Development of an identified serotonergic neuron in the antennal lobe of the moth and effects of reduction in serotonin during construction of olfactory glomeruli

Each olfactory (antennal) lobe of the moth Manduca sexta contains a single serotonin (5-HT) immunoreactive neuron whose processes form tufted arbors in the olfactory glomeruli. To extend our present understanding of the intercellular interactions involved in glomerulus development to the level of an individual, identified antennal lobe neuron, we first studied the morphological development of the 5-HT neuron in the presence and absence of receptor axons. Development of the neuron's glomerular tufts depends, as it does in the case of other multiglomerular neurons, on the presence of receptor axons. Processes of the 5-HT neuron are excluded from the region in which the initial steps of glomerulus construction occur and thus cannot provide a physical scaffolding on which the array of glomeruli is organized. Because the neuron's processes are present in the antennal lobe neuropil throughout postembryonic development, 5-HT could provide signals that influence the pattern of development in the lobe. By surgically producing 5-HT-depleted antennal lobes, we also tested the importance of 5-HT in the construction of olfactory glomeruli. Even in the apparent absence of 5-HT, the glomerular array initiated by the receptor axons was histologically normal, glial cells migrated to form glomerular borders, and receptor axons formed terminal branches in their normal region within each glomerulus. In some cases, 5-HT-immunoreactive processes from abnormal sources entered the lobe and formed the tufted intraglomerular branches typical of most antennal lobe neurons, suggesting that local cues strongly influence the branching patterns of developing antennal lobe neurons. © 1995 John Wiley & Sons, Inc.     

Neither barium nor calcium prevents the inhibition by Bacillus thuringiensis δ-endotoxin of sodium- or potassium gradient-dependent amino acid accumulation by tobacco hornworm midgut brush border membrane vesicles

Rapid filtration assays were used to determine the effects of barium, calcium and an insecticidal δ-endotoxin from Bacillus thuringiensis on sodium and potassium ion gradient dependent phenylalanine accumulation by brush border membrane vescles from the larval midgut of the tobacco hornworm (Manduca sexta). Neither barium nor calcium had a significant effect on sodium ion gradient dependent phenylalanine accumulation by the membrane vesicles. Both barium and calcium inhibited potassium ion gradient dependent phenylalanine accumulation by the membrane vesicles. B. thuringiensis δ-endotoxin inhibited both sodium and potassium ion gradient dependent phenylalanine accumulation by the vesicles. Inhibition of both sodium and potassium ion gradient dependent phenylalanine accumulation increased similarly with increasing δ-endotoxin inhibition of either sodium or potassium dependent phenylalanine accumulation by the vesicles.     

Studies on vitellogenin from the tobacco hornworm,Manduca sexta

In the tobacco hornworm moth, Manduca sexta, vitellogenin (Vg) is a very high-density (1.29 g/ml) phosphoglycolipoprotein containing 13% lipids, 3% carbohydrates, and 0.6% protein-bound phosphorus. Vitellogenin (M_r～500,000) has two apoproteins designated apoVg-l (M_r 177,000 ± 3,600) and apoVg-ll (M_r45,000 ± 5,000). ApoVg-l and apoVg-II can be dissociated with 6 M guanidine HCI and separated from each other by gel permeation chromatography. Immunoblotting experiments using antibodies against the apoproteins showed that apoVg-l and apoVg-II antigens were immunologically distinct polypeptides. Antibodies against Vg reacted only with apoVg-l. Antibodies against Vg and apoVg-l reacted with Vg in double immunodiffusion experiments, whereas antibodies against apoVg-II did not. These results suggest that in the native Vg molecule, apoVg-II is positioned inside the molecule away from the aqueous environment. Only apoVg-I contained covalently bound carbohydrate as shown by fluorescein isothiocyanateconjugated concanavalin A, periodate-Schiff reagent, and in vivo labeling with ³H-Man. In vivo labeling with ³²P-inorganic phosphate and chemical determination showed that apoproteins of both Vg and vitellin contain covalently bound phosphate groups.     

Juvenile hormone regulates the titer of a hemolymph factor that enhances ecdysone synthesis by insect (Manduca sexta) prothoracic glands in vitro

The hemolymph of last instar Manduca sexta larvae contains a protein factor that enhances ecdysone synthesis by prothoracic glands in vitro. The titer of the factor fluctuates during development in a pattern that suggests that it is regulated by juvenile hormone (JH). In untreated control larvae, the titer drops from 2.17 U ml^?1 on day 1 to 0.27 U ml^?1 on day 3. When larvae were treated with (7S)-hydroprene (a JH analog), the titer remained elevated (2.09 U ml^?1 on day 3). JH I, however, was ineffective in preventing the precommitment drop in the titer of the factor. After pupal commitment, the titer of the factor increases in untreated larvae from 0.84 U ml^?1 on day 5 to 1.62 U ml^?1 on day 7. This increase was blocked when the sources of JH (the corpora allata) were removed on day 5 by head ligation. When head-ligated day 5 larvae were treated with either (7S)-hydroprene or JH I, the titer of the factor was driven to a level (1.88 U ml^?1 and 2.05 U ml^?1, respectively) that was not significantly different from that found in untreated day 7 larvae (1.62 U ml^?1). The combined results indicate the titer of the hemolymph factor is regulated by JH.     

Differentiation of roles of chemosensory organs in food discrimination among host and non-host plants by larvae of the tobacco hornworm, Manduca sexta

ABSTRACT. The contributions of olfactory and gustatory organs in food plant discrimination were examined in larvae of Manduca sexta (Johan.) (Lepidoptera, Sphingidae). Larvae, from which various chemosensory organs had been removed surgically, were tested for feeding preferences for a host, tomato ( Lycopersicon esculentum ); a weakly acceptable non-host, rape ( Brassica napus ); and an unacceptable non-host canna ( Canna generalis ), using a two-choice disc bioassay.
Removal of all known chemosensory organs resulted in failure to show discriminatory behaviour in a strictly chemosensory bioassay, indicating that all external chemosensory organs have been accounted for. The involvement of non-chemosensory organs results in residual discrimination for leaves by individuals with total chemosensory ablations.
Larvae possessing either olfactory or gustatory organs still exhibit normal preferences for tomato over rape. Gustatory (but not olfactory) organs are required for larvae to show normal preferences for tomato over canna; in fact, olfactory organs do not appear to participate in this decision.
To examine which if any of the plant species is being selected in two-choice tests, larvae were given a choice between each leaf species and a 'neutral' substance (wet filter paper). Both olfactory and gustatory organs are required for normal preferences for tomato, but either alone will suffice for rape. Only gustation is needed to select canna, and participation of either the epipharyngeal sensilla or a single medial sensillum styloconicum is sufficient to elicit complete rejection behaviour.
We conclude that, in larvae of M. sexta , the complement of chemosensory organs needed for food plant discrimination varies with the plant species sampled. Evidence is presented exposing a potential artefact of ablation experiments; extirpation of one sensory organ may affect the functioning of others nearby, even though they may appear normal by visual inspection.     

Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [³⁵S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of M_r ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ～8.6). Electrophoresis under denaturing conditions reveals a subunit M_r ≈ 50,000 while the native protein has an apparent M_r ～ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.     

Using nutritional indices to study LOX3-dependent insect resistance

Induced resistance to biotic attackers is thought to be mediated by responses elicited by jasmonic acid (JA), a subset of which are lipoxygenase 3 (LOX3) dependent. To understand the importance of LOX3-mediated insect resistance, we analysed the performance of Manduca sexta larvae on wild-type (WT) and on isogenic Nicotiana attenuata plants silenced in NaLOX3 expression and JA signalling, and we used Waldbauer nutritional indices to measure the pre- and post-ingestive effects. LOX3-mediated defenses reduced larval growth, consumption and frass production. These defenses reduced how efficiently late-instar larvae converted digested food to body mass (ECD). In contrast, LOX3-mediated defenses decreased approximate digestibility (AD) in early instar larvae without affecting the ECD and total food consumption. Larvae of all instars feeding on defended WT plants behaviourally compensate for their reduced body mass by consuming more food per unit of body mass gain. We suggest that larvae feeding on plants silenced in NaLOX3 expression (as-lox) initially increase their AD, which in turn enables them to consume more food in the later stages and consequently, to increase their ECD and efficiency of conversion of ingested food (ECI). We conclude that N. attenuata's oxylipin-mediated defenses are important for resisting attack from M. sexta larvae, and that Waldbauer nutritional assays reveal behavioural and physiological counter responses of insects to these plant defenses.     

Jasmonic acid signalling and herbivore resistance traits constrain regrowth after herbivore attack in Nicotiana attenuata

Because traits conferring resistance on herbivores can reduce fitness-associated traits, trade-offs may occur between tolerance and resistance responses. We examined these trade-offs in genotypes of Nicotiana attenuata that were transformed to silence trypsin proteinase inhibitor (TPI) production (AS-Natpi), an antiherbivore defense associated with (14%) reductions in seed production, and the jasmonate signal cascade that elicits these defenses (AS-Nalox3), by measuring stalk and axillary branch growth and seed production after two defoliation regimes and Manduca sexta larval attack to bottom or middle and top stalk leaves. Larval attack and defoliation at middle and top leaves depressed seed production and increased axillary branching more than at bottom leaves. AS-Nalox3 and AS-Natpi plants produced significantly longer (two- to fourfold) branches than did wild-type (WT) plants, results that are consistent with resource-based trade-offs between resistance and regrowth. Methyl jasmonate (MeJA) treatment of AS-Nalox3 plants restored WT branch growth, suggesting that jasmonic acid (JA) signalling suppresses regrowth and contributes to apical dominance. These results are consistent with the existence of JA- and resource-mediated trade-offs between regrowth and herbivore resistance traits.     

Separation of insect hemolymph proteins by cascade-mode multi-affinity chromatography.

The hemolymph of the adult female Manduca sexta was fractionated by cascade-mode multi-affinity chromatography (CASMAC) on a main-line tandem column chain containing Zn(2+)-TED, T-gel, Ni(2+)-DPA, and phenylsepharose and a side-line column containing Zn(2+)-DPA. The technique separated some of the previously described major hemolymph proteins, and yielded a number of fractions with simple composition. Some of these fractions contained only less abundant proteins of Manduca hemolymph. Thus, it appears that CASMAC would be a very useful fractionation technique for purification and characterization of the minor proteins of insect hemolymph.     

Immunolocalization of a proton V-ATPase in ovarian follicles of the tobacco hornworm Manduca sexta

A monoclonal antibody, directed against an H⁺ translocating V-ATPase of the midgut of Manduca sexta, has been used for immunolocalization studies in ovarian follicles and testes of Manduca sexta. In testes, no distinct staining above background levels was observed. In vitellogenic follicles, V-ATPase immunoreactivity first appears in the cytoplasm of the trophocytes and then in the oocyte, but by far the strongest reaction is present in the region of the oolemma during endocytosis. All types of follicle cells surrounding both the oocyte and the trophocyte compartments show a distinct positive reaction. In the cylindrical follicle cells surrounding the oocyte, the immunoreactivity is clearly restricted to the basal part. Our results suggest an important role for V-ATPase in vitellogenin uptake in Manduca, similar to that suggested on electro-physiological grounds in Hyalophora cecropia. © 1995 Wiley-Liss, Inc.     

Variability in odor-modulated flight by moths

Based on previous studies of odor-modulated flight where track parameter data was lumped and averaged, the speed and orientation of the moths' movement along their flight tracks have been said to be controlled to maintain certain “preferred” values. The results from our fine-scaled analysis of this behavior show that none of the track parameters typically measured are held constant. The moths' speed along the flight track is modulated substantially and predictably: fastest along the straight legs and slowest around the turns. In addition, about half of the individuals studied progressively reduced the peak speed along the straight legs as they approached the pheromone source. While most of the track legs between the turns were directed upwind, their orientations were widely distributed, indicating no preferred direction. Small fluctuations of orientation along some straight legs suggest corrective maneuvers to stabilize flight direction about an internal set point. The visual inputs hypothesized to control steering and speed, transverse and longitudinal image flow, changed continuously during upwind flight in pheromone, but no regular relationship between them was observed. We found that the orientation of the longitudinal body axis and the direction of thrust (course angle) were only rarely coincident during upwind flight to the odor source, suggesting that moths receive sensory input which differs quantitatively from that calculated by conventional methods. Our results strongly suggest that the long-accepted hypothetical mechanisms of control for this behavior do not operate in the manner in which they have been proposed. Accepted: 11 July 1997     

pfaB products determine the molecular species produced in bacterial polyunsaturated fatty acid biosynthesis

Insect blood (hemolymph) contains prophenoloxidase, a proenzyme that is activated to protective phenoloxidase when the insect is damaged or challenged with microorganisms. The Gram-negative bacterium Photorhabdus luminescens kills the lepidopteron insect Manduca sexta by using a variety of toxins. We screened P. luminescens and Photorhabdus asymbiotica cosmid libraries in an Escherichia coli host against previously activated M. sexta hemolymph phenoloxidase and identified three overlapping cosmid clones from P. luminescens and five from P. asymbiotica that suppressed the activity of the enzyme both in vitro and in vivo . Genome alignments of cosmid end sequences from both species confirmed that they contained orthologous loci. We examined one of the cosmids from P. luminescens in detail: it induced the formation of significantly fewer melanotic nodules, proliferated faster within the insect host and was significantly more virulent towards fifth-stage larvae than E. coli control bacteria. Insertional mutagenesis of this cosmid yielded 11 transposon mutants that were no longer inhibitory. All of these were insertions into a single 5.5-kb locus, which contained three ORFs and was homologous to the maltodextrin phosphorylase locus of E. coli . The implications of this novel inhibitory factor of insect phenoloxidase for Photorhabdus virulence are discussed.     

Organization of the antennal motor system in the sphinx moth Manduca sexta

The antennae of the sphinx moth Manduca sexta are multimodal sense organs, each comprising three segments: scape, pedicel, and flagellum. Each antenna is moved by two systems of muscles, one controlling the movement of the scape and consisting of five muscles situated in the head capsule (extrinsic muscles), and the other system located within the scape (intrinsic muscles) and consisting of four muscles that move the pedicel. At least seven motoneurons innervate the extrinsic muscles, and at least five motoneurons innervate the intrinsic muscles. The dendritic fields of the antennal motoneurons overlap one another extensively and are located in the neuropil of the antennal mechanosensory and motor center. The density of motoneuronal arborizations is greatest in the lateral part of this neuropil region and decreases more medially. None of the motoneurons exhibits a contralateral projection. The cell bodies of motoneurons innervating the extrinsic muscles are distributed throughout an arching band of neuronal somata dorsal and dorsolateral to the neuropil of the antennal mechanosensory and motor center, whereas the cell bodies of motoneurons innervating the intrinsic muscles reside mainly among the neuronal somata situated dorsolateral to that neuropil. Received: 30 March 1996 / Accepted: 23 June 1996     

Absorption and storage of phosphorus by larval Manduca sexta

The role of phosphorus (P) in numerous important biological structures, coupled with the observation that P-content of many insect foods is disproportionately low, suggests that P may be a critical nutrient for growing insects — however, the few studies examining the effects of dietary P on insect performance have generally found only weak relationships. This mismatch may be reconciled by understanding the physiological mechanisms by which insects handle P. Here we describe P processing by larvae of Manduca sexta. When given un-manipulated leaves of a common host plant, Datura wrightii, fifth-instar larvae retained about 85% of P consumed; when given P-enriched leaves larvae retained only 25% of P consumed. Analysis of gut concentrations of P at four sites along the digestive tract, and in leaves and feces, indicates that the rectum is the primary site of P transport between the gut and body and that differences in P retention may be accounted for by differential rates of rectal P transport. Larvae given P-enriched leaves also showed an eightfold increase in the concentration of P in the hemolymph, primarily as α-glycerophosphate — but only a 12% increase in the concentration of P in body tissues, suggesting that hemolymph plays a central role in storage and buffering of P.