Experience-dependent plasticity in the chemosensory system of the Egyptian cotton leafworm Spodoptera littoralis

Männliche Nachtfalter besitzen eine bereits angeborene hohe Empfindlichkeit für die Sexualpheromone arteigener Weibchen. Verhaltensreaktionen auf das Weibchenpheromon können bei Männchen der Spezies Spodoptera littoralis dennoch durch eine sehr kurze, einmalige Vorexponierung mit dem Pheromon weiter verstärkt werden (Anderson et al. 2003, 2007). Diese Empfindlichkeitssteigerung wurde in zwei Zeitfenstern nach der Vorexponierung beobachtet: es kam zu einem nach 15 Minuten gemessenen Kurzzeiteffekt und einem Langzeiteffekt, der bis zu 51 Stunden anhielt. Parallel dazu erhöhte sich die Pheromon-Empfindlichkeit der Neurone in den primären olfaktorischen Zentren, den Antennalloben (AL), 27 Stunden nach der Vorexponierung mit dem Pheromon. In der vorliegenden Arbeit wurden Geschmacksreize für die Vorexponierung und den darauffolgenden Test verwendet: die Empfindlichkeit für Zucker in den gustatorischen Rezeptorneuronen auf den Antennen von Spodoptera littoralis Männchen wurde 24 Stunden nach einer kurzen Vorbehandlung mit Zucker bzw. Chinin mithilfe von extrazellulären elektrophysiologischen Ableittechniken gemessen, um die neurobiologischen Vorgänge zu studieren, die der in einer vorhergehenden Studie beobachteten erfahrungsabhängigen Plastizität im Verhalten zugrunde liegen. In einer zweiten Serie von Experimenten wurde die Empfindlichkeit für das Weibchenpheromon nach kurzer Vorexponierung mit Geschmacksreizen getestet: die Empfindlichkeitsschwelle zentraler olfaktorischer Neurone im Antennallobus für das Sexualpheromon zwischen Zucker-exponierten und naiven Tieren wurde mithilfe von intrazellulären Ableitmethoden verglichen. Ergebnisse einer parallel durchgeführten Verhaltensstudie hatten darauf hingewiesen, dass Geschmackserfahrung auch die Empfindlichkeit für das Pheromon steigert. Die Ergebnisse der vorliegenden Arbeit deuten darauf hin, dass neuronale Plastizität innerhalb des Geschmackssystems nicht auf der peripheren sensorischen Ebene stattfindet, da keine Unterschiede in der Empfindlichkeit gustatorischer Rezeptorneurone für Saccharose zwischen naiven und vorexponierten Männchen gefunden wurde. Die neuronale Basis der intra-modalen Plastizität befindet sich demnach vermutlich im zentralen Nervensystem, es ist bisher jedoch unbekannt, ob zentrale Geschmacksneurone ihre Antworteigenschaften überhaupt erfahrungsabhängig ändern, oder ob die im Verhalten beobachtete Effekte anders verursacht wurden. Außerdem scheint sich die neuronale Grundlage des auf Verhaltensebene beobachteten, Sinnesmodalitäten überkreuzenden Effekts der gustatorischen Vorbehandlung nicht auf Ebene des Antennallobus zu befinden, da sich die Empfindlichkeit der AL-Neurone für das weibliche Sexualpheromon in naiven und mit Zucker vorbehandelten Männchen nicht unterschied. Die nicht-assoziativen Lernprozesse, die durch die Präexponierung verursacht werden laufen demnach vermutlich auf höheren Verarbeitungsebenen im Protocerebrum, wie den Pilzkörpern und dem lateralen Protocerebrum, ab, wo multimodale Sinneseindrücke verschaltet und gemeinsam verarbeitet werden.Male moths innately have a high sensitivity for female–produced sex pheromones. In the noctuid moth species Spodoptera littoralis, behavioural responses to the pheromone can nevertheless still be increased by previous brief experience with the sex pheromone, through neuronal plasticity (Anderson et al. 2003, 2007). This increase in sensitivity could be observed in two time windows after exposure: a short-term effect occurred within 15 minutes and a long-term effect lasted up to 51 hours. In parallel, an increase in sensitivity of neurons within the primary olfactory centre, the antennal lobe (AL), was observed 27 h after pre-exposure with pheromone. In the present study gustatory stimuli were used for pre-exposure and test: the sugar sensitivity of antennal gustatory receptor neurons (GRNs) as a function of pre-exposure with sucrose or quinine, was measured by means of extracellular electrophysiological recording techniques to study the neurobiological mechanisms underlying the behavioural plasticity that had been observed in a prior study. In a second series of experiments the sensitivity for the female-emitted sex pheromone was tested after brief pre-exposure with gustatory stimuli: the sensitivity of central olfactory neurons within the antennal lobe for the sex pheromone was compared in sucrose-exposed and naïve males using intracellular recording techniques. Results of a behavioural study conducted in parallel, had revealed a positive effect of brief gustatory pre-exposure onto pheromone sensitivity. The results of the present study indicate neuronal plasticity within the gustatory pathway not to occur at the peripheral sensory level, since no difference in GRN sensitivity for sucrose was observed between naïve and pre-exposed males. Thus, the neuronal basis of the “intra-modal” plasticity must be located at some level within the central nervous system, but we do not know by now if central gustatory neurons even change their response characteristics after pre-exposure, or if the behavioural effect is caused differently. In addition, the neuronal basis of the “cross-modal” effect of pre-exposure does not seem to be located at the antennal lobe level, since the sensitivity of AL neurons for the female sex pheromone did not differ in naïve and gustatory pre-exposed males. The non-associative learning processes caused by taste pre-exposure are more likely located at higher processing levels within the protocerebrum, like the mushroom bodies and the lateral protocerebrum, where multi-modal sensory input is integrated and processed together

Brief Exposure to Sensory Cues Elicits Stimulus-Nonspecific General Sensitization in an Insect

The effect of repeated exposure to sensory stimuli, with or without reward is well known to induce stimulus-specific modifications of behaviour, described as different forms of learning. In recent studies we showed that a brief single pre-exposure to the female-produced sex pheromone or even a predator sound can increase the behavioural and central nervous responses to this pheromone in males of the noctuid moth Spodoptera littoralis. To investigate if this increase in sensitivity might be restricted to the pheromone system or is a form of general sensitization, we studied here if a brief pre-exposure to stimuli of different modalities can reciprocally change behavioural and physiological responses to olfactory and gustatory stimuli. Olfactory and gustatory pre-exposure and subsequent behavioural tests were carried out to reveal possible intra- and cross-modal effects. Attraction to pheromone, monitored with a locomotion compensator, increased after exposure to olfactory and gustatory stimuli. Behavioural responses to sucrose, investigated using the proboscis extension reflex, increased equally after pre-exposure to olfactory and gustatory cues. Pheromone-specific neurons in the brain and antennal gustatory neurons did, however, not change their sensitivity after sucrose exposure. The observed intra- and reciprocal cross-modal effects of pre-exposure may represent a new form of stimulus-nonspecific general sensitization originating from modifications at higher sensory processing levels

Proboscis extension reflex (PER) responses to sucrose (SUC) of males pre-exposed to gustatory and olfactory stimuli.

<p><b>A</b>) pre-exposure to SUC and QUI, test with 0.03 M SUC. <b>B</b>) pre-exposure to SUC ipsi- or contralateral antenna, test with 0.03 M SUC. <b>C</b>) pre-exposure to PHE, test with 0.03 M SUC. <b>D</b>) responses of males to SUC after a non-specific mechanical stimulus, test with 0.1 M SUC. Columns show the percentage of males extending the proboscis when one of their antennae was contacted with a toothpick soaked with a SUC solution. Within each frame (A, B, C, D), the percentage of PER responses were significantly different between columns with different letters (Chi-Square, p<0.05). Numbers at the bottom of bars indicate numbers of tested males. Sensitivity to SUC was intra-modally increased by pre-exposure to SUC and QUI, and cross-modally increased by pre-exposure to PHE.</p

Trajectometry analyses of the walking pathways of PHE pre-exposed males confronted with different concentrations of PHE.

<p>Kinetic parameters describing different characteristics of the movement of the males on the locomotion compensator were calculated: <b>A</b>) “latency” (time until males activated), <b>B</b>) “walked distance” (displacement in any direction), <b>C</b>) “walked time” (active time) and <b>D</b>) “mean speed” (“walked distance” divided by “walked time”). The numbers of analyzed trajectories are n = 7 and n = 19 for C(hex) and PHE 0.1FE; n = 18 and n = 25 for C(hex) and PHE 0.25FE; n = 18 and n = 26 for C(hex) and PHE 0.5FE, respectively. The boundaries of the box indicate the 25th and 75th percentiles, whiskers indicate the 90th and 10th percentiles and black dots show outliers. Full and dashed lines within the box mark the median and mean respectively. Median tests (p<0.05) were carried out to reveal differences between different tested concentrations and pre-exposures. Only “walked distance” and “mean speed” increased in PHE pre-exposed male moths, but were not dependent on pheromone concentration. “Latency” and “walked time” changed with pheromone concentration but not after pre-exposure.</p

Behavioural responses to linalool (A: LIN), geraniol (B: GER) and different concentrations of female pheromone extracts (C and D: PHE) of males pre-exposed to LIN, GER and PHE.

<p>Grey and black columns show the percentage of males walking towards and against the source, respectively. The length of the whole column (grey+black) shows their activity level. Within each frame (A, B, C, D), the percentage of PER responses were significantly different between columns with different letters (Chi-Square, p<0.05). The number of tested males is n = 30 for each column. Circular diagrams show the mean angle of individual males (the stimulus is situated at 0°). Asterisks in circular diagrams show groups of insects that showed non-uniform distributions (Rayleigh test, p<0.05). Sensitivity to LIN and GER did not vary with any kind of pre-exposure. Sensitivity to PHE increased with both LIN and GER pre-exposure.</p

Proboscis extension reflex (PER) responses to different test-concentrations of sucrose (SUC) of males pre-exposed to sucrose (1 M SUC) and quinine (0.1 M QUI).

<p>Each point represents the percentage of males extending their proboscis when their antennae were contacted with different concentrations of SUC. The number of tested males is n = 40 for each data point. Dashed boxes enclose values that do not differ significantly (Chi-Square tests, p<0.05). Sensitivity to SUC was higher in males pre-exposed to SUC and QUI, showing a stronger difference with naïve males at low doses.</p

Cumulative frequency curves of response thresholds for the main pheromone compound (<i>Z,E</i>-9,11-14:OAc) of intracellularly-recorded MGC neurons in naïve (C_(H20)) and pre-exposed (SUC) males.

<p>No significant differences between the sensitivity of naïve and pre-exposed males were found, as revealed by a G-Test comparing the percentages of AL neurons responding at different thresholds. Note the bimodal distribution of thresholds in both groups: only few neurons with an intermediate threshold were found (flat curve between 10⁻⁵ and 10⁻² ng doses).</p

Behavioural responses to different concentrations of female pheromone extracts (PHE) of males pre-exposed to PHE on a locomotion compensator.

<p>Grey and black columns show the percentage of males walking towards and against the source, respectively. The length of the whole column (grey+black) shows their activity level. Within each frame (<b>A</b>, <b>B</b>, <b>C</b>, <b>D</b>), locomotor activity was significantly different between columns with different lower case letters (Chi-Square, p<0.05), and orientation levels differed significantly between columns with different numbers (Chi-Square, p<0.05). The number of tested males is n = 30 for each column. Circular diagrams show the mean angle of individual males (the stimulus is situated at 0°). Asterisks in circular diagrams show groups of insects that showed non-uniform distributions (Rayleigh test, p<0.05). Pheromone responses increased with the tested dose, but more pre-exposed than naïve males responded independently of the tested concentration.</p