathogens affecting evolution of immunity in an insect[END_REF].

Furthermore, in addition of paying the usual immune activation costs, immune-challenged mothers were also found to pay a cost to TGIP when producing and transferring immune products to the offspring [START_REF] Moreau | Trans-generational immune priming is constrained by the maternal immune response in an insect[END_REF]. However, the division of TGIP costs between mothers and their offspring might depend on the mechanisms through which the immune protection of the offspring is achieved, which is currently unknown.

In insects, bacterially immune-challenged females produced eggs containing enhanced level of antibacterial activity [START_REF] Dubuffet | Transgenerational immune priming protects the eggs only against gram-positive bacteria in the mealworm beetle[END_REF][START_REF] Moreau | Trans-generational immune priming is constrained by the maternal immune response in an insect[END_REF][START_REF] Sadd | Facultative but persistent trans-generational immunity via the mother's eggs in bumblebees[END_REF][START_REF] Trauer-Kizilelma | Impact of transgenerational immune priming on the defence of insect eggs against parasitism[END_REF][START_REF] Zanchi | Relationship between maternal transfer of immunity and mother fecundity in an insect[END_REF]. This TGIP effect on eggs may result from two non-exclusive mechanisms, which should have different fitness outcomes for mothers and their progeny. On the one hand, primed eggs might be passively supplied with antibacterial substances from the mother [START_REF] Berry | Maternal Direction of Oogenesis and Early Embryogenesis in Insects[END_REF]. Therefore, eggs should exhibit strong levels of antibacterial activity soon after being laid. This antibacterial activity may then persist or decline during incubation, as the antibacterial substances are metabolized. Since the mother mostly ensures the egg immune protection, they may pay a significant cost to TGIP, which may take the form of a reduced fecundity [START_REF] Moreau | Trans-generational immune priming is constrained by the maternal immune response in an insect[END_REF]. However, as eggs do not need to spend resources for their defense, they are not expected to pay a cost to TGIP. On the other hand, immune-challenged mothers may elicit the activation of immune genes in the developing embryo as insect eggs were shown to activate their own immune response [START_REF] Freitak | The maternal transfer of bacteria can mediate trans-generational immune priming in insects[END_REF][START_REF] Jacobs | Egg survival is reduced by grave-soil microbes in the carrion beetle, Nicrophorus vespilloides[END_REF][START_REF] Jacobs | Endogenous egg immune defenses in the yellow mealworm beetle (Tenebrio molitor)[END_REF][START_REF] Jacobs | Immune competence in insect eggs depends on the extraembryonic serosa[END_REF]. The egg immune response could either be induced by the maternal transfer of bacteria or bacterial fragments into the egg [START_REF] Freitak | The maternal transfer of bacteria can mediate trans-generational immune priming in insects[END_REF][START_REF] Knorr | Translocation of bacteria from the gut to the eggs triggers maternal transgenerational immune priming in Tribolium castaneum[END_REF][START_REF] Salmela | Transfer of immunity from mother to offspring is mediated via egg-yolk protein vitellogenin[END_REF] or by epigenetic mechanisms such as DNA methylation or histone modification [START_REF] Castro-Vargas | Methylation on RNA: A Potential Mechanism Related to Immune Priming within But Not across Generations[END_REF][START_REF] Sysoev | Global changes of the RNA-bound proteome during the maternal-tozygotic transition in Drosophila[END_REF]. In this case, eggs should exhibit temporal increase in level of antibacterial activity with time after oviposition as the immune response of the embryo is activated. Because immune responses are costly [START_REF] Moret | Survival for immunity: the price of immune system activation for bumblebee workers[END_REF], eggs raising their own immune activity are expected to pay a cost to TGIP too. Indeed, eggs have limited resources and nutrients do develop into larvae and those consumed by the immune response could be then missing for the developing embryo. As resource shortage in insect eggs affects the development of the embryo and the survival of the resulting larvae [START_REF] Geister | Adult nutrition and butterfly fitness: effects of diet quality on reproductive output, egg composition, and egg hatching success[END_REF], resources used by the egg immune response may reduce hatching success rate, extend incubation time or decrease survival of young larvae.

Here, we used the mealworm beetle, Tenebrio molitor, to first decipher the mechanisms through which immune-challenged mothers protect their eggs. We then tested the fitness consequences that these mechanisms may have on egg success when exposed or not to the same bacterial pathogen used to immune challenge the mother. To this purpose, we characterized the temporal dynamics of antibacterial activity in eggs laid by immunechallenged mothers with different Gram-positive bacterial pathogens. Changes in antibacterial activity levels with time after oviposition should reveal the main process involved for egg protection. In particular, increased levels of antibacterial activity with time post-oviposition should indicate that eggs are producing themselves their antibacterial substances. By contrast, maintained or decreased levels of antibacterial activity with time post-oviposition would indicate that the mother is mostly providing antibacterial substances to the eggs, although both mechanisms are likely not exclusive. In addition, to estimate fitness costs associated to egg immune protection, we compared the success of eggs of control and immune-challenged mothers with different Gram-positive bacteria in terms of egg size, hatching rate and incubation time when exposed or not to the same bacterial pathogen that immune challenged the mother. When eggs successfully hatched, we further compared the survival of the resulting larvae under food privation, while prolonging the infectious treatment to which they were exposed at the egg stage. We expected that primed eggs and young larvae should respectively show improved hatching success rate and survival when exposed to the bacterial infection. Furthermore, in pathogen free condition, we did not expect to see any cost of TGIP resulting from a passive transfer of antibacterial substances from the mother. Conversely, eggs protected through the activation of their own immune response were expected to exhibit lower hatching success rate and/or to show prolonged incubation time. In addition, larvae that successfully hatched were expected to suffer from a lower survival to starvation.

Material and Methods

Insect cultures

Virgin adult beetles of controlled age (10 ± 2 days post-emergence) were obtained from pupae taken at random from an outbred stock culture maintained in standard laboratory conditions at the University of Burgundy, Dijon, France. All the experimental insects were reared and maintained in an insectary at 24 ± 2°C, 70% RH in permanent darkness, and supplied ad libitum with bran flour and water, supplemented by apple, unless specified. All the insects were weighed to the nearest 1 mg with an OHAUS balance (discovery series, DU114C) before use in the experiments. We conducted two separate experiments to study the impact of TGIP on produced eggs. The first experiment aimed to characterize the temporal dynamics of antibacterial activity in eggs after being laid by immune-challenged females with two Gram-positive bacteria: Arthrobacter globiformis and Bacillus thuringiensis. B. thuringiensis is a known entomopathogenic bacterium of beetles, whereas A. globifomis is opportunistic. The second experiment tested the effect of maternal priming with either A. globifomis or B. thuringiensis on egg size, egg hatching success rate, egg incubation time and survival to starvation of the resulting larvae, while exposed or not to the same bacteria that was used for the maternal challenge. These experiments used these two Gram-positive bacteria because antibacterial activity found in T. molitor eggs was shown to be active against Gram-positive bacteria only [START_REF] Dubuffet | Transgenerational immune priming protects the eggs only against gram-positive bacteria in the mealworm beetle[END_REF].

Temporal dynamics of antibacterial activity in eggs

Groups of 40 virgin females were anaesthetized for 10 min on ice and injected with a 5-µL suspension of either inactivated A. globiformis or inactivated B. thuringiensis at a concentration of 10 8 cells per mL of phosphate buffer saline solution (PBS 10 mM, pH 7.4).

An additional group of 40 females was treated in the same way but without bacteria, as a procedural control for the effect of the injection. Immediately after their immune treatment, the females were paired with a virgin and immunologically naïve male of the same age and allowed to produce eggs in a Petri dish supplied with wheat flour, apple and water in standard laboratory conditions (24 ± 2°C, 70% RH; permanent darkness). Males were removed from the Petri dishes two days after being paired with the females. Only the eggs produced at the third day after maternal priming were collected to test their antibacterial activity, because females protect most of their eggs from day 2 to day 8 after immunization [START_REF] Zanchi | Relationship between maternal transfer of immunity and mother fecundity in an insect[END_REF]. Three eggs per female were collected and randomly allocated to be tested for their antibacterial activity at either day 1 (between 1 and 24 hours after being laid), day 3 (between 48 and 72 hours after being laid), day 5 (between 96 and 120 hours after being laid) or day 7 (between 144 and 168 hours after being laid) after being laid. Hence, within each immune treatment group, 3 eggs of 10 females independently contributed to each age point of the eggs.

When reaching their allocated age (1, 3, 5 or 7 days after being laid), eggs were snap frozen into liquid nitrogen and stored at -80°C, awaiting measurement of their antibacterial activity using a standard zone of inhibition assay [START_REF] Zanchi | Relationship between maternal transfer of immunity and mother fecundity in an insect[END_REF] against A. globiformis. Previous work showed that egg antibacterial activity of immune-challenged mothers was only active against Gram-positive bacteria [START_REF] Dubuffet | Transgenerational immune priming protects the eggs only against gram-positive bacteria in the mealworm beetle[END_REF]. In addition, since antibacterial activity of a same egg extract tested on A. globiformis and B. thuringiensis were highly positively correlated, zone inhibition tests, in this study, were only done on A. globiformis, which also offers the best sensitivity to any antimicrobial activity developed in the mealworm beetle [START_REF] Dubuffet | Transgenerational immune priming protects the eggs only against gram-positive bacteria in the mealworm beetle[END_REF].

Egg size, egg hatching rate, egg incubation time and larval survival

Assessment of primed egg success in terms of egg size, egg hatching success rate, egg incubation time and larval survival while exposed or not to the same bacterial pathogen that immune challenged mothers used 40 immune-challenged females with A. globiformis, 40 immune-challenged females with B. thuringiensis and 60 control females, all treated and allowed to produce eggs as described above. Three eggs per female were stored at -80°C for later measurement of their size. Egg size was estimated by measuring egg area under microscope (magnification x20) SMZ1500 connected to a camera and a computer running NIS-Elements-AR. Egg size is expected to reveal difference in egg quality, with larger eggs believed to be of better quality [START_REF] Capinera | Yolk and yolk depletion of gypsy moth eggs: implications for population quality 1, 2[END_REF]. Three additional eggs per female were isolated into 12-wells micro-titer plates. Within each maternal immune treatment group, eggs were randomly assigned for being exposed to the living bacterium used to challenge their mother or to pathogen free condition. Eggs of control mothers were randomly divided into three equal groups allocated to exposure to living A. globiformis, living B. thuringiensis and free pathogen condition. Hatching success and incubation time (time required from the moment the egg has been laid to the moment it hatched) were recorded. Four days after hatching, young larvae were transferred into a new well to measure their survival while starved. Larvae resulting from eggs exposed to bacterial pathogen were further exposed to the same bacterium as the one used at the egg stage. Survival to starvation is expected to reveal difference in body condition post hatching. Larvae with the best body condition at hatching are expected to show the longest survival to starvation. Survival was recorded for 31 days post-hatching.

Bacterial cultures for maternal treatment and offspring exposure

All the bacteria used in this study were obtained from the Pasteur Institute: A. globiformis (CIP105365) and B. thuringiensis (CIP53.1) and cultured as described in [START_REF] Dubuffet | Transgenerational immune priming protects the eggs only against gram-positive bacteria in the mealworm beetle[END_REF] and [START_REF] Dhinaut | Immune priming specificity within and across generations reveals the range of pathogens affecting evolution of immunity in an insect[END_REF]. Briefly, bacteria were grown overnight at 28°C in liquid Broth medium (10 g bacto-tryptone, 5 g yeast extract, 10 g NaCl in 1000 mL of distilled water, pH 7.5). To perform the maternal immune treatment, the bacteria were then inactivated in 0.5% formaldehyde prepared in PBS for 30 minutes, rinsed three times in PBS, and their concentration adjusted to 10 8 bacteria per mL using a Neubauer improved cell counting chamber. The success of the inactivation was tested by plating a sample of the bacterial solution on sterile Broth medium with 1% of bacterial agar and incubated at 28°C for 24 hours [START_REF] Dhinaut | Immune priming specificity within and across generations reveals the range of pathogens affecting evolution of immunity in an insect[END_REF]. Aliquots were kept at -20°C until use. Maternal treatment was done by injection of 5 µL of the bacterial suspension through the pleural membrane between the second and third abdominal tergites using sterile glass capillaries that had been pulled out to a fine point with an electrode puller (Narashige PC-10). For egg and larvae bacterial treatments, overnight cultures (20 mL) were centrifuged at 3500 g at 4°C for 30 min. The supernatant was discarded and the bacteria pellet was used to spread them, using a small paintbrush, on the bottom of wells hosting eggs or larvae.

Egg antibacterial activity

Antibacterial activity of the eggs was measured using a standard zone of inhibition assay [START_REF] Dubuffet | Transgenerational immune priming protects the eggs only against gram-positive bacteria in the mealworm beetle[END_REF][START_REF] Zanchi | Relationship between maternal transfer of immunity and mother fecundity in an insect[END_REF]. Samples were thawed on ice, and egg extracts were prepared by mashing eggs into an acetic acid solution (0.05%, 5 μl per egg). After centrifugation (3500 g, 2 min, 4°C) 2 µL of the supernatant was used to measure antibacterial activity on zone of inhibition plates seeded with A. globiformis. An overnight culture of the bacterium was added to Broth medium containing 1% agar to achieve a final concentration of 10 5 cells per mL. Six millilitres of this seeded medium was then poured into a Petri dish and allowed to solidify. Sample wells were made using a Pasteur pipette fitted with a ball pump.

Two microlitres of sample solution was added to each well, and a positive control (Tetracycline: Sigma-Aldrich, St Louis, MO, USA, T3383; 2.5 mg.mL -1 in absolute ethanol) was included on each plate [START_REF] Zanchi | Differential expression and costs between maternally and paternally derived immune priming for offspring in an insect[END_REF]. Plates were then incubated overnight at 28 °C. The diameter of inhibition zones was then measured for each sample. As there was slight variation among plates for inhibition zones resulting from tetracycline addition, the diameter of the zone of inhibition of each sample was adjusted to take into account such a between plate variation. For this, the Petri dish in which the inhibition zone resulting from the tetracycline positive control was the largest was used as reference. Then, for each sample, inhibition zone was multiplied by the ratio between the inhibition zone due to tetracycline in the reference Petri dish and that due to tetracycline in the Petri dish in which the sample was measured [START_REF] Dhinaut | Immune priming specificity within and across generations reveals the range of pathogens affecting evolution of immunity in an insect[END_REF].

Statistics

Variation in egg antibacterial according to maternal immune treatment and time after oviposition was analyzed using a Generalized Linear Model (GLM) fitted with a gamma distribution and a Log link function. Differences between each female treatment at each time point after oviposition (or egg age) were highlighted by estimating the degree of overlap of the 95% confidence intervals (CI), following recommendations by [START_REF] Cumming | Inference by eye: confidence intervals and how to read pictures of data[END_REF]. The difference was considered significant when the 95% CI did not overlap on more than half of their length (P < 0.05, Fig. 1).

Data on egg number and egg size were analyzed using univariate analyses of variance (ANOVAs), according to maternal immune treatment, mother body mass and female identity (when required) as main effects.

Data on egg hatching success and egg incubation time were respectively analyzed using a binary logistic regression analysis and an ANOVA, according to maternal immune treatment, egg bacterial exposure, mother body mass and female identity. For these analyses, the best statistical models were searched using a backward stepwise procedure from a full model that included all the main effects and the interaction term between maternal treatment and egg bacterial exposure.

Larval survival was analysed by using a Cox regression analysis, according to maternal immune treatment, larvae bacterial exposure, mother body mass and female identity.

The best statistical model was searched using a backward stepwise procedure from a full model including all the main effect and the interaction between maternal immune treatment and larvae bacterial exposure.

All statistical analyses used IBM® SPSS® Statistics 19 for Macintosh.

Results

Temporal dynamics of antibacterial activity in eggs

Overall, eggs of immune-challenged females with either A. globiformis or B. thuringiensis exhibited higher levels of antibacterial activity than those from control females (GLM χ² = 154.84, df = 2, N = 432, P < 0.001, Fig. 1). Egg antibacterial activity in primed eggs was also dependent on egg age (χ²3, 420 =21.74, P = <0,001, Fig. 1). However, pattern of co-variation between egg antibacterial activity and egg age highly differed according to the bacteria used to immune challenge mothers (Maternal treatment*Egg age χ²6, 420 = 55.52, P < 0.001, Fig. 1). Indeed, egg antibacterial activity of B. thuringiensis-immunized females increased with egg age. Antibacterial activity of 1-day old eggs is significantly lower than that of 3, 5 or 7-days old eggs (Fig. 1). By contrast, antibacterial activity of 1, 3 and 5-days old eggs from immunechallenged females with A. globiformis was significantly higher than that of 7-days old eggs (Fig. 1). Egg antibacterial activity from immune-challenged mothers either by B. thuringiensis or A. globiformis was significantly dependent on mother body mass (χ²1, 420 = 16.09, P = 0.001), but not on mother identity (χ²1, 420 = 0.25, P = 0.61).

Egg size, hatching rate, incubation time and larval survival

Number of eggs produced by females was independent of the maternal immune treatment and female body mass (Table 1). Conversely egg size was dependent on the maternal immune treatment (Table 1). Immune-challenged mothers with A. globiformis produced the largest eggs, whereas immune-challenged mothers with B. thuringiensis produced the smallest (Fig.

2).

When correcting for mothers body mass and identity, hatching success was about 8 to 12 times (see odd ratios in Table 2) higher for eggs of immune-challenged mothers with respectively either A. globiformis and B. thuringiensis than for eggs of control mothers (Table 2, Fig. 3). Egg bacterial exposure with either A. globiformis or B. thuringiensis did not explain any variation in hatching rate either as main effect or in interaction with the maternal immune treatment (Table 2). Hatching success of eggs produced by large females tended to decrease than that of eggs produced by small females in the experiment that used A. globiformis (Table 2A). Female identity explained part of the variation in egg hatching success too (Table 2).

Variation in egg incubation time could not be significantly explained by the statistical models comprising maternal immune treatment, egg bacterial exposure, mother body mass and mother identity as main effects either when comparing eggs of control mothers with those of challenged mothers with A. globiformis (Global model: F4, 141 = 1.43 P = 0.226) or those of challenged mothers with B. thuringiensis (Global model: F4, 136 = 2.03 P = 0.094). However, for the data from the experiment that used B. thurigiensis as bacterial pathogen for priming and egg bacterial exposure, removing maternal immune treatment from the latter statistical model (this factor was the one that explained the less variation in incubation time: F1, 136 = 0), succeeded in explaining variation in egg incubation time (Global model: F3, 137 = 2.73 P = 0.047), essentially because of the egg bacterial exposure treatment (F41 137 = 7.19 P = 0.008).

Eggs exposed to the bacterium B. thuringiensis exhibited a slight prolonged incubation time (mean in days ± 95% IC: 9.10 ± 0.12) than non-exposed ones (mean in days ± 95% IC: 8.9 ± 0.08). A similar procedure for the analysis of the data from the experiment that used A. globiformis failed to improve variance explanation (Global model: F3, 132 = 1.25 P = 0.295).

In the experiment using A. globiformis for the maternal treatment and offspring bacterial exposure, survival of larvae was only explained by the bacterial exposure treatment, which imposed a 1.6-folds survival reduction of the larvae (Table 3A, Fig. 4A). In the experiment that used B. thuringiensis, larval exposure to the bacterium imposed a 2.7-folds survival reduction (Table 3B, Fig. 4B). However, larvae hatched from eggs of immune-challenged mothers with B. thuringiensis exhibited improved survival, irrespectively of the bacterial exposure treatment (Table 3B). Mother identity significantly explained survival of newborn larvae, but not mother body mass (Table 3B).

Discussion

Our results show that eggs produced by immune-challenged females of the mealworm beetle, T. molitor, with different bacterial pathogens exhibited contrasted patterns of variation in antibacterial activity with time post-oviposition. In particular, primed eggs of immunechallenged mothers with an opportunistic bacterial pathogen, such as A. globiformis, exhibited strong levels of antibacterial activity soon after being laid, which was however significantly lower by day 7 post-oviposition (Fig. 1). Such a pattern of variation in antibacterial activity with egg age is consistent with the hypothesis that mothers passively supply their eggs with antibacterial substances, which are then metabolized as eggs develop. By contrast, primed eggs of immune-challenged mothers with a known entomopathogenic bacteria of T. molitor, such as B. thuringiensis [START_REF] Du Rand | Determination of insecticidal toxicity of three species of entomopathogenic spore-forming bacterial isolates against Tenebrio molitor L. (Coleoptera: Tenebrionidae)[END_REF] exhibited very low levels of antibacterial activity soon after being laid. Antibacterial activity then increased to reach a plateau from the third day post-oviposition to hatching (Fig. 1). Such a pattern of variation clearly suggests that antimicrobial substances are synthesized in the eggs and are not directly provided by the mother. The production of immune effectors by the egg themselves is probably not surprising as eggs in some insect species can activate their own immune response [START_REF] Freitak | The maternal transfer of bacteria can mediate trans-generational immune priming in insects[END_REF][START_REF] Gorman | Bacterial challenge stimulates innate immune responses in extra-embryonic tissues of tobacco hornworm eggs[END_REF][START_REF] Jacobs | Immune competence in insect eggs depends on the extraembryonic serosa[END_REF][START_REF] Tingvall | The GATA factor Serpent is required for the onset of the humoral immune response in Drosophila embryos[END_REF]. This is also true in T. molitor [START_REF] Jacobs | Endogenous egg immune defenses in the yellow mealworm beetle (Tenebrio molitor)[END_REF]. However, while our results clearly suggest that antibacterial substances, which are proteins [START_REF] Dubuffet | Transgenerational immune priming protects the eggs only against gram-positive bacteria in the mealworm beetle[END_REF], are produced by the eggs, we are unable to state whether the transcripts at the origin of these antibacterial proteins are synthesized in the eggs too or produced and transferred by mothers to the eggs. Further work is needed to decipher the complete mechanisms of egg immune protection. Nevertheless, we found evidence that the mechanisms through which eggs become immune protected after a maternal immune challenge are pathogen-dependent. The existence of these different mechanisms suggests that at least one of them may have specifically evolved from a tight coevolutionary history between T. molitor immune system and one of the pathogen used in our experiment. Compared to A. globiformis, B. thuringiensis is a known bacterial entomopathogen of T. molitor [START_REF] Du Rand | Determination of insecticidal toxicity of three species of entomopathogenic spore-forming bacterial isolates against Tenebrio molitor L. (Coleoptera: Tenebrionidae)[END_REF], characterised by a strong ability to persist in the environment between insect generations, by forming endospores (Jurat-Fuentes and Jackson, 2012). It is therefore tempting to speculate that the mechanism of egg protection after a maternal infection with B. thuringiensis could be specific to that range of bacterial pathogens, whereas the one involved in the response to a maternal infection with A. globiformis could be more generic. However, further studies are required to confirm or refute such a hypothesis. These mechanisms were assumed to have different implications about fitness costs associated to egg immune protection resulting from the maternal immune challenge. Indeed, mothers were expected to mostly support the cost of the egg immune protection when producing and transferring immune effectors to the eggs. Conversely, eggs were expected to mostly support the cost of their immune protection when induced to produce themselves their immune effectors. However, our results did not reveal any evidence of cost associated to the egg immune protection for mothers, whatever the mechanisms involved. Indeed, control and immune-challenged females, with either A. globiformis or B. thuringiensis, produced a similar number of eggs. This suggests that immune-challenged females did not pay an immediate cost on fecundity to the immune challenge it-self and for supplying immune effectors to the eggs.

The latter is particularly true for immune-challenged females with A. globiformis, which, despite directly supplying antibacterial substances to their eggs, and producing larger eggs, did not show any reduced fecundity. It is nevertheless possible that while fecundity was not affected at this particular egg-laying sequence of the female beetles, it could be reduced during subsequent egg-laying sequences as previously found [START_REF] Moreau | Trans-generational immune priming is constrained by the maternal immune response in an insect[END_REF].

Furthermore, provisioning eggs with antibacterial substances could be done at the expense of the own immune response of the mother [START_REF] Moreau | Trans-generational immune priming is constrained by the maternal immune response in an insect[END_REF], which was not estimated in this study.

We found that the maternal immune challenge had no negative effect on egg success either. On the contrary, eggs of immune-challenged mothers by either A. globiformis or B. thuringiensis even exhibited higher hatching success rates than those of control mothers, and this, whether eggs were exposed or not to the maternal bacterial pathogen. This result confirms a recent observation in Manduca sexta, where the hatching success of eggs of immune-challenged mothers was improved [START_REF] Rosengaus | Pathogen-induced maternal effects result in enhanced immune responsiveness across generations[END_REF]. In addition, primed eggs did not exhibit longer incubation time, which could have been expected when eggs are using limited resources to produce their own immune effectors. Here, eggs of immune-challenged mothers by B. thuringiensis, which we found producing themselves their own antibacterial substances, did not traded-off their development against their immunity. This suggests that primed eggs might be sufficiently provisioned with the necessary metabolites, such as carbohydrates, proteins and lipids [START_REF] Kinsella | Metabolic patterns of the fatty acids of Periplanteta americana (L.) during its embryonic development[END_REF][START_REF] Pant | Changes in carbohydrates and lipids during embryonic development of Antheraea mylitta (Lepidoptera)[END_REF], to develop normally and even exhibit higher hatching success. While incubation time of control and primed eggs exposed to A. globiformis was not affected, it was slightly increased in control and primed eggs exposed to B. thuringiensis. This result shows that eggs, primed or not, could somehow specifically sense the presence of pathogens, which presumably affects their development.

However, the mechanisms through which the eggs respond to the presence of certain bacterial pathogens remain unknown.

While exposure to living bacteria did not affect egg success, it strongly reduced the early survival performance of the larvae. However, survival of larvae of immune-challenged mothers with B. thuringiensis was higher than survival of those of control mothers whether they were exposed or not to the living bacterium. Such a survival improvement was not found among larvae of immune-challenged mothers with A. globiformis. This confirms that the priming response leading to enhanced immunity of the eggs and then of the larvae is dependent on the bacterial pathogens that previously immune challenged the mothers. Again, in pathogen free conditions, B. thuringiensis-primed larvae performed better than control larvae, suggesting that larvae pay no cost to TGIP during their early life. In this study, larvae were not fed and therefore could not reach the adult stage. Previous studies have evidenced that the offspring of immune-challenged mothers with various bacterial pathogens, including B. thuringiensis, are paying a cost to TGIP by exhibiting a prolonged larval developmental time [START_REF] Zanchi | Differential expression and costs between maternally and paternally derived immune priming for offspring in an insect[END_REF][START_REF] Dhinaut | Immune priming specificity within and across generations reveals the range of pathogens affecting evolution of immunity in an insect[END_REF]. Longer developmental time is costly because it reduced competitive ability for food under higher densities [START_REF] Koella | A genetic correlation between age at pupation and melanization immune response of the yellow fever mosquito Aedes aegypti[END_REF] and should delay access to reproduction. Moreover, it could increase the probability of mortality [START_REF] Bell | The costs of reproduction and their consequences[END_REF], especially in insects like T. molitor, which exhibits cannibalism on juveniles [START_REF] Ichikawa | Larval cannibalism and pupal defense against cannibalism in two species of Tenebrionid beetles[END_REF]. Costs of TGIP on larval development of the primed offspring were evidenced in other insect models, such as flour beetles [START_REF] Roth | Paternally derived immune priming for offspring in the red flour beetle, Tribolium castaneum[END_REF] or mosquitoes [START_REF] Lorenz | Maternal environment shapes the life history and susceptibility to malaria of Anopheles gambiae mosquitoes[END_REF]. In M. sexta, adult offspring derived from immune-challenged parents have a lower fecundity than those derived from control parents [START_REF] Trauer | Parental Legacy in Insects: Variation of Transgenerational Immune Priming during Offspring Development[END_REF]. This suggests that, while TGIP may show no cost early in life of the offspring and may even provide benefits beyond enhanced immunity to the current pathogenic threat, it is nevertheless costly and its costs appear later in life of the offspring. Such long-term trade-offs are classical in the study of maternal effects [START_REF] Clark | Prenatal influences on reproductive life history strategies[END_REF][START_REF] Lindström | Early development and fitness in birds and mammals[END_REF][START_REF] Qvarnström | Maternal effects, paternal effects and sexual selection[END_REF], to which TGIP belongs. Hence TGIP may have strong consequences on individual life-history trajectories, which evolutionary and ecological impacts on population dynamics and life history evolution are still underexplored.

To summarize, this study reports three novel findings about TGIP in the eggs of the mealworm beetle T. molitor following a maternal bacterial immune challenge. First, we found that enhanced antibacterial activity in eggs of immune-challenged mothers relies on different mechanisms that are dependent on the bacterial pathogen challenging the mother. On the one hand, females produce and transfer antibacterial substances to the eggs and, on the other hand, eggs are allowed to produce them-selves their antibacterial substances. Whether these various mechanisms, affecting the temporal dynamics of egg immunity have evolved from relatively specific coevolutionary history between the insect immune system and pathogens remain unknown. Second, TGIP provided fitness benefits to the offspring beyond enhanced of immunity as it increased egg hatching success and early larval survival while exposed or not to the infection by the bacteria that previously immune challenged the mother. This suggests that TGIP does not only enhance immunity of the offspring but also implies the provision of extra metabolites or nutrients to the offspring, increasing their individual quality. Third, we failed to evidence any immediate fitness cost associated to TGIP for the eggs and early larval stages, whereas numerous previous studies have evidence fitness costs for the offspring later in life [START_REF] Lorenz | Maternal environment shapes the life history and susceptibility to malaria of Anopheles gambiae mosquitoes[END_REF][START_REF] Roth | Paternally derived immune priming for offspring in the red flour beetle, Tribolium castaneum[END_REF][START_REF] Trauer | Parental Legacy in Insects: Variation of Transgenerational Immune Priming during Offspring Development[END_REF][START_REF] Zanchi | Differential expression and costs between maternally and paternally derived immune priming for offspring in an insect[END_REF][START_REF] Dhinaut | Immune priming specificity within and across generations reveals the range of pathogens affecting evolution of immunity in an insect[END_REF]. This suggests that early life of primed offspring appear critical in the optimization of life history trajectory of the beetles exposed to pathogens exhibiting high probability of persistence between insect generations.

Fig. 1 .

 1 Fig. 1.

Table 1 .

 1 Univariate analyses of egg number and egg size produced by control and immune-challenged T. molitor females with either A. globiformis or Bacillus thuringiensis (Maternal treatment), controlling for mother body mass and mother identity (when required). Values where P ≤ 0.05 are given in bold.

	Dependent variables Source of variation	F	d.f.	P
	Egg number	Model	0.98 3, 167	0.405
		Maternal treatment	0.76 2, 167	0.467
		Mother body mass	1.64 1, 167	0.203
	Egg Size	Model	9.60 4, 481 < 0.001
		Maternal treatment 11.68 2, 481 < 0.001
		Mother body mass 12.91 1, 481 < 0.001
		Mother identity	6.37 1, 481	0.012

Table 3 .

 3 Cox regression survival analyses for the young larvae hatched from eggs produced by control and immune-challenged T. molitor females either with (A) A. globiformis or (B) B. thuringiensis ('Maternal treatment') when exposed or not to the same bacteria used for the maternal treatment ('Larvae exposure'), controlling for mother body mass and mother identity. The "simple" contrast was used for maternal immune treatment (hatching rate of eggs of control mothers was used as baseline) and egg bacterial exposure (hatching rate of eggs unexposed to bacteria was used as baseline). Values where P ≤ 0.05 are given in bold.

	Variables in the best model	B	S.E. Wald d.f.	P	Odd ratio
	Larvae exposure	0.48 0.18 7.20	1	0.007	1.62
	Variables not in the best model	Score d.f.	P
	Maternal treatment	0.32	1	0.570
	Mother identity	0.20	1	0.652
	Mother body mass	0.87	1	0.350
	Maternal treatment* Larvae exposure 0.45	1	0.504
	B Maternal immune challenge and larvae exposure with B. thuringiensis
	Variables in the best model	B	S.E. Wald d.f.	P	Odd ratio
	Maternal treatment	-3.07 0.74 17.42 1 < 0.001	0.05
	Larvae exposure	1.00 0.20 24.70 1 < 0.001	2.72
	Mother identity	0.01 0.004 16.09 1 < 0.001	0.985
	Maternal priming* Larvae exposure	0.63 0.38 2.77	1	0.096	1.88
	Variables not in the best model	Score d.f.	P
	Mother body mass	0.001	1	0.976

A Maternal immune challenge and larvae exposure with A. globiformis

Acknowledgements

We thank L. Madeline and M. Heriche for technical assistance, and B. O'Driscoll for proofreading our Manuscript. also thank the two anonymous reviewers for their helpful comments.

Funding

This study was funded by the ANR (ANR-14-CE02-0009) to YM. JD was supported by a Doctoral grant from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche.

Competing interests

The authors declare no competing interests Table 2.

Binary logistic regression analysis of hatching rate of eggs produced by control and immunechallenged T. molitor females either with (A) A. globiformis ('Maternal treatment': control females n = 40; challenged females n = 40) or (B) Bacillus thuringiensis (Maternal treatment': control females n = 40; challenged females n = 40) when or not to the same bacteria used for the maternal treatment ('Egg exposure'), controlling for mother body mass and mother identity. The "simple" contrast was used for maternal immune treatment (hatching rate of eggs of control mothers was used as baseline) and egg bacterial exposure (hatching rate of eggs unexposed to bacteria was used as baseline). Values where P ≤ 0.05 are given in bold.


Julien Signoles

Online Runtime Verification Competitions: How To Possibly Deal With Their Issues (Position Paper)

Keywords: online runtime verification, tool competitions, benchmarks

This short paper presents a compilation of feedback about online runtime verification competitions from an active contestant. In particular, it points out several issues and how they could possibly be fixed.

Introduction

CRV, the runtime verification competition, was created in 2014 [START_REF] Bartocci | First international Competition on Runtime Verification: rules, benchmarks, tools, and final results of CRV[END_REF], then organized again in 2015 [START_REF] Falcone | Second international competition on runtime verification[END_REF] and 2016 [START_REF] Reger | Third international competition on runtime verification[END_REF]. Each year, the competition was split in three categories: the C track, the Java track and the offline track. The number of competitors used to be low in each track. The C track was even cancelled in 2016 because the E-ACSL tool [START_REF] Kosmatov | A lesson on runtime assertion checking with Frama-C[END_REF] was eventually the only contestant. Then the organizers decided to take a break and to not organize the competition this year (2017). I think that it is an appropriate moment to summarize what could be learned from the first editions in order to try to improve the next ones. This paper is an attempt in that direction for the online tracks (C and Java). Yet some ideas could also apply to the offline track. It is based on my own experience from my participation in the C track with the E-ACSL tool in 2014 (2nd place) and 2015 (winner). It is also based on informal discussions with a few other contestants and organizers. The main goal of this paper is to encourage discussions between the competition organizers and the tool developers in order to improve the next editions of any online runtime verification competition.

Areas of Improvement

This section introduces several areas of improvement. First, Section 2.1 introduces the competition goals, while Section 2.2 discusses the scoring method. Then, Section 2.3 and Section 2.4 respectively describe the wide variety of properties and input languages targeted by CRV. Next, Section 2.5 details benchmark issues. Finally, Section 2.6 deals with a few organization concerns.

Competition Goals

The official goals of the competition organizers are multiple [START_REF] Bartocci | First international Competition on Runtime Verification: rules, benchmarks, tools, and final results of CRV[END_REF]:

• stimulate the runtime verification tool development;

• produce shared runtime verification benchmarks;

• discuss measures for comparing tools;

• compare different aspects of tools with different benchmarks and criteria;

• enhance runtime verification tool visibility.

All these goals are perfectly fine and were actually shared by almost all contestants, even if possibly with different priorities. For instance some of them were more interested in measuring runtime and memory overhead, some others preferred to focus on tool expressiveness, while others would like to experiment with new benchmarks. However it is often difficult to kill several birds with a single stone and the risk is high to kill no bird at all. In particular, organizers and contestants have only a limited amount of time to devote to the competition. For instance if contestants spend a lot of time to create new benchmarks, they have almost no time left to run benchmarks from the other contestants. Indeed it is always important to remember that tool developers do not participate if it requires too much efforts for not enough benefits. However, if they do not invest enough effort, then the organization team may quickly be demotivated and would not organize the competition anymore. Though removing motivating goals is always annoying. A workaround could be to just fix priorities between them, so organizers and contestants would reach the most important goals but could just skip a few others according to their available time.

Scoring

Fixing priorities could also help to improve tool evaluation. Indeed runtime verification tools are usually evaluated according to three main criteria:

• efficiency: what is the time and memory overhead of the instrumented program with respect to the input program?

• expressiveness: what properties is the tool able to verify?

• correctness: does the tool solve correctly the input problem?

The current scoring methodology of CRV tries to take into account these three criteria by including a correctness score, a time overhead score and a memory score in a same formula [START_REF] Bartocci | First international Competition on Runtime Verification: rules, benchmarks, tools, and final results of CRV[END_REF], while correctness is measured by a score of 0 for every unanswered problem. It leads to a final score which mixes all these criteria. However it is difficult (or even impossible) to interpret such a result: is tool A better than tool B because it is more time efficient or more expressive? We just do not know. A practical solution could be to compute two different scores, one for expressiveness and another one for efficiency, while unsoundness should result in any case to very low (or even negative) scores.

Properties

The above-mentioned question of expressiveness is particularly important because runtime verification tools may verify very broad kinds of properties, including but not limited to various timed or temporal properties, informal flow properties, concurrency properties, integer and memory overflows, memory errors and functional properties. It seems unlikely that a single tool is able to tackle all of them. It usually focuses on one single class of properties even if it could possibly encode a few more. But comparing tools which verify different properties is dubious, even if the target programming language is the same. Consequently creating several categories corresponding to different properties is necessary. However, the usual low number of contestants is probably an evidence that there are not enough tools to create numerous categories. That is certainly a tough issue which should be carefully dealt with. One workaround could be to split the online tracks into two or three big families of properties (e.g., for C programs, temporal and timed properties on one hand, and undefined behaviors in another hand). The properties which do not belong to any track could not be addressed but they could possibly be grouped together in a free unrated categories to make known that some tools are able to deal with them.

Input Languages

Online runtime verification tools analyze at runtime a program written in a programming language (either C or Java in the first CRV editions) in order to check the validity of properties written in a specification language. Sometimes these properties are even left implicit, for instance when checking the absence of C undefined behaviors. Specifying the programming language is necessary but usually easy: it is enough to indicate the exact supported version(s) and add a pointer to a reference document (e.g the norm ISO C99 [14] for C or the Java Language Specification for Java SE 7 [START_REF] Gosling | The Java Language Specification, Java SE 7 Edition[END_REF]).

Specifying the specification language is more complicated because there is nowadays no de facto standard for most classes of properties, apart from JML [START_REF] Leavens | JML: A Notation for Detailed Design[END_REF] for Java and ACSL [START_REF] Baudin | ACSL: ANSI/ISO C Specification Language[END_REF] for C when interested in functional properties. For instance, there are many temporal logic languages and, even for one particular language like LTL [START_REF] Pnueli | The temporal logic of programs[END_REF], there are several input languages expressing properties as logic formulae or Büchi automata. Currently most tools support their own input specification language and there are only a few translators from one language to the other. Given that situation, I think that the present CRV choice is fine. It consists in providing both an informal specification and a formal one expressed in any specification language as long as the expected property is clear enough.

Benchmarks

There is no competition without benchmarks. Though there is currently no widespread benchmarks for runtime verification tools. One of the underlying goal of CRV was actually to create them. Consequently, taking inspiration from the software verification competition (SV-COMP) [START_REF] Beyer | Competition on software verification[END_REF], CRV was split in three different phases:

1. collecting benchmarks;

training tools and submitting monitors;

3. evaluating results.

The two first steps were performed by the contestants while the latter was performed by the organizers. In particular, the contestants should provide the benchmarks of their choice (either 3, or 5 benchmarks, depending on the competition edition). Each benchmark must contain a program package including the source code and the way to compile and run it, as well as a specification package including an English description of the property to be verified, one logical representation in any specification language, instrumentation information based on a notion of events and traces and the expected verdict [START_REF] Bartocci | First international Competition on Runtime Verification: rules, benchmarks, tools, and final results of CRV[END_REF]. Depending on the edition, the submitted results consist in an instrumented code (CRV 2014) or a way to generate it (CRV 2015 and 2016). Even if it is globally fine, I see some issues with this approach.

Too Strong Assumptions

First, it makes some implicit assumptions about runtime verification tool design which are not always true. Indeed it assumes 1. explicit notions of events and traces; and 2. a clear separation between the monitor and the instrumented system.

While events and traces are at the heart of runtime verification tools [START_REF] Falcone | A tutorial on runtime verification[END_REF][START_REF] Reger | What Is a Trace? A Runtime Verification Perspective[END_REF], they are not necessarily made explicit. In particular, runtime assertion checkers and memory debuggers usually consider program states as events and the successive states of the program at runtime as traces, but they almost never exhibit them. Also, automatic pruning of states in order to observe only a few memory locations and make the verification efficient may be seen as part of the tool's necessary features [START_REF] Jakobsson | Fast as a Shadow, Expressive as a Tree: Optimized Memory Monitoring for C[END_REF]Section 5]. Consequently, the events of interest (that is, the set of memory locations to be observed) should not be part of the inputs to be provided and property 1 should not be assumed.

Also assumption 2 does usually not hold for inline verification tools which weave the monitor directly in the input program. Indeed, in that case, the instrumented system contains both the input program and the inline monitor. Consequently the input program should not be preinstrumented. It is also much better to produce as result the ways to generate the output programs, otherwise it may eventually lead to some manual modifications of the monitor or the instrumented system before submission.

Benchmark Origins

A second issue of this competition is that the contestants must provide all the benchmarks. That is indeed very time consuming and Section 2.1 already explained what are the probable consequences in term of team participation. It may also lower benchmark quality since contestants have possibly not enough time to prepare them accurately. They could also contain bias in order to match the tool capabilities of the benchmark author. Still a competition needs benchmarks and, when there is none, they must be created at some point. It would certainly be too much effort to let the organization committee deal with their creation alone. A potential trade-off could be to have a few benchmarks proposed by the organizers, some proposed by the contestants and some others coming from past editions. In addition to solving the time issue, the benchmarks of lower quality would disappear from edition to edition. It would also make visible the progress of each tool by illustrating which benchmarks are successfully/efficiently tackled on year y which were only imperfectly/slowly handled in year y -1.

Organization

As already pointed out, one of the major issue of such competitions is the lack of time of both organizers and tool developers. It causes unexpected delays, a low number of submitted benchmarks of various degrees of quality, and an even lower number of final submissions. That eventually leads to demotivating both organizers and contestants. A few solutions have been proposed in the previous sections. However, if implemented, some work previously done by contestants would now be done by organizers. For instance, from Section 2.5, the organizers must provide new benchmarks and also select additional ones from past editions. It would require extra work for the organization committee. To solve this drawback, a few contestants could be partly involved in the competition organization. Also one could consider to split the organization in different parts. This way, an organizer could be only partially involved and so (s)he would have more free time to do something else, including becoming a competition contestant. Below is one organization proposal.

• The steering committee defines the competition policy, frequency and goals, follows the competition from year to year and provides advice. It also chooses the organization committee of the next edition.

• The organization committee leads one particular edition. In particular, it sets the exact rules and evaluates the submissions.

• The track committee for every track (or a single competition committee shared by all tracks) is composed by one contestant of each registered team and a subset of the organization committee. It chooses benchmarks (in addition to the contestant benchmarks), either from past editions or newly created. It also validates contestant benchmarks, asks for clarification and fixes contentions.

Related Work

Tool competitions have been organized in several computational domains for a long time. We limit our related work to competitions related to program analysis and provers. CRV is directly inspired by SV-COMP [START_REF] Beyer | Software Verification with Validation of Results (Report on SV-COMP 2017)[END_REF] organized since 2012. SV-COMP aims at a comparative evaluation of fully-automated software verifiers, mainly model checkers. SV-COMP 2017 targeted C programs only and was split into several categories and sub-categories. Categories tackled different properties (e.g. termination or overflow), while sub-categories reflect different types of programs.

Having many categories and sub-categories is certainly possible because of the large number of contestants (32 in 2017). The 2017 edition also required contestants to provide witness automata as proof of validity or invalidity.

Similarly to SV-COMP, termCOMP [START_REF] Giesl | Termination Competition (term-COMP 2015)[END_REF] has also different categories depending on the type of properties to be verified (worst case execution time, certified properties or termination) and sub-categories depending on the target systems (reactive systems, Java or C programs). In 2017, 17 tools entered into the competition which contained 4 categories and 20 sub-categories (with between 2 and 5 contestants in each of them).

VerifyThis [START_REF] Huisman | Verifythis 2012 -A program verification competition[END_REF] is a 2-day event organized during international conferences or workshops that focuses on semi-automatic mathematical proof guided by tools (instead of fully-automatic verification). The competition is more about comparing teams' skills than evaluating tools. Accordingly, the criteria of evaluation are correctness, completeness and elegance of solutions, which are fully different from the current CRV criteria.

The RERS grey-box challenge [START_REF] Howar | The RERS grey-box challenge 2012: Analysis of event-condition-action systems[END_REF] focuses on verifying specific properties (reachability and LTL properties) on specific program patterns. Consequently they only address a few programs (for instance, C programs whose all variables are of type int). It is worth noting that this competition has two different rankings, one numerical ranking and one conceptual ranking. The latter emphasizes the challenge character of the employed methods and is manually computed by the organizers.

The SAT [START_REF] Balyo | Sat competition 2016: Recent developments[END_REF], SMT-COMP [START_REF] Barrett | 6 years of SMT-COMP[END_REF] and CASC [START_REF] Sutcliffe | The CADE ATP System Competition -CASC[END_REF] competitions respectively focus on comparing SAT solvers, SMT solvers and automated theorem provers (ATPs). Each of them takes benefit of uniform common formats supported by every contestant tool (e.g. TPTP for ATPs). This way, comparison of tool expressiveness is made easier. Cuoq et al. [START_REF] Cuoq | Benchmarking static analyzers[END_REF] explain how static analyzers should be benchmarked while highlighting issues that competition benchmarks should avoid. Some of their conclusions are similar to ours.

Conclusion

CRV, the runtime verification competition, was created in 2014. It suffers from a lack of active contestants and has not been organized this year. This paper explains a few of its issues regarding the online runtime verification tracks. It also proposes possible solutions. While I do not pretend that it would instantaneously solve all of them, I hope that this discussion paper provides a point of view from an active contestant that could be useful for competition organizers.

Acknowledgment

I would like to thank Zakaria Chihani for his feedback, as well as the anonymous reviewers from their helpful advice. They significantly contribute to improve the quality of this paper. This work has received funding for the S3P project from French DGE and BPIFrance.