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Effects of zinc and female aging on nymphal life history in a grasshopper from polluted sites
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Journal of Insect Physiology 54 (2008) 41–50 www.elsevier.com/locate/jinsphys
Effects of zinc and female aging on nymphal life history in a grasshopper from polluted sites Maria Augustyniaka,, Agnieszka Babczyn´skaa, Micha" Koz"owskia, Tomasz Sawczyna, Micha" Augustyniakb a
Department of Animal Physiology and Ecotoxicology, University of Silesia, Bankowa 9, PL 40-007 Katowice, Poland b Faculty of Earth Sciences, University of Silesia, Bedzinska 60, PL 41-200 Sosnowiec, Poland Received 20 March 2007; received in revised form 3 August 2007; accepted 6 August 2007
Abstract Insect reproduction is influenced by various factors, including food quality and quantity, temperature, population density and female age. Contamination, including heavy metals, may disturb reproductive processes. The aim of this work was to assess interactions between effects of aging in female Chorthippus brunneus and environmental pollution on their reproduction measured in number of laid eggs. We also compared basic developmental parameters (number of hatchlings, body mass, embryonic developmental rate) in grasshopper nymphs additionally exposed to zinc during diapause. Aging grasshoppers from heavily polluted areas (Olkusz and Szopienice) lay significantly fewer eggs than insects from the reference site (Pilica). Zinc application caused the decrease in hatching success and duration of embryogenesis in insects from each site. This suggests a cumulative effect of female age, pollutants and additional stressing factors. The intensity of this process differed between populations. In insects from the reference site, it was shown in a moderate degree. In insects from Szopienice, an additional stressor exerted a weaker effect than in insects from Pilica. In grasshoppers from Olkusz, we found the strongest decrease of hatching percentage and increase in duration of embryogenesis after zinc intoxication. This may indicate that the population from Olkusz exists at the limit of its energetic abilities. r 2007 Elsevier Ltd. All rights reserved. Keywords: Grasshoppers; Aging; Development; Diapause; Zinc
1. Introduction It is generally accepted that an energetic potential, defined as the total amount of energy accessible for an individual through its whole life, for each species is limited. Moreover, we expect that insects exposed to additional environmental stressing factors have to bear much higher costs of detoxification (Stone et al., 2001; Wilczek, 2005). The energetic expenditures caused by settling new, heavily polluted habitats, may involve the production of additional enzymes for detoxification, inactivation, storage or excretion of toxins. That is why many species inhabiting polluted regions start mating and reproducing later, and hence their brood is less numerous compared to those from relatively unpolluted areas (Fisher, 1981; Schmidt et al., Corresponding author. Tel.: +48 32 2587737; fax: +48 32 2587737.
E-mail address: [email protected] (M. Augustyniak). 0022-1910/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2007.08.002
1992; Devkota and Schmidt, 1999). An energetic deficit may be a serious problem especially for old insects, still able to reproduce, living in heavily polluted environments. We know that aging female insects lay fewer eggs. The decrease in the number of egg pods, as well as number of eggs in each egg pod, is caused also by low temperature, food deprivation or excessive density of a population (Cherrill and Begon, 1989; Willott and Hassall, 1998). The fecundity of insects is also negatively affected by environmental toxins, including heavy metals (Devkota and Schmidt, 1999). However, the issue of aging insects in polluted sites remains to be elucidated. The problem of developmental abilities of old females’ offspring, especially this originating from polluted habitats, still needs investigation. Thus, the following hypotheses were proposed: (i) The development of insects of a given species follows the same pattern, irrespective of mother’s age and
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M. Augustyniak et al. / Journal of Insect Physiology 54 (2008) 41–50
exposure to environmental toxins, because the potential the offspring inherits is unchangeable and does not depend on the factors mentioned above. (ii) Long lasting (several hundreds years) contact with toxin, as it is at post-industrial areas, might have led to the selection of individuals that are able to cope more effectively with neutralization than insects not subjected to such pre-adaptation. This may be also reflected in the offspring’ development. (iii) The development of insects living in heavily polluted areas is abnormal because of cumulative effects of female aging and environmental pollution. The settlement of polluted areas will probably cause enhanced aging of the insects and severely decrease their fecundity and quality of eggs as aging progresses. The question of aging has been widely investigated. According to current theory, aging results from an increase of pro-oxidative factors and a gradual weakening of the antioxidant systems (Le Bourg, 2001; Sohal, 2002; Sohal et al., 2002). Previous reports stress that some heavy metals, widely present in the post-industrial environment, may stimulate free radicals production. Heavy metals may enhance oxidative stress and force the organism to synthesize additional peptides and enzymes involved in free radical scavenging, accelerating the aging processes (Augustyniak and Migula, 1996, 2000). This indicates that aging of an organism in a heavy metal polluted environment will be faster than in an environment free of additional stressing factors. On the other hand, we cannot exclude the possibility that some populations chronically exposed to metals, have developed energy-efficient mechanisms to perform at least some of the vital functions. Analysis of the connections between the kind of metal, its dose, the time of exposure, and the stage of diapause during ontogenesis of a specific insect species cannot be neglected. During this sensitive stage of life, insects are mostly deprived of avoidance strategies and can be exposed to toxins in soil and soil solution. Among the insects that inhabit dumps of industrial origin there are various grasshopper species which lay egg pods directly in the soil, diapause in this stage and undergo embryogenesis in such conditions (Devkota and Schmidt, 1999). Zinc is an important biogenic metal (Kim et al., 1999; Takeda, 2000; Zirpel and Parks, 2001; Maret, 2005). It plays structural, catalytic and regulatory functions in an organism. It is necessary for signal transduction, DNA replication, transcription and protein synthesis. Zinc is crucial for proper actions of several hundreds of enzymes. It forms the so-called ‘zinc-finger’ protein domains and controls the architecture of protein complexes. Zinc is also essential for regular functions of the nervous system. Since zinc plays various roles in metabolism, we may expect that any disturbance of its homeostasis may cause improper growth and morphogenesis (Takeda, 2000; Maret, 2005). However, as with other metals, zinc can be toxic when its concentration exceeds its physiological limits. We know
that zinc toxicity of may lead to unexpected neuronal death with the features of either apoptosis or necrosis (Kim et al., 1999; Zirpel and Parks, 2001). The aim of this work was to assess a possible influence of either age of female grasshoppers or the degree of environmental pollution of their habitat on fecundity. Among grasshopper populations chosen for this study, two originated from areas located around metal smelters. Both sites are strongly polluted with heavy metals, especially with zinc. Thus, in this work, we compare basic developmental parameters (quantity, body mass and embryonic development rate) of grasshopper nymphs that developed from the eggs laid by aging females, and then exposed to additional zinc intoxication during diapause. The zinc concentrations we used are similar to the concentration of zinc in humus layer at natural habitat of the insects. 2. Material and methods 2.1. Insects Chorthippus brunneus (Thunberg) is a representative of the Acrididae family. This hemimetabolic insect inhabits mainly sunny, open grassy areas such as meadows, farms, fields and pastures. C. brunneus overwinters in the egg stage. Like other Acrididae, C. brunneus is characterized by high fecundity. In optimal conditions, a female may lay an egg pod containing 10 or more eggs a day. The number of egg pods decreases with female’s age of, when the temperature is too low, during food deprivation periods or when the population gets overcrowded (Moriarty, 1969; Monk, 1985; Cherrill and Begon, 1989; Willott and Hassall, 1998). 2.1.1. Study sites and insect collection The insects were collected at three sites. Two sites were in the close vicinity of metal smelters (Olkusz and Szopienice), and the third was far from industrial plants (Pilica). In Olkusz, the main sources of pollution are the miningmetallurgic smelter ‘Boleslaw’, mining plants ‘Pomorzany’, ‘Olkusz’ and ‘Boleslaw’, all situated close to each other. Within a distance of 1 km from the Szopienice site, there is a mining-metallurgic smelter ‘Szopienice’ which is one of biggest emitters of particles containing compounds of zinc, lead, cadmium and other heavy metals. The concentration of zinc in soil at the Szopienice site reaches up to 2.1 mg g 1; therefore exceeding, by 10 times, the permissible soil concentration (Kucharski, 1993). The reference area was localized far from industrial pollutants, near Pilica town. For each site, the pollution index (PI) was calculated, which, for Szopienice, Olkusz and Pilica was equal to 22.7, 12.1 and 0.53, respectively. A detailed description of pollution and types of vegetation at each site are published in Augustyniak and Migula (2000), Stone et al. (2001), Łaszczyca et al. (2004) and Augustyniak et al. (2005). Male and female insects were collected in July as soon as the majority of insects reached sexual maturity.
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The insects caught were used to initiate the laboratory culture. 2.1.2. Experimental set-up In the laboratory, insects were weighed. Mean body mass for females from Pilica, Olkusz and Szopienice was 0.24, 0.22 and 0.20 g, respectively, while male grasshoppers from Pilica, Olkusz and Szopienice weighed on average 0.085, 0.08 and 0.07 g, respectively. The initial maternal culture of grasshoppers was reared at controlled laboratory conditions (temperature 30 1C71.5; 46% RH; photoperiod 12:12 h). Cubic cages (30 cm 30 cm 30 cm), where the insects were kept (each cage contained 25 pairs) were equipped with little watering pots, flasks with grass and a container filled with wet sand, where females could lay egg pods. The watering pots and flasks with grass were replaced daily. As soon as the females started laying eggs, the egg pods were collected once every 2 days and placed in sterilized, wet sand, washed previously with Hogland’s solution (Jalil et al., 1994). Once a week, all egg pods were gathered and classified into the same ‘age group’. This way 7 ‘age groups’ were obtained (named I–VII): the first one originated from the youngest and the last one from the oldest females. The experiment was terminated when the old females stopped laying egg pods. Egg pods were kept at 25 1C for 2 weeks to assure proper embryogenesis (Moriarty, 1969; Monk, 1985; Cherrill and Begon, 1989). After this period, all pods laid by the females from each site (and from each ‘age groups’) were again randomly classified into one of two experimental groups named Zn-1000, Zn-10,000 and one control group. The experimental groups varied in the concentration of zinc in the sand where the egg pods were transferred to (1000 and 10,000 mg Zn g 1 dry weight of sand, respectively). The egg pods from the control groups were kept in zinc-unpolluted sand. Each group consisted of 10 egg pods. The egg pods were then placed at 4 1C for 12 weeks to induce diapause (Moriarty, 1969; Monk, 1985). Summarizing, the egg pods were divided into groups according to the site of parent’s collection (Pilica, Szopienice and Olkusz). Within the groups, ‘age groups’ were selected based on the age of female at the moment of egg laying (I–VII). Additionally, each ‘age group’ was divided into experimental groups differing in the concentration of zinc in sand in which the egg pods were kept during their diapause. After diapause, the egg pods were placed into incubators (temperature 30 1C71.5; photoperiod 12:12). Each egg pod was placed in a separate container. As soon as the hatching had begun the nymphs were counted, and at the end of this process the pods were dissected and undeveloped eggs (as well as dead nymphs) were counted. Since each egg pod was placed in a separate container, it was possible to count the total number of eggs in each egg pod, as well as the number of nymphs hatched from each egg pod. Based on these data, the number of eggs per pod (E) and the
43
percentage hatchlings per pod (K) were calculated. Additionally, the mass of hatchlings (M) and the duration of embryonic development (T), defined as the period between the end of diapause and the day of hatching, were assessed. 2.1.3. Statistical procedures The results for parameters M and T were reported as mean values7CI (confidence interval). Hatchlings per pod (K) were expressed in %. Since the egg pods were placed separately in containers, we were able to assess the hatching efficiency (%) for each egg pod. For these data, statistical procedures were performed. Normality was checked using the Kolmogorov–Smirnov test. Homogeneity of variances was tested using the Levene test. Duration of embryonic development (T) and the mass of hatchlings (M) did not show the normal distribution, so the data were log-transformed and then analyzed within the experimental groups using the Tuckey’s test (ANOVA/MANOVA) for unequal sample size (Table 1). For each parameter, the analyses of main effects (site, treatment, ‘age groups’) were done. When necessary, we used a simplified classification of independent variables for these analyses. This was the case when there was a lack of data (especially in Zn-exposed groups) or when the number of values differed between groups. That is why for the variable ‘age groups’ the groups were called: cohorts: Y—young (the offspring of young females), M—middleage (the offspring of middle-aged females) and O—old (the offspring of old females), created as the result of joining I/II, III/IV and V/VI groups, respectively (Table 2). 3. Results 3.1. Number of eggs per pod The highest mean number of eggs per pod (E) for females from each study site was found at the beginning of the reproductive period. The number decreased along with the aging of female grasshoppers. However, the rate of change differed in insects from various sites (Tables 1 and 2, Fig. 1). The egg pods laid by insects from the reference site (Pilica) during the first 3 weeks contained nearly the same number of eggs (about seven per egg pod). Beginning with the fourth week, an average number of eggs per pod decreased to about five. This number of eggs was laid by insects from Pilica until the end of the reproductive period. Insects from Szopienice during the first week produced pods containing the highest number of eggs (on average 8.65), but the decrease of egg number per pod (to five eggs) started no later than in the second week and lasted until the V week. During the next week, the number decreased even more, to 2.5 eggs per pod in the VII week (Table 1; Fig. 1). Grasshoppers from Olkusz during first 2 weeks laid pods containing up to seven eggs per pod. Beginning on the III week, a dramatic fall in the number of eggs per pod was observed. It lasted until the VI week (on average 1.87 eggs
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Table 1 Developmental parameters in grasshopper C. brunneus from control and zinc-exposed egg pods laid by females collected at variously polluted sites: Pilica, Szopienice and Olkusz Age groups
E
Control
Zn-1000
Zn-10,000
K (%)
M (mg)
T (days)
K (%)
M (mg)
T (days)
K (%)
M (mg)
T (days)
61a 60a 66a 38b 49ab 13b X
4.7ab (4.5, 4.9) 4.4b (3.9, 4.9) 5.0a (4.7, 5.4) 6.0a (4.8, 7.5) 4.1b (3.9, 4.4) 4.4b (4.0, 4.8) X
12.0b (12.0, 12.0) 16.7a (15.9, 17.7) 12.1b (11.4, 12.7) 12.2b (9.9, 15.1) 10.0c (10.0, 10.0) 14.5ab (11.7, 17.9) X
68ab 59b 91a 86a 52b X X
4.4ab (4.1, 4.6) 4.1b (3.8, 4.5) 4.8a (4.6, 5.0) 4.7ab (4.3, 5.0) 4.2ab (4.0, 4.5) X X
14.3a (13.3, 15.3) 13.7ab (12.3, 15.3) 12.1bc (11.7, 12.5) 12.7ab (11.0, 14.5) 10.1c (9.9, 10.3) X X
20a 20a 22a 1b X X X
4.2a 4.7a 4.3a 4.3a X X X
(3.9, (4.3, (3.9, (4.1,
4.6) 5.0) 4.8) 4.5)
16.9a 21.3a 18.0a 17.0a X X X
(14.2, (18.8, (16.9, (17.0,
Szopienice I II III IV V VI VII
8.65a 5.60b 5.40b 3.95c 4.47bc 3.50c 2.50c
46b 70a 72a 75a 65a X X
4.3a 4.0a 4.7a 4.6a 4.5a X X
(4.3, (3.5, (4.3, (4.4, (1.1,
4.6) 4.6) 5.2) 4.9) 4.9)
14.6a (13.0, 16.3) 13.8a (13.2, 14.4) 12.8ab (12.1, 13.5) 11.0bc (10.8, 11.2) 10.6c (9.9, 11.3) X X
36b 86a 85a 71a 12b 16b X
4.8a 4.3a 4.8a 4.6a 4.5a 5.2a X
15.1a (13.8, 16.6) 12.5b (11.8, 13.1) 13.3b (12.0, 14.8) 11.5b (10.9, 12.1) 12.0b (12.0, 12.0) 12.0b (12.0, 12.0) X
25a 25a 22a 9a 27a X X
4.8a 4.4a 3.8a 4.3a 4.0a X X
(4.3, (4.2, (3.1, (4.2, (3.7,
5.4) 4.6) 4.8) 4.4) 4.2)
17.4a 14.4a 15.2a 14.5a 14.5a X X
(15.4, 19.7) (13.6, 15.3) (13.4, 17.3) (8.5, 24.7) (11.9, 17.7)
Olkusz I II III IV V VI VII
6.95a 6.57ab 3.82bc 2.42c 2.07c 1.87c X
30b 41b 90a 84a 16b X X
4.7a 5.1a 6.1a 5.1a 5.7a X X
(4.3, (5.0, (5.6, (4.1, (5.1,
5.2) 5.2) 6.7) 6.5) 5.7)
12.0b (12.0, 12.0) 15.0a (14.9, 15.1) 12.3b (11.7, 12.9) 10.2c (9.9, 10.4) 12.0b (11.8, 12.1) X X
50a 42a 40a X X X X
5.5a (5.1, 6.1) 6.2a (4.4, 8.8) 5.3a (3.1, 9.1) X X X X
15.2a (13.5, 17.0) 10.7b (10.3, 11.0) 11.0ab (10.2, 11.9) X X X X
X X X 16 X X X
X X X 4.2 (4.2, 4.2) X X X
(4.6, (4.0, (4.6, (3.8, (4.4, (5.0,
5.0) 4.7) 5.0) 5.7) 4.6) 5.4)
20.3) 24.1) 19.2) 17.0)
X X X 15.0 (15.0, 15.0) X X X
Note: E, mean number of eggs per pod; K, hatchlings per pod (%); M, hatchling weight (mean; confidence interval—CI); T, duration of embryogenesis (mean; CI). M and T were log-transformed prior to analysis. a, b, c Different letters indicate significant differences between ‘age groups’ within experimental group.
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7.07a 7.85a 6.97a 4.45c 6.15b 5.02bc 4.82c
M. Augustyniak et al. / Journal of Insect Physiology 54 (2008) 41–50
Pilica I II III IV V VI VII
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per pod). In the VII week, grasshoppers from this site ceased eggs laying. Main effects analysis showed that the number of eggs per pod was strongly influenced by the age of a female (variable: ‘age group’) and the habitat where the females were collected (variable: site). Interaction between these two variables was also significant (Table 2, Fig. 1).
3.2. Hatchlings per pod The highest hatching percentage from egg pods (K) from control groups was found for egg pods laid in III (Pilica, Olkusz) or IV (Szopienice) week of the experiment. This means that the egg pods were laid by middle-aged females (Table 1). The application of lower Zn concentration (Zn1000 group) during diapause did not cause any significant changes in grasshoppers from Pilica. In grasshoppers from polluted sites K parameter was decreased significantly only in cases of egg pods laid by middle-aged females from Olkusz (Fig. 2). Intoxication with a higher Zn concentraTable 2 Analysis of variance (ANOVA/MANOVA) for number of eggs (E), hatchlings weight (M), and duration of embryogenesis (T), in C. brunneus from Olkusz, Szopienice and Pilica (with cohorts and site as a categorical factors) Source of variation d.f. E
M
F Cohorts (1) Site (2) 12
2 2 4
p
F
T p
F
p
7.710 o0.001 8.756 o0.001 10.851 o0.000001 4.224 o0.01 6.288 o0.01 0.613 0.545 3.195 o0.01 1.895 0.1210 0.488 0.745
9
3.3. Hatchling weight The analysis of main effects revealed that the weight (M) of hatching nymphs strongly depended on the original female age; original habitat of the insects seemed less significant (Tables 1 and 2). The application of higher Zn concentrations caused a decrease in the weight of newly hatched grasshoppers only in the case of the offspring of middle-aged females from Pilica. In the remaining cases,
a a
b mean number of eggs per pod
tion (Zn-10,000 group) caused a significant decrease in the percentage of nymphs hatched from egg pods laid by middle-aged and old females from all study sites; the strongest effect was observed for insects from Olkusz (Fig. 2). In insects from the reference site (Pilica), we observed a strong decrease in K value in Zn-10,000 group, in which K reached 20% during the first three weeks. No individuals hatched from egg pods laid by females from Pilica during V–VII weeks, which were kept in sand contaminated with a higher Zn concentration (Tables 1 and 3). A similar effect was found in grasshoppers from Szopienice, where hatching percentage at Zn-10,000 decreased to 25%. However, in this case, hatching ceased no earlier than in the VI ‘age group’. The strongest response to the additional stressing factor was observed in grasshoppers from Olkusz. In the Zn-1000 group, the last hatching took place in egg pods classified in the III ‘age group’. Diapause in sand contaminated with the higher Zn concentration caused almost a total lack of hatching, apart from IV ‘age group’ (Tables 1 and 3). Thus, at least in case of grasshoppers from Olkusz, we observed an adverse effect of both female age and an additional stress factor.
a
8
ab
7 6
45
ab
5
a b
a a
b
b
a
b
4
b
3
ab
a c
2
b
b
1 0
I
II
III
Pilica
IV cohorts Szopienice
V
VI
VII
Olkusz
Fig. 1. Mean number of eggs per pod (E) in grasshopper C. brunneus from Pilica (reference site), Szopienice and Olkusz (polluted sites) collected during whole reproductive period. The same letters indicate homogenous groups within site (separately for each ‘age group’).
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3.4. Duration of embryogenesis
Pilica
hatchlings per pod [%]
100 **
80 * 60
*
40 *
* **
20 0 Y
M
O
cohorts
Duration of embryogenesis (T) for grasshoppers developing from eggs laid during subsequent weeks, unlike hatchling weight, strongly depends on female age at the time of egg laying. The original habitat appeared insignificant (Table 2). We found a regular pattern in the duration of embryogenesis which was shorter in grasshoppers developing from egg pods laid by older females than in insects from eggs laid by young females. In the contrary, longer embryogenesis was observed in Zn-intoxicated egg pods, especially after exposure to a higher zinc concentration, in all ‘age groups’ from all study sites in comparison with appropriate control groups. The effect was strongest in grasshoppers from Pilica (Table 1, Fig. 4).
Szopienice
4. Discussion
hatchlings per pod [%]
100 *
80
** 60 40 * **
20 0 Y
M
O
cohorts Olkusz 100
*
hatchlings per pod [%]
** 80 60 40 *
20
**
0 Y
M control
Zn-1000
O Zn-10000
Fig. 2. Hatchlings per pod (K) in grasshopper C. brunneus from Pilica (reference site), Szopienice and Olkusz (polluted sites) for egg pods laid by young (Y), middle-aged (M) and old female (O) additionally exposed to zinc during diapause (1000 or 10,000 mg Zn g 1). Stars indicate statistically significant differences between experimental groups within cohorts.
Zn did not cause a significant change in the body mass of the nymphs (Fig. 3). Statistical analysis within ‘age groups’ showed that significant differences refer only to grasshoppers from Pilica (control and Zn-1000 groups; Table 1).
The number of eggs per pod (E) laid by aging females decreased gradually in insects from polluted sites, in accordance with observations of other authors (Moriarty, 1969; Monk, 1985; Cherrill and Begon, 1989). However, in the case of insects from Pilica (reference site) the agedependent decrease in the egg number per egg pod was mild and gradual. In addition, we showed that insects from polluted sites laid fewer eggs than individuals from the reference site. This effect was more pronounced in grasshoppers from Olkusz. Apart from a lower number of eggs, the period of egg lying in these females was shorter than in insects from the other two sites (Tables 1 and 2, Fig. 1). The results may be surprising since, during the whole reproductive period, the females were fed with the same grass, without any additional pollutants. Thus, if the adaptation to polluted environments depends exclusively on a physiological shift in energy allocation, the majority of processes should return to the basic level shortly after removal of the stress factor. This means that the strategy of egg lying we observed for the three populations might be genetically coded and inherited. During the aging process the equilibrium between pro-oxidants and antioxidants (including antioxidative enzymes) becomes disturbed and shifts towards pro-oxidants. Free radical generation rate increases with age, causing the enhancement of reactive oxygen species (ROS) to attack such molecules as proteins, lipids, DNA. In such situations, an organism is not able to repair all its macromolecule damage. This accumulation of damage leads to aging and death (Canesi and Viarengo, 1997; Le Bourg, 2001; Sohal, 2002). Reactive oxygen species, including free radicals, appear spontaneously during natural metabolic processes, however various factors, like heavy metals, may contribute to the increase of their concentration in cells. We suggest that the dramatic fall in the number of eggs per pod with age, in the case of grasshoppers from Olkusz, is not caused by additional factors stimulating ROS generation. We did not consider, however, toxic effects on the females during their life in natural environment before the start of the experiment
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Table 3 Results of w2 test for hatching nymphs from the egg pods classified to the categories according to the number of eggs No. of eggs per pod
Factors Age group 2
1 2 3 4 5 6 7 8 9 10 11 12 13
Site 2
2
Zn treatment
w
p
w
p
w
p
w
p
w2
p
w2
p
222.90
o0.000001
14.3 11.0 46.6 30.9 62.8 35.1 48.2 55.9 49.6 47.7 25.2
0.014 0.525 o0.001 0.155 o0.001 0.513 0.238 0.049 0.644 0.037 0.193
112.49
o0.000001
o0.000001
0.659
0.820 0.007 0.407 o0.001 o0.001 o0.001 0.002 0.302 0.036 o0.001 0.175 0.172 0.329
130.52
0.8
0.9 17.8 9.3 51.9 79.7 53.7 44.4 27.1 41.6 55.5 19.9 5.0 2.2
9.6 16.3 6.7 17.2 35.9 22.9 41.8 43.8 44.9 31.9 11.4 5.0 6.7
0.022 0.012 0.667 0.142 0.002 0.196 0.004 0.008 0.016 0.129 0.325 0.172 0.155
(from hatching to catching last stage nymphs). Such influences are difficult to measure. We may assume, however, that xenobiotics that had entered an organism in its natural habitat, were biodegraded or bound, excreted or deposed in specific body and cell compartments. Thus, even if any amount of metal entered the body of grasshoppers from Olkusz, it was probably neutralized and inactivated and should not influence the amount of free radicals present in the body. Our results show that insects from polluted meadows (especially from Olkusz) realize their reproductive functions mainly when they are very young. From our analyses, we conclude that the effects of environmental pollution (but not xenobiotics themselves) and female aging may interact (Table 2). In the second stage of our studies, we compared selected developmental parameters in nymphs. We found the highest percentage of hatchlings per pod (K) in cohorts III and IV. Then the success of hatching gradually decreased (Tables 1 and 3; Fig. 2). This suggests that female fitness is best in the middle of their reproduction period. It is noteworthy that in zinc-polluted sand (10,000 mg g 1) there was practically no hatching from egg pods laid by females from Olkusz, while for females from Szopienice, percentages of hatching were higher than in the respective group from the reference site (Table 1; Zn10,000 group). It must be stressed here that the zinc concentration of 10,000 mg g 1 was at the same level as in the humus layer in Olkusz and Szopienice sites (Augustyniak and Migula, 2000; Stone et al., 2001; Łaszczyca et al., 2004; Augustyniak et al., 2005). It should be remembered though, that the bioavailability of the metal in the sand in our experiment and in the soil in the natural habitat may differ. However, the bioavailability assessment was not performed in this study. The negative effects of Cd, Pb and Hg on the hatching rate in various grasshopper species were studied by
2
Devkota and Schmidt (1999). These authors, however, did not consider the origin of the insects, hence neglecting possible effects of pre-exposure to environmental toxins. The mechanism of metal activity in the organism is well recognized and documented. Metals after entering the body easily bind to sulphydryl (–SH) groups of important proteins, including enzymes of various metabolic pathways. This may result in decrease in ATP synthesis and cause an energetic crisis. Moreover, the necessity to resynthesize the pool of inactivated proteins, necessary for proper functions of the organism, requires additional energy (Augustyniak and Migula, 2000). This might be the reason for lower numbers of hatched nymphs in zincexposed groups. However, the problem is more complex, since the intensity of this phenomenon differed between the populations. Especially in the case of the insects from Olkusz, to a less extent from Pilica, and the least—from Szopienice, the effects of females aging and toxicity of zinc seem to sum (see groups Zn-1000 and Zn-10,000; Table 1). The weakest effect found in the grasshoppers from Szopienice raises a question about mechanisms of adaptation to heavy metals in individuals of this population. The difference in body mass of hatchlings (M) was influenced mainly by female age; their habitat was less important. Also exposure to zinc, with one exception, appeared insignificant for this parameter (Tables 1 and 2, Fig. 3). Willott and Hassall (1998) measured hatchling body mass in relation to the temperature changes during the development period in various grasshopper species and stated that, within the 25–35 1C range weight of the hatchlings was similar in all experimental groups. The highest body weight was found in nymphs from Olkusz. Such results suggest that grasshoppers from Olkusz may compensate for a lower number of eggs per pod (E) by improved egg quality and increasing the amount of yolk. It is possible that grasshoppers from each population have to
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48
* **
Pilica *
5
duration of embryogenesis [day]
hatchling weight [mg]
6
*
4 3 2 1
20 16
* **
Pilica
* **
* **
12 8 4 0
0
Y
Y
M
M
O
O
cohorts
cohorts Szopienice duration of embryogenesis [day]
Szopienice
hatchling weight [mg]
6 5 4 3 2
20 16 12 8 4 0
1
Y
M
Y
M
O
Olkusz duration of embryogenesis [day]
cohorts Olkusz 6
hatchling weight [mg]
O
cohorts
0
5 4 3 2
20 16 12 8 4 0 Y
1
M cohorts control
0 Y
M Zn-1000
Zn-10000
O
cohorts control
Zn-1000
O
Zn-10000
Fig. 3. Hatchling weight (M) in grasshopper C. brunneus from Pilica (reference site), Szopienice and Olkusz (polluted sites) for egg laid by young (Y), middle-aged (M) and old female (O) additionally exposed to zinc during diapause (1000 or 10,000 mg Zn g 1). Stars indicate statistically significant differences between experimental groups within cohorts.
reach a minimal body mass before they start their lives outside the egg. In other cases, hatching is impossible. In our other works, we assessed the degree of DNA damage in brains of grasshoppers from Pilica and Olkusz using
Fig. 4. Duration of embryogenesis (T) in grasshopper C. brunneus from Pilica (reference site), Szopienice and Olkusz (polluted sites) for egg laid by young (Y), middle-age (M) and old female (O) additionally exposed to zinc during diapause (1000 or 10,000 mg Zn g 1). Stars indicate statistically significant differences between experimental groups within cohort.
a comet assay. Then we compared the results with metal brain concentration (and distribution) obtained by microPIXE mapping (Augustyniak et al., 2006a). Here, we exposed the egg pods to two zinc concentrations: 100 and 1000 mg Zn g 1. The increase in DNA damage as well as zinc concentration in the grasshopper brain were found in the group that diapaused in soil supplemented with the
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lower Zn concentration. In grasshoppers intoxicated with a higher concentration of zinc (1000 mg Zn g 1), the amount of DNA damage and Zn concentration in the brain was similar to the control values. It is worth stressing that Ca2+ concentration in the grasshopper brain was proportional to the Zn concentration in sand where the egg pods were kept. A response like this may be connected to the frequency of apoptosis and necrosis in insects exposed to additional toxic factor (in this case, zinc) (Augustyniak et al., 2006a). It is known that apoptosis, programmed cell death, is connected to an increase of DNA fragmentation, but also with an increase of Ca2+ concentration in cells (Panayiotidis et al., 1999; Zirpel and Parks, 2001; Woz´niak and Blasiak, 2003). Thus, an increase of Ca2+ concentration in the brain cells of grasshoppers exposed to Zn shows that apoptosis and/or necrosis may appear more often (Augustyniak et al., 2006a). Therefore, the death of embryos in groups intoxicated with zinc might have resulted from indirect Zn activity: through the disturbance of Ca2+ ion homeostasis. Both the age of the female at the time of egg laying and Zn concentration in the sand where diapause took place significantly influenced the duration of embryogenesis. The habitat factor seemed insignificant (Table 2). Egg pod exposure to 10,000 mg Zn g 1 sand resulted in a significant increase in duration of embryogenesis (T). Devkota and Schmidt (1999) showed a similar effect, caused by Cd, Hg and Pb, in grasshoppers reared continuously under laboratory conditions. Willott and Hassall (1998) stated that this parameter is also very sensitive to temperature change. Our results point out that another important factor affects embryogenesis. The shortening of embryogenesis in grasshoppers hatching from eggs laid by older females is equally surprising and difficult to explain. From our study, we conclude that the duration of embryonic development is exceptionally sensitive to the age of female at the moment of oviposition. The same factor appeared to be important also for the activity of selected detoxifying enzymes in offspring. In our other paper, we show that activity of glutathione reductase in I-stage nymphs hatched from eggs laid by old females from Pilica and Olkusz was significantly lower than in hatchlings developed from eggs laid by young females. In grasshoppers from Szopienice, we found an opposite tendency. Also glutathione concentration in offspring of old females from Pilica and Olkusz was lower than in insects hatched from eggs laid by young grasshoppers, while in insects from Szopienice the tripeptide concentration did not depend on female age (Augustyniak et al., 2006b). Therefore, differences connected to adaptations to metal-polluted environments are observed at various levels. 5. Conclusions In grasshoppers from the reference site (Pilica), the effects of female aging and additional metal stress applied during diapause sum. Pre-exposure may change the pattern. The grasshoppers from Szopienice have probably developed a form of defense against stress caused by heavy
49
metals: the application of higher zinc concentration during diapause exerted a weaker effect than in insects from the reference site. The mechanism was not found in grasshoppers from Olkusz, despite their similar pre-exposure to zinc in their habitat. This may indicate that the population exists at the limit of its energetic abilities. Acknowledgments This work was supported by the Grant (No. 2 P04G 006 27) of Polish National Committee for Scientific Research. References Augustyniak, M., Migula, P., 1996. Patterns of glutathione S-transferase activity as a biomarker of exposure to industrial pollution in the grasshopper Chorthippus brunneus (Thunberg). Studia Societatis Scientiarum Torunensis 4, 9–15. Augustyniak, M., Migula, P., 2000. Body burden with metals and detoxifying abilities of the grasshopper—Chorthippus brunneus (Thunberg) from industrially polluted areas. In: Merkert, B., Friese, K. (Eds.), Trace Elements—Their Distribution and Effects in the Environment. Elsevier Science, Amsterdam, pp. 423–454. Augustyniak, M., Babczynska, A., Migula, P., Wilczek, G., Łaszcyca, P., Kafel, A., Augustyniak, M., 2005. Joint effects of dimethoate and heavy metals on metabolic responses in a grasshopper (Chorthippus brunneus) from a heavy metals pollution gradient. Comparative Biochemistry and Physiology C 141, 412–419. Augustyniak, M., Juchimiuk, J., Przyby"owicz, W.J., Mesjasz-Przyby"owicz, J., Babczyn´ska, A., Migula, P., 2006a. Zinc-induced DNA damage and the distribution of metals in the brain of grasshoppers by the comet assay and micro-PIXE. Comparative Biochemistry and Physiology C 144, 242–251. Augustyniak, M., Babczyn´ska, A., Szulin´ska, E., Witas, I., 2006b. Effects of female aging and metal pollution on glutathione-dependent enzymes in Chorthippus brunneus nymphs. Toxicology Letters 164S, 153–154. Canesi, L., Viarengo, A., 1997. Age-related differences in glutathione metabolism in mussel tissues (Mytilus edulis L.). Comparative Biochemistry and Physiology 116B, 217–221. Cherrill, A.J., Begon, M., 1989. Timing of life cycles in a seasonal environment: the temperature-dependence of embryogenesis and diapause in a grasshopper (Chorthippus brunneus Thunberg). Oecologia 78, 237–241. Devkota, B., Schmidt, G.H., 1999. Effects of heavy metals (Hg2+, Cd2+, Pb2+) during the embryonic development of Acridid grasshoppers (Insecta, Caelifera). Archives of Environmental Toxicology 36, 405–414. Fisher, Z., 1981. Energy budget of grasshopper Chorthippus sp. in industrial environment. Polish Ecological Studies 7, 5–14. Jalil, A., Selles, F., Clarke, M.J., 1994. Growth and cadmium accumulation in two durum wheat cultivars. Communications in Soil Science and Plant Analysis 25, 2597–2611. Kim, Y.-H., Kim, E.Y., Gwag, B.J., Sohn, S., Koh, J.-Y., 1999. Zincinduced cortical neuronal death with features of apoptosis and necrosis: mediation by free radicals. Neuroscience 89, 175–182. Kucharski, R., 1993. Zanieczyszczenie gleb i ros´ lin jadalnych i paszowych na terenie Wojewo´dztwa Katowickiego. In: Jankowski, A.T. (Ed.), Aktualne Problemy Ekologiczne Regionu Go´rnos´ la˛ skiego. WNoZ USl, Katowice, pp. 62–68. Łaszczyca, P., Augustyniak, M., Babczyn´ska, A., Bednarska, K., Kafel, A., Migula, P., Wilczek, G., Witas, I., 2004. Profiles of detoxifying enzymatic activity in earthworms from zinc, lead and cadmium polluted areas near Olkusz (Poland). Environment International 30, 901–910.
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Le Bourg, E., 2001. Oxidative stress, aging and longevity in Drosophila melanogaster. FEBS Letters 498, 183–186. Maret, W., 2005. Zinc coordination environments in proteins determine zinc functions. Journal of Trace Elements in Medicine and Biology 19, 7–12. Monk, K.A., 1985. Effect of habitat on the life history strategies of some British grasshoppers. Journal of Animal Ecology 54, 163–177. Moriarty, F., 1969. The laboratory breeding and embryonic development of Chorthippus brunneus Thunberg (Orthoptera: Acrididae). Proceedings of the Royal Society of London (A) 44, 25–34. Panayiotidis, M., Tsolas, O., Galaris, D., 1999. Glucose oxidase-produced H2O2 induces Ca2+-dependent DNA damage in human peripherial blood lymphocytes. Free Radical Biology and Medicine 26, 548–556. Schmidt, G.H., Ibrahim, N.M.M., Abdallah, D.M., 1992. Long-term effects of heavy metals in food on developmental stages of Aiolopus thalassinus (Saltatoria: Acrididae). Archives of Environmental Contamination and Toxicology 23, 375–382. Sohal, R.S., 2002. Role of oxidative stress and protein oxidation in the aging process. Free Radical Biology and Medicine 33, 37–44.
Sohal, R.S., Mockett, R.J., Orr, W.C., 2002. Mechanisms of aging: an appraisal of the oxidative stress hypothesis. Free Radical Biology and Medicine 33, 575–586. Stone, D., Jepson, P., Kramarz, P., Laskowski, R., 2001. Time to death response in carabid beetles exposed to multiple stressors along a gradient of heavy metal pollution. Environmental Pollution 113, 239–244. Takeda, A., 2000. Movement of zinc and its functional significance in the brain. Brain Research Reviews 34, 137–148. Wilczek, G., 2005. Apoptosis and biochemical biomarkers of stress in spiders from industrially polluted areas exposed to high temperature and dimethoate. Comparative Biochemistry and Physiology 141, 194–206. Willott, S.J., Hassall, M., 1998. Life-history responses of British grasshoppers (Orthoptera: Acrididae) to temperature change. Functional Ecology 12, 232–241. Woz´niak, K., Blasiak, J., 2003. In vitro genotoxicity of lead acetate: induction of single and double DNA strand breaks and DNA-protein cross-links. Mutation Research 535, 127–139. Zirpel, L., Parks, T.N., 2001. Zinc inhibition of group ImGluR-mediated calcium homeostasis in auditory neurons. Journal of the Association for Research in Otolaryngology 2, 180–187.
Journal of Insect Physiology 54 (2008) 41–50 www.elsevier.com/locate/jinsphys
Effects of zinc and female aging on nymphal life history in a grasshopper from polluted sites Maria Augustyniaka,, Agnieszka Babczyn´skaa, Micha" Koz"owskia, Tomasz Sawczyna, Micha" Augustyniakb a
Department of Animal Physiology and Ecotoxicology, University of Silesia, Bankowa 9, PL 40-007 Katowice, Poland b Faculty of Earth Sciences, University of Silesia, Bedzinska 60, PL 41-200 Sosnowiec, Poland Received 20 March 2007; received in revised form 3 August 2007; accepted 6 August 2007
Abstract Insect reproduction is influenced by various factors, including food quality and quantity, temperature, population density and female age. Contamination, including heavy metals, may disturb reproductive processes. The aim of this work was to assess interactions between effects of aging in female Chorthippus brunneus and environmental pollution on their reproduction measured in number of laid eggs. We also compared basic developmental parameters (number of hatchlings, body mass, embryonic developmental rate) in grasshopper nymphs additionally exposed to zinc during diapause. Aging grasshoppers from heavily polluted areas (Olkusz and Szopienice) lay significantly fewer eggs than insects from the reference site (Pilica). Zinc application caused the decrease in hatching success and duration of embryogenesis in insects from each site. This suggests a cumulative effect of female age, pollutants and additional stressing factors. The intensity of this process differed between populations. In insects from the reference site, it was shown in a moderate degree. In insects from Szopienice, an additional stressor exerted a weaker effect than in insects from Pilica. In grasshoppers from Olkusz, we found the strongest decrease of hatching percentage and increase in duration of embryogenesis after zinc intoxication. This may indicate that the population from Olkusz exists at the limit of its energetic abilities. r 2007 Elsevier Ltd. All rights reserved. Keywords: Grasshoppers; Aging; Development; Diapause; Zinc
1. Introduction It is generally accepted that an energetic potential, defined as the total amount of energy accessible for an individual through its whole life, for each species is limited. Moreover, we expect that insects exposed to additional environmental stressing factors have to bear much higher costs of detoxification (Stone et al., 2001; Wilczek, 2005). The energetic expenditures caused by settling new, heavily polluted habitats, may involve the production of additional enzymes for detoxification, inactivation, storage or excretion of toxins. That is why many species inhabiting polluted regions start mating and reproducing later, and hence their brood is less numerous compared to those from relatively unpolluted areas (Fisher, 1981; Schmidt et al., Corresponding author. Tel.: +48 32 2587737; fax: +48 32 2587737.
E-mail address: [email protected] (M. Augustyniak). 0022-1910/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2007.08.002
1992; Devkota and Schmidt, 1999). An energetic deficit may be a serious problem especially for old insects, still able to reproduce, living in heavily polluted environments. We know that aging female insects lay fewer eggs. The decrease in the number of egg pods, as well as number of eggs in each egg pod, is caused also by low temperature, food deprivation or excessive density of a population (Cherrill and Begon, 1989; Willott and Hassall, 1998). The fecundity of insects is also negatively affected by environmental toxins, including heavy metals (Devkota and Schmidt, 1999). However, the issue of aging insects in polluted sites remains to be elucidated. The problem of developmental abilities of old females’ offspring, especially this originating from polluted habitats, still needs investigation. Thus, the following hypotheses were proposed: (i) The development of insects of a given species follows the same pattern, irrespective of mother’s age and
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M. Augustyniak et al. / Journal of Insect Physiology 54 (2008) 41–50
exposure to environmental toxins, because the potential the offspring inherits is unchangeable and does not depend on the factors mentioned above. (ii) Long lasting (several hundreds years) contact with toxin, as it is at post-industrial areas, might have led to the selection of individuals that are able to cope more effectively with neutralization than insects not subjected to such pre-adaptation. This may be also reflected in the offspring’ development. (iii) The development of insects living in heavily polluted areas is abnormal because of cumulative effects of female aging and environmental pollution. The settlement of polluted areas will probably cause enhanced aging of the insects and severely decrease their fecundity and quality of eggs as aging progresses. The question of aging has been widely investigated. According to current theory, aging results from an increase of pro-oxidative factors and a gradual weakening of the antioxidant systems (Le Bourg, 2001; Sohal, 2002; Sohal et al., 2002). Previous reports stress that some heavy metals, widely present in the post-industrial environment, may stimulate free radicals production. Heavy metals may enhance oxidative stress and force the organism to synthesize additional peptides and enzymes involved in free radical scavenging, accelerating the aging processes (Augustyniak and Migula, 1996, 2000). This indicates that aging of an organism in a heavy metal polluted environment will be faster than in an environment free of additional stressing factors. On the other hand, we cannot exclude the possibility that some populations chronically exposed to metals, have developed energy-efficient mechanisms to perform at least some of the vital functions. Analysis of the connections between the kind of metal, its dose, the time of exposure, and the stage of diapause during ontogenesis of a specific insect species cannot be neglected. During this sensitive stage of life, insects are mostly deprived of avoidance strategies and can be exposed to toxins in soil and soil solution. Among the insects that inhabit dumps of industrial origin there are various grasshopper species which lay egg pods directly in the soil, diapause in this stage and undergo embryogenesis in such conditions (Devkota and Schmidt, 1999). Zinc is an important biogenic metal (Kim et al., 1999; Takeda, 2000; Zirpel and Parks, 2001; Maret, 2005). It plays structural, catalytic and regulatory functions in an organism. It is necessary for signal transduction, DNA replication, transcription and protein synthesis. Zinc is crucial for proper actions of several hundreds of enzymes. It forms the so-called ‘zinc-finger’ protein domains and controls the architecture of protein complexes. Zinc is also essential for regular functions of the nervous system. Since zinc plays various roles in metabolism, we may expect that any disturbance of its homeostasis may cause improper growth and morphogenesis (Takeda, 2000; Maret, 2005). However, as with other metals, zinc can be toxic when its concentration exceeds its physiological limits. We know
that zinc toxicity of may lead to unexpected neuronal death with the features of either apoptosis or necrosis (Kim et al., 1999; Zirpel and Parks, 2001). The aim of this work was to assess a possible influence of either age of female grasshoppers or the degree of environmental pollution of their habitat on fecundity. Among grasshopper populations chosen for this study, two originated from areas located around metal smelters. Both sites are strongly polluted with heavy metals, especially with zinc. Thus, in this work, we compare basic developmental parameters (quantity, body mass and embryonic development rate) of grasshopper nymphs that developed from the eggs laid by aging females, and then exposed to additional zinc intoxication during diapause. The zinc concentrations we used are similar to the concentration of zinc in humus layer at natural habitat of the insects. 2. Material and methods 2.1. Insects Chorthippus brunneus (Thunberg) is a representative of the Acrididae family. This hemimetabolic insect inhabits mainly sunny, open grassy areas such as meadows, farms, fields and pastures. C. brunneus overwinters in the egg stage. Like other Acrididae, C. brunneus is characterized by high fecundity. In optimal conditions, a female may lay an egg pod containing 10 or more eggs a day. The number of egg pods decreases with female’s age of, when the temperature is too low, during food deprivation periods or when the population gets overcrowded (Moriarty, 1969; Monk, 1985; Cherrill and Begon, 1989; Willott and Hassall, 1998). 2.1.1. Study sites and insect collection The insects were collected at three sites. Two sites were in the close vicinity of metal smelters (Olkusz and Szopienice), and the third was far from industrial plants (Pilica). In Olkusz, the main sources of pollution are the miningmetallurgic smelter ‘Boleslaw’, mining plants ‘Pomorzany’, ‘Olkusz’ and ‘Boleslaw’, all situated close to each other. Within a distance of 1 km from the Szopienice site, there is a mining-metallurgic smelter ‘Szopienice’ which is one of biggest emitters of particles containing compounds of zinc, lead, cadmium and other heavy metals. The concentration of zinc in soil at the Szopienice site reaches up to 2.1 mg g 1; therefore exceeding, by 10 times, the permissible soil concentration (Kucharski, 1993). The reference area was localized far from industrial pollutants, near Pilica town. For each site, the pollution index (PI) was calculated, which, for Szopienice, Olkusz and Pilica was equal to 22.7, 12.1 and 0.53, respectively. A detailed description of pollution and types of vegetation at each site are published in Augustyniak and Migula (2000), Stone et al. (2001), Łaszczyca et al. (2004) and Augustyniak et al. (2005). Male and female insects were collected in July as soon as the majority of insects reached sexual maturity.
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The insects caught were used to initiate the laboratory culture. 2.1.2. Experimental set-up In the laboratory, insects were weighed. Mean body mass for females from Pilica, Olkusz and Szopienice was 0.24, 0.22 and 0.20 g, respectively, while male grasshoppers from Pilica, Olkusz and Szopienice weighed on average 0.085, 0.08 and 0.07 g, respectively. The initial maternal culture of grasshoppers was reared at controlled laboratory conditions (temperature 30 1C71.5; 46% RH; photoperiod 12:12 h). Cubic cages (30 cm 30 cm 30 cm), where the insects were kept (each cage contained 25 pairs) were equipped with little watering pots, flasks with grass and a container filled with wet sand, where females could lay egg pods. The watering pots and flasks with grass were replaced daily. As soon as the females started laying eggs, the egg pods were collected once every 2 days and placed in sterilized, wet sand, washed previously with Hogland’s solution (Jalil et al., 1994). Once a week, all egg pods were gathered and classified into the same ‘age group’. This way 7 ‘age groups’ were obtained (named I–VII): the first one originated from the youngest and the last one from the oldest females. The experiment was terminated when the old females stopped laying egg pods. Egg pods were kept at 25 1C for 2 weeks to assure proper embryogenesis (Moriarty, 1969; Monk, 1985; Cherrill and Begon, 1989). After this period, all pods laid by the females from each site (and from each ‘age groups’) were again randomly classified into one of two experimental groups named Zn-1000, Zn-10,000 and one control group. The experimental groups varied in the concentration of zinc in the sand where the egg pods were transferred to (1000 and 10,000 mg Zn g 1 dry weight of sand, respectively). The egg pods from the control groups were kept in zinc-unpolluted sand. Each group consisted of 10 egg pods. The egg pods were then placed at 4 1C for 12 weeks to induce diapause (Moriarty, 1969; Monk, 1985). Summarizing, the egg pods were divided into groups according to the site of parent’s collection (Pilica, Szopienice and Olkusz). Within the groups, ‘age groups’ were selected based on the age of female at the moment of egg laying (I–VII). Additionally, each ‘age group’ was divided into experimental groups differing in the concentration of zinc in sand in which the egg pods were kept during their diapause. After diapause, the egg pods were placed into incubators (temperature 30 1C71.5; photoperiod 12:12). Each egg pod was placed in a separate container. As soon as the hatching had begun the nymphs were counted, and at the end of this process the pods were dissected and undeveloped eggs (as well as dead nymphs) were counted. Since each egg pod was placed in a separate container, it was possible to count the total number of eggs in each egg pod, as well as the number of nymphs hatched from each egg pod. Based on these data, the number of eggs per pod (E) and the
43
percentage hatchlings per pod (K) were calculated. Additionally, the mass of hatchlings (M) and the duration of embryonic development (T), defined as the period between the end of diapause and the day of hatching, were assessed. 2.1.3. Statistical procedures The results for parameters M and T were reported as mean values7CI (confidence interval). Hatchlings per pod (K) were expressed in %. Since the egg pods were placed separately in containers, we were able to assess the hatching efficiency (%) for each egg pod. For these data, statistical procedures were performed. Normality was checked using the Kolmogorov–Smirnov test. Homogeneity of variances was tested using the Levene test. Duration of embryonic development (T) and the mass of hatchlings (M) did not show the normal distribution, so the data were log-transformed and then analyzed within the experimental groups using the Tuckey’s test (ANOVA/MANOVA) for unequal sample size (Table 1). For each parameter, the analyses of main effects (site, treatment, ‘age groups’) were done. When necessary, we used a simplified classification of independent variables for these analyses. This was the case when there was a lack of data (especially in Zn-exposed groups) or when the number of values differed between groups. That is why for the variable ‘age groups’ the groups were called: cohorts: Y—young (the offspring of young females), M—middleage (the offspring of middle-aged females) and O—old (the offspring of old females), created as the result of joining I/II, III/IV and V/VI groups, respectively (Table 2). 3. Results 3.1. Number of eggs per pod The highest mean number of eggs per pod (E) for females from each study site was found at the beginning of the reproductive period. The number decreased along with the aging of female grasshoppers. However, the rate of change differed in insects from various sites (Tables 1 and 2, Fig. 1). The egg pods laid by insects from the reference site (Pilica) during the first 3 weeks contained nearly the same number of eggs (about seven per egg pod). Beginning with the fourth week, an average number of eggs per pod decreased to about five. This number of eggs was laid by insects from Pilica until the end of the reproductive period. Insects from Szopienice during the first week produced pods containing the highest number of eggs (on average 8.65), but the decrease of egg number per pod (to five eggs) started no later than in the second week and lasted until the V week. During the next week, the number decreased even more, to 2.5 eggs per pod in the VII week (Table 1; Fig. 1). Grasshoppers from Olkusz during first 2 weeks laid pods containing up to seven eggs per pod. Beginning on the III week, a dramatic fall in the number of eggs per pod was observed. It lasted until the VI week (on average 1.87 eggs
44
Table 1 Developmental parameters in grasshopper C. brunneus from control and zinc-exposed egg pods laid by females collected at variously polluted sites: Pilica, Szopienice and Olkusz Age groups
E
Control
Zn-1000
Zn-10,000
K (%)
M (mg)
T (days)
K (%)
M (mg)
T (days)
K (%)
M (mg)
T (days)
61a 60a 66a 38b 49ab 13b X
4.7ab (4.5, 4.9) 4.4b (3.9, 4.9) 5.0a (4.7, 5.4) 6.0a (4.8, 7.5) 4.1b (3.9, 4.4) 4.4b (4.0, 4.8) X
12.0b (12.0, 12.0) 16.7a (15.9, 17.7) 12.1b (11.4, 12.7) 12.2b (9.9, 15.1) 10.0c (10.0, 10.0) 14.5ab (11.7, 17.9) X
68ab 59b 91a 86a 52b X X
4.4ab (4.1, 4.6) 4.1b (3.8, 4.5) 4.8a (4.6, 5.0) 4.7ab (4.3, 5.0) 4.2ab (4.0, 4.5) X X
14.3a (13.3, 15.3) 13.7ab (12.3, 15.3) 12.1bc (11.7, 12.5) 12.7ab (11.0, 14.5) 10.1c (9.9, 10.3) X X
20a 20a 22a 1b X X X
4.2a 4.7a 4.3a 4.3a X X X
(3.9, (4.3, (3.9, (4.1,
4.6) 5.0) 4.8) 4.5)
16.9a 21.3a 18.0a 17.0a X X X
(14.2, (18.8, (16.9, (17.0,
Szopienice I II III IV V VI VII
8.65a 5.60b 5.40b 3.95c 4.47bc 3.50c 2.50c
46b 70a 72a 75a 65a X X
4.3a 4.0a 4.7a 4.6a 4.5a X X
(4.3, (3.5, (4.3, (4.4, (1.1,
4.6) 4.6) 5.2) 4.9) 4.9)
14.6a (13.0, 16.3) 13.8a (13.2, 14.4) 12.8ab (12.1, 13.5) 11.0bc (10.8, 11.2) 10.6c (9.9, 11.3) X X
36b 86a 85a 71a 12b 16b X
4.8a 4.3a 4.8a 4.6a 4.5a 5.2a X
15.1a (13.8, 16.6) 12.5b (11.8, 13.1) 13.3b (12.0, 14.8) 11.5b (10.9, 12.1) 12.0b (12.0, 12.0) 12.0b (12.0, 12.0) X
25a 25a 22a 9a 27a X X
4.8a 4.4a 3.8a 4.3a 4.0a X X
(4.3, (4.2, (3.1, (4.2, (3.7,
5.4) 4.6) 4.8) 4.4) 4.2)
17.4a 14.4a 15.2a 14.5a 14.5a X X
(15.4, 19.7) (13.6, 15.3) (13.4, 17.3) (8.5, 24.7) (11.9, 17.7)
Olkusz I II III IV V VI VII
6.95a 6.57ab 3.82bc 2.42c 2.07c 1.87c X
30b 41b 90a 84a 16b X X
4.7a 5.1a 6.1a 5.1a 5.7a X X
(4.3, (5.0, (5.6, (4.1, (5.1,
5.2) 5.2) 6.7) 6.5) 5.7)
12.0b (12.0, 12.0) 15.0a (14.9, 15.1) 12.3b (11.7, 12.9) 10.2c (9.9, 10.4) 12.0b (11.8, 12.1) X X
50a 42a 40a X X X X
5.5a (5.1, 6.1) 6.2a (4.4, 8.8) 5.3a (3.1, 9.1) X X X X
15.2a (13.5, 17.0) 10.7b (10.3, 11.0) 11.0ab (10.2, 11.9) X X X X
X X X 16 X X X
X X X 4.2 (4.2, 4.2) X X X
(4.6, (4.0, (4.6, (3.8, (4.4, (5.0,
5.0) 4.7) 5.0) 5.7) 4.6) 5.4)
20.3) 24.1) 19.2) 17.0)
X X X 15.0 (15.0, 15.0) X X X
Note: E, mean number of eggs per pod; K, hatchlings per pod (%); M, hatchling weight (mean; confidence interval—CI); T, duration of embryogenesis (mean; CI). M and T were log-transformed prior to analysis. a, b, c Different letters indicate significant differences between ‘age groups’ within experimental group.
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7.07a 7.85a 6.97a 4.45c 6.15b 5.02bc 4.82c
M. Augustyniak et al. / Journal of Insect Physiology 54 (2008) 41–50
Pilica I II III IV V VI VII
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per pod). In the VII week, grasshoppers from this site ceased eggs laying. Main effects analysis showed that the number of eggs per pod was strongly influenced by the age of a female (variable: ‘age group’) and the habitat where the females were collected (variable: site). Interaction between these two variables was also significant (Table 2, Fig. 1).
3.2. Hatchlings per pod The highest hatching percentage from egg pods (K) from control groups was found for egg pods laid in III (Pilica, Olkusz) or IV (Szopienice) week of the experiment. This means that the egg pods were laid by middle-aged females (Table 1). The application of lower Zn concentration (Zn1000 group) during diapause did not cause any significant changes in grasshoppers from Pilica. In grasshoppers from polluted sites K parameter was decreased significantly only in cases of egg pods laid by middle-aged females from Olkusz (Fig. 2). Intoxication with a higher Zn concentraTable 2 Analysis of variance (ANOVA/MANOVA) for number of eggs (E), hatchlings weight (M), and duration of embryogenesis (T), in C. brunneus from Olkusz, Szopienice and Pilica (with cohorts and site as a categorical factors) Source of variation d.f. E
M
F Cohorts (1) Site (2) 12
2 2 4
p
F
T p
F
p
7.710 o0.001 8.756 o0.001 10.851 o0.000001 4.224 o0.01 6.288 o0.01 0.613 0.545 3.195 o0.01 1.895 0.1210 0.488 0.745
9
3.3. Hatchling weight The analysis of main effects revealed that the weight (M) of hatching nymphs strongly depended on the original female age; original habitat of the insects seemed less significant (Tables 1 and 2). The application of higher Zn concentrations caused a decrease in the weight of newly hatched grasshoppers only in the case of the offspring of middle-aged females from Pilica. In the remaining cases,
a a
b mean number of eggs per pod
tion (Zn-10,000 group) caused a significant decrease in the percentage of nymphs hatched from egg pods laid by middle-aged and old females from all study sites; the strongest effect was observed for insects from Olkusz (Fig. 2). In insects from the reference site (Pilica), we observed a strong decrease in K value in Zn-10,000 group, in which K reached 20% during the first three weeks. No individuals hatched from egg pods laid by females from Pilica during V–VII weeks, which were kept in sand contaminated with a higher Zn concentration (Tables 1 and 3). A similar effect was found in grasshoppers from Szopienice, where hatching percentage at Zn-10,000 decreased to 25%. However, in this case, hatching ceased no earlier than in the VI ‘age group’. The strongest response to the additional stressing factor was observed in grasshoppers from Olkusz. In the Zn-1000 group, the last hatching took place in egg pods classified in the III ‘age group’. Diapause in sand contaminated with the higher Zn concentration caused almost a total lack of hatching, apart from IV ‘age group’ (Tables 1 and 3). Thus, at least in case of grasshoppers from Olkusz, we observed an adverse effect of both female age and an additional stress factor.
a
8
ab
7 6
45
ab
5
a b
a a
b
b
a
b
4
b
3
ab
a c
2
b
b
1 0
I
II
III
Pilica
IV cohorts Szopienice
V
VI
VII
Olkusz
Fig. 1. Mean number of eggs per pod (E) in grasshopper C. brunneus from Pilica (reference site), Szopienice and Olkusz (polluted sites) collected during whole reproductive period. The same letters indicate homogenous groups within site (separately for each ‘age group’).
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46
3.4. Duration of embryogenesis
Pilica
hatchlings per pod [%]
100 **
80 * 60
*
40 *
* **
20 0 Y
M
O
cohorts
Duration of embryogenesis (T) for grasshoppers developing from eggs laid during subsequent weeks, unlike hatchling weight, strongly depends on female age at the time of egg laying. The original habitat appeared insignificant (Table 2). We found a regular pattern in the duration of embryogenesis which was shorter in grasshoppers developing from egg pods laid by older females than in insects from eggs laid by young females. In the contrary, longer embryogenesis was observed in Zn-intoxicated egg pods, especially after exposure to a higher zinc concentration, in all ‘age groups’ from all study sites in comparison with appropriate control groups. The effect was strongest in grasshoppers from Pilica (Table 1, Fig. 4).
Szopienice
4. Discussion
hatchlings per pod [%]
100 *
80
** 60 40 * **
20 0 Y
M
O
cohorts Olkusz 100
*
hatchlings per pod [%]
** 80 60 40 *
20
**
0 Y
M control
Zn-1000
O Zn-10000
Fig. 2. Hatchlings per pod (K) in grasshopper C. brunneus from Pilica (reference site), Szopienice and Olkusz (polluted sites) for egg pods laid by young (Y), middle-aged (M) and old female (O) additionally exposed to zinc during diapause (1000 or 10,000 mg Zn g 1). Stars indicate statistically significant differences between experimental groups within cohorts.
Zn did not cause a significant change in the body mass of the nymphs (Fig. 3). Statistical analysis within ‘age groups’ showed that significant differences refer only to grasshoppers from Pilica (control and Zn-1000 groups; Table 1).
The number of eggs per pod (E) laid by aging females decreased gradually in insects from polluted sites, in accordance with observations of other authors (Moriarty, 1969; Monk, 1985; Cherrill and Begon, 1989). However, in the case of insects from Pilica (reference site) the agedependent decrease in the egg number per egg pod was mild and gradual. In addition, we showed that insects from polluted sites laid fewer eggs than individuals from the reference site. This effect was more pronounced in grasshoppers from Olkusz. Apart from a lower number of eggs, the period of egg lying in these females was shorter than in insects from the other two sites (Tables 1 and 2, Fig. 1). The results may be surprising since, during the whole reproductive period, the females were fed with the same grass, without any additional pollutants. Thus, if the adaptation to polluted environments depends exclusively on a physiological shift in energy allocation, the majority of processes should return to the basic level shortly after removal of the stress factor. This means that the strategy of egg lying we observed for the three populations might be genetically coded and inherited. During the aging process the equilibrium between pro-oxidants and antioxidants (including antioxidative enzymes) becomes disturbed and shifts towards pro-oxidants. Free radical generation rate increases with age, causing the enhancement of reactive oxygen species (ROS) to attack such molecules as proteins, lipids, DNA. In such situations, an organism is not able to repair all its macromolecule damage. This accumulation of damage leads to aging and death (Canesi and Viarengo, 1997; Le Bourg, 2001; Sohal, 2002). Reactive oxygen species, including free radicals, appear spontaneously during natural metabolic processes, however various factors, like heavy metals, may contribute to the increase of their concentration in cells. We suggest that the dramatic fall in the number of eggs per pod with age, in the case of grasshoppers from Olkusz, is not caused by additional factors stimulating ROS generation. We did not consider, however, toxic effects on the females during their life in natural environment before the start of the experiment
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Table 3 Results of w2 test for hatching nymphs from the egg pods classified to the categories according to the number of eggs No. of eggs per pod
Factors Age group 2
1 2 3 4 5 6 7 8 9 10 11 12 13
Site 2
2
Zn treatment
w
p
w
p
w
p
w
p
w2
p
w2
p
222.90
o0.000001
14.3 11.0 46.6 30.9 62.8 35.1 48.2 55.9 49.6 47.7 25.2
0.014 0.525 o0.001 0.155 o0.001 0.513 0.238 0.049 0.644 0.037 0.193
112.49
o0.000001
o0.000001
0.659
0.820 0.007 0.407 o0.001 o0.001 o0.001 0.002 0.302 0.036 o0.001 0.175 0.172 0.329
130.52
0.8
0.9 17.8 9.3 51.9 79.7 53.7 44.4 27.1 41.6 55.5 19.9 5.0 2.2
9.6 16.3 6.7 17.2 35.9 22.9 41.8 43.8 44.9 31.9 11.4 5.0 6.7
0.022 0.012 0.667 0.142 0.002 0.196 0.004 0.008 0.016 0.129 0.325 0.172 0.155
(from hatching to catching last stage nymphs). Such influences are difficult to measure. We may assume, however, that xenobiotics that had entered an organism in its natural habitat, were biodegraded or bound, excreted or deposed in specific body and cell compartments. Thus, even if any amount of metal entered the body of grasshoppers from Olkusz, it was probably neutralized and inactivated and should not influence the amount of free radicals present in the body. Our results show that insects from polluted meadows (especially from Olkusz) realize their reproductive functions mainly when they are very young. From our analyses, we conclude that the effects of environmental pollution (but not xenobiotics themselves) and female aging may interact (Table 2). In the second stage of our studies, we compared selected developmental parameters in nymphs. We found the highest percentage of hatchlings per pod (K) in cohorts III and IV. Then the success of hatching gradually decreased (Tables 1 and 3; Fig. 2). This suggests that female fitness is best in the middle of their reproduction period. It is noteworthy that in zinc-polluted sand (10,000 mg g 1) there was practically no hatching from egg pods laid by females from Olkusz, while for females from Szopienice, percentages of hatching were higher than in the respective group from the reference site (Table 1; Zn10,000 group). It must be stressed here that the zinc concentration of 10,000 mg g 1 was at the same level as in the humus layer in Olkusz and Szopienice sites (Augustyniak and Migula, 2000; Stone et al., 2001; Łaszczyca et al., 2004; Augustyniak et al., 2005). It should be remembered though, that the bioavailability of the metal in the sand in our experiment and in the soil in the natural habitat may differ. However, the bioavailability assessment was not performed in this study. The negative effects of Cd, Pb and Hg on the hatching rate in various grasshopper species were studied by
2
Devkota and Schmidt (1999). These authors, however, did not consider the origin of the insects, hence neglecting possible effects of pre-exposure to environmental toxins. The mechanism of metal activity in the organism is well recognized and documented. Metals after entering the body easily bind to sulphydryl (–SH) groups of important proteins, including enzymes of various metabolic pathways. This may result in decrease in ATP synthesis and cause an energetic crisis. Moreover, the necessity to resynthesize the pool of inactivated proteins, necessary for proper functions of the organism, requires additional energy (Augustyniak and Migula, 2000). This might be the reason for lower numbers of hatched nymphs in zincexposed groups. However, the problem is more complex, since the intensity of this phenomenon differed between the populations. Especially in the case of the insects from Olkusz, to a less extent from Pilica, and the least—from Szopienice, the effects of females aging and toxicity of zinc seem to sum (see groups Zn-1000 and Zn-10,000; Table 1). The weakest effect found in the grasshoppers from Szopienice raises a question about mechanisms of adaptation to heavy metals in individuals of this population. The difference in body mass of hatchlings (M) was influenced mainly by female age; their habitat was less important. Also exposure to zinc, with one exception, appeared insignificant for this parameter (Tables 1 and 2, Fig. 3). Willott and Hassall (1998) measured hatchling body mass in relation to the temperature changes during the development period in various grasshopper species and stated that, within the 25–35 1C range weight of the hatchlings was similar in all experimental groups. The highest body weight was found in nymphs from Olkusz. Such results suggest that grasshoppers from Olkusz may compensate for a lower number of eggs per pod (E) by improved egg quality and increasing the amount of yolk. It is possible that grasshoppers from each population have to
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48
* **
Pilica *
5
duration of embryogenesis [day]
hatchling weight [mg]
6
*
4 3 2 1
20 16
* **
Pilica
* **
* **
12 8 4 0
0
Y
Y
M
M
O
O
cohorts
cohorts Szopienice duration of embryogenesis [day]
Szopienice
hatchling weight [mg]
6 5 4 3 2
20 16 12 8 4 0
1
Y
M
Y
M
O
Olkusz duration of embryogenesis [day]
cohorts Olkusz 6
hatchling weight [mg]
O
cohorts
0
5 4 3 2
20 16 12 8 4 0 Y
1
M cohorts control
0 Y
M Zn-1000
Zn-10000
O
cohorts control
Zn-1000
O
Zn-10000
Fig. 3. Hatchling weight (M) in grasshopper C. brunneus from Pilica (reference site), Szopienice and Olkusz (polluted sites) for egg laid by young (Y), middle-aged (M) and old female (O) additionally exposed to zinc during diapause (1000 or 10,000 mg Zn g 1). Stars indicate statistically significant differences between experimental groups within cohorts.
reach a minimal body mass before they start their lives outside the egg. In other cases, hatching is impossible. In our other works, we assessed the degree of DNA damage in brains of grasshoppers from Pilica and Olkusz using
Fig. 4. Duration of embryogenesis (T) in grasshopper C. brunneus from Pilica (reference site), Szopienice and Olkusz (polluted sites) for egg laid by young (Y), middle-age (M) and old female (O) additionally exposed to zinc during diapause (1000 or 10,000 mg Zn g 1). Stars indicate statistically significant differences between experimental groups within cohort.
a comet assay. Then we compared the results with metal brain concentration (and distribution) obtained by microPIXE mapping (Augustyniak et al., 2006a). Here, we exposed the egg pods to two zinc concentrations: 100 and 1000 mg Zn g 1. The increase in DNA damage as well as zinc concentration in the grasshopper brain were found in the group that diapaused in soil supplemented with the
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lower Zn concentration. In grasshoppers intoxicated with a higher concentration of zinc (1000 mg Zn g 1), the amount of DNA damage and Zn concentration in the brain was similar to the control values. It is worth stressing that Ca2+ concentration in the grasshopper brain was proportional to the Zn concentration in sand where the egg pods were kept. A response like this may be connected to the frequency of apoptosis and necrosis in insects exposed to additional toxic factor (in this case, zinc) (Augustyniak et al., 2006a). It is known that apoptosis, programmed cell death, is connected to an increase of DNA fragmentation, but also with an increase of Ca2+ concentration in cells (Panayiotidis et al., 1999; Zirpel and Parks, 2001; Woz´niak and Blasiak, 2003). Thus, an increase of Ca2+ concentration in the brain cells of grasshoppers exposed to Zn shows that apoptosis and/or necrosis may appear more often (Augustyniak et al., 2006a). Therefore, the death of embryos in groups intoxicated with zinc might have resulted from indirect Zn activity: through the disturbance of Ca2+ ion homeostasis. Both the age of the female at the time of egg laying and Zn concentration in the sand where diapause took place significantly influenced the duration of embryogenesis. The habitat factor seemed insignificant (Table 2). Egg pod exposure to 10,000 mg Zn g 1 sand resulted in a significant increase in duration of embryogenesis (T). Devkota and Schmidt (1999) showed a similar effect, caused by Cd, Hg and Pb, in grasshoppers reared continuously under laboratory conditions. Willott and Hassall (1998) stated that this parameter is also very sensitive to temperature change. Our results point out that another important factor affects embryogenesis. The shortening of embryogenesis in grasshoppers hatching from eggs laid by older females is equally surprising and difficult to explain. From our study, we conclude that the duration of embryonic development is exceptionally sensitive to the age of female at the moment of oviposition. The same factor appeared to be important also for the activity of selected detoxifying enzymes in offspring. In our other paper, we show that activity of glutathione reductase in I-stage nymphs hatched from eggs laid by old females from Pilica and Olkusz was significantly lower than in hatchlings developed from eggs laid by young females. In grasshoppers from Szopienice, we found an opposite tendency. Also glutathione concentration in offspring of old females from Pilica and Olkusz was lower than in insects hatched from eggs laid by young grasshoppers, while in insects from Szopienice the tripeptide concentration did not depend on female age (Augustyniak et al., 2006b). Therefore, differences connected to adaptations to metal-polluted environments are observed at various levels. 5. Conclusions In grasshoppers from the reference site (Pilica), the effects of female aging and additional metal stress applied during diapause sum. Pre-exposure may change the pattern. The grasshoppers from Szopienice have probably developed a form of defense against stress caused by heavy
49
metals: the application of higher zinc concentration during diapause exerted a weaker effect than in insects from the reference site. The mechanism was not found in grasshoppers from Olkusz, despite their similar pre-exposure to zinc in their habitat. This may indicate that the population exists at the limit of its energetic abilities. Acknowledgments This work was supported by the Grant (No. 2 P04G 006 27) of Polish National Committee for Scientific Research. References Augustyniak, M., Migula, P., 1996. Patterns of glutathione S-transferase activity as a biomarker of exposure to industrial pollution in the grasshopper Chorthippus brunneus (Thunberg). Studia Societatis Scientiarum Torunensis 4, 9–15. Augustyniak, M., Migula, P., 2000. Body burden with metals and detoxifying abilities of the grasshopper—Chorthippus brunneus (Thunberg) from industrially polluted areas. In: Merkert, B., Friese, K. (Eds.), Trace Elements—Their Distribution and Effects in the Environment. Elsevier Science, Amsterdam, pp. 423–454. Augustyniak, M., Babczynska, A., Migula, P., Wilczek, G., Łaszcyca, P., Kafel, A., Augustyniak, M., 2005. Joint effects of dimethoate and heavy metals on metabolic responses in a grasshopper (Chorthippus brunneus) from a heavy metals pollution gradient. Comparative Biochemistry and Physiology C 141, 412–419. Augustyniak, M., Juchimiuk, J., Przyby"owicz, W.J., Mesjasz-Przyby"owicz, J., Babczyn´ska, A., Migula, P., 2006a. Zinc-induced DNA damage and the distribution of metals in the brain of grasshoppers by the comet assay and micro-PIXE. Comparative Biochemistry and Physiology C 144, 242–251. Augustyniak, M., Babczyn´ska, A., Szulin´ska, E., Witas, I., 2006b. Effects of female aging and metal pollution on glutathione-dependent enzymes in Chorthippus brunneus nymphs. Toxicology Letters 164S, 153–154. Canesi, L., Viarengo, A., 1997. Age-related differences in glutathione metabolism in mussel tissues (Mytilus edulis L.). Comparative Biochemistry and Physiology 116B, 217–221. Cherrill, A.J., Begon, M., 1989. Timing of life cycles in a seasonal environment: the temperature-dependence of embryogenesis and diapause in a grasshopper (Chorthippus brunneus Thunberg). Oecologia 78, 237–241. Devkota, B., Schmidt, G.H., 1999. Effects of heavy metals (Hg2+, Cd2+, Pb2+) during the embryonic development of Acridid grasshoppers (Insecta, Caelifera). Archives of Environmental Toxicology 36, 405–414. Fisher, Z., 1981. Energy budget of grasshopper Chorthippus sp. in industrial environment. Polish Ecological Studies 7, 5–14. Jalil, A., Selles, F., Clarke, M.J., 1994. Growth and cadmium accumulation in two durum wheat cultivars. Communications in Soil Science and Plant Analysis 25, 2597–2611. Kim, Y.-H., Kim, E.Y., Gwag, B.J., Sohn, S., Koh, J.-Y., 1999. Zincinduced cortical neuronal death with features of apoptosis and necrosis: mediation by free radicals. Neuroscience 89, 175–182. Kucharski, R., 1993. Zanieczyszczenie gleb i ros´ lin jadalnych i paszowych na terenie Wojewo´dztwa Katowickiego. In: Jankowski, A.T. (Ed.), Aktualne Problemy Ekologiczne Regionu Go´rnos´ la˛ skiego. WNoZ USl, Katowice, pp. 62–68. Łaszczyca, P., Augustyniak, M., Babczyn´ska, A., Bednarska, K., Kafel, A., Migula, P., Wilczek, G., Witas, I., 2004. Profiles of detoxifying enzymatic activity in earthworms from zinc, lead and cadmium polluted areas near Olkusz (Poland). Environment International 30, 901–910.
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Sohal, R.S., Mockett, R.J., Orr, W.C., 2002. Mechanisms of aging: an appraisal of the oxidative stress hypothesis. Free Radical Biology and Medicine 33, 575–586. Stone, D., Jepson, P., Kramarz, P., Laskowski, R., 2001. Time to death response in carabid beetles exposed to multiple stressors along a gradient of heavy metal pollution. Environmental Pollution 113, 239–244. Takeda, A., 2000. Movement of zinc and its functional significance in the brain. Brain Research Reviews 34, 137–148. Wilczek, G., 2005. Apoptosis and biochemical biomarkers of stress in spiders from industrially polluted areas exposed to high temperature and dimethoate. Comparative Biochemistry and Physiology 141, 194–206. Willott, S.J., Hassall, M., 1998. Life-history responses of British grasshoppers (Orthoptera: Acrididae) to temperature change. Functional Ecology 12, 232–241. Woz´niak, K., Blasiak, J., 2003. In vitro genotoxicity of lead acetate: induction of single and double DNA strand breaks and DNA-protein cross-links. Mutation Research 535, 127–139. Zirpel, L., Parks, T.N., 2001. Zinc inhibition of group ImGluR-mediated calcium homeostasis in auditory neurons. Journal of the Association for Research in Otolaryngology 2, 180–187.