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

Citation: Tabunoki, H., Gorman, M. J., Dittmer, N. T., & Kanost, M. R. (2016). Superoxide dismutase 2 knockdown leads to defects in locomotor activity, sensitivity to paraquat, and increased cuticle pigmentation in Tribolium castaneum. Scientific Reports, 6, 8. doi:10.1038/srep29583Insects 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

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

Citation: Tabunoki, H., Gorman, M. J., Dittmer, N. T., & Kanost, M. R. (2016). Superoxide dismutase 2 knockdown leads to defects in locomotor activity, sensitivity to paraquat, and increased cuticle pigmentation in Tribolium castaneum. Scientific Reports, 6, 8. doi:10.1038/srep29583Insects 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

Cuticular protein with a low complexity sequence becomes cross-linked during insect cuticle sclerotization and is required for the adult molt

Citation: Mun, S., Noh, M. Y., Dittmer, N. T., Muthukrishnan, S., Kramer, K. J., Kanost, M. R., & Arakane, Y. (2015). Cuticular protein with a low complexity sequence becomes cross-linked during insect cuticle sclerotization and is required for the adult molt. Scientific Reports, 5, 11. doi:10.1038/srep10484In the insect cuticle, structural proteins (CPs) and the polysaccharide chitin are the major components. It has been hypothesized that CPs are cross-linked to other CPs and possibly to chitin by quinones or quinone methides produced by the laccase2-mediated oxidation of N-acylcatechols. In this study we investigated functions of TcCP30, the third most abundant CP in protein extracts of elytra (wing covers) from Tribolium castaneum adults. The mature TcCP30 protein has a low complexity and highly polar amino acid sequence. TcCP30 is localized with chitin in horizontal laminae and vertically oriented columnar structures in rigid cuticles, but not in soft and membranous cuticles. Immunoblot analysis revealed that TcCP30 undergoes laccase2-mediated cross-linking during cuticle maturation in vivo, a process confirmed in vitro using recombinant rTcCP30. We identified TcCPR27 and TcCPR18, the two most abundant proteins in the elytra, as putative crosslinking partners of TcCP30. RNAi for the TcCP30 gene had no effect on larval and pupal growth and development. However, during adult eclosion, similar to 70% of the adults were unable to shed their exuvium and died. These results support the hypothesis that TcCP30 plays an integral role as a cross-linked structural protein in the formation of lightweight rigid cuticle of the beetle

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

Abstract 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

Annotation and expression analysis of cuticular proteins from the tobacco hornworm, Manduca sexta

International audienc

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

Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae

Citation: Gorman, M.J., Sullivan, L.I., Nguyen, T.D.T., Dai,H., Arakane,Y., Dittmer, N.T.,…Kanost, M.R. (2012). Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae. Insect Biochemistry and Molecular Biology, 42(3), 193-202.Laccase-2 is a highly conserved multicopper oxidase that functions in insect cuticle pigmentation and tanning. In many species, alternative splicing gives rise to two laccase-2 isoforms. A comparison of laccase-2 sequences from three orders of insects revealed eleven positions at which there are conserved differences between the A and B isoforms. Homology modeling suggested that these eleven residues are not part of the substrate binding pocket. To determine whether the isoforms have different kinetic properties, we compared the activity of laccase-2 isoforms from Tribolium castaneum and Anopheles gambiae. We partially purified the four laccases as recombinant enzymes and analyzed their ability to oxidize a range of laccase substrates. The predicted endogenous substrates tested were dopamine, Nacetyldopamine (NADA), N-b-alanyldopamine (NBAD) and dopa, which were detected in T. castaneum previously and in A. gambiae as part of this study. Two additional diphenols (catechol and hydroquinone)and one non-phenolic substrate (2,20-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) were also tested. We observed no major differences in substrate specificity between the A and B isoforms. Dopamine, NADA and NBAD were oxidized with catalytic efficiencies ranging from 51 to 550 min 1 mM 1. These results support the hypothesis that dopamine, NADA and NBAD are endogenous substrates for both isoforms of laccase-2. Catalytic efficiencies associated with dopa oxidation were low, ranging from 8 to 30 min 1 mM 1; in comparison, insect tyrosinase oxidized dopa with a catalytic efficiency of 201 min 1 mM 1. We found that dopa had the highest redox potential of the four endogenous substrates, and this property of dopa may explain its poor oxidation by laccase-2. We conclude that laccase-2 splice isoforms are likely to oxidize the same substrates in vivo, and additional experiments will be required to discover any isoform-specific functions

Analysis of chitin-binding proteins from Manduca sexta provides new insights into evolution of peritrophin A-type chitin-binding domains in insects

International audienc

Complement activation and increased anaphylatoxin receptor expression are associated with cortical grey matter lesions and the compartmentalised inflammatory response of multiple sclerosis

BackgroundThe extent of cortical pathology is an important determinant of multiple sclerosis (MS) severity. Cortical demyelination and neurodegeneration are related to inflammation of the overlying leptomeninges, a more inflammatory CSF milieu and with parenchymal microglia and astroglia activation. These are all components of the compartmentalised inflammatory response. Compartmentalised inflammation is a feature of progressive MS, which is not targeted by disease modifying therapies. Complement is differentially expressed in the MS CSF and complement, and complement receptors, are associated with demyelination and neurodegeneration.MethodsTo better understand if complement activation in the leptomeninges is associated with underlying cortical demyelination, inflammation, and microglial activation, we performed a neuropathological study of progressive MS (n = 22, 14 females), neuroinflammatory (n = 8), and non-neurological disease controls (n = 10). We then quantified the relative extent of demyelination, connective tissue inflammation, complement, and complement receptor positive microglia/macrophages.ResultsComplement was elevated at the leptomeninges, subpial, and within and around vessels of the cortical grey matter. The extent of complement C1q immunoreactivity correlated with connective tissue infiltrates, whilst activation products C4d, Bb, and C3b associated with grey matter demyelination, and C3a receptor 1+ and C5a receptor 1+ microglia/macrophages closely apposed C3b labelled cells. The density of C3a receptor 1+ and C5a receptor 1+ cells was increased at the expanding edge of subpial and leukocortical lesions. C5a receptor 1+ cells expressed TNFα, iNOS and contained puncta immunoreactive for proteolipid protein, neurofilament and synaptophysin, suggesting their involvement in grey matter lesion expansion.InterpretationThe presence of products of complement activation at the brain surfaces, their association with the extent of underlying pathology and increased complement anaphylatoxin receptor positive microglia/macrophages at expanding cortical grey matter lesions, could represent a target to modify compartmentalised inflammation and cortical demyelination.</p

Proteomic and Transcriptomic Analyses of Rigid and Membranous Cuticles and Epidermis from the Elytra and Hindwings of the Red Flour Beetle, <i>Tribolium castaneum</i>

The insect cuticle is a composite biomaterial made up primarily of chitin and proteins. The physical properties of the cuticle can vary greatly from hard and rigid to soft and flexible. Understanding how different cuticle types are assembled can aid in the development of novel biomimetic materials for use in medicine and technology. Toward this goal, we have taken a combined proteomics and transcriptomics approach with the red flour beetle, <i>Tribolium castaneum</i>, to examine the protein and gene expression profiles of the elytra and hindwings, appendages that contain rigid and soft cuticles, respectively. Two-dimensional gel electrophoresis analysis revealed distinct differences in the protein profiles between elytra and hindwings, with four highly abundant proteins dominating the elytral cuticle extract. MALDI/TOF mass spectrometry identified 19 proteins homologous to known or hypothesized cuticular proteins (CPs), including a novel low complexity protein enriched in charged residues. Microarray analysis identified 372 genes with a 10-fold or greater difference in transcript levels between elytra and hindwings. CP genes with higher expression in the elytra belonged to the Rebers and Riddiford family (CPR) type 2, or cuticular proteins of low complexity (CPLC) enriched in glycine or proline. In contrast, a majority of the CP genes with higher expression in hindwings were classified as CPR type 1, cuticular proteins analogous to peritrophins (CPAP), or members of the Tweedle family. This research shows that the elyra and hindwings, representatives of rigid and soft cuticles, have different protein and gene expression profiles for structural proteins that may influence the mechanical properties of these cuticles