Enhanced bioaccumulation and toxicity of Fenpropathrin by polystyrene nano(micro)plastics in the model insect, silkworm (Bombyx mori)

Abstract

Background

Nano(micro)plastics (NMPs) and agrochemicals are ubiquitous pollutants. The small size and physicochemical properties of NMPs make them potential carriers for pollutants, affecting their bioavailability and impact on living organisms. However, little is known about their interactions in terrestrial ecosystems. This study investigates the adsorption of Fenpropathrin (FPP) onto two different sizes of polystyrene NMPs and examines their impacts on an insect model, silkworm Bombyx mori. We analyzed the systemic effects of acute exposure to NMPs and FPP, individually and combined, at organismal, tissue, cellular, and gut microbiome levels.

Results

Our results showed that NMPs can adsorb FPP, with smaller particles having higher adsorption capacity, leading to size-dependent increases in the bioaccumulation and toxicity of FPP. These effects led to higher mortality, reduced body weight, delayed development, and decreased cocoon production in silkworms. Additionally, the pollutants caused physical and oxidative damage to the midgut and altered gene expression related to juvenile hormone (JH) and silk protein synthesis. The gut microbiome analysis revealed significant changes and reduced abundance of potentially beneficial bacteria. Thus, the aggravated toxicity induced by NMPs was size-dependent, with smaller particles (NPs) having a greater impact.

Conclusions

This study demonstrates the role of NMPs as carriers for contaminants, increasing their bioavailability and toxicity in terrestrial ecosystems. These findings have significant implications for ecosystem health and biodiversity.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-025-03120-8.

Introduction

Plastic is an essential material widely used in various aspects of life, with global production expected to reach 400.3 million metric tons in 2022 and 445.25 million metric tons by 2025 [1]. However, the accumulation of nanoplastics (< 100 nm) and microplastics (< 5 mm), collectively known as NMPs, represents a growing environmental concern [2, 3]. Primary NMPs are intentionally manufactured for products like cosmetics and paints, while secondary NMPs result from the degradation of larger plastic waste [4–6]. The physicochemical characteristics of NMPs, including their size, shape, color, type, charge, density, and gravity, influence their mobility, behavior, and fate in the environment [7–9]. When ingested, inhaled, or in contact with skin, NMPs can internalize cells, disrupt homeostasis, and induce toxicity [5, 10–12]. Numerous studies have investigated the toxicological effects of single exposures to NMPs on various organisms in aquatic and terrestrial ecosystems [2, 10, 13–16]. However, an important concern with NMPs is their tendency to interact with persistent organic pollutants, such as pesticides, heavy metals, pathogens, and other nanomaterials. These interactions lead to coronas forming (adsorption of substances on NMPs), which impact the particles’ transportation, uptake, distribution, and toxicity [7, 17–21]. In aquatic ecosystems, NMPs act as carriers for the adsorbed pollutants and their negative impacts on the growth, development, behavior, reproduction, and mortality of various aquatic organisms has been widely documented [17–20]. However, the current state of NMPs acting as carriers of environmental pollutants in terrestrial ecosystems remains enigmatic.

Pesticides are widely used to manage agricultural and household pests [22, 23]. Pyrethroids, a major group of synthetic pesticides, are widely known for their effectiveness, broad-spectrum range, and affordability. However, the extensive use of these pesticides has resulted in pest resistance and economic losses [24, 25]. Among the pyrethroids, fenpropathrin (FPP) is commonly found in China and used in agriculture and households to control arthropod pests [26–28]. It primarily targets the nervous system, affecting voltage-sensitive sodium, calcium, and chloride channels, leading to symptoms like restlessness, hyperactivity, incoordination, paralysis, and eventual death [27]. Due to the potential toxicity of FPP to non-target organisms, the Agency for Toxic Substances and Disease Registry (ATSDR) and the European Food Safety Authority (EFSA) have established Maximum Residue Levels (MRLs) and an Acceptable Daily Intake (ADI). The ADI is set at 0-0.03 mg/kg, indicating the amount that can be safely consumed daily over a lifetime without significant health risks [29, 30]. This determination is based on a No-Observed-Adverse-Effect Level (NOAEL) of 3 mg/kg identified in various animal toxicity studies, which help assess its safety and biological effects [29, 30]. Pyrethroid insecticides, known for their hydrophobicity, readily adsorb onto NMPs due to their high octanol-water partition coefficient [7, 31]. NMPs have high surface area, hydrophobicity, and strong binding affinity, which enhance their capacity to sorb pyrethroid insecticides [32]. Contaminants adsorbed onto NMPs can be released back into the environment or organisms through desorption. The interaction/adsorption of contaminants with NMPs depends on their physicochemical characteristics, such as size, surface area, roughness, color, shape, polymer type, functional groups, aggregation efficiency, and environmental parameters [2, 7, 18, 33, 34]. Smaller-sized particles (NPs) have a higher surface area-to-volume ratio, better suspension, and stronger hydrophobicity. This enables them to adsorb more contaminants compared to larger particles (MPs) [35, 36]. Additionally, NPs can easily penetrate cell membranes, facilitating the transport of adsorbed contaminants. As a result, there is an increased bioaccumulation and bioavailability of these contaminants in organisms [36–38].

The presence of pesticide residues, including FPP, in agroecosystems and residential areas poses significant risks to human health, biodiversity, and the environment [26, 39–41]. Furthermore, polystyrene (PS) NMPs are prevalent environmental pollutants that interact with pesticides, forming coronas that enhance residue transfer and bioavailability. However, there is limited research on the co-exposure impacts of these pollutants on terrestrial organisms, which highlights the need for further study. The silkworm, Bombyx mori, is an ideal model organism for this research due to its economic importance, simple biology, short life cycle, high offspring count, ease of rearing, and clear genetic background [42–45]. Considering the coexistence of NMPs and pesticides in the ecosystem, it is crucial to investigate the adsorption of FPP on NMPs and their interactive effects on biota in different environments [26–28]. Given the capacity of NMPs to carry certain contaminants, we hypothesized that the adsorption of FPP on NMPs could enhance its toxicity by serving as vectors to facilitate its internalization and bioaccumulation/bioavailability, with NPs potentially exerting a greater effect than MPs on silkworms. To test the hypothesis, we first investigated the potential adsorption of FPP on NMPs of two different sizes: NP of 50–100 nm and MP of 5–5.9 μm. We then evaluated the carrier effects (accumulation/bioavailability) of these NMPs on pesticides in silkworm tissues. Next, using a multitier approach, we assessed the toxicity of FPP induced by different-sized particles at various levels: organism level (survival, development, and productive performance), tissue level (histopathology of the midgut, accumulation in gut, fat body, and silk gland), cellular level (gene expression changes, oxidative stress), and gut microbial community level. This study presents new evidence on how PS-NMPs and FPP interact and impact animal health in the terrestrial ecosystem. It also emphasizes assessing the risks of coexisting emerging pollutants to non-target organisms, including humans.

Materials and methods

Chemical materials

Polystyrene (PS) particles (non-functionalized) used in this study were purchased from MACKLIN^® in Shanghai, China. The NPs had particle sizes ranging from 50 to 100 nm, while the MPs ranged between 5 and 5.9 μm. The physicochemical characteristics of these particles, including size, shape, and chemical composition, were determined using scanning electron microscopy (SEM, Hitachi SU8000, Japan) and pyrolysis with gas chromatography-mass spectrometry (Py-GC/MS) (Fig. 1A and B) [46]. The particle count was calculated (Table S1), following the method described in our previous study [46]. The pesticide Fenpropathrin (CAS#39515-4-41-8, analytical standard, purity 99%) was purchased from Shanghai Yuanye Biotechnology Co. Ltd (Fig. 1C and D). The stock solution of the pesticide (100 µg/mL) was prepared in methanol to ensure its solubility. However, to minimize the influence of methanol on the experimental system, its final concentration in all exposure solutions was reduced to less than 0.1% (v/v), a level considered negligible and unlikely to impact interactions between nanoparticles (NMPs) and FPP or to affect bioavailability and toxicity. Additionally, control groups without FPP were exposed to the same concentration of methanol to account for any solvent-related effects, ensuring that the observed outcomes were solely due to FPP and NMPs. The experimental solutions were prepared using double distilled water (purified twice to remove impurities) to avoid any potential solvent-related interference with the biological system.

Fig. 1.

Chemical materials and experimental setup. Physicochemical characteristics of polystyrene nanoplastics (NPs) (A) and microplastics (MPs) (B) were evaluated using SEM, TEM, and ImageJ software. (C) Molecular and (D) 3D structure of the pesticide Fenpropathrin (FPP). (E) Exposure of silkworm larvae to single and combined contaminants, and evaluation of toxicological implications at multiple levels

Susceptibility of silkworms to Fenpropathrin (FPP)

The silkworm culture (Jingsong × Haoyue) was maintained under controlled conditions in our laboratory [42]. To determine the susceptibility of silkworms to FPP, we exposed the fifth instar larvae on the first day with an average biomass of 0.78 ± 0.056 g per individual to a silkworm diet amended with gradient concentrations of FPP (0, 1.0, 2.0, 4.0 8.0, and 16 µg/mL). Mortality was assessed visually by observing the inability of the silkworms to respond to gentle stimuli over a 24 h period. After determining the baseline susceptibility of silkworms to FPP, we then investigated the interaction between FPP and NMPs to understand how adsorption onto plastic particles could affect its bioavailability and toxicity.

FPP adsorption on NMPs

To investigate the adsorption of FPP, a sublethal concentration (2.5 µg/mL) of pesticides and various concentrations of NMP solutions (0, 0.5, 5, and 50 µg/mL) were prepared using double distilled water in 50 mL conical flasks, with three replicates for each of the four concentration groups. These flasks were incubated at room temperature with constant mixing for 36 h on a shaker (100 rpm). The duration of incubation was based on previous studies that investigated the adsorption of pesticides onto NMPs [47–49]. Afterward, the liquid samples were centrifuged for 10 min at 8000 rpm (25 ℃). Supernatants were collected and filtered through a 0.22 μm filter membrane to measure the fraction of FPP that was present in the solution. Meanwhile, the pellets containing NMPs were resuspended in acetonitrile, ultrasonicated for 30 min to separate the adsorbed FPP from the NMPs, and filtered through a filter membrane [47]. FPP concentrations were determined using an Agilent 8890-7000D mass spectrometry (GC-MS/MS 8890-7000D, Agilent, USA). GC separation was performed using an Agilent HP-5 MS capillary column (30 m × 0.25 mm × 0.25 μm). The following oven conditions were applied: 60 °C, held for 1 min; ramped from 60 °C to 170 °C at 40 °C/min; and then from 170 °C to 310 °C at 20 °C/min, held for 3 min. The inlet temperature was set to 280 °C. A flow rate of 3.0 mL/min was maintained with 99.999% pure helium as the carrier gas for chromatographic analysis. An injection volume of 1 µL was analyzed in splitless mode under high-pressure conditions (3.788 psi). The triple quadrupole mass spectrometer operated in electron impact (EI) ionization mode with a 25-eV ionization voltage. The interface (transfer line to the tandem MS), ion source, and quadrupole temperatures were maintained at 280 °C and 130 °C, respectively. Multiple reaction monitoring (MRM) mode was used for target detection. Working solutions containing pesticide concentrations of 0.5, 1, 2.5, 5, 10, 50, and 100 µg/L were prepared to generate calibration curves. Minimum analyte concentrations in spiked blank samples that induced MRM traces with signal-to-noise ratios (S/N) of 3 and 10, respectively, were used to calculate the limit of detection (LOD) and limit of quantification (LOQ) values. In this study, the LOD and LOQ were determined to be 1 µg/L and 2.5 µg/L, respectively. The recovery rate was measured using three different concentrations (10.0, 20.0, and 100.0 µg/L) according to the method described above, yielding a recovery rate of more than 84% in this study. Building on the findings of FPP adsorption onto NMPs, we tested the combined effects of these contaminants on silkworms to investigate how this adsorption influences toxicity and bioaccumulation in a living organism.

Exposure of silkworm larvae to single and combined NMPs and FPP contaminants

To investigate the single and combined effects of NMPs and FPP contaminants on silkworms, healthy fifth instar larvae (freshly molted) were divided into different groups (n = 50 × 3 per group) (Fig. 1E) and had their weight measured on an analytical balance before exposure to contaminants [50]. Based on the susceptibility assay, we chose a pesticide concentration (5 µg/mL) that is moderately toxic. This concentration falls within the range of maximum residue limits of FPP in fruit and vegetables [51]. We then incubated it with NMPs at gradient concentrations (0.5, 5, and 50 µg/mL) as outlined in the adsorption assay. These concentrations are environmentally relevant and have been detected in terrestrial ecosystems, ranging from 0.1 µg/g to 5.0 µg/g [52]. The silkworms were fed an artificial diet that was amended with contaminants at the desired concentrations by thoroughly mixing the contaminant solutions into the feed. The silkworms were allowed to feed on this contaminated diet for 24 h, after which any uneaten food was removed to ensure accurate dosing. The combined exposure groups included NP 0.5 + FPP, NP 5.0 + FPP, NP 50 + FPP, MP 0.5 + FPP, MP 5.0 + FPP, and MP 50 + FPP. Meanwhile, a parallel control (CTR) and single exposure groups including FPP (5 µg/mL), NP 0.5, NP 5.0, NP 50, MP 0.5, MP 5.0, and MP 50 were also maintained under the same conditions. The exposure occurred in a controlled environment with a 12-hour light/dark cycle [42].

Effects of single and combined exposure to contaminants on the survival, development, and productive performance of silkworms

After exposing silkworm larvae to single and combined PS-NMPs and FPP contaminants, the number of deceased and surviving individuals was recorded daily across all groups for seven days. We selected this timeframe to align with the normal developmental window of silkworms, as their fifth instar stage typically lasts for seven days under healthy conditions, ensuring an accurate assessment of treatment effects. The primary aim of this experiment was to determine how these exposures affect silkworm survival, development, and productivity. Survival analysis was conducted using the Kaplan-Meier (log-rank) test, and the resulting survival curves were generated in GraphPad Prism [53], enabling us to compare survival rates across the different groups. By linking the survival data to developmental and productive outcomes, we ensured a comprehensive assessment of the impact of contaminants.

The larval body weight of 50 individuals from each group was measured to assess the impact on growth. Higher mortality in NP 50 + FPP and MP 50 + FPP groups led to their exclusion from further experiments, ensuring that developmental and productivity assessments focused on groups with survivable doses. The development time of the fifth instar larvae, which typically lasts seven days under normal conditions for healthy silkworms, was monitored until the prepupal stage in all treatments and compared to the control group. Productive performance was then assessed through key metrics, including, cocooning rate, whole cocoon weight, and cocoon shell weight to establish a link between survival and production outcomes. These metrics were determined using the methods described previously [54] (see SI for detailed methodology).

Accumulation of FPP in different tissues of silkworms

Based on the observed survival rates, we hypothesized that NMPs may enhance the bioavailability and tissue accumulation of FPP, which may explain the increased mortality in the combined exposure groups. To explore the mechanisms behind the enhanced toxicity observed in the combined exposure groups, we used a sublethal concentration (2.5 µg/mL), a dose that does not cause death but may still affect physiological functions, in our subsequent experiments to ensure an adequate number of insects for analysis. This sublethal concentration allowed us to determine the bioaccumulation of FPP in different tissues (gut, fat body, and silk gland) and examine the interaction between NMPs and pesticide uptake. The capability of polystyrene particles to pass through the biological membranes and accumulate in different tissues has already been demonstrated for silkworms [46, 55]. Now, to determine the carrier capacity of NMPs (5 µg/mL) for FPP in the silkworm body, the bioavailable fraction of pesticide was determined in different tissues. After the corresponding experimental exposures, nine larvae were randomly selected from each group. The larvae were dissected under sterile conditions on ice and the tissues of three individuals were pooled as one replicate (n = 3 replicates). After weighing, the samples were homogenized in acetonitrile, sonicated, and centrifuged (10 min, 8000 rpm, 25 ℃). Supernatants were collected and filtered through a 0.22 μm filter. The FPP fraction was quantified using the GC-MS/MS analysis as described above. This approach provided a precise measurement of FPP bioaccumulation, emphasizing the potential role of NMPs as carriers in increasing pesticide toxicity.

Histopathological examination

To explore the structural impacts of contaminants at the tissue level, we conducted histopathological examinations of the silkworm midguts from each treatment group after 24 h of exposure to a sublethal concentration (2.5 µg/mL). Midguts from three individuals in each group were dissected, fixed in a 4% paraformaldehyde solution for 24 h at 4 °C, and rinsed with PBS. Subsequently, the tissues were dehydrated using an increasing concentration of ethanol and xylene. The tissues were embedded in paraffin wax, cut into thin Sects. (5–10 μm thick) using a microtome, and stained with hematoxylin and eosin (H & E). The stained tissues were examined under a light microscope (Nikon Eclipse, Japan). For SEM analysis, the tissues were washed with 0.75% NaCl and then fixed with 2.5% glutaraldehyde overnight at 4 °C, followed by 1% osmium tetraoxide treatment for 1–2 h at room temperature (25 ± 2 °C). After fixation, the tissues were dehydrated with ethanol and acetone, dried, mounted, and imaged using scanning electron microscopy (Hitachi Regulus8100). The H & E figures were opened in SlideViewer (v 2.5.0.143918) to measure midgut thickness across different treatment groups. This dual approach enabled us to identify both microscopic and ultrastructural changes, ensuring thorough assessments of tissue damage across the exposed groups.

Analysis of enzymatic activity

After observing tissue damage through histopathological analysis, we investigated oxidative stress responses to evaluate the cellular-level disruptions caused by contaminants. We measured the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) in gut tissues to assess oxidative stress responses. The gut tissues from the CTR and treatment groups were homogenized in ice-cold PBS, and the supernatants were collected by centrifugation at 10,000 × g for 10 min at 4 °C [46]. Enzyme assays were conducted using commercial kits for total protein content (BCA Protein Assay Kit Tiangen PA115, Tiangen Biotech), SOD (Total SOD Activity Detection Kit WST-8 method, S0101 Beyotime Biotechnology, China), CAT (Catalase Assay Kit Serial No: A007-1 Jiancheng Bioengineering Institute, Nanjing, China), and GST (Glutathione S-transferase Activity Detection Kit D799611-0050, Sangon Biotech, China). The detection assays were conducted following the manufacturer’s instructions, ensuring quality control through the use of at least four biological replicates per treatment group. The enzyme activity data shed light on the physiological stress that silkworms experience when exposed to contaminants, establishing a connection between these responses and their overall survival and development.

Determination of juvenile hormone (JH) titer

Juvenile hormone (JH) is a crucial insect hormone that plays a vital role in regulating the development and physiology of insects. To investigate the mechanism of developmental toxicity induced by FPP and NMPs-FPP complex, the level of JH was determined 24 h post-exposure. The hemolymph was collected from individuals in the CTR and treatment groups by cutting the second pair of abdominal prolegs with sharp scissors, and it was stored in 1.5 mL tubes. The tubes were immediately centrifuged at 10,000 rpm for 5 min at 4 °C, and the supernatant was taken for subsequent analysis. The Insect JH ELISA Kit (RJ23126, Renjie BioTech. Co. Ltd. Shanghai) was used to determine the titer of JH according to the manufacturer’s instructions. This analysis was designed to link JH level alterations to developmental toxicity observed in exposed silkworms, providing a mechanistic basis for delayed growth or disrupted maturation.

Gene expression analysis

To understand the molecular responses underlying the observed toxic effects, we extracted the RNA from the gut tissues, silk gland, and head dissected from the CTR and treatment groups. The Promega Eastep^® Super Extraction Kit (Beijing Biotec. Co. Ltd) was used for RNA extraction, following the manufacturer’s instructions. After quality assessments, the RNA was reverse-transcribed using the HiScript^® II Q RT SuperMix R223-01 (Vazyme Biotech Co., Ltd) to synthesize cDNA. qPCR and transcript abundance analyses were performed as previously described [46]. The primer sequences used in this study are listed in Table S2. Gene expression patterns were analyzed to correlate molecular responses with physiological changes observed in the survival, growth, and development metrics, establishing a deeper mechanistic understanding of contaminant toxicity.

DNA extraction, high-throughput sequencing, and gut microbiome analysis

To study the effects of single and combined exposures of PS-NMPs and FPP, the guts (5 replicates) from six groups, namely CTR, NP (5.0 µg/mL), MP (5.0 µg/mL), FPP (2.5 µg/mL), NP + FPP, and MP + FPP, were extracted under sterile conditions after exposure for 24 h. The DNeasy Blood and Tissue Kit (Qiagen, Germany) was used to extract total DNA from the gut tissues [53]. The primer set 515FmodF_806RmodR (F-GTGYCAGCMGCCGCGGTAA, R-GGACTACNVGGGTWTCTAAT) was used to amplify the 16 S rRNA gene (V4 region). Illumina high-throughput sequencing analysis was performed to decipher the structure and composition of the gut microbiota of the CTR and treatment groups. Subsequent bioinformatic analysis, including trimming and filtering to obtain effective reads, amplicon sequence variant (ASV) analysis, alpha and beta diversity analysis, as well as composition analyses, were performed as described previously [42].

Statistical analysis

To validate the observed effects across all experiments, lethal concentrations of FPP were determined in silkworms using Probit analysis, and the mortality curve was generated in GraphPad Prism. The one-way analysis of variance (ANOVA) with Tukey’s post hoc multiple comparison test was used to compare the means between the control (CTR) and treatment groups. The independent samples t-test was used to compare the means between the two groups. Results are presented as the mean ± standard deviation, and differences with a P-value < 0.05 were considered statistically significant. A normal distribution test was performed before data analysis.

Results

FPP exhibited significant adsorption on NMPs

The GC-MS/MS approach was used to detect and quantify the FPP content adsorbed on PS-NMPs (Fig. 2A and B). A significantly higher amount of FPP (1.01 µg/mL) was present in the solution with no NMPs, which gradually decreased with the increasing concentration of NMPs, measuring 0.15 ± 0.03 SD µg/mL and 0.28 ± 0.027 SD µg/mL at 50 µg/mL of NPs and MPs, respectively (F _{(3, 9)} = 302.22, P < 0.0001, Fig. 2C). This removal of FPP from the solution indicates its adsorption on NMPs (Fig. 2C). To confirm the adsorption, the amount of FPP taken up by NMPs was measured in the solution resuspended from the pellets. Consistently, a significant amount of FPP was detected, which increased with the increasing NMPs concentration and confirmed their adsorption on NMPs (F _{(2, 6)} = 354.55, P < 0.0001, Fig. 2D). Notably, the removal/adsorption capacity of NPs was significantly higher (37.14–60.74%) than that of MPs under the tested condition. This difference can be attributed to the greater number of NPs compared to MPs (Table S1) and their higher surface area-to-volume ratio, which provides a larger surface area available for pesticide adsorption.

Fig. 2.

Adsorption of Fenpropathrin (FPP) on NMPs. (A) GC-MS/MS chromatograph of FPP. (B) Mass spectrograph of the constituents of FPP. (C) FPP content in the solution with and without the presence of NMPs, and (D) content of FPP adsorbed on NMPs. The data is presented as mean values ± standard deviation. Differences of variance across different concentrations were estimated using one-way ANOVA (Tukey’s post-hoc test) and between the two groups using the student t-test. Different letters indicate significant differences (P < 0.05) between groups; t-test: *P < 0.05, **P < 0.01, ***P < 0.001

NMPs increased the toxicity of FPP, negatively impacting silkworm survival and growth

First, we determined the susceptibility of silkworms to FPP and the medium lethal concentration (LC₅₀ = 7.5 µg/mL, Fig. S1). Then, considering the adsorption of FPP and the carrier capacity of NMPs, we used a moderately toxic pesticide concentration (5 µg/mL). We observed the survival and larval growth of silkworms after exposure to single and combined NMPs and FPP contaminants (Fig. 3). Single exposures to NMPs had no impact on the survival and larval growth of silkworms (P > 0.05). However, both FPP and NMPs-FPP complexes caused a significant decrease in survival rates in a concentration-dependent manner (log-rank test χ² = 51.58, P < 0.0001). Importantly, this decrease in survival rates was more pronounced for the NPs-FPP complex (Fig. 3A, Table S3) than the MPs-FPP complex (Fig. 3B Table S4), which can be attributed to their higher adsorption of the pesticides compared to MPs (Fig. 2D). Additionally, NP particles are more likely to internalize and cause increased cellular damage than MPs. Similarly, individuals in both the FPP and NMPs-FPP complexes were smaller in size and had significantly reduced body weight (Fig. 3C and D). The decrease in body weight in the combined exposure groups was influenced by NMP concentrations and size, with the data indicating that NPs causing stronger impacts than MPs under the tested conditions.

Fig. 3.

Survivorship and body weight change after single and combined exposures of NMPs and FPP contaminants. Survival probability was assessed using Kaplan-Meier survival analysis and survival curves were generated for silkworms exposed to (A) NPs and FPP, both single and combined contaminants, and (B) MPs and FPP, also as single and combined contaminants. The body weight changes of silkworms exposed to NPs and FPP single and combined contaminants are presented in (C) and for MPs and FPP single and combined contaminants in (D). The body weights changes across different groups were compared using one-way ANOVA with Tukey’s post hoc test for multiple comparisons. Different letters indicate statistically significant differences across different treatments (P < 0.05) and the error bars represent the standard deviation

NMPs increased the bioaccumulation/bioavailability of FPP in silkworm tissues in a size-dependent manner

To understand the mechanisms of enhanced toxicity of FPP by NMPs, we determined the bioavailable fraction of the pesticide in different tissues, including the gut, fat body, and silk gland (Fig. 4). In the control group, FPP was not detected in any of the tested tissues of the silkworm. In the FPP treatment group, a small amount of FPP was detected in the gut tissues and fat body, but no detectable FPP was present in the silk gland (Fig. 3). However, when NMPs and FPP were combined, there was a significant increase in the bioaccumulation of the pesticide in silkworm tissues compared to the CTR or pesticide alone (P < 0.05). The highest concentration of FPP was detected in the gut tissues (16.82-fold), fat body (43.01-fold), and silk gland of the NP + FPP treatment group, followed by the MP + FPP treated counterparts (gut = 1.64-fold, and fat body = 3.21-fold increase in gut and fat body of the CTR group, respectively. The increased bioaccumulation of the pesticide in silkworm tissues supports the potential of PS-NMPs to act as carriers for FPP. This may partially explain the enhanced toxicity observed in the combined exposure groups, especially in the NP + FPP group where higher levels of toxicant was detected compared to the MP + FPP group.

Fig. 4.

Bioaccumulation of FPP in silkworm tissues. The FPP content in the gut (A), fat body (B), and silk gland (C) was determined ng/g of tissues when exposed to pesticide alone (FPP) or in the presence of NMPs and compared with the CTR group. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test for multiple comparisons. Different letters indicate statistically significant differences across different treatments (P < 0.05) and the error bars represent the standard deviation

NMPs exacerbated the histopathological changes in the silkworm midgut induced by FPP

To further understand the toxicity mechanism, we visualized the ultrastructural changes of the peritrophic membrane (PM) after exposure to contaminants (Fig. 5). The PM is considered the first midgut barrier that protects the midgut epithelial cells from physical damage caused by xenobiotics. Its structural integrity is positively related to the resistance of insects against xenobiotics. Our SEM analysis showed that the PM of CTR and single NMPs exposure groups had a smooth, orderly surface with no obvious damage to the chitin microfibrils (Fig. 5A-F). However, the PM surface structure of the FPP-treated individuals was rough, and the microfibrils were broken and had abnormal trenches (Fig. 5B). Notably, these damages were further exacerbated in the combined exposure groups, resulting in a more disrupted physical defense (Fig. 5D and F). Furthermore, the examination of the histopathological section of the midgut revealed that the midgut matrix (black arrows) in both the control group and the group exposed to single NMPs was intact (black arrows) (Fig. 5G, I, and K). The goblet cells (red arrows) and columnar cells (blue arrows) appeared morphologically healthy and were arranged in an alternating pattern (Fig. 5G, I, and K). In contrast, in the FPP and NMPs-FPP complexes, the cell layer of the intestinal wall was damaged, and some epithelial cells disintegrated (Fig. 5H, J, and L). Additionally, it resulted in changes to the cell morphology, primarily affecting goblet and columnar cells (Fig. 5H, J, and L). In line with these observations, the midgut thickness was significantly shorter in the FPP and NMPs-FPP complexes, especially in the NP + FPP complex compared to the CTR and single NMPs exposure groups (F _{(5, 84)} = 37.51, P < 0.0001, Fig. S2). These results suggest that the presence of NMPs may exacerbate the pathological changes in the midgut cells of silkworms caused by FPP. Additionally, the combined exposure to NP + FPP appeared to cause more pronounced damage compared to the groups exposed to MP + FPP or FPP alone. Based on the results of the ultrastructural and histopathological analysis, the physical damage to the midgut could be a contributing factor to the increased toxicity observed in the combined exposure groups.

Fig. 5.

Histopathology examination of the midgut of the 5th instar silkworm larvae exposed to single and combined NMPs and FPP contaminants. The surface structure of the peritrophic matrix (PM) was examined using SEM for both the control group (A) and the contaminants-exposed groups (B-F). Pathological changes of the midgut were observed after acute exposure to contaminants, including (G) control, (H) FPP, (I) NP, (J) NP + FPP, (K) MP, and (L) MP + FPP. The midgut matrix is indicated by black arrows, goblet cells by red arrows, and columnar cells by blue arrows

FPP and NMPs-FPP complexes induced oxidative stress

We assayed the antioxidant and detoxification enzymes and the expression of their related genes to determine the oxidative stress induced by single and combined exposures to NMPs and FPP. In the single exposure groups of NP or MP groups, no significant effects were observed on the antioxidant and detoxification activities and were comparable with the control group, indicating that short-term (24 h) exposure to low concentrations (0.5 and 5.0 µg/mL) did not induce oxidative stress in silkworms. However, FPP and PS-NMPs-FPP complex increased the level of SOD, especially in the combined exposure groups (Fig. 6A). Consistently, the expression of the BmSOD gene exhibited upregulation in the FPP and NMPs-FPP complex, particularly in the combined exposure groups (Fig. 6B). On the other hand, CAT activity and its expression (BmCAT) in the FPP and NMPs-FPP complex exhibited the opposite trend and was significantly downregulated, especially in the NP 5.0 + FPP group (Fig. 6C and D). Furthermore, detoxification represented by GST activity was also induced with significant upregulation of the BmGST gene in the FPP and NMPs-FPP complex (Fig. 6E and F). These results suggest that FPP and the NMPs-FPP complex may induce oxidative stress in silkworms, as evidenced by the upregulation of antioxidant (SOD) and detoxification (GST) defense mechanisms. However, the observed inhibition of CAT activity implies a potential disruption in the balance of antioxidant enzymes, which could contribute to oxidative damage.

Fig. 6.

Effects of exposure to single and combined NMPs and FPP on the antioxidant and detoxification mechanisms of silkworms. The activities of antioxidant enzymes and the expression of their corresponding genes were measured. (A) SOD, (B) BmSOD, (C) CAT, and (D) BmCAT. Additionally, the relative level of the detoxification enzyme GST (E) and its expression (F) were examined across the control group (CTR) and treatment groups. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test for multiple comparisons. Different letters indicate statistically significant differences across different treatments (P < 0.05). Error bars represent the standard deviation, and scatter plot dots are included to show biological variability

FPP and NMPs-FPP complexes significantly delayed silkworm development

Exposure to contaminants can affect the development of insects and the duration of their life cycle. In comparison to the control group (CTR) or the single exposure of NMPs, the developmental time of fifth-instar silkworms was significantly delayed by at least 1.13 d (27 h) in both the group exposed to FPP and the group exposed to a combination of contaminants (F _{(9, 290)} = 24.72, P < 0.0001) (Fig. 7A). Notably, the NP 5.0 + FPP group experienced an extended delay of two days before they began spinning the cocoon. To understand the molecular mechanism behind the delayed developmental time in the exposure groups of contaminants, we determined the level of JH III in the hemolymph as well as the transcriptional changes of JH-responsive genes. Since the lowest concentration (0.5 µg/mL) of NMPs combined with FPP had no significant effect on the pesticide’s toxicity and was comparable to FPP single exposure application, we only measured the JH titer in the single and combined exposure groups of NMPs at the relatively higher concentration (5.0 µg/mL) and compared it with the control or FPP treated groups. The results showed that the level of JH was significantly increased by 1.3-fold, 1.44-fold, and 1.32-fold in the FPP, NP + FPP, and MP + FPP exposure groups, respectively (Fig. 7B). Correspondingly, the gene expression profiles of Met-1, Met-2, and Kr-h1 were also significantly upregulated in the aforementioned groups (Fig. 7C). Furthermore, the relative expression of JH degradation gene (JHE) was significantly downregulated in the FPP (1.3-fold) and the combined exposure groups (2.7-3.4-fold) (Fig. 7C). These findings suggest that FPP and its combined exposure with NMPs may contribute to transcriptional changes in JH-responsive genes, potentially leading to elevated levels of JH III, which could impact larval development.

Fig. 7.

Effects of FPP and NMPs-FPP complexes on the silkworm development (A), JH level (B), and expression of JH-responsive genes (C). Differences of variance across different groups were estimated using one-way ANOVA (Tukey’s post-hoc test). Significant differences (P < 0.05) between the groups are indicated using different letters

FPP and NMPs-FPP complexes significantly reduced silk production

To investigate the impact of exposure to single and combined contaminants, NMPs and FPP, on the productivity of silkworms, we measured the cocooning rate and cocoon weight (Fig. 8A and B). In the groups exposed to only NP or MP, we did not observe any significant effects on the rate of cocooning or the weight of the cocoons (both whole cocoon and cocoon shell weight). These groups showed similar results to the control group. However, when silkworms were exposed to FPP, there was a significant reduction in the rate of cocooning (F _{(9, 20)} = 84.77, P < 0.0001, Fig. 8A). This reduction was even more pronounced in the groups exposed to combined NMPs and FPP (NMPs + FPP complexes). Furthermore, this reduction was exacerbated as the concentration of NMPs combined with FPP increased, particularly in the NP 5.0 + FPP group, followed by the MP 5.0 + FPP group. Besides, in the FPP and NMPs-FPP complexes, the larvae exhibited a delay in spinning and produced smaller cocoons (Fig. S3). The weight of both the whole cocoon and the cocoon shell was also reduced (Fig. 8B). Once again, this detrimental impact on cocoon production was more prominent in the groups exposed to the combined NMPs and FPP. The silk gland is an organ that synthesizes and secretes silk substances. To better understand how exposure to contaminants affects cocoon production, we examined the transcript profiles of the genes that regulate the synthesis of silk proteins, such as sericin and fibroin. Like the previous endpoints, acute exposure to NMPs had no significant effect on the expression of genes related to silk protein synthesis. However, their expression (Fib-h, Fib-l, p25, and Ser-2) was downregulated by exposure to FPP (Fig. 8C). This downregulation was significantly more pronounced in the combined exposure groups, particularly in the NP + FPP complex (Fig. 8C). Overall, these findings indicate that FPP alone may reduce silk protein synthesis. Additionally, combined exposure to NMPs and FPP appears to aggravate this effect, with the NP-FPP complex potentially having a more severe impact.

Fig. 8.

Effects of FPP and NMPs-FPP complexes on the cocooning rate (A), cocoon weight (B), and the expression profile of the genes regulating the synthesis of silk protein (C). Differences of variance across different groups were estimated using one-way ANOVA (Tukey’s post-hoc test). Significant differences (P < 0.05) between the groups are indicated using different letters

FPP and NMPs-FPP complexes altered silkworm gut microbiota

Gut microbiota homeostasis is critical for basic physiological functions such as digestion, immunity, and host disease resistance. After exposure to single and combined FPP and NMPs contaminants, high-throughput sequencing was used to investigate the configuration (structure and composition) of the silkworm gut microbiota. Table S5 contains the statistic on the amplicon library, including sequencing reads, minimum and maximum sequence length, as well as the number of base pairs. Meanwhile, Fig. S4 shows the amplicon sequence variants (ASVs) shared among all groups (58 ASVs), overlapped between groups, and unique to a specific group. Alpha diversity indices (species richness and community diversity) were increased in the combined exposure groups (Fig. 9A-D), with no significant difference among the single exposure groups compared to the control group (Fig. 9). Similar results were observed in the β-diversity analysis using non-metric multidimensional scaling (NMDS) where FPP and its combined exposure with NMPs clustered separately and showed significant differences compared to the control and single NMPs exposure groups (R² = 0.52, P = 0.001, Fig. 9E). Concurrent with the previous endpoint, more pronounced differences were observed in the microbial diversity of the NP + FPP exposure group, emphasizing the importance of particle size and its carrier effects. Furthermore, the distinct microbiota structure in the groups exposed to contaminants can be attributed to changes in the abundance of key bacterial taxa at various taxonomic levels (phylum to genus/species). Generally, Proteobacteria is the most abundant phylum in the gut of healthy silkworms. In our study, its abundance was significantly decreased in both the NP + FPP (35.53%) and MP + FPP (56.22%) treatment groups as compared to the control (80.45%) or FPP (84.75%) treatment group (P < 0.05, Fig. 9F). On the other hand, these treatment groups had a higher abundance of Firmicutes (NP + FPP = 55.78%, MP + FPP = 27.6%) and Bacteroides (NP + FPP = 6.03%, MP + FPP = 11.09%) compared to the single exposure groups (Firmicutes < 20.2% and Bacteroides < 3.6%). Similarly, changes in gut microbiota composition at the genus level exhibited significant differences upon contaminants exposure (Fig. S5). The combined exposure led to a decrease in the abundance of Sphingomonas (up to 4.2%) and Brevundimonas (up to 9.5%) from 34.2% to 33.44% for Sphingomonas, and 29.9% and 26.92% for Brevundimonas in the control and FPP treated groups, respectively (Fig. 9G). Meanwhile, it increased the abundance of Weissella up to 54.94% in the NP + FPP and 16.7% in MP + FPP compared to 4.43% in the control and 3.09% in the FPP-exposed group. The gut microbiota composition in the individuals exposed to single NMPs contaminants was largely comparable to the control group. Overall, NMPs seemed to exacerbate the disruption of dominant microbiota abundance induced by FPP. Additionally, the exacerbation caused by smaller particles (NPs) appeared more pronounced than that caused by larger particles (MPs), suggesting a greater imbalance in the silkworm gut microbiota.

Fig. 9.

Effects of FPP and NMPs-FPP complexes on the structure and composition of the silkworm gut microbiota. The alpha diversity indices, ACE (A), Chao 1 (B), Shannon (C), and Simpson (D), were used to assess species richness and diversity within the microbiota. Beta diversity is represented by NMDS (E), where each point in the NMDS plot corresponds to a specific sample from our dataset. The spatial arrangement of these points reflects the similarities/dissimilarities in community composition among the samples, with FPP and its combined exposure with NMPs clustered separately and showed significant differences compared to the control and single. The stress value (Stress = 0.043) indicates an excellent fit for the two-dimensional representation of our high-dimensional data. The relative abundance of key taxa at the phylum and genus levels is shown in (F) and (G), respectively. Different letters indicate significant differences calculated using one-way ANOVA with Tukey’s post hoc test for multiple comparisons

Discussion

To date, the effects of NMPs on the bioaccumulation of pesticides and the potential health risks they pose to terrestrial organisms, particularly insects, remain largely unknown. NMPs, with their hydrophobic nature and large surface area to volume ratio, can serve as carriers for hydrophobic pollutants like pesticides and may facilitate their transfer to organisms [31, 34, 56]. However, the role of NMPs as pesticide carriers has been rarely investigated, especially in terrestrial ecosystems. Given their ubiquity, these contaminants inevitably enter the environment and may increase toxicity to non-target organisms. One proposed pathway is the “Trojan Horse” effect, in which NMPs act as carriers for the transport of toxic substances to organisms. The carrier effects of NMPs are linked to their adsorption properties, which influence transport across biological and environmental compartments, potentially increasing exposure and adverse effects on organisms [32, 35]. Our study found that nano-sized particles (NPs) adsorbed a significantly higher amount of FPP than micro-sized particles (MPs) (Fig. 2), due to their larger surface area to volume ratio and shorter diffusion pathway, which enhance contaminant adsorption and rapid exchange [35, 56]. Polystyrene NMPs exhibit high adsorption capacity for hydrophobic contaminants, owing to the benzene rings in their polymer structure, which increase chain distance and facilitate contaminant attachment and integration [47, 57].

Our results showed that acute exposure (24 h) to NMP contaminants at low concentrations (0.5 and 5.0 µg/mL) did not affect the silkworms at any of the tested endpoints. This aligns with a previous study on white seabass Atractoscion nobilis, which observed similar results when exposed to PS-MPs at environmentally relevant concentrations for a short term [58]. However, it was observed that FPP alone exhibited obvious toxicity across all monitored endpoints, and this toxicity was further aggravated in a dose- and size-dependent manner by NMPs. The increased toxicity observed in the combined exposure groups can be attributed to the adsorption ability of contaminants on NMPs (Fig. 2), which can alter their accumulation and the risk of exposure to organisms [2, 20, 31, 56]. Based on previous studies, it has been found that PS-NMPs tend to accumulate in various tissues of the silkworm, including the gut lumen, midgut epithelium, malpighian tubules, and hemocytes [46, 55]. This accumulation suggests that NMPs can transfer the adsorbed FPP to different organs and tissues of the silkworm. As hypothesized, significantly higher amounts of FPP were found in silkworm tissues (gut, fat body, and silk gland) when using NMPs-FPP complexes (Fig. 4). Thus, these particles can function as carriers, transporting FPP into various matrices. The phenomenon can enhance the accumulation of contaminants in the tissues and organelles of the exposed organism and increase their toxicity [35, 36, 47]. Although the concentrations (by weight) of the particles administered were the same, the difference in size meant that silkworms treated with NP + FPP were exposed to a higher number of plastic particles than those treated with MP + FPP (Table S1) [46]. This is why the NPs carried a significantly higher amount of FPP than the MPs. Additionally, smaller particles (NPs) can more easily penetrate cell membranes and accumulate in tissues [36–38]. For example, the accumulation of 100 nm-sized particles in zebrafish intestines was greater than 5 μm-sized particles at the same concentration (500 µg/L) [37]. Consequently, the increased toxicity observed in the combined exposure groups can be attributed to the greater bioaccumulation/bioavailability of the pesticide in silkworms.

Pesticide poisoning is a serious concern in sericulture. It has been reported to have negative effects on the development, survival, productive performance (cocoon production and quality), endocrine function, and metabolism of the silkworms [26, 59–61]. In this study, exposure to FPP and NMPs-FPP complexes had adverse effects on the survival and development of the silkworms (Fig. 3). The exposure to contaminants resulted in smaller individuals, which in turn affected cocoon production, including the cocooning rate, whole cocoon weight, and cocoon shell weight. As expected, the combined exposure had synergistic effects, with NP + FPP causing more severe impacts than the MP + FPP complex. In line with our findings, previous studies have demonstrated that NMPs accumulate contaminants in different tissues of organisms in a size-dependent manner, thereby exacerbating their toxicity [34–36, 62]. For example, the combined exposure of organochlorine pesticides with PS-NPs resulted in stronger toxicity, as well as reduced body length and life span of Caenorhabditis elegans [35]. Similarly, PS-MPs increased the accumulation of Cu in the tissues of Zebrafish, specifically in the liver and gut. This resulted in an aggravation of toxicity dependent on the size of the particles [36]. In addition to particle size, the surface charge of plastic also affects the adsorption and carrier capacity of pollutants on NMPs. In a recent study, Zhang et al., (2024) demonstrated that the presence of differentially charged (positive and negative) NPs increased the toxicity of perfluoroalkyl acid (F-53B), leading to detrimental effects on the larval growth, emergence, oxidative stress, and inflammation of aquatic insects Chironomus kiinensis. The toxicity of F-53B was found to be more strongly enhanced by positively charged particles than by negatively charged particles. This enhanced toxicity was attributed to the greater adsorption and increased bioaccumulation of F-53B in positively charged NPs compared to negatively charged NPs [34].

Moreover, pesticide exposure can delay silkworm development [61, 63]. In this study, FPP exposure resulted in a delay in the development of silkworms (Fig. 7). Furthermore, we observed that this effect was even more pronounced when FPP was combined with NMPs. Specifically, the combination of NP + FPP had a stronger effect compared to MP + FPP. JH plays a crucial role in regulating the growth and development of silkworms (Li et al., 2019). The delayed development observed in our study was directly linked to the JH content measured in the hemolymph. Alongside the JH contents, the expression of JH synthesis-related genes (Met-1, Met-2, and Kr-h1) was upregulated, while the expression of the JH-degrading gene (JHE) was downregulated. Both JH synthesis and degradation-related genes are vital for maintaining JH balance in insect hemolymph [64], and any changes in their expression due to xenobiotics can lead to an imbalance that affects silkworm development [61]. These results suggest that FPP and NMP + FPP complexes impaired the endocrine function, resulting in altered JH content and delayed silkworm development. Furthermore, the enhanced negative effects of FPP by NMPs on economic indicators (cocoon production), can be attributed to diminished silk protein synthesis due to a decrease in the expression of genes in the silk gland. The synthesis of silk protein (fibroin and sericin) in silkworms is controlled by multiple genes in the silk gland. These genes determine the process of cocoon formation [59]. In our study, we observed a downregulation of Fib-h, Fib-l, p25, and Ser-2 expression when exposed to FPP (Fig. 8). This downregulation was notably more pronounced in the combined exposure groups, especially in the NP + FPP complex. Thus, exposure to FPP and NMPs + FPP significantly reduces cocoon production in silkworms due to the downregulation of key silk protein genes.

The silkworm midgut serves important functions such as digestion, nutrient absorption, and acts as a barrier against contaminants entering the hemocoel. Studies have shown that NMPs carrying contaminants or pathogens can be released into organisms and cause combined toxicity in the intestine [18, 65]. Our histological examination revealed that FPP, as well as its co-exposure with NMPs, caused physical damage and morphological changes to the PM, goblet cells, and columnar cells (Fig. 5). The PM acts as the first line of defense against bacteria and toxic materials, preventing direct contact with the midgut epithelium [66]. Any injury to the PM can increase its permeability, making the silkworm more susceptible to xenobiotic toxicity [66]. The morphological changes observed in the midgut can be attributed to the dysfunction of the gut epithelial cells, which in turn impact the growth and development of the silkworm [67]. Overall, the histopathological changes observed in the midgut exposed to the combined NMPs + FPP were more severe, especially in the NP + FPP complex (Fig. 5). These changes may have played a role in making silkworms more susceptible to these contaminants.

The activation of the oxidative stress pathway is considered one of the key mechanisms involved in xenobiotic-mediated hazardous effects [18, 19, 68]. Among the various antioxidant enzymes, CAT and SOD are considered the primary biomarkers of xenobiotic-mediated oxidative stress [19, 68, 69]. They play a crucial role in protecting against the harmful effects of reactive oxygen species (ROS) by breaking them down into harmless substances [70]. The detoxification enzyme GST is also used as an early biomarker for exposure to environmental contaminants [19]. In our study, we found that the expression of the BmSOD gene and the levels of SOD were increased when exposed to both FPP and NMPs + FPP together. This increase was most noticeable in the combined exposure groups. Similarly, we also observed an increase in GST activity and BmGST gene expression in both FPP and the NMPs-FPP complexes. Such induction of oxidative stress by environmental contaminants has been observed and found to be further enhanced by the presence of MPs [36]. On the other hand, a significant decrease was observed in CAT activity and BmCAT expression, specifically in the NP 5.0 + FPP group. These results confirmed that FPP-induced oxidative stress, when combined with NMPs, has a synergistic effect that ultimately enhances its toxicity. Moreover, the inhibitory effects on CAT activity may have resulted from the activation of the downstream MAPK (mitogen-activated protein kinase) pathway that suppressed the CAT activity [71–73]. Suppressed antioxidant enzyme activity is observed when contaminants induce excessive ROS, leading to severe oxidative damage and the loss of compensatory mechanisms [71, 73]. Furthermore, it has been observed that the extent of contaminants-induced oxidative stress caused by NMPs is dependent on their size, concentration, and polymer type [2, 7, 74]. Once again, smaller-sized particles exert a stronger impact on oxidative stress and the antioxidant defense system [46, 75].

The gut microbiota is important to maintain the host’s health by modulating physiological functions such as digestion, immunity, and host disease resistance [42, 76–78]. The toxic effects of NMPs or pesticides can be related to changes in the gut microbial community of the silkworm [53, 79]. FPP and its combined exposure with NMPs caused dysbiosis in the silkworm gut microbiota (Fig. 9). Correspondingly, the combined exposure of NPs had a stronger impact compared to MP + FPP. This dysbiosis can be attributed to the damage inflicted on the midgut and the disruption of its functions by these contaminants. Consequently, this creates a favorable environment for the excessive growth of bacteria, leading to an increase in species richness and community diversity. The phenomenon of increased species richness and microbiota diversity has been observed in silkworms exposed to phoxim or guadipyr pesticides, resulting from the destruction of the intestinal environment [79, 80]. Besides, the exposure altered the level of certain key taxa, reducing their abundance while increasing the abundance of others. This change in abundance may be linked to pathological effects on silkworms. For example, the silkworm gut is dominated by the phylum Proteobacteria, which includes several important bacterial species involved in silkworm metabolism [42, 44, 53, 81]. This phylum was significantly reduced in the combined exposure groups. Notably, the ingestion of NMPs + FPP contaminants significantly reduced the abundance of Sphingomonas and Brevundimonas. These bacteria are known for their potential to promote health and contribute to metabolic processes, such as pesticide resistance, nutrient supplementation, and adaptation to environmental stressors, for their insect hosts [82–84]. On the other hand, the increased abundance of Weisella species can pose a risk as opportunistic pathogen, especially in individuals under stressed conditions [85]. Overall, our results demonstrated that FPP and NMPs + FPP complexes had a detrimental impact on the gut microbiota of silkworms, leading to adverse effects in the host.

While our study provided novel insights into the enhanced bioaccumulation and toxicity of FPP in silkworms when combined with PS-NMPs, certain limitations must be acknowledged to contextualize our findings and guide future research. Firstly, our study focused solely on FPP, a widely used pesticide that represents just one agrochemical in a broad category. Many other agrochemicals may interact with NMPs in ecosystems. Secondly, we examined only one polymer type (PS) in two different sizes; however, numerous other polymer types exist in various shapes and sizes. Lastly, we only investigated the effects of these pollutants on silkworms, which, while a valuable model organism, may not fully capture the complexity of soil-dwelling organisms. Therefore, future studies exploring the interactions between NMPs and agrochemicals should include other classes of pesticides (e.g., organophosphates, carbamates, neonicotinoids), additional plastic types (e.g., polyethylene, polypropylene, polyvinyl chloride, polyester), and various shapes (e.g., fibers, fragments, films, foams). Furthermore, it is essential to investigate their effects on other terrestrial and aquatic organisms to elucidate the broader implications of these interactions for environmental health and biodiversity.

Conclusion

This study investigated the risks associated with the role of NMPs as carriers of environmental contaminants. The data collected through a multi-tier approach demonstrated that the pesticide FPP was adsorbed on NMPs. When silkworms were exposed to FPP-loaded NMPs, the pesticide accumulated in their tissues in a size-dependent manner. This study observed that the formation of the NMPs-FPP complex altered the pesticide’s toxicity, with indications of enhanced toxicity across different levels of biological organization, including organism level (survival, development, and productive performance), tissue level (histopathology of the midgut, accumulation in the gut, fat body, and silk gland), cellular level (gene expression changes, oxidative stress), and gut microbiota level in the silkworms. Furthermore, small-sized NPs particles could easily enter cells and organelles, and NPs loaded with pollutants appeared to induce greater toxicity as evidenced by the monitored endpoints, compared to larger-sized MPs. Given the complex interactions between NMPs and environmental pollutants, future research focused on exploring combined pollution risks of NMPs and other emerging contaminants would provide more comprehensive insights into their safety and potential environmental impacts.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

A. M. Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project Administration, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. Z. Q. Data curation. Y. L. Investigation. X. L. Methodology. J. I. Methodology, Investigation. X. S. Investigation. J. H. Validation. N. Z. Investigation. C. S. Validation. Y. S. Supervision, Conceptualization, Funding acquisition, Writing – review & editing. The manuscript was written collaboratively by all the authors, and the final version has been approved by all of them.

Funding

This work was supported by grants from the National Natural Science Foundation of China (Grant No. 32250410276), Zhejiang Provincial Natural Science Foundation of China (LZ22C170001), and China Agriculture Research System of MOF and MARA (Grant No. CARS-18-ZJ0302).

Data availability

The raw sequencing data generated during the study have been deposited in the NCBI Sequence Read Archive (SRA) with the accession number PRJNA1128369.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.