Bioaccumulation of Mercury and Its Effects on Survival, Development and Webweaving in the Funnel-Web Spider Agelena labyrinthica (Araneae: Agelenidae) Jin Liu, Jin Gao, Yueli Yun, Zhiyong Hu & Yu Peng
Bulletin of Environmental Contamination and Toxicology ISSN 0007-4861 Bull Environ Contam Toxicol DOI 10.1007/s00128-013-0966-y
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Author's personal copy Bull Environ Contam Toxicol DOI 10.1007/s00128-013-0966-y
Bioaccumulation of Mercury and Its Effects on Survival, Development and Web-weaving in the Funnel-Web Spider Agelena labyrinthica (Araneae: Agelenidae) Jin Liu • Jin Gao • Yueli Yun • Zhiyong Hu Yu Peng
•
Received: 30 July 2012 / Accepted: 31 January 2013 Ó Springer Science+Business Media New York 2013
Abstract This study investigated the bioaccumulation and effects of mercury (Hg) in funnel-web spiders, Agelena labyrinthica, following exposure to sublethal concentrations of Hg(NO3)2 in their drinking water. The results showed that the Hg content in adult A. labyrinthica increased rapidly with the number of days exposed to the Hg(NO3)2 solution, and the mortality of adult spiders within 30 days increased with increased concentrations of Hg(NO3)2 in the drinking water. The total developmental duration of A. labyrinthica exposed to Hg(NO3)2 was significantly longer than in the control spiders, but there were no significant differences in the total developmental duration of spiders among the three treatment groups (exposed to 10, 20 and 50 mg/L Hg(NO3)2 solution). We also compared the web-weaving of the control and treated spiders, and found no significant differences in shape, structure, color, or size of the webs between the control and treated spiders; however, there was a significant difference in web placement between the treatment and control groups. The spiders in the control group appeared to have an episodic-like memory, choosing to weave their five webs in the same corner in the five time periods allowed. Keywords Araneae Mercury Bioaccumulation Survival Developmental duration Web-weaving
J. Liu Y. Yun Z. Hu Y. Peng (&) College of Life Sciences, Hubei University, Wuhan 430062, People’s Republic of China e-mail:
[email protected] J. Gao Hubei Testing Centre of Agricultural Products Quality and Safety, Wuhan 430070, People’s Republic of China
Many reports have indicated that spiders (Wilczek et al. 2003; Chen et al. 2011) and spider webs (Shao et al. 2006) can be used as biological indicators of heavy-metal contamination. Spiders are sensitive, at the community level, to habitat structure and type (Jung et al. 2008), making them effective indicators for comparing the biodiversity of various habitats, and for monitoring and assessing the effect of anthropogenic disturbances such as heavy-metal contamination on diversity (Wheater et al. 2000; Jung et al. 2008; Babczyn´ska et al. 2011). Jung et al. (2008) found that the assemblage of spider species was very different between a site heavily polluted with metals and an unpolluted site. Heavy metals can be absorbed from the soil through the roots of plants, and can also be absorbed through the leaves in gases or dust collected from the atmosphere, and then enter herbivorous animals such as cicadas, locusts and sawflies (Thornton 1991). When the carnivorous spiders are exposed to environmental pollutants, growth and reproduction can be sharply reduced due to an increased metabolic detoxification effort (Hendrickx et al. 2003; Wilczek et al. 2003; Chen et al. 2011). To date, most studies have focused on ground-dwelling spiders (Araneae), and few on web-weaving spiders. Ground-dwelling spiders have regularly been used as biological indicators of heavy-metal contamination (Laing et al. 2002), because heavy metals can alter the community structure of soil invertebrates, and in particular affect ground-dwelling spiders such as the wolf spider (Pardosa astrigera L. Koch) (Fountain et al. 2007; Jung et al. 2008). These spiders accumulate more heavy metals in their systems because they frequently hunt on the ground (Wilczek et al. 2004; Jung and Lee 2011). Agelena labyrinthica, a funnel-web spider, plays an important role in the control of agricultural pests. The spiders build flat surface webs
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connected to funnel-shaped retreats similar to labyrinths, which are typically constructed among short grasses and short vegetation. The spiders usually occur in areas near forests and in short vegetation, as well as in wet grasslands (Zhao 1993). In this study, we investigated the bioaccumulation of mercury (Hg) and the effects of Hg on survival, developmental duration, and web-weaving in the funnel-web spider A. labyrinthica. We exposed the spiders to Hg by using sublethal concentrations of Hg(NO3)2 solutions as their drinking water.
Materials and Methods Individuals of A. labyrinthica used in the experiment were collected in brushy vegetation on the Hubei University campus, Wuhan (114°310 N, 30°520 E), Hubei Province, China, in 2011. The spiders were kept in individual cylindrical glass tubes (diameter 2 cm, height 12 cm), with a layer of sponge (1.5 cm thick) moistened with distilled water on the bottom of the tube. The tubes were plugged with cotton. The spiders were kept in environmental chambers at 24°C and relative humidity of 60 %–80 % under a light: dark cycle of 14:10 h (lights turned on at 08:00). We fed the spiders with adults of Drosophila melanogaster and Tendipes sp. every 2 days. The standard Hg solution used in the experiment was purchased from the NCATN (National Center of Analysis and Testing for Nonferrous Metals and Electronic Materials, Beijing, China). We diluted this standard to compose a series of Hg(NO3)2 solutions corresponding to 10, 20 and 50 mg/L. In the Hg accumulation test, adults of A. labyrinthica were provided with a 10 mg/L Hg(NO3)2 solution instead of distilled water as their drinking water, according to Chen et al. (2011). After the spiders were exposed to the Hg(NO3)2 solution for 5, 10, 15 or 20 days, the Hg contents in their bodies were determined. The spiders were dissolved in a mixture of 8 mL 68 % HNO3 and 2 mL 30 % H2O2 and placed in a drying oven at 95 ± 2°C for 2 h, then heated to 135 ± 2°C for 3 h. When the volume of the solutions was reduced to approximately 1 mL, they were cooled to room temperature and made up to a volume of 50 mL with distilled water. The Hg content in the spiders’ bodies was analyzed with a dual-channel atomic fluorescence photometer (AFS-930, Jitian Instrument Co., Beijing, China). All reagents used were guaranteed reagent grade, and all the glassware was immersed in 3 mol/L nitric acid for 24 h before use. The Hg content of certified reference materials (GBW-10024) was (40 ± 7)lg/kg, and the mean test result was 35 lg/kg. The minimum detection level was 0.2 lg/kg, and the Hg content of the blanks was \0.1 lg/L.
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Third-instar spiderlings were reared individually in cylindrical glass tubes (diameter 2 cm, height 12 cm). A total of 200 individuals were randomly divided into four groups (50 in each group): one control group, provided with distilled water as their drinking water and fed adult fruit flies, D. melanogaster; and three treatment groups provided with different concentrations (10, 20 or 50 mg/L) of Hg(NO3)2 solution as their drinking water and fed the same number of fruit flies as the control group. The moistened sponges were replaced every 2 days. Molts were recorded when exuviae were observed in the tube, and the time between molts was used as our measure of developmental duration. Mortality of the spiderlings was calculated after 30 days. To investigate the effect of Hg on the web-weaving of A. labyrinthica, individual adult females were provided with a 10 mg/L Hg(NO3)2 solution instead of distilled water, and 15 days later they were placed in a web-weaving chamber made of glass (Cuboid: length 22 cm, width 6 cm, and height 20 cm). After the spider finished weaving a web, we checked the shape, structure, color, and size of the web and recorded the web placements (top or bottom corner of the chamber). Because chemical traces could be left in the chambers by previous spiders, the webs were removed and the test chambers were cleaned and dried before the next trial. Another, paired adult female that had been given distilled water as a control, was also allowed to weave a web. Each experimental treatment consisted of 10 spiders, and was repeated five times. We considered that a spider appeared to have an episodic-like memory if it wove the five webs in the same corner in the five time periods. Then, we compared the choice of web placement between the control and the Hg-treated spiders. Data are expressed as mean ± SE. The differences in Hg contents and development times were compared using Duncan’s multiple range test (SAS Institute 1997). The data were tested for homogeneity of variance, using Levene’s test of equality of error variances. The differences in mortality of spiders exposed to different concentrations of the Hg(NO3)2 solution were compared by the Mann– Whitney U and Kruskal–Wallis tests. Fisher’s exact test was used to test the difference in episodic-like memory for choosing web placement between the treatment and control groups. All statistical analyses were performed with SPSS for Windows (version 14.0, SPSS Inc., Chicago, IL, USA).
Results and Discussion The accumulation of Hg in A. labyrinthica increased rapidly with the number of days exposed to the Hg(NO3)2 solution, and the Hg content was highest in spiders exposed for 20 days (Fig. 1). Jung et al. (2005) reported that the
Author's personal copy Bull Environ Contam Toxicol
Concentration of Hg (µg/kg )
600
a
500 400 300 200
b c c
100 0 5
10
15
20
Exposure time (days) Fig. 1 Mercury (Hg) content in adult A. labyrinthica exposed to 10 mg/L Hg(NO3)2 solution for different periods (n = 6). Different letters above columns indicate significant differences among treatments (p \ 0.05)
40
a
30
Mortality (%)
initial levels of heavy metals in unexposed spiders were negligible, and they did not detect Hg initially in the spiders. In their experiment, the saturation level of Hg (ca. 3 lg/body) was achieved within 2 weeks of exposure (10 mM HgCl2). These observations indicate that dietary exposure is the main route of accumulation of heavy metals in spiders (Jung et al. 2005; van Ooik et al. 2007); and in the food-chain transfer process, the Hg concentration has been shown to increase with increasing trophic level. For spiders exposed to different concentrations of the Hg(NO3)2 solution for 30 days, mortality increased with the increase in concentration of the solution (Fig. 2). The concentration of the solution significantly affected the mortality of the spiders (Kruskal–Wallis text: v2 = 13.212, df = 3, p = 0.004) (Table 1). However, there was no significant difference in mortality between the control and the group treated with 10 mg/L Hg(NO3)2 solution (p = 0.458), or between the 20 mg/L and 50 mg/L Hg(NO3)2 treatment groups (p = 0.240). Mortality differed significantly among the other four groups (p \ 0.05) (Table 1; Fig. 2). In contrast to these findings, when P. astrigera (Araneae: Lycosidae), a ground-dwelling spider, was exposed to a HgCl2 solution of 2.715 mg/L, no mortality was observed during the exposure period (Jung et al. 2005). This may indicate that ground-dwelling spiders are more tolerant of Hg than funnel-web spiders. There was also increased mortality with increased metal concentration in insects when exposed to metals (Mousavi et al. 2003; Hayford and Ferrington 2005). With the exception of the 4th instar, all other stages showed significant increases in developmental time at one or more Hg exposure levels. The total duration of development from the 3rd to 6th instar of the control spiders was 50.0 ± 0.52 days, significantly shorter than the durations of the Hg treatment groups (Table 2). However, there were
ab 20
c
10
c 0
0
10
20
50
Concentration of Hg(NO3) 2 (mg/L) Fig. 2 Mortality of A. labyrinthica exposed to different concentrations of Hg(NO3)2 (n = 50). Groups sharing the same letters above columns were not significantly different from one another at the 0.05 probability level Table 1 Statistical results for mortality data of A. labyrinthica after 30 days exposure at different concentrations of Hg(NO3)2 Concentrations of Hg(NO3)2 (mg/L)
Mann–Whitney U test N
Z
p
0–10
6
-0.903
0.458
0–20
6
-2.333
0.026
0–50
6
-2.791
0.004
10–20
6
-2.056
0.041
10–50
6
-2.508
0.009
20–50
6
-1.212
0.240
no significant differences in developmental duration among the three treatment groups [exposed to 10, 20 and 50 mg/L Hg(NO3)2 solution, respectively]. Our results showed that Hg had a similar general effect in delaying development as in the studies on P. astrigera and heavy metals (Jung et al. 2005; Chen et al. 2011). Similarly, developmental delays have been observed in insects exposed to metals (Mousavi et al. 2003; Hayford and Ferrington 2005). There were no significant differences in shape, structure, color or size of the web between the treated spiders (exposed to 10 mg/L Hg(NO3)2 solution) and the control group (data not shown). However, there was a significant difference in choosing web placement between the treatment and control groups (Fisher’s exact test: p = 0.011). Of the 10 spiders in the control group, 6 (60 %) chose to weave their five webs in the same corner in all five time periods allowed (Table 3). The spiders in the control group appeared to have an episodic-like memory. Other animals species have been shown to act similarly. Scrub jays, for example, appear to have an episodic-like memory (Clayton and Dickinson 1998; Davies et al. 2012). In contrast, the Hg-treated spiders exposed to the 10 mg/L Hg(NO3)2
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Author's personal copy Bull Environ Contam Toxicol Table 2 The developmental duration (d) of Agelena labyrinthica exposed to different concentrations of Hg(NO3)2 Developmental stages
Concentration of Hg(NO3)2 (mg/L) 0 (control)
10
20
50
3rd instar
10.6 ± 0.51b
12.2 ± 0.73b
12.6 ± 0.81b
13.0 ± 0.71a
4th instar
10.8 ± 0.58a
11.4 ± 0.81a
12.2 ± 1.02a
12.8 ± 1.56a
5th instar
13.2 ± 0.37b
16.0 ± 1.34a
16.6 ± 1.21a
17.2 ± 1.07a
6th instar
15.4 ± 0.75b
18.8 ± 1.71b
19.6 ± 1.81b
20.2 ± 1.53a
Total
50.0 ± 1.52b
58.4 ± 2.88a
61.0 ± 2.91a
63.2 ± 2.42a
For the same developmental stage, numbers with different letters denote a significant difference (p \ 0.05) Table 3 The placements of webs woven by the spiders from the control and Hg-treated groups Replications
Control
Hg-treated
Top corner
Bottom corner
Top corner
Bottom corner
1
0
5
1
4
2
4
1
2
3
3
5
0
2
3
4
0
5
3
2
5
1
4
1
4
6
5
0
2
3
7
1
4
2
3
8
0
5
3
2
9
5
0
2
3
10
1
4
3
2
solution did not have such an episodic-like memory; i.e., none of them wove its five webs in the same corner. The extent of Hg contamination in the environment will directly impact the growth and development of funnel-web spiders. Heavy metals occur naturally in soils and rock formations, but may also occur in fertilizers and pesticides, as a result of which they may be taken up by plants and eaten by invertebrates, thereby affecting the latter’s survival and development (Massadeh et al. 2008; Sepehr and Hilliker 2009). As predatory invertebrates, spiders are generally recognized as macro concentrators of metals (Wilczek et al. 2003), and as secondary consumers, they ingest considerable amounts of various xenobiotics, including heavy metals. The amount of ingested metals depends on the hunting activity of the spiders and on the body composition of their prey. On the other hand, the specificity of metal excretion and storage in intracellular granules, rather than the quality of food, has been reported as responsible for their high metal loads (Wilczek and Babczynska 2000). This form of metal storage is known also from other invertebrate species (Babczyn´ska et al. 2011). In summary, Hg was bioaccumulated in A. labyrinthica, resulting in high mortalities and delays in the development
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of spiderlings. Hg exposure at a level of 10 mg/L in the drinking water of adult female spiders caused a significant behavioral response by changing the web placement. These results show that the funnel-web spider A. labyrinthica has good potential as an indicator of contamination by Hg, and possibly other metals, in the terrestrial landscape. Acknowledgments This study was supported by the National Natural Science Fund of China (31071895), the Natural Science Fund of Hubei Province (2011CDB071) and the Key Scientific and Technological Projects of Wuhan (No: 201120722216-3). We are grateful to the anonymous reviewer and the associate editor for their suggestions and improvements to this manuscript. We also thank Dr. Janet W. Reid (Biological consulting and editing services, JWR Associates, New York, USA) for helping us edit the English.
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