Efflux patterns for organic molecules from the CNS of the tobacco hornworm. Manduca sexta

The efflux of nicotine and its CNS metabolites from the nerve cord of the nicotine-insensitive tobacco hornworm is simpler (two efflux components) than from the nerve cord of the nicotine-sensitive cockroach (three components). This paper looks at the question of whether this disparity is merely a fortuitous species difference, or whether it may have regulatory significance. It is found that Manduca and Periplaneta cords display strikingly similar tripartite efflux patterns of leucine efflux, and that this pattern also characterises efflux of three extracellular markers from Manduca CNS. Efflux of the alkaloid, atropine, and the basic amino acid, arginine, from Manduca CNS follow different patterns from both nicotine/metabolite efflux and the other substances tested. These data support the notion that the simple efflux pattern for nicotine/metabolites in Manduca reflects the activity of special physiological processes, and is not simply imposed by factors such as tissue geometry.     

Uptake and metabolism of nicotine by the CNS of a nicotine-resistant insect,the tobacco hornworm (Manduca sexta)

Penetration of the CNS by nicotine occurred equally rapidly in the nicotine-insensitive Manduca cord and the nicotine-sensitive Periplaneta cord, ruling out the possibility that lowered permeability renders Manduca insensitive. Although a saturable concentrative component of nicotine uptake was found in the Manduca cord, it was difficult to examine this component rigorously, because, except at high concentrations, the CNS metabolises the bulk of the nicotine that is taken up. The CNS metabolites of nicotine are water-soluble compounds. They are special first by virtue of the fact that they are formed in the CNS itself and secondly because their chromatographic characteristics are different from mammalian nicotine metabolites (which are not formed by nervous tissue). When subjected to hydrolysis, the metabolites acted like conjugates. Periplaneta CNS also metabolised nicotine, but much less extensively than Manduca. It is speculated that enzymic detoxification of dietary neurotoxins may be a necessary function of the insect CNS, since insects have no anatomical equivalent of a hepatic-portal system for detoxifying ingested compounds before they reach the blood-brain interface.     

A putative nicotine pump at the metabolic blood–brain barrier of the tobacco hornworm

In mammals, P-glycoprotein immunostaining at the blood–brain barrier has implicated the multidrug pump in the restricted movement of many cytotoxic agents into the central nervous system (NCS). Since many insects require as sophisticated blood–brain barrier system to protect their CNS from plant-derived neurotoxins, we have investigated the possibility that a P-glycoprotein homolog constitutes a component of the insect blood–brain barrier. We have used the nicotine-resistant tobacco hornworm (Manduca sexta) to address this issue. Manduca has been previously shown, in physiological studies, to have an alkaloid (nicotine/morphine/atropine) pump at its excretory malpighian tubules. We show (1) that the tubules are P-glycoprotein immunopositive, (2) that Manduca has a metabolic blood–brain barrier for nicotine, (3) that the barrier co-localizes with P-glycoprotein immunostaining, and (4) that detoxifying enzymes as well as the nicotine pump are likely to account for the metabolic blood–brain to nicotine. These findings may provide insights on two major fronts, the troublesome problem of multi-insecticide resistance, a phenomenon that parallels multidrug resistance in tumor cells, and the problem of tolerance to addictive neuroactive drugs like nicotine or morphine. 1994 John Wiley & Sons, Inc.     

Functional analysis of Scr during embryonic and post-embryonic development in the cockroach, Periplaneta americana

The cockroach, Periplaneta americana represents a basal insect lineage that undergoes the ancestral hemimetabolous mode of development. Here, we examine the embryonic and post-embryonic functions of the hox gene Scr in Periplaneta as a way of better understanding the roles of this gene in the evolution of insect body plans. During embryogenesis, Scr function is strictly limited to the head with no role in the prothorax. This indicates that the ancestral embryonic function of Scr was likely restricted to the head, and that the posterior expansion of expression in the T1 legs may have preceded any apparent gain of function during evolution. In addition, Scr plays a pivotal role in the formation of the dorsal ridge, a structure that separates the head and thorax in all insects. This is evidenced by the presence of a supernumerary segment that occurs between the labial and T1 segments of RNAiScr first nymphs and is attributed to an alteration in engrailed (en) expression. The fact that similar Scr phenotypes are observed in Tribolium but not in Drosophila or Oncopeltus reveals the presence of lineage-specific variation in the genetic architecture that controls the formation of the dorsal ridge. In direct contrast to the embryonic roles, Scr has no function in the head region during post-embryogenesis in Periplaneta, and instead, strictly acts to provide identity to the T1 segment. Furthermore, the strongest Periplaneta RNAiScr phenotypes develop ectopic wing-like tissue that originates from the posterior region of the prothoracic segment. This finding provides a novel insight into the current debate on the morphological origin of insect wings.     

Sucrose-binding lectin in regenerating cockroach (Periplaneta americana) legs: Its purification from adult hemolymph

Previously, we purified Periplaneta lectin from the hemolymph of adult Periplaneta americana (American cockroach) (Kubo and Natori Eur. J. Biochem. 168, 75–82, 1987). Immunoblotting analysis using antibody against Periplaneta lectin showed that the cockroach hemolymph contains another lectin that cross reacted immunologically with Periplaneta lectin. We have purified this new lectin (regenectin) to homogeneity. Affinity purified antibody against regenectin cross reacted with Periplaneta lectin. Thus, Periplaneta lectin and this new lectin were found to be different lectins sharing common antigenicity. After leg amputation, this new lectin was found to appear transiently at a specific stage of regeneration as revealed by immunoblotting, suggesting that it plays a role in the regeneration process.     

Purification and characterization of a small cationic protein from the tobacco hornworm Manduca sexta

The prophenoloxidase (proPO) activation system is an important defense mechanism in arthropods, and activation of proPO to active phenoloxidase (PO) involves a serine proteinase cascade. Here, we report the purification and characterization of a small cationic protein CP8 from the tobacco hornworm, Manduca sexta, which can stimulate proPO activation. BLAST search showed that Manduca CP8 is similar to a fungal proteinase inhibitor-1 (AmFPI-1), an inducible serine proteinase inhibitor-1 (ISPI-1), and other small cationic proteins with unknown functions. However, we showed that Manduca CP8 did not inhibit proteinase activity, but stimulated proPO activation in plasma. When small amount (0.1 μg) of purified native CP8 or BSA was added to cell-free plasma samples and incubated for 20 min, low PO activity was observed in both groups. But significantly higher PO activity was observed in the CP8-group than in the BSA-group when more proteins (0.5 μg) were added and incubated for 20 min. However, when the plasma samples were incubated with proteins for 30 min, high PO activity was observed in both the CP8 and BSA groups regardless of the amount of proteins added. Moreover, when PO in the plasma was pre-activated with Micrococcus luteus, addition of CP8 did not have an effect on PO activity, and CP8/bacteria mixture did not stimulate PO activity to a higher level than did BSA/bacteria. These results suggest that CP8 helps activate proPO more rapidly at the initial stage. CP8 mRNA was specifically expressed in fat body and its mRNA level decreased when larvae were injected with saline or bacteria. However, CP8 protein concentration in hemolymph did not change significantly in larvae injected with saline or microorganisms.     

Competition Between Entomopathogenic and Free-Living Bactivorous Nematodes in Larvae of the Weevil Diaprepes abbreviatus

Field and laboratory experiments were conducted to determine the degree to which free-living, bactivorous nematodes (FLBN) are able to competitively displace entomopathogenic nematodes (EPN) from insect cadavers. Two hundred larvae of the insect Diaprepes abbreviatus were buried at regular intervals during 2 years in experimental plots that were untreated or treated twice annually with Steinernema riobrave. Larvae were recovered after 7 days, and nematodes emerging from cadavers during the next 30 days were identified. The monthly prevalence of FLBN was directly related to that of S. riobrave (r = 0.38; P = 0.001) but was not related to the prevalence of the endemic EPN, S. diaprepesi, Heterorhabditis zealandica, H. indica, or H. bacteriophora (r = 0.02; P = 0.80). In a second experiment, treatment of small field plots with S. riobrave increased the prevalence of insect cadavers in which only FLBN were detected compared to untreated controls (30% vs. 14%; P = 0.052), and increased numbers of FLBN per buried insect by more than 10-fold. In the laboratory, sand microcosms containing one D. abbreviatus larva were treated with (i) the FLBN, Pellioditis sp.; (ii) S. riobrave; (iii) S. riobrave + Pellioditis; or (iv) neither nematode. Insect mortality was higher in the presence of both nematodes (57%) than when S. riobrave was alone (42%) (P = 0.01). An average of 59.2 Pellioditis sp. g^-1 insect body weight emerged in the presence of S. riobrave, whereas 6.2 nematodes g^-1 insect were recovered in the absence of the EPN (P = 0.01). Pellioditis sp. reduced the number of S. riobrave per cadaver by 84%; (P = 0.03), and per available insect by 82% (P = 0.001), compared to S. riobrave alone. Population size of S. diaprepesi was not affected by Pellioditis sp. in experiments of the same design. Faster development (P = 0.05) and nutrient appropriation within the insect cadaver by S. diaprepesi compared to S. riobrave may increase the fitness of the former species to compete with Pellioditis sp. The results of these studies demonstrate the potential of FLBN to regulate population densities of EPN and to dampen estimates of EPN-induced mortality of insect pests in the field.     

Integrated modeling of protein-coding genes in the Manduca sexta genome using RNA-Seq data from the biochemical model insect

The genome sequence of Manduca sexta was recently determined using 454 technology. Cufflinks and MAKER2 were used to establish gene models in the genome assembly based on the RNA-Seq data and other species' sequences. Aided by the extensive RNA-Seq data from 50 tissue samples at various life stages, annotators over the world (including the present authors) have manually confirmed and improved a small percentage of the models after spending months of effort. While such collaborative efforts are highly commendable, many of the predicted genes still have problems which may hamper future research on this insect species. As a biochemical model representing lepidopteran pests, M. sexta has been used extensively to study insect physiological processes for over five decades. In this work, we assembled Manduca datasets Cufflinks 3.0, Trinity 4.0, and Oases 4.0 to assist the manual annotation efforts and development of Official Gene Set (OGS) 2.0. To further improve annotation quality, we developed methods to evaluate gene models in the MAKER2, Cufflinks, Oases and Trinity assemblies and selected the best ones to constitute MCOT 1.0 after thorough crosschecking. MCOT 1.0 has 18,089 genes encoding 31,666 proteins: 32.8% match OGS 2.0 models perfectly or near perfectly, 11,747 differ considerably, and 29.5% are absent in OGS 2.0. Future automation of this process is anticipated to greatly reduce human efforts in generating comprehensive, reliable models of structural genes in other genome projects where extensive RNA-Seq data are available.     

Herbivore-induced ethylene suppresses a direct defense but not a putative indirect defense against an adapted herbivore

Herbivory induces both direct and indirect defenses in plants; however, some combinations of these defenses may not be compatible. The jasmonate signal cascade activated both direct (nicotine accumulations) and indirect (mono- and sesquiterpene emissions) whole-plant defense responses in the native tobacco Nicotiana attenuata Torr. Ex Wats. Nicotine accumulations were proportional to the amount of leaf wounding and the resulting increases in jasmonic acid (JA) concentrations. However, when larvae of the nicotine-tolerant herbivore, Manduca sexta, fed on plants or their oral secretions were applied to leaf punctures, the normal wound response was dramatically altered, as evidenced by large (4- to 10-fold) increases in the release of (i) volatile terpenoids and (ii) ethylene, (iii) increased (4- to 30-fold) accumulations of endogenous JA pools, but (iv) decreased or unchanged nicotine accumulations. The ethylene release, which was insensitive to inhibitors of induced JA accumulation, was sufficient to account for the attenuated nicotine response. Applications of ethylene and ethephon suppressed the induced nicotine response and pre-treatment of plants with a competitive inhibitor of ethylene receptors, 1-methylcyclopropene, restored the full nicotine response. This ethylene burst, however, did not inhibit the release of volatile terpenoids. Because parasitoids of Manduca larvae are sensitive to the dietary intake of nicotine by their hosts, this ethylene-mediated switching from direct to a putative indirect defense may represent an adaptive tailoring of a plant's defense response. Received: 13 June 1999 / Accepted: 21 August 1999     

A model for the genetic control of differential adhesion in insect epidermis

Adhesive relations among cells are believed to play a major role in determining patterns of serial homology, of intercalary regeneration, and of neuronal connectivity. Models for the genetic control of adhesion during development can provide a framework for further analysis of these phenomena. Investigators studying development of Drosophila have proposed that differentiation of segments and of imaginal discs is controlled by a set of bistable “selector genes”. In each region the settings of the selector genes form a binary “word” which determines the properties of cells in the region, including their adhesiveness. I have made an explicit proposal for the relation between binary words and adhesiveness, by assuming that active selector genes repress synthesis of “adhesor” macromolecules, which promote adhesion. This hypothesis correctly predicts the relative cohesiveness of cells in four pupal tissues of the moth Manduca. Works of cohesion and adhesion among the four cell types are deduced from published results of grafting experiments by modelling insect epidermis as a viscoelastic fluid.Further comparisons between deductions from the genetic and fluid models suggest that selector genes, or the adhesor molecules they regulate, interact within single cells in determining adhesiveness between cells. From a specific version of the genetic model I deduce that pairwise interactions between selector genes or adhesor molecules can determine many, though not all, of the relative works of adhesion between unlike cells in Manduca. The genetic and fluid models thus provide a set of working hypotheses for predicting patterns of intercellular adhesion in insect epidermis and for analyzing results of experiments designed to test such predictions.     

Regulation of the mitochondrial adenine nucleotide pool size

A mechanism by which normal adult rat liver mitochondria may regulate the matrix adenine nucleotide content was studied in vitro. If mitochondria were incubated with 1 mm ATP at 30 ° C in 225 mm sucrose, 2 mm K₂HPO₄, 5 mm MgCl₂, and 10 mm Tris-Cl (pH 7.4), the adenine nucleotide pool size increased at a rate of 0.44 ± 0.02 nmol/mg mitochondrial protein/min. The rate of adenine nucleotide accumulation under these conditions was concentration dependent and specific for ATP or ADP; AMP was not taken up. The rate of net ADP uptake was 50–75% slower than that for ATP. The K_m values for net uptake of ATP and ADP were 2.08 and 0.36 mm, respectively. Adenine nucleotide uptake was stoichiometrically dependent on Mg²⁺ and stimulated by inorganic phosphate. Net uptake was inhibited by n-ethylmaleimide, or mersalyl, but not by n-butylmalonate. Nigericin inhibited net uptake, but valinomycin did not. In the presence of uncouplers, net uptake was not only inhibited, but adenine nucleotide efflux was observed instead. Like uptake, uncoupler-induced efflux of adenine nucleotides was inhibited by mersalyl, indicating that a protein was required for net flux in either direction. Carboxyatractyloside, bongkrekic acid, or respiratory substrates reduced the rate of adenine nucleotide accumulation, however, this did not appear to be a direct inhibition of the transport process, but rather was probably related indirectly to an increase in the matrix $\text{ATP}\text{ADP}$ ratio. The collective properties of the transport mechanism(s) for adenine uptake and efflux were different from those which characterize any of the known transport systems. It is proposed that uptake and efflux operate to regulate the total matrix adenine nucleotide pool size: a constant pool size is maintained if the rates of uptake and efflux are equal. Transient alterations in the relative rates of uptake and efflux may occur in response to hormones or other metabolic signals, to bring about net changes in the pool size.     

Short-term nicotine exposure induces long-lasting modulation of gustatory plasticity in Caenorhabditis elegans

Nicotine administration induces many effects on animal behavior. In wild-type Caenorhabditis elegans, gustatory plasticity results in reduced chemotaxis toward NaCl of otherwise attractive concentrations after pre-exposure to 100 mM NaCl in the absence of food. However, acute nicotine administration during a 15 min pre-exposure period inhibits gustatory plasticity, whereas chronic nicotine administration during worm development facilitates the plasticity. To investigate the relationship between the duration of nicotine administration and its effects, we exposed worms to nicotine for various periods during development. The modulatory effect of nicotine on gustatory plasticity was gradually switched from inhibition to facilitation with increased duration of nicotine administration. Moreover, inhibition of plasticity was sustained after relatively short-term chronic administration, with effects lasting for 45 h after the removal of nicotine. Similar to the acute inhibitory effect after 15 min nicotine pre-exposure, the inhibitory effect after short-term chronic administration was dependent on the nicotinic acetylcholine receptor subunit genes lev-1 and unc-29, and genes involved in serotonin biosynthesis bas-1 and tph-1. The impaired inhibition in bas-1 and tph-1mutants was recovered by exogenous serotonin, demonstrating that serotonin plays an important role in the long-lasting inhibitory effects of short-term chronic nicotine exposure.     

Effects of Abscisic Acid and Cytoplasmic pH on Potassium and Chloride Efflux in Arabidopsis thaliana Seedlings

The effects of ABA, isobutyric acid (IBA) and nicotine on K⁺and Cl⁺ efflux were studied in Arabidopsis thaliana seedlings,and the role of pH_cyt, and E_m in the regulation of the effluxof these ions was discussed. The data show that treatments withIBA and nicotine influenced in opposite directions the effluxof either K⁺ or Cl^–: K⁺ efflux was increased by nicotineand reduced in the presence of IBA, whereas Cl^– effluxwas stimulated by IBA and decreased by nicotine treatment. Underall the conditions tested ABA induced cytoplasmic acidificationand inhibition of K⁺ and Cl^– net efflux. Experiments aimedto estimate the individual contribution of pH_cyt and E_m in modulatingK^– efflux indicated that, within the range of acidic pH_cytvalues, a regulation of K⁺ efflux was imposed by pH_cyt on thecontrol exerted by E_m, the efflux being inhibited by lower pH_cytvalues. Conversely, in the alkaline side of pH_cyt K⁺ effluxseemed linked only to the E_m values. These results are consistentwith the hypothesis that the decrease in K⁺ efflux observedin non-stomatal tissues in the presence of ABA may be mediatedby the cytoplasmic acidification induced by the hormone. (Received August 6, 1996; Accepted January 19, 1997)     

A Characterization of the Manduca sexta Serotonin Receptors in the Context of Olfactory Neuromodulation

Neuromodulation, the alteration of individual neuron response properties, has dramatic consequences for neural network function and is a phenomenon observed across all brain regions and taxa. However, the mechanisms underlying neuromodulation are made complex by the diversity of neuromodulatory receptors expressed within a neural network. In this study we begin to examine the receptor basis for serotonergic neuromodulation in the antennal lobe of Manduca sexta. To this end we cloned all four known insect serotonin receptor types from Manduca (the Ms5HTRs). We used phylogenetic analyses to classify the Ms5HTRs and to establish their relationships to other insect serotonin receptors, other insect amine receptors and the vertebrate serotonin receptors. Pharmacological assays demonstrated that each Ms5HTR was selective for serotonin over other endogenous amines and that serotonin had a similar potency at all four Ms5HTRs. The pharmacological assays also identified several agonists and antagonists of the different Ms5HTRs. Finally, we found that the Ms5HT1A receptor was expressed in a subpopulation of GABAergic local interneurons suggesting that the Ms5HTRs are likely expressed heterogeneously within the antennal lobe based on functional neuronal subtype.     

A new form of antibiosis in nicotiana

Species belonging to the genus Nicotiana, section Repandae, imparted high levels of mortality to Manduca sexta, the tobacco hornworm, a tobacco-associated insect which is not susceptible to the toxic effects of nicotine. One hundred per cent mortality was observed after 24 hr following topical application of 1 mg of crude exudate material. The nicotinoid antibiosis component of the leaf exudate was shown to be different from conventional nicotinoid alkaloids. Ten micrograms of the active alkaloidal material caused 87–100% mortality within 24 hr. The nicotine derivative produced by these plants is thus able to circumvent the insect's physiological defence against nicotine.     

Blood-to-brain influx transport of nicotine at the rat blood–brain barrier: Involvement of a pyrilamine-sensitive organic cation transport process

Nicotine is the most potent neural pharmacological alkaloid in tobacco, and the modulation of nicotine concentration in the brain is important for smoking cessation therapy. The purpose of this study was to elucidate the net flux of nicotine transport across the blood–brain barrier (BBB) and the major contributor to nicotine transport in the BBB. The in vivo brain-to-blood clearance was determined by a combination of the rat brain efflux index method and a rat brain slice uptake study, and the blood-to-brain transport of nicotine was evaluated by in vivo vascular injection in rats and a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13 cells) as an in vitro model of the rat BBB. The blood-to-brain nicotine influx clearance was obtained by integration plot analysis as 272 μL/(min g brain), and this value was twofold greater than the brain-to-blood efflux clearance (137 μL/(min g brain)). Thus, it is suggested that the net flux of nicotine transport across the BBB is dominated by blood-to-brain influx transport. In vivo blood-to-brain nicotine transport was inhibited by pyrilamine. [³H]Nicotine uptake by TR-BBB13 cells exhibited time-, temperature-, and concentration-dependence with a K_m value of 92 μM. Pyrilamine competitively inhibited nicotine uptake by TR-BBB13 cells with a K_i value of 15 μM, whereas substrates and inhibitors of organic cation transporters had little effect. These results suggest that pyrilamine-sensitive organic cation transport process(es) mediate blood-to-brain influx transport of nicotine at the BBB, and this is expected to play an important role in regulating nicotine-induced neural responses.