Stéphane Debernard

Characterization of an Antennal Carboxylesterase from the Pest Moth Spodoptera littoralis Degrading a Host Plant Odorant

Background Carboxyl/cholinesterases (CCEs) are highly diversified in insects. These enzymes have a broad range of proposed functions, in neuro/developmental processes, dietary detoxification, insecticide resistance or hormone/pheromone degradation. As few functional data are available on purified or recombinant CCEs, the physiological role of most of these enzymes is unknown. Concerning their role in olfaction, only two CCEs able to metabolize sex pheromones have been functionally characterized in insects. These enzymes are only expressed in the male antennae, and secreted into the lumen of the pheromone-sensitive sensilla. CCEs able to hydrolyze other odorants than sex pheromones, such as plant volatiles, have not been identified. Methodology In Spodoptera littoralis, a major crop pest, a diversity of antennal CCEs has been previously identified. We have employed here a combination of molecular biology, biochemistry and electrophysiology approaches to functionally characterize an intracellular CCE, SlCXE10, whose predominant expression in the olfactory sensilla suggested a role in olfaction. A recombinant protein was produced using the baculovirus system and we tested its catabolic properties towards a plant volatile and the sex pheromone components. Conclusion We showed that SlCXE10 could efficiently hydrolyze a green leaf volatile and to a lesser extent the sex pheromone components. The transcript level in male antennae was also strongly induced by exposure to this plant odorant. In antennae, SlCXE10 expression was associated with sensilla responding to the sex pheromones and to plant odours. These results suggest that a CCE-based intracellular metabolism of odorants could occur in insect antennae, in addition to the extracellular metabolism occurring within the sensillar lumen. This is the first functional characterization of an Odorant-Degrading Enzyme active towards a host plant volatile.

Degradation of pheromone and plant volatile components by a same odorant-degrading enzyme in the cotton leafworm, Spodoptera littoralis.

Background Odorant-Degrading Enzymes (ODEs) are supposed to be involved in the signal inactivation step within the olfactory sensilla of insects by quickly removing odorant molecules from the vicinity of the olfactory receptors. Only three ODEs have been both identified at the molecular level and functionally characterized: two were specialized in the degradation of pheromone compounds and the last one was shown to degrade a plant odorant. Methodology Previous work has shown that the antennae of the cotton leafworm Spodoptera littoralis, a worldwide pest of agricultural crops, express numerous candidate ODEs. We focused on an esterase overexpressed in males antennae, namely SlCXE7. We studied its expression patterns and tested its catalytic properties towards three odorants, i.e. the two female sex pheromone components and a green leaf volatile emitted by host plants. Conclusion SlCXE7 expression was concomitant during development with male responsiveness to odorants and during adult scotophase with the period of male most active sexual behaviour. Furthermore, SlCXE7 transcription could be induced by male exposure to the main pheromone component, suggesting a role of Pheromone-Degrading Enzyme. Interestingly, recombinant SlCXE7 was able to efficiently hydrolyze the pheromone compounds but also the plant volatile, with a higher affinity for the pheromone than for the plant compound. In male antennae, SlCXE7 expression was associated with both long and short sensilla, tuned to sex pheromones or plant odours, respectively. Our results thus suggested that a same ODE could have a dual function depending of it sensillar localisation. Within the pheromone-sensitive sensilla, SlCXE7 may play a role in pheromone signal termination and in reduction of odorant background noise, whereas it could be involved in plant odorant inactivation within the short sensilla.

Identification of steroid hormone signaling pathway in insect cell differentiation

Abstract.To dissect the steroid hormone signaling pathway involved in insect cell morphological differentiation, we extended the application of the doublestranded RNA-mediated interference (dsRNAi) method to the epidermal IAL-PID2 cell line from Plodia interpunctella Lepidoptera. We first demonstrated that dsRNA was capable of efficiently blocking the steroid hormone 20-hydroxyecdysone (20E) inducibility of proteins that belong to the nuclear receptor superfamily, including the ecdysone receptor (EcR), its partner Ultraspiracle (USP), the insect homolog of the vertebrate retinoid X receptor and the HR3 transcription factor. We then showed that inhibiting the 20E induction of EcR, USP or HR3 proteins prevented the increased synthesis of β tubulin and consequently the morphological transformation of cells. Thanks to this functional approach, we have shown, for the first time, the participation of EcR, USP and HR3 in a 20E signaling pathway that directs morphological differentiation in insect cells by regulating β tubulin expression.

Cell cycle profiles of EcR, USP, HR3 and B cyclin mRNAs associated to 20E‐induced G2 arrest of Plodia interpunctella imaginal wing cells

Using the IAL‐PID2 cell line established from pupally committed imaginal wing discs of Plodia interpunctella, we have investigated the dynamics of cellular and molecular events involved in the G2/M arrest. We have first cloned a cDNA sequence named PIUSP‐2 that likely encodes a homologue of the Ultraspiracle‐2 isoform of Manduca sexta. When the IAL‐PID2 cells were exposed to a 8 h 20E treatment applied at different times of the cell cycle, an optimal period of sensitivity of cells to 20E, in inducing G2 arrest, was determined at the S/G2 transition. Using cDNA probes specifically designed from Plodia B cyclin (PcycB), ecdysone receptor B1‐isoform (PIEcR‐B1) and HR3 transcription factor (PHR3), we provide evidence that the 20E‐induced G2 arrest was correlated to a high induction of PHR3, PIEcR‐B1, PIUSP‐2 mRNAs at the S/G2 transition and a decrease in PcycB mRNA level at the end of G2 phase.

Periodic expression of an ecdysteroid-induced nuclear receptor in a lepidopteran cell line (IAL-PID2)

A set of DNA primers was designed within the DNA-binding domain of the Manduca hormone receptor 3 (MHR3) cDNA. These primers were used in RT-PCR to isolate a 204 bp cDNA fragment from IAL-PID2 cells exposed to 10(-6) M 20-hydroxyecdysone (20E) for 12 h. The amino acid sequence deduced from the cDNA fragment presented 100% identity with the zinc finger domain of Manduca hormone receptor 3 (MHR3), Galleria hormone receptor 3 (GHR3) and Choristoneura hormone receptor 3 (CHR3). This cDNA fragment was used as a probe on total RNA from IAL-PID2 cells exposed to 20E and hybridized to mRNA, the size of which was close to 4.5 kb and named Plodia hormone receptor 3 (PHR3). Kinetics of induction of PHR3 mRNA were similar to that of HR3 genes but varied according to the position of cells in their cell cycle. The non-steroidal ecdysone agonist, RH-5992 induced the expression of PHR3 at lower concentrations than 20E. From sequence similarity, mRNA size, 20E and RH-5992 inducibilities, we conclude that PHR3 transcript could encode a Plodia hormone receptor 3 involved in the genetic signalling cascade of 20E. Thanks to its periodic expression, this putative orphan nuclear receptor could serve as a suitable cellular marker for studying changes of epidermal cell sensitivity to 20E during the cell cycle.

Synchronization of Plodia interpunctella lepidopteran cells and effects of 20-hydroxyecdysone.

We have investigated the molecular and cellular mechanisms involved in the control of insect cell cycle by 20‐hydroxyecdysone (20E) using the IAL‐PID2 cell line established from imaginal wing discs of Plodia interpunctella. We first defined conditions for use of hydroxyurea, a reversible inhibitor of DNA synthesis, in order to synchronize the IAL‐PID2 cells in their division cycle. A high degree of synchrony was reached when cells were exposed to two consecutive hydroxyurea treatments at 1 mm for 36 h spaced 16 h apart. Under these conditions, flow cytometry analysis demonstrated that 20E at 10−6 m induced an inhibition of cell growth by an arrest of 90% of the cells in G2/M phase. Using cDNA probes specifically designed from E75 and HR3 nuclear receptors of Plodia interpunctella, we showed that PiE75 and PHR3 were highly induced by 20E through S and G2 phases with maximal enhancement just before the G2/M arrest of cells. These findings suggest that PiE75 and PHR3 could be involved in a 20E‐induced genetic cascade leading to G2/M arrest.

A TRP channel is expressed in Spodoptera littoralis antennae and is potentially involved in insect olfactory transduction.

The molecular characterization of post‐receptor actors involved in insect olfactory transduction has yet to be understood. We have investigated the presence of a Transient Receptor Potential (TRP) channel in the peripheral olfactory system of the moth Spodoptera littoralis. A cDNA encoding a Lepidopteran TRP channel (TRPγ) was identified by analysis of a male‐antennal EST database and subsequently cloned by RACE PCR. In adult males, the TRPγ transcript was detected in antennae, at the base of olfactory sensilla. Moreover, TRPγ was observed in antennae in both pupal and adult stages. This work is the first step in understanding the involvement of TRPγ in signalling pathways involved in the development and function of the insect olfactory system.

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.

Molecular Characterization of a Phospholipase C β Potentially Involved in Moth Olfactory Transduction

To clarify the role of phospholipase C (PLC) in insect olfactory transduction, we have undertaken its molecular identification in the moth Spodoptera littoralis. From the analysis of a male antennal expressed sequence tag library, we succeeded in cloning a full-length cDNA encoding a PLC that belongs to the cluster of PLC-beta subtypes. In adult males, the PLC-beta transcript was located predominantly in brain and antennae where its presence was detected in the olfactory sensilla trichodea. Moreover, PLC-beta was expressed in antennae at the beginning of the pupal stage, then reached a maximum at the end of this stage and was maintained at this level during the adult period. Taken together, these results provided molecular evidence for the putative participation of a PLC-beta in signaling pathways responsible for the establishment and the functioning of insect olfactory system.

The Ecdysteroids' Effects in the Control of Cell Proliferation and Differentiation

In insects, the steroid hormone 20-hydroxyecdysone (20E) plays a critical role in the control of cellular proliferation and differentiation. The cells show different responses to 20E according to the concentration to which they are exposed. The 20E at 10 M induces an inhibition of growth by a blockage of cells in G2/M and long term morphological transformation. The establishment of 20E responsive cell lines provided potentialities to investigate the molecular events responsible for these cellular responses as well as theirs dynamics through the cell cycle. In the Plodia interpunctella IAL-PID2 cell line, an optimal period of sensitivity of cells to 20E, in inducing G2/M arrest, was preferentially located at the transition S/G2 with a high induction of EcR, USP and HR3 mRNAs and a decrease in the expression level of B-cyclin at the end of G2 phase. On the other hand, the 20E-induced cytoskeleton rearrangement was accompanied by a redistribution of cytoplasmic microtubules which was concomitant with an increase in the β tubulin mRNA amount. The use of RNAi technique allowed to demonstrate that inhibiting the induction of EcR, USP and HR3 suppressed the 20E effects on the synthesis of B-cyclin and β tubulin then consequently prevented the arrest and the transformation o`f IAL-PID2 cells. This functional approach revealed that 20E was able to regulate the cellular differentiation and proliferation by acting on regulators of cell cycle and cytoskeleton proteins through a genomic signaling pathway involving EcR, USP and HR3.