domain: sdv sde

text:
Allah A H Abd

Nicolas Mohammed

Lucie S Desneux

Yinjun Monticelli

Xueyan Fan

Raul N C Shi

Xiwu Guedes

Gao

Mohammed

Nicolas Desneux
email: nicolas.desneux@sophia.inra.fr

Lucie S Monticelli

Fan Yinjun

Xueyan Shi

Raul N C Guedes

Xiwu Gao
email: gaoxiwu@263.net.cn

Potential for insecticide-mediated shift in ecological dominance between two competing aphid species

Keywords: Intraspecific competition, interspecific competition, Rhopalosiphum padi, Sitobion avenae, imidacloprid, dominance shift

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

Introduction

Resource-sharing organisms are the subject of competition when the shared resource is limited forcing their interaction, what may take place among individuals of the same species resulting in intraspecific competition, or among individuals of different species leading to interspecific competition (Reitz and Trumble, 2002;[START_REF] Van Veen | Apparent competition, Quantitative food webs, and the structure of phytophagous insect communities[END_REF][START_REF] Villemereuil | Consumer functional responses under intraand inter-specific interference competition[END_REF][START_REF] Barabás | The effect of intra-and interspecific competition on coexistence in multispecies communities[END_REF]Naselli et al. 2016;Zhao et al., 2017).

Competition is an important community structuring phenomenon in nature [START_REF] Iwabuchi | Competitive outcomes between herbivorous consumers can be predicted from their stoichiometric demands[END_REF][START_REF] Soares | The role of competition in structuring ant communities: A review[END_REF] but impact of pesticides on competition processes has been scarcely studied in agroecosystems. The few studies available suggest that such anthropic compounds could be important in shaping arthropod communities associated with various crops or crop-related systems [START_REF] Oliveira | Competition between insecticide-susceptible and -resistant populations of the maize weevil, Sitophilus zeamais[END_REF][START_REF] Cordeiro | Insecticide-mediated shift in ecological dominance between two competing species of grain beetles[END_REF]. For example community structure may be compromised as population recovery may be delayed after pesticide application(s), and species dominance may shift favoring secondary pest outbreaks [START_REF] Liess | Intraspecific competition delays recovery of population structure[END_REF][START_REF] Guedes | Pesticide-induced stress in arthropod pests for optimized integrated pest management programs[END_REF]2017) The outcome of competition varies with the competing species ranging from potentially negative impact on both species [START_REF] Oliveira | Competition between insecticide-susceptible and -resistant populations of the maize weevil, Sitophilus zeamais[END_REF][START_REF] Moon | Direct and indirect interactions[END_REF][START_REF] Jaworski | Apparent competition between major pests reduces pest population densities on tomato crop, but not yield loss[END_REF], or on the weaker competitors [START_REF] Paini | Competitive exclusion of a worldwide invasive pest by a native. Quantifying competition between two phytophagous insects on two host plant species[END_REF][START_REF] Bompard | Sharing a predator: can an invasive alien pest affect the predation on a local pest?[END_REF][START_REF] Jaworski | Preference and prey switching in a generalist predator attacking local and invasive alien pests[END_REF][START_REF] Tuelher | Competition between the phytophagous stink bugs Euchistus heros and Piesodorus guildinii in soybeans[END_REF], to not affecting either or even favoring at least one of them [START_REF] Mallqui | Competition between the bean weevils Acanthoscelides obtectus and Zabrotes subfasciatus in common beans[END_REF]. Regardless, intraspecific competition is usually considered more important and stronger than interspecific competition [START_REF] Moon | Direct and indirect interactions[END_REF][START_REF] Villemereuil | Consumer functional responses under intraand inter-specific interference competition[END_REF][START_REF] Arco | Effects of intra-and interspecific competition on the sensitivity of Daphnia magna populations to the fungicide carbendazim[END_REF], but both need to be considered in any given scenario to assess their relative impacts. Furthermore, initial evidence is suggestive that variation in intraspecific competition can change the outcome of interspecific competition among aphids [START_REF] Hazell | Intraspecific heritable variation in life-history traits can alter the outcome of interspecific competition among insect herbivores[END_REF].

Several environmental factors, both biotic and abiotic, may affect arthropod competitive interactions [START_REF] Gergs | Chemical and natural stressors combined: from cryptic effects to population extinction[END_REF][START_REF] Duan | Disentangling effects of abiotic factors and biotic interactions on cross-taxon congruence in species turnover patterns of plants, moths and beetles[END_REF][START_REF] Jordan | Abiotic and biotic factors regulating inter-Kingdom.engagementbetween insects and microbe activity on vertebrate remains[END_REF].

Temperature, light, relative humidity and rainfall among others, are regarded as important abiotic factors for competition [START_REF] Marchioro | Development and survival of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) as a function of temperature: effects on the number of generations in tropical and subtropical regions[END_REF][START_REF] Savopoulou-Soultani | Abiotic factors and insect abundance[END_REF][START_REF] Marchioro | Biotic factors are more important than abiotic factors in regulating the abundance of Plutella xylostella L., in Southern Brazil[END_REF]. Nonetheless anthropogenic abiotic stressors are also potentially important for species competition, but they are frequently neglected in such framework [START_REF] Guedes | Pesticide-induced stress in arthropod pests for optimized integrated pest management programs[END_REF][START_REF] Guedes | Sublethal exposure, insecticide resistance, and community stress[END_REF]Zhao et al., 2017). Pesticides, and particularly insecticides, are representatives of such stressors, whose distribution in agroecosystems is ubiquitous and deserves attention [START_REF] Desneux | Diaeretiella rapae limits Myzus persicae populations following applications of deltamethrin in oilseed rape[END_REF]2007;[START_REF] Sánchez-Bayo | Impacts of Agricultural Pesticides on Terrestrial Ecosystems[END_REF][START_REF] Passos | Lethal, sublethal and transgenerational effects caused by insecticides on Macrolophus basicornis, predator of Tuta absoluta[END_REF], as they may promote species outbreaks through changing the arthropod community structure associated with such environments [START_REF] Biondi | Using organic-certified rather than synthetic pesticides may not be safer for biological control agents: selectivity and side effects of 14 pesticides on the predator Orius laevigatus[END_REF][START_REF] Lu | Widespread adoption of Bt cotton and insecticide decrease promotes biocontrol services[END_REF][START_REF] Gao | Local crop planting systems enhance insecticide-mediated displacement of two invasive Leafminer Fly[END_REF][START_REF] Guedes | Pesticide-induced stress in arthropod pests for optimized integrated pest management programs[END_REF][START_REF] Guedes | Sublethal exposure, insecticide resistance, and community stress[END_REF]Zhao et al., 2017).

The relevance of pesticides on competition goes beyond lethal effects, as sublethal stresses are likely as important or even more important drivers of competition than actual mortality [START_REF] Cordeiro | Insecticide-mediated shift in ecological dominance between two competing species of grain beetles[END_REF][START_REF] Gao | Local crop planting systems enhance insecticide-mediated displacement of two invasive Leafminer Fly[END_REF][START_REF] Guedes | Sublethal exposure, insecticide resistance, and community stress[END_REF]Zhao et al., 2017).

Frequent and widespread use of pesticides in crops lead to an almost chronic exposure to sublethal concentrations of pesticides in organisms inhabiting such habitats [START_REF] Boone | Interactions of an insecticide with competition and pond drying in amphibian communities[END_REF]Semlitsch, 2002, Pisa et al., 2017). However, this issue only received only limited attention [START_REF] Knillmann | Interspecific competition delays recovery of Daphnia spp. populations from pesticide stress[END_REF][START_REF] Cordeiro | Insecticide-mediated shift in ecological dominance between two competing species of grain beetles[END_REF], even less in the case of systemic pesticides despite their prevalent use in agriculture nowadays (which actually is increasing due to their versatile use, [START_REF] Miao | Sub-lethal effects of four neonicotinoid seed treatments on the demography and feeding behaviour of the wheat aphid Sitobion avenae[END_REF][START_REF] Wang | Sublethal and transgenerational effects of short-term and chronic exposures to the neonicotinoid nitenpyram in the aphid Aphis gossypii[END_REF][START_REF] Zhang | Effects of imidacloprid and clothianid in seed treatments on wheat aphids and their natural enemies on winter[END_REF]. The systemic insecticides and specially neonicotinoids have been the target of concerns because of their harmful effects on non-target species and their broad agriculture use [START_REF] Damalas | Pesticide Exposure, Safety Issues, and Risk Assessment Indicators[END_REF][START_REF] Wu | Sub-Lethal Effects of Pesticide Residues in Brood Comb on Worker Honey Bee (Apismellifera) Development and Longevity[END_REF]Zhu et al., 2017). However, we still lack a thorough understanding of complex interactions taking place in agroecosystems in which neonicotinoids are frequently applied, despite that it appears essential for potentially improving pest management programs and minimizing environmental impact of crop protection methods [START_REF] Chailleux | Natural enemymediated indirect interactions among prey species: current theories and potential for enhancing biocontrol services in agro-ecosystems[END_REF][START_REF] Bebber | Range expanding pests and pathogens in a warming world[END_REF][START_REF] Mohammed | Impact of imidacloprid and natural enemies on cereal aphids: Integration or ecosystem service disruption?[END_REF].

Systemic pesticides, notably imidacloprid, have been largely used to target the wheat pest species, particularly the bird cherry-oat aphid Rhopalosiphum padi L. and the English grain aphid Sitobion avenae F. [START_REF] Tang | Establishment of baseline toxicity data to different insecticides for Aphis craccivora Koch and Rhopalosiphum maidis (Fitch) (Homoptera: Aphididae) by Glass Tube Residual Film Technique[END_REF][START_REF] Chagnon | Risks of large-scale use of systemic insecticides to ecosystem functioning and services[END_REF][START_REF] Simon-Delso | Systemic insecticides (neonicotinoids and fipronil): trends, uses, mode of action and metabolites[END_REF][START_REF] Mohammed | Impact of imidacloprid and natural enemies on cereal aphids: Integration or ecosystem service disruption?[END_REF] which are two key pests on wheat worldwide [START_REF] Duan | Life cycle and population genetics of bird cherry-oat aphids Rhopalosiphum padi in China: an important pest on wheat crops[END_REF][START_REF] Luo | Indirect effects of chemical hybridization agent SQ-1 on clones of the wheat aphid Sitobion avenae[END_REF][START_REF] Ali | Key aphid natural enemies showing positive effects on wheat yield through biocontrol services in northern China[END_REF][START_REF] Ortiz-Martinez | The effect of landscape context on the biological control of Sitobion avenae: temporal partitioning response of natural enemy guilds[END_REF]). However, the relative impact of this compound in both these aphid species and their interaction remains unknown.

Therefore, the present study aimed to assess the impact of the systemic (neonicotinoid) insecticide imidacloprid on intra and interspecific competition between R. padi and S. avenae.

The laboratory experiments were carried out by using varying combinations of both aphid species. The goal was to test whether imidacloprid would mediate and thus affect the intra and interspecific competitive interaction between both aphid pest species.

Materials and Methods

Biological materials

Wheat seeds used in competition experiments were provided by the Zhuozhou Experimental Station of the China Agricultural University (Hebei, China). Seeds were planted in plastic pots (10 cm in diameter and 9 cm high), and all of the pots were filled with fertile soil (granularly (mm): 4.0-8.0; pH: 5.5-6.5; N mg/kg: 300-600; P2O5 mg/Kg: 150-300; K2O mg/kg: 300-550; humidity: % 45-60; organic matter % ≥ 30). The soil was purchased from the Fangjie Huahui Yingyang Tu Company (Beijing, China). Five days after germination, the seedlings were thinned to 10 healthy wheat seedlings per pot. The colonies of cherry-oat (R. padi) and grain aphids (S. avenae) were obtained from laboratory insect cultures and maintained at the Toxicology Lab of the Department of Entomology, College of Plant Protection, China Agricultural University (Beijing, China). Aphid populations of both species were reared from single parthenogenetic adults and held in the laboratory for several years without insecticide exposure (Lu and Gao, 2009;Lu et al., 2009;[START_REF] Lu | Comparative studies on acetylcholinesterase characteristics between the aphids, Sitobion avenae and Rhopalosiphum padi[END_REF]. The aphid populations used as stock cultures for the laboratory experiments were reared on insecticidefree wheat seedling in rearing cages (30 cm in width, 30 cm in length and 30 cm in height) within growth chamber (23 -25 C°, 60-70% RH, and 16:8 L:D). The competition experiments were carried out under the same environmental conditions.

Insecticide

The neonicotinoid insecticide imidacloprid was used in the competition experiments as the abiotic agent of stress. The commercial formulation of imidacloprid (Bi Chong Lin, 10 %) used was purchased from the Jiang Su Kesheng Company (Jiangsu, China). This formulation is one of the main formulations registered and used in China at the manufacture's label rate for aphid control in the wheat field (40-70 g a.i. ha -1 ). Concentration-mortality bioassays with this insecticide and formulation were carried out to assess its toxicity to both aphid species allowing to recognize the sublethal range based on the LC5. The commercial formulation of imidacloprid was diluted in distilled water for use in the experiments described below.

Insecticide bioassay

Imidacloprid toxicity was assessed with apterous adult aphids by using the leaf dip method [START_REF] Guo | Sublethal and transgenerational effects of chlorantraniliprole on biological traits of the diamondback moth, Plutella xylostella[END_REF][START_REF] Liu | Widespread resistance of the aphid Myzus persicae to pirimicarb across China, and insights on ace2 mutation frequency in this species[END_REF]. Six serial dilutions (mg/L) of imidacloprid were prepared for treating the wheat seedlings (5 days old), which were dipped into the desired concentrations for 10 seconds; the seedlings were subsequently removed from the solution, whose excess was adsorbed off with clean, dry filter paper pieces. The treated seedlings were then transferred to plastic Petri dishes lined with moistened filter paper (to keep humidity on seedling roots) and placed under room temperature. Twenty apterous adults of either aphid species were used for each insecticide concentration using three replicates per concentration and species. The aphids in the control group fed on seedlings dipped in distilled water. The insects were then maintained under laboratory conditions at 23-25 °C temperature, 60-70% relative humidity, and 16:8 h [L:D] photoperiod. Insect mortality was recorded after 24 h exposure and the aphids were recognized as dead when failing to exhibit movement after a gentle touch with a camel's hair brush. The results obtained were subjected to probit analysis and the respective LC5 was thus estimated. The estimated LC5 of each aphid species was further checked against the control insects in an additional 24 h exposure bioassay where mortality was again recorded as detailed above, but using 12 replicates. These estimated LC5 were subsequently used to in the competition experiments.

Imidacloprid and intraspecific competition

The intraspecific (single species) competition experiments were carried out under the same experimental conditions detailed above. Three densities of either aphid species (5, 15, 30 apterous adults) were inoculated on each pot containing wheat seedlings treated or not with imidacloprid at 6.86 mg/l a.i. (= LC5), following a three-way factorial arrangement (2 species x 3 densities x 2 insecticidal treatment conditions) in a completely randomized design. The insecticide was sprayed to the seedlings at the desired concentration always using distilled water for the dilutions; only distillated water was used to spray the seedlings of the control pots and insects. The aphid species were released in each experimental unit 24 hours after spraying. The pots were subsequently covered with cylindrical transparent plastic bag (9 cm in diameter, and 21 cm high, with 0.5 mm mesh from top) to prevent the aphids from escaping. The number of live aphids in each experimental unit was recorded after 7 days.

Each treatment was replicated 12 times.

Imidacloprid and interspecific competition

Two experiments of interspecific competition between the cherry-oat and the grain aphids were performed. The first followed a treatment series placing the two competing species in three even densities (5:5, 15:15, and 30:30). The second set followed a uneven treatment series using the following density combinations 30:0, 25:5, 20:10, 15:15, 10: 20, 5:25 and 0:30. Both experiments were performed as described for the intraspecific experiments established in a three-way factorial arrangement in a completely randomized design with 12 replications. The number of aphids of each species was recorded in each replicate and treatment 7 days after the start of the experiments.

Statistical analyses

The mortality results from the concentration-mortality bioassays were subjected to probit analyses using the software PoloPlus 2.0 (LeOra software, 2006) to allow the assessment of the imidacloprid toxicity to each aphid species, which also allowed the estimates of the LC5 and LC50 ' s. The toxicity curves were considered as significantly different when the confidence limits (95%) at their LC5 and LC50 values did not overlap [START_REF] Prabhaker | Compatibility of two systemic neonicotinoids, imidacloprid and amethoxam, with various natural enemies of agricultural pests[END_REF].

The instantaneous rate of population growth (ri), a strong surrogate estimator of the intrinsic rate of population growth (rm) [START_REF] Stark | Population-level effects of pesticides and other toxicants on arthropods[END_REF], was calculated using the formula ri= [Ln(Nj /Ni )]/Dt, where Nj and Ni are the final and initial number of live insects (in each cage), respectively, and Dt is the duration of the experiment (i.e., 7 days). The following statistical analyses were performed using R version 3.3.3. After checking the assumptions of normality and homoscedasciticy, three-way analyses of variance using generalized linear model were carried out to test the impact of the initial aphid density (intra and interspecific competition) and the presence of insecticide on the final insect population and on the instantaneous rate of population growth (ri). The results of interspecific competition were subjected to regression analyses with initial insect density of both aphid species as independent variables and their final density and rate of population growth as dependent variables. The regressions models were obtained using the curve-fitting procedure of the software TableCurve 3D (Systat, San Jose, CA, USA). The significant regression models were selected based on the criteria of parsimony, high F-value, and sudden increase in R 2 with model complexity. The residual distributions were checked to validate parametric assumptions.

Results

Concentration-mortality response

The results of the leaf dip bioassays of imidacloprid toxicity to adult aphids enabled estimating the toxicity of the neonicotinoid to both aphid species; it proved to be about 3-fold less toxic to the grain aphid S. avenae than to the cherry-oat aphid R. padi (Table 1). LC5 values for S. avenae and R. padi were 10.22 and 3.49 ppm, respectively (Table 1), with an average LC5 for both species of 6.86 ppm (this average value was used when both species were exposed to the insecticide simultaneously i.e. during the inter-specific competition experiments). Control mortality was < 5% in all replicates.

Imidacloprid and intra-specific competition

The presence of insecticide had a negative impact on the final population density and ri of both aphid species 7 days after treatment (F1,132 = 1030.10, P < 0.001 and F1,132 = 547.14, P < 0.001 respectively), as were the effects of insect species and density combinations (F1,132 > 35.35, P < 0.001). The cherry-oat aphid R. padi exhibited steady increase in final density with increase in initial density, as did the wheat aphid S. avenae although always at lower densities (Fig. 1A). Such trend was however inverted when the insects were exposed to imidacloprid and the density of R. padi became slightly lower than S. avenae for the whole range of initial densities considered (Fig. 1B). The same trends were observed when the rate of population growth was considered, with the cherry-oat aphid maintaining higher growth without insecticide exposure (Fig. 2A), but the reverse taking place when imidacloprid was used (Fig. 2B). At the highest density of each species, there was a convergence in population growth of both species exhibiting similar values (Fig. 2B).

Imidacloprid and inter-specific competition

The regression analyses assessing the final aphid populations based on their competing initial densities subjected or not to imidacloprid exposure indicated a contrasting pattern between R. padi and S. avenae. Without imidacloprid R. padi was significantly affected by its own initial density and that of S. avenae, although at a smaller degree (Fig. 3). The final population densities of R. padi reached levels above 500 insects per experimental unit at initial conspecific densities as low as 20 insects/unit and such high densities took place even with up densities of the competing S. avenae as high as 30 insects/unit. In contrast, the final density of S, avenae was much lower reaching 300 insects/unit just at its highest initial densities and just with little or non-existent heterospecific competition by R. padi (Fig. 3). Both outcomes indicate interference between competing species, but with a stronger effect of R. padi, which is the dominant competitor when facing S. avenae.

Imidacloprid exposure allowed for a very different scenario imposing a drastic stress on the competition between R. padi and S. avenae. The high final populations of R. padi observed without imidacloprid did not occur with contamination by this insecticide reaching final peak of populations barely extending to 200 insects/unit and only at its highest initial density (i.e., 30 insects/unit) (Fig. 3). In contrast, the performance of S. avenae suffered little change with imidacloprid contamination, although its final population levels were slightly reduced (Fig. 3). Again, the effect of heterospecific competition was significant for both species, as indicated by the respective regression models where the initial density of both species was necessary to reliably estimate the final population densities of both R. padi and S. avenae, although the influence of conspecifics was always stronger (Fig. 3).

The effect of intraspecific competition on the population growth of both competing aphid species was even more revealing than that of the final populations although the general trends were similar. Again R. padi was a much stronger competitor without imidacloprid contamination maintaining high rates of population growth at initial conspecific densities lower than 5 insects/unit with only limited effect of S. avenae. The opposite is true for S. avenae, which was more drastically affected by R. padi and reached only intermediary rates of population growth (≤ 35 insects/ day) even at highest S. avenae density (Fig. 3).

Imidacloprid nullified the species interference in the intraspecific competition (i.e., heterospecific initial density was not necessary for the regression models) and favored higher population growth of S. avenae rather than the previously dominant R. padi shift their respective status as competitors (Fig. 3).

Discussion

Anthropic contaminants such as pesticides are considered as one of the factors threating ecosystem sustainability, notably through interfering with direct and indirect biotic relationships in arthropod communities [START_REF] Boone | Interactions of an insecticide with competition and pond drying in amphibian communities[END_REF][START_REF] Knillmann | Interspecific competition delays recovery of Daphnia spp. populations from pesticide stress[END_REF][START_REF] Cordeiro | Insecticide-mediated shift in ecological dominance between two competing species of grain beetles[END_REF][START_REF] Biondi | Life stagedependent susceptibility of Aphytis melinus DeBach (Hymenoptera: Aphelinidae) to two pesticides commonly used in citrus orchards[END_REF][START_REF] Guedes | Pesticide-induced stress in arthropod pests for optimized integrated pest management programs[END_REF][START_REF] Guedes | Sublethal exposure, insecticide resistance, and community stress[END_REF][START_REF] Xiao | Sublethal effects of imidacloprid on the predatory seven-spot ladybird beetle Coccinella septempunctata (Coleoptera: Coccinellidae)[END_REF]. We demonstrated that the presence of pesticide on wheat plants modulated both intraspecific and interspecific competitions involving the cherry-oat aphid R. padi and the wheat aphid

S. avenae.

The performance of each species under intraspecific competition conditions diverged with and without imidacloprid exposure, with R. padi suffering more with the insecticide, which greatly suppressed the population of this species while exhibiting only milder impact on S. avenae. Imidacloprid exposure relaxed intraspecific competition leading to increased rate of population growth under this condition reverting the trend of reduced population growth with increased initial density from the insecticide-free environment. In addition, imidacloprid exposure also led to a shift in the outcome of interspecific competition between R. padi and S. avenae compromising the dominance of the former species in insecticide-free plants, while favoring the prevalence of the latter species, the wheat aphid, under imidacloprid exposure. The mutual interference between species observed without imidacloprid contamination was virtually nullified when the insecticide was applied. This finding is also suggestive of the importance of interspecific competition in shaping the associated community in contrast with the usual emphasis given to intraspecific competition [START_REF] Kaplan | Interspecific interactions in phytophagous insects revisited: a quantitative assessment of competition theory[END_REF][START_REF] Moon | Direct and indirect interactions[END_REF][START_REF] Villemereuil | Consumer functional responses under intraand inter-specific interference competition[END_REF].

Differential (acute) toxicity

The susceptibility of both aphid species to imidacloprid was different, what is likely a contributing factor for the observed outcome of competition (Zhao et al., 2017). The concentration-mortality bioassays performed indicated that R. padi is about 3-fold more susceptible to imidacloprid than S. avenae. Therefore, imidacloprid exposure should lead to higher mortality of the former species than of the latter, but this expectation is based solely in a mortality assessment while the demographic impact of the compound is of greater importance although frequently neglected [START_REF] Stark | Population-level effects of pesticides and other toxicants on arthropods[END_REF][START_REF] Guedes | Pesticide-induced stress in arthropod pests for optimized integrated pest management programs[END_REF], allowing even the recognition of potential hormesis-like stimulatory effects that may take place [START_REF] Guedes | Insecticide-induced hormesis and arthropod pest management[END_REF]. For example, low imidacloprid concentrations can exert stimulatory effects on reproduction and immature development in the soybean aphid Aphis glycines [START_REF] Qu | Sublethal and hormesis effects of imidacloprid on the soybean aphid Aphis glycines[END_REF]. Regardless, a range of factors may determine such differential susceptibility, including distinct rates of insecticide penetration, detoxification activity, and even minute differences in the target site of insecticide action, but S. avenae seems to exhibit higher insecticide detoxification activity than R. padi, as suggested by previous studies [START_REF] Lu | Comparative studies on acetylcholinesterase characteristics between the aphids, Sitobion avenae and Rhopalosiphum padi[END_REF][START_REF] Yang | Assessment of sublethal and transgenerational effects of pirimicarb on the wheat aphids Rhopalosiphum padi and Sitobion avenae[END_REF][START_REF] Lu | Inhibiting effects of common insecticides on carboxylesterase activity in Sitobion avenae and Rhopalosiphum padi (Hemiptera: Aphididae) and their synergism to beta-cypermethrin[END_REF].

Imidacloprid impact on intraspecific competition

Intraspecific competition did occur in both aphid species, R. padi and S. avenae, when no insecticide was present and as already suggested by field results from [START_REF] Jarosik | Comparison of field population growths of three cereal aphid species on winter wheat[END_REF]. In contrast, when aphids were exposed to the imidacloprid intraspecific competition was relaxed and the aphids exhibited higher population with higher initial population. Indeed, low initial densities afford lower mating opportunities reducing later competition [START_REF] Huston | A general hypothesis of species diversity[END_REF], what was enhanced with higher initial densities particularly under imidacloprid contamination.

Nonetheless, population growth was highest at the lowest initial densities of both species in without imidacloprid contamination indication that densities as low as 5 insects/pot of 10 seedlings, while the highest density likely approached the carrying capacity of the experimental units used.

When the intraspecific competition of both aphid species are compared, the prevalence of R. padi over S. avenae without imidacloprid contamination was nullified in the presence of the insecticide. The higher acute toxicity of imidacloprid to R. padi causing higher mortality in this species than in S. avenae is a likely cause for such outcome, but sublethal and transgenerational effects are also arguably playing a relevant role for this observed outcome [START_REF] Yang | Assessment of sublethal and transgenerational effects of pirimicarb on the wheat aphids Rhopalosiphum padi and Sitobion avenae[END_REF]. An aided concern is the fact that the relaxation of intraspecific competition with imidacloprid exposure affords higher changes of survival and selection for insecticide resistance in both species [START_REF] Cordeiro | Insecticide-mediated shift in ecological dominance between two competing species of grain beetles[END_REF][START_REF] Guedes | Pesticide-induced stress in arthropod pests for optimized integrated pest management programs[END_REF][START_REF] Guedes | Sublethal exposure, insecticide resistance, and community stress[END_REF]. Nonetheless, the strength of selection will probably be stronger for R. padi since it is the most susceptible species (and thus subjected to stronger selection pressure for resistance).

Imidacloprid impact on interspecific competition

The sublethal insecticide concentrations are expected to interfere with not only intra-, but also with interspecific competition [START_REF] Liess | Culmination of low-dose pesticide effects[END_REF][START_REF] Cordeiro | Insecticide-mediated shift in ecological dominance between two competing species of grain beetles[END_REF][START_REF] Guedes | Pesticide-induced stress in arthropod pests for optimized integrated pest management programs[END_REF].

Our study also provides support for this notion and consistent with our results of intraspecific competition for both aphid species. Again imidacloprid contamination relaxed competition reducing the final population and population growth of both species, but much more so of when under competition, which is suggested by the results without insecticide contamination, are suppressed with imidacloprid exposure, unlike suggested observations with whiteflies, leafminers and thrips [START_REF] Sun | Competitive displacement between two invasive whiteflies: insecticide application and host plant effects[END_REF][START_REF] Gao | Local crop planting systems enhance insecticide-mediated displacement of two invasive Leafminer Fly[END_REF]Zhao et al., 2017). Again, both lethal and sublethal effects on each aphid species are likely playing a role for the observed outcome [START_REF] Cordeiro | Insecticide-mediated shift in ecological dominance between two competing species of grain beetles[END_REF]Xiaon et al., 2015).

Both species were able to co-exist under competition, regardless of sublethal imidacloprid contamination. However, the insecticide contamination shifted the species dominance greatly compromising the prevalence of R. padi over S. avenae. This result consistent with several earlier reports about the interactions among sap feeder arthropods under natural colonization [START_REF] Pascual | Intra-and interspecific competition between biotypes B and Q of Bemisia tabaci (Hemiptera: Aleyrodidae) from Spain[END_REF][START_REF] Qureshi | Interactions among three species of cereal aphids simultaneously infesting wheat[END_REF][START_REF] Umina | Competitive interactions among four pest species of Earth Mites (Acari: Penthaleidae)[END_REF][START_REF] Paini | Competitive exclusion of a worldwide invasive pest by a native. Quantifying competition between two phytophagous insects on two host plant species[END_REF][START_REF] Tapia | Experimental evidence for competitive exclusion of Myzus persicae nicotianae by Myzus persicae s.s. (Hemiptera: Aphididae) on sweet pepper, Capsicum annuum (Solanaceae)[END_REF][START_REF] Sun | Competitive displacement between two invasive whiteflies: insecticide application and host plant effects[END_REF]Zhao et al., 2017).

These reports showed that all competing species can be negatively affected by competition, and the weaker competitors will be more affected. In addition, these reports also suggest that pesticide application may induce a shift in competitive potential of competing species, possibility demonstrated by [START_REF] Cordeiro | Insecticide-mediated shift in ecological dominance between two competing species of grain beetles[END_REF] in density-dependent and concentrationdependent experiments with grain beetles.

The findings reported here showing a shift in dominance between competing aphid species when subjected to imidacloprid-contaminated plants is consistent with the intermediate disturbance hypotheses. This hypothesis was earlier developed to explain the maximization of species diversity under intermediate levels of disturbance able to reduce the abundance of the competitively dominant species (Connell, 1978;Shea et al., 2004). The rational and hypothesis was more recently used to in the context of insecticide disturbance mediating competitive interactions [START_REF] Cordeiro | Insecticide-mediated shift in ecological dominance between two competing species of grain beetles[END_REF][START_REF] Guedes | Pesticide-induced stress in arthropod pests for optimized integrated pest management programs[END_REF], context equality applicable to the present study with the competing aphids R. padi and S. avenae. This is the case because a low imidacloprid concentration (LC5) favored the weaker competing species compromising the dominance of the stronger competitor, R. padi in our case. High concentrations of imidacloprid would likely greatly compromise both species, while very low concentrations would not affect them, but intermediate concentrations would potentially allow higher diversity and a shift in ecological dominance, as reported here. The extended use of imidacloprid will likely change the associated arthropod community and may favor secondary pest outbreaks, particularly of S. avenae, what is a management concern and should be considered when designing pest management programs. The regression models predicting the reported outcomes are indicated in each plot.

Figure captions Fig. 1 .Fig. 2 .

 captions12 Figure captions

Fig. 3 .

 3 Fig. 3. Filled contour plots showing the effect of conspecific and heterospecific

Fig. 4 .

 4 Fig. 4. Filled contour plots showing the effect of conspecific and heterospecific

Fig. 1 .Fig

 1 Fig. 1.

Table 1 .

 1 Relative toxicity of imidacloprid to two aphid species, Rhopalosiphum padi and Sitobion avenae, after 24 hours post treatment.

	Species	n* Slope ±	χ 2	df	LC5 (ppm)	LC50 (ppm)
			SE
						Confidence Limits 95%
	R. padi	30	2.55±0.26 6.21	13 3.49 (2.20-4.81) 15.46 (12.91-
		0	5				18.19)
	S.	30	2.50±0.27 7.36	13 10.22 (6.93-	46.50 (39.53-
	avenae	0	7			13.39)	55.81)

* n: Number of insects per each experiment.

Acknowledgements

This study was financially supported by the Modern Agro-industry Technology Research System (CARS-05).

wheat. Pest Management Science. 2:1141-1149. Zhao X., Reitz S. R., Yuan H., Lei Z., Paini D. R., and Gao Y.(2017). Pesticide-mediated interspecific competition between local and invasive thrips pests. Sci. Rep. 7: 40512. DOI: 10.1038/srep40512. Zhu Y. C. Yao J., Adamczyk J., and Luttrell R., (2017).Feeding toxicity and impact of imidacloprid formulation and mixtures with six representative pesticides at residue concentrations on honey bee physiology (Apis mellifera).PLoS ONE. 12: e0178421.

Author Contributions


Olivier Camuzard

Sabine Santucci-Darmanin

Georges F Carle

Dr Valérie Pierrefite-Carle

Role of autophagy in osteosarcoma

Keywords: autophagy, osteosarcoma, survival, cell death

Osteosarcoma (OS) is the most common primary bone tumour in children and adolescents. It is a highly aggressive tumor with a tendency to spread to the lungs, which are the most common site of metastasis. Advanced osteosarcoma patients with metastasis share a poor prognosis. Despite the use of chemotherapy to treat OS, the 5-year overall survival rate for patients has remained unchanged at 65-70% for the past 20 years. In addition, the 5-year survival of patients with a metastatic disease is around 20%, highlighting the need for novel therapeutic targets.

Autophagy is an intracellular degradation process which eliminates and recycles damaged proteins and organelles to improve cell lifespan. In the context of cancer, numerous studies have demonstrated that autophagy is used by tumor cells to repress initial steps of carcinogenesis and/or support the survival and growth of established tumors. In osteosarcoma, autophagy appears to be deregulated and could also act both as a pro or anti-tumoral process. In this manuscript, we aim to review these major findings regarding the role of autophagy in osteosarcoma.

Introduction

Autophagy is an intracellular degradation pathway identified more than 50 years ago.

The 2016 Nobel prize award to Pr Ohsumi for his work on autophagy renewed interest for the involvement of this process in various fields and autophagy is now recognized as a critical process in bone homeostasis [START_REF] Pierrefite-Carle | Autophagy in bone: Self-eating to stay in balance[END_REF].

Although autophagy's role in cancer is complex and context-dependent, pharmacological modulation of this process is emerging in clinical trials. However, understanding the role of autophagy within the tumor and its microenvironment is an essential prerequisite.

Osteosarcoma is an aggressive cancer mainly occurring in children and young adults. Osteosarcoma treatment relies on chemotherapy and surgery, leading to a 70% 5-year survival in patients with a non-metastatic disease. Nevertheless, the 5year survival of patients with a metastatic disease is around 20%, emphasizing the importance to develop new therapeutic strategies. As autophagy modulation could be part of these new options, the aim of the present review is to summarize the major findings regarding the role of autophagy in osteosarcoma.

Autophagy, a major degradative pathway

Autophagy is an intracellular degradation process which eliminates and recycles damaged proteins and organelles [START_REF] Mizushima | Autophagy: process and function[END_REF][START_REF] Levine | Autophagy in the pathogenesis of disease[END_REF]. An isolation membrane, called a phagophore, is formed in the cytosol to sequester the damaged material. After elongation and closure of the phagophore, a double-membrane vesicle called an autophagosome is generated. Autophagosomes then fuse with lysosomes to generate autolysosomes, in which the content is degraded and recycled. Basal autophagy exists in all eukaryotic cells and exerts a quality control function. Autophagy can also be stimulated by various stresses such as starvation or hypoxia to sustain cell survival [START_REF] Mizushima | Autophagy: process and function[END_REF][START_REF] Levine | Autophagy in the pathogenesis of disease[END_REF]. Although autophagy is essentially a prosurvival mechanism, overactivated autophagy can lead to cell death [START_REF] Fulda | Cell death by autophagy: emerging molecular mechanisms and implications for cancer therapy[END_REF]. In addition to its classical degradation role, autophagy was recently shown to participate in some secretion processes [START_REF] Ponpuak | Secretory autophagy[END_REF].

Autophagy is a complex process regulated by the coordinated action of more than 30 autophagy-related proteins (ATG) (Figure 1). Autophagy initiation is mediated by the UNC-51-like kinase (ULK1) complex which is negatively regulated by the mammalian target of rapamycin (mTOR) in nutrient-rich conditions [START_REF] Kim | AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1[END_REF]. Upon starvation, mTOR is inactivated, leading to ULK-1 activation and autophagy induction [START_REF] Hosokawa | Nutrientdependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy[END_REF]. The Class III PI3K complex I is then recruited to initiate autophagosome formation. Elongation and closure of the phagophore requires two complexes containing ubiquitin-like proteins and their respective conjugation machineries [START_REF] Ohsumi | Two ubiquitin-like conjugation systems essential for autophagy[END_REF]. The first complex mediates the covalent conjugation of ATG12 to ATG5 due to the action of ATG7 and ATG10 enzymes. The ATG12-ATG5 conjugate then binds to ATG16 to form the ATG12-ATG5/ATG16 complex which is essential for autophagosome biogenesis. The second ubiquitin-like conjugation system allows the conjugation of phosphatidylethanolamine (PE) to the microtubule-associated light chain 3 (LC3 or ATG8). Nascent LC3 is first processed by the protease ATG4, activated by the ATG7 enzyme, transferred to the ATG3 conjugating enzyme, and then conjugated with PE [START_REF] Shpilka | Ubiquitin-like proteins and autophagy at a glance[END_REF]. LC3-PE (LC3-II) is an integral membrane protein present in autophagosomes and is used as an autophagosome marker.

The cellular material targeted to degradation is sequestered by autophagosomes through the action of selective autophagy receptors such as SQSTM1/p62, NBR1, NDP52 or Optineurin. Due to a specific amino acid sequence binding to ATG8 protein family members (LC3-interacting region or LIR motif), these receptors mediate the cargo delivery to autophagosomes [START_REF] Mancias | Mechanisms of Selective Autophagy in Normal Physiology and Cancer[END_REF][START_REF] Khaminets | Ubiquitin-Dependent And Independent Signals In Selective Autophagy[END_REF].

Autophagy in bone

Bone is a complex organ in which several cell types act in a coordinated manner.

Bone remodeling starts by the resorption of old mineralized bone matrix by osteoclasts (OC) followed by de novo bone formation by osteoblasts (OB).

Osteocytes (OST), the multifunctional mechanosensing cells, which are embedded within the bone matrix, orchestrate this remodeling process.

Autophagy is involved in OB differentiation [START_REF] Liu | Suppression of autophagy by FIP200 deletion leads to osteopenia in mice through the inhibition of osteoblast terminal differentiation[END_REF][START_REF] Nollet | Autophagy in osteoblasts is involved in mineralization and bone homeostasis[END_REF] , survival [START_REF] Zhang | Acidic pH environment induces autophagy in osteoblasts[END_REF]15] and function, i.e.

bone matrix mineralization [START_REF] Nollet | Autophagy in osteoblasts is involved in mineralization and bone homeostasis[END_REF][START_REF] Li | Defective autophagy in osteoblasts induces endoplasmic reticulum stress and causes remarkable bone loss[END_REF]. In OC, autophagy is also required for differentiation [START_REF] Wang | Osteoclast precursor differentiation by MCPIP via oxidative stress, endoplasmic reticulum stress, and autophagy[END_REF][START_REF] Chung | Beclin-1 is required for RANKL-induced osteoclast differentiation[END_REF][START_REF] Xiu | Chloroquine reduces osteoclastogenesis in murine osteoporosis by preventing TRAF3 degradation[END_REF] and several autophagic proteins are involved in bone resorption [START_REF] Deselm | Autophagy proteins regulate the secretory component of osteoclastic bone resorption[END_REF]. OST, which are long-lived key regulators of bone remodelling, are also highly dependent on autophagy for their survival and function [START_REF] Onal | Suppression of autophagy in osteocytes mimics skeletal aging[END_REF][START_REF] Piemontese | Low bone mass and changes in the osteocyte network in mice lacking autophagy in the osteoblast lineage[END_REF]. Finally, several lines of evidence suggest that an autophagy defect could be related to some bone pathologies such as osteoporosis [START_REF] Camuzard | Sex-specific autophagy modulation in osteoblastic lineage: a critical function to counteract bone loss in female[END_REF], Paget's disease of bone [START_REF] Daroszewska | A point mutation in the ubiquitin-associated domain of SQSMT1 is sufficient to cause a Paget's disease-like disorder in mice[END_REF], or osteopetrosis [START_REF] Ochotny | The R740S mutation in the V-ATPase a3 subunit results in osteoclast apoptosis and defective early-stage autophagy[END_REF] .

Autophagy in cancer

In the context of cancer, numerous studies have demonstrated that autophagy is used by tumor cells as a highly dynamic mechanism to repress initial steps in carcinogenesis and/or support the survival and growth of established tumors [START_REF] Amaravadi | Recent insights into the function of autophagy in cancer[END_REF][START_REF] White | Deconvoluting the context-dependent role for autophagy in cancer[END_REF] (Figure 2). In the early stages of tumorigenesis, autophagy may be tumor suppressive by (i) limiting chromosomal instability, (ii) restricting oxidative stress which can act as an oncogenic stimulus, (iii) preventing intratumoral necrosis and local inflammation. In advanced cancers, tumor-promoting functions of autophagy rely on survival in response to stresses due to cancer progression, metastasis and treatments [START_REF] White | Deconvoluting the context-dependent role for autophagy in cancer[END_REF]. Tumor cells use autophagy to maintain mitochondrial function and homeostasis required by their high energy demand of unrestrained proliferation. In addition, autophagy-mediated secretion can play a role in tumor microenvironment modification [START_REF] Kraya | Identification of secreted proteins that reflect autophagy dynamics within tumor cells[END_REF]. Autophagy has also been involved in the generation of a dormancy state which protects cells from chemotherapeutic stress [START_REF] Shimizu | IGF2 preserves osteosarcoma cell survival by creating an autophagic state of dormancy that protects cells against chemotherapeutic stress[END_REF]. Moreover, autophagy was shown to be required in the maintenance of stem cell properties, particularly in cancer stem cells (CSC) [START_REF] Pan | Autophagic control of cell "stemness[END_REF]. Autophagy is also required for the motility and invasion of metastatic tumor cells by the promotion of focal adhesion disassembly in an autophagic dependent manner via paxilline degradation [START_REF] Sharifi | Autophagy Promotes Focal Adhesion Disassembly and Cell Motility of Metastatic Tumor Cells through the Direct Interaction of Paxillin with LC3[END_REF] . Lastly, autophagy can also be used by cancer cells to modify host antitumor immunosurveillance and to inhibit adaptative and innate immune responses [START_REF] Viry | Hijacker of the Antitumor Immune Response: Autophagy Is Showing Its Worst Facet[END_REF] .

Hence, autophagy exerts context-dependent roles in cancer and most clinical trials, which target established tumors, were performed to inhibit autophagy [START_REF] Levy | Targeting autophagy in cancer[END_REF]. These trials, associating autophagy inhibitors with conventional treatments, led to encouraging results but further research is needed to define the safety and utility of this approach in each cancer type [START_REF] Levy | Targeting autophagy in cancer[END_REF]. In osteosarcoma, a clinical trial combining the autophagy inhibitor hydroxychloroquine with gemcitabine and docetaxel is presently in progress [START_REF] Gemcitabine | Docetaxel, and Hydroxychloroquine in Treating Participants With Recurrent or Refractory Osteosarcoma -Full Text View -ClinicalTrials[END_REF].

Autophagy in osteosarcoma

Autophagy is deregulated in osteosarcoma

Numerous genomic and epigenetic studies have revealed a striking genomic complexity and heterogeneity in osteosarcoma tumors. Several heritable genetic predisposition syndromes with germline mutations in the tumor suppressor genes such as TP53 and RB1 or in RecQL4 gene are associated with osteosarcoma [START_REF] Kansara | Translational biology of osteosarcoma[END_REF][START_REF] Gianferante | Germline and somatic genetics of osteosarcoma -connecting aetiology, biology and therapy[END_REF]. In addition, altered candidate driver genes with biological evidence for a role in osteosarcoma development have been identified in osteosarcoma somatic genomes [START_REF] Kansara | Translational biology of osteosarcoma[END_REF] .

Among the tumor suppressors frequently inactivated in osteosarcoma, several ones have been demonstrated to be involved in autophagy regulation [START_REF] Liu | Autophagy and cancer--issues we need to digest[END_REF][START_REF] Maiuri | Control of autophagy by oncogenes and tumor suppressor genes[END_REF] (Figure 3).

Indeed, RB1, ARF, WIF1, PTEN and TSSC3 were shown to trigger autophagy through various mechanisms [START_REF] Jiang | The RB-E2F1 pathway regulates autophagy[END_REF][START_REF] Pimkina | ARF induces autophagy by virtue of interaction with Bcl-xl[END_REF][41][START_REF] Chen | ATM-mediated PTEN phosphorylation promotes PTEN nuclear translocation and autophagy in response to DNA-damaging agents in cancer cells[END_REF][START_REF] Zhao | TSSC3 promotes autophagy via inactivating the Src-mediated PI3K/Akt/mTOR pathway to suppress tumorigenesis and metastasis in osteosarcoma, and predicts a favorable prognosis[END_REF]. TP53 exhibit a dual role in autophagy regulation, nuclear TP53 inducing autophagy through transcriptional regulation and cytoplasmic TP53 acting as a master autophagy repressor [START_REF] Maiuri | Control of autophagy by oncogenes and tumor suppressor genes[END_REF]. RECQL4 was also recently shown to exert a dual regulation of autophagy [START_REF] Duan | RecQL4 regulates autophagy and apoptosis in U2OS cells[END_REF]. In addition, several oncogenes activated in osteosarcoma such as IGF2, H19, COPS3 and RUNX2 also regulate autophagy [START_REF] Shimizu | IGF2 preserves osteosarcoma cell survival by creating an autophagic state of dormancy that protects cells against chemotherapeutic stress[END_REF][START_REF] Xu | Overexpression of long noncoding RNA H19 promotes invasion and autophagy via the PI3K/AKT/mTOR pathways in trophoblast cells[END_REF][START_REF] Zhang | Tumor-derived exosomes induce N2 polarization of neutrophils to promote gastric cancer cell migration[END_REF][START_REF] Tandon | The role of Runx2 in facilitating autophagy in metastatic breast cancer cells[END_REF]. Finally, different studies evidenced that the mTOR pathway was overactivated in osteosarcoma, suggesting autophagy inhibition [START_REF] Hu | mTOR signaling in osteosarcoma: Oncogenesis and therapeutic aspects (Review)[END_REF][START_REF] Ding | mTOR: An attractive therapeutic target for osteosarcoma?[END_REF]. Taken together, these data indicate that numerous autophagy regulators are affected in osteosarcoma, suggesting that this critical process is deregulated.

Nevertheless, autophagy can be induced in OS and the molecular mechanisms involved in autophagy triggering have been reviewed elsewhere [START_REF] O'farrill | Autophagy in osteosarcoma[END_REF].

The present review aims at updating our current knowledge regarding the complex pro and antitumoral role of autophagy in OS. Indeed, autophagy can either promote cell survival [START_REF] Shimizu | IGF2 preserves osteosarcoma cell survival by creating an autophagic state of dormancy that protects cells against chemotherapeutic stress[END_REF][START_REF] Zhang | Tumor-derived exosomes induce N2 polarization of neutrophils to promote gastric cancer cell migration[END_REF][START_REF] Akin | A novel ATG4B antagonist inhibits autophagy and has a negative impact on osteosarcoma tumors[END_REF][START_REF] Zhang | Knockdown of autophagy-related protein 6, Beclin-1, decreases cell growth, invasion, and metastasis and has a positive effect on chemotherapyinduced cytotoxicity in osteosarcoma cells[END_REF][START_REF] Min | Autophagy as a potential target for sarcoma treatment[END_REF] or contributes to cell death [START_REF] Li | Cell apoptosis, autophagy and necroptosis in osteosarcoma treatment[END_REF]. Moreover, autophagy can be involved in chemosensitivity or chemoresistance during osteosarcoma therapy [START_REF] Li | Cell apoptosis, autophagy and necroptosis in osteosarcoma treatment[END_REF]. In the next paragraphs, we will review several studies illustrating both aspects of autophagy role in osteosarcoma.

Autophagy as a protumoral process in osteosarcoma

In normal conditions, autophagy is a prosurvival pathway that preserves organelle function, prevents cellular waste product toxicity, and produces energy and substrates for survival. In this context, tumors can use autophagy to survive in a hostile microenvironment and to increase growth and aggressiveness.

Autophagy role in OS tumor cells

The protumoral role of autophagy has been demonstrated in several osteosarcoma cell lines by the knockdown of key autophagy genes. It has been shown that ATG4B, the cysteine proteinase activating LC3, is crucial for osteosarcoma development in the Saos-2 cell model [START_REF] Akin | A novel ATG4B antagonist inhibits autophagy and has a negative impact on osteosarcoma tumors[END_REF]. Indeed, treatment with an ATG4B chemical inhibitor results in autophagy deficiency and leads to a decreased proliferation in vitro and tumor growth in vivo [START_REF] Akin | A novel ATG4B antagonist inhibits autophagy and has a negative impact on osteosarcoma tumors[END_REF]. Similarly, using a siRNA approach in three osteosarcoma cell lines (HOS, MG-63, and U2OS), Zhang et al. have shown that BECN1 knockdown induces a decrease in cell growth and invasion in vitro and reduces metastasis development in vivo [START_REF] Zhang | Knockdown of autophagy-related protein 6, Beclin-1, decreases cell growth, invasion, and metastasis and has a positive effect on chemotherapyinduced cytotoxicity in osteosarcoma cells[END_REF].

Autophagy role in treatment resistance

In addition to the role of autophagy in osteosarcoma survival and progression, numerous studies have demonstrated its role in treatment resistance [START_REF] Li | Cell apoptosis, autophagy and necroptosis in osteosarcoma treatment[END_REF][START_REF] He | Molecular mechanisms of chemoresistance in osteosarcoma (Review)[END_REF]. First, several studies demonstrated that chemotherapy-resistant OS cells exhibit increased autophagy [START_REF] Jiang | Autophagic degradation of FOXO3a represses the expression of PUMA to block cell apoptosis in cisplatin-resistant osteosarcoma cells[END_REF][START_REF] Mukherjee | The dynamic role of autophagy and MAPK signaling in determining cell fate under cisplatin stress in osteosarcoma cells[END_REF][START_REF] Guan | Overexpression of caveolin-1 reduces Taxol resistance in human osteosarcoma cells by attenuating PI3K-Akt-JNK dependent autophagy[END_REF]. Then, different factors known to mediate drug resistance such as HMGB1, HSP90AA1 or GFRA1, exert their effects through autophagy induction [START_REF] Huang | HMGB1 promotes drug resistance in osteosarcoma[END_REF][START_REF] Xiao | HSP90AA1-mediated autophagy promotes drug resistance in osteosarcoma[END_REF][START_REF] Kim | GFRA1 promotes cisplatin-induced chemoresistance in osteosarcoma by inducing autophagy[END_REF]. Moreover, several microRNAs (miRNAs) inhibiting autophagy are downregulated in OS, participating in treatment resistance. miRNAs are small noncoding RNAs that regulate gene expression by binding to the 3′ untranslated region of their target mRNAs, leading to their translational suppression or degradation. MiRNAs are now recognized as major contributors in cancer development and treatment resistance, including in OS [START_REF] Chen | Drug resistance-related microRNAs in osteosarcoma: Translating basic evidence into therapeutic strategies[END_REF]. In addition, miRNAs were shown to regulate autophagy [START_REF] Chen | Regulation of Autophagy by MiRNAs and Their Emerging Roles in Tumorigenesis and Cancer Treatment[END_REF],

and several miRNAs target autophagy genes such as Atg5 [START_REF] Gu | LncRNA DICER1-AS1 promotes the proliferation, invasion and autophagy of osteosarcoma cells via miR-30b/ATG5[END_REF] or Atg16L1 [START_REF] Chen | MicroRNA-410 regulates autophagy-related gene ATG16L1 expression and enhances chemosensitivity via autophagy inhibition in osteosarcoma[END_REF].

MiR-410, which directly decreases the autophagy gene ATG16L1 expression, is markedly downregulated in human osteosarcoma tissues [START_REF] Chen | MicroRNA-410 regulates autophagy-related gene ATG16L1 expression and enhances chemosensitivity via autophagy inhibition in osteosarcoma[END_REF]. MiR-30a, which targets BECN1, is significantly reduced in doxorubicin-resistant OS cells [START_REF] Xu | MicroRNA-30a downregulation contributes to chemoresistance of osteosarcoma cells through activating Beclin-1-mediated autophagy[END_REF].

Chemoresistance of osteosarcoma tumor cells to doxorubicin is also associated with the downregulation of miR-143 expression, leading to autophagy activation with upregulation of ATG2B, Bcl-2 and LC3-II protein levels [67]. Similarly, miR-140-5p, which suppresses autophagy through HMGN5, is downregulated in OS and associated with patient's chemoresistance [START_REF] Meng | MicroRNA-140-5p regulates osteosarcoma chemoresistance by targeting HMGN5 and autophagy[END_REF]. More recently, it has been shown that some long non-coding RNAs are involved in autophagy activation in OS through miRNA targeting [START_REF] Gu | LncRNA DICER1-AS1 promotes the proliferation, invasion and autophagy of osteosarcoma cells via miR-30b/ATG5[END_REF][START_REF] Liu | LncRNA SNHG15 contributes to proliferation, invasion and autophagy in osteosarcoma cells by sponging miR-141[END_REF].

Finally, autophagy inhibition through BECN1 or Barkor/ATG14 knockdown results in an increased sensitivity of OS cells to chemotherapy [START_REF] Zhang | Knockdown of autophagy-related protein 6, Beclin-1, decreases cell growth, invasion, and metastasis and has a positive effect on chemotherapyinduced cytotoxicity in osteosarcoma cells[END_REF][START_REF] Zhao | Silencing of Barkor/ATG14 sensitizes osteosarcoma cells to cisplatin induced apoptosis[END_REF].

Autophagy involvement in treatment resistance can be mediated through various mechanisms. The protective effect can be mediated by ROS reduction, as it was described for radioresistance of OS cells [START_REF] Feng | Hypoxia-induced autophagy as an additional mechanism in human osteosarcoma radioresistance[END_REF]. Treatment resistance can also involve the degradation of apoptosis-promoting proteins. This is the case in cisplatinresistant OS cell lines exhibiting autophagic degradation of FOXO3A, which leads to decreased PUMA expression and apoptosis inhibition [START_REF] Jiang | Autophagic degradation of FOXO3a represses the expression of PUMA to block cell apoptosis in cisplatin-resistant osteosarcoma cells[END_REF]. Autophagy was also shown to participate in the maintenance of a dormancy-like state protecting OS cells from chemotherapeutic drugs [START_REF] Shimizu | IGF2 preserves osteosarcoma cell survival by creating an autophagic state of dormancy that protects cells against chemotherapeutic stress[END_REF]. Finally, Ma et al. have shown in osteosarcoma tissues that a weak level of the autophagy substrate p62, which correlates with an active autophagic flux, may be associated with higher metastatic and chemoresistant rates in osteosarcoma patients, suggesting a protumoral effect of autophagy [START_REF] Ma | Expression and Clinical Implication of Autophagy-Associated Protein p62 in Osteosarcoma[END_REF].

Considering the role of autophagy in OS treatment resistance, many studies combined the use of anti-cancer agents with autophagy inhibitors such as chloroquine, 3-methyladenine or spautin-1 (specific and potent autophagy inhibitor-1). These autophagy inhibitors enhance OS cell death induced by cisplatin [START_REF] Shen | Chloroquine blocks the autophagic process in cisplatin-resistant osteosarcoma cells by regulating the expression of p62/SQSTM1[END_REF],

doxorubicin [START_REF] Schott | The autophagy inhibitor spautin-1, either alone or combined with doxorubicin, decreases cell survival and colony formation in canine appendicular osteosarcoma cells[END_REF], bone-targeted gallium compound KP46 [START_REF] Kubista | Distinct activity of the bone-targeted gallium compound KP46 against osteosarcoma cells -synergism with autophagy inhibition[END_REF], and increase in vivo tumor regression following photodynamic therapy [START_REF] Yu | Autophagy inhibitor enhance ZnPc/BSA nanoparticle induced photodynamic therapy by suppressing PD-L1 expression in osteosarcoma immunotherapy[END_REF] or mTOR inhibition [START_REF] Zhu | The anti-cancer activity of the mTORC1/2 dual inhibitor XL388 in preclinical osteosarcoma models[END_REF].

Autophagy role in OS cancer stem cells

Autophagy was also shown to participate in the maintenance of cancer stem cell (CSC) properties [START_REF] Boya | Autophagy in stem cells: repair, remodelling and metabolic reprogramming[END_REF]. Regarding OS, very few studies investigated the role of autophagy in CSC. Zhang et al. have used the CD271 marker to isolate CSC from two human OS cell lines. CD271+ OS cells showed a higher autophagy activity than CD271-OS cells under hypoxia and low nutrient condition. Autophagy deficiency in the CD271+ cells decreased the stemness marker expression, restored chemotherapeutics sensitivity and restricted the advantage of CD271+ OS cells in terms of tumorigenesis in vivo [START_REF] Zhang | Defective autophagy leads to the of stem-like features of CD271+ osteosarcoma cells[END_REF]. More recently, calpain-6 was demonstrated to control OS CSC fate by promoting autophagy and preventing senescence [START_REF] Andrique | Calpain-6 controls the fate of sarcoma stem cells by promoting autophagy and preventing senescence[END_REF].

Autophagy as an antitumoral in osteosarcoma

While the cytoprotective role of autophagy is well described, particularly in response to cancer treatment, its role in cell death has long been controversial. However, several evidences now demonstrate autophagy implication in cell death, including in osteosarcoma.

Indeed, autophagy can result in an autophagic cell death, defined as an autophagymediated cell death that can be suppressed by the inhibition of the autophagic pathway [START_REF] Fulda | Cell death by autophagy: emerging molecular mechanisms and implications for cancer therapy[END_REF][START_REF] Doherty | Life, death and autophagy[END_REF]. Such an autophagic cell death was observed in vitro in WT and doxorubicin-resistant U2OS cells after treatment with the bisindolic alkaloid voacamine [START_REF] Meschini | The plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells[END_REF]. Voacamine induced an apoptosis-independent cell death associated with autophagy stimulation, and knockdown of key autophagy genes resulted in decreased cell death [START_REF] Meschini | The plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells[END_REF].

In addition, autophagy can also promote apoptosis or necroptosis through various mechanisms [START_REF] Doherty | Life, death and autophagy[END_REF]. This can be achieved, for example, through the degradation of anti-apoptotic and cell survival factors [START_REF] Doherty | Life, death and autophagy[END_REF]. In this context, treatment of U2OS or Saos-2 osteosarcoma cell lines with Riccardin (RD), a naturally occurring macrocyclic bisbibenzyl, triggers both autophagy and apoptosis and pharmacological or genetic autophagy inhibition decreased RD-mediated cell death [START_REF] Wang | Riccardin D induces cell death by activation of apoptosis and autophagy in osteosarcoma cells[END_REF]. Similarly, celastrol, a triterpene from traditional chinese medicine induces JNK activation and ROS generation, resulting in apoptosis and autophagy in MG-63 osteosarcoma cells [START_REF] Li | Celastrol induces apoptosis and autophagy via the ROS/JNK signaling pathway in human osteosarcoma cells: an in vitro and in vivo study[END_REF]. Pharmacological autophagy inhibition diminished caspase-3 and PARP cleavage, suggesting that autophagy promoted apoptosis.

Although a large number of studies demonstrate that chemotherapy triggers a cytoprotective autophagy, some of them indicate that autophagy can sensitize OS cells to chemotherapy. Inhibition of camptothecin-induced autophagy was shown to decrease cytotoxicity in DLM8 OS cells and to increase cytotoxicity in K7M3 OS cells [START_REF] Hollomon | Knockdown of autophagy-related protein 5, ATG5, decreases oxidative stress and has an opposing effect on camptothecin-induced cytotoxicity in osteosarcoma cells[END_REF]. Similarly, autophagy inhibition led to opposite effect in LM7 and CCH-OS-D or K7M3 OS cells [START_REF] O'farrill | Autophagy in osteosarcoma[END_REF]. In this last report, the authors showed that the expression of phosphorylated heat shock protein 27 (HSP27) after chemotherapy, predicts the effect of autophagy inhibition on OS cell survival or death.

Interestingly, a recent study of Livingston et al. demonstrated that the presence of LC3B+ puncta, an autophagosome marker, is an independent prognostic biomarker of improved survival following neoadjuvant chemotherapy [START_REF] Livingston | Analysis of HSP27 and the Autophagy Marker LC3B+ Puncta Following Preoperative Chemotherapy Identifies High-Risk Osteosarcoma Patients[END_REF]. This work, which was performed in tumor specimens isolated from 260 osteosarcoma patients, suggests that autophagy could be beneficial in this context.

Conclusion

Autophagy is a prosurvival pathway used by OS tumor cells to increase their proliferation and development, resist to cancer treatments and preserve a pool of CSC within the tumor. Nevertheless, autophagy can also be antitumoral in OS and lead to cell death but several general questions remains to be answered: which signal turns prosurvival autophagy into a death process ? Is there an autophagy threshold level required for cell death induction ? We only start to address these issues which are essential to understand the role of autophagy in OS and to use this pathway as a potential weapon against this cancer.

Legends to figures

Figure 1. Autophagy mechanism. The autophagy mechanism and the different molecular complexes involved in the process are presented. In response to different stimuli such as mTORC1 inactivation, autophagy is initiated through the action of the ULK1 complex and the class III PI3K complex. A phagophore is generated in the cytosol to isolate damaged organelles, aggregates and proteins. The ATG12 conjugation system and the LC3 conjugation system are then involved in the elongation and closure of the phagophore, leading to autophagosome formation.

Finally, the autophagosome fuses with lysosomes to degrade the material which will then be recycled.

Figure 2 .

 2 Figure 2. Autophagy role in cancer. In early stages of tumor development,

Figure 3 .

 3 Figure 3. Potential autophagy deregulation in osteosarcoma

Acknowledgements

The MATOs team is supported by grants from the France Cancer association, Fondation ARC pour la recherche sur le cancer and Cancéropôle PACA. We apologize to the authors that we were unable to cite due to space limitations.