Full length articleAntioxidative status, immunological responses, and heat shock protein expression in hepatopancreas of Chinese mitten crab, Eriocheir sinensis under the exposure of glyphosate
Introduction
Glyphosate, an organophosphorus herbicide with broad-spectrum activity, has been extensively used worldwide in agricultural crops including corn, soybean, cotton, rice, and trees in orchards and groves over the past 40 years [1]. In 2014, approximately 125,000 tons of glyphosate were used in the USA and 825,000 tons worldwide [2]. The main action of glyphosate is the inhibition of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate acid pathway. Because of the absence of the shikimate pathway in nontarget organisms, glyphosate is considered to have low toxicity to animals [3]. However, glyphosate is known to be resistant to degradation because of the inert CP linkage in the molecule [4]. To date, several studies have demonstrated that glyphosate, or glyphosate-based herbicide, is toxic to nontarget aquatic organisms including microorganisms [5,6], aquatic plants [7], invertebrates [8], amphibians [9], and fishes [[10], [11], [12], [13]]. However, there are no reports on economically important crustaceans such as crabs.
The Chinese mitten crab, Eriocheir sinensis, is one of the most important freshwater species widely bred in China. Rice-crab co-culture is a high-benefit ecobreeding pattern that has been extensively developed in east China in recent decades [14]. In the meantime, China has become the top producer of glyphosate with extensive use in nonagricultural areas such as aquatic ponds, for invasive plants, or for weed control. Glyphosate and its degradation products have been measured in both surface and ground water with a concentration ranging from 2 to 430 μg/L in the USA due to the rain wash and runoff from agricultural and urban land [1]. However, few data are available on glyphosate in the environment in China. Because of its relatively long half-life in water (most commonly 45–60 days) [15] and repeated application in practice, the residue of glyphosate in aquatic water could reach up to 0.765 mg/L within 1 day after spaying in a commercial pond [16]. That exceeds the maximum detection concentration in the water of the USA and Europe and the median lethal concentration of most aquatic species [[17], [18], [19], [20]]. As a bottom dweller, crabs are more sensitive to the xenobiotics such as pesticides in water. These animals could be used as bioindicators for monitoring the state of pollution in an aquatic environment [21]. Therefore, the use of glyphosate may be an obstacle to both the crab breeding industry and ecological environment.
Herbicides are proved to cause oxidative stress in aquatic animals by the excessive production of reactive oxygen species (ROS). ROS may cause damage to DNA and biological macromolecules to induce cell injury [22]. As a consequence, the breakdown of the equilibration between antioxidant defence and generation of ROS could be the main reason for the toxic effect of herbicide to non-target organisms. In the first line of oxidative defence, superoxide dismutase (SOD) and peroxidase (POD) are the main enzymes to catalyse the conversion of the superoxide anion into hydrogen peroxide and the hydrogen peroxide into water and molecular oxygen. Thus, the activities of SOD and POD in different tissues were proposed as indicators of pollutant-mediated oxidative stress [12,23]. However, herbicides are generally considered to suppress the immune system in fishes, mussels, and crustaceans by tissue damage, enzyme inhibition, and decreased resistance to pathogens [[24], [25], [26]]. Therefore, two oxidative stress parameters, SOD and POD, and three immune-related enzymes, acid phosphatase (ACP), alkaline phosphatase (AKP) and phenoloxidase (PO), were detected to evaluate the potential toxic effect of glyphosate to E. sinensis in this study.
The recognised effect of organophosphorus in both invertebrates and vertebrates is the inhibition of the enzyme acetylcholinesterase (AChE), which is responsible for terminating the transmission of the nerve impulse [22]. The detection of AChE in different tissues was applied as a sensitive biomarkers for the assessment of several herbicides exposure to fish and shrimps [27,28]. In this study, we also detected the variation of AChE in hepatopancreas of E. sinensis.
Heat shock proteins (HSPs) commonly exist in cells from both eukaryotic and prokaryotic organisms with highly conservative evolution. It is proved that HSPs have important biomolecular functions including molecular chaperone, antioxidation, cell apoptosis, and immune response [29]. Several studies on the response of HSPs to the exposure of heavy metal and pesticides in aquatic animals have been reported [30,31]. To our knowledge, no information about the responses of HSPs to glyphosate exposure to crustaceans has been provided from any scholar. However, the variation of HSPs may provide a better explanation about the mechanism of antioxidation and immune response during a herbicide exposure.
Hepatopancreas is an important tissue in crustacean for metabolism and detoxication. The impaired activity of hepatopancreas marker enzymes such as ACP, AKP, SOD and POD is directly related to the degree of hepatopancreas damage in E. sinensis induced by toxins including pesticides [32]. From our previous study, glyphosate induced evident immunosuppression by haemocyte composition alteration, immune-related enzyme inhibition, and phagocytic activity decline, as well as haemocyte DNA damage [33]. In the current work, we investigated several oxidative stress and immunological response parameters in the hepatopancreas to evaluate the toxic effect of glyphosate to the important commercial species, E. sinensis.
Section snippets
Animals and chemicals
Adult experimental crabs, Eriocheir sinensis (Crustacean: Decapoda: Grapsidae), with average weight of 104.4 ± 8.7 g, were collected from a commercial farm in Jiangsu Province. Crabs were acclimated to laboratory conditions for 2 weeks before the beginning of the experiment. A recirculation system containing filtered freshwater and ultraviolet-treated PVC tubes as shelters in 150 cm × 100 cm × 120 cm glass aquariums was utilised in this period. The system had controlled light (12 h light:12 h
Effect of glyphosate exposure on antioxidative stress and immunological responses
Both concentration and time affected the SOD activity and there is interaction between glyphosate concentrations and exposure time (F = 5.728, P < 0.05), as well as other parameters. The SOD activities in hepatopancreas of E. sinensis were induced first at concentrations of 44 mg/L and 98 mg/L after 6 h exposure, and reached a peak at 12 h. Then, concentrations dropped dramatically at 24 h and kept decreasing until 96 h. At that time point, SOD activities in treatment of 9.8 mg/L, 44 mg/L, and
Discussion
Despite the numerous reports on the presence of glyphosate in the aquatic ecosystem and its toxicology to aquatic species, information about the toxic effects to macrocrustaceans is scarce, particularly in a co-culture system where glyphosate is commonly used. In the current study, several parameters associated with oxidative stress and immune response were measured to investigate the potential toxic effect of E. sinensis induced by hepatopancreas injury for the first time.
A novel research from
Conclusion
In this study, a prominent toxic effect of glyphosate on the Chinese mitten crab E. sinensis was detected for the antioxidative and immunological enzyme inhibition and AChE reduction in hepatopancreas, even at concentrations of 4.4 mg/L and 9.8 mg/L. Meanwhile, the protective response depending on the promotion of mRNA expression of HSP 60, HSP 70, and HSP 90 in hepatopancreas was induced to mitigate the oxidative stress. Additionally, parameters of HSP expression, especially in HSP 70, were
Acknowledgments
The current study was financially supported through a grant from the general science research programme of Sichuan Province (Grant No. 17ZB0405).
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