Evidence of red sensitive photoreceptors in Pygopleurus israelitus (Glaphyridae: Coleoptera) and its implications for beetle pollination in the southeast Mediterranean.

A very well-documented case of flower-beetle interaction is the association in the Mediterranean region between red bowl-shaped flowers and beetles of the family Glaphyridae. The present study examines the visual mechanisms by which Pygopleurus israelitus (Glaphyridae: Scarabaeoidea: Coleoptera) would perceive the colors of flowers they visit by characterizing the spectral sensitivity of its photoreceptors. Our measurements revealed the presence of three types of photoreceptors, maximally sensitive in the UV, green and red areas of the spectrum. Using color vision space diagrams, we calculated the distribution of beetle-visited flower colors in the glaphyrid and honeybee color space and evaluated whether chromatic discrimination differs between the two types of pollinators. Respective color loci in the beetle color space are located on one side of the locus for green foliage background, whereas in the honeybee the flower color loci surround the locus occupied by green foliage. Our results represent the first evidence of a red sensitive photoreceptor in a flower-visiting coleopteran species, highlighting Glaphyridae as an interesting model group to study the role of pollinators in flower color evolution.

Neurobiology of polarization vision in the locust Schistocerca gregaria.

The polarization pattern of the blue sky serves as an important reference for spatial orientation in insects. To understand the neural mechanisms involved in sky compass orientation we have analyzed the polarization vision system in the locust Schistocerca gregaria. As in other insects, photoreceptors adapted for the detection of sky polarization are concentrated in a dorsal rim area (DRA) of the compound eye. Stationary flying locusts show polarotactic yaw-torque responses when illuminated through a rotating polarizer from above. This response is abolished after painting the DRAs. Central stages of the polarization vision system, revealed through tracing studies, include dorsal areas in the lamina and medulla, the anterior lobe of the lobula, the anterior optic tubercle, the lateral accessory lobe and the central complex. Physiological analysis of polarization-sensitive (POL) neurons has focussed on the optic tubercle and on the central complex. Each POL neuron was maximally excited at a certain e-vector (phimax) and was maximally inhibited at an e-vector perpendicular to phimax. The neurons had large visual fields, and many neurons received input from both eyes. The neuronal organization of the central complex suggests a role as a spatial compass within the locust brain.

Behavioral analysis of polarization vision in tethered flying locusts.

For spatial navigation many insects rely on compass information derived from the polarization pattern of the sky. We demonstrate that tethered flying desert locusts (Schistocerca gregaria) show e-vector-dependent yaw-torque responses to polarized light presented from above. A slowly rotating polarizer (5.3 degrees s(-1)) induced periodic changes in yaw torque corresponding to the 180 degrees periodicity of the stimulus. Control experiments with a rotating diffuser, a weak intensity pattern, and a stationary polarizer showed that the response is not induced by intensity gradients in the stimulus. Polarotaxis was abolished after painting the dorsal rim areas of the compound eyes black, but remained unchanged after painting the eyes except the dorsal rim areas. During rotation of the polarizer, two e-vectors (preferred and avoided e-vector) induced no turning responses: they were broadly distributed from 0 to 180 degrees but, for a given animal, were perpendicular to each other. The data demonstrate polarization vision in the desert locust, as shown previously for bees, flies, crickets, and ants. Polarized light is perceived through the dorsal rim area of the compound eye, suggesting that polarization vision plays a role in compass navigation of the locust.

Anatomy and physiology of neurons with processes in the accessory medulla of the cockroach Leucophaea maderae.

The accessory medulla (AMe), a small neuropil in the insect optic lobe, has been proposed to serve a circadian pacemaker function analogous to the role of the suprachiasmatic nucleus in mammals. Building upon considerable knowledge of the circadian system of the cockroach Leucophaea maderae, we investigated the properties of AMe neurons in this insect with intracellular recordings combined with dye injections. Responses of neurons with processes in the AMe to visual stimuli, including stationary white light, moving objects, and polarized light were compared with the responses of adjacent medulla tangential neurons. Neurons with processes in the AMe and additional ramifications in the medulla strongly responded to stationary light stimuli and might, therefore, be part of photic entrainment pathways to the clock. Accessory medulla neurons lacking significant processes in the medulla but with projections to the midbrain or to the contralateral optic lobe, in contrast, responded weakly or not at all to light and, thus, seem to be part of the clock's output pathway. Two types of commissural neurons with tangential arborizations in both medullae were sensitive to polarized light, suggesting a role of these neurons in celestial navigation. Sidebranches in the AMae of one of the two cell types are discussed with respect to a possible involvement of the AMe in polarization vision. Finally, neurons responding to movement stimuli did not arborize in the AMe. The results show that the AMe receives photic input and support a role of this neuropil in circadian timekeeping functions.

Candidates for extraocular photoreceptors in the cockroach suggest homology to the lamina and lobula organs in beetles.

Using light- and electron microscopic methods, we describe two novel putative extraocular photoreceptor organs in the optic lobes of the cockroaches Leucophaea maderae and Blaberus craniifer. The lamina organ is an elongated structure distal to the first optic chiasm, adjacent to the anterior edge of the lamina. The lobula organ is situated on the anterior distal surface of the lobula. In cross sections through the pigment-free organs, cell bodies are arranged in a closed or open circle and are interconnected by desmosomes. They send protrusions with rhabdom-like microvilli into a common, central space apparently filled with extracellular matrix. A different cell type gives rise to electron-dense lamellae, which also extend into the central space and partly join to form a common lamellar bundle. Axonal processes extend from the microvillar cells and run along the outer surface of the organs to the neighboring optic neuropils. The organs receive multiple efferent innervation from neurosecretory axons. Both organs show strong immunostaining with an antiserum against Arabidopsis cryptochrome 2 that is associated with the lamellated structure in the central lumen. The specific features of the organs suggest that they are homologous to similar organs in the optic lobe of beetles and may serve a role as extraocular photoreceptors for light entrainment of the circadian system.

Parallel organization in honey bee mushroom bodies by peptidergic Kenyon cells.

Antisera against the neuromodulatory peptides, Phe-Met-Arg-Phe-NH(2)-amide (FMRFamide) and gastrin cholecystokinin, demonstrate that the mushroom bodies of honey bees are subdivided longitudinally into strata. Three-dimensional reconstructions demonstrate that these strata project in parallel through the entire pedunculus and through the medial and vertical lobes. Immunostaining reveals clusters of immunoreactive cell bodies within the calyx cups and immunoreactive bundles of axons that line the inside of the calyx cup and lead to strata. Together, these features reveal that immunoreactive strata are composed of Kenyon cell axons rather than extrinsic elements, as suggested previously by some authors. Sorting amongst Kenyon cell axons into their appropriate strata already begins in the calyx before these axons enter the pedunculus. The three main concentric divisions of each calyx (the lip, collar, and basal ring) are divided further into immunoreactive and immunonegative zones. The lip neuropil is divided into two discrete zones, the collar neuropil is divided into five zones, and the basal ring neuropil is divided into four zones. Earlier studies proposed that the lip, collar, and basal ring are represented by three broad bands in the lobes: axons from adjacent Kenyon cell dendrites in the calyces are adjacent in the lobes even after their polar arrangements in the calyces have been transformed to rectilinear arrangements in the lobes. The universality of this arrangement is not supported by the present results. Although immunoreactive zones are found in all three calycal regions, immunoreactive strata in the lobes occur mainly in the two bands that were ascribed previously to the collar and the basal ring. In the lobes, immunoreactive strata are visited by the dendrites of efferent neurons that carry information from the mushroom bodies to other parts of the brain. Morphologically and chemically distinct subdivisions through the pedunculus and lobes of honey bees are comparable to longitudinal subdivisions demonstrated in the mushroom bodies of other insects, such as the cockroach Periplaneta americana. The functional and evolutionary significance of the results is discussed.

Histamine-immunoreactive neurons in the brain of the cockroach Leucophaea maderae.

Histamine is the neurotransmitter of insect photoreceptor cells but has also been found in a small number of interneurons in the insect brain. In order to investigate whether the accessory medulla (AMe), the putative circadian pacemaker of the cockroach Leucophaea maderae receives direct visual input from histaminergic photoreceptors, we analyzed the distribution of histamine-like immunoreactivity in the optic lobe and midbrain of the cockroach. Intense immunostaining was detected in photoreceptor cells of the compound eye, which terminated in the first optic neuropil, the lamina, and in a distal layer of the medulla, the second optic neuropil. Histamine immunostaining in parts of the AMe, however, originated from a centrifugal neuron of the midbrain. Within the midbrain 21-23 bilaterally symmetric pairs of cell bodies were stained. Most areas of the brain were innervated by one or more of these neurons, but the protocerebral bridge and the mushroom bodies were devoid of histamine immunoreactivity. The branching patterns of most histamine-immunoreactive neurons could be reconstructed individually. While the majority of identified neurons arborized in both brain hemispheres, five cells were local neurons of the antennal lobe. A comparison with other insect species shows striking similarities in the position of certain histamine-immunoreactive neurons, but considerable variations in the presence and branching patterns of others. The data suggest a role for histamine in a non-photic input to the circadian system of the cockroach.

Regulation of cyclic GMP elevation in the developing antennal lobe of the Sphinx moth, Manduca sexta.

In the moth, Manduca sexta, 3',5'-guanosine monophosphate (cGMP) is transiently elevated during adult development in about 100 neurons of the antennal lobe. We demonstrate that nearly all of these neurons are local interneurons of the lateral cluster I, that their capacity to show a strong cGMP response during development is regulated by the steroid hormone 20-hydroxyecdysone, and that in a subpopulation of these neurons cGMP elevation seems to be controlled directly by the gaseous messenger molecule nitric oxide (NO). Treatment with the acetylcholine esterase inhibitor eserine, antennal nerve transection, and electrical stimulation of the antennae suggest that NO/cGMP signaling during development is an activity-dependent process. Besides input from the antennae, input from the central brain and the ventral ganglia is involved in upregulating cGMP in the antennal-lobe neurons. Possible sources are centrifugal aminergic neurons, since application of serotonin and histamine enhances the GMP signal in local interneurons. Comparing the time course of cGMP elevation with events occurring during development leads us to the hypothesis that the NO/cGMP signaling pathway might be involved in synapse formation of a subset of antennal-lobe neurons.

Development of dopamine-immunoreactive neurons associated with the antennal lobes of the honey bee, Apis mellifera.

This report examines the development of the dopaminergic system in the primary antennosensory centres (antennal lobes) of the brain of the honey bee, Apis mellifera, and the effects of dopamine on neurite outgrowth of antennal-lobe neurons in vitro. Antibodies raised against dopamine were used to follow the development of a small population of dopamine-immunoreactive neurons that invade the antennal lobes during metamorphic adult development. Immunopositive somata associated with the antennal lobes were first detected at stage 2 of the nine stages of metamorphic adult development, but processes of these neurons within the antennal-lobe neuropil did not exhibit immunostaining until pupal stage 3. Severe depletion of primary sensory input to the right antennal lobe early in metamorphic adult development or removal of the right antenna from newly emerged bees did not alter the expression of dopamine immunoreactivity in the antennal-lobe neuropil. The presence of dopamine in developing antennal lobes was confirmed by using high performance liquid chromatography with electrochemical detection. Levels of dopamine were significantly higher at pupal stage 4 than at all other stages examined. This surge in dopamine levels coincided with rapid growth and compartmentalisation of the antennal-lobe neuropil. Exogenously applied dopamine (50 microM) enhanced the growth of antennal-lobe neurons in vitro, but only in cells derived from pupae at stage 5 of metamorphic adult development. The early appearance of dopamine-immunoreactive neurons and the effects of dopamine on stage 5 antennal-lobe neurons in vitro support the view that dopamine plays a role in the developing brain of the honey bee.

Immunocytochemistry of GABA in the central complex of the locust Schistocerca gregaria: identification of immunoreactive neurons and colocalization with neuropeptides.

The central complex is a highly organized neuropil structure in the insect brain and plays a role in motor control and visual orientation. We describe the distribution of gamma-aminobutyric acid (GABA) immunostaining in the central complex of the locust Schistocerca gregaria in an effort to analyze inhibitory neural circuits within this brain area. Antisera against GABA and the GABA-synthesizing enzyme glutamic acid decarboxylase resulted in identical patterns of immunostaining. Cell counts revealed about 100 bilateral pairs of GABA-immunoreactive neurons with arborizations in the central complex. Five types of immunostained neurons could be identified through reconstruction of the staining pattern, comparison with individually stained neurons, and double labeling experiments with Neurobiotin-injected neurons. All of these GABA-immunostained neurons are tangential neurons that connect the lateral accessory lobes to distinct layers of the central body. Three types of immunostained neurons (TL2, TL3, TL4) invade the lower division of the central body, and two additional types of neurons (TU1, TU2) have ramifications in layers I and II of the upper division of the central body. Double-labeling experiments with peptide antisera suggest that peptides related to Phe-Met-Arg-Phe-NH2/bovine pancreatic polypeptide and Dip-allatostatin might act as cotransmitters with GABA in TL4 neurons of the lower division and (Dip-allatostatin only) in TU2 neurons of the upper division of the central body. The high conservation in the pattern of GABA immunostaining in all insect species investigated so far suggests that GABA plays an essential role in the basic neural circuitry of the central complex in insects.

Organization of the circadian system in insects.

The circadian systems of different insect groups are summarized and compared. Emphasis is placed on the anatomical identification and characterization of circadian pacemakers, as well as on their entrainment, coupling, and output pathways. Cockroaches, crickets, beetles, and flies possess bilaterally organized pacemakers in the optic lobes that appear to be located in the accessory medulla, a small neuropil between the medulla and the lobula. Neurons that are immunoreactive for the peptide pigment-dispersing hormone (PDH) arborize in the accessory medulla and appear to be important components of the optic lobe pacemakers. The neuronal architecture of the accessory medulla with associated PDH-immunoreactive neurons is best characterized in cockroaches, while the molecular machinery of rhythm generation is best understood in fruit flies. One essential component of the circadian clock is the period protein (PER), which colocalizes with PDH in about half of the fruit fly's presumptive pacemaker neurons. PER is also found in the presumptive pacemaker neurons of beetles and moths, but appears to have different functions in these insects. In moths, the pacemakers are situated in the central brain and are closely associated with neuroendocrine functions. In the other insects, neurons associated with neuroendocrine functions also appear to be closely coupled to the optic lobe pacemakers. Some crickets and flies seem to possess central brain pacemakers in addition to their optic lobe pacemakers. With respect to neuronal organization, the circadian systems of insects show striking similarities to the vertebrate circadian system.

Immunocytochemical demonstration of locustatachykinin-related peptides in the central complex of the locust brain.

The central complex, a highly ordered neuropil area in the insect brain, plays a role in motor control and spatial orientation. To further elucidate the neurochemical architecture of this brain area, we have investigated the distribution and morphology of neurons containing locustatachykinin I/II-related substances in the central complex of the locust Schistocerca gregaria. The central complex is innervated by at least 66 locustatachykinin I/II-immunoreactive neurons, which belong to two sets of tangential neurons and four sets of columnar neurons. These neurons give rise to immunostaining in the protocerebral bridge, in several layers of the upper division of the central body, and in all layers except layer 5 of the lower division of the central body. Double-label experiments show colocalization of immunoreactivity for both locustatachykinin I/II and octopamine in tangential neurons of the protocerebral bridge. A pair of tangential neurons of the lower division of the central body exhibits both locustatachykinin I/II and gamma-aminobutyric acid (GABA) immunoreactivity. A set of 16 columnar neurons of the lower division of the central body shows colocalized immunoreactivity for locustatachykinin II, leucokinin, and substance P. This study reveals novel features of the anatomical organization of the locust central complex and suggests a prominent role for locustatachykinin-related peptides as neuromediators and cotransmitters within this brain area.

Movement-sensitive, polarization-sensitive, and light-sensitive neurons of the medulla and accessory medulla of the locust, Schistocerca gregaria.

Several lines of evidence suggest that the accessory medulla of orthopteroid insects is implicated in the control of circadian rhythms. To investigate the role of this brain area in more detail, anatomical and physiological properties of accessory-medulla neurons of the locust were studied by intracellular recordings combined with Lucifer dye injections. The responses of these neurons to visual stimuli were compared with visual responses of adjacent tangential neurons of the medulla. Principal neurons of the accessory medulla showed weak tonic excitations to stationary light stimuli, but they were not sensitive to movement stimuli or to different e-vector orientations of polarized light. These neurons connected the accessory medulla to the lamina, the anterior medulla, and to several areas in the midbrain including the superior protocerebrum and the posterior optic tubercle. A second class of neurons had tangential arborizations in the medulla, a few sidebranches in the accessory medulla, and projections to the lamina or to the contralateral optic lobe. Several of these neurons were sensitive to polarized light. Finally, a third class of neurons had tangential arborizations in the medulla and axonal projections to the lobula and to the lateral protocerebrum. These neurons showed phasic responses to light and nondirectional selective responses to motion stimuli. The results show that neurons of the accessory medulla receive photic input and support an involvement of this neuropil in circadian timekeeping functions. The possible role of the accessory medulla in polarization vision is discussed.

Neuroarchitecture of the lower division of the central body in the brain of the locust (Schistocerca gregaria).

We have investigated the anatomical organization of the lower division of the central body in the brain of the locust Schistocerca gregaria. Bodian preparations, Golgi impregnations, and intracellular filling with Lucifer yellow have revealed that the lower division of the central body is organized into six horizontal layers and sixteen vertical columns. Neurons of the lower division of the central body have been classified into five types of tangential neuron (TL1-TL5) and two types of columnar neuron (CL1, CL2). TL1-TL4 neurons ramify in specific layers in the lower division of the central body and in the lateral triangle (TL1, TL2 neurons), the median olive (TL3 neurons), or the dorsal shell (TL4 neurons) of the lateral accessory lobe. TL5 neurons ramify in the protocerebral bridge, in the lateral accessory lobe, and in all layers of the lower division of the central body. The two types of columnar neurons have arborizations in the protocerebral bridge and in the lower division of the central body and project to the lateral triangle of the lateral accessory lobe (CL1 neurons) or to the lower subunit of the nodulus (CL2 neurons). Possible functional implications for the processing of neuronal information in the central complex are discussed.

Neuroanatomy and immunocytochemistry of the median neuroendocrine cells of the subesophageal ganglion of the tobacco hawkmoth, Manduca sexta: immunoreactivities to PBAN and other neuropeptides.

The median neuroendocrine cells of the subesophageal ganglion, important components of the neuroendocrine system of the tobacco hawkmoth, Manduca sexta, have not been well investigated. Therefore, we studied the anatomy of these cells by axonal backfills and characterized their peptide immunoreactivities. Both larvae and adults were examined, and developmental changes in these neuroendocrine cells were followed. Processes of the median neuroendocrine cells project to terminations in the corpora cardiaca via the third and the ventral nerves of this neurohemal organ, but the ventral nerve of the corpus cardiacum is the principal neurohemal surface for this system. Cobalt backfills of the third cardiacal nerves revealed lateral cells in the maxillary neuromere and a ventro-median pair in the labial neuromere. Backfills of the ventral cardiacal nerves revealed two ventro-median pairs of cells in the mandibular neuromere and two ventro-median triplets in the maxillary neuromere. The efferent projections of these cells are contralateral. The anatomy of the system is basically the same in larvae and adults. The three sets of median neuroendocrine cells are PBAN- and FMRFamide-immunoreactive, but only the mandibular and maxillary cells are proctolin-immunoreactive. During metamorphosis, the mandibular and maxillary cells also acquire CCK-like immunoreactivity and the labial cells become SCP- and sulfakinin-immunoreactive. Characteristics of FMRFamide-like immunostaining suggest that the median neuroendocrine cells may contain one or more of the FLRFamides that have been identified in M. sexta. The mandibular and maxillary neuroendocrine cells appear to produce the same set of hormones, and a somewhat different set of hormones is produced by the labial neuroendocrine cells. Two pairs of interneurons immunologically related to the neurosecretory cells are associated with the median maxillary neuroendocrine cells. These cells are PBAN-, FMRFamide-, SCP-, and sulfakinin-immunoreactive and project to arborizations in the brain and all ventral ganglia. These interneurons appear to have extensive modulatory functions in the CNS.

Distribution of Dip-allatostatin I-like immunoreactivity in the brain of the locust Schistocerca gregaria with detailed analysis of immunostaining in the central complex.

The distribution and morphology of neurons containing allatostatin-related substances in the brain of the locust Schistocerca gregaria was investigated using an antiserum against Diploptera punctata allatostatin I (Dip-allatostatin I, APSGAQRLYGFGL-amide). In each brain hemisphere, about 550 neurons in the midbrain and 500 neurons in the optic lobe exhibit Dip-allatostatin I-like immunoreactivity, including about eight lateral neurosecretory cells with processes to the retrocerebral complex. All major brain areas except the antennal lobe, the mushroom body, and large parts of the lamina, are innervated by Dip-allatostatin I-immunoreactive processes. Immunostaining in the central complex was studied in detail. The central complex is innervated by more than 260 Dip-allatostatin I-immunoreactive neurons belonging to six different cell types, four sets of tangential neurons and two sets of columnar neurons. These neurons give rise to intense immunostaining in the protocerebral bridge, in several layers of the upper division of the central body, and in the dorsalmost layer of the lower division of the central body. Double-label experiments show colocalization of Dip-allatostatin I- and serotonin-like immunoreactivities in one type of columnar and one type of tangential neurons of the central complex. The similar patterns of Dip-allatostatin I- and galanin message-associated peptide-like immunoreactivities result from cross-reactivity of the anti-galanin message-associated peptide antiserum with Dip-allatostatin I. The results provide further insight into the anatomical and neurochemical organization of the locust central complex and suggest a prominent neuroactive role for Dip-allatostatin I-related peptides in this brain area.

Immunocytochemical mapping of serotonin and neuropeptides in the accessory medulla of the locust, Schistocerca gregaria.

Accumulating evidence suggests that pigment-dispersing hormone-immunoreactive neurons with ramifications in the accessory medulla of the insect brain are involved in circadian pacemaking functions. We have used immunocytochemical techniques to investigate the neurochemical organization of the accessory medulla in the locust Schistocerca gregaria. Local neurons with arborizations largely restricted to the accessory medulla are immunoreactive with antisera against serotonin, Manduca sexta allatotropin, and Diploptera punctata allatostatin 7. Projection neurons with arborizations in the accessory medulla and fibers to the lamina and/or several areas in the midbrain including the posterior optic tubercles, the inferior and the superior protocerebrum show Phe-Met-Arg-Phe (FMRF)amide-, gastrin/cholecystokinin-, crustacean cardioactive peptide-, and substance P immunoreactivities. A unique neuron with tangential ramifications in the medulla and lamina and varicose terminals in the accessory medulla contains a peptide related to locustatachykinin I/II. Double-label experiments show colocalization of pigment-dispersing hormone-immunoreactivity with substances related to gastrin/cholecystokinin, FMRFamide, substance P, or crustacean cardioactive peptide in certain projection neurons of the accessory medulla. The results suggest that neuropeptides and biogenic amines play major neuroactive roles in the accessory medulla of the locust. The abundance and extensive colocalization of neuropeptides in the locust accessory medulla is discussed with respect to the possible involvement of this brain area in circadian pacemaking functions.

Immunocytochemical characterization of the accessory medulla in the cockroach Leucophaea maderae.

Several lines of evidence suggest that pigment-dispersing hormone-immunoreactive neurons with ramifications in the accessory medulla are involved in the circadian system of insects. The present study provides a detailed analysis of the anatomical and neurochemical organization of the accessory medulla in the brain of the cockroach Leucophaea maderae. We show that the accessory medulla is compartmentalized into central dense nodular neuropil surrounded by a shell of coarse fibers. It is innervated by neurons immunoreactive to antisera against serotonin and the neuropeptides allatostatin 7, allatotropin, corazonin, gastrin/cholecystokinin, FMRFamide, leucokinin I, and pigment-dispersing hormone. Some of the immunostained neurons appear to be local neurons of the accessory medulla, whereas others connect this neuropil to various brain areas, including the lamina, the contralateral optic lobe, the posterior optic tubercles, and the superior protocerebrum. Double-label experiments show the colocalization of immunoreactivity against pigment-dispersing hormone with compounds related to FMRFamide, serotonin, and leucokinin I. The neuronal and neurochemical organization of the accessory medulla is consistent with the current hypothesis for a role of this brain area as a circadian pacemaking center in the insect brain.