Introduction

Silkworm (Bombyx mori) belongs to Lepidoptera (Bombycidae) and is an economically important insect. It was domesticated from wild B. mori over 5700 years ago [1]. B. mori is a complete metamorphosis insect with four stages of development of about 50 days: egg, larva, pupa, and adult. The adult insect lays about 500 eggs per animal and is designated as the model insect of Lepidoptera since 2002 [2]. In 2003, the complete genome of the insect was sequenced, and the sequence information has been widely used in genetics, developmental biology, ecology, toxicology, cell biology, and biochemistry studies [3].

Titanium dioxide nanoparticles (TiO2 NPs) are widely used as an additive in the food industry, cosmetics, electronics, coatings, medical products, and animal husbandry [4–7]. In animal husbandry, TiO2 NPs are used to promote animal growth [8] and protein synthesis [9]. Previous studies found that TiO2 NPs can improve the growth and antioxidation capacity of plants [10, 11] and enhance carbon and nitrogen metabolism and increase the resistance of spinach [11]. In Drosophila melanogaster, ingestion of TiO2 NPs up to a dose of 200 μg/mL during the larval stage did not affect the development or survivorship [12]. Zhang et al. found that feeding B. mori with 5 mg/L of TiO2 NPs resulted in increased weight and leaf-silk conversion, improved activity of the midgut proteases for more efficient nutrient uptake, and increased protein synthesis and metabolism [13]. Previous studies have shown that more than 90 % of B. mori proteins were allocated to the silk gland and the ovaries for silking and oviposition [14]. Increased silking has been observed after TiO2 NPs feeding [13]. However, whether the feeding will affect the reproduction activity of B. mori remains unclear.

In the present study, gonad development, oviposition, and expression of reproduction-related genes were investigated after feeding B. mori with a low concentration of TiO2 NPs (5 mg/L) to understand the effect of TiO2 NPs on fecundity.

Materials and Methods

Insect and Chemicals

The larvae of B. mori strain (Suju, Minghu) maintained in our laboratory were reared on mulberry leaves under a 12-h light/12-h dark cycle. TiO2 NPs were prepared via the controlled hydrolysis of titanium tetrabutoxide. The detailed 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 methylcellulose (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 was 9.28 mV [16].

Method for Suspending TiO2 NPs

TiO2 NPs powder was dispersed into 0.5 % (w/v) HPMC, and the mixture containing TiO2 NPs was treated with ultrasonication for 30 min and mechanically stirred for 5 min to suspend the compound.

TiO2 NPs Feeding

Mulberry leave were soaked in 5 mg/L TiO2 NPs. The soaked leaves were dried naturally at room temperature and used to feed the 5th instar larvae continuously from exuviation till mounting, three times a day. The control larvae were fed with the leaves soaked in water. Female and male B. mori in the TiO2 NPs treatment and control groups were reared separately, and each treatment was repeated three times with 30 larvae.

Sample Preparation and Total RNA Isolation

The larvae were dissected at mature silkworm after being fed with TiO2 NPs, and the ovaries and testes were collected and stored at −80 °C. Total RNA was extracted from the samples using TRIzol reagent (Takara Bio Inc., Dalian, China) and then treated with DNases to remove the potentially contaminated genomic DNA. The integrity and quality of the RNA were assessed by formaldehyde agarose gel electrophoresis, and the concentration was quantitated spectrophotometrically at 260 and 280 nm.

Histopathological Evaluation

The ovaries and testes were histopathologically examined using the standard laboratory procedures. The samples were embedded in paraffin, sliced to thin sections (5-μm thick), and mounted onto glass slides. After hematoxylin-eosin staining, the sections were evaluated by a histopathologist unaware of the treatments, using a light microscope (Nikon U-III Multi-point Sensor System, Nikon, Tokyo, Japan).

RT-qPCR Analysis

The primers specific for the twelve genes of interest are listed in Table 1. Reverse transcription polymerase chain reaction (RT-qPCR) was performed on a ViiA 7 PCR machine (ABI Applied Biosystems, Foster City, CA, USA) using the SYBR Premix Ex Taq kit (Takara Bio Inc., Dalian, China) in a total of reaction of 20 μL under conditions as follows: denaturation at 95 °C for 1 min, followed by 45 cycles of denaturing at 95 °C for 5 s, annealing at 55 °C for 10 s, and extension at 72 °C for 10 s.

Table 1 Primers of twelve genes selected for RT-qPCR analysis
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Statistical Analysis

All the data were averaged from three independent measurements and expressed as mean ± SD. One-way analysis of variance (ANOVA) was carried out to compare the differences of means among the multi-groups. Dunnett’s test was performed to compare the data with 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 Analysis

Effect of TiO2 NPs Feeding on Gonad Development

In order to investigate the effect of TiO2 NPs on gonad development, ovaries and testes of mature silkworm were dissected and examined under a microscope. As shown in Fig. 1a, the morphology of the ovaries and testes was normal in control B. mori and B. mori fed with TiO2 NPs, but the sizes of the ovaries and testes were larger in TiO2-NPs-fed B. mori. Histopathological observations showed that the animals in the control and treatment groups had differentiating sperm and egg cells with normal morphology and thin wall (Fig. 1b). After the TiO2 NPs treatment, the ovaries showed denser oocyte differentiation and formation compared with those of the control (Fig. 1b). However, no significant difference was observed in testes (Fig. 1b), indicating that TiO2 NPs exhibited stronger impact on ovaries. Gonad development is also regulated by endocrine hormones secreted under external stimuli. After TiO2 NPs treatment, gonadosomatic indexes (GSIs) were significantly higher than in the control (Fig. 2) with a 20.03 % increase in male and 24.89 % increase in female. These results were consistent with the data in Fig. 1 and showed that TiO2 NPs promote not only the growth of gonad but also the differentiation and formation of eggs in the ovary.

Fig. 1
figure 1

Effects of TiO2 NPs on the gonadal development (a) and histophysiological examination of the gonad of mature silkworm (b)

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Fig. 2
figure 2

Effect of TiO2 NPs on the gonadosomatic index of mature silkworm. Gonadosomatic index (GSI = gonad wet weight (g)/total body wet weight (g) × 100) of mature silkworm (n = 10). Each group was analyzed in triplicate, and the results are shown as mean ± standard error. Significant differences between groups were analyzed using one-way ANOVA and Tukey’s multiple comparison test. Asterisks (**) indicate significant differences compared to the control values (p < 0.01)

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Effects of TiO2 NPs Feeding on Fecundity

In order to study the effect of TiO2 NPs feeding on fecundity, the size of pupae, ovipositor of virgin moth, and oviposition were compared between the B. mori fed with TiO2 NPs and those without TiO2 NPs feeding (Fig. 3). As shown in Fig. 3a, the 3-day-old pupae had normal morphology and color in the control and treated groups. However, they were slightly larger after feeding with TiO2 NPs with body weight increases of 0.23 and 0.17 g for female and male pupae, respectively. These data are consistent with the data in Fig. 1, indicating that TiO2 NPs treatment increased the absorption and accumulation of nutrients in B. mori. The ovipositors of virgin moths are shown in Fig. 3b. As shown, there were more densely distributed eggs in the ovipositors of TiO2-NPs-fed animals than those of the control, suggesting that TiO2 NPs feeding is beneficial for egg formation in the adult stage. In order to determine the cumulative effect of TiO2 NPs feeding on oviposition, fecundity was investigated (Table 2) and 51 more eggs per insect and 0.34 × 10−4 g more mass per egg were found after TiO2 NPs feeding (Table 2). However, fertilization rates were not different between the two groups (P > 0.05). The morphology of the eggs is shown in Fig. 3c. The density of eggs was higher in insects fed with TiO2 NPs than in the control. These data suggested that TiO2 NPs not only increase the nutrient accumulation and transformation during the reproductive development but also improve the oviposition ability in B. mori.

Fig. 3
figure 3

Effects of TiO2 NPs on the growth of 3-day-old pupae (a), the ovipositor of virgin moth (b), and the oviposition of B. mori (c)

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Table 2 Effects of TiO2 NPs on the oviposition of B. mori
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Effects of TiO2 NPs Feeding on Expression of Gonad Development-Related Genes

To investigate the effects of TiO2 NPs on transcription and expression of gonad development-related genes, the transcript levels of these genes in the ovaries and testes were determined by the RT-qPCR method using actin 3 as the housekeeping gene (Fig. 4). The TiO2-NPs-induced upregulated genes in the ovaries are shown in Fig. 4a, including vitellogenin (Vg), Ovo, Ovo-781, Ovo-498, Otu, Sxl-S, and Sxl-L, with 6.77-, 2.00-, 5.33-, 2.99-, 2.43-, 1.73-, and 2.30-folds increase, respectively. Vg, Ovo, Ovo-781, Ovo-498, and Otu are related to nutrition metabolism in ovarian development [17, 18]. The upregulation of these genes following TiO2 NPs feeding indicates that the compound can increase nutrient accumulation in the ovary for the development of eggs. Sxl-S and Sxl-L are the self-splicing regulatory genes, and their upregulation is an indication that TiO2 NPs facilitate the self-modification of female transcription factors. The TiO2-NPs-induced upregulated genes in the testes are shown in Fig. 4b, including Achi-L, Aly, and Vlg with 3.55-, 5.78-, and 2.36-folds increase, respectively. The three genes are related to energy metabolism and gamete differentiation in the testis, and their upregulation suggests that TiO2 NPs treatment can provide sufficient energy for better gamete formation [19]. The above results show that TiO2 NPs can upregulate the transcription of genes related to reproductive development of gonads and could be responsible for the reproductive ability of B. mori.

Fig. 4
figure 4

Analysis of transcriptional levels of the gonad development genes in the ovary (a) and testis (b) of mature silkworm after TiO2 NPs feeding. All gene expressions were normalized with actin 3. Each test sample contained five individuals and each sample was run in triplicate. Asterisks indicate significant differences compared to the control values (*p < 0.05, **p < 0.01)

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Discussion

Effects of TiO2 NPs on the Biological Environment

As a new additive, TiO2 NPs are widely used in bleaching, food additive, cosmetics, air purification fields, and biological research [20–24]. The safety and efficacy of TiO2 NPs in various biological environments have been extensively studied. In recent years, TiO2 NPs have been investigated through whole body exposure, dermal exposure, gastric lavage, inhalation, and feeding using animal models in efforts to define their impacts on various biological and physiological metabolisms for potential biological applications [25–27]. In fact, the different impacts of the compound are resulted from different organ concentration of TiO2 NPs and different effects on the biological processes. High concentration of TiO2 NPs is toxic to the reproductivity of CD-1 mice and the fruit fly, Drosophila [28]. Low concentration of TiO2 NPs is not biologically toxic, and instead, it can promote the growth and differentiation of vertebrate cells. Fabian et al. found that TiO2 NPs at the dose of 5 mg/kg body weight is not toxic to tissue of male Wistar rats [29], which is consistent with what was reported by Umbreit et al. [30]. In addition, a trace amount of TiO2 NPs incorporated into orthopedic materials is shown to promote the self-repair and regeneration of bones [31]. In cell engineering, a TiO2-doped glass-microsphere-fortified surface is found to be a suitable surface for cell adhesion and growth [32].

Feeding of B. mori with TiO2 NPs is found to increase the metabolism of proteins and carbohydrates to meet the energy demand for growth and development. It also increases the antioxidation capacity and resistance to pesticide and virus damage [13, 33, 34]. Previous reports have demonstrated that TiO2 NPs feeding can increase the amount of silk production and improve the silk quality. In this study, for the first time, we found that TiO2 NPs feeding can promote the growth and development of the gonad (Figs. 1 and 2), increase differentiation and formation of eggs (Fig. 1b), and improve the ability to oviposition (Fig. 3 and Table 2). These findings provide a new clue for the use of low concentration of TiO2 NPs to improve the reproductive capacity of organisms.

Possible Mechanism Underlying the Effect on Reproductive Ability

Vitellogenin (Vg) is a complex precursor of yolk protein. It is involved in energy reserves in embryonic development and is synthesized in the liver of vertebrates, which is an equivalent of fat body in insects [35, 36]. The Vg gene is an excellent biomarker for reproductivity and is widely used in determination of the reproductive ability in insects with rich yolk proteins in egg [37]. Previous reports have indicated that the development of B. mori ovary is regulated by nutrient accumulation and energy metabolism [37]. Xu et al. pointed out that the development of B. mori ovary is associated with the transfer of yolk proteins from the fat body to the ovary. 4-NP (4-nonylphenol) a known endocrine-disrupting chemical is a persistent environmental contaminant. When exposed to 4-NP, the expression of the Vg gene was downregulated, leading to the reduced translocation of yolk proteins and abnormal development of egg and sperm cells in gonad and subsequently reduced gonadosomatic index, fecundity, and hatching rate [37]. The expression level of the Vg gene in the ovary was 6.77-fold more in the TiO2-NPs-fed animals than in the control (Fig. 4a), suggesting that the compound may promote the development and growth of reproductive gland and improve the fecundity in B. mori.

The Ovo and Otu genes are closely associated with ovarian development. As a transcriptional factor, Ovo is involved in energy metabolism. Depletion of Ovo expression by RNA interference did not change the fertilization rate greatly in B. mori eggs with a slight reduction in the hatching rate and remarkable reduction in egg production by about 15 % [17, 18]. Data in Fig. 4a show that the expressions of Ovo, Ovo-781, Ovo-498, and Otu in the ovary of TiO2-NPs-fed animals were 2.00-, 5.33-, 2.99-, and 2.43-folds that of the control, respectively, indicating that these genes work together with Vg to regulate nutrient accumulation in the ovary to provide adequate nutrition for the development of the egg. Sxl has an important role in the regulation of sex differentiation in B. mori. The female-specific Sxl+ mRNA encodes a full-length Sxl+ protein that functions to autoregulate the splicing of its own pre-mRNA, as well as the pre-mRNA of the tra + gene. The alternative slicing of the Sxl gene is important in regulating the development of the ovary [38]. Data in Fig. 4a show that the expressions of two subtypes of Sxl-S and Sxl-L of the Sxl gene in the ovary of TiO2-NPs-fed animals increased by 1.73- and 2.30-folds, respectively, comparing with those in the control. These data and results in Fig. 1b all show that upregulation of the Sxl gene after TiO2 NPs feeding promotes the alternative splicing and is conducive to the differentiation and formation of eggs.

In the testes of TiO2-NPs-fed B. mori, Achi-L, Aly, and Vlg were 3.55-, 5.78-, and 2.36-folds higher than those of the control, respectively (Fig. 4b), while the differentiation and formation of sperm cells were not different between the two treatments (Fig. 1b), nor the fertilization rate (Table 2), although the testis GSI was higher in TiO2-NPs-fed B. mori than in the control (Fig. 2). These results indicate that increased expressions of Achi-L, Aly, and Vlg following TiO2 NPs are related to the growth of the testis, but not related to the differentiation and formation of germ cells. More studies are needed to define the sensitivity of ovary and testis to TiO2 NPs.

The Significance to Improve the Reproductivity of B. mori

B. mori is an important social insect. The number and quality of the eggs are important for the silk industry [3, 37]. Our work shows that TiO2 NPs can increase the amount of eggs and improve the reproductivity of the B. mori. This suggests that as a B. mori feed additive, TiO2 NPs can increase the raw cocoon production [14] and also the parent silkworm rearing. These findings broaden the application of TiO2 NPs as additive in silk production.

In summary, our work has demonstrated that TiO2 NPs feeding upregulate the expression of genes related to reproductive systems and promote the development and growth of the reproductive gland in B. mori. It consequently facilitates the differentiation and formation of gametes in the ovary and increases the amount of produced eggs and fecundity in the insect.