Immunocytochemical localization of peptidergic neurons and neurosecretory cells in the neuro-endocrine system of the Colorado potato beetle with antisera to vertebrate regulatory peptides

Summary A large number of antisera to regulatory vertebrate peptides was tested immunocytochemically on the nervous system of the Colorado potato beetle to further characterize the peptidergic cells of the neuro-endocrine system and to reveal cells participating in endocrine control mechanisms. Neurons, neurosecretory cells, axons and axon terminals were revealed by antisera to ACTH, gastrin, CCK, -endorphin, -endorphin, 1-MSH, insulin, motilin, human calcitonin, growth hormone, somatostatin, CRF, ovine prolactin and rat prolactin. Together with previously described results these findings demonstrate that at least 19 different peptidergic cell types are present in the Colorado potato beetle. Several of these cell types are identical with the known neurosecretory cells, while others have not been identified before.The functions of the immunoreactive neurons are as yet unclear, although in two cases the localization of these cells gives some clues. Thus the lateral neurosecretory cells, which are immunoreactive with antisera to -endorphin and ovine prolactin, may regulate corpus allatum activity, whereas a CRF immunoreactive substance seems to be used as neurotransmitter by antennal receptors. These immunocytochemical findings do not imply that the immunoreactive substances are evolutionarily related to the vertebrate peptides to which the antisera were raised. It is postulated that if the part of the substance recognized by a certain antiserum is functionally important for the insect, which should be so if the insect peptide is evolutionarily related to its vertebrate homologue, the antiserum should reveal homologous cells in different insect species. The consequence of this hypothesis is, that if an antiserum does not reveal homologous neurons in different insect species, the immunologically demonstrated substance is probably of little physiological importance, and will not be related evolutionarily to the vertebrate analogue. The positive immunocytochemical results in the Colorado potato beetle are discussed in relation to these considerations.     

A proctolin-like peptide and its immunocytochemical localization in the Colorado potato beetle,Leptinotarsa decemlineata

Summary Evidence is presented that neurons in the adult Colorado potato beetle contain a proctolin-like substance. By use of immunocytochemical methods the location of immunoreactive neurons in the central and stomatogastric nervous systems is described. No such neurons were found in the proto- and deutocerebrum or optic lobe. Few immunoreactive neurons are present in the tritocerebrum and numerous proctolin-immunoreactive neurons occur in all ventral ganglia and in the frontal ganglion. Two groups of neurosecretory cells in the suboesophageal ganglion contain a proctolin-immunoreactive substance. In these cells this material is co-localized with a bovine pancreatic polypeptide/FMRF amide-like substance and with a vasopressin/vasotocin/oxytocin-like substance. Proctolin-immunoreactive axon terminals were found on the musculature of the fore- and hindgut and of the vas deferens, and on some segmental muscles. Furthermore, proctolin-immunoreactive neurosecretory axon terminals were found in the corpus cardiacum. The proctolin-like substance may therefore function both as a neurotransmitter/neuromodulator and as a neurohormone. The presence of a proctolin-like substance was also demonstrated with a sensitive bioassay. On fractionation of extracts of the nervous systems of Leptinotarsa decemlineata with high performance liquid chromatography most of the proctolin-like bioactive material comigrated with authentic proctolin. This shows that a proctolin-like substance in this insect is very similar to, if not identical with, the known pentapeptide proctolin.     

Immunocytochemical localization of peptidergic cells in the neuro-endocrine system of the Colorado potato beetle,Leptinotarsa decemlineata,with antisera against vasopressin,vasotocin and oxytocin

Summary Antisera against vasopressin, vasotocin, oxytocin, neurophysin-1 and neurophysin-2 were used to investigate immunocytochemically the presence of neurons containing substances antigenically related to these peptides in the nervous system of the Colorado potato beetle. Ten different antisera were used, four against vasopressin, three against oxytocin and one against vasotocin, neurophysin-1, and neurophysin-2. Immunoreactivity was shown by all antisera except those against the neurophysins. The vasopressin antisera all gave different results. One antiserum revealed only a single neuron pair, whereas others revealed in addition one or two other different cell groups. The oxytocin antisera likewise revealed different neurons. The fixation procedure influenced the outcome of the immunocytochemical reaction. Immunoreactivity as revealed by vasopressin, vasotocin and oxytocin antisera is often co-localized in the same neurons; solid phase adsorptions showed that this is due to cross-reactivity of the antisera. Some of the immunoreactive neurons are identical to those recently described to contain a bovine pancreatic polypeptide/FMRFamide-like peptide. This co-localization is probably not due to a cross-reaction. These findings indicate the presence of several vasopressin-like and oxytocin-like substances which in the Colorado potato beetle all have a different degree of immunocytochemical resemblance to vasopressin and oxytocin.     

Immunocytochemical localization of peptidergic cells in the neuro-endocrine system of the Colorado potato beetle, Leptinotarsa decemlineata, with antisera against vasopressin, vasotocin and oxytocin

Antisera against vasopressin, vasotocin, oxytocin, neurophysin-1 and neurophysin-2 were used to investigate immunocytochemically the presence of neurons containing substances antigenically related to these peptides in the nervous system of the Colorado potato beetle. Ten different antisera were used, four against vasopressin, three against oxytocin and one against vasotocin, neurophysin-1, and neurophysin-2. Immunoreactivity was shown by all antisera except those against the neurophysins. The vasopressin antisera all gave different results. One antiserum revealed only a single neuron pair, whereas others revealed in addition one or two other different cell groups. The oxytocin antisera likewise revealed different neurons. The fixation procedure influenced the outcome of the immunocytochemical reaction. Immunoreactivity as revealed by vasopressin, vasotocin and oxytocin antisera is often co-localized in the same neurons; solid phase adsorptions showed that this is due to cross-reactivity of the antisera. Some of the immunoreactive neurons are identical to those recently described to contain a bovine pancreatic polypeptide/FMRFamide-like peptide. This co-localization is probably not due to a cross-reaction. These findings indicate the presence of several vasopressin-like and oxytocin-like substances which in the Colorado potato beetle all have a different degree of immunocytochemical resemblance to vasopressin and oxytocin.     

FMRFamide- and pancreatic polypeptide-like immunoreactivity of endocrine cells in the midgut of a mosquito

Immunocytochemical surveys of midguts from female mosquitoes, Aedes aegypti, reveal that half of the estimated 500 endocrine cells in a midgut contain a substance recognized by antisera to bovine pancreatic polypeptide and a molluscan peptide, FMRFamide (phenylalanine-methionine-arginine-phenylalanine-amide). With light microscopy the cells resemble an endocrine type because of their basal position in the epithelium, conical shape, and, in some instances, apical extensions to the lumen. At the ultrastructural level, the immunoreactive substance is contained specifically within the secretory granules of such cells. Immunoreactive cells are distributed exclusively in the midgut region where blood is stored, and ingestion of vertebrate blood reduces the number of such cells and the intensity of reaction in others. These two facts suggest that a blood meal stimulates release of the immunoreactive substance from the cells. Since the immunocytochemical localization is supplemented by a demonstrated secretory response, the cells are considered to be peptidergic endocrine cells.     

Immunocytochemical localization of neurons in the nervous system of the Colorado potato beetle with antisera against FMRFamide and bovine pancreatic polypeptide

Summary Particular neurons in the nervous system of the Colorado potato beetle, Leptinotarsa decemlineata, are recognized by antisera against bovine pancreatic polypeptide and FMRFamide. Both antisera react with the same neurons. Solid phase absorptions showed that antiserum against bovine pancreatic polypeptide cross-reacts with FMRFamide, whereas antiserum against FMRFamide cross-reacts with bovine pancreatic polypeptide. Some of the immunoreactive neurons have axons branching extensively within the neuropile, which suggests that the peptide is used as transmitter. In the corpus cardiacum, a neurohaemal organ in insects, numerous immunoreactive axon terminals are present. Here, the peptide material is presumably released as a hormone.     

Immunocytochemical localization of a diuretic hormone of the beetle Tenebrio molitor,Tenmo-DH(37), in nervous system and midgut

Although the mealworm Tenebrio molitor inhabits very dry environments, it has at least two diuretic peptides, which increase fluid secretion by the free portions of the Malpighian tubules. Unlike other insect corticotropin-releasing factor (CRF)-related peptides isolated to date, these are non-amidated peptides. The immunocytochemical localization of Tenmo-DH(37) was investigated using antisera raised against this hormone. Immunoreactive neurosecretory cells were found in the brain and abdominal ganglia with immunoreactive processes projecting to the peripheral nervous system. Intense staining of the neurohaemal release site, the corpora cardiaca, was observed. In addition, neurosecretory cells immunoreactive to Tenmo-DH(37) were found in the posterior midgut and a network of immunoreactive nerve processes extended over the surface of the midgut. Tenmo-DH(37) is widely distributed and its staining pattern resembles that found for other, amidated CRF-related diuretic peptides.     

Immunohistological demonstration of a CRF-like material in the central nervous system of the annelid Dendrobaena

Summary CSF is the most recently isolated and synthetized hypothalamic releasing factor in vertebrates. The use of an antiserum raised against CRF made it possible to reveal, in an oligochaetous annelid, immunoreactive cells in the suboesophageal ganglion and in all ganglia of the ventral nerve cord. Ten cells were numbered in each ganglion and each presents a pattern which is identical from one somite to the next. It thus appears that in the case of CRF as well as other vertebrate neuropeptides, there exist immunologically related neurosecretory products in the central nervous systems of some invertebrates.     

Immunocytochemical demonstration of peptidergic neurons in the central and peripheral nervous systems of the flatworm Microstomum lineare with antiserum to FMRF-amide

Summary The central nervous system (CNS) and the peripheral nervous system (PNS) of the flatworm Microstomum lineare were studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method, with the use of antisera to the molluscan cardioactive peptide FMRF-amide. FMRF-amide immunoreactive perikarya and nerve fibres are observed in the CNS and the PNS. In the CNS, immunoreactive perikarya and nerve fibres occur in the brain, in the epithelial lining and the mesenchymal surroundings of the ciliated pits, and positive fibres in the longitudinal nerve cords. In the PNS, immunoreactive fibre bundles with variocosities occur in the pharyngeal nerve ring, in symmetrical groups of perikarya on each side of the pharynx, and in the mouth area. Positive perikarya and meandering nerve fibres appear in the intestinal wall. A few immunoreactive cells and short nerve processes are observed at the male copulatory organ and on both sides of the vagina. Some immunoreactive peptidergic cells do not correspond to cells previously identified by histological techniques for neurosecretory cells. The distribution of immunoreactivity suggests that the FMRF-amide-like substance in CNS and PNS in this worm has roles similar to those of the brain-gut peptides in vertebrates. The status of FMRF-amide-like peptides as representatives of an evolutionarily old family of peptides is confirmed by the positive immunoreaction to anti-FMRF-amide in this primitive microturbellarian.     

Immunohistochemical investigation of urotensins in the caudal spinal cord of four species of elasmobranchs and the lamprey,Lampetra japonica

Summary In the four species of elasmobranchs examined (Triakis scyllia, Heterodontus japonicus, Scyliorhinus torazame, Dasyatis akajei), all identifiable caudal neurosecretory cells and their corresponding neurohemal areas showed urotensin II (UII)-immunoreactivity with varied intensity. To localize urotensin I (UI) in the caudal neurosecretory system of the dogfish, Triakis scyllia, h-CRF (1–20) antiserum that cross-reacts with UI was used in place of UI antiserum. CRF/UI-immunoreactivity was demonstrated in the neurosecretory cells and neurohemal areas. A considerable number of neurons showed both UII- and CRF/UI-immunoreactivities, suggesting that UII and UI are produced in the same neurosecretory cells. However, some neurons exhibited UII-immunoreactivity, but no CRF/UI-immunoreactivity. Cells immunoreactive only to CRF antiserum were not detected. At least two populations of neurons exist in the dogfish caudal neurosecretory system: (i) cells immunoreactive for both CRF/UI and UII, and (ii) cells immunoreactive for UII. The dorsal cells of the lamprey, Lampetra japonica, did not react with either UII or CRF antiserum.     

Monoclonal antibody immunohistochemistry in the American cockroach identifies a novel neuropeptidergic system similar to,but distinct from this insect's (FM)RFamide-like system

A monoclonal antibody to peptidergic neurons in the neuroendocrine system of the Colorado potato beetle immunohistochemically labels neuropeptide-like substances throughout the cephalic neurosecretory system of the American cockroach, Periplaneta americana. The dictyopteran antigen shows a histological distribution similar to that of the neuropeptide-like material which we have described earlier using (FM)RFamide specific antibodies. It was conclusively demonstrated, particularly by means of a convenient double labelling procedure, that the (FM)RFamide- and the coleopteran neuropeptide-like antigens are not localized in the same neuronal structures, although both neurochemicals are in close vicinity to one another. The relative abundance of the immunoreactive products in neurons throughout the cockroach nervous system and retrocerebral complex, in addition to its apparently homologous distribution in species of different insect orders, suggests an important role of this material in insect neuro(endocrine)-physiology.     

A double sequential immunofluorescence method demonstrating the co-localization of urotensins I and II in the caudal neurosecretory system of the teleost,Gillichthys mirabilis

Summary A double immunofluorescence method was devised to localize simultaneously urotensin-I (UI) and -II (UII) immunoreactivities in the caudal neurosecretory system of the goby, Gillichthys mirabilis. In a sequential fashion, sections of the posterior spinal cord and urophysis were treated with antiserum to corticotropin-releasing factor (CRF) that cross-reacts with UI, fluorescein-conjugated sheep anti-rabbit IgG, biotinylated anti-UII and rhodamine-conjugated avidin. UI and UII immunoreactivities appeared to coexist in some neurons and in most fibers and urophysial tissue; the remainder of the fibers and urophysis and the majority of neurons were immunoreactive for CRF/ UI only. No convincing evidence of immunoreactivity for UII only was found. A few nonreactive cells were seen, but these may not be neurosecretory neurons. The two immunoreactive cell types were not segregated topographically, and the intensity of perikaryal immunofluorescence for CRF/UI was variable. To explain these results a hypothesis that all caudal neurosecretory cells may synthesize both UI and UII and that immunoreactive differences may reflect different states of cellular activity, is suggested. This sequential double immunofluorescence method offers several advantages over other techniques and is especially useful for co-localization studies when primary antisera from different species are not available.     

Localization of CRF immunoreactivity in the central nervous system of three vertebrate and one insect species

Summary By use of a specific antiserum against synthetic ovine corticotropin-releasing factor (CRF) in the peroxidase-antiperoxidase (PAP) immunocytochemical procedure (Vandesande and Dierickx 1976), CRF-like antigenic determinants were demonstrated in the central nervous system of a human fetus, the Wistar rat, the frog Rana ridibunda, and the American cockroach Periplaneta americana. The immunoreactive CRF-producing cells occur mainly in the nucleus paraventricularis of the rat, while in Rana ridibunda these cells occur in the nucleus praeopticus. Immunoreactive CRF-containing fibres were also visualized. Very clear CRF-immunoreactive products were observed in the brain as well as the corpora cardiaca (CC) and corpora allata (CA) of the cockroach Periplaneta americana. ACTH-immunoreactivity was also demonstrated in the brain-CC-CA complex of this insect. Double immunohistochemical staining (Vandesande 1983) also revealed that both the CRFand ACTH-like substances occur in different neurosecretory neurons and nerve fibres. These results suggest that the antigenic determinants of CRF are very similar in vertebrates and insects bespeaking their very long evolutionary history.     

Immunocytochemical demonstration of proopiomelanocortin- and other opioid-related substances and a CRF-like peptide in the gut of the american cockroach, Periplaneta americana L

Using the peroxidase-antiperoxidase technique, we showed the presence of peptides which are immunologically resembling mammalian corticotropin releasing hormone (CRF)-, adrenocorticotropic hormone (ACTH)-, beta-endorphin (beta-END)-, alpha-melanocyte stimulating hormone (alpha-MSH)-, methionine-enkephalin (met-ENK)- and leucine enkephalin (leu-ENK)- like immunoreactivity in hundreds to thousands of endocrine cells and nerve fibers in the midgut of the American cockroach Periplaneta americana. In the cockroach hindgut no immunoreactive cell bodies could be observed, although nerve fibers were clearly noticed to be recognized by antisera to CRF, ACTH1-24, ACTH11-24 and beta-END. Nothing is exactly known as to the function(s) of the demonstrated materials, but one can speculate that these numerous immunoreactive cells, might have important paracrine and/or endocrine functions in the insect physiology.     

Comparative immunocytochemical study of release sites of insulin,glucagon and AKH-like products in Locusta migratoria,Periplaneta americana,and Carausius morosus

Summary Insulin, glucagon and adipokinetic hormone antisera were applied to the corpora cardiaca, perisympathetic organs, neurohemal areas and peripheral neurosecretory cells of three insect species, the locust Locusta migratoria, the cockroach Periplaneta americana, and the stick insect Carausius morosus. The neurohemal part of the corpora cardiaca was shown to be immunoreactive to both insulin and glucagon antisera while the glandular cells reacted to adipokinetic hormone antisera. The perisympathetic organs seem to be devoid of these three substances, but certain peripheral neurohemal areas contained AKH and glucagon immunoreactive products. The latter were found to originate in the peripheral neurosecretory cells.     

Immunocytochemical observations of corticotropin-releasing-factor-containing neurons in the rat hypothalamus with special reference to neuronal communication

Synapses between neurons with corticotropin-releasing-factor-(CRF)-like immunoreactivities and other immunonegative neurons in the hypothalamus of colchicine-treated rats, especially in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) were observed by immunocytochemistry using CRF antiserum. The immunoreactive nerve cell bodies and fibers were numerous in both the PVN and the SON. The CRF-containing neurons had synaptic contacts with immunonegative axon terminals containing a large number of clear synaptic vesicles alone or combined with a few dense-cored vesicles. We also found CRF-like immunoreactive axon terminals making synaptic contacts with other immunonegative neuronal cell bodies and fibers. And since some postsynaptic immunonegative neurons contained many large neurosecretory granules, they are considered to be magnocellular neurosecretory cells. These findings suggest that CRF functions as a neurotransmitter and/or modulator in addition to its function as a hormone.     

Isolation and immunochemical characterization of neuropeptides from the midgut of the Colorado potato beetleLeptinotarsa decemlineata

The midgut of the Colorado potato beetle showed endocrine cells immunopositive to monoclonals like MAC-18, MAC-3 and polyclonals to FMRF-amide and AKH-241. Extraction and HPLC-fractionation of the midgut extracts after partial purification and characterization showed immunopositive reaction to the monoclonals and also showed myotropic activity stimulating the potato beetle gut. Molecular mass of the two active peptides isolated were 22 and 26 kDa respectively. Both these peptides could be immunocytochemically demonstrated in the brain neurosecretory cells of the red cotton bug,Dysdercus cingulatus and the castor semilooper,Achaea janata as well.     

Immunocytochemical localization of prolactin-like antigenic determinants in the neuroendocrine system of the honeybee (Apis mellifica)

In the brain of the adult worker bee (Apis mellifica) prolactin-like (PRL) immunoreactive cells were localized in the lateral neurosecretory cell region and the subesophageal ganglion by means of the PAP procedure. These cells emit nerve fibers which pass through the neuropile of the brain to the corpora cardiaca where a great number of immunoreactive axon terminals is present. Test with antisera against rat pituitary prolactin and human luteinizing hormone were negative. These results indicate that hPRL material is produced in neurosecretory cells of the bee brain and transferred via axons to the corpora cardiaca for storage and subsequent release into haemolymph.     

Neural control of the corpus allatum in the Colorado potato beetle, Leptinotarsa decemlineata: An electron microscope study utilizing the in vitro tannic acid Ringer incubation method

The release of material from neurosecretory synapses in the corpora allata of the Colorado potato beetle, Leptinotarsa decemlineata was visualised by an electron microscope procedure which involved tissue incubation in tannic acid. Using morphometry, the frequency of exocytosis phenomena was quantified in beetles kept under two different photoregimes. The number of exocytosis phenomena in the neurosecretory synapses in the corpora allata of beetles kept under short days was significantly higher than that of beetles reared under long-day conditions. In addition, the corpus allatum gland cells appeared to be more richly innervated by neurosecretory synapses under short-day than under long-day conditions. Previous studies using the in vitro radiochemical assay showed that the corpus allatum activity of short-day beetles is at least partly restrained by neurally mediated factors. The present morphological data strongly imply that this corpus allatum inhibitory substance is released from the neurosecretory synapses.     

Resistance potential of colorado potato beetle (Coleoptera: Chrysomelidae) to novaluron

Novaluron (Rimon 10 EC), a novel insect growth regulator, could play an important role in future management programs for Colorado potato beetle, Leptinotarsa decemlineata (Say). Studies were conducted to determine the potential of Colorado potato beetle to develop resistance to novaluron before its widespread use in Colorado potato beetle management. Second instars of an imidacloprid-resistant Colorado potato beetle strain exhibited reduced susceptibility (2.5-fold) to novaluron. The toxicity of novaluron to this strain was synergized by S,S,S-tributyl phosphorotrithioate (DEF) but not by piperonyl butoxide (PBO), suggesting that esterase-based detoxification mechanisms were responsible for novaluron resistance. Bioassays with treated potato foliage found that a single low- or medium-rate novaluron application was highly persistent under field conditions, resulting in up to 85% mortality of second instars 5 wk after treatment. Thus, intense selection pressure for novaluron-resistant Colorado potato beetle may continue long after population densities have been reduced below an economic threshold level. In a national survey, the susceptibility of second instars to a novaluron diagnostic dose was determined for 27 different field populations collected from six Canadian provinces in summer 2003. Despite no previous exposure to novaluron, mortalities at the diagnostic dose ranged from 55 to 100%. Although novaluron has several characteristics that should delay resistance development in insect pests, these results highlight the need for judicious use of the compound in management of Colorado potato beetle.