Distinct Projections of Two Populations of Olfactory Receptor Axons in the Antennal Lobe of the Sphinx Moth Manduca sexta

The central projections of olfactory receptor cells associatedwith two distinct types of antennal sensilla in the sphinx mothManduca sexta were revealed by anterograde staining. In bothsexes, receptor axons that arise from sexually isomorphic, type-IItrichoid sensilla (and possibly some basiconic sensilla) projectto the spheroidal glomeruli in the ipsilateral antennal lobe.Each axon terminates in one glomerulus. Axons from a limitedregion of the antenna project to glomeruli throughout the lobe,arguing against strict topographic mapping of antennal receptorcells onto the array of glomeruli. Axons of sex-pheromone-selectivereceptor cells in the male-specific type-I trichoid sensillaproject exclusively to the sexually dimorphic macroglomerularcomplex (MGC). Axons from sensilla on the dorsal surface ofthe antenna are biased toward the medial MGC and those fromventral sensilla, toward the lateral MGC. Some receptor-cellaxons branch before reaching the MGC, but their terminals arealways confined to one of the two main glomerular divisionsof the MGC, the cumulus and toroid. These findings confirm thatprimary-afferent information about pheromonal and non-pheromonalodors is segregated in the antennal lobe and suggest that thereis a functional correspondence between particular olfactoryreceptor cells and specific glomeruli. Chem. Senses 20: 313–323,1995.     

Nitric Oxide Upregulates Proteasomal Protein Degradation in Neurons

Nitric oxide (NO) is involved in many neuronal functions such as neuromodulation and intracellular signaling. Recent studies have demonstrated that nitric oxide is involved in regulation of proteasomal protein degradation. However, its role in neuronal protein degradation still remains unclear. In our study, we investigated the influence of endogenous nitric oxide production in this process. We have shown that nitric oxide synthase blockade prevents decline of the Ub^G76V-GFP fluorescence (GFP-based proteasomal protein degradation reporter) in neuronal processes of the cultured hippocampal neurons. It suggests that nitric oxide may regulate ubiquitin-dependent proteasomal protein degradation in neurons. Also, we have confirmed that the NO synthesis blockade alone significantly impairs long-term potentiation, and demonstrated for the first time that simultaneous blockade of the NO and proteins synthesis leads to the long-term potentiation amplitude rescue to the control values. Obtained results suggest that nitric oxide is involved in the protein degradation in proteasomes in physiological conditions.     

Antennal receptive fields of pheromone-responsive projection neurons in the antennal lobes of the male sphinx moth Manduca sexta

Stimulation of the antenna of the male moth, Manduca sexta, with a key component of the female's sex pheromone and a mimic of the second key component evokes responses in projection neurons in the sexually dimorphic macroglomerular complex of the antennal lobe. Using intracellular recording and staining techniques, we studied the antennal receptive fields of 149 such projection neurons. An antennal flagellum was stimulated in six regions along its proximo-distal axis with one or both of the pheromone-related compounds while activity was recorded in projection neurons. These neurons fell mainly into two groups, based on their responses to the two-component blend: neurons with broad receptive fields that were excited when any region of the flagellum was stimulated, and neurons selectively excited by stimulation of the proximal region of the flagellum. Projection neurons that were depolarized by stimulation of one antennal region were not inhibited by stimulation of other regions, suggesting absence of antennotopic center-surround organization. In most projection neurons, the receptive field was determined by afferent input evoked by only one of the two components. Different receptive-field properties of projection neurons may be related to the roles of these neurons in sensory control of the various phases of pheromone-modulated behavior of male moths. Accepted: 30 January 1998     

Opposing Effects of Nitric Oxide on Different Connexins Expressed in the Vascular System

Gap junctions—clusters of intercellular channels built by connexins (Cx)—are thought to be important for vascular cell functions such as differentiation, control of tone, or growth. In the vascular system, gap junctions can be formed by four different connexins (Cx37, Cx40, Cx43 and Cx45). The permeability of these connexin-formed gap junctions determines the amount of intercellular coupling and can be modulated by several vasoactive substances such as prostacyclin or nitric oxide (NO). We demonstrate here that NO has specific effects on certain connexins. Using two different techniques—injection of a fluorescent dye in single cells as well as detection of the de novoformation of gap junctions by a flow cytometry based technique—we found that NO decreases the functional coupling in Cx37 containing gap junctions whereas it increases the de novoformation of gap junctions containing Cx40. We conclude that NO, in addition to its known vasomotor effects, has a novel role in controlling intercellular coupling resulting in opposing effects depending on the specific connexin expressed in the cells.     

Odorant-Dependent Generation of Nitric Oxide in Mammalian Olfactory Sensory Neurons

The gaseous signalling molecule nitric oxide (NO) is involved in various physiological processes including regulation of blood pressure, immunocytotoxicity and neurotransmission. In the mammalian olfactory bulb (OB), NO plays a role in the formation of olfactory memory evoked by pheromones as well as conventional odorants. While NO generated by the neuronal isoform of NO synthase (nNOS) regulates neurogenesis in the olfactory epithelium, NO has not been implicated in olfactory signal transduction. We now show the expression and function of the endothelial isoform of NO synthase (eNOS) in mature olfactory sensory neurons (OSNs) of adult mice. Using NO-sensitive micro electrodes, we show that stimulation liberates NO from isolated wild-type OSNs, but not from OSNs of eNOS deficient mice. Integrated electrophysiological recordings (electro-olfactograms or EOGs) from the olfactory epithelium of these mice show that NO plays a significant role in modulating adaptation. Evidence for the presence of eNOS in mature mammalian OSNs and its involvement in odorant adaptation implicates NO as an important new element involved in olfactory signal transduction. As a diffusible messenger, NO could also have additional functions related to cross adaptation, regeneration, and maintenance of MOE homeostasis.     

In Vivo Expression of Inducible Nitric Oxide Synthase in Cerebellar Neurons

Abstract: In the CNS, nitric oxide (NO) functions as both neuromodulator and neurotoxic agent. In vivo neuronal expression of NO synthase (NOS) has been attributed to constitutive NOS—both the neuronal and the endothelial types. The other class of NOS—the inducible NOS (iNOS)—is known to mediate toxic effects of NO in various tissues. In this study, we show for the first time that direct intracerebellar injection of endotoxin and cytokine (lipopolysaccharide and interferon-γ) induced in vivo neuronal expression of the iNOS gene, as demonstrated by fluorescent in situ hybridization and immunohistochemical staining analyzed by confocal laser-scanning microscopy. This raises the possibility that neuronal iNOS might contribute significantly to the vulnerability of the brain to various insults.     

Structure-activity studies on adipokinetic hormones in Manduca sexta.

Structure-activity studies were performed for adipokinetic hormone (AKH) in Manduca sexta. Seven naturally occurring and four synthetic peptides of the red pigment concentrating hormone (RPCH)/AKH family were tested in larvae of M. sexta for activation of glycogen phosphorylase in fat body. pGlu at the N-terminal was found to be important for activity of peptides; however, Manduca AcGly1AKH is partially active. The amino acids at all positions appear to be of importance for activity, with the possible exception of the two serine residues in positions six and seven. Generally, the more amino acids are exchanged, the less the peptide will bind to the receptor. In M. sexta a beta-bend appears not to be important for the binding of peptides. Peptides ten amino acids long appear to be more active than shorter ones.     

Effects of ingestion of some non-host plant secondary compounds on larvae of Manduca sexta

The behavior of nonirradiated and radiation-sterilized laboratory-adapted Mediterranean fruit flies, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), was observed on apples hung on guava trees in outdoor cages in Hawaii. Oviposition and resting behavior, and physical encounters between flies were recorded. The numbers of nonirradiated females observed resting on fruit were reduced several fold by the presence of irradiated females, either alone or with irradiated males, but not by irradiated males alone. Similarly, the number and duration of nonirradiated females observed ovipositing was reduced by the presence of irradiated females. In control cages (all nonirradiated flies), females averaged 255.9±15.0 (SE) seconds on fruit, while nonirradiated and irradiated females in the mixed (treatment) cage averaged 157±19.8 seconds and 77.5±7.5 seconds on fruit, respectively. Irradiated females behaved skittishly on fruit and frequently engaged in physical encounters with other flies. By reducing oviposition of normal females, irradiated females may help suppress wild populations in sterile-insect release programs.

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Influence de la présence de femelles irradiées sur la ponte de Ceratitis capitata

Résumé Les comportements de ponte et de repos et le nombre de rencontres de mouches irradiées ou non, d'une souche de laboratoire de C. capitata ont été examinés sur des pommes suspendues sur des goyaviers dans des cages, hors du laboratoire, à Hawaï. Le nombre de femelles non-irradiées stationnant sur les fruits était réduit de plusieurs fois par la présence de femelles irradiées, soit seules, soit avec des mâles irradiés, mais non par la présence exclusive de mâles irradiés. De même, le nombre de femelles non-irradiées en train de pondre, ainsi que la durée des pontes étaient réduits par la présence de femelles irradiées. Dans les cages témoins (sans mouche irradiée), les femelles restent en moyenne 255,9±15,0 sec (SE) sur les fruits, tandis que les femelles nonirradiées et irradiées mélangées dans les cages traitées, séjournent respectivement 157±19,8 et 77,5±7,5 sec sur les fruits. Les femelles irradiées se sont comportées capricieusement sur les fruits et ont souvent eu des contacts physiques avec les autres mouches. En réduisant la ponte des femelles normales, les femelles irradiées peuvent contribuer à supprimer les populations sauvages dans les programmes de lâchers d'insectes stériles.

     

Eclosion Hormone Stimulates Cyclic GMP Levels in Manduca sexta Nervous Tissue via Arachidonic Acid Metabolism with Little or No Contribution from the Production of Nitric Oxide

The neuropeptide eclosion hormone acts directly on the nervous system of the tobacco hornworm, Manduca sexta, to trigger ecdysis behavior at the end of each molt. Previous studies have shown that the action of eclosion hormone is mediated via the intracellular messenger cyclic GMP. In the present study we have investigated the mechanisms involved in the eclosion hormone-stimulated increases in cyclic GMP. No stimulation of guanylate cyclase was seen in homogenized nervous tissue, suggesting that eclosion hormone does not directly stimulate a membrane-bound form of guanylate cyclase. Nitric oxide synthase inhibitors, N-methylarginine and nitroarginine, had no effect on eclosion hormone-stimulated cyclic GMP levels. By contrast, 4-bromophenacyl bromide, an inhibitor of arachidonic acid release, and nordihydroguaiaretic acid, an inhibitor of arachidonic acid metabolism, almost completely abolished the eclosion hormone-stimulated cyclic GMP increase. We hypothesize that eclosion hormone receptors are coupled to a lipase, activation of which causes the release of arachidonic acid. Either the arachidonic acid directly stimulates the soluble guanylate cyclase or further metabolism of arachidonic acid yields compounds that activate guanylate cyclase.     

Intracellular calcium in prothoracic glands of Manduca sexta

Cytosolic free calcium was measured in individual prothoracic gland cells of Manduca larvae with Fura-2. During the last larval instar there was no correlation between intracellular calcium concentration and ecdysteroid secretion by the glands. The addition of prothoracicotropic hormone (PTTH) from brains of Manduca larvae to prothoracic glands in vitro resulted in a significant increase in the calcium concentration of the gland cells. The effect of PTTH was inhibited by the inorganic calcium channel antagonists, cadmium, lanthanum and nickel, and by the antagonist of T-type calcium channels, amiloride, whereas all the other antagonists tested failed to block the action of PTTH. TMB-8, an inhibitor of intracellular calcium mobilization, did not reduce the PTTH-induced rise in calcium, which suggests that IP(3)-dependent intracellular calcium stores are not involved in the calcium-mediated stimulation of ecdysteroid synthesis. Moreover, PTTH is thought to increase intracellular calcium in prothoracic glands of Manduca by influencing calcium channels in the plasma membrane.     

Structural studies on the neutral glycosphingolipids of Manduca sexta

Glycosphingolipids (GSLs) have been implicated as playing major roles in cellular interactions and control of cell proliferation in muticellular organisms. Moreover GSLs and other sphingolipids such as sphingomyelins, ceramides and sphingosines serve a variety of roles in signal transduction. Hence, identification of structures of GSLs in different biota will shed light in understanding their physiological role. During this study, the major glycosphingolipid component present in the extracts of stage-12 and stage-17/18 metamorphosing adults of Manduca sexta was identified as mactosyl ceramide. We report the isolation of several ceramide disaccharides, a ceramide trisaccharide and a ceramide tetrasaccharide. The GSL structures were confirmed by high-resolution mass spectrometry and tandem mass spectrometry. The identity of the monosaccharides was proved using exoglycosidases. The predominant sphingosine chain-length varied from C-14 (tetradecasphing-4-enine) to C-16 (hexadecasphing-4-enine) in these GSLs. Sphingosines of both chain lengths were accompanied by their doubly unsaturated counterparts tetradecasphinga-4,6-diene and hexadecasphinga-4,6-diene. It is also interesting to note the presence of tetradecasphinganine and hexadecasphinganine in minute amounts in the form of a GSL in the extracts of M. sexta. The varying degrees of unsaturation in the sphingosine moiety of GSLs in M. sexta may be biologically significant in insect metamorphosis. The ceramide trisaccharides and ceramide tetrasaccharide belong to the arthro-series, The observation of fucose in the M. sexta GSLs is the first report of the presence of fucose in an arthroseries GSL.     

Effects of stress on the hemolymph juvenile hormone binding protein titers of Manduca sexta

External stressors disrupt physiological homeostasis; in insects, the response to stress may result in delayed development as the animal attempts to restore homeostasis before proceeding with its complex life cycle. Previous studies have demonstrated that exposure to stress leads to increased levels of the juvenile hormone (JH), a hormone responsible for maintaining the insect larval state. In Manduca sexta, JH is transported to target tissue by a high-affinity binding protein, hemolymph JH binding protein (hJHBP). Since JH titers are elevated in stressed Manduca, we examined levels of hJHBP to better understand (1) the role of JH in regulating hJHBP levels and (2) the hJHBP-regulated bioavailability of hormone at the target site. Fourth stadium Manduca (48 h post-ecdysis) were exposed for 24h to various stressors including nutritional deprivation, microbial infection, cutaneous injury, episodic movement, and temperature elevation. Insects raised on diets lacking nutritional content exhibited mean hJHBP levels that were less than half (45%) those of control insects. Similarly, insects injected with Escherichia coli demonstrated a 47% reduction in hJHBP titers. Cutaneous injury, episodic movement, and temperature elevation lowered hJHBP levels by 47%, 43%, and 38%, respectively. Total hemolymph protein concentration was not affected. After a stress event (injury), a 50% reduction in abundance of fat body hJHBP mRNA was observed within 4h; hJHBP levels did not drop until 24h after injury. Stress in the fourth stadium was manifest in fifth instars, with 100% of the injured insects displaying an extended larval stadium or failing to pupate. Computational modeling of the JH-hJHBP interaction indicates that unbound JH doubles in stressed insects. These results indicate that in response to stress larval hJHBP titers are significantly reduced, increasing JH bioavailability at the target site and thereby impacting development and survival of the insect. Treatment of unstressed insects with physiological doses of JH I did not affect hJHBP levels, suggesting that elevated JH levels were not solely responsible for the observed down-regulation in stressed insects.     

Ectonucleotide diphosphohydrolase activities in hemocytes of larval Manduca sexta

In this work, we describe the ability of living hemocytes from an insect (Manduca sexta, Lepidoptera) to hydrolyze extracellular ATP. In these intact cells, there was a low level of ATP hydrolysis in the absence of any divalent metal (8.24 +/- 0.94 nmol of Pi/h x 10(6) cells). The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 15.93 +/- 1.74 nmol of Pi/h x 10(6) cells. Both activities were linear with cell density and with time for at least 90 min. The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.33 mM MgCl2. This stimulatory activity was not observed when Ca2+ replaced Mg2+. The apparent Km values for ATP-4 and Mg-ATP2- were 0.059 and 0.097 mM, respectively. The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.6 and 7.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced by an increase of pH. These ecto-ATPase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, sodium fluoride, tartrate, and levamizole. To confirm the observed hydrolytic activities as those of an ecto-ATPase, we used an impermeant inhibitor, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), as well as suramin, an antagonist of P2-purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents. Interestingly, lipopolysaccharide, a component of cell walls of gram-negative bacteria that increase hemocyte aggregation and phagocytosis, increased the Mg2+-dependent ecto-ATPase activity in a dose-dependent manner but did not modify the Mg2+-independent ecto-ATPase activity.     

The biochemical basis of antimicrobial responses in Manduca sexta

Innate immunity is essential for the wellbeing of vertebrates and invertebrates. Key components of this defense system include pattern recognition receptors that bind to infectious agents, extra-and intra-cellular proteins that relay signals, as well as molecules and cells that eliminate pathogens. We have been studying the defense mechanisms in a biochemical model insect, Manduca sexta. In this insect, hemolin, peptidoglycan recognition proteins, β-1,3-glucan recognition proteins and C-type lectins detect microbial surface molecules and induce immune responses such as phagocytosis, nodulation, encapsulation, melanization and production of antimicrobial peptides. Some of these responses are mediated by extracellular serine proteinase pathways. The proteolytic activation of prophenoloxidase （proPO） yields active phenoloxidase （PO） which catalyzes the formation of quinones and melanin for wound healing and microbe killing. M. sexta hemolymph proteinase 14 （HP 14） precursor interacts with peptidoglycan or β-1,3-glucan, autoactivates, and leads to the activation of other HPs including HP21 and proPO-activating proteinases （PAPs）. PAP-1, -2 and -3 cut proPO to generate active PO in the presence of two serine proteinase homologs. Inhibition of the proteinases by serpins and association of the proteinase homologs with bacteria ensure a localized defense reaction. M. sexta HP1, HP6, HP8, HP17 and other proteinases may also participate in proPO activation or processing of spatzle and plasmatocyte spreading peptide.     

Depletion of Intracellular Glutathione Increases Susceptibility to Nitric Oxide in Mesencephalic Dopaminergic Neurons

Using primary neuronal cultures, we investigated the effects of GSH depletion on the cytotoxic effects of glutamate and NO in dopaminergic neurons. Intracellular GSH was depleted by 24-h exposure to L-buthionine-[S,R]-sulfoximine (BSO), an irreversible inhibitor of GSH synthase. BSO exposure caused concentration-dependent reduction of the viability of both dopaminergic and nondopaminergic neurons. In contrast, 24-h exposure of cultures to glutamate or NOC18, an NO-releasing agent, significantly reduced the viability of nondopaminergic neurons without affecting that of dopaminergic neurons. Pretreatment with N-acetyl-L-cysteine for 24 h ameliorated the NOC18-induced toxicity in nondopaminergic neurons. In dopaminergic neurons, sublethal concentrations of BSO reduced intracellular GSH content and markedly potentiated glutamate- and NOC18-induced toxicity. These results suggested that glutamate toxicity was enhanced in dopaminergic neurons by suppression of defense mechanisms against NO toxicity under conditions of GSH depletion. Under such conditions, free iron plays an important role because BSO-enhanced NO toxicity was ameliorated by the iron-chelating agent, deferoxamine. These results suggest that GSH plays an important role in the expression of NO-mediated glutamate cytotoxicity in dopaminergic neurons. Free iron may be related to enhanced NO cytotoxicity under GSH depletion.     

Protease inhibitors of Manduca sexta expressed in transgenic cotton

Summary To explore the effectiveness of insect derived protease inhibitors in protecting plants against insect feeding, anti-trypsin, anti-chymotrypsin and anti-elastase protease inhibitor (PI) genes from Manduca sexta L. were expressed in transgenic cotton (Gossypium hirsutum L.). From 198 independent transformants, 35 elite lines were further analyzed. Under the control of the 35S promoter of CaMV, PI accumulated to approximately 0.1% of total protein, depending on the tissue analyzed. Using cell-flow cytometry, DNA content/ nuclei of transgenic and non-transformed cotton were identical. On cotton plants expressing PIs, fecundity of Bemisia tabaci (Genn.), the sweetpotato whitefly, was reduced compared to controls. Expression of these protease inhibitors may reduce the developmental rate of B. tabaci and other insects, and provide a strategy for cotton protection.