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Highlights • Endo- and exogenous stimuli activate inflammasomes. • Inflammasome activation contributes to innate and adaptive immune responses. • Inflammasome activation induces IL-1β secretion. • IL-1β promotes Th17 cell differentiation via IL-1/IL-1R signaling. • Th17 responses are associated with inflammasome activation. Abstract Immune sensing of exogenous molecules from microbes (e.g., pathogen-associated molecular patterns) and nonmicrobial molecules (e.g., asbestos, alum, and silica), as well as endogenous damage-associated molecular patterns (e.g., ATP, uric acid crystals, and amyloid A) activates innate immunity by inducing immune-related genes, including proinflammatory cytokines, which further facilitate the development of adaptive immunity. The roles of transcriptional responses downstream of immune sensing have been widely characterized in informing adaptive immunity; however, few studies focus on the effect of post-translational responses on the modulation of adaptive immune responses. Inflammasomes activated by the previously described endo- and exogenous stimuli autocatalytically induce intracellular pro-caspase-1, which cleaves the inactive precursors of interleukin-1β (IL-1β) and IL-18 into bioactive proinflammatory cytokines. IL-1β and IL-18 not only contribute to the host defense against infections by activating phagocytes, such as monocytes, macrophages, dendritic cells, and neutrophils, but also induce T-helper 17 (Th17)- and Th1-mediated adaptive immune responses. In synergy with IL-6 and IL-23, IL-1β activates IL-1 receptor (IL-1R) signaling to drive the differentiation of IL-17-producing Th17 cells, which not only play critical roles in host protective immunity to infections of bacteria, fungi, and certain viruses but also participate in the pathology of inflammatory disorders and tumorigenesis. Consequently, targeting inflammasomes and IL-1/IL-1R signaling may effectively improve the treatment of Th17-associated disorders, such as autoinflammatory diseases and cancers, thereby providing novel insights into drug development. [ABSTRACT FROM AUTHOR]

Abstract: Activation of prophenoloxidase and synthesis of antimicrobial peptides (AMPs) are two important innate immune mechanisms in insects. In the current study, we investigated immune responses activated by three major bacterial components, lipopolysaccharide (LPS) (including rough mutants of LPS), lipoteichoic acid (LTA), and peptidoglycan (PG), in the larvae of a lepidopteran insect, Manduca sexta. We found that two DAP (diaminopimelic acid)-type PGs from Escherichia coli and Bacillus subtilis were much more potent than LPS and LTA from the respective bacteria as well as a Lysine-type PG in activation of prophenoloxidase in M. sexta larval plasma in vitro. Transcription levels of AMP genes, such as Attacin, Lebocin and Moricin genes, in the hemocytes and fat body of larvae were significantly induced by smooth LPS (TLR4grade) and rough mutants of LPS (TLRgrade™), synthetic lipid A, LTA, and PG. LPS from E. coli and LTA from B. subtilis activated AMP expression to significantly higher levels than PGs from the respective bacterial strains, and smooth LPS were more potent than lipid A and rough mutants of LPS in activation of AMP expression. Our results demonstrated for the first time that LTA can activate AMP expression, and different moieties of LPS may synergistically activate AMP expression in M. sexta. [ABSTRACT FROM AUTHOR]

Abstract: The unprecedented, stereospecific interconversion of cis- and trans-γ,δ-epoxy α,β-unsaturated ester systems has been realized, which involves the palladium-catalyzed stereospecific alkoxy or hydroxy substitution reaction with double inversion of configuration at the γ-position as the key step. The new methodology is not only applicable to various disubstituted and trisubstituted epoxy unsaturated esters, but also these interconversions proceed with an extremely high degree of stereoselectivity and efficiency. [Copyright &y& Elsevier]

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

Abstract: Phagocytosis is an important innate immune response against microbial infections and an effective mechanism to eliminate apoptotic cells. In vertebrates, phagocytes such as macrophages and dendritic cells are involved in phagocytosis. We demonstrate here that insect hemocytes have distinct functions in phagocytosis of foreign particles and self dead cells. Plasmatocytes from the tobacco hornworm Manduca sexta were major hemocytes involved in phagocytosis of non-self microsphere beads, whereas granulocytes were apparently the only hemocytes that phagocytose self dead cells. We also showed that M. sexta immulectin-2, a pattern recognition receptor that protects larvae from bacterial infection, has an opsonic activity in phagocytosis. Immulectin-2 bound to the surface of granulocytes from the naïve larvae, but more immulectin-2 bound to plasmatocytes when larvae were injected with microsphere beads. Coupling of immulectin-2 onto microsphere beads enhanced in vitro phagocytosis of the beads. Our results suggest that insect hemocytes can have specialized functions similar to vertebrate phagocytes in phagocytosis, and pattern recognition receptors may function as opsonins to enhance phagocytosis. [Copyright &y& Elsevier]

Abstract: An effective innate immune response against parasites in insects is encapsulation followed by melanization. In cellular encapsulation, formation of capsules requires plasmatocytes and granulocytes, the two most abundant hemocytes in lepidopteran insects. We have developed an in vitro encapsulation assay to elucidate functions of immulectins in encapsulation. Immulectins are members of the C-type (calcium-dependent) lectin superfamily, and they function as humoral pattern recognition receptors in innate immunity of the tobacco hornworm Manduca sexta. We demonstrate that coating of immulectins to agarose beads enhanced cellular encapsulation in vitro. Immulectin-1 enhanced encapsulation but not melanization. Encapsulation occurred with isolated hemocytes that lack plasmatocytes when agarose beads were coated with immulectins. However, plasmatocytes were required for aggregation of encapsulated beads. We also showed that inhibitors of serine proteinases such as para-aminophenylmethylsulfonyl fluoride (p-APMSF) inhibited cellular encapsulation of immulectin-coated agarose beads. Our results suggest that humoral pattern recognition receptors may enhance cellular encapsulation and melanization. [Copyright &y& Elsevier]

Abstract: In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including hemocyte surfaces. In this study, we showed that in the naïve larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of hemocytes with surface proPO/PO increased at 12h post-injection, but dropped to the normal level at 24h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these hemocytes. The number of melanized hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated hemocytes in the presence of cell-free plasma. Our results suggest that activation of hemocyte surface proPO may initiate melanization, leading to the systemic melanization of hemocyte capsules. [Copyright &y& Elsevier]

Abstract: Lectins interact with carbohydrates. They can function as pattern recognition receptors and play an important role in the innate immune system of animals. Previously, we have isolated two calcium-dependent (C-type) lectins, named immulectin-1 and -2, from the tobacco hornworm Manduca sexta. Both immulectin-1 and -2 stimulate prophenoloxidase activation in plasma. Here, we describe isolation and cDNA cloning of a novel member of immulectins, immulectin-3 (IML-3). IML-3, like immulectin-1 and -2, contains tandem carbohydrate-recognition domains (CRDs). The cDNA clone encoding IML-3 is 3802bp long, with an open reading frame of 930bp. This cDNA clone has an extremely long noncoding region at the 3′ end that contains eight polyadenylation signal sequences. Northern analysis showed that a 5.0kb IML-3 transcript was present in the fat body of control larvae (injected with saline) but not in the fat body of larvae injected with bacteria. However, a much more abundant 3.1kb transcript was induced in the fat body of bacteria-injected larvae. IML-3 mRNA was not detected in hemocytes of control or bacteria-injected larvae. Recombinant IML-3 was expressed in bacteria and purified. It specifically bound to immobilized lipopolysaccharide (LPS) and lipoteichoic acid from bacteria, and to laminarin, a -1, 3-glucan. Binding of IML-3 to immobilized LPS was competed by excess free LPS. More importantly, IML-3 contains an anti-death-like motif in the carboxyl-terminal CRD. Endogenous IML-3 was detected in the cytoplasm of hemocytes, and FITC-labeled recombinant IML-3 was translocated from hemolymph into hemocytes. Coating of IML-3 onto agarose beads enhanced encapsulation of the beads. [Copyright &y& Elsevier]

In insects, encapsulation followed by melanization is a major defense mechanism against metazoan parasites. However, insects must recognize and differentiate nonself before they mount an immune response. Recognition of pathogens in insects is accomplished by a set of pattern recognition receptors (PRRs). Binding of PRRs to pathogens is linked to a variety of immune responses including phagocytosis, nodule formation, encapsulation, and prophenoloxidase activation. So far, little is known about how recognition of pathogens by PRRs triggers different immune responses. In this article, we report that immulectin-2, a C-type lectin, enhances encapsulation and melanization processes in the tobacco hornworm, Manduca sexta. Coating of agarose beads with recombinant carboxyl-terminal carbohydrate-recognition domain (CRD2-II) of immulectin-2 enhanced encapsulation of the beads in vitro by hemocytes and melanization of the beads in vivo in M. sexta larvae. Recombinant CRD2-II also directly bound to granular cells and oenocytoids, but not to plasmatocytes or spherule cells. Immulectin-2 in hemolymph of M. sexta larvae bound to the surface of a nematode, Caenorhabditis elegans, and recombinant CRD2-II directly bound to C. elegans and a human filarial nematode, Brugia malayi. Binding of CRD2-II to C. elegans enhanced melanization of the nematode in vivo. Our results suggest that binding of immulectin-2 to the surface of parasites can trigger encapsulation and melanization responses in M. sexta. [Copyright &y& Elsevier]

We previously reported the isolation of a lipopolysaccharide (LPS)-specific immulectin-2 from the tobacco hornworm, Manduca sexta [J. Biol. Chem. 275 (2000) 37373]. Immulectin-2 is a C-type lectin that is present at a constitutively low level in hemolymph of naive larvae, and its synthesis is induced after injection of Gram-negative bacteria or LPS. Immulectin-2 contains two carbohydrate recognition domains. It binds to LPS and stimulates prophenoloxidase activation in plasma. In this paper, we focus on properties of carbohydrate recognition domain-2 of immulectin-2 and the biological functions of immulectin-2 in immune responses. The carboxyl-terminal carbohydrate recognition domain (CRD2) of immulectin-2 was able to bind bacterial LPS. Binding of recombinant CRD2 to LPS stimulated activation of prophenoloxidase in plasma. Injection of antiserum against immulectin-2 into M. sexta larvae inhibited clearance of a Gram-negative bacterial pathogen, Serratia marcescens, and decreased survival of infection. These results suggest that immulectin-2 plays an important role in the immune system of M. sexta, and helps to protect the animal from Gram-negative bacterial infections. [Copyright &y& Elsevier]

In insects, the prophenoloxidase activation system is a defense mechanism against parasites and pathogens. Recognition of parasites or pathogens by pattern recognition receptors triggers activation of a serine proteinase cascade, leading to activation of prophenoloxidase-activating proteinase (PAP). PAP converts inactive prophenoloxidase (proPO) to active phenoloxidase (PO), which then catalyzes oxidation of phenolic compounds that can polymerize to form melanin. Because quinone intermediates and melanin are toxic to both hosts and pathogens, activation of proPO must be tightly regulated and localized. We report here purification and cDNA cloning of serine proteinase homologs (SPHs) from the tobacco hornworm, Manduca sexta, which interact with PAP-1 in proPO activation. Two SPHs were co-purified from plasma of M. sexta larvae with immulectin-2, a C-type lectin that binds to bacterial lipopolysaccharide. They contain an amino-terminal clip domain connected to a carboxyl-terminal serine proteinase-like domain. PAP-1 alone cannot efficiently activate proPO, but a mixture of SPHs and PAP-1 was much more effective for proPO activation. Immulectin-2, proPO and PAP-1 in hemolymph bound to the immobilized recombinant proteinase-like domain of SPH-1, indicating that a complex containing these proteins may exist in hemolymph. Since immulectin-2 is a pattern recognition receptor that binds to surface carbohydrates on pathogens, such a protein complex may localize activation of proPO on the surface of pathogens. SPH, which binds to immulectin-2, may function as a mediator to recruit proPO and PAP to the site of infection. [Copyright &y& Elsevier]

The diamondback moth, Plutella xylostella , is the first insect to develop resistance to Bacillus thuringiensis (Bt) in the field. To date, little is known about the molecular mechanism of the interaction between Bt and midgut immunity in P. xylostella. Here, we report immune responses in the P. xylostella midgut to Bt strain Bt8010 using a combined approach of transcriptomics and quantitative proteomics. Many genes in the Toll, IMD, JNK and JAK-STAT pathways and antimicrobial peptide genes were activated at 18 h post-infection. In the prophenoloxidase (PPO) cascade, four serpin genes were activated, and the PPO1 gene was suppressed by Bt8010. Inhibition of the two PPO proteins was observed at 18 h post-infection. Feeding Bt8010-infected larvae recombinant PPOs enhanced their survival. These results revealed that the Toll, IMD, JNK and JAK-STAT pathways were triggered and participated in the immune defence of the midgut against Bt8010, while the PPO cascade was inhibited and played an important role in this process. • The Toll, IMD, JNK and JAK-STAT pathways were activated in midgut by Bt8010. • Midgut attempted to trigger the PPO cascade, which was eventually inhibited by Bt8010. • Two recombinant PPOs improved the survival of Bt8010-infected larvae. • The PPO cascade plays an important role in the defence of midgut against Bt8010. [ABSTRACT FROM AUTHOR]

Insect gut immunity plays a key role in defense against microorganism infection. The knowledge of insect gut immunity has been obtained mostly from Drosophila melanogaster . Little is known about gut immunity in the diamondback moth, Plutella xylostella (L.), a pest destroying cruciferous crops worldwide. In this study, expressions of the immune-related genes in the midgut of P . xylostella orally infected with Staphylococcus aureus , Escherichia coli and Pichia pastoris were profiled by RNA-seq and qRT-PCR approaches. The results revealed that the Toll, IMD, JNK and JAK-STAT pathways and possibly the prophenoloxidase activation system in P . xylostella could be activated by oral infections, and moricins, gloverins and lysozyme2 might act as important effectors against microorganisms. Subsequent knock-down of IMD showed that this gene was involved in regulating the expression of down-stream genes in the IMD pathway. Our work indicates that the Toll, IMD, JNK and JAK-STAT pathways may synergistically modulate immune responses in the P . xylostella midgut, implying a complex and diverse immune system in the midgut of insects. [ABSTRACT FROM AUTHOR]

Abstract: ML (MD-2 (myeloid differentiation factor-2)-related Lipid-recognition) is a conserved domain identified in MD-2, MD-1, NPC2 (Niemann-Pick disease type C2), and mite major allergen protein from animals, plants, and fungi. Vertebrate members of the ML family proteins, such as NPC2 and MD-2, play important roles in lipid metabolism and immune signaling pathway. MD-2 is an essential co-receptor in the lipopolysaccharide (LPS)/Toll-like receptor 4 (TLR4) signaling pathway. Insects contain multiple ML genes, arbitrarily named md-2- or npc2-like genes. However, whether insect ML genes have functions similar to vertebrate md-2 is unknown. In Drosophila melanogaster, there are eight npc2 genes (npc2a–h), and they can be further divided into three subgroups based on the numbers of cysteine residues (6, 7 and 8 Cys) in the mature proteins. The purpose of this study is to investigate whether any Drosophila npc2 genes may have functions in immune signaling pathways. We chose npc2a, npc2e and npc2h genes representing the three subgroups for this study. We showed that recombinant NPC2a, NPC2e and NPC2h not only bound to LPS and lipid A, but also bound to peptidoglycan (PG) and lipoteichoic acid (LTA), a property that has not been reported previously for vertebrate NPC2 or MD-2. More importantly, we showed that over-expression of NPC2a and NPC2e activated diptericin promoter reporter in S2 cells stimulated by PG, suggesting that NPC2e and NPC2a may play a role in the immune deficiency (Imd) pathway. This is the first in vitro study about NPC2 proteins in innate immunity of D. melanogaster. [Copyright &y& Elsevier]

Abstract: Insects produce a group of antimicrobial peptides (AMPs) in response to microbial infections. Most AMPs are synthesized as inactive precursors/pro-proteins and require proteolytic processing to generate small active peptides. Here we report identification and functional analysis of two lebocin-related proteins (Leb-B and Leb-C) from the tobacco hornworm, Manduca sexta. The mRNA levels of Leb-B and Leb-C increased significantly in larval fat body and hemocytes after injection of Escherichia coli, Micrococcus luteus and Saccharomyces cerevisiae. Western blotting using rabbit polyclonal antibody to Leb-B showed accumulation of large protein(s) and small peptide(s) in larval hemolymph after microbial injection. This result and the presence of RXXR motifs in the deduced amino acid sequences led to our postulation that Leb-B/C may be inactive precursors that are processed in larval hemolymph to generate short active peptides. To test this hypothesis, we expressed and purified full-length and various fragments of Leb-B and Leb-C as thioredoxin (TRX) fusion proteins. We found that fusion proteins could be cleaved by induced larval plasma, and the cleavage sites were determined by protein sequencing. Antibacterial activity of peptide fragments was also verified using synthetic peptides, and active M. sexta lebocin peptides were located at the N-termini of Leb-B/C, which are different from Bombyx mori lebocins 1–4 that are located close to the C-termini. In addition, we found that synthetic Leb-B22–48 peptide not only had higher antibacterial activity but also caused agglutination of E. coli cells. Our results provide valuable information for studying processing of lebocin precursors in lepidopteran insects. [Copyright &y& Elsevier]

Abstract: Insects produce a variety of antimicrobial peptides (AMPs). Induction of insect AMP genes is regulated by the Toll and IMD (immune deficiency) pathways via NF-κB and GATA factors. Little is known about species-specific regulation of AMP genes. In this report, we showed that activities of most Manduca sexta and Drosophila melanogaster AMP gene promoters were regulated in a species-specific manner in Drosophila (Dipteran) S2 cells and Spodoptera frugiperda (Lepidopteran) Sf9 cells. A κB-GATA element (22 bp) from M. sexta moricin (MsMoricin) promoter could significantly increase activities of Drosophila AMP gene promoters in S2 cells, and an MsMoricin promoter activating element (MPAE) (140 bp) could increase activity of drosomycin promoter specifically in Sf9 cells. However, κB and GATA factors alone were not sufficient for MsMoricin gene activation, suggesting that other co-regulators may be required to fully activate AMP genes. Our results suggest that induction of insect AMP genes may require a transcription complex composed of common nuclear factors (such as NF-κB and GATA factors) and species-related co-regulators, and it is the co-regulators that may confer species-specific regulation of AMP genes. In addition, we showed that activity of Drosophila drosomycin promoter could be activated cooperatively by the inserted exogenous κB-GATA element and the endogenous κB element. These findings revealed an approach of engineering AMP genes with enhanced activities, which may lead to broad applications. [Copyright &y& Elsevier]

Abstract: Activation of the prophenoloxidase (proPO) system and synthesis of antimicrobial peptides (including lysozyme) are two key defense mechanisms in arthropods. Activation of proPO involves a cascade of serine proteinases that eventually converts proPO to active phenoloxidase (PO). However, a trade-off between lysozyme/antibacterial activity and PO activity has been observed in some insects, and a mosquito lysozyme can inhibit melanization. It is not clear whether lysozyme can inhibit PO activity and/or proPO activation. In this study, we used in vitro assays to investigate the role of lysozyme in proPO activation in the tobacco hornworm Manduca sexta. We showed that lysozymes from M. sexta, human milk and hen egg white did not inhibit PO activity in the pre-activated naïve plasma of M. sexta larvae, but significantly inhibited proPO activation in the naïve plasma. Western blot analysis showed that direct incubation of M. sexta lysozyme with the naïve plasma prevented conversion of proPO to PO, but stimulated degradation of precursor proteins for serine proteinase homolog-2 (SPH2) and proPO-activating proteinase-1 (PAP1), two key components required for proPO activation. Far-western blot analysis showed that M. sexta lysozyme and proPO interacted with each other. Altogether, our results suggest that lysozymes may inhibit the proPO activation system by preventing conversion of proPO to PO via direct protein interaction with proPO. [Copyright &y& Elsevier]

Abstract: Hybrid antibacterial peptide CecropinAD (CAD) is a linear cationic peptide that has potent antimicrobial properties without hemolytic activity. To explore a new approach to express the hybrid peptide CAD in the methylotrophic yeast Pichia pastoris, the cDNA sequence encoding CAD was obtained by recursive PCR (rPCR) and cloned into the vector pPICZα-A. The Sac I-linearized recombinant plasmid pPICZα-CAD was transformed into P. pastoris GS115 by electroporation. Expression of recombinant CAD was induced for 96h with 1.0% methanol at 28°C, pH 5.0. The recombinant CAD was purified by two steps of reversed-phase HPLC and 1.8mg pure active CAD was obtained from 100ml culture. Tricine-SDS-PAGE and mass spectrometry analyses demonstrated that the molecular weight of the purified CAD was 3.8kDa. Analysis of circular dichroism (CD) revealed that CAD mainly has α-helixes in the presence of 10mM phosphate buffer (pH 7.2), 50% TFE/water solution (pH 2.0), or 30mM SDS (pH 10.8). FACScan analysis showed that the antibacterial mechanism of CAD is to act on the cell membrane to disrupt bacterial cell structure. Antimicrobial assays demonstrated that recombinant CAD has a broad spectrum of anti-microbial property against fungi, as well as Gram-positive and Gram-negative bacteria, but does not have hemolytic activity against human erythrocytes. Our results suggest that recombinant antimicrobial peptide CAD may serve as an attractive candidate for the development of therapeutic antimicrobial drugs. [Copyright &y& Elsevier]

Abstract: Immulectin-3 (IML-3) is a C-type lectin from the tobacco hornworm Manduca sexta that contains a motif (NWGV) similar to the BH1 motif (NWGR) of the mammalian galectin-3. IML-3 is synthesized in fat body and secreted into hemolymph, but can be translocated into hemocytes. In this study, we showed that IML-3 was predominantly localized to the nucleus of hemocytes and some metaphase, anaphase and telophase hemocytes from M. sexta larvae injected with bacterial lipopolysaccharide (LPS). IML-3 was detected in the membrane and soluble extracts of hemocytes, suggesting that it may be translocated into hemocytes via receptor-mediated endocytosis. To investigate the role of IML-3 translocation to the nucleus, we expressed recombinant wild-type IML-3 and a deletion mutant ΔIML-3 that has the NWGV motif deleted in Drosophila S2 cells. We found that recombinant wild-type IML-3, but not ΔIML-3, was localized to the nucleus of some S2 cells and also detected in the nuclear extract. Expression of recombinant wild-type IML-3, but not ΔIML-3 or GFP, increased the number of proliferating S2 cells. Our results suggest that nuclear translocation of IML-3 may stimulate hemocyte proliferation. [Copyright &y& Elsevier]

Abstract: Genomic and cDNA sequences of a Toll receptor were cloned from the tobacco hornworm, Manduca sexta. M. sexta Toll (MsToll) gene contains six introns and seven exons. The full-length cDNA of MsToll is 3495bp with an open reading frame of 2892bp, which encodes a protein of 963 amino acids. MsToll is a typical single-pass transmembrane protein containing characteristic domain architecture of Toll and Toll-like receptors, including an extracellular domain composing of leucine-rich repeats (LRRs) and a cytoplasmic TIR domain. The deduced amino acid sequence of MsToll is most similar to Apis mellifera Toll (27% identity). Reverse-transcription polymerase chain reaction (RT-PCR) showed that MsToll was expressed in hemocytes, fat body, Malpighian tubule, midgut and epidermis. Real-time PCR analysis showed that MsToll mRNA in hemocytes was significantly induced by Escherichia coli, as well as by yeast (Saccharomyces cerevisiae) and Micrococcus lysodeikticus. However, MsToll transcript in fat body was not induced by these microorganisms. Immunohistochemistry assay showed that MsToll was detected on hemocytes. The TIR domain of MsToll also has high similarity to those of vertebrate TLR4 and zebra fish TLR (35–39% identity). Our results suggest that MsToll may play a role in innate signaling in response to E. coli infections. [Copyright &y& Elsevier]

Abstract: For the waveguide grating photocoupler with novel structure [Yu XQ, Zhang X, Wong KS, Xu GB, Xu XG, Ren Y, et al. A fabrication of coupling grating in the polymeric waveguide by using two-photon initiated photopolymerization. Mater Lett 2004;58:3879–83. ; Yu XQ, Zhang X, Xu GB, Zhao HP, He W, Shao ZS, et al. Fabrication of grating waveguide and coupling grating using two-photon initiated photopolymerization. Chem J Chin Univ 2004;25(10):1931–3 (in Chinese). ], the electric fields of the TE guided wave and the TE radiating wave are obtained by solving the Helmholtz equation in the spatial rectangular coordinates. And then the relations between the loss coefficient and the different structure parameters of the waveguide and grating are analyzed by using the mode coupling theories, and their corresponding numerical simulation results are given. In the end the result obtained for this novel structure and that for conventional rectangle structure are compared, and the difference and the sameness are obtained. [Copyright &y& Elsevier]

Abstract: C-type lectins are calcium-dependent carbohydrate binding proteins, and animal C-type lectins participate in innate immunity and cell–cell interactions. In the fruit fly Drosophila melanogaster, more than 30 genes encode C-type lectin domains. However, functions of Drosophila C-type lectins in innate immunity are not well understood. This study is to investigate whether two Drosophila C-type lectins, CG33532 and CG33533 (designated as DL2 and DL3, respectively), are involved in innate immune responses. Recombinant DL2 and DL3 were expressed and purified. Both DL2 and DL3 agglutinated Gram-negative Escherichia coli in a calcium-dependent manner. Though DL2 and DL3 are predicted to be secreted proteins, they were detected on the surface of Drosophila hemocytes, and recombinant DL2 and DL3 also directly bound to hemocytes. Coating of agarose beads with recombinant DL2 and DL3 enhanced their encapsulation and melanization by Drosophila hemocytes in vitro. However, hemocyte encapsulation was blocked when the lectin-coated beads were pre-incubated with rat polyclonal antibody specific for DL2 or DL3. Our results suggest that DL2 and DL3 may act as pattern recognition receptors to mediate hemocyte encapsulation and melanization by directly recruiting hemocytes to the lectin-coated surface. [Copyright &y& Elsevier]

Phenoloxidase (PO) is a key enzyme implicated in several defense mechanisms in insects and crustaceans. It is converted from prophenoloxidase (proPO) through limited proteolysis by prophenoloxidase-activating proteinase (PAP). We previously isolated PAP-1 from integument and PAP-2 from hemolymph of the tobacco hornworm, Manduca sexta. Here, we report the purification, characterization, and regulation of PAP-3 from the hemolymph. Similar to M. sexta PAP-2, PAP-3 consists of two amino-terminal clip domains followed by a carboxyl-terminal catalytic domain, whereas PAP-1 contains only one clip domain at its amino-terminus. Purified PAP-3 cleaved proPO at Arg51 and generated a low level of PO activity. However, the enzyme efficiently activated proPO when M. sexta serine proteinase homolog-1 and -2 were present. These proteinase-like proteins associate with immulectin-2, a pattern-recognition receptor for lipopolysaccharide. M. sexta PAP-3 was inhibited by recombinant serpin-1J, which formed an SDS-stable complex with the enzyme. PAP-3 mRNA was detected at a low level in the fat body or hemocytes of naive larvae, but was elevated in insects that had been challenged with bacteria. These data, along with our previous results on PAP-1 and PAP-2, indicate that proPO activation by PAPs is a tightly regulated process. Individual PAPs could play different roles during immune responses and developmental processes. [Copyright &y& Elsevier]

Abstract An important innate immune response in Drosophila melanogaster is the production of antimicrobial peptides (AMPs). Expression of AMP genes is mediated by the Toll and immune deficiency (IMD) pathways via NF-κB transcription factors Dorsal, DIF and Relish. Dorsal and DIF act downstream of the Toll pathway, whereas Relish acts in the IMD pathway. Dorsal and DIF are held inactive in the cytoplasm by the IκB protein Cactus, while Relish contains an IκB-like inhibitory domain at the C-terminus. NF-κB factors normally form homodimers and heterodimers to regulate gene expression, but formation of heterodimers between Relish and DIF or Dorsal and the specificity and activity of the three NF-κB homodimers and heterodimers are not well understood. In this study, we compared the activity of Rel homology domains (RHDs) of Dorsal, DIF and Relish in activation of Drosophila AMP gene promoters, demonstrated that Relish-RHD (Rel-RHD) interacted with both Dorsal-RHD and DIF-RHD, Relish-N interacted with DIF and Dorsal, and overexpression of individual RHD and co-expression of any two RHDs activated the activity of AMP gene promoters to various levels, suggesting formation of homodimers and heterodimers among Dorsal, DIF and Relish. Rel-RHD homodimers were stronger activators than heterodimers of Rel-RHD with either DIF-RHD or Dorsal-RHD, while DIF-RHD-Dorsal-RHD heterodimers were stronger activators than either DIF-RHD or Dorsal-RHD homodimers in activation of AMP gene promoters. We also identified the nucleotides at the 6th and 8th positions of the 3' half-sites of the κB motifs that are important for the specificity and activity of NF-κB transcription factors. Highlights • Relish-N interacted with DIF and Dorsal, suggesting formation of NF-κB homodimers and heterodimers. • Homodimers and heterodimers of DIF, Dorsal and Relish RHDs activated antimicrobial peptide gene promoters. • Relish-RHD homodimers were stronger activators than heterodimers of Relish-RHD with DIF-RHD or Dorsal-RHD. • DIF-RHD-Dorsal-RHD heterodimers were stronger activators than DIF-RHD or Dorsal-RHD homodimers. • The nucleotides at the 6th and 8th positions of NF-κB motifs are important for specificity and activity of NF-κB factors. [ABSTRACT FROM AUTHOR]

Abstract Drosophila melanogaster possesses a sophisticated and effective immune system composed of humoral and cellular immune responses, and production of antimicrobial peptides (AMPs) is an important defense mechanism. Expression of AMPs is regulated by the Toll and IMD (immune deficiency) pathways. Production of AMPs can be systemic in the fat body or a local event in the midgut and epithelium. So far, most studies focus on systemic septic infection in adult flies and little is known about AMP gene activation after ingestion of killed bacteria. In this study, we investigated activation of AMP genes in the wild-type w 1118 , MyD88 and Imd mutant flies after ingestion of heat-killed Escherichia coli and Staphylococcus aureus. We showed that ingestion of E. coli activated most AMP genes, including drosomycin and diptericin , in the first to third instar larvae and pupae, while ingestion of S. aureus induced only some AMP genes in some larval stages or in pupae. In adult flies, ingestion of killed bacteria activated AMP genes differently in males and females. Interestingly, ingestion of killed E. coli and S. aureus in females conferred resistance to septic infection by both live pathogenic Enterococcus faecalis and Pseudomonas aeruginosa , and ingestion of E. coli in males conferred resistance to P. aeruginosa infection. Our results indicated that E. coli and S. aureus can activate both the Toll and IMD pathways, and systemic and local immune responses work together to provide Drosophila more effective protection against infection. Highlights • Feeding killed E. coli in Drosophila activated most AMP genes in larvae and pupae. • Feeding S. aureus activated only some AMP genes in some larval stages or in pupae. • Feeding killed bacteria activated AMP genes differently in adult females and males. • Feeding E. coli and S. aureus triggered both the Toll and IMD pathways. • Feeding bacteria protects flies from septic infection by E. faecalis and P. aeruginosa. [ABSTRACT FROM AUTHOR]

Abstract Wolbachia are endosymbiotic bacteria present in a wide range of invertebrates. Although their dramatic effects on host reproductive biology have been well studied, little is known about the effects of Wolbachia on the learning and memory capacity (LMC) of hosts, despite their distribution in the host nervous system, including brain. In this study, we found that Wolbachia infection significantly enhanced LMC in both Drosophila melanogaster and D. simulans. Expression of LMC-related genes was significantly increased in the head of D. melanogaster infected with the w Mel strain, and among these genes, crebA was up-regulated the most. Knockdown of crebA in Wolbachia -infected flies significantly decreased LMC, while overexpression of crebA in Wolbachia -free flies significantly enhanced the LMC of flies. More importantly, a microRNA (miRNA), dme-miR-92b , was identified to be complementary to the 3'UTR of crebA. Wolbachia infection was correlated with reduced expression of dme-miR-92b in D. melanogaster , and dme-miR-92b negatively regulated crebA through binding to its 3'UTR region. Overexpression of dme-miR-92b in Wolbachia -infected flies by microinjection of agomirs caused a significant decrease in crebA expression and LMC, while inhibition of dme-miR-92b in Wolbachia -free flies by microinjection of antagomirs resulted in a significant increase in crebA expression and LMC. These results suggest that Wolbachia may improve LMC in Drosophila by altering host gene expression through a miRNA-target pathway. Our findings help better understand the host-endosymbiont interactions and, in particular, the impact of Wolbachia on cognitive processes in invertebrate hosts. Graphical abstract Image 1 Highlights • Wolbachia infection improves the learning and memory capacity (LMC) of Drosophila. • Wolbachia infection increases the expression of crebA. • Wolbachia infection decreases the expression of dme-miR-92b. • CrebA is negatively regulated by dme-miR-92b. • Wolbachia improves LMC likely by altering crebA expression through dme-miR-92b. [ABSTRACT FROM AUTHOR]

Abstract Antimicrobial peptides (AMPs) are important effector molecules of insect humoral immunity, and expression of AMPs is mainly regulated by the Toll and immune deficiency (IMD) pathways. FoxO, a key downstream regulator of the insulin-like signaling (ILS) pathway, has been recently reported to be involved in the regulation of AMPs in Drosophila melanogaster. In the present study, we investigated AMP gene expression and the regulation pathway controlled by the starvation in the silkworm Bombyx mori. We discovered that antibacterial activity in the hemolymph of B. mori larvae was increased by starvation, and expression of AMP genes (BmCecB6 , BmAtta1 , BmLeb3 and BmDefB) as well as the ILS target genes (FoxO , InR and Brummer) were strongly activated in the fat body by starvation. Moreover, phosphorylation of Akt kinase was reduced in the Bm-12 cells after starvation, suggesting that the ILS pathway was inhibited by starvation. We then showed that more FoxO protein was present in the cytoplasm than in the nucleus of Bm-12 cells under normal conditions, but more FoxO was detected in the nucleus after cells were starved for 8 h, indicating that FoxO was activated by starvation. In summary, our results indicated that starvation can activate AMP gene expression in B. mori via the ILS/FoxO signaling pathway. Graphical abstract Image 1 Highlights • Transcription factor FoxO can be activated by starvation in Bombyx mori. • Overexpression of an active form FoxOCA can induce promoter activity of BmDefB. • RNAi of insulin receptor (InR) decreases the insulin-like signal (ILS) and activates BmDefB. • Starvation upregulates the expression of AMPs via the ILS/FoxO signaling pathway. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) is a common component of the outermost cell wall in Gram-negative bacteria. In mammals, LPS serves as an endotoxin that can be recognized by a receptor complex of TLR4 (Toll-like receptor 4) and MD-2 (myeloid differentiation-2) and subsequently induce a strong immune response to signal the release of tumor necrosis factor (TNF). In Drosophila melanogaster , no receptors for LPS have been identified, and LPS cannot activate immune responses. Here, we report a protein, BmEsr16, which contains an ML (MD-2-related lipid-recognition) domain, may function as an LPS receptor in the silkworm Bombyx mori . We showed that antibacterial activity in the hemolymph of B. mori larvae was induced by Escherichia coli , peptidoglycan (PGN) and LPS and that the expression of antimicrobial peptide genes was also induced by LPS. Furthermore, both the expression of BmEsr16 mRNA in the fat body and the expression of BmEsr16 protein in the hemolymph were induced by LPS. Recombinant BmEsr16 bound to LPS and lipid A, as well as to PGN, lipoteichoic acid, but not to laminarin or mannan. More importantly, LPS-induced immune responses in the hemolymph of B. mori larvae were blocked when the endogenous BmEsr16 protein was neutralized by polyclonal antibody specific to BmEsr16. Our results suggest that BmEsr16 may function as a key accessory protein for LPS signaling in B. mori . [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) are produced by the stimulated humoral immune system. Most mature AMPs contain less than 50 amino acid residues. Some of them are generated from proproteins upon microbial challenges. Here, we report the antimicrobial activities of a proline-rich proprotein, named SlLebocin1 (SlLeb1), from the tobacco cutworm Spodoptera litura . SlLebocin1 cDNA contains a 477-bp open reading frame (ORF). It is mainly expressed in hemocytes and the midgut in naïve larvae. The transcript level was significantly induced in hemocytes but repressed in the midgut and fat body by bacterial challenges. The proprotein contains 158 amino acids with 3 RXXR motifs that are characteristic of some Lepidopteral lebocin proproteins. Four peptides corresponding to the predicted processed fragments were synthesized chemically, and their antimicrobial activities against two Gram-negative and two Gram-positive bacterial strains were analyzed. The peptides showed differential antimicrobial activities. For Escherichia coli and Bacillus subtilis , only the C-terminal fragment (124–158) showed strong inhibitory effects. For Staphylococcus aureus , all peptides showed partial inhibitions. None of them inhibited Serratia marcescens . Bacterial morphologies were examined by the scanning electron microscopy and confocal laser scanning microscopy. The antimicrobial peptides either disrupted cellular membrane or inhibited cell division and caused elongated/enlarged morphologies. The results may provide ideas for designing novel antibiotics. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) are a family of proteins that contain characteristic modules of carbohydrate-recognition domains (CRDs) and they possess the binding activity to ligands in a calcium-dependent manner. CTLs play important roles in animal immune responses, and in insects, they are involved in opsonization, nodule formation, agglutination, encapsulation, melanization, and prophenoloxidase activation, as well as in maintaining gut microbiome homeostasis. In this review, we will summarize insect CTLs, compare the properties of insect CTLs with vertebrate CTLs, and focus mainly on the domain organization and functions of insect CTLs in innate immunity. [ABSTRACT FROM AUTHOR]

Insects rely completely on the innate immune system to sense the foreign bodies and to mount the immune responses. Germ-line encoded pattern recognition receptors play crucial roles in recognizing pathogen-associated molecular patterns. Among them, β-1,3-glucan recognition proteins (βGRPs) and gram-negative bacteria-binding proteins (GNBPs) belong to the same pattern recognition receptor family, which can recognize β-1,3-glucans. Typical insect βGRPs are comprised of a tandem carbohydrate-binding module in the N-terminal and a glucanase-like domain in the C-terminal. The former can recognize triple-helical β-1,3-glucans, whereas the latter, which normally lacks the enzymatic activity, can recruit adapter proteins to initiate the protease cascade. According to studies, insect βGRPs possess at least three types of functions. Firstly, some βGRPs cooperate with peptidoglycan recognition proteins to recognize the lysine-type peptidoglycans upstream of the Toll pathway. Secondly, some directly recognize fungal β-1,3-glucans to activate the Toll pathway and melanization. Thirdly, some form the ‘attack complexes’ with other immune effectors to promote the antifungal defenses. The current review will focus on the discovery of insect βGRPs, functions of some well-characterized members, structure-function studies and their potential application. [ABSTRACT FROM AUTHOR]

Growth-blocking peptide (GBP) is an insect cytokine that stimulates plasmatocyte adhesion, thereby playing a critical role in encapsulation reaction. It has been previously demonstrated that GBP-binding protein (GBPB) is released upon oenocytoid lysis in response to GBP and is responsible for subsequent clearance of GBP from hemolymph. However, current knowledge about GBPB is limited and the mechanism by which insects increase GBPB levels to inactivate GBP remains largely unexplored. Here, we have identified one GBP precursor ( HaGBP precursor ) gene and two GBPB (namely HaGBPB1 and HaGBPB2 ) genes from the cotton bollworm, Helicoverpa armigera . The HaGBP precursor was found to be predominantly expressed in fat body, whereas HaGBPB1 and HaGBPB2 were mainly expressed in hemocytes. Immunological analyses indicated that both HaGBPB1 and HaGBPB2 are released from hemocytes into the plasma during the wandering stage. Additionally, 20-hydroxyecdysone (20E) treatment or bead challenge could promote the release of HaGBPB1 and HaGBPB2 at least partly from oenocytoids into the plasma. Furthermore, we demonstrate that the N-terminus of HaGBPB1 is responsible for binding to HaGBP and suppresses HaGBP-induced plasmatocyte spreading and encapsulation. Overall, this study helps to enrich our understanding of the molecular mechanism underlying 20E mediated regulation of plasmatocyte adhesion and encapsulation via GBP-GBPB interaction. [ABSTRACT FROM AUTHOR]

The encapsulation reaction in invertebrates is analogous to granuloma formation in vertebrates, and this reaction is severely compromised when ecdysone signaling is blocked. However, the molecular mechanism underlying the encapsulation reaction and its regulation by ecdysone remains obscure. In our previous study, we found that the C-type lectin HaCTL3, from the cotton bollworm Helicoverpa armigera , is involved in anti-bacterial immune response, acting as a pattern recognition receptor (PRR). In the current study, we demonstrate that HaCTL3 is involved in defense against parasites and directly binds to the surface of nematodes. Our in vitro and in vivo studies indicate that HaCTL3 enhances hemocytic encapsulation and melanization, whereas H. armigera β-integrin (Haβ-integrin), located on the surface of hemocytes, participates in encapsulation. Additionally, co-immunoprecipitation experiments reveal HaCTL3 interacts with Haβ-integrin, and knockdown of Haβ-integrin leads to reduced encapsulation of HaCTL3-coated beads. These results indicate that Haβ-integrin serves as a hemocytic receptor of HaCTL3 during the encapsulation reaction. Furthermore, we demonstrate that 20-hydroxyecdysone (20E) treatment dramatically induces the expression of HaCTL3, and knockdown of the 20E receptor (EcR)/ultraspiracle (USP), abrogates this response. Overall, this study provides the first evidence of the presence of a hemocytic receptor (Haβ-integrin), that interacts with the PRR HaCTL3 to facilitate encapsulation reaction in insects and demonstrates the regulation of this process by the steroid hormone ecdysone. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) depend on the carbohydrate-recognition domain (CRD) to recognize carbohydrates by a Ca 2+ -dependent mechanism. In animals, CTLs play critical roles in pathogen recognition, activation of the complement system and signaling pathways. Immulectins (Dual-CRD CTLs) in lepidopteran are involved in recognizing pathogens. However, little is known about the immune-related functions of insect single-CRD CTLs. Here, we reported the characterization of C-type lectin-S3 ( CTL-S3 ), a single-CRD CTL from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S3 gene is 672 bp, which encodes a putative protein of 223 amino acids. CTL-S3 gene was expressed in a variety of tissues. Levels of CTL-S3 mRNA in fertilized eggs and whole larvae were elevated upon bacterial challenges. CTL-S3 was secreted to larval hemolymph. The recombinant protein (rCTL-S3) binds to bacterial cell wall components and bacteria. CTL-S3 inhibited the growth of Bacillus subtilis and caused agglutination of Staphylococcus aureus . More importantly, CTL-S3 facilitated the rapid clearance of Escherichia coli and Staphylococcus aureus from the body cavity of larvae. Taken together, our results suggested that CTL-S3 may function as an opsonin in larval hemolymph to enhance the clearance of pathogens. [ABSTRACT FROM AUTHOR]

Most studies on the contribution of the altered immune response by endoparasitoid have been restricted to the interactions between Ichneumonoidea and their hosts, while effects of parasitism by Chalcidoidea on the hosts have rarely been characterized except some wasps such as Pteromalidae. Endoparasitoid Tetrastichus brontispae Ferrière, belonging to Eulophidae (Hymenoptera), has a great potential to control some Coleopteran beetles such as Octodonta nipae , one invasive species in southern China. However, the physiological mechanism underlying the escape from the melanotic encapsulation in O. nipae pupae has not been demonstrated. In the present study, effects of parasitism on the immune function of its pupal host O. nipae were investigated. The combining results that granulocytes and plasmatocytes could phagocytize bacteria from 2 to 48 h and granulocytes, plasmatocytes and oenocytoids were prophenoloxidase/phenoloxidase positive hemocytes indicated that granulocytes, plasmatocytes and oenocytoids were the main immunocompetent hemocytes in O. nipae pupae. Parasitism by T. brontispae resulted in a significant increase in the percentage of hemocytes viability and spreading at 96 h, growing percentage of granulocytes at 24 h but no effects on the total hemocyte counts, and an enhanced phenoloxidase activity only at 12 and 72 h while a significantly longer melanization time of the hemolymph at 96 h following parasitism. These results indicate that mixtures of systemic active and local active regulation are used for T. brontispae to escape host encapsulation in O. nipae pupae. The present study contributes to the understanding of the diversity of virulence strategies used by parasitoids. [ABSTRACT FROM AUTHOR]

Galectins (S-type lectins) are an ancient family of lectins with the β-galactoside binding activity. In mammals, galectins play essential roles in many biological processes, such as development, immune homeostasis and tumor progression. However, few studies have been devoted to their functions in insects. Here, we characterized the only dual-CRD galectin in the silkworm Bombyx mori (BmGalectin-4). BmGalectin-4 cDNA possesses an open reading frame of 1089 bp, which encodes a putative galectin of 363 amino acids containing tandem carbohydrate recognition domains (CRDs). BmGalectin-4 was expressed in various tissues but the protein was most abundant in fertilized eggs. Its transcript level in fertilized eggs was upregulated upon bacterial challenge. Recombinant BmGalectin-4 purified from Escherichia coli bound to bacterial cell wall components and bacterial cells. In addition, the recombinant protein induced bacterial agglutination, but did not have antibacterial activity against selected microorganisms. Taken together, our results suggest that BmGalectin-4 may function as a pattern recognition receptor primarily in silkworm fertilized eggs. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) represent a large family of proteins that can bind carbohydrate moieties normally in a calcium-dependent manner. CTLs play important roles in mediating cell adhesion and the recognition of pathogens in the immune system. In the present study, we have identified 23 CTL genes in domestic silkworm Bombyx mori . CTL-domain proteins (CTLDPs) are classified into three groups based on the number of carbohydrate-recognition domains (CRDs) and the domain architectures. These include twelve CTL-S (Single-CRD), six immulectins (Dual-CRD) and five CTL-X (CRD with other domains). We studied their phylogenetic features, analyzed the conserved residues, predicted tertiary structures, and examined the tissue expression profile and immune inducibility. Through bioinformatics analysis, we have putatively identified ten secretory and two cytoplasmic CTL-S; four secretory and two cytoplasmic immulectins; one secretory, one cytoplasmic and three transmembrane forms of CTL-X. Most B . mori CTLDPs form monophyletic groups with orthologs from Lepidoptera, Diptera, Coleoptera and Hymenoptera species. Immulectins of B . mori and Manduca sexta evolved from common ancestor genes perhaps due to gene duplication events of CTL-S ancestor genes. Homology modeling revealed that the overall structures of B . mori CTL domains are analogous to those of humans with a variable loop region. We examined the expression profile of CTLDP genes in naïve and immune-stimulated tissues. The expression and induction of CTLDP genes were related to the tissues and microorganisms. Together, our gene identification, sequence comparison, phylogenetic analysis, homology modeling and expression analysis laid a good foundation for the further studies of B . mori CTLDPs and comparative genomics. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) are a large family of Ca 2+ -dependent carbohydrate-binding proteins recognizing various glycoconjugates and functioning primarily in immunity and cell adhesion. We have identified 34 CTLDP (for CTL-domain protein) genes in the Manduca sexta genome, which encode proteins with one to three CTL domains. CTL-S1 through S9 (S for simple) have one or three CTL domains; immulectin-1 through 19 have two CTL domains; CTL-X1 through X6 (X for complex) have one or two CTL domains along with other structural modules. Nine simple CTLs and seventeen immulectins have a signal peptide and are likely extracellular. Five complex CTLs have both an N-terminal signal peptide and a C-terminal transmembrane region, indicating that they are membrane anchored. Immulectins exist broadly in Lepidoptera and lineage-specific gene duplications have generated three clusters of fourteen genes in the M. sexta genome, thirteen of which have similar expression patterns. In contrast to the family expansion, CTL-S1∼S6, S8, and X1∼X6 have 1:1 orthologs in at least four lepidopteran/dipteran/coleopteran species, suggestive of conserved functions in a wide range of holometabolous insects. Structural modeling suggests the key residues for Ca 2+ -dependent or independent binding of certain carbohydrates by CTL domains. Promoter analysis identified putative κB motifs in eighteen of the CTL genes, which did not have a strong correlation with immune inducibility in the mRNA or protein levels. Together, the gene identification, sequence comparisons, structure modeling, phylogenetic analysis, and expression profiling establish a solid foundation for future studies of M. sexta CTL-domain proteins. [ABSTRACT FROM AUTHOR]

Significance of microRNA-mediated posttranscriptional regulation has been appreciated ever since its discovery. In the tobacco hornworm Manduca sexta , 164 conserved and 16 novel microRNAs have been identified experimentally (Zhang et al., 2012, 2014). To extend the list of microRNAs in this lepidopteran model species and further explore their possible regulatory roles, we constructed and sequenced small RNA libraries of M. sexta fat body, hemocytes and midgut, since transcriptomes of these tissues from the 5th instar larvae had been studied quite extensively. Each library represented a mixture of the same tissues from larvae that were naïve or induced by three different pathogens. From a total of 167 million reads obtained, we identified two new variants of conserved miR-281 and miR-305 and six novel microRNAs. Abundances of all microRNAs were normalized and compared to reveal their differential expression in these three tissues. Star strands of ten microRNAs were present at higher levels than the corresponding mature strands. From a list of tissue-specific transcripts, we predicted target sites in 3′-UTRs using preferentially expressed microRNA groups in each tissue and suggested possible regulatory roles of these microRNAs in energy metabolism, insecticide resistance, and some mitochondrial and immune gene expression. Examining manifold targets, microRNA regulations were suggested of multiple physiological processes. This study has enriched our knowledge of M. sexta microRNAs and how microRNAs potentially coordinate different physiological processes. [ABSTRACT FROM AUTHOR]

Highlights: [•] A Toll receptor with 2 TIR domains (HcToll-2) was identified from H. cumingii. [•] The LRR domain of HcToll-2 could bind bacteria, LPS and PGN. [•] HcToll-2 could regulate the expression of AMPs. [ABSTRACT FROM AUTHOR]

Abstract: Wolbachia are endosymbionts that infect many insect species. They can manipulate the host's reproduction to increase their own maternal transmission. Cytoplasmic incompatibility (CI) is one such manipulation, which is expressed as embryonic lethality when Wolbachia-infected males mate with uninfected females. However, matings between males and females carrying the same Wolbachia strain result in viable progeny. The molecular mechanisms of CI are currently not clear. We have previously reported that the gene Juvenile hormone-inducible protein 26 (JhI-26) exhibited the highest upregulation in the 3rd instar larval testes of Drosophila melanogaster when infected by Wolbachia. This is reminiscent of an interaction between Wolbachia and juvenile hormone (JH) pathway in flies. Considering that Jhamt gene encodes JH acid methyltransferase, a key regulatory enzyme of JH biosynthesis, and that methoprene-tolerant (Met) has been regarded as the best JH receptor candidate, we first compared the expression of Jhamt and Met between Wolbachia-infected and uninfected fly testes to investigate whether Wolbachia infection influence the JH signaling pathway. We found that the expressions of Jhamt and Met were significantly increased in the presence of Wolbachia, suggesting an interaction of Wolbachia with the JH signaling pathway. Then, we found that overexpression of JhI-26 in Wolbachia-free transgenic male flies caused paternal-effect lethality that mimics the defects associated with CI. JhI-26 overexpressing males resulted in significantly decrease in hatch rate. Surprisingly, Wolbachia-infected females could rescue the egg hatch. In addition, we showed that overexpression of JhI-26 caused upregulation of the male accessory gland protein (Acp) gene CG10433, but not vice versa. This result suggests that JhI-26 may function at the upstream of CG10433. Likewise, overexpression of CG10433 also resulted in paternal-effect lethality. Both JhI-26 and CG10433 overexpressing males resulted in nuclear division defects in the early embryos. Finally, we found that Wolbachia-infected males decreased the propensity of the mated females to remating, a phenotype also caused by both JhI-26 and CG10433 overexpressing males. Taken together, our results provide a working hypothesis whereby Wolbachia induce paternal defects in Drosophila probably by interaction with the JH pathway via JH response genes JhI-26 and CG10433. [Copyright &y& Elsevier]

Abstract: Parasitoid wasps inject venom into the host to protect their offspring against host immune responses. In our previous study, we identified a calreticulin (CRT) in Pteromalus puparum venom. In this study, we expressed the wild-type and the coiled-coil domain deletion mutant P. puparum calreticulins (PpCRTs) in Escherichia coli and prepared polyclonal antibody in rabbit against PpCRT. Western blot analysis showed that PpCRT protein was not only present in the venom but also in all the tissues tested. Real time PCR results indicated that PpCRT mRNA was highly expressed in the venom gland. The transcript level of PpCRT in the venom gland was peaked at 2 days post-eclosion, while the PpCRT protein in the venom was maintained at a constant level. Both recombinant wild-type and mutant PpCRT proteins could bind to the surface of P. puparum eggs. Recombinant PpCRT inhibited hemocyte spreading and cellular encapsulation of the host Pieris rapae in vitro, and the coiled-coil domain is important for the inhibitory function of PpCRT. Immunocytochemistry results showed that PpCRT entered P. rapae hemocytes, and the coiled-coil domain played a role in this process. After injection of recombinant PpCRT into P. rapae pupae, real time PCR results showed that PpCRT inhibited transcript levels of host encapsulation-related genes, including calreticulin and scavenger receptor genes. In conclusion, our results suggest that P. puparum venom protects its offspring against host cellular immune responses via its functional component PpCRT to inhibit the expression of host cellular response-related genes. [Copyright &y& Elsevier]

Highlights: [•] Litopenaeus vannamei Toll-interacting protein (LvTollip) were cloned and characterized. [•] The expression of LvTollip was altered in response to microbial infection. [•] In Drosophila S2 cells, LvTollip localized to the membrane and cytoplasm. [•] In Drosophila S2 cells, LvTollip significantly inhibited the promoter activities of Penaeidin4. [•] Silencing of LvTollip using dsRNA-mediated RNA interference increased the expression levels of penaeidin-4 in the gill. [Copyright &y& Elsevier]