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.

BmINR and BmAC6 genes involve in diapause regulation via the insulin/IGF signaling pathway in the silkworm (Bombyx mori).

Diapause of the silkworm (Bombyx mori) is an important ecological adaptation strategy regulated by multiple signaling pathways. As an evolutionarily conserved signaling pathway, the insulin/IGF signaling (IIS) pathway is essential in regulating lifespan, energy accumulation, and stress resistance in diapause insects. However, the regulatory mechanism of IIS on diapause in B. mori is still not fully understood. To investigate the role of the IIS pathway in regulating diapause, we first analyzed the transcription levels of the insulin receptor (BmINR) and its downstream gene adenylate cyclase 6 (BmAC6). The diapause-terminated eggs of a bivoltine strain QiuFeng (V2-QF) were incubated at 25 °C in natural room light for preparing diapause egg producers (DEPs) and at 17 °C in total darkness for preparing non-diapause egg producers (NDEPs), respectively. Then we investigated the effects of BmINR and BmAC6 on diapause phenotype and expression of diapause-related genes by RNA interference (RNAi) and overexpression techniques. The results showed that the mRNA expression levels of BmINR and BmAC6 in the head and ovary of NDEPs were higher than those in DEPs during the early and middle pupal stages. Furthermore, when BmINR was knocked down in the NDEPs, approximately 14.43% of eggs were in light red color and subsequently changed into gray-purple color after 48 h post-oviposition, then stayed in a diapause state. On the other hand, overexpression of BmINR or BmAC6 via recombinant baculoviruses did not cause any obvious phenotypic alterations in NDEPs, but it upregulated the expression of genes related to carbohydrate metabolism, which provides energy for embryonic growth and development. Therefore, it can be concluded that BmINR and BmAC6 genes regulate embryonic diapause in bivoltine B. mori.

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.

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.

Bombyx mori miR-2845 represses the expression of fibroin light chain gene both in vitro and in vivo.

To study the regulatory function of Bombyx mori (B. mori) miRNAs (bmo-miR) on the expression of fibroin light chain gene (BmFib-L), the 3'UTR of BmFib-L mRNA was used as the target for online prediction of miRNAs from miRBase using RNAhybrid Software, and miR-2845 was screened out. First, the expression profiles of miR-2845 and BmFib-L in larvae of the 5th instar were analyzed by Real-time quantitative PCR (RT-qPCR). Then recombinant plasmids (pcDNA3.0-pre-miR-2845 and pGL3.0-BmFib-L) were constructed to use for the expression of miR-2845 and BmFib-L 3'UTR, respectively. Cellular-level functional verification of miR-2845 on BmFib-L was carried out using multiple experimental methods (including dual luciferase reporter vectors, artificially synthesized mimics and inhibitors, and target site mutations). Finally, in vivo functional verification was performed by injecting the recombinant vector in 5th instar larvae. BmFib-L expression levels were detected using RT-qPCR in the posterior silk glands (PSG) of the injected larvae. Results showed that the expression of miR-2845 increased between the 1st and 5th day in 5th instar larvae, but began to decline on the 5th day, while the expression of the target gene BmFib-L increased sharply. This suggests that miR-2845 and BmFib-L expression levels show opposing trends, implying a negative regulatory relationship. In BmN cells, miR-2845 significantly down-regulated the expression of BmFib-L; the inhibitory effect of miR-2845 on BmFib-L was disappeared after mutation of the targeting site on 3'UTR of BmFib-L; in individuals, miR-2845 significantly down-regulated BmFib-L expression levels. Our results provide new experimental data for clarifying the molecular regulation mechanism of silk protein expression.

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.

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.

Semaphorin-1a-like gene plays an important role in the embryonic development of silkworm, Bombyx mori.

Fuyin-lethal red egg (Fuyin-lre) is a red egg mutant discovered from the germplasm resource Fuyin of Bombyx mori. The embryo of Fuyin-lre stops developing at the late stage of gastrulation due to chromosome structural variation. In this work, precise mutation sites at both ends of the mutated region were determined, and two inserted sequences with lengths of 1232 bp and 1845 bp were obtained at both ends of the mutation region. Interestingly, a bmmar1 transposon was detected in the inserted 1845 bp sequence. Bmmar1 possesses features of the Tcl/mariner superfamily of transposable elements (TEs), which belongs to class II TEs that use a DNA-mediated "cut and paste" mechanism to transpose. This finding suggests that Fuyin-lre mutation might be related to the "cut and paste" action of bmmar1. The mutation resulted in the deletion of 9 genes in the mutation region, of which the red egg gene re (BMSK0002766) did not affect embryonic development of B. mori, and the BMSK0002765 gene was unexpressed during the early stage of embryonic development. The RNA interference results of the remaining 7 genes suggest that the semaphorin-1a-like gene (BMSK0002764) had a major contribution to the embryonic lethality of Fuyin-lre.

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.

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.

The role of N6-methyladenosine modification on diapause in silkworm (Bombyx mori) strains that exhibit different voltinism.

N6-methyladenosine (m6 A) plays a key role in regulating gene expression in myriad organisms. Diapause is an important plastic phenotype that allows insects to survive under specific environmental conditions. However, the diapause molecular mechanism remains unknown. In this study, we analyzed the phylogenetics of genes related to the m6 A modification complex in the silkworm (Bombyx mori) based on identified sequences from other organisms. We detected the expression of these genes during different developmental phases from four strains with different voltinism. We also determined total m6 A content in cells treated with different diapause hormone concentrations or eggs exposed to hydrochloric acid. Our data revealed that m6 A-modification-related gene expression and m6 A content were greater in diapause-destinated compared to nondiapause-destined strains. Our findings suggest that m6 A modification may provide significant epigenetic regulation of diapause-related genes in the silkworm.

The silkworm (Bombyx mori) neuropeptide orcokinin is involved in the regulation of pigmentation.

The natural colorful cuticles of insects play important roles in many physiological processes. Pigmentation is a physiological process with a complex regulatory network whose regulatory mechanism remains unclear. Bombyx mori pigmentation mutants are ideal materials for research on pigmentation mechanisms. The purple quail-like (q-lp) and brown quail-like (q-lb) mutants originated from plain silkworm breeds 932VR and 0223JH respectively exhibit similar cuticle pigmentation to that of the quail mutant. The q-lp mutant also presents a developmental abnormality. In this study, genes controlling q-lp and q-lb mutants were located on chromosome 8 by positional cloning. Then the neuropeptide gene orcokinin (OK) was identified to be the major gene responsible for two quail-like mutants. The B. mori orcokinin gene (BommoOK) produces two transcripts, BommoOKA and BommoOKB, by alternative splicing. The CRISPR/Cas9 system and orcokinin peptides injection were used for further functional verification. We show a novel function of BommoOKA in inhibiting pigmentation, and one mature peptide of orcokinin A, OKA_type2, is the key factor in pigmentation inhibition. These results provide a reference for studying the function of orcokinin and are of theoretical importance for studying the regulatory mechanism of pigmentation.

Bom-miR-2805 upregulates the expression of Bombyx mori fibroin light chain gene in vivo.

MicroRNAs (miRs) are inner regulatory RNAs mainly by regulating expression of genes at the posttranscriptional level. To investigate the regulatory function of Bombyx mori (B. mori) fibroin protein genes, the mRNA 3'-untranslated region (3'-UTR) of fibroin light chain gene (BmFib-L) was used as the target and one miRNA, miR-2805 was predicted by using the Software. miR-2805 expression plasmid pcDNA3.0[ie1-egfp-pre-miR-2805-SV40] and BmFib-L 3'-UTR plasmid pGL3.0[A3-luc-Fib-L-3'-UTR-SV40] were constructed, respectively. The mentioned plasmids were cotransfected in BmN cells, and the regulatory function of miR-2805 on BmFib-L was detected by assay of dual luciferase activities, as well as synthesized mimic and inhibitor of miR-2805. The results revealed that miR-2805 significantly downregulated the expression of BmFib-L in BmN cells. To validate the function of miR-2805 in vivo, cultured silk glands or larvae were injected with solution containing pcDNA3.0[ie1-egfp-SV40], pcDNA3.0[ie1-egfp-pre-miR-2805-SV40], mimic, inhibitor respectively. BmFib-L expression was analyzed by quantitative reverse transcription polymerase chain reaction using total RNAs extracted from silk glands. The results showed that miR-2805 significantly upregulated the expression of BmFib-L in both cultured tissues and individuals. To find out how miR-2805 differentially regulates BmFib-L expression in cells and tissues or individuals, we analyzed the expression level of transcription factors (TFs) involved in expression of silk protein genes. The results showed that miR-2805 upregulated the expression of TFs BmAwh and Bmdimm. These results suggest that miR-2805 may up-regulate the expression of BmFib-L interaction with BmAwh and/or Bmdimm in vivo. These findings are beneficial to clarify the molecular mechanism of miRNAs in regulating B. mori silk protein biosynthesis.

Expression profile of several genes on ecdysteroidogenic pathway related to diapause in pupal stage of Bombyx mori bivoltine strain.

Ecdysone is involved in regulation of embryonic diapause in the silkworm, Bombyx mori. However, its mechanism still remains unclear. To explore the role of ecdysteroidogenic pathway (EP) genes in diapause process of bivoltine B. mori, the eggs of "Qiufeng", a bivoltine strain, were used as the study materials and arranged into diapause eggs producers (DEPs) and non-diapause eggs producers (NDEPs), respectively. The differential expression of EP genes between two groups was analysed during the early pupal stage. The expression of Shadow was significantly increased in the NDEPs in day-3 pupae and reached the peak simultaneously, indicating that Shadow was in coincidence with diapause process. To validate this hypothesis, a repression of Shadow by RNA interference was performed in day-2 pupae of NDEPs. The expression of Shadow was downregulated by RNAi, and ßFtz-F1, a downstream gene of EP, was also decreased. Furthermore, the genes encoding the kynurenine-synthetase were upregulated in the ovary, and Brown, AdenoK which link Shadow to the kynurenine-synthase gene were also upregulated in the fat body. The progeny eggs appeared a light purple colour at 48 h after oviposition, revealing a certain tendency to diapause. We speculate that inhibition of Shadow upregulates 3-hydroxy-kynurenine synthesis by increasing the expression of Brown and AdenoK. In addition, Shadow was cloned, and expressed in E. coli for further functional study of Shadow protein. Our study provided insight into the role of EP genes in the process of diapause of B. mori.

Expression analysis and functional identification of several genes related to diapause in Bombyx mori.

Diapause is an important characteristic of insects used to adapt to extreme changes in environmental conditions. Embryonic diapause of the bivoltine silkworm (Bombyx mori) is determined by environmental conditions experienced by the mother while in the embryo stage. If they are incubated at 25°C with natural light, their progenies will be diapause-destined. If they are incubated at 17°C in darkness, their progenies will be non-diapause-destined. The molecular mechanism of diapause remains unknown. In the present study, we analyzed two downregulated genes (BGIBMGA003835, BGIBMGA012335) and two upregulated genes (BGIBMG012996, BGIBMG002426) related to carbohydrate metabolism, verified differentially expressed in ovaries and heads of 1-day-old fifth-instar larvae to 6-day pupae by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). In line with published data, the expression level of these genes in larvae were generally lower than in pupae. We further analyzed the expression levels of the four genes in BmN cells that had been treated with various concentrations of diapause hormone (DH). It demonstrated that the expression of these genes was affected by DH. Knockdown of the selected genes in non-diapause-destined female pupae changed the fate of the progeny from non-diapause- to daipause-destined, as seen by the appearance of diapause eggs. Our study provides insight into the molecular mechanism of diapause in B. mori.

In vitro expression and functional characterization of NPA motifs in aquaporins of Nosema bombycis.

Nosema bombycis contains functional aquaporins (NbAQPs), which are key targets for exploring the mechanism of N. bombycis infection; however, the regulation of these NbAQPs remains unknown. The two highly conserved asparagine-proline-alanine sequences (NPA motifs) play important roles in AQP biogenesis. As part of this study, we constructed a series of NbAQP mutants (NbAQP_NPA1, NbAQP_NPA2, and NbAQP_NPA1,2) and expressed them in BmN cells. The results showed that mutations in either NPA motif or in both NPA motifs did not affect NbAQP expression in vitro. After expression in Xenopus laevis oocytes, those injected with wild-type NbAQP rapidly expanded, whereas oocytes injected with NbAQP_NPAs did not significantly change in size. The associated water permeability (pf) of NbAQP_NPAs was significantly reduced five-six times compared to that of wild-type NbAQP. These results indicated that NPA motifs are necessary for the water channel function of AQPs in N. bombycis. The present study shows for the first time that the NbAQP NPA motif has an impact on the water permeability of aquaporin in N. bombycis, thereby providing a platform for further research into the mechanisms underlying the regulation of NbAQP expression.

Inhibition of expression of BmNPV cg30 by bmo-miRNA-390 is a host response to baculovirus invasion.

Bombyx mori larvae exhibit in vivo defensive reactions immediately after invasion by a virus. One of these defense systems is to express appropriate microRNAs (miRNAs) to respond to the infection. A novel Bombyx mori-encoded miRNA, bmo-miR-390, was identified previously by high-throughput sequencing. Based on bioinformatic predictions, the Bombyx mori nuclear polyhedrosis virus cg30 gene (BmNPV-cg30) is one of the target genes of bmo-miR-390. In this study, expression vectors with an enhanced green fluorescence protein (EGFP) or a luciferase (luc) reporter gene together with bm-miR-390 or the cg30 3' UTR were constructed and used to co-transfect BmN cells. Using a dual luciferase reporter (DLR) assay, we found that bmo-miR-390 significantly downregulates the expression of BmNPV-cg30 (P < 0.05) in vitro. Moreover, artificially synthesized bmo-miR-390 mimics enhanced the regulatory effect of bmo-miR-390, while an inhibitor eliminated the inhibitory effect. These results show for the first time that bmo-miR-390 can effectively downregulate the expression of BmNPV-cg30 in BmNPV-infected BmN cells.

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.