ngrA-dependent natural products are required for interspecies competition and virulence in the insect pathogenic bacterium Xenorhabdus szentirmaii.

Xenorhabdus species are symbionts of entomopathogenic nematodes and pathogens of susceptible insects. Nematodes enter insect hosts and perforate the midgut to invade the haemocoel where Xenorhabdus bacteria are released transitioning to their pathogenic stage. During nematode invasion microbes from the insect gut translocate into the haemocoel. Different species of nematodes carrying specific strains of Xenorhabdus can also invade the same insect. Xenorhabdus species thereby compete for nutrients and space with both related strains and non-related gut microbes. While Xenorhabdus species produce diverse antimicrobial compounds in complex media, their functions in insect hosts are not well understood. We show that Xenorhabdus szentirmaii produced ngrA-dependent antibiotics that were active against both gut-derived microbes and Xenorhabdus nematophila whereas antibiotics of X. nematophila were not active against X. szentirmaii. X. nematophila growth was inhibited in co-cultures with wild-type X. szentirmaii in medium that mimics insect haemolymph. An antibiotic-deficient strain of X. szentirmaii was created by inactivating the ngrA gene that encodes the enzyme that attaches the 4' phosphopantetheinyl moiety to non-ribosomal peptide synthetases involved in antibiotic biosynthesis. X. nematophila growth was not inhibited in co-cultures with the ngrA strain. The growth of X. nematophila was suppressed in Manduca sexta co-injected with wild-type X. szentirmaii and X. nematophila. In contrast, growth of X. nematophila was not suppressed in M. sexta co-injected with the ngrA strain. Two unique compounds were detected by MALDI-TOF MS analysis in haemolymph infected with the wild-type but not with the ngrA strain. Finally, killing of M. sexta was delayed in insects infected with the ngrA strain. These findings indicate that in the insect host X. szentirmaii produces ngrA-dependent products involved in both interspecies competition and virulence.

Innate and Learned Olfactory Responses in a Wild Population of the Egg Parasitoid Trichogramma (Hymenoptera: Trichogrammatidae).

Parasitoid insects face the fundamental problem of finding a suitable host in environments filled with competing stimuli. Many are deft sensors of olfactory cues emitted by other insects and the plants they live on, and use these cues to find hosts. Using olfactory cues from host-plants is effective because plants release volatile organic compounds (VOCs), in response to herbivory or oviposition, that contain information about the presence of hosts. However, plant-produced cues can also be misleading because they are influenced by a variety of stimuli (abiotic variation, infection and multiple sources of induction via herbivory or oviposition). Flexible behavior is one strategy that parasitoids may use to cope with variation in olfactory cues. We examine the innate and learned responses of a natural population of wasp egg parasitoids (Trichogramma deion and Trichogramma sathon) using a series of laboratory and field Y-olfactometer experiments. Wasps typically attack eggs of the hawkmoth Manduca sexta and Manduca quinquemaculata on native Datura wrightii plants in the southwestern United States. We show that Trichogramma wasps responded innately to VOCs produced by D. wrightii and could distinguish plants recently attacked by M. sexta from non-attacked plants. Furthermore, adult Trichogramma wasps were able to learn components of the VOC blend given off by D. wrightii, though they did not learn during exposure as pupae. By further exploring the behavioral ecology of a natural population of Trichogramma, we gain greater insight into how egg parasitoids function in tri-trophic systems.

Elastic proteins in the flight muscle of Manduca sexta.

The flight muscles (DLM1) of the Hawkmoth, Manduca sexta are synchronous, requiring a neural spike for each contraction. Stress/strain curves of skinned DLM1 showed hysteresis indicating the presence of titin-like elastic proteins. Projectin and kettin are titin-like proteins previously identified in Lethocerus and Drosophila flight muscles. Analysis of Manduca muscles with 1% SDS-agarose gels and western blots showed two bands near 1 MDa that cross-reacted with antibodies to Drosophila projectin. Antibodies to Drosophila kettin cross-reacted to bands at ∼500 and ∼700 kDa, but also to bands at ∼1.6 and ∼2.1 MDa, that had not been previously observed in insect flight muscles. Mass spectrometry identified the 2.1 MDa protein as a product of the Sallimus (sls) gene. Analysis of the gene sequence showed that all 4 putative Sallimus and kettin isoforms could be explained as products of alternative splicing of the single sls gene. Both projectin and sallimus isoforms were expressed to higher levels in ventrally located DLM1 subunits, primarily responsible for active work production, as compared to dorsally located subunits, which may act as damped springs. The different expression levels of the 2 projectin isoforms and 4 sallimus/kettin isoforms may be adaptations to the specific requirements of individual muscle subunits.

N-(18-hydroxylinolenoyl)-L-glutamine: a newly discovered analog of volicitin in Manduca sexta and its elicitor activity in plants.

Plants attacked by insect herbivores release a blend of volatile organic compounds (VOCs) that serve as chemical cues for host location by parasitic wasps, natural enemies of the herbivores. Volicitin, N-(17-hydroxylinolenoyl)-L-glutamine, is one of the most active VOC elicitors found in herbivore regurgitants. Our previous study revealed that hydroxylation on the 17th position of the linolenic acid moiety of N-linolenoyl-L-glutamine increases by more than three times the elicitor activity in corn plants. Here, we identified N-(18-hydroxylinolenoyl)-L-glutamine (18OH-volicitin) from larval gut contents of tobacco hornworm (THW), Manduca sexta. Eggplant and tobacco, two solanaceous host plants of THW larvae, and corn, a non-host plant, responded differently to this new elicitor. Eggplant and tobacco seedlings emitted twice the amount of VOCs when 18OH-volicitin was applied to damaged leaf surfaces compared to N-linolenoyl-L-glutamine, while both these fatty acid amino acid conjugates (FACs) elicited a similar response in corn seedlings. In both solanaceous plants, there was no significant difference in the elicitor activity of 17OH- and 18OH-volicitin. Interestingly, other lepidopteran species that have 17OH-type volicitin also attack solanaceous plants. These data suggest that plants have developed herbivory-detection systems customized to their herbivorous enemies.

Molecular model of a soluble guanylyl cyclase fragment determined by small-angle X-ray scattering and chemical cross-linking.

Soluble guanylyl/guanylate cyclase (sGC) converts GTP to cGMP after binding nitric oxide, leading to smooth muscle relaxation and vasodilation. Impaired sGC activity is common in cardiovascular disease, and sGC stimulatory compounds are vigorously sought. sGC is a 150 kDa heterodimeric protein with two H-NOX domains (one with heme, one without), two PAS domains, a coiled-coil domain, and two cyclase domains. Binding of NO to the sGC heme leads to proximal histidine release and stimulation of catalytic activity. To begin to understand how binding leads to activation, we examined truncated sGC proteins from Manduca sexta (tobacco hornworm) that bind NO, CO, and stimulatory compound YC-1 but lack the cyclase domains. We determined the overall shape of truncated M. sexta sGC using analytical ultracentrifugation and small-angle X-ray scattering (SAXS), revealing an elongated molecule with dimensions of 115 Å × 90 Å × 75 Å. Binding of NO, CO, or YC-1 had little effect on shape. Using chemical cross-linking and tandem mass spectrometry, we identified 20 intermolecular contacts, allowing us to fit homology models of the individual domains into the SAXS-derived molecular envelope. The resulting model displays a central parallel coiled-coil platform upon which the H-NOX and PAS domains are assembled. The ß1 H-NOX and α1 PAS domains are in contact and form the core signaling complex, while the α1 H-NOX domain can be removed without a significant effect on ligand binding or overall shape. Removal of 21 residues from the C-terminus yields a protein with dramatically increased proximal histidine release rates upon NO binding.

Structural insight into the novel iron-coordination and domain interactions of transferrin-1 from a model insect, Manduca sexta.

Transferrins function in iron sequestration and iron transport by binding iron tightly and reversibly. Vertebrate transferrins coordinate iron through interactions with two tyrosines, an aspartate, a histidine, and a carbonate anion, and conformational changes that occur upon iron binding and release have been described. Much less is known about the structure and functions of insect transferrin-1 (Tsf1), which is present in hemolymph and influences iron homeostasis mostly by unknown mechanisms. Amino acid sequence and biochemical analyses have suggested that iron coordination by Tsf1 differs from that of the vertebrate transferrins. Here we report the first crystal structure (2.05 Å resolution) of an insect transferrin. Manduca sexta (MsTsf1) in the holo form exhibits a bilobal fold similar to that of vertebrate transferrins, but its carboxyl-lobe adopts a novel orientation and contacts with the amino-lobe. The structure revealed coordination of a single Fe3+ ion in the amino-lobe through Tyr90, Tyr204, and two carbonate anions. One carbonate anion is buried near the ferric ion and is coordinated by four residues, whereas the other carbonate anion is solvent exposed and coordinated by Asn121. Notably, these residues are highly conserved in Tsf1 orthologs. Docking analysis suggested that the solvent exposed carbonate position is capable of binding alternative anions. These findings provide a structural basis for understanding Tsf1 function in iron sequestration and transport in insects as well as insight into the similarities and differences in iron homeostasis between insects and humans.

Effects of Manduca sexta allatostatin and an analogue on the peach-potato aphid Myzus persicae (hemiptera: aphididae) and degradation by enzymes in the aphid gut.

The oral toxicity of the C-type allatostatin, Manduca sexta allatostatin (Manse-AS) and the analogue δR³Î´R5Manse-AS, where R residues were replaced by their D-isomers, were tested against the peach-potato aphid Myzus persicae by incorporation into an artificial diet. Both peptides had significant dose-dependent effects on mortality, growth, and fecundity compared with control insects. The analogue, δR³Î´R5Manse-AS, had an estimated LC50 of 0.31 µg/µl diet and was more potent than Manse-AS (estimated LC50 of 0.58 µg/µl diet). At a dose of 0.35 µg δR³Î´R5Manse-AS/µl diet, 76% of the aphids were dead after 6 days and all were dead after 10 days. In comparison, three times the dose of Manse-AS was required to achieve 74% mortality after 8 days and 98% mortality after 16 days. The degradation of both peptides by extracts prepared from the gut of M. persicae was investigated. The estimated half-life of Manse-AS, when incubated with the gut extract from M. persicae, was 31 min. Degradation was due to a cathepsin L-like cysteine protease, carboxypeptidase-like activity, endoprotease activity with glutamine specificity, pyroglutamate aminopeptidase activity, and possibly trypsin-like proteases. The half-life of the δR³Î´R5 Manse-AS analogue was enhanced (73 min) with the D-isomers of R appearing to prevent cleavage around the R residues by cathepsin L-like cysteine proteases or from trypsin-like proteases. The greater stability of the analogue may explain its increased potency in M. persicae. This work demonstrates the potential use of Manse-AS and analogues, with greater resistance to enzymatic attack, in aphid control strategies.

Changes in composition and levels of hemolymph proteins during metamorphosis of Manduca sexta.

The tobacco hornworm, Manduca sexta, is a lepidopteran model species widely used to study insect biochemical processes. Some of its larval hemolymph proteins are well studied, and a detailed proteomic analysis of larval plasma proteins became available in 2016, revealing features such as correlation with transcriptome data, formation of immune complexes, and constitution of an immune signaling system in hemolymph. It is unclear how the composition of these proteins may change in other developmental stages. In this paper, we report the proteomes of cell-free hemolymph from prepupae, pupae on day 4 and day 13, and young adults. Of the 1824 proteins identified, 907 have a signal peptide and 410 are related to immunity. Drastic changes in abundance of the storage proteins, lipophorins and vitellogenin, for instance, reflect physiological differences among prepupae, pupae, and adults. Considerably more proteins lacking signal peptide are present in the late pupae, suggesting that plasma contains relatively low concentrations of intracellular components released from remodeling tissues during metamorphosis. The defense proteins detected include 43 serine proteases and 11 serine protease homologs. Some of these proteins are members of the extracellular immune signaling network found in feeding larvae, and others may play additional roles and hence confer new features in the later life stages. In summary, the proteins and their levels revealed in this study, together with their transcriptome data, are expected to stimulate focused explorations of humoral immunity and other physiological systems in wandering larvae, pupae, and adults of M. sexta and shed light upon functional and comparative genomic research in other holometabolous insects.

Peptides based on the reactive center loop of Manduca sexta serpin-3 block its protease inhibitory function.

One innate immune response in insects is the proteolytic activation of hemolymph prophenoloxidase (proPO), regulated by protease inhibitors called serpins. In the inhibition reaction of serpins, a protease cleaves a peptide bond in a solvent-exposed reactive center loop (RCL) of the serpin, and the serpin undergoes a conformational change, incorporating the amino-terminal segment of the RCL into serpin ß-sheet A as a new strand. This results in an irreversible inhibitory complex of the serpin with the protease. We synthesized four peptides with sequences from the hinge region in the RCL of Manduca sexta serpin-3 and found they were able to block serpin-3 inhibitory activity, resulting in suppression of inhibitory protease-serpin complex formation. An RCL-derived peptide with the sequence Ser-Val-Ala-Phe-Ser (SVAFS) displayed robust blocking activity against serpin-3. Addition of acetyl-SVAFS-amide to hemolymph led to unregulated proPO activation. Serpin-3 associated with Ac-SVAFS-COO- had an altered circular dichroism spectrum and enhanced thermal resistance to change in secondary structure, indicating that these two molecules formed a binary complex, most likely by insertion of the peptide into ß-sheet A. The interference of RCL-derived peptides with serpin activity may lead to new possibilities of "silencing" arthropod serpins with unknown functions for investigation of their physiological roles.

De novo molecular modeling and biophysical characterization of Manduca sexta eclosion hormone.

Eclosion hormone (EH) is an integral component in the cascade regulating the behaviors culminating in emergence of an insect from its old exoskeleton. Little is known regarding the EH solution structure; consequently, we utilized a computational approach to generate a hypothetical structure for Manduca sexta EH. The de novo algorithm exploited the restricted conformational space of disulfide bonds (Cys14-Cys38, Cys18-Cys34, and Cys21-Cys49) and predicted secondary structure elements to generate a thermodynamically stable structure characterized by 55% helical content, an unstructured N-terminus, a helical C-terminus, and a solvent-exposed loop containing Trp28 and Phe29. Both the strain and pseudo energies of the predicted peptide compare favorably with those of known structures. The 62-amino acid peptide was synthesized, folded, assayed for activity, and structurally characterized to confirm the validity of the model. The helical content is supported by circular dichroism and hydrogen-deuterium exchange mass spectrometry. Fluorescence emission spectra and acrylamide quenching are consistent with the solvent exposure predicted for Trp28, which is shielded by Phe29. Furthermore, thermodynamically stable conformations that deviated only slightly from the predicted Manduca EH structure were generated in silico for the Bombyx mori and Drosophila melanogaster EHs, indicating that the conformation is not species-dependent. In addition, the biological activities of known mutants and deletion peptides were rationalized with the predicted Manduca EH structure, and we found that, on the basis of sequence conservation, functionally important residues map to two conserved hydrophobic clusters incorporating the C-terminus and the first loop.

Identification of ecdysis-triggering hormone from an epitracheal endocrine system.

Developing insects repeatedly shed their cuticle by means of a stereotyped behavior called ecdysis, thought to be initiated by the brain peptide eclosion hormone. Here an ecdysis-triggering hormone, Mas-ETH, is described from the tobacco hornworm Manduca sexta. Mas-ETH contains 26 amino acids and is produced by a segmentally distributed endocrine system of epitracheal glands (EGs). The EGs undergo a marked reduction in volume, appearance, and immunohistochemical staining during ecdysis, at which time Mas-ETH is found in the hemolymph. Injection of EGs extract or synthetic Mas-ETH into pharate larvae, pupae, or adults initiates preecdysis within 2 to 10 minutes, followed by ecdysis. Sensitivity to injected Mas-ETH appears much earlier before ecdysis and occurs with shorter latency than that reported for eclosion hormone. The isolated central nervous system responds to Mas-ETH, but not to eclosion hormone, with patterned motor bursting corresponding to in vivo preecdysis and ecdysis. Mas-ETH may be an immediate blood-borne trigger for ecdysis through a direct action on the nervous system.

The three-dimensional structure and recognition mechanism of Manduca sexta peptidoglycan recognition protein-1.

Peptidoglycan recognition proteins (PGRPs) recognize bacteria through their unique cell wall constituent, peptidoglycans (PGs). PGRPs are conserved from insects to mammals and all function in antibacterial defense. In the tobacco hornworm Manduca sexta, PGRP1 and microbe binding protein (MBP) interact with PGs and hemolymph protease-14 precursor (proHP14) to yield active HP14. HP14 triggers a serine protease network that produces active phenoloxidase (PO), Spätzle, and other cytokines to stimulate immune responses. PGRP1 binds preferentially to diaminopimelic acid (DAP)-PGs of Gram-negative bacteria and Gram-positive Bacillus and Clostridium species than Lys-PGs of other Gram-positive bacteria. In this study, we synthesized DAP- and Lys-muramyl pentapeptide (MPP) and monitored their associations with M. sexta PGRP1 by surface plasmon resonance. The Kd values (0.57 µM for DAP-MPP and 45.6 µM for Lys-MPP) agree with the differential recognition of DAP- and Lys-PGs. To reveal its structural basis, we produced the PGRP1 in insect cells and determined its structure at a resolution of 2.1 Å. The protein adopts a fold similar to those from other PGRPs with a classical L-shaped PG-binding groove. A unique loop lining the shallow groove suggests a different ligand-binding mechanism. In summary, this study provided new insights into the PG recognition by PGRPs, a critical first step that initiates the serine protease cascade.

Pyrosequence analysis of expressed sequence tags for Manduca sexta hemolymph proteins involved in immune responses.

The tobacco hornworm Manduca sexta is widely used as a model organism to investigate the biochemical basis of insect physiological processes but little transcriptome information is available. To get a broad view of the larval hemolymph proteins, particularly those related to immunity, we synthesized and sequenced cDNA fragments from a mixture of eight total RNA samples: fat body and hemocytes from larvae injected with killed bacteria, fat body, hemocytes, integument and trachea from naïve larvae, and fat body and hemocytes from wandering larvae. Using massively parallel pyrosequencing, we obtained 95,458 M. sexta expressed sequence tags (ESTs) at an average size of 185bp per read. A majority of the sequences (69,429 reads) could be assembled into 7231 contigs with an average size of 300bp, 1178 of which had significant similarity with Drosophila genes from various functional groups. Only approximately 8% (606) of the contigs matched known M. sexta cDNA sequences, representing 186 of the 375 unique NCBI entries. The remaining 6625 contigs represented newly discovered cDNA segments from this well studied biochemical model insect. A search of the 7231 contigs using Tribolium castaneum, Drosophila melanogaster, and Bombyx mori immunity-related sequences revealed 424 cDNA contigs with significant similarity (E-value <1 x 10(-5)). These included 218 previously unknown M. sexta sequences coding for putative defense molecules such as pattern recognition receptors, serine proteinases, serpins, Spätzle, Toll-like receptors, intracellular signaling molecules, and antimicrobial peptides.

Structural studies on the neutral glycosphingolipids of Manduca sexta.

Glycosphingolipids (GSLs) have been implicated as playing major roles in cellular interactions and control of cell proliferation in muticellular organisms. Moreover GSLs and other sphingolipids such as sphingomyelins, ceramides and sphingosines serve a variety of roles in signal transduction. Hence, identification of structures of GSLs in different biota will shed light in understanding their physiological role. During this study, the major glycosphingolipid component present in the extracts of stage-12 and stage-17/18 metamorphosing adults of Manduca sexta was identified as mactosyl ceramide. We report the isolation of several ceramide disaccharides, a ceramide trisaccharide and a ceramide tetrasaccharide. The GSL structures were confirmed by high-resolution mass spectrometry and tandem mass spectrometry. The identity of the monosaccharides was proved using exoglycosidases. The predominant sphingosine chain-length varied from C-14 (tetradecasphing-4-enine) to C-16 (hexadecasphing-4-enine) in these GSLs. Sphingosines of both chain lengths were accompanied by their doubly unsaturated counterparts tetradecasphinga-4,6-diene and hexadecasphinga-4,6-diene. It is also interesting to note the presence of tetradecasphinganine and hexadecasphinganine in minute amounts in the form of a GSL in the extracts of M. sexta. The varying degrees of unsaturation in the sphingosine moiety of GSLs in M. sexta may be biologically significant in insect metamorphosis. The ceramide trisaccharides and ceramide tetrasaccharide belong to the arthro-series, The observation of fucose in the M. sexta GSLs is the first report of the presence of fucose in an arthroseries GSL.

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.

Localization of a Drosophila melanogaster homolog of the putative juvenile hormone esterase binding protein of Manduca sexta.

A putative juvenile hormone esterase (JHE) binding protein, P29, was isolated from the tobacco hornworm Manduca sexta [J. Biol. Chem. 275(3), 1802-1806]. A homolog of P29 was identified in Drosophila melanogaster by sequence alignment. This gene, CG3776 was cloned, recombinant DmP29 expressed in Escheriscia coli and two anti-DmP29 antisera raised. In vitro binding of the P29 homolog to Drosophila JHE was confirmed. P29 mRNA and an immunoreactive protein of 25 kDa were detected in Drosophila larvae, pupae and adults. The predicted size of the protein is 30 kDa. Drosophila P29 is predicted to localize to mitochondria (MitoProt; 93% probability) and has a 6 kDa N-terminal targeting sequence. Subcellular organelle fractionation and confocal microscopy of Drosophila S2 cells confirmed that the immunoreactive 25 kDa protein is present in mitochondria but not in the cytosol. Expression of P29 without the predicted N-terminal targeting sequence in High Five cells showed that the N-terminal targeting sequence is shorter than predicted, and that a second, internal mitochondrial targeting signal is also present. An immunoreactive protein of 50 kDa in the hemolymph does not result from alternative splicing of CG3776 but may result from dimerization of P29. The function of P29 in mitochondria and the possible interaction with JHE are discussed.

Ligand-binding specificity of an invertebrate (Manduca sexta) putative cellular retinoic acid binding protein.

Intracellular lipid-binding proteins (iLBPs) are small cytoplasmic proteins that specifically interact with hydrophobic ligands. Fatty acid-binding proteins (FABPs), cellular retinoic acid-binding proteins (CRABPs) and cellular retinol-binding proteins (CRBPs) belong to the iLBP family. A recently identified insect (Manduca sexta) iLBP has been reported to possibly represent an invertebrate CRABP mimicking the role of CRABPs in vertebrate organisms. The presence in this protein of the characteristic binding triad residues involved in the interaction with ligand carboxylate head groups, a feature pertaining to several FABPs and to CRABPs, and the close phylogenetic relationships with both groups of vertebrate heart-type FABPs and CRBPs/CRABPs, makes it difficult to assign it to either FABPs or CRABPs. However, its negligible interaction with retinoic acid and high affinity (K(d) values in the 10(-8) M range) for fatty acids have been established by means of direct and competitive binding assays. As shown by phylogenetic analysis, the M. sexta iLBP belongs to a wide group of invertebrate iLBPs, which, besides being closely related phylogenetically, share distinctive features, such as the conservation of chemically distinct residues in their amino acid sequences and the ability to bind fatty acids. Our results are in keeping with the lack of cellular retinoid-binding proteins in invertebrates and with their later appearance during the course of chordate evolution.

Calcium is not required for immulectin-2 binding, but protects the protein from proteinase digestion.

Mammalian C-type lectins are calcium-dependent carbohydrate-binding proteins. They serve as cell adhesion molecules in cell-cell interactions, or function as pattern-recognition receptors in innate immunity. Calcium is a direct ligand for carbohydrate binding in mammalian C-type lectins such as mannose-binding proteins and macrophage mannose receptor. In the tobacco hornworm Manduca sexta, a group of lectins named immulectins have been discovered. Each immulectin contains dual carbohydrate-recognition domains. Previously, we showed that immulectin-2 (IML-2) binds to a bacterial lipopolysaccharide, and agglutination of Escherichia coli cells by IML-2 is calcium dependent. In this study, we demonstrated that IML-2 bound to bacterial lipid A, smooth and rough mutants of lipopolysaccharide, lipoteichoic acid and peptidoglycan, as well as to fungal mannan and beta-1, 3-glucan (laminarin and curdlan). Binding of IML-2 to microbial components was calcium independent, and was increased by addition of spermine, a polyamine. In addition, plasma IML-2 bound to mannan-agarose independent of calcium. But trypsin digestion of IML-2 was inhibited in the presence of calcium. Our results suggest that calcium is not required for IML-2 binding but protects IML-2 from trypsin digestion.

Structure-activity relationship of ETH during ecdysis in the tobacco hornworm, Manduca sexta.

In insects, ecdysis or shedding of the old cuticle, consists of a series of behaviors that are regulated by the coordinated actions of a number of neuropeptides, one of which is ecdysis triggering hormone (ETH). ETH acts directly on central pattern generators of the abdominal ganglia to trigger onset of pre-ecdysis behaviors, as well as indirectly to activate release of eclosion hormone, thereby inducing onset of ecdysis behaviors through a cGMP-mediated mechanism. We assessed the minimal C-terminal amino acids required for biological activity of ETH, by assessing: (i) onset of pre-ecdysis and ecdysis behaviors in vivo, after injection of peptide analogs, (ii) onset of fictive pre-ecdysis and ecdysis motor patterns in vitro, as recorded extracellularly, after incubation of the CNS with the peptide analogs, and (iii) accumulation of cGMP within cells of the abdominal ganglia, as assessed immunohistochemically. Amidation of ETH at the C-terminus was required to elicit a biological response in vivo and in vitro, as well as an accumulation of cGMP within the CNS. The five amino acid amidated C-terminus of ETH (NIPRMamide) was the minimal moiety able to induce a robust pre-ecdysis response in vivo and in vitro, while a seven amino acid core (NKNIPRMa) was required for induction of ecdysis, including accumulation of cGMP immunoreactivity within the CNS. Analogs smaller than 12 amino acids in length were only active at very high concentrations in vivo, suggesting that smaller fragments might be susceptible to hemolymph degradation. Some alanine substitutions or removal of internal amino acids altered the activity of ETH, as well as the time of onset of ecdysis behaviors, suggesting that internal amino acids play a role in maintaining proper folding of the peptide for successful binding or activity at the ETH receptor.

Alanine substitution and deletion analogues of Manduca sexta allatostatin: structure-activity relationship on the spontaneous contractions of the foregut of larval Lacanobia oleracea.

Manduca sexta allatostatin (Manse-AS) is a 15-residue non-amidated peptide with a blocked N-terminus and a disulphide bridge between the cysteine residues at positions 7 and 14. Analogues of Manse-AS were used to examine the structural requirements of Manse-AS for inhibitory activity on spontaneous foregut contractions of larval tomato moth (Lacanobia oleracea). Breaking the disulphide bond between C(7) and C(14) by reduction reduced the potency of the peptide, suggesting that the conformation of Manse-AS is important for its biological activity. When either of the cysteine residues were replaced with alanine the Manse-AS analogue had no measurable bioactivity. Alanine substitution at Q(6) was as potent as Manse-AS, all other alanine substitution analogues (R(5), Y(8), F(9), N(10), P(11), I(12) and S(13)), were myoinhibitory but less potent than native Manse-AS to varying degrees. Analogues with alanine substitution at amino acids with aromatic side chains (Y(8) and F(9)) were the least active. Amino-terminal deletion analogues Manse-AS(6-15) and Manse-AS(7-15) were inactive whereas Manse-AS(5-15) was fully active but not as potent as Manse-AS. The results show that amino acid residues both inside and outside the disulphide ring are important for biological activity.