From phyllosphere to insect cuticles: silkworms gather antifungal bacteria from mulberry leaves to battle fungal parasite attacks.

BACKGROUND: Bacterial transfers from plants to insect herbivore guts have been well investigated. However, bacterial exchanges between plant phyllospheres and insect cuticles remain unclear, as does their related biological function. RESULTS: Here, we report that the cuticular bacterial loads of silkworm larvae quickly increased after molting and feeding on the white mulberry (Morus alba) leaves. The isolation and examination of silkworm cuticular bacteria identified one bacterium Mammaliicoccus sciuri that could completely inhibit the spore germination of fungal entomopathogens Metarhizium robertsii and Beauveria bassiana. Interestingly, Ma. sciuri was evident originally from mulberry leaves, which could produce a secreted chitinolytic lysozyme (termed Msp1) to damage fungal cell walls. In consistency, the deletion of Msp1 substantially impaired bacterial antifungal activity. Pretreating silkworm larvae with Ma. sciuri cells followed by fungal topical infections revealed that this bacterium could help defend silkworms against fungal infections. Unsurprisingly, the protective efficacy of ΔMsp1 was considerably reduced when compared with that of wild-type bacterium. Administration of bacterium-treated diets had no negative effect on silkworm development; instead, bacterial supplementation could protect the artificial diet from Aspergillus contamination. CONCLUSIONS: The results of this study evidence that the cross-kingdom transfer of bacteria from plant phyllospheres to insect herbivore cuticles can help protect insects against fungal parasite attacks. Video Abstract.

The Mulberry SPL Gene Family and the Response of MnSPL7 to Silkworm Herbivory through Activating the Transcription of MnTT2L2 in the Catechin Biosynthesis Pathway.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, as unique plant transcription factors, play important roles in plant developmental regulation and stress response adaptation. Although mulberry is a commercially valuable tree species, there have been few systematic studies on SPL genes. In this work, we identified 15 full-length SPL genes in the mulberry genome, which were distributed on 4 Morus notabilis chromosomes. Phylogenetic analysis clustered the SPL genes from five plants (Malus × domestica Borkh, Populus trichocarpa, M. notabilis, Arabidopsis thaliana, and Oryza sativa) into five groups. Two zinc fingers (Zn1 and Zn2) were found in the conserved SBP domain in all of the MnSPLs. Comparative analyses of gene structures and conserved motifs revealed the conservation of MnSPLs within a group, whereas there were significant structure differences among groups. Gene quantitative analysis showed that the expression of MnSPLs had tissue specificity, and MnSPLs had much higher expression levels in older mulberry leaves. Furthermore, transcriptome data showed that the expression levels of MnSPL7 and MnSPL14 were significantly increased under silkworm herbivory. Molecular experiments revealed that MnSPL7 responded to herbivory treatment through promoting the transcription of MnTT2L2 and further upregulating the expression levels of catechin synthesis genes (F3'H, DFR, and LAR).

Transcriptome analysis of resistant and susceptible mulberry responses to Meloidogyne enterolobii infection.

BACKGROUND: Mulberry (Morus alba L.) is an important sericulture crop; however, root-knot nematode infection seriously limits its production. Understanding the mechanism of interaction between mulberry and nematode is important for control of infection. RESULTS: Using sequencing and de novo transcriptome assembly, we identified 55,894 unigenes from root samples of resistant and susceptible mulberry cultivars at different stages after infection with the nematode Meloidogyne enterolobii; 33,987 of these were annotated in the Nr, SWISS-PROT, KEGG, and KOG databases. Gene ontology and pathway enrichment analyses of differentially expressed genes (DEGs) revealed key genes involved in hormone metabolic processes, plant hormone signal transduction, flavonoid biosynthesis, phenylpropanoid biosynthesis, and peroxisomal and photosynthetic pathways. Analysis of key trends in co-expression networks indicated that expression of unigenes 0,015,083, 0,073,272, 0,004,006, and 0,000,628 was positively correlated with resistance to M. enterolobii. Unigene 0015083 encodes tabersonine 16-O-methyltransferase (16OMT), which is involved in alkaloid biosynthesis. Unigene 0073272 encodes a transcription factor contributing to nitric oxide accumulation during plant immune responses. Unigenes 0,004,006 and 0,000,628 encode ERF and MYB transcription factors, respectively, involved in plant hormone signaling. We verified the accuracy of transcriptome sequencing results by RT-qPCR of 21 DEGs. CONCLUSIONS: The results of this study increase our understanding of the resistance mechanisms and candidate genes involved in mulberry-M. enterolobii interaction. Thus, our data will contribute to the development of effective control measures against this pathogen.

Morus alba: Host reaction for Meloidogyne javanica, biological nematicides assessment and study of these relationships with yield and quality of leaves, cocoon and health of the silkworm.

Root-knot nematodes cause damage to several crops and the importance of each species can vary according with the crop and the agricultural region. In Brazil, Meloidogyne javanica is one of the most important nematode species parasitizing mulberry. To define management strategies, it is important to know if the crop species is damaged by the parasitism of the nematode and the best choices for control, as the use of nematicides. Biological nematicides have been extensively used in Brazil, but no information regarding its efficiency to control M. javanica in mulberry is available. Besides, it is not known if biological nematicides could improve the quality of leaves or if they alter the nutrient composition of leaves, which could interfere in the development of the silkworms that are feed with these leaves or in the quality of the silk produced. With the aim to address these questions, we propose a study that will start in the phenotyping of the main Brazilian mulberry cultivars to Meloidogyne species, passing through the test of efficiency of biological nematicides in the control of M. javanica in mulberry cultivar Miura, evaluation of the amount and quality of leaves produced and, using these leaves to feed silkworms, in the analyzes of the impact of these diet in the health of silkworms, and in the production and quality of the silk.

Analysis of the Glyphodes pyloalis larvae immune transcriptome in response to parasitization by its endoparasitoid, Aulacococentrum confusum.

The interaction between a host and its parasitoid is one of the most fascinating relationships of insects. Immune-related genes play crucial roles in this association. Nevertheless, until now, identification of these genes on a large scale has not received much attention. To gain insight into the parasitic effects of the endoparasitoid Aulacocentrum confusum (Hymenoptera: Braconidae) on Glyphodes pyloalis (Lepidoptera: Pyralidae) larva, which is a destructive pest of mulberry (Morus alba L.) trees in China, we presented a transcriptome dataset for uncovering immune-related genes in parasitized G. pyloalis larvae. In total, 91,118,138 and 92,778,814 clean reads were obtained from parasitized and healthy host larvae, respectively, and de novo assembly generated 57,122 unigenes. The transcriptional profile of G. pyloalis larvae was remarkably influenced by parasitism. A total of 3259 differentially expressed genes (DEGs) were identified in parasitized and nonparasitized G. pyloalis larvae and 55 genes related to immune response were screened from these DEGs. Among the 55 DEGs, 37 genes were significantly upregulated, and 18 genes were downregulated. qRT-PCR validated the sequencing results and revealed that the expression levels of selected immune-related genes depended on the parasitization and duration after parasitization. Knocking down the C type lectin gene (CTL) changed the expression of serine proteinase, serine protease inhibitor, antimicrobial peptide, prophenoloxidase activating enzymes and peroxiredoxin in G. pyloalis larvae, suggesting CTL can modulate the immune response after parasitization by A. confusum females. The present study provides a foundation for revealing the molecular mechanisms of immune response in G. pyloalis larvae when they are parasitized by A. confusum and promotes the development of novel biological control practices for G. pyloalis.

A determining factor for insect feeding preference in the silkworm, Bombyx mori.

Feeding preference is critical for insect adaptation and survival. However, little is known regarding the determination of insect feeding preference, and the genetic basis is poorly understood. As a model lepidopteran insect with economic importance, the domesticated silkworm, Bombyx mori, is a well-known monophagous insect that predominantly feeds on fresh mulberry leaves. This species-specific feeding preference provides an excellent model for investigation of host-plant selection of insects, although the molecular mechanism underlying this phenomenon remains unknown. Here, we describe the gene GR66, which encodes a putative bitter gustatory receptor (GR) that is responsible for the mulberry-specific feeding preference of B. mori. With the aid of a transposon-based, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system, the GR66 locus was genetically mutated, and homozygous mutant silkworm strains with truncated gustatory receptor 66 (GR66) proteins were established. GR66 mutant larvae acquired new feeding activity, exhibiting the ability to feed on a number of plant species in addition to mulberry leaves, including fresh fruits and grain seeds that are not normally consumed by wild-type (WT) silkworms. Furthermore, a feeding choice assay revealed that the mutant larvae lost their specificity for mulberry. Overall, our findings provide the first genetic and phenotypic evidences that a single bitter GR is a major factor affecting the insect feeding preference.

Silkworms suppress the release of green leaf volatiles by mulberry leaves with an enzyme from their spinnerets.

In response to herbivory, plants emit a blend of volatile organic compounds that includes green leaf volatiles (GLVs) and terpenoids. These volatiles are known to attract natural enemies of herbivores and are therefore considered to function as an indirect defense. Selection should favor herbivores that are able to suppress these volatile emissions, and thereby make themselves less conspicuous to natural enemies. We tested this possibility for silkworms, which were observed to leave secretions from their spinnerets while feeding on mulberry leaves. When we ablated the spinnerets of silkworms, no secretions were observed. Leaves infested by intact silkworms released smaller amounts of GLVs than leaves infested by ablated silkworms, indicating that the spinneret secretion suppressed GLV production. This difference in GLV emissions was also reflected in the behavioral response of Zenillia dolosa (Tachinidae), a parasitoid fly of silkworms. The flies laid fewer eggs when exposed to the volatiles from intact silkworm-infested leaves than when exposed to the volatiles from ablated silkworm-infested leaves. We identified a novel enzyme in the secretion from the spinneret that is responsible for the GLV suppression. The enzyme converted 13(S)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid, an intermediate in the biosynthetic pathway of GLVs, into its keto-derivative in a stereospecific manner. Taken together, this study shows that silkworms are able to feed on mulberry in a stealthy manner by suppressing GLV production with an enzyme in secretions of their spinnerets, which might be a countermeasure against induced indirect defense by mulberry plants.

Potential of Visible and Near-Infrared Hyperspectral Imaging for Detection of Diaphania pyloalis Larvae and Damage on Mulberry Leaves.

Mulberry trees are an important crop for sericulture. Pests can affect the yield and quality of mulberry leaves. This study aims to develop a hyperspectral imaging system in visible and near-infrared (NIR) region (400⁻1700 nm) for the rapid identification of Diaphania pyloalis larvae and its damage. The extracted spectra of five region of interests (ROI), namely leaf vein, healthy mesophyll, slight damage, serious damage, and Diaphania pyloalis larva at 400⁻1000 nm (visible range) and 900⁻1700 nm (NIR range), were used to establish a partial least squares discriminant analysis (PLS-DA) and least-squares support vector machines (LS-SVM) models. Successive projections algorithm (SPA), uninformation variable elimination (UVE), UVE-SPA, and competitive adaptive reweighted sampling were used for variable selection. The best models in distinguishing between leaf vein, healthy mesophyll, slight damage and serious damage, leaf vein, healthy mesophyll, and larva, slight damage, serious damage, and larva were all the SPA-LS-SVM models, based on the NIR range data, and their correct rate of prediction (CRP) were all 100.00%. The best model for the identification of all five ROIs was the UVE-SPA-LS-SVM model, based on visible range data, which had the CRP value of 97.30%. In summary, visible and near infrared hyperspectral imaging could distinguish Diaphania pyloalis larvae and their damage from leaf vein and healthy mesophyll in a rapid and non-destructive way.

Expression plasticity and evolutionary changes extensively shape the sugar-mimic alkaloid adaptation of nondigestive glucosidase in lepidopteran mulberry-specialist insects.

During the co-evolutionary arms race between plants and herbivores, insects evolved systematic adaptive plasticity to minimize the chemical defence effects of their host plants. Previous studies mainly focused on the expressional plasticity of enzymes in detoxification and digestion. However, the expressional response and adaptive evolution of other fundamental regulators against host phytochemicals are largely unknown. Glucosidase II (GII), which is composed of a catalytic GIIα subunit and a regulatory GIIß subunit, is an evolutionarily conserved enzyme that regulates glycoprotein folding. In this study, we found that GIIα expression of the mulberry-specialist insect was significantly induced by mulberry leaf extract, 1-deoxynojirimycin (1-DNJ), whereas GIIß transcripts were not significantly changed. Moreover, positive selection was detected in GIIα when the mulberry-specialist insects diverged from the lepidopteran order, whereas GIIß was mainly subjected to purifying selection, thus indicating an asymmetrically selective pressure of GII subunits. In addition, positively selected sites were enriched in the GIIα of mulberry-specialist insects and located around the 1-DNJ-binding sites and in the C-terminal region, which could result in conformational changes that affect catalytic activity and substrate-binding efficiency. These results show that expression plasticity and evolutionary changes extensively shape sugar-mimic alkaloids adaptation of nondigestive glucosidase in lepidopteran mulberry-specialist insects. Our study provides novel insights into a deep understanding of the sequestration and adaptation of phytophagous specialists to host defensive compounds.

Proteomics Provides Insight into the Interaction between Mulberry and Silkworm.

Mulberry leaves have been selected as a food source for the silkworm (Bombyx mori) for over 5000 years. However, the interaction mechanisms of mulberry-silkworm remain largely unknown. We explore the interaction between mulberry and silkworm at the protein level. Total proteins were extracted from mulberry leaves and silkworm feces on day 5 of the fifth larval instar and analyzed on shotgun liquid chromatography-tandem mass spectrometry, respectively. In total, 2076 and 210 foliar proteins were identified from mulberry leaves and silkworm feces, respectively. These proteins were classified into four categories according to their subcellular location: chloroplast proteins, mitochondrial proteins, secretory-pathway proteins, and proteins of other locations. Chloroplast proteins accounted for 68.3% in mulberry leaves but only 23.2% in the feces. In contrast, secretory-pathway proteins had low abundance in mulberry leaves (7.3%) but were greatly enriched to the largest component in the feces (60.1%). Most of the foliar secretory-pathway proteins in the feces were found to be resistant to silkworm feeding by becoming involved in primary metabolite, proteinase inhibition, cell-wall remodeling, redox regulation, and pathogen-resistant processes. On the contrary, only six defensive proteins were identified in the fecal chloroplast proteins including two key proteins responsible for synthesizing jasmonic acid, although chloroplast proteins were the second largest component in the feces. Collectively, the comparative proteomics analyses indicate that mulberry leaves not only provide amino acids to the silkworm but also display defense against silkworm feeding, although the silkworm grows very well by feeding on mulberry leaves, which provides new insights into the interactions between host-plant and insect herbivores.

Complete mitochondrial genome of the mulberry white caterpillar Rondotia menciana (Lepidoptera: Bombycidae).

The mulberry white caterpillar, Rondotia menciana, belongs to the lepidopteran family Bombycidae, in which the domestic silkworm, Bombyx mori is included. In this study, we describe the complete mitochondrial genome of R. menciana in terms of general genomic features and characteristic features found in the A+T-rich region. The 15,364 bp long genome consisted of a typical set of genes (13 protein-coding genes [PCGs], 2 rRNA genes, and 22 tRNA genes) and 1 major non-coding A+T-rich region, with the typical arrangement found in Lepidoptera. Twelve of the 13 PCGs started with typical ATN codons, except for the COI, which began with CGA and twelve of 13 PCGs had complete stop codons, except for the COII, which ended with a single T. The 360 bp long A+T-rich region harbored the conserved sequence blocks typically found in lepidopteran insects. Additionally, the A+T-rich region of R. menciana contained one tRNA(Met)-like structure, which had a proper anticodon and secondary structure.

Overexpression of host plant urease in transgenic silkworms.

Bombyx mori and mulberry constitute a model of insect-host plant interactions. Urease hydrolyzes urea to ammonia and is important for the nitrogen metabolism of silkworms because ammonia is assimilated into silk protein. Silkworms do not synthesize urease and acquire it from mulberry leaves. We synthesized the artificial DNA sequence ureas using the codon bias of B. mori to encode the signal peptide and mulberry urease protein. A transgenic vector that overexpresses ure-as under control of the silkworm midgut-specific P2 promoter was constructed. Transgenic silkworms were created via embryo microinjection. RT-PCR results showed that urease was expressed during the larval stage and qPCR revealed the expression only in the midgut of transgenic lines. Urea concentration in the midgut and hemolymph of transgenic silkworms was significantly lower than in a nontransgenic line when silkworms were fed an artificial diet. Analysis of the daily body weight and food conversion efficiency of the fourth and fifth instar larvae and economic characteristics indicated no differences between transgenic silkworms and the nontransgenic line. These results suggested that overexpression of host plant urease promoted nitrogen metabolism in silkworms.

Flavonoids from the cocoon of Rondotia menciana.

Two flavonol glycosides along with four known flavonoids were isolated from the cocoon of the mulberry white caterpillar, Rondotia menciana (Lepidoptera: Bombycidae: Bombycinae), a closely related species of the domesticated silkworm Bombyx mori, both of which feed on leaves of mulberry (Morus alba). The two glycosides were characterized as quercetin 3-O-ß-d-galactopyranosyl-(1→3)-ß-d-galactopyranoside and kaempferol 3-O-ß-d-galactopyranosyl-(1→3)-ß-d-galactopyranoside, based on spectroscopic data and chemical evidence. The flavonol galactosides found in the cocoon were not present in the host plant, nor in the cocoon of the silkworm, B. mori. Notably, flavonol glucosides, which are the main constituents of cocoon flavonoids in B. mori mori, were not found in the R. menciana cocoon. The present result strongly suggests that R. menciana is quite unique in that they predominantly use an UDP-galactosyltransferase for conjugation of dietary flavonoids, whereas UDP-glucosyltransferases are generally used for conjugation of plant phenolics and xenobiotics in other insects.

Silk hydrogels from non-mulberry and mulberry silkworm cocoons processed with ionic liquids.

Matrices based on silk fibroin from the non-mulberry silkworm Antheraea mylitta and the mulberry silkworm Bombyx mori have demonstrated good applicability in regenerative medicine. However, the cocoons of A. mylitta are underutilized in part due to their lack of solubility in traditional organic solvents. Therefore, the present work investigates the solubilization and processing of degummed fibers obtained from the cocoons of both silkworm species into hydrogels using ionic liquids (ILs). The developed hydrogels exhibited a rubbery consistency, viscoelastic behavior and rapid degradation in the presence of protease XIV. Scanning electron and confocal microscopy images suggest that human adipose stem cells (hASCs) are able to adhere to and migrate at different levels within the hydrogel structures. Moreover, the MTS assay demonstrated the maintenance of cell metabolic activity for up to 28 days, while DNA quantification showed that hASCs were able to proliferate on the seeded hydrogels. The findings indicate that complete IL removal from the fabricated hydrogels results in a positive hASCs cellular response. Thus the present approach provides a unique opportunity to broaden the processability and application of silk fibroin obtained from A. mylitta cocoons for regenerative medicine, namely cartilage regeneration.

Invited review nonmulberry silk biopolymers.

The silk produced by silkworms are biopolymers and can be classified into two types--mulberry and nonmulberry. Mulberry silk of silkworm Bombyx mori has been extensively explored and used for century old textiles and sutures. But for the last few decades it is being extensively exploited for biomedical applications. However, the transformation of nonmulberry silk from being a textile commodity to biomaterials is relatively new. Within a very short period of time, the combination of load bearing capability and tensile strength of nonmulberry silk has been equally envisioned for bone, cartilage, adipose, and other tissue regeneration. Adding to its advantage is its diverse morphology, including macro to nano architectures with controllable degradation and biocompatibility yields novel natural material systems in vitro. Its follow on applications involve sustained release of model compounds and anticancer drugs. Its 3D cancer models provide compatible microenvironment systems for better understanding of the cancer progression mechanism and screening of anticancer compounds. Diversely designed nonmulberry matrices thus provide an array of new cutting age technologies, which is unattainable with the current synthetic materials that lack biodegradability and biocompatibility. Scientific exploration of nonmulberry silk in tissue engineering, regenerative medicine, and biotechnological applications promises advancement of sericulture industries in India and China, largest nonmulberry silk producers of the world. This review discusses the prospective biomedical applications of nonmulberry silk proteins as natural biomaterials.

Comparing the rheology of mulberry and "wild" silkworm spinning dopes.

Lepidoperan silks provide a superb opportunity for comparative studies of spinning and fiber characteristics. Comparing the four species, Bombyx mori (China), Actias selene (India), Antheraea yamamai (Japan), Gonometa postica (Africa), allows us to examine differences on the family, species, and race levels. Measured rheological properties were consistent with phylogenetic relationships and in the context of resource allocation and gland morphology. We propose that the thorough domestication of the mulberry silkworm B. mori for high silk yield has resulted in a compensatory optimization for spinning efficiency. This is in stark contrast to the wild silkworms, where Saturnids appear to minimize their energetic input toward silk output and G. postica seems to balance both. We conclude that comparative studies provide valuable baseline information for future biomimetic applications and modeling, as well as illuminating biologically important details of silk processing.

Effect of sweet wormwood Artemisia annua crude leaf extracts on some biological and physiological characteristics of the lesser mulberry pyralid, Glyphodes pyloalis.

The lesser mulberry pyralid, Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is a monophagous and dangerous pest of mulberry that has been recently observed in Guilan province, northern Iran. In this study, the crude methanol extract of sweet wormwood Artemisia annua L. (Asterales: Asteracaea) was investigated on toxicity, biological and physiological characteristics of this pest under controlled conditions (24 ± 1 °C, 75 ± 5% RH, and 16:8 L:D photoperiod). The effect of acute toxicity and sublethal doses on physiological characteristics was performed by topical application. The LC50 and LC20 values on fourth instar larvae were calculated as 0.33 and 0.22 gram leaf equivalent/ mL, respectively. The larval duration of fifth instar larvae in LC50 treatment was prolonged (5.8 ± 0.52 days) compared with the control group (4.26 ± 0.29 days). However larval duration was reduced in the LC20 treatment. The female adult longevity in the LC50 dose was the least (4.53 ± 0.3 days), while longevity among controls was the highest (9.2 ± 0.29 days). The mean fecundity of adults after larval treatment with LC50 was recorded as 105.6 ± 16.84 eggs/female, while the control was 392.74 ± 22.52 eggs/female. The percent hatchability was reduced in all treatments compared with the control. The effect of extract in 0.107, 0.053, 0.026 and 0.013 gle/mL on biochemical characteristics of this pest was also studied. The activity of α-amylase and protease 48 hours post-treatment was significantly reduced compared with the control. Similarly lipase, esterase, and glutathione S-transferase activity were significantly affected by A. annua extract.

Partial biochemical characterization of alpha- and beta-glucosidases of lesser mulberry pyralid, Glyphodes pyloalis Walker (Lep.: Pyralidae).

The Lesser Mulberry Pyralid, Glyphodes pyloalis, is an important pest of mulberry. This pest feeds on mulberry leaves, and causes some problems for the silk industries in the north of Iran. The study of digestive enzymes is highly imperative to identify and apply new pest management technologies. Glucosidases have an important role in the final stages of carbohydrate digestion. Some enzymatic properties of alpha- and beta-glucosidases from midgut and salivary glands of G. pyloalis larvae were determined. The activities of alpha- and beta-glucosidase in the midgut and salivary glands of 5th instar larvae were obtained as 0.195, 1.07, 0.194 and 0.072 micromol(-1) min(-1) mg protein(-1), respectively. Activity of alpha- and beta-glucosidase from whole body of larval stages was also determined. Data showed that the highest activity of alpha- and beta-glucosidase was observed in the 5th larval stage, 0.168 and 0.645 micromol(-1) min(-1) mg protein(-1), respectively and the lowest activity in the 2nd larval stage, 0.042 and 0.164 micromol(-1) min(-1) mg protein(-1), respectively. Results showed that the optimal pH for alpha- and beta-glucosidase activity in midgut and salivary glands were 7.5, 5.5, 8-9 and 8-9 respectively. Also, the optimal temperature for alpha- and beta-glucosidase activity in the midgut was obtained as 45 degrees C. The addition of CaCl(2) (40 mM) decreased midgut beta-glucosidase activity whereas alpha-glucosidase activity was significantly increased at this concentration. The alpha-glucosidase activity, in contrast to beta-glucosidase, was enhanced with increasing in concentration of EDTA. Urea (4 mM) and SDS (8 mM) significantly decreased digestive beta-glucosidase activity. Characterization studies of insect glucosidases are not only of interest for comparative investigations, but also understanding of their function is essential when developing methods of insect control such as the use of enzyme inhibitors and transgenic plants to control insect pest.

Population fluctuation of soil and plant parasitic nematodes at Khangabok Wangbal Government silkfarm, Wangbal, Thoubal district Manipur, India.

The population fluctuation of nematodes around the rhizospheric regions of mulberrry plants at Khangabok Wangbal Government Silk farm, Wangbal, Thoubal District, Manipur, India was studied in relation to environmental factors like soil moisture content, soil pH, soil temperature, rainfall and moisture content of air for a consecutive period of three years, 2006-2008. During 2006, nematode population was highest in the month of May with very high rainfall (174.2 mm). Positive correlation of nematode population was found with soil temperature, soil pH, rainfall and relative humidity and negative correlation with soil moisture. During 2007, nematode population was highest in the month of May with least soil moisture and highest rainfall (15.1 p.c. and 190.6 mm). Nematode population had positive correlations with soil moisture, temperature, pH, rainfall and relative humidity of air. During 2008, nematode population was highest in the month of April with highest soil temperature of 24.8 degrees C, 66.0 p.c. moderate relative humidity and 21.0 mm rainfall. There were positive correlation with soil temperature and pH, and negative correlation with soil moisture, rainfall and relative humidity. Lowest nematode population was found during January (2006), and during December (2007, 2008) there were negligible rain and sometimes no rainfall at all. Among all dorylaimids, tylenchids, aphelenchids and mononchids, Helicotylenchus sp. pertain the most numerous nematode species in all the three years and seasons.

Non-mulberry silk gland fibroin protein 3-D scaffold for enhanced differentiation of human mesenchymal stem cells into osteocytes.

This study investigates the potential of three-dimensional (3-D) scaffolds of wild non-mulberry tropical tasar silk gland fibroin protein as substratum for osteogenic differentiation of human mesenchymal stem cells (hMSCs). The novelty of the study lies in the fabrication of scaffolds from non-bioengineered silk fibroin directly extracted from the glands of non-mulberry tropical tasar silkworms using sodium dodecyl sulfate dissolution protocol and its osteogenic application using single- and double-seeding methods. The scaffolds were mechanically robust and showed homogenous pore distribution within the scaffold. hMSCs were seeded on the scaffolds and were cultured for up to 28days under static conditions in osteogenic media. Osteogenic differentiation of hMSCs seeded on fibroin scaffolds resulted in extensive mineralization with the formation of large calcium nodules, higher alkaline phosphatase activity and intense von Kossa staining. Real-time studies revealed higher transcript levels for osteopontin (OS) and bone sialoprotein (IBSP) under double-seeded conditions as compared to single-seeded scaffolds. Histological analysis showed the development of osteoblastic cells and large calcified nodules. The development and spreading of nuclei and actin filaments on fibroin matrices were revealed through confocal studies. The results suggest the suitability of non-mulberry silk-fibroin protein 3-D scaffolds as natural biomaterial for potential in vitro bone-tissue engineering applications.