Effects of Chromium Exposure on the Gene Expression of the Midgut in Silkworms, Bombyx mori.

Chromium is a severe heavy metal pollutant with significant environmental risks. The effects of Chromium on the digestion of Bombyx mori (silkworms) are of particular importance due to their ecological and economic significance. Herein, RNA sequencing was conducted on nine midgut samples from silkworms exposed to control, 12 g/kg and 24 g/kg Chromium chemical diets. Comparative transcriptomics revealed that under moderate Chromium exposure, there was a significant increase in up-regulated genes (1268 up-regulated to 857 down-regulated), indicating a stimulation response. At higher stress levels, a weakened survival response was observed, with a decrease in up-regulated genes and an increase in down-regulated genes (374 up-regulated to 399 down-regulated). A notable shift in cellular responses under medium chromium exposure was exposed, signifying the activation of crucial metabolic and transport systems and an elevation in cellular stress and toxicity mechanisms. The observation of up-regulated gene expression within xenobiotic metabolism pathways suggests a heightened defense against Chromium-induced oxidative stress, which was primarily through the involvement of antioxidant enzymes. Conversely, high-dose Chromium exposure down-regulates the folate biosynthesis pathway, indicating biological toxicity. Two novel genes responsive to pressure were identified, which could facilitate future stress adaptation understanding. The findings provide insights into the molecular mechanisms underlying silkworms' digestion response to Chromium exposure and could inform its biological toxicity.

Transcriptome analysis reveals immune and metabolic regulation effects of Poria cocos polysaccharides on Bombyx mori larvae.

Poria cocos polysaccharides (PS) have been used as Chinese traditional medicine with various pharmacological effects, including antiviral, anti-oxidative, and immunomodulatory activities. Herein Bombyx mori silkworm was used as a model animal to evaluate the immunomodulatory effects of PS via detecting the changes of innate immune parameters and explore the underlying molecular mechanism of the immunoregulatory effect of PS using Illumina HiSeq Xten platform. The results presented here demonstrated that a hemocoel injection of PS significantly enhanced the cellular immunity of silkworm, including hemocyte phagocytosis, microaggregation, and spreading ability. A total of 335 differentially expressed genes (DEGs) were screened, including 214 upregulated genes and 121 downregulated genes by differential expression analysis. Gene annotation and enrichment analyses showed that many DEGs related to immune signal recognition, detoxification, proPO activation, carbohydrate metabolism, and lipid metabolism were significantly upregulated in the treatment group. The Kyoto Encyclopedia of Genes and Genomes-based Gene Set Enrichment Analysis also revealed that the more highly expressed gene sets in the PS treatment silkworm were mainly related to immune signal transduction pathways and energy metabolism. In addition, the activity of four enzymes related to immunity and energy metabolism-including phenoloxidase, glucose-6-phosphate dehydrogenase, hexokinase, and fatty acid synthetase-were all significantly increased in the larvae injected with PS. We performed qRT-PCR to examine the expression profile of immune and metabolic-related genes, which further verified the reliability of our transcriptome data and suggested that PS can regulate the immunity of silkworm by enhancing the cellular immunity and modulating the expression levels of genes related to immune responses and physiological metabolism. These findings will lay a scientific foundation for the use of PS as an immunomodulator in disease prevention in human beings or animals.

Foliar supplementation of ascorbic acid modulates biochemical performance of silkworm, Bombyx mori under thermal stress.

Dietary supplementation of ascorbic acid was found to be effective in modifying the composition of essential biomolecules. A relative investigation on effects of exogenous dietary supplementation of 0.2% ascorbic acid on the fifth instar larvae of silkworm, Bombyx mori exposed to a high thermal stress of range 40 ± 2 °C was carried out in the lab-set conditions. The observed elevation in various biomolecules, viz., DNA, RNA, protein, lipids, and carbohydrates were quantified in both the thermal stress-induced test groups and in the control, set aside. The test results so obtained were proven to be statistically significant. The present study reveals that foliar supplementation of ascorbic acid has been effective in positively-modulating the biochemical performance in larvae exposed to thermal stress. Moreover, the study also uncovers the possibilities of ascorbic acid as a potential candidate, capable of facilitating the production of good quality cocoons, from larvae exposed to thermal stress.

Molecular cloning, expression, and characterization of BmSOD3 in silkworm (Bombyx mori).

Superoxide dismutases (SODs) play an essential role in eliminating excess reactive oxygen species and maintaining the redox balance of the immune system. To study the function of BmSOD3 in silkworm, 543-bp full-length complementary DNA-encoding BmSOD3 was cloned from silkworm. The BmSOD3 amino acids were compared to their homologs, and several highly conserved regions were analyzed. We also carried out phylogenetic analyses of the SOD gene. Our results showed that the BmSOD3 gene belonged with the ecCu/Zn SOD gene. The BmSOD3 gene was transformed into the pET28a vector for functional expression in Escherichia coli. The sodium salt-polyacrylamide gel electrophoresis results showed that the molecular weight of recombinant BmSOD3 was about 22 kDa. The recombinant protein BmSOD3 was purified to detect its properties. After purification analyses, the enzyme activity showed Cu/Zn SOD activity, and the specific activity of the purified enzyme was 0.51 U/mg. The BmSOD3 transcripts showed tissue-specific expression in the midgut and malpighian tubule. The immune microarray data for BmSOD3 showed an expression signal that had a strong response to the induction of four pathogens (Bacillus bombyseptieus, Beauveria bassiana, E. coli, and nuclear polyhedrosis virus), particularly after infection for 24 h, which indicates that the BmSOD3 gene plays a key role in response to bacterial, fungal, and viral invasion. The fusion protein also showed antibacterial activity against E. coli in vitro. Thus, the fusion protein BmSOD3 exhibits antibacterial activity and may be used in production to combat diseases caused by bacteria in silkworm.

Transcriptome analysis reveals gene expression changes of the fat body of silkworm (Bombyx mori L.) in response to selenium treatment.

A comparative transcriptome analysis was conducted to investigate the gene expression changes in the fat body of silkworm after treatment with different concentrations (50 µM and 200 µM) of selenium (Se). 912 differential expression genes (DEGs) (371 up-regulated and 541 down-regulated) and 1420 DEGs (1078 up-regulated and 342 down-regulated) were identified in silkworm fat body treated with 50 µM and 200 µM of Se, respectively. In case of 50 µM group, DEGs were mainly enriched in the peroxisome pathway and fatty acid metabolism pathway, and later were associated with antioxidant defense and nutrition regulation. After 200 µM Se-treatment, DEGs were mainly located in the glycerolipid metabolism and arachidonic acid metabolism pathways, which further encoded detoxification related genes. Furthermore, 32 candidate DEGs from these pathways had been selected to confirm the RNA-seq data. Among these DEGs, 14 genes were up-regulated in the 50 µM Se-treated group (only three genes in the 200 µM Se-treated group) which were involved in lipid metabolism and antioxidant defense, and 13 up-regulated genes (only two genes were up-regulated in the 50 µM Se-treated group) were involved in detoxification of the 200 µM Se-treated group. These changes showed that lower concentration of Se could regulate the nutrition and promote antioxidation pathways; whereas, high levels of Se promoted the detoxification of silkworm. These findings can be helpful to understand the possible mechanisms of Se action and detoxification in silkworm and other insects.

Applying microarray-based technique to study and analyze silkworm (Bombyx mori) transcriptomic response to long-term high iron diet.

To investigate the biological processes affected by long-term iron supplementation, newly hatched silkworms were exposed to high iron mulberry diet (10 and 100 ppm) and its effect on silkworm transcriptom was determined. The results showed that the silkworm was responsive to iron by increasing iron concentration and ferritin levels in the hemolymph and by regulating the expression of many other genes. A total of 523 and 326 differentially expressed genes were identified in 10 and 100 ppm Fe group compared to the control, respectively. Of these genes, 249 were shared between in both the 10 ppm and 100 ppm Fe group, including 152 up-regulated and 97 down-regulated genes. These shared genes included 19 known Fe regulated, 24 immune-related, 12 serine proteases and serine proteases homologs, 41 cuticular and cuticle genes. Ten genes (carboxypeptidases A, serine protease homologs 85, fibrohexamerin/P25, transferrin, sex-specific storage-protein 2, fungal protease inhibitor F, insect intestinal mucin, peptidoglycan recognition protein B, cuticle protein CPH45, unknown gene) were involved in the regulation of iron overload responses.

A novel PCR-based technology for rapid and non-sequencing authentication of Bombyx batryticatus using species-specific primers.

A novel PCR technology was developed to detect short DNA fragments using species-specific primers for rapid and non-sequencing authentication of Bombyx batryticatus based on differences in the mitochondrial genome. Three specifically designed primer reactions were established to target species for the reliable identification of their commercial products. They were confirmed to have a high inter-species specificity and intra-species stability. The limit of detection was estimated as 1 ng of genomes for Beauveria bassiana and 100 pg for Bombyx mori and Metarhizium anisopliae. Furthermore, validation results demonstrated that raw materials and their processed products can be conveniently authenticated with good sensitivity and precision using this newly proposed approach. In particular, when counterfeits were assayed, these primer sets performed well, whereas COI barcoding technology did not. These could also assist in the discrimination and identification of adulterates of other animal-derived medicines in their pulverized and processed forms and even in complexes.

Extraordinary Mechanical Properties of Composite Silk Through Hereditable Transgenic Silkworm Expressing Recombinant Major Ampullate Spidroin.

Spider dragline silk is a remarkable material that shows excellent mechanical properties, diverse applications, biocompatibility and biodegradability. Transgenic silkworm technology was used to obtain four types of chimeric silkworm/spider (termed composite) silk fibres, including different lengths of recombinant Major ampullate Spidroin1 (re-MaSp1) or recombinant Major ampullate Spidroin2 (re-MaSp2) from the black widow spider, Latrodectus hesperus. The results showed that the overall mechanical properties of composite silk fibres improved as the re-MaSp1 chain length increased, and there were significant linear relationships between the mechanical properties and the re-MaSp1 chain length (p < 0.01). Additionally, a stronger tensile strength was observed for the composite silk fibres that included re-MaSp1, which only contained one type of repetitive motif, (GA)n/An, to provide tensile strength, compared with the silk fibres that includedre-MaSp2, which has the same protein chain length as re-MaSp1 but contains multiple types of repetitive motifs, GPGXX and (GA)n/An. Therefore, the results indicated that the nature of various repetitive motifs in the primary structure played an important role in imparting excellent mechanical properties to the protein-based silk fibres. A silk protein with a single type of repetitive motif and sufficiently long chains was determined to be an additional indispensable factor. Thus, this study forms a foundation for designing and optimizing the structure of re-silk protein using a heterologous expression system.

Isolation and functional characterization of the pheromone biosynthesis activating neuropeptide receptor of Chinese oak silkworm, Antheraea pernyi.

Insect pheromone biosynthesis activating neuropeptide (PBAN) controls the synthesis and actuating of sex pheromones of female adult. In the current examination, the full-length cDNA encoding the PBAN receptor was cloned from the pheromone gland (PG) of Antheraea pernyi (AntpePBANR). The AntpePBANR displayed the characteristic seven transmembrane areas of the G protein-coupled receptor (GPCR) and was closely related to the PBANR from Bombyx mori and Manduca sexta in the phylogenetic tree. The AntpePBANR expressed in mammalian cell lines were enacted by AntpePBAN in a concentration-dependent manner. AntpePBANR activation resulted in the calcium mobilization but did not activate the cAMP elevation pathway. Cells expressing AntpePBANR were profoundly responsive to Antpe-γ-SGNP (suboesophageal ganglion neuropeptides) and Antpe-DH (diapause hormone), different individuals from FXPRLamide (X = T, S or V) family in A. pernyi. Deletion of residues in the C-terminal hexapeptide (FSPRLamide) proved that P, R and L played the key parts in initiating the AntpePBANR, the amination to the last C terminal residues which can also likewise impact the activation of AntpePBAN receptor altogether. The mRNA of the AntpePBANR gene demonstrated the most noteworthy transcript levels in pheromone gland followed by fat body.

Clone and functional analysis of Seryl-tRNA synthetase and Tyrosyl-tRNA synthetase from silkworm, Bombyx mori.

Aminoacyl-tRNA synthetases are the key enzymes for protein synthesis. Glycine, alanine, serine and tyrosine are the major amino acids composing fibroin of silkworm. Among them, the genes of alanyl-tRNA synthetase (AlaRS) and glycyl-tRNA synthetase (GlyRS) have been cloned. In this study, the seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) genes from silkworm were cloned. Their full length are 1709 bp and 1868 bp and contain open reading frame (ORF) of 1485 bp and 1575 bp, respectively. RT-PCR examination showed that the transcription levels of SerRS, TyrRS, AlaRS and GlyRS are significantly higher in silk gland than in other tissues. In addition, their transcription levels are much higher in middle and posterior silk gland than in anterior silk gland. Moreover, treatment of silkworms with phoxim, an inhibitor of silk protein synthesis, but not TiO2 NP, an enhancer of silk protein synthesis, significantly reduced the transcription levels of aaRS and content of free amino acids in posterior silk gland, therefore affecting silk protein synthesis, which may be the mechanism of phoxim-silking disorders. Furthermore, low concentration of TiO2 NPs showed no effect on the transcription of aaRS and content of free amino acids, suggesting that TiO2 NPs promotes silk protein synthesis possibly by increasing the activity of fibroin synthase in silkworm.

Identification and characterization of an atypical 2-cys peroxiredoxin from the silkworm, Bombyx mori.

Peroxiredoxins (Prxs) play an important role in the protection of insects against the toxicity of reactive oxygen species. Here, we identified and characterized a novel, atypical 2-cysteine (Cys) peroxiredoxin (BmPrx3) from an expressed sequence tag database in a lepidopteran insect, Bombyx mori. The BmPrx3 cDNA contained an open reading frame of 684 bp that encodes a 228-amino-acid protein with a calculated molecular mass of 25 kDa. Sequence comparison revealed that BmPrx3 belongs to the atypical 2-Cys Prxs. Quantitative real-time PCR revealed that BmPrx3 can be detected in all tissues and developmental stages. Recombinant BmPrx3 purified from Escherichia coli exhibited antioxidant activity that removed hydrogen peroxide and protected DNA from oxidative damage. Disc diffusion and viability assays revealed that recombinant BmPrx3 increased bacterial survival under H2 O2 -mediated oxidative stress. In addition, quantitative real-time PCR analysis indicated that BmPrx3 transcription levels were significantly increased in response to various oxidative stresses. Furthermore, BmPrx3 transcription levels in the midgut were regulated by bacterial infection. Taken together, these results suggest that BmPrx3 acts as an antioxidant enzyme to protect the silkworm from various oxidative stresses.

Plant phenolics are detoxified by prophenoloxidase in the insect gut.

Plant phenolics are a group of important secondary metabolites that are toxic to many animals and insects if ingested at high concentrations. Because most insects consume plant phenolics daily, they have likely evolved the capacity to detoxify these compounds. Here, we used Drosophila melanogaster, Bombyx mori and Helicoverpa armigera as models to study the metabolism of plant phenolics by prophenoloxidases. We found that insect foreguts release prophenoloxidases into the lumen, and that the survival of prophenoloxidase-deletion mutants was impaired when fed several plant phenolics and tea extracts. Using l-DOPA as a model substrate, biochemical assays in large Lepidopteran insects demonstrated that low levels of l-DOPA are rapidly metabolized into intermediates by phenoloxidases. Feeding with excess l-DOPA showed that the metabolic intermediate 5,6-dihydroxyindole reached the hindgut either by passing directly through the midgut, or by transport through the hemolymph. In the hindgut, 5,6-dihydroxyindole was further oxidized by prophenoloxidases. Intermediates exerted no toxicity in the hemocoel or midgut. These results show that plant phenolics are not toxic to insects unless prophenoloxidase genes are lost or the levels of phenolics exceed the catalytic activity of the gut prophenoloxidases.

Molecular Characterization of Endoplasmic Reticulum Oxidoreductin 1 from Bombyx mori.

We isolated a complementary DNA (cDNA) clone encoding endoplasmic reticulum oxidoreductin 1 (bERO1, a specific oxidant of protein disulfide isomerase (PDI)) from Bombyx mori. This protein has a putative open reading frame (ORF) of 489 amino acids and a predicted size of 57.4 kDa. Although bERO1 protein shares less than 57% amino acid sequence homology with other reported ERO1s, it contains two conserved redox active motifs, a Cys-X-X-X-X-Cys motif of N-terminal and Cys-X-X-Cys-X-X-Cys motif of C-terminal. Both motifs are typically present in ERO1 protein family members. The bEro1 mRNA expression was highest in posterior silk gland on the sixth day of the 5th instar larvae. Expression of bEro1 mRNA also markedly increased during endoplasmic reticulum (ER) stress induced by stimulation with antimycin, calcium ionophore A23187, dithiothreitol, H2O2, monencin, and tunicamycin. In addition, expression levels of bEro1 exactly coincided with that of bPdi. This is the first result suggesting that bERO1 plays an essential role in ER quality control through the combined activities of bERO1 and bPDI as a catalyst of protein folding in the ER and sustaining cellular redox homeostasis.

Fluorescence imaging for a noninvasive in vivo toxicity-test using a transgenic silkworm expressing green fluorescent protein.

In drug development, the toxicity of candidate chemicals must be carefully examined in an animal model. Here we developed a live imaging technique using silkworms for a noninvasive toxicity test applicable for drug screening. Injection of carbon tetrachloride, a tissue-injuring chemical, into transgenic silkworms expressing green fluorescent protein (GFP) induced leakage of GFP from the tissues into the hemolymph. The leakage of GFP was suppressed by pre-administration of either cimetidine, a cytochrome P450 inhibitor, or N-acetyl cysteine, a free-radical scavenger. The transgenic silkworm was made transparent by feeding a diet containing chemicals that inhibit uric acid deposition in the epithelial cells. In the transparent silkworms, GFP fluorescence in the fat body could be observed from outside the body. Injection of salicylic acid or iron sulfate, tissue-injuring chemicals, into the transparent silkworms decreased the fluorescence intensity of the GFP in the fat body. These findings suggest that the transparent GFP-expressing silkworm model is useful for evaluating the toxicity of chemicals that induce tissue injury.

Comparative analyses of the cholinergic locus of ChAT and VAChT and its expression in the silkworm Bombyx mori.

The cholinergic locus, which encodes choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), is specifically expressed in cholinergic neurons, maintaining the cholinergic phenotype. The organization of the locus is conserved in Bilateria. Here we examined the structure of cholinergic locus and cDNA coding for ChAT and VAChT in the silkworm, Bombyx mori. The B. mori ChAT (BmChAT) cDNA encodes a deduced polypeptide including a putative choline/carnitine O-acyltransferase domain and a conserved His residue required for catalysis. The B. mori VAChT (BmVAChT) cDNA encodes a polypeptide including a putative major facilitator superfamily domain and 10 putative transmembrane domains. BmChAT and BmVAChT cDNAs share the 5'-region corresponding to the first and second exon of cholinergic locus. Polymerase chain reaction analyses revealed that BmChAT and BmVAChT mRNAs were specifically expressed in the brain and segmental ganglia. The expression of BmChAT was detected 3 days after oviposition. The expression level was almost constant during the larval stage, decreased in the early pupal stage, and increased toward eclosion. The average ratios of BmChAT mRNA to BmVAChT mRNA in brain-subesophageal ganglion complexes were 0.54±0.10 in the larvae and 1.92±0.11 in adults. In addition, we examined promoter activity of the cholinergic locus and localization of cholinergic neurons, using a baculovirus-mediated gene transfer system. The promoter sequence, located 2kb upstream from the start of transcription, was essential for cholinergic neuron-specific gene õexpression. Cholinergic neurons were found in several regions of the brain and segmental ganglia in the larvae and pharate adults.

Superoxide dismutases, SOD1 and SOD2, play a distinct role in the fat body during pupation in silkworm Bombyx mori.

One way that aerobic biological systems counteract the generation of reactive oxygen species (ROS) is with superoxide dismutase proteins SOD1 and SOD2 that metabolize superoxide radicals to molecular oxygen and hydrogen peroxide or scavenge oxygen radicals produced by the extensive oxidation-reduction and electron-transport reactions that occur in mitochondria. We characterized SOD1 and SOD2 of Bombyx mori isolated from the fat body of larvae. Immunological analysis demonstrated the presence of BmSOD1 and BmSOD2 in the silk gland, midgut, fat body, Malpighian tubules, testis and ovary from larvae to adults. We found that BmSOD2 had a unique expression pattern in the fat body through the fifth instar larval developmental stage. The anti-oxidative functions of BmSOD1 and BmSOD2 were assessed by exposing larvae to insecticide rotenone or vasodilator isosorbide dinitrate, which is an ROS generator in BmN4 cells; however, exposure to these compounds had no effect on the expression levels of either BmSOD protein. Next, we investigated the physiological role of BmSOD1 and BmSOD2 under environmental oxidative stress, applied through whole-body UV irradiation and assayed using quantitative RT-PCR, immunoblotting and microarray analysis. The mRNA expression level of both BmSOD1 and BmSOD2 was markedly increased but protein expression level was increased only slightly. To examine the differences in mRNA and protein level due to UV irradiation intensity, we performed microarray analysis. Gene set enrichment analysis revealed that genes in the insulin signaling pathway and PPAR signaling pathway were significantly up-regulated after 6 and 12 hours of UV irradiation. Taken together, the activities of BmSOD1 and BmSOD2 may be related to the response to UV irradiation stress in B. mori. These results suggest that BmSOD1 and BmSOD2 modulate environmental oxidative stress in the cell and have a specific role in fat body of B. mori during pupation.

The initial uridine of primary piRNAs does not create the tenth adenine that Is the hallmark of secondary piRNAs.

PIWI-interacting RNAs (piRNAs) silence transposons in animal germ cells. PIWI proteins bind and amplify piRNAs via the "Ping-Pong" pathway. Because PIWI proteins cleave RNAs between target nucleotides t10 and t11-the nucleotides paired to piRNA guide positions g10 and g11-the first ten nucleotides of piRNAs participating in the Ping-Pong amplification cycle are complementary. Drosophila piRNAs bound to the PIWI protein Aubergine typically begin with uridine (1U), while piRNAs bound to Argonaute3, which are produced by Ping-Pong amplification, often have adenine at position 10 (10A). The Ping-Pong model proposes that the 10A is a consequence of 1U. We find that 10A is not caused by 1U. Instead, fly Aubergine as well as its homologs, Siwi in silkmoth and MILI in mice, have an intrinsic preference for adenine at the t1 position of their target RNAs; during Ping-Pong amplification, this t1A subsequently becomes the g10A of a piRNA bound to Argonaute3.

Cloning of cDNA encoding a Bombyx mori homolog of human oxidation resistance 1 (OXR1) protein from diapause eggs, and analyses of its expression and function.

To better understand the molecular mechanisms of diapause initiation, we used the sensitive cDNA subtraction (selective amplification via biotin- and restriction-mediated enrichment) method and isolated a novel gene expressed abundantly in diapause eggs of the silkworm, Bombyx mori, which encodes a homolog of the human oxidation resistance 1 (OXR1) protein. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analyses confirmed that BmOXR1 mRNA and its 140-kDa protein were differentially expressed in diapause eggs compared to non-diapause eggs. OXR1 double-stranded RNA (dsRNA) was injected into diapause-destined eggs before the cellular blastoderm stage, and 4days later, when untreated eggs reached the diapause stage, the OXR1 protein disappeared; however, these eggs remained in diapause, suggesting that BmOXR1 is not essential for diapause initiation and/or maintenance. To further investigate the in vivo function of BmOXR1 apart from its role in diapause, we overexpressed BmOXR1 in Drosophila melanogaster. The fruit fly male adult life-span was significantly extended in the 50%-survival time when adults were reared on diets both with and without H2O2 solution under 25°C incubation. These results suggest that BmOXR1 functions in D. melanogaster via a possible antioxidant effect. As BmOXR1 was expressed mainly in the nuclei of D. melanogaster cells, the mechanism underlying its antioxidation effect appears to be different from that in humans where it is expressed mainly in the mitochondria. Taken together, these results suggest that BmOXR1 might serve as an antioxidant regulator during the early diapause stage.