2025 Volume 50 Issue 1 Pages 23-32
The indispensability of biometals nickel, copper, and selenium in pharmaceutical, agricultural, and other industrial applications, coupled with their release from mining processes, has made them potent environmental contaminants, especially when present in aquatic ecosystems at levels above the essential range. The toxicity of these biometals in fish embryogenesis, including their toxicity levels, was studied using medaka embryos. Test solutions (0.001–10 ppm) of the biometals, along with an isotonic solution as a control, were introduced into the embryos using a nanosecond pulsed electric field application. The exposed embryos were cultured at 25 ± 1°C and microscopically observed daily for 14 days in an isotonic solution. Developmental abnormalities and toxicity were observed during the 14-day observation period. All biometals caused some abnormalities in developing embryos at all concentrations. Major abnormalities included delayed development; deformities such as curvature of bones or spines; abnormal formation of the hearts, eyes, and circulatory systems; and mortality. The toxicity of the biometals was significantly different (p < 0.05) from that of the control. Gene expression analysis revealed that 4747, 1961, and 1952 genes were affected by copper, nickel, and selenium, respectively. Copper affected the highest number of genes and caused the highest toxicity. These results indicate that nickel, copper, and selenium can cause toxicity in developing fish embryos at concentrations ranging from 0.01 ppb to 10 ppm. Therefore, there is a need to constantly monitor the levels of these biometals, particularly in aquatic ecosystems, to preserve aquatic life.
Nickel (Ni), copper (Cu), and selenium (Se) are among the essential metals often required by living organisms in trace amounts for certain biological functions and are hence termed as biometals (Ghosh et al., 2016; Lavado et al., 2019). The lack or excess of these biometals, owing to concentration imbalances in living systems, can cause detrimental pathological conditions or metabolic disorders. The maintenance of appropriate concentration levels of these biometals is crucial for the proper functioning of living systems, particularly in humans (Osredkar, 2011; Ghosh et al., 2016). Zdrojewicz et al. (2016) reported that adequate levels of Ni in the human body increase hormonal activity and facilitate lipid metabolism. According to Osredkar (2011), Cu has antioxidant and pro-oxidant activities, is involved in the production of hemoglobin, myelin, and melanin, and ensures the proper functioning of various critical enzymes in the human body. Se also provides antioxidant protection to the vascular endothelium, low-density lipoproteins, DNA, and chromosomes, serves as an immunomodulator, and facilitates the conversion of thyroxine to triiodothyronine during thyroid hormone biosynthesis (Batta, 2017).
Despite their beneficial roles, these biometals may cause certain pathological conditions, particularly when their levels are above essential or normal ranges. According to Zdrojewicz et al. (2016), the harmful effects of excessive Ni include genotoxicity, teratogenicity, immunotoxicity, carcinogenicity, and blood-related disorders. Zdrojewicz et al. (2016) further stated that increasing incidences of allergies to Ni have caused European regulations to reduce the permitted levels of Ni in Ni-containing products. Vetchý et al. (2018) reported that hepatic accumulation of Cu can cause severe chronic liver disease and idiopathic toxicosis. Excess Se in the human body produces oxygen radicals and causes apoptotic cell death through the induction of oxidation and cross-linking of protein thiol groups, which might adversely affect the function of critical proteins (Batta, 2017).
These biometals are included in trace amounts in some pharmaceutical formulations, such as hematinics, and in nutritional or food supplements to help maintain their appropriate concentrations in the human body. In addition, they have numerous industrial and agricultural applications (Zhou et al., 2017). Their use in pharmaceutical, agricultural, and other industrial applications, coupled with their release from mining processes, has made them potent environmental contaminants. They may be present in industrial wastes, such as wastewater, mining waste, pharmaceutical waste, and agricultural waste, and may be released into the environment, especially aquatic ecosystems, at levels above essential amounts, which may be detrimental to aquatic animals such as fish.
Verma (2012) reported that excess Ni in aquatic environments affects the behavior, survival, growth, and reproduction of aquatic animals. Al-Attar (2007) demonstrated that Ni exposure for 3 weeks reduced the serum levels of sodium and chloride and osmolality and increased those of glucose, cholesterol, total protein, albumin, amylase, alanine aminotransferase, and aspartate aminotransferase in Oreochromis niloticus. Yokota et al. (2019) also reported that high concentrations of Ni nanoparticles and NiCl2 led to tissue damage in the gills, digestive tract, and liver of Danio rerio and that the damage was largely characterized by epithelial degeneration and necrosis in the gills, esophagus, and intestines. According to Malhotra et al. (2020), exposure to low (180 µg/L), medium, and high (560, 1000, and 3200 µg/L, respectively) concentrations of Cu induced changes in the morphology of winter flounder fish. The gills of fish, especially freshwater fish, serve as an exposure pathway to acute Cu toxicity in the concentration range of 0.01–0.02 ppm (Senior et al., 2020). The USEPA (2021) reported that chronic exposure of fish and aquatic invertebrates to Se causes reproductive impairments, such as larval deformity and mortality, and affects juvenile growth and mortality.
These studies indicate that despite their essential roles, biometals can exhibit toxic effects on both humans and aquatic animals. However, information on their toxicity in aquatic animal embryogenesis is limited, and there are very few reports regarding methods for incorporating biometal solutions into embryos for toxicity studies. Therefore, in this study, we investigated the developmental toxicity of Ni, Cu, and Se in aquatic organisms using Japanese medaka embryos as a model. Initial attempts to evaluate embryonic development faced challenges with poor penetration of the eggshell chorion. To address this, we developed nanosecond pulsed electric field (nsPEF) technology to introduce chemical substances into the embryo (Tominaga et al., 2010; Kono et al., 2015; Yamaguchi et al., 2018; Nishiyama et al., 2021). The nsPEF method is an electroporation method in which several thousand volts are discharged in nanoseconds, as opposed to the conventional electroporation methods wherein several hundred volts are discharged in milliseconds. Using this technique, the permeability of the medaka egg membrane is temporarily altered, which facilitates easy and quantifiable introduction of chemical substances. Previous studies have shown that this technique can be used to introduce chemical substances without adversely affecting the development of medaka embryos. Additionally, we explored the toxicity mechanisms of these biometals to understand their impact on aquatic animals, particularly fish.
Two types each of nickel salts, NiCl2 (CAS RN®: 7718-54-9, 95% purity) and Ni(NO3)2 (CAS RN®: 13478-00-7, 97% purity); copper salts, CuSO4.5H2O (CAS RN®: 7758-99-8, 99.5% purity) and CuCl2 (CAS RN®: 7758-99-8, 99.5% purity); selenium salts, SeO2 (CAS RN®: 7446-08-4, 97% purity) and Na2SeO4 (CAS RN®: 13410-01-0, 97% purity); as well as NaCl (CAS RN®: 7647-14-5, 99% purity) and KCl (CAS RN®: 7447-40-7, 99% purity) were obtained from FUJIFILM Wako Pure Chemical Industries, Osaka, Japan.
Preparation and measurement of exposure test solutionsStock solutions of approximately 100 ppm NiCl2, Ni(NO3)2, CuSO4.5H2O, CuCl2, SeO2, and Na2SeO4 were prepared and adjusted to pH 7 using an HCl or NaOH solution, as required. The concentrations of stock solutions were measured using a Microwave Plasma Atomic Emission Spectrophotometer (4100MP-AES, Agilent Technologies, Santa Clara, CA, USA). Stock solutions of each metal salt were serially diluted to obtain exposure test solutions at concentrations of 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, and 10 ppm. The concentrations of the diluted solutions were measured using the same analytical techniques as for the stock solutions. The diluent was an isotonic salt solution that contained 28 mM NaCl, 2.7 mM KCl, and 1.8 mM CaCl2, with the pH adjusted to 7.3 using a 1 N NaHCO3 solution. This isotonic solution was also used as the control.
Test animalsJapanese medaka (d-rR strain), which had been maintained for more than 10 years in our laboratory and fed Artemia nauplii twice daily, were used for this study. The glass tank of the animals was maintained under a photoperiod of 16:8 hr light: dark cycle, and the temperature of the culture water was 25 ± 1°C. Embryos spawned by each adult female medaka were carefully collected 6 hr after fertilization, and the viable ones were used for the exposure test. Fertilized and viable embryos were examined under a digital microscope VHX-900F (KEYENCE, Osaka, Japan).
Exposure and toxicity assessmentExposure test solutions of the respective metal salts were introduced into the embryos according to the methods described by Tominaga et al. (2010), Kono et al. (2015), and Yamaguchi et al. (2018). In summary, exposure test solutions of the respective metal salts (nominal concentrations of 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, and 10 ppm were used for NiCl2, Ni(NO3)2, CuSO4, CuCl2, SeO2, and Na2SeO4) were introduced into embryos using nsPEF in an isotonic solution. Embryos were immersed in the respective metal salt solutions for 2 hr after injection, following which they were washed in an isotonic solution after immersion, individually transferred into a 96-well plate (one embryo per well) containing an isotonic solution, cultured, and observed microscopically in an isotonic solution for 14 days using a digital microscope VHX-900F (KEYENCE, Osaka, Japan). Each group consisted of 12 medaka embryos, with one plate per chemical, and this experiment was performed in triplicate.
During the 14-day observational period, the embryos were maintained at 25 ± 1°C, and the isotonic solution was changed daily. Developmental abnormalities and mortality were determined through microscopic observations, and daily records of abnormalities and mortality were maintained. Embryos that died within 1 day of nsPEF treatment were excluded from the evaluation and were considered to have died from electrical effects, whereas those that died after 1 day of nsPEF treatment were considered to have died owing to salt exposure. Embryos that did not hatch within 14 days of nsPEF application were termed delayed embryos, whereas those with abnormal formation of the heart, blood circulatory system, and curvature of bones were considered malformed or deformed embryos. Mortality, malformation (deformities), and delay rates were obtained from the number of dead, malformed (deformed), and delayed populations, respectively, of the total number of embryos used for the test. These observational methods and endpoints were in accordance with those described in our previous study (Uchida et al., 2023). In addition, calcein staining was performed on the hatchlings that showed spinal curvature. For calcein staining, 100 µL of staining solution (1 mL isotonic solution mixed with 20 µL of a 1% calcein solution and 20 µL of a 10% MS222 solution) was added to medaka (100 µL isotonic solution) immediately after hatching and incubated in the dark for 15 min. After staining, the medaka were washed with an isotonic solution and transferred to glass slides. A stereomicroscope (VB-S20, KEYENCE) with a filter for GFP (bandpass 500–560 nm) was attached and fluorescence was observed (excitation 440–460 nm).
Gene expression analysisWe performed gene expression analysis on medaka embryos treated with 1 ppm CuCl2, NiCl2, or SeO2 via injection using nsPEF. The treated samples were collected at 2 days post fertilization (dpf) and stored for further analyses. This procedure was repeated several times, and 50 samples were collected. The recovered embryos were rapidly frozen in liquid nitrogen and stored at −80°C until analysis.
Total RNA was isolated from medaka eggs using the RNeasy Micro Kit (Qiagen, Hilden, Germany) as described previously (Tominaga et al., 2010; Yamaguchi et al., 2018, 2020). The quantity and purity of total RNA were examined photometrically by measuring the A260:A280 nm and A260:A230 nm ratios using a Q5000 spectrophotometer (Tomy Seiko Co., Ltd.; Tokyo, Japan) and electrophoretically analyzed using the RNA 6000 Nano LabChip Kit and Agilent Bioanalyzer 2100 (Agilent Technologies; Santa Clara, CA, USA). RNA samples with RNA integrity numbers (RIN, as defined by Agilent Technologies) >9.0 were subjected to mRNA purification using the Oligotex-dT30 <Super> mRNA Purification Kit (TAKARA BIO Inc., Shiga, Japan). A cDNA library was prepared from mRNA (1 µg) using Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The constructed library was then subjected to PCR amplification, purification, and size selection (200 bp). The selected PCR products were subjected to emulsion PCR using an Ion PGM Hi-Q OT2 Kit (Thermo Fisher Scientific). The emulsion PCR products were sequenced on Ion PGM using Ion 318 Chip kit v2 (200 bp, 5M reads) (Thermo Fisher Scientific).
In the analysis, the genes whose expression levels were at least three times greater or less than one-third of those in the nsPFE-treated control were considered to have been affected by exposure to metals. Bioinformatics analyses were performed using CLC Genomics Workbench software version 10.1.1 (Qiagen, Hilden, Germany).
Statistical analysisStatistical analyses were performed using SPSS Statistics ver. 28.0 (IBM, Tokyo, Japan). Levene’s test was used to assess all data to ensure the homogeneity of variance across treatments. When assumptions were made, the data were analyzed using parametric one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison tests. If heterogeneity was found, the data were analyzed using the nonparametric Kruskal–Wallis test, followed by Dunn’s multiple comparison test.
Table 1 shows the actual concentrations of the stock solutions of all metal salts. The percentage deviation between the nominal and measured (actual) concentrations ranged from 0.2% to 2.2%. This indicated a deviation <20%, suggesting that the solutions were within the required concentrations for use as exposure test solutions.
| Concentration (ppm) of metal salts | |||
|---|---|---|---|
| Metal compound | Nominal | Measured (Actual) | Deviation (%) |
| CuCl2 | 100 | 102.2 | 2.2 |
| CuSO4 | 100 | 98.7 | 1.3 |
| NiCl2 | 100 | 100.3 | 0.3 |
| Ni(NO3)2 | 100 | 101.1 | 1.1 |
| SeO2 | 100 | 99.8 | 0.2 |
| Na2SeO4 | 100 | 101.1 | 1.1 |
After a 2 hr exposure to each metal at a concentration of 10 ppm without the use of the nsPEF method, the incidence of abnormalities was not significantly different from that in the control group (data not shown). However, when the nsPEF method was used, at 14 days of exposure, the highest mortality rate noted across different exposure concentrations was 8.3% for Cu, 8.7% for Ni, and 4.5% for Se compared to the 2.8% rate for the control (Fig. 1). Malformation occurred in 13.3% of the controls, whereas it reached up to 59.6%, 43.8%, and 62.2% in the Cu-, Ni-, and Se-treated groups (Fig. 1). These observations revealed an increased incidence of abnormalities (Fig. 1) in the metal-treated groups. The incidence of all abnormalities was in the order of Cu > Se > Ni, indicating that the toxicity of Cu was the highest among the biometals. Fig. 2 (A–C) shows the various abnormal characteristics induced by specific metals (delayed development, bone curvature, abnormal eye formation, curved spines and inability to swim upwards, and abnormal heart formation) in medaka embryos during the 14-day observation period. Developmental delays, spine abnormalities, and heart abnormalities were common to all the metal-treated groups, whereas eye abnormalities were characteristic of the Ni- and Cu-treated groups. Fig. 3 shows bone-formation abnormalities caused by biometals in medaka embryos, as confirmed using the calcein staining method. Staining of the bones with calcein revealed straight vertebral formation in the controls. In contrast, metal-exposed individuals exhibited spinal dysplasia and defects, which suggested that these osteogenic abnormalities were responsible for the curvature of the vertebrae (Fig. 3).
Statistical analysis of the effects of various concentrations of biometal ions injected using high electric field on the development of Medaka embryos. (A) Cu (CuCl2 and CuSO4), (B) Ni (NiCl2 and Ni(NO3)2), (C) Se (SeO2 and Na2SeO4). a, significant difference vs control (-) p < 0.05, b, significant difference vs control (+) p < 0.05, c, significant difference vs control (-) p < 0.01, d, significant difference vs control (+) p < 0.01.
Developmental abnormalities identified in the biometal exposure group.
Embryos were injected with the respective concentrations of metal ion salts, and incubated at 25°C. Photographs were taken at the indicated dpf. N = 12 per group.
A: Cu
a: Individuals with delays in spine formation; b: Abnormal eye formation; c: Individuals with curved tail; d: Individuals whose spines were curved and who could not swim up; e: Individuals with abnormal heart formation.
B: Ni
a: Individuals that died during hatching; b: Individuals with delays in eye and heart formation; c: Individuals with large curved spines; d: Individuals that had abnormalities in eye formation; e: An abnormality was seen in eye formation, and the hatched individual.
C: Se
a: Individuals with delays in spine formation; b: Individuals with abnormal heart formation; c: Individuals with curved spines at the tail; d: Individuals whose spines are curved and cannot swim up; e: Individuals with abnormalities in the formation of the head and spine.
Identification of abnormal bone formation in Medaka embryos owing to biometal ion exposure using the calcein staining method. The calcein-stained spine of a normal individual showed complete spine-formation in the tail, whereas the spines of the individuals exposed to the metal ions were observed to have curvature and defects owing to dysplasia, especially in the tail regions.
Gene expression analysis revealed the number of genes whose expression levels changed upon the exposure of the embryos to biometal solutions. The expression levels of 4747, 1961, and 1952 genes were altered upon exposure to Cu, Ni, and Se, respectively (Fig. 4). Fig. 4A shows that individual exposure to Ni and Se increased the expression levels of 1329 and 1339 genes, respectively. Among these, 301 genes showed increased expression levels in both the Ni- and Se-treated groups. Cu exposure caused an increase in the expression of 37 genes, of which eight also exhibited increased expression in the Ni-treated group and eight other genes showed increased expression in the Se-treated group. However, none of the genes showed increased expression in all the three metal-treated groups (Fig. 4A).
Numbers of genes that showed increased or decreased expression levels upon metal ion exposure. (A) Genes with increased expression levels; (B) Genes with decreased expression levels
Cu exposure resulted in a reduction in the expression of 4010 genes, whereas Ni and Se exposure caused a reduction in the expression of 623 and 613 genes, respectively (Fig. 4B). Overall, 393 genes were commonly downregulated by both Cu and Ni, 378 were commonly downregulated by both Cu and Se, and 161 were commonly downregulated by both Ni and Se. Additionally, there were 13 genes whose expression levels were reduced upon exposure to each of the three biometals (Fig. 4B). Table 2 lists some of the common genes that showed decreased expression levels upon exposure to biometals.
| Gene name | Change in expression levels |
|---|---|
| furin mRNA for furin endoprotease | -7.84 |
| neuropeptide Y (NPY), mRNA | -∞ |
| UCA mRNA for MHC class I antigen | -∞ |
| transcription factor Sp3-like, (LOC101157986), transcript variant X1, mRNA | -5.06 |
All the metals, at concentration ranges of 0.01 ppb to 10 ppm, exhibited certain degrees of toxicity or resulted in abnormalities in the medaka embryos. The abnormalities caused by the metals affected organs such as the eyes and heart and also resulted in bone and blood disorders. Common abnormalities included delayed hatching and defects in spine and heart formation. Hatched embryos with spine defects, especially curved spines, could not swim. The most significant observation was that the extent or degree of toxicity or abnormalities caused by the biometals was not concentration dependent, contrary to the findings of Ololade and Oginni (2010), Verma (2012), and Rudel et al. (2013). The differences in the concentration dependency of the metals between this study and other studies probably resulted from differences in the developmental stages (i.e., embryos for this study vs adult fish for the other studies) and the route and duration of exposure of the test models (Hamilton, 2004).
Although studies on the embryological developmental toxicities of Ni, Cu, and Se are limited, researchers have reported the toxicity of these biometals to aquatic animals, especially fish, in freshwater environments. Verma (2012) indicated that exposure of M. lamarrei and M. dayanum to Ni concentrations of 65.77 and 416.47 ppm, respectively, led to increased aggression and loss of balance, respectively. Ololade and Oginni (2010) also observed a decrease in blood parameters such as erythrocyte and leukocyte counts, hematocrit, and hemoglobin levels upon the exposure of Clarias gariepinus to Ni and concluded that hematological parameters can be used as indicators of Ni-related stress in fish upon exposure to elevated levels of Ni. Furthermore, exposure of Oryzias melastigma to Ni at concentrations above 0.13 ppm can reportedly cause malformations (Liu et al., 2021). According to Woody and O’Neal, (2012) and Senior et al., (2020), chronic exposure of fish to sublethal concentrations of Cu can cause impairment in reproduction and growth and that Cu is acutely toxic to freshwater fish at concentrations ranging from 0.01 to 0.02 ppm, which fall in the range of concentrations of biometals used in this study. In the present study, the Cu concentration taken up in the embryo was considered to be less than 0.01 ppm, leading to developmental rather than lethal effects being observed in this study. Furthermore, the results of this study confirmed previous findings that Cu exposure causes spine curvature and malformations in O. melastigma, exposure to Cu concentrations >0.8 ppm results in more than 60% morphological abnormalities, and spine curvature is a common effect of Cu exposure (Wang et al., 2020). Lemly (2002) reported that Se toxicity in Belews Lake, North Carolina, USA, resulted in reduced production of viable eggs owing to ovarian pathology, post-hatch mortality owing to bioaccumulation of Se in eggs, and teratogenic deformities of the spine, head, mouth, and fins. The findings of the USEPA (2021), which also included reproductive impairments such as larval deformity, juvenile growth, and mortality upon exposure of fish to Se, are consistent with the findings of the current study. It has also been reported that exposure of medaka to 50 µM selenium causes reduced hatchability and malformation (Kupsco and Schlenk, 2014). The findings of this study support this previous result, and the methods used are considered to be very sensitive because the effects can be ascertained even at low concentrations. Therefore, the aforementioned findings, along with results of the present study, suggest that Ni, Cu, and Se are toxic to fish and may have significant effects on embryogenesis.
The number of genes affected by a particular biometal corresponded to the degree of toxicity caused by that biometal (Fig. 4). The findings wherein the genes were commonly affected by two biometals, such as Cu, and Ni or Ni and Se, or all three biometals suggested that the biometals involved may have similar toxic effects on embryos. Table 2 shows some common genes that showed decreased expression levels upon exposure of the embryos to biometals. Oryzias latipes encodes a proprotein conversion enzyme called furin A. It has also been reported that knockout of the furin A gene in medaka results in malformation of the spine (Murata and Kinoshita, 2015). These results are consistent with the abnormalities that appear in the spines of medaka embryos upon exposure to biometals. The results of this study suggest that the biosynthesis of furin A is suppressed by biometals, thereby suppressing the activation of proteins and leading to embryonic malformation. Oryzias latipes neuropeptide Y (NPY) and its mRNA (Table 2) were associated with NPY, a peptide neurotransmitter, and their expression levels were confirmed to be reduced. NPY is widely present in the hypothalamus, cerebral cortex, and hippocampus and is involved in the regulation of hypothalamic hormones, appetite, and memory. It has also been reported to be involved in stress responses by functioning as a catecholamine cotransmitter in the peripheral nervous system (Munefumi, 1997). Oryzias latipes UCA mRNA for MHC class I antigens (Table 2) encodes the main histogenic gene complex (MHC) required for immune responses. MHC are classified as class I or II and play a role in presenting antigens to Class II molecules bind to foreign amperes (derived from bacteria and viruses) taken up by endocytosis to present antigens, such as Class II molecules and extracellular antigens, to helper T cells. In contrast, class I antigens bind to the proteins produced in cells (tumor-specific antigens) to present antigens to killer T cells as class I molecules and intracellular antigens. Killer T cells are antigen-reactive to virus-infected cells and cancer cells presented by Class I molecules and prevent the proliferation of viruses and cancer cells (Kajikawa and Kasahara, 2009).These results confirmed that the expression of Oryzias latipes neuropeptide Y (NPY) mRNA and Oryzias latipes UCA mRNA for MHC class I antigen genes is related to stress and immune responses. This meant that the defects in these genes not only resulted in abnormalities such as spine and angioplasty but also in endogenous effects such as immune dysfunction. However, many genes with changes in expression levels common or unique to these biometals were not clearly identified. For example, the gene responsible for abnormalities in eye formation upon exposure to Ni has not yet been identified. It is possible that the abnormalities in eye formation may have resulted from defects in several genes; hence, future research is required to resolve this uncertainty.
ConclusionThis study showed that the introduction of metal ions into medaka embryos via nsPEF application is possible; hence, nsPEF can be used to evaluate biometal toxicity in aquatic animals. Compared to that in conventional methods (fertilized embryos of zebrafish and medaka), abnormalities can be detected after a short exposure period, and the effects on early development can be evaluated. Each biometal showed different degrees of toxicity to medaka embryos, with the degree of toxicity being independent of concentration. Among the three, Cu showed the highest degree of toxicity based on gene expression analysis. However, Ni showed greater toxicity than Cu with respect to eye formation abnormalities. The results of this study indicate that biometals may be toxic to fish, particularly during embryogenesis.
Part of this research was supported by the Matching Planner Program "Exploratory Test" from JST and Grants-in-Aid for Scientific Research (B) (no. 23H03562) from the Japan Society for the Promotion of Science (JSPS), Japan. We would like to thank Editage (www.editage.jp) for English language editing.
Conflict of interestThe authors declare that there is no conflict of interest.
