Mating-Induced Differential Peptidomics of Neuropeptides and Protein Hormones in Agrotis ipsilon Moths.

In many insects, mating induces drastic changes in male and female responses to sex pheromones or host-plant odors. In the male moth Agrotis ipsilon, mating induces a transient inhibition of behavioral and neuronal responses to the female sex pheromone. As neuropeptides and peptide hormones regulate most behavioral processes, we hypothesize that they could be involved in this mating-dependent olfactory plasticity. Here we used next-generation RNA sequencing and a combination of liquid chromatography, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and direct tissue profiling to analyze the transcriptome and peptidome of different brain compartments in virgin and mated males and females of A. ipsilon. We identified 37 transcripts encoding putative neuropeptide precursors and 54 putative bioactive neuropeptides from 23 neuropeptide precursors (70 sequences in total, 25 neuropeptide precursors) in different areas of the central nervous system including the antennal lobes, the gnathal ganglion, and the corpora cardiaca-corpora allata complex. Comparisons between virgin and mated males and females revealed tissue-specific differences in peptide composition between sexes and according to physiological state. Mated males showed postmating differences in neuropeptide occurrence, which could participate in the mating-induced olfactory plasticity.

Rice Sucrose Partitioning Mediated by a Putative Pectin Methyltransferase and Homogalacturonan Methylesterification.

Homogalacturonan (HG) is the main component of pectins. HG methylesterification has recently emerged as a key determinant controlling cell attachment, organ formation, and phyllotaxy. However, whether and how HG methylesterification affects intercellular metabolite transport has rarely been reported. Here, we identified and characterized knockout mutants of the rice (Oryza sativa) OsQUA2 gene encoding a putative pectin methyltransferase. Osqua2 mutants exhibit a remarkable decrease in the degree of methylesterification of HG in the culm-sieve element cell wall and a markedly reduced grain yield. The culm of Osqua2 mutant plants contains excessive sucrose (Suc), and a 13CO2 feeding experiment showed that the Suc overaccumulation in the culm was caused by blocked Suc translocation. These and other findings demonstrate that OsQUA2 is essential for maintaining a high degree of methylesterification of HG in the rice culm-sieve element cell wall, which may be critical for efficient Suc partitioning and grain filling. In addition, our results suggest that the apoplastic pathway is involved in long-distance Suc transport in rice. The identification and characterization of the OsQUA2 gene and its functionality revealed a previously unknown contribution of HG methylesterification and provided insight into how modification of the cell wall regulates intercellular transport in plants.

Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification.

High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter::GUS lines in hybrid aspen (Populus tremula × tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification.

AUXIN BINDING PROTEIN1 links cell wall remodeling, auxin signaling, and cell expansion in arabidopsis.

Cell expansion is an increase in cell size and thus plays an essential role in plant growth and development. Phytohormones and the primary plant cell wall play major roles in the complex process of cell expansion. In shoot tissues, cell expansion requires the auxin receptor AUXIN BINDING PROTEIN1 (ABP1), but the mechanism by which ABP1 affects expansion remains unknown. We analyzed the effect of functional inactivation of ABP1 on transcriptomic changes in dark-grown hypocotyls and investigated the consequences of gene expression on cell wall composition and cell expansion. Molecular and genetic evidence indicates that ABP1 affects the expression of a broad range of cell wall-related genes, especially cell wall remodeling genes, mainly via an SCF(TIR/AFB)-dependent pathway. ABP1 also functions in the modulation of hemicellulose xyloglucan structure. Furthermore, fucosidase-mediated defucosylation of xyloglucan, but not biosynthesis of nonfucosylated xyloglucan, rescued dark-grown hypocotyl lengthening of ABP1 knockdown seedlings. In muro remodeling of xyloglucan side chains via an ABP1-dependent pathway appears to be of critical importance for temporal and spatial control of cell expansion.

Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose.

Cell wall hemicelluloses and pectins are O-acetylated at specific positions, but the significance of these substitutions is poorly understood. Using a transgenic approach, we investigated how reducing the extent of O-acetylation in xylan affects cell wall chemistry, plant performance and the recalcitrance of lignocellulose to saccharification. The Aspergillus niger acetyl xylan esterase AnAXE1 was expressed in Arabidopsis under the control of either the constitutively expressed 35S CAMV promoter or a woody-tissue-specific GT43B aspen promoter, and the protein was targeted to the apoplast by its native signal peptide, resulting in elevated acetyl esterase activity in soluble and wall-bound protein extracts and reduced xylan acetylation. No significant alterations in cell wall composition were observed in the transgenic lines, but their xylans were more easily digested by a ß-1,4-endoxylanase, and more readily extracted by hot water, acids or alkali. Enzymatic saccharification of lignocellulose after hot water and alkali pretreatments produced up to 20% more reducing sugars in several lines. Fermentation by Trametes versicolor of tissue hydrolysates from the line with a 30% reduction in acetyl content yielded ~70% more ethanol compared with wild type. Plants expressing 35S:AnAXE1 and pGT43B:AnAXE1 developed normally and showed increased resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis, probably due to constitutive activation of defence pathways. However, unintended changes in xyloglucan and pectin acetylation were only observed in 35S:AnAXE1-expressing plants. This study demonstrates that postsynthetic xylan deacetylation in woody tissues is a promising strategy for optimizing lignocellulosic biomass for biofuel production.

Brief sensory experience differentially affects the volume of olfactory brain centres in a moth.

Experience modifies behaviour in animals so that they adapt to their environment. In male noctuid moths, Spodoptera littoralis, brief pre-exposure to various behaviourally relevant sensory signals modifies subsequent behaviour towards the same or different sensory modalities. Correlated with a behavioural increase in responses of male moths to the female-emitted sex pheromone after pre-exposure to olfactory, acoustic or gustatory stimuli, an increase in sensitivity of olfactory neurons within the primary olfactory centre, the antennal lobe, is found for olfactory and acoustic stimuli, but not for gustatory stimuli. Here, we investigated whether anatomical changes occurring in the antennal lobes and in the mushroom bodies (the secondary olfactory centres) possibly correlated with the changes observed in behaviour and in olfactory neuron physiology. Our results showed that significant volume changes occurred in glomeruli (olfactory units) responsive to sex pheromone following exposure to both pheromone and predator sounds. The volume of the mushroom body input region (calyx) also increased significantly after pheromone and predator sound treatment. However, we found no changes in the volume of antennal lobe glomeruli or of the mushroom body calyx after pre-exposure to sucrose. These findings show a relationship of antennal lobe sensitivity changes to the pheromone with changes in the volume of the related glomeruli and the output area of antennal lobe projection neurons elicited by sensory cues causing a behavioural change. Behavioural changes observed after sucrose pre-exposure must originate from changes in higher integration centres in the brain.

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.

Mating-induced transient inhibition of responses to sex pheromone in a male moth is not mediated by octopamine or serotonin.

In the male moth, Agrotis ipsilon, mating induces a transient inhibition of behavioural and central nervous responses to sex pheromone. Newly mated males are not attracted to sex pheromone, and the sensitivity of their antennal lobe (AL) neurons is lower than in virgin males. This rapid transient olfactory inhibition prevents them from re-mating unsuccessfully until they have refilled their sex glands. We hypothesized that this olfactory 'switch off' might be controlled by neuromodulators such as biogenic amines. To test our hypothesis, we studied the effects of octopamine (OA) and serotonin (5-hydroxytryptamine, 5-HT) on the coding properties of pheromone-sensitive AL neurons in virgin and newly mated males. We show that AL neuron sensitivity increased in newly mated males after injection of OA or 5-HT, but only OA treatment affected certain response characteristics of AL neurons in virgin males. Whereas all measured AL neuron response characteristics were different between virgin and newly mated males, amine treatment in newly mated males restored only the latency and spike frequency, but not the duration of excitatory and inhibitory phases, which were initially found in virgin males. Additionally, we investigated the behavioural effects of OA and 5-HT treatments in virgin and mated males. Although OA and 5-HT enhanced the general flight activity of newly mated males, amine treatments did not restore the behavioural pheromone response of mated moths. Altogether, these results show that, although biogenic amines modulate the olfactory system in moths, OA and 5-HT are probably not involved in the post-mating inhibition of responses to sex pheromone in A. ipsilon males.

The role of contact chemoreception in the host location process of an egg parasitoid.

Taste allows insects to detect palatable or toxic foods, identify a mate, and select appropriate oviposition sites. The gustatory system strongly contributes to the survival and reproductive success of many species, yet it is rarely studied in insect parasitoids. In order to locate and assess a host in which they will lay their eggs, female wasps actively search for chemical cues using their sensory organs present mainly on the antennae. In this paper, we studied the role of antennal taste sensilla chaetica in the perception of contact semiochemicals in Trissolcus brochymenae (Hymenoptera: Platygastridae), an egg parasitoid of the brassicaceae pest Murgantia histrionica (Heteroptera: Pentatomidae). Methanolic extracts obtained from male and female hosts elicited action potentials in taste neurons housed in antennal sensilla chaetica, indicating that these sensilla are involved in the perception of non volatile host kairomones. In behavioural assays, wasp females displayed an intense searching behaviour in open arenas treated with host extracts, thus confirming that these kairomones are soluble in polar solvents. We further investigated the extracts by Gas Chromatography-Mass Spectrometry (GC-MS) and found that they contain several compounds which are good candidates for these contact kairomones. This study contributes to better understanding contact chemoreception in egg parasitoids and identifying gustatory receptor neurons involved in the host location process.

Perception of cuticular hydrocarbons by the olfactory organs in Periplaneta americana (L.) (Insecta: Dictyoptera).

Despite the importance of cuticular hydrocarbons (CHs) in insect chemical communication, direct proof that they are detected and recognized by insects by contact or by olfactory receptors are rare. In Periplaneta americana, CHs induce aggregation. The aim of our study was to investigate how CHs are detected by P. americana antennae. Using solid phase microextraction and gas chromatography, the three main CHs of the species profile were identified in the volatiles emitted by these insects. Gas chromatography coupled to electroantennography recordings demonstrated that the antennae responded to these three CHs. Furthermore, CHs had an attraction effect in Y-olfactometer bioassays when presented at high concentrations. As CHs can be perceived by P. americana, at least from a short distance, they could play a role in attracting conspecifics during aggregation processes, in addition to inducing aggregation when direct contact is possible.

Involvement of the G-protein-coupled dopamine/ecdysteroid receptor DopEcR in the behavioral response to sex pheromone in an insect.

Most animals including insects rely on olfaction to find their mating partners. In moths, males are attracted by female-produced sex pheromones inducing stereotyped sexual behavior. The behaviorally relevant olfactory information is processed in the primary olfactory centre, the antennal lobe (AL). Evidence is now accumulating that modulation of sex-linked behavioral output occurs through neuronal plasticity via the action of hormones and/or catecholamines. A G-protein-coupled receptor (GPCR) binding to 20-hydroxyecdysone, the main insect steroid hormone, and dopamine, has been identified in Drosophila (DmDopEcR), and was suggested to modulate neuronal signaling. In the male moth Agrotis ipsilon, the behavioral and central nervous responses to pheromone are age-dependent. To further unveil the mechanisms of this olfactory plasticity, we searched for DopEcR and tested its potential role in the behavioral response to sex pheromone in A. ipsilon males. Our results show that A. ipsilon DopEcR (named AipsDopEcR) is predominantly expressed in the nervous system. The corresponding protein was detected immunohistochemically in the ALs and higher brain centers including the mushroom bodies. Moreover, AipsDopEcR expression increased with age. Using a strategy of RNA interference, we also show that silencing of AipsDopEcR inhibited the behavioral response to sex pheromone in wind tunnel experiments. Altogether our results indicate that this GPCR is involved in the expression of sexual behavior in the male moth, probably by modulating the central nervous processing of sex pheromone through the action of one or both of its ligands.