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.

The Development of New Methods to Stimulate the Production of Antimicrobial Peptides in the Larvae of the Black Soldier Fly Hermetia illucens.

(1) The global population is projected to reach a staggering 9.8 billion people by the year 2050, leading to major concerns about food security. The necessity to increase livestock production is inevitable. The black soldier fly (BSF) is known for its ability to consume a wide range of organic waste, and BSF larvae have already been used as a partial substitute for fishmeal. In contrast, the use of antibiotics in livestock feed for growth promotion and prophylaxis poses a severe threat to global health owing to antimicrobial resistance. Insect antimicrobial peptides (AMPs) have shown the potential to rapidly disrupt target bacterial membranes, making bacterial resistance to AMPs a less likely concern. (2) In this study, we explored various methods for stimulating AMP synthesis in BSF larvae and found that thermal injury effectively induced the production of various AMP types. Additionally, we investigated the activation of innate immune response pathways that lead to AMP production following thermal injury. (3) Interestingly, thermal injury treatment, although not involving bacteria, exhibited a similar response to that observed following Gram-positive bacterial infection in eliciting the expression of AMP genes. (4) Our findings offer support for the industrial use of BSF to enhance livestock production and promote environmental health.

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.

Temporal changes in the physical and mechanical properties of beetle elytra during maturation.

Changes in physical properties of Tenebrio molitor and Tribolium castaneum elytra (hardened forewings) were studied to understand how the development of microstructure and chemical interactions determine cuticle mechanical properties. Analysis of these properties supports a model in which cuticular material is continuously secreted from epidermal cells to produce an extracellular matrix so that the outermost layers mature first. It is hypothesized that enzymatic crosslinking and pigmentation reactions along with dehydration help to stabilize the protein-chitin network within the initial layers of cuticle shortly after eclosion. Mature layers are proposed to bear most of the mechanical loads. The frequency dependence of the storage modulus and the tan δ values decreased during the beginning of maturation, reaching constant values after 48 h post-eclosion. A decrease of tan δ indicates an increase in crosslinking of the material. The water content declined from 75% to 31%, with a significant portion lost from within the open spaces between the dorsal and ventral cuticular layers. Dehydration had a less significant influence than protein crosslinking on the mechanical properties of the elytron during maturation. When Tribolium cuticular protein TcCP30 expression was decreased by RNAi, the tan δ and frequency dependence of E' of the elytron did not change during maturation. This indicates that TcCP30 plays a role in the crosslinking process of the beetle's exoskeleton. This study was inspired by previous work on biomimetic multicomponent materials and helps inform future work on creating robust lightweight materials derived from natural sources. STATEMENT OF SIGNIFICANCE: Examination of changes in the physical properties of the elytra (hardened forewings) of two beetle species advanced understanding of how the molecular interactions influence the mechanical properties of the elytra. Physical characterization, including dynamic mechanical analysis, determined that the outer portion of the elytra matured first, while epidermal cells continued to secrete reactive components until the entire structure reached maturation. RNA interference was used to identify the role of a key protein in the elytra. Suppression of its expression reduced the formation of crosslinked polymeric components in the elytra. Identifying the molecular interactions in the matrix of proteins and polysaccharides in the elytra together with their hierarchical architecture provides important design concepts in the development of biomimetic materials.

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.

Phenotypic analyses, protein localization, and bacteriostatic activity of Drosophila melanogaster transferrin-1.

Transferrin-1 (Tsf1) is an extracellular insect protein with a high affinity for iron. The functions of Tsf1 are still poorly understood; however, Drosophila melanogaster Tsf1 has been shown to influence iron distribution in the fly body and to protect flies against some infections. The goal of this study was to better understand the physiological functions of Tsf1 in D. melanogaster by 1) investigating Tsf1 null phenotypes, 2) determining tissue-specific localization of Tsf1, 3) measuring the concentration of Tsf1 in hemolymph, 4) testing Tsf1 for bacteriostatic activity, and 5) evaluating the effect of metal and paraquat treatments on Tsf1 abundance. Flies lacking Tsf1 had more iron than wild-type flies in specialized midgut cells that take up iron from the diet; however, the absence of Tsf1 had no effect on the iron content of whole midguts, fat body, hemolymph, or heads. Thus, as previous studies have suggested, Tsf1 appears to have a minor role in iron transport. Tsf1 was abundant in hemolymph from larvae (0.4 µM), pupae (1.4 µM), adult females (4.4 µM) and adult males (22 µM). Apo-Tsf1 at 1 µM had bacteriostatic activity whereas holo-Tsf1 did not, suggesting that Tsf1 can inhibit microbial growth by sequestering iron in hemolymph and other extracellular environments. This hypothesis was supported by detection of secreted Tsf1 in tracheae, testes and seminal vesicles. Colocalization of Tsf1 with an endosome marker in oocytes suggested that Tsf1 may provide iron to developing eggs; however, eggs from mothers lacking Tsf1 had the same amount of iron as control eggs, and they hatched at a wild-type rate. Thus, the primary function of Tsf1 uptake by oocytes may be to defend against infection rather than to provide eggs with iron. In beetles, Tsf1 plays a role in protection against oxidative stress. In contrast, we found that flies lacking Tsf1 had a typical life span and greater resistance to paraquat-induced oxidative stress. In addition, Tsf1 abundance remained unchanged in response to ingestion of iron, cadmium or paraquat or to injection of iron. These results suggest that Tsf1 has a limited role in protection against oxidative stress in D. melanogaster.

Superoxide dismutase 6 is required during metamorphosis for the development of properly movable legs in Tribolium castaneum.

The body form of holometabolous insects dramatically transforms from larval to adult stages during metamorphosis that occurs in the pupal stage. The larval disorganization and then new adult tissues are built up at this time. In motoneuron, larval neuronal cells degenerate, and new adult neurons are remodeled. Finally, adult neurons reconnect to new adult muscles. However, the factors that control metamorphosis have not yet been fully elucidated. Here, we show that an antioxidant enzyme, Tribolium castaneum superoxide dismutase 6 (TcSOD6), is secreted into the haemolymph and is required for proper movable legs during metamorphosis. TcSOD6 has a unique domain architecture and is mainly expressed in the pupal stage. The depletion of TcSOD6 expression in the pupa inhibits normal axon development and results in adults that display dysfunctional leg motions, suggesting that SOD6 expression is required for the development of properly movable legs. Therefore, we speculate that TcSOD6 might participate in some of the processes for larval neurons to be remodelled to new adult functions in the legs during metamorphosis, providing new insight into the evolution of SOD functions.

cDNA Cloning and Partial Characterization of the DJ-1 Gene from Tribolium castaneum.

The DJ-1 gene is highly conserved across a wide variety of organisms and it plays a role in anti-oxidative stress mechanisms in cells. The red flour beetle, Tribolium castaneum, is widely used as a model insect species because it is easy to evaluate gene function in this species using RNA interference (RNAi). The T. castaneum DJ-1 (TcDJ-1) sequence is annotated in the T. castaneum genome database; however, the function and characteristics of the TcDJ-1 gene have not been elucidated. Here, we investigated the cDNA sequence of TcDJ-1 and partially characterized its function. First, we examined the TcDJ-1 amino acid sequence and found that it was highly conserved with sequences from other species. TcDJ-1 mRNA expression was higher in the early pupal and adult developmental stages. We evaluated oxidant tolerance in TcDJ-1 knockdown adults using paraquat and found that adults with TcDJ-1 knockdown exhibited increased sensitivity to paraquat. Our findings show that TcDJ-1 has an antioxidant function, as observed for DJ-1 from other insects. Therefore, these results suggest that TcDJ-1 protects against oxidative stress during metamorphosis.

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.

Development of a new method for collecting hemolymph and measuring phenoloxidase activity in Tribolium castaneum.

OBJECTIVE: Hemolymph plays many important roles in the physiology of an insect throughout its lifetime; however, for small-bodied insects, studies are lacking because of the difficulties encountered while collecting hemolymph. The objective of our study was to develop a method to collect hemolymph plasma from various stages of Tribolium castaneum and to evaluate phenoloxidase activity in the plasma samples. We first designed a procedure for easily and quickly collecting clear hemolymph plasma from T. castaneum. RESULTS: By using this method, we collected approximately 5 µl plasma from 30 individuals at the larval, pupal or adult stages. And then, we studied the expression of phenoloxidase by performing western blot analysis of the plasma samples and found that phenoloxidase is present in hemolymph in each developmental stage. We also measured phenoloxidase activity in control plasma and plasma treated with Gram-positive bacteria, Micrococcus luteus. Phenoloxidase activity was greater in some of the M. luteus-treated plasma samples compared with control samples. Thus, we developed a method to collect hemolymph plasma that is suitable for studies of phenoloxidase activity.

Expression and Characterization of Manduca sexta Stress Responsive Peptide-1; an Inducer of Antimicrobial Peptide Synthesis.

In response to stress conditions such as wounding or infections in insects, several short peptides are processed to act as cytokines that induce AMP gene expression. To study their structure-activity relationship, immune inducibility, tissue specificity, stress responsiveness, and development relatedness, we chemically synthesized Manduca sexta stress response peptide-1, a 25-residue peptide with one disulfide bond (SRP1: FGVRVGTCPSGYVRRGTFCFPDDDY). Upon injection of the SRP1 into naïve larvae, several antimicrobial peptide genes were expressed at higher levels. The mRNA levels of SRP1 increased significantly in hemocytes and fat body after larvae were challenged with a mixture of bacteria and ß-1,3-glucan. The expression patterns of SRP1 and its target genes are somewhat different from SRP2's, suggesting overlapping yet distinct functions. We elucidated the 3D structure of SRP1 in solution by two-dimensional 1H-1H NMR spectroscopy. The tertiary structure of SRP1 consists of two short ß-strands at Y12-R15 and F18-F20, one type-II ß-turn at R15-F18 in its well-defined core and is stabilized by a covalent disulfide bond between C8 and C19. The conformational ensemble of SRP1 from extensive atomistic simulation in explicit solvent (with 3.0 µs total effective sampling) shows high consistency with experimental intramolecular NOEs of the core region. The SRP1 core adopts a fold similar to the carboxyl-terminal subdomain of epidermal growth factor (EGF), suggesting that SRP1 may interact with EGF receptor-like molecules to trigger its biological function.

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.