In the insect antennal lobe different types of local interneurons mediate complex excitatory and inhibitory interactions between the glomerular pathways to structure the spatiotemporal representation of odors. Mass spectrometric and immunohistochemical studies have shown that in local interneurons classical neurotransmitters are likely to colocalize with a variety of substances that can potentially act as cotransmitters or neuromodulators. In the antennal lobe of the cockroach Periplaneta americana, gamma-aminobutyric acid (GABA) has been identified as the potential inhibitory transmitter of spiking type I local interneurons, whereas acetylcholine is most likely the excitatory transmitter of nonspiking type IIa1 local interneurons. This study used whole-cell patch clamp recordings combined with single-cell labeling and immunohistochemistry to test if the GABAergic type I local interneurons and the cholinergic type IIa1 local interneurons express allatotropin and tachykinin-related neuropeptides (TKRPs). These are two of the most abundant types of peptides in the insect antennal lobe. GABA-like and choline acetyltransferase (ChAT)-like immunoreactivity were used as markers for GABAergic and cholinergic neurons, respectively. About 50% of the GABA-like immunoreactive (-lir) spiking type I local interneurons were allatotropin-lir, and ∼ 40% of these neurons were TKRP-lir. About 20% of nonspiking ChAT-lir type IIa1 local interneurons were TKRP-lir. Our results suggest that in subpopulations of GABAergic and cholinergic local interneurons, allatotropin and TKRPs might act as cotransmitters or neuromodulators. To unequivocally assign neurotransmitters, cotransmitters, and neuromodulators to identified classes of antennal lobe neurons is an important step to deepen our understanding of information processing in the insect olfactory system.

In contrast to other eukaryotes, which manufacture lipoic acid, an essential cofactor for several vital dehydrogenase complexes, within the mitochondrion, we show that the plastid (apicoplast) of the obligate intracellular protozoan parasite Toxoplasma gondii is the only site of de novo lipoate synthesis. However, antibodies specific for protein-attached lipoate reveal the presence of lipoylated proteins in both, the apicoplast and the mitochondrion of T. gondii. Cultivation of T. gondii-infected cells in lipoate-deficient medium results in substantially reduced lipoylation of mitochondrial (but not apicoplast) proteins. Addition of exogenous lipoate to the medium can rescue this effect, showing that the parasite scavenges this cofactor from the host. Exposure of T. gondii to lipoate analogues in lipoate-deficient medium leads to growth inhibition, suggesting that T. gondii might be auxotrophic for this cofactor. Phylogenetic analyses reveal the secondary loss of the mitochondrial lipoate synthase gene after the acquisition of the plastid. Our studies thus reveal an unexpected metabolic deficiency in T. gondii and raise the question whether the close interaction of host mitochondria with the parasitophorous vacuole is connected to lipoate supply by the host.

The red flour beetle Tribolium castaneum is an emerging insect model organism representing the largest insect order, Coleoptera, which encompasses several serious agricultural and forest pests. Despite the ecological and economic importance of beetles, most insect olfaction studies have so far focused on dipteran, lepidopteran, or hymenopteran systems.

Aedes aegypti is a model species in which the endogenous regulation of odor-mediated host seeking behavior has received some attention. Sugar feeding and host seeking in female A. aegypti are transiently inhibited following a blood meal. This inhibition is partially mediated by short neuropeptide F (sNPF). The paired antennal lobes (ALs), as the first processing centers for olfactory information, has been shown to play a significant role in the neuropeptidergic regulation of odor-mediated behaviors in insects. The expression of sNPF, along with other peptides in the ALs of A. aegypti, indicate parallel neuromodulatory systems that may affect olfactory processing. To identify neuropeptides involved in regulating the odor-mediated host seeking behavior in A. aegypti, we use a semi-quantitative neuropeptidomic analysis of single ALs to analyze changes in the levels of five individual neuropeptides in response to different feeding regimes. Our results show that the level of sNPF-2, allatostatin-A-5 (AstA-5) and neuropeptide-like precursor-1-5 (NPLP-1-5), but not of tachykinin-related-peptides and SIFamide (SIFa), in the AL of female mosquitoes, changes 24 h and 48 h post-blood meal, and are dependent on prior access to sugar. To assess the role of these neuropeptides in modulating host seeking behavior, when systemically injected individually, sNPF-2 and AstA-5 significantly reduced host seeking behavior. However, only the injection of the binary mixture of the two neuropeptides lead to a host seeking inhibition similar to that observed in blood fed females. We conclude that modulation of the odor mediated host seeking behavior of A. aegypti is likely regulated by a dual neuropeptidergic pathway acting in concert in the ALs.

Insects depend on their olfactory sense as a vital system. Olfactory cues are processed by a rather complex system and translated into various types of behavior. In holometabolous insects like the red flour beetle Tribolium castaneum, the nervous system typically undergoes considerable remodeling during metamorphosis. This process includes the integration of new neurons, as well as remodeling and elimination of larval neurons.

The paired antennal lobes were long considered the sole primary processing centers of the olfactory pathway in holometabolous insects receiving input from the olfactory sensory neurons of the antennae and mouthparts. In hemimetabolous insects, however, olfactory cues of the antennae and palps are processed separately. For the holometabolous red flour beetle Tribolium castaneum, we could show that primary processing of the palpal and antennal olfactory input also occurs separately and at distinct neuronal centers. While the antennal olfactory sensory neurons project into the antennal lobes, those of the palps project into the paired glomerular lobes and the unpaired gnathal olfactory center. Here we provide an extended analysis of the palpal olfactory pathway by combining scanning electron micrographs with confocal imaging of immunohistochemical staining and reporter expression identifying chemosensory and odorant receptor-expressing neurons in the palpal sensilla. In addition, we extended the anatomical characterization of the gnathal olfactory center by 3D reconstructions and investigated the distribution of several neuromediators. The similarities in the neuromediator repertoire between antennal lobes, glomerular lobes, and gnathal olfactory center underline the role of the latter two as additional primary olfactory processing centers.

A multitude of potential neurotransmitters and neuromodulators, including peptides, have been detected in the antennal lobe (AL), the first synaptic relay of the central olfactory pathway in the insect brain. However, the functional role of neuropeptides in this system has yet to be revealed. An important prerequisite to understanding the role of neuropeptides is to match the functionally different cell types in the AL with their peptide profiles by using electrophysiological recordings combined with immunocytochemical studies and/or single-cell mass spectrometry. The olfactory system of Periplaneta americana is particularly well suited to accomplish this goal because several physiologically distinct neuron types can be unequivocally identified. With the aim to analyze the neuropeptide inventory of the P. americana AL, this study is an essential step in this direction. First, we systematically analyzed different parts of the AL by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry to obtain the complete set of neuropeptides present. Altogether, 56 ion signals could be assigned to products of 10 neuropeptide genes (allatostatins A, B, C, SIFamide, allatotropin, FMRFamide-related peptides [myosuppressin, short neuropeptides F, extended FMRFamides], crustacean cardioactive peptide, tachykinin-related peptides). In a second step, a combination of immunocytochemistry and mass spectrometric profiling of defined AL compartments was used to reveal the spatial distribution of neuropeptide-containing cells. Finally, we demonstrated the feasibility of MALDI-TOF mass spectrometric profiling of single AL neurons, which is an important precondition for combining electrophysiology with peptide profiling at the single-cell level.

Beetles comprise about 400,000 described species, nearly one third of all known animal species. The enormous success of the order Coleoptera is reflected by a rich diversity of lifestyles, behaviors, morphological, and physiological adaptions. All these evolutionary adaptions that have been driven by a variety of parameters over the last about 300 million years, make the Coleoptera an ideal field to study the evolution of the brain on the interface between the basic bauplan of the insect brain and the adaptions that occurred. In the current study we concentrated on the paired antennal lobes (AL), the part of the brain that is typically responsible for the first processing of olfactory information collected from olfactory sensilla on antenna and mouthparts. We analyzed 63 beetle species from 22 different families and thus provide an extensive comparison of principal neuroarchitecture of the AL. On the examined anatomical level, we found a broad diversity including AL containing a wide range of glomeruli numbers reaching from 50 to 150 glomeruli and several species with numerous small glomeruli, resembling the microglomerular design described in acridid grasshoppers and diving beetles, and substructures within the glomeruli that have to date only been described for the small hive beetle, Aethina tumida. A first comparison of the various anatomical features of the AL with available descriptions of lifestyle and behaviors did so far not reveal useful correlations. In summary, the current study provides a solid basis for further studies to unravel mechanisms that are basic to evolutionary adaptions of the insect olfactory system.

Chemoreception is based on the senses of smell and taste that are crucial for animals to find new food sources, shelter, and mates. The initial step in olfaction involves the translocation of odorants from the periphery through the aqueous lymph of the olfactory sensilla to the odorant receptors most likely by chemosensory proteins (CSPs) or odorant binding proteins (OBPs).

The fruitfly, Drosophila, is dependent on its olfactory sense in food search and reproduction. Processing of odorant information takes place in the antennal lobes, the primary olfactory center in the insect brain. Besides classical neurotransmitters, earlier studies have indicated the presence of a few neuropeptides in the olfactory system. In the present study we made an extensive analysis of the expression of neuropeptides in the Drosophila antennal lobes by direct profiling using matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry and immunocytochemistry. Neuropeptides from seven different precursor genes were unambiguously identified and their localization in neurons was subsequently revealed by immunocytochemistry. These were short neuropeptide F, tachykinin related peptide, allatostatin A, myoinhibitory peptide, SIFamide, IPNamide, and myosuppressin. The neuropeptides were expressed in subsets of olfactory sensory cells and different populations of local interneurons and extrinsic (centrifugal) neurons. In some neuron types neuropeptides were colocalized with classical neurotransmitters. Our findings suggest a huge complexity in peptidergic signaling in different circuits of the antennal lobe.

Nitric oxide (NO) is a gaseous messenger molecule formed during conversion of L: -arginine into L: -citrulline by the enzyme NO synthase (NOS), which belongs to a group of NADPH diaphorases. Because of its gaseous diffusion properties, NO differs from classical neurotransmitters in that it is not restricted to synaptic terminals. In target cells, NO activates soluble guanylyl cyclase leading to an increase in cGMP levels. In insects, this NO/cGMP-signalling pathway is involved in development, memory formation and processing of visual, olfactory and mechanosensory information. We have analysed the distribution of putative NO donor and target cells in the central complex, a brain area involved in sky-compass orientation, of the locust Schistocerca gregaria by immunostaining for L: -citrulline and cGMP. Six types of citrulline-immunostained neurons have been identified including a bilateral pair of hitherto undescribed neurons that connect the lateral accessory lobes with areas anterior to the medial lobes of the mushroom bodies. Three-dimensional reconstructions have revealed the connectivity pattern of a set of 18 immunostained pontine neurons of the central body. All these neurons appear to be a subset of previously mapped NADPH-diaphorase-positive neurons of the central complex. At least three types of central-complex neurons show cGMP immunostaining including a system of novel columnar neurons connecting the upper division of the central body and the lateral triangle of the lateral accessory lobe. Our results provide the morphological basis for further studies of the function of the labelled neurons and new insights into NO/cGMP signalling.

Toxin complex (Tc) proteins termed TcaABC, TcdAB, and TccABC with insecticidal activity are present in a variety of bacteria including the yersiniae.

Invertebrate tachykinin-related peptides (TKRPs) comprise a group of signaling molecules having sequence similarities to mammalian tachykinins. A growing body of evidence has demonstrated the presence of TKRPs in the central nervous system of insects. In this investigation, we used an antiserum against locustatachykinin-II to reveal the distribution pattern of these peptides in the brain of the moth Heliothis virescens. Immunolabeling was found throughout the brain of the heliothine moth. Most of the roughly 500 locustatachykinin-II immunoreactive cell bodies, that is, ca. 400, were located in the protocerebrum, whereas the rest was distributed in the deutocerebrum, tritocerebrum, and the gnathal ganglion. Abundant immunoreactive processes were located in the same regions. Labeled processes in the protocerebrum were especially localized in optic lobe, central body, lateral accessory lobe, superior protocerebrum, and lateral protocerebrum, while those in the deutocerebrum were present exclusively in the antennal lobe. In addition to brain interneurons, four pairs of median neurosecretory cells in the pars intercerebralis with terminal processes in the corpora cardiaca and aorta wall were immunostained. No sexual dimorphism in immunoreactivity was found. Comparing the data obtained here with findings from other insect species reveals considerable differences, suggesting species-specific roles of tachykinin-related peptides in insects.

Lepidopterans like the giant sphinx moth Manduca sexta are known for their conspicuous sexual dimorphism in the olfactory system, which is especially pronounced in the antennae and in the antennal lobe, the primary integration center of odor information. Even minute scents of female pheromone are detected by male moths, facilitated by a huge array of pheromone receptors on their antennae. The associated neuropilar areas in the antennal lobe, the glomeruli, are enlarged in males and organized in the form of the so-called macroglomerular complex (MGC). In this study we searched for anatomical sexual dimorphism more downstream in the olfactory pathway and in other neuropil areas in the central brain. Based on freshly eclosed animals, we created a volumetric female and male standard brain and compared 30 separate neuropilar regions. Additionally, we labeled 10 female glomeruli that were homologous to previously quantitatively described male glomeruli including the MGC. In summary, the neuropil volumes reveal an isometric sexual dimorphism in M. sexta brains. This proportional size difference between male and female brain neuropils masks an anisometric or disproportional dimorphism, which is restricted to the sex-related glomeruli of the antennal lobes and neither mirrored in other normal glomeruli nor in higher brain centers like the calyces of the mushroom bodies. Both the female and male 3D standard brain are also used for interspecies comparisons, and may serve as future volumetric reference in pharmacological and behavioral experiments especially regarding development and adult plasticity. J. Comp. Neurol. 517:210-225, 2009. (c) 2009 Wiley-Liss, Inc.

Several studies showed adult persisting neurogenesis in insects, including the red flour beetle Tribolium castaneum, while it is absent in honeybees, carpenter ants, and vinegar flies. In our study, we focus on cell proliferation in the adult mushroom bodies of T. castaneum. We reliably labelled the progenies of the adult persisting mushroom body neuroblasts and determined the proliferation rate under several olfactory conditions within the first week after adult eclosion. We found at least two phases of Kenyon cell proliferation in the early adult beetle. Our results suggest that the generation of Kenyon cells during the first three days after adult eclosion is mainly genetically predetermined and a continuation of the developmental processes (nature), whereas from day four on proliferation seems to be mainly dependent on the odour environment (nurture). Considering that the mushroom bodies are linked to learning and memory, neurogenesis in the mushroom bodies is part of the remodelling of neuronal circuits leading to the adaption to the environment and optimization of behaviour.

DURING METAMORPHOSIS, THE TRANSITION FROM THE LARVA TO THE ADULT, THE INSECT BRAIN UNDERGOES CONSIDERABLE REMODELING: new neurons are integrated while larval neurons are remodeled or eliminated. One well acknowledged model to study metamorphic brain development is the sphinx moth Manduca sexta. To further understand mechanisms involved in the metamorphic transition of the brain we generated a 3D standard brain based on selected brain areas of adult females and 3D reconstructed the same areas during defined stages of pupal development. Selected brain areas include for example mushroom bodies, central complex, antennal- and optic lobes. With this approach we eventually want to quantify developmental changes in neuropilar architecture, but also quantify changes in the neuronal complement and monitor the development of selected neuronal populations. Furthermore, we used a modeling software (Cinema 4D) to create a virtual 4D brain, morphing through its developmental stages. Thus the didactical advantages of 3D visualization are expanded to better comprehend complex processes of neuropil formation and remodeling during development. To obtain datasets of the M. sexta brain areas, we stained whole brains with an antiserum against the synaptic vesicle protein synapsin. Such labeled brains were then scanned with a confocal laser scanning microscope and selected neuropils were reconstructed with the 3D software AMIRA 4.1.