Effects of zinc exposure on the reproduction of Spodoptera litura Fabricius (Lepidoptera: Noctuidae)

ARTICLE IN PRESS Ecotoxicology and Environmental Safety 72 (2009) 2130–2136

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Effects of zinc exposure on the reproduction of Spodoptera litura Fabricius (Lepidoptera: Noctuidae) Yinghua Shu, Yuanyuan Gao, Hongxia Sun, Zhiwen Zou, Qiang Zhou , Guren Zhang State Key Laboratory of Biological Control and Institute of Entomology, Sun Yat-sen University, Guangzhou 510275, China

a r t i c l e in f o

a b s t r a c t

Article history: Received 3 June 2008 Received in revised form 2 March 2009 Accepted 11 June 2009 Available online 3 July 2009

Reproductive toxicity of Zn to insects was investigated in this study. By exposing phytophagous insect Spodoptera litura Fabricius to Zn in artificial diets of larvae, we investigated the effects of Zn on reproduction at ecological and molecular levels. A significantly shorter period of laying eggs was observed in S. litura exposed to 300–750 mg Zn/kg. The oviposition rate, fecundity and hatchability of female adults treated with 750 mg Zn/kg were significantly lower than those of the controls (31.43%, 20.95% and 52%, respectively, compared to the control). The Zn accumulation and vitellin (Vn) content in eggs were tested by inductively coupled plasma-atomic emission spectrometry and Bradford combining Western-blot, respectively. The results showed that Zn accumulated in the eggs, which has affected the weight and Vn content of eggs with significant negative correlations. The down-regulated expression levels of vitellogenin (Vg) mRNA were detected by real-time polymerase chain reaction (RT-PCR): the relative quantity of Vg mRNA was less than half of the controls at higher than 450 mg Zn/kg wet weight. These results indicated that excess Zn made expression of Vg gene down-regulated and caused poor accumulation of egg yolk, which led to a reduction in egg numbers and failure of eggs to hatch. & 2009 Elsevier Inc. All rights reserved.

Keywords: Real-time PCR Spodoptera litura Vitellogenin Vitellin Zinc

1. Introduction Zinc (Zn) is one of the essential trace elements for animal nutrition, having structural, catalytic and regulatory functions in an organism (Takeda, 2000; Maret, 2005). Zn is a crucial element for the proper action of over 300 enzymes and controls the architecture of protein complexes. It is also necessary in signal transduction via metallothioneins, thioneins, DNA replication, transcription and protein synthesis (Augustyniak et al., 2006). Furthermore, Zn plays an essential role in embryogenesis. This function has depended on a large body of phenomenological information available on the effects of its deficiency (Vallee and Falchuk, 1993), or studies of its presence in eggs and embryos (Falchuk and Montorzi, 2001). Thus, any alteration of Zn homeostasis may disrupt proper morphogenesis and growth of an organism (Takeda, 2000; Maret, 2005). Zn is potentially toxic when its concentration in an organism seriously exceeds physiological limits, which has been studied in some insect species. When insects inhabited in Zn-contaminated environment, chironomid larvae and ground beetles Pterostichus oblongopunctatus Fabricius, Poecilus cupreus L. had body abnormalities (Janssens de Bisthoven et al., 1992; Martinez et al., 2001,

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E-mail address: [email protected] (Q. Zhou). 0147-6513/$ - see front matter & 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2009.06.004

 2002, 2004; Maryanski et al., 2002; Lagisz, 2008). When insects feed on Zn-contaminated food, insects had lower development rates, for example Aglais urticae L. larvae (Lindqvist, 1994), ¨ Trichoplusia ni Hubner (Larsen et al., 1994), P. oblongopunctatus (Zygmunt et al., 2006) and Issoria lathonia L. (Noret et al., 2007). The high concentrations of Zn resulted in a lower survival rate for Priosotoma minuta Tullberg adults (Nursita et al., 2005). To our knowledge, the effect of Zn on reproduction of the insect has been less investigated yet. It was only observed in P. minuta (Nursita et al., 2005) and grasshoppers Chorthippus brunneus Thunberg (Augustyniak et al., 2008). Although two reports have investigated reproductive toxicity of zinc to insect, too little attention has been directed to the potentially deleterious mechanism of Zn at molecular levels, i.e. effects of Zn on vitellogenin (Vg) of insect. A potential mechanism for heavy metals affecting reproduction in oviparous animals is inhibition of Vg synthesis and yolk protein accumulation. The reduced reproduction in marine copepods following dietary exposure to metals was due to a disturbance of vitellogenesis (Hook and Fisher, 2001a, b, 2002). This is also supported by the fact that cadmium (Cd) uptake via ingestion of food resulted in a reduced accumulation of yolk protein (lipovitellin) in blue crabs Callinectes sapidus Rathbun (Lee and Noone, 1995). Significantly decreased Vg was observed in the hemolymph and ovaries of Oncopeltus fasciatus Dallas feed on Cd-contaminated drinking water (Cervera et al., 2005), which was also in line with above hypothesis. The dietary Zn specifically

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affected reproduction of Daphnia magna Straus through accumulation of Zn in particular target sites, possibly cells or tissues where Vg synthesis or processing occur (De Schamphelaere et al., 2004). However, the data were still scarce for insects. We aim to provide more evidence for the inhibition mechanism of heavy metals affecting on the production or processing of insect Vg. In the present study, the common cutworm Spodoptera litura Fabricius larvae were exposed to the control and five Zn concentrations, i.e. 150, 300, 450, 600, 750 mg Zn/kg wet weight artificial diet (termed mg Zn/kg). The adults that succeeded to emergence were used to investigate the effects of Zn on their reproduction. The reproductive parameters of S. litura including the period of laying eggs, the oviposition rate, fecundity and hatchability were observed. Special attention was taken to detect Zn concentrations in eggs by inductively coupled plasma-atomic emission spectrometry (ICP-AES) in order to study effects of Zn accumulation on the quality of eggs such as weight and Vn consent of eggs. Furthermore, the expression of the Vg gene in S. litura female adults was also investigated in the present study. The purpose of this study was to provide how Zn exposure affects the reproduction of insects and explore the potential mechanism of excess Zn with reproductive toxicity to insects.

2. Materials and methods 2.1. Insects rearing and treatments Eggs of S. litura, the larvae of which feed on a standard artificial diet for various generations (Chen et al., 2000), were provided by the Insectarium in the Institute of Entomology, Sun Yat-sen University. Upon hatching, the first instar larvae were fed on the artificial diet treated with Zn by adding different doses of ZnCl2 (Merck, Darmstadt, Germany). The final concentrations of Zn in the diets were 0 (control), 150, 300, 450, 600, and 750 mg Zn/kg. The rearing was carried out at constant conditions of 27 1C, 65% relative humidity and a 12-h dark/12-h light photoperiod in a climatic chamber. Pupae and adults from each concentration were kept under the same conditions.

2.2. Experimental design Approximately 1000 larvae per concentration were divided into five groups. Each group was kept in a 200 ml plastic-box with adequate fresh artificial diets. When adults emerged, adults of the similar size and weight were removed from the larvae boxes. Five pairs of adults of each group were transferred to a special adult jar. After about 2 days, each pair was transferred to plastic boxes and observed the duration of oviposition (the time from female adults start laying eggs to the end of ovipositing). Another five pairs of adults from each group were kept in special oviposition jars. The eggs laid in the adult jars were counted every day to determine the oviposition rate (the average number of eggs laid per female and day) and fecundity (the average number of eggs laid per female). In addition, the eggs of five pairs of adults from each group were used to investigate the hatching rate (the percentage of hatched larvae from eggs). The rest of the adults were used in the following experiments.

2.3. Zn accumulation in eggs All the eggs of 10 female adults (five replicates for each of the six concentrations), were vacuum dried at 60 1C for 24 h in Pyrex test tubes. Hundred milligrams of samples (dry weight) were digested in 10 ml of boiling nitric acid (65%) and 1 ml concentrated perchloric acid (BAKER ANALYZED reagent; Baker, Deventer, Holland) (Xia et al., 2005). When the fume was white and the solution was completely clear, the samples were cooled to room temperature. After filtrated by filter paper, the clear solution was transferred to a volumetric flask which was then filled to 50 ml with deionized water. Zn concentrations were estimated by an inductively coupled plasma-atomic emission spectrometer (ICP-AES, Thermo Jarrell Ash Company, USA). Concentrated nitric acid and perchloric acid were used as the blank control. Zn concentrations in eggs were calculated as follows: concentration of Zn ¼ (C  50)/100 mg, where C is the Zn concentration detected by the ICP-AES.

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2.4. Purification and detection of Vn in eggs 2.4.1. Preparation of polyclonal antibody According to the procedures described by Gong et al. (1980) and Nose et al. (1997), 100 mg of fresh eggs were homogenized on ice with 100 ml of 0.4 M NaCl in an all-glass microhomogenizer. The homogenate was centrifuged at 10,000  g for 10 min at 4 1C. The sample buffer was added to the supernatant (80/10 ml supernatant), then mixed well. The mixture was incubated overnight at 4 1C. The next day, the mixture was centrifuged (5000  g for 10 min), the supernatant was discarded, and the pellet was suspended in 0.4 M NaCl. The sample was separated by electrophoresis on 10% polyacrylamide gels. Native polyacrylamide gel electrophoresis was conducted using a Bio-Rad mini PROTEANs3 Cell Electrophoresis apparatus at 100 V for 2 h. The gels were stained lightly with Coomassie blue, and single major Vn bands were excised and processed to obtain polyclonal anti-S. litura Vn antiserum from female New Zealand white rabbits. 2.4.2. Detection of Vn in eggs of females exposed to Zn One thousand eggs of paired adults laid in each concentration (five replicates per concentration) were weighed, and then purified according to the method described in Section 2.4.1. The total content of Vn in eggs was determined by the Bradford protein assay using bovine serum albumin (BSA) as the standard (Bradford, 1976). Hundred micrograms of fresh eggs of paired adults laid in each concentration were homogenized in PBS (PBS; 137 mM NaCl; 2.7 mM KCl; 8 mM Na2HPO4; 1.5 mM KH2PO4; pH 7.5) followed by centrifugation for 20 min at 12,000  g and 4 1C. The supernatants were freeze-dried. An equal amount of the freeze-dried total protein (20 mg) extracted from eggs was separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Optitran BA-S83, Schleicher and Schuell, Keene, NH, USA) in buffer (25 mM Tris, pH 8.3; 192 mM glycine; 10% methanol) by a semi-dry technique using the Trans-Blots SD semi-dry transfer cell (Bio-Rad, USA) at 12 V for 40 min. The membrane was blocked with 5% fat-free milk for 2 h, then incubated for 2 h with rabbit anti-Vn diluted 1:1000 by PBST (0.2% Tween-20 in PBS). The membrane was washed three times with PBST (15 min per wash) then incubated for 1 h with goat anti-rabbit immunoglobulin G–horseradish peroxidase (IgG–HRP) (Sigma, USA) diluted 1:2000 with PBST. The membrane was then again washed with PBST for three times (15 min per wash) and the color was developed with 3,30 ,5,50 -tetramethylbenzidene (Promega, USA) (Towbin et al., 1979). The bands of Western blots were imaged and densitometric analysis was performed using the GeneGenius Match systems (Syngene, USA) with Gene Tools from Syngene software. The relative content of Vn in the total protein extracted from eggs was calculated as the densitometric value of treatment/that of the control*100 (%). 2.5. Real-time polymerase chain reaction (RT-PCR) for quantification of Vg mRNAs Total RNA was extracted from the fat body of five female adults at 24 h old (quantification by RT-PCR showed that the expression of Vg mRNA in fat body of 24 h-old adult was the highest level, data not published) from each concentration with Trizol reagent according to the manufacturer’s specifications (Invitrogen, Carlsbad, CA, USA). Next, the total RNA was treated with DNA-Free (Ambion, USA) according to the manufacturer’s specifications to eliminate DNA contamination. According to the manufacturer’s instructions, 1 mg of total RNA was used for the first strand synthesis of cDNA in 10 ml of total volume using the PrimeScriptTM cDNA synthesis system (TaKaRa, Japan) with Oligo dT Primer and Random 6 mers as the primer. Gene-specific primers were designed in terms of the S. litura Vg gene (EU095334): QVgS (50 -CCA GTT GGT TTG GTC AGT GTT G-30 ) and QVgR (50 -TCG TAG TTG ATG GAG CGG TTG C-30 ). The primers of housekeeping gene b-actin as endogenous control, QActinS (50 -TGA GAC CTT CAA CTC CCC CG-30 ) and QActinR (50 -GCG ACC AGC CAA GTC CAG AC-30 ), were also synthesized. The qPCR was run on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with SYBR Premix Ex Taq (Perfect Real Time) Kit (TaKaRa, Japan) under the following thermal program: one cycle of 95 1C for 10 s, 40 cycles of 95 1C for 5 s and 60 1C for 30 s. After qPCR, the homogeneity of the PCR product was confirmed by a melting curve analysis. The ratios of Vg/b-actin levels of gene expression were calculated. The relative copy number of Vg mRNA was calculated according to the 2–DDCT method (Livak and Schmittgen, 2001). The threshold cycle value difference (DCT) between Vg mRNA and b-actin RNA of each reaction was used to normalize the level of total RNA. The assay was repeated three times with separately extracted total RNA samples. Three replicates were performed for each reaction to account for intra-experiment variation. 2.6. Statistical analysis Statistical analysis was performed by the use of the SAS 8.1 program (SAS Institute Inc., 1989, USA). Differences among treatments in the following matters were evaluated by analysis of variance (ANOVA) and Tukey’s Studentized range test (P ¼ 0.05) through the PROC GLM program: the duration of oviposition, oviposition rate, fecundity, egg hatching rate, Zn accumulation in eggs, the weight

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of eggs and Vn content in eggs. The correlations of Zn accumulation and weight of eggs as well as Zn accumulation and Vn content of eggs were analyzed by the correlation analysis of SAS. The relationships between Zn concentration in eggs and the Zn concentration in the artificial diet were analyzed with regression analysis.

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3. Results

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3.1. Effects of zinc exposure on reproduction of S. litura

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The parameters with relation to the reproduction of S. litura, including the duration of oviposition, oviposition rate and fecundity, as well as hatchability of eggs, were all affected by Zn exposure. The details were described in Figs. 1–3, respectively. A reduction in the duration of oviposition was observed in Fig. 1. When insects were exposed to 150 mg Zn/kg, the period of laying eggs was 5.4 days, slightly shorter than the control (5.571 days). However, insects exposed to 300–750 mg Zn/kg kept reproducing for about 4 days, which was 1–2 days shorter than control. The shortest time (3.670.55 days) to keep reproducing was observed in insects fed on the diet treated with the highest concentration of Zn (750 mg Zn/kg). For insects treated with 150 mg Zn/kg, the oviposition rate (305.61738.09 eggs/female/day) was more than the control (266.13734.56 eggs/female/day). But the oviposition rate of the other treatments decreased along with the increase of Zn concentrations in the diets (Fig. 2). In particular, much lower oviposition rates for insects treated with 600 and 750 mg Zn/kg were observed, which was 49.41% and 31.43% compared to control, respectively. The fecundity declined following the increase of Zn concentration in the artificial diets (Fig. 2). When insects were exposed to 150 mg Zn/kg, the fecundity (1592.737323.28 eggs/female) was almost as much as control (1596.807321.02 eggs/female). Whereas fecundities of other treatments were significantly less than the control, which were 60.00% (300 mg Zn/kg), 36.67% (450 mg Zn/kg), 27.66% (600 mg Zn/kg) and 20.95% (750 mg Zn/kg) compared to control. A reduction in the hatching rate of eggs was observed with the increase of Zn concentration in the artificial diet (Fig. 3). Of those females exposed to 150 mg Zn/kg, the hatching rate (88.2672.47%) had little difference compared with the control (90.2672.46%). But the hatching rates of the insects treated with higher Zn concentration diets were significantly lower than control: for insects exposed to 750 mg Zn/kg, almost half of the eggs failed to hatch (52%).

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Fig. 1. Effect of Zn on the duration of oviposition of S. litura. The bars denote the average days (+SD) of oviposition in five replicates per concentration. Values followed by the same capital or small letter within a column were not significantly different (ANOVA df ¼ 24, F ¼ 7.86, Pr ¼ 0.0002o0.01).

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0 Oviposition rate Fecundity Zinc concentration in the artificial diets (mg Zn/kg) Fig. 2. Effect of Zn on the oviposition rate and fecundity of S. litura. The bars represent the average (+SD) number of eggs in three replicates per concentration; Values followed by the same capital or small letter in a group were not significantly different. The results of SAS analysis was (df ¼ 12, F ¼ 3.27, Pr ¼ 0.0428o0.05) and (ANOVA df ¼ 12, F ¼ 5.27, Pr ¼ 0.0086o0.01) for oviposition rate and fecundity, respectively.

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3.2. Zn accumulation and Vn content in eggs The Zn concentrations in eggs of all treatments were significantly higher than control, and this increased with the increase of Zn concentrations in diets. The appropriate equation (R2 ¼ 0.9437) has been simulated in Fig. 4. When exposed to 600 and 750 mg Zn/kg, insects accumulated Zn twice as much as the control (814733.16 mg Zn/kg) in eggs, but the difference between two was not significant. Similarly, when insects were exposed to 150 and 300 mg Zn/kg, whose accumulations of Zn in eggs were almost half of the control; little difference was observed in two treatments. The weight of their eggs decreased with Zn concentrations for the insects fed on the artificial diets treated with higher concentrations of Zn (450, 600 and 750 mg Zn/kg) (Fig. 5). In contrast, the weights of the eggs produced by insects treated with 150 and 300 mg Zn/kg were noticeably more than

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Fig. 3. Effect of Zn on the hatching rate of eggs produced by S. litura. The bars represent the average (+SD) percentage of the eggs hatched in five replicates per concentration; Values followed by the same letter within a column were not significantly different (ANOVA df ¼ 24, F ¼ 79.83, Pro0.0001).

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0.225 Vn concentration in the eggs (mg/g)

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Fig. 4. Zn accumulations in eggs of S. litura. The symbols represent the means and SD in five replicates per concentration; the difference between treatments and control was also analyzed (ANOVA df ¼ 24, F ¼ 74.47, Pro0.0001).

Fig. 6. Effect of Zn on Vn content in eggs of S. litura. The bars denote the average (+SD) Vn content extracted from 1000 eggs produced by females in three replicates per concentration, and the same letter means no significant difference (ANOVA df ¼ 12, F ¼ 79.94, Pro0.0001).

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Fig. 5. Effect of Zn on the weight of eggs produced by S. litura. The bars denote the average (+SD) weight of 1000 eggs produced by females in five replicates per concentration. Values followed by the same letter within a column were not significantly different (ANOVA df ¼ 24, F ¼ 198.23, Pro0.0001).

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the control, being 1.31 and 1.23 times as heavy as control (0.04770.001 g/1000 eggs), respectively. Next, the change in Vn of eggs was consistent with the change in their weight (Fig. 6). The Vn contents in eggs produced by insects fed on the diets with 150 and 300 mg Zn/kg were, respectively, 1.87 and 1.65 times that of control (0.1037 0.006 mg Vn/g eggs). Nevertheless, their Vn contents in eggs were less than the control when exposed to higher concentrations of Zn (600 and 750 mg Zn/kg). The Vn content in eggs produced by the insects was further investigated by using Western-blot with densitometric image analysis. Except for the band of eggs produced by insects treated with 150 mg Zn/kg, the others weakened gradually with the increase of Zn concentrations in the diets (Fig. 7). The small amount of Vn in the total protein extracted from eggs of the 750 mg Zn/kg treated insect was detected, which was only 14.38% of control. However, the Vn content in eggs produced by insects fed on the diet treated with 150 mg Zn/kg was more than the control, which was in agreement with Vn content in eggs determined by Bradford (1976). After exposure to different concentrations of Zn, the accumulations of Zn in eggs evidently affected the weight and Vn content of eggs. There were significant negative correlations in two (R ¼ 0.69, Po0.0001 for weight of eggs; R ¼ 0.68, Po0.002 for Vn content of eggs).

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Fig. 7. Effect of Zn on Vn content in the total protein extracted from eggs of S. litura.

3.3. Vg expression in fat body of S. litura female adults Compared to the control, the expression level of Vg mRNA in treated insects decreased with the increase of Zn concentrations in the diet (Fig. 8). There was a significantly lower expression level in the insects fed on diets treated with high concentrations of Zn (300–750 mg Zn/kg): the relative quantities of Vg mRNA were 0.419 for 450 mg Zn/kg and 0.403 for 600 mg Zn/kg. Less Vg mRNA was detected in the fat body of insects treated with 750 mg Zn/kg, at less than a quarter of that for the control.

4. Discussion The phytophagous insect S. litura Fabricius is a worldwide terrestrial pest. Insects living in terrestrial environments are mainly exposed to Zn and other heavy metals through their food ¨ ¨ anen, ¨ (Heliovaara and Vais 1993). In natural conditions, S. litura larvae feeds mainly on crops such as cotton, soybean, groundnut, tobacco, and then pupates in the soil of farmland (Etman and Hooper, 1979; Matsuura and Naito, 1997; Qin et al., 2004). For our

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Relative Quality of Vitellogenin

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1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

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Fig. 8. Effect of Zn on Vg expression in fat body of S. litura. The bars represent the average (7SD) of Vg expression in the fat body, values on the y-axis denote the relative quantities of Vg, and the same letter means no significant difference (Pro0.05).

experiment, as we considered that a preferable artificial diet was applied in S. litura for several years (Chen et al., 2000), we chose a pathway, Zn-contaminated artificial diets. In the field, the mean and maximal concentrations of Zn in the surface soil of farmland in the Pearl River Delta ranges from 166.9 to 963.0 mg Zn/kg dry weight (Chai et al., 2004). Zn is also readily accumulated by plants and stored in their stems and leaves. The concentrations of Zn in the leaf or stem of some cabbages grown in Zn-rich soil are recorded as high as 1297 and 1141.8 mg Zn/kg dry weight, for the average concentration of Zn in vegetables being 808.7 mg Zn/kg wet weight (Wu et al., 1996). According to the Zn levels in soil and plants, the Zn concentrations used in the present study ranged from 150 to 750 mg Zn/kg, in which the lowest concentration was little lower than the lowest Zn concentration of farmland in the Pearl River Delta, and the highest concentration was also a little lower than the average concentration of plants. S. litura have a no-feeding stage for pupae and adults, which requires female larvae to take up adequate nutrition and storage energies from food ready to pupation, emergence and reproducing offspring. All female adults used in this experiment possessed similar size and weight. It was considered that growth of S. litura female adults was not affected by dietary Zn. According to Kooijman (2000), effects of heavy metal contaminated food on animal would be reflected in both reproduction and growth effects. Hence, the dietary Zn specifically acted on the reproductive physiology of S. litura. In the present work, the impairing effects of high Zn concentrations were observed in the reproduction of S. litura. The results showed that reproduction-related parameters of S. litura were severely affected by high Zn exposure including the duration of oviposition (Fig. 1), the oviposition rate and the fecundity (Fig. 2), as well as the hatching rate of eggs (Fig. 3). The lowest Zn concentration that caused a significant decrease in the above parameters was 300 mg Zn/kg. In particular, the number of eggs produced by insects exposed to 750 mg Zn/kg contaminated diets decreased drastically compared to the control (31.43% for the oviposition rate and 20.95% for fecundity). Similar investigation on grasshoppers showed that the number of eggs laid by aging females decreased gradually in insects exposed to Zn (Augustyniak et al., 2008). This was also in accordance with the observations in springtails P. minuta, which suffered a reduction in adult survival and reproduction at high concentrations of Zn (Nursita et al., 2005). In our experimental conditions, the hatching rate of eggs significantly decreased at high concentrations of Zn (Fig. 3). When insects were exposed to 750 mg Zn/kg, almost half

of the eggs failed to hatch. The failure of hatching was due to the decline in the quality of eggs. Zn accumulated in eggs increased with the increase of Zn concentrations in the artificial diets, following a dose–response relationship (Fig. 4). Along with the increase of Zn concentration in eggs, a significant decrease in the weight and Vn content of eggs was observed in insects exposed to higher Zn (450, 600 and 750 mg Zn/kg) (Figs. 5 and 6). The sites where excess Zn accumulated may be damaged, which was observed in brain of grasshoppers, being the increase in DNA damage as well as Zn concentration in the grasshopper C. brunneus brain (Augustyniak et al., 2006). Excess Zn has significantly negative effects on quality of eggs through impairing the conversion of energy reserves and resources into offspring, which may be an important factor in the failure of eggs to hatch. A significant down-regulated expression of Vg mRNA in fat body was observed in S. litura fed on diets treated with higher Zn concentrations. The Vg mRNA level in insects exposed to 750 mg Zn/kg was less than a quarter that of the control (Fig. 8), which resulted in the decreased content of Vg processed to Vn. Therefore, an obvious decrease of Vn content was detected in eggs of S. litura exposed to higher Zn concentrations (600 and 750 mg Zn/kg) (Figs. 6 and 7). In S. litura, excess Zn caused impaired reproduction, which was due to the down-regulated expression of Vg gene and poor accumulation of egg yolk. This may lead to a reduction in egg numbers and failure of eggs to hatch. The above results were in agreement with suggestions of De Schamphelaere et al. (2004), who showed that the target sites where accumulation of dietary Zn would lead to reproductive toxicity are the most likely sites where production of Vg occurs or where Vg is further processed to yolk protein. In insects, Vg synthesis is believed to occur in the fat body. Vg is then transferred via the hemolymph into the oocytes where it is further processed to Vn (Raikhel and Dhadialla, 1992; Sappington and Raikhel, 1998; Snigirevskaya and Raikhel, 2005). Potential sites of toxic accumulation of Zn in S. litura were most probably the fat body (inhibition of Vg production) or the oocytes (inhibition of processing of Vg to Vn). The previous study showed that fat body of S. litura accumulated a mass of Zn (Xia et al., 2005). Furthermore, this work indicated that eggs could accumulate most Zn too. The exact mechanism by which inhibition of Vg production or processing by Zn takes place is unknown. De Schamphelaere et al. (2004) summarized two hypotheses for the inhibition of Vg synthesis by heavy metal. One hypothesis suggested that inhibition of vitellogenesis was due to binding of metals to enzymes involved in vitellogenesis, a process which could occur in the Vg production (fat body) or processing (oocytes) stage of the Vn. An alternative hypothesis showed that there was the reduced mitochondrial energy production and decreased glycogen content in the storage cells, where in this state would be less able to properly carry out their Vg synthesizing function. Augustyniak et al., (2006) suggested that the presence of DNA damages in brain was connected to apoptosis, programmed cell death. This in turn would be expected to disrupted nervous system, thereby leading to reduced hormone secretion and DNA repair, and thus ultimately inhibiting the synthesis and secretion of Vg. In order to investigate the reproductive effect in more detail, other additional reproductive traits of the S. litura, i.e. the changes in hormone levels or enzyme activities of insects, were worthy of study. The effects of the low concentration of Zn (150 mg Zn/kg) on reproduction were minor. The impairment by Zn on the duration of oviposition, the fecundity and Vg mRNA levels of insects exposed to 150 mg Zn/kg was not obvious, whereas it made the oviposition rate and Vn content in eggs increase significantly. It is

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well known that Zn is the major structural component of a variety of enzymes and transcription factors (Vallee and Falchuk, 1993). These gene regulatory proteins of Zn are most active during periods of increased proliferation and differentiation, such as that which occurs during vitellogenesis. According to Falchuk and Montorzi (2001), Vg is a metallo-protein that contains Zn and calcium. When Vg is processed to Vn in oocytes, Zn is also transported in plasma by, and is taken into the oocyte bound to, Vg. When Zn concentration in diets that was not beyond body burden of insect, insects were able to take up and storage moderate Zn. Those Zn may be available on the reproductive adults, whereas excess Zn was deleterious. The quadratic equation of Zn accumulation in eggs (y ¼ 2E05x2+1.351x+849.38, R2 ¼ 0.9437) available simulated above state (Fig. 4). The increase of weight and Vn content in eggs was observed in insects fed on low concentrations of Zn (150 mg Zn/kg) further emphasized it. Therefore, a moderate concentration of Zn might induce the synthesis of Vg and accumulation of Vn in eggs. This led to the enhancement of quality and quantity of eggs which was the basis for a rapid growth of a pest population. Perhaps, a moderate concentration of Zn was a possible stimulator of a pest outbreak. The mechanism by which Zn inhibited the synthesis and accumulation of Vg in insects was complicated. It was clearly needed for us to investigate endocrine-disruptive activity of excess Zn. Changes caused by excess Zn in nutrition element for reproduction was worthy of study. In addition, the effects of low Zn concentration on insect with long-term exposure were also clearly needed in future research.

5. Conclusion The present study has revealed multiple effects of higher Zn on S. litura reproductive characteristics which are considered as major fitness components, i.e. the duration of oviposition, the number and the hatching rate of eggs. When insects were exposed to Zn, eggs of insect could accumulate a mass of Zn, which led to the decrease in quality of eggs such as weight of eggs and Vn content. Vg gene expression inhibition in female adult fat body and Vn protein less accumulation in eggs was detected in insect exposed to higher Zn concentration. Overall, our results thus indicate that Zn specifically targets reproduction in S. litura through accumulation in particular target sites, possibly cells or tissues where vitellogenin synthesis (fat body) or processing occur (oocytes). Our data also indicate that excess Zn made expression of Vg gene down-regulated and caused poor accumulation of egg yolk, leading to a reduction in egg numbers and failure of eggs to hatch.

Acknowledgments This work was supported by the National 973 project of China (2006CB102001), the National Natural Science Foundation of China (Grant No. 30771458) and the Doctoral Fund of Ministry of Education of China (Grant No. 20070558029). References  Augustyniak, M., Babczynska, A., Koz"owski, M., Sawczyn, T., Augustyniak, M., 2008. Effects of zinc and female aging on nymphal life history in a grasshopper from polluted sites. J. Insect Physiol. 54, 41–50. Augustyniak, M., Juchimiuk, J., Przyby"owicz, W.J., Mesjasz-Przyby"owicz, J.,  Babczynska, A., Migula, P., 2006. Zinc-induced DNA damage and the distribution of metals in the brain of grasshoppers by the comet assay and micro-PIXE. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 144, 242–251.

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