Insect Salivary Glands Research Articles

The aminergic and peptidergic innervation of insect salivary glands

Insect salivary glands are glands associated with nutrient intake whose secretions are generally involved in the digestion and lubrication of food. They are under the control of neuroactive substances and may be innervated from several sources including the suboesophageal ganglion, the stomatogastric nervous system and the unpaired median nerves. Both amines and peptides have been suggested to play roles in the control of insect salivation, as indicated by their association with terminals on salivary glands, their effects in salivary gland bioassays and their ability to alter second messenger levels and ion channel conformations. Serotonin and dopamine appear to be the most prominent amines associated with insect salivary glands. Either one or both of these amines are found associated with the salivary glands of the locust, stick insect, cockroach, cricket, dragonfly, mosquito, adult moth and kissing bug. Their roles, although not fully elucidated, appear to be in the control of salivary secretion. Several peptides, including members of the FMRFamide-related family of peptides, are also found associated with insect salivary glands. Sources of peptidergic innervation are as varied as those for aminergic innervation, but information regarding the physiological role of these peptides is lacking. The relevance of the different levels of complexity of salivary gland innervation, which range from the absence of innervation in some species (blowfly) to the presence of several distinct sources in others (locust, cockroach), is not well understood. This review serves to consolidate what is known of the phenotype of salivary neurones in relation to the control of salivation.

Nitric Oxide Binding and Crystallization of Recombinant Nitrophorin I, a Nitric Oxide Transport Protein from the Blood-Sucking Bug Rhodnius prolixus

A nitric oxide transport protein (nitrophorin I) from the salivary glands of the blood-sucking bug Rhodnius prolixus has been expressed as an insoluble form in Escherichia coli, reconstituted with heme, and characterized with respect to NO binding kinetics and equilibria. NO binding and absorption spectra for recombinant nitrophorin I were indistinguishable from those of the insect-derived protein. The degree of NO binding, the rate of NO release, and the Soret absorption maxima for nitrophorin I were all pH dependent. The NO dissociation constant rose 9-fold over the pH range 5.0-8.3, from 0.19 x 10(-6) to 1.71 x 10(-6). The NO dissociation rate rose 2500-fold between pH 5.0 and pH 8.3, from 1.2 x 10(-3) to 3.0 s(-1). Thus, the NO association rate must also be pH dependent and reduced at pH 5.0 by approximately 280-fold. These factors are consistent with nitrophorin function: NO storage in the apparent low pH of insect salivary glands and NO release into the tissue of the insect's host, where vasodilation is induced. The reversible nature of NO binding, which does not occur with most other heme proteins, and the apparent kinetic control of NO release are discussed. We also report crystals of nitrophorin I that are suitable for structure determination by X-ray crystallography. The most promising crystal form contains two protein molecules in the asymmetric unit and diffracts beyond 2.0 A resolution.

Insect nuclear receptors: A developmental and comparative perspective

The appearance of puffs on the polytene chromosomes of insect salivary glands incubated with 20-hydroxyecdysone provided the first demonstration that steroids act directly at the gene transcriptional level to bring about subsequent cellular changes (Becker, 1959; Clever and Karlson, 1960). Despite that auspicious beginning, learning about the molecular mechanisms that underlie the hormonal regulation of insect development was impeded for many years by the difficulty associated with isolating and identifying rare regulatory factors from limited tissue sources. The advent of recombinant DNA methodology and powerful techniques such as the polymerase chain reaction (PCR) along with the recognition that many important endocrine factors are structurally conserved across a wide range of species has, however, all but eliminated the technical obstacles once facing the insect endocrinologist trying to isolate and study these regulatory molecules. This review will discuss recent progress and recall some earlier experiments concerning the molecular basis of hormonal action in insects focusing primarily on the members of the nuclear hormone receptor superfamily in Drosophila melanogaster. Two members of this family comprise the functional ecdysteroid receptor and at least a dozen other “orphans” have been identified in Drosophila for which no cognate ligand has yet been found. Many of these orphans are regulated by ecdysteroids. A discussion of juvenile hormone binding proteins that are not family members has been included because of their potential impact on nuclear receptor function. As receptor homologues have been identified in other insects, several general ideas concerning insect hormonal regulation have begun to emerge and these will be examined from a comparative point of view.

Transmission ofSpiroplasma citriLines and Their Ability to Cross Gut and Salivary Gland Barriers Within the Leafhopper VectorCirculifer tenellus

Spiroplasma citri lines all derived from a single phytopathogenic strain, BR3-3X, but differing in subculturing or maintenance history, were tested for transmission to a susceptible plant host after injection into the leafhopper vector Circulifer tenellus and for their ability to cross both the gut and salivary gland barriers in the insect. BR3-T (a line maintained in turnips by leafhopper transmission) was detected in leafhoppers 18 days after injection and in plants exposed to injected leafhoppers. Plants exposed to leafhoppers injected with BR3-P (a line subcultured in artificial medium more than 130 times) and BR3-G (a line maintained in periwinkle over 8 years by graft transmission) did not become diseased. However, like BR3-T, both of these lines were detected in leafhoppers 18 days after injection. In a separate experiment, a fourth line, BR3-M, that had been passed in liquid culture 43 times, retained insect transmissibility. After leafhoppers fed on suspensions of spiroplasmas, BR3-T was readily recovered from the hemolymph after a 6-wk latent period, whereas BR3-P was rarely recovered and BR3-G was never recovered. Similarly, BR3-T was recovered from solutions fed upon by injected insects after a 14-day latent period, whereas BR3-P was rarely recovered and BR3-G was never recovered. Thus, although all lines multiply within the hemocoel, the ability to traverse insect gut and salivary gland barriers has been lost in BR3-G and significantly impaired in BR3-P.

Relationship between latency and period for 5-hydroxytryptamine-induced membrane responses in the Calliphora salivary gland.

Following stimulation with a calcium-mobilizing agonist there is often a distinct latency (L) preceding the onset of the first calcium spike. In the continued presence of the agonist, repetitive spikes appear separated by a variable period (P). The relationship between L and P has been investigated in an insect salivary gland responding to 5-hydroxytryptamine (5-HT). Both L and P were found to decrease as the concentration of 5-HT was increased over its physiological range of 1-10 nM. Lowering the concentration of external calcium from 1 x 10(-3) M to 1 x 10(-5) M increased both P and L. However, the effect on L was apparent only at low levels of 5-HT. Reducing the content of the internal stores by repeated stimulation in a calcium-free medium resulted in a progressive prolongation of L. On the other hand, the effect of L decreased when glands were stimulated repetitively in normal calcium-containing medium. All these results are consistent with a hypothesis that calcium plays a critical role in determining the kinetics of calcium release during both L and P. An important component seems to be the entry of external calcium, which sets the stage for calcium release by loading up the internal stores. As these stores fill up with calcium, the Ins(1,4,5)P3 receptors will initiate a calcium spike once they become sensitized to the ambient level of Ins(1,4,5)P3.

Nitric oxide synthase activity from a hematophagous insect salivary gland

The salivary glands of the hematophagous insect, Rhodnius prolixus, contain a nitrosylhemeprotein that dissociates its ligand, NO, to the host tissues while the insect is searching for a blood meal. We now report a salivary nitric oxide synthase activity in this insect. The activity is dependent on NADPH, FAD, tetrahydrobiopterin, calmodulin, Ca 2+, and converts arginine to citrulline while producing vasorelaxing activity. Molecular sieving indicates a molecular weight of 185 kDa, coeluting with a diaphorase activity. Results indicate similarity of this insect activity to the vertebrate constitutive NO synthase, suggesting NO synthesis is an evolutionary old biological pathway.

A Reappraisal of the Oil-Gap Technique for the Measurement of Transtubular Potentials in Insect Epithelia

ABSTRACT Transtubular potentials of insect Malpighian tubules and salivary glands are commonly measured in unperfused tubules in which the two ends are separated by means of an insulating oil gap. The validity of this method has been examined, using Malpighian tubules of a tenebrionid beetle, Onymacris plana. The measured interelectrode potential was found to depend not only on the transtubular potential but also on potentials within the oil gap and at the open end of the tubule, and upon the core resistance, the length constant and a small but finite electrical leak beneath the insulating oil. In perfused tubules of O. plana, the apparent transtubular potential recorded at the collecting end (analogous to the oil gap) was indistinguishable from that previously measured with the oil-gap technique. However, it was invariably lower than that recorded by an intraluminal pipette. The results demonstrate that the interelectrode potential observed with the oil-gap technique is a poor measure of the true transtubular potential.

Receptor activation and inositol lipid hydrolysis in neural tissues.

Recent advances in our knowledge of the biochemistry, pharmacology, and cell biology of ligand-induced, stimulated turnover of inositol lipids in such diverse tissues as insect salivary glands, platelets, lymphocytes, and a number of exocrine glands have greatly altered our understanding of intracellular events leading to secretion, contraction, chemotaxis, and other cellular responses. Much of what has been learned regarding ligand-stimulated turnover of inosito1 lipids has come from nonneural systems, so that the presumption that signal transduction via this mechanism mediates significant steps in neural function must still be considered inferential. This reservation has been a major theme of a recent comprehensive review (Hawthorne, 1986). Nevertheless, that the brain contains large amounts of the substrates and enzymes of inositol lipid turnover and its associated second messenger systems and, further, that it is enriched with receptors that are linked to stimulated inositol lipid turnover argue strongly that the phosphoinositides indeed play an important role in brain function. There exist several general reviews on stimulated inositol lipid turnover (Bemidge, 1984; Bemdge and Irvine, 1984; Nishizuka, 1984, 1986; Hokin, 1985; Abdel-Latif, 1986; Downes, 1986; Williamson, 1986), including several that emphasize the nervous system (Downes, 1982, 1983; Fisher and Agranoff, 1986; Hawthorne, 1986; Nahorski et al., 1986). The present contribution emphasizes developments of neurochemical relevance since a review on the subject in this journal 8 years ago (Hawthorne and Pickard, 1979).

Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate.

Activation of receptors for a wide variety of hormones and neurotransmitters leads to an increase in the intracellular level of calcium. Much of this calcium is released from intracellular stores but the link between surface receptors and this internal calcium reservoir is unknown. Hydrolysis of the phosphoinositides, which is another characteristic feature of these receptors, has been implicated in calcium mobilization. The primary lipid substrates for the receptor mechanism seem to be two polyphosphoinositides, phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2), which are rapidly hydrolysed following receptor activation in various cells and tissues. The action of phospholipase C on these polyphosphoinositides results in the rapid formation of the water-soluble products inositol 1,4-bisphosphate (Ins1,4P2) and inositol 1,4,5-trisphosphate (Ins1,4,5P3). In the insect salivary gland, where changes in Ins1,4P2 and Ins1,4,5P2 have been studied at early time periods, increases in these inositol phosphates are sufficiently rapid to suggest that they might mobilize internal calcium. We report here that micromolar concentrations of Ins1,4,5P3 release Ca2+ from a nonmitochondrial intracellular Ca2+ store in pancreatic acinar cells. Our results strongly suggest that this is the same Ca2+ store that is released by acetylcholine.

Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol.

The agonist-dependent hydrolysis of inositol phospholipids was investigated by studying the breakdown of prelabelled lipid or by measuring the accumulation of inositol phosphates. Stimulation of insect salivary glands with 5-hydroxytryptamine for 6 min provoked a rapid disappearance of [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and [3H]phosphatidylinositol 4-phosphate (PtdIns4P) but had no effect on the level of [3H]phosphatidylinositol (PtdIns). The breakdown of PtdIns(4,5)P2 was associated with a very rapid release of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which reached a peak 5 1/2 times that of the resting level after 5 s of stimulation. This high level was not maintained but declined to a lower level, perhaps reflecting the disappearance of PtdIns(4,5)P2. 5-Hydroxytryptamine also induced a rapid and massive accumulation of inositol 1,4-bisphosphate [Ins(1,4)P2]. The fact that these increases in Ins(1,4,5)P3 and Ins(1,4)P2 precede in time any increase in the level of inositol 1-phosphate or inositol provides a clear indication that the primary action of 5-hydroxytryptamine is to stimulate the hydrolysis of PtdIns(4,5)P2 to yield diacylglycerol and Ins(1,4,5)P3. The latter is then hydrolysed by a series of phosphomonoesterases to produce Ins(1,4)P2, Ins1P and finally inositol. The very rapid agonist-dependent increases in Ins(1,4,5)P3 and Ins(1,4)P2 suggests that they could function as second messengers, perhaps to control the release of calcium from internal pools. The PtdIns(4,5)P2 that is used by the receptor mechanism represents a small hormone-sensitive pool that must be constantly replenished by phosphorylation of PtdIns. Small changes in the size of this small energy-dependent pool of polyphosphoinositide will alter the effectiveness of the receptor mechanism and could account for phenomena such as desensitization and super-sensitivity.

Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides.

The formation of inositol phosphates in response to agonists was studied in brain slices, parotid gland fragments and in the insect salivary gland. The tissues were first incubated with [3H]inositol, which was incorporated into the phosphoinositides. All the tissues were found to contain glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, which were identified by using anion-exchange and high-resolution anion-exchange chromatography, high-voltage paper ionophoresis and paper chromatography. There was no evidence for the existence of inositol 1:2-cyclic phosphate. A simple anion-exchange chromatographic method was developed for separating these inositol phosphates for quantitative analysis. Stimulation caused no change in the levels of glycerophosphoinositol in any of the tissues. The most prominent change concerned inositol 1,4-bisphosphate, which increased enormously in the insect salivary gland and parotid gland after stimulation with 5-hydroxytryptamine and carbachol respectively. Carbachol also induced a large increase in the level of inositol 1,4,5-trisphosphate in the parotid. Stimulation of brain slices with carbachol induced modest increase in the bis- and tris-phosphate. In all the tissues studied, there was a significant agonist-dependent increase in the level of inositol 1-phosphate. The latter may be derived from inositol 1,4-bisphosphate, because homogenates of the insect salivary gland contain a bisphosphatase in addition to a trisphosphatase. These results suggest that the earliest event in the stimulus-response pathway is the hydrolysis of polyphosphoinositides by a phosphodiesterase to yield inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate, which are subsequently hydrolysed to inositol 1-phosphate and inositol. The absence of inositol 1:2-cyclic phosphate could indicate that, at very short times after stimulation, phosphatidylinositol is not catabolized by its specific phosphodiesterase, or that any cyclic derivative liberated is rapidly hydrolysed by inositol 1:2-cyclic phosphate 2-phosphohydrolase.

Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands.

1. The effect of Li+ on the agonist-dependent metabolism of [3H]inositol has been studied in rat brain, rat parotid and the insect salivary gland. 2. When brain or parotid slices were incubated in the presence of [3H]inositol, Li+ was found to amplify the ability of agonists such as carbachol, phenylephrine, histamine, 5-hydroxytryptamine and Substance P to elevate the amount of label appearing in the inositol phosphates. 3. A different approach was used with the insect salivary gland, which was prelabelled with [3H]inositol. After washing out the label, the subsequent release of [3H]inositol induced by 5-hydroxytryptamine was greatly decreased by Li+. During Li+ treatment there was a large accumulation of [3H]inositol 1-phosphate. 4. This ability of Li+ to greatly amplify the agonist-dependent accumulation of myo-inositol 1-phosphate offers a novel technique for identifying those receptors that function by hydrolysing phosphatidylinositol. 5. The therapeutic action of Li+ may be explained by this inhibition of myo-inositol 1-phosphatase, which lowers the level of myo-inositol and could lead to a decrease in the concentration of phosphatidylinositol, especially in those neurons that are being stimulated excessively. This alteration in phosphatidylinositol metabolism may serve to reset the sensitivity of those multifunctional receptors that generate second messengers such as Ca2+, cyclic GMP and the prostaglandins.

Portasomes as Coupling Factors in Active Ion Transport and Oxidative Phosphorylation

SYNOPSIS. We propose that particles, 7–15 nm in diameter, observed on the apical plasma membranes of cation transporting cells of insect midgut, salivary glands, and Malpighian tubules are modified F1-F coupling complexes such as those found on phosphorylating membranes of mitochondria, chloroplasts, and bacteria. We suggest the generic term, portasome, to describe all of these particles and point out that they are located on the side of the membrane which is electronegative and has the low cation concentration, i.e. , on the input side in each case. Biophysical evidence identifies the portasome bearing membrane as the ion transporting membrane in several insect epithelia, some of which exhibit ion modulated ATPase activity. The activity of a K+-modulated ATPase from Manduca sexta midgut is increased in portasome enriched plasma membrane fractions. We propose that portasomes orient the scalar hydrolysis of negatively charged MgATP2− to less negatively charged MgADP thereby eliminating the attraction of MgATP2− to K+ with the result that the K+ ions are ejected to the opposite side of the portasome bearing membrane. This mechanism explains the coupling of the scalar hydrolysis of ATP to the vectorial active transport of K+ which leads to the establishment of a K+ electrochemical gradient. The reverse process, but with an H+ ionophore replacing a K+ ionophore in the portasome, would provide a mechanism for coupling the vectorial flow of H+, driven by a proton electrochemical gradient, to scalar ATP synthesis and thereby provide a mechanism for oxidative phosphorylation. Electrogenic active potassium ion transport would appear to have evolved from oxidative phosphorylation.

Diameter of the cell-to-cell junctional membrane channels as probed with neutral molecules.

The cell-to-cell channels in the junctions of an insect salivary gland and of insect and mammalian cells in culture were probed with fluorescent molecules-neutral linear oligosaccharides, neutral branched glycopeptides, and charged linear peptides. From the molecular dimensions of the largest permeants and smallest impermeants the permeation-limiting channel diameter was obtained: 16 to 20 angstroms for the mammalian cells and 20 to 30 angstroms for the insect cells.

Changes in cyclic AMP and cyclic GMP concentrations during the action of 5-hydroxytryptamine on an insect salivary gland.

Salivary glands from adult blowflies (Calliphora erythrocephala Meigen) were studied in vitro. The time course of changes in cyclic AMP content of the glands was followed at different concentration of 5-hydroxytryptamine. There was an immediate biphasic rise and fall in cyclic AMP content, following by a slower rise and subsequent gradual decline. The initial rise preceded the onset of fluid secretion by the glands. Rises in cyclic AMP content were inhibited by compound RMI 12330 A (an adenylate cyclase inhibitor) and were halted after about 15-20s if the glands were deprived of Ca2+. Theophylline (a phosphodiesterase inhibitor) abolished the decline phase of the fast response, Losses of cyclic AMP from the glands either to the bathing medium or to the saliva were small and could not account for the rapid fall found. Evidence is presented that cyclic GMP is not involved in the process of initiating secretion in the blowfly salivary gland.

Preliminary measurements of intracellular calcium in an insect salivary gland using a calcium-sensitive microelectrode

A calcium-sensitive microeletrode was used to measure free intracellular calcium in salivary gland cells of Calliphora during stimulation with 5-hydroxytryptamine (5-HT). The resting level of calcium was approximately 10 −7M or less but increased in a dose-dependent way sometimes to levels in excess of 10 −6M. The onset of the calcium signal was closely related to changes in membrane and transepithelial potential. This calcium response was greatly reduced when the extracellular calcium concentration was reduced from 10 −3 to 10 −4M. This dependence on external calcium is consistent with previous observations that 5-HT acts to increase the permeability of the basal plasma membrane to calcium. These observations indicate that an increase in the intracellular level of calcium is an early event associated with the onset of fluid secretion in this insect salivary gland.

Ion-selective electrode studies on the effects of 5-hydroxytryptamine on the intracellular level of potassium in an insect salivary gland.

Potassium-sensitive microelectrodes were used to measure the intracellular level of potassium in Calliphora salivary glands. During stimulation with 1 X 10(-8) M 5-HT, the potassium concentration increased from a resting level of 133.2 to 139.0 mM. When the external concentration of potassium was reduced below 2mM there was a rapid loss of potassium which could be partially reversed by 5-HT. During 5-HT stimulation, the addition of ouabain had no effect on a number of parameters including the rate of fluid secretion, the ionic composition of the saliva and the intracellular level of potassium. The possibility that potassium enters the cells passively is discussed.

Differential inhibitory effect of 5-fluorouridine on RNA labelling in dipteran salivary gland cells

In Chironomus salivary glands, 5-fluorouridine inhibits labelling of ribosomal RNA. The drug does not prevent labelling of other types of RNA-like heterodisperse messenger-like RNA, 75-S RNA of Balbiani ring origin and 4-S RNA, which furthermore are exported to cytoplasm. The potential use of this drug for the study of RNA metabolism in insect salivary glands can be compared with the use of low doses of actinomycin D in cultured mammalian cells.

Ultrastructural Analysis of the Salivary Glands of Gromphadorhina Portentosa (Schaum)

The structure of insect salivary glands has been extensively investigated during the past decade; however, none have attempted scanning electron microscopy (SEM) in ultrastructural examinations of these secretory organs. This study correlates fine structure by means of SEM cryofractography with that of thin-sectioned epoxy embedded material observed by means of transmission electron microscopy (TEM).Salivary glands of Gromphadorhina portentosa were excised and immediately submerged in cold (4°C) paraformaldehyde-glutaraldehyde fixative1 for 2 hr, washed and post-fixed in 1 per cent 0s04 in phosphosphate buffer (4°C for 2 hr). After ethanolic dehydration half of the samples were embedded in Epon 812 for TEM and half cryofractured and subsequently critical point dried for SEM. Dried specimens were mounted on aluminum stubs and coated with approximately 150 Å of gold in a cold sputtering apparatus.Figure 1 shows a cryofractured plane through a salivary acinus revealing topographical relief of secretory vesicles.

Mobility restriction in vivo of the heavy ribosomal subunit in a secretory cell.

Analysis in insect (Chironomus tentans) salivary gland cells of labeled RNA as a function of time after precursor injection and its distance to the nuclear membrane, cytoplasmic zone analysis, could previously be used to demonstrate the presence of short-lasting gradients in newly labeled ribosomal RNA. Since the gradients were sensitive to puromycin, they are likely to be a result of diffusion restriction due to an engagement of the subunits into polysomes. In the present paper the possibility was explored of recording gradients that were caused by labeled subunits in puromycin-resistant associations, which, in all probability, involve the endoplasmic reticulum. It was found that labeled 28 S and 5 S RNA but not 18 S RNA were present in radioactivity gradients lasting for at least 2 days but less than 6 days. The gradients also remained during inhibition of RNA synthesis by actinomycin, and they were completely resistant to puromycin whether given in vivo or in vitro. The semipermanent gradients formed fhere offer a unique parameter for the in vivo study of conditions for formation and maintenance of heavy subunits in puromycin-resistant bonds. An explanation for these and previous results is that the light subunit, although restricted in movement by engagement to polysomes, is nevertheless free to exchange and spread between rounds of translation, whereas at least part of the heavy subunit population is bound to the endoplasmic reticulum and less free to spread. These results offer a good in vivo correlate to previous results on in vitro exchangeability of subunits.