Genetic Variability and Changes of Elemental Concentrations in Cells of Tetrix tenuicornis ( Orthoptera : Tetrigidae ) from Polluted and Unpolluted Areas

GRZYWACZ B., WARCHA£OWSKA-LIWA E, BANACH Z., PYZAE. 2012. Genetic variability and changes of elemental concentrations in cells of Tetrix tenuicornis (Orthoptera: Tetrigidae) from polluted and unpolluted areas. Folia biologica (Kraków) 60: 17-25. Genetic variability between populations of the orthopteran insect Tetrix tenuicornis, collected from six locations in Poland, was assayed by using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method. The results show that insects in a population frommetal polluted areas in Boles3aw have reduced genetic variability in contrast to five other populations located in unpolluted areas. The insects from polluted sites also showed significant changes in elemental concentrations in nerve and muscle cells, measured by X-ray spectroscopy, when compared to insects of the same species collected from unpolluted sites.

Environmental effects have been detected by determining how different environmental conditions influence mutation rates, the stability of an organism's development, and how genes interact with the environment to produce phenotypes (HOFF-MAN & PARSONS 1997).This is especially important for species which invade heavily polluted environments, such as heavy metal processing industrial areas, and persist in stable populations under these conditions.Such organisms accumulate large amounts of heavy metals in tissues but are able to develop efficient mechanisms of detoxification and other adaptations for life in a polluted environment.It is known that heavy metals are genotoxic, neurotoxic and affect many physiological and cellular processes in all organisms which have been studied so far (ROSS et al. 2002;BON-ACKER et al. 2005;FLOREA & BÜSSELBERG 2006).They affect development, fertility and survival depending on their concentrations in the environment, interactions and influences of other environmental factors (BOROWSKA et al. 2004).
The present study is a continuation of our research on heavy metal genotoxicity and cytotoxicity in T. tenuicornis from populations living in contaminated and uncontaminated sites.Individuals collected from polluted areas accumulates heavy metals that leads to whole body concentrations of zinc (Zn), lead (Pb), copper (Cu), and cadmium (Cd) that are 4.03, 4.32, 1.5 and 41.73 times higher, respectively, than in insects of the same species from unpolluted areas.A cytogenetic analysis of this species, intoxicated by heavy metals, also showed several anomalies in chromosome number and their morphology during mitosis (WARCHA£OWSKA-OELIWA et al. 2005).
In the present study, random amplified DNA polymorphism (RAPD) revealed by the polymerase chain reaction method (RAPD-PCR) have been applied to investigate effects of heavy metals at the population level by studying the phenetic structure of T. tenuicornis populations from polluted and unpolluted sites.The RAPD method has been used by other authors to generate molecular markers and analyze genetic and phenetic vari-abilities in different organisms, including humans, fungi and plants (WILLIAMS et al. 1991;LARK et al. 1992;KOLLER et al. 1993;STILES et al. 1993) and insects (BLACK et al. 1992;ZHOU et al. 2000;SHARMA et al. 2003;KIM & SAPPINGTON 2004;SESARINI & REMIS 2008).It has already been applied in ecotoxicology (WOLF et al. 2004) to diagnose the genotoxicity of copper (ATIENZAR et al. 2001), lead and other metals (ROSS et al. 2002), benzo[a]pirene (ATIENZAR et al. 1999(ATIENZAR et al. , 2002a(ATIENZAR et al. , 2002b)), and UV or X rays (ATIENZAR et al. 2000;JONES & KORTENKAMP 2000).This technique is useful for the preliminary screening of populations for genotoxic effects and for determining the phenetic/genetic diversity in natural populations exposed to pollutants (WOLF et al. 2004).Moreover, the RAPD-PCR method needs very small quantities of DNA, explores many loci, and neither cloning nor sequencing is necessary in comparison with other molecular techniques used for genomic characterization (FRAGA et al. 2005).
Since populations of T. tenuicornis are stable in contaminated sites, the insects seem to tolerate heavy metal toxicity, however, the physiological changes at the cellular level in these insects are unknown.So, in addition to the study at the population level, we examined heavy metal intoxication at the cellular level by comparing concentrations of major elements in cells of the nervous and muscle systems.Any changes in the concentration of sodium (Na), potassium (K), chloride (Cl), magnesium (Mg), sulphur (S) and phosphorus (P) disrupt the physiology of these cells.In an earlier study we found that changes in concentrations of elements in cells of different tissues are correlated with heavy metal accumulation in these tissues (TYLKO et al. 2005).To evaluate the effects of heavy metals on neurons and muscles, we carried out an Xray microanalysis of selected elements important for cell metabolism in insects collected from populations from polluted and unpolluted areas.
Heavy metals are easily absorbed through the body surface or via the digestive tract and accumulated in specific tissues where they are immobilized, or are present in blood affecting different cell types.In a previous study we found that heavy metals influence the elemental composition of cells, even those protected by the blood-brain barrier and which are not the main targets for heavy metals, such as neurons (TYLKO et al. 2005).Cells affected by heavy metals include immunocompetent cells, decreasing the functioning of the immune system and as a consequence the survival of the animal.In the housefly, a model organism for metal toxicity, exposure to heavy metals during development under laboratory conditions significantly decreases the number and alters the function of haemocytes, i.e. insect blood cells (BOROWSKA et al. 2004).Elemental changes in other cells, such as neurons and muscle cells, lead to serious dysfunctions of the nervous system and muscles (TYLKO et al. 2005) affecting animal behaviour, survival and longevity.
There are organisms however, that are able to develop a tolerance to heavy metal toxicity.This has been observed mostly in plants (CHIANG et al. 2006) and fungi (ZAFAR et al. 2006), but also reported in animals (BARSYTE et al. 2001).Such species are especially informative in relation to the adaptive processes enabling survival in environments altered as a result of industrialization.
The aim of our study was to carry out a diagnostic analysis of RAPD band similarity of T. tenicornis populations from heavy metal polluted and unpolluted sites as well as to examine the concentration of Na, K, Cl, Mg, S and P in neurons and muscles of insects originating from these populations in order to investigate their physiological condition.
Genomic DNA was extracted from one leg of each insect using a QIAamp TM DNA Dneasy Tissue Kit (Qiagen TM Germany) according to the manufacturer's protocol.The RAPD assay was performed in 20 Fl volumes containing: 2 Fl of DNA template, 2 Fl 10 x PCR buffer (Qiagen), 0.8 Fl of MgCl 2 , 0.4 Fl of deoxynucleotide triphosphates (dNTPs), 0.2 Fl of Taq DNA polymerase (Qiagen).Amplification was performed using a MJ research Minicycler.The PCR reaction started with an initial denaturation for 4 min at 94°C, followed by 40 cycles for: 1 min at 94°C, 1 min at 35°C and 2.5 min at 72°C.After amplification, the solutions were stored at 4°C until electrophoresis and visualization.RAPD reactions on the DNA of each individual was repeated three times.As a result of this procedure identical band patterns were obtained indicating the reliability of the method.RAPD PCR products were mixed with a loading buffer and separated by electrophoresis in 2% agarose gel with ethidium bromide in TBE (Trisborate-EDTA) at 50 V for 3 h.Specific bands were detected using fluorescent or silver tags and UV transillumination.
Twenty 10-nucleotide primers, used in other studies of orthopteran species genomes (JIANG & LU 2003;LI & ZHENG 2003;SESARINI & REMIS 2008), were initially screened using ten samples of DNA from each population of T. tenuicornis.Primers were synthesized in the Institute of Biochemistry and Biophysics of the Polish Academy of Sciences in Poland.Their sequences are given in Table 1.
The Bio1D++ software (Vilbert Lourmat, France) was used to calculate intra-and interpopulation relationships on the basis of the similarity of DNA band patterns obtained with the RAPD methods, according to NEI and LI'S (1979) and Jackard's (SNEATH 1957;SOKAL & MICHEN-DER 1958) similarity coefficients, i.e. S = 2N AB / (N A + N B ) where: N AB is the number of shared bands in both individuals A and B; N A and N B are the number of bands in individual A and B, respectively, and C j = j / x+ y -j, where: j is the number of shared bands in both individuals A and B; x is the number of bands in individual A, and y is the number of bands in individual B. Dendrograms were constructed on the basis of the similarity values in the matrix using the unweighted pair group match average (UPGMA).The UPGMA algorithm is a phenetic distance method (NEI 1987;PAGE & HOLMES 1998;GRAUR & LI 2000) employing a sequential clustering algorithm.The results of DNA electrophoresis were entered into the database as 0 or 1 meaning absence or presence of a band, respectively.

X-ray microanalysis
We analysed 40 and 32 muscle samples and 33 and 36 brain samples of T. tenuicornis from We³ecz and Boles³aw, respectively, according to the methods described by TYLKO et al. (2005).Analyses were carried out to estimate the elemental concentrations of Na, K, Cl, Mg, S and P in the brain cells and muscle fibres of legs.
Insects were sacrificed by freezing at -30 o C for 10 min, covered with 2-methylcellulose (Agar Scientific Inc.) and plunged into isopentane cooled to -140 o C with liquid nitrogen.Brains and legs were cut into 30 m sections using a cryostat.Next, the sections were lyophilized in -30 o C under a vacuum of 2 10 -2 Torr for 4 h at -30 o C and left in the vacuum, in room temperature, overnight.After lyophilization they were covered with a 20 nm carbon layer to prevent a charging effect (JEOL JEE-4C, JVG-N1, Japan) and 24 quantitative Xray microanalyses of each tissue (in pointed mode) were performed by means of EDXMA using a scanning electron microscope (SEM) (JEOL JSM-5410, Japan) equipped with a NORAN 30 mm 2 Si(Li) detector (679A-3SES, Noran Instruments Inc).The following conditions of analyses were used: 100 s of live time, at 10 keV of accelerating voltage and probe current 0.3 nA measured on pure aluminium.Quantitative analyses were based on calibration curves obtained on gelatin standards containing mineral salts (NaCl, MgSO 4 , K 2 PO 4 ) (TYLKO et al. 2004).Spectra were obtained from leg muscle fibres and brain cells, analysed on the basis of the "top-hat" filtering method combined with least-squares fitting (STATHAM 1977), and net counts of collected X-ray quanta were calculated.The detection limits for elements were estimated on the basis of the GOLDSTEIN and YAKOWITZ (1975) equation.For statistical analysis of normality the Kolomogorov-Smirnov test was used followed by an analysis of variance

RAPD-PCR analysis
The twenty selected primers were used to examine the level of polymorphism detectable in the six populations of T. tenuicornis.Clear amplification was produced by only 7 primers while 13 primers produced either a smear or no amplification at all.Thus, all insects were examined using seven primers: S4, S8, S18, S20, S83, S97, S397 (see Table 1).They produced the highest number of scorable bands that would be able to differentiate between populations.Fig. 1, as an example, shows the electrophoretic pattern using primer S83.All amplifications were repeated at least twice in order to confirm the reproducible amplification of scored and compiled into a data matrix.The marked changes observed in RAPD profiles (disappearance and/or appearance of bands in comparison with negative control) were evaluated.These primers amplified a total of 0-7 bands in the molecular weight range of approximately 100-1031 bp based on the MassRuler TM Low Range DNA Ladder, ready-to-use, molecular marker.It was very hard to find bands which were characteristic for individuals in 6 populations.Of all primers tested, primer S83 yielded clear, sharp, polymorphic band patterns, and hence was used for comparative analysis of T. tenuicornis.Of all primers tested, both S83 (Fig. 1) and S4 (Fig. 2) yielded clear, sharp, polymorphic band patterns.The primer S83 was more informative than S4, which uniformly amplified a few bands and of minimum molecular weight of 200 bp in most of the samples in each population.

Elemental concentrations in cells
The results of X-ray microanalysis showed significant differences in all measured elements in leg muscle fibres of insects collected in Boles³aw when compared with samples from unpolluted sites.The concentrations of Na, Cl and S were significantly reduced by 79%, 30% and 6%, respectively, in muscle cells of insects collected from Boles³aw compared to those originating from We³ecz.In contrast, the concentrations of K, P and Mg in muscle fibres were significantly higher, by 172%, 52% and 46%, respectively, in the insects from Boles³aw (Fig. 4A).
Cells in the brain of insects collected from contaminated and uncontaminated sites showed similar concentrations of all measured elements except S and Cl, which had lower concentrations by 25% and 19%, respectively, in insects originating from Boles³aw as compared to samples from the unpolluted We³ecz site (Fig. 4B).These differences were statistically significant.

Discussion
In this study, natural populations of the geophile orthopteran species T. tenuicornis from polluted and unpolluted sites were examined at the population level in terms of diagnostic analysis of RAPD-PCR bands, as well as at the cellular level by examination of the concentration of physiologically important elements in excitable cells such as muscle fibers and brain cells.In these cells any changes in Na, K, Cl and Mg ion concentrations affect the resting potential of cell membranes and cell excitation.In turn, changes in concentration of S and P affect protein synthesis, such as metallothioneins important for metal binding, and cell metabolism.
The present data, gives evidence that support the RAPD analysis in determinig variability within a population.In a previous study T. tenuicornis was identified as a species typical for Paleoarctic xerotherms (WARCHA£OWSKA-OELIWA et al. 2005), including habitats polluted with heavy metals,  1991).We have already found that this species accumulates heavy metals and shows chromosomal aberration, however, the whole body concentration of heat shock protein Hsp70, known as a biomarker of heavy metal intoxication, was lower in insects living in contaminated as compared to clean habitats (WARCHA£OWSKA-OELIWA et al. 2005).The decrease in Hsp70 concentration in tis-sues exposed to heavy metals may be the result of adaptation to this environment and/or the occurrence of other mechanisms of resistance to heavy metal toxicity, rather than the synthesis of stress proteins such as Hsp70, in this species.
In the present study, we examined two additional parameters in this species from polluted and unpolluted areas by assessing the genetic diversity of its populations and concentrations of important elements for cell metabolism in nerve and muscle cells to evaluate the physiological condition of insects from polluted sites.
In the first step we analysed the level of genetic diversity, which represents a new approach in ecotoxicology, by using seven primers.After genera- tion of clear, reproducible bands, their patterns were analyzed in order to characterize DNA polymorphism in six T. tenuicornis populations.The results show that band pattern are different in each population.Individuals from the contaminated Boles³aw site were less polymorphic than specimens from uncontaminated sites in We³ecz, Jas³o, Grabowiec, £uczyce and ¯urawica.There were found two genetic clusters among 30 individuals from six populations of T. tenuicornis by using RAPD method.First cluster concluded individuals from polluted areas from Boles³aw and the second one contained specimens of five populations from unpolluted areas.NEI and LI'S (1979) similarity coefficient indicated that the within population similarity was the highest in samples from Boles³aw and lower in We³ecz, Jas³o, Grabowiec, £uczyce and ¯urawica.However, interpopulation comparisons showed that similarity coefficients between the populations from Boles³aw and Jas³o, as well as between Boles³aw and Grabowiec or Boles³aw and £uczyce were higher than that of the populations from We³ecz and Grabowiec or We³ecz-£uczyce.In general, these results suggest that metal pollution in Boles³aw affects the genetic variability in this population.Molecular data presented differences at the DNA level between T. tenuicornis from polluted and unpolluted ares.
The population-genetic and evolutionary effects of contaminant exposure have been studied by other authors (e.g.BELFIORE & ANDERSON 1988;BICKHAM et al. 2000;CRONIN & BICKHAM 1998;ROSS et al. 2002;MULLER et al. 2007), and the RAPD-PCR technique has been applied in both plants and animals.This analysis has also wide applications in the study of genetic diversity between and among populations (e.g.APOSTOL et al. 1996;BLOK et al. 1997;CENIS 1993, TABERNER et al. 1997;GELETA et al. 2007), including ecotoxicological studies (NADIG et al. 1998;KRANE et al. 1999;BICKHAM et al. 2000;ROSS et al. 2002).
It has been reported, however, that populations exposed to heavy metal pollution may either increase or decrease their genetic variation.For example, a reduction of genetic diversity has been observed in prawns and isopods exposed to a mixture of heavy metals including zinc, lead, cadmium, copper and manganese present in the marine environment surrounding a lead smelter (ROSS et al. 2002).A decrease of polymorphism may occur as a result of selection for toxicant resistance but also depends on the species and in addition may be affected by other factors.In turn, new mutations may increase genetic variability and it is known that heavy metals have mutagenic effects on DNA and may indirectly decrease growth and reproduction and increase mortality in populations (THEODORAKIS & SHUGART 1998).Changes in genetic variability may also be a consequence of adaptation to a contaminated environment (BICK-HAM & SMOLEN 1994), genetic drift, gene flow and/or selection in microevolutionary process which determine the pattern of genetic structure.
The observed genotypic differences between populations of the same species may be important in determining the physiological or ecological significance of altered gene frequencies in populations from polluted and unpolluted sites.Using the RAPD method for the evaluation of DNA polymorphism, it is possible to detect population genetic responses to chemical exposure.Several studies on aquatic organisms have shown higher genetic variability in populations originating from an environment with a low contamination level in comparison with populations living in a highly contaminated environment (RONG & YIN 2004;MAES et al. 2005).
Toxic compounds affect organisms, but their survival depends not only on the presence or absence of environmental toxicants but also on population demographic properties (BICKHAM et al. 2000).Beside toxicants, genetic variability may also be altered by natural factors such as temperature and migration.Gene flow is increased as a result of migration of individuals between populations as well as hybridization and nucleotide substitution (BICKHAM et al. 2000).
The results showed that the RAPD-PCR technique is a useful method in ecotoxicological studies for the preliminary detection of phenetic/genetic variability within and between populations of T. tenuicornis.This study is the first report on the use of a DNA marker for the analysis of populations of T. tenuicornis from polluted and unpolluted areas.
The reduced genetic diversity of T. tenuicornis populations exposed to heavy metals seems to be a result of selective pressure, bottlenecks, and/or genetic drift on this species leading to the development of tolerance and resistance to these substances in the environment.However, future studies using more populations exposed and unexposed to high levels of heavy metals, using amplified fragment length polymorphism (AFLP) markers and microsatellite markers are needed to confirm the mechanisms of decreasing polymorphism in populations living under pressure of environmental pollutants.
Significant differences in element composition were detected between T. tenuicornis individuals collected from polluted and unpolluted ecosystems.The orthopterans collected in polluted sites near Boles³aw accumulate 1.5, 4.03, 4.32 and 41.73 times more Cu, Zn, Pb and Cd, respectively, than insects from unpolluted sites (WAR- CHA£OWSKA-OELIWA et al. 2005).This affects the concentrations of vital elements in their cells, ex-amined in the present study, especially in cells which are not protected by specialized barriers such as muscle fibers.We found that concentrations of Na, Mg, P, S, Cl and K were significantly altered in muscle cells.The highest increase and decrease in concentration was in the case of K (by 172%) and Na (by 79%), which must affect the plasma membrane potential and excitability of muscle fibers.In contrast, cells in the brain, protected by the haemolymph/brain barrier, did not show significant changes in concentrations of the elements studied, except for S and Cl.The increase of concentrations of K, Mg and decrease of Na and Cl in muscle fibers of T. tenuicornis which has colonized zinc-lead mine spoils, suggests an influence of heavy metals on ion pumps and channels involved in regulation of ion concentrations in cytoplasm and in extracellular space.The concentration of Na + and K + ions inside cells is regulated by Na + /K + -ATPase and it is known that its structure and function is affected by heavy metals which catalyze cleavage of the Na + /K + -ATPase subunits (GOLDSHLEGER & KARLISH 1997).In turn the increase of P and decrease of S concentrations indicate a possible increase of ATP production and decrease of protein synthesis, respectively.
Effects of heavy metals such as Zn, Cu, Pb and Cd on concentrations of Na, Mg, P, S, Cl and K in cells have also been studied in another insect species, the housefly, under laboratory conditions (TYLKO et al. 2005).In this study a similar increase of K and P and decrease of Na, P and S concentrations in muscle cells of imagines was found after treating larvae with Cd with low (3 Fg/g) or high (50 Fg/g) concentrations in the rearing medium (TYLKO et al. 2005).Since the effects of other heavy metals including Cu, Zn and Pb on the same elements were different, the changes in the elemental concentrations detected in the present study may result from the toxicity of cadmium.
In the present studies, we were able to show that T. tenuicornis living in the polluted environment in Boles³aw has established a stable, genetically uniform population, however, heavy metal accumulated by these insects affects their genetic material and physiological processes but the toxic effects of heavy metals do not significantly increase their mortality, probably due to the tolerance developed by T. tenuicornis.

Fig. 3 .
Fig. 3.The diagrams of the cluster analysis of RAPD pattern similarity matrix of the studied T. tenuicornis populations fromBoles³aw, We³ecz, Jas³o, Grabowiec, £uczyce, and ¯urawica.The UPGMA method was used for analysis.

Fig. 4 .
Fig. 4. The elemental composition of the muscle fibres (A) and nerve cells of the brain (B) of the insects collected from different sites, contaminated (Boles³aw) and uncontaminated (We³ecz), as mean ± SE.Asterisks indicate significant differences between groups at P<0.05.
Genetic Variability and Changes of Elemental Concentrations in Cells of T. tenuicornis