The adult insect brain is composed of neuropils present in most taxa. However, the relative size, shape, and developmental timing differ between species. This diversity of adult insect brain morphology has been extensively described while the genetic mechanisms of brain development are studied predominantly in Drosophila melanogaster. However, it has remained enigmatic what cellular and genetic mechanisms underlie the evolution of neuropil diversity or heterochronic development. In this perspective paper, we propose a novel approach to study these questions. We suggest using genome editing to mark homologous neural cells in the fly D. melanogaster, the beetle Tribolium castaneum, and the Mediterranean field cricket Gryllus bimaculatus to investigate developmental differences leading to brain diversification. One interesting aspect is the heterochrony observed in central complex development. Ancestrally, the central complex is formed during embryogenesis (as in Gryllus) but in Drosophila, it arises during late larval and metamorphic stages. In Tribolium, it forms partially during embryogenesis. Finally, we present tools for brain research in Tribolium including 3D reconstruction and immunohistochemistry data of first instar brains and the generation of transgenic brain imaging lines. Further, we characterize reporter lines labeling the mushroom bodies and reflecting the expression of the neuroblast marker gene Tc-asense, respectively.Electronic supplementary materialThe online version of this article (doi:10.1007/s00427-016-0542-7) contains supplementary material, which is available to authorized users.

Neuropeptides are a highly diverse group of signaling molecules that affect a broad range of biological processes in insects, including development, metabolism, behavior, and reproduction. In the central nervous system, neuropeptides are usually considered to act as neuromodulators and cotransmitters that modify the effect of "classical" transmitters at the synapse. The present study analyzes the neuropeptide repertoire of higher cerebral neuropils in the brain of the red flour beetle Tribolium castaneum. We focus on two integrative neuropils of the olfactory pathway, the antennal lobes and the mushroom bodies. Using the technique of direct peptide profiling by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, we demonstrate that these neuropils can be characterized by their specific neuropeptide expression profiles. Complementary immunohistological analyses of selected neuropeptides revealed neuropeptide distribution patterns within the antennal lobes and the mushroom bodies. Both approaches revealed consistent differences between the neuropils, underlining that direct peptide profiling by mass spectrometry is a fast and reliable method to identify neuropeptide content.

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SIFamides form a group of highly conserved neuropeptides in insects, crustaceans, and chelicerates. Beyond their biochemical commonalities, the neuroanatomical distribution of SIFamide in the insect nervous system also shows a remarkable degree of conservation. Thus, expression of SIFamide has been found to be restricted to four neurons of the pars intercerebralis in different holometabolous species. By means of immunohistological stainings, we here show that in Manduca sexta, those four cells are complemented by additional immunoreactive cells located in the vicinity of the mushroom body calyx. Immunopositive processes form arborizations throughout the brain, innervating major neuropils like the antennal lobes, the central complex, and the optic neuropils.