Nanoplastic Exposure at Predicted Environmental Concentrations Induces Activation of Germline Ephrin Signal Associated with Toxicity Formation in the Caenorhabditis elegans Offspring

In nematode Caenorhabditis elegans, exposure to polystyrene nanoparticles (PS-NPs) at predicted environmental concentrations can cause induction of transgenerational toxicity. However, the underlying mechanisms for toxicity formation of PS-NP in the offspring remain largely unknown. In this study, based on high-throughput sequencing, Ephrin ligand EFN-3 was identified as a target of KSR-1/2 (two kinase suppressors of Ras) in the germline during the control of transgenerational PS-NP toxicity. At parental generation (P0-G), exposure to 0.1–10 μg/L PS-NP caused the increase in expression of germline efn-3, and this increase in germline efn-3 expression could be further detected in the offspring, such as F1-G and F2-G. Germline RNAi of efn-3 caused a resistance to transgenerational PS-NP toxicity, suggesting that the activation of germline EFN-3 at P0-G mediated transgenerational PS-NP toxicity. In the offspring, Ephrin receptor VAB-1 was further activated by the increased EFN-3 caused by PS-NP exposure at P0-G, and RNAi of vab-1 also resulted in resistance to transgenerational PS-NP toxicity. VAB-1 acted in both the neurons and the germline to control toxicity of PS-NP in the offspring. In the neurons, VAB-1 regulated PS-NP toxicity by suppressing expressions of DBL-1, JNK-1, MPK-1, and GLB-10. In the germline, VAB-1 regulated PS-NP toxicity by increasing NDK-1 and LIN-23 expressions and decreasing EGL-1 expression. Therefore, germline Ephrin ligand EFN-3 and its receptor VAB-1 acted together to mediate the formation of transgenerational PS-NP toxicity. Our data highlight the important role of activation in germline Ephrin signals in mediating transgenerational toxicity of nanoplastics at predicted environmental concentrations in organisms.


Introduction
Over the past two decades, the usage of plastic products has increased drastically, which has led to the occurrence of plastic pollution in the amount of millions of tons per year [1]. The sources of plastic waste are associated with product wear, management policy, and human consumption. In the environment, microplastics and nanoplastics can be formed from plastic waste via weathering degradation, oxidative degradation, and biological degradation by microorganisms [2,3]. Nanoplastics are those plastic particles with sizes less than 100 nm, derived from bulk plastics through degradation or direct environmental release [4,5]. Largely due to their very small size, nanoplastic particles have the potential to easily cross some biological barriers to target different tissues in organisms [6]. The polystyrene nanoparticle (PS-NP) is a commonly examined nanoplastic. Exposure to nanoplastics, such as PS-NP, can result in different aspects of toxicity in organisms, such as embryonic toxicity, neurotoxicity, immunotoxicity, and metabolic dysfunction [7][8][9]. In addition, due to high hydrophobicity, PS-NPs exhibit strong affinity towards other pollutants The 20 nm PS-NPs were purchased from Janus New-Materials Co. (Nanjing, China). The analysis of transmission electron microscopy (TEM, JEOL Ltd., Tokyo, Japan) indicated the spherical morphology of PS-NPs ( Figure S1). The size of the PS-NPs was 20.55 ± 3.1 nm, and the zeta potential of PS-NPs was −5.298 ± 0.697 mV based on dynamic light scattering (DLS) assay. The FTIR spectrum and Raman spectrum of PS-NPs have been reported previously [23,28].

C. elegans Maintenance
Information for used C. elegans strains is provided in Table S1. Strain maintenance was performed as previously described [32].
To perform PS-NP exposure, L1-larvae population needed to be prepared. For this aim, gravid hermaphrodite nematodes were treated with lysis solution containing 0.45 M NaOH and 2% HOCl. After release of eggs from the body, the collected eggs were transferred onto the surface of new NGM plates containing Escherichia coli OP50 as the food source. The eggs were allowed to develop into the synchronized L1-larvae.

Exposure
To examine the transgenerational PS-NP toxicity, the nematodes were exposed to PS-NP suspensions at a concentration of 0.1-10 µg/L from L1-larvae to adult day-3 (approximately for 6-day) at P0-G [33]. The PS-NP suspensions were refreshed daily throughout the exposure process. In the PS-NP suspensions, E. coli OP50 was added to the final concentration of~4 × 10 6 colony-forming unit (CFU). Before the exposure, sonication was conducted for PS-NP suspensions for 30 min at 40 kHz and 100 W. From the first filial generation (F1-G), the nematodes were allowed to develop on normal NGM plates fed with E. coli OP50 and without PS-NP exposure.

Endpoints
Both inhibition in locomotion behavior and suppression in reproductive capacity could be observed in the offspring of PS-NPs exposed nematodes [23]. Thus, locomotion behavior reflected by body bend and head thrash and brood size were used as endpoints for assessment of transgenerational PS-NPs toxicity. The frequencies of body bend and head thrash were counted as the changes of direction for bending at mid-body and posterior bulb (y-axis), respectively, if we considered the direction of swimming for nematodes as x-axis [34]. Brood size was measured as the total number of offspring produced beyond the egg stage [35]. For each exposure, 50 nematodes were examined for assay of locomotion, and 30 nematodes were examined for assay of brood size.

Transcriptional Expression Analysis
The reagent Trizol was used to exact total RNA of adult nematodes. The obtained RNA was assessed for quality using a Nanodrop One based on OD260/280 ratio, and then used for cDNA synthesis using a Gradient MasterCycler (Eppendorf, Hamburg, Germany). In the SYBR Green master mix for real-time polymerase chain reaction (RT-PCR), alterations in gene expression were analyzed using a StepOnePlus real-time PCR instrument. The comparative Ct method was used for quantifying the gene expression after normalization with expression of reference gene tba-1. To analyze the expression of certain genes in the gonad, we isolated the intact gonad. Thirty gonads were used for each treatment. Three replicates were performed. Table S2 shows the related primer information.

RNA Interference (RNAi)
To inhibit gene expression, RNAi knockdown was performed by feeding L1-larvae with E. coli HT115 expressing dsRNA of certain genes [36]. Progeny of nematodes on RNAi plates were used for PS-NP exposure. Nematodes fed with HT115 expressing empty vector of L4440 were used as control [37]. RNAi efficiency was determined by qRT-PCR ( Figures S2 and S3). RNAi efficiency of ksr-1 and ksr-2 in the germline was reported previously [28]. To perform the tissue-specific RNAi knockdown of genes, strains of DCL569 and TU3401 were used as germline and neuronal RNAi knockdown tools, respectively (Table S1) [28].
Using RNAs isolated from these three groups of samples, mRNA libraries were prepared for Illumina HiSeqTM 2000 sequencing. Using Fast QC, the quality of reads was examined. Dysregulation of genes was assessed by fold change analysis and statistical significance.

Construct Generation and Transgene
To determine the interaction of EFN-3 and VAB-1, Pmex-5-efn-3 was constructed. The cDNA of efn-3 was subcloned into pPD95_77 with Pmex-5 promoter to obtain the Pmex-5-efn-3. The transgene was performed by the co-injection of constructs (50 µg/mL) and marker construct (Pdop-1::rfp, 50 µg/mL) in the gonad. After cultivation for 3-4 days, transgenic nematodes were picked out and selected on new NGM plates for related experiments. Primers for construction generation are shown in Table S3.

Data Analysis
Statistical tests were performed with SPSS v19.0 software. The significant differences among treatment groups were determined by one-way or two-way ANOVA (for multi-factor comparison) followed by post-hoc test. A p-value of <0.01 (**) was deemed statistically significant. Statistical significance between curves for transgenerational analysis was determined by Kaplan-Meier survival analysis, followed by the log-rank test.
After PS-NP exposure, ksr-2 knockout further dysregulated expressions of daf-18 and sod-3 in insulin signaling pathway (Table S5). It has been reported that the signaling cascade from daf-2 encoding insulin receptor to sod-3 encoding target of DAF-16 transcriptional factor in the insulin signaling pathway does not function in the germline to control the toxicity of nanoplastics [40].

Identification of Ephrin Ligand EFN-3 as Candidate Downstream Target of KSR-1/2 in Controlling Transgenerational PS-NP Toxicity
Besides some dysregulated stress-response-related genes indicated above, we further found that both ksr-1 RNAi and ksr-2 RNAi could increase the expression of efn-3 encoding an Ephrin ligand in 1 µg/L PS-NP exposed nematodes (Tables S3 and S4). After 0.1-10 µg/L PS-NP exposure, the germline efn-3 expression was further increased ( Figure 1A). In addition, after 1 µg/L PS-NP exposure, this increase in germline efn-3 expression could be observed at F1-G and F2-G ( Figure 1B).
Using locomotion and brood size as the endpoints, germline RNAi of efn-3 conferred a resistance to the transgenerational PS-NP toxicity ( Figure 1C,D). These observations suggested the requirement of germline Ephrin ligand EFN-3 in modulating transgenerational PS-NP toxicity. After the PS-NP exposure, the curves of efn-3 (RNAi) showed a significant difference (p < 0.01) compared to DCL569 (L4440). A total of 30 intact gonads were used for the qRT-PCR assay. ** p < 0.01 vs control or P0 (control) (if not specifically indicated).
Using locomotion and brood size as the endpoints, germline RNAi of efn-3 conferred a resistance to the transgenerational PS-NP toxicity ( Figure 1C,D). These observations suggested the requirement of germline Ephrin ligand EFN-3 in modulating transgenerational PS-NP toxicity.

Tissue-Specific Activity of VAB-1 in Controlling Transgenerational PS-NPs Toxicity
In nematodes, VAB-1 can be expressed in neurons and reproductive tissues [42,43]. Both germline and neuronal RNAi of vab-1 caused the resistance to transgenerational PS-NP toxicity as reflected by the endpoints of locomotion behavior and brood size ( Figure  5A,B). That is, VAB-1 could function in both the germline and neurons to regulate the toxicity induction of PS-NPs in the offspring.

Tissue-Specific Activity of VAB-1 in Controlling Transgenerational PS-NPs Toxicity
In nematodes, VAB-1 can be expressed in neurons and reproductive tissues [42,43]. Both germline and neuronal RNAi of vab-1 caused the resistance to transgenerational PS-NP toxicity as reflected by the endpoints of locomotion behavior and brood size ( Figure 5A,B). That is, VAB-1 could function in both the germline and neurons to regulate the toxicity induction of PS-NPs in the offspring.

Tissue-Specific Activity of VAB-1 in Controlling Transgenerational PS-NPs Toxicity
In nematodes, VAB-1 can be expressed in neurons and reproductive tissues [42,43]. Both germline and neuronal RNAi of vab-1 caused the resistance to transgenerational PS-NP toxicity as reflected by the endpoints of locomotion behavior and brood size ( Figure  5A,B). That is, VAB-1 could function in both the germline and neurons to regulate the toxicity induction of PS-NPs in the offspring.

Identification of Potential Targets of Germline VAB-1 in Controlling PS-NP Toxicit
In the germline, some molecular signals were also raised to be required for ling PS-NP toxicity [49,50]. Among these signals, EGL-1 is a BH3 protein go germline cell death, WRT-3 is a Hedgehog ligand, LIN-23 is an E3 ubiquitin liga 12 is a component of hemidesmosomes, NHL-2 is a miRISC cofactor, and NDK-1 i H1 homolog. In PS-NP exposed nematodes, expressions of nhl-2, pat-12, and wr not affected by germline RNAi of vab-1 ( Figure 6B). In contrast, after PS-NP expos 23 and ndk-1 expressions were significantly decreased by germline RNAi of vab-1, 1 expression was increased by germline RNAi of vab-1 ( Figure 6B).

Discussion
In nematodes, during the control of transgenerational toxicity of pollutants, d forms of epigenetic regulations play important functions. For example, histone m tion-or demethylation-related molecular signals are associated with transgene toxicity induction of different pollutants, such as arsenite, CuO nanopartic

Identification of Potential Targets of Germline VAB-1 in Controlling PS-NP Toxicity
In the germline, some molecular signals were also raised to be required for controlling PS-NP toxicity [49,50]. Among these signals, EGL-1 is a BH3 protein governing germline cell death, WRT-3 is a Hedgehog ligand, LIN-23 is an E3 ubiquitin ligase, PAT-12 is a component of hemidesmosomes, NHL-2 is a miRISC cofactor, and NDK-1 is NM23-H1 homolog. In PS-NP exposed nematodes, expressions of nhl-2, pat-12, and wrt-3 were not affected by germline RNAi of vab-1 ( Figure 6B). In contrast, after PS-NP exposure, lin-23 and ndk-1 expressions were significantly decreased by germline RNAi of vab-1, and egl-1 expression was increased by germline RNAi of vab-1 ( Figure 6B).

Discussion
In nematodes, during the control of transgenerational toxicity of pollutants, different forms of epigenetic regulations play important functions. For example, histone methylationor demethylation-related molecular signals are associated with transgenerational toxicity induction of different pollutants, such as arsenite, CuO nanoparticles, and nanoplastics [26,51,52]. In addition, alterations in certain long non-coding RNAs also mediate the transgenerational toxicity induction of multiwalled carbon nanotubes [35]. Recently, we further found that certain miRNAs, such as mir-38, are also involved in controlling transgenerational PS-NP toxicity [28]. In the germline, the mir-38 regulated transgenerational PS-NP toxicity by inhibiting NDK1-KSR-1/2 axis. However, the underlying mechanism for how decreases in germline KSR-1/2 mediated the PS-NP toxicity induction in the offspring remains largely unclear. For this reason, we performed high-throughput sequencing for ksr-1(RNAi) and ksr-2(RNAi) nematodes after PS-NP exposure. Nevertheless, among the dysregulated stress-response-related genes caused by germline RNAi ksr-1 and ksr-2 after PS-NP exposure, we did not observe their function in controlling transgenerational toxicity ( Figure S4). These observations implied that, although these dysregulated stress-responserelated genes can be expressed in the germline, they may be not able to exert their function in regulating stress responses to PS-NP in this tissue.
For the underlying molecular mechanisms of germline KSR-1/2 in controlling transgenerational toxicity, we assumed that the KSR-1/2 may affect certain secretory ligands in the germline so as to regulate the induction of PS toxicity in the offspring. In this study, we further found that the expression of Ephrin ligand EFN-3 was increased by germline RNAi of ksr-1 or ksr-2 in PS-NP exposed nematodes (Tables S4 and S5). The EFN-3 expression showed a pattern of transgenerational increase after PS-NP exposure ( Figure 1A,B), and the efn-3(RNAi) nematodes showed resistance to transgenerational PS-NP toxicity ( Figure 1C,D). Moreover, in the germline, EFN-3 could function downstream of KSR-1/2 to control transgenerational PS-NP toxicity ( Figure 2). These observations suggested that, during the control of transgenerational toxicity, germline mir-38 suppressed the function of Ephrin signaling by inhibiting NDK1-KSR1/2 axis. That is, EFN-3 is one of the downstream secretory targets of KSR-1/2 in the germline. Previous studies have indicated the function of EFN-3 in the control of epidermal organization [41]. Our data here further demonstrate the function of germline EFN-3 in regulating stress responses. In nematodes, there are four Ephrin ligands (EFN-1-4). Nevertheless, EFN-1, EFN-2 and EFN-4 are not expressed in the germline (https://wormbase.org, accessed on 1 October 2022). In addition, PS-NP (0.1-10 µg/L) exposure did not affect the expression of efn-1, efn-2, and efn-4 (data not shown).
The activation of Ephrin receptor VAB-1 by ligand EFN-3 has been confirmed by biochemical analysis [41]. In C. elegans, Ephrin ligands (such as EFN-1 and EFN-2) can function together with receptor VAB-1 in regulating cellular organization during development [41]. In this study, we also observed the transgenerational increase in VAB-1 expression after PS-NP exposure ( Figure 3A,B), and the vab-1(RNAi) nematodes also exhibited resistance to transgenerational PS-NP toxicity ( Figure 3C,D). Therefore, Ephrin ligand EFN-3 acted together with its receptor VAB-1 to regulate transgenerational toxicity of PS-NP in nematodes ( Figure 6C).
Several lines of evidence were further raised in this study to suggest the potential involvement of transgenerational communication of Ephrin signaling in controlling transgenerational toxicity of PS-NP. On the one hand, we found that RNAi of vab-1 encoding Ephrin receptor at F1-G could inhibit the induction of transgenerational toxicity in the PS-NP-exposed transgenic strain overexpressing germline EFN-3 ( Figure 4A,B). On the other hand, in PS-NP exposed nematodes, germline overexpression of EFN-3 could increase VAB-1 expression at F1-G ( Figure 4C). These observations suggested that the increase in germline Ephrin ligand EFN-3 caused by PS-NP exposure mediated the transgenerational toxicity by functioning upstream of its receptor VAB-1 in the offspring. Recently, we also found that several germline insulin ligands (INS-3, INS-39, and DAF-28) and germline Wnt ligand (LIN-44) could be activated by PS-NP exposure, which then potentially mediates the transgenerational toxicity induction by activating or inhibiting corresponding receptors in the offspring [22,53].
VAB-1 is mainly expressed in neurons and reproductive tissues [42,43]. Our tissuespecific activity analysis on VAB-1 indicated that VAB-1 acted in both neurons and germline to affect PS-NP toxicity in the offspring ( Figure 6C). This implied that, after the activation in the germline at P0-G, the EFN-3 could regulate transgenerational PS-NP toxicity by further activating receptor VAB-1 in neurons and germline, respectively, in the offspring. During development, VAB-1 has been found to be required for neuronal morphogenesis, neuronal regeneration, and axon guidance [54][55][56]. In addition, VAB-1 is also involved in the control of oocyte meiotic maturation and germline apoptosis [57,58].
In the germline, we further identified LIN-23, NDK-1, and EGL-1 as the targets of Ephrin receptor VAB-1 during the control of PS-NP toxicity ( Figure 6C). In PS-NP-exposed nematodes, germline RNAi of vab-1 caused the decrease in expressions of ndk-1 and lin-23, and the increase in egl-1 expression ( Figure 6B). This suggested that, in PS-NP-exposed nematodes, germline RNAi of vab-1 induced two different downstream molecular responses. In C. elegans, E3 ubiquitin ligase LIN-23 exhibits the function of cell division limitation [59], which is helpful for understanding the function of Ephrin signaling pathway and the observed reduction in brood size in PS-NP exposed nematodes. In C. elegans, EGL-1 is required for controlling stress responses, and egl-1 RNAi caused a susceptibility to toxicants or stresses, such as PS-NP and pathogen infection [48,60]. Moreover, we observed the increase in NDK-1 expression in PS-NP exposed nematodes with germline RNAi of vab-1 ( Figure 6B). Recently, we found that the decreased germline mir-38 could increase KSR-1/2 expression by suppressing the function of NDK-1 [28]. This suggested that, once the germline mir-38 was decreased by PS-NP exposure at P0-G, this could drive the suppression of NDK-1-KSR-1/2-EFN-3 signaling cascade transgenerationally. These findings are useful for explaining the observed toxicity in the offspring, such as those at both F1-G and F2-G [23].

Conclusions
Together, we here investigated the role of germline Ephrin signal in regulating transgenerational PS-NP toxicity using C. elegans as an animal model. Activation of Ephrin ligand EFN-3 could mediate the induction of transgenerational toxicity of PS-NP at predicted environmental concentrations by acting downstream of KSR-1/2, two kinase suppressors of Ras, in the germline. The activated germline EFN-3 by PS-NP exposure induced toxicity in the offspring by activating corresponding Ephrin receptor VAB-1 in both neurons and germline. In the neurons and the germline, VAB-1 controlled PS-NP toxicity by activating and/or inhibiting certain downstream targets. Our results are helpful for understanding how toxicity is induced in offspring (such as F1-G and F2-G) after PS-NP exposure at P0-G. Nevertheless, considering the examined C. elegans are hermaphrodites, the role and the underlying mechanism of germline Ephrin signals in regulating transgenerational PS-NP toxicity still need to be further elucidated in mammal models.

Conflicts of Interest:
The authors declare that they have no known competing financial interest or personal relationship that could have appeared to influence the work reported in this paper.