Effects of microplastics on reproductive characteristics and mechanisms of the marine rotifer Brachionus plicatilis

Microplastic pollution, especially secondary microplastics (MPs), poses a significant threat to marine ecosystems. Despite its prevalence, the impact of natural-aged MPs on marine organisms, hindered by collection challenges, remains poorly understood. This study focused on 1–3 μm natural-aged MPs collected from Japan's coastal sea, investigating their effects on the rotifer Brachionus plicatilis sensu stricto and its reproductive mechanisms. Rotifers exposed to varying MP concentrations (0, 20, and 200 particles/mL) over 14-day batch cultures exhibited reduced population growth and fertilization rates. Down-regulation of reproductive genes and up-regulation of oxidative stress-related genes were observed, indicating MP-induced disruptions. Enhanced activities of superoxide dismutase and acetylcholinesterase and elevated malondialdehyde levels further emphasized oxidative stress. These findings underscore the detrimental impact of MPs on rotifer reproductivity, shedding light on the underlying mechanisms.

parameters.Additionally, exposure to the microbeads resulted in the up-regulation of reactive oxygen species (ROS) production and the induction of multiple antioxidant-related enzymes in the experimental plankton 16 .Similarly, negative effects on growth rate, mortality, and reproduction were documented in the freshwater cladoceran Daphnia magna upon exposure to nanosized polystyrene beads 17 .In a previous study involving copepods, a decrease in fecundity was measured in test groups exposed to artificial MPs with diameters of 0.5 μm and 6 μm 18 .Furthermore, it has been established that the adverse effects of the MPs on rotifers are contingent upon the size and concentration of the particles 16,18,19 , with life history experiments demonstrating that high concentrations of small-sized MPs (0.07 μm) decrease rotifer survival and reproduction, prolong the time to maturation, and reduce body size at maturation, while large-sized MPs (0.7 and 7 μm) do not significantly affect rotifer life-history traits 19 .Moreover, studies have demonstrated that ingested MPs can disrupt the endocrine system and influence physiological functions, development, and reproduction in aquatic animals [20][21][22] .
While Vroom et al. 23 has provided clear evidence of the adverse impacts of MPs on the survival and reproduction of animal plankton, the majority of these studies have utilized artificial primary MPs, such as polystyrene microbeads.There is a scarcity of experiments that utilize MPs found in the marine environment.It has been shown that aged MPs are ingested by a greater number of individuals and at higher rates than pristine plastics in copepod species, indicating the importance of considering aging and weathering effects when investigating the dynamics and effects of plastic ingestion by marine organisms in laboratory settings.Therefore, the present study aims to investigate the effects of marine MPs directly collected from the ocean on the reproduction of marine zooplankton, the euryhaline rotifer Brachionus plicatilis as well as elucidate the underlying mechanisms.

Rotifer reproduction
No significant differences were observed in the rotifer reproductive parameters, such as population growth rate (r), mixis rate, and fertilization rate (Table 1), although, visible variations in population changes were observed between the control group and the experimental groups.The experimental rotifers exposed to MPs exhibited a declining trend in both asexual and sexual reproduction.Specifically, the total population of rotifers exposed to MPs at 20 particles/mL (MP20) showed a decrease during the rearing experiment.Furthermore, the population of female rotifers without eggs (?♀) was lower in the MP20 group compared to the control group.Similarly, the MP20 group displayed a lower growth trend in the population of female rotifers with amictic female eggs (♀♀).Additionally, the population of mictic female rotifers with male eggs (♂♀) exposed to MPs at 200 particles/mL (MP200) showed a decreasing trend compared to the control rotifers.Notably, the number of mictic female rotifers with resting eggs (R♀) in the MP20 group exhibited a decreased pattern compared to the control group (Fig. 1).

Relative gene expression
Gene expression analysis was conducted to determine the antioxidant metabolism of the experimental rotifer.The expression levels of the following target genes were assessed, and the obtained results were descripted in Fig. 2A: CAT , CuZnSOD, CYP (Clan 3), GSTo1 and GSTs2, and MnSOD.Significant differences in mRNA expression were observed for most of the genes related to antioxidant metabolism, except for CYP (Clan 3).In the MP20 group, there was a significant upregulation in gene expression for CAT , CuZnSOD, GSTo1, GSTs2, and MnSOD.Specifically, CAT gene expression was upregulated by 1.2-fold, CuZnSOD by 1.5-fold, GSTo1 by 1.6-fold, GSTs2 by 2.8-fold, and MnSOD by 1.3-fold.In the MP200 group, there was a significant upregulation in gene expression for CuZnSOD, GSTo1, GSTs2, and MnSOD.Nevertheless, several gene expressions were upregulated such as CuZnSOD by 1.1-fold, GSTo1 by 2.6-fold, GSTs2 by 6.0-fold, and MnSOD by 1.1-fold.
To assess oxidative stress in the experimental rotifer, we examined the gene expression levels of AChE, HSC70 like 1, and NADPH oxidase 5 and the results were represented in Fig. 2B.In the MP20 group, there was a significant increase in gene expression of HSC70 like 1, which was upregulated by 1.6-fold.On the other hand, the MP200 group showed a significant decrease in the gene expression of NADPH oxidase 5, which was downregulated by 0.8-fold.
The lipid metabolism of the experimental rotifer was assessed by examining the gene expression levels of ACC and mitochondrial CYP what results were confirmed in Fig. 2C.In the MP20 group, there was a significant increase in gene expression for both ACC and mitochondrial CYP compared to the control group.ACC gene expression was upregulated by 1.1-fold, while mitochondrial CYP gene expression was upregulated by 1.6-fold.In contrast, in the MP200 group, there was a significant decrease in gene expression of ACC compared to the control group, with a downregulation of 0.9-fold.

MP20 Control MP200
Population growth rate (r) 0.   examining the gene expression levels of several genes associated with asexual reproduction, including Cathepsin L, Septin-2, and Sirtuin-2, as well as those associated with sexual reproduction, such as cdc42P, histone H2A, P300 CREB, and vasa.In the MP20 group, the expression of the Cathepsin L gene was downregulated by 0.6-fold and Septin-2 by 0.2-fold, both of which are related to asexual reproduction.Additionally, the gene expression of cdc42P was downregulated by 0.9-fold and P300 CREB by 0.3-fold, both related to sexual reproduction.However, the expression of Sirtuin-2, which is involved in both asexual and sexual reproduction, was significantly upregulated by 1.25-fold compared to the control group.In the MP200 group, the expression of Cathepsin L was downregulated by 0.3-fold, Septin-2 by 0.2-fold, cdc42P by 0.6-fold, histone H2A by 0.8-fold, and P300 CREB by 0.3-fold.

Generated ROS levels
After 6 days of exposure, the intracellular levels of ROS were found to be significantly decreased (p < 0.05) in all the MP groups tested, as shown in Fig. 3A.The experimental rotifers exhibited ROS levels that were approximately 83.5%, and 79.1% of its level in the control group.

Relative MDA levels and enzymatic activities (AChE, SOD, and CAT)
The relative levels of MDA and enzymatic activities associated with oxidative stress and antioxidant responses in experimental rotifers exposed to different concentrations of MPs revealed significant differences (p < 0.05), except for relative CAT activity.
Regarding oxidative stress biomarkers, the MDA level showed a significant upregulation in the MPs groups, reaching 169.4% in the MP20 group and 116.5% in the MP200 group (Fig. 3B).Furthermore, the relative activity of AChE was significantly upregulated in the MPs groups, measuring 136.1% in the MP20 group and 150.0% in the MP200 group.
Regarding antioxidant response biomarkers, the activity of SOD was significantly upregulated in the MPs groups, reaching 133.2% in the MP20 group and 118.4% in the MP200 group (Fig. 3C).

Discussion
Monogonont rotifers reproduce through cyclical parthenogenesis, which involves frequent asexual reproduction and sporadic sexual reprodcution 14,[24][25][26][27][28][29][30][31] .The transition from asexual (amictic) to sexual (mictic) reproduction is influenced by a range of internal and external factors, including genotype, diet, population density, photoperiod, temperature, and salinity 29 .Notably, the reproductive physiology of amictic and mictic females varies significantly.Additionally, their sizes and the types of eggs they produce are distinct from one another [27][28][29] .Furthermore, their mechanisms for oogenesis differ, as amictic females produce diploid eggs while mictic females produce haploid eggs 24 .Consequently, these distinctions in reproductive physiology lead to differential responses to various environmental conditions in amictic and mictic females 19,29,30 .In this experiment, no statistically significant differences were observed in the reproductive parameters of rotifers tested, while there was a noticeable decline in the population density of female rotifers with amictic female eggs (♀♀), mictic female rotifers with male eggs (♂♀), and the number of mictic female rotifers with resting eggs (R♀).These findings indicate that both sexual and asexual reproductions were influenced by the presence of marine MPs.Analysis of relative gene expression revealed significant downregulation of genes associated with asexual reproduction, such as Cathepsin L and Septin-2, which play roles in asexual female development and yolk processing 24,32,33 .Additionally, genes related to sexual reproduction, including cdc42P, histone H2A, and P300 CREB, which are involved in gametogenesis and chromosome segregation, were also significantly downregulated 32,34,35 .Furthermore, the conserved germ cell-specific gene vasa, responsible for germ cell formation, and crucial for gametogenesis and embryogenesis, showed decreased expression in the MP200 group compared to the control group [36][37][38] .Conversely, the aging-related gene Sirtuin-2, known to be associated with slowing aging in mammals, exhibited upregulation in the MP20 group [39][40][41] .Even if this study could not observe aging patterns of rotifers related to MP exposure through individual cultures, the difference may exist to lead maximum population density.www.nature.com/scientificreports/Previous studies have established a link between oxidative stress and sperm abnormalities, highlighting the adverse impact of elevated oxidative stress levels on mitochondrial membrane potential, electron transport chain-related male fertility, and sperm quality [42][43][44] .In the context of our experiment, the observed reduction in the prevalence of reproductive patterns in rotifers, such as female rotifers bearing amictic female eggs (♀♀), mictic female rotifers producing male eggs (♂♀), and mictic female rotifers forming resting eggs (R♀), is likely attributed to elevated oxidative stress levels and the consequent accumulation of oxidative stress.
ROS can be generated in the process of normal metabolism, while heightened in response to toxic substances and environmental stress, leading to detrimental effects on organismal growth 21,45 .Excessive ROS production results in oxidative stress, causing cellular dysfunction and mortality 46,47 .It has been known that MPs induce oxidative stress and disrupt antioxidant systems in organisms 16 .Therefore, assessing the antioxidant response is crucial for elucidating the toxic mechanisms by which MPs exerts its harmful effects on rotifers 48 .In our experiment, rotifers exposed to marine MPs exhibited lower relative ROS levels compared to the control group, contrary to our expectations.However, biomarkers indicating damage caused by ROS, such as relative MDA levels, relative AChE, and SOD activity, were significantly higher in the MP-exposed group compared to the control group.Also, in the analysis of relative gene expression, the gene HSC70 like1, associated with oxidative stress, was significantly upregulated in the control group, and all genes related to antioxidant metabolism (CAT , CuZnSOD, CYP (Clan 3), GSTo1, GSTs2, and MnSOD) were upregulated regardless of MP concentration.Upon analyzing these patterns, the diminished levels of ROS in rotifers from the marine MPs group, in contrast to the control group, can be ascribed to two plausible explanations.It is conceivable that rotifers actively synthesized enzymes, such as SOD, which are involved in the mitigation of reactive oxygen species, thereby accounting for the observed trend.Alternatively, this distinction could stem from a diminishment in metabolic activity within the rotifers.These findings serve as compelling evidence that rotifers exposed to marine MPs underwent oxidative stress.
Exposure of aquatic animals to contaminants, including MPs, not only increases ROS production but also triggers the subsequent activation of antioxidant mechanisms [49][50][51] .It has been known that the extent of oxidative damage depends on the effectiveness of an organism's antioxidant defense system 52 , including SOD, CAT, CYP, glutathione (GSH), and glutathione peroxidase (GPx), effectively eliminates ROS [53][54][55] .Previous studies investigating the impact of MPs exposure on organisms have presented evidence that heightened activities of SOD and CAT effectively alleviate the harmful consequences of ROS 56,57 .In the present study, the observed reduction in ROS levels in the MP-exposed groups can be attributed to the enhanced enzymatic activity of SOD, a key component of the antioxidant metabolism.

Conclusion
Our experiment provides valuable insights into the impact of marine MP exposure on organisms, particularly rotifers.The findings reveal that marine MPs induce oxidative stress and disrupt the antioxidant defense systems in rotifers, as evidenced by the significant alterations in biomarkers associated with ROS and antioxidant activity.Despite the unexpected lower relative ROS levels in MP-exposed rotifers compared to the control group, biomarkers indicating oxidative damage were significantly higher in the MP-exposed group.These results suggest that rotifers exposed to marine MPs experience oxidative stress, potentially impairing their reproductive patterns and overall fitness.Further investigations are necessary to elucidate the specific mechanisms by which MPs induce oxidative stress and impair reproductive processes in rotifers.In this study, a comparison between marine MPs and artificial MP beads was not conducted.However, future investigations should include such a comparison to examine the actual toxicity of marine MPs on marine organisms.As MP pollution continues to be a global concern, further research in this area is vital for a comprehensive understanding of its ecological implications and potential threats to aquatic ecosystems.

Marine microplastics
To investigate the impact of marine MPs on rotifer reproductivity and elucidate the underlying molecular mechanisms, we conducted a comprehensive sampling effort of marine MPs in the coastal sea of the Kyushu area, Japan (32°46.49′N129°43.80′E).Employing a subsequent filtration with mixed cellulose ester membrane filter (Membrane filter, pore size 1.00 μm and 3.00 μm, Advantec MFS Inc., Japan) enabled us to obtain a purified subset of MPs, specifically targeting particles approximately 3 μm (1-3 μm of size distribution) in diameter.The size of marine MPs were chosen to match the size of the feed phytoplankton (2 to 4 μm 58 ), which could potentially have a greater impact on the experimental rotifers through their digestive system, as they are filter feeders.To determine the accurate size distribution of marine MPs filtered, we measured the size of randomly selected 30 particles with an inverted microscope (ECLIPSE Ts2, Nikon Instruments Inc., New York, USA) (Fig. 4).

Target species
We employed the marine rotifer B. plicatilis sensu stricto (NH1L strain) with an average lorica length of 275 μm 59 .The rotifers used in this study were derived from populations of B. plicatilis (NH17L strain) that have been maintained at the Aquaculture Biology Laboratory, Nagasaki University, Japan, for over two decades.During the experiment, the rotifers were maintained in sterilized seawater at a salinity of 22 parts per thousand (ppt) and a water temperature of 25 °C in a controlled incubator (MIR-254S-PJ, PHCbi, Tokyo, Japan) under complete darkness.The culture medium was prepared by diluting natural seawater with distilled water (RFP841AA Pure Water Purifier, Adventec Manufacturing Inc., Ontario, Canada), followed by filtration (0.45 μm Membrane filter, Nihon Millipore K.K., Tokyo, Japan) to remove small particles and autoclaving (at 121 °C for 20 min).Additionally, the rotifers were fed on cultivated microalgae cells of Nannochloropsis oculata ad libitum.

Population growth of the rotifer
To assess the impact of marine MPs on rotifer reproductivity, we initiated batch cultures of rotifers which were maintained at 22 ppt and 25 °C with feeding of N. oculata at 7 × 10 6 cells/mL.From the one of these cultures, female rotifers carrying amictic eggs were pipetted out and inoculated into a prepared 15 mL glass bottle at a density of 1 individual per 1 mL.The experimental rotifers were subjected to specific concentrations of marine MPs.A concentration of 20 particles/mL, denoted as MP20, was selected to represent a realistic high level of MPs in marine environments 60,61 , thereby facilitating an assessment of the current impact of marine microplastics on local ecosystems.Additionally, a concentration of 200 particles/mL, denoted as MP200, was employed to assess potential future effects arising from the ongoing and escalating accumulation of plastic waste in marine ecosystems.These concentrations were maintained over a 14-day period during the experimental exposure of rotifers to marine MPs.The culture experiment with rotifers was carried out with three replicate trials.Throughout the culture period, we daily counted the total number of rotifers and female rotifers at various reproductive stages, including female rotifers without eggs (?♀), amictic female rotifers carrying female eggs (♀♀), mictic female rotifers carrying male eggs (♂♀), and fertilized mictic females carrying resting eggs (R♀).
We estimated the effects of MPs on sexual and asexual reproductivity by calculating population growth rate, mixis rate, and fertilization rate using the following equations.
In the equation for population growth (r), t represents the culture day, N t and N 0 denote the total number of female rotifers on day t, and N 0 , respectively.

Rotifer incubation for the assessment of associated biomarkers
To investigate the impact of marine MPs on rotifers, we conducted an incubation experiment using the same rotifer strain and experimental conditions employed in the population growth study.The rotifers were exposed to three different concentrations of marine MPs: 0 (control), 20, and 200 particles/mL, over a 6-day period.This incubation was carried out in 300 mL bottles, each containing a density of 10 rotifers per mL, and the experiment was conducted for 6 days, replicated in triplicate.This experimental setup was meticulously designed to ensure a sufficient population of rotifers for subsequent assessments.These assessments included analyzing gene expression associated with oxidative stress, encompassing the generation of reactive oxygen species (ROS), antioxidant metabolism, and reproduction, which were all analyzed using RT-qPCR.It also allowed for the measurement of ROS levels and the evaluation of the relative activities of key enzymes engaged in antioxidant defense, specifically superoxide dismutase (SOD), catalase (CAT), and acetylcholinesterase (AChE).Additionally, it facilitated the quantification of the relative amount of malondialdehyde (MDA).www.nature.com/scientificreports/

Relative gene expression
For RNA extraction, approximately 6000 rotifers were sampled and NucleoSpin® RNA kit (Takara Bio Inc, Shiga, Japan) was used following the manufacturer's instructions.The extracted RNA underwent treatment with a TURBO DNA-free™ kit (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) to eliminate genomic DNA contamination.The quality and quantity of RNA were assessed spectrophotometrically at 230 nm, 260 nm, and 280 nm using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA).Subsequently, complementary DNA (cDNA) synthesis was carried out utilizing the PrimeScript™ II 1st strand cDNA Synthesis Kit (Takara Bio Inc, Shiga, Japan), and the resulting cDNA was stored at − 20 °C until further analysis.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed using 1 μg of cDNA template, 0.5 μL of forward and reverse primers (100 μM), and 10 μL of TB Green Premix Ex Taq (2 ×) (Takara Bio Inc, Shiga, Japan), making a total volume of 20 μL.The RT-qPCR assay was carried out on a QuantStudio 1 Real-Time PCR System (Thermo Fisher Scientific, Waltham, Massachusetts, USA) with the following thermal cycling conditions: an initial denaturation at 94 °C for 4 min, followed by 43 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. Melting curve analysis was performed to confirm the amplification of specific products, involving cycles at 95 °C for 10 s, 55 °C for 1 min, and 80 cycles at 55 °C for 10 s, with a 0.5 °C increment per cycle 62 .The genes subjected to analysis can be located in Table 2, while their corresponding primer sequences were designed based on several previous studies [62][63][64][65][66] .The elongation factor 1-α (EF1-α) gene was selected as a reference due to its stable expression across and within the experimental groups.The transcriptional levels were calculated using the 2 (−ΔΔCt) method 67 .All experiments were conducted in triplicate to ensure reliability and reproducibility of the results.

Investigation of reactive oxygen species levels and enzymatic/biomarker activities
In addition to gene expression analysis, we also measured ROS levels and assessed the relative activities of key enzymes involved in antioxidant defense, namely superoxide dismutase (SOD), catalase (CAT), and acetylcholinesterase (AChE).Furthermore, we quantified the relative amount of malondialdehyde (MDA), which serves as a biomarker of lipid peroxidation and oxidative damage.These measurements allowed us to evaluate the oxidative stress status and the potential impact of MPs on enzymatic and biomarker activities in rotifers.
The levels of ROS were determined in rotifers exposed to marine MPs using a fluorogenic probe called 2′,7′-dichlorofluorescein diacetate (H2DCFDA) from Sigma-Aldrich.This probe undergoes oxidation by ROS, resulting in the production of fluorescent 2′,7′-dichlorofluorescein (DCF).The fluorescence intensity was measured using a Cytation™ 3 multimode plate reader (BioTek Instruments, Winooski, Vermont, USA) with excitation and emission wavelengths set at 485 nm and 520 nm, respectively.To prepare the samples, approximately 5000 rotifers were homogenized in a buffer solution containing 0.32 M sucrose, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM MgCl 2 , and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) (pH 7.4, adjusted with NaOH and HCl) using a Branson Sonifier 150 (Eerson Electiric Co., St. Louis, Missouri, USA).The homogenate was then centrifuged at 10,000×g for 20 min at 4 °C, and the resulting supernatant was transferred to new microtubes for further analysis.The reaction mixture consisted of 170 μL of phosphate-buffered saline, 20 μL of H2DCFDA probe, and 10 μL of the prepared sample.The mixture was incubated at 25 °C for 30 min in a 96-well black microplate (Nunc A/S, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA).Subsequently, the fluorescence was measured using a multimode plate reader (BioTek Cytation™ 3, Winooski, Vermont, USA) with excitation and emission wavelengths set at 485 nm and 520 nm, respectively.The obtained data were normalized to the total protein content and expressed relative to the control (100%).Total protein was quantified using the Bradford assay (Bio-Rad Protein Assay, Bio-Rad Laboratories, Hercules, California, USA) (Bradford, 1976).All experiments were performed in triplicate.

Malondialdehyde levels
The amount of Malondialdehyde (MDA) was quantified using a colorimetric method based on the reaction between MDA and thiobarbituric acid.A lipid peroxidation (MDA) assay kit from Sigma-Aldrich was employed for the measurement of MDA activity.In brief, the samples (approximately 4000 individuals) were homogenized on ice using 200 μL of MDA lysis buffer and sonication.The resulting homogenate was then centrifuged at 13,000×g for 10 min at 4 °C, and the supernatant was collected in new tubes for subsequent analysis, following the manufacturer's instructions.The absorbance of the samples was measured at an optical density of 532 nm.

Acetylcholinesterase activity
Acetylcholinesterase (AChE) participates in the hydrolysis of acetylthiocholine, leading to the production of thiocholine.The activity of AChE was determined using a Colorimetric AChE assay kit obtained from Abcam (Cambridge, UK).Briefly, approximately 4000 rotifers were homogenized in 300 μL of precooled phosphate buffer (0.1 M, pH 8.0) using sonication, following the method described in previous studies 62,68 .The resulting homogenate was then centrifuged at 3000×g for 30 min at 4 °C, and the supernatant was transferred to new tubes for subsequent analysis, as per the manufacturer's instructions.The activity of the enzyme was measured using 5,5-dithiobis (2-nitrobenzoic acid) at an absorbance of 410 nm.synthesis.The measurement of SOD activity was conducted using a colorimetric SOD activity assay kit (Abcam, Cambridge, UK) following the provided manufacturer's instructions.To perform the assay, approximately 4000 rotifers were homogenized on ice using 150 μL of 0.1 M Tris/HCl buffer (pH 7.4) containing 0.5% Triton X-100, 5 mM β-mercaptoethanol, and 0.1 mg/mL PMSF through sonication.The resulting homogenate was centrifuged at 14,000×g for 5 min at 4 °C, and the supernatant was transferred to a clean tube for further analysis according to the manufacturer's instructions.The activity of SOD was measured by monitoring the absorbance at 440 nm.

Catalase activity
Catalase (CAT) activity was assessed through a CAT-H   www.nature.com/scientificreports/ The measurement of enzyme activities was conducted using a commercially available enzyme kit, following standard laboratory protocols.Colorimetric signals were detected using a multimode plate reader (BioTek Cytation™ 3, Winooski, VT, USA) at the appropriate wavelengths for each enzyme.To account for variations in sample protein content, all enzyme activities were normalized to total protein and expressed relative to the control group (set as 100%).The quantification of total protein was carried out using the Bradford assay 69 .All experiments were performed in triplicate to ensure reproducibility and accuracy of the results (Supplementary Fig. S1).

Statistical analysis
Statistical analysis was conducted on the measured values of various rotifer population growth parameters, including population growth rate (r), mixis rate, and fertilization rate, as well as the relative enzymatic/biomarker activities such as relative gene expression, ROS levels, MDA level, AChE, SOD, and CAT activities in the experimental rotifer.To investigate the relationship between MPs and the growth of the rotifer population, an independent t-test (n = 3) was performed.This test compared the growth parameters of the experimental rotifer in the presence and absence of MPs to identify any significant differences.Furthermore, to assess the effect of MPs on the relative enzymatic/biomarker activities, including relative gene expression, ROS levels, MDA level, AChE, SOD, and CAT activities, a one-way ANOVA was conducted (n = 3).Prior to the ANOVA, Levene's test was used to confirm the homogeneity of variances.For post-hoc analysis, the Tukey HSD test was employed when the p-value was found to be less than 0.05.The statistical program SPSS (Sigma Stat 3.0, SPSS, Chicago, U.S.A) was utilized for all the statistical analyses.
Figure 2D illustrates the gene expression patterns associated with asexual and sexual reproduction in response to different concentrations of MPs.The reproductive behavior of the experimental rotifers was assessed by

Figure 2 .
Figure 2. Effects of marine microplastics at different concentrations of 0 particles/mL (control), 20 particles/ mL (MP20), 200 particles/mL (MP200) on the relative gene expression of rotifers.The relative gene expression related to antioxidant metabolism (A), oxidative stress (B), lipid metabolism (C), and reproduction (D) of the rotifers was analyzed in the present study.Superscript alphabet letters were used to indicate significant differences among the experimental groups (a > b > c, Tukey's test, p < 0.05, n = 3).

Figure 4 .
Figure 4. Microscopic view of microplastic particles collected from coastal sea of Kyushu area, illustrating the diversity in size and shape.Scale bar = 500 nm.

Table 2 .
GenBank accession numbers and primer sets employed in this study for assessing the associated biomarkers associated with oxidative/antioxidant activity, lipid metabolism, and rotifer reproduction.