Flight duration and flight muscle ultrastructure of unfed hawk moths.

Flight muscle breakdown has been reported for many orders of insects, but the basis of this breakdown in insects with lifelong dependence on flight is less clear. Lepidopterans show such muscle changes across their lifespans, yet how this change affects the ability of these insects to complete their life cycles is not well documented. We investigated the changes in muscle function and ultrastructure of unfed aging adult hawk moths (Manduca sexta). Flight duration was examined in young, middle-aged, and advanced-aged unfed moths. After measurement of flight duration, the main flight muscle (dorsolongitudinal muscle) was collected and histologically prepared for transmission electron microscopy to compare several measurements of muscle ultrastructure among moths of different ages. Muscle function assays revealed significant positive correlations between muscle ultrastructure and flight distance that were greatest in middle-aged moths and least in young moths. In addition, changes in flight muscle ultrastructure were detected across treatment groups. The number of mitochondria in muscle cells peaked in middle-aged moths. Many wild M. sexta do not feed as adults; thus, understanding the changes in flight capacity and muscle ultrastructure in unfed moths provides a more complete understanding of the ecophysiology and resource allocation strategies of this species.

Control of moth flight posture is mediated by wing mechanosensory feedback.

Flying insects rapidly stabilize after perturbations using both visual and mechanosensory inputs for active control. Insect halteres are mechanosensory organs that encode inertial forces to aid rapid course correction during flight but serve no aerodynamic role and are specific to two orders of insects (Diptera and Strepsiptera). Aside from the literature on halteres and recent work on the antennae of the hawkmoth Manduca sexta, it is unclear how other flying insects use mechanosensory information to control body dynamics. The mechanosensory structures found on the halteres, campaniform sensilla, are also present on wings, suggesting that the wings can encode information about flight dynamics. We show that the neurons innervating these sensilla on the forewings of M. sexta exhibit spike-timing precision comparable to that seen in previous reports of campaniform sensilla, including haltere neurons. In addition, by attaching magnets to the wings of moths and subjecting these animals to a simulated pitch stimulus via a rotating magnetic field during tethered flight, we elicited the same vertical abdominal flexion reflex these animals exhibit in response to visual or inertial pitch stimuli. Our results indicate that, in addition to their role as actuators during locomotion, insect wings serve as sensors that initiate reflexes that control body dynamics.

The skeletomuscular system of the larva of Drosophila melanogaster (Drosophilidae, Diptera): a contribution to the morphology of a model organism.

The morphological features of the third instar larva of the most important insect model, Drosophila melanogaster, are documented for the first time using a broad spectrum of modern morphological techniques. External structures of the body wall, the cephaloskeleton, and the musculature are described and illustrated. Additional information about other internal organs is provided. The systematic implications of the findings are discussed briefly. Internal apomorphic features of Brachycera and Cyclorrhapha are confirmed for Drosophila. Despite the intensive investigations of the phylogeny of the megadiverse Diptera, evolutionary reconstructions are still impeded by the scarcity of anatomical data for brachyceran larvae. The available morphological information for the life stages of three insect model organisms -D. melanogaster (Diptera, Drosophilidae), Manduca sexta (Lepidoptera, Sphingidae) and Tribolium castaneum (Coleoptera, Tenebrionidae) - is addressed briefly. The usefulness of a combination of traditional and innovative techniques for an optimized acquisition of anatomical data for different life stages is highlighted.

Localization of a GABA transporter to glial cells in the developing and adult olfactory pathway of the moth Manduca sexta.

Glial cells have several critical roles in the developing and adult olfactory (antennal) lobe of the moth Manduca sexta. Early in development, glial cells occupy discrete regions of the developing olfactory pathway and processes of gamma-aminobutyric acid (GABA)ergic neurons extend into some of these regions. Because GABA is known to have developmental effects in a variety of systems, we explored the possibility that the glial cells express a GABA transporter that could regulate GABA levels to which olfactory neurons and glial cells are exposed. By using an antibody raised against a characterized high-affinity M. sexta GABA transporter with high sequence homology to known mammalian GABA transporters (Mbungu et al. [1995] Arch. Biochem. Biophys. 318:489-497; Umesh and Gill [2002] J. Comp. Neurol. 448:388-398), we found that the GABA transporter is localized to subsets of centrally derived glial cells during metamorphic adult development. The transporter persists into adulthood in a subset of the neuropil-associated glial cells, but its distribution pattern as determined by light-and electron-microscopic-level immunocytochemistry indicates that it could not serve to regulate GABA concentration in the synaptic cleft. Instead, its role is more likely to regulate extracellular GABA levels within the glomerular neuropil. Expression in the sorting zone glial cells disappears after the period of olfactory receptor axon ingrowth, but may be important during ingrowth if GABA regulates axon growth. Glial cells take up GABA, and that uptake can be blocked by L-2,4-diaminobutyric acid (DABA). This is the first molecular evidence that the central glial cell population in this pathway is heterogeneous.

Atomic force microscopy imaging of Bacillus thuringiensis Cry1 toxins interacting with insect midgut apical membranes.

Atomic force microscopy was used to image Bacillus thuringiensis (Bt) toxins interacting with their natural targets, Manduca sexta midgut brush border membranes (BBMs), as well as with dipalmitoylphosphatidylcholine-dioleoylphosphatidylcholine (DPPC-DOPC) solid-supported lipid bilayers. In lipid bilayers, Cry1Aa formed structures 30-60 nm wide and 3-7 nm high, mostly at the interface of domains formed by the two different lipids or at the edge of DOPC-enriched domains. BBM vesicles, in the absence of toxin, formed flat membrane fragments of up to 25 microm(2) and 4.2 nm high, with irregular embedded structures. After incubation with Cry1Aa, Cry1Ac and Cry1C, which are active against M. sexta, new structures, 35 nm wide and 5.1-6.7 nm high, were observed in some membrane fragments, sometimes only in particular regions. Their density, which reached a plateau within 4 h, was toxin- and concentration-dependent. The structures formed by Cry1Ac were often grouped into dense, two-dimensional arrangements. No such specific interactions were observed with Cry1Ba, which is inactive against M. sexta. This study provides the first visual demonstration of specific interactions of Bt toxins with insect midgut BBMs at the nanometric scale. The observed structures likely represent the protein complexes forming functional Bt pores in target membranes.

Influence of the biophysical and biochemical environment on the kinetics of pore formation by Cry toxins.

The effect of Bacillus thuringiensis toxins on the permeability of the luminal membrane of Manduca sexta midgut columnar epithelial cells is strongly influenced by several biophysical and biochemical factors, including pH, ionic strength, and divalent cations, suggesting an important role for electrostatic interactions. The influence of these factors can differ greatly, however, depending on the toxin being studied, even for closely related toxins such as Cry1Ac and Cry1Ca. In the present study, the possibility of using temperature changes as a tool for controlling the rate and extent of pore formation in midgut brush border membrane vesicles was evaluated. Lowering temperature gradually decreased the rate of pore formation, but had little effect on the permeability of vesicles previously incubated with toxin at room temperature. The formation of new pores, following incubation of the vesicles with toxin, could thus be almost abolished by rapidly cooling the vesicles to 2 degrees C. Using this approach, changes in the rate of pore formation could be more easily distinguished from alterations in the properties of the pores formed, thus allowing a more detailed analysis of the kinetics and mechanism of pore formation.

Expression of Cry1Ac cadherin receptors in insect midgut and cell lines.

Cadherin-like proteins have been identified as putative receptors for the Bacillus thuringiensis Cry1A proteins in Heliothis virescens and Manduca sexta. Immunohistochemistry showed the cadherin-like proteins are present in the insect midgut apical membrane, which is the target site of Cry toxins. This subcellular localization is distinct from that of classical cadherins, which are usually present in cell-cell junctions. Immunoreactivity of the cadherin-like protein in the insect midgut was enhanced by Cry1Ac ingestion. We also generated a stable cell line Flp-InT-REX-293/Full-CAD (CAD/293) that expressed the H. virescens cadherin. As expected, the cadherin-like protein was mainly localized in the cell membrane. Interestingly, toxin treatment of CAD/293 cells caused this protein to relocalize to cell membrane subdomains. In addition, expression of H. virescens cadherin-like protein affects cell-cell contact and cell membrane integrity when the cells are exposed to activated Cry1Ab/Cry1Ac.

Activity affects dendritic shape and synapse elimination during steroid controlled dendritic retraction in Manduca sexta.

Insect metamorphosis is a compelling example for dendritic and synaptic remodeling as larval and adult behaviors place distinct demands on the CNS. During the metamorphosis of the moth, Manduca sexta, many larval motoneurons are remodeled to serve a new function in the adult. During late larval life, steroid hormones trigger axonal and dendritic regression as well as larval synapse elimination. These regressive events are accompanied by stereotypical changes in motor behavior during the so-called wandering stages. Both normally occurring changes in dendritic shape and in motor output have previously been analyzed quantitatively for the individually identified motoneuron MN5. This study tested whether activity affected steroid-induced dendritic regression and synapse disassembly in MN5 by means of chronically implanted extracellular electrodes. Stimulating MN5 in vivo in intact, normally developing animals during a developmental period when it usually shows no activity significantly slowed the regression of high-order dendrites. Both physiological and anatomical analysis demonstrated that reduced dendritic regression was accompanied by a significant reduction in larval synapse disassembly. Therefore, steroid-induced alterations of dendritic shape and synaptic connectivity are modified by activity-dependent mechanisms. This interaction might be a common mechanism for rapid adjustments of rigid, inflexible, hormonal programs.

Analysis of the properties of Bacillus thuringiensis insecticidal toxins using a potential-sensitive fluorescent probe.

A potential-sensitive fluorescent probe, 3,3'-dipropylthiadicarbocyanine iodide, was used to analyze, at pH 7.5 and 10.5, the effects of Bacillus thuringiensis toxins on the membrane potential generated by the efflux of K(+) ions from brush border membrane vesicles purified from the midgut of the tobacco hornworm, Manduca sexta. Fluorescence levels were strongly influenced by the pH and ionic strength of the media. Therefore, characterization of the effects of the toxins was conducted at constant pH and ionic strength. Under these conditions, the toxins had little effect on the fluorescence levels measured in the presence or absence of ionic gradients, indicating that the ionic selectivity of their pores is similar to that of the intact membrane. Valinomycin greatly increased the potential generated by the diffusion of K(+) ions although membrane permeability to the other ions used to maintain the ionic strength constant also influenced fluorescence levels. In the presence of valinomycin, active toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C and Cry1E) efficiently depolarized the membrane at pH 7.5 and 10.5.

Differential effects of pH on the pore-forming properties of Bacillus thuringiensis insecticidal crystal toxins.

The effect of pH on the pore-forming ability of two Bacillus thuringiensis toxins, Cry1Ac and Cry1C, was examined with midgut brush border membrane vesicles isolated from the tobacco hornworm, Manduca sexta, and a light-scattering assay. In the presence of Cry1Ac, membrane permeability remained high over the entire pH range tested (6.5 to 10.5) for KCl and tetramethylammonium chloride, but was much lower at pH 6.5 than at higher pHs for potassium gluconate, sucrose, and raffinose. On the other hand, the Cry1C-induced permeability to all substrates tested was much higher at pH 6.5, 7.5, and 8.5 than at pH 9.5 and 10.5. These results indicate that the pores formed by Cry1Ac are significantly smaller at pH 6.5 than under alkaline conditions, whereas the pore-forming ability of Cry1C decreases sharply above pH 8.5. The reduced activity of Cry1C at high pH correlates well with the fact that its toxicity for M. sexta is considerably weaker than that of Cry1Aa, Cry1Ab, and Cry1Ac. However, Cry1E, despite having a toxicity comparable to that of Cry1C, formed channels as efficiently as the Cry1A toxins at pH 10.5. These results strongly suggest that although pH can influence toxin activity, additional factors also modulate toxin potency in the insect midgut.

Flight-motor-driven respiratory air flow in the hawkmoth Manduca sexta.

Intratracheal pressure during tethered flight was analyzed at the anterior spiracles and mesoscutellar air sacs in the hawkmoth Manduca sexta using electronic pressure sensors. CO(2) emission from the anterior spiracles and the posterior thoracic and abdominal spiracles was measured using a URAS gas analyzer with a split-specimen chamber. Experiments were accompanied by photocell recordings of the wingbeat. The structural differences between the mesothoracic and metathoracic spiracles are described. Deformations of the lateral thorax and their effect upon the spiracles were observed under stroboscopic light. During shivering, ventilation pulses are generated by the flight muscles reminiscent of an autoventilation mechanism with tidal air flow. During steady flight, however, a unidirectional airstream arises with a mean negative (subatmospheric) pressure at the first (mesothoracic) spiracles and a mean positive pressure in the mesoscutellar air sacs. As a result of this pressure difference during flight, CO(2) is emitted only at the posterior spiracles. The suction force for the inspiration flow at the anterior spiracles is generated by the flight apparatus as a result of prevention of inspiration through the posterior thoracic spiracles. During the downstroke, the volume of the thoracic air sacs increases, while the posterior thoracic spiracles are automatically enclosed in the subalar cleft below the wing hinge and are probably closed. During the upstroke, the air sac volume decreases and the moth expires through the open posterior spiracles.

Identification of the cellular target for eclosion hormone in the abdominal transverse nerves of the tobacco hornworm, Manduca sexta.

The isolated abdominal central nervous system of Manduca sexta undergoes an increase in cyclic GMP (cGMP) when exposed to the insect peptide eclosion hormone (EH) before pupal ecdysis. Previously, cGMP immunocytochemistry revealed that the EH-stimulated increase in cGMP was contained in numerous filamentous processes within the transverse nerve associated with each abdominal ganglion. These processes seemed to be the axons of neurosecretory cells projecting to this neurohemal organ. In the present paper, we now show that the EH-stimulated cGMP is not present in neurosecretory terminals. There is no colocalization of the EH-stimulated cGMP with immunoreactivity of two peptides, known to be present in axons in the transverse nerves. Furthermore, there is no colocalization of EH-stimulated cGMP with the synaptic vesicle protein, synaptotagmin. The neurosecretory axons are localized to a narrow band at the anterior margin of the transverse nerve, whereas the cellular elements showing an EH-stimulated cGMP increase are primarily present in the posterior region. There are two cell types in this region: a granular and a nongranular type. The cGMP immunoreactivity seems to be contained within the nongranular type. During adult development, the cells of the posterior compartment spread in a thin layer between the transverse and dorsal nerves, become positive for myosin immunoreactivity between pupal stages 5 and 8, and seem to form the adult ventral diaphragm muscles. We conclude that the EH-sensitive filaments in the transverse nerves of Manduca are most likely to be intrinsic cells that subsequently develop into the ventral diaphragm muscles of the adult.

Isolation and characterization of an apically sorted 41-kDa protein from the midgut of tobacco hornworm (Manduca sexta).

Immunocytochemical localization and sorting properties of a newly purified 41-kDa protein (MsM41) were investigated in an insect, the tobacco hornworm Manduca sexta. The protein purified from midgut homogenates of feeding fifth-stadium larvae was found exclusively in this tissue on Western blots. Presence of MsM41 protein was indicated in both anterior and posterior regions of the midgut during the whole fifth stadium. However, in the posterior region an additional 39-kDa protein was also detected during the feeding period of the last larval stage. Upon light-microscopic examination immunoreactivity was localized in the columnar cells, while the goblet, endocrine and regenerative cells remained unlabeled. Distribution of the label during the feeding period was different in the anterior and posterior regions. In the anterior region immunoreactivity was localized only to the brush border membrane of columnar cells, while in the posterior region some cytoplasmic structures identified as large trans-Golgi vesicles, endoplasmic reticulum and small secretory vesicles were also labeled. Large, apical extrusions remained immunonegative. In vitro translation confirmed that our protein was expressed only in the posterior region of the midgut. The primary translation product was a 39-kDa protein. Putative post-translational modifications yielded the 41-kDa form, which was then secreted apically. Its presence in the region of the anterior part microvilli was probably due to the countercurrent flux of the ectoperitrophic fluid.

Evidence for a diazepam-binding inhibitor (DBI) benzodiazepine receptor-like mechanism in ecdysteroidogenesis by the insect prothoracic gland.

The diazepam-binding inhibitor (DBI) is a 10-kDa highly evolutionarily conserved multifunctional protein. In mammals, one of DBI's functions is in the activation of steroid hormone biosynthesis via binding to a specific outer mitochondrial membrane receptor (benzodiazepine receptor, BZD) and promoting cholesterol transport to the inner membrane. In this work, a multitiered approach was utilized to study the role of this receptor-like activity in ecdysteroidogenesis by larval insect prothoracic glands (PGs). First, both DBI protein and messenger RNA (mRNA) levels were correlated with peak PG ecdysteroid production. In vitro ecdysteroid production was stimulated by the diazepam analogue FGIN 1-27 and inhibited anti-DBI antibodies. The DBI protein was found distributed throughout PG cells, including regions of dense mitochondria, supposed subcellular sites of ecdysteroid synthesis. Finally, a potential mitochondrial BZD receptor in PG cells was demonstrated by photoaffinity labeling. These results suggest an important role for the insect DBI in the stimulation of steroidogenesis by prothoracic glands and indicate that a pathway for cholesterol mobilization leading to the production of steroid hormones appears to be conserved between arthropods and mammals.

Analysis of the large aqueous pores produced by a Bacillus thuringiensis protein insecticide in Manduca sexta midgut-brush-border-membrane vesicles.

An osmotic swelling assay utilising carboxyfluorescein self-quenching to measure intravesicular volume changes was adapted to investigate permeability changes induced by the Bacillus thuringiensis Cry1Ac delta-endotoxin in Manduca sexta midgut-brush-border-membrane vesicles (BBMV). This assay provides a more quantitative analysis of Cry-toxin-induced BBMV permeability changes, extending our previously published protocol which employed a light-scattering signal to monitor delta-endotoxin activity [Carroll, J. & Ellar, D. J. (1993) Eur. J. Biochem. 214, 771-778]. The fluorescence signal changes, supported by electron microscopy of the BBMV, demonstrated that Cry1Ac altered the membrane permeability for large non-electrolyte solutes. With this approach Cry1Ac was observed to induce or form pores freely permeant for raffinose (1.14 nm diameter) and using non-electrolytes of increasing size the pores were estimated to have a limiting diameter of approximately 2.4-2.6 nm under alkaline pH conditions.

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.

Distribution of acetylcholinesterase activity in the deutocerebrum of the sphinx moth Manduca sexta.

We have used a cytochemical technique to investigate the distribution of acetylcholinesterase (AChE) activity in the deutocerebrum of the brain of the sphinx moth Manduca sexta. To distinguish between extra- and intracellular pools of the enzyme, some brains were treated prior to histochemical staining with echothiophate, an irreversible AChE inhibitor which penetrates cell membranes very slowly and, therefore, inhibits only extracellular AChE. In the antennal nerve, fascicles of presumably mechanosensory fibers show echothiophate-insensitive AChE activity. They bypass the antennal lobe and project to the antennal mechanosensory and motor center of the deutocerebrum. In the antennal lobe, fibers in the coarse neuropil, cell bodies in the lateral cell group, and all glomeruli exhibit AChE activity. In most ordinary glomeruli, echothiophate-sensitive AChE activity is concentrated in the outer cap regions, corresponding to the terminal arborizations of olfactory afferents. A previously unrecognized glomerulus in the ventro-median antennal lobe shows uniform and more intense AChE-specific staining that the other glomeruli. No AChE activity appeared to be associated with male-specific pheromone-sensitive afferents in the macroglomerular complex. About 67 interneurons with somata in the lateral cell group of the antennal lobe show echothiophate-insensitive AChE activity. These neurons seem to be members of two types of antennal-lobe projection neurons with fibers passing through the outer-antennocerebral tract to the protocerebrum. AChE-stained arborizations of these neurons appear to invade all glomeruli, including three distinguishable subunits of the male-specific macroglomerular complex. In echothiophate-treated animals, the projections of one of these types of fiber form large terminals in the lateral horn of protocerebrum, which partly protrude into the adjacent glial cell layer. The results suggest that extracellularly accessible AChE is associated with ordinary olfactory receptor terminals but apparently not with pheromone-sensitive afferents. Intracellular AChE appears to be present in antennal mechanosensory fibers and in two types of olfactory projection neurons of the antennal lobe. The study provides further evidence for cholinergic neurotransmission of most antennal afferents. The AChE-containing interneurons might be cholinergic as well or use the enzyme for functions unrelated to hydrolysis of acetylcholine.