Microplastics (MPs, <5 mm in size) mainly come from the daily life of people or from the aging, weathering, and broken of the large plastic pieces (Cole et al., 2011). Due to their small volume, MPs are difficult to remove by sewage treatment completely (Hamidian et al., 2021), which make them able to flow into inland rivers and eventually into marine ecosystems (Wang et al., 2021a). In recent years, many studies have been made on the occurrence of MPs in the environment and their effects on marine organisms, including physical damage, behavior change, tissue lesions, oxidative stress, and gene damage (Huang W. et al., 2021). MPs can also be used as a carrier for some pollutants in the environment and carry the pollutants into the body when organisms ingest MPs, which may cause more serious damages to organisms (Gu et al., 2020).

With the increasing discoveries of MPs in rivers and lakes, MPs have been increasingly viewed as a serious health concern to freshwater ecosystems (Shang et al., 2020). China is a major country in traditional aquaculture (Su et al., 2020), and freshwater aquaculture products account for more than half of the total production in 2020 (Fisheries Administration Ministry of Agriculture Rural Affairs, 2021). In recent years, MPs have been found in major rivers and lakes in China such as Yangtze River, Yellow River, Pearl River, Poyang Lake, Dongting Lake, and Taihu Lake (Fu and Wang, 2019). Especially in Taihu Lake, the concentration of MPs has reached an astonishing 25,800 items/m³ (Su et al., 2016), which even greatly exceeds the Yellow Sea (0.33 items/m³) and South China Sea (2,569 items/m³) (Cai et al., 2018; Wang et al., 2018). In addition to the contamination of MPs in freshwater aquaculture water sources, various freshwater aquaculture patterns, such as cycled-water, cement-pool, net-cage, rice-field, and fish-light, can release MPs into aquaculture waters (Sahu et al., 2016; Lv et al., 2020). Due to the multiple sources of MPs, freshwater aquaculture objects are inevitably subjected to MP pollution. However, less is known on the effects of MPs on freshwater species, especially economic freshwater fish.

In recent years, the harm of MPs to the immune system of fish by affecting the expression of immune genes in fish has been revealed. Tumor necrosis factor-α (TNF-α), interleukin (IL), and interferon (IFN) are important factors in the immune system, which play the role of activating immune cells, regulating immune cells, and activating antiviral cells, respectively (Samuel, 2001; Whyte, 2007). Huang et al. (2020b) showed that the expressions of TNF-α, IL-6, and IFN in the liver of the guppy Poecilia reticulata were significantly increased after 28 days of exposure to MPs. What is more, MPs not only affect the expression of IFN at the gene level but also increase the secretion of the protein corresponding to IFN (Jin et al., 2018). Luo et al. (2021) found that MPs induced the expression of increased IL-8 compared with the control group. The expression changes of these proinflammatory biomarkers under the MPs make them become the important index on the evaluation of fish welfare.

Hypoxia, a common phenomenon in aquaculture, which can be caused by temperature rise, eutrophication, high-density aquaculture, and water pollution (Wang M. et al., 2021), often occurs at the bottom of the water body, leading to behavioral change (Xu et al., 2006), metabolic obstruction (Obirikorang et al., 2020), and decreased immunity (Ngoepe et al., 2020) of aquaculture objects. What is more, hypoxia can aggravate the negative effects of other environmental stressors on aquatic animals (Hu et al., 2016; Somo et al., 2020), thus reducing the production efficiency of aquaculture. With the deepening of research, the hypoxia-inducible factor (HIF) is found to be the main transcription factor regulating the hypoxia signaling pathway in vertebrates (Wang C. et al., 2020). HIF-1 is widely used in the study of hypoxia (Xu et al., 2021), and the α subunit of HIF-1 is most sensitive to hypoxia compared with the β subunit (Abdel-Tawwab et al., 2019). Although this subunit has a short half-life in cells, it is difficult to be hydrolyzed under hypoxia and accumulates in large quantities in cells, which can activate the hypoxia signaling pathway (He et al., 2019). At present, the HIF-1α gene sequence has been successfully cloned from an aquaculture object, and its function in the hypoxia signaling pathway has been found (Lin et al., 2021). In addition, other studies have shown that HIF-1α plays an important role in regulating inflammation, but this effect has only been found in higher vertebrates such as human cells and mouse cells (Li et al., 2020; Pena et al., 2020).

On the one hand, MPs are newly emerging environmental pollutants in freshwater aquaculture, which have been confirmed the biological accumulation of aquaculture species (Lv et al., 2020). On the other hand, hypoxia is a traditional inhibitor of aquaculture and often occurs in an aquatic pond (Xu et al., 2006). However, the effect of these two simultaneous factors at the bottom of the pond on benthic objects is unknown.

The yellow catfish Pelteobagrus fulvidraco is a unique freshwater aquaculture species in China, which is widely loved for its delicious meat. Its production reached 565,477 tons in 2020 (Fisheries Administration, Ministry of Agriculture and Rural Affairs, People's Republic of China, 2021). Hypoxia occurs frequently in yellow catfish aquaculture due to the intensive, high-density, and high-feeding farming, while MPs were also detected from yellow catfish in a reservoir (Zhang et al., 2017; Wang M. et al., 2021). However, this research on yellow catfish mainly focuses on breeding and nutrition (Wang et al., 2021b; Zhao et al., 2021), while the information on the complex effects of MPs and hypoxia on its physiology and ecology is very scarce. In contrast, compared with other freshwater aquaculture fish, their scaleless bodies make them more vulnerable to MPs and their immune function more important (Feng et al., 2019), and the demersal habits of yellow catfish make them more susceptible to MPs and hypoxia. Therefore, we aimed to study the specific growth rate (SGR) and immune-associated genes, i.e., HIF-1α, TNF-α, IL-8, and IFN of yellow catfish under combined exposure of hypoxia and MPs. We concluded that (1) MPs cause the cellular immunological stress of yellow catfish and (2) hypoxia aggravates the negative effects of MPs on immune parameters of yellow catfish at a high concentration.

Microplastics have become a hot topic since Thompson et al. (2004) discovered them. But there is still less research on MPs in freshwater organisms than in marine organisms. The Qinghai-Tibet Plateau is the birthplace of many rivers (e.g., the Yangtze River, the Yellow River, and the Lancang River). However, Jiang et al. (2019) found that the water bodies of the Qinghai-Tibet Plateau contained MPs, and the Yangtze River would receive more than 3.1 × 10⁵ tons MPs each year (Lebreton et al., 2017). Some studies have carried out the toxicological effects of MPs on tilapia (Oreochromis niloticus), but the size of MP particles (0.1 and 5 μm, respectively) and the exposure concentration (100 and 50 μg/L, respectively) are far from the results of the environmental investigation (Ding et al., 2018; Huang Y. et al., 2021). Based on Fu and Wang (2019), in Chinese freshwater ecosystem, the highest MP concentration is 25,800 items/m³, and the smallest size of MPs is just about 20 μm. Therefore, this study can provide environmentally relevant insights on MPs in the field of freshwater based on the real size and concentration in the environment.

The SGR was an important index to measure the economic benefits of fisheries, and the larger the value was, the greater of fish would be. Malinich et al. (2018) found that MPs had no impact on the growth of the fathead minnow Pimephales promelas, and a similar result was detected from the brown trout Salmo trutta (Jakubowska et al., 2020). However, MPs could induce growth inhibition in the common carp Cyprinus carpio. The reason for the difference may be the different exposure methods (waterborne or foodborne). In this study, yellow catfish did not exhibit a significant difference between MP treatments, and it might be associated with the smooth and spherical shape of MPs, which made them easily expel from the intestine (Mazurais et al., 2015). Hypoxia could inhibit the growth of juvenile fish, which had been reported previously (Yang et al., 2013; Campbell and Rice, 2014), and yellow catfish showed a significant decrease in SGR under hypoxia for 8 weeks (Yang et al., 2015). Under hypoxia, although there was no statistical difference, a minimal lower change could be seen compared with normoxia in only 15 days of exposure, which indicated that the hypoxia may impact the growth of yellow catfish regardless of the presence or absence of MPs.

Hypoxia can increase the expression of HIF-1α in organisms has been confirmed in a large number of studies (Kelly et al., 2020; Wang M. et al., 2021; Xu et al., 2021). HIF-1 is a heterodimer composed of HIF-1α and HIF-1β (Semenza, 1998). Among them, HIF-1β is insensitive to the changes in O₂ availability, while HIF-1α can accumulate in large quantities in vivo due to its difficulty in degradation under hypoxia, activating the hypoxia signaling pathway (Uchida et al., 2004). Activated hypoxia signaling pathways modulate other biological processes associated with increased O₂ release or decreased O₂ consumption, such as the regulations of erythropoietin, glucose transporter, glycolytic enzyme, and expression of vascular endothelial growth factor (Semenza, 1999), allowing the organism to adapt to new environmental changes. This could also explain why the expression of HIF-1α changes significantly under hypoxia compared with normoxia. As shown in Figure 4A, both low (0.115 μg/L) and high (11.5 μg/L) concentration MPs could increase the expression of HIF-1α under normoxia. A similar result was obtained in the microcrustacean Daphnia pulex (Liu Z. et al., 2020), despite nanoplastics were used in their experiments, which might be more biotoxic than MPs (Browne et al., 2008). It was possible that HIF-1α could also be used as an indicator for toxicological tests of contaminants as well as other cytokines, such as the heat shock proteins (Varo et al., 2019; Jaikumar et al., 2021). Although this study did not detect the presence and quantity of MPs in gill tissues, it could be speculated from previous studies that the increased expression of HIF-1α might be related to the accumulation of MPs in gill tissues, which would influence the normal respiration (Lu et al., 2016; Wang et al., 2019; Huang et al., 2020a). However, in this study, we did not find that MPs increased the expression of HIF-1α under hypoxia treatment, indicating that MPs did not exert biological toxicity under hypoxia. As we know, fish will head out of the water to utilize the oxygen in the air when they live in the hypoxic water environment (Juca-Chagas, 2004), and this behavior is defined as “floating head.” During the experiment, in the hypoxia group, “floating head” was recorded. This behavior could decrease the amount of water fish filtered, reduce the number of MPs getting into fish, and thus ease the negative effects of MPs. Oxygen was necessary for the survival of oxygen-consuming organisms, and organisms had already developed an effective defense mechanism in the face of the occasional lack of oxygen during survival (Bickler and Buck, 2007). However, in the face of emerging environmental pollutants such as MPs, yellow catfish seemingly did not find an effective solution to resist the impacts of MPs, but hypoxia can hold back the effects of MPs on HIF-1α.

According to previous studies, harmful substances in the environment, such as MPs, heavy metals, pesticides, and antibiotics, could cause the inflammatory response of organisms (Liu H. et al., 2020; Maselli et al., 2020; Pirsaheb et al., 2020; Wang Y. et al., 2021). However, inflammatory responses in fish were often closely related to cytokines such as TNF, IL, and IFN (Falcão et al., 2021). TNF-α is an important immune factor that is heavily induced in injury, inflammation, and wound responses and activates macrophages, enhancing their phagocytosis and clearance to pathogens (Secombes et al., 2001). TNF-α has been shown to induce the expression of other genes involved in the immune response, such as IL-1β, IL-8, and COX2. IL-8, the first known chemokine, mainly attracts the movement of neutrophils, T-lymphocytes, and basophils in the body (Whyte, 2007). IFN is a secreted protein that induces antiviral activity in vertebrates and regulates apoptosis and cellular immunity (Samuel, 2001). Three factors interact to enhance the resistance of organisms to the external environment. Therefore, the transcription of these three types of immune factors was detected to comprehensively evaluate the immune status of yellow catfish.

In this study, the expressions of the three immune factors showed different changes under different MP concentrations. Under normoxia, the expressions of TNF-α were significantly increased with the presence of MPs. This suggested that MPs activated immune mechanisms in yellow catfish, which was due to the activation of the NF-κB signaling pathway, leading to the increased expression of related signaling factors (Wu et al., 2020; Yang et al., 2020). The same result was also obtained by Luo et al. (2021) who studied zebrafish (Danio rerio). Compared with normoxia, the expression of TNF-α was higher under hypoxia with 0 MPs, although this increase was not significant. Martinez et al. (2020) have found that hypoxia increased the expression level of TNF-α, and it was similar to our results. Under hypoxia, the relative expression of TNF-α was lower than those under normoxia in 0.115 μg/L MP treatment. But in 11.5 μg/L MP treatment, TNF-α was higher than those in 0.115 μg/L MPs under hypoxia. It is explained that hypoxia could reduce the negative effects of MPs on yellow catfish to some extent. The reasons for this result might be the reduced uptake of MPs by fish (direct reason) and the regulation of HIF-1α (indirect reason).

In contrast, the relative expression of IL-8 in normoxia was not influenced by the MPs, and it might be due to the size of MPs. Based on the experiment in Danio rerio, 0.5 μm MPs could decrease the relative expression of IL-8, but 50 μm MPs did not have the same negative effects (Jin et al., 2018), and the fact that smaller MPs have more serious negative effects has been proved by Browne et al. (2008). Under hypoxia, the variation tendency of IL-8 was the same as the normoxia condition which mainly showed an increase at 0.115 μg/L MPs and a decrease at 11.5 μg/L MPs, although the decrease at 11.5 μg/L was significant. In the study by Zhang et al. (2019) on the immune effects of di-2-ethylhexyl phthalate (DEHP, a kind of phthalic acid ester, PAEs) on yellow catfish P. fulvidraco, it was also observed that the relative expression of IL-8 increased at low concentration DEHP and decreased at high concentration DEHP. We held the opinion that this variation tendency of IL-8 was unique to yellow catfish exposed to environmental pollutants. However, the relative expression of IL-8 in 11.5 μg/L MPs in hypoxia was lower than that in normoxia, which explained that the composite effects of hypoxia and high concentration MPs could inhibit the expression of IL-8. And the interactive effects of hypoxia and MPs were observed in this study, which indicated that hypoxia aggravates the inhibition of hypoxia in the expression of IL-8. But this interaction did not have the same inhibition in 0.115 μg/L MPs under hypoxia, which might be related to the decrease of MP intake.

As a cytokine, IFN plays an important role in the immunity of the body, apoptosis, and antivirus (Samuel, 2001). Previous studies have shown that MPs can increase the relative expression of IFN in zebrafish (Danio rerio) and guppies (Poecilia reticulata) (Jin et al., 2018; Huang et al., 2020b). In this study, we also saw an increase in the relative expression of IFN in 0.115 μg/L MP treatment under normoxia, although this increase was not significant. It might be related to the inflammatory response caused by MPs. However, in the 11.5 μg/L MP group, the expression of IFN was significantly reduced under normoxia, which had not been found before. As mentioned above, it might be related to the difference of the species. Previous studies have shown that hypoxia could cause a significant increase in the expression of IFN (Niklasson et al., 2011; Chen et al., 2017), and the same result was obtained in this study. Based on the result of two-way ANOVA, a significant interaction of hypoxia and MPs was observed. This concentration-dependent interaction induced the inhibition in the expression of IFN.

According to the PCA, two principal components accounted for 75.77% of the total composition. PC1 separated MP treatment from non-MP treatment, accounting for 43.17 of the total variances, while PC2 separated normoxic treatment from hypoxic treatment, accounting for 32.60% of the total variances, indicating that MP treatment had a greater impact on yellow catfish. Significant positive correlation between TNF-α and HIF-1α, and this relationship had been discovered in mouse cells and human cells (van Uden et al., 2008; Li et al., 2020). We also found that there was a relationship between TNF-α and HIF-1α in fish, which could make up the gap between HIF-1α and TNF-α in fish. When inflammation occurred in the body, TNF-α induced a large number of macrophages to accumulate in the inflammatory site, consumed a large amount of oxygen, resulting in hypoxia in the inflammatory part and the production of a large number of HIF-1α (Cummins et al., 2016). In contrast, TNF-α could activate the NF-κB signaling pathway to produce NF-κB factor, and several subsequent NF-κB subunits bind to the promoter of HIF-1α, thereby inducing HIF-1α production in the body (Lin and Simon, 2016; Warbrick and Rabkin, 2019). In addition, HIF-1α plays a crucial role in the initiation, regulation, and coordination of cell responses during inflammation (Frede et al., 2007).

This study filled the gap in understanding the effects of MPs on benthic fish under hypoxia. In this study, MPs were able to induce immune responses on yellow catfish. Hypoxia seemed to alleviate the effects of MPs at 0.115 μg/L on yellow catfish to some extent, but the interaction between hypoxia and MPs aggravated the negative effects of MPs on the expression of immune parameters on yellow catfish at 11.5 μg/L MPs. Thus, attention should be paid to the harm of MPs to aquaculture fish species, and the occurrence of hypoxia in water bodies should be reduced as far as possible. To form a systematic understanding, future studies should focus on the molecular pathway of immune cells in fish exposed to MPs and hypoxia.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines Institute of Hydrobiology, Chinese Academy of Sciences.

LL: formal analysis, investigation, methodology, data curation, writing—original draft, and review and editing. RX: formal analysis, investigation, methodology, visualization, and writing—review and editing. LJ: investigation and methodology. EX and MH: investigation and writing—review and editing. MW, JW, and BL: investigation and methodology. LZ: funding acquisition, supervision, validation, and writing—review and editing. YW: conceptualization, supervision, validation, and writing—review and editing. All authors contributed to the article and approved the submitted version.

This study was supported by the China Agriculture Research System of MOF and MARA (CARS-46) and the Subei Science and Technology Project of Jiangsu Province (Grant Nos.: SZ-HA2019017 and SZ-HAJH202005).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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