The distribution of pancreatic polypeptide in the nervous system and gut of the blowfly,Calliphora vomitoria (Diptera)

Summary The distribution of a neuropeptide, previously shown to have the same or a very similar amino acid composition as vertebrate pancreatic polypeptide (PP), has been studied in the nervous system and gut of the blowfly, Calliphora vomitoria. Neurones immunoreactive to a bovine PP antiserum occur in the thoracic and abdominal ganglionic components of the central nervous system, in addition to the brain and suboesophageal ganglion. Pancreatic polypeptide appears to be relayed from its cells of origin to a neurohaemal organ in the dorsal sheath of the thoracic ganglion. PP immunoreactivity is also found in cells of the hypocerebral ganglion of the stomatogastric nervous system and in associated nerve fibres. The mid-gut contains PP-positive material in flask-shaped cells of its epithelial lining.     

Mapping of enkephalin-related peptides in the nervous system of the blowfly,Calliphora vomitoria,and their co-localization with cholecystokinin (CCK)- and pancreatic polypeptide (PP)-like peptides

Summary The distribution of enkephalin-like immunoreactive material has been studied in the CNS of C. vomitoria. The presence of both Met- and Leu-enkephalin-related peptides is suggested by differential immunostaining with a variety of antisera. Comparisons made between certain of the enkephalin-immunoreactive perikarya, nerve fibres and terminals with cells in corresponding positions as evidenced in previously published neuroanatomical studies of the dipteran brain have suggested specific enkephalinergic pathways. As examples, one Met-enkephalin-immunoreactive neuron appears to link the lobula with the dorsal protocerebrum, and a group of Leu-enkephalin cells in the pars intercerebralis appear to have arborisations in both the central body (fan-shaped body) and the tritocerebral neuropil around the oesophageal foramen. Neuronal pathways of this type indicate that the enkephalin-like peptides of the fly brain are functioning as neurotransmitters and/or neuromodulators. In the thoracic ganglia, symmetrically arranged cells, immunoreactive to both Met- and Leu-enkephalin antisera, are positioned ventrally in pairs on either side of the mid-line in a sagittal plane. Very little immunoreactive material is observed in the neuropil, however, and the source of the accumulation of Leu-enkephalin-immunoreactivity in the dorsal neural sheath is not certain. It is suggested that this material, in contrast to that present in areas of the brain, acts as a neurohormone and that it may have a physiological role following its release into the haemolymph. The enkephalin-like immunoreactive material of certain neurons identified within the brain and thoracic ganglion shows a complex pattern of co-existence with pancreatic polypeptide- and gastrin/cholecystokinin-like peptides.     

Distribution and functional significance of Leu-callatostatins in the blowfly Calliphora vomitoria

The Leu-callatostatins are a series of four neuropeptides isolated from nervous tissues of the blowfly Calliphora vomitoria that show C-terminal sequence homology to the allatostatins of cockroaches. The allatostatins have an important role in the reproductive processes of insects as inhibitors of the synthesis and release of juvenile hormone from the corpus allatum. In this study, the distribution of the Leu-callatostatin-immunoreactive neurones and endocrine cells has been mapped in C. vomitoria and, in contrast to the cockroach allatostatins, it has been shown that there is no cytological basis to suggest that the dipteran peptides act as regulators of juvenile hormone. Although occurring in various neurones in the brain and thoracico-abdominal ganglion, there is no evidence of Leu-callatostatin-immunoreactive pathways linking the brain to the corpus allatum, or of immunoreactive terminals in this gland. Three different types of functions for the Leu-callatostatins are suggested by the occurrence of immunoreactive material in cells and by the pathways that have been identified. (1) A role in neurotransmission or neuromodulation appears evident from immunoreactive neurones in the medulla of the optic lobes, and from immunoreactive material in the central body and in descending interneurones in the suboesophageal ganglion that project to the neuropile of the thoracico-abdominal ganglion. (2) Leu-callatostatin neurones directly innervate muscles of the hindgut and the heart. Immunoreactive fibres from neurones of the abdominal ganglion pass by way of the median abdominal nerve to ramify extensively over several areas of the hindgut. Physiological experiments with synthetic peptides show that the Leu-callatostatins are potent inhibitors of peristaltic movements of the ileum. Leu-callatostatin 3 is active at 10^-16 to 10^-13 M. This form or regulatory control over gut motility appears to be highly specific since the patterns of contraction in other regions are unaffected by these peptides. (3) Evidence that the Leu-callatostatins act as neurohormones comes from the presence of varicosities in axons passing through the corpus cardiacum (but not the corpus allatum) and also from material in extraganglionic neurosecretory cells in the thorax. Fibres from these peripheral neurones are especially prominent over the large nerve bundles supplying the legs. There are also a considerable number of Leu-callatostatin-immunoreactive endocrine cells in a specific region of the midgut. The conclusion from this study is that although conservation of the structure of the allatostatin-type of peptides is evident through a long period of evolution it cannot be assumed that all of their functions have also been conserved. Several different types of functions for the Leu-callatostatins of the blowfly are proposed in this study, but there is no evidence to suggest a role in the regulation of juvenile hormone synthesis and release.     

Serotonin and gastrin/cholecystokinin-like immunorcactive neurons in the larval retrocerebral complex of the blowfly Calliphora erythrocephala

Summary The presence and distribution of neurons immunoreactive against antibodies to serotonin (5-HT) and gastrin/cholecystokinin (gastrin/CCK) has been studied in the larval retrocerebral complex of the blowfly Calliphora erythrocephala, a composite structure which consists of the corpus cardiacum, the corpus allatum, the thoracic gland and a portion of the cephalic aorta. Immunoreactive material was found in all these elements except in the corpus allatum. Six to eight cell bodies in the corpus cardiacum and four to eight cell bodies in the thoracic gland were 5-HT immunoreactive (5-HTi). These 5-HTi cell bodies send processes to the neuropil of the corpus cardiacum and to neurohemal sites in the cephalic aorta, corpus cardiacum and ventral part of the thoracic gland. Six to eight cell bodies in the corpus cardiacum and four to six cell bodies in the thoracic gland reacted with antibodies against gastrin/CCK. These cell bodies send processes to the neuropil of the corpus cardiacum and to neurohemal sites in the corpus cardiacum and the cephalic aorta in a pattern resembling that of the 5-HTi fibers. Additional gastrin/CCK-like immunoreactive fibers were shown to come from the central nervous system via the two nervi corporis cardiaci. An electron-microscopical analysis was performed to analyze further the morphological features revealed by the light-microscopic immunocytochemical technique. This confirmed the existence of neurosecretory-like terminals among the gland cells of the thoracic glands and the existence of neurohemal sites in several regions of the larval retrocerebral complex. Some functional aspects of the retrocerebral complex are discussed on the basis of the presented data.     

Distribution and functional significance of Met-enkephalin-Arg6-Phe7- and Met-enkephalin-Arg6-Gly7-Leu8-like peptides in the blowfly Calliphora vomitoria

Summary Neuronal pathways immunoreactive to antisera against the extended-enkephalins, Met-enkephalin-Arg⁶-Phe⁷ (Met-7) and Met-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-8), have been identified in the brain of the blowfly Calliphora vomitoria. Co-localisation with other enkephalins in certain neurons suggests that a precursor similar to preproenkephalin A exists in insects and that differential enzymatic processing occurs as in vertebrates. Co-localisations of the extended-enkephalin-like peptides with other vertebrate-type peptides, including cholecystokinin and pancreatic polypeptide, also occur. The enkephalinergic pathways are specific, comprising a few groups of highly characteristic neurons and areas of neuropil. Of special interest is the finding that parts of the antennal chemosensory and the optic lobe visual systems contain Met-8 immunoreactive neurons. Within the median neurosecretory cell groups, some of the giant neurons show immunoreactivity to Met-8 and others to both Met-8 and Met-7. Fibres from these cells project to the corpus cardiacum and also to the suboesophageal ganglion, where arborisations occur in the tritocerebral neuropil. Co-localisation studies of these cells have shown that at certain terminals, one particular type of peptide is the dominant neuroregulator, whilst at other terminals, within the same cell, a different co-synthesised peptide predominates. Several groups of lateral neurosecretory cells show clearly defined enkephalinergic pathways, most of which have connections with the central body. The complex patterns of immunoreactivity seen in terminals in the different parts of the central body, suggest an important role for the enkephalin-like peptides in the integration of multimodal sensory inputs. The physiological functions of the extended-enkephalin-like peptides in the brain of Calliphora is still unknown, but the anatomical evidence suggests they may have a role similar to that in mammals, where they are thought to control aspects of feeding behaviour.     

Inhibition of pupariation and adult development in Calliphora vomitoria treated with ivermectin

Puparium formation and adult development in Calliphora vomitoria L. (Diptera: Calliphoridae) are seriously disrupted after treating final-stage larvae with ivermectin. Fifty % of larvae treated topically with 1.01 g ivermectin 2 days before anticipated pupariation fail to form puparia, while the same response requires 2.24 g given one day before pupariation. Permanent larvae so produced remain alive for up to 2 weeks. Of the larvae that pupariate, there is disruption of adult development, leading to larval-adult intermediates. Only 0.1 g ivermectin induces such disruption in 50% of the treated larvae.The effects on puparium formation and adult development are independent. Inhibition of pupariation can be explained in terms of a disturbance of neuromuscular events leading up to pupariation. The effect on adult development is more difficult to explain. This work shows that ivermectin has great potential in clarifying the sequence of events leading up to puparation in cyclorrhaphous Diptera.

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Résumé L'ivermectine est un insecticide qui a été développé dans le but d'éradiquer les parasites chez les bestiaux. Mais ce produit élimine également beaucoup d'insectes herbivores aussi bien en expériences de laboratoire qu'à l'extérieur. Cet insecticide est effectif dans les synapses entre les nerfs et les muscles, là où la substance chimique GABA est active. Cependant lorsque l'ivermectine est administrée aux larves de mouches à viande, Calliphora vomitoria L., elle produit des réactions différentes.En conditions normales, les larves, une fois qu'elles se sont nourries, errent sans but, deux ou trois jours avant de se transformer en nymphes. Si on administre une dose de 1,01 g d'ivermectine aux larves deux jours avant leur transformation anticipée, 50% d'entr'elles n'accompliront pas leur métamorphose. Elles resteront en permanence à l'état larvaire. Mais, si on leur administre une dose un jour seulement avant la transformation anticipée, il faut alors qu'elle de 2,24 g pour pouvoir empêcher la métamorphose de s'opérer. Ces doses ne tuent pas les larves. Si le traitement est donné le jour même de la transformation, celle ci s'opère alors normalement.Cependant un certain nombre de larves se métamorphosent en nymphes malgré leur traitement par ivermectine, mais beaucoup d'entr'elles n'atteindront jamais le stade adulte; elles resteront á un stade intermédiaire entre celui de nymphe et celui d'adulte. La dose nécéssaire pour empêcher le développement de l'adulte est de 0,1 g d'ivermectine. Il semble que les deux effets soient indépendants l'un de l'autre.L'effet produit par l'ivermectine sur la transformation de larve en nymphe peut s'expliquer par le fait que le traitement soit donné avant la métamorphose, affectant le comportement normal par une perte de mobilité mais non de paralysie. Par contre, l'effet sur le développement des adultes est plus difficile à expliquer. Néanmoins, l'ivermectine pourrait être employée très utilement dans les travaux de recherches sur la métamorphose des diptères.

     

Localisation of pancreatic polypeptide (PP)-like immunoreactive material in neurones of the brain of the blowfly,Calliphora erythrocephala (Diptera)

Summary The brain of the blowfly, Calliphora erythrocephala, has been studied by means of the peroxidase-antiperoxidase immunocytochemical method, with the use of antibodies to bovine pancreatic polypeptide (BPP). A number of immunoreactive neurones have been localised, some corresponding to neurones previously identified tentatively as neurosecretory. This finding is further evidence that biologically active peptides, previously considered to be vertebrate, also exist in invertebrates. It also supports the concept of their evolutionary origin in nervous tissue.     

Immunocytochemical demonstration of S-antigen (arrestin) in the brain of the blowfly Calliphora vicina

S-Antigen (arrestin)-immunoreaction can be considered as a marker for retinal and extraretinal photoreceptors in both vertebrate and invertebrate species. The present immunocytochemical study with the blowfly Calliphora vicina revealed S-antigen immunoreaction in retinal photoreceptors and various groups of neurons bilaterally distributed in the optic lobes and in the proto-, deuto- and tritocerebrum. S-Antigen-immunoreactive processes and terminal formations were found in the lower division of the central body complex and in the neuropil of the mushroom body. Also neuropil regions of the optic lobe, the lamina, medulla and lobula displayed S-antigen-immunoreactive fibers which were arranged in different patterns. These immunocytochemical data suggest that extraocular photoreceptors may be located in various parts of the blowfly brain. They provide a structural basis for further experiments which are needed to identify definitely these elements as extraretinal photoreceptors.     

Light- and electron-microscopic immunocytochemistry of peptidergic neurons innervating thoracico-abdominal neurohaemal areas in the blowfly

Summary Ventral thoracic neurosecretory cells (VTNCs) of the blowflies, Calliphora erythrocephala and C. vomitoria, innervating thoracic neuropil and the dorsal neural sheath of the thoracico-abdominal ganglion have been shown to be immunoreactive to a variety of mammalian peptide antisera. In the neural sheath the VTNC terminals form an extensive neurohaemal network that is especially dense over the abdominal ganglia. The same areas are invaded by separate, ut overlapping serotonin-immunoreactive (5-HT-IR) projections derived from neuronal cell bodies in the suboesophageal ganglion. Immunocytochemical studies with different antisera, applied to adjacent sections at the lightmicroscopic level, combined with extensive cross-absorption tests, suggest that the perikarya of the VTNCs contain co-localized peptides related to gastrin/cholecystokinin (CCK), bovine pancreatic polypeptide (PP), Met- and Leuenkephalin and Met-enk-Arg⁶-Phe⁷ (Met-enk-RF). Electron-microscopic immunogold-labeling shows that some of the terminals in the dorsal sheath react with several of the individual peptide antisera, whilst others with similar cytology are non-immunoreactive. In the same region, separate terminals with different cytological characteristics contain 5-HT-IR. Both 5-HT-IR and peptidergic terminals are localized outside the cellular perineurium beneath the acellular permeable sheath adjacent to the haemocoel. Hence, we propose that various bioactive substances may be released from thoracic neurosecretory neurons into the circulating haemolymph to act on peripheral targets. The same neurons may also interact by synaptic or modulatory action in the CNS in different neuropil regions of the thoracic ganglion.     

Localisation of sulfakinin neuronal pathways in the blowfly Calliphora vomitoria

The distribution of neurones immunoreactive to antisera raised against the undecapeptide C-terminal fragment of drosulfakinin II (DrmSKII), Asp-Gln-Phe-Asp-Asp-Tyr(SO₃H)-Gly-His-Met-Arg-Phe-NH₂, has been studied in the blowfly Calliphora vomitoria. Antisera were preabsorbed with combinations of the parent antigen, the tetrapeptide Phe-Met-Arg-Phe-NH₂ and cholecystokinin, the vertebrate sulfated octapeptide (CCK-8), Asp-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-Phe-NH₂, in order to ensure specificity for the sulfakinin peptides of C. vomitoria (the nonapeptide callisulfakinin I is identical to drosulfakinin I and callisulfakinin II differs from DrmSK II only by the presence of -Glu³-Glu⁴- in place of -Asp³-Asp⁴-). Only four pairs of sulfakinin-immunoreactive neurones have been visualised in the entire nervous system. These occur in the brain: two pairs of cells situated medially in the caudo-dorsal region close to the roots of the ocellar nerve and two other pairs at the same level but positioned more laterally. Despite the small number of sulfakinin-immunoreactive cells, there are extensive projections to many areas of neuropile in the brain and the thoracic ganglion. The pathway of the medial sulfakinin cells extends into each of the three thoracic ganglia and a metameric arrangement of sulfakinin neuronal projections is also seen in the abdominal ganglia. Neither the dorsal neural sheath of the thoracic ganglion, nor the abdominal nerves contain sulfakinin-immunoreactive material. These observations suggest that the sulfakinins of the blowfly function as neurotransmitters or neuromodulators. They do not appear to have a direct role in gut physiology, as has been shown by in vitro bioassays for the sulfakinins of orthopterans and blattodeans. In addition to the neurones that display specific sulfakinin immunoreactivity, other cells within the brain and thoracic ganglion are immunoreactive to cholecystokinin/gastrin antisera. There are, therefore, at least two types of dipteran neuropeptides with amino acid sequences that are similar to the vertebrate molecules cholecystokinin and gastrin.     

The haemocytes of Calliphora erythrocephala (Meig.) (Diptera)

Summary The haemocytes of larvae and young pupae of Calliphora erythrocephala are studied by phase contrast and electron microscopy and three cell lineages are distinguished: plasmatocytes, thrombocytoids and oenocytoids. The plasmatocytes show important modifications during larval development and at the time of histolysis, which are described and discussed in relation to the function of these cells in the physiology of Calliphora. The thrombocytoids, haemocytes which had not been recorded so far, are characterized by a strong tendency to fragmentation, this process leading to the formation of the anucleated cytoplasmic fragments and the naked nuclei referred to by earlier authors. The ability of the cell fragments, which retain normal cytological characteristics, to agglutinate and form intricate networks, is discussed in relation to haemostasis in Calliphora.The ultrastructural study of the haemocyte accumulations in the vicinity of the posterior part of the dorsal vessel reveals the basic organization of haemocytopoetic tissue, as described recently in orthopteran insects. The functional importance of this tissue in the production of haemocytes is demonstrated by X-irradiation and ligation experiments in larvae of Calliphora.

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Résumé L'étude en microscopie en contraste de phase et au microscope électronique permet de distinguer dans le sang circulant de larves et de jeunes pupes de Calliphora erythrocephala trois lignées cellulaires: les plasmatocytes, les thrombocytoïdes et les oenocytoïdes. Les plasmatocytes, numériquement les plus importants, présentent au cours du développement larvaire et chez les pupes des modifications considérables, qui sont décrites et discutées en rapport avec la fonction évidente de ces hémocytes chez Calliphora. Les thrombocytoïdes, inconnus dans la littérature, se caractérisent par une forte tendance à la fragmentation, qui aboutit à la formation des «fragments cytoplasmiques anucléés» et des «noyaux nus» signalés par divers auteurs. Les phénomènes d'agglutination des «fragments cytoplasmiques anucléés», dont les caractères cytologiques restent normaux, sont discutés en rapport avec le problème de l'hémostase chez cet insecte.L'étude ultrastructurale des accumulations hémocytaires autour du vaisseau dorsal dans la partie postérieure de l'abdomen montre une organisation de base comparable à celle décrite dans les organes hématopoïétiques des Insectes Orthoptères. L'importance fonctionelle de ce tissu hématopoïétique de Calliphora dans la production des hémocytes au cours de la vie larvaire est démontrée par des irradiations de ce tissu et par des ligatures de la partie postérieure de l'abdomen.

     

Cholinergic neurons in the lamina ganglionaris of the blowfly Calliphora erythrocephala

Summary The distribution of putative cholinergic neurons in the lamina of the blowfly Calliphora erythrocephala was studied by immunocytochemical and histochemical methods. Three different antibodies directed against the AChsynthesizing enzyme, choline acetyltransferase (ChAT), revealed a cholinergic population of fibres running parallel to the laminar cartridges, which have branch-like structures at the distal lamina border. Cell bodies in the chiasma next to the lamina border were also labelled by the anti-ChAT antibodies. Monopolar cell bodies in the nuclear layer were faintly labelled. The distribution of the acetylcholine hydrolyzing enzyme, acetylcholine esterase (AChE), was revealed by histochemical staining and was similar to the ChAT immunocytochemistry. The arrangement of ChAT positive fibres in transverse and longitudinal sections and the distribution of AChE stained fibres indicate that the amacrine cells of the lamina are cholinergic cells.We dedicate this work to Prof. F. Zettler who passed away in fall 1988: K.-H. Datum, I. Rambold     

Ultrastructure of the myocardial cell and its membrane systems in the adult fly Calliphora erythrocephala (Insecta: Diptera)

Summary Calliphora erythrocephala has cross-striated cardiac muscle cells with A, I and Z-bands. The diameters of the myosin and actin filaments are 200–250 Å and 85 Å respectively and the length of the myosin filaments (A-band) is approximately 1.5 . Usually 8–10 actin filaments surround each myosin filament.The myocardial cells show a well-developed membrane system and interior couplings. A perforated sheet of SR envelopes the myofibrils at the A-band, dilates into flattened cisternae at both A-I band levels before it merges into a three-dimensional net-work between the actin filaments of the I-bands and between the dense bodies of the discontinuous Z-discs. The T-system consists of broad flattened tubules running between the myofibrils at the A-I band levels forming dyads with the SR-cisternae. Longitudinal connections between the transverse (T-) tubules often occur.It is suggested that this well-developed SR may be an adaptation to facilitate a rapid contraction/relaxation frequency by an effective Ca²⁺ uptake.     

Postembryonic development of serotonin-immunoreactive neurons in the central nervous system of the blowfly,Calliphora erythrocephala

Summary The postembryonic development of serotonin-immunoreactive (5-HTi) neurons was studied in the optic lobe of the blowfly. In the adult fly there are 24 5-HTi neurons invading each optic lobe. The perikarya of two of these neurons are situated in the dorso-caudal part of the protocerebrum (LBO-5HT neurons; large bilateral optic lobe 5-HTi neurons). The cell bodies of the remaining 22 neurons are located anteriorly at the medial base of the medulla (2 innervating the lobula, LO-5HT neurons; and 20 neurons innervating the medulla, ME-5HT neurons). The two central neurons (LBO-5HT neurons) are derived from metamorphosing larval neurons, while the ME- and LO-5HT neurons are imaginai optic lobe neurons differentiating during pupal development.The 5-HTi neurons of the optic lobe seem to have different ancestors. The LBO-5HT neurons are probably derived from segmental protocerebral neuroblasts, whereas the ME-and LO-5HT neurons are most likely derived from the inner optic anlage. The first 5-HTi fibers to reach the imaginal optic lobes are seen in the late third instar larva and are derived from the LBO-5HT neurons. The first ME- and LO-5HT neurons become immunoreactive at 24 h (10%) pupal development. At about 96 h (40%) of pupal development all the 5-HTi neurons of the optic lobes have differentiated and attained their basic adult morphology. The further development mainly entails increase in volume of arborizations and number of finer processes. The differentiation and outgrowth of 5-HTi processes follows that of, e.g., columnar neurons in the optic lobe neuropils. Hence, 5-HTi processes invade neuropil relatively late in the differentiation of the optic lobe.     

Distribution and functional significance of Met-enkephalin-Arg6-Phe7-and Met-enkephalin-Arg6-Gly7-Leu8-like peptides in the blowflyCalliphora vomitoria

Summary Neuronal pathways in the retrocerebral complex and thoracico-abdominal ganglionic mass of the blowflyCalliphora vomitoria have been identified immunocytochemically with antisera against the extended-enkephalins, Met-enkephalin-Arg⁶-Phe⁷ (Met-7) and Met-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-8). Neurons of the hypocerebral ganglion, immunoreactive to Met-8, have axons in the crop duct nerve and terminals in muscles of the crop and its duct. Certain neurons of the hypocerebral ganglion are also immunoreactive to Met-7, and axons from these cells innervate the heart. Met-8 immunoreactive nerve terminals invest the cells of the corpus allatum. The source of this material is believed to ve a single pair of lateral neurosecretory cells in the brain. There is no Met-7 immunoreactive material in the corpus allatum. In the corpus cardiacum neither Met-7 nor Met-8 immunoreactivity is present in the cells. However, in the neuropil of the gland certain fibres, with their origins elsewhere, do contain Met-8 immunoreactivity. The most prominent neurons in the thoracic ganglion are the Met-7 immunoreactive ventral thoracic neurosecretory cells, axons from which project to neurohaemal areas in the dorsal neural sheath and also, via the ventral connective, to the brain. Co-localisation studies show that the perikarya of these cells are immunoreactive to antisera raised against several vertebrate-type peptides, such as Met-7, gastrin/cholecystokinin and pancreatic polypeptide. However, their axons and terminals show varying amounts of the peptides, suggesting differential transport and utilisation. Only a few cells in the thoracic ganglion are immunoreactive to Met-8 antisera. These lie close to the nerve bundles suppling the legs. In the abdominal ganglion, Met-8 immunoreactive neurons project to the muscles of the hindgut. This study suggests that the extended enkephalin-like peptides ofCalliphora may have a variety of different roles: as neurotransmitter or neuromodulator substances; in the direct innervation of effector organs; and as neurohormones.     

Ocellar interneurones in the blowfly Calliphora erythrocephala: Morphology and central projections

Summary The morphology and central projections of first-order ocellar interneurones were analysed in the blowfly, Calliphora erythrocephala after cobalt and horseradish-peroxidase labelling. Three classes of interneurones can be distinguished on the basis of axon diameters: large, medium and small neurones. In total there are 12 large, 10 medium and an unknown number of small interneurones. These interneurones connect the fused first-order ocellar neuropil (underlying the three ocelli) with various areas of the central nervous system. The large neurones terminate in three subregions of the posterior slope (ocellar foci); the medium neurones arborise in several regions of the lateral protocerebrum, in the posterior slope, the lobula, the ventral medulla, and in the pro- and mesothoracic ganglia. The thin fibers arborise in all the above regions (except in the thoracic ganglia), and in addition in the neuropil dorsal to the oesophagus and antero-ventral to posterior slope (tritocerebrum). The anatomy of the ocellar pathway in C. erythrocephala is compared with those in other studied insects. Possible interactions between ocellar interneurones and other pathways are discussed.     

Immunofluorescent localization of insulin-like material in the median neurosecretory cells of the blowfly,Calliphora vomitoria (Diptera)

Summary Immunocytochemical staining has shown that the median neurosecretory cells (MNC) of the brain of the blowfly, Calliphora vomitoria, contain an insulin-like material which cross reacts with antibodies to bovine insulin. There are 24–26 paraldehyde fuchsin-positive MNC of which only 6–8 show the specific insulin-like immunoreactivity.Dr. M.C. Thorndyke, Department of Zoology, Bedford College, London University, kindly provided us with fluorescence facilities, and we are also grateful to him for helpful discussions on the immunofluorescence procedure     

Incorporation of calliphorin into the cuticle of the developing blowfly,Calliphora vicina

Summary The stage-specific appearance of calliphorin in cuticles of Calliphora vicina was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The fate of the protein, injected into last instar larvae, was pursued by autoradiography of histological sections. Fractionation of sclerotized pupal cuticle in buffer-soluble, urea-soluble and NaOH-soluble fractions shows that calliphorin forms covalent and non-covalent links with other cuticle components. Calliphorin traverses the epidermal cells and enters the cuticle in an undegraded state and appears to be an important constituent of the sclerotizing system.