domain: sdv sde

text:
Marianne A Soares
email: marianne_bio@hotmail.com

Geraldo A Carvalho

Mateus R Campos

Luis C Passos

Marcelo M Haro

Anne-Violette Lavoir

Antonio Biondi

Lucia Zappalà

Nicolas Desneux

Detrimental sublethal effects hamper the effective use of natural and chemical pesticides in combination with a key natural enemy of Bemisia tabaci on tomato

Keywords: biological control, ecotoxicology, integrated pest management, predatory mirids, whitefly

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

INTRODUCTION

The silverleaf whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) (MED -biotype Q), is one of the most devastating pests of several crops, and is of global economic importance. [START_REF] Oliveira | History, current status, and collaborative research projects for Bemisia tabaci[END_REF][START_REF] Barro | Bemisia tabaci: a statement of species status[END_REF] The whitefly is highly polyphagous, feeding on and damaging ∼ 500 host plants, including ornamentals, vegetables, legumes, cotton, and corn. [START_REF] Barro | Bemisia tabaci: a statement of species status[END_REF][START_REF] Horowitz | Management of Bemisia tabaci whiteflies, in The Whitefly, Bemisia tabaci (Homoptera: Aleyrodidae) Interaction with Geminivirus-Infected Host Plants[END_REF][START_REF] Quintela | Reproduction of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) B biotype in maize fields (Zea mays L.) in Brazil[END_REF][START_REF] Taggar | Host plant resistance in Vigna sp. towards whitefly, Bemisia tabaci (Gennadius): a review[END_REF] This herbivore can cause damage to plants through direct phloem feeding; however, the greatest threat is due to the transmission of plant pathogens during feeding. [START_REF] Navas-Castillo | Emerging virus diseases transmitted by whiteflies[END_REF][START_REF] Gilbertson | Role of the insect supervectors Bemisia tabaci and Frankliniella occidentalis in the emergence and global spread of plant viruses[END_REF] In addition, B. tabaci excretes 'honeydew' while feeding on the plant, favoring the growth of opportunistic fungi, reducing plant photosynthesis, and consequently causing yield losses. [START_REF] Barro | Bemisia tabaci: a statement of species status[END_REF][START_REF] Boykin | Is agriculture driving the diversification of the Bemisia tabaci species complex (Hemiptera: Sternorrhyncha: Aleyrodidae)?: Dating, diversification and biogeographic evidence revealed[END_REF] Management of B. tabaci populations and the plant diseases transmitted by this polyphagous insect represents a challenge for farmers and researchers. [START_REF] Ellsworth | IPM for Bemisia tabaci: a case study from North America[END_REF] Difficulty in controlling whiteflies results from their rapid population growth, ability to develop resistance to conventional insecticides, and their biological development characteristics in that all life stages (eggs, nymphs and adults) remain protected in the abaxial leaf surface of the host plant. [START_REF] Oliveira | History, current status, and collaborative research projects for Bemisia tabaci[END_REF][START_REF] Horowitz | Management of Bemisia tabaci whiteflies, in The Whitefly, Bemisia tabaci (Homoptera: Aleyrodidae) Interaction with Geminivirus-Infected Host Plants[END_REF] Tomato production systems usually require the use of insecticides to control B. tabaci, and in most cases, this tool is used in an inadequate manner. [START_REF] Palumbo | Insecticidal control and resistance management for Bemisia tabaci[END_REF] This practice has triggered resistance to several conventional insecticides with different modes of action in various B. tabaci populations. [START_REF] Liang | Short-term and transgenerational effects of the neonicotinoid nitenpyram on susceptibility to insecticides in two whitefly species[END_REF] Cases of resistance are related to modulators of nicotinic acetylcholine receptors (e.g., neonicotinoids), molecules that act on the nerves and muscles of insects (e.g., pyrethroids, organophosphosphates compounds, and diamides) and a wide variety of growth-regulator insecticides. [START_REF] Palumbo | Insecticidal control and resistance management for Bemisia tabaci[END_REF][START_REF] Roditakis | Current status of insecticide resistance in Q biotype Bemisia tabaci populations from Crete[END_REF][START_REF] Bass | The global status of insect resistance to neonicotinoid insecticides[END_REF][START_REF] Dângelo | Insecticide resistance and control failure likelihood of the whitefly Bemisia tabaci (MEAM1; B biotype): a Neotropical scenario[END_REF] The appropriate use of zoophytophagus predators is a safe alternative to pesticides for controlling pests in tomato crops. Additionally, these predators can be combined with other natural enemy insects (e.g., parasitoids), natural pathogens (e.g., fungi, nematodes, bacteria, and viruses) or selective insecticides (e.g., the diamide chemical group). [START_REF] Lykouressis | Predation rates of Macrolophus pygmaeus (Hemiptera: Miridae) on different densities of eggs and nymphal instars of the greenhouse whitefly Trialeurodes vaporariorum (Homoptera: Aleyrodidae)[END_REF][START_REF] Bonato | Spatiotemporal distribution of Macrolophus caliginosus in protected tomato crop (Heteroptera: Miridae)[END_REF][START_REF] Perdikis | Nymphal development on plant vs. leaf with and without prey for two omnivorous predators: Nesidiocoris tenuis (Reuter, 1895)(Hemiptera: Miridae) and Dicyphus errans (Wolff, 1804)(Hemiptera: Miridae)[END_REF][START_REF] Passos | Physiological susceptibility of the predator Macrolophus basicornis (Hemiptera: Miridae) to pesticides used to control of Tuta absoluta (Lepidoptera: Gelechiidae)[END_REF][START_REF] Passos | Lethal, sublethal and transgenerational effects of insecticides on Macrolophus basicornis, predator of Tuta absoluta[END_REF][START_REF] Pérez-Valencia | Why can a predator increase its consumption of prey when it is released along with a parasitoid?[END_REF][START_REF] Pérez-Hedo | Defensive plant responses induced by Nesidiocoris tenuis (Hemiptera: Miridae) on tomato plants[END_REF] Among the natural enemies studied for the control of Solanaceae pests, are the predators Macrolophus pygmaeus (Rambur) and Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae), which are considered to be efficient naturally occurring and/or commercialized agents for biological control against several herbivorous arthropods in Europe, Asia, and Africa. [START_REF] Zappalà | Natural enemies of the south American moth, Tuta absoluta, in Europe, North Africa and Middle East, and their potential use in pest control strategies[END_REF][START_REF] Jaworski | Apparent competition between major pests reduces pest population densities on tomato crop, but not yield loss[END_REF][START_REF] Biondi | Ecology, worldwide spread, and management of the invasive South American tomato pinworm, Tuta absoluta: past, present, and future[END_REF][START_REF] Mansour | Occurrence, biology, natural enemies and management of Tuta absoluta in Africa[END_REF] These predators are able to feed on various small pests (e.g., whiteflies, aphids, mites, and lepidopterans), [START_REF] Jaworski | Preference and prey switching in a generalist predator attacking local and invasive alien pests[END_REF][START_REF] Sylla | Life-history traits of Macrolophus pygmaeus with different prey foods[END_REF][START_REF] Bouagga | Comparative biocontrol potential of three predatory mirids when preying on sweet pepper key pests[END_REF] move over the trichomes of plants to a satisfactory degree, and females lay their eggs endophytically in tomato leaves. [START_REF] Wheeler | Hemiptera: Heteroptera) specialists of sticky plants: adaptations, interactions, and ecological implications[END_REF] Because of possible mortality (acute toxicity) and several sublethal physiological and behavioral effects of non-selective insecticides in beneficial arthropods, [START_REF] Desneux | The sublethal effects of pesticides on beneficial arthropods[END_REF][START_REF] Wanumen | Impact of feeding on contaminated prey on the life parameters of Nesidiocoris tenuis (Hemiptera: Miridae) adults[END_REF] active compounds derived from plants may be an alternative to other types of pesticides for use in integrated pest management (IPM). [START_REF] Campolo | Citrus peel essential oil nanoformulations to control the tomato borer, Tuta absoluta: chemical properties and biological activity[END_REF][START_REF] Chaieb | Chemical composition and aphicidal potential of Citrus aurantium peel essential oils[END_REF][START_REF] Hatt | Aromatic plants of east asia to enhance natural enemies towards biological control of insect pests, a review[END_REF][START_REF] Benelli | Insecticidal activity of the essential oil from Schizogyne sericea (Asteraceae) on four insect pests and two non-target species[END_REF][START_REF] Petrović | Assessment of toxicity and biochemical response of Tenebrio molitor and Tribolium confusum exposed to Carum carvi essential oil[END_REF][START_REF] Moreno | Bioactivity of compounds from Acmella oleracea against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and selectivity to two non-target species[END_REF] Botanical pesticides may be used as isolated substances or complex mixtures, and their range of action includes their use as insecticides, fungicides, nematicides, and bactericides. [START_REF] Isman | A renaissance for botanical insecticides?[END_REF] However, before including new products in IPM programs, it is necessary to know the effects of these botanical insecticides on target and non-target species. Approaches that use population models can provide useful population dynamics predictions based on individual-level parameters. [START_REF] Bacci | Seasonal variation in natural mortality factors of Tuta absoluta (Lepidoptera: Gelechiidae) in open-field tomato cultivation[END_REF][START_REF] Drobnjaković | Life history traits and population growth of Encarsia formosa Gahan (Hymenoptera: Aphelinidae) local population from Serbia[END_REF] Thus, the harmful effects of insecticides on population levels of pests and natural enemies can be estimated based on the life history parameters of these organisms. [START_REF] Stark | Reproductive potential: its influence on the susceptibility of a species to pesticides[END_REF][START_REF] Stark | Estimating susceptibility of biological control agents to pesticides: influence of life history strategies and population structure[END_REF][START_REF] Biondi | Do biopesticides affect the demographic traits of a parasitoid wasp and its biocontrol services through sublethal effects?[END_REF][START_REF] Amarasekare | Testing the selectivity of pesticide effects on natural enemies in laboratory bioassays[END_REF][START_REF] Jam | Sublethal effects of imidacloprid and pymetrozine on the functional response of the aphid parasitoid, Lysiphlebus fabarum[END_REF][START_REF] Zhang | Demographic analysis of progeny fitness and timing of resurgence of Laodelphax striatellus after insecticides exposure[END_REF] This study explored the potential of a commercial product based on borax plus citrus oil (BCO) combined with the predator N. tenuis as a tool for whitefly control. The objectives of this study were to: (i) provide information on the population growth of B. tabaci following exposure to BCO and a standard synthetic insecticide (lambda-cyhalothrin) in the presence and absence of N. tenuis; (ii) determine the influence of the botanical insecticide and conventional insecticide on population growth in N. tenuis; and (iii) evaluate possible sublethal effects on the foraging behavior and predation rate of N. tenuis following exposure to BCO and lambda-cyhalothrin.

MATERIALS AND METHODS

Biological materials

Bioassays were performed at the National Agronomic Research Institute (INRA; Sophia-Antipolis) in France under controlled laboratory conditions (25 ± 2 °C, 75 ± 10% relative humidity (RH) and 16:8 h light/dark photoperiod). Tomato and tobacco plants (Solanum lycopersicum cv. Marmande and Nicotiana tabacum var. Wild) were cultivated in a climatic chamber (25 ± 2 °C, 75 ± 5% RH and 16:8 h light/dark photoperiod) on a commercial substrate (Tour-nesol®, Nice, France) in plastic pots (2 L), without receiving pesticide applications.

Bemisia tabaci (biotype Q) was maintained on tobacco plants in a climatized room (25 ± 2 °C, 75 ± 5% RH and 116:8 h light/dark photoperiod). Whiteflies were kept on two tobacco plants (20 and 40 days old) in an anti-aphid cage made by covering a wooden cage (100 × 100 × 75 cm 3 ) with an insect-proof net (50 mesh). Plants were irrigated three times a week. Once a month, a new tobacco plant was offered to the colony and the oldest plant was discarded.

The predator N. tenuis and an alternative prey Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) were supplied by Koppert Biological Systems (Almeria, Spain). Predators were kept on tobacco plants (∼ 40 days old) in a plastic cage (60 × 60 × 40 cm 3 ) covered by an insect-proof net (50 mesh). Nesidiocoris tenuis was fed on E. kuehniella eggs. Adult predators were transferred to a new plant once a week, on which females could lay eggs over a period of 1 week. Thus, predators of a similar instar were kept together inside the anti-aphid cage to minimize cannibalism by larger insects. [START_REF] Ovchinnikov | Factors determining larval cannibalistic behavior in invasive and native populations of the multicolored Asian ladybird, Harmonia axyridis[END_REF]

Insecticides

Prev-Am® (borates tetrasodium salts and 60 g of orange oil per liter; ORO AGRI Internacional Ltda., Gennevilliers, France), referred to as BCO in this study, is produced from sweet orange peel oil and is used as an alternative pest control agent in agroecological systems. To evaluate the demographic parameters of B. tabaci and N. tenuis, bioassays were performed using three BCO concentrations, namely, the maximum concentration recommended to control whiteflies in the field (0.2352 g a.i. L -1 ), half of the maximum concentration (0.1176 g a.i. L -1 ), and 10 % of the maximum concentration (0.0235 g a.i. L -1 ). The predation bioassay was performed using only the maximum concentration of this product.

Karate Zeon® 100 EC (Syngenta International Ltda., Milano, Italy) is a commercial formulation of the pyrethroid insecticide lambdacyhalothrin, which is widely used to control pests in agricultural systems. It was used as a positive control at the maximum recommended concentration to control whiteflies (0.0237 g a.i. L -1 ) in the bioassays of demographic parameters and predation behavior.

The given concentration were obtained by diluting Karate Zeon® and Prev-Am® in distilled water, which alone served as negative control.

Estimating the demographic parameters of Bemisia tabaci and Nesidiocoris tenuis

The bioassays aimed to determine the mean offspring and the population growth of B. tabaci (ten treatments, of which five had added predators and five had no predators) and N. tenuis (five treatments). In both bioassays, 20 replicates were performed per treatment.

Adults of B. tabaci and N. tenuis were exposed to dry residues of lambda-cyhalothrin and BCO on tomato leaves. Tomato leaves with fully expanded leaflets (∼ 15 cm 2 , composed of five leaflets) were immersed in BCO (in one of the three concentrations: 0.2352, 0.1176 and 0.0235 g a.i. L -1 ), lambda-cyhalothrin (0.0237 g a. i. L -1 ) or distilled water for 5 s. The leaves were left to dry for 1 h and placed inside a system composed of overlapping plastic cups, as an experimental unit proposed by Biondi et al. [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] The plastic cup (700 mL, length: 15 cm) had a hole in the bottom center to allow the leaf stem to reach a second plastic cup (350 mL, length: 11 cm) below that contained water. Subsequently, a fine mesh net was fixed in the upper opening of the first upper cup to permit ventilation and prevent insects from escaping.

To evaluate the demographic parameters of B. tabaci, four pairs of whiteflies (∼ 2 days old) were maintained in each experimental unit. In treatments comprising B. tabaci plus predator, one N. tenuis female [START_REF] Kim | Lifecycle, biology, and descriptions of greenhouse biological control agent, Nesidiocoris tenuis (Reuter, 1895) (Hemiptera: Miridae)[END_REF] (∼2 days old) was also added. Additionally, to evaluate the demographic parameters of N. tenuis, two pairs [START_REF] Kim | Lifecycle, biology, and descriptions of greenhouse biological control agent, Nesidiocoris tenuis (Reuter, 1895) (Hemiptera: Miridae)[END_REF] of predators (∼8 days old) were maintained on tomato leaves in the experimental unit described above.

Eleven days after the beginning of each bioassay, living offspring of the whiteflies and predators at each stage of life cycle were counted under a stereomicroscope (×40 magnification). The evaluation time was that proposed by Walthall and Stark. [START_REF] Walthall | Comparison of two population-level ecotoxicological endpoints: the intrinsic (rm) and instantaneous (ri) rates of increase[END_REF] 2.4 Effects of insecticides on the foraging behavior and predation rate of Nesidiocoris tenuis Bioassays to determine behavioral response and predation rate of N. tenuis were performed using B. tabaci (second instar) nymphs as prey, and were performed with three treatments (BCO, lambda-cyhalothrin and untreated control). Twenty replicates were performed for each treatment.

Untreated tomato leaves were previously offered to B. tabaci adults for 24 h so the insects could lay eggs. After 13 days, excess nymphs were removed, leaving 150 whitefly nymphs (second instar) in each replicate.

Nesidiocoris tenuis females 49 (∼2 days old) were kept on treated tomato leaves, and starved for 24 h. Leaves were treated with a BCO or lambda-cyhalothrin solutions, both at the maximum concentrations recommended for the control of whiteflies, as previously described. Distilled water was used as a negative control.

Predators were subsequently isolated in Petri dishes (10 cm diameter × 2 cm height) containing an untreated tomato leaflet infested with B. tabaci nymphs in an agar-water solution (1%). Petri dishes were closed with Teflon® film to prevent insects escaping.

Predator activity was recorded for 10 min per replicate. Thus, five actions were simultaneously recorded in real time: walking, cleaning, feeding on plants, preying on B. tabaci and resting. Time spent on each action was recorded using ETHOWATCHER® software. [START_REF] Junior | ETHOWATCHER: validation of a tool for behavioral and video-tracking analysis in laboratory animals[END_REF] The second step was to evaluate the predation rate of N. tenuis. Thus, after the behavioral assay, predators were kept in the Petri dish described above, and their predation rate (i.e., the number of preyed on whitefly nymphs) was recorded 12 and 24 h after the start of the experiment.

Statistical analyses

Data were subjected to tests of normality (Shapiro-Wilk) [START_REF] Shapiro | An analysis of variance test for normality (complete samples)[END_REF] and homoscedasticity (Bartlett). [START_REF] Bartlett | Properties of sufficiency and statistical tests[END_REF] Subsequently, the number of B. tabaci and N. tenuis nymphs at each instar was subjected to an analysis of variance (PROC ANOVA) and Tukey's test to detect the differences between the treatments (P < 0.05) (PROC GLM).

The demographic parameters of B. tabaci and N. tenuis were evaluated using the instantaneous rate of increase (r i ), based on the equation proposed by Walthall and Stark [START_REF] Walthall | Comparison of two population-level ecotoxicological endpoints: the intrinsic (rm) and instantaneous (ri) rates of increase[END_REF] as follows:

r i = ln N N0

Δt

where N f is the final number of living insects, N 0 is the initial number of insects, and Δt is the time interval (days) from the beginning to the end of the laboratory experiments. [START_REF] Stark | Reproductive potential: its influence on the susceptibility of a species to pesticides[END_REF][START_REF] Walthall | Comparison of two population-level ecotoxicological endpoints: the intrinsic (rm) and instantaneous (ri) rates of increase[END_REF] Subsequently, the population growth of B. tabaci and N. tenuis was estimated according to the equation below:

dN dt =rN K-N K
Where N is the initial population size, which represents the initial number of B. tabaci or N. tenuis used in each experimental unit. r represents the per capita growth rate (capacity of each B. tabaci or N. tenuis to produce descendants). In earlier trials, the carrying capacity of the arenas was determined, based on the approximate number of insects in natural colonized plants, which was represented by the K value; (K-N) is the unused capacity. Growth curves were adjusted using Kaplan-Meier estimators from the non-parametric procedure (PROC LIFETEST). Similarities between the timeresponse curves were tested in paired comparisons (χ 2 log-rank test) between the curves.

Canonical variate analysis (CVA) of predator behaviors (walking, cleaning, plant feeding, preying, and resting) when subjected to different treatments was performed to recognize possible differences and the main behavior contributing to the observed differences (PROC CANDISC with the Distance statement).

Finally, differences in the predation rates between times and among treatments were analyzed by Generalized Linear Models (GLM's) following a Poisson distribution. All analyses were performed in SAS v. 9.2 (SAS Institute, Cary, NC, USA), except for the predation rate analyses, which were performed using 'R' 3.4.4 (R Foundation for Statistical Computing, Vienna, Austria).

RESULTS

Estimating the demographic parameters of Bemisia tabaci

In the absence of N. tenuis, whitefly females laid more eggs on treatments exposed to distilled water and the minimum concentration of BCO (Table 1). Bemisia tabaci females reduced their oviposition when exposed to half-maximum and maximum concentrations of BCO. The lowest number of eggs was reported for the lambda-cyhalothrin treatment. The number of first instar nymphs found in the distilled water treatment did not differ significantly from the insects exposed to the minimum concentration of BCO. The number of nymphs found at the maximum concentrations of BCO and lambda-cyhalothrin treatments was lower than with other treatments. For second instar nymphs, the highest number of offspring was found for insects in distilled water and the minimum BCO concentration; the lambda-cyhalothrin treatment had the lowest number of nymphs. At the third instar, there were no significant differences in the numbers of offspring for insects exposed to minimum and half the recommended BCO concentrations. Finally, the lowest number of nymphs was found in the lambdacyhalothrin treatment (Table 1).

In the presence of N. tenuis, the lowest number of B. tabaci eggs was obtained in the control and lambda-cyhalothrin treatments. Regarding the first instar of B. tabaci, the lowest number of nymphs was observed in the distilled water and lambda-cyhalothrin treatments. For the second instar of B. tabaci, the largest number of descendants was found in treatments containing the half and maximum concentrations of BCO. In addition, a lower number of B. tabaci nymphs was observed in the distilled water and lambda-cyhalothrin treatments. With respect to the third instar, the largest number of B. tabaci nymphs was observed at the half-maximum concentration of BCO. Additionally, no significant differences were observed between treatments composed of distilled water, maximum BCO concentration and lambda-cyhalothrin (Table 1).

The treatment consisting solely of B. tabaci, B. tabaci + BCO (minimum concentration), and B. tabaci + BCO (half concentration) resulted in the greatest population growth capacity. Bemisia tabaci + N. tenuis resulted in fewer B. tabaci nymphs. However, the lowest number of B. tabaci offspring was found on tomato leaves treated with lambda-cyhalothrin (Fig. 1).

Estimation of demographic parameters for

Nesidiocoris tenuis

For the three BCO concentrations and lambda-cyhalothrin, the number of N. tenuis nymphs that emerged from tomato leaves was significantly lower than for insects exposed in the untreated control. Emergence of first and fourth instar nymphs was significantly lower in BCO treatments (three concentrations) than in the untreated control. With respect to second instar nymphs, the three concentrations of BCO also reduced emergence. The half and maximum recommended concentrations of BCO were more toxic for the third instar nymphs. Nesidiocoris tenuis adults did not reproduce when exposed to lambda-cyhalothrin (Table 2).

Although the three BCO concentrations reduced the mean number of offspring of N. tenuis, analysis of the demographic parameters showed positive population growth for predators exposed to BCO treated leaves (Fig. 2). In addition, the increase in the population of predators exposed to the minimum BCO concentration was similar to that for the untreated control. However, the same was not observed for insects exposed to lambda-cyhalothrin, which did not show any capacity for population growth following exposure to the synthetic insecticide (Fig. 2).

Effects of insecticides on the foraging behavior and predation rate of Nesidiocoris tenuis

CVA indicated significant global differences in the behavior of the predator insects when subjected to different treatments (Wilks ⊗ = 0.61; F = 2.93; df (num/den) = 10/106; P = 0.002) (Table 3).

The CVA diagram suggests that the lambda-cyhalothrin and BCO treatments affected predator behavior compared with the untreated control (Fig. 3). The first axis (P = 0.002) explained 98% of the observed differences (Table 3). Higher canonical loads were observed in cleaning and predation behavior on the first axis, which were responsible for most of the divergence observed between the treatments.

N. tenuis individuals exposed to BCO and lambda-cyhalothrin predated fewer B. tabaci nymphs than those in the untreated control. Moreover, the interaction between time factors and treatments showed significant differences in the reduction in the predation rate (χ 2 = 8.42, df = 2, P = 0.015). The lowest number of predated B. tabaci nymphs was observed for predators exposed to lambda-cyhalothrin, and the highest number was observed for predators treated with distilled water (χ 2 = 67.40, df = 2, P < 0.001). In all treatments, predators preyed more during the first evaluation period (1-12 h) than during the second (12-24 h) (χ 2 = 1443.62, df = 1, P < 0.001) (Fig. 4).

DISCUSSION

Insecticides have been widely used in tomato and other crops due to their effective pest control and rapid action against susceptible populations of B. tabaci. [START_REF] Naranjo | Economic injury levels for Bemisia tabaci (Homoptera: Aleyrodidae) in cotton: impact of crop price, control costs, and efficacy of control[END_REF] However, environmentally safe approaches (e.g., combining natural enemies and selective products) should be prioritized to avoid a build-up of insecticide resistance. [START_REF] Liang | Short-term and transgenerational effects of the neonicotinoid nitenpyram on susceptibility to insecticides in two whitefly species[END_REF][START_REF] Guedes | Pesticide-induced stress in arthropod pests for optimized integrated pest management programs[END_REF][START_REF] Wang | Selectivity of novel and traditional insecticides used for management of whiteflies on the parasitoid Encarsia formosa[END_REF] In our study, the B. tabaci population was able to increase when exposed to dry residues of BCO on tomato leaves. Furthermore, a seemingly additive relationship between this botanical insecticide and N. tenuis was observed, although a greater decrease in whiteflies nymphs was observed in the treatment where N. tenuis acted alone. In addition, the insecticide lambda-cyhalothrin provided the greatest control of B. tabaci. By contrast, predators exposed to lambda-cyhalothrin exhibited the greatest reduction in population growth and predation rate. The behavioral characteristics of N. tenuis were affected when predators were exposed to BCO and lambda-cyhalothrin. Most toxicological studies involving botanical insecticides are based on the acute mortality of target species and natural enemies. [START_REF] Regnault-Roger | Essential oils in insect control: low-risk products in a high-stakes world[END_REF] However, after exposure to harmful compounds, insects can compensate their individual mortality in the population dynamics. [START_REF] Stark | Estimating susceptibility of biological control agents to pesticides: influence of life history strategies and population structure[END_REF][START_REF] Mcnair | Is there a place for ecotoxicology[END_REF] Thus, insects are able to show logistic growth in the population, even after the mortality of some individuals. In our study, B. tabaci adults were able to reproduce and, consequently, increase their population even after exposure to BCO. The alternative hypothesis is as follows: (i) whiteflies are more susceptible to BCO during the nymphal stadium than during the egg and adult stages; (ii) serosal cells present in eggs could protect the embryo from BCO toxic compounds; 59 and (iii) BCO does not have a large residual effect by contact against B. tabaci. By contrast, whitefly exhibited a decline in demographic parameters after exposure to lambda-cyhalothrin, which was also observed for N. tenuis.

Nesidiocoris tenuis is an effective predator of B. tabaci on tomato plants in greenhouses and open fields. [START_REF] Calvo | Release rate for a pre-plant application of Nesidiocoris tenuis for Bemisia tabaci control in tomato[END_REF] Release of this predator in tomato crops may result in > 80% B. tabaci control. [START_REF] Calvo | Predation by Nesidiocoris tenuis on Bemisia tabaci and injury to tomato[END_REF] Despite the benefits provided by N. tenuis in terms of pest control, in some cases, use of insecticides and botanical insecticides is necessary in agricultural systems, especially when pests reach the economic damage threshold in crops. [START_REF] Zappalà | Efficacy of sulphur on Tuta absoluta and its side effects on the predator Nesidiocoris tenuis[END_REF][START_REF] Madbouni | Compatibility of insecticides and fungicides with the zoophytophagous mirid predator Nesidiocoris tenuis[END_REF] Our results showed that BCO and lambda-cyhalothrin caused a significant reduction in the emergence of N. tenuis nymphs. However, the sublethal effect on predator reproduction when exposed to both products cannot be considered similar. Nesidiocoris tenuis exposed to BCO showed an increase in its population growth curve. The same was not observed for lambda-cyhalothrin, which compromised the reproduction of the predators. BCO did not reduce the longevity of N. tenuis, as reported for lambda-cyhalothrin, after exposure to dry residues of these products. [START_REF] Soares | Botanical insecticide and natural enemies: a potential combination for pest management against Tuta absoluta[END_REF] Another important generalist predator, Orius laevigatus (Fieber) (Hemiptera: Anthocoridae), did not have its longevity altered when exposed to BCO (at field concentration), suggesting that BCO has no effect on this biological trait in these predators. In turn, the number of O. laevigatus offspring was reduced after exposure to BCO. [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] However, no effects were observed on the survival and reproduction of adult females of the scale insect parasitoid Anagyrus pseudococci (Girault) (Hymenoptera: Encyrtidae) and the tomato leafminer parasitoid Bracon nigricans Szépligeti (Hymenoptera: Braconidae) when exposed to dry BCO residues. [START_REF] Biondi | Do biopesticides affect the demographic traits of a parasitoid wasp and its biocontrol services through sublethal effects?[END_REF][START_REF] Mansour | Evaluating side effects of newer insecticides on the vine mealybug parasitoid Anagyrus sp. near pseudococci, with implications for integrated pest management in vineyards[END_REF] D-Limonene (the primary component of BCO) is a monocyclic terpene produced by plants as a secondary metabolite and is often abundant in citrus peel. [START_REF] Ciriminna | Limonene: a versatile chemical of the bioeconomy[END_REF] Interestingly, the mode of action of D-limonene-based biopesticides is similar to that described for lambda-cyhalothrin, which acts on the nervous systems of insects. [START_REF] Desneux | Dose-response relationship in lethal and behavioural effects of different insecticides on the parasitic wasp Aphidius ervi[END_REF][START_REF] Desneux | Effects of sub-lethal and lethal doses of lambda-cyhalothrin on oviposition experience and host-searching behaviour of a parasitic wasp, Aphidius ervi[END_REF][START_REF] Malacrinò | Fumigant and repellent activity of limonene enantiomers against Tribolium confusum du Val[END_REF][START_REF] Mossa | Green pesticides: essential oils as biopesticides in insectpest management[END_REF] This property could explain the results of our bioassays, in which both commercial products had detrimental effects on the behavioral response of N. tenuis. The undesirable sublethal effects on the predator behavioral response may lead to reductions in the reproductive rate, host search capacity and prey capture. [START_REF] Soares | Botanical insecticide and natural enemies: a potential combination for pest management against Tuta absoluta[END_REF][START_REF] Desneux | Dose-response relationship in lethal and behavioural effects of different insecticides on the parasitic wasp Aphidius ervi[END_REF][START_REF] Müller | Impacts of sublethal insecticide exposure on insects-facts and knowledge gaps[END_REF][START_REF] Soares | Side effects of insecticides commonly used against Tuta absoluta on the predator Macrolophus basicornis[END_REF] In a behavioral follow-up study, we offered Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) as prey for N. tenuis, and the predators spent more time walking and resting after exposure to BCO during the assessments. [START_REF] Soares | Botanical insecticide and natural enemies: a potential combination for pest management against Tuta absoluta[END_REF] Moreover, the predation rate was lower compared to that of the untreated insects. Despite the results described above, in the same report, lambdacyhalothrin was more harmful to N. tenuis than BCO. [START_REF] Soares | Botanical insecticide and natural enemies: a potential combination for pest management against Tuta absoluta[END_REF] The services provided by natural enemies during crop protection may be direct, by predation and/or parasitism, and indirect, by inhibiting the presence of herbivores, because these insects can recognize the chemical signals released by predators and/or parasitoids. [START_REF] Pérez-Hedo | Defensive plant responses induced by Nesidiocoris tenuis (Hemiptera: Miridae) on tomato plants[END_REF][START_REF] Nomikou | Herbivore host plant selection: whitefly learns to avoid host plants that harbour predators of her offspring[END_REF][START_REF] Messelink | Generalist Predators, Food Web Complexities and Biological Pest Control in Greenhouse Crops[END_REF][START_REF] Desneux | Temporal synchrony mediates the outcome of indirect effects between prey via a shared predator[END_REF] Consequently, the importance of using harmless insecticides can be emphasized because, even with lower predation rates, the presence of predators is indirectly beneficial for tomato crops. In our results, N. tenuis had the highest predation rate in B. tabaci nymphs during the first evaluation, due to the fasting time (24 h) before exposure to prey in the experimental areas. Furthermore, both compounds promoted a reduction in the number of B. tabaci nymphs preyed on by N. tenuis. The same products decreased the total predation rate of N. tenuis over T. absoluta in a laboratory bioassay. [START_REF] Soares | Botanical insecticide and natural enemies: a potential combination for pest management against Tuta absoluta[END_REF] The undesirable interference in predatory behavior after insecticide exposure can be characterized by the: (i) repellent effect, (ii) anti-alimentary properties, and (iii) interruption in the ability to locate prey. [START_REF] Desneux | The sublethal effects of pesticides on beneficial arthropods[END_REF][START_REF] Müller | Impacts of sublethal insecticide exposure on insects-facts and knowledge gaps[END_REF] We suggest that hypothesis (iii) is applicable here because both commercial products could trigger random movements and increase insect restlessness. [START_REF] He | Lethal effect of imidacloprid on the coccinellid predator Serangium japonicum and sublethal effects on predator voracity and on functional response to the whitefly Bemisia tabaci[END_REF] In summary, despite the smaller effect of BCO on the demographic parameters of B. tabaci, use of this type of botanical insecticide should be prioritized instead of lambda-cyhalothrin in IPM programs. BCO could act simultaneously with N. tenuis against B. tabaci, which was not noted for predators exposed to lambdacyhalothrin. Bemisia tabaci was exposed via residual contact with dry residues; however, direct topical spraying on nymphs and adults may provide more effective pest control. Consequently, direct topical spraying could have additional negative effects on the predator. Thus, to achieve the maximum potential of both control methods, BCO should be employed at first aiming to reduce the B. tabaci population. Since this commercial product is considered to have low persistence in agronomic environments (∼ 7 days, according to the manufacturer), N. tenuis could be released as a second line of defenze on tomato crops. It may also be worth evaluating the sublethal effects of BCO on N. tenuis under commercial conditions, as this predator could avoid treated tomato plants, thus reducing the damage caused to its biological characteristics by pesticides.

Figure 1

 1 Figure 1 Curves and box plots of estimated population curves of Bemisia tabaci exposed to dry residues of borax plus citrus oil (BCO; at 10, 50 and 100% label concentration), lambda-cyhalothrin (treated control) and distilled water (untreated control). Box plots indicate the median and dispersion (lower and upper quartiles and outliers) population growth. Box plots with different lower-case letters are significantly different by pairwise comparison in χ 2 log-rank test (P ≤ 0.05). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2 Figure 3

 23 Figure 2 Curves and box plots of estimated population curves of Nesidiocoris tenuis exposed to dry residues of borax plus citrus oil (BCO; at 10, 50 and 100% label concentration), lambda-cyhalothrin (treated control) and distilled water (untreated control). Box plots indicate median and dispersion (lower and upper quartiles and outliers) population growth. Box plots with different lower case letters are significantly different by pairwise comparison in χ 2 logrank test (P ≤ 0.05). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

 4 Figure 4 Mean (± SE) consumption of Nesidiocoris tenuis females preying upon Bemisia tabaci after exposure to dry residues of borax plus citrus oil (BCO; 100% label concentration), lambda-cyhalothrin (treated control) and distilled water (untreated control), at two times after exposure to the treatments (0-12 h and 12-24 h), and total consumption after 24 h. Bars followed by different capital letters are significantly different among treatments, and those followed by different lower case letters are significantly different between the two consumption times (GLM with Poisson distribution, followed by Tukey's test, P ≤ 0.05). [Color figure can be viewed at wileyonlinelibrary.com]

Table 1

 1 Mean (± SE) number of eggs laid and emerged individuals reaching the first, second or third instar in the progeny of Bemisia tabaci exposed to dry residues of borax plus citrus oil (at 10, 50 and 100% label concentration), lambda-cyhalothrin (treated control) and distilled water (untreated control)

				Absence of Nesidiocoris tenuis*
	Treatment		Eggs	First instar	Second instar	Third instar
	B. tabaci +	Distilled water	325.9 ± 12.1 a	38.3 ± 2.8 a	308.9 ± 9.1 a	55.6 ± 4.9 a
		BCO 10%	300.1 ± 10.2 a	37.4 ± 2.9 a	274.1 ± 6.9 ab	40.4 ± 4.0 b
		BCO 50%	221.4 ± 13.9 b	26.8 ± 2.5 b	246.6 ± 14.9 b	37.2 ± 3.4 b
		BCO 100%	117.4 ± 14.3 c	5.9 ± 0.9 c	167.7 ± 12.1 c	14.2 ± 1.8 c
		Lambda-cyhalothrin	1.3 ± 0.5 d	0.1 ± 0.1 c	1.1 ± 0.4 d	1.1 ± 0.4 d
	F 4,95		139.58	67.69	150.05	43.81
	P		<0.001	<0.001	<0.001	<0.001
				Presence of Nesidiocoris tenuis*
			Eggs	First instar	Second instar	Third instar
	B. tabaci +	Distilled water	17.1 ± 3.0 c	1.6 ± 0.3 c	27.1 ± 4.2 c	1.5 ± 0.4 c
		BCO 10%	68.4 ± 7.0 b	5.7 ± 0.8 b	104.1 ± 10.3 a	11.4 ± 2.1 b
		BCO 50%	109.9 ± 11.0 a	20.6 ± 1.6 a	103.5 ± 7.5 a	26.2 ± 1.5 a
		BCO 100%	88.1 ± 12.2 ab	8.0 ± 1.0 b	66.5 ± 9.5 b	5.2 ± 1.4 c
		Lambda-cyhalothrin	1.4 ± 0.4 c	0.5 ± 0.2 c	1.3 ± 0.4 c	1.2 ± 0.4 c
	F 4, 95		32.55	71.26	38.49	61.84
	P		<0.001	<0.001	<0.001	<0.001

*Values followed by the same letter within a column are not significantly different by Tukey's HSD test (P ≤ 0.05).

  Values followed by the same letter within a column are not significantly different by Tukey's HSD test (P ≤ 0.05).

	F 4,95	30.81	57.43	48.09	32.32
	P	<0.001	<0.001	<0.001	<0.001

*

Table 3

 3 Canonical loadings (between canonical structures) of the canonical axes for the Nesidiocoris tenuis foraging behavior previously exposed to dry residues of borax plus citrus oil (at 10, 50 and 100% label concentration), lambda-cyhalothrin (treated control) and distilled water (untreated control)

		Canonical axes
	Predator behavior	First	Second
	Resting	-0.55	0.47
	Preying	0.68	0.08
	Plant feeding	0.73	-0.34
	Walking	-0.37	0.15
	Cleaning	-0.99	-0.92
	F appr.	2.93	0.11
	Proportion	0.98	0.01
	P	0.002*	0.974
	Eigenvalue	0.61	0.01

Bold type indicates the main contributors of each axis. *Significant axes.

ACKNOWLEDGEMENTS

The authors would like to thank Philippe Bearez, Edwige Amiens-Desneux and Christiane Metay-Merrien from INRA for technical assistance and the Coordination of Superior Level Staf Improvement -Capes (grant number: 88881.132785/2016-2101), Minas Gerais State Foundation for Research (FAPEMIG) and National Council for Scientifc and Technological Development (CNPq) for provide funding to MAS (Ph.D. fellowship), the project EUCLID (H2020-SFS-2014, grant number: 633999) for funding to ND, and the project STomP (ARIMnet2, grant agreement: 618127) for funding to ND, A-VL, AB and LZ. ND and MC were supported by the IPM Innovation Lab (AID-OAA-14-000018).


Marie-Caroline Certain

Marie-Camille Chaumais

Xavier Jaïs

Laurent Savale

Andrei Seferian

Florence Parent

Marjolaine Georges

Nicolas Favrolt

Arnaud Bourdin

Clément Boissin


domain: sdv sdv.bv sde

text:
Fatma Aridhi
email: aridhifatma@gmail.com

Hajer Sghaier
email: sghaier.hajer@hotmail.com

Allyzée Gaitanaros
email: allyzee.gaitanaros@hotmail.fr

Ayda Khadri
email: khadriayda@yahoo.fr

Samira Aschi-Smiti
email: aschi-smitisamira.smiti@planet.tn

Renaud Brouquisse
email: renaud.brouquisse@inrae.fr

Nitric oxide

Keywords: Hypoxia, Legume, Nitrogen-fixing symbiosis, Phytoglobin NO respiration, Salt stress ARA Acetylene reducing activity BNF Biological nitrogen fixation cPTIO 2-[4-carboxyphenyl]-4,4,

Introduction

Nitric oxide (NO) is a reactive gaseous molecule with a broad spectrum of regulatory functions in plant growth and development [START_REF] Besson-Bard | New insights into nitric oxide signaling in plants[END_REF], and response to biotic [START_REF] Hichri | Nitric oxide: a multifaceted regulator of the nitrogen-fixing symbiosis[END_REF][START_REF] Thalineau | Cross-regulation between N metabolism and nitric oxide (NO) signaling during plant immunity[END_REF]) and abiotic stresses, including salinity and hypoxia [START_REF] Blokhina | Oxidative metabolism, ROS and NO under oxygen deprivation[END_REF][START_REF] Simontacchi | Plant survival in a changing environment: the role of ritric oxide in plant responses to abiotic stress[END_REF]. Under salt stress, enhancement of NO production is accompanied by the exclusion of Na + and retention of K + , through increased membrane H + -ATPase and H + -PPase activities [START_REF] Zhang | Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na + /H + antiport in the tonoplast[END_REF][START_REF] Wang | Ethylene and nitric oxide are involved in maintaining ion homeostasis in Arabidopsis callus under salt stress[END_REF][START_REF] Zhao | Nitric oxide is required for melatonin-enhanced tolerance against salinity stress in rapeseed (Brassica napus L) seedlings[END_REF]. NO induces the expression of genes related to osmotic adjustment, heatshock protein, and ROS-scavenging processes [START_REF] Uchida | Effects of hydrogen peroxide and nitric oxide on both salt and heat stress tolerance in rice[END_REF][START_REF] Kopyra | Nitric oxide stimulates seed germination and counteracts the inhibitory effect of heavy metals and salinity on root growth of Lupinus luteus[END_REF]. It contributes to the maintenance of cellular redox homeostasis by controlling the NADPH level [START_REF] Liu | Glucose-6-phosphate dehydrogenase plays a pivotal role in nitric oxide-involved defense against oxidative stress under salt stress in red kidney bean roots[END_REF]) and by inducing antioxidant enzyme activities [START_REF] Tanou | Hydrogen peroxideand nitric oxide-induced systemic antioxidant prime-like activity under NaCl-stress and stress free conditions in citrus plants[END_REF]. Overall speaking, NO protects plants by helping them to control water status, maintain ionic homeostasis and reduce oxidative damage imposed during salt stress [START_REF] Molassiotis | NO says more than 'YES' to salt tolerance salt priming and systemic nitric oxide signaling in plants[END_REF][START_REF] Simontacchi | Plant survival in a changing environment: the role of ritric oxide in plant responses to abiotic stress[END_REF].

On the other hand, increased NO production is a hallmark of plant response to flooding and hypoxia [START_REF] Blokhina | Oxidative metabolism, ROS and NO under oxygen deprivation[END_REF][START_REF] Gupta | The anoxic plant mitochondrion as a nitrite: NO reductase[END_REF]. Hypoxia compromises mitochondrial respiration and leads to an insufficiency in ATP for energy-demanding processes [START_REF] Bailey-Serres | Flooding stress: acclimations and genetic diversity[END_REF]. In these conditions, NO contributes to the recycling of NADH and the synthesis of ATP through the setting up of an alternative respiration called "phytoglobin (Phytogb)-NO respiration" [START_REF] Stoimenova | Nitritedriven anaerobic ATP synthesis in barley and rice root mitochondria[END_REF][START_REF] Igamberdiev | Plant mitochondrial function during anaerobiosis[END_REF][START_REF] Gupta | The anoxic plant mitochondrion as a nitrite: NO reductase[END_REF]. NO is also essential for the development of lysigenous aerenchyma that enhance O 2 diffusion along with the roots [START_REF] Wany | Nitric oxide is essential for the development of aerenchyma in wheat roots under hypoxic stress[END_REF]. Furthermore, the signaling role of NO in the perception and the response to hypoxia was recently elucidated in Arabidopsis thaliana [START_REF] Hartman | Ethylene-mediated nitric oxide depletion pre-adapts plants to hypoxia stress[END_REF]). An increase in NO production, therefore, appears to be a common response to both salt stress and hypoxia.

Exposure of nitrogen-fixing legumes to high salt concentrations resulted in a rapid decrease in nitrogenase activity and nodule respiration, and this decrease was compensated by raising pO 2 [START_REF] Serraj | Salt stress induces a decrease in the oxygen uptake of soybean nodules and their permeability to oxygen diffusion[END_REF][START_REF] Serraj | Effects of salinity and nitrogen source on growth and nitrogen fixation in alfalfa[END_REF]. Similarly, by comparing nodule respiration of various legumes under NaCl treatments, [START_REF] Delgado | Effects of salt stress on growth and nitrogen fixation by pea, faba-bean, common bean and soybean plants[END_REF] concluded that under low salinity the supply of O 2 to nodules is the limiting factor of the biological nitrogen fixation (BNF). These observations suggest that under salt stress, the nodulated roots of legumes could also face hypoxia and that this could limit BNF.

To test this hypothesis, using the Medicago sativa legume model, we analyzed BNF, NO production and energy state in the nodulated roots of plants grown in the presence of low to average salinity and subjected to short-term flooding. Our data show that, under moderate salt stress as under flooding, a nitrate reductase (NR)-dependent NO production is induced which participates to the maintenance of the energy state of the root system.

Materials and methods

Biological material and growth conditions

Seeds of Medicago sativa (var. Siriver; obtained from Espave Vert Tunesie, Tunis, Tunesia) were sterilized and germinated as previously described [START_REF] Del Giudice | Nitric oxide is required for an optimal establishment of the Medicago truncatula-Sinorhizobium meliloti symbiosis[END_REF].

Five-day-old germinated seeds were transplanted in pots (2 plants/pot) filled with 250 cm 3 of B5 sand (0.6-1.6 mm diameter). Plants growth conditions and nutrient solution are described in [START_REF] Sghaier | Effects of combined salt and flooding stresses on the growth and physiological behavior of alfalfa (Medicago sativa L.)[END_REF]. To set salinity conditions, nutrient solution also contained either 0.2 mM KNO 3 (Control, Ctrl), 0.2 mM KNO 3 and 20 mM NaCl (Treatment 20, T20), or 0.9 mM KNO 3 and 50 mM NaCl (Treatment 50, T50). Plants were inoculated 7 days after transplanting with Ensifer meliloti 2011 bacteria [START_REF] Sghaier | Effects of combined salt and flooding stresses on the growth and physiological behavior of alfalfa (Medicago sativa L.)[END_REF]).

In the first set of experiments (experiment 1), at 28 days post-inoculation (dpi), half of the Ctrl, T20 and T50 plants were subjected to flooding for 5 days by submerging pots in their respective nutrient solutions to sand level (Fig. S1a). Non-flooded plants were watered with nutrient solutions at 28 and 31 dpi (Fig. S1b). At 32 dpi, nodulated roots were harvested and either used for growth parameters analysis (9 plants/assay), N 2 -fixing capacity (2 plants/assay), NO production (2 plants/assay), or immediately frozen in liquid N 2 , ground in powder and stored at -80 °C until analysis of protein and adenine nucleotide contents (2 plants/assay).

In the second set of experiments (experiment 2), at 28 dpi, the plants were transferred either into syringes filled with 35 ml nutrient solution and traversed by a humidified airflow (normoxia, Fig. S2a andb) or into glass tubes filled with 40 ml nutrient solution previously flushed with a mixture of 4.5-95.5%: O 2 -N 2 (hypoxia, Fig. S2c andd). The plants (2 plants/syringe or tube) were cultured either in normoxia or hypoxia for 5 days. To compensate for the evaporation, syringes and tubes were supplemented daily with nutrient solution. One day before harvest, nutrient solutions were supplemented with either 0.1 mM 2-[4-carboxyphenyl]-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a scavenger of NO, or 1 mM sodium tungstate (Tg), an inhibitor of NR, or water (control). At 32 dpi, nodulated root was promptly excised, and used for the analysis of NO production (3 × 2 plants/assay), or immediately frozen in liquid N 2 , ground in powder and stored at -80 °C until adenine nucleotide measurements (3 × 2 plants/assay).

Vegetative growth analysis

Root systems were washed in distilled water, blotted on filter paper and weighted. Dry weights were measured after drying of samples for 48 h at 70 °C.

Biological nitrogen-fixing capacity

BNF capacity of nodulated roots was determined in vivo by measuring the acetylene reducing activity (ARA) as previously described [START_REF] Pierre | Involvement of papain and legumain proteinase in the senescence process of Medicago truncatula nodules[END_REF].

Measurement of NO production

Segments (2 cm-long) of nodulated roots were incubated in the dark, at 23 °C, in 5 mL tubes containing 2 mL of detection medium (10 mM Tris-HCl pH 7.5, 10 mM KCl) in the presence of 10 μM 4,5-diaminofluorescein (DAF-2, Coger, with excitation at 495 nm and emission at 515 nm) fluorescent probe. NO production and controls were carried out as in [START_REF] Horchani | Both plant and bacterial nitrate reductases contribute to nitric oxide production in Medicago truncatula nitrogen-fixing nodules[END_REF]. In addition, to test the specificity of the DAF-2 probe, NO production was also analysed with the Cu(II) fluorescein (CuFL) fluorescent probe (Strem Chemicals, with excitation at 495 nm and emission at 515 nm), which is known to react rapidly and specifically with NO itself [START_REF] Lim | Visualization of nitric oxide in living cells by a copper-based fluorescent probe[END_REF].

Extraction and measurement of adenine nucleotides and proteins

Adenine nucleotides were extracted essentially as in [START_REF] Horchani | Both plant and bacterial nitrate reductases contribute to nitric oxide production in Medicago truncatula nitrogen-fixing nodules[END_REF]. All extraction steps were carried out at 4 °C. Frozen material (40-60 mg) was crushed in liquid nitrogen with 300 mL of perchloric acid solution, containing 7% (v/v) HClO 4 and 25 mM Na 2 EDTA, with a mortar and pestle. After thawing, the extract was taken and the mortar was rinsed with 200 mL of perchloric acid solution, which was then pooled with the extract. The sample was centrifuged for 5 min at 13,000g. The supernatant was quickly and carefully neutralized at pH 5.6-6.0 using a 2 M KOH-0.3 M MOPS solution. KClO 4 precipitate was discarded by centrifugation (5 min, 13,000g). Adenine nucleotides of the supernatant were measured in a Xenius spectrofluorimeterluminometer (Safas, Monaco) using the ATPlite one-step assay system (ATPLT1STP-0509; Perkin-Elmer) according to the manufacturer's instructions. Proteins were extracted and quantified on clarified extracts as in [START_REF] Horchani | Does the source of nitrogen affect the response of subterranean clover to prolonged root hypoxia?[END_REF].

Results

Salinity and flooding affect nodulated roots nitrogen-fixing capacity and energy state, and increase NO production

Salt and flooding treated M. sativa roots were first analysed for their biomass and nodule production. As reported in Table S1, root system dry weight is not affected by the two treatments. However, T20 and T50 salt treatments decrease by 38 and 52%, respectively, the number of nodules per plant, indicating that salt stress affects nodulation. On its side, flooding treatment has no effect on the number of nodules per plant. It can be noted that, regardless of the treatment, the protein content of the root system remains unchanged (Fig. S3), suggesting that, as a whole, the root system is not senescent and is still functional.

The effects of salinity and flooding on M. sativa BNF capacity were assessed by measuring the ARA of nodules (Fig. 1a). As compared with control nodules in normoxia, T20 and T50 treatments result in an 18 and 75% decrease in ARA, respectively. When compared to normoxia, flooding also results in a 33 and 52% decrease in ARA for control and T20 nodules, respectively, but is without additional effect for T50 nodules.

To test the effects of salinity and flooding on the energy state, we analyzed the ATP/ADP ratio (experiment 1). ATP/ADP ratio of control roots is 7.4 (Fig. 1b), which is in agreement with previous analysis in either roots or nodules [START_REF] Brouquisse | Study of glucose starvation in excised maize root tips[END_REF][START_REF] Horchani | Both plant and bacterial nitrate reductases contribute to nitric oxide production in Medicago truncatula nitrogen-fixing nodules[END_REF]. ATP/ADP ratio is not modified in T20 (7.0), but significantly decreased in T50 roots (6.5), indicating that average but not low salt stress impairs the energy status of the nodulated roots. After a 5-day flooding period, ATP/ADP ratios decrease close to 5.0, 4.5 and 4.2 in Ctrl, T20 and T50 treated roots, respectively (Fig. 1b). These results indicate that the saline and flooding stresses, independently of each other, affect the energy state of the nodulated roots and that their combined effect is partly cumulative.

In roots and nodules under hypoxia, NO production has been associated with the establishment of a Phytogb-NO respiration, whose function is to maintain the energy state of the tissues when O 2 concentration decreases [START_REF] Igamberdiev | Plant mitochondrial function during anaerobiosis[END_REF][START_REF] Horchani | Both plant and bacterial nitrate reductases contribute to nitric oxide production in Medicago truncatula nitrogen-fixing nodules[END_REF]. We checked whether NO production could be involved in the maintenance of the root system energy state under each stress. As compared with control, T20 and T50 treatments caused a 1.8 and 3.8fold increase in NO production (Fig. 1c). Similarly, flooding induced a 1.3 to 2.7-fold increase in NO production compared to normoxic conditions. As a control, NO production was also measured with Cu(II) fluorescein probe with similar results (Fig. S3), indicating that measurements with DAF-2 are reliable and relevant in this context. Thus, NO production was increased by both saline and flooding treatments, suggesting (1) that the effects of salinity and flooding on NO production and energy state could result from hypoxic environment, and (2) that Phytogb-NO respiration could be activated in response to both saline and flooding/ hypoxia treatments.

NO production contributes to the maintenance of energy state under both salinity and hypoxia

To test the above hypothesis, we analyzed the effects of cPTIO (a NO scavenger) and Tg (a NR and Phytogb-NO respiration inhibitor) on ATP/ADP ratio and NO production in the root system of control (0) and T50 (50) treated plants submitted (H) or not (N) to hypoxia. To this end, we used a specific hydroponic experimental setup where the root system was incubated in nutrient solution supplemented or not with the inhibitors (experiment 2, Fig. S2). Compared to the control (0-N), NR inhibition by Tg results in a 43, 33 and 41% decrease of ATP/ADP ratio in 0-H, 50-N and 50-H roots, respectively (Table 1). Similarly, NO scavenging by cPTIO results in a 34, 37 and 37% decrease of ATP/ADP ratio in 0-H, 50-N, and 50-H roots, respectively, compared to control conditions. cPTIO and Tg has no significant effect on the ATP/ADP ratio in 0-N roots. Treatment with Tg triggers a strong decrease in NO production in 0-H, 50-N and 50-H treated root systems, but not in 0-N treated root system (Table 1). In the presence of cPTIO, NO production is totally abolished. Taken together, these data indicate that both the functioning of NR and the production of NO are necessary to maintain root and nodule energy state under salinity and hypoxia, alone or combined together.

Discussion

The present data show that nodulation, but not root growth, of M. sativa plants is affected by average salinity (Table S1). This is in line with literature data reporting that alfalfa is a moderately salt-tolerant legume [START_REF] Bruning | Symbiotic nitrogen fixation in legumes: perspectives for saline agriculture[END_REF] and that its growth is significantly affected only from NaCl concentrations above 80 mM [START_REF] Rogers | Diversity in the genus Melilotus for tolerance to salinity and waterlogging[END_REF][START_REF] Rogers | The influence of NaCl salinity and hypoxia on aspects of growth in Trifolium species[END_REF][START_REF] Rogers | Salinity and waterlogging tolerance amongst accessions of messina (Melilotus siculus)[END_REF]. However, salinity is known to inhibit BNF more rapidly than growth in legumes [START_REF] Bruning | Symbiotic nitrogen fixation in legumes: perspectives for saline agriculture[END_REF]. In our study, ARA is the more inhibited as the salt concentration increases (Fig. 1). Under low salt stress, the supply of O 2 to nodules was suggested to be the limiting factor for BNF [START_REF] Bergersen | Root nodules of legumes: Structure and functions[END_REF][START_REF] Delgado | Effects of salt stress on growth and nitrogen fixation by pea, faba-bean, common bean and soybean plants[END_REF][START_REF] Serraj | Salt stress induces a decrease in the oxygen uptake of soybean nodules and their permeability to oxygen diffusion[END_REF]. This inhibition would be due to the fact that the salt stress disrupts the symplastic connections between nodule cells, decreases the permeability of the nodules to O 2 and increases its critical O 2 pressure, which would have the effect of inhibiting the energy metabolism and the regeneration of ATP. These interpretations were supported by [START_REF] Serraj | Effects of salinity and nitrogen source on growth and nitrogen fixation in alfalfa[END_REF] and [START_REF] Serraj | Effects of salinity and nitrogen source on growth and nitrogen fixation in alfalfa[END_REF] who showed that the BNF of common bean, soybean and alfalfa submitted to 50-100 mM NaCl concentrations is partly inhibited compared to that of control plants when measured at 21% O 2 , but that the inhibition is reversed by increasing pO 2 . Considered together, these studies suggest that the decrease in ARA under salt stress is due to an O 2 limitation within the nodules leading to a reduction in energy metabolism.

Short-term effects of flooding on BNF have been little studied. However, in Trifolium repens [START_REF] Pugh | The effect of waterlogging on nitrogen fixation and nodule morphology in soil-grown white clover (Trifolium repens L.)[END_REF] and soybean [START_REF] Sánchez | Production of nitric oxide and nitrosylleghemoglobin complexes in soybean nodules in response to flooding[END_REF]) nodulated roots, a short period of flooding (6-7 days) resulted in a complete or half inhibition of ARA, respectively. Similarly, our data show that in M. sativa nodulated roots, ARA is inhibited after 5 days of flooding (Fig. 1), confirming that flooding inhibits ARA presumably through flooding-associated O 2 shortage. In the root system of many plant species, including legumes, submitted to flooding or hypoxia, the induction of a Phytogb-NO respiration occurs, allowing cell energy status retention [START_REF] Dordas | Expression of a stress-induced hemoglobin affects NO levels produced by alfalfa root cultures under hypoxic stress[END_REF][START_REF] Gupta | The anoxic plant mitochondrion as a nitrite: NO reductase[END_REF][START_REF] Berger | Pathways of nitric oxide metabolism and operation of phytoglobins in legume nodules: missing links and future directions[END_REF]. The Phytogb-NO respiration cycle involves (1) the reduction of NO 3 -by NR (2) the translocation of NO 2 -from the cytosol to the mitochondrial matrix, (3) the reduction of NO 2 -to NO via the mitochondrial electron transfer chain (ETC) allowing ATP regeneration, and finally (4) the passive diffusion of NO to the cytosol where it is oxidized back to NO 3 -by Phytogb [START_REF] Gupta | The anoxic plant mitochondrion as a nitrite: NO reductase[END_REF]. Functional legume nodules are naturally characterized by a microoxic environment, and the existence of such a Phytogb-NO respiration was also evidenced in M. truncatula nodules [START_REF] Horchani | Both plant and bacterial nitrate reductases contribute to nitric oxide production in Medicago truncatula nitrogen-fixing nodules[END_REF]. In these nodules, a hypoxic treatment triggers an increase in NO production, which is abolished by the addition of either cPTIO or Tg, and the energy state (ATP/ADP) is significantly inhibited when nodules are incubated in the presence of Tg [START_REF] Horchani | Both plant and bacterial nitrate reductases contribute to nitric oxide production in Medicago truncatula nitrogen-fixing nodules[END_REF]). In our study, under either salt or hypoxic treatment, or both, the use of Tg and cPTIO leads to a reduction of the ATP/ADP ratio and to an inhibition of NO production (Table 1) indicating that both NR activity and NO production are necessary to maintain the energy state. Taken together our data first confirm that in M. sativa nodulated roots under flooding/hypoxia, NO production is involved in maintaining energy state presumably through the establishment of a Phytogb-NO respiration. Second, our study shows that NR-dependent NO production is also involved in maintaining energy state under salt stress, which argues that salt treatment triggers a hypoxic stress in the nodulated roots. Third, this study strongly suggests that the functioning of Phytogb-NO respiration is induced in nodulated roots under salt stress.

Fig. 1 a

 1 Fig.1a Nitrogen-fixing activity, b energy state and c NO production of Medicago sativa nodulated roots submitted to saline and hypoxic treatments. T20 and T50 refer to 20 and 50 mM NaCl treatments, respectively. Nitrogen-fixing activity is measured through acethylene reducing activity (ARA). Energy state is evaluated through ATP/ADP ratio. The values are mean ± SE (n = 3 for ATP/ADP ratio; n = 4 for ARA and NO production) of 4 independent experiments. Values followed by different letters are significantly different according to oneway ANOVA analysis followed by a Tukey test (P < 0.05)

Acknowledgements

This work was supported by INRAE and by the Comité Mixte Franco-Tunisien pour la Coopération Universitaire (PHC Utique, Grant no. 17G0904).

Author contributions statement RB and SA-S planned and designed the research. FA, HS, AG and RB performed experiments and conducted fieldwork. AK, SA-S and RB interpreted the data. RB wrote the manuscript.