Elsevier

Science of The Total Environment

Volume 666, 20 May 2019, Pages 944-955
Science of The Total Environment

Cadmium-induced oxidative stress, histopathology, and transcriptome changes in the hepatopancreas of freshwater crayfish (Procambarus clarkii)

https://doi.org/10.1016/j.scitotenv.2019.02.159Get rights and content

Highlights

  • Cd induced oxidative stress to P. clarkii hepatopancreas.

  • P. clarkii hepatopancreas could accumulate Cd in a time- and dose-dependent manner.

  • Cd exposure caused severe histological alterations to P. clarkii hepatopancreas.

  • GO terms and KEGG pathways were significantly enriched after Cd treatment.

  • DEGs related to oxidation-reduction, immune response, ion homeostasis, etc. were identified.

Abstract

Cadmium (Cd) is a common contaminant in environment. Crayfish are considered suitable for indicating the impact of heavy metals on the environment. However, there is limited information on the mechanisms causing damage to the hepatopancreas of Procambarus clarkii exposed to Cd. We exposed adult male P. clarkii to 2.0, 5.0, and 10.0 mg/L Cd for 24, 48, and 72 h to explore Cd toxicity. Afterwards, we measured bioaccumulations in the hepatopancreas and determined malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST). Additionally, the hepatopancreas histopathology was analyzed and the transcriptome analysis of the P. clarkii hepatopancreas under Cd stress was conducted. The results revealed that hepatopancreas could accumulate Cd in a time- and dose-dependent manner. Cd induced significant changes in MDA content and antioxidant enzyme activity. Severe histological alterations were observed in crayfish hepatopancreas. After 72 h exposure to 2.0, 5.0, and 10.0 mg/L Cd, transcriptome analysis identified 1061, 747, and 1086 differentially expressed genes (DEGs), respectively. Exposure to 5.0 mg/L Cd inhibited heme binding, tetrapyrrole binding, iron ion binding and activity of oxidoreductase and sulfotransferase, while exposure to 10.0 mg/L Cd enhanced the export of matters from nucleus. In the hepatopancreas treated with 10.0 mg/L Cd, pathways related to diseases and immune system were significantly enriched. Meanwhile, 31, 31, 24, 7, and 12 identified DEGs were associated with the oxidation-reduction process, immune system, ion homeostasis, digestion and absorption, and ATPases, respectively. Our study provides comprehensive information for exploring the toxic mechanisms of Cd and candidate biomarkers for aquatic Cd risk evaluation.

Introduction

Heavy metal pollution in the aquatic environment has gained increasing attention over the past years (Kouba et al., 2010; Goretti et al., 2016). As a type of non-biodegradable pollutant, cadmium (Cd) has caused particular concern with respect to the environment and human health across the world (Wang et al., 2018). The toxic effects of Cd can be bio-amplified and accumulated in the food chain (Devi et al., 1996; Fowler, 2009). Furthermore, Cd can induce adverse changes, including morphological disruptions, biochemical alterations, and physiological dysfunctions in organisms (Lei et al., 2011; Sfakianakis et al., 2015; Huo et al., 2017). It was also revealed that Cd could cause reactive oxygen species (ROS) accumulation, hence resulting in injury to biological functions and cellular structures (Van Dyk et al., 2007; Sun et al., 2016).

The red swamp crayfish P. clarkii is a commercially important freshwater cultured species. Due to its wide distribution, long lifecycle, and relatively simple anatomy, it has been used as a typical bioindicator of toxic pollutants in studies on aquatic environments (Vioque-Fernándeza et al., 2009; Brittle et al., 2016). The hepatopancreas, as a multifunctional and vital organ for crustaceans, has been reported to play a critical role in metabolism, nutrient absorption, and immune function (Rőszer, 2014). In addition, the hepatopancreas is referred to as a critical target organ for many types of environmental stresses (Wang et al., 2008; Clark et al., 2013; Xie et al., 2014). The hepatopancreas and bioaccumulation ratio between hepatopancreas and abdominal muscle of P. clarkii exposed to heavy metals have been used to indicate the pollution of these metal contaminants (Alcorlo et al., 2006; Kouba et al., 2010; Goretti et al., 2016).

As a highly powerful technique to identify differentially expressed genes (DEGs), high-throughput RNA-sequencing (RNA-seq) has been used to identify functional genes (Liu et al., 2018a). Furthermore, the development and application of RNA-seq have facilitated rapid, comprehensive, and detailed analysis of the genomes and transcriptomes of non-model organisms (Collins et al., 2008). In recent years, the RNA-seq approach has also been used to study the genetic responses of host organisms to Cd (Poynton et al., 2011; Zhang et al., 2017; Zhou et al., 2018). Likewise, this technology enables us to conduct transcriptome analysis of the freshwater crayfish P. clarkii, hence further revealing how Cd affects crayfish at the molecular level.

Despite excellent studies on Cd toxicity (Martín-Díaz et al., 2006; Al Kaddissi et al., 2012, Al Kaddissi et al., 2014), the toxic mechanisms of Cd in the P. clarkii hepatopancreas deserve further investigation. In the present study, the P. clarkii hepatopancreas was used to investigate the molecular and physiological effects of acute Cd exposure. The molecular responses, including malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST), in the hepatopancreas of freshwater crayfish P. clarkii were assessed. Furthermore, histopathology sections of hepatopancreas were made to investigate the effects of acute Cd exposure on tissue. In order to have a deeper insight into the molecular mechanisms of crayfish responding to Cd, we analyzed the transcriptome data of the hepatopancreas in adult male crayfish after acute Cd exposure and identified the differentially expressed genes (DEGs). The main goal of this study is to assess the effects of Cd on the P. clarkii hepatopancreas from the aspects of accumulation, oxidative stress, histopathology, and transcriptome changes, and reveal the mechanisms of Cd toxicity in crayfish.

Section snippets

Test chemical

Cadmium chloride (CAS: No. 10108–64-2) was obtained from Sigma (St. Louis, MO, USA). For making the stock solution, cadmium chloride was dissolved in deionized water.

Crayfish

Live and apparently healthy adult male crayfish (21.6 ± 4.4 g in wet weight; 11.4 ± 1.0 cm in length) were collected from an aquatic product market of Harbin, China. The crayfish were fed once a day with red worms, but the feeding was stopped during exposure. They were acclimatized to laboratory conditions in glass aquaria

Cd bioaccumulation in the hepatopancreas of P. clarkii

As exhibited in Table S2, due to the adsorption on the aquarium walls and the accumulation in crayfish, Cd concentrations in water decreased slightly as the time went by. After exposure for 72 h, the mean (arithmetic) measured Cd concentrations were 0, 2.04 ± 0.04, 4.80 ± 0.51, and 9.75 ± 0.26 mg/L, respectively.

Cd concentrations measured in hepatopancreas were shown in Table S3. Cd bioaccumulation in hepatopancreas was time- and dose-dependent in all the experiments. At 24 h, a significant

Discussion

This study was designed to study the response of P. clarkii to Cd and explore the mechanisms inducing injury to the hepatopancreas of P. clarkii challenged with Cd. In previous articles, antioxidant enzymes, histological variations, and related genes in the hepatopancreas of P. clarkii after exposure to Cd have been researched (Al Kaddissi et al., 2012, Al Kaddissi et al., 2014). On the basis of these reports, we investigated the transcriptome analysis, together with bioaccumulation,

Conclusions

In conclusion, the toxicological effects of Cd on the P. clarkii hepatopancreas under acute exposure were explored and discussed in this study. Hepatopancreas could accumulate Cd in a time- and dose-dependent manner. Biochemical and molecular biomarkers, including MDA, SOD, CAT, and GST, showed significant variations after challenge with Cd, indicating that they can act as rapid, effective, and sensitive diagnostic parameters for the biomonitoring of toxic pollutants. Additionally, Cd caused

Acknowledgements

This work was financially supported by the Open Project Program of the State Key Lab of Urban Water Resource and Environment (No. ESK201701).

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