Introduction
Due to its affordability, durability, and excellent malleability, plastic has been mass-produced since the 1940s and has gradually become an indispensable part of daily life (Iqbal et al., Reference Iqbal, Zhao, Yin, Zhao, Xie, Khan, Zhao, Nazar, Li and Du2023; Mitrano and Wohlleben, Reference Mitrano and Wohlleben2020). However, during use, plastic waste has revealed issues of low degradability and low recycling rates, leading to its continuous accumulation in the environment (Balabantaray et al., Reference Balabantaray, Singh, Pandey, Chaturvedi and Sharma2023; Geyer et al., Reference Geyer, Jambeck and Law2017). These plastic wastes degrade into smaller plastic particles under physical, chemical, and biological factors, and plastic particles with a particle diameter of smaller than 5 mm are collectively referred to as microplastics (MPs) (Khan et al., Reference Khan, Tariq, Alabbosh, Rehman, Jalal, Khan, Farooq, Li, Iqbal, Ahmad, Khan and Du2024; Payanthoth et al., Reference Payanthoth, Mut, Samanta, Li and Jung2024; Zhang et al., Reference Zhang, Luo, Xu, Yao, Fan, Mao, Song, Yang, Pan and Khattak2023). MPs can be categorised based on polymer types, such as polystyrene, polyethylene, polypropylene, polyamide, and polyethylene terephthalate (Devi et al., Reference Devi, Hansa, Gupta, Syam, Upadhyay, Kaur, Lajayer and Sharma2023). Numerous studies have increasingly reported the presence of various types of MPs in terrestrial, marine, and freshwater ecosystems (Dissanayake et al., Reference Dissanayake, Kim, Sarkar, Oleszczuk, Sang, Haque, Ahn, Bank and Ok2022; Sharma et al., Reference Sharma, Bhardwaj, Thakur and Saini2023; Talbot and Chang, Reference Talbot and Chang2022). Terrestrial ecosystems, including agricultural lands, accumulate MPs through multiple pathways, such as plastic mulching, wastewater irrigation, biosolid applications, and atmospheric deposition. While marine and freshwater environments also serve as major sinks, agricultural lands represent a critical recipient of MPs with potential ecological consequences (Jin et al., Reference Jin, Tang, Lyu, Wang, Gillmore and Schaeffer2022). A study from Malaysia revealed that wastewater generated by washing machines during laundry contains MPs at concentrations ranging from 6.9 to 183 mg/m3 (Praveena et al., Reference Praveena, Syahira Asmawi and Chyi2020). These MPs, primarily polyester fibers and polyamide, accumulate in sewage sludge, a significant portion of which is ultimately applied to agricultural fields (De Falco et al., Reference De Falco, Di Pace, Cocca and Avella2019; Zhang et al., Reference Zhang, Xie, Liu, Zhong, Qian and Gao2020). In addition, the accumulation of agricultural plastic films (mainly polyethylene) in farmlands further exacerbates the contamination of agricultural ecosystems by MPs (Huang et al., Reference Huang, Liu, Jia, Yan and Wang2020). MPs in the environment can enter organisms through ingestion and respiration (Bradney et al., Reference Bradney, Wijesekara, Palansooriya, Obadamudalige, Bolan, Ok, Rinklebe, Kim and Kirkham2019). Therefore, investigating the ecotoxicity of PA-MPs and polyethylene microplastics (PE-MPs) is beneficial for improving the management of MPs pollution in agricultural production. Compared to PA-MPs, the toxic effects of PE-MPs have received more attention (Djouina et al., Reference Djouina, Vignal, Dehaut, Caboche, Hirt, Waxin, Himber, Beury, Hot, Dubuquoy, Launay, Duflos and Body-Malapel2022, Reference Djouina, Waxin, Dubuquoy, Launay, Vignal and Body-Malapel2023). However, polyamide has a higher density than polyethylene, making it more likely to accumulate in soil (Choi and Kim, Reference Choi and Kim2023; Zheng et al., Reference Zheng, Wang, Li, Xiong and Wu2024). Studies on the toxic effects of PA-MPs primarily focus on their sublethal effects. For instance, PA-MPs have been reported to induce oxidative stress in Drosophila melanogaster, alter gene expression in Chironomus riparius, and affect feeding and lipid accumulation in Calanus finmarchicus (Khosrovyan et al., Reference Khosrovyan, Doria, Kahru and Pfenninger2022; Zhong et al., Reference Zhong, Jin, Tang, Xu, Liu and Shen2022).
The silkworm Bombyx mori (Lepidoptera: Bombycidae), an important economic insect, has been cultivated in China for thousands of years (Hu et al., Reference Hu, Zhu and Chen2023; Wang et al., Reference Wang, Sun, Ma, Liu, Xia and Chen2022). Due to prolonged domestication and indoor rearing, silkworms have become highly sensitive to their surroundings, thus they are often used as model organisms in toxicological research (Abdelli et al., Reference Abdelli, Peng and Keping2018; Chen et al., Reference Chen, Zhang, Ye, Wu, Cao and Zhou2022). The complete sequencing of the silkworm genome was accomplished as early as 2008, significantly advancing subsequent research on genome functions (Xia et al., Reference Xia, Guo, Zhang, Li, Xuan, Li, Dai, Li, Cheng, Li, Cheng, Jiang, Becquet, Xu, Liu, Zha, Fan, Lin, Shen, Jiang, Jensen, Hellmann, Tang, Zhao, Xu, Yu, Zhang, Li, Cao, Liu, He, Zhou, Liu, Zhao, Ye, Du, Pan, Zhao, Shao, Zeng, Wu, Li, Pan, Li, Yin, Li, Wang, Zheng, Wang, Zhang, Li, Yang, Lu, Nielsen, Zhou, Wang, Xiang and Wang2009). Additionally, silkworms are easy to rear, have a short life cycle, and present no ethical concerns (Abdelli et al., Reference Abdelli, Peng and Keping2018). These advantages make silkworms a reliable model organism widely used for identifying the toxicological mechanisms of harmful substances, such as pesticides, heavy metals, and other toxic agents (Li et al., Reference Li, Li, Wang, Mao, Chen, Lu, Qu, Fang and Li2020; Liu et al., Reference Liu, Liang, Yang, Shi, Lu, Wang, Wang, Xia and Ma2021; Tang et al., Reference Tang, Xiao, Long, Li, Peng, Zhu, He, Lou and Zhu2021). Meanwhile, as an important model organism in terrestrial ecosystems, silkworms are also utilised for environmental monitoring and pollution assessment (Parenti et al., Reference Parenti, Binelli, Caccia, Della Torre, Magni, Pirovano and Casartelli2020). This is attributed to its sensitivity to environmental pollutants and its essential role in terrestrial ecosystems. Given the widespread presence of MPs in terrestrial ecosystems, analysing their ecological toxicity is crucial (Muhammad et al., Reference Muhammad, Zhang, He, Shen, Zhu, Xiao, Qian, Sun and Shao2024; Wu et al., Reference Wu, Zhang, Chen, Ye, Cao, Hu and Zhou2023). Therefore, the silkworm can be used to assess the ecological toxicity of MPs, novel environmental pollutants, which is of great significance for understanding their impact on terrestrial ecosystems.
The age-stage, two-sex life table is an effective tool for analyzing insect growth, development, reproduction, and population dynamics (Wang et al., Reference Wang, Li, Yuan, Li, Wang and Chi2017). Compared to traditional life tables that focus solely on female populations, the age-stage, two-sex life table incorporates data on male populations as well as developmental stage-specific information (Birch, Reference Birch1948; Hull et al., Reference Hull, Chen, Li, Wang, Ma, Huang and Huang2017). This tool is currently applied in various research, such as the effects of different temperatures on insect development, the effects of various host plants on insect growth, and the effects of environmental pollutants on insect development (Hull et al., Reference Hull, Chen, Li, Wang, Ma, Huang and Huang2017; Jiang et al., Reference Jiang, Yang, Zhang, Chen, Hu, Chen, Zhang and Prager2023; Yang et al., Reference Yang, Qi, Wang, Zhou, Zhao, Dong, Li, Li and Chaudhury2022). In addition, the age-stage, two-sex life table is not limited to studying the development of parental insects but can also be used for the research of insect offspring. Analysing the population dynamics of insect offspring through life table studies enables the assessment of the transgenerational effects of toxic substances (Chi et al., Reference Chi, Mingyuan, Fengshou, Jun, Xiaohu, Bing, Changbin, Tian, Yongquan and Xingang2021; Shen et al., Reference Shen, Liu, Mou, Ma, Li, Song, Tang, Han and Zhao2021; Tamilselvan et al., Reference Tamilselvan, Kennedy and Suganthi2021). For instance, the insecticide broflanilide has been shown to suppress the population growth of Spodoptera litura Fabricius in subsequent generations (Shen et al., Reference Shen, Liu, Mou, Ma, Li, Song, Tang, Han and Zhao2021). The insecticide tolfenpyrad negatively affects Coccinella septempunctata and has a lasting impact on its offspring (Chi et al., Reference Chi, Mingyuan, Fengshou, Jun, Xiaohu, Bing, Changbin, Tian, Yongquan and Xingang2021). The insecticide spinetoram influences the population growth of Plutella xylostella by reducing its development and reproduction (Tamilselvan et al., Reference Tamilselvan, Kennedy and Suganthi2021).
This study aims to assess the transgenerational effects of polyamide microplastics (PA-MPs) on the silkworm Bombyx mori, focusing on their potential impacts on development, reproduction, and silk yield. Using the age-stage, two-sex life table approach, we investigate whether PA-MPs exposure in the parental generation affects the offspring’s growth and population parameters. Additionally, we examine whether PA-MPs particle size influences the severity of these effects, providing insights into the potential ecotoxicity of PA-MPs in terrestrial ecosystems.
Materials and methods
Morphological characterization of PA-MPs
According to the experimental purpose, two PA-MPs with different particle sizes were selected for this experiment. Both powdered PA-MPs were purchased from Macklin Biochemical Technology Co., Ltd (Shanghai, China). According to the supplier’s description, the particle sizes of the two PA-MPs are 75 – 150 μm and 37 – 75 μm, with CAS number 63428-83-1. The Product number are P816278 and P823113. A trace amount of PA-MPs powder was applied directly onto conductive adhesive, followed by gold sputter-coating using a Quorum SC7620 sputter coater, and finally, the sample morphology was captured with a scanning electron microscope (SEM, Hitachi, Japan). The particle size of the PA-MPs was subsequently measured and analysed using ImageJ software.
The silkworm strain and rearing conditions
The silkworm strain 306 was provided by the School of Life Sciences at Jiangsu University. The silkworm eggs were incubated at 26 – 27°C and 80% relative humidity. The silkworm larvae were reared on fresh mulberry leaves under conditions of 26 ± 1 C, 75 – 80% relative humidity, and a 12:12 hours light/dark cycle until the fifth instar.
Exposure of F0 generation silkworms to PA-MPs
PA-MPs were pretreated before the experiment as follows: the PA-MPs powder was added to a beaker containing 0.1% Tween 20 solution (Sangon Biotech, Shanghai, China). The mixture was stirred with a magnetic stirrer for 10 minutes, and then subjected to ultrasonication for 30 min to achieve uniform dispersion of PA-MPs particles in the 0.1% Tween 20 solution. The prepared 1 g/L PA-MPs suspension was used for the exposure experiment on F0 generation silkworms. Ultrapure water was used as a blank control, and 0.1% Tween 20 solution served as a solvent control. Each treatment group included 30 silkworms with three replicates. Fresh mulberry leaves were immersed in PA-MPs suspensions of different particle sizes for 30 seconds, then slowly removed and air-dried at room temperature. The treated leaves were fed to silkworms on the first day of the fifth instar for five consecutive days. The silkworms were observed and counted after 120 hours of exposure to PA-MPs. The statistical indicators included larval weight, pupal weight, cocoon weight, egg laying amount and cocoon shell weight.
Transgenerational effects of PA-MPs on F1 generation silkworms
After exposure to PA-MPs-treated mulberry leaves, the F0 generation silkworms were reared until the eggs were layed, and the eggs from each treatment group were collected for further analysis. Fifty eggs were randomly selected from each treatment group and the larvae were hatched after acid treatment to break diapause and synchronise hatching. F1 generation silkworm larvae were reared until cocooning and eclosion. The number of surviving silkworms in each treatment group was counted daily until the adults mated, laid eggs, and died naturally. The whole experiment was repeated three times and finally, the duration of the silkworms in different stages was recorded. Based on age-stage, two-sex life table analyses, we used TWOSEX-MSChart software to analyse the raw data of the F1 generation silkworm life table (Chi et al., Reference Chi, Kavousi, Gharekhani, Atlihan, Özgökçe M, Güncan, Gökçe, Smith, Benelli, Guedes, Amir-Maafi, Shirazi, Taghizadeh, Maroufpoor, Xu, Zheng, Ye, Chen, You, Fu, Li, Shi, Hu, Zheng, Luo, Yuan, Zang, Chen, Tuan, Lin, Wang, Gotoh, Shaef Ullah, Botto-Mahan, De Bona, Bussaman, Gabre, Saska, Schneider, Ullah and Desneux2023). The usage guidelines of software refer to the website https://www.faas.cn/cms/sitemanage/index.shtml?siteId=810640925913080000 (Fujian Academy of Agricultural Sciences, China). The calculated life history parameters included the age-stage specific survival rate (sxj, where x is age and j is the stage, the probability that a newly hatched individual survives from egg to age x and stage j), the age-stage life expectancy (exj, life expectancy of an individual with age x and stage j), the age-specific survival rate (lx, the probability of a newly laid egg surviving to age x), the age-specific fecundity (mx, age-specific fecundity of the cohort at age x), the age-specific maternity (lxmx), the age-stage specific fecundity (fxj) and the reproductive value (vxj, the contribution of individuals of age x and stage y to the future population). In addition, we also calculated the population parameters, including the net reproductive rate (R 0, the total number of offspring that an average individual can produce during its entire life cycle), finite rate of increase (λ, the population growth rate as time approaches infinity and population reaches the stable age-stage distribution), intrinsic rate of increase (r), and mean generation time (T, the period that a population increases to R 0-fold of its size at stable age-stage distribution).
Statistical analysis
The data represent the mean values of three independent experiments, expressed as mean ± SD (standard deviation). Data analysis was performed using GraphPad Prism 8.0 software, with one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test for group comparisons. Different letters indicate significant differences (p < 0.05).
The raw life table data of F1 generation silkworms were analysed using the TWOSEX-MSChart software, and the graphs were created using GraphPad Prism 8.0 software. The population parameters are shown as mean ± SE (standard error) and were calculated using a bootstrap technique (100 000×) with a non-parametric method. Different letters indicate significant differences, which were calculated by a paired bootstrap test using the TWOSEX-MSChart program (p < 0.05) (Wang et al., Reference Wang, Li, Yuan, Li, Wang and Chi2017). The formula is described below.
The age-specific survival rate (lx) is calculated as:
\begin{equation*}{l_x} = \mathop{\sum}\limits_{j = 1}^k {s_{xj}}\end{equation*}The age-specific fecundity (mx) is calculated as:
\begin{equation*}{m_x} = \frac{{\mathop {\sum} \limits_{j = 1}^k {s_{xj}}\,{f_{xj}}}}{{\mathop{ \sum }\limits_{j = 1}^k {s_{xj}}}}\end{equation*}The age-stage life expectancy (exj) is calculated as:
\begin{equation*}{e_{xj}} = \mathop {\sum} \limits_{i = x}^\infty \mathop {\sum} \limits_{y = 1}^m s'_{iy}\end{equation*}The reproductive value (vxj) is calculated as:
\begin{equation*}{v_{xj}} = \frac{{{e^{r\left( {x + 1} \right)}}}}{{{s_{xj}}}}\mathop {\sum} \limits_{i = 1}^\infty {e^{ - r\left( {i + 1} \right)}}\mathop {\sum }\limits_{y = j}^m s'_{ij}\,{f_{iy}}\end{equation*}The net reproductive rate (R 0) is calculated as:
\begin{equation*}{R_0} = \mathop {\sum} \limits_{x = 0}^\infty {l_x}{m_x}\end{equation*}The finite rate of increase (λ) is calculated as:
\begin{equation*}\mathop {\sum }\limits_{x = 0}^\infty \left\{ {{\lambda ^{ - \left( {x + 1} \right)}}\mathop{ \sum }\limits_{j = 1}^m {f_{xj}}{s_{xj}}} \right\} = 1\end{equation*}The intrinsic rate of increase (r) is calculated as:
\begin{equation*}\mathop{ \sum }\limits_{x = 0}^\infty {e^{ - r\left( {x + 1} \right)}}{l_x}{m_x} = 1\end{equation*}The mean generation time (T) is calculated as:
\begin{equation*}T = \frac{{\ln {R_0}}}{r}\end{equation*}Results
Morphological characterization of PA-MPs
The SEM images of PA-MPs are shown in fig. 1. Both PA-MPs with different particle sizes exhibit irregular, granular shapes with rough, uneven surfaces. By estimating the particle size of PA-MPs using ImageJ software, we found that most of the 75 – 150 μm PA-MPs were around 100 μm (fig. 1A), while most of the 37 – 75 μm PA-MPs were around 50 μm (fig. 1B). Thus, in subsequent experiments, the two PA-MPs used will be referred to as PA 100 μm and PA 50 μm.
Figure 1. The scanning electron microscope (SEM) examination of polyamide microplastics (PA-MPs) used in this study. PA 100 μm (A) and PA 50 μm (B).
Effects of PA-MPs on the development, reproduction, and silk yield of silkworms
After 120 hours of exposure to PA-MPs, the biological indicators of each group were statistically analysed (table 1). As shown in fig. 2, the indicators of the 0.1% Tween 20 group did not show significant changes compared to the control group (p > 0.05), indicating that the 0.1% Tween 20 solution did not affect the growth and development of the silkworms. After ingesting PA-MPs, no mortality was observed in the silkworms, but their growth, development, and reproduction were significantly inhibited. This indicates that PA-MPs have sublethal effects on silkworms. In the larval stage, the larval weight (F = 6.016; df = 3, 8; p < 0.05) of silkworms in the PA 100 μm group was significantly lower compared to the control group, while no significant difference was observed between the PA 50 μm group and the control group (p > 0.05) (fig. 2A). In the pupal stage, only the pupal weight (F= 4.436; df = 3, 8; p < 0.05) and cocoon weight (F = 4.522; df = 3, 8; p < 0.05) of silkworms in the PA 100 μm group were significantly lower than those of the control group (fig. 2B and C). In the adult stage, the egg laying amount (F = 18.49; df = 3, 8; p < 0.001) in both the PA 100 μm group and the PA 50 μm group was significantly lower compared to the control group (fig. 2D).
Figure 2. Polyamide microplastics (PA-MPs) inhibited the development, reproduction, and silk yield of F0 generation silkworms. Statistics on the larval weight (A), pupal weight (B), cocoon weight (C), egg laying amount, and (D) and cocoon shell weight (E). Control: ultrapure water. Solvent control: 0.1% Tween 20 solution. Date are shown as mean ± SD (standard deviation), n = 3. Different letters indicate significant differences between different treatments using ANOVA and tukey’s multiple comparison test (p < 0.05).
Table 1 Polyamide microplastics (PA-MPs) inhibited the development, reproduction, and silk yield of silkworms
Date are shown as mean ± SD (standard deviation), n = 3. Different letters within the same row indicate significant differences between different treatments using ANOVA and Tukey’s multiple comparison test (p < 0.05).
Abbreviation: df, degrees of freedom.
Furthermore, exposure to PA-MPs may have also affected the development of silk glands, ultimately impacting silk yield (fig. 2). As shown in fig. 2E, the cocoon shell weight (F = 4.855; df = 3, 8; p < 0.05) in the PA 100 μm group was significantly lower compared to the control group, while no significant difference between the PA 50 μm group and the control group (p > 0.05). This suggests that the sublethal effects of PA-MPs also impact silk yield in silkworms. Meanwhile, the biological indicators in the PA 100 μm group showed greater reductions compared to the PA 50 μm group, indicating that the ingestion of larger-sized PA-MPs may cause more severe damage to silkworms.
Effects of PA-MPs on developmental duration and total longevity of the F1 generation silkworms
An increasing number of studies have shown that the sublethal effects of MPs on organisms also include transgenerational effects, which further affect the development of offspring. We recorded the duration of each stage and the total longevity of the F1 generation silkworms (table 2). Compared to the control group (12.23 ± 0.17 days and 22.46 ± 0.23 days, respectively), the egg stage (F = 22.36; df = 3, 8; p < 0.001) and larval stage (F = 26.07; df = 3, 8; p < 0.001) durations of the PA 100 μm group (13.44 ± 0.19 days and 24.47 ± 0.26 days, respectively) and the PA 50 μm group (13.32 ± 0.12 days and 23.80 ± 0.27 days, respectively) were significantly prolonged. During the pupal stage (F = 16.10; df = 3, 8; p < 0.001), the duration in the PA 100 μm group (16.18 ± 0.20 days) was significantly longer than that in the control group (14.95 ± 0.27 days), while there was no significant difference between the PA 50 μm group (15.47 ± 0.18 days) and the control group (p > 0.05).
Table 2 Development time of different life stages of the progeny (F1) from fifth instar larvae of F0 generation silkworms treated with polyamide microplastics (PA-MPs)
Date are shown as mean ± SD (standard deviation), n = 3. Different letters within the same row indicate significant differences between different treatments using ANOVA and Tukey’s multiple comparison test (p < 0.05).
Abbreviation: df, degrees of freedom.
However, in the adult stage, we found that the duration of male adults (F = 20.81; df = 3, 8; p < 0.001) and female adults (F = 16.28; df = 3, 8; p < 0.001) in the PA 100 μm group (9.18 ± 0.23 days and 10.49 ± 0.23 days, respectively) was significantly reduced compared to the control group (11.05 ± 0.24 days and 12.05 ± 0.20 days, respectively). Finally, the total longevity of the PA 100 μm group (56.16 ± 0.27 days) (F = 5.806; df = 3, 8; p < 0.05) was significantly shorter compared to the control group (57.11 ± 0.36 days), while there was no significant difference between the PA 50 μm group (56.99 ± 0.23 days) and the control group (p > 0.05). This indicates that after silkworms ingest PA-MPs, the transgenerational effects prolong the developmental duration of the offspring while shortening the lifespan of adults and the total longevity. Furthermore, we also found that the effects of PA-MPs on F1 generation silkworms increase with particle size.
Effects of PA-MPs on life history parameters of the F1 generation silkworms
To more accurately analyse the effects of PA-MPs on the F1 generation silkworms, we calculated the life history parameters of the F1 generation silkworms. Analysis of the age-stage specific survival rate (sxj) showed that the PA 100 μm and PA 50 μm groups exhibited more pronounced stage overlap compared to the control (fig. 3). This may be attributed to the transgenerational effects of PA-MPs, which disrupt the population development process of the silkworm offspring. Among all life stages, the differences in the sxj values between the PA-MPs treatment groups and the control group were greatest during the larval and pupal stages. The age-stage life expectancy (exj) is shown in fig. 4. The exj values for the PA 100 μm and PA 50 μm groups were significantly lower than those of the control group, with values of 46.16, 49.72, and 51.8 days, respectively. This suggests that PA-MPs affected the development of silkworm offspring and shortened their expected lifespan. Additionally, we also calculated the age-specific survival rate (lx), age-specific fecundity (mx), age-stage specific fecundity (fxj), and age-specific maternity (lxmx). As shown in fig. 5, the lx values for each life stage in the control group were higher than those in the PA 100 μm and PA 50 μm groups. In the control group, the maximum value of fxj was 37, occurring on day 47. However, the maximum values of fxj in the PA 100 μm and PA 50 μm groups were 20.133 and 30.667, occurring on day 50 and day 47, respectively. Furthermore, the maximum lxmx value in the control group was also higher than that in the PA 100 μm and PA 50 μm groups. Finally, PA-MPs also reduced the reproductive value (vxj) of the F1 generation silkworms (fig. 6). In the control group, the highest vxj value for females was 51.326 on day 47. The peak vxj values for the PA 100 μm and PA 50 μm groups were 33.548 and 40.681, occurring in females on day 48 and day 47, respectively. Our results indicate that PA-MPs prolonged the developmental time of the F1 generation silkworms during the larval and pupal stages, reduced the lifespan of adults, and decreased reproduction. Meanwhile, the extent of this effect on silkworms was still influenced by the particle size of the PA-MPs.
Figure 3. The age-stage specific survival rate (sxj) of the progeny (F1) from fifth instar larvae of F0 generation silkworms treated with polyamide microplastics (PA-MPs). (A) Control group. (B) 0.1% Tween 20 group. (C) PA 100 μm group. (D) PA 50 μm group.
Figure 4. The age-stage life expectancy (exj) of the progeny (F1) from fifth instar larvae of F0 generation silkworms treated with polyamide microplastics (PA-MPs). (A) Control group. (B) 0.1% Tween 20 group. (C) PA 100 μm group. (D) PA 50 μm group.
Figure 5. The age-specific survival rate (lx), the age-specific fecundity (mx), the age-specific maternity (lxmx) and the age-stage specific fecundity (fxj) of the progeny (F1) from fifth instar larvae of F0 generation silkworms treated with polyamide microplastics (PA-MPs). (A) Control group. (B) 0.1% Tween 20 group. (C) PA 100 μm group. (D) PA 50 μm group.
Figure 6. The reproductive value (vxj) of the progeny (F1) from fifth instar larvae of F0 generation silkworms treated with polyamide microplastics (PA-MPs). (A) Control group. (B) 0.1% Tween 20 group. (C) PA 100 μm group. (D) PA 50 μm group.
Effects of PA-MPs on population parameters of the F1 generation silkworms
Besides the life history parameters of the F1 generation silkworms, we also analysed the population parameters. The results are shown in table 3, including the net reproductive rate (R 0), finite rate of increase (λ), intrinsic rate of increase (r), and mean generation time (T). Compared to the control group (60.190 ± 2.488 and 0.081 ± 0.001, respectively), both the PA 100 μm group (29.824 ± 2.224 and 0.067 ± 0.002, respectively) and the PA 50 μm group (42.266 ± 2.486 and 0.074 ± 0.002, respectively) showed significant reductions in R 0 and r values. However, only the PA 100 μm group (1.066 ± 0.004 and 50.411 ± 0.211, respectively) showed significant reductions in λ and T values compared to the control group (1.084 ± 0.003 and 51.173 ± 0.237, respectively). The above results indicate that PA-MPs also affect the population parameters of the F1 generation silkworms, and the effect caused by large-sized PA-MPs was greater.
Table 3 Population parameters of the progeny (F1) from fifth instar larvae of F0 generation silkworms treated with polyamide microplastics (PA-MPs)
Date are shown as mean ± SE (standard error). Different letters within the same row indicate significant differences using a paired bootstrap test by TWOSEX-MS Chart program (p < 0.05).
Abbreviations: R 0, net reproductive rate; λ, finite rate of increase; r, intrinsic rate of increase; T, mean generation time.
Discussion
With the widespread use of plastic mulching and wastewater irrigation in agricultural activities, MPs pollution in agricultural lands has become increasingly severe (Jin et al., Reference Jin, Tang, Lyu, Wang, Gillmore and Schaeffer2022). Previous study has shown that MPs ranging from 0.03 to 1 mm accounted for 48.73% and 59.81% of the shallow and deep soils in Chinese farmlands, respectively (Liu et al., Reference Liu, Lu, Song, Lei, Hu, Lv, Zhou, Cao, Shi, Yang and He2018). In the tree-planted soils of Yunnan, 95% of the MPs sampled were in the size range of 0.05–1 mm (Zhang and Liu, Reference Zhang and Liu2018). In this study, the particle sizes of the two PA-MPs used are consistent with those reported, and they exhibit fragmented and granular shapes (fig. 1). Although most MPs in the environment are fibrous and fragmented, the granular shapes are also common forms of MPs in the environment (Liu et al., Reference Liu, Lu, Song, Lei, Hu, Lv, Zhou, Cao, Shi, Yang and He2018; Xu et al., Reference Xu, Zhang, Gu, Shen, Yin, Aamir and Li2020). We chose these two different particle sizes of PA-MPs for the experiment to further investigate the effect of particle size on their toxicity, based on the study of their ecotoxicity. Multiple studies have shown that MPs of different particle sizes cause varying degrees of harm to organisms (Geyer et al., Reference Geyer, Jambeck and Law2017; Bringer et al., Reference Bringer, Thomas, Prunier, Dubillot, Bossut, Churlaud, Clérandeau, Le Bihanic and Cachot2020). Research by Bringer et al. (Reference Bringer, Thomas, Prunier, Dubillot, Bossut, Churlaud, Clérandeau, Le Bihanic and Cachot2020) demonstrated that PE-MPs affect the development of Crassostrea gigas, with smaller-sized PE-MPs exhibiting greater toxicity. In contrast, studies by Geyer et al. (Reference Geyer, Jambeck and Law2017) revealed that larger-sized polyethylene microspheres increase the mortality rate of Palaemonetes pugio. In summary, current research is yet to provide a definitive conclusion regarding the effect of particle size on MPs toxicity, and further evidence is required. In this study, larger-sized PA-MPs caused more severe sublethal and transgenerational effects on silkworms. This may be because larger particles can cause mechanical damage to the digestive tract or lead to starvation, while smaller particles are more likely to be excreted from the body (Bucci et al., Reference Bucci, Tulio and Rochman2020). Besides mechanical damage, the toxic mechanisms of MPs also include altering biological indicators, inducing oxidative stress, inducing gene expression, and modifying behavioral characteristics, among other effects (Bergami et al., Reference Bergami, Bocci, Vannuccini, Monopoli, Salvati, Dawson and Corsi2016; Jeong et al., Reference Jeong, Won, Kang, Lee, Hwang, Hwang, Zhou, Souissi, Lee and Lee2016). Our current results only demonstrate the sublethal and transgenerational effects of PA-MPs on silkworms. Investigating the specific toxic mechanisms of PA-MPs will be the direction of our future research.
The ingestion of MPs by animals in the environment is typically a prolonged process (Devi et al., Reference Devi, Hansa, Gupta, Syam, Upadhyay, Kaur, Lajayer and Sharma2023). The study by Liu et al. (Reference Liu, Yu, Cai, Wu, Zhang, Huang and Zhao2019) showed that long-term exposure to MPs in Daphnia pulex led to slowed growth and reproductive impairments. This is similar to our results. In this study, after 120 hours of exposure to PA-MPs, the development, reproduction, and silk yield of silkworms were significantly inhibited (fig. 2). During the fifth instar, silkworm larvae begin to consume large amounts of food and reach their critical body weight between days 3.5 and 4 (Sun et al., Reference Sun, Zhang, Chen, Yan, Hong, Xu, Chen and Sun2025). Consuming enough food is the basis for normal development in silkworms. The accumulation of MPs in the intestines can cause pseudo-satiation and intestinal damage (Anbumani and Kakkar, Reference Anbumani and Kakkar2018). This may reduce the food intake of silkworms, leading to slow development. This also explains the significant reduction in the larval weight and pupal weight of silkworms after 120 hours of exposure to PA-MPs (fig. 2A and B). Meanwhile, the egg-laying amounts of silkworms significantly decreased, indicating that their reproduction is also affected by PA-MPs (fig. 2D). The decrease in reproductive output is attributed to the reduced feeding behavior, which results in a decreased allocation of energy to reproduction (Mukherjee et al., Reference Mukherjee, Ogonowski, Schür, Jarsén and Gorokhova2016). Additionally, PA-MPs significantly reduced the silk yield of the silkworms (fig. 2E). The silk gland is the organ responsible for silk production in silkworms, and its development is closely related to silk yield (Tang et al., Reference Tang, Liu, Shi, Chen, Kang, Wang and Zhao2020). Silk gland development begins during the embryonic stage, but during the fifth instar of the larval stage, silk gland cells undergo extensive DNA replication, providing the basis for the large-scale transcription of silk protein genes (Hou et al., Reference Hou, Sun, Wu, Cheng and Liu2019). Therefore, the ingestion of PA-MPs by fifth-instar silkworm larvae affects their subsequent development. This explains the significant decline in the silk yield of larvae and the reproduction of adults. In the study by Wang et al. (Reference Wang, Li, Zhao, Mu, Wang, Wang, Xue, Qi and Wu2021), polystyrene microplastics (PS-MPs) also reduced the feeding rate of bees, subsequently leading to a decrease in their body weight. However, the accumulation or metabolism of PA-MPs within silkworms after ingestion remains unclear, and the mechanisms underlying their sublethal effects require further investigation.
Besides inhibiting growth and development, inducing oxidative stress, and regulating gene expression, MPs also have transgenerational effects. (Liao et al., Reference Liao, Gao, Junaid, Liu, Kong, Chen, Pan, Zheng, Ai, Chen and Wang2023; Lu et al., Reference Lu, Kumar, Melvin, Ziajahromi, Neale and Leusch2023; Tu et al., Reference Tu, Deng, Di, Lin, Chen, Li, Tian and Zhang2023). This underscores the need to focus research on the offspring produced by parent organisms exposed to MPs. The developmental process of offspring can reflect their adaptability to MPs and the residual toxicity of MPs (Yu et al., Reference Yu, Luk and Liao2021). For example, Lu et al. (Reference Lu, Kumar, Melvin, Ziajahromi, Neale and Leusch2023) conducted a metabolomic study on two generations of Chironomus tepperi and found that the effects of PE-MPs on the parent generation did not carry over to the next generation. However, Liao et al. (Reference Liao, Gao, Junaid, Liu, Kong, Chen, Pan, Zheng, Ai, Chen and Wang2023) discovered that PS-MPs reduced the survival rate of F1 Daphnia magna, and this reproductive toxicity persisted until the F3 generation. The age-stage, two-sex life table enables us to assess the population dynamics of offspring exposed to PA-MPs, providing insights into the transgenerational effects of PA-MPs on silkworms (Chi et al., Reference Chi, Mingyuan, Fengshou, Jun, Xiaohu, Bing, Changbin, Tian, Yongquan and Xingang2021). To the best of our knowledge, no studies have yet utilised the two-sex life table to investigate the transgenerational effects of MPs. Therefore, we used the TWOSEX-MSChart software to calculate their life history parameters and population parameters. In this study, PA-MPs significantly prolonged the larval and pupal stages of the F1 generation silkworms, while shortening their adult lifespan and total longevity (table 2). Additionally, PA-MPs reduced the life history parameters (figs. 3–6) and population parameters (table 3) of the F1 generation silkworms. The above results indicate that the effects of parental exposure to PA-MPs extend to their offspring. The most affected stages were the larval and pupal stages of the offspring, with significantly prolonged durations, whereas the adult lifespan and total longevity were shortened. Meanwhile, more pronounced stage overlaps were observed in the offspring of the treatment groups, indicating disrupted population development in the offspring, with more individuals delaying the overall developmental progression. The reduction and delay in the maximum vxj value further suggest that the reproduction of the offspring was also affected by PA-MPs. PA-MPs exhibit significant transgenerational effects on silkworms, affecting the development and reproduction of their offspring. Moreover, these effects are still related to particle size, with larger PA-MPs causing more severe harm. The current results do not clarify the mechanism underlying the transgenerational effects of PA-MPs on silkworms. Further, multigenerational studies are needed to investigate whether silkworm populations develop adaptability to PA-MPs over successive generations and to determine the associated costs of such adaptation.
In summary, our results indicate that ingestion of 1 g/L PA-MPs does not cause lethal effects on silkworms. However, the sublethal effects of PA-MPs impact the development, reproduction, and silk yield of F0 generation silkworms. Meanwhile, the transgenerational effects extend the developmental duration of F1 silkworms, shorten the adult lifespan and total longevity, and suppress reproduction. These effects hinder the propagation of silkworm populations, ultimately leading to a decline in population size. Furthermore, larger PA-MPs exert more pronounced effects on silkworms. Our study provides evidence for the ecotoxicity of PA-MPs and offers a foundation for future research on the impact of PA-MPs on terrestrial ecosystems.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (31861143051 and 31872425), key research and development program of Zhenjiang (H2023039), key research and development program of Taizhou (TS202443), and Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX23_3743).
Conflict of interest
The authors declare that they have no conflict of interest in the content of this article.
