Insights from the past: the work of Hans von Alten on the evolution of brain structure, ecological adaptation, and cognition in hymenopteran species.

In his treatise on arthropod brains, Hans von Alten (1910) focuses on a specific functional group of insects-the flying Hymenoptera-which exhibit a spectrum of lifestyles ranging from solitary to social. His work presents a distinctive comparative neuro-anatomical approach rooted in an eco-evolutionary and eco-behavioral background. We regard his publication as an exceptionally valuable source of information and seek to inspire the research community dedicated to the study of the insect brain to explore its insights further, even after more than 110 years. We have translated and annotated his work, expecting it to engage researchers not just with its remarkable drawings but also with its substantive content and exemplary research strategy. The present text is designed to complement von Alten's publication, situating it within the temporal context of nineteenth-century and early twentieth-century studies, and to draw connections to contemporary perspectives, especially concerning a central brain structure: the mushroom body.

Ring-shaped odor coding in the antennal lobe of migratory locusts.

The representation of odors in the locust antennal lobe with its >2,000 glomeruli has long remained a perplexing puzzle. We employed the CRISPR-Cas9 system to generate transgenic locusts expressing the genetically encoded calcium indicator GCaMP in olfactory sensory neurons. Using two-photon functional imaging, we mapped the spatial activation patterns representing a wide range of ecologically relevant odors across all six developmental stages. Our findings reveal a functionally ring-shaped organization of the antennal lobe composed of specific glomerular clusters. This configuration establishes an odor-specific chemotopic representation by encoding different chemical classes and ecologically distinct odors in the form of glomerular rings. The ring-shaped glomerular arrangement, which we confirm by selective targeting of OR70a-expressing sensory neurons, occurs throughout development, and the odor-coding pattern within the glomerular population is consistent across developmental stages. Mechanistically, this unconventional spatial olfactory code reflects the locust-specific and multiplexed glomerular innervation pattern of the antennal lobe.

A unified platform to manage, share, and archive morphological and functional data in insect neuroscience.

Insect neuroscience generates vast amounts of highly diverse data, of which only a small fraction are findable, accessible and reusable. To promote an open data culture, we have therefore developed the InsectBrainDatabase (IBdb), a free online platform for insect neuroanatomical and functional data. The IBdb facilitates biological insight by enabling effective cross-species comparisons, by linking neural structure with function, and by serving as general information hub for insect neuroscience. The IBdb allows users to not only effectively locate and visualize data, but to make them widely available for easy, automated reuse via an application programming interface. A unique private mode of the database expands the IBdb functionality beyond public data deposition, additionally providing the means for managing, visualizing, and sharing of unpublished data. This dual function creates an incentive for data contribution early in data management workflows and eliminates the additional effort normally associated with publicly depositing research data.

Insect neuroscience, like any field in the natural sciences, generates vast amounts of data. Currently, only a fraction are publicly available, and even less are reusable. This is because insect neuroscience data come in many formats and from many species. Some experiments focus on what insect brains look like (morphology), while others focus on how insect brains work (function). Some data come in the form of high-speed video, while other data contain voltage traces from individual neurons. Sharing is not as simple as uploading the raw files to the internet. To get a clear picture of how insect brains work, researchers need a way to cross-reference and connect different experiments. But, as it stands, there is no dedicated place for insect neuroscientists to share and explore such a diverse body of work. The community needs an open data repository that can link different types of data across many species, and can evolve as more data become available. Above all, this repository needs to be easy for researchers to use. To meet these specifications, Heinze et al. developed the Insect Brain Database. The database organizes data into three categories: species, brain structures, and neuron types. Within these categories, each entry has its own profile page. These pages bring different experiments together under one heading, allowing researchers to combine and compare data of different types. As researchers add more experiments, the profile pages will grow and evolve. To make the data easy to navigate, Heinze et al. developed a visual search tool. A combination of 2D and 3D images allow users to explore the data by anatomical location, without the need for expert knowledge. Researchers also have the option to upload their work in private mode, allowing them to securely share unpublished data. The Insect Brain Database brings data together in a way that is accessible not only to researchers, but also to students, and non-scientists. It will help researchers to find related work, to reuse existing data, and to build an open data culture. This has the potential to drive new discoveries combining research across the whole of the insect neuroscience field.

A reversal in sensory processing accompanies ongoing ecological divergence and speciation in Rhagoletis pomonella.

Changes in behaviour often drive rapid adaptive evolution and speciation. However, the mechanistic basis for behavioural shifts is largely unknown. The tephritid fruit fly Rhagoletis pomonella is an example of ecological specialization and speciation in action via a recent host plant shift from hawthorn to apple. These flies primarily use specific odours to locate fruit, and because they mate only on or near host fruit, changes in odour preference for apples versus hawthorns translate directly to prezygotic reproductive isolation, initiating speciation. Using a variety of techniques, we found a reversal between apple and hawthorn flies in the sensory processing of key odours associated with host fruit preference at the first olfactory synapse, linking changes in the antennal lobe of the brain with ongoing ecological divergence. Indeed, changes to specific neural pathways of any sensory modality may be a broad mechanism for changes in animal behaviour, catalysing the genesis of new biodiversity.

Mutagenesis of odorant coreceptor Orco fully disrupts foraging but not oviposition behaviors in the hawkmoth Manduca sexta.

The hawkmoth Manduca sexta and one of its preferred hosts in the North American Southwest, Datura wrightii, share a model insect-plant relationship based on mutualistic and antagonistic life-history traits. D. wrightii is the innately preferred nectar source and oviposition host for M. sexta Hence, the hawkmoth is an important pollinator while the M. sexta larvae are specialized herbivores of the plant. Olfactory detection of plant volatiles plays a crucial role in the behavior of the hawkmoth. In vivo, the odorant receptor coreceptor (Orco) is an obligatory component for the function of odorant receptors (ORs), a major receptor family involved in insect olfaction. We used CRISPR-Cas9 targeted mutagenesis to knock out (KO) the MsexOrco gene to test the consequences of a loss of OR-mediated olfaction in an insect-plant relationship. Neurophysiological characterization revealed severely reduced antennal and antennal lobe responses to representative odorants emitted by D. wrightii In a wind-tunnel setting with a flowering plant, Orco KO hawkmoths showed disrupted flight orientation and an ablated proboscis extension response to the natural stimulus. The Orco KO gravid female displayed reduced attraction toward a nonflowering plant. However, more than half of hawkmoths were able to use characteristic odor-directed flight orientation and oviposit on the host plant. Overall, OR-mediated olfaction is essential for foraging and pollination behaviors, but plant-seeking and oviposition behaviors are sustained through additional OR-independent sensory cues.

Inverse resource allocation between vision and olfaction across the genus Drosophila.

Divergent populations across different environments are exposed to critical sensory information related to locating a host or mate, as well as avoiding predators and pathogens. These sensory signals generate evolutionary changes in neuroanatomy and behavior; however, few studies have investigated patterns of neural architecture that occur between sensory systems, or that occur within large groups of closely-related organisms. Here we examine 62 species within the genus Drosophila and describe an inverse resource allocation between vision and olfaction, which we consistently observe at the periphery, within the brain, as well as during larval development. This sensory variation was noted across the entire genus and appears to represent repeated, independent evolutionary events, where one sensory modality is consistently selected for at the expense of the other. Moreover, we provide evidence of a developmental genetic constraint through the sharing of a single larval structure, the eye-antennal imaginal disc. In addition, we examine the ecological implications of visual or olfactory bias, including the potential impact on host-navigation and courtship.

Synaptic Spinules in the Olfactory Circuit of Drosophila melanogaster.

Here we report on ultrastructural features of brain synapses in the fly Drosophila melanogaster and outline a perspective for the study of their functional significance. Images taken with the aid of focused ion beam-scanning electron microscopy (EM) at 20 nm intervals across olfactory glomerulus DA2 revealed that some synaptic boutons are penetrated by protrusions emanating from other neurons. Similar structures in the brain of mammals are known as synaptic spinules. A survey with transmission EM (TEM) disclosed that these structures are frequent throughout the antennal lobe. Detailed neuronal tracings revealed that spinules are formed by all three major types of neurons innervating glomerulus DA2 but the olfactory sensory neurons (OSNs) receive significantly more spinules than other olfactory neurons. Double-membrane vesicles (DMVs) that appear to represent material that has pinched-off from spinules are also most abundant in presynaptic boutons of OSNs. Inside the host neuron, a close association was observed between spinules, the endoplasmic reticulum (ER) and mitochondria. We propose that by releasing material into the host neuron, through a process triggered by synaptic activity and analogous to axonal pruning, synaptic spinules could function as a mechanism for synapse tagging, synaptic remodeling and neural plasticity. Future directions of experimental work to investigate this theory are proposed.

Olfactory coding from the periphery to higher brain centers in the Drosophila brain.

BACKGROUND: Odor information is processed through multiple receptor-glomerular channels in the first order olfactory center, the antennal lobe (AL), then reformatted into higher brain centers and eventually perceived by the fly. To reveal the logic of olfaction, it is fundamental to map odor representations from the glomerular channels into higher brain centers. RESULTS: We characterize odor response profiles of AL projection neurons (PNs) originating from 31 glomeruli using whole cell patch-clamp recordings in Drosophila melanogaster. We reveal that odor representation from olfactory sensory neurons to PNs is generally conserved, while transformation of odor tuning curves is glomerulus-dependent. Reconstructions of PNs reveal that attractive and aversive odors are represented in different clusters of glomeruli in the AL. These separate representations are preserved into higher brain centers, where attractive and aversive odors are segregated into two regions in the lateral horn and partly separated in the mushroom body calyx. CONCLUSIONS: Our study reveals spatial representation of odor valence coding from the AL to higher brain centers. These results provide a global picture of the olfactory circuit design underlying innate odor-guided behavior.

Bacterial Community and PHB-Accumulating Bacteria Associated with the Wall and Specialized Niches of the Hindgut of the Forest Cockchafer (Melolontha hippocastani).

A characterization of the bacterial community of the hindgut wall of two larval and the adult stages of the forest cockchafer (Melolontha hippocastani) was carried out using amplicon sequencing of the 16S rRNA gene fragment. We found that, in second-instar larvae, Caulobacteraceae and Pseudomonadaceae showed the highest relative abundances, while in third-instar larvae, the dominant families were Porphyromonadaceae and Bacteroidales-related. In adults, an increase of the relative abundance of Bacteroidetes, Proteobacteria (γ- and δ- classes) and the family Enterococcaceae (Firmicutes) was observed. This suggests that the composition of the hindgut wall community may depend on the insect's life stage. Additionally, specialized bacterial niches hitherto very poorly described in the literature were spotted at both sides of the distal part of the hindgut chamber. We named these structures "pockets." Amplicon sequencing of the 16S rRNA gene fragment revealed that the pockets contained a different bacterial community than the surrounding hindgut wall, dominated by Alcaligenaceae and Micrococcaceae-related families. Poly-ß-hydroxybutyrate (PHB) accumulation in the pocket was suggested in isolated Achromobacter sp. by Nile Blue staining, and confirmed by gas chromatography-mass spectrometry analysis (GC-MS) on cultured bacterial mass and whole pocket tissue. Raman micro-spectroscopy allowed to visualize the spatial distribution of PHB accumulating bacteria within the pocket tissue. The presence of this polymer might play a role in the colonization of these specialized niches.

The Circuitry of Olfactory Projection Neurons in the Brain of the Honeybee, Apis mellifera.

In the honeybee brain, two prominent tracts - the medial and the lateral antennal lobe tract - project from the primary olfactory center, the antennal lobes (ALs), to the central brain, the mushroom bodies (MBs), and the protocerebral lobe (PL). Intracellularly stained uniglomerular projection neurons were reconstructed, registered to the 3D honeybee standard brain atlas, and then used to derive the spatial properties and quantitative morphology of the neurons of both tracts. We evaluated putative synaptic contacts of projection neurons (PNs) using confocal microscopy. Analysis of the patterns of axon terminals revealed a domain-like innervation within the MB lip neuropil. PNs of the lateral tract arborized more sparsely within the lips and exhibited fewer synaptic boutons, while medial tract neurons occupied broader regions in the MB calyces and the PL. Our data show that uPNs from the medial and lateral tract innervate both the core and the cortex of the ipsilateral MB lip but differ in their innervation patterns in these regions. In the mushroombody neuropil collar we found evidence for ALT boutons suggesting the collar as a multi modal input site including olfactory input similar to lip and basal ring. In addition, our data support the conclusion drawn in previous studies that reciprocal synapses exist between PNs, octopaminergic-, and GABAergic cells in the MB calyces. For the first time, we found evidence for connections between both tracts within the AL.

Synaptic circuitry of identified neurons in the antennal lobe of Drosophila melanogaster.

In Drosophila melanogaster olfactory sensory neurons (OSNs) establish synapses with projection neurons (PNs) and local interneurons within antennal lobe (AL) glomeruli. Substantial knowledge regarding this circuitry has been obtained by functional studies, whereas ultrastructural evidence of synaptic contacts is scarce. To fill this gap, we studied serial sections of three glomeruli using electron microscopy. Ectopic expression of a membrane-bound peroxidase allowed us to map synaptic sites along PN dendrites. Our data prove for the first time that each of the three major types of AL neurons is both pre- and postsynaptic to the other two types, as previously indicated by functional studies. PN dendrites carry a large proportion of output synapses, with approximately one output per every three input synapses. Detailed reconstructions of PN dendrites showed that these synapses are distributed unevenly, with input and output sites partially segregated along a proximal-distal gradient and the thinnest branches carrying solely input synapses. Moreover, our data indicate synapse clustering, as we found evidence of dendritic tiling of PN dendrites. PN output synapses exhibited T-shaped presynaptic densities, mostly arranged as tetrads. In contrast, output synapses from putative OSNs showed elongated presynaptic densities in which the T-bar platform was supported by several pedestals and contacted as many as 20 postsynaptic profiles. We also discovered synaptic contacts between the putative OSNs. The average synaptic density in the glomerular neuropil was about two synapses/µm(3) . These results are discussed with regard to current models of olfactory glomerular microcircuits across species.

Drosophila Avoids Parasitoids by Sensing Their Semiochemicals via a Dedicated Olfactory Circuit.

Detecting danger is one of the foremost tasks for a neural system. Larval parasitoids constitute clear danger to Drosophila, as up to 80% of fly larvae become parasitized in nature. We show that Drosophila melanogaster larvae and adults avoid sites smelling of the main parasitoid enemies, Leptopilina wasps. This avoidance is mediated via a highly specific olfactory sensory neuron (OSN) type. While the larval OSN expresses the olfactory receptor Or49a and is tuned to the Leptopilina odor iridomyrmecin, the adult expresses both Or49a and Or85f and in addition detects the wasp odors actinidine and nepetalactol. The information is transferred via projection neurons to a specific part of the lateral horn known to be involved in mediating avoidance. Drosophila has thus developed a dedicated circuit to detect a life-threatening enemy based on the smell of its semiochemicals. Such an enemy-detecting olfactory circuit has earlier only been characterized in mice and nematodes.

Central projections of antennular chemosensory and mechanosensory afferents in the brain of the terrestrial hermit crab (Coenobita clypeatus; Coenobitidae, Anomura).

The Coenobitidae (Decapoda, Anomura, Paguroidea) is a taxon of hermit crabs that includes two genera with a fully terrestrial life style as adults. Previous studies have shown that Coenobitidae have evolved a sense of spatial odor localization that is behaviorally highly relevant. Here, we examined the central olfactory pathway of these animals by analyzing central projections of the antennular nerve of Coenobita clypeatus, combining backfilling of the nerve with dextran-coupled dye, Golgi impregnations and three-dimensional reconstruction of the primary olfactory center, the antennular lobe. The principal pattern of putative olfactory sensory afferents in C. clypeatus is in many aspects similar to what have been established for aquatic decapod crustaceans, such as the spiny lobster Panulirus argus. However, there are also obvious differences that may, or may not represent adaptations related to a terrestrial lifestyle. In C. clypeatus, the antennular lobe dominates the deutocerebrum, having more than one thousand allantoid-shaped subunits. We observed two distinct patterns of sensory neuron innervation: putative olfactory afferents from the aesthetascs either supply the cap/subcap region of the subunits or they extend through its full depth. Our data also demonstrate that any one sensory axon can supply input to several subunits. Putative chemosensory (non-aesthetasc) and mechanosensory axons represent a different pathway and innervate the lateral and median antennular neuropils. Hence, we suggest that the chemosensory input in C. clypeatus might be represented via a dual pathway: aesthetascs target the antennular lobe, and bimodal sensilla target the lateral antennular neuropil and median antennular neuropil. The present data is compared to related findings in other decapod crustaceans.

Decoding odor quality and intensity in the Drosophila brain.

To internally reflect the sensory environment, animals create neural maps encoding the external stimulus space. From that primary neural code relevant information has to be extracted for accurate navigation. We analyzed how different odor features such as hedonic valence and intensity are functionally integrated in the lateral horn (LH) of the vinegar fly, Drosophila melanogaster. We characterized an olfactory-processing pathway, comprised of inhibitory projection neurons (iPNs) that target the LH exclusively, at morphological, functional and behavioral levels. We demonstrate that iPNs are subdivided into two morphological groups encoding positive hedonic valence or intensity information and conveying these features into separate domains in the LH. Silencing iPNs severely diminished flies' attraction behavior. Moreover, functional imaging disclosed a LH region tuned to repulsive odors comprised exclusively of third-order neurons. We provide evidence for a feature-based map in the LH, and elucidate its role as the center for integrating behaviorally relevant olfactory information.

Drosophila FoxP mutants are deficient in operant self-learning.

Intact function of the Forkhead Box P2 (FOXP2) gene is necessary for normal development of speech and language. This important role has recently been extended, first to other forms of vocal learning in animals and then also to other forms of motor learning. The homology in structure and in function among the FoxP gene members raises the possibility that the ancestral FoxP gene may have evolved as a crucial component of the neural circuitry mediating motor learning. Here we report that genetic manipulations of the single Drosophila orthologue, dFoxP, disrupt operant self-learning, a form of motor learning sharing several conceptually analogous features with language acquisition. Structural alterations of the dFoxP locus uncovered the role of dFoxP in operant self-learning and habit formation, as well as the dispensability of dFoxP for operant world-learning, in which no motor learning occurs. These manipulations also led to subtle alterations in the brain anatomy, including a reduced volume of the optic glomeruli. RNAi-mediated interference with dFoxP expression levels copied the behavioral phenotype of the mutant flies, even in the absence of mRNA degradation. Our results provide evidence that motor learning and language acquisition share a common ancestral trait still present in extant invertebrates, manifest in operant self-learning. This 'deep' homology probably traces back to before the split between vertebrate and invertebrate animals.

Comparative neuroanatomy of the antennal lobes of 2 homopteran species.

We compared the morphology of the primary olfactory center, the antennal lobe (AL), in 2 homopteran insects, Hyalesthes obsoletus Signoret (Homoptera: Cixiidae) and Scaphoideus titanus Ball (Homoptera: Cicadomorpha). The comparison between the ALs of the 2 species is particularly interesting considering that, although both use volatile cues to locate their host plants, their feeding behavior differs considerably: specifically, H. obsoletus is a highly polyphagous species, whereas S. titanus is strictly monophagous (on grapevine). Our investigation of the AL structure using immunocytochemical staining and antennal backfills did not reveal any sexual dimorphism in either the size of the ALs or in the size of individual glomeruli for either species. Instead, the AL of H. obsoletus displayed numerous and well-delineated glomeruli (about 130 in both sexes) arranged in a multilayered structure, whereas the smaller AL of S. titanus contained fewer than 15 glomerular-like structures. This difference is likely to reflect the comparatively reduced olfactory abilities in S. titanus, probably as a consequence of the reduced number of volatiles coming from the single host plant. Instead, in H. obsoletus, the ability to distinguish among several host plants may require a more complex olfactory neuronal network.

The antennal lobe of Libellula depressa (Odonata, Libellulidae).

Here we describe the antennal lobe of Libellula depressa (Odonata, Libellulidae), identified on the basis of the projections of the afferent sensory neurons stemming from the antennal flagellum sensilla. Immunohistochemical neuropil staining as well as antennal backfills revealed sensory neuron terminal arborizations covering a large portion of the antennal lobe. No clear glomerular structure was identified, thus suggesting an aglomerular antennal lobe condition as previously reported in Palaeoptera. The terminal arbors of backfilled sensory neurons do, however, form spherical knots, probably representing the connections between the few afferent neurons and the antennal lobe interneurons. The reconstruction revealed that the proximal part of the antennal nerve is divided into two branches that innervate two spatially separated areas of the antennal lobe, an anterioventral lobe and a larger posteriodorsal lobe. Our data are consistent with the hypothesis that one tract of the antennal nerve of L. depressa contains olfactory sensory neurons projecting into one of the sublobes, while the other tract contains thermo-hygroreceptive neurons projecting into the other sublobe.

The Digital Bee Brain: Integrating and Managing Neurons in a Common 3D Reference System.

The honeybee standard brain (HSB) serves as an interactive tool for relating morphologies of bee brain neurons and provides a reference system for functional and bibliographical properties (http://www.neurobiologie.fu-berlin.de/beebrain/). The ultimate goal is to document not only the morphological network properties of neurons collected from separate brains, but also to establish a graphical user interface for a neuron-related data base. Here, we review the current methods and protocols used to incorporate neuronal reconstructions into the HSB. Our registration protocol consists of two separate steps applied to imaging data from two-channel confocal microscopy scans: (1) The reconstruction of the neuron, facilitated by an automatic extraction of the neuron's skeleton based on threshold segmentation, and (2) the semi-automatic 3D segmentation of the neuropils and their registration with the HSB. The integration of neurons in the HSB is performed by applying the transformation computed in step (2) to the reconstructed neurons of step (1). The most critical issue of this protocol in terms of user interaction time - the segmentation process - is drastically improved by the use of a model-based segmentation process. Furthermore, the underlying statistical shape models (SSM) allow the visualization and analysis of characteristic variations in large sets of bee brain data. The anatomy of neural networks composed of multiple neurons that are registered into the HSB are visualized by depicting the 3D reconstructions together with semantic information with the objective to integrate data from multiple sources (electrophysiology, imaging, immunocytochemistry, molecular biology). Ultimately, this will allow the user to specify cell types and retrieve their morphologies along with physiological characterizations.

Physiological and morphological characterization of local interneurons in the Drosophila antennal lobe.

The Drosophila antennal lobe (AL) has become an excellent model for studying early olfactory processing mechanisms. Local interneurons (LNs) connect a large number of glomeruli and are ideally positioned to increase computational capabilities of odor information processing in the AL. Although the neural circuit of the Drosophila AL has been intensively studied at both the input and the output level, the internal circuit is not yet well understood. An unambiguous characterization of LNs is essential to remedy this lack of knowledge. We used whole cell patch-clamp recordings and characterized four classes of LNs in detail using electrophysiological and morphological properties at the single neuron level. Each class of LN displayed unique characteristics in intrinsic electrophysiological properties, showing differences in firing patterns, degree of spike adaptation, and amplitude of spike afterhyperpolarization. Notably, one class of LNs had characteristic burst firing properties, whereas the others were tonically active. Morphologically, neurons from three classes innervated almost all glomeruli, while LNs from one class innervated a specific subpopulation of glomeruli. Three-dimensional reconstruction analyses revealed general characteristics of LN morphology and further differences in dendritic density and distribution within specific glomeruli between the different classes of LNs. Additionally, we found that LNs labeled by a specific enhancer trap line (GAL4-Krasavietz), which had previously been reported as cholinergic LNs, were mostly GABAergic. The current study provides a systematic characterization of olfactory LNs in Drosophila and demonstrates that a variety of inhibitory LNs, characterized by class-specific electrophysiological and morphological properties, construct the neural circuit of the AL.

Distinct mechanisms produce functionally complementary actions of neuropeptides that are structurally related but derived from different precursors.

Many bioactive neuropeptides containing RFamide at their C terminus have been described in both invertebrates and vertebrates. To obtain insight into the functional logic of RFamide signaling, we investigate it here in the feeding system of Aplysia. We focus on the expression, localization, and actions of two families of RFamide peptides, the FRFamides and FMRFamide, in the central neuronal circuitry and the peripheral musculature that generate the feeding movements. We describe the cloning of the FRFamide precursor protein and show that the FRFamides and FMRFamide are derived from different precursors. We map the expression of the FRFamide and FMRFamide precursors in the feeding circuitry using in situ hybridization and immunostaining and confirm proteolytic processing of the FRFamide precursor by mass spectrometry. We show that the two precursors are expressed in different populations of sensory neurons in the feeding system. In a representative feeding muscle, we demonstrate the presence of both FRFamides and FMRFamide and their release, probably from the processes of the sensory neurons in the muscle. Both centrally and in the periphery, the FRFamides and FMRFamide act in distinct ways, apparently through distinct mechanisms, and nevertheless, from an overall functional perspective, their actions are complementary. Together, the FRFamides and FMRFamide convert feeding motor programs from ingestive to egestive and depress feeding muscle contractions. We conclude that these structurally related peptides, although derived from different precursors, expressed in different neurons, and acting through different mechanisms, remain related to each other in the functional roles that they play in the system.