A conserved asparagine residue stabilizes iron binding in Manduca sexta transferrin-1.

Transferrin 1 (Tsf1) is an insect-specific iron-binding protein that is abundant in hemolymph and other extracellular fluids. It binds iron tightly at neutral pH and releases iron under acidic conditions. Tsf1 influences the distribution of iron in the body and protects against infection. Elucidating the mechanisms by which Tsf1 achieves these functions will require an understanding of how Tsf1 binds and releases iron. Previously, crystallized Tsf1 from Manduca sexta was shown to have a novel type of iron coordination that involves four iron-binding ligands: two tyrosine residues (Tyr90 and Tyr204), a buried carbonate anion, and a solvent-exposed carbonate anion. The solvent-exposed carbonate anion was bound by a single amino acid residue, a highly conserved asparagine at position 121 (Asn121); thus, we predicted that Asn121 would be essential for high-affinity iron binding. To test this hypothesis, we analyzed the iron-binding and -release properties of five forms of recombinant Tsf1: wild-type, a Y90F/Y204F double mutant (negative control), and three Asn121 mutants (N121A, N121D and N121S). Each of the Asn121 mutants exhibited altered spectral properties, confirming that Asn121 contributes to iron coordination. The N121D and N121S mutations resulted in slightly lower affinity for iron, especially at acidic pH, while iron binding and release by the N121A mutant was indistinguishable from that of the wild-type protein. The surprisingly minor consequences of mutating Asn121, despite its high degree of conservation in diverse insect species, suggest that Asn121 may play a role that is essential in vivo but non-essential for high affinity iron binding in vitro.

Serine Protease Networks Mediate Immune Responses in Extra-Embryonic Tissues of Eggs in the Tobacco Hornworm, Manduca sexta.

The melanization and Toll pathways, regulated by a network of serine proteases and noncatalytic serine protease homologs (SPHs), have been investigated mostly in adult and larval insects. However, how these innate immune reactions are regulated in insect eggs remains unclear. Here we present evidence from transcriptome and proteome analyses that extra-embryonic tissues (yolk and serosa) of early-stage Manduca sexta eggs are immune competent, with expression of immune effector genes including prophenoloxidase and antimicrobial peptides. We identified gene products of the melanization and Toll pathways in M. sexta eggs. Through in vitro reconstitution experiments, we demonstrated that constitutive and infection-induced serine protease cascade modules that stimulate immune responses exist in the extra-embryonic tissues of M. sexta eggs. The constitutive module (HP14b-SP144-GP6) may promote rapid early immune signaling by forming a cascade activating the cytokine Spätzle and regulating melanization by activating prophenoloxidase (proPO). The inducible module (HP14a-HP21-HP5) may trigger enhanced activation of Spätzle and proPO at a later phase of infection. Crosstalk between the two modules may occur in transition from the constitutive to the induced response in eggs inoculated with bacteria. Examination of data from two other well-studied insect species, Tribolium castaneum and Drosophila melanogaster, supports a role for a serosa-dependent constitutive protease cascade in protecting early embryos against invading pathogens.

Proteomic analysis of pharate pupal molting fluid from the tobacco hornworm, Manduca sexta.

The insect cuticle is a key component of their success, being important for protection, communication, locomotion, and support. Conversely, as an exoskeleton, it also limits the size of the insect and must be periodically molted and a new one synthesized, to permit growth. To achieve this, the insect secretes a solution of chitinases, proteases and other proteins, known collectively as molting fluid, during each molting process to break down and recycle components of the old cuticle. Previous research has focused on the degradative enzymes in molting fluid and offered some characterization of their biochemical properties. However, identification of the specific proteins involved remained to be determined. We have used 2D SDS-PAGE and LC/MS-based proteomic analysis to identify proteins in the molting fluid of the tobacco hornworm, Manduca sexta, undergoing the larval to pupal molt. We categorized these proteins based on their proposed functions including chitin metabolism, proteases, peptidases, and immunity. This analysis complements previous reported work on M. sexta molting fluid and identifies candidate genes for enzymes involved in cuticle remodeling. Proteins classified as having an immune function highlight potential for molting fluid to act as an immune barrier to prevent infections during the cuticle degradation and ecdysis processes. Several proteins known to function in melanin synthesis as an immune response in hemolymph were present in molting fluid. We demonstrated that the bacterium Micrococcus luteus and the entomopathogenic fungus Beauveria bassiana can stimulate activation of phenoloxidase in molting fluid, indicating that the recognition proteins, protease cascade, and prophenoloxidase needed for melanin synthesis are present as a defense against infection during cuticle degradation. This analysis offers insights for proteins that may be important not only for molting in M. sexta but for insects in general.

A mechanistic analysis of bacterial recognition and serine protease cascade initiation in larval hemolymph of Manduca sexta.

Serine protease cascades have evolved in vertebrates and invertebrates to mediate rapid defense responses. Previous biochemical studies showed that in hemolymph of a caterpillar, Manduca sexta, recognition of fungi by ß-1,3-glucan recognition proteins (ßGRP1 and ßGRP2) or recognition of bacteria by peptidoglycan recognition protein-1 (PGRP1) and microbe binding protein (MBP) results in autoactivation of hemolymph protease-14 precursor (proHP14). HP14 then activates downstream members of a protease cascade leading to the melanization immune response. ProHP14 has a complex domain architecture, with five low-density lipoprotein receptor class A repeats at its amino terminus, followed by a Sushi domain, a Sushi domain variant called Wonton, and a carboxyl-terminal serine protease catalytic domain. Its zymogen form is activated by specific proteolytic cleavage at the amino-terminal end of the protease domain. While a molecular mechanism for recognition and triggering the response to ß-1,3-glucan has been delineated, it is unclear how bacterial recognition stimulates proHP14 activation. To fill this knowledge gap, we expressed the two domains of M. sexta MBP and found that the amino-terminal domain binds to diaminopimelic acid-peptidoglycan (DAP-PG). ProHP14 bound to both the carboxyl-terminal domain (MBP-C) and amino-terminal domain (MBP-N) of MBP. In the mixture of DAP-PG, MBP, and larval plasma, inclusion of an HP14 fragment composed of LDLa repeats 2-5 (LDLa2-5) or MBP-C significantly reduced prophenoloxidase activation, likely by competing with the interactions of the full-length proteins, and suggesting that molecular interactions involving these regions of proHP14 and MBP take part in proHP14 activation in response to peptidoglycan. Using a series of N-terminally truncated versions of proHP14, we found that autoactivation required LDLa2-5. The optimal ratio of PGRP1, MBP, and proHP14 is close to 3:2:1. In summary, proHP14 autoactivation by DAP-type peptidoglycan requires binding of DAP-PG by PGRP1 and the MBP N-terminal domain and association of the LDLa2-5 region of proHP14 with the MBP C-terminal domain. These interactions may concentrate the proHP14 zymogen at the bacterial cell wall surface and promote autoactivation.

Phylogenetic and sequence analyses of insect transferrins suggest that only transferrin 1 has a role in iron homeostasis.

Iron is essential to life, but surprisingly little is known about how iron is managed in nonvertebrate animals. In mammals, the well-characterized transferrins bind iron and are involved in iron transport or immunity, whereas other members of the transferrin family do not have a role in iron homeostasis. In insects, the functions of transferrins are still poorly understood. The goals of this project were to identify the transferrin genes in a diverse set of insect species, resolve the evolutionary relationships among these genes, and predict which of the transferrins are likely to have a role in iron homeostasis. Our phylogenetic analysis of transferrins from 16 orders of insects and two orders of noninsect hexapods demonstrated that there are four orthologous groups of insect transferrins. Our analysis suggests that transferrin 2 arose prior to the origin of insects, and transferrins 1, 3, and 4 arose early in insect evolution. Primary sequence analysis of each of the insect transferrins was used to predict signal peptides, carboxyl-terminal transmembrane regions, GPI-anchors, and iron binding. Based on this analysis, we suggest that transferrins 2, 3, and 4 are unlikely to play a major role in iron homeostasis. In contrast, the transferrin 1 orthologs are predicted to be secreted, soluble, iron-binding proteins. We conclude that transferrin 1 orthologs are the most likely to play an important role in iron homeostasis. Interestingly, it appears that the louse, aphid, and thrips lineages have lost the transferrin 1 gene and, thus, have evolved to manage iron without transferrins.

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.

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.

Hemolymph protease-5 links the melanization and Toll immune pathways in the tobacco hornworm, Manduca sexta.

Proteolytic activation of phenoloxidase (PO) and the cytokine Spätzle during immune responses of insects is mediated by a network of hemolymph serine proteases (HPs) and noncatalytic serine protease homologs (SPHs) and inhibited by serpins. However, integration and conservation of the system and its control mechanisms are not fully understood. Here we present biochemical evidence that PO-catalyzed melanin formation, Spätzle-triggered Toll activation, and induced synthesis of antimicrobial peptides are stimulated via hemolymph (serine) protease 5 (HP5) in Manduca sexta Previous studies have demonstrated a protease cascade pathway in which HP14 activates proHP21; HP21 activates proPAP2 and proPAP3, which then activate proPO in the presence of a complex of SPH1 and SPH2. We found that both HP21 and PAP3 activate proHP5 by cleavage at ESDR176*IIGG. HP5 then cleaves proHP6 at a unique site of LDLH112*ILGG. HP6, an ortholog of Drosophila Persephone, activates both proHP8 and proPAP1. HP8 activates proSpätzle-1, whereas PAP1 cleaves and activates proPO. HP5 is inhibited by Manduca sexta serpin-4, serpin-1A, and serpin-1J to regulate its activity. In summary, we have elucidated the physiological roles of HP5, a CLIPB with unique cleavage specificity (cutting after His) that coordinates immune responses in the caterpillar.

Iron binding and release properties of transferrin-1 from Drosophila melanogaster and Manduca sexta: Implications for insect iron homeostasis.

Transferrins belong to an ancient family of extracellular proteins. The best-characterized transferrins are mammalian proteins that function in iron sequestration or iron transport; they accomplish these functions by having a high-affinity iron-binding site in each of their two homologous lobes. Insect hemolymph transferrins (Tsf1s) also function in iron sequestration and transport; however, sequence-based predictions of their iron-binding residues have suggested that most Tsf1s have a single, lower-affinity iron-binding site. To reconcile the apparent contradiction between the known physiological functions and predicted biochemical properties of Tsf1s, we purified and characterized the iron-binding properties of Drosophila melanogaster Tsf1 (DmTsf1), Manduca sexta Tsf1 (MsTsf1), and the amino-lobe of DmTsf1 (DmTsf1N). Using UV-Vis spectroscopy, we found that these proteins bind iron, but they exhibit shifts in their spectra compared to mammalian transferrins. Through equilibrium dialysis experiments, we determined that DmTsf1 and MsTsf1 bind only one ferric ion; their affinity for iron is high (log K' = 18), but less than that of the well-characterized mammalian transferrins (log K' ~ 20); and they release iron under moderately acidic conditions (pH50 = 5.5). Iron release analysis of DmTsf1N suggested that iron binding in the amino-lobe is stabilized by the carboxyl-lobe. These findings will be critical for elucidating the mechanisms of Tsf1 function in iron sequestration and transport in 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.

Inhibition of immune pathway-initiating hemolymph protease-14 by Manduca sexta serpin-12, a conserved mechanism for the regulation of melanization and Toll activation in insects.

A network of serine proteases (SPs) and their non-catalytic homologs (SPHs) activates prophenoloxidase (proPO), Toll pathway, and other insect immune responses. However, integration and conservation of the network and its control mechanisms have not yet been fully understood. Here we present evidence that these responses are initiated through a conserved serine protease and negatively regulated by serpins in two species, Manduca sexta and Anopheles gambiae. We have shown that M. sexta serpin-12 reduces the proteolytic activation of HP6, HP8, proPO activating proteases (PAPs), SPHs, and POs in larval hemolymph, and we hypothesized that these effects are due to the inhibition of the immune pathway-initiating protease HP14. To test whether these changes are due to HP14 inhibition, we isolated a covalent complex of HP14 with serpin-12 from plasma using polyclonal antibodies against the HP14 protease domain or against serpin-12, and confirmed formation of the complex by 2D-electrophoresis, immunoblotting, and mass spectrometry. Upon recognition of bacterial peptidoglycans or fungal ß-1,3-glucan, the zymogen proHP14 became active HP14, which formed an SDS-stable complex with serpin-12 in vitro. Activation of proHP21 by HP14 was suppressed by serpin-12, consistent with the decrease in steps downstream of HP21, proteolytic activation of proPAP3, proSPH1/2 and proPO in hemolymph. Guided by the results of phylogenetic analysis, we cloned and expressed A. gambiae proSP217 (an ortholog of HP14) and core domains of A. gambiae serpin-11 and -17. The recombinant SP217 zymogen became active during expression, with cleavage between Tyr394 and Ile395. Both MsHP14 and AgSP217 cleaved MsSerpin-12 and AgSRPN11 at Leu*Ser (P1*P1') and formed complexes in vitro. ProPO activation in M. sexta plasma increased after recombinant AgSP217 had been added, indicating that it may function in a similar manner as the endogenous initiating protease HP14. Based on these data, we propose that inhibition of an initiating modular protease by a serpin may be a common mechanism in holometabolous insects to regulate proPO activation and other protease-induced immune responses.

Comparative analysis of seven types of superoxide dismutases for their ability to respond to oxidative stress in Bombyx mori.

Insects are well adapted to changing environmental conditions. They have unique systems for eliminating reactive oxygen species (ROS). Superoxide dismutase (SOD) is a key enzyme that plays a primary role in removing ROS. Bombyx mori is a lepidopteran insect, whose body size is larger than the model insect Drosophila melanogaster, which enabled us to more easily examine gene expression at the tissue level. We searched B. mori SOD (BmSOD) genes using genome database, and we analyzed their function under different type of oxidative stress. Consequently, we identified four new types of BmSODs in addition to the three types already known. Two of the seven types had a unique domain architecture that has not been discovered previously in the SOD family, and they were expressed in different tissues and developmental stages. Furthermore, these BmSODs responded differently to several kinds of stressors. Our results showed that the seven types of BmSODs are likely to play different roles in B. mori; therefore, B. mori could be used to distinguish the functions of each SOD for resistance to oxidative stress that changes with the environmental conditions.

The Manduca sexta serpinome: Analysis of serpin genes and proteins in the tobacco hornworm.

Members of the serpin superfamily of proteins occur in animals, plants, bacteria, archaea and some viruses. They adopt a variety of physiological functions, including regulation of immune system, modulation of apoptosis, hormone transport and acting as storage proteins. Most members of the serpin family are inhibitors of serine proteinases. In this study, we searched the genome of Manduca sexta and identified 32 serpin genes. We analyzed the structure of these genes and the sequences of their encoded proteins. Three M. sexta genes (serpin-1, serpin-15, and serpin-28) have mutually exclusive alternatively spliced exons encoding the carboxyl-terminal reactive center loop of the protein, which is the site of interaction with target proteases. We discovered that MsSerpin-1 has 14 splicing isoforms, including two undiscovered in previous studies. Twenty-eight of the 32 M. sexta serpins include a putative secretion signal peptide and are predicted to be extracellular proteins. Phylogenetic analysis of serpins in M. sexta and Bombyx mori indicates that 17 are orthologous pairs, perhaps carrying out essential physiological functions. Analysis of the reactive center loop and hinge regions of the protein sequences indicates that 16 of the serpin genes encode proteins that may lack proteinase inhibitor activity. Our annotation and analysis of these serpin genes and their transcript profiles should lead to future advances in experimental study of their functions in insect biochemistry.

Manduca sexta serpin-12 controls the prophenoloxidase activation system in larval hemolymph.

Insect prophenoloxidase activation is coordinated by a serine protease network, which is regulated by serine protease inhibitors of the serpin superfamily. The enzyme system also leads to proteolytic processing of a Spätzle precursor. Binding of Spätzle to a Toll receptor turns on a signaling pathway to induce the synthesis of defense proteins. Previous studies of the tobacco hornworm Manduca sexta have revealed key members of the protease cascade, which generates phenoloxidase for melanogenesis and Spätzle to induce immunity-related genes. Here we provide evidence that M. sexta serpin-12 regulates hemolymph protease-14 (HP14), an initiating protease of the cascade. This inhibitor, unlike the other serpins characterized in M. sexta, has an amino-terminal extension rich in hydrophilic residues and an unusual P1 residue (Leu429) right before the scissile bond cleaved by a target protease. Serpins with similarities to serpin-12, including Drosophila Necrotic, were identified in a wide range of insects including flies, moths, wasps, beetles, and two hemimetabolous species. The serpin-12 mRNA is present at low, constitutive levels in larval fat body and hemocytes and becomes more abundant after an immune challenge. We produced the serpin-12 core domain (serpin-12ΔN) in insect cells and in Escherichia coli and demonstrated its inhibition of human cathepsin G, bovine α-chymotrypsin, and porcine pancreatic elastase. MALDI-TOF analysis of the reaction mixtures confirmed the predicted P1 residue of Leu429. Supplementation of larval plasma samples with the serpin-12ΔN decreased prophenoloxidase activation elicited by microbial cells and reduced the proteolytic activation of the protease precursors of HP6, HP8, PAPs, and other serine protease-related proteins. After incubation of plasma stimulated with peptidoglycan, a 72 kDa protein appeared, which was recognized by polyclonal antibodies against both serpin-12 and HP14, suggesting that a covalent serpin-protease complex formed when serpin-12 inhibited HP14. Together, these data suggest that M. sexta serpin-12 inhibits HP14 to regulate melanization and antimicrobial peptide induction.

The immune properties of Manduca sexta transferrin.

Transferrins are secreted proteins that bind iron. The well-studied transferrins are mammalian serum transferrin, which is involved in iron transport, and mammalian lactoferrin, which functions as an immune protein. Lactoferrin and lactoferrin-derived peptides have bactericidal activity, and the iron-free form of lactoferrin has bacteriostatic activity due to its ability to sequester iron. Insect transferrin is similar in sequence to both serum transferrin and lactoferrin, and its functions are not well-characterized; however, many studies of insect transferrin indicate that it has some type of immune function. The goal of this study was to determine the specific immune functions of transferrin from Manduca sexta (tobacco hornworm). We verified that transferrin expression is upregulated in response to infection in M. sexta larvae and determined that the concentration of transferrin in hemolymph increases from 2 µM to 10 µM following an immune challenge. It is also present in molting fluid and prepupal midgut fluid, two extracellular fluids with immune capabilities. No immune-induced proteolytic cleavage of transferrin in hemolymph was observed; therefore, M. sexta transferrin does not appear to be a source of antimicrobial peptides. Unlike iron-saturated lactoferrin, iron-saturated transferrin had no detectable antibacterial activity. In contrast, 1 µM iron-free transferrin inhibited bacterial growth, and this inhibition was blocked by supplementing the culture medium with 1 µM iron. Our results suggest that M. sexta transferrin does not have bactericidal activity, but that it does have a bacteriostatic function that depends on its iron sequestering ability. This study supports the hypothesis that insect transferrin participates in an iron withholding strategy to protect insects from infectious bacteria.

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta.

Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.

Superoxide dismutase 2 knockdown leads to defects in locomotor activity, sensitivity to paraquat, and increased cuticle pigmentation in Tribolium castaneum.

Insects can rapidly adapt to environmental changes through physiological responses. The red flour beetle Tribolium castaneum is widely used as a model insect species. However, the stress-response system of this species remains unclear. Superoxide dismutase 2 (SOD2) is a crucial antioxidative enzyme that is found in mitochondria. T. castaneum SOD2 (TcSOD2) is composed of 215 amino acids, and has an iron/manganese superoxide dismutase domain. qRT-PCR experiments revealed that TcSOD2 was present through all developmental stages. To evaluate TcSOD2 function in T. castaneum, we performed RNAi and also assessed the phenotype and antioxidative tolerance of the knockdown of TcSOD2 by exposing larvae to paraquat. The administration of paraquat resulted in significantly higher 24-h mortality in TcSOD2 knockdown larval groups than in the control groups. The TcSOD2 knockdown adults moved significantly more slowly, had lower ATP content, and exhibited a different body color from the control groups. We found that TcSOD2 dsRNA treatment in larvae resulted in increased expression of tyrosinase and laccase2 mRNA after 10 days. This is the first report showing that TcSOD2 has an antioxidative function and demonstrates that T. castaneum may use an alternative antioxidative system when the SOD2-based system fails.

Characterization and regulation of expression of an antifungal peptide from hemolymph of an insect, Manduca sexta.

Insects secrete antimicrobial peptides as part of the innate immune response. Most antimicrobial peptides from insects have antibacterial but not antifungal activity. We have characterized an antifungal peptide, diapausin-1 from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). Diapausin-1 was isolated by size exclusion chromatography from hemolymph plasma of larvae that were previously injected with a yeast, Saccharomyces cerevisiae. Fractions containing activity against S. cerevisiae were analyzed by SDS-PAGE and MALDI-TOF MS/MS and found to contain a 45-residue peptide that was encoded by sequences identified in M. sexta transcriptome and genome databases. A cDNA for diapausin-1 was cloned from cDNA prepared from fat body RNA. Diapausin-1 is a member of the diapausin family of peptides, which includes members known to have antifungal activity. The M. sexta genome contains 14 genes with high similarity to diapausin-1, each with 6 conserved Cys residues. Diapausin-1 was produced as a recombinant protein in Escherichia coli. Purified recombinant diapausin-1 was active against S. cerevisiae, with IC50 of 12 µM, but had no detectable activity against bacteria. Spores of some plant fungal pathogens treated with diapausin-1 had curled germination tubes or reduced and branched hyphal growth. Diapausin-1 mRNA level in fat body strongly increased after larvae were injected with yeast or with Micrococcus luteus. In addition, diapausin-1 mRNA levels increased in midgut and fat body at the wandering larval stage prior to pupation, suggesting developmental regulation of the gene. Our results indicate that synthesis of diapausin-1 is part of an antifungal innate immune response to infection in M. sexta.

Initiating protease with modular domains interacts with ß-glucan recognition protein to trigger innate immune response in insects.

The autoactivation of an initiating serine protease upon binding of pattern recognition proteins to pathogen surfaces is a crucial step in eliciting insect immune responses such as the activation of Toll and prophenoloxidase pathways. However, the molecular mechanisms responsible for autoactivation of the initiating protease remains poorly understood. Here, we investigated the molecular basis for the autoactivation of hemolymph protease 14 (HP14), an initiating protease in hemolymph of Manduca sexta, upon the binding of ß-1,3-glucan by its recognition protein, ßGRP2. Biochemical analysis using HP14 zymogen (proHP14), ßGRP2, and the recombinant proteins as truncated forms showed that the amino-terminal modular low-density lipoprotein receptor class A (LA) domains within HP14 are required for proHP14 autoactivation that is stimulated by its interaction with ßGRP2. Consistent with this result, recombinant LA domains inhibit the activation of proHP14 and prophenoloxidase, likely by competing with the interaction between ßGRP2 and LA domains within proHP14. Using surface plasmon resonance, we demonstrated that immobilized LA domains directly interact with ßGRP2 in a calcium-dependent manner and that high-affinity interaction requires the C-terminal glucanase-like domain of ßGRP2. Importantly, the affinity of LA domains for ßGRP2 increases nearly 100-fold in the presence of ß-1,3-glucan. Taken together, these results present the first experimental evidence to our knowledge that LA domains of an insect modular protease and glucanase-like domains of a ßGRP mediate their interaction, and that this binding is essential for the protease autoactivation. Thus, our study provides important insight into the molecular basis underlying the initiation of protease cascade in insect immune responses.