Introduction

Silkworm (Bombyx mori) is a member of the family Bombycidae within the order Lepidoptera. It has been domesticated for 5,700 years. Mulberry leaves are the major food source for B. mori [1]. Sericulture is a traditional industry in China. The world famous “Silk Road” has made great contributions to the world’s economy. China is a major silk production country, and its annual production of raw silk accounts for more than 78 % of the total raw silk in the world [2]. However, due to the long breeding cycle needed for the traditional breeding of B. mori varieties and the immaturity of the transgenic technology, it is difficult to satisfactorily meet the demand for increasing silk production only by the way of traditional breeding and improvement of the varieties of B. mori. Therefore, there is an urgent need to find a new and quick way for effectively promoting the development of sericulture industry.

Titanium dioxide nanoparticles (TiO2 NPs) have been widely used in many areas [3, 4], such as drug delivery [5], personal care [6], bacterial adhesion, and degermation [7]. They have also been used as food additives for improving the growth of livestock [8] and for increasing protein synthesis in animals [9]. TiO2 NPs have been demonstrated to promote carbon and nitrogen metabolisms [10–14]. Therefore, it is worthy to conduct an in-depth study for finding out whether they can improve fibroin synthesis in B. mori or not. In this study, we analyzed the gene expression profiles in larvae of B. mori fed with TiO2 NPs based on the digital gene expression (DGE) and studied the changes in fibroin synthesis after feeding the larvae with TiO2 NPs. The results will provide a useful and important guidance for enhancing fibroin synthesis in B. mori.

Materials and Methods

Insect and Chemicals

The larvae of a B. mori (strain: Suju × minghu) maintained in our laboratory were reared on mulberry leaves under 12 h light/12 h dark cycle. TiO2 NPs were prepared via the controlled hydrolysis of titanium tetrabutoxide. The details of the synthesis and characterization of TiO2 NPs were described in our previous reports [15, 16]. The average particle size of the TiO2 NPs powder suspended in 0.5 % (w/v) hydroxypropyl methycellulose (HPMC) K4 M solvent after 24-h incubation ranged from 5 to 6 nm. The mean hydrodynamic diameter of TiO2 NPs in HPMC solvent ranged between 208 and 330 nm (mainly 294 nm), and the zeta potential after 24-h incubation was 9.28 mV [16].

Method for Dissolving TiO2 NPs

TiO2 NPs powder was dispersed onto the surface of 0.5 % (w/v) HPMC, and then the solutions containing TiO2 NPs were treated using an ultrasonic technique for 30 min and mechanically vibrated for 5 min.

TiO2 NPs Feeding

Mulberry leave were soaked in 5 μg ml−1 TiO2 NPs. The soaked leaves were dried naturally at room temperature and used to feed the 5th instar larvae continuously from the newly exuviated larvae till mounting three times a day. The controlled larvae were fed with the mulberry leaves soaked in water. All the larvae were maintained at the temperature of 25 ± 0.5 °C and relative humidity (RH) of 75 ± 5 %. Each treatment was replicated three times with 30 larvae.

DGE Library Preparation, Sequencing, and Tag Mapping

DGE was performed on the silk gland samples collected at the 5th instar of larvae of B. mori fed with 5 μg ml−1 TiO2 NPs for 72 h. Sequence tag preparation was done with Illumina’s Digital Gene Expression Tag Profiling Kit according to the manufacturer’s protocol. A tag library was further amplified by PCR for 15 cycles, and the 95-bp fragments were purified by 6 % Tris-borate-EDTA (TBE) polyacrylamide gel electrophoresis. The single-stranded molecules were attached to the Illumina sequencing chip for sequencing. Sequencing-received raw image data were transformed by base calling into sequence data, called raw data or raw reads (see supplementary material for detailed methodology).

To map the DGE tags, the sequenced raw data were filtered to remove low-quality tags (tags with unknown nucleotide “N”), empty tags (no tag sequence between the adaptors), and tags with only one copy number (which might be resulted from sequencing errors). All the possible CATG + 17 nucleotide tags were created by using the B. mori genomic database and other NCBI data. All clean tags were mapped to the reference sequences, and only 1 bp mismatch was allowed. Clean tags mapped to reference sequences from multiple genes were filtered. The remaining clean tags were designated as unambiguous clean tags. The number of unambiguous clean tags for each gene was calculated and normalized to TPM [17, 18]. To compare the differences in gene expression, the tag frequency in each DGE library was statistically analyzed according to the method described by Audic and Claverie [19]. The false discovery rate (FDR) was used to determine the threshold P value in multiple tests. A FDR < 0.001 and an absolute value of the log2 ratio > 1 were used as the thresholds to determine significant differences in gene expression.

Total RNA Isolation, RT-PCR, and RT-qPCR Analysis

The larvae were dissected at 48, 96, 144, and 192 h, respectively, after being fed with TiO2 NPs. Posterior silk gland (PSG), midgut, and hemolymph were collected and stored at −80 °C. Total RNA was extracted from the silk gland using Trizol reagent (Takara, Dalian, China) and then treated with DNases to remove the potentially contaminated genomic DNA residues. The quality of the RNA was quantitated spectrophotometrically at 260 and 280 nm. cDNA was synthesized using PrimeScript RT-PCR kit (Takara) as described previously [20]. PCR amplification was performed using ExTaq HS polymerase (Takara) with primers and appropriate cycles. PCR products were analyzed on 1 % agarose gels.

The specific primers for the 11 genes of interest were listed in Table 1. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed on a ViiA 7 (ABI Applied Biosystems, Foster City, CA, USA) using the SYBR Premix ExTaq Kit in a total of 20 μl reaction system under the conditions as follows: denaturation at 95 °C for 1 min, followed by 45 cycles at 95 °C for 5 s, 55 °C for 10 s, and 72 °C for 10 s.

Table 1 The detail sequences of the primers for the genes selected for RT-qPCR analysis
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Western Blot Analysis

The silk gland samples were homogenized in lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). The samples were centrifuged at 15,800 × g for 10 min, and the supernatants were collected. Protein quantitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Western blot were carried out according to Gu et al. [21, 22]. The primary antibodies against α-Tubulin (Cell Signaling, Danvers, MA, USA) at 1:1,000 and Fib-L (Genscript, Piscataway, NJ, USA) at 1:5,000 were used, and the secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Beyotime, China) at 1:5,000 and goat anti-mouse IgG (Beyotime) at 1:1,000, respectively.

Histopathological Evaluation of Silk Gland

Histopathological photomicrographs of the PSG sections were prepared from the 5th instar B. mori after feeding with TiO2 NPs for 72 h. All histopathologic examinations were performed using the standard laboratory procedures. The silk gland was embedded in paraffin blocks, then sliced to thin section (5-μm thickness), and placed onto glass slides. After hematoxylin–eosin staining, the stained sections were evaluated by a histopathologist unaware of the treatments, using a light microscope (Nikon U-III Multipoint Sensor System, Japan).

Determination of Protease Activity

The larvae at 5th instar were electrically stimulated at 80 V, making them to reject their intestinal fluids, which were then used for protease activity assay according to the method reported previously [23, 24]. 25 μl of the intestinal fluid was diluted in 5 ml of deionized water and used as the crude enzyme. l ml of 1 % casein (pH 1.5) was used as the substrate and mixed with l ml of the crude enzyme. After incubation in a water bath at 28 °C for 20 min, 3.5 ml of protein precipitation solution (120 mmol l−1 TAC, 170 mmol l−1 NaAc, and 25 mmol l−1 HAC) was added, mixed well, and settled for 10 min to fully precipitate the undecomposed proteins. The mixture was centrifuged at 3,000 rpm for 20 min, and the supernatant was taken to measure OD275. For the control, the intestinal fluid was firstly mixed with the protein precipitation solution and then with the crude enzyme. One unit of enzyme activity was defined as the amount of enzyme needed to increase 0.01 at OD275 per min.

Determination of Contents of Amino Acids

The contents of amino acids in hemolymph were measured using Hitachi L-8800 high speed amino acid analyzer [25]. The hemolymph samples were loaded through an auto-sampler into the guard column for preseparation. The samples entered the separation column through which they were separated and then mixed in a mixer with acetone ninhydrin solution which was absorbed by the pump. The mixture was sent to reaction column where a deep blue solution was formed after the reaction completed. The separated blue solution was measured by a photoelectric colorimeter for the contents of amino acids in the hemolymph. The data were uploaded directly into the computer-aided workstation, and the contents of amino acids in the hemolymph were calculated.

Statistical Analysis

All the data were averaged from three independent measurements for each sample and expressed as mean ± SD. One-way analysis of variance (ANOVA) was carried out to compare the differences of mean data among the multigroups. Dunnett’s test was performed to compare each set data with the data of the control group. Statistical significance for all tests was judged at a probability level of 0.05 and 0.01 (P < 0.05; P < 0.01).

Results and Analyses

DGE Analysis of Gene Expression Profiles in Silk Gland

A total of 4,741 expressed genes were detected in the samples. Among them, 306 genes were differentially expressed, of which 137 were upregulated and 169 were downregulated. Among the genes involved in fibroin synthesis, 106 genes were significantly altered, 97 genes were upregulated and nine genes were downregulated (Table 2). Clustering based on similarity in gene expression revealed the difference in the transcriptional profiles of all differentially expressed genes in the TiO2 NPs-fed and control groups. Evaluating the physiological relevancies of gene expression profiles revealed that the most meaningfully enriched biological functions of the differentially expressed genes were associated with carbohydrate metabolism, lipid metabolism, transcription, translation, protein synthesis, protein processing, and protein transport (Table S1). The numbers of the known genes that were differentially expressed in the silk gland of B. mori fed with the TiO2 NPs were listed in Table 2.

Table 2 The numbers of known genes known differently expressed in the silk gland of the TiO2 NPs-fed larvae
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Quantitative Validation of the Data Acquired with DGE

Nine genes which are closely related to protein synthesis and hydrolysis in the silk gland were further analyzed by RT-qPCR (Table 3). Among them, aspartylglucosaminidase, the cathepsin L in Tribolium castaneum, and similar to SPRY domain-containing SOCS box protein 3, the enzymes involved in protein hydrolysis and degradation, were downregulated in silk gland of B. mori fed with TiO2 NPs (P < 0.05; Table 3). Lysyl-tRNA synthetase, cuticular protein glycine-rich 10, splicing factor arginine/serine-rich 6, serine protease inhibitor 28, and aspartate aminotransferase are protein synthetase and modifying enzymes. They were significantly upregulated in silk gland of B. mori fed with TiO2 NPs (P < 0.05) (Table 3). SGF-1 is an important transcription factor for fibroin synthesis [26, 27] and its expression was significantly upregulated in silk gland of B. mori fed with TiO2 NPs (Table 3). Results of RT-qPCR for all nine genes assayed were consistent with the data obtained from the DGE analysis in terms of either upregulation or downregulation. These results indicate that feeding of B. mori with TiO2 NPs results in upregulation of transcription of the genes related to fibroin synthesis and, in turn, might lead to an increase of fibroin synthesis.

Table 3 Comparison between fold difference with RT-qPCR and DGE assay in TiO2 NPs-treated group
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Expression Level of Fib-L in Silk Gland

RT-qPCR and semi-qPCR methods were used to measure the transcriptional levels of Fib-L, the gene encoding light chain of fibroin, at different time points after the TiO2 NPs feeding (Fig. 1). Using Actin 3 as internal reference, the transcription levels of Fib-L were determined by RT-qPCR. The results showed that the mRNA levels of Fib-L were 1.30-folds, 1.29-folds, 3.17-folds (P < 0.05), and 5.93-folds (P < 0.01) higher at 48, 96, 144, and 192 h after feeding with TiO2 NPs as compared with those at the corresponding time points in the control group (Fig. 1a). While the extent of the increases in the level of Fib-L was smaller at 48 h after feeding, it became remarkable at 144 and 192 h after feeding with TiO2 NPs.

Fig. 1
figure 1

The analysis of transcriptional level of Fib-L in silk gland of B. mori after being fed with TiO2 NPs. (a): RT-qPCR analysis; (b) Semi-qPCR; and (c) The quantitative optical density analysis of semi-qPCR. All data were normalized to the corresponding optical density of the reference gene, Actin 3. The data shown are representative of at least three independent experiments and were analyzed by Dunnett's test (*, p<0.05; **, p<0.01)

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Semi-qPCR method was also used to measure the transcription levels of Fib-L (Fig. 1b). In comparison with those of the control, the transcriptional levels of Fib-L were increased by 1.00-folds, 1.21-folds, 1.76-folds (P < 0.05), and 1.36-folds (P < 0.05), respectively, at the corresponding time points after feeding (Fig. 1c), indicating that the two PCR methods give the consistent results and that the results obtained are reliable.

The expression levels of Fib-L protein were analyzed by Western blot (Fig. 2a). Compared with those in the control group, feeding larvae with TiO2 NPs caused the increases in Fib-L protein levels by 1.28-, 1.38-, 1.27- and 1.51-folds, respectively, at the indicated time points with the highest increase occurring at 192 h after feeding (Fig. 2b). Together, these data indicate that the TiO2 NPs feeding can increase the expression of genes related to the synthesis of silk proteins not only at the mRNA level but also at protein level.

Fig. 2
figure 2

The expression of Fib-L protein in silk gland of B. mori fed with TiO2 NPs. (a) Western blot result; (b) The gray value analysis of western blot. All data were normalized to those of the reference protein, α-Tubulin. The data shown are representative of at least three independent experiments

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Histopathological Evaluation

Histopathological photomicrographs of the PSG section were shown in Fig. 3. Although both the control and TiO2 NPs-fed silk gland samples showed normal architecture with thin walls and full gland lumen, the gland lumen of the control was not fully filled with proteins (Fig. 3a), while that of TiO2 NPs-treated B. mori was full of proteins (Fig. 3b). These results showed that the TiO2 NPs feeding increased the protein content in the gland lumen and facilitated fibroin synthesis.

Fig. 3
figure 3

Histophysiological examination of the posterior silk gland tissue at 72 h fed with TiO2 NPs. (a) Control; (b) TiO2 NPs. Yellow arrow indicates proteins in the lumen of the posterior silk gland

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Protease Activity in Midgut

The amino acids used for fibroin synthesis come from mulberry leaves, which are digested by the proteases present in the B. mori digestion system, absorbed in the midgut and stored in the body for fibroin synthesis [28, 29]. The protease activity was assayed in midgut of the larvae after being fed with TiO2 NPs for different time points (Fig. 4). The results showed that the protease activities in the TiO2 NPs-fed larvae at 48, 96, 144, and 192 h were 2.44-folds (P < 0.05), 1.77-folds (P < 0.05), 2.46-folds (P < 0.01), and 1.33-folds higher than those at the corresponding time points of the control group, respectively, (Fig. 4). In the TiO2 NPs-fed larvae, the protease activities were increased and then decreased over feeding periods from 48 to 192 h, with the highest activity at 96 h. In the control, the highest protease activities were at 192 h. Proteases are the major digestive enzymes in the midgut, and their activities there directly affect the absorption and utilization efficiency of the proteins derived from mulberry leaves [30]. Thus, the increased protease activities in the midgut of B. mori following TiO2 NPs feeding would be of great significance for improving the absorption and utilization of proteins in the mulberry leaves.

Fig. 4
figure 4

The activities of proteases in the midgut of B. mori fed with TiO2 NPs. The data shown are representative of at least three independent experiments and were. analyzed by Dunnett's test (*, p<0.05; **, p<0.01)

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Amino Acid Contents in the Hemolymph

Fibroin mainly contains four amino acids, namely, glycine (GLY), alanine (ALA), serine (SER), and tyrosine (TYR), which account for 90 % of the total silk protein. Among them, GLY and ALA account for 42.80 % and 32.40 % of silk protein, respectively [31]. To demonstrate the effect of TiO2 NPs feeding on the transport of amino acids, the contents of these four amino acids in the hemolymph were determined after continuously feeding B. mori with TiO2 NPs (Table 4).

Table 4 The contents of four main amino acids in the hemolymph of B. mori fed with TiO2 NPs
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It has been showed that the proteins intake from mulberry leaves are mainly used for the synthesis of body proteins in the earlier period of the 5th instar, while these uptake proteins are mainly used for the synthesis of fibroin in the later period of the 5th instar stage [29, 32]. The contents of main amino acids for the synthesis of fibroin were increased in the earlier period of the 5th instar. For example, the levels of GLY and ALA of TiO2 NPs-fed larvae were 5.03 % and 5.52 % higher than those in control at 48 h, respectively. Thereafter, the contents of these two amino acids declined in the later period. By 192 h, they were declined by 2.72 % and 3.79 %, respectively (Table 4 ). These data indicated that during the earlier period, more amino acids were absorbed and stored in the body from the digested mulberry leaves and were used for fibroin synthesis in the later periods, demonstrating that TiO2 NPs feeding can improve the transport of these amino acids.

Discussion

DGE Data Analysis

Silk gland is an organ of B. mori specifically for fibroin synthesis. Morphologically, this gland can be divided into three parts, i.e. the anterior silk gland (ASG), the middle silk gland (MSG), and PSG. The fibroin is mainly synthesized in the PSG [29]. In order to study the effect of TiO2 NPs feeding on the transcription of the genes in the PSG of larvae at the 5th instar, DGE libraries were prepared and analyzed.

Protein synthesis is a process during which transamination of amino acids takes place with the consumption of cellular energy and the utilization of carbon skeletons. Carbon skeletons and cellular energy derived from carbohydrate metabolism provide both the precursors and energy required for fibroin synthesis [33]. Our DGE data showed that 20 genes were significantly upregulated in B. mori after being fed with TiO2 NPs (Table 2). For example, ß-galactosidase with a log2 value of 1.19 in DGE data (Table S1), an enzyme that hydrolyzes lactose into glucose and galactose, is involved in the digestion and absorption of lactose [34] and may improve the digestion and absorption of lactose. ß-Fructofuranosidase with a log2 value of 2.57 in DGE data (Table S1) is a fructosyltransferase converting disaccharides into optical monosaccharides [35] and may increase the ability of B. mori to convert disaccharides into monosaccharides. Fructose-1, 6-bisphosphatase with a log2 value of 1.13 in DGE data (Table S1) converts fructose-l, 6-diphosphate to fructose-6-phosphate, a key step in gluconeogenesis [36], indicating a likely increased gluconeogenesis following the TiO2 NPs feeding. The upregulation of these three genes indicates that feeding B. mori with TiO2 NPs may result in the increased synthesis and utilization of carbohydrates.

The precursors for the biosynthesis of carbohydrates and proteins are also derived from lipid metabolism pathways, which are closely linked to carbohydrate and protein metabolism pathways [37]. DGE data showed that 13 genes involved in the lipid metabolism were also differentially expressed and their expression levels were all upregulated after the TiO2 NPs feeding (Table 2). For instance, lipase 1-like with a log2 value of 1.27 in DGE data (Table S1) is a lipase catalyzing the hydrolysis of its natural substrates, lipids, to form fatty acids, glycerol, and mono- or di-esters [38], indicating that lipid hydrolysis might be enhanced. Integument esterase 1 with a log2 value of 2.03 in DGE data (Table S1) is an esterase hydrolyzing lipids into fatty acids and alcohols [39], showing that more ester molecules could be hydrolyzed to acids and alcohols. The upregulation of these two genes indicates that TiO2 NPs feeding may provide more precursor molecules available for fibroin synthesis.

Fibroin synthesis is complicatedly regulated by a number of biochemical processes at different levels [40]. Among them, aspartylglucosaminidase with a log2 value of −3.59 in DGE data (Table 3) is a hydrolase, which hydrolyzes aspartate [41], showing that the hydrolysis of aspartate may be reduced. The cathepsin L with a log2 value of −8.11 in DGE data (Table 3) in T. castaneum is a protease in the hydrolyzed tissues [42, 43], indicating a probably reduced hydrolysis of tissue proteins. Similar to SPRY domain-containing SOCS box protein 3 with a log2 value of −1.24 in DGE data (Table 3) has been shown to be involved in the ubiquitination of protein, a process during which ubiquitin is transferred a protein and “labels” it. The changes in the expression of this protein will change the binding of ubiquitin to the target protein to be degraded [44, 45] and may lead to a reduced degradation of proteins via ubiquitination. Thus, the downregulation of these three genes would result in a reduced protein metabolism.

Lysyl-tRNA synthetase with a log2 value of 1.21 in DGE data (Table 3) is responsible for selecting the correct lysine and distributing it to lysyl-tRNAs for protein synthesis in the ribosomes [46] and may result in an increase in lysyl-tRNA and a more efficient transfer of lysine. Cuticular protein glycine-rich 10 with a log2 value of 2.07 in DGE data (Table 3) is involved in glycine synthesis in epidermis [47], suggesting that more glycine molecules are synthesized in epidermis. Splicing factor arginine/serine-rich 6 with a log2 value of 2.07 in DGE data (Table 3) can shear and modify arginine and serine to make them active [48], suggesting that more arginine/serine molecules can be sheared or modified. Serine protease inhibitor 28 with a log2 value of 2.14 in DGE data (Table 3) is an effective inhibitor of serine proteases [49], suggesting that the degradation of serine might be reduced, and thus, the level of serine will be maintained. Aspartate aminotransferase with a log2 value of 1.12 in DGE data (Table 3) is a transaminase actively involved in the transfer of amidogen in aspartic acid synthesis [50], suggesting that during the synthesis of aspartic acid, the transfer of amidogen might be increased. The upregulation of these genes may lead to an increased protein synthesis. These RT-qPCR analyses are consistent with the DGE analysis and further demonstrate that feeding B. mori with TiO2 NPs could increase the transcription of genes related to fibroin synthesis.

Expression of Fib-L and SGF-1 Genes Important for Fibroin Synthesis

Silk gland is an important organ specifically for the synthesis of fibroin in B. mori [51, 52]. Fibroin is a complex protein, with a basic structural unit comprising of fibroin heavy chain (Fib-H), Fib-L, and P25 protein [53] at a molar ratio of 6:6:1 [54]. A previous study showed that these fibroin components of fibroin are mainly expressed in the PSG [55]. The Fib-L gene was highly expressed in the silk gland in various stages of the larvae, particularly in the 5th instar stage, the full appetite staged during which a large amount of mulberry leaves is consumed by larvae. By contrast, its expression was almost completely shut down during various periods of dormancy and the metamorphic period [26]. These expression patterns are regulated by the developmentally specific transcription factors and the tissue-specific factors [26]. After feeding B. mori with TiO2 NPs, the major specific transcription factor, SGF-1, was not detected in the DGE analysis. However, in RT-qPCR results, its level in the TiO2 NPs-fed larvae was about 2.21-folds that of the control (Table 3). SGF-1 is an important transcriptional factor that specifically regulates the expression of genes that promote fibroin synthase in PSG. SGF-1 was thought to be required initially for the development of silk gland and, subsequently, for the utilization in the transcription control of genes encoding silk protein [26, 27]. Its upregulation induced by feeding with TiO2 NPs indicates that this compound can increase the transcription and expression of the genes that promote the synthesis of silk protein. In addition, histological study indicated that proteins in the gland lumen of TiO2 NPs-treated larvae were more abundant than those of the control (Fig. 3). This is a line of clear evidence that TiO2 NPs feeding increases the synthesis of silk proteins.

Protease Activity and Amino Acid Contents

When B. mori are fed with the mulberry leaves, they digested the mulberry leaves in their midgut, where the mulberry leaf-derived amino acids are absorbed and then transported mainly to the hemocoele [56]. The protease activity in midgut of B. mori fed with TiO2 NPs showed that there was an increase as compared with that in the control (Fig. 4), suggesting that the TiO2 NPs-fed larvae have a higher efficiency of protein absorption and transport. Previous studies have shown that a majority of amino acids in the larvae need to be transported to its silk gland for fibroin synthesis at 0–72 h of the 5th instar [29, 32, 57]. In this study, it was found that in larvae at the 5th instar, higher levels of amino acids were present in hemolymph, and higher contents of amino acids were detected in the larvae at the first 72 h after feeding with TiO2 NPs. After feeding for 48 h, the contents of amino acids in the TiO2 NPs-fed larvae began to decline gradually and were lower than those in the control at 192 h after the feeding (Table 4). 72 h after reaching the 5th instar, there were an increased transport of amino acid and an increased utilization of amino acids, which may be associated with the increased fibroin synthesis. The changes in the contents of amino acids imply that TiO2 NPs feeding has resulted in a higher efficiency of transport and utilization of amino acids.

In summary, in this study, the differential expression profiles of genes and proteins in the silk gland of B. mori fed without and with TiO2 NPs were analyzed and compared using DGE, RT-qPCR, semi-qPCR, and Western blot analysis. These analyses revealed that feeding B. mori with TiO2 NPs resulted in: (a) upregulation of important genes involved in the synthesis and utilization of both carbohydrates and lipids; (b) downregulation of several genes involved in hydrolysis and degradation of proteins; (c) upregulation of genes involved in the synthesis of four amino acids (GLY, ALA, SER, and TYR), the key amino acid precursors for fibroin synthesis; (d) increased expression of Fib-L, a key component of fibroin, and SGF-1, an important transcriptional factor involved in the regulation of fibroin synthesis; and (e) increased protease activity and higher contents of amino acids in hemolymph of B. mori, making the amino acids amply available for fibroin synthesis. The combined actions of these effects caused by TiO2 NPs feeding lead to increased fibroin synthesis in silk gland. These findings indicate that TiO2 NPs feeding can significantly improve the efficiency of absorption and utilization of amino acids from the feed and thus could be a new way to increase the fibroin synthesis in B. mori. Thus, the nanomaterials, such as TiO2 NPs, may be applied for effectively promoting the development of sericulture industry in the future.