A positive feedback mechanism in the Manduca sexta prophenoloxidase activation system

In Manduca sexta, pathogen recognition triggers a branched serine proteinase cascade which generates active phenoloxidase (PO) in the presence of a proPO-activating proteinase (PAP) and two noncatalytic serine proteinase homologs (SPHs). PO then catalyzes the production of reactive compounds for microbe killing, wound healing, and melanin formation. In this study, we discovered that a minute amount of PAP1 (a final component of the proteinase pathway) caused a remarkable increase in PO activity in plasma from na?ve larvae, which was significantly higher than that from the same amounts of PAP1, proPO and SPHs incubated in vitro. The enhanced proPO activation concurred with the proteolytic activation of HP6, HP8, PAP1, SPH1, SPH2 and PO precursors. PAP1 cleaved proSPH2 to yield bands with mobility identical to SPH2 generated in vivo. PAP1 partially hydrolyzed proHP6 and proHP8 at a bond amino-terminal to the one cut in the PAP1-added plasma. PAP1 did not directly activate proPAP1. These results suggest that a self-reinforcing mechanism is built into the proPO activation system and other plasma proteins are required for cleaving proHP6 and proHP8 at the correct site to strengthen the defense response, perhaps in the early stage of the pathway activation.     

Molecular patterning mechanism underlying metamorphosis of the thoracic leg in Manduca sexta

The tobacco hornworm Manduca sexta, like many holometabolous insects, makes two versions of its thoracic legs. The simple legs of the larva are formed during embryogenesis, but then are transformed into the more complex adult legs at metamorphosis. To elucidate the molecular patterning mechanism underlying this biphasic development, we examined the expression patterns of five genes known to be involved in patterning the proximal-distal axis in insect legs. In the developing larval leg of Manduca, the early patterning genes Distal-less and Extradenticle are already expressed in patterns comparable to the adult legs of other insects. In contrast, Bric-a-brac and dachshund are expressed in patterns similar to transient patterns observed during early stages of leg development in Drosophila. During metamorphosis of the leg, the two genes finally develop mature expression patterns. Our results are consistent with the hypothesis that the larval leg morphology is produced by a transient arrest in the conserved adult leg patterning process in insects. In addition, we find that, during the adult leg development, some cells in the leg express the patterning genes de novo suggesting that the remodeling of the leg involves changes in the patterning gene regulation.     

Juvenile hormone esterase activity from Manduca sexta corpora allata in vitro

Juvenile hormone esterase (JHE) activity released by the corpora allata (CA) into incubation media (CA-JHE) was titered daily during the course of the last (fifth [V]) larval stadium of Manduca sexta. This CA-JHE activity was relatively low during the early last stadium up to the time of commitment (V4), then rose rapidly to a peak on V6. Activity declined sharply almost to precommitment levels by V8, before rising to a second peak on the first day of the pupal phase (P0). This pattern of activity is distinct from that of hemolymph JHE activity, which peaks just prior to wandering on V4 and again just prior to pupation (V9). Although the CA-JHE and hemolymph-JHE possess different temporal patterns of activity, isoelectric focusing, gel electrophoresis, and initial studies with selected inhibitors suggest that the enzymes responsible for the CA-JHE and hemolymph-JHE activities are similar, but not identical, in nature. Exposure of the V6 CA in vitro to JH II (0.1 μM) or fluoromevalonolactone (FMev; 0.1 mM) produced an approximate fivefold increase and 60% decrease in JH acid synthesis, respectively. Conversely, the same treatments resulted in an inhibition (JH II) and stimulation (FMev) of CA-JHE activity. These observations suggest that JH may be involved in the direct positive feedback regulation of postwandering larval CA and that the CA-JHE may also be integrally related to this positive feedback mechanism.     

The biochemical basis of antimicrobial responses in Manduca sexta

Innate immunity is essential for the wellbeing of vertebrates and invertebrates. Key components of this defense system include pattern recognition receptors that bind to infectious agents, extra-and intra-cellular proteins that relay signals, as well as molecules and cells that eliminate pathogens. We have been studying the defense mechanisms in a biochemical model insect, Manduca sexta. In this insect, hemolin, peptidoglycan recognition proteins, β-1,3-glucan recognition proteins and C-type lectins detect microbial surface molecules and induce immune responses such as phagocytosis, nodulation, encapsulation, melanization and production of antimicrobial peptides. Some of these responses are mediated by extracellular serine proteinase pathways. The proteolytic activation of prophenoloxidase （proPO） yields active phenoloxidase （PO） which catalyzes the formation of quinones and melanin for wound healing and microbe killing. M. sexta hemolymph proteinase 14 （HP 14） precursor interacts with peptidoglycan or β-1,3-glucan, autoactivates, and leads to the activation of other HPs including HP21 and proPO-activating proteinases （PAPs）. PAP-1, -2 and -3 cut proPO to generate active PO in the presence of two serine proteinase homologs. Inhibition of the proteinases by serpins and association of the proteinase homologs with bacteria ensure a localized defense reaction. M. sexta HP1, HP6, HP8, HP17 and other proteinases may also participate in proPO activation or processing of spatzle and plasmatocyte spreading peptide.     

Broad-spectrum antimicrobial activity of the reactive compounds generated in vitro by Manduca sexta phenoloxidase

Although quinone production and melanin formation are widely recognized as an integral part of the insect defense system, experimental evidence is lacking that the proteolytic activation of prophenoloxidase participates in the direct killing of invading microbes-active phenoloxidase generates quinones that polymerize to form melanin. Here, we report the antimicrobial effect of reactive intermediates produced in phenoloxidase-catalyzed reactions. After being treated with Manduca sexta phenoloxidase and dopamine, Escherichia coli and Bacillus subtilis ceased to grow, whereas the growth of Pichia pastoris was slightly affected. Microscopic analysis showed melanin deposition on cell surface, aggregation of bacteria, and loss of cell mobility. Viability tests revealed major decreases in the bacterial colony counts and, since the decrease remained significant after dispersion of the cell clumps, the reactive compounds were surmised to have aggregated and killed E. coli and B. subtilis cells. Under the experimental conditions, 60-94% of the Gram-negative bacteria (E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium) and 52-99% of the Gram-positive bacteria (Bacillus cereus, B. subtilis, Micrococcus luteus, and Staphylococcus aureus) were killed. In the presence of phenoloxidase, dopamine or 5,6-dihydroxyindole (DHI) exhibited much higher antibacterial activity than L-dopa, N-acetyldopamine (NADA) or N-beta-alanyldopamine (NBAD) did, suggesting that DHI and its oxidation products were cytotoxic. The antifungal activity of DHI was detected using P. pastoris, Saccharomyces cerevisiae, Candida albicans, and Beauveria bassiana. These results established that prophenoloxidase activation is an integral component of the insect defense system involving a multitude of enzymes (e.g. proteinases, oxidases, and dopachrome conversion enzyme (DCE)), which immobilizes and kills invading microorganisms.     

Mandibular premotor interneurons of larval Manduca sexta

Simultaneous intracellular recordings were made from interneurons and from closer or opener mandibular motor neurons in the isolated suboesophageal ganglion of the larva of Manduca sexta. This article describes various morphologically and physiologically distinguishable premotor spiking interneurons which make direct excitatory connections with the motor neurons. In addition, two presumptive non-spiking interneurons make excitatory and inhibitory connections respectively with opener motor neurons. Both classes of interneurons receive excitatory and inhibitory sensory inputs from the mouthparts. Their circuitry and functions are discussed.Abbreviations A anterior - AP action potential - CEC circumoesophageal connective - Cl-MN closer motor neuron - EPSP excitatory postsynaptic potential - IN interneuron - IPSP inhibitory postsynaptic potential - MdN mandibular nerve - MN motor neuron - MxN maxillary nerve - O-MN opener motor neuron - PSP postsynaptic potential     

Characterization of tyrosine hydroxylase from Manduca sexta

In insects, 3,4-dihydroxyphenylalanine (DOPA) is required for tanning of newly formed cuticle and the production of melanin during some types of immune responses. DOPA is produced by the hydroxylation of tyrosine, and this reaction can be catalyzed by two types of enzymes: tyrosine hydroxylase (TH) and phenoloxidase (PO). TH is required for cuticle tanning in Drosophila melanogaster and for cuticle pigmentation in other insect species, but additional functions of TH have been uncertain. In contrast, an immune function for PO has been well documented. The goal of this study was to characterize TH from Manduca sexta with a focus on its possible contribution to cuticle tanning and immune-associated melanization. We cloned a full-length TH cDNA, purified recombinant TH, and confirmed that MsTH and MsPO have tyrosine hydroxylating activity. To determine possible functions, we analyzed TH expression profiles. TH mRNA and protein were present in eggs at the stage when the pharate larval cuticle begins to tan and also in the integument of molting larvae. The amount of TH in the integument was correlated with the degree of cuticle tanning. Unlike PO, which was found to be constitutively expressed by hemocytes and was present in plasma, TH was upregulated in hemocytes and the fat body in response to an immune challenge and remained intracellular. These data suggest that TH is required for cuticle tanning and immunity in M. sexta. Based on the collective information from many studies, we propose a model in which TH is a major producer of the DOPA required for both cuticle tanning and immune-associated melanization.     

Intracellular calcium in prothoracic glands of Manduca sexta

Cytosolic free calcium was measured in individual prothoracic gland cells of Manduca larvae with Fura-2. During the last larval instar there was no correlation between intracellular calcium concentration and ecdysteroid secretion by the glands. The addition of prothoracicotropic hormone (PTTH) from brains of Manduca larvae to prothoracic glands in vitro resulted in a significant increase in the calcium concentration of the gland cells. The effect of PTTH was inhibited by the inorganic calcium channel antagonists, cadmium, lanthanum and nickel, and by the antagonist of T-type calcium channels, amiloride, whereas all the other antagonists tested failed to block the action of PTTH. TMB-8, an inhibitor of intracellular calcium mobilization, did not reduce the PTTH-induced rise in calcium, which suggests that IP(3)-dependent intracellular calcium stores are not involved in the calcium-mediated stimulation of ecdysteroid synthesis. Moreover, PTTH is thought to increase intracellular calcium in prothoracic glands of Manduca by influencing calcium channels in the plasma membrane.     

Pupal cuticle formation by Manduca sexta epidermis in vitro: Patterns of ecdysone sensitivity

During the larval-pupal transformation, various regions of the epidermis of Manduca sexta larvae have previously been found to require different lengths of exposure to the prothoracic glands in order to form pupal cuticle. To distinguish between requirements for differing threshold concentrations of ecdysone and those for differing durations of exposure to ecdysone, wandering stage larval epidermis was cultured in Grace's medium. When most of the thick larval cuticle was removed, the epidermis responded to concentrations of β-ecdysone of 1.0 μ/ml or greater for 4 days by forming cysts which later formed tanned pupal cuticle. No fat body or protein supplement was required. When the larval integument was explanted intact, similar requirements for cuticle formation and for tanning were found. All regions of the fifth abdominal segment required similar concentrations of β-ecdysone (0.4–0.6 μg/ml) for 4 days for 50% to form pupal cuticle, but gin trap epidermis required the least exposure to a threshold concentration of ecdysone (1.5 days in 0.9 μg/ml). The anterior dorsal intersegmental region required about 0.5 day longer, followed by the posterior intersegmental and the dorsal intrasegmental regions. Thus, the duration of exposure seemed more important. About 1 day longer of exposure to ecdysone was required for subsequent tanning of the new cuticle than for cuticle formation, yet tanning of the cuticle did not occur with prolonged exposure to ecdysone.     

An in vitro analysis of ecdysteroid-elicited cell death in the prothoracic gland of Manduca sexta

The prothoracic glands of the tobacco hornworm, Manduca sexta, secrete the precursor of the insect molting hormone and normally undergo programmed cell death (PCD) during pupal-adult metamorphosis, between days 5 and 6 after pupation. This phenomenon can be elicited prematurely in vitro by the addition of 20-hydroxyecdysone (20E) to the gland cultures. To induce nuclear condensation in vitro in the glands from day-1 pupae, the effective dose range of 20E is 0.7-7 micrograms/ml and the minimum exposure period is 24 h. Prothoracic glands from different stages of pupal-adult development express different responsiveness to exogenous ecdysteroids. By utilizing terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) and the apoptotic DNA laddering method together with transmission electron microscopy, it has been demonstrated that the ecdysteroid-induced cell death of the prothoracic glands occurs via not only apoptosis but also autophagy, i.e., the induced dying cells show both severe nuclear fragmentation and autophagic vacuole formation, characteristics typical of apoptotic and autophagic cell death. The composite data indicate that ecdysteroids regulate directly both apoptotic and autophagic mechanisms of PCD of the prothoracic glands.     

Beta-cyclodextrin facilitates cholesterol efflux from larval Manduca sexta fat body and midgut in vitro

The ability of 2-hydroxypropyl-β-cyclodextrin (HPβCD) and methyl-β-cyclodextrin (MβCD) to promote cholesterol efflux from [³H]cholesterol-labeled larval Manduca sexta fat body and midgut was tested. In fat body, both β-cyclodextrins induced a two-phase efflux of cholesterol. The first rapid phase depended on cyclodextrin concentration and was more rapid for MβCD than for HPβCD. The second, slower, phase was independent of cyclodextrin concentration and type. In midgut, only the concentration-dependent phase was observed; the rate constants are approximately 85% slower than for fat body. In both cases, a low activation energy for transfer was observed, consistent with a collision mechanism where cyclodextrin interacts directly with cholesterol in plasma membrane to affect transfer. In fat body, the second slower phase is suggestive of a second pool of exchangeable cholesterol and most likely represents transfer of cholesterol from internal membranes or different lateral domains of the plasma membrane. The lack of this second phase in midgut suggests that midgut has only a single pool of exchangeable cholesterol. Although the rates are somewhat different, the overall kinetic pattern for cyclodextrin-mediated cholesterol transfer in insect fat body closely resembles that for vertebrate cells, while the single pool behavior of the midgut is not found in vertebrate cells.     

Developmental expression of Manduca sexta hemolin.

Hemolin is hemolymph protein that is a member of the immunoglobulin superfamily. Its induced expression after bacterial infection suggests that it functions in the immune response. In this paper, we describe the expression of the Manduca sexta hemolin gene at certain developmental stages in the absence of microbial challenge. Hemolin was present at a very low level in hemolymph of naive larvae until the beginning of the wandering stage prior to pupation, when its concentration in hemolymph increased dramatically. At the same time, hemolin could be found in the fluid contained in the midgut lumen. The appearance of hemolin mRNA in fat body and midgut at the beginning of the wandering stage correlated with the presence of hemolin in the hemolymph and midgut lumen. Hemolin was present in hemolymph through the pupal and adult stages. Hemolin was also present in newly deposited eggs, and persisted in eggs throughout embryonic development. A hemolin cDNA isolated from an adult fat body library had the same sequence as those previously obtained from larval libraries. Hemolin purified from hemolymph of bacteria-injected larvae, from hemolymph of naive wandering stage larvae and adult moths, and from midgut fluid of wandering stage larvae had the same apparent mass, which was consistent with the mass predicted from the hemolin cDNA sequence. Hemolin from hemolymph of wandering stage larvae did not contain any detectable carbohydrate, but hemolin from the hemolymph of bacteria-injected larvae and from naive adult moths was associated with carbohydrate, although of different amounts and composition. These results suggest that a single hemolin gene is developmentally regulated and is also induced when insects are exposed to microbial infection. M. sexta hemolin apparently lacks post-translational covalent glycosylation, but instead is associated under some conditions with non-covalently bound carbohydrates. Arch.     

Juvenile hormone and ovarian growth in Manduca sexta

In Manduca sexta the major size increase of ovarian follicles is accomplished by two processes: (1) vitellogenesis in which follicular volume and dry weight increase simultaneously, and (2) hydration in which absorption of water by the oocyte accounts for an 80% increase in volume prior to chorion formation. Vitellogenic growth occurs in both a slow and rapid phase. Rapid vitellogenic growth is initiated only by follicles of a threshold size (1 mm) and is a juvenile hormone (JH)-dependent event. In the absence of JH follicles grow to 1 mm and then degenerate.     

Protease inhibitor controls prophenoloxidase activation in Manduca sexta

Prophenoloxidase from the hemolymph of tobacco hornworm Manduca sexta can be activated by a specific activating enzyme found in the cuticle. Inhibition studies with benzamidine, diisopropyl phosphofluoridate and p-nitrophenyl-p'-guanidinobenzoate indicate that the activating enzyme is a trypsin-like serine protease. An endogenous protease inhibitor, isolated from the hemolymph of Manduca larvae, inhibits the prophenoloxidase activation mediated by this enzyme. These results indicate that the probable physiological role of endogenous protease inhibitor is to control the undesired activation of prophenoloxidase in the hemolymph.     

Ectonucleotide diphosphohydrolase activities in hemocytes of larval Manduca sexta

In this work, we describe the ability of living hemocytes from an insect (Manduca sexta, Lepidoptera) to hydrolyze extracellular ATP. In these intact cells, there was a low level of ATP hydrolysis in the absence of any divalent metal (8.24 +/- 0.94 nmol of Pi/h x 10(6) cells). The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 15.93 +/- 1.74 nmol of Pi/h x 10(6) cells. Both activities were linear with cell density and with time for at least 90 min. The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.33 mM MgCl2. This stimulatory activity was not observed when Ca2+ replaced Mg2+. The apparent Km values for ATP-4 and Mg-ATP2- were 0.059 and 0.097 mM, respectively. The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.6 and 7.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced by an increase of pH. These ecto-ATPase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, sodium fluoride, tartrate, and levamizole. To confirm the observed hydrolytic activities as those of an ecto-ATPase, we used an impermeant inhibitor, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), as well as suramin, an antagonist of P2-purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents. Interestingly, lipopolysaccharide, a component of cell walls of gram-negative bacteria that increase hemocyte aggregation and phagocytosis, increased the Mg2+-dependent ecto-ATPase activity in a dose-dependent manner but did not modify the Mg2+-independent ecto-ATPase activity.     

Water homeostasis by wild larvae of Manduca sexta

Modulation of faecal water loss is the principal mechanism by which larval Lepidoptera maintain water homeostasis in the laboratory. Is this also true of larvae in nature? We observed 12 fifth‐instar larvae of Manduca sexta feeding on Datura wrightii in the Sonoran Desert, U.S.A. The two main sources of water stress were: evaporative water loss across the cuticle, which appeared to be promoted by increasing body temperatures and decreasing relative humidities during daytime observation periods; and attacks by tachinid flies, which prompted caterpillars to defaecate large quantities of water and to regurgitate digestive fluid onto themselves. In both cases, caterpillars responded by producing drier faecal pellets. A subset of caterpillars consumed water‐rich flower buds of D. wrightii, which led to the production of comparatively wet faecal pellets. These data demonstrate that larval water balance in nature is affected by a variety of biotic and abiotic factors and that larvae respond to these perturbations by modulating the loss of water in the faeces.     

Fine structural studies of Johnston's organ in the tobacco hornworm moth,Manduca sexta (Johannson)

There are approximately 650 scolopidial units in Johnston's organ of the tobacco hornworm moth, Manduca sexta. These sensory units exhibit greater complexity than noted previously (Gray, '60; Howse, '65; Uga and Kuwabara, '65; Moeck, '68; Ong, '69; Schmidt, '69). Each scolopale is innervated by a bipolar neuron whose dendrite terminals are modified into three ciliary structures. The largest of these differentiates near the tip into a multitubular structure. The remaining two cilia are structurally similar along the entire length of the scolopale shaft. From each of their bases, a collagen-like structure differentiates into numerous microtubules which extend proximally into individual channels of dendritic cytoplasm. A third channel, with a less developed root apparatus, was apparent for the largest cilium. Preliminary evidence suggests a proprioceptive function for this structure rather than an auditory one.     

Plant acids modulate chemosensory responses in Manduca sexta larvae

Abstract.The effects of three plant acids on the gustatory response of Manduca sexta larvae were examined. Two different categories of response were recorded in the styloconic sensilla of different individuals in the presence of salt: the gustatory response to salt was reduced especially at the lower pHs; the deterrent cell fired particularly at the lower pHs. In some individuals neither response was seen except at the lowest pHs. Additional experiments indicated that the effect was due to pH and not to specific effects of any of the acids. Ascorbic acid was tested with the nutrient carbohydrates glucose and inositol, and was found consistently to reduce the gustatory responses to these compounds. The data indicate that plant acids at natural concentrations could influence food acceptability.