Foodplant selection and induction of feeding preference among host and non-host plants in larvae of the tobacco hornworm Manduca sexta

Ten host plant (Solanaceae) and twelve non-host plant species were tested as foodplants for first instar larvae of the tobacco hornworm, Manduca sexta. Only nine host and three non-host plant species elicited feeding and supported growth up to fifth instar. The range of acceptability suggested that plants be divided into hosts, acceptable non-hosts, and unacceptable non-hosts. Using the two-choice feeding preference test we found that the initial preference for hosts was preserved when larvae were reared on hosts, but was less strong or absent for larvae reared on acceptable non-hosts. Thus oligophagy in the tobacco hornworm is not induced, but must be inherited.Newly-hatched first instar larvae and fifth instar larvae showed a preference hierarchy among both hosts and non-hosts. Fifth instar larvae reared separately on two different host species showed slightly different preference hierarchies among hosts. The preference for the rearing plant was increased and also two other host species changed positions in hierarchies.Feeding preferences of larvae reared on hosts or acceptable non-hosts were determined using plant combinations of host vs. host, host vs. acceptable non-host, and acceptable non-host vs. acceptable non-host. Induction of feeding preference was found in all three of these categories. This shows that induction of feeding preference in the tobacco hornworm is not restricted to host plant species.The degree to which feeding preferences were induced ranged from very strong to undetectable and dependend on the plant species paired. The strength of induction in the tobacco hornworm was found to correlate inversely with taxonomic relatedness of the plant species paired. Analysis of induction data from the literature revealed a similar correlation for other lepidopteran species.

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Résumé Vingt-deux espèces de plantes, dont 10 planteshôtes (Solanées), ont été testés comme plantes alimentaires pour des chenilles de ler stade de Manduca sexta. Sur cet ensemble, seulement 12 plantes (dont 9 plantes hôtes) induisaient la prise de nourriture et permettaient la croissance jusqu'au 5ème stade. La diversité des résultats suggère que les plantes pouvaient être classées en hôtes, non-hôtes acceptables et non-hôtes refusés. En utilisant le test du choix alimentaire préférentiel entre deux rondelles de feuilles, les chenilles néonates de ler stade ont préféré nettement les plantes-hôtes aux autres. Cette préférence initiale pour les plantes-hôtes était préservée quand les cheniles étaient élevées sur plantes-hôtes, mais devenait moins nette ou disparaissait pour des chenilles élevées sur d'autres plantes acceptées. Ainsi l'oligophagie ches M. sexta n'est pas induite, mais doit être héritée.Les chenilles néonates, aussi bien que celles de 5ème stade, présentent des préférences hiérarchisées parmi les plantes hôtes ou non. La seule frontiere nette observée était entre espèces de plantes acceptables ou non. Les hiérarchies préférentielles des chenilles du 5ème stade différaient légèrement lors-qu'elles avaient été élevées sur deux plantes-hôtes différentes. La différence essentielle était l'observation d'une préférence accrue pour l'espèce ayant servi à l'élevage, mais deux autres plantes-hôtes changaient aussi de position hiérarchique.La cause de ces changements de préférence a été approfondie, les chenilles étant élevées sur des feuilles de chaque espèce acceptable (hôte ou non). Leurs préférences alimentaires ont été définies en utilisant des combinaisons diverses (hôte x hôte, hôte x non-hôte acceptable, non-hôte acceptable x non-hôte acceptable). L'induction de la préférence alimentaires a été obtenue dans ces trois associations. Ceci montre que l'induction des choix alimentaires chez M. sexta n'est pas limitée aux plantes-hôtes. Le degré d'induction de la préférence alimentaire variait de très fort à indécelable; il dépendait de l'association examinée. La source de la variabilité de cette induction a été examinée en fonction de la relation entre la force de l'induction et les rapports taxonomiques des plantes associées. La relation obervée était inversée pour M. sexta. L'examen des données de la littérature ont révélé une relation du même type pour les autres espèces de Lépidoptères.

     

Cloning and expression analysis of midgut chymotrypsin-like proteinases in the tobacco hornworm

Digestion of proteins in the midgut of lepidopteran larvae relies on different trypsin and chymotrypsin isoforms. In this study we describe three chymotrypsin-like proteinases (CTLP2-4) from the larval midgut of Manduca sexta, which are closely related to CTLP1 and less closely related to another chymotrypsin (CT), two previously described proteinases present in the larval midgut of M. sexta. CTLP1-4 fit perfectly into a novel subgroup of insect CTLPs by sequence similarity and by the replacement of GP by SA in the highly conserved GDSGGP motif. When we examined CTLP expression in different tissues, most of the proteinases were predominantly expressed in the anterior and median midgut, while some were found in the Malpighian tubules. When we examined CTLP expression at different physiological states, we observed that the CTLP mRNA amounts did not differ considerably in feeding and starving larvae except for CTLP2, whose mRNA dropped significantly upon starvation. During moulting, however, the mRNA amounts of all CTLPs dropped significantly. When we immunologically examined CTLP amounts, mature proteinases were only detectable in the gut lumen of feeding and re-fed larvae, but not in that of starving or moulting larvae, suggesting that CTLP secretion is suspended during starvation or moult.     

Wild species of Nicotiana as a new source of tobacco resistance to the tobacco hornworm Manduca sexta

Sixteen species of Nicotiana were examined for resistance to second instar larvae of the tobacco hornworm, Manduca sexta (L.). The tests were designed so as to discriminate between antibiosis and nonpreference. High levels of antibiosis resistance were observed in several wild species such as N. gossei and species in the Repandae section. Several other species, such as N. excelsior, showed antibiosis that appeared to be due to a different mechanism than the alkaloid-trichome exudate based resistance of the above species. The data indicate that these species of Nicotiana may be used as a new source of resistance to larvae of M. sexta.

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D'espèces sauvages de Nicotiania comme nouvelle source de résistance du tabac à Manduca sexta

Résumé La résistance aux chenilles de second stade de Manduca sexta L. a été examinée chez seize espèces de Nicotiana. Les essais ont été conçus pour distinguer absence d'appétance et antibiose. Plusieurs espèces sauvages comme N. gossei et les espèces de la section Repandae ont présenté un niveau élevé d'antibiose. D'autres espèces, comme N. excelsior ont présenté une antibiose vraisemblablement due à un mécanisme différent de la résistance des espèces précédentes provoquée par un exsudat alcaloïde des trichomes. Les résultats indiquent que ces espèces de Nicotiana peuvent être utilisées comme nouvelle source de résistance aux chenilles de M. sexta.

     

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     

The role of destruxins in the pathogenicity of 3 strains of Metarhizium anisopliae for the tobacco hornworm Manduca sexta

Three out of 4 isolates of the Deuteromycete Metarhizium anisopliae were pathogenic for larvae of the tobacco hornworm, Manduca sexta. The most virulent isolate (ME1) grew sparsely in the insect prior to death and caused paralysis of its host. The other 2 pathogenic isolates killed Manduca larvae more slowly, grew profusely in the haemolymph and did not induce symptoms of toxicosis. Toxicosis is apparently due to the production by the fungus of several cyclodepsipeptide toxins, destruxins (DTX). ME1 produced large quantities of DTX in vitro, while other isolates produced less. Destruxin A (DTX A) was recovered from the haemolymph of paralysed, diseased insects infected with ME1, but not with other isolates. It is suggested that DTX may have a pathogenic role, when the toxins are active in causing disease, or an aggressive role, when they facilitate the establishment of the pathogen.     

Feeding responses to solanaceous allelochemicals by larvae of the tobacco hornworm, Manduca sexta

Feeding responses of the oligophagous tobacco hornworm to allelochemicals prevalent in their host plants were determined in food choice-tests using filter paper discs laced with a test solution or water (control). Six solanaceous alkaloids, tomatine, tomatidine, solanine, solanocapsine, atropine and nicotine, were tested and only tomatine and solanocapsine were found to influence preference behavior. Solanocapsine (5 mM) deters feeding whereas tomatine (1 mM) stimulates feeding slightly. No synergistic effect of either tomatine or tomatidine with sucrose was found.The responses to tomatine are affected by previous feeding experience. Tomatine slightly stimulates feeding in larvae reared on tomato (Lycopersicon esculentum), but slightly deters feeding in larvae reared on Jerusalem cherry (Solanum pseudocapsicum). Such induced preference is absent for the other alkaloids tested, which indicates that these alkaloids do not by themselves induce preferences for the plants containing them.The non-alkaloid allelochemicals, chlorogenic acid, rutin, and 2-tridecanone also influenced food choice behavior. Chlorogenic acid is slightly stimulatory at its natural concentration (1mM), but strongly deterrent at higher concentrations. Rutin stimulates feeding in a concentration-dependent manner. Its activity must be due to the glycosylated structure, because both the aglycone (quercetin) and the sugar moiety (rutinose) are neutral. Removal of the glucose part of rutin, as in quercitrin, results in feeding deterrent activity. 2-Tridecanone is neutral at its concentration in cultivated tomato (1 mM), but strongly deterrent and toxic at higher concentrations. Preference behavior is not affected by solanesol, GABA, and a mixture of host plant compounds stimulatory for anothe solanaceous-specific feeder, the Colorado potato beetle (Leptinotarsa decemlineata).We conclude that the prevalent solanaceous alkaloids and other allelochemicals tested do not play important roles in food selection of the tobacco hornworm, although some may make small contributions.

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Résumé Des experiences de choix de chenilles oligophages de M. sexta ont été réalisees avec des disques de papier filtre imbiles d'eau ou de solutions des substances allélochimiques dominantes dans les plantes consommées. Sur les six alcaloïdes de solanées examinés: tomatine, tomatidine, solanine, solanocapsine, atropine et nicotine, seuls la tomatine et la solanocapsine ont influé sur le choix; la solanocapsine (5 mM) empêche la prise de nourriture, tandis que la tomatine (1 mM) la stimule légèrement. Aucun effet synergique de la tomatine ou de la tomatidine n'a été observé en présence de sucrose.La réponse à la tomatine est modifiée par la prise de nourriture antérieure. Elle stimule légèrement l'alimentation de chenilles élevées sur tomates (Lycopersicon esculentum), mais dissuade légèrement les chenilles élevées sur Solanum pseudocapsicum. II n'y a pas d'action induite semblable avec les autres alcalïdes examinés, ce qui indique que ces alcaloïdes ne peuvent pas induire par eux-mêmes de préférences pour les plantes qui les contiennent.Des substances allélochimiques non-alcaloïdes: acide chlorogénique, rutine, et 2-tridécanone, influent aussi sur le comportement de choix alimentaire. L'acide chlorogénique est légèrement stimulant à sa concentration naturelle (1 mM), mais fortement dissuasif aux concentrations supérieures. La rutine stimule la prise de nourriture en fonction de sa concentration. Son activité doit être due à sa structure glucosylate, puisqu'aussi bien l'aglycone (quercitine) que la moiteé sucrée (rutinose) sont neutres. La suppression de la partie glucose de la rutine, comme dans le cas de la quercitine, a un effet dissuasif. A sa concentration dans la tomate cultivée (1 mM), le 2-tridécanone est neutre, mais il est fortement dissuasif et toxique à des concentrations supérieures.Le comportement de choix n'est pas modifié par le solanésol, le GABA, et par un mélange de composés végétaux stimulant un consommateur spécifique de solanées, comme le doryphore (Leptinotarsa decemlineata).Nous pouvons conclure que les principaux alcaloïdes et autres substances allélochimiques des solanées que nous avons examinés n'interviennent pas d'une façon importante, mais peuvent avoir une influence secondaire, dans les choix alimentaires de Manduca sexta.

     

Microstructure of feeding by tobacco hornworm caterpillars, Manduca sexta

The microstructure of the feeding activity of tobacco hornworm caterpillars (Manduca sexta Johansson) on tomato leaf was examined by means of an automated cafeteria. In this device each activity of the caterpillar generates a characteristic slow electrical change which can be recorded. The apparatus is therefore both accurate and sensitive. Examination of the activity records indicated that larger animals ate more than smaller ones by increasing both bite frequency and the lengths of meals. Meal frequency did not increase. Correlations amongst a variety of measures indicated that there was regulation of feeding both between and within meals.     

Diet-induced food preference by Manduca sexta larvae: acceptable non-host plants elicit a stronger induction than host plants

Diet-induced changes in food preference by fifth instar larvae of the tobacco hornworm, Manduca sexta (Johan.) (Lepidoptera, Sphingidae), were examined. Two groups of larvae with different diet experience were used: larvae reared on a host or on an acceptable non-host plant species. Each group of larvae was offered a choice between leaf discs from each rearing plant species (2-plant choice test) and food preference was measured as the consumption of one plant species relative to that of the other plant species. Diet-induced changes in preferences were larger with the host versus acceptable non-host plant pairs Solanum pseudocapsicum (L.) versus Raphanus sativus (L.), Lycopersicon esculentum (Mill.) versus Vigna sinensis (Savi), and Datura innoxia (L.) versus V. sinensis than with the host versus host plant pairs L. esculentum versus Capsicum annuum (L.) and L. esculentum versus D. innoxia. To examine how much the food preference had been altered for each test plant species alone, two other groups of larvae were offered a choice between leaf discs from a single plant species and filter paper discs laced with distilled water (1-plant choice test). Larvae preferred the dietary plant species more strongly than the non-dietary plant species in tests using the following plant species: for C. annuum with C. annuum and L. esculentum as diets, for V. sinensis with V. sinensis and L. esculentum or D. innoxia as diets, and for R. sativus with R. sativus and S. pseudocapsicum as diets. The preference for the hosts L. esculentum and D. innoxia did not change significantly after rearing larvae on different hosts or on an acceptable non-host. Thus, diet-induction by M. sexta larvae results in an enhancement of preference for the dietary plant species which is much stronger with acceptable non-hosts than with hosts.     

Heat sensitivity and protein synthesis during heat-shock in the tobacco hornworm,Manduca sexta

Summary Fifth instar larvae of the tobacco hornworm,Manduca sexta, tolerate 1-h exposures to temperatures as high as 42°C. Above 42°C, survival declines rapidly to 18% at 44°C and 0% at 48°C. As in other insects, the heat-shock response ofManduca sexta involves the induction of synthesis of heat-shock proteins very similar in size to theDrosophila heat-shock proteins (84, 73, 71, 27, 25, 23, and 22 kd). In the epidermis, heat-shock protein synthesis peaks at 42°C, correlating with the heat sensitivity of both the tissue itself and the intact larva. Some heat-shock proteins have different isoelectric forms depending on tissue. Also, the heat-shock proteins are synthesized over a wider range of temperatures in the imaginal discs and the fat body as compared to the epidermis. In contrast to dipteran insects,Manduca sexta does not exhibit a strong repression of non-heat-shock protein synthesis under tolerable conditions.Abbreviations TCA trichloroacetic acid - PAGE polyacrylamide gel electrophoresis - AZT arbitrary Zeitgeber time - kd kilodaltons     

RearingTrichogramma nubilale [Hymenoptera: Trichogrammatidae] on eggs of the tobacco hornworm,Manduca sexta [Lepidoptera: Sphingidae]

The fresh frozen egg of the tobacco hornworm (TBH)Manduca sexta (L.), is an efficient and superior host for mass production ofTrichogramma nubilale Ertle & Davis. Each host egg may produce 10.7±2.8 (n=7) large, robust, and activeT. nubilale. The proportion of ♀♀ stabilized at 80–90% with 69.9±26.6 (n=8) ovarian eggs per female. As many as 3 ♀♀ were observed ovipositing simultaneously into a single TBH egg. Superparasitism (>10 progeny) should be avoided because it may cause nutritional or space limitations on the development of effectiveT. nubilale.      

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.     

Site of synthesis and phylogenetic distribution of a hemolymph trophic factor of the tobacco hornworm,Manduca sexta

Summary Identification of fith instar larvalManduca sexta fat body and epidermis as sites of synthesis of a hemolymph protein (hemolymph trophic factor or HTF) was achieved using in vitro³H-leucine incorporation into protein and subsequent immunoprecipitation of tissue homogenates. Fat body is the primary site of HTF synthesis with a maximal rate on Day 1; epidermis is a secondary site with peak synthesis on Day 0. In vitro radiolabelling followed by TCA precipitation of general protein of fat body and epidermal homogenates suggest that fat body actively elaborates protein on Days 0–5 with peak rates on Days 1 and 4, while epidermis is active on Days 0–5 with a peak rate on Day 3. Based on Anti-HTF ELISA estimates, HTF [500 to 1000 μg/ml] was found in the hemolymph of representatives of the insect orders Blattodea, Hemiptera, Orthoptera, and Lepidoptera and in the class Crustacea, but not in the class Merostomata. These studies suggest a possible fundamental role for HTF among modern arthropods in cuticular deposition involving both epidermis and fat body. The physiological role of HTF is undetermined.     

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.     

K<Superscript>+</Superscript> transport in the caterpillar intestine epithelium: role of osmolytes for the K<Superscript>+</Superscript>-secretory capacity of the tobacco hornworm midgut

The midgut of the tobacco hornworm, Manduca sexta, actively secretes potassium ions. This can be measured as short-circuit current (I_sc) with the midgut mounted in an Ussing chamber and superfused with a high-K⁺ saline containing as its major osmolyte 166 mM sucrose. Iso-osmotic substitution of sucrose by non-metabolisable compounds (mannitol, urea, NaCl and the polyethylene glycols 200, 400 and 600) led to a dramatic, though reversible, drop in the current. Acarbose, a specific inhibitor of invertase (sucrase) in vertebrates and insects, had no detectable influence on I_sc. Unexpectedly, after replacing sucrose iso-osmotically with the saccharides glucose, fructose, trehalose or raffinose, the K⁺ current could no longer be supported. However, all osmolytes smaller than sucrose (except for NaCl), metabolisable or not, initiated an immediate, quite uniform but transient, increase in I_sc by about 20%, before its eventual decline far below the control value. Hypo-osmotic treatment by omission of sucrose also transiently increased the K⁺ current. Small osmolytes substituted for sucrose caused no transient I_sc stimulation when the epithelium had been challenged before with hypo-osmolarity; however, the eventual decline in I_sc could not be prevented. Our data seem inconsistent with a role of sucrose as energiser or simple osmolyte. Rather, we discuss here its possible role as analogous to that of sucrose in lower eukaryotes or plants, as an extra- and/or intracellular compatible osmolyte that stabilises structure and/or function of the proteins implicated in K⁺ transport.Communicated by G. Heldmaier     

Hormonally-regulated differential expression of the two duplicated insecticyanin genes,ins-a and ins-b,during development of the tobacco hornworm,Manduca sexta

The effects of juvenile hormone (JH) and 20-hydroxyecdysone (20E) on the developmental expression of the two insecticyanin genes, ins-a and ins-b, were investigated with two gene-specific probes. Removal of the corpora allata (-CA, source of JH) clearly delayed and down-regulated the epidermal expression of these genes but enhanced their expression in the fat body during the early development of the fifth instar. Application of JH I to the -CA larvae at the time of head capsule slippage completely restored the normal epidermal expression pattern of the two genes in the early fifth instar, then INS-a mRNA declined prematurely whereas INS-b mRNA remained similar to that in the intact larvae. By contrast, in the fat body of -CA larvae, the exogenous JH had little effect on the levels of INS-a mRNA, but enhanced expression of INS-b mRNA relative to intact larvae. Culture of epidermis from day 1 fifth instar larvae with 40 ng/ml 20E for up to 24 h accelerated the loss of INS-a mRNA without affecting the levels of INS-b mRNA. Both mRNAs declined in isolated larval abdomens over a 24 h period, and this decline was slowed by 1 g methoprene (a JH analog). Together these results indicate that JH controls the levels of the two mRNAs in both the epidermis and fat body, with additional factors involved in regulating these genes in the fat body during the molt and in the epidermis during the growth phase.     

Molecular cloning of a multidomain cysteine protease and protease inhibitor precursor gene from the tobacco hornworm (Manduca sexta) and functional expression of the cathepsin F-like cysteine protease domain

A Manduca sexta (tobacco hornworm) cysteine protease inhibitor, MsCPI, purified from larval hemolymph has an apparent molecular mass of 11.5 kDa, whereas the size of the mRNA is very large (9 kilobases). MsCPI cDNA consists of a 9,273 nucleotides that encode a polypeptide of 2,676 amino acids, which includes nine tandemly repeated MsCPI domains, four cystatin-like domains and one procathepsin F-like domain. The procathepsin F-like domain protein was expressed in Escherichia coli and processed to its active mature form by incubation with pepsin. The mature enzyme hydrolyzed Z-Leu–Arg–MCA, Z-Phe–Arg–MCA and Boc–Val–Leu–Lys–MCA rapidly, whereas hydrolysis of Suc–Leu–Tyr–MCA and Z-Arg–Arg–MCA was very slow. The protease was strongly inhibited by MsCPI, egg-white cystatin and sunflower cystatin with K_i values in the nanomolar range. When the MsCPI tandem protein linked to two MsCPI domains was treated with proteases, it was degraded by the cathepsin F-like protease. However, tryptic digestion converted the MsCPI tandem protein to an active inhibitory form. These data support the hypothesis that the mature MsCPI protein is produced from the MsCPI precursor protein by trypsin-like proteases. The resulting mature MsCPI protein probably plays a role in the regulation of the activity of endogenous cysteine proteases.     

The relative insensitivity of Manduca sexta larvae to non-host plant secondary compounds

We tested the deterrent effects of non-host plant secondary compounds on Manduca sexta Johan. larvae, in choice tests. Treatment chemicals were applied, at approximately natural concentrations to either glass fiber or tobacco leaf disks. Of 30 compounds tested on glass fiber disks, with artificial diet reared larvae, 6 were deterrent. When the 6 deterrent chemicals were tested on tobacco leaf disks only one was still deterrent. No deterrent effects were observed when tobacco reared larvae were tested with chemicals applied to tobacco leaf disks. The results suggest that maintenance of restricted host range in M. sexta larvae relies not on avoidance of non-hosts due to deterrence but on attraction and stimulation to feed on hosts.

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Résumé Nous avons examiné au moyen des expériences de choix, les effects dissuasifs des composés secondaires de plantes non-hôtes des chenilles de M. sexta Johan. Des disques de fibres de verre ou des rondelles de feuilles de tabac ont été traités chimiquement, aux concentrations à peu près naturelles. Sur les 30 substances examinées sur les disques de fibres de verre, 6 ont été répulsives pour des chenilles élvées sur aliment artificiel. Quand l'examen est effectué avec des rondelles de feuilles de tabac, seul un produit est encore répulsif. Aucun effet répulsif n'est observé quand des chenilles élevées sur tabac sont mises en présence de substances associées à des rondelles de feuille de tabac. Ces résultats suggèrent que le maintien d'un spectre d'hôtes limité chez les chenilles de M. sexta ne repose pas sur l'évitement des plantes non-hôtes par suite de leurs substantes dissuasives, mais sur l'attraction et la stimulation provoquées par les hôtes.

     

Trehalose-hydrolysing enzymes of Metarhizium anisopliae and their role in pathogenesis of the tobacco hornworm,Manduca sexta

Trehalose is the main haemolymph sugar in most insects including the tobacco hornworm, Manduca sexta, and is potentially a prime target for an invading pathogenic fungus. There was considerably more trehalose-hydrolysing activity in the haemolymph of caterpillars infected with Metarhizium anisopliae than in controls. This appeared to be due primarily to additional isoforms; one of which could also hydrolyse maltose and was designated an alpha-glucosidase. A comparable isoform was identified in in vitro culture of the fungus, supporting a fungal origin for the in vivo enzyme. The in vitro fungal enzyme, alpha-glucosidase-1 (alpha-gluc-1), was purified to homogeneity and partially characterised. A study with the trehalase inhibitor trehazolin and C14 trehalose suggested that extracellular hydrolysis is important for fungal mobilisation of trehalose. Haemolymph glucose increases significantly during mycosis of tobacco hornworm larvae by M. anisopliae, consistent with the hydrolysis of trehalose by extracellular fungal enzymes. The implications for the host insect are discussed.