Elsevier

Science of The Total Environment

Volume 438, 1 November 2012, Pages 210-217
Science of The Total Environment

Response of the common cutworm Spodoptera litura to zinc stress: Zn accumulation, metallothionein and cell ultrastructure of the midgut

https://doi.org/10.1016/j.scitotenv.2012.06.065Get rights and content

Abstract

By exposing the common cutworm Spodoptera litura Fabricius larvae to a range of Zinc (Zn) stress, we investigated the effects of dietary Zn on Zn accumulation, metallothionein (MT), and on the ultrastructure of the midgut. The techniques we used were inductively coupled plasma-atomic emission spectrometer (ICP-AES), real-time PCR combined with cadmium-hemoglobin total saturation, and transmission electron microscopy (TEM), respectively. There was a significant dose–response relationship between the Zn accumulations in the midgut of the larvae and the Zn concentrations in the diet. Furthermore, both MT content and MT gene expression in the midgut were significantly induced in the 50–500 mg Zn/kg treatments, and were significantly positively correlated with the Zn accumulations in the midgut. When S. litura larvae were fed with the diet treated with 500 mg Zn/kg, Zn accumulation and MT content in the midgut was 4450.85 mg Zn/kg and 372.77 mg/kg, respectively, thereafter there was a little increase; the level of MT gene expression was maximal, thereafter there was a sharp decrease. TEM showed that numerous electron-dense granules (EDGs) and vacuoles appeared in the cytoplasm of the midgut cells, their number and size being closely correlated with the Zn accumulations in the midgut. Moreover, the nuclei were strongly influenced by Zn stress, evidenced by chromatin condensation and irregular nuclear membranes. Therefore, after being exposed to Zn in the threshold (500 mg Zn/kg) range, S. litura larvae could accumulate Zn in the midgut, which led to the induction of MT and changes in cell ultrastructure (mainly the presence of EDGs). The induction of MT and precipitation of Zn in EDGs may be the effective detoxification mechanisms by which the herbivorous insect S. litura defends itself against heavy metals.

Graphical abstract

When the herbivorous insect Spodoptera litura Fabricius larvae were fed on the artificial diet with different concentrations of Zn, amounts of Zn could be accumulated in larval midgut, which significantly led to the increase of metallothionein (MT) content, the induction of MT gene expression, and the presence of numerous granules with different types (Type A (black arrow), B granule (white arrow)).

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Highlights

► A dose–response was observed between Zn accumulations in S. litura larvae midgut and Zn concentrations in diet. ► Metallothionein in midgut of S. litura larvae was positively correlated with Zn accumulations in the midgut. ► Numerous electron-dense granules and vacuoles appeared in cytoplasm of the midgut from Zn stress treatments. ► MT induction and EDGs are the effective detoxification mechanisms for S. litura larvae to protect against heavy metals.

Introduction

Zinc (Zn) is a ubiquitous and essential trace element in biological systems; the Zn ion is a catalytic component of many enzymes and plays a structural role in a large number of proteins and transcription factors (Maret, 2005). However, Zn can be toxic when its concentration exceeds physiological limits. With the rapid development of industrialization and urbanization, contamination with heavy metals such as Zn has become a global problem. It is estimated that 1.372 million tons of Zn per year are emitted into the environment globally, and much of the Zn can be accumulated in farmland soil. For an example, the mean and maximal concentrations of Zn in the surface soil of farmland in the Pearl River Delta in Southern China are 166.9 and 963.0 mg Zn/kg, respectively, while the background is 47.9 mg Zn/kg (Chai et al., 2004). Zn is easily accumulated by plants, and through them it can be passed on to higher trophic levels such as herbivorous insects. This is of concern because heavy metals ingested by insects pose potential risks to their physiological process (Mulder et al., 2005).

In nature, herbivorous insects are mainly exposed to heavy metals through their host plants, whereas aquatic and soil-dwelling insects are directly exposed to heavy metal ions in the water or soil. Aquatic insects can protect against heavy metals by excreting elements from the hemolymph, across the respiratory surfaces, into the external medium. Soil-dwelling insects which survive in heavy metal polluted areas often present detoxification mechanisms, including the induction and activation of metal-binding proteins (e.g. metallothioneins (MT)) (Hensbergen et al., 2000, Hensbergen et al., 2001, Choi et al., 2008), and the precipitation of heavy metals in storage structures recognized ultrastructurally as intracellular electron-dense granules (EDGs) (Hopkin and Martin, 1982, Prosi and Dallinger, 1988, Pawert et al., 1996, Köhler et al., 1996, Köhler, 2002, Ballan-Dufrançais, 2002, Pigino et al., 2005). In contrast, relatively little is known regarding the mechanisms by which herbivorous insects defend against heavy metals. It is known that both soil-dwelling insects and herbivorous insects mainly store heavy metals in digestive, storage or excretory organs, such as the midgut. As well as soil-dwelling insects, the midgut of herbivorous insects acts as a barrier between the food pulp and the internal environment (the hemolymph) of the organism (Ballan-Dufrançais, 2002, Pigino et al., 2005). However, whether herbivorous insects have similar heavy metal detoxification mechanisms as soil-dwelling insects is poorly understood, and is worthy of further study.

The common cutworm, Spodoptera litura Fabricius (Lepidoptera: Noctuida), is a herbivorous pest affecting numerous economically important crops worldwide. The larvae feed on over 112 cultivated plants including vegetables, cotton, soybean, groundnut and tobacco, and then pupate in the farmland soil (Qin et al., 2004). Therefore, S. litura are exposed to two sources of heavy metals: host plants and soil. Experimental studies have shown that S. litura larvae can accumulate potentially toxic nickel (Ni) ions in their midgut when they are fed with a diet treated with Ni (Sun et al., 2007, Sun et al., 2008), and the toxic effects of Zn stress on S. litura have been studied using Zn-treated diet imitating natural conditions (Xia et al., 2005, Shu et al., 2009).

The detoxification mechanisms by which S. litura defends against Zn stress have not yet been recognized. Therefore the aim of this study was to obtain new information about the Zn detoxification mechanisms of S. litura through the investigation of: (i) Zn accumulation, (ii) MT content and MT gene expression, and (iii) ultrastructural changes, including the presence of EDGs, in the midgut.

Section snippets

Insect rearing and zinc treatments

Eggs of S. litura were provided by the Insectarium of the Institute of Entomology, Sun Yat-sen University, Guangzhou, China. Upon hatching, the 1st instar larvae were fed with an artificial diet, to which had been added different doses of ZnCl2 (Merck, Darmstadt, Germany). There were 6 Zn concentrations in the diet: 0 (control), 50, 100, 150, 500 and 1000 mg Zn/kg wet weight artificial diet (termed mg Zn/kg).

About 600 larvae per concentration were divided into three groups, and each group (about

Zn accumulations in midgut

Zn accumulations in the midgut of S. litura larvae increased with the increase of Zn concentrations in the diet. A significant dose–response relationship was observed between them, and the quadratic equation was simulated (Fig. 1). When larvae were fed a diet with only 50 mg Zn/kg, the Zn accumulations in the midgut was 843.35 mg Zn/kg, which was not significantly different from the control (592.88 mg Zn/kg). In contrast, at each of the 4 higher levels of dietary Zn (100, 150, 500 and 1000 mg Zn/kg),

Discussion

The insect midgut is the main site of absorption, and plays an important role in ionic regulation and mineral accumulation (Ballan-Dufrançais, 2002). When essential metals are assimilated in excess, or when non-essential metals enter the digestive tract, the midgut can accumulate metals, and act as a barrier between the food in the lumen and the internal environment of the insect (i.e. the hemolymph) (Pigino et al., 2005). In the present study, we have demonstrated that Zn was accumulated in

Conclusions

After being exposed to Zn stress in the threshold (500 mg Zn/kg) range, S. litura larvae were able to accumulate Zn in the midgut, which significantly induced MT and caused changes in the ultrastructure of the midgut cells, particularly the presence of numerous granules. Therefore, the midgut of the S. litura larvae was the main site of Zn accumulation and storage, and served as a barrier to prevent excessive Zn from entering the internal environment. The induction of MT and precipitation of Zn

Acknowledgments

We thank Dr. Sarah L. Poynton, Johns Hopkins University Medical School, Baltimore, USA for a critical reading of the manuscript. We also thank the reviewers and the associate editor for providing valuable suggestions to improve the manuscript. This research was financially supported by the National Natural Science Foundation of China (31170506, 31200308), the Research Fund for the Doctoral Program of Higher Education (20104404110003), the Ministry of Agriculture Science and Technology of

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