Exorista sorbillans (Diptera: Tachinidae) parasitism shortens host larvae growth duration by regulating ecdysone and juvenile hormone titers in Bombyx mori (Lepidoptera: Bombycidae).

The tachinid fly, Exorista sorbillans, is a notorious ovolarviparous endoparasitoid of the silkworm, Bombyx mori, causing severe damage to silkworm cocoon industry. Silkworm larvae show typically precocious wandering behavior after being parasitized by E. sorbillans; however, the underlying molecular mechanism remains unexplored. Herein, we investigated the changes in the levels of 20-hydroxyecdysone (20E) and juvenile hormone (JH) titer, and they both increased in the hemolymph of parasitized silkworms. Furthermore, we verified the expression patterns of related genes, which showed an upregulation of 20E signaling and biosynthesis genes but a significant downregulation of ecdysone oxidase (EO), a 20E inactivation enzyme, in parasitized silkworms. In addition, related genes of the JH signaling were activated in parasitized silkworms, while related genes of the JH degradation pathway were suppressed, resulting in an increase in JH titer. Notably, the precocious wandering behavior of parasitized silkworms was partly recoverable by silencing the transcriptions of BmCYP302A1 or BmCYP307A1 genes. Our findings suggest that the developmental duration of silkworm post parasitism could be shortened by regulation of 20E and JH titers, which may help silkworm to resist the E. sorbillans infestation. These findings provide a basis for deeper insight into the interplay between silkworms and E. sorbillans and may serve as a reference for the development of a novel approach to control silkworm myiasis.

Hydrogen sulfide treatment retrieves the inhibition of growth and development characteristics in silkworm (Bombyx mori) via phosphoacetyl glucosamine mutase gene knock down.

Phosphoacetyl glucosamine mutase (PGM) is the key gene for glycolysis of important metabolic pathways in silkworm, and H2 S (7.5 µM) can promote the growth and development of silkworm. Herein, we used body cavity injection of small-interfering RNA (siRNA) to interfere with the PGM gene in H2 S-treated silkworms. After RNA interference (RNAi), we investigated the growth and development of the silkworm. H2 S treatment could significantly recover the inhibition of body weight, cocoon weight, cocoon shell weight, and cocoon shell ratio by knocking down PGM gene in silkworm, without significant effects on eggs laying and production, and then analyzed the mRNA expression of PGM gene. The interference of siRNA significantly decreased the expression of targeted PGM gene and was concentrated in 48 h followed by gradual recovery. Three interference fragments also showed different interference effects, and siRNA of PGM-3 exerted the highest interference effect to the target gene expression. Fat body had the highest mRNA expression of PGM gene, and the best interference effect was observed after siRNA injection. The results showed that the gene based on H2 S treatment may have an important impact on the growth and development of silkworm by affecting its metabolic pathway.

Evaluation of the Metabolic Effects of Hydrogen Sulfide on the Development of Bombyx mori (Lepidoptera: Bombycidae), Using Liquid Chromatography-Mass Spectrometry-Based Metabolomics.

Hydrogen sulfide (H2S) is a highly poisonous gas with an unpleasant smell of rotten eggs. Previous studies of H2S have primarily focused on its effects on mammalian nervous and respiratory systems. In this study, silkworm developmental parameters and changes in metabolites in response to H2S exposure were investigated using a hemolymph metabolomic approach, based on liquid chromatography-mass spectrometry (LC-MS). The developmental parameters, body weight, cocoon weight, cocoon shell weight, and cocoon shell ratio, were noticeably increased following H2S exposure, with the greatest effects observed at 7.5-µM H2S. Metabolites upregulated under H2S exposure (7.5 µM) were related to inflammation, and included (6Z, 9Z, 12Z)-octadecatrienoic acid, choline phosphate, and malic acid, while hexadecanoic acid was downregulated. Identified metabolites were involved in biological processes, including pyrimidine, purine, and fatty acid metabolism, which are likely to affect silk gland function. These results demonstrate that H2S is beneficial to silkworm development and alters metabolic pathways related to spinning function and inflammation. The present study provides new information regarding the potential functions of H2S in insects and metabolic pathways related to this phenomenon.

Overview of research on Bombyx mori microRNA.

MicroRNAs (miRNAs) constitute some of the most significant regulatory factors involved at the post-transcriptional level after gene expression, contributing to the modulation of a large number of physiological processes such as development, metabolism, and disease occurrence. This review comprehensively and retrospectively explores the literature investigating silkworm, Bombyx mori L. (Lepidoptera: Bombicidae), miRNAs published to date, including discovery, identification, expression profiling analysis, target gene prediction, and the functional analysis of both miRNAs and their targets. It may provide experimental considerations and approaches for future study of miRNAs and benefit elucidation of the mechanisms of miRNAs involved in silkworm developmental processes and intracellular activities of other unknown non-coding RNAs.

Selenium Treatment Alleviates the Inhibition Caused by Nep-L Gene Knockdown in Silkworm (Bombyx mori).

Recent studies have emphasized the beneficial effects of 50 µM selenium (Se) on the growth and development of the silkworm, Bombyx mori; however, less is known about its underlying mechanism. To unravel the effect of 50 µM Se on the silkworms with neutral endopeptidase 24.11-like gene (NEP-L) knockdown, we injected small interfering RNA (siRNA) into the body cavity of silkworms. Phenotypic characteristics, mRNA expression of the Nep-L gene, and enriched Se content were evaluated in silkworms from each treatment group. After injecting Nep-L siRNA, the body weight, cocoon quality (cocoon weight, cocoon shell weight, and cocoon shell ratio), and egg production of silkworms were significantly reduced, without any significant effect on egg laying number. However, Se treatment could significantly alleviate the inhibition of body weight, and cocoon quality, without significant effects on egg laying number and production. In addition, the gene knockdown increased Se content in the B. mori. On the molecular level, the targeted Nep-L gene was inhibited significantly by siRNA interference, essentially with the strongest effect at 24 h after RNAi, followed by steady recovery. Among the three fragments, the siRNA of Nep-L-3 was the most effective in interfering with target gene expression. Nep-L gene showed the highest expression in Malpighian tubules (MTs). Both at the phenotypic and genotypic levels, our results show that Nep-L knockdown can exert a significant inhibitory effect on silkworms, and 50 µM Se can reverse the negative effect, which provides a practical prospect for strengthening the silkworm food industry.

Transcriptional regulation of the gene for prothoracicotropic hormone in the silkworm, Bombyx mori.

Prothoracicotropic hormone (PTTH) is one of key players in regulation of insect growth, molting, metamorphosis, diapause, and is expressed specifically in the two pairs of lateral PTTH-producing neurosecretory cells in the brain. Analysis of cis-regulatory elements of the PTTH promoter might elucidate the regulatory mechanism controlling PTTH expression. In this study, the PTTH gene promoter of Bombyx mori (Bom-PTTH) was cloned and sequenced. The cis-regulatory elements in Bom-PTTH gene promoter were predicted using Matinspector software, including myocyte-specific enhancer factor 2, pre-B-cell leukemia homeobox 1, TATA box, etc. Transient transfection assays using a series of fragments linked to the luciferase reporter gene indicated that the fragment spanning -110 to +33 bp of the Bom-PTTH promoter showed high ability to support reporter gene expression, but the region of +34 to +192 bp and -512 to -111 bp repressed the promoter activity in the BmN and Bm5 cell lines. Electrophoretic mobility shift assays demonstrated that the nuclear protein could specifically bind to the region spanning -124 to -6 bp of the Bom-PTTH promoter. Furthermore, we observed that the nuclear protein could specifically bind to the -59 to -30 bp region of the Bom-PTTH promoter. A classical TATA box, TATATAA, localized at positions -47 to -41 bp, which is a potential site for interaction with TATA box binding protein (TBP). Mutation of this TATA box resulted in no distinct binding band. Taken together, TATA box was involved in regulation of PTTH gene expression in B. mori.

The discovery approaches and detection methods of microRNAs.

MicroRNAs (miRNAs) are small, highly conserved, non-coding RNAs that regulate gene expression of target mRNAs through cleavage or translational inhibition. Computer-based approaches for miRNA gene identification are being considered as indispensable in miRNAs research. Similarly, experimental approaches for detection of miRNAs are crucial to the testing and validating of computational algorithms. The detection of miRNAs in tissues or cells can supply valuable information for investigating the biological function of these molecules. Selective and highly sensitive detection methods will pave the way for extended understanding of miRNA function within organisms. In this review, we summarize the various computational methods for identification of miRNAs as well as the methodologies that have been developed to detection miRNAs.

Genomic analysis of silkworm microRNA promoters and clusters.

MicroRNAs (miRNAs) are endogenous single-stranded RNAs of 18-22 nt in length, which can regulate the complementary mRNAs at the post-transcriptional level by cleavage or repression of translation of the target mRNAs. Studies have shown that the majority of animal miRNAs are transcribed from independent transcription units, and someare transcribed together with their host genes. However, the nature of the primary transcript of intergenic miRNAs remains unknown. Silkworm (Bombyx mori) miRNAs are representative of those of the Lepidoptera insects and many of them are conserved in Caenorhabditis elegans and other animal species. To date, little is known about the transcriptional regulation of silkworm miRNA genes. We performed the genomic analysis on the silkworm miRNA transcripts around the promoter region including the transcription start site (TSS) and the TATA-box, and on the organization of the miRNA cluster. In 73 pre-miRNAs from the silkworm 131 promoters were detected via a bioinformatics approach. Among them the portion of non-conserved promoters is greater than that of the conserved ones. The genomic organization of pre-miRNAs of the silkworm was globally analyzed and it was determined that 11 of them we reorganized into five clusters. Sequence alignment showed that paralogs existed for some of the miRNAs in the cluster. These results may increase the understanding of the specific sequences upstream of the pre-miRNAs and of the functional implications of miRNA clusters in the silkworm.

Differential expression of microRNA-2b with potential target coding P25 in the fifth instar larvae posterior silk gland of the silkworm.

MicroRNAs (miRNAs) are a class of non-protein coding small RNAs that regulate a gene expression at the post-transcriptional level. Using in silico screening, we found that the 3'-untranslated regions of the P25 gene mRNA are perfectly complementary to nucleotides 2-8 at the 5' end of the miRNA-2b (miR-2b). The expression of miR-2b and the P25 gene in posterior silk gland of the fifth instar larval silkworm was investigated using real-time PCR detection method. The results indicated that expression of the P25 gene was very high in the posterior silk gland during the fifth instar larvae, whereas a level of miR-2b sharply decreased until reaching the lowest one on the 8th day. The expression patterns of miR-2b and P25 gene indicate that miR-2b might act as a fine-tuning regulator of expression of the P25 gene at the post-transcriptional level.

Cordyceps cicadae polysaccharides inhibit human cervical cancer hela cells proliferation via apoptosis and cell cycle arrest.

The present study presented the extraction and purification of polysaccharides from artificially cultured Cordyceps cicadae and wild Cordyceps cicadae by pre-soaking ultrasonic water extraction. The effects of different concentrations of polysaccharides on proliferation and cytotoxicity of Hela cells were detected by MTT and LDH methods. The results showed that the proliferation of Hela cells was inhibited by polysaccharides treatment (25 µg/mL-1600 µg/mL). The results of flow cytometry further confirmed that polysaccharides blocked the cell cycle in the S phase and promoted apoptosis. RT-qPCR and Western Blot were used to study the mRNA and protein expression of genes related to cell cycle and apoptosis signaling pathway. The results showed that polysaccharides treatment inhibited the expression of Cyclin E, Cyclin A and CDK2 and up regulated the expression of P53. Further, activation of Caspase cascade reaction, up regulation of death receptor, and the ratio of pro-apoptotic factor/anti-apoptotic factors, thus caused the cell cycle arrest and induced the apoptosis. The above research results lay a foundation for extending the anti-cancer effects of natural plant resources with low toxicity and high efficiency.

Transcriptome Analysis Reveals the Gene Expression Changes in the Silkworm (Bombyx mori) in Response to Hydrogen Sulfide Exposure.

Hydrogen sulfide (H2S) has been recognized for its beneficial influence on physiological alterations. The development (body weight) and economic characteristics (cocoon weight, cocoon shell ratio, and cocoon shell weight) of silkworms were increased after continuous 7.5 µM H2S treatment. In the present study, gene expression changes in the fat body of silkworms at the 5th instar larvae in response to the H2S were investigated through comparative transcriptome analysis. Moreover, the expression pattern of significant differentially expressed genes (DEGs) at the 5th instar larvae was confirmed by quantitative real-time PCR (qRT-PCR) after H2S exposure. A total of 1200 (DEGs) was identified, of which 977 DEGs were up-regulated and 223 DEGs were down-regulated. Most of the DEGs were involved in the transport pathway, cellular community, carbohydrate metabolism, and immune-associated signal transduction. The up regulated genes under H2S exposure were involved in endocytosis, glycolysis/gluconeogenesis, the citrate cycle (TCA cycle), and the synthesis of fibroin, while genes related to inflammation were down-regulated, indicating that H2S could promote energy metabolism, the transport pathway, silk synthesis, and inhibit inflammation in the silkworm. In addition, the expression levels of these genes were increased or decreased in a time-dependent manner during the 5th instar larvae. These results provided insight into the effects of H2S on silkworms at the transcriptional level and a substantial foundation for understanding H2S function.

A 14-amino acids deletion in BmShadow results to non-moult on the 2nd instar in the bivoltine silkworm, Bombyx mori.

The Bombyx mori Shadow gene (BmShadow) belongs to the superfamily of cytochrome P450 genes. To elucidate the function of the BmShadow gene and its association with diapause, we employed the CRISPR/Cas9 system to knock out the BmShadow gene in the bivoltine strain Qiufeng. The mutant (BmShadow-/-) was obtained in G2, exhibiting a 42-base deletion corresponded exactly to the amino acids regions from positions 155 to 168. The larvae of BmShadow-/- cannot moult at the pre-moulting stage of the 2nd instar. When the BmShadow-/- larvae were fed with 20E analogue at the late stage of the 2nd instar, they were rescued and developed into the 3rd instar. Rescue experiments indicated that the 20E concentration of BmShadow-/- larvae was significantly lower than that in WT larvae, and the 20E concentration of BmShadow-/- larvae which fed 20E analogue was restored to normal levels. Interestingly, the BmShadow-/- larvae could not moult on the 1st instar when they hatched from eggs after being stored at 5 °C for 40 days or after hibernation, suggesting that the 20E transported from the mother was partially consumed in the diapause maintenance phase. Our study confirmed that BmShadow is involved in 20E synthesis and a 14-amino acids region from position 155 to 168 was essential for its function, also there appears to be no other compensation pathway in vivo, which offered an important potential target locus for the control of silkworm development and the biological control of agricultural and forestry pests.

Computational identification and characteristics of novel microRNAs from the silkworm (Bombyx mori L.).

MicroRNAs (miRNAs) are a class of non-protein coding small RNAs that regulate expression of genes at post-transcriptional levels. Increasing evidence has shown that miRNAs play multiple roles in biological processes, including development, cell proliferation and apoptosis. Based on the conservation of miRNAs sequence, using a computational homology search based on genomic survey sequence analysis, a total of 16 novel miRNAs were identified and characteristics such as family and evolutionary conservation have been described. By using these newly identified miRNAs, the mRNA database of silkworm was blasted and 21 potential targets of miRNAs were detected. Most of these miRNA targeted genes were predicted to encode transcription factors. The semi-stem loop RT-PCR based assays were performed and found that silkworm miRNAs have diverse expression patterns during development.

Molecular cloning and characterization of hatching enzyme-like gene in the silkworm, Bombyx mori.

Hatching is the important process for the life of the metazoan, in which hatching enzyme (HE) plays a key role. In this paper, we cloned the full-length sequence of hatching enzyme-like cDNA from bluish-silkworm-eggs of Bombyx mori (BmHEL) by the method of in silico cloning, SMART cDNA synthesis and RACE-PCR technique. The BmHEL is 974 bp in length, and contains an ORF of 885 bp, encoding 294 amino acids residues. The deduced amino acid sequence of BmHEL has 30.3-47.1% identities to that of HE identified in the other species. Two similar signature sequences of HE gene family harbor in the BmHEL. The BmHEL gene structure is 6-exon-5-intron, and a promoter region with high scores has been predicted, which harbors some basal elements and some embryo-development related transcription factor binding sites. In the silkworm eggs at different developmental stages during incubation, the BmHEL transcripts can be detected and keep at a low level during the early stages, increase dramatically since 7th day of incubation, and reach to the maximum on 9th day. Change of BmHEL transcripts is in accordance with the process of embryo development and hatching, indicated that it plays an important role in these processes. Moreover, BmHEL transcript can be detected in the midgut and testis at larval stage, suggested that BmHEL may have other biological functions. To the best of our knowledge, this is the first report on HE gene in the Lepidoptera insects and will be helpful to provide a molecular basis for understanding the complicated mechanism underlying silkworm hatching.

Effect of Dietary Selenium Supplementation on Growth and Reproduction of Silkworm Bombyx mori L.

The effects of selenium (Se) on the growth and reproduction of the Lepidoptera insect, the silkworm, Bombyx mori L were investigated. Initially, the silkworms were divided into eight groups (150 larvae/group) on the basis of feeding with mulberry leaves saturated with different concentrations of Se (25, 50, 100, 125, 150, 175, and 200 µM) and control from the first day of the fourth instar larvae. After feeding, growth and reproductive performance of B. mori L. were investigated with standard techniques used in sericulture. After the data analysis, 50 µM of Se was recognized as the optimal level which positively influenced the growth and production, with prolonged stage of larvae, increased larval, cocoon, and pupal weights, and enhanced number of eggs laid by the female moth as compared to the control group. On the contrary, 200 µM of Se treatment displayed toxic to silkworm and induced significant decrease in the growth, cocoon production, and reproduction. The weight of the cocoon shell, the cocoon shell ratio, number of eggs produced, and fertilization ratio in all the Se-treated groups were lower than the control group. The present study indicated that lower levels of (50 µM) of Se can promote the larval and pupal growth of the B. mori L. resulting in the higher yield of cocoon crop and significantly influencing the fecundity, while high concentration was toxic to silkworm. Our data supply the novel application of Se which could be highly beneficial to sericulture farmers.

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.

Protein-DNA interactions in the promoter region of the gene encoding diapause hormone and pheromone biosynthesis activating neuropeptide of the cotton bollworm, Helicoverpa armigera.

Diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) are two crucial neuropeptides which regulate insect development and sex pheromone biosynthesis respectively. These peptides are encoded by a single gene, termed DH-PBAN gene. In this study, we characterized the promoter of the DH-PBAN gene in Helicoverpa armigera (Har). Transient transfection assays using a series of stepwise deletion fragments linked to the luciferase reporter gene indicate that the promoter contains multiple regulator domains that can activate and repress reporter gene expression. The fragment spanning -467 to -371 bp of the DH-PBAN promoter is an activator domain of transcription, whereas the region from -965 to -534 bp represses the promoter activity in the insect cell line BmN. Electrophoretic mobility shift assays demonstrate that at least two nuclear protein factors from the nuclear protein extracts of H. armigera suboesophageal ganglion, Har-DHMBP-1 and-2 (DH-modulator-binding protein) can specifically bind to the activating region. Furthermore, we characterized in detail that the nuclear protein factor Har-DHMBP-3 can specifically bind to a classical E-box, CAGCTG localized at positions -360 to -355 bp, a potential site for interaction with basic helix-loop-helix transcription factors. Mutation of this E-box results in a significant reduction of the promoter activity, suggesting it can modulate the previously identified activator domain. Taken together, multipartite cis-elements and transcription factors in the DH-PBAN promoter are involved in regulation of the gene expression.

Characterization of CIb1 gene promoter from silkworm, Bombyx mori.

The hemolymph chymotrypsin inhibitor b1 (CIb1) of silkworm, Bombyx mori, plays an important role in innate immunity. In order to study its encoding gene CIb1, five heterogeneous promoter fragments of 844 bp, 682 bp, 516 bp, 312 bp and 82 bp in length were cloned from genomic DNA of the p50 silkworm strain. Characterization of the CIb1 promoter was performed in vitro using the firefly luciferase gene as reporter. The results showed that CIb1 promoter fragments have transcription activities in the B. mori ovary-derived BmN cell line. The 82 bp fragment (-72 to +10 nt) containing the eukaryotic core promoter elements revealed a basic transcription activity. The Bm1 element, upstream the transcription initiation site, showed a positive regulation function to the CIb1 promoter. CIb1 promoter-like fragments from the genomic DNA of the tetra hybrid silkworm SujuxMinghu provided a natural deletion model for the study of the CIb1 promoter. In vitro analysis indicated that the 132 bp fragment from -517 nt to -386 nt upstream of the transcription initiation site strongly suppressed the transcription activity of the CIb1 promoter, suggesting that the 132 bp fragment harbours strong negative cis-acting elements. Infection of Bombyx mori nucleopolyhedrovirus (BmNPV) increased the activity of the CIb1 promoter, having provided another evidence to the function of CIb1 in the innate immunity of silkworm.

Transcriptome sequencing reveals potential mechanisms of diapause preparation in bivoltine silkworm Bombyx mori (Lepidoptera: Bombycidae).

In the bivoltine strain of the silkworm, Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause ability is determined by the environmental condition such as temperature and lightness that mothers experience during their own embryonic development. Diapause preparation is a crucial phase of this process; diapause-destined individuals undergo a series of preparatory events before the entry into developmental arrest. However, the molecular regulatory mechanisms of diapause preparation have largely remained unknown. In the present study, we sequenced the transcriptome of bivoltine silkworm Qiufeng's ovaries resulted in laying of diapause destined or non-diapause eggs, using high-throughput RNA-Seq technology. Differential expression analyses identified 183 genes with higher expression, and 106 with lower expression under diapause-inducing conditions. GO and KEGG analysis revealed that the enrichment of several functional terms related to peroxisome, glycerolipid metabolism, steroid biosynthesis, longevity regulating pathway - multiple species, three signaling transductions, insect hormone biosynthesis, and cytoskeleton components. We conducted a detailed comparison of transcript profile data of ovaries from diapause-inducing and non-diapause conditions, the results imply up-regulation of peroxisomal metabolism, triacylglycerides accumulation, cryoprotectant production, and ecdysteroid biosynthesis in diapause-inducing group. Differential expression of genes related to actin cytoskeleton implies the occurrence of shifts in cellular structure and composition between diapause-inducing and non-diapause-inducing groups. The Hippo and FOXO signaling pathways may play an important role in preparing for entering diapause. This study provides an insight into the molecular events of insect diapause, in particular for the preparatory phase.

bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro.

Based on bioinformatic analysis, we selected two novel microRNAs (miRNAs), bmo-miR-0001 and bmo-miR-0015, from high-throughput sequencing of the Bombyx mori larval posterior silk gland (PSG). Firstly, we examined the expression of bmo-miR-0001 and bmo-miR 12 different tissues of the 5th instar Day-3 larvae of the silkworm. The results showed that the expression levels of both bmo-miR-0001 and bmo-miR-0015 were obviously higher in the PSG than in other tissues, implying there is a spatio-temporal condition for bmo-miR-0001 and bmo-miR-0015 to regulate the expression of BmFib-L. To test this hypothesis, we constructed pri-bmo-miR-0001 expressing the plasmid pcDNA3.0 and pri-bmo-miR-0015 expressing the plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-0015-SV40]. Finally, the BmN cells were harvested and luciferase activity was detected. The results showed that luciferase activity was reduced significantly (P<0.05) in BmN cells co-transfected by pcDNA3.0 [ie1-egfp-pri-bmo-miR-0001-SV40] or pcDNA3.0 with pGL3.0 [A3-luc-Fib-L-3'UTR-SV40], suggesting that both bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of BmFib-L in vitro.