Optical inactivation of a proprioceptor in an insect by non-genetic tools.

BACKGROUND: The specific role of sensory organs in locomotor pattern generation is traditionally investigated by means of mechanical ablation in arthropods that currently do not allow genetic manipulation. Mechanical ablation is irreversible, and may lead to injury discharges and changes in the structural integrity of the cuticle. NEW METHOD: Here, we present a new method to temporarily or permanently deprive parts of an insect nervous system of sensory feedback from leg proprioceptors by means of blue light application. We illuminated campaniform sensilla (CS) with a blue LED (420-480 nm) or a 473 nm laser at different light intensities to optically eliminate sensory and motor neuron responses to mechanical stimulation. RESULTS: We were able to eliminate all stimulus-evoked responses of CS. Individual CS groups were precisely and selectively inactivated without affecting nearby proprioceptors, using an optical fiber (Ø 200 µm) to guide the light. Our results demonstrated that lower light intensities significantly increase the required exposure time, but also the chance for recovery, thus making the effect reversible. COMPARISON WITH EXISTING METHODS: In contrast to mechanical ablation, optical inactivation of individual sensory organs is non-invasive and does not affect the behavioral state of the animal, nor does it induce escape behavior. This is especially relevant in non-model system experimental animals where optogenetic manipulation cannot be used, due to a lack of established methods of access. CONCLUSION: Our results show that the proposed method is a reliable alternative to mechanical ablation and can be successfully applied to the CS, as it fulfills all requirements regarding selectivity, efficiency, and reproducibility.

Identification of mature peptides from pban and capa genes of the moths Heliothis peltigera and Spodoptera littoralis.

By transcriptome analysis, we identified PBAN and CAPA precursors in the moths Spodoptera littoralis and Heliothis peltigera which are among the most damaging pests of agriculture in tropical and subtropical Africa as well as in Mediterranean countries. A combination of mass spectrometry and immunocytochemistry was used to identify mature peptides processed from these precursors and to reveal their spatial distribution in the CNS. We found that the sites of expression of pban genes, the structure of PBAN precursors and the processed neuropeptides are very similar in noctuid moths. The sequence of the diapause hormone (DH; tryptopyrokinin following the signal peptide), however, contains two N-terminal amino acids more than expected from comparison with already published sequences of related species. Capa genes of S. littoralis and H. peltigera encode, in addition to periviscerokinins, a tryptopyrokinin showing sequence similarity with DH, which is the tryptopyrokinin of the pban gene. CAPA peptides, which were not known from any noctuid moth so far, are produced in cells of abdominal ganglia. The shape of the release sites of these hormones in H. peltigera represents an exceptionally derived trait state and does not resemble the well-structured abdominal perisympathetic organs which are known from many other insects. Instead, axons of CAPA cells extensively ramify within the ventral diaphragm. The novel information regarding the sequences of all mature peptides derived from pban and capa genes of H. peltigera and S. littoralis now enables a detailed analysis of the bioactivity and species-specificity of the native peptides, especially those from the hitherto unknown capa genes, and to explore their interactions with PBAN/DH receptors.