Instar Larvae Of Bombyx Mori Research Articles

Effect of ethanolic plant extractives on cephalic neuroendocrine system of BmNPV inoculated 5th instar larvae of Bombyx mori L. (Lepidoptera, Bombycidae)

In this research 3000, 5000 and 8000ppm concentration of ethanolic extractives of leaves of Eupatorium odaratum, Hyptis suaveolens and fruits of Aegle marmelos were tested on mulberry silkworm fifth instar larvae of PMxCSR2 inoculated with Bombyx mori Nuclear Polyhedrosis Virus (BmNPV). Extractives of A. marmelos, H. suaveolens and E. odaratum showed promising results against BmNPV of silkworm larvae @8000 ppm. Total improvement occurs in the function of neurosecretory cells (NSC), A1 and A2 cells of median neurosecretory cells (MNC) group. Also found that, NCC I and II, corpus cardiacum (CC) lobes and fine branching of NCA I over corpus allatum (CA) showed strongly stained PF positive granules of NSM due to the subsequent treatment of given ethanolic plant extractives which reduced BmNPV infection about (30 - 40%).

Studies on influence of various stages of mulberry leaf in the growth and cocoonic parameters of silkworm Bombyx mori (L.)

The objective of this work was to determine the effects of different stages of mulberry leaves on growth performance of treated larvae of Bombyx mori L. (Lepidoptera, Bombycidae) and characters of the resulted cocoons were evaluated. Results showed that the food consumption rate and assimilation rate were greatest for larvae fed on tender and over matured mulberry leaves as compared to larvae fed on mature leaves. Assimilation and conversion efficiencies were very high in the larvae grown on matured mulberry leaves (0.189 and 1.99% respectively). Moreover, the larvae fed on matured mulberry leaves showed higher growth rate, cocoon weight and shell ratio. Among the three different growth stages of leaves, the matured leaves were found to be the best food for final instar larvae of Bombyx mori L. which promotes maximum larval growth and it gains to quality cocoon and raw silk production.

Zinc oxide nanoparticles induce oxidative stress, genotoxicity, and apoptosis in the hemocytes of Bombyx mori larvae

The expanded uses of zinc oxide nanoparticles (ZnO-NPs) have grown rapidly in the field of nanotechnology. Thus, the increased production of nanoparticles (NPs) increases the potential risks to the environment and occupationally exposed humans. Hence, safety and toxicity assessment including genotoxicity of these NPs is indispensable. In the present study, we have evaluated the genotoxic effect of ZnO-NPs on 5th larval instar of Bombyx mori after feeding on mulberry leaves treated with ZnO-NPs at concentrations 50 and 100 μg/ml. Moreover, we evaluated its effects on total and different hemocyte count, antioxidant potential and catalase activity on the hemolymph of treated larvae. Results showed that ZnO-NPs at concentrations of 50 and 100 µg/ml have significantly decreased the total hemocyte count (THC) and different hemocyte count (DHC) except the number of oenocytes as they were significantly increased. Gene expression profile also showed up-regulation of GST, CNDP2 and CE genes suggesting increase in antioxidant activity and alteration in cell viability as well as cell signaling.

Proliferative and preparative cell divisions in wing discs of the last larval instar are regulated by different hormones and determine the size and differentiation of the wing of Bombyx mori

Through investigating the two different enhanced cell division stages, we tried to clarify the switch from the growth to differentiation in the wing disc of the last larval instar of Bombyx mori. The response to insulin and 20E in vitro was stage specific. Bmmyc expression in V1 wing discs showed differences after being cultured with and without insulin. Bmmyc expression in V5 wing discs also showed differences after being cultured with and without 20E. Cell cycle–related genes, BmE2F1 and BmcycE, were upregulated with insulin or 20E in cultured wing discs of V1 or V5, respectively. Bmwnt1 and Bmras1 showed upregulation with 20E in cultured wing discs. Bmwnt1 showed upregulation with insulin in cultured wing discs, but Bmras1 did not show clear upregulation with insulin treatment. In contrast, Bmdpp showed upregulation with insulin, but did not show clear upregulation with 20E. The addition of PI3K or TOR inhibitors inhibited the upregulation of Bmmyc expression that was upregulated with insulin or 20E. The upregulation of Bmmyc and Bmwnt1 with insulin or 20E was inhibited with the addition of Myc or Wnt inhibitors, respectively. Genes related to matrix metalloprotease showed upregulation with 20E, and the upregulation was inhibited by the addition of Myc or Wnt inhibitors. From the present results, we concluded that cell division during the feeding stage occurred through PI3K/TOR cascade, and that at the wandering stage occurred through ecdysone and PI3K/TOR cascade; the former is for growth and the latter for differentiation.

Thermal tolerance role of novel polyamine, caldopentamine, identified in fifth instar Bombyx mori.

Silkworms have limited ability to regulate their body temperature; therefore, environmental changes, such as global warming, can adversely affect their viability. Polyamines have shown protection to various organisms against heat stress. This study evaluated the qualitative and quantitative changes in heat-stressed Bombyx mori larvae polyamines. Fifth instar Bombyx mori larvae were divided into two groups; control group,reared at room temperature, i.e., 28 ± 2°C, and the heat shock group, exposed to 40°C. Dansylation of the whole worm polyaminesand subsequent thin-layer chromatography revealed the presence of components with the same Rf value as dansyl-putrescine, spermidine, and spermine. The dansyl-putrescine, spermidine, and spermine polyamines were identified by mass spectrometric analyses. After heat shock, the thin-layer chromatography of the whole-larvae tissue extracts showed qualitative and quantitative changes in dansylated polyamines. A new polyamine, caldopentamine, was identified, which showed elevated levels in heat-stressed larvae. This polyamine could play a role in helping the larvae tolerate various stress, including thermal stress. No significant changes in silk fiber's economic and mechanical properties were observed in our study. This study indicated that PA, caldopentamine, supplementation could improve heat-stress tolerance in Bombyx mori.

Spermidine alleviates heat shock and promotes the growth of Bombyx mori

Silkworms have limited ability to regulate their body temperature upon exposure to varying atmospheric temperatures. Environmental changes, such as global warming, adversely affect silkworm viability. Spermidine, a polyamine, protects various organisms against heat stress. This study aimed to evaluate the effect of spermidine on the thermotolerance of Bombyx mori larvae. 5th instar Bombyx mori larvae were divided into the control and spermidine groups and reared at 28 ± 1 °C and 80%–85% relative humidity. To induce heat stress, the larvae were exposed to various temperatures 32 °C, 36 °C, and 40 °C for 1 h on day 5 and subsequently allowed to recover at 28 ± 1 °C. Growth characteristics were evaluated by examining larval viability and quantifying proteins and carbohydrates. The thermotolerance of the spermidine group was higher than that of the control group at 40 °C heat stress conditions. The spermidine feeding increased the protein content and reduced the carbohydrate content significantly under heat stress condition. For the first time, this study demonstrated that spermidine alleviated thermal stress by enhancing the nutritional indices and antioxidant potential of the Bombyx mori larvae. A significant increase in economic properties was observed in spermidine fed groups. Thus, foliar feeding of spermidine to B. mori larvae markedly improved silkworm thermotolerance.

Encapsulation, release and insecticidal activity of Pongamia pinnata (L.) seed oil

Pongamia pinnata (L.) seed oil is effective for its insecticidal and larvicidal activities. However, its low aqueous solubility, high photosensitivity, and high volatility restrict its application for the control of agricultural pests. Encapsulation can be an effective technique to overcome such hindrances. Therefore, P. pinnata oil (PO) was extracted from its seeds and analyzed for karanjin content (3.18%) by GC-MS/MS as the marker compound. Micro-encapsulation (MC) of PO was prepared by interfacial polymerization between isocyanates and polyamine and tested for insecticidal and larvicidal activities. Bioassay of the developed formulations was tested in-vitro against 2nd instar larvae of Bombyx mori (Bivoltine hybrid) and in-vivo insecticidal bio-efficacy was tested against aubergine aphid (Aphis gossypii G.) and whitefly (Bemisia tabaci G.). Various properties of micro-capsules viz., stability, size, oil content and release kinetics were examined. Average diameter of capsules (1 μm) with Zeta potential (-16 mV) was indicated by the Dynamic Light Scattering (DLS) instrument. Existence of PO in the microcapsules was confirmed by an optical microscopic study. Spectroscopic analysis revealed 87.4% of PO was encapsulated in polyurea shell. The shelf-life (T10), half-life (T50), and expiry-life (T90) of polyurea coated capsules were 11.4, 75.3 and 250.0 h, respectively. Polyurea coated PO capsule formulation showed evidence of in-vitro toxicity against 2nd instar larvae of B. mori (LC50= 1.1%; LC90 = 5.9%). The PO formulation also exhibited 67.0–71.8% and 62.4–74.8% control of aphid and whitefly population in aubergine at 4.0% dose following 7–14 days after application. The study unveiled its significance in developing controlled release herbal formulations of P. pinnata as an alternative to harmful conventional synthetic insecticides for crop protection.

Comprehensive analysis of differentially expressed proteins in the male and female Bombyx mori larval instars exposed to thermal stress

Gender sensitivity to ambient heat, despite well known in insect species, how it manifests during young and late larval instars of Bombyx mori is still unclear. To uncover this cryptic feature, different instars male and female larvae were subjected to varied thermal stress separately and sex-stage specific expression of proteins was investigated. Interestingly, heat shock proteins(HSPs) 90 and 70 were expressed differently in all the instars and also between male and female larvae as confirmed by immunoblot assay. Besides up- and downregulation of few HSPs and other normal proteins, discreet expression of protein was noticed in the two-dimensional gel electrophoresis of male than female larvae which were identified as HSP70 by mass spectrometry. Furthermore, quantitative polymerase chain reaction results show 3.98- and 4.86-fold higher levels of Bmhsp70 and Bmhsp90 transcripts in male and female larvae, respectively, as a response to 40°C heat shock (HS) treatment. Conversely, in spite of the massive production of HSPs due to HS at 45°C, all the larvae were found dead, which is a strong proof of concept for autophagy. Comparatively, female larvae HS at 40°C succeed to spin cocoons with increased weight and silk contents than non-HS larvae. Comprehensively, in the present study, we have noticed a strong correlation for the first time that Bmhsp90 and Bmhsp70 genes expressions due to thermal stress are not only sex specific but also explicit preferential and coordinated action on survivability and biosynthetic potential of the silkworm, B. mori larvae during different instars.

The influence of infection of Beauveria bassiana (Bals) Vuill, a fungal species (Family: Clavicipitaceae) on quality of the cocoons of spinned by the larval instars of Bombyx mori (L) (Race: PMx CSR2

The influence of infection of Beauveria bassiana (Bals) Vuill, a fungal species (Family: Clavicipitaceae) on quality of the cocoons of spinned by the larval instars of Bombyx mori (L) (Race: PMx CSR2

The Quality of the Cocoons of Spinned by the Larval Instars of Bombyx mori (L) (Race: PM x CSR2) Inoculated with Beauveria Bassiana (Bals.) Vuill

The Quality of the Cocoons of Spinned by the Larval Instars of Bombyx mori (L) (Race: PM x CSR2) Inoculated with Beauveria Bassiana (Bals.) Vuill

Role of BmDredd during Apoptosis of Silk Gland in Silkworm, Bombyx mori.

Silk glands (SGs) undergo massive apoptosis driven degeneration during the larval-pupal transformation. To better understand this event on molecular level, we investigated the expression of apoptosis-related genes across the developmental transition period that spans day 4 in the fifth instar Bombyx mori larvae to day 2 pupae. Increases in the expression of BmDredd (an initiator caspase homolog) closely followed the highest BmEcR expression and resembled the expression trend of BmIcE. Simultaneously, we found that BmDredd expression was significantly higher in SG compared to other tissues at 18 h post-spinning, but reduced following injection of the apoptosis inhibitor (Z-DEVD-fmk). Furthermore, BmDredd expression correlated with changes of caspase3-like activities in SG and RNAi-mediated knockdown of BmDredd delayed SG apoptosis. Moreover, caspase3-like activity was increased in SG by overexpression of BmDredd. Taken together, the results suggest that BmDredd plays a critical role in SG apoptosis.

Direct and indirect effects of RNA interference against pyridoxal kinase and pyridoxine 5′-phosphate oxidase genes in Bombyx mori

Vitamin B6 comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5′-phosphate is a coenzyme involved in a high diversity of biochemical reactions. Humans and animals obtain B6 vitamers from diet, and synthesize pyridoxal 5′-phosphate by pyridoxal kinase and pyridoxine 5′-phosphate oxidase. Currently, little is known on how pyridoxal 5′-phosphate biosynthesis is regulated, and pyridoxal 5′-phosphate is supplied to meet their requirement in terms of cofactor. Bombyx mori is a large silk-secreting insect, in which protein metabolism is most active, and the vitamin B6 demand is high. In this study, we successfully down-regulated the gene expression of pyridoxal kinase and pyridoxine 5′-phosphate oxidase by body cavity injection of synthesized double-stranded small interfering RNA to 5th instar larvae of Bombyx mori, and analyzed the gene transcription levels of pyridoxal 5′-phosphate dependent enzymes, phosphoserine aminotransferase and glutamic-oxaloacetic transaminase. Results show that the gene expression of pyridoxal kinase and pyridoxine 5′-phosphate oxidase has a greater impact on the gene transcription of enzymes using pyridoxal 5′-phosphate as a cofactor in Bombyx mori. Our study suggests that pyridoxal 5′-phosphate biosynthesis and dynamic balance may be regulated by genetic networks.

A Critical Assessment of Bombyx mori Haemolymph Extract on Staphylococcus aureus an In vitro and In silico Approach

The newly reemerging drug-resistant Gram-positive pathogens require the discovery of new drug targets and the development of new therapeutics. We focus on antimicrobial proteins which inhibits growth of Staphylococcus aureus. In the present study fifth instar Bombyx mori larvae was used and infected with S. aureus by intrahaemocoelic injection of bacterial sample. The haemolymph was collected from the healthy and infected larvae after 24 h of infection and stored at -4°C in eppendorf tubes until use. Haemolymph extract was prepared and antimicrobial activity was done by the classical well diffusion method. Haemolymph extract prepared form infected larvae exhibit maximum zone of inhibition on S. aureus when compared with healthy haemolymph extract. Bioinformatics tools were used to find out the molecular interaction and binding mode of antimicrobial peptides from B. mori such as Moricin and Cecropin on three different drug target proteins Glycerol phosphate lipoteichoic acid synthase (PDB: 2W5Q), ABC transporter (PDB: 1P99) and DNA Gyrase (PDB: 2XCO) from S. aureus. Hex Docking software was used for the docking studies. The interaction mode and binding results can be represented in terms of docking energy. The best binding energies were obtained from the docking of DNA gyrase with Moricin and Cecropin (-702.13 Kcal/mol and -639.39 Kcal/mol) respectively.

A comprehensive study of the changes in ecdysteroid levels during the feeding phase of fifth instar larvae of the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

The present study describes changes in haemolymph ecdysteroid levels during the feeding phase of fifth instar larvae of Bombyx mori. A small and sharp peak in ecdysteroid level was recorded on day 4 when larvae had already reached the critical weight (2.76 ± 0.11 g) for metamorphosis. Food deprivation of larvae from day 4 resulted in an increase in the ecdysteroid level. Injection of insulin in both 1 day (before reaching critical weight) and 4 day (after reaching critical weight) old fed larvae resulted in an increase in the ecdysteroid level. However, when deprived of food only the haemolymph ecdysteroid levels of 4 day old larvae respond to an injection of insulin. The hormone level was also increased by treating 1day old larvae with a juvenile hormone analogue, but again, like insulin, only the fed larvae were affected by this treatment. Neck- and thorax-ligated 4 day old larvae had decreased ecdysteroid levels compared to both fed and food deprived larvae. However, a significant increase in ecdysteroid level was recorded in both neck- and thorax-ligated larvae injected with insulin. The injection of 20-hydroxyecdysone into 4 day old larvae resulted in an increase in the level of this substance in the haemolymph of fed and food deprived larvae, but the level was significantly higher in neck- and thorax-ligated larvae. The data, thus, indicates that ecdysteroid levels vary both in relation to the critical weight of the larvae and its nutritional status, and insulin might also play an important role in regulating the level of ecdysteroid.

Preparation of Porous Scaffolds from Silk Fibroin Extracted from the Silk Gland of Bombyx mori (B. mori)

In order to use a simple and ecofriendly method to prepare porous silk scaffolds, aqueous silk fibroin solution (ASF) was extracted from silk gland of 7-day-old fifth instar larvae of Bombyx mori (B. mori). SDS-page analysis indicated that the obtained fibroin had a molecular weight higher than 200 kDa. The fabrication of porous scaffolds from ASF was achieved by using the freeze-drying method. The pore of porous scaffolds is homogenous and tends to become smaller with an increase in the concentration of ASF. Conversely, the porosity is decreased. The porous scaffolds show impressive compressive strength which can be as high as 6.9 ± 0.4 MPa. Furthermore, ASF has high cell adhesion and growth activity. It also exhibits high ALP activity. This implies that porous scaffolds prepared from ASF have biocompatibility. Therefore, the porous scaffolds prepared in this study have potential application in tissue engineering due to the impressive compressive strength and biocompatibility.

Screening of aqueous plant extracts against Beauveria Bassiana infection to 5th instar larvae of Bombyx mori L.

Screening of aqueous plant extracts against Beauveria Bassiana infection to 5th instar larvae of Bombyx mori L.

Transcription profiling of eight cytochrome P450s potentially involved in xenobiotic metabolism in the silkworm, Bombyx mori

Cytochrome P450 (CYP) is a multifunctional super gene family, some of which play an important role in pesticide resistance. In this research, we used a real-time quantitative RT-PCR method, and a novel strategy, to measure the transcriptional level per gene copy using an exogenous RNA reference and DNA reference. The transcription levels of eight P450 genes in different tissues of fifth instar Bombyx mori larvae and their responses to pesticides (dichlorvos and deltamethrin) and sodium fluoride (NaF) were investigated. The results show different levels and patterns of expression of the different P450s in the various tissues observed. The P450s can be induced by pesticide and fluoride, but their responses to each are different. The results of this research are helpful in studying the tissue-specific expression of P450s in B. mori, and in developing new pesticide resistant silkworm varieties.

Stage-specific regulation of juvenile hormone biosynthesis by ecdysteroid in Bombyx mori

In the penultimate (4th) instar larvae of Bombyx mori, juvenile hormone (JH) synthesis by corpora allata (CA) fluctuates. When diet containing 20-hydroxyecdysone (20E) was fed, JH synthetic activity of the CA was first stimulated as the ecdysteroid titer increased, then suppressed slightly by the higher molting concentration of ecdysteroids (>250ng/ml). The overall JH biosynthetic activity was modulated by the expression of JH biosynthetic enzymes in the CA: primarily JH acid O-methyltransferase (JHAMT), isopentenyl diphosphate isomerase, and farnesyl diphosphate synthase 1. After the last (5th) larval ecdysis, the artificially increased high ecdysteroid level due to the 20E diet activated JH synthesis by the CA, which required intact nervous connections with the brain. A factor(s) from the 20E-activated brain controls mainly JHAMT and HMG Co-A reductase expression to stimulate the JH synthesis. In the normal last instar larvae, the ecdysteroid titer declines so that these activation mechanisms are absent; therefore the decline of the ecdysteroid titer after the final larval ecdysis is one of the factors which induces the cessation of the JH synthesis by CA.

Investigation of the influence of the juvenile hormone analogue fenoxycarb on major hemolymph proteins of the silkworm Bombyx mori during the last larval instar

Fenoxycarb 0-ethyl N-(2-(4-pheoxyphenoxy)-ethyl) carbamate is the most potent juvenile hormone analogue (JHA) against a variety of insect species including the silkworm Bombyx mori. In this study, we aimed to demonstrate the effects of this juvenile hormone on major hemolymph proteins of Bombyx mori during the fifth larval instar, by using fenoxycarb and clarifying the interactions between these proteins and the juvenile hormone. Topical applications of fenoxycarb to the fifth instar larvae of Bombyx mori were performed immediately after the fourth ecdysis (on day 0) and at the beginning of the prepupal period (on day 6). Fenoxycarb application on day 0 increased the length of instar in both sexes and induced high total protein content in the hemolymph. When fenoxycarb application was performed on day 6, there was no difference in the length of larval instar and a similar total protein amount was reached with control. Detected hemolymph proteins are grouped as follows by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE): storage proteins (72 kDa-76 kDa), 30kDa proteins, lipophorin (230 kDa), and vitellogenins (178 kDa and 42 kDa). Our results suggest that major hemolymph proteins have different sensitivities to fenoxycarb and that application days and sex are more important factors to get response.

Quantitative analysis of expression of six BmGST genes in silkworm, Bombyx mori

Glutathione S-transferases (GSTs) are a multifunctional super gene family, some of which play an important role in insecticide resistance. In this research, we used a real-time quantitative RT-PCR method, and a novel strategy, to measure the transcriptional level per gene copy using an exogenous RNA reference and DNA reference. The transcription levels of six BmGST genes in different tissues of fifth instar Bombyx mori larvae and their responses to insecticide and fluoride were investigated. The results show different levels and patterns of expression of the different BmGSTs in the various tissues observed. The BmGSTs can be induced by insecticide and fluoride, but their responses to each are different. The results of this research are helpful in studying the tissue-specific expression of BmGSTs in Bombyx mori, and in developing new pesticide resistant silkworm varieties.