LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes,fibH, fibL and fhx,in the silk gland of the silkworm,Bombyx mori

In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors.     

Carbonic anhydrase generates a pH gradient in Bombyx mori silk glands

Silk is a protein of interest to both biological and industrial sciences. The silkworm, Bombyx mori, forms this protein into strong threads starting from soluble silk proteins using a number of biochemical and physical cues to allow the transition from liquid to fibrous silk. A pH gradient has been measured along the gland, but the methodology employed was not able to precisely determine the pH at specific regions of interest in the silk gland. Furthermore, the physiological mechanisms responsible for the generation of this pH gradient are unknown.In this study, concentric ion selective microelectrodes were used to determine the luminal pH of B. mori silk glands. A gradient from pH 8.2 to 7.2 was measured in the posterior silk gland, with a pH 7 throughout the middle silk gland, and a gradient from pH 6.8 to 6.2 in the beginning of the anterior silk gland where silk processing into fibers occurs. The small diameter of the most anterior region of the anterior silk gland prevented microelectrode access in this region. Using a histochemical method, the presence of active carbonic anhydrase was identified in the funnel and anterior silk gland of fifth instar larvae. The observed pH gradient collapsed upon addition of the carbonic anhydrase inhibitor methazolamide, confirming an essential role for this enzyme in pH regulation in the B. mori silk gland. Plastic embedding of whole silk glands allowed clear visualization of the morphology, including the identification of four distinct epithelial cell types in the gland and allowed correlations between silk gland morphology and silk stages of assembly related to the pH gradient.B. mori silk glands have four different epithelial cell types, one of which produces carbonic anhydrase. Carbonic anhydrase is necessary for the mechanism that generates an intraluminal pH gradient, which likely regulates the assembly of silk proteins and then the formation of fibers from soluble silk proteins. These new insights into native silk formation may lead to a more efficient production of artificial or regenerated silkworm silk fibers.     

GC/MS-based metabolomic studies reveal key roles of glycine in regulating silk synthesis in silkworm,Bombyx mori

Metabolic profiling of silkworm, especially the factors that affect silk synthesis at the metabolic level, is little known. Herein, metabolomic method based on gas chromatography-mass spectrometry was applied to identify key metabolic changes in silk synthesis deficient silkworms. Forty-six differential metabolites were identified in Nd group with the defect of silk synthesis. Significant changes in the levels of glycine and uric acid (up-regulation), carbohydrates and free fatty acids (down-regulation) were observed. The further metabolomics of silk synthesis deficient silkworms by decreasing silk proteins synthesis using knocking out fibroin heavy chain gene or extirpating silk glands operation showed that the changes of the metabolites were almost consistent with those of the Nd group. Furthermore, the increased silk yields by supplying more glycine or its related metabolite confirmed that glycine is a key metabolite to regulate silk synthesis. These findings provide important insights into the regulation between metabolic profiling and silk synthesis.     

Sternal gland structures in males of bean flower thrips,Megalurothrips sjostedti,and Poinsettia thrips,Echinothrips americanus,in comparison with those of western flower thrips,Frankliniella occidentalis (Thysanoptera: Thripidae)

Sternal pores are important features for identification of male thrips, especially within the subfamily Thripinae. They vary in shape, size and distribution even between species of one genus. Their functional role is speculated to be that of sex- and/or aggregation pheromone production. Yet, sexual aggregations are not reported in Echinothrips americanus, known to have sternal pores, while we observed aggregations in Megalurothrips sjostedti, previously reported to lack them.We examined the sternal glands and pores of the thripine species E. americanus and M. sjostedti males, in comparison with those of Frankliniella occidentalis using light microscopy, as well as scanning and transmission electron microscopy. Pore plates of F. occidentalis were ellipsoid and medial on sternites III–VII, while in E. americanus they were distributed as multiple micro pore plates on sternites III–VIII. In M. sjostedti they appeared as an extremely small pore in front of the posterior margin of each of sternites IV–VII. Pore plate and pore plate area were distributed similarly on sternites III–VII in F. occidentalis. However, in E. americanus the total pore plate area increased significantly from sternites III to VIII. Ultrastructure of cells associated with sternal glands showed typical characteristics of gland cells that differ in size, shape and number. The function of sternal glands is further discussed on the basis of morphological comparisons with other thrips species.     

TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi

Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields.     

CYP341B14: A cytochrome P450 involved in the specific epoxidation of pheromone precursors in the fall webworm Hyphantria cunea

Two of the four sex pheromone components in the fall webworm Hyphantria cunea (Lepidoptera: Arctiidae), cis-9,10-epoxy-(3Z,6Z)-3,6-henicosadiene and cis-9,10-epoxy-(3Z,6Z)-1,3,6-henicosatriene, possess an epoxy ring within their molecules. These compounds have been suggested to be biosynthesized from dietary linolenic acid via the following enzymatic reactions; chain elongation, terminal desaturation (in the case of the latter component), decarboxylation, and epoxidation. The last step of this biosynthesis, epoxidation, is known to occur specifically in the sex pheromone gland of females. We identified the enzyme involved in the epoxidation of pheromone precursors by focusing on cytochromes P450, which are known to catalyze the oxidation of various compounds. Three P450-like sequences (Hc_epo1, Hc_epo2, and Hc_epo3) were identified in the cDNA library prepared from the sex pheromone gland of H. cunea. Among these clones, only Hc_epo1 was specifically expressed in the pheromone gland. The full-length sequence of Hc_epo1 contained an ORF of 1527 bp, which encoded a protein of 509 amino acids with a predicted molecular weight of 57.9 kDa. The deduced Hc_epo1 amino acid sequence possessed the characteristics of P450. A phylogenetic analysis of the sequence indicated that Hc_epo1 belonged to the CYP341B clade in the CYP341 family. Therefore, it was named CYP341B14. A subsequent functional assay using Sf-9 cells transiently expressing CYP341B14 demonstrated that this P450 protein was able to specifically epoxidize a (Z)-double bond at the 9th position in the pheromone precursor, (3Z,6Z,9Z)-3,6,9-henicosatriene.     

The silkworm glutathione S-transferase gene noppera-bo is required for ecdysteroid biosynthesis and larval development

Insect molting and metamorphosis are tightly controlled by ecdysteroids, which are important steroid hormones that are synthesized from dietary sterols in the prothoracic gland. One of the ecdysteroidogenic genes in the fruit fly Drosophila melanogaster is noppera-bo (nobo), also known as GSTe14, which encodes a member of the epsilon class of glutathione S-transferases. In D. melanogaster, nobo plays a crucial role in utilizing cholesterol via regulating its transport and/or metabolism in the prothoracic gland. However, it is still not known whether the orthologs of nobo from other insects are also involved in ecdysteroid biosynthesis via cholesterol transport and/or metabolism in the prothoracic gland. Here we report genetic evidence showing that the silkworm Bombyx mori ortholog of nobo (nobo-Bm; GSTe7) is essential for silkworm development. nobo-Bm is predominantly expressed in the prothoracic gland. To assess the functional importance of nobo-Bm, we generated a B. mori genetic mutant of nobo-Bm using TALEN-mediated genome editing. We show that loss of nobo-Bm function causes larval arrest and a glossy cuticle phenotype, which are rescued by the application of 20-hydroxyecdysone. Moreover, the prothoracic gland cells isolated from the nobo-Bm mutant exhibit an abnormal accumulation of 7-dehydrocholesterol, a cholesterol metabolite. These results suggest that the nobo family of glutathione S-transferases is essential for development and for the regulation of sterol utilization in the prothoracic gland in not only the Diptera but also the Lepidoptera. On the other hand, loss of nobo function mutants of D. melanogaster and B. mori abnormally accumulates different sterols, implying that the sterol utilization in the PG is somewhat different between these two insect species.     

Manduca sexta hemolymph protease-1, activated by an unconventional non-proteolytic mechanism,mediates immune responses

Tissue damage or pathogen invasion triggers the auto-proteolysis of an initiating serine protease (SP), rapidly leading to sequential cleavage activation of other cascade members to set off innate immune responses in insects. Recently, we presented evidence that Manduca sexta hemolymph protease-1 zymogen (proHP1) is a member of the SP system in this species, and may activate proHP6. HP6 stimulates melanization and induces antimicrobial peptide synthesis. Here we report that proHP1 adopts an active conformation (*) to carry out its function, without a requirement for proteolytic activation. Affinity chromatography using HP1 antibodies isolated from induced hemolymph the 48 kDa proHP1 and also a 90 kDa band (detected by SDS-PAGE under reducing conditions) containing proHP1 and several serpins, as revealed by mass spectrometric analysis. Identification of tryptic peptides from these 90 kDa complexes included peptides from the amino-terminal regulatory part of proHP1, indicating that proHP1* was not cleaved, and that it had formed a complex with the serpins. As suicide inhibitors, serpins form SDS-stable, acyl-complexes when they are attacked by active proteases, indicating that proHP1* was catalytically active. Detection of M. sexta serpin-1, 4, 9, 13 and smaller amounts of serpin-3, 5, 6 in the complexes suggests that it is regulated by multiple serpins in hemolymph. We produced site-directed mutants of proHP1b for cleavage by bovine blood coagulation factor Xa at the designed proteolytic activation site, to generate a form of proHP1b that could be activated by Factor Xa. However, proHP1b cut by Factor Xa failed to activate proHP6 and, via HP6, proHP8 or proPAP1. This negative result is consistent with the suggestion that proHP1* is a physiological mediator of immune responses. Further research is needed to investigate the conformational change that results in conversion of proHP1 to active proHP1*.     

Cadherin AdCad1 in Alphitobius diaperinus larvae is a receptor of Cry3Bb toxin from Bacillus thuringiensis

Bacillus thuringiensis (Bt) Cry proteins are used as components of biopesticides or expressed in transgenic crops to control diverse insect pests worldwide. These Cry toxins bind to receptors on the midgut brush border membrane and kill enterocytes culminating in larval mortality. Cadherin proteins have been identified as Cry toxin receptors in diverse lepidopteran, coleopteran, and dipteran species. In the present work we report a 185 kDa cadherin (AdCad1) from larvae of the lesser mealworm (Alphitobius diaperinus) larvae as the first identified receptor for Cry3Bb toxin. The AdCad1 protein contains typical structural components for Cry toxin receptor cadherins, including nine cadherin repeats (CR9), a membrane-proximal extracellular domain (MPED) and a cytosolic region. Peptides corresponding to the CR9 and MPED regions bound Cry3Bb toxin with high affinities (23 nM and 40 nM) and significantly synergized Cry3Bb toxicity against A. diperinus larvae. Silencing of AdCad1 expression through RNA interference resulted in highly reduced susceptibility to Cry3Bb in A. diperinus larvae. The CR9 peptide fed with toxin to RNAi-treated larvae restored Cry3Bb toxicity. These results are evidences that AdCad1 is a functional receptor of Cry3Bb toxin and that exogenously fed CR9 peptide can overcome the effect of reduced AdCad1expression on Cry3Bb toxicity to larvae.     

Mechanisms of nodule-specific melanization in the hemocoel of the silkworm,Bombyx mori

In the insect immune system, nodules are known to be a product of the cellular response against microorganisms and may be a preferential target for melanization. However, the mechanism of nodule-preferential melanization remains to be explored. In this study, we identified several mechanisms of nodule-preferential melanization by analyzing congregation and the activation of several factors involved in the prophenoloxidase (proPO)-activating system in the silkworm, Bombyx mori. Microorganism-binding assays revealed that B. mori larval plasma have an effective invading microorganism-surveillance network consisting of at least six pattern-recognition receptors (PRRs). We also found that a hemolymph serine proteinase, BmHP14, can bind to Saccharomyces cerevisiae. Pull-down assays showed that PRR C-type lectins form protein complexes with serine proteinase homologs, BmSPH1 and BmSPH2, which leads to the activated forms of BmSPH1 and BmSPH2 being gathered on microorganisms and trapped in nodules. Immunostaining analysis revealed that most factors in the proPO-activating system and some factors in the triggering system for antimicrobial peptide production exist in the granules of hemocytes which can gather in nodules. Western blot analysis showed that factors in the proPO-activating system are congregated in formed nodules by their concentration in plasma and aggregating hemocytes.     

Analyses of α-amylase and α-glucosidase in the malaria vector mosquito,Anopheles gambiae,as receptors of Cry11Ba toxin of Bacillus thuringiensis subsp. jegathesan

Bacillus thuringiensis subsp. jegathesan produces Cry11Ba crystal protein with high toxicity to mosquito larvae. The Cry11Ba toxicity is dependent on its receptors on mosquito larval midgut epithelial cells. Previously, a cadherin-like protein (AgCad2), aminopeptidase (AgAPN2) and alkaline phosphatase (AgALP1) were reported to be involved in regulation of Cry11Ba toxicity on Anopheles gambiae larvae. Here, the cDNAs encoding α-amylase (AgAmy1) and α-glucosidase (Agm3) were cloned from A. gambiae larva midgut. Both are glycophosphatidylinositol (GPI) anchored proteins on brush border membranes (BBMV). Immunohistochemistry revealed their localization on different regions of the larval midgut. AgAmy1 and Agm3 bound Cry11Ba with high affinity, 37.6 nM and 21.1 nM respectively. Cry11Ba toxicity against A. gambiae larvae was neutralized by both AgAmy1 and Agm3. The results provide evidence that both AgAmy1 and Agm3 function as receptors of Cry11Ba in A. gambiae.     

Midgut aminopeptidase N isoforms from Ostrinia nubilalis: Activity characterization and differential binding to Cry1Ab and Cry1Fa proteins from Bacillus thuringiensis

Aminopeptidase N (APN) isoforms from Lepidoptera are known for their involvement in the mode of action of insecticidal Cry proteins from Bacillus thuringiensis. These enzymes belong to a protein family with at least eight different members that are expressed simultaneously in the midgut of lepidopteran larvae. Here, we focus on the characterization of the APNs from Ostrinia nubilalis (OnAPNs) to identify potential Cry receptors. We expressed OnAPNs in insect cells using a baculovirus system and analyzed their enzymatic activity by probing substrate specificity and inhibitor susceptibility. The interaction with Cry1Ab and Cry1Fa proteins (both found in transgenic insect-resistant maize) was evaluated by ligand blot assays and immunocytochemistry. Ligand blots of brush border membrane proteins showed that both Cry proteins bound mainly to a 150 kDa-band, in which OnAPNs were greatly represented. Binding analysis of Cry proteins to the cell-expressed OnAPNs showed that OnAPN1 interacted with both Cry1Ab and Cry1Fa, whereas OnAPN3a and OnAPN8 only bound to Cry1Fa. Two isoforms, OnAPN2 and OnAPN3b, did not interact with any of these two proteins. This work provides the first evidence of a differential role of OnAPN isoforms in the mode of action of Cry proteins in O. nubilalis.     

Pirfenidone inhibits cryoablation induced local macrophage infiltration along with its associated TGFb1 expression and serum cytokine level in a mouse model

PurposeTo investigate the effects of pirfenidone (PFD) on post-cryoablation inflammation in a mouse model.Materials and methodsIn this IACUC-approved study, eighty Balb/c mice were randomly divided into four groups (20/group): sham + vehicle, sham + PFD, cryoablation + vehicle, and cryoablation + PFD. For cryoablation groups, a 20% freeze rate cryoablation (20 s to less than −100 °C) was used to ablate normal muscle in the right flank. For sham groups, the cryoprobe was advanced into the flank and maintained for 20 s without ablation. PFD or vehicle solution was intraperitoneally injected (5 mg/kg) at days 0, 1, 2, 3, and then every other day until day 13 after cryoablation. Mice were euthanized at days 1, 3, 7, and 14. Blood samples were used for serum IL-6, IL-10, and TGFβ1 analysis using electrochemiluminescence and ELISA assays, respectively. Immunohistochemistry-stained ablated tissues were used to analyze macrophage infiltration and local TGFβ1 expression in the border region surrounding the cryoablation-induced coagulation zone.ResultsCryoablation induced macrophage infiltration and increased TGFβ1 expression in the border of the necrotic zone, and high levels of serum IL-6, peaking at days 7 (70.5 ± 8.46/HPF), 14 (228 ± 18.36/HPF), and 7 (298.67 ± 92.63), respectively. Animals receiving PFD showed reduced macrophage infiltration (35.5 ± 16.93/HPF at day 7, p < 0.01) and cytokine levels (60.2 ± 7.6/HPF at day 14, p < 0.01). PFD also significantly reduced serum IL-6 levels (p < 0.001 vs. all non-PFD groups).ConclusionsPFD mitigates cryoablation induced muscle tissue macrophage infiltration, increased IL-6 levels, and local TGFβ1 expression in a small animal model.     

Immunological response induced by cryoablation against murine H22 hepatoma cell line in vivo

ObjectivesTo describe immunological consequences induced by cryoablation against H22 cells in vivo.MethodsAdult BALB/c mice underwent subcutaneous implantation of H22 cells. All of them were assigned into three groups randomly: group A (false surgery), group B (cryoablation) and group C (cryoablation plus Freund's adjuvant). Animals were sacrificed 1, 2 and 3 weeks after treatment. Serum IFN-γ and IL-4, Th1/Th2 in spleens and cytotoxicity were detected.ResultsCompared with that of group A, (1) INF-γ of group B was higher, but IL-4 was lower; cryoablation plus Freund's adjuvant enhanced these effects. (2) Th1/Th2 rose significantly in both group B and group C. (3) Strong cytolytic activity against H22 cells of group B and group C was found on day 7, 14 and 21.ConclusionsOur study showed a marked shift toward Th1 and IFN-γ expression after cryoablation, with an immuno-stimulatory effect against murine H22 hepatoma Cell.