TmDorX2 positively regulates antimicrobial peptides in Tenebrio molitor gut, fat body, and hemocytes in response to bacterial and fungal infection.

Dorsal, a member of the nuclear factor-kappa B (NF-κB) family of transcription factors, is a critical downstream component of the Toll pathway that regulates the expression of antimicrobial peptides (AMPs) against pathogen invasion. In this study, the full-length ORF of Dorsal was identified from the RNA-seq database of the mealworm beetle Tenebrio molitor (TmDorX2). The ORF of TmDorX2 was 1,482 bp in length, encoding a polypeptide of 493 amino acid residues. TmDorX2 contains a conserved Rel homology domain (RHD) and an immunoglobulin-like, plexins, and transcription factors (IPT) domain. TmDorX2 mRNA was detected in all developmental stages, with the highest levels observed in 3-day-old adults. TmDorX2 transcripts were highly expressed in the adult Malpighian tubules (MT) and the larval fat body and MT tissues. After challenging the larvae with Staphylococcus aureus and Escherichia coli, the TmDorX2 mRNA levels were upregulated 6 and 9 h post infection in the whole body, fat body, and hemocytes. Upon Candida albicans challenge, the TmDorX2 mRNA expression were found highest at 9 h post-infection in the fat body. In addition, TmDorX2-knockdown larvae exposed to E. coli, S. aureus, or C. albicans challenge showed a significantly increased mortality rate. Furthermore, the expression of 11 AMP genes was downregulated in the gut and fat body of dsTmDorX2-injected larvae upon E. coli challenge. After C. albicans and S. aureus challenge of dsTmDorX2-injected larvae, the expression of 11 and 10 AMPs was downregulated in the gut and fat body, respectively. Intriguingly, the expression of antifungal transcripts TmTenecin-3 and TmThaumatin-like protein-1 and -2 was greatly decreased in TmDorX2-silenced larvae in response to C. albicans challenge, suggesting that TmDorX2 regulates antifungal AMPs in the gut in response to C. albicans infection. The AMP expression profiles in the fat body, hemocytes, gut, and MTs suggest that TmDorX2 might have an important role in promoting the survival of T. molitor larvae against all mentioned pathogens.

Manduca sexta hemolymph protease-2 (HP2) activated by HP14 generates prophenoloxidase-activating protease-2 (PAP2) in wandering larvae and pupae.

Melanization is a universal defense mechanism of insects against microbial infection. During this response, phenoloxidase (PO) is activated from its precursor by prophenoloxidase activating protease (PAP), the terminal enzyme of a serine protease (SP) cascade. In the tobacco hornworm Manduca sexta, hemolymph protease-14 (HP14) is autoactivated from proHP14 to initiate the protease cascade after host proteins recognize invading pathogens. HP14, HP21, proHP1*, HP6, HP8, PAP1-3, and non-catalytic serine protease homologs (SPH1 and SPH2) constitute a portion of the extracellular SP-SPH system to mediate melanization and other immune responses. Here we report the expression, purification, and functional characterization of M. sexta HP2. The HP2 precursor is synthesized in hemocytes, fat body, integument, nerve and trachea. Its mRNA level is low in fat body of 5th instar larvae before wandering stage; abundance of the protein in hemolymph displays a similar pattern. HP2 exists as an active enzyme in plasma of the wandering larvae and pupae in the absence of an infection. HP14 cleaves proHP2 to yield active HP2. After incubating active HP2 with larval hemolymph, we detected higher levels of PO activity, i.e. an enhancement of proPO activation. HP2 cleaved proPAP2 (but not proPAP3 or proPAP1) to yield active PAP2, responsible for a major increase in IEARpNA hydrolysis. PAP2 activates proPOs in the presence of a cofactor of SPH1 and SPH2. In summary, we have identified a new member of the proPO activation system and reconstituted a pathway of HP14-HP2-PAP2-PO. Since high levels of HP2 mRNA were present in integument and active HP2 in plasma of wandering larvae, HP2 likely plays a role in cuticle melanization during pupation and protects host from microbial infection in a soil environment.

The Toll Signaling Pathway in the Chinese Oak Silkworm, Antheraea pernyi: Innate Immune Responses to Different Microorganisms.

The Toll pathway is one of the most important signaling pathways regulating insect innate immunity. Spatzle is a key protein that functions as a Toll receptor ligand to trigger Toll-dependent expression of immunity-related genes. In this study, a novel spatzle gene (ApSPZ) from the Chinese oak silkworm Antheraea pernyi was identified. The ApSPZ cDNA is 1065 nucleotides with an open reading frame (ORF) of 777 bp encoding a protein of 258 amino acids. The protein has an estimated molecular weight of 29.71 kDa and an isoelectric point (PI) of 8.53. ApSPZ is a nuclear and secretory protein with no conserved domains or membrane helices and shares 40% amino acid identity with SPZ from Manduca sexta. Phylogenetic analysis indicated that ApSPZ might be a new member of the Spatzle type 1 family, which belongs to the Spatzle superfamily. The expression patterns of several genes involved in the Toll pathway were examined at different developmental stages and various tissues in 5th instar larvae. The examined targets included A. pernyi spatzle, GNBP, MyD88, Tolloid, cactus and dorsalA. The RT-PCR results showed that these genes were predominantly expressed in immune-responsive fat body tissue, indicating that the genes play a crucial role in A. pernyi innate immunity. Moreover, A. pernyi infection with the fungus Nosema pernyi and the gram-positive bacterium Enterococcus pernyi, but not the gram-negative bacterium Escherichia coli, activated the Toll signaling pathway. These results represent the first study of the Toll pathway in A. pernyi, which provides insight into the A. pernyi innate immune system.

Transcriptomic response of Manduca sexta immune tissues to parasitization by the bracovirus associated wasp Cotesia congregata.

During oviposition, Cotesia congregata parasitoid wasps inject into their host, Manduca sexta, some biological factors such as venom, ovarian fluid and a symbiotic polydnavirus (PDV) named Cotesia congregata bracovirus (CcBV). During parasitism, complex interactions occur between wasp-derived factors and host targets that lead to important modifications in host physiology. In particular, the immune response leading to wasp egg encapsulation is inhibited allowing wasp survival. To date, the regulation of host genes during the interaction had only been studied for a limited number of genes. In this study, we analysed the global impact of parasitism on host gene regulation 24 h post oviposition by high throughput 454 transcriptomic analyses of two tissues known to be involved in the host immune response (hemocytes and fat body). To identify specific effects of parasitism on host transcription at this time point, transcriptomes were obtained from non-treated and parasitized larvae, and also from larvae injected with heat-killed bacteria and double stimulated larvae that were parasitized prior to bacterial challenge. Results showed that, immune challenge by bacteria leads to induction of certain antimicrobial peptide (AMP) genes in M. sexta larvae whether they were parasitized or not prior to bacterial challenge. These results show that at 24 h post oviposition pathways leading to expression of AMP genes are not all inactivated suggesting wasps are in an antiseptic environment. In contrast, at this time point genes involved in phenoloxidase activation and cellular immune responses were globally down-regulated after parasitism in accordance with the observed inhibition of wasp egg encapsulation.

Dual function of a bee (Apis cerana) inhibitor cysteine knot peptide that acts as an antifungal peptide and insecticidal venom toxin.

Inhibitor cysteine knot (ICK) peptides exhibit ion channel blocking, insecticidal, and antimicrobial activities, but currently, no functional roles for bee-derived ICK peptides have been identified. In this study, a bee (Apis cerana) ICK peptide (AcICK) that acts as an antifungal peptide and as an insecticidal venom toxin was identified. AcICK contains an ICK fold that is expressed in the epidermis, fat body, or venom gland and is present as a 6.6-kDa peptide in bee venom. Recombinant AcICK peptide (expressed in baculovirus-infected insect cells) bound directly to Beauveria bassiana and Fusarium graminearum, but not to Escherichia coli or Bacillus thuringiensis. Consistent with these findings, AcICK showed antifungal activity, indicating that AcICK acts as an antifungal peptide. Furthermore, AcICK expression is induced in the fat body and epidermis after injection with B. bassiana. These results provide insight into the role of AcICK during the innate immune response following fungal infection. Additionally, we show that AcICK has insecticidal activity. Our results demonstrate a functional role for AcICK in bees: AcICK acts as an antifungal peptide in innate immune reactions in the body and as an insecticidal toxin in venom. The finding that the AcICK peptide functions with different mechanisms of action in the body and in venom highlights the two-pronged strategy that is possible with the bee ICK peptide.

Rmcystatin3, a cysteine protease inhibitor from Rhipicephalus microplus hemocytes involved in immune response.

The Rhipicephalus microplus tick is responsible for losses in the livestock production estimated in 2 billions USD. Despite its economical importance the knowledge in tick's physiology is sparse. In order to contribute to this scenario we describe the characterization of a cysteine proteinase inhibitor named Rmcystatin-3. Purified recombinant Rmcystatin-3 was able to inhibit cathepsin L (Ki = 2.5 nM), BmCl1 (Ki = 1.8 nM) and cathepsin B (Ki = 136 nM). Western blot and quantitative PCR analysis revealed the presence of Rmcystatin-3 in fat body, salivary gland but mainly in hemocytes. The mRNA levels of Rmcystatin-3 during bacterial challenge are drastically down-regulated. In order to define the Rmcystatin-3 possible role in tick immunity, the cystatin gene was knockdown by RNA interference with and without Escherichia coli infection. Our results showed that the Rmcystatin-3 silenced group was more immune competent to control bacterial infection than the group injected with non-related dsRNA. Taking together, our data strongly suggested an important role of Rmcystatin-3 in tick immunity.

A short-type peptidoglycan recognition protein from the silkworm: expression, characterization and involvement in the prophenoloxidase activation pathway.

Recognition of invading microbes as non-self is the first step of immune responses. In insects, peptidoglycan recognition proteins (PGRPs) detect peptidoglycans (PGs) of bacterial cell wall, leading to the activation of defense responses. Twelve PGRPs have been identified in the silkworm, Bombyx mori, through bioinformatics analysis. However, their biochemical functions are mostly uncharacterized. In this study, we found PGRP-S5 transcript levels were up-regulated in fat body and midgut after bacterial infection. Using recombinant protein isolated from Escherichia coli, we showed that PGRP-S5 binds to PGs from certain bacterial strains and induces bacteria agglutination. Enzyme activity assay confirmed PGRP-S5 is an amidase; we also showed it is an antibacterial protein effective against both Gram-positive and -negative bacteria. Additionally, we demonstrated that specific recognition of PGs by PGRP-S5 is involved in the prophenoloxidase activation pathway. Together, these data suggest the silkworm PGRP-S5 functions as a pattern recognition receptor for the prophenoloxidase pathway initiation and as an effecter to inhibit bacterial growth as well. We finally discussed possible roles of PGRP-S5 as a receptor for antimicrobial peptide gene induction and as an immune modulator in the midgut.

Transcriptome responses of insect fat body cells to tissue culture environment.

Tissue culture is performed to maintain isolated portions of multicellular organisms in an artificial milieu that is outside the individual organism and for considerable periods of time; cells derived from cultured explants are, in general, different from cells of the corresponding tissue in a living organism. The changes in cultured tissues that precede and often explain the subsequent cell proliferation of explant-derived cells have been partially studied, but little is known about the molecular and genomic basis of these changes. Comparative transcriptomics of intact and cultured (90 hours in MGM-450 insect medium) Bombyx mori tissues revealed that fewer genes represented a larger portion of the transcriptome of intact fat body tissues than of cultured fat body tissues. This analysis also indicated that expression of genes encoding sugar transporters and immune response proteins increased during culture and that expression of genes encoding lipoproteins and cuticle proteins decreased during culture. These results provide support for hypotheses that cultured tissues respond immunologically to surgery, adapt to the medium by accelerating sugar uptake, and terminate their identity as part of an intact organism by becoming independent of that organism.

Effect of anti-fat body antibodies on reproductive capacity of mosquito Anopheles stephensi and transmission blocking of Plasmodium vivax.

Effect of anti-mosquito-fat body antibodies on the development of the malaria parasite, Plasmodium vivax has been studied by feeding Anopheles stephensi mosquitoes with infected blood supplemented with serum from immunized rabbits. Immunogenic polypeptides were identified by western blot. Mosquitoes that ingested anti-fat body antibodies along with infectious blood meal had significantly fewer oocysts than the mosquitoes in the control group. Effect of anti-mosquito fat body antibodies on fecundity, hatchability, mortality and engorgement of mosquitoes has also been reported. A significant reduction in fecundity and hatchability was observed, however, effect on mortality and engorgement was variable and statistically insignificant. Results indicated that fat body antibodies have the potential to disrupt reproductive physiology of malaria vector An. stephensi.

Drosophila Ras/MAPK signalling regulates innate immune responses in immune and intestinal stem cells.

Immune signalling pathways need to be tightly regulated as overactivation of these pathways can result in chronic inflammatory diseases and cancer. NF-κB signalling and associated innate immune pathways are crucial in the first line of defense against infection in all animals. In a genome-wide RNAi screen for modulators of Drosophila immune deficiency (IMD)/NF-κB signalling, we identified components of the Ras/MAPK pathway as essential for suppression of IMD pathway activity, even in the absence of an immune challenge. Downregulation of Ras/MAPK activity mimics the induction of innate immune responses by microbial patterns. Conversely, ectopic Ras/MAPK pathway activation results in the suppression of Drosophila IMD/NF-κB signalling. Mechanistically, we show that the Ras/MAPK pathway acts by inducing transcription of the IMD pathway inhibitor Pirk/Rudra/PIMS. Finally, in vivo experiments demonstrate a requirement for Ras/MAPK signalling in restricting innate immune responses in haemocytes, fat body and adult intestinal stem cells. Our observations provide an example of a pathway that promotes cell proliferation and has simultaneously been utilized to limit the immune response.

[Immune system of the blowworm Calliphora vicina (diptera, calliphoridae) as a source of medicines].

Study of mechanisms of cellular and humoral immunity of larvae of the blowworm Calliphora vicina has revealed three groups of pharmacologically active substances perspective for use in medicine--alloferons, allostatins, and antimicrobial peptides. Alloferons--the family of peptides of C. vicina stimulating selectively the cytotoxic activity of natural killers of the evolutionary ancient group of immunocompetent cells playing the key role in the system of mammalian antiviral and antitumor immunity. Alloferons are used in medicine for treatment of herpes-viral infections and viral hepatitis B. Allostatins-synthetic peptides combining structural characteristics of alloferons and some mammalian immunologically active proteins. Allostatins, like alloferons, produce stimulating effects on cytotoxic activity of natural killers and interferon production, but differ from them by pronounced adjuvant properties--the ability to enhance immune recognition of alien antigens. At present, allostatins are used to increase resistance of skin and mucosae to viral infections; in future, they might also find use in immunotherapy of cancer and other diseases. Another protein and peptide group perspective for use in medicine serve as antimicrobial peptides in immune response of larvae C. vicina. The study of the agent containing defensins, cecropins, diptericins, and praline-enriched C. vicina peptides shows the drugs of this type to be of great interest as the tool for treatment and prevention of infections caused by antibiotic-resistant bacteria.

Characterization of Kunitz-type protease inhibitor purified from hemolymph of Galleria mellonella larvae.

We characterized a Kunitz-type protease inhibitor (Gm KTPI) obtained from the hemolymph of Galleria mellonella larvae immunized with Escherichia coli. The structural analysis of the cloned cDNA showed that it consists of 56 residues derived from the precursor of 75 amino acids. The peptide was constitutively produced in the fat bodies, but not in the midgut nor the integument of larvae. In our analysis of stage-dependent expression, its transcript was detected within the midgut, the fat bodies and the integument of the prepupae, which undergo tissue remodeling. The inhibition assays showed that Gm KTPI was capable of inhibiting only the trypsin-like activity of the larval midgut extracts. Furthermore, it was determined that Gm KTPI induced the activation of extracellular signal-regulated kinase (ERK) in the fat bodies and integument cells, and this kinase is known to perform a central role in cell proliferation signaling. Its effect on ERK activation was also verified in a control experiment using a human endothelial cell culture. Collectively, it was suggested that Gm KTPI might be responsible for the protection of other tissues against proteolytic attack by trypsin-like protease(s) from larval midgut during metamorphosis, and might play a role in the proliferation of cells in the fat body and integument.

Plasmatocyte-spreading peptide (PSP) plays a central role in insect cellular immune defenses against bacterial infection.

Insect hemocytes (blood cells) are a central part of the insect's cellular response to bacterial pathogens, and these specialist cells can both recognize and engulf bacteria. During this process, hemocytes undergo poorly characterized changes in adhesiveness. Previously, a peptide termed plasmatocyte-spreading peptide (PSP), which induces the adhesion and spreading of plasmatocytes on foreign surfaces, has been identified in lepidopteran insects. Here, we investigate the function of this peptide in the moth Manduca sexta using RNA interference (RNAi) to prevent expression of the precursor protein proPSP. We show that infection with the insect-specific bacterial pathogen Photorhabdus luminescens and non-pathogenic Escherichia coli induces proPSP mRNA transcription in the insect fat body but not in hemocytes; subsequently, proPSP protein can be detected in cell-free hemolymph. We used RNAi to silence this upregulation of proPSP and found that the knock-down insects succumbed faster to infection with P. luminescens, but not E. coli. RNAi-treated insects infected with E. coli showed a reduction in the number of circulating hemocytes and higher bacterial growth in hemolymph as well as a reduction in overall cellular immune function compared with infected controls. Interestingly, RNAi-mediated depletion of proPSP adversely affected the formation of melanotic nodules but had no additional effect on other cellular responses when insects were infected with P. luminescens, indicating that this pathogen employs mechanisms that suppress key cellular immune functions in M. sexta. Our results provide evidence for the central role of PSP in M. sexta cellular defenses against bacterial infections.

Bacterial, but not baculoviral infections stimulate Hemolin expression in noctuid moths.

Lepidopteran larvae are regularly infected by baculoviruses during feeding on infected plants. The differences in sensitivity to these infections can be substantial, even among closely related species. For example, the noctuids Cotton bollworm (Helicoverpa zea) and Tobacco budworm (Heliothis virescens), have a 1000-fold difference in sensitivity to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection. Recent data were interpreted to indicate that the lepidopteran immunoglobulin protein, Hemolin, is synthesized upon viral injection and therefore to participate in anti-viral responses. To investigate whether Hemolin synthesis is affected by a natural viral infection, specific transcription in fat bodies and hemocytes of H. zea and H. virescens larvae was monitored following per os infection with the baculovirus HzSNPV (H. zea single nucleopolyhedrovirus). Both moths showed the same expression pattern as seen in uninfected animals and coincided with ecdysone responses, previously known to induce Hemolin expression. In contrast, injection of lyophilized Micrococcus luteus resulted in increased Hemolin expression supporting a role for Hemolin as an immuno-responsive protein in these species. The combined data are consistent with the suggestion that while Hemolin seems to participate in the response to virus infection in the superfamily Bombycoidea, this is not true in the Noctuoidea.

The DExD/H-box helicase Dicer-2 mediates the induction of antiviral activity in drosophila.

Drosophila, like other invertebrates and plants, relies mainly on RNA interference for its defense against viruses. In flies, viral infection also triggers the expression of many genes. One of the genes induced, Vago, encodes a 18-kilodalton cysteine-rich polypeptide. Here we provide genetic evidence that the Vago gene product controlled viral load in the fat body after infection with drosophila C virus. Induction of Vago was dependent on the helicase Dicer-2. Dicer-2 belongs to the same DExD/H-box helicase family as do the RIG-I-like receptors, which sense viral infection and mediate interferon induction in mammals. We propose that this family represents an evolutionary conserved set of sensors that detect viral nucleic acids and direct antiviral responses.

A novel ML protein from Manduca sexta may function as a key accessory protein for lipopolysaccharide signaling.

Lipopolysaccharide (LPS) present on the outer membrane of Gram-negative bacteria is one of the most important pathogen-associated molecular patterns and a potent elicitor in innate immunity. In human, TLR4 (Toll-like receptor 4) and MD-2 (myeloid differiation-2) form a receptor complex to transduce the LPS signal into cells. However, in invertebrates, receptors that recognize LPS have not been determined. Here we report the purification, characterization and cDNA cloning of an ML (MD-2-related lipid-recognition) protein from the tobacco hornworm Manduca sexta. The full-length cDNA of this M. sexta ML protein, named MsML-1, is 532bp with an open reading frame of 456bp that encodes a polypeptide of 151 amino acids containing an ML domain. MsML-1 is a secreted glycoprotein and its mRNA is expressed in fat body and hemocytes. The expression level of MsML-1 mRNA in fat body and hemocytes as well as MsML-1 protein in hemolymph are not induced by immune challenge. Recombinant MsML-1 protein specifically binds to LPS from several Gram-negative bacteria and LPS Re mutant, as well as to lipid A, but not to KDO (2-keto-3-deoxyoctonate). Our results suggest that MsML-1 may function as a key accessory protein for LPS signaling in M. sexta against Gram-negative bacterial infection.

Studies on the role of protein kinase A in humoral immune response of Galleria mellonella larvae.

Protein kinase A (PKA) activity was detected in the fat body of Galleria mellonella larvae by a non-radioactive method using a specific peptide substrate-kemptide. The enzyme activity was stimulated by cAMP and its analogues: BzcMP, 8-Chl-cAMP and 8-Br-cAMP in concentrations of 1-4muM. Cyclic GMP was not effective in PKA activation. A two-fold increase in PKA activity was detected in the fat body of G. mellonella LPS-challenged larvae. Selective, membrane-permeable PKA inhibitors, H89 and Rp-8-Br-cAMPS, inhibited protein kinase A activity in the fat body of G. mellonella larvae in vitro and in vivo. The inhibition of PKA activity in vivo was correlated with a considerable lowering of haemolymph antibacterial activity and a decrease in lysozyme content in the fat body of immune challenged larvae. The use of phospho-motif antibodies recognising PKA phosphorylation consensus site allowed identification of four potential PKA phosphorylation substrates of 79, 45, 40 and 36kDa in G. mellonella fat body.

Immune stimulation in the silkworm, Bombyx mori L., by CpG oligodeoxynucleotides.

Synthetic ODNs containing unmethylated CpG dinucleotides are known to stimulate immune responses in vertebrates, but so far the effect has not been studied in insects. In this report, we describe an induction of immune response following injection of oligodeoxynucleotides (ODNs) into the insect hemocoel. The fifth instar silkworm (Bombyx mori L.) larvae were injected with several synthetic ODNs containing variable number of unmethylated CpG motifs, heat-denatured genomic DNA of B. mori itself, or intact genomic DNA to observe a new induction pattern in the insect immune mechanism. When the induction of immune response was examined based on the expression rates of genes for antibacterial peptides such as attacin and cecropin, we could confirm that it was triggered upon injection of ODNs. The expression was, however, neither dependent on numbers of CpG motifs nor methylation of CpGs in ODNs. Furthermore, it was confirmed that the presence of CpG in ODN was not involved in the induction pattern of insect immunity caused by ODNs, although it has been reported that vertebrates respond in a specific manner against invading ODNs containing CpG dinucleotides. In addition, insect immunity was not stimulated by injection of intact DNA from host. In contrast, the injection of denatured genomic DNA provoked the host immune reaction. Taken together, our data suggest that foreignness of ODNs or DNA might be a key factor in the induction of insect immunity.

Serpent regulates Drosophila immunity genes in the larval fat body through an essential GATA motif.

Insects possess a powerful immune system, which in response to infection leads to a vast production of different antimicrobial peptides. The regulatory regions of many immunity genes contain a GATA motif in proximity to a kappaB motif. Upon infection, Rel proteins enter the nucleus and activate transcription of the immunity genes. High levels of Rel protein-mediated Cecropin A1 expression previously have been shown to require the GATA site along with the kappaB site. We provide evidence demonstrating that the GATA motif is needed for expression of the Cecropin A1 gene in larval fat body, but is dispensable in adult fat body. A nuclear DNA-binding activity interacts with the Cecropin A1 GATA motif with the same properties as the Drosophila GATA factor Serpent. The GATA-binding activity is recognized by Serpent-specific antibodies, demonstrating their identity. We show that Serpent is nuclear in larval fat body cells and haemocytes both before and after infection. After overexpression, Serpent increases Cecropin A1 transcription in a GATA-dependent manner. We propose that Serpent plays a key role in tissue-specific expression of immunity genes, by priming them for inducible activation by Rel proteins in response to infection.