Tobacco Hornworm Moth Research Articles

Biosynthesis of conjugated olefinic systems in the sex pheromone gland of female tobacco hornworm moths, Manduca sexta (L.)

Deuterium-labelled fatty acid putative precursors of the 16-carbon aldehydic sex pheromones of Manduca sexta (L.) were applied to the sex pheromone glands of females. After 24 h in vivo incubation, and injection of pheromone biosynthesis activating neuropeptide into the abdomens of treated females, glands were excised, extracted, and the extracts analyzed by gas chromatography-mass spectrometry. Incubation of glands with [16, 16, 16- 2H 3]hexadecanoic acid resulted in the label being incorporated into seven of the eight 16-carbon aldehydes found in the gland, including monoenes, dienes, and trienes. Similarly, incubation with [16, 16, 16- 2H 3](Z)-11-hexadecenoic acid led to incorporation of the label into ( Z)-11-hexadecenal and both conjugated diene and triene aldehydes. However, incubation with [13, 13, 14, 14, 15, 15, 16, 16, 16- 2H 9]( Z)-11-hexadecenoic acid resulted in incorporation of the label into only ( Z)-11-hexadecenal, ( E, Z)- and ( E, E)-10, 12-hexadecadienal, indicating interference of the deuterium isotope in the desaturation process leading to the conjugated triene system. Incubation with the similarly labelled ( E)-11-hexadecenoic acids gave labelled ( E)-11-hexadecenal in both cases, but none of the other unsaturated pheromone aldehydes. The results indicate that fatty acids are the precursors of the aldehydic pheromones in this species and that hexadecanoate is the precursor of the 16-carbon monoenes, dienes, and trienes. The conjugated dienes and trienes are formed via desaturation/isomerization of ( Z)-11-hexadecenoate.

PBAN regulation of pheromone biosynthesis in female tobacco hornworm moths,Manduca sexta (L.)

AbstractManduca sexta females that were decapitated produced no pheromone during the scotophase following decapitation, indicating that they were free of pheromone biosynthesis activating neuropeptide (PBAN). When deuterated hexadecanoic or (Z)‐11‐hexadecenoic acid was applied to the sex pheromone glands of decapitated or intact females of the same age, and allowed to incubate in vivo for 24 h, deuterium labeled Δ‐11‐ and Δ‐10, 12‐unsaturated 16‐carbon fatty acids were produced in both types of females. Injection of PBAN into intact or decapitated females 23 h after application of labeled acids had no effect on the production of unsaturated labeled fatty acids. However, deuterium labeled aldehydes were produced only in females that were injected with PBAN. Therefore, in this species, PBAN activates the process by which fatty acyl precursors in the pheromone gland are converted into the pheromonal aldehydes. © 1995 Wiley‐Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.

Correlation between glycerolipids and pheromone aldehydes in the sex pheromone gland of female tobacco hornworm moths, Manduca sexta (L.)

AbstractAnalysis by TLC and HPLC revealed that the triacylglycerols comprise the most abundant lipid class in the sex pheromone glands of Manduca sexta females. Also, conjugated olefinic acyl analogs of the major pheromone aldehydes occur principally in the triacylglycerols. The amount of triacylglycerols with conjugated diene acyl moieties significantly decreased when the period of pheromone production was extended by 7 h beyond the normal period of pheromone production by 3 injections of pheromone biosynthesis activating neuropeptide (PBAN) at 3 h intervals. This decrease indicates that the triacylglycerols stored in the gland may serve as major sources of pheromone precursors in the biosynthesis of the sex pheromone aldehydes. Furthermore, analysis of pheromone aldehydes and triacylglycerols in the gland from moths treated with PBAN showed that the proportions of the triacylglycerols with conjugated diene moieties were closely correlated with the proportions of aldehydes found in the same gland. This correlation suggests that the proportions of fatty acids bound to certain triacylglycerols regulates the proportions of aldehydes in biosynthesis of the pheromone blend in M. sexta. © 1995 Wiley‐Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.

Diuretic activity of Mas‐DP II, an identified neuropeptide from Manduca sexta: An in vivo and in vitro examination in the adult moth

AbstractMas‐DP II, a recently identified 30 amino acid diuretic peptide isolated from the tobacco hornworm moth, Manduca sexta, was tested for its ability to increase fluid excretion in adult M. sexta, and for the ability to elevate the rate of fluid secretion from isolated Malpighian tubules cultured in vitro. Mas‐DP II was found to increase fluid weight loss from decapitated adult moths in a dose‐dependent manner; weight loss increased significantly at doses as low as 5 ng for female moths and 25 ng for male moths. Male moths injected with large doses of Mas‐DP II continued to exhibit increased rates of fluid loss up to 4 h post‐injection. In vitro, Mas‐DP II stimulated fluid secretion from isolated Malpighian tubules at concentrations as low as 4 nM for tubules from both male and female moths. © 1994 Wiley‐Liss, Inc.

A cuticle-degrading proteinase from the moulting fluid of the tobacco hornworm, Manduca sexta

Moulting fluid of pharate adult tobacco hornworm moths, Manduca sexta, contains a novel cuticle-degrading proteinase, designated as MFP-1. The enzyme has been purified using heparin affinity chromatography and partially characterized. Before purification MFP-1 is associated with a large complex having an apparent native molecular mass > 669 kDa. After purification MFP-1 has a molecular mass of 41 kDa. The p I of the enzyme is 5.54. MFP-1 can be classified as generally trypsin-like on the basis of its substrate specificity and inhibition by soybean trypsin inhibitor. The enzyme's preferred substrate, Tos-Gly-Pro-Arg- p NA, its inhibition by hirudin, and its affinity for herapin, all indicate that MFP-1 has some characteristics in common with the vertebrate blood-clotting enzyme thrombin. MFP-1 is probably a serine protease, since it is inhibited by both DFP and PMSF (specific inhibitors of serine proteinases). However, the enzyme was also inhibited by a number of agents that effect cysteine proteinases. Purified MFP-1 degrades Manduca cuticle in vitro. We suggest that the enzyme may act as the first step in the degradation of the cuticle during the moulting process.

Fatty acyl pheromone precursors in the sex pheromone gland of female tobacco hornworm moths, Manduca sexta (L.)

The total fatty acyl composition, including free fatty acids and fatty acyl moieties in lipids, of sex pheromone glands and selected other body tissues of Manduca sextra females was determined by capillary gas chromatographic and mass spectroscopic analysis. Fatty acyl analogs of eleven pheromone aldehydes were found in the epidermal cells of the pheromone gland. While the common fatty acyl groups, hexadecanoate, (Z)-9-hexadecenoate, octadecanoate, (Z)-9-octadecenoate, (Z,Z)-9,12-octadecadienoate and (Z,Z,Z)-9,12,15-octadecatrienoate, were detected in other body tissues of females as well as in the epidermal cells and cuticle of the pheromone gland, uncommon fatty acid pheromone analogs were not found in significant amounts in tissues other than the epidermal cells of the pheromone gland. Quantitative analysis of extracts revealed that the fatty acyl analogs of pheromone aldehydes were present continuously in pheromone glands of females from about 3 or 4 days prior to eclosion from the pupal stage until the adults were 8 days old. While injection of pheromone biosynthesis activating neuropeptide into the abdomen of the females at any age, even in the late pupal stage, resulted in pheromone production, the quantities of the bound fatty acyl components in the pheromone gland were not measurably changed.

Developmental regulation of ecdysteroid receptors in the nervous system of Manduca sexta.

The technique of steroid hormone autoradiography has been used to study the cellular distribution of ecdysteroid binding sites in the ventral nervous system of the tobacco hornworm moth, Manduca sexta. The ligand was 26-[125I]iodoponasterone. Tissue was examined from the subesophageal ganglia, thoracic ganglia, and abdominal ganglia of larvae at two times during the larval-pupal transient: the 2nd day of wandering and the prepupal stage. The patterns of neuronal binding seen were compared with those found in earlier autoradiographic studies of hormone binding in tissue sampled on the 1st day of wandering, in the pharate adult, and in the 4-day-old moth (Fahrbach and Truman, '89). The pattern of binding was reproducible but dependent upon developmental stage: whereas only a subset of neurons exhibited nuclear accumulation of radiolabeled ecdysteroids on the 1st day of wandering, less than 24 hours later nearly every neuron in the ventral nervous system was labeled. A limited pattern of binding, however, was seen again in the prepupal nervous system. Thus, the insect nervous system is able to use a single hormone both as a general cue for metamorphic development and as a single targeted to stage-specific subsets of neurons by alternating periods of ubiquitous expression of receptor with periods during which the capacity to bind the steroid hormone is highly restricted.

Identification of neuroendocrine cells producing a diuretic hormone in the tobacco hornworm moth, Manduca sexta

Separate antisera were raised to the N- and C-terminal half of the diuretic hormone from Manduca sexta. Antisera against the two halves of this peptide recognized the same cells in M. sexta, and preabsorption of the antisera with the peptides used as antigens abolished the immunoreactivity, confirming their specificity. The antisera reacted with two median neurosecretory cells on each side of the protocerebral groove in larvae, and with a group of about 80 small median neurosecretory cells in the adult, as well as their axons to, and their axon terminals in, the corpora cardiaca. During the early pupal stages, small cells, which are possibly derived from a common neuroblast, differentiate into immunoreactive neurosecretory cells, which explains the large increase in cell numbers in the adult. In the sleepy sulphur butterfly, Eurema nicippe, homologous median neurosecretory cells in the adult were immunoreactive with both antisera.

Effects of starvation and sucrose feeding on corpora allata of last instar larvae of Manduca sexta: in vitro activity, size, and cell number

Abstract Larvae of the tobacco hornworm moth Manduca sexta starved for the first 3 days of the last (fifth) stadium undergo a supernumerary moult. If they are provided with sucrose during the starvation period, they develop into normal pupae although pupation is delayed. The activities of the corpora allata (CA) from normal, starved, and sucrose fed larvae were followed through the fifth stadium with a radiochemical assay for Juvenile Hormone (JH) biosynthesis. An attempt was made to correlate CA‐activity with CA cell number, size, and protein content.In CA of normally fed larvae the rate of JH synthesis declined to undetectable levels by day 4 which was also the time of exposure of the dorsal vessel. In CA of starved larvae, the rate of JH synthesis at first decreased but began to increase on day 3 and reached a peak value by day7, at which time head capsule slippage occurred. In CA of sucrose fed larvae, the rate of biosynthesis declined as in normal larvae but the decline was extended over a longer period. Exposure of the dorsal vessel was delayed in the same manner and occurred on days 7–9. The major JH in all cases was JH‐II.The CA comprise c. 150 cells in the early fifth stadium, and this number remained constant during the fifth stadium in all three feeding regimens. In normal larvae, CA size and protein content increased several‐fold during the stadium whereas in starved and sucrose‐fed larvae they increased slowly and in agreement with the altered timing of developmental events. In none of the groups was the CA activity pattern correlated with morphometric changes of the CA. The rates of JH biosynthesis were not closely correlated with published JH titre curves. The in vivo mechanisms for regulation of JH production remain to be elucidated.

11-cis retinal restores visual function in vitamin A-deficient Manduca

Larvae of the tobacco hornworm moth Manduca sexta were reared on either a carotenoid-supplemented or a carotenoid-deficient diet. The former yields fortified adults with normal visual function, whereas visual sensitivity and rhodopsin content are reduced by 2-4 log units in the compound eyes of the deprived moths reared on the latter. We characterized the retinoids of fortified retinas and investigated the recovery of visual function in deprived moths that were provided with retinaldehyde as a source of photopigment chromophore. Retinoids were identified and measured by high-performance liquid chromatography (HPLC). Fortified retinas contained mainly 3-hydroxyretinaldehyde (R3); 11-cis R3 predominated in dark-adaptation, all-trans in light-adaptation, indicating that R3 is the photopigment chromophore. No retinoids could be measured in deprived eyes. Retinaldehyde (R1) was delivered to the retinas of deprived moths by "painting" solutions of 11-cis or all-trans R1 in dimethylsulfoxide (DMSO) on the corneal surfaces of the compound eyes or on the head capsule between the eyes. 11-cis R1 induced rapid recovery: during 3 days, sensitivity rose to within a log unit of that measured from fortified animals. By 7 days, sensitivity was close to normal. Although rhodopsin and P-face particle densities of photoreceptor membranes increased, neither rose to the levels found in fortified animals. All-trans R1 induced only a slight increase in sensitivity that could have resulted from some nonspecific isomerization of the all-trans to the 11-cis isomer; we found no evidence for a retinal isomerase that functions in darkness. Small amounts of R3 were measured in recovering retinas, indicating some conversion of R1 to R3.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth and differentiation of the larval midgut epithelium during molting in the moth, Manduca sexta

The number of epithelial cells comprising larval midgut of the tobacco hornworm moth, Manduca sexta increases 200-fold in development from the first to the fifth instar. We have examined larvae periodically before and during molting to follow epithelial cell proliferation and differentiation. The midgut epithelium in Manduca sexta consists predominantly of columnar and goblet cells. These are arranged in a characteristic pattern with each goblet cell surrounded by a single layer of 4–6 columnar cells (Hakim et al., (1988)). While undifferentiated basal stem cells are infrequently seen in intermolt larvae, just prior to the period when external signs of molting are visible, their number increases and mitotic figures become common. Proliferation continues for several hours and then these stem cells differentiate following a pattern similar to that seen during embryogenesis (Hakim et al., (1988)). Here, however, the newly differentiating cells become intercalated among the mature differentiated cells already present in the epithelium. Since the pattern of individual goblet cells surrounded by a reticulum of columnar cells is maintained after the addition of new cells, the midgut epithelium of molting larvae appears to be a useful model for studying pattern formation in development.

Isolation of a functional antigen-Ia complex.

The helper T-cell recognition of globular protein antigens requires that the antigen be processed and presented by an I-region associated (Ia)-expressing antigen-presenting cell (APC). Processing involves the uptake of antigen into an intracellular, proteolytic, acidic compartment; release of peptide fragments containing the T-cell antigenic determinant; association of these peptides with Ia; and presentation of these complexes on the cell surface for recognition by the specific T cells. The molecular mechanisms by which processed antigenic peptides associate with Ia within the APC are poorly understood. To date, functional antigen-Ia complexes have not been isolated from cells that have processed native antigens, although the resolution of the structure of a major histocompatibility complex (MHC) class I protein indicates that peptide is bound in a groove between two alpha-helical regions of the molecule and synthetic peptides have been demonstrated to bind purified MHC both in detergent solution and incorporated into planar membranes, where the MHC-peptide complexes function to activate specific T cells. Here we demonstrate that Ia purified from APCs that have processed the native globular protein antigen cytochrome c, when incorporated into lipid membranes, stimulates cytochrome c-specific T cells in the absence of exogenous antigenic peptide. The T-cell response to Ia purified from cytochrome c-pulsed APCs shows the same MHC restriction and antigen fine specificity as the response to antigen-pulsed APCs. Indeed, T-cell recognition of pigeon cytochrome c (Pc) shows a well documented high-affinity heteroclitic cross-reaction to insect cytochromes c-namely, those of Drosophila melanogaster (DMc) and tobacco hornworm moth (THMc). The same heteroclitic response is observed when purified Ia from unpulsed cells, incorporated into lipid membranes, is used to present antigenic peptides of Pc and of THMc. Significantly, Ia purified from APCs that have processed DMc is approximately 50-fold more active in stimulating specific T cells compared to Ia purified from APCs that have processed Pc. The peptide-Ia complex isolated here may provide the necessary material for analysis of the physiochemical properties of the processed form of the antigen that is produced by the APC and associates with Ia.

Hormonally Mediated Modifications of Neuronal Structure, Synaptic Connectivity, and Behavior During Metamorphosis of the Tobacco Hornworm,Manduca sexta

During insect metamorphosis, each life stage (larva, pupa, adult) exhibits a largely distinct behavioral repertoire. This turnover in behaviors is accompanied by extensive remodelling of the nervous system, which is controlled hormonally. A fundamental question raised by metamorphosis is how hormonal effects on the structure and function of individual neurons ultimately produce changes in behavior. We have examined this question in the tobacco hornworm moth, Manduca sexta. During the larval-pupal transformation of Manduca, the motoneurons innervating muscles of the abdominal prolegs (larval locomotory appendages) undergo an extensive dendritic regression that is triggered by ecdysteroid hormones. Over the same time period, behaviors involving the prolegs weaken and disappear. In this review we describe experiments examining the relationship between hormonally mediated proleg motoneuron regression, and the elimination of the proleg behaviors. In the case of a simple monosynaptic sensory-to-motoneuron reflex, and a peptide-activated rhythmic ecdysis motor pattern, we suggest that the ecdysteroid-mediated regression of the motoneuron arbors contributes to the developmental loss of the behaviors. In a third example, involving the activity of another class of motoneurons during peptide-activated pre-ecdysis motor patterns, the behavioral changes apparently originate at the interneuronal rather than motoneuronal level. We conclude that the hormonally mediated dendritic regression of neurons, and resultant modifications of their synaptic connections, is one of the mechanisms that contributes to the elimination of outmodedbehaviors during metamorphosis.

Ultrastructure of carotenoid deprivation in photoreceptors of Manduca sexta: myeloid bodies and intracellular microvilli

The ultrastructural effects of carotenoid (vitamin A) deprivation were studied in the adult photoreceptors of the tobacco hornworm moth Manduca sexta. Moths were reared on a “deprived” diet, which lacked the carotenoid sources of the photopigment chromophore, 3-hydroxy retinal, or on a control, “fortified” diet, containing ample carotenoid. The latter supported normal levels of visual function, whereas visual pigment and sensitivity were reduced by at least 3 log units in moths reared on the deprived diet. Myeloid bodies, massed cisternae of hypertrophied smooth endomembrane, filled the cytoplasm in the receptors of deprived animals. The myeloid bodies assumed various configurations that included lamellate stacks of parallel cisternae, and tubular networks in a “paracrystalline” form. Freeze-fracture preparations of myeloid membranes revealed a high density of P-face particles. Vacuoles containing microvilli similar to those of the rhabdomere were also present in deprived photoreceptors. We suggest that the elaboration of smooth endoplasmic reticulum as myeloid bodies in chromophore-deprived photoreceptors may stem from the hypertrophy of a biochemical system for processing the chromophore or the interruption of the intracellular pathway that normally carries visual pigment to the rhabdomere.

Sex‐specific neuronal respecification during the metamorphosis of the genital segments of the tobacco hornworm moth Manduca sexta

At metamorphosis, the terminal abdominal segments of larvae of the moth Manduca sexta transform into either male or female genitalia. At the start of this transformation, the larval muscles degenerate but their remains may persist to form the scaffolding on which the new adult muscles differentiate. The survival and subsequent orientation of larval muscle remnants is determined by the sex of the individual and is independent of motor innervation at the start of metamorphosis. Many of the larval motoneurons persist through metamorphosis and innervate the skeletal muscle of the adult. The survival of particular motoneurons is also sex-dependent and correlated with the survival of its respective muscle remnant. No new skeletal motoneurons arise postembryonically, so all of the adult skeletal muscle motoneurons are derived from preexisting larval skeletal muscle motoneurons. The fates during metamorphosis are more complex for the visceral muscle motoneurons. Those innervating the adult hindgut of both sexes are identical and are derived from the larval hindgut motoneurons. Other hindgut motoneurons in the larva switch targets during metamorphosis and come to innervate the oviduct in adult females or perish in adult males. Other regions of the reproductive tract become innervated by adult-specific cells that differentiate during metamorphosis. These cells come from distinct lineages in males and females.

A pheromone-degrading aldehyde oxidase in the antennae of the moth Manduca sexta

Antennae of the tobacco hornworm moths Manduca sexta contain an aldehyde oxidase (AOX) that oxidizes aldehydes to carboxylic acids. The enzyme, which is distinguishable from aldehyde-oxidizing activities in other tissues, is secreted into the receptor lymph that bathes the primary olfactory dendrites. First detectable about 3 d before eclosion, AOX levels increase through the first day after eclosion. This parallels the development of the antennal responsiveness to bombykal (a male attractant aldehydic pheromone produced by female M. sexta) and trans-2-hexenal (an aldehyde commonly found in leaves). The AOX is about 60% more abundant in antennae of males than in antennae of females. The antennal AOX is a dimer with Mr of 295 kDa and is capable of oxidizing a variety of aldehydes. Of all aldehydes examined, the pheromone bombykal was the best substrate with an apparent Km of 5 microM, whereas the next best substrate, benzaldehyde, had an apparent Km of 255 microM. Using kinetic parameters estimated in vitro and the assumption of first-order kinetics, the half-life of bombykal in sensilla was estimated to be about 0.6 msec. The affinity of the antennal AOX for bombykal, its location in the receptor lymph, and its pattern of developmental expression all suggest that it plays a role in modulating the sensitivity of adult M. sexta to aldehyde odors and, in particular, the sensitivity of males to the pheromone bombykal.

Identification of a pheromone blend attractive to Manduca sexta (L.) males in a wind tunnel

AbstractAnalyses of solvent rinses of the external surfaces of pheromone glands excised from calling female tobacco hornworm moths, Manduca sexta (L.), revealed the presence of the following compounds: (Z)‐9‐hexadecenal, (Z)‐11‐hexadecenal, (E)‐11‐hexadecenal, hexadecanal, (E,Z)‐10,12‐hexadecadienal, (E,E)‐10,12‐hexadecadienal, (E,E,Z)‐10,12,14‐hexadecatrienal, (E,E,E,)‐10,12,14‐hexadecatrienal, (Z)‐11‐octadecenal, (Z)‐13‐octadecenal, octadecanal, and (Z,Z)‐11,13‐octadecadienal. The two trienals were identified by mass and PMR spectral analyses and by ozonolyses, and their structures were confirmed by synthesis. In a wind tunnel male tobacco hornworm moths exhibit the same behaviors in response to a synthetic blend of all of the components, the gland rinse, or a calling female. Both (E,Z)‐10,12‐hexadecadienal and (E,E,Z)‐10,12,14‐hexadecatrienal are required to stimulate males to complete the characteristic behavioral sequence: anemotaxis, approaching and touching the pheromone source, and bending their abdomens in apparent copulatory attempts. The other components of the blend may play more subtle roles.

Calling Behavior of Manduca sexta (L.) (Lepidoptera: Sphingidae) with Notes on the Morphology of the Female Sex Pheromone Gland

The calling behavior of the female tobacco hornworm moth, Manduca sexta (L.), is described and correlated with pheromone release. The distribution of calling times over the first seven evenings after eclosion is reported; the females tended to start calling earlier and for a longer period the older they were. During observations over 96 h of continuous darkness, four regularly spaced periods of calling were seen with a mean interval of 25.4 h—a finding consistent with the existence of a circadian rhythm. Electroantennogram bioassays of parts of the female abdominal tip confirm the location of the female sex pheromone gland. The highest pheromonal activity was found in the region of the ventral membrane of the ninth segment, an area previously identified on histological evidence. The gland was examined with light and scanning electron microscopy. It is a modification of the ventral membrane of the ninth abdominal segment and consists of a monolayer of cuboid to columnar cells lining the cuticle. The surface of the intersegmental membrane is covered by small spines, some of which appear to have droplets at their tips. A second area of glandular appearance also was found underlying the anal papillae of the ninth segment.

Enhanced T cell responses to antigenic peptides targeted to B cell surface Ig, Ia, or class I molecules.

The helper T cell recognition of soluble globular protein antigens requires that the proteins be processed by an APC, releasing a peptide that is transported to and held on the APC surface where it is recognized by the specific T cell in conjunction with Ia. When cellular processing functions are blocked, APC lose their ability to present native antigens while retaining the capacity to activate T cells when provided with a cognate peptide fragment that contains the T cell antigenic determinant. In this report, we show that a peptide fragment of the soluble globular protein antigen tobacco hornworm moth cytochrome c, residues 92-103 containing an additional NH2-terminal cysteine residue (THMcCys92-103), is effectively presented by B cells to an I-Ek-restricted, THMc-specific T cell hybrid when covalently coupled to antibodies specific for B cell surface Ig, Ia (Ak), or class I (Kk). Maximal activation of the T cells to the THMcCys92-103-antibody conjugates is achieved with 1/100-1/1,000th of the peptide required using unconjugated THMcCys92-103 or THMcCys92-103 coupled to nonspecific antibody. The T cell response to the peptide antibody conjugates is MHC restricted, but unlike native cytochrome c-antibody conjugates, THMcCys92-103-antibody conjugates do not require processing and can be presented by paraformaldehyde-fixed B cells. The THMcCys92-103-antibody conjugate are nearly as effective when incubated with B cells, and the unbound conjugates washed away before addition of T cells as when continuously present in culture with T cells and B cells, indicating that the active peptide antibody conjugate is associated at the B cell surface. The presentation of THMcCys92-103 coupled to monovalent Fab fragments of rabbit anti-Ig antibodies is less effective than that of the peptide coupled to bivalent antibody when either live or fixed B cells are APC, indicating that the avidity for the APC surface afforded by bivalent binding may be important in the conjugate's antigenicity. The results presented here indicate that a T cell-antigenic peptide, covalently coupled to a larger antibody molecule, can be readily recognized by an Ia-restricted helper T cell in the absence of processing. Moreover, the ability of the peptide to bind to B cell surfaces greatly augments the peptide's antigenicity, even when the binding is to structures distinct from the Ia molecule required for T cell activation.

CDNA cloning and deduced amino acid sequence of microvitellogenin, a female specific hemolymph and egg protein from the tobacco hornworm, Manduca sexta.

Microvitellogenin is a female-specific yolk protein from the tobacco hornworm moth Manduca sexta. A cDNA library was constructed from poly(A)+ RNA isolated from adult female fat body. cDNA clones of mRNA for microvitellogenin were isolated by using antiserum against microvitellogenin. Northern blot analysis of poly(A)+ RNA isolated from different life stages and sexes reveals that mRNA coding for microvitellogenin is only present in adult female fat body. Immunoprecipitation of the protein product translated from hybrid selected mRNA indicates that the cDNA clone is specific for microvitellogenin. The complete nucleotide sequence of the 834-base pair cDNA insert has been determined by the dideoxy chain termination method. The cDNA sequence predicts that microvitellogenin is a protein of 232 residues with a calculated molecular weight of 26,201. The cDNA also predicts an amino-terminal extension of 17 residues which are not present in the mature form. This sequence appears to be a signal peptide. A comparison of the translated amino acid sequence with the sequences in the National Biomedical Foundation protein library did not establish any sequence homology with other known proteins.