Sex Speci c Effects of PET-MPs On Drosophila Lifespan

In recent years, as an emerging pollutant, microplastic pollution is gradually becoming a research hotspot. Microplastics are ubiquitous in the entire ecological environment. Organisms can be exposed to microplastics via inhalation or ingestion. In view of the widespread of microplastics pollution, the impact of microplastics on biology should be further investigation. In previous experiments, we have conducted research on the physiology of Drosophila exposed to PET Microplastics (PET-MPs). However, under long-term exposure, will PET-MPs affect the lifespan of Drosophila? Our experimental results indicate that for ANOVA analysis, there are signicant differences between males and females (F (1, 895) =68.19, p<0.001), between PET-MPs concentration (F (3, 895) = 8.11, p<0.001). There are also signicant interactions between sex and Microplastic concentration (F (3, 895) = 4.00, p<0.01). For Cox and log rank test, 1g/L of PET-MPs prolongs the lifespan of male ies. The reason of this phenomenon may be the hormesis effect.


Introduction
Microplastics are plastic fragments with a diameter of less than 5 mm. Since 2004, British scientists rst proposed the concept of microplastic (Thompson et al., 2004), the researchers on microplastic pollution are gradually deepening. Previous studies have illustrated the observations of microplastic deposition in the atmosphere (Chen et al., 2020), water (Sharma and Chatterjee, 2017), soil (Rillig and Lehmann, 2020), as well as common foods such as oysters, mussels, sh, sea salt, honey (De-la-Torre, 2020).
Microplastics even exist in the atmospheric sediments of remote mountainous (French Pyrenees) (Allen et al., 2019) and in the frozen layers of the Antarctic (Kelly et al., 2020). These researches are conveying an important message to us: microplastics pollution are ubiquitous around the world, and we must pay attention to the potential effects of microplastics pollution.
Humans can be exposed to microplastics via inhalation and ingestion. Will microplastics affect the human body? It has been reported that nylon ock workers are much higher exposed to harmful bers than the general person. Microplastic bers may irritate the lungs, causing lung volume decreased (van Dijk et al., 2021). Kelly and Wright predicted that the intake of microplastics may wreck immune cells, induce cell necrosis and tissue in ammation (Wright and Kelly, 2017). And nanoplastics may cause the more serious impact. The potential health risks of nanoplastic can be assessed the same as engineered nanoparticles (Chain, 2016). Research studies revealed that the size and hydrophobicity of nanoparticles enable them to pass through the blood-brain barrier and placenta into the lungs and intestines (Seltenrich, 2015), and cause changes in endogenous metabolites and the intestinal microbial community (Bergin and Witzmann, 2013;Cui et al., 2019). The impact of microplastics on human health has not yet been determined. In this stage, continuous experiment is required to have a comprehensive understanding on the potential hazards of microplastics.
We previously explored the effects of polyethylene terephthalate microplastics (PET-MPs) on the physiology of Drosophila. Our ndings demonstrated that PET-MPs can affect the number of eggs laid by female ies and the TG content of male ies (Shen et al., 2021). Under the long-term exposure, can animals regulate immune system and offset the impact of PET-MPs. Based on the question, we investigated the effect of PET-MPs on the ies' lifespan. The results showed that microplastics had no signi cant negative effect on the lifespan of female ies. To our surprise, the lifespan of male ies increased at the concentration of 1g/L. This is an interesting experimental phenomenon.

Drosophila Culture and Life Span Assays
The Drosophila melanogaster wild-type stock Canton-S (Bloomington Stock Center) was maintained on a 12 h:12 h light: dark cycle at the temperature of 25°C, humidity of 60% and light intensity of 500 Lux.
Enclosing adult ies were collected over a 24-hour period and mated for 48 hours before sorting into male vials or female vials. Life span experiments were conducted with a density of 40 ies per vial on sugar − yeast − agar (SYA) medium. Flies were transferred to new vials every other day and the number of deaths was counted.

Preparation of PET-MPs suspensions
PET-MPs of 2µm were used in this experiment. The ethanol and water were prepared to facilitate particle distribution in the 1:1 ratio. That is, suspend 1 g, 10 g and 20 g PET-MPs into 60 ul ethanol and 60 ul H2O.
So as to make it evenly mixed in the food, magnetic stirring was conducted for 2 hours at rst, and before the food was nished, ultrasonic vibration was executed for 30 minutes to make it evenly dispersed.
When the food temperature is cooled to 60°C, 1 g/L, 10 g/L or 20 g/L PET-MPs was added to 1 L of food respectively. The 60ul ethanol and 60ul H2O was add for the control group.

Statistical Analysis
Statistical analyses were performed using GraphPad Prism v. 6 and SPSS 25.0. 2-way ANOVA was conducted on gender and microplastic concentration analysis. The mean, median, maximum, minimum, Log rank tests and COX proportional hazards analysis were performed on survival curves.

Results
In our experiment, we investigated the effect of PET-MPs of different concentration on the ies' life span.
Simple effect result presented that for Control, 1g/L, 10g/L group, at the same concentration, the mean lifespan between female and male presence signi cant difference (Table 2). Under the same sex, 10 g/L PET-MPs caused 20.41 % decrease in female mean life span, and 20 g/L PET-MPs caused 16.0 % decrease in male mean life span (Table 3). But ANOVA is a signi cant test based on the mean of the sample (Fisher, 1992). Therefore, we conducted Cox analysis and log rank test to analyze under the same sex, the signi cance of microplastics on the lifespan of ies. "sex1" indicates female ,2 is male. Food indicates the concentration of PET-MPs. 1 is control, 2 is 1g/L, 3 is10 g/l, 4 is 20 g/L. "sex1" indicates female ,2 is male. Food indicates the concentration of PET-MPs. 1 is control, 2 is 1g/L, 3 is10 g/l, 4 is 20 g/L.
For log rank test, our result indicated that PET-MPs had no effect on the lifespan of female ies (Fig. 1A).
However, it is interesting that 1g/L PET-MPs prolongs the lifespan of male ies (P < 0.05) (Fig. 1B) ( Table 4). The media life span of male ies was augmented by 9.52 %. At the concentration of 20 g/L, the average life span of male ies was reduced by 17.39 %, but the p > 0.05. KM is a non-parametric test. while Cox is a semi-parametric test with higher test effect. Therefore, we choose Cox to verify the experimental conclusions (David, 1972). For Cox analysis. the results were consistent with log rank test (Table 5, Table 6).

Discussion
Before the experiment, we surmised the PET-MPs to increase mortality. But the experimental results, PET-MPs did not cause high mortality, which is different from our envision. One explanation of the result could be that the chemical properties of PET is highly inert and it has been designed for food packaging purposes (Zimmermann et al., 2019). The previous reports highlighted PET-MPs do not negatively affect the survival, metabolism and feeding activity of Gammarus pulex (Weber et al., 2018). There was no signi cant effect on the head capsule lengths weight HSP70 level in Chironomus riparius exposed to PET micro bers (Setyorini et al., 2021). Another potential explanation is related to peritrophic matrix (PM). PM, mainly composed of chitin and protein (Lehane, 1997), is a four-layered membrane secreted by Drosophila cardia (King, 1988). PM acts as physical barrier between innergut epithelial cell and food, and can protect innergut epithelial cells against mechanical damage caused by granular food (Hegedus et al., 2009). It has been reported that PET-MPs are densely packaged in the digestive tract of Gammarus pulex, indicating they may be covered in the PM and subsequently processed (Weber et al., 2018). According to this hypothesis, we speculated that the PM protects the ies from the damage of PET-MPs to the digestive system. Thus, no negatively effect was observed on ies exposed to PET-MPs. The possible explanation for the result could be due to the puri cation effect of microplastics. Drosophila exposed to 1µm of Polystyrene uorescent microplastics, cleared them in 24 h (Matthews et al., 2021). The diameter of ies' gut is 160µm (Buchon et al., 2013), and the larger particles are easy to form intestinal obstructions. The size of the microplastics used in our experiment is 2µm. We surmised that the microplastics may be excreted smoothly from ies' intestines.
An interesting phenomenon was found in our experiment. The exposure to 1 g/L PET-MPs increased the lifespan of the ies. The possible reason for this phenomenon is hormetic effects. Slight stress would disturb the homeostasis of the organism, forcing the animal to make adaptive response. Slight stress can improve functional capacity of the organism and even prolong longevity: this phenomenon is called hormesis (Minois and Rattan, 2003). Exposure to mild stress, such as hypergravity (Le Bourg and Minois, 1999), heat shock (Sørensen et al., 2007), low dose radiation (TG, 2003), cold shocks (Le Bourg, 2007), can slightly extend the life of ies. We speculated microplastics can also be seen as a mild stress.
Exposure to PET-MPs induces faster growth in the giant snail Achatina reticulata (De Felice et al., 2021). Low concentration of MPs can stimulate the activity of SOD, and activate the antioxidant system, while SOD activity and the destruction of oxidant system at high concentrations (Trestrail et al., 2020). In our previous study, PET-MPs enhanced the spontaneous activity of ies. We speculated that microplastics can induce the adaptive responses of ies and enhance biological performance to extend the lifespan of ies.
Interestingly, the phenomenon of prolonging life only appeared in male ies. Sex speci c hormetic in Drosophila is very common. The bene cial effects of mild stress often occur in male ies. For example, Despite no negative effects of PET-MPs on the ies' lifespan in this experiment, we still cannot ignore microplastics potential risks. Plastic products contain additives such as antimicro-bial agents, heat stabilizers, plasticizers, ameretardants, ame retardants, UV stabilizers, pigments, llers, and to provide speci c properties (Lithner, 2011), while pure plastic powder was chosen in our experiment. Studies have shown that at the highest leachate of recyclable plastics, the survival of barnacle Amphibalanus amphitrite larvae was signi cantly lowered (Li et al., 2016). In addition, due to higher surface area, microplastics are easier to accumulate persistent organic pollutants (POPs) such as uorobenzenes(CBS), per uorochemicals (PFCS), poly uorobiphenyls (PCB), heavy metals, viruses, bacteria, and so on (Mato et al., 2001). A recent study indicated that MPs can aggravate the toxicity of Cadmium and induce an enhancement of gene silencing in somatic tissues of Drosophila (Zhang et al., 2020). Therefore, evaluating the synergistic effects of MPs interaction with additives and environmental pollutants is critical in future studies.

Declaration of competing interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper. Figure 1