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The effects of chromium VI on the fitness and on the β-tubulingenes during in vivo development of the nematodeSteinernema feltiae

Stephen Boyle⁎, Thomais Kakouli-DuarteMolecularEcologyandNematodeResearchGroup,Department of Science andHealth, Institute of TechnologyCarlow, KilkennyRoad, Carlow, Ireland

A R T I C L E I N F O

⁎ Corresponding author. Tel.: +353 59 9170554E-mail addresses: stepboyles@yahoo.com (

0048-9697/$ – see front matter © 2008 Elsevidoi:10.1016/j.scitotenv.2008.05.024

A B S T R A C T

Article history:Received 16 January 2008Received in revised form 12May 2008Accepted 13 May 2008Available online 18 July 2008

The entomopathogenic nematode (EPN), Steinernema feltiae, is a commonly occurringnematode in the soil in Ireland. Consequently, we have conducted investigations as to theutility of this species as a candidate organism for the detection of chromium in Irish soils.These experiments have demonstrated that S. feltiae can survive and reproduce in thepresence of high concentrations of chromium VI. It was observed that concentrations ashigh as 1000 ppm have little effect on the ability of this organism to produce large numbersof progeny. Nematodes were not observed to reproduce above 1800 ppm. However, anincrease in development times for the nematode in vivo was noted at concentrations of400 ppm upwards.This paper also illustrates the effects upon the β-tubulin genes within nematodepopulations exposed to chromium VI in vivo. DNA sequencing has shown that elevatedlevels of variations occur among the population treatments, although these variations donot appear to be dependent upon chromium concentration.These findings constitute this organism appropriate for further investigation for thedevelopment of sub-lethal end points and biomarkers for the detection and biomonitoringof chromium VI contamination in soil.

© 2008 Elsevier B.V. All rights reserved.

Keywords:NematodeEPNChromium VISoil pollutionBioindicatorBiomarkerMutagen

1. Introduction

Hexavalent chromium (chromium VI) exists in soils naturallyand is the sixthmost abundant element in the Earth's crust. It isalso present as a result of human practices, and mostly fromthose practices associated with industry. Chromium occursnaturally in Irish agricultural soils in concentrations between 5and 250 mg/kg (McGrath et al., 2001). Studies have suggestedthat hexavalent chromium contamination adversely affects thelifespanof fish (Perez-Benito, 2006), can initiate behavioural andbiological changes inearthworms (SivakumarandSubbhuraam,2005), and cells of algae are known to suffer serious morpholo-gical andbiochemical alterations (Devars el al., 1998; Rai andRai,1998; Okamoto et al., 2001).

.S. Boyle), thomae.kakouli

er B.V. All rights reserved

Nematodes are among the world's most numerous andubiquitous organisms and there is an increasing interest intheir use as bioindicators or as biomarkers for soil and aquaticcontaminants (review — Bongers and Ferris, 1999). Theauthors put forward seven points explaining why nematodesmake very good bioindicators, including their rapid responseto environmental disturbance and enrichment, and theirubiquity. More recently, a long ranging study establishedthat complex nematode communities are adversely affectedby the presence of heavy metals and other microelements inthe soil (Bakonyi et al., 2003). Interestingly, and in the contextof the work presented here, the authors reported thatchromium continued to have adverse affects among nema-tode communities over the entire experimental period,

@itcarlow.ie (T. Kakouli-Duarte).

.

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despite chromium failing to display phytotoxic effects afterthe first year of the trials. This proposes that nematodecommunities may serve as greater indicators of chromiumpollution than several species of agricultural plants.

Besides the marine nematode Monhystera disjuncta (Vran-ken and Heip, 1986) and species of the genus Panagrellus(Samoiloff et al., 1980; Haight et al., 1982; Sherry et al., 1997),Caenorhabditis elegans is clearly the most widely used nema-tode for toxicological studies. This free living soil dweller hasproven to be effective as a test organism for the presence ofmany different types of contaminants (Anderson et al., 2003).Various approaches have been employed in these studiesincluding the use of transgenic bioluminescence strains(Lagido et al., 2001), online computer monitoring systems(Gerhardt et al., 2002), detection of ion-biomolecule interac-tions via atomic absorption spectrophotometry (AAS; Tataraet al., 1997), and standard methods such as aquatic toxicitytesting (Williams and Dusenbery, 1990) and aquatic acutetoxicity testing (Ura et al., 2002). Although wide ranging, thesemethods are still heavily focused on one organism. Ifscientists are to pursue the understanding of the effects oftoxicants on the environment and to develop systems thatobserve and measure such effects, then it is imperative thatadditional nematode species are studied.

One such additional species is the entomopathogenicnematode (EPN) Steinernema feltiae. This nematode is aubiquitous species in Ireland (Griffin et al., 1991; Dillon et al.,1999) and can be easily isolated from soils (Bedding andAkhurst, 1975). It is a relatively small nematode and is anobligate and lethal parasite of insects (Reid and Hominick,1992; Burnell and Stock, 2000). It is used as a biocontrolorganism in horticulture and agriculture and its life cycle andphysiology has been extensively studied. In the area ofecotoxicology, however, it has been largely ignored, with theexception of a study by Jaworska and Gorczyca (2000), inwhichthe effects of metal ions on S. feltiae suggested a decrease in itsability to infect and reproduce in Galleria mellonella afterexposure to lead, cadmium, zinc and copper.

Entomopathogenic nematode fitness is a physiologicalbarometer that may be tested using a number of biologicalcharacteristics as parameters. The ability to produce largenumbers of progeny and the ability to infect host larvaeeffectively have been employed as fitness tests to determinethe effects of age (Yoder et al., 2004), application method(Perez et al., 2004), temperature during development (Haziret al., 2001) and production methods (Grewal et al., 1999) ondifferent species of EPN. In all cases there were significanteffects on most species. However, these EPN traits were rarelyemployed to detect the effects of contaminants, and no usefulmolecular or DNA markers exist for the determination or thedetection of the effects of chromium VI on soil dwellingnematodes.

It has previously been reported that chromium VI is apotentially powerful mutagen. Hexavalent chromium hasbeen extensively shown to induce general environmentaltoxicity, as well as more specific effects of acute and chronicnature such as neurotoxicity, dermatoxicity, genotoxicity,carcinogeneticity, and immunotoxicity, (von Burg and Liu,1983; Barceloux, 1999). It is believed that chromium inflictsmost damage during reduction of chromium VI to chromium

III, a process considered to be initiated in the cell byglutathione (Bose et al., 1992; Stearns and Wetterhahn, 1994;Moghaddas et al., 1995).

Important genetic loci for normal cellular function, such asthe β-tubulin genes, may be potential candidates for thedevelopment of ecotoxicological molecular markers, and thusdeserve further investigation. The β-tubulin genes areinvolved in many crucial cellular processes and in nematodesthere can be as many as five different functional specificisotypes in existence (Gogonea et al., 1999). PCR primers for theamplification of these genes from S. feltiae were developed byBoyle (2007), and as a result different fragments can beaccessed to assess the levels of DNA change or damageinferred by toxicants such as chromium.

In this paper we demonstrate that S. feltiae exhibits a highdegree of resistance to chromium VI and propose it as acandidate organism to detect the effects of chromium VIcontamination in the environment. Suitable sub-lethal end-points were found to be the nematode reproductive potentialand the rate of nematode development in the presence of thetoxicant. We also investigate the occurrence of increased DNAvariations between populations of a strain of S. feltiae culturedin the presence of chromium.

2. Materials and methods

2.1. Culturing of nematodes

An Irish isolate of S. feltiae [strain 12(1); Boyle, 2007] wascultured at room temperature in G. mellonella and the resultinginfective juveniles (IJ) were collected and subsequentlywashed with double-distilled deionised H2O. IJ were allowedto settle to the bottom of 50 ml sterile disposable centrifugetubes (Sarstedt) and water was aspirated using a Pasteurpipette. Clean H2O was then added to the nematodes. Thisprocedure was repeated at least three times, or until clear H2Owas achieved after IJ settled to the bottom of the tubes.

2.2. Exposure of IJ to chromium VI in H2O

Experiments were carried out in an effort to determine thelevels of toxicity of chromium VI to the non-feeding IJ stage.

Concentrations of chromium VI were prepared in H2O fromsodium dichromate (Merck) ranging from 200 ppm to3000 ppm in increments of 200 ppm. 1 ml of each was placedinto a separate well of two 24 well plates. A nematodesuspension was prepared containing approximately 300nematodes in 10 μl, and 10 μl of this suspension was addedto each treatment well of the 24 well plates. Exposureexperiments were replicated three times, comprising a totalof 48 treatments. Controls comprised of S. feltiae IJ incubatedwith treatments but in H2O only. All pipette tips were flushedwith 2% Triton X-100 directly before use so as to minimisenematodes adhering to the inside of the tip. All treatmentswere incubated at room temperature (20–22 °C) for 24 h in theabsence of direct light.

After incubation each treatment replicate was mixed bygently ‘flushing’ the nematode suspension in and out of apipette tip. 20 μl was taken and alive and dead nematodes

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were counted visually using a light stereoscope. Six countswere performed per treatment in this manner and meanswere calculated.

2.3. Exposure of nematodes to chromium VI during in vivodevelopment

Last instar larvae of thewaxmoth, (G. mellonella) wereweighedto ensure that all larvae were in the 250–350 mg weight range.Each was injected with approximately 10–15 IJ of S. feltiaestrain 12(1). After larval deaths, which occurred approximately24 h after injections, the cadavers were weighed again and theweights were recorded. Sodium dichromate was then injectedinto larvae to give concentrations of chromium VI from200 ppm to 3000 ppm per cadaver weights at increments of200 ppm. Treatmentswere replicated five times. Controlswerealso prepared where S. feltiae strain 12(1) was injected intolarvae but with no subsequent chromium injections, and aseparate set of controls was prepared where larvae wereinjected with only 10 μl of sterile double-distilled deionisedH2O. All controls consisted of five replicates each.

All waxmoth larvae cadavers were placed individually ontoseparate White traps (White, 1927), labelled with associatedrecorded weights and Cr VI concentrations and stored at roomtemperature (20 °C–22 °C) in the absence of direct light.

2.4. IJ emergence times

Time taken for IJ to emerge was assessed by recording thetime, in days, at which IJ were first observed in the H2Oreservoirs at the bottom of theWhite traps. This was achievedusing a light stereoscope and observations were performedonce a day. In this case, the observations were carried out at 11am each morning of the trials.

2.5. Total IJ numbers (reproductive potential)

The following procedure is taken from that described byRolston(2004). Briefly, 7 days after emergence was first observed thecontents of theWhite trapswere decanted into 50ml centrifugetubes (Sarstedt) andwashed aspreviously described. After threewashes (or more if needed) centrifuge tubes with nematodeswere filled to a total volumeof 50mlwith sterile double-distilledand deionised H2O. This nematode suspension was homoge-nised by inversions and six aliquots of 10 μl volumeswere takenand pipetted onto a clean Petri dish. Nematodes present in eachaliquot were counted visually using a light stereoscope andresultswere recorded. The approximate total count of IJ presentin the White trap reservoirs was calculated and, using theweights recorded at the point of sodium dichromate injections,the approximate nematode count per mg of cadaver (hostinsect) was calculated.

2.6. Extraction of DNA

Nematodes were collected from the reproductive potentialtrials for DNA extraction. The quantity of nematodes availablevaried, but there were generally adequate amounts collectedfor extraction. Those that yielded lesser amounts, such as the1600 ppm treatment, produced enough nematodes after

extended emergence and harvesting. Generally, approxi-mately 50–150 mg of fresh or frozen nematode pellet wascrushed in a sterile mortar and pestle. 1.5 ml of lysis buffer(0.1M Tris–HCl, pH 8.5; 0.05M EDTA; 0.2MNaCl; 1% SDS; 100 μg/ml Proteinase K) was added and mixed with the nematodepaste by gentle swirling. Using a pipette this mixture wasremoved to a clean, sterile 50 ml polypropylene centrifugetube and incubated overnight at 37 °C. An equal volume ofbuffer saturated phenol was added and the solution wasextracted for 10 min by gentle rolling back and forth on abench. The solution was centrifuged for 15min at 10,000 g andthe upper aqueous phase carefully removed and placed into anew clean, sterile 50 ml polypropylene centrifuge tube. Anequal volume of phenol/chloroform (1:1) was added to theaqueous phase and extracted and centrifuged as before. Thistime the upper aqueous phase was removed and placed intosterile micro-centrifuge tubes. An equal volume of chloro-form/iso-amyl-alcohol was added to the aqueous phase andsolutions were extracted and centrifuged as before, only thistime with a micro-centrifuge. The upper aqueous phase wasremoved, placed into a fresh micro-centrifuge tube and DNAwas precipitated from solution by the addition of 2 M sodiumacetate (to a final concentration of 0.2 M) and 2 volumes of 95%molecular biology grade ethanol (pre-chilled to −20 °C).Solutions were mixed thoroughly and incubated at −20 °Covernight. The sample was then centrifuged for 15 min at14,000 g and the supernatant was discarded. The resultingDNA pellet was washed with 70% ethanol and air dried in aDNA-clean hood at room temperature for up to 1 h. The pelletwas re-suspended in 300 μl TE buffer (Tris-EDTA pH 8; 10 MmTris–HCl; 1 Mm EDTA). The re-suspended pellet was treatedwith a RNase solution (2 mg/ml) in a 1:100 (v/v) ratio. This wasincubated at room temperature for 1 h. Following incubationthe solution was extracted with phenol/chloroform andchloroform/iso-amyl-alcohol as previously described, andsodium acetate/ethanol precipitations and 70% ethanolwashes were performed also as previously described. Thefinal ethanol washed DNA pellet was air dried at roomtemperature in a DNA-clean hood for 2 h and re-suspendedin sterile double-distilled and deionised H2O and, either usedimmediately, or stored either at 4 °C or −20 °C for future use.

2.7. PCR amplification of the β-tubulin genes

Two primers that were designed for the amplification of β-tubulin genes from S. feltiae (Boyle, 2007) were used for thisstudy. The sequences of these primers are as follows:Tub1AfwdSpc; 5′ GCG GTA ATC AGA TCG GTT C 3′: Tub4Ar-evspc; 5′ GCG GTC GGG GTA TTC TTC GCG 3′. PCR conditionswere: 5 mM final concentration of MgCl2, 0.2 mM finalconcentration of dNTP (0.05 mM each of dTTP, dGTP, dCTP,dATP), 50 pmol of each primer, 50–100 ng template DNA, 1.25 Uof Taq DNA polymerase (GoTaq Polymerase, Promega), a 1Xfinal concentration of associated PCR buffer, and steriledouble-distilled H2O to a final volume of 50 μl. Thermocyclingconditions were as follows: an initial denaturation step of94 °C for 5min, followed by 35 cycles of 94 °C for 1min, 57 °C for30 s, and 72 °C for 1min, with a final extension step of 72 °C for5 min. Results were visualised by electrophoresis using 1.5%agarose stained with 0.5 μg/ml ethidium bromide.

Fig. 1 –LC50 test results on S. feltiae IJ in the presence of varying chromium VI concentrations in H2O over a 24 hour period.

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Samples to be sequenced were processed and sent accord-ing to specific contract sequencer requirements (Qiagen,Gemany) and results were returned as a zip file via email.

2.8. Statistical analyses

Statistical analyses were conducted using SPSS (Version 12.0.1for Windows) and Microsoft Excel XP Professional 2002(Version 5.1 for Windows). Specifically, tests of significant fitof data to the normal distribution were determined by theKolmogorov–Smirnov test (SPSS). The significance of datafrom IJ exposure to chromium VI experiments, and theemerging IJ number experiments, were tested using theKruskal–Wallace Test for non-parametric data (SPSS). Pairwiseanalysis was performed on data from the emerging IJ numbersexperiment using the Mann–Whitney U test (SPSS). Correla-tions between time of emergence and chromium VI concen-trations, and between emerging numbers and chromium VIconcentrations were tested using the Kendall's Tau-b test fornon-parametric data (SPSS). Means, standard deviations,

Fig. 2 –Mean of emerging numbers of S. feltiae IJ from host insectconcentrations of chromium VI.

standard errors and charts were calculated using MicrosoftExcel.

For the sequence analysis experiments, DNA sequenceswere analysed using ClustalW (Pearson and Lipman, 1988) andMEGA, Version 3.1. Tests to investigate the null hypothesis ofthe neutrality of observed DNA mutations were performedusing Tajima's neutrality test (Tajima, 1989).

3. Results

3.1. Fitness trials

There were significant differences in nematode survivalbetween treatments where IJ were exposed to differentconcentrations of chromium VI (Fig. 1; Kruskal–Wallace Test:Pb0.01 confidence interval, H=43.419, df=15). However,although IJ deathswere observed in the presence of chromiumVI from concentrations of 400 ppm, survival rates did notappear to be radically reduced until at the relatively high

s (Galleria mellonella) contaminated with increasing

Fig. 3 –Mean number of days taken for S. feltiae IJ's to emerge from host insects (Galleria mellonella) contaminated withincreasing concentrations of chromium VI.

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concentration of 1000 ppm and higher. At 1600 ppm survivalrates dropped to below 20% and remained so with increasingchromium VI concentrations. Interestingly, above 1600 ppmthere was a slight increase in survival numbers. This was notmaintained after 2400 ppm chromium VI.

When S. feltiae was exposed to varying levels of chromiumVI in vivo (during the life cycle inside the host insect) resultsonce again indicated a high level of resistance (Fig. 2; Kruskal–Wallace Test: Pb0.01 confidence interval, H=55.60, df=15).Nematodes appeared to develop and reproduce in numberscomparable to the controls up to concentrations of 1000 ppm(Mann–Whitney U: PN0.05). There was an increase in numbersof IJ emerging compared to the controls in concentrations upto 600 ppm, and a reduction in emerging numbers comparedto the controls at 800 ppm and higher. Neither of thesevariations was statistically significant however (Mann–Whit-ney U: PN0.05). Pairwise analysis of the data revealed thatconcentrations of 1600 ppm chromium VI and above resultedin a reduction in IJ numbers compared to the controls that wasstatistically significant (Mann–Whitney U: Pb0.05). At concen-trations of 2000 ppm and higher there was no nematodeemergence. In cases where no emergence was observed, therewas substantial eventual colonisation of the host cadavers byother microorganisms that resulted in the eventual degrada-tion of the cadaver.

A clear increase innematodeemergence timeswasobservedwith increasing chromiumVI concentrations (Fig. 3). There wasa significant correlationbetweenthe times taken for IJ to emergeand the concentrations of chromium (Fig. 3; Kendall's Tau-bcorrelation coefficient=−0.452, Pb0.001), and between numbersemerging and the concentrations of chromium (Fig. 2; Kendall'sTau-b correlation coefficient=−0.599, Pb0.001).

3.2. β-tubulin gene analysis

Variations were observed among fragments of the β-tubulingenes amplified and sequenced from DNA isolated fromemerging IJ from the described treatments (Fig. 4). Althoughthe variations were not as many as one would have expected,

they were sufficient to result in a rejection of the nullhypothesis of neutrality (Tajima, 1989). The estimates of θare 0.006477 per nucleotide based on S (the number ofsegregating sites) and 0.0026 based on π (the average pairwisedifference). The estimates are significantly different fromeachother, and therefore are not consistent with neutrality(Tajima's D statistic of −1.7113; Pb0.01).

4. Discussion

From the results presented in this paper it is apparent that theentomopathogenic nematode S. feltiae exhibits considerableresistance to hexavalent chromium. A previous study reportedthat metal ions such as lead, cadmium, zinc and copper hadobservable negative effects on S. feltiae IJ when they wereexposed to them in H2O for 96 h (Jaworska and Gorczyca, 2000).The authors also reported that manganese had a positiveeffect on the IJ resulting in improved infectivity and reproduc-tion potential after exposure. In the experiments reported inthis paper there was also a slight increase in nematodereproduction potential compared to the controls between200 ppmand 600 ppm (Fig. 2). This finding is interesting in thatthe supposed contaminant appears to actually aid theorganism at certain concentrations, although because statis-tical significance was not observed this statement must bemade with caution.

Members of the order Rhabditida, of which S. feltiae, is amember, are believed to display a generally high level ofresistance to toxicants due to the close symbiotic relationshipswith bacteria that results in regular contact with potentiallytoxic end products (Bongers and Ferris, 1999). In addition, the IJof steinernematid species possesses a thick cuticle and a closedalimentary canal. Such characteristics are believed to conferresistance to extreme environmental conditions and to preventtheentryofpathogensandreducewater lossduringexistence insoils (Poinar, 1990). S. feltiae IJ are also not affected by osmoticstresses created while in H2O unlike S. feltiae adults (personalobservations). It is quite possible that these adaptations also

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serve to protect the IJ from toxicants such as chromium VI.However, it would be prudent to note that there were also highchromiumVI resistance levels detected for S. feltiaeduring the invivo exposure trials (Fig. 2). This may indicate that although thespecific adaptations of an S. feltiae IJ confer protective advan-tageswhile in the soil, theremay in fact be other factors atwork

Fig. 4 –DNA sequence alignment of β-tubulin gene fragments. Fragpopulations that were cultured in the presence of varying concentrchromiumVIpresent insourcesamples, andnumbers inbrackets re

that allow survival during and after heavymetal exposurewhilein development within a suitable host insect.

Once an IJ invades a targeted host insect it moults to afourth stage juvenile and begins feeding. Such developmentspresumably increase the vulnerability of the nematode to arange of deleterious factors as a result of the loss of the

ments were PCR amplified from DNA isolated from S. feltiaeations of chromiumVI. Sequence titles reflect concentrations ofpresent replicatenumbersusedassources forDNAsequencing.

Fig. 4 (continued).

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impermeable cuticle and the resumption of food intake. Yetdespite this, S. feltiae can still produce offspring relativelynormally in the presence of 1000 ppm chromium VI. In theseexperiments IJ production was observed even at concentra-tions as high as 1800 ppm, although reduced reproductive

potential was observed at these higher concentrations. Con-centrations of 200 ppm or less were observed as being highlytoxic to the host insect G. mellonella, (personal observations).This fact necessitated a particular technique in order tointroduce chromium VI to developing nematodes; chromium

Fig. 4 (continued).

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could only be injected into the host after death occurred due toIJ infection. In cases where chromium was injected first,almost instant death of the insects occurred, creating a lessthan ideal environment for IJ colonisation. S. feltiae performsbest when host death occurs as a result of proliferation of thenematode symbiotic bacteria (Poinar and Thomas, 1966).

The effects of chromium on nematodes have so far notreceived much attention apart from work by Bakonyi et al.(2003) and Nagy et al. (2004). These studies showed a markeddecrease in nematode density and taxon richness, a reductionin the maturity index (MI), as well as the structure index (SI),and the predator and omnivore (P+O) ratio, in response to

Fig. 4 (continued).

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heavy metals. Measurements with these parameters sug-gested strongly that chromium was one of the more deleter-ious of the heavy metal contaminants and that its effects canpersist over a number of years.

Williams and Dusenbery (1990) investigated the LC50values of various heavy metals, including chromium, on C.elegans and reported a value of 156 ppm chromium at 24 hexposure. Their results suggest that chromium is among theheavy metals that are most deleterious to nematodes, as theauthors reported that salts such as zinc, cadmium and arsenichave higher LC50 values than chromium. They also suggest, in

the context of the present study, that C. elegans possesses amuch lower resistance to chromium than does S. feltiae, assurvival in the results presented here only begins to reduceconsiderably after approximately 1200 ppm (Fig. 1). Therefore,if in situ nematodes were to be employed to obtain a moreaccurate indication of the effects of chromium in theenvironment and the soil biota, C. elegans would be mostlikely discounted as an indicator as it cannot survive inconcentrations that are often found naturally in Irish soils.Thus, the higher resistance of S. feltiae to chromium wouldallow for the use of natural field populations of this nematode

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to be employed as “on site” bioindicators, compared to C.elegans test nematodes which are largely used as “off site”indicator organisms and in a laboratory setting.

Themolecular effects of chromiumhave not been assessedfor nematodes. In the experiments reported here β-tubulingene fragments from S. feltiae progeny emerging from thereproductive potential experiments were PCR amplified andsequenced. A number of variationswere observed between theβ-tubulin genes from the treatments although theywerenot asnumerous as expected. In the study reported here thevariations occurring were observed to appear at differentsites among the fragment in different sequencing experi-ments. Additionally, variations were inconsistent amongreplicates further supporting the hypothesis that chromiumwill inducemutations in a randomnature rather than affectingparticular codons or base-pairs. Consequently these variationscannot be detected using site specific techniques such asRestriction Fragment Length Polymorphism (RFLP) analysis.Furthermore, the levels of variations did not appear to beconsistent with increasing concentrations of chromium VI,ruling out the utilisation of the variations in a quantitativetechnique that could ascertain the levels of chromium VIcontamination relative to DNA change.

The apparently slight effect of chromium VI on the β-tubulin gene structure in S. feltiae was surprising, given theimportance and activity of these genes and their proteins incell division, cellular communication and other crucial cellprocesses. However, although there appeared to be a lowdetection rate for variations within the β-tubulin genes ofprogeny that developed in the presence of chromium, thismay not imply that these variations did not occur. Althoughthere is evidence of sperm specific β-tubulin genes (Rudolphet al., 1987) the functions (or isotypes) of the β-tubulin genesstudied here are unknown. It is quite possible that themajority of mutations occurred in somatic cells of S. feltiaeindividuals during chromium exposure but not in germlinecells. Themutations that were detectedmaywell be a result ofgermline mutations that confer little disadvantage to theprogeny. The delay in the development and emergence ofprogeny at higher chromium concentrationsmay be a result ofa recovery period among the population, as selection removesthose affected by damaging somatic cell DNA variations, andallows progeny with increased tolerance to develop andeventually thrive. Indeed, population genetic theory showsthat deleterious mutations will be removed from a populationby natural selection (Clark et al., 1981; Cronin and Bickham,1998). It would be informative to know in how manygenerations deleterious mutations would be removed fromthe genome of S. feltiae. Although this is currently not known,it would constitute a very interesting avenue for investigationto understand the molecular mechanisms of exposure to, andrecovery from, chromium in this nematode.

Interestingly, a study of the mitochondrial DNA of thenematode Pellioditis marina from a heavy metal contaminatedsite also reported reduced population development with areduced genetic diversity among the population (Derycke et al.,2007). The authors put forward a number of factors that mayhave resulted in such a limited genetic effect such as too low atoxicant concentration, the selective neutrality of the chosenmarkers and the limited duration of their experiments.

In these experiments long term trials were not conductedeither. It would therefore be a worthy study in the future toapply β-tubulin gene sequence analysis to nematodes isolatedfrom long term chromium contaminated soils to investigaterecurrent or increased germline cell DNA effects.

5. Conclusions

It is clear that developing populations of S. feltiae demonstrateconsiderable resistance to chromium VI in vivo. This wasapparent from the development and production of largenumbers of IJ in insect hosts prepared with concentrations ofup 1000 ppm chromium VI. The reduction in nematodeemerging numbers and increasing intervals of time of nema-tode emergence, with increasing chromiumVI concentrations,do suggest that there is an effect however. At concentrationsbetween 1000 ppm and 1800 ppm there was evidence ofemergence but this ability was observed to reduce at eachintervening interval. Beyond a chromium VI concentration of1800 ppm, nematodes were not observed to survive initialinfection.

The fact that this organism can survive exposure to highlevels of such an environmentally relevant contaminantconstitutes it as a candidate species for chromium relatedenvironmental protection activities. This may be achieved byA): the establishment of sub-lethal end points, which takeinto account effects undetected with acute toxicity measure-ments, and B): the development of molecular markers forchromium detection. To date the β-tubulin genes show littlepredictable variations that may be used as molecular mark-ers, although variations have been observed whose rateof occurrence is higher than the rate that may have beenexpected after intra-species, and indeed, intra-population,analysis.

As S. feltiae appears to be far more resistant to chromiumVIthan C. elegans, it is possible that S. feltiae also exhibits higherchromium VI resistance levels than those exhibited by othernematodes. An organism that can survive in the presence of atoxicant holds good bioindication potential for the toxicanteffects, if tangible molecular or physiological changes occurthat can be detected and employed for quantitative and/orqualitative measurements.

S. feltiae is commonly found in Ireland and can be easilyisolated from the soil and cultured in the laboratory. A highlevel of chromium resistance coupled with measurablebiomarkers, genetic or otherwise, that demonstrate bioavail-able chromium would be a valuable tool for environmentalrisk assessment.

Acknowledgments

This project had been funded by the Environmental Protec-tion Agency (EPA) Ireland under the ERTDI PostdoctoralFellowship Programme, Ref No: 2005-FS-28-M1. The authorswould like to thank Dr. Finbarr Horgan at Teagasc, Oak Park,Ireland and PatMurphy of the Institute of Technology Carlow,Ireland, for their kind assistance during the preparation ofthis manuscript.

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R E F E R E N C E S

Anderson G, Cole R, Williams P. Assessing behavioural toxicitywith Caenorhabditis elegans. Environ Toxicol Chem2003;25:1235–40.

Bakonyi G, Nagy P, Kadar I. Long term affects of heavy metals andmicroelements on nematode assemblages. Toxicol Lett2003;140–141:391–401.

Barceloux D. Chromium. Clin Toxicol 1999;37:172–94.Bedding R, Akhurst R. A simple technique for the detection of

insect parasitic nematodes in the soil. Nematologica1975;21:109–10.

Bongers T, Ferris H. Nematode community structure as abioindicator in environmental monitoring. Trends Ecol Evol1999;14:224–8.

Bose R, Moghaddas S, Gelerinter E. Long-lived chromium(IV) andchromium(V) metabolites in the chromium(VI)-glutathionereaction; NMR, ESR,HPLC, and kinetic characterization. InorgChem 1992;31:1987–94.

Boyle, S.,An investigation on the intra specific genetic variationand genetic structure of Irish and exotic Steinernema feltiae(Nematoda: Steinernematidiae). PhD Thesis. Dept. Science andHealth, Institute of Technology, Carlow, Kilkenny Road, Co.Carlow, Ireland. 2007.

Burnell A, Stock P. Heterorhabditis, Steinernema and their bacterialsymbionts— lethal pathogens of insects. Nematol 2000;2:31–42.

Clark A, Feldman M, Christiansen F. The estimation of epistasis incomponents of fitness in experimental populations ofDrosophila melanogaster. Heredity 1981;46:321–46.

CroninM, Bickham J. A population genetic analysis of the potentialfor a crude oil spill to induce heritable mutations and impactnatural populations. Ecotoxicology 1998;7:259–78.

Derycke S, Hendrickx F, Backeljau T, D'Hondt S, Camphijn L, VincxM, Moens T. Effects of sublethal abiotic stressors on populationgrowth and genetic diversity of Pellioditis marina (Nematoda)from the Westerschelde estuary. Aquat Toxicol 2007;82:110–9.

Devars S, Hernandez R, Moreno-Sánchez R. Enhanced heavymetaltolerance in two strains of photosynthetic Euglena gracilis bypre-exposure to mercury or cadmium. Arch Environ ContamToxicol 1998;34:128–35.

Dillon A, Griffin C, Downes M. Hylobius Interest Group ActivityReport No. 2. Second Meeting of the Hylobius Interest Group.Maynooth: National University of Ireland; 1999.

Gerhardt A, Schmidt S, Hoss S. Measurement of movementpatterns of Caenorhabditis elegans (Nematoda) withMultispecies Freshwater Biomonitor ® (MBF)— a potential newmethod to study a behavioural toxicity parameter of nema-todes in sediments. Environ Pollut 2002;120:513–6.

Gogonea C, Gogonea V, Ali Y, Merz Jr K, Siddiqui SS. Compuationalprediction of the three-dimensional structure for theCaenorhabditis elegans tubulin family. J Mol Graph Model1999;17:90–100.

Grewal P, Converse V, Georgis R. Influence of production andbioassay methods on infectivity if two ambush foragers(Nematoda: Steinernematidae). J Invertebr Pathol 1999;73:40–4.

Griffin C, Moore J, Downes M. Occurrence of insect-parasiticnematodes (Heterorhabditidae, Steinernematidae) in theRepublic of Ireland. Nematologica 1991;37:92–100.

Haight M, Mudry T, Pasternak J. Toxicity of seven heavy metalson Panagrellus silusiae: The efficacy of the free-livingnematode as an in vivo toxicological bioassay. Nematologica1982;28:1–11.

Hazir S, Stock P, Kaya H, Koppenhofer A, Keskin N. Developmentaltemperature effects on five geographic isolates of theentomopathogenic nematode Steinernema feltiae (Nematoda:Steinernematidae). J Invertebr Pathol 2001;77:243–50.

Jaworska M, Gorczyca A. The effects of metal ions on mortality,pathogenicity and reproduction of entomopathogenic

nematodes Steinernema feltiae Filipjev {Rhabditida,Steinernematidae). Pol J Environ Stud 2000;11:517–9.

Lagido C, Pettitt J, Porter A, Paton G, Glover L. Development andapplication of bioluminescent Caenorhabditis elegans asmulticellular eukaryotic biosensors. FEBS Lett 2001;493:36–9.

McGrath D, Carton O, Diamond S, O'Sullivan A, Murphy W, RogersP, Parle P, Byrne E. Soil, herbage, feed and water. Investigationof animal health problems at Askeaton, Co. Limerick.Johnstown Castle Estate, Wexford, Ireland: EPA; 2001.

Moghaddas S, Gelerinter E, Bose R. Mechanisms of formation anddecomposition of hypervalent chromium metabolites inglutathione-Cr(VI) reaction. J Inorg Biochem 1995;57:135–46.

Nagy P, Bakonyi G, Bongers T, Kadar I, Fabian M, Kiss I. Effects ofmicroelements on soil nematode assemblages seven yearsafter contaminating an agricultural field. Sci Total Environ2004;320:131–43.

Okamoto O, Pinto E, Latorre L, Bechara E, Colepicolo P.Antioxidant modulation in response to metal-inducedoxidative stress in algal chloroplasts. Arch Environ ContamToxicol 2001;40:18–24.

Pearson W, Lipman D. Improved tools for biological sequencecomparisons. PNAS 1988;85:2444–8.

Perez-Benito J. Effects of chromium (VI) and vanadium V on thelifespan of fish. J Trace Elem Med Biol 2006;20:161–70.

Perez E, Lewis E, Shapiro-Ilan D. Effect of application method onfitness of entomopathogenic nematodes emerging at differenttimes. J Nem 2004;36:534–9.

Poinar G. Taxonomy and biology of Steinernematidae andHeterorhabditidae, pp. 23–61. In: Gaugler R, Kaya HK, editors.Entomopathogenic nematodes in biological control. NewDelhi:CRC Press, Boca Raton Pvt. Ltd.; 1990. p. 251–80.

Poinar G, Thomas G. Significance of Achromobacter nematophilis(Achromobacteraceae: Eubacteriales) in the development ofthe nematode, DD-136, (Neoaplectana sp. Steinernematidae).Parasitology 1966;56:385–90.

Rai P, Rai L. Algal response to heavy metals with special referenceto mechanism of toxicity and tolerance. In: Verma BN,Kargupta AN, Goyal SK, editors. Advances in Phycology. APCPublications; 1998.

Reid A, Hominick W. Restriction fragment length polymorphismswithin the ribosomal DNA repeat unit of Britishentompathogenic nematodes (Rhabditida: Steinernematidae).Parsitology 1992;105:317–23.

Rolston, A.,Distribution, relatedness, fitness and behaviour ofentomopathogenic nematodes from Bull Island, Dublin. PhDthesis, 2004. National University of Ireland, Maynooth, Ireland.

Rudolph J, Kimble M, Hoyle H, Subler M, Raff E. Three Drosophilabeta tubulin sequences: a developmentally regulated isoform(beta 3), the testis-specific isoform (beta 2), and anassembly-defective mutation of the testis-specific isoform(B2t8) reveal both an ancient divergence in metazoan isotypesand structural constraints for beta tubulin function. Mol CellBiol 1987;7:2231–42.

Samoiloff M, Schulz S, Jordan Y, Denich K, Arnott E. A rapid simplelong-term toxicity assay for aquatic contaminants using thenematode Panagrellus redivivus. Can J Fish Aquat Sci1980;37:1167–74.

Sherry J, Scott B, Dutka B. Use of various acute, sublethal and earlylife stage tests to evaluate toxicity of refinery effluents. EnvironToxicol Chem 1997;16:2249–57.

Sivakumar S, Subbhuraam V. Toxicity of chromium(III) andchromium(VI) to the earthworm Eisenia fetida. EcotoxicolEnviron Saf 2005;62:93–8.

Stearns D, Wetterhahn K. Reaction of Chromium(VI) withAscorbate Produces Chromium(V), Chromium(IV), andCarbon-Based Radicals. Chem Res Toxicol 1994;7:219–30.

Tajima F. Statistical methods to test for nucleotide mutationhypothesis by DNA polymorphism. Genetics1989;123:585–95.

67S C I E N C E O F T H E T O T A L E N V I R O N M E N T 4 0 4 ( 2 0 0 8 ) 5 6 – 6 7

Tatara C, Newman M, McCloskey J, Williams P. Predicting relativemetal toxicity with ion characteristics: Caenorhabditis elegansLC50. Aquat Toxicol 1997;39:279–90.

Ura K, Kai T, Sakata S, Iguchi T, Arizono K. Aquatic acute toxicitytesting using the nematodes Caenorhabditis elegans. J Health Sci2002;48:583–6.

von Burg R, Liu D. Chromium and hexavalent chromium. J ApplToxicol 1983;13:225–30.

Vranken G, Heip C. Toxicity of copper, mercury and lead to amarine nematode. Mar Pollut Bull 1986;17:453–7.

White G. A method for obtaining infective nematode larvae fromcultures. Science 1927;66:302.

Williams P, Dusenbery D. Aquatic toxicity testing using thenematode Caenorhabditis elegans. Environ Toxicol Chem1990;9:1285–90.

Yoder C, Grewal P, Taylor A. Rapid age-related changes in infectionbehaviour of entomopathogenic nematodes. J Parasitol2004;90:1229–34.