Marie-Cécile Dufour

Plasticity of olfactory-guided behaviour and its neurobiological basis: lessons from moths and locusts

The sense of smell plays an important role in guiding the behaviour of many animals including insects. The attractiveness of a volatile is not only dependent on the nature of the chemical, but might change with the physiological status (e.g., age/hormone or mating status) or environmental conditions (e.g., photoperiod or temperature) of the individual. Here we summarize our studies focused on the plasticity of olfactory‐guided behaviour and its neurobiological basis linked with the physiological status in Lepidoptera and migratory locusts. In moths and locusts, age and juvenile hormone changed the behavioural responses to pheromones. In moths, mating had an effect on pheromone responses in males and plant odour responses in females. In all cases of behavioural plasticity studied, we found changes in the sensitivity of olfactory interneurons in the antennal lobe, whereas the peripheral system does not seem to show any plasticity in that context. Changes in the central nervous system were slow under the influence of juvenile hormone (days) or fast after mating (minutes). The olfactory system seems thus to adapt to the physiological or environmental situation of an animal to avoid a waste of energy. We discuss possible mechanisms underlying the observed plasticity.

Transient post-mating inhibition of behavioural and central nervous responses to sex pheromone in an insect

Mating is costly for both male and female insects and should therefore only occur if it is likely to be successful. Within one scotophase, which is the dark period of the light cycle, male moths can only produce one single spermatophore, which is transferred to the female during mating. Remating within the same scotophase would thus be unsuccessful. We tested the hypothesis that newly mated males of the moth Agrotis ipsilon have developed an energy-saving strategy based on the transient inhibition of their sexual behaviour, thus avoiding unsuccessful remating. Agrotis ipsilon males do not copulate more than once during the same scotophase. Moreover, newly mated males do not respond behaviourally to the female sex pheromone although electroantennograms showed that their peripheral olfactory system is fully functional. However, intracellular recordings of antennal lobe neurons showed that the sensitivity for the synthetic sex pheromone blend decreased as compared with that of unmated males. Both the sexual behaviour and the sensitivity of the antennal lobe neurons were restored when tested during the next scotophase. Our results show a fast, transient neuronal plasticity that ‘switches off’ the olfactory system, which could prevent males from mating unsuccessfully.

Attraction of the grapevine moth to host and non-host plant parts in the wind tunnel: effects of plant phenology, sex, and mating status

In the European grapevine moth, Lobesia botrana (Denis and Schiffermüller) (Lepidoptera: Tortricidae), it has recently been shown that volatiles emitted from the main host plant, grapevine, are attractive to adult females. Here, using wind tunnel experiments, we tested the attractiveness of various grapevine, Vitis vinifera L. (Vitaceae), plant parts at different phenological stages, including ripe berries infested with the pathogenic fungus Botrytis cinerea (Persoon: Fries) (Sclerotiniaceae) to laboratory‐reared virgin and mated male and female moths. We also tested the attractiveness of the non‐host plant, tansy [Tanacetum vulgare (L.) (Asteraceae)], as flowers and flower volatiles were previously shown to be attractive to L. botrana females in the field. Mated female moths were the only adults to exhibit upwind orientation to grape plant parts in the flight tunnel. The most attractive parts of the grapevine plants were leaves, flower buds, and ripe berries. No attraction was observed to flowers. This corresponds to the phenological stages available when females of the 2–3 generations during a year are present in the field. No attraction was observed to leaves and flowers of tansy.

Adult neurogenesis in a moth brain

In both vertebrates and invertebrates, neurogenesis not only occurs during development but also persists throughout adult life. So far, adult neurogenesis has been detected in a few insect orders but not in Lepidoptera. In the moth, Agrotis ipsilon, the sensitivity of antennal lobe interneurons of males to sex pheromone is age‐ and juvenile hormone‐dependent in accordance with changes in the behavioral response. As a first step to understand this neuronal plasticity, we tested the hypothesis that adult neurogenesis could occur in the moth brain using 5‐bromo‐2′‐deoxyuridine injections on newly born and sexually mature moths. Our results show that persistent neurogenesis occurs in mushroom bodies of adult males and females of A. ipsilon. Two clusters of one to four neuroblasts in each brain hemisphere continue to divide in adult moths and give rise to small clusters of Kenyon cells in the cortex of the mushroom body calyces. Neurogenesis was observed in both newly born and sexually mature males. There was a clear increase in the number of newly born cells in brains as the time increased after the treatment that was performed soon after emergence. When treatments were performed in mature 3‐day‐old adults, neurogenesis was still detected in brains dissected 3 hours after treatment but was hardly visible 2 days later. Adult neurogenesis was also detected in the optic lobes but not in the antennal lobes. We hypothesize that the newly born neurons could play a role in the central nervous plasticity of olfactory processing in the adult moth A. ipsilon. J. Comp. Neurol. 495:635–643, 2006.

Age-dependent plasticity of sex pheromone response in the moth, Agrotis ipsilon: Combined effects of octopamine and juvenile hormone

Male moths use sex pheromones to find their mating partners. In the moth, Agrotis ipsilon, the behavioral response and the neuron sensitivity within the primary olfactory centre, the antennal lobe (AL), to sex pheromone increase with age and juvenile hormone (JH) biosynthesis. By manipulating the JH level, we previously showed that JH controls this age-dependent neuronal plasticity, and that its effects are slow (within 2 days). We hypothesized that the hormonal effect might be indirect, and one neuromodulator candidate, which might serve as a mediator, is octopamine (OA). Here, we studied the effects of OA and an OA receptor antagonist, mianserin, on behavioral and AL neuron responses of mature and immature males during stimulation with sex pheromone. Our results indicate that, although OA injections enhanced the behavioral pheromone response in mature males, OA had no significant effect on behavior in immature males. However, mianserin injections decreased the behavioral response in mature males. AL neuron sensitivity increased after OA treatment in immature males, and decreased after mianserin treatment in mature males. Determination of OA levels in ALs of immature and mature males did not reveal any difference. To study the possible interactive effects of JH and OA, the behavioral pheromone response was analyzed in JH-deprived mature males injected with OA, and in immature males injected with fenoxycarb, a JH agonist, and mianserin. Results show that both JH and OA are necessary to elicit a behavioral response of A. ipsilon males to sex pheromone.

Assessment of fungicide resistance and pathogen diversity in Erysiphe necator using quantitative real-time PCR assays.

BACKGROUND Management of grapevine powdery mildew Erysiphe necator Schw. requires fungicide treatments such as sterol demethylation inhibitors (DMIs) or mitochondrial inhibitors (QoIs). Recently, reduction in the efficacy of DMIs or QoIs was reported in Europe and the United States. The aim of the present study was to develop real-time qPCR tools to detect and quantify several CYP51 gene variants of E. necator: (i) A versus B groups (G37A) and (ii) sensitive versus resistant to sterol demethylase inhibitor fungicides (Y136F). RESULTS The efficacy of the qPCR tools developed was better than the CAPS method, with a limit of 2 pg for E necator DNA, 0.06 ng for genetic group A and 1.4 ng for the DMI-resistant allele. The detection limits of qPCR protocols (LOD) ranged from 0.72 to 0.85%, and the quantification limits (LOQ) ranged from 2.4 to 2.85% for the two alleles G47A and Y136F respectively. The application of qPCR to field isolates from French vineyards showed the presence of DMI-resistant and/or QoI-resistant alleles in French pathogen populations, linked to genetic group B. CONCLUSION The real-time PCR assay developed in this study provides a potentially useful tool for efficient quantification of different alleles of interest for fungicide monitoring and for population structure of E. necator.

Diversity and fitness of Plasmopara viticola isolates resistant to QoI fungicides

The effectiveness of Quinone outside Inhibitor (QoI) fungicides against grape downy mildew in European vineyards has significantly decreased in the last decade. One nucleotide polymorphism, G143A in the cytochrome b gene of Plasmopara viticola, is involved in resistance to QoIs. Previous genetic examination on the mitochondrial genomes showed four major haplotypes (IR, IS, IIR, IIS) coexisting in European vineyards. A resistant allele (G143A) was present in IR and IIR haplotypes. The purpose of the present study was to estimate the diversity of the different mitochondrial haplotypes and their distribution in QoI-resistant populations before evaluating the potential cost of the resistant mutation G143A in P. viticola population. From 2000 to 2004, the frequencies of resistant isolates ranged from 0% to 23.25% with an average of 4.64 % among the populations examined. To evaluate the fitness of sensitive and resistant isolates, a comparison of different biological parameters including latent period, spore production and infection frequency was performed, enabling a fitness index (FI) to be determined. Resistant isolates exhibited greater infection frequency than sensitive isolates, whereas no significant difference was found in sporulation ability and latent period between sensitive and resistant isolates. To further investigate competitiveness among isolates, an assay including two resistant isolates in different proportion with a sensitive isolate was conducted on eight asexual growing cycles in the absence of a QoI fungicide. The competitiveness of resistant isolates varied according to their fitness parameters, suggesting that there is no noticeable cost of QoI resistance in controlled conditions in Plasmopara viticola.

Is the rapid post-mating inhibition of pheromone response triggered by ecdysteroids or other factors from the sex accessory glands in the male moth Agrotis ipsilon?

In many animals, male copulation is dependent on the detection and processing of female-produced sex pheromones, which is generally followed by a sexual refractory post-ejaculatory interval (PEI). In the male moth, Agrotis ipsilon, this PEI is characterized by a transient post-mating inhibition of behavioral and central nervous responses to sex pheromone, which prevents males from re-mating until they have refilled their reproductive tracts for a potential new ejaculate. However, the timing and possible factors inducing this rapid olfactory switch-off are still unknown. Here, we determined the initial time delay and duration of the PEI. Moreover, we tested the hypothesis that the brain, the testis and/or the sex accessory glands (SAGs) could produce a factor inducing the PEI. Lastly, we investigated the possible involvement of ecdysteroids, hormones essential for development and reproduction in insects, in this olfactory plasticity. Using brain and SAG cross-injections in virgin and newly-mated males, surgical treatments, wind tunnel behavioral experiments and EIA quantifications of ecdysteroids, we show that the PEI starts very shortly after the onset of copulation, and that SAGs contain a factor, which is produced/accumulated after copulation to induce the PEI. Moreover, SAGs were found to be the main source of ecdysteroids, whose concentration decreased after mating, whereas it increased in the haemolymph. 20-Hydroxyecdysone (20E) was identified as the major ecdysteroid in SAGs of A. ipsilon males. Finally, 20E injections did not reduce the behavioral pheromone response of virgin males. Altogether our data indicate that 20E is probably not involved in the PEI.

Pinus pinaster Knot: A Source of Polyphenols against Plasmopara viticola

Pine knot extract from Pinus pinaster byproducts was characterized by UHPLC-DAD-MS and NMR. Fourteen polyphenols divided into four classes were identified as follows: lignans (nortrachelogenin, pinoresinol, matairesinol, isolariciresinol, secoisolariciresinol), flavonoids (pinocembrin, pinobanksin, dihydrokaempferol, taxifolin), stilbenes (pinosylvin, pinosylvin monomethyl ether, pterostilbene), and phenolic acids (caffeic acid, ferulic acid). The antifungal potential of pine knot extract, as well as the main compounds, was tested in vitro against Plasmopara viticola. The ethanolic extract showed a strong antimildew activity. In addition, pinosylvins and pinocembrin demonstrated significant inhibition of zoospore mobility and mildew development. These findings strongly suggest that pine knot is a potential biomass that could be used as a natural antifungal product.

Comparison of the impact of two molecules on plant defense and on efficacy against Botrytis cinerea in the vineyard: a plant defense inducer (Benzothiadiazole) and a fungicide (Pyrimethanil)

Grapevine is subject to diseases that affect yield and wine quality caused by various pathogens including Botrytis cinerea. To limit the use of fungicides, an alternative is to use plant elicitors such as benzothiadiazole (BTH). We investigated the effect of a fungicide (Pyrimethanil) and an elicitor (benzothiadiazole) on plant defenses. Applications for two consecutive years in the vineyard significantly reduced gray mold. Two and seven days after treatments, the expressions of 48 genes involved in defenses showed differential modulation (up- or down-regulation) depending on treatment. Some genes were identified as potential markers of protection and were linked to an increase in total polyphenols (TP) in leaves. Surprisingly, the fungicide also induced the expression of defense genes and increased the polyphenol content. This suggests that BTH acts as an efficient elicitor in the vineyard and that Pyrimethanil may act, in part, as a defense-inducing agent on the vine.