Developmental attenuation of the pre-ecdysis motor pattern in the tobacco hornworm,Manduca sexta

Summary At the culmination of each molt, the larval tobacco hornworm exhibits a pre-ecdysis behavior prior to shedding its old cuticle at ecdysis. Both pre-ecdysis and ecdysis behaviors are triggered by the peptide, eclosion hormone (EH). Pre-ecdysis behavior consists of rhythmic abdominal compressions that loosen the old larval cuticle. This behavior is robust at larval molts, but at the larval-pupal molt the only comparable behavior consists of rhythmic dorso-ventral flexions of the anterior body. These flexions appear to be an attenuated version of the larval pre-ecdysis behavior because (1) they show the same EH dependence, and (2) the motor patterns recorded from EH treated, deafferented larval and pupal preparations are similar except that the pupal pattern is much weaker. Both patterns are characterized by rhythmic, synaptically-driven bursts of action potentials in motoneurons MN-2 and MN-3, which occur synchronously in all segments. However, the synaptic drive to the motoneurons and their resultant levels of activity are reduced during the pupal pre-ecdysis motor pattern, especially in posterior abdominal segments. Although the dendritic arbors of both motoneurons regress somewhat during the larval-pupal transformation, this does not appear to be the primary source of diminished synaptic drive because regression is greatest in the segments in which synaptic inputs remain the strongest. The developmental weakening of the pre-ecdysis motor pattern thus may be due to changes at the interneuronal level.Abbreviations A2, A3... abdominal segments 2, 3, etc. - ALE anterior lateral external muscle - day L3 third day of the 5th larval instar - day P0 the day of pupal ecdysis - DN _a anterior branch of the dorsal nerve - EH eclosion hormone - HPLC high performance liquid chromatography - TP tergopleural muscle     

Parasitism of an insect Manduca sexta L. alters feeding behaviour and nutrient utilization to influence developmental success of a parasitoid

The effects of macronutrient balance on nutrient intake and utilization were examined in Manduca sexta larvae parasitized by Cotesia congregata. Insects fed an artificial diet having constant total macronutrient, but with varied ratios of protein and carbohydrate, with altered diet consumption in response to excesses and deficiencies of the individual macronutrients. Bivariate plots of protein and carbohydrate consumption for non-parasitized larvae demonstrated a curvilinear relationship between points of nutrient intake for the various diets, and the larvae grew best on carbohydrate-biased diets. The relationship was linear for parasitized larvae with the growth uniform across diets. On protein-biased diets, the larvae regulated the nitrogen content, containing similar amounts of nitrogen regardless of consumption. Efficiency of nitrogen conversion in non-parasitized larvae was greatest on carbohydrate-biased diets, while nitrogen conversion by parasitized larvae was greatest with intermediate nutrient ratios. Accounting for carbohydrate consumption, the lipid content decreased as dietary carbohydrate increased, but parasitized larvae contained significantly less lipid. The total biomass of parasites developing in individual host larvae was positively correlated with host protein consumption, but the individual parasites were similar in size. Parasitism influences host nutrient consumption in a manner that achieves uniform host growth under diverse nutritional regimes, thereby constraining blood nutrient concentrations within limits suitable for parasite growth and development.     

Effects of chlorogenic acid-and tomatine-fed caterpillars on the behavior of an insect predator

If generalist insect predators are a selective force contributing to patterns of feeding specialization by insect herbivores, then predators should be deterred from eating allelochemical-fed prey. The attack and feeding behaviors of naive predators (Podisus maculiventris stinkbugs) reared on control caterpillars (Manduca sexta) fed plain diet were compared to experienced predators reared on caterpillars fed tomato allelochemicals. Tomatine-fed prey were found more quickly by both naive and tomatine-experienced predators, and chlorogenic acid-experienced predators were more stimulated to begin searching for prey. However, experienced predators were less likely to attack both chlorogenic acidfed and tomatine-fed caterpillars than were naive predators. These results indicate that allelochemical-fed prey were easier for predators to locate, but allelochemical-containing prey often deterred predation by experienced predtors.     

Expression of two different isoforms of fasciclin II during postembryonic central nervous system remodeling in <Emphasis Type="Italic">Manduca sexta</Emphasis>

Insect metamorphosis serves as a useful model to investigate postembryonic development in the central nervous system, because the transformation between larval and adult life is accompanied by a remodeling of neural circuitry. Most changes are controlled by ecdysteroids, but activity-dependent mechanisms and cell surface signals also play a role. This immunocytochemical study investigates the expression patterns of two isoforms of the neural cell adhesion molecule, fasciclin II (FasII), during postembryonic ventral nerve cord remodeling in the moth, Manduca sexta. Both the expression of the glycosyl-phosphatidylinositol (GPI)-linked isoform and the transmembrane isoform of Manduca FasII (TM-MFasII) are regulated in a stereotyped spatio-temporal pattern. TM-MFasII is expressed in a stage-specific manner in a subset of neurons. Subsets of central axons express high levels during outgrowth supporting a functional role for TM-FasII during pathfinding. Dendritic localization is not found at any stage of metamorphosis, suggesting no homophilic interactions of TM-MFasII during central synapse development. GPI-MFasII is expressed in a stage-specific manner, most likely only in glial cells. The larval and adult stages show almost no GPI-MFasII expression, whereas during pupal life, positive GPI-MFasII labeling is present around synaptotagmin-negative tracts or commissures, so that either homophilic stabilization of glial boundaries or heterophilic neuron-glial interactions possibly stabilize the axons within their tracts. GPI-MFasII expression is not co-localized with synaptotagmin-positive central terminals, rendering a role for synapse development unlikely. Neither isoform is expressed in all neurons of a specific class at any developmental stage, indicating that MFasII functions are restricted to specific subsets of neurons or to individual neurons. The support of the German Science Foundation (Du 331/4–1) and of Arizona State University to C.D. is greatly appreciated.     

Eicosanoids mediate insect hemocyte migration

Hemocyte migration toward infection and wound sites is an essential component of insect defense reactions, although the biochemical signal mechanisms responsible for mediating migration in insect cells are not well understood. Here we report on the outcomes of experiments designed to test the hypotheses that (1) insect hemocytes are able to detect and migrate toward a source of N-formyl-Met-Leu-Phe (fMLP), the major chemotactic peptide from Escherichia coli and (2) that pharmaceutical modulation of eicosanoid biosynthesis inhibits hemocyte migration. We used primary hemocyte cultures prepared from fifth-instar tobacco hornworms, Manduca sexta in Boyden chambers to assess hemocyte migration toward buffer (negative control) and toward buffer amended with fMLP (positive control). Approximately 42% of negative control hemocytes migrated toward buffer and about 64% of positive control hemocytes migrated toward fMLP. Hemocyte migration was inhibited (by >40%) by treating hornworms with pharmaceutical modulators of cycloxygenase (COX), lipoxygenase and phospholipase A2 (PLA2) before preparing primary hemocyte cultures. The influence of the COX inhibitor, indomethacin, and the glucocorticoid, dexamethasone, which leads to inhibition of PLA2, was expressed in a dose-dependent way. The influence of dexamethasone was reversed by injecting arachidonic acid (precursor to eicosanoid biosynthesis) into hornworms before preparing primary hemocyte cultures. The saturated fatty acid, palmitic acid, did not reverse the inhibitor effect. These findings support both our hypotheses, first that insect hemocytes can detect and respond to fMLP, and second, that insect hemocyte migration is mediated by eicosanoids.     

Cuticle degrading proteases from insect moulting fluid and culture filtrates of entomopathogenic fungi

Insects degrade their own cuticle during moulting, a process which is catalysed by a complex mixture of enzymes. Entomopathogenic fungi infect the insect host by penetration of the cuticle, utilizing enzymatic and/or physical mechanisms. Protein is a major component of insect cuticle and a major recyclable resource for the insect and, therefore, represents a significant barrier to the invading fungus. To this end, both insects and entomopathogenic fungi produce a variety of cuticle degrading proteases. The aim of this paper is to review these proteases and to highlight their similarities, with particular reference to the tobacco hornworm, Manduca sexta, and the entomopathogenic fungus, Metarhizium anisopliae     

Feeding behaviour and nutrient selection in an insect <Emphasis Type="Italic">Manduca sexta</Emphasis> L. and alterations induced by parasitism

Manduca sexta L. larvae exhibit broad food acceptance with regard to nutrient content during the first 3 days of the last stadium. Larvae fed diets with a constant combined level of casein and sucrose, but variable ratios, display a linear relationship between protein and carbohydrate intake. Larvae grow best on a diet with equal nutrients, but will consume an excess of one nutrient in order to obtain an adequate amount of the other, as nutrient ratio shifts. Parasitized larvae feed similarly, but the nutrient ratio does not affect growth. Unparasitized larvae regulate intake of protein and carbohydrate when offered choices of protein-biased and carbohydrate-biased diets having combined nutrient levels of 120 g/l, but with variable ratios. Larvae normally consume equal amounts of nutrients, regardless of ratio, and grow similarly. As combined nutrient level is reduced in one diet, larvae abandon regulation and feed randomly. Parasitized larvae offered choice diets with 120 g/l combined nutrients do not regulate nutrient intake. Consumption of nutrients varies widely, but growth is unaffected. Larvae offered choices of diets having equal amounts of casein and sucrose but variable fat (corn oil), fail to regulate fat intake, although both unparasitized and parasitized larvae prefer a diet containing higher fat.     

Behavioral responses to host foodplants of two populations of the insect parasitoid Cotesia congregata (Say)

To test the hypothesis that natural enemy populations differ in their behavioral responses to plants or to plant allelochemicals, we compared two populations of the gregarious larval endoparasitoid, Cotesia congregata (Say) (Hymenoptera: Braconidae) that differed in their historical and present exposure to tobacco. The major hosts for both populations were Manduca sexta L. and M. quinquemaculata (Haworth) (Lepidoptera: Sphingidae), but these hosts were typically encountered on tobacco by parasitoids in one population (Upper Marlboro) and on tomato by parasitoids in another population (Wye). Early in the season, Wye parasitoids preferred to oviposit in M. sexta on tomato rather than on tobacco and Upper Marlboro parasitoids showed no preference; neither population showed any preference later in the season. Neither of the strains originating from the two populations showed a landing preference for tobacco or tomato in flight chamber trials, but Upper Marlboro parasitoids searched longer on tobacco than on tomato, and Wye parasitoids searched longer on tomato. When nicotine solutions were applied to tobacco leaf, searching responses of Upper Marlboro parasitoids were enhanced by 0.001–1.0% nicotine, and searching responses of Wye parasitoids were decreased by 0.01–1.0% nicotine. We speculate that population differences in searching responses to tobacco and nicotine may explain the differential parasitism responses found early in the season.     

Inositol-trisphosphate-dependent calcium currents precede cation currents in insect olfactory receptor neurons in vitro

Specialized olfactory receptor neurons in insects respond to species-specific sex pheromones with transient rises in inositol trisphosphate and by opening pheromone-dependent cation channels. These channels resemble cation channels which are directly or indirectly Ca²⁺-dependent. But there appear to be no internal Ca²⁺ stores in the outer dendrite where the olfactory transduction cascade is thought to start. Hence, it remains to be determined whether an influx of external Ca²⁺ precedes pheromone-dependent cation currents. Patch clamp measurements in cultured olfactory receptor neurons from Manduca sexta reveal that a transient inward current precedes pheromone-dependent cation currents. A transient inositol trisphosphate-dependent Ca²⁺ current, also preceding cation currents with the characteristics of pheromone-dependent cation currents, shares properties with the transient pheromone-dependent current. These results match the biochemical measurements with the electrophysiological data obtained in insect olfactory receptor neurons.Abbreviations ORNs Olfactory receptor neurons - IP₃ Inositol-1,4,5-trisphosphate - I_t Transient pheromone-dependent current - I_ir Transient IP₃-dependent current     

Expression and in vitro activation of Manduca sexta prophenoloxidase-activating proteinase-2 precursor (proPAP-2) from baculovirus-infected insect cells

Prophenoloxidase activation is a component of the immune system in insects and crustaceans. We recently purified and cloned a new prophenoloxidase-activating proteinase (PAP-2) from hemolymph of the tobacco hornworm Manduca sexta [J. Biol. Chem. 278, 3552-3561]. As the terminal component of a putative serine proteinase cascade, this enzyme activates prophenoloxidase (proPO) via limited proteolysis. To purify and study the activating proteinase for PAP-2 from this insect, we expressed the zymogen of PAP-2 (proPAP-2) in insect cells infected by a recombinant baculovirus that harbors the cDNA. To facilitate the purification of proPAP-2, we modified a commercial vector (pFastBac1) by inserting a synthetic DNA fragment encoding a hexahistidine sequence, allowing fusion of the affinity tag to the carboxyl terminus of a protein. After Spodoptera frugiperda Sf21 cells were infected by the virus, recombinant proPAP-2 was efficiently secreted into the media at a concentration of 5.9 microg/ml under the optimal conditions. After ammonium sulfate precipitation, the proenzyme was purified to near homogeneity by affinity chromatography on Ni(2+)-NTA agarose. Western blot analysis indicated that the recombinant proPAP-2 has a mobility slightly lower than that of the zymogen from M. sexta hemolymph. The molecular mass and isoelectric point of proPAP-2 were determined to be 47,573+/-11Da and 6.6, respectively. After the purified proenzyme was added to hemolymph from induced M. sexta larvae, it was rapidly activated by an unknown proteinase in the presence of peptidoglycan.     

Purification of an active, oligomeric chitin synthase complex from the midgut of the tobacco hornworm

Chitin formation depends on the activity of a family II glycosyltransferase known as chitin synthase, whose biochemical and structural properties are largely unknown. Previously, we have demonstrated that the chitin portion of the peritrophic matrix in the midgut of the tobacco hornworm, Manduca sexta, is produced by chitin synthase 2 (CHS-2), one of two isoenzymes encoded by the Chs-1 and Chs-2 genes (also named Chs-A and Chs-B), and that CHS-2 is located at the apical tips of the brush border microvilli. Here we report the purification of the chitin synthase from the Manduca midgut as monitored by its activity and immuno-reactivity with antibodies to the chitin synthase. After gel permeation chromatography, the final step of the developed purification protocol, the active enzyme eluted in a fraction corresponding to a molecular mass between 440 and 670 kDa. Native PAGE revealed a single, immuno-reactive band of about 520 kDa, thrice the molecular mass of the chitin synthase monomer. SDS-PAGE and immunoblotting indicated finally that an active, oligomeric complex of the chitin synthase was purified. In summary, the chitin synthase from the midgut of Manduca may prove to be a good model for investigating the enzymes' mode of action.     

Ultrastructure of an excitatory synapse

Summary The ultrastructure of the afferent synapse in hair cells of the lateral line-canal organ was studied using different fixation and staining techniques. Glutaraldehyde-fixed tissue without post-osmication, contrasted by section staining with uranyl acetate and lead citrate, was compared with (a) osmium tetroxide-fixed tissue followed by the same staining procedure, and with (b) glutaraldehyde-fixed tissue, block-impregnated with phosphotungstic acid (PTA). The results reveal a pronounced heterogeneity in the composition of the synaptic body, reflecting regional differences in chemical affinity to the fixatives and staining agents. It is proposed that the intracleft substance, the synaptic structure defined by the PTA staining technique, is actually due to the glutaraldehyde fixation procedure and is apparently the outer leaflet of the postsynaptic membrane. A special technique that allows alternate sections of a series to be differentially stained for electron microscopy is proposed.This research was carried out at the King Gustaf V Research Institute, Stockholm, Sweden     

Fictive chewing activity in motor neurons and interneurons of the suboesophageal ganglion of Manduca sexta larvae

Alternating antiphasic rhythmic activity was observed in opener and closer mandibular motor neurons in the isolated suboesophageal ganglion of the larva of Manduca sexta (Lepidoptera: Sphingidae). This was interpreted provisionally as fictive chewing; the pattern is similar to that seen in semiintact animals but of lower frequency. Additionally, a variety of associated rhythmic activities were observed in suboesophageal interneurons. These could be classified into several different physiological types by their activity patterns in relation to the chewing cycle. Some of these neurons can modulate the rhythm when injected with current. It seems likely that they are part of or associated with a central pattern generator circuit for chewing.Abbreviations A anterior - CEC circumoesophageal connective - Cl-MN closer motor neuron - IN interneuron - MdN mandibular nerve - MN motor neuron - O-MN opener motor neuron     

The serosa of Manduca sexta (Insecta, Lepidoptera): ontogeny, secretory activity, structural changes, and functional considerations

In Manduca sexta, the blastoderm forms successively and becomes immediately cellularized as the cleavage energids reach the surface of the oocyte. Presumptive serosal cells are large and contain 2 or 4 large polyploid nuclei; presumptive embryonic cells are small and mononuclear. All parts of the blastoderm participate in the uptake and digestion of yolk material. About 10 h post-oviposition, the blastoderm breaks at the amnioserosal fold and the extraembryonic part closes above the germ band and constitutes the serosa (12 h post-oviposition, i.e. 10% development completed). At once, the serosa starts to secrete a cuticle consisting of an epi- and a lamellated endocuticle. Detachment of the serosal cuticle, 22h post-oviposition, is reminiscent of apolysis of larval cuticle. Thereafter, the serosa deposits a membranous structure, the serosal membrane. The sercretory process lasts from 23h to 44h post-oviposition. At first a fine granular layer, then an amorphous, spongy-like, fibrillar layer is secreted via microvilli. This persisting membrane is tough, rubbery and very elastic. It may serve to bolster the serosa during katatrepsis (48h post-oviposition) and later embryonic movements. After detachment of the serosal membrane, 44h post-oviposition, a distinct subcellular reorganization of the serosa takes place. The nuclei become still larger and more irregular. Uptake of yolk granules, but not of lipid droplets, ceases, although interaction of serosa and yolk cells are intense. Serosal cells include many mitochondria, large areas of rER, besides some sER, increasing amounts of lysosomal bodies and prominent Golgi complexes. Most conspicuous is the assembly of spindle-shaped, electron-lucent vesicles below the apical surface. These vesicles may contain metabolic products which are released into the peripheral space. The studies show that the serosa assumes changing functions during embryogenesis: digestion of yolk substances, synthesis of a serosal cuticle and a serosal membrane, which may have a protective function, and excretion.     

Antiviral, anti-parasitic, and cytotoxic effects of 5,6-dihydroxyindole (DHI), a reactive compound generated by phenoloxidase during insect immune response

Phenoloxidase (PO) and its activation system are implicated in several defense responses of insects. Upon wounding or infection, inactive prophenoloxidase (proPO) is converted to active PO through a cascade of serine proteases and their homologs. PO generates reactive compounds such as 5,6-dihydroxyindole (DHI), which have a broad-spectrum antibacterial and antifungal activity. Here we report that DHI and its spontaneous oxidation products are also active against viruses and parasitic wasps. Preincubation of a baculovirus stock with 1.25 mM DHI for 3 h near fully disabled recombinant protein production. The LC₅₀ for lambda bacteriophage and eggs of the wasp Microplitis demolitor were 5.6 ± 2.2 and 111.0 ± 1.6 ??M, respectively. The toxicity of DHI and related compounds also extended to cells derived from insects that serve as hosts for several of the aforementioned pathogens. Pretreatment of Sf9 cells with 1.0 mM DHI for 4 h resulted in 97% mortality, and LC₅₀ values of 20.3 ± 1.2 ??M in buffer and 131.8 ± 1.1 ??M in a culture medium. Symptoms of DHI toxicity in Sf9 cells included DNA polymerization, protein crosslinking, and lysis. Taken together, these data showed that proPO activation and DHI production is strongly toxic against various pathogens but can also damage host tissues and cells if not properly controlled.     

Differences between larval and pupal hemocytes of the tobacco hornworm, Manduca sexta, determined by monoclonal antibodies and density centrifugation

Insect hemocytes play a major role in developmental processes where they disassociate and rebuild metamorphosing tissues while undergoing physiological changes themselves. We identified hemocyte changes from the last larval to the beginning of the pupal stage of the tobacco hornworm, Manduca sexta. Larval and pupal hemocytes behaved differently in a 40% Percoll density gradient. Larval granular cells were found in almost all density layers, pupal granular cells were abundant in high density layers; larval plasmatocytes occurred in dense layers, pupal plasmatocytes became enriched in less dense layers of the gradient. Using a panel of monoclonal antibodies generated against purified hemocytes, several different antibody binding patterns were identified. Quantitative differences in staining intensities were observed more often than qualitative changes, e.g. a loss or a gain of staining. Both phenomena were related to both plasmatocytes and granular cells. The distribution of the corresponding antigens in tissues was tested on cross sections of larvae and pupae as well as in Western blot analyses using organ homogenates. Several antibodies were specific for hemocytes only, among which two antibodies bound to molecules of the hematopoietic organ. Other antibodies had an additional reactivity to other tissues, mainly to the basal lamina.     

The vacuolar-type ATPase from insect plasma membrane: immunocytochemical localization in insect sensilla

Summary This comparative immunocytochemical investigation provides evidence that the electrogenic potassium pump of insect sensilla is a vacuolar-type proton ATPase energizing potassium-proton antiport, as was shown recently for the electrogenic potassium pump in the larval midgut of the sphinx moth Manduca sexta. Antennal sensilla of the saturniid moth Antheraea pernyi were probed with antibodies to the midgut vacuolar-type ATPase. The monoclonal antibodies recognized their epitopes in the native and SDS-denatured state, and bound specifically to the subunit with the relative molecular mass (M_r) of 67000 (antibody 86-3) or to the subunits of M_r 28000 and 16000 (antibody 47-5). Both antibodies labelled the apical region of the auxiliary cells, as was demonstrated by immunofluorescence microscopy. Immunogold-electron microscopy localized the binding sites of the 47-5 antibody in the highly folded apical plasma membranes of the auxiliary cells. Labelling was selective and was detected in all types of examined sensilla (S. trichodea, S. styloconica, S. coeloconica). These findings are in agreement with the current view that an electrogenic potassium pump is situated in the apical plasma membrane of the auxiliary cells and that the pump is involved in driving the receptor current. They support the hypothesis that a proton-motive force generated by a vacuolar-type ATPase provides an alternative to the classical Na⁺/K⁺-ATPase to energize animal plasma membranes.