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European Journal of Soil Biology 61 (2014) 80e89

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European Journal of Soil Biology

journal homepage: http: / /www.elsevier .com/locate/ejsobi

Original article

Effects of metal pollution on survival and physiological responsesin Carabus (Chaetocarabus) lefebvrei (Coleoptera, Carabidae)

F. Talarico a, P. Brandmayr a, P.G. Giulianini c, F. Ietto a, A. Naccarato b, E. Perrotta a,A. Tagarelli b, A. Giglio a,*

aDipartimento di Biologia, Ecologia e Scienze della Terra, University of Calabria, Via P. Bucci, I-87036 Arcavacata di Rende, ItalybDipartimento di Chimica e Tecnologie Chimiche, University of Calabria, Via P. Bucci, I-87036 Arcavacata di Rende, ItalycDipartimento di Scienze della Vita, University of Trieste, Via Giorgieri 5, I-34127 Trieste, Italy

a r t i c l e i n f o

Article history:Received 27 November 2013Received in revised form22 January 2014Accepted 6 February 2014Available online 25 February 2014Handling editor: Stefan Schrader

Keywords:Ground beetleLife cycleBioaccumulationImmunoecology

* Corresponding author. Tel.: þ39 0984 492982; faE-mail address: anita.giglio@unical.it (A. Giglio).

http://dx.doi.org/10.1016/j.ejsobi.2014.02.0031164-5563/� 2014 Elsevier Masson SAS. All rights res

a b s t r a c t

The aim of this study was to evaluate the effects of heavy metal pollution on life cycle traits andphysiological responses of Carabus (Chaetocarabus) lefebvrei Dejean 1826. The bioaccumulation factorrevealed that As and Hg were bioaccumulated in adults collected in the field close to an urban solid wastesite, a source of heavy metal pollution. The ultrastructure of Malpighian tubules was investigated as amarker of adult detoxification capability. The metal contents recorded in adults emerging from labora-tory rearing indicate that the uptake of metals or their transfer along the food chain is related to theconcentrations in soil and food. The levels of elements such as B, Cr and Cu in the beetle body are closelyrelated to their amounts in the soil. Some elements are accumulated with respect to the levels in food, inthe following rank order: Hg > Cr > B > Be > Pb > V ¼ Zn ¼ As. Larval growth and survival do not varysignificantly with the metal concentrations recorded in soil and food. The immune response, indicated byphenoloxidase enzyme activity, is highly sensitive to environmental pollution in the preimaginal stagesand thus can be used as an early warning parameter to assess the sublethal effects of heavy metalpollution.

� 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

Metals introduced into the environment as a result of humanactivity have been implicated in ecotoxicological effects on thenatural environment. Terrestrial invertebrates living in close con-tact with the soil, such as arthropods, gastropod molluscs andearthworms, have been studied as soil pollution indicators [1e3].Data on the effects of exposure to pollution have shown that thebiological responses of the organism depend on the pollutantconcentration and its physical and chemical properties, as well ason the pedologic properties of soil. On the other hand, the toler-ance, adaptation or sublethal responses to pollution are closelyrelated to the trophic level of the species, its physiological prop-erties (metal uptake, elimination and immobilization ability) andits life stages [1,2,4,5]. The responses to metal pollution have beeninvestigated from the molecular to the ecosystem level. At the celllevel, metal sequestration involves cytoplasmic processes such as

x: þ39 0984 492986.

erved.

the formation of inclusion bodies and the induction of metal-binding proteins (e.g. metallothioneins) [6]. At the organism level,the high concentration of metals in the habitat may affect thebehaviour [7], survival, reproduction rate and growth [8,9].

Carabid beetles are well known ecologically and taxonomicallyand they are sensitive to environmental changes [10e12]. Severalspecies have been used to evaluate metal contamination in thefield as non-specialized predators and second-order consumers inthe food web. Many researchers have shown that they have goodpotential as biological indicators of bioaccumulation [2,9,13e18]and toxicant effects (toxicological and ecological indicators) [19e24]. However, compared with other terrestrial invertebrates,carabid beetles seem to be poor accumulators of heavy metals dueto their efficient detoxification mechanisms. Therefore, the bodyconcentration of a contaminant cannot be used as an indicator ofexposure levels, and toxic effects of metals on individuals are notexpected. Nevertheless, recent studies have shown that metals canaffect physiological traits of an organism even if it is able toimmobilize and remove the pollutant [24]. Hence, further studiesare needed to provide data on carabid beetle reactions to pollutantexposure.

F. Talarico et al. / European Journal of Soil Biology 61 (2014) 80e89 81

The aim of the study was to analyze the impacts of heavy metalson different ecological and biological levels from the food web levelto the enzymatic level. The model organism was Carabus (Chaeto-carabus) lefebvrei Dejean, 1826 since this wingless and low-mobileground beetle cannot escape from the sub-optimal environmentalconditions [25]. Moreover, this species is helicophagous and thuspotentially exposed to pollutants accumulated in tissues of theprey. In this study, the biological impact of heavy metals on thisspecies was assessed both in the field and in the laboratory. In fieldconditions, metal bioaccumulation and the ultrastructure of Mal-pighian tubules (a marker of detoxification ability by excretion)were tested in adults inhabiting a sampling area close to an urbanmunicipal solid waste dump. Laboratory rearing was performed toanalyze the transfer of metals through the trophic levels and to testthe effects of metal toxicity on survival and developmental timeand on immune function, indicated by phenoloxidase enzymeactivity.

2. Materials and methods

2.1. Study area

The study area was a site for the disposal of solid urban wastelocated about 1.5 km from the town of Decollatura (39�03’1300N,16�2204000 E, 1410 m a.s.l.; Calabria, southern Italy) (Fig. 1). The area,unused for about 10 years, was covered with a thick layer of locallyobtained inert material. It was completely lacking in cover vege-tation and was without any controlled channelling of rainfall. As aresult, surface run-off has eroded large rills, with consequentexposure of waste materials, mainly along their sidewalls. There isno system of collection or stocking of the resulting percolate, norany systems to reduce biogases from decomposition of the organicfraction of the waste. In these conditions, the pollution hazard ishigh for the area surrounding the site, with possible impacts on thelocal ecosystem. The lithology of the area consists of a substrate ofmetamorphic rocks belonging to the Unità di Bagni [26,27],

Fig. 1. Map of Calabria shows the study area. P ¼ sampling area lo

composed of grey phyllitic schists with quarzitic intercalations. It isa lithoid complex with strong resistance to erosion and lowpermeability. The lithoid substrate is covered by a substantial layerof detritic deposit originating from weathering of the crystallinecomplex of the basement, composed of phyllitic clasts in a sandy-loam matrix with a clayey component. The sandy-loam textureprovides high permeability and good drainage of the horizons.Conifers and broad-leaved trees such as oak, pine, alder, poplar andchestnut are present in the surrounding area.

A survey of metal concentrations in the soil of forests sur-rounding the urban waste site was performed. Sampling was con-ducted in April 2009 and soil cores were taken to a depth of about20 cm from the field close to the urban waste site (Fig. 1). Fivesamples were collected 40 m apart (site P, about 500 m2) in theforest located on the slope next to the disposal site, 20e50 m fromthe site boundary. The sampling in the polluted area was plannedon the basis of the potential transport of pollutants from the urbanwaste disposal site by means of both infiltrating waters and surfaceflow.

The control site (site C), similar in lithological and vegetationalcomposition, was chosen in a relatively clean habitat (pine forest)30 km north-east of the polluted site (39�190 N, 16�70 E, 900e1000 m a.s.l.; San Fili, Calabria, southern Italy) (Fig. 1).

2.2. Animal rearing

To investigate the effects of exposure to heavy metals on the lifehistory parameters of C. lefebvrei, we hand-collected males andfemales from sites P and C in early spring 2010. In the laboratory,the beetles were separated by site (polluted and control), sexed andkept in groups (males and females) in 10 L plastic boxes filled to adepth of 6 cmwith moistened humus from the capture areas. Aftercopulation, males were removed from the boxes to reduce thedisturbance to females, which readily laid eggs. To expose the F1generation to experimental treatments, we immediately trans-ferred single eggs into 150 mL glass jars filled to a depth of 5 cm

cated in the slop down of the urban waste; C ¼ control site.

F. Talarico et al. / European Journal of Soil Biology 61 (2014) 80e8982

with moistened humus from the adult capture areas (sites P and C).Egg production and larval developmental timewere recorded everytwo days for both the polluted and control rearing groups. Allspecimens were reared with a light regime of L/D¼ 15/9 h, 70% RH,a day/night room temperature of 23/18 �C, and a diet of snails (Helixpomatia). The animals from the polluted and control groups wereused for survival and development tests, chemical and morpho-logical analyses and enzymatic assays.

2.3. Chemical analyses of soil and animals

Metal concentrations in animals and in soil were measured byinductively coupled plasma-mass spectrometry (ICP-MS). The de-terminations were carried out with an Elan DRC-e ICP-MS in-strument (PerkinElmer SCIEX, Canada) and the sample deliverysystem consisted of a PerkinElmer model AS-93 Plus autosamplerwith peristaltic pump and a cross-flow nebulizer with a Scott typespray chamber. The ICP torch was a standard torch (Fassel typetorch) with platinum injector. A solution containing Rh, Mg, Pb, Baand Ce (10 mg/L, Merck) was used to optimize the instrument interms of sensitivity, resolution and mass calibration. A multi-element solution of Ag, Al, As, Ba, Be, Bi, Ca, Cd, Co, Cr, Cs, Cu,Fe, Ga, In, K, Li, Mg, Mn, Na, Ni, Pb, Rb, Se, Sr, Tl, V, U and Zn(100 mg/L, Merck) and single element solutions of B, Mo and Hg(100 mg/L, Merck) were used to prepare aqueous calibrationstandard solutions after appropriate dilution. An Anton PaarMultiwave 3000 with programmable power control (maximumpower 1400 W) and XF100 rotor (operating pressure up to 120 barmaximum; operating temperature 260 �C maximum; constructionmaterial PTFE-TFM for the liner) was used for the microwavedigestion of the samples.

Preparation of each soil sample involved separation from theorganic component, drying at 50 �C for 24 h, grinding in a me-chanical mill with agate spheres, and solubilisation of 100 mg ofpowder in a mixture of hydrofluoric acid (HF, 6 mL), nitric acid(HNO3, 5 mL) and perchloric acid (HClO4, 3 mL). The resulting so-lution was put in a microwave oven with the following minerali-zation programme: 60 min at 1200 W; 30 min at 600 W; 15 mincooling. The mineralized sample was brought to a volume of100 mL by adding ultra-pure water and then analyzed with the ICP-MS. The quantitative determination of elements was carried outwith external standards. Eight-point calibration curves coveringthe range 0.1e2000 mg/L were used. Standard solutions were pre-pared by diluting the above-mentioned multi-element solutions.

Heavy metal concentrations in animals were measured in: i)adults (n ¼ 5) from site P; ii) emerging adults reared on contami-nated soil from site P (n ¼ 5) and on uncontaminated control soilfrom site C (n ¼ 5); iii) snails (n ¼ 5) (H. pomatia) from the popu-lation used as food for the larval stages. All specimens were dried at105 �C and each animal was directly weighed in the liner of themicrowave system. The digestionwas performed by adding 3 mL ofHNO3, 3 mL of H2O2 and 1 mL of H2O to each sample. The operatingconditions for the microwave digestion systemwere the following:720 W for 20 min. After digestion the extracts were quantitativelytransferred into a graduated polypropylene test tube and dilutedwith ultrapure water to 50 mL.

Metal accumulation in carabids from site P was estimated as thebioaccumulation factor under field conditions (BAF), i.e. the ratiobetween the metal concentration in the beetle body (in mg KgL1

dry weight) and that in the soil (in mg KgL1 dry weight). The BAFwas used to classify C. lefebvrei as a macroconcentrator (BAF > 2),microconcentrator (1 < BAF < 2) or deconcentrator (BAF < 1) asproposed by Dallinger [28].

To assess the metal uptake by contact or food routes in thelaboratory, we compared the concentration of each metal: a)

between emerging adult beetles reared on soil from sites P(polluted) and C (control) used as substrate, and b) betweenemerging adult beetles and their prey (H. pomatia specimens usedas food for the larval stages). The increase of pollutant concentra-tions along the trophic chainwas estimated as the biomagnificationfactor under laboratory conditions, i.e. the ratio between the con-centrations of chemicals in predators (C. lefebvrei adults emergingfrom laboratory rearing) and in their prey.

2.4. Transmission electron microscopy

An ultrastructural analysis of the adult hindgut was performedto evaluate decontamination processes by means of discharge ofabsorbed metal into the lumen of the digestive tract. Malpighiantubules from polluted and control adults were removed from thealimentary canal and immediately immersed in 2.5% glutaralde-hyde, 1% paraformaldehyde and 7.5% picric acid in 0.1 M cacodylatebuffer, pH 7.4, with 1.5% sucrose, and were fixed for 2 h at 4 �C. Theywere then rinsed in cacodylate buffer, post-fixed with 1% osmiumtetroxide in 0.1 M cacodylate buffer for 1 h at 4 �C and rinsed in thesame buffer. Dehydration in a graded acetone series was followedby embedding in Epon resin (Electron Microscopy Sciences, Inc.).Thin sections, cut with a Leica Ultracut UCT ultramicrotome, werestainedwith uranyl acetate and lead citrate and viewedwith a ZeissEM10 electron microscope at 60 kV. For transmission electronmicroscopy, images were acquired with a Veleta e 2k _ 2k side-mounted TEM CCD Camera (Olympus, Germany) provided withan iTEM imaging platform and saved in JPEG format. Measurementswere taken with Image-Pro Plus version 4.5 software (Media Cy-bernetics) on digitised images and processed as means � standarderror.

2.5. Phenoloxidase (PO) assay

To establish the toxic responses at the molecular level, weassessed the effects of metals on the immune response bymeasuring the amount of phenoloxidase (PO) in hemolymph. Theanimals were CO2 anesthetized before hemolymph collection. He-molymph was collected from pupae and larvae by puncturing thesoft cuticle (29-gauge needle) between the second and third dorsalabdominal segment at the level of the hemolymphatic vessel.Adults were punctured at the ventral level of the pro-mesothoraxarticulation. The first droplet of hemolymph was collected. 5 mL ofwhole hemolymph were diluted with 95 mL of 10 mM sterilephosphate buffered saline (PBS, Sigma) and centrifuged at 5000 g at4 �C for 5 min. 40 mL of hemolymph-buffer supernatant were takenand mixed with 160 mL of DL-DOPA (3,4-dihydroxy-DL-phenylala-nine, Sigma; 3 mg/mL phosphate buffer) in a microtiter plate. POactivity at 25 �C was measured at 492 nm for 30 min in 5-min in-tervals using a plate reader (Sirio S, SEAC). Enzyme activity wasexpressed as absorbance units representing an absorbance per mL ofhemolymph. The animals used for these analyses were last (3rd)instar larvae, 10-day-old pupae and 10-day-old adults reared onpolluted and control soil.

2.6. Statistical analyses

All statistical analyses were performed using R version 2.13.1software (R Development Core Team 2011).

Differences in individual survival were assessed using therearing soil as the variable (polluted soil from site P versus controlsoil from site C). For the survival analysis, a numerical value wasgiven to each stage: from egg¼ 1 stage equivalent to adult¼ 6 stageequivalent. Both exponential and Weibull parametric models withcensoring were chosen because some individuals outlived the

F. Talarico et al. / European Journal of Soil Biology 61 (2014) 80e89 83

experiments. After ANOVA comparison, the distribution showingthe error structure with the minimum error deviance was chosen.

For calculation of the metal transfer from soil fraction to beetles,the metal concentrations of emerging animals reared on the humusfrom the control and polluted sites were checked for normalitywith the ShapiroeWilk test and the homogeneity of variance waschecked with the Bartlett test. The differences were assessed bynon-parametric statistics, i.e. ManneWhitneyeWilcoxon test, sincethe null hypothesis of the ShapiroeWilk and/or the Bartlett testcould not be rejected. Due to the small sample size (n ¼ 5), we alsoran the Welch two sample t-test and we took into account onlydifferences that were significant in both the parametric and non-parametric tests. The box and whiskers plots were drawn withthe boxplot command.

Differences in metal concentrations between food (snail) andbeetles reared on humus from the adult capture areas (sites P andC) were assessed by non-parametric statistics, i.e. KruskaleWallisfollowed by pairwise comparisons using the Wilcoxon rank sumtest with Bonferroni adjustment.

Differences in plasmatic PO activity were assessed between F1larvae, pupae and adults reared on humus from the adult captureareas (sites P and C). The increase of absorbance units per min islinear within 30 min and the slope of the calculated linearregression represents the Vmax of the enzymatic activity. Theabsorbance units at different times were plotted for each life stageand their regression lines were calculated. Differences in slopes ofregression lines showing the PO activity as absorbance units permin were tested by analysis of covariance (ANCOVA).

3. Results

3.1. Trace elements in the soils

It can be derived from the chemical analyses that at site Pseveral heavy metal concentrations are above the limits for publicgreen areas as established by current Italian legislation, e.g. Be, Hg,V and Zn, (Table 1). Even at the control area (C) Cr is close to thelimits. However, the naturally high concentration of Be, Tl and V inthe soil in the study area can be attributed to outcropping surfacelayers.

Table 1a) Heavy metal content (mg kg�1 dry weight; mean � SE) found in the soil samplesfrom the study sites P and C (see Fig. 1). b) Limits specified in the Italian guideline onthe admitted concentration of the heavy metals in soil.

Metals a) b)

P (n ¼ 5) C (n ¼ 3) Limits for publicgreen use(Law 152/06)

Limits forindustrial use(Law 152/06)

Ag 0.92 � 0.05 0.20 � 0.02 # #As 0.61 � 0.24 7.99 � 1.16 20 50B 25.02 � 5.97 5.42 � 0.30 # #Be 5.16 � 0.35 0.55 � 0.05 2 10Bi 0.60 � 0.05 12.50 � 2.30 # #Cd 0.94 � 0.34 0.05 � 0.005 2 15Co 12.23 � 0.85 16.31 � 1.54 20 250Cr 65.58 � 7.95 118.98 � 13.84 150 800Cu 29.44 � 2.16 20.36 � 1.87 120 600Hg 1.66 � 0.93 0.34 � 0.10 1 5Mo 1.09 � 0.10 0.03 � 0.004 # #Ni 28.65 � 3.51 46.82 � 4.03 120 500Pb 32.65 � 1.88 3.67 � 0.60 100 1000Sr 76.88 � 8.29 19.67 � 0.61 # #Tl 1.30 � 0.16 0.24 � 0.02 1 10V 178.10 � 10.31 57.17 � 6.56 90 250Zn 157.31 � 10.21 36.09 � 2.04 150 1500

#, No legal requirement.

3.2. Metal analyses in invertebrates

The trace element levels found in specimens from the C. lefebvreipopulation living in the forest are the same as those in the forestsoil except Ag and Bi (Table 2). The BAF values for adults from site Pshow that this species is a macroconcentrator for As (BAF ¼ 61.07)and a microconcentrator for Hg (BAF ¼ 1.50).

Laboratory rearing was carried out to evaluate metal uptakefrom direct contact with humus during the growth phase and fromfood. The metal concentrations are compared in experimental an-imals (control and polluted groups) and in snails used as food forthe larval stages (Fig. 2). Significantly higher concentrations of B, Cdand Cu were recorded in carabids reared on humus from site P thanin those reared on humus from site C (ManneWhitneyeWilcoxontest, p < 0.05 and Welch two sample t-test, p ¼ 0.05 for B; ManneWhitneyeWilcoxon test, p < 0.01 and Welch two sample t-test,p < 0.05 for Cd and Cu). Moreover, the concentrations of Cd, Co, Cu,Sr and Tl are significantly lower in reared carabids than in snailsused as food for the larval stages (Fig. 2; KruskaleWallis rank sumtest, p < 0.02; pairwise comparisons using Wilcoxon rank sum test,p ¼ <0.048). On the other hand, we recorded significantly highervalues of Hg in beetles than in snails (KruskaleWallis rank sum test,p ¼ 0.014; pairwise comparisons using Wilcoxon rank sum test,p¼ 0.024), indicating a clear biomagnification for this element. Thistrend is confirmed by the biomagnification values resulting fromtrophic transfer (Table 3). The biomagnification factors of accu-mulated metals increase in emerging beetles in the following or-der: Hg > Cr > B > Be > Pb > V ¼ Zn ¼ As. The differences inbiomagnification values between polluted and control beetles arerelated to the metal concentrations in the substrate.

3.3. Ultrastructure of Malpighian tubules

The numerous Malpighian tubules of C. lefebvrei adults open inthe posterior end of the midgut and are about 70 mm in diameter.The tubulewall consists of a single layer of cuboid cells surroundinga central lumen; the cells have a large nucleus about 10 mm indiameter resting on the basal lamina (Fig. 3A). Tracheoles are foundalong the length of the tubules. Cross sections reveal that the cellshave numerous well-developed infoldings of the plasma mem-brane at the basal surface (Fig. 3B) and a brush border at the lumensurface consisting of regular closely packed microvilli (Fig. 3A).Basal infoldings and apical microvilli (about 0.26 mm in diameter)are in close association with many elongated mitochondria about

Table 2Heavy metal content in the body (mg kg�1 dry weight; mean � SE, n ¼ 5) ofC. lefebvrei (BF) adult from site P. BAF: bioaccumulation factor under field conditionsindicating the ratio between the metal concentration in the beetle body and that inthe soil (mg kgL1 dry weight) from site P (Table 1a).

Metals BF BAF

As 37.01 � 20.35 61.07B 2.04 � 0.53 0.082Be 0.002 � 0.001 0.0004Cd 0.01 � 0.003 0.01Co 0.02 � 0.01 0.002Cr 0.34 � 0.03 0.005Cu 4.49 � 0.35 0.15Hg 2.49 � 0.88 1.50Mo 0.02 � 0.004 0.02Ni 0.12 � 0.02 0.004Pb 0.02 � 0.01 0.0006Sr 0.79 � 0.25 0.01Tl 0.003 � 0.0003 0.01V 0.07 � 0.01 0.0004Zn 34.87 � 2.77 0.22

Fig. 2. Heavy metal content in C. lefebvrei adults emerging from the laboratory rearing on soil from P (poll.: polluted) and C sites (ctrl) and feed with snails; Snails: H. pomatiaspecimens from the population used as food (mg kg�1 dry weight; mean � SE, n ¼ 5).

F. Talarico et al. / European Journal of Soil Biology 61 (2014) 80e8984

3 mm in length. In adults from site P (Figs. 3CeF and 4AeE), themost conspicuous feature of these cells is the presence of largelysosomes with dense dark metal precipitation on their surface(Figs. 3C and 4A,B) and spherical laminated concretions (Figs. 3DeFand 4E). Both structures arise from the Golgi complex (Fig. 4A) orthe rough endoplasmic reticulum (Fig. 3E, F). The laminate con-cretions are surrounded by a membrane and are 0.53 � 0.01 mm indiameter (n ¼ 88) (Fig. 3E, F and 4E). Some cells show clumping ofthe nuclear chromatin (Fig. 3C) and mitochondrial swelling withloss of cristae (Fig. 4BeD).

Table 3Biomagnification factor (BMF) measured as the ratio between the concentration of metalfrom laboratory rearing (polluted: beetles reared on soil from site P, control: beetles rearethe larval stage).

As B Be Cd Co Cr Cu

Polluted 0.96 1.70 1.25 0.15 0.46 0.54 0.36Control 0.68 0.20 1.39 0.01 0.54 1.82 0.18

3.4. Development and survival tests

The metal contents of the soil used as substrate in rearing testsdid not affect the survival of the beetles. The KaplaneMeier sur-vivorship plot of the death time in days from hatching shows thelongest tail for the polluted soil (Fig. 5a). The ANOVA comparison ofthe exponential and Weibull parametric models revealed that theWeibull model (scale 0.657, hazard decreases with age) is a sig-nificant improvement over the exponential model (p ¼ 2.51e-15).The mean ages at death are 22.03 � 1.13 and 22.78 � 1.20 days for,

in the body (mg/kg dry weight; mean � SE; see Fig. 2) of C. lefebvrei adults emergingd on soil from site) and H. pomatia (specimens from the population used as food for

Hg Mo Ni Pb Sr Tl V Zn

2.23 0.55 0.50 1.08 0.005 0.28 1.05 1.033.60 0.64 0.82 1.72 0.003 0.49 1.06 0.97

Fig. 3. Transmission electron micrographs of cross sections through the proximal segment of the Malpighian tubules. (A) Cells having brush border formed by regular closely packedmicrovilli and many elongated mitochondria in the control adults. (B) Detail of numerous well-developed basal infoldings of the plasma membrane in control adults. (C) Cells ofMalpighian tubules in the animals from site P show clumping of nuclear chromatin. Some aggregates of dense dark material (D, arrowheads) are evident in the cytoplasm as well asenlarged laminated concretions (E, F) surrounded by membrane. bl: basal lamina, c: laminate concretions; in: basal infoldings, m: mitochondria, mi: microvilli n: nucleus Bars 5 mm(A), 1 mm (B), 2 mm (C, D), 0.5 mm (E, F).

F. Talarico et al. / European Journal of Soil Biology 61 (2014) 80e89 85

respectively, the control and polluted soil, while those predictedwith the Weibull model are 25.32 and 26.48 days. The survivorshipanalysis shows that the mean stages at death are 2.47 � 0.10 and2.39 � 0.10 stage equivalent for, respectively, the control andpolluted soil, while those predicted with the Weibull model are2.85 and 2.78 stage equivalent (Fig. 5b).

3.5. Phenoloxidase (PO) assay

The PO activity at different stages varies between animals rearedon control or polluted soil (Fig. 6). In the larval stage, animals rearedon polluted soil have significantly lower PO activity than thosereared on control soil (ANCOVA: F3,269, p < 0.001; nCTRL ¼ 19,nPolluted ¼ 20). Pupae from polluted soil also have significantlylower PO activity than controls (ANCOVA: F3,199, p < 0.05;

nCTRL ¼ 16, nPolluted ¼ 13), whereas the enzymatic activity does notdiffer significantly between adults reared on control (n ¼ 8) orpolluted soil (n ¼ 21).

4. Discussion

Our results show that C. lefebvrei has the ability to regulate thelevels of various metals in the body and thus can avoid metalpoisoning in both the adult and larval stages. Carabid beetles usu-ally have the lowest body concentrations of metals among animalscollected in metal-contaminated areas [2,10]. Nevertheless, inC. lefebvrei adults exposed to metal-contaminated habitats, wefound a set of metals that were also present in the forest soildownhill of a municipal solid urban waste site. The high bio-accumulation values recorded for As (BAF ¼ 61.07) and Hg

Fig. 4. Transmission electron micrographs of cross sections through the proximal segment of the Malpighian tubules of animals from site P. The cells show lysosomes containselectron-dense metal precipitation (arrowheads) (A and B), mitochondrial swelling with loss cristae (B, C, D; arrows) and laminate concretions (E; c). g: Golgi complexes, m:mitochondria, rer: rough endoplasmic reticulum. Bars 1 mm (A, B, D, E), 0.5 mm (C).

Fig. 5. KaplaneMeier survival distributions of the animal reared on different soils showing the day at death (a) and the stage at death (b).

F. Talarico et al. / European Journal of Soil Biology 61 (2014) 80e8986

Fig. 6. Changes of phenoloxidase activity at different stages recorded as absorbanceunits per min in relation to rearing soil, the regression lines are shown.

F. Talarico et al. / European Journal of Soil Biology 61 (2014) 80e89 87

(BAF ¼ 1.50), two biologically toxic elements, suggest that thisspecies is a good indicator of As and Hg in the environment. TheMalpighian tubules are the most suitable structure to evaluateheavy metal hazards as biomarkers for risk assessment in envi-ronments [28e33] due to their involvement in excretion andosmoregulation. Our ultrastructural analysis of the cell tubules ofC. lefebvrei adults exposed to pollution in the field shows thatpotentially toxic metals can be safely stored in intracellular com-partments, such as lysosomes, in an insoluble and physiologicallyinactive form [28,29]. Moreover, strong ultrastructural variationsoccurring in mitochondria and nuclei are markers of cellular injury.Mitochondrial swelling reflects the entry of solutes and water intothe matrix due to metals such as Cd, Hg and As acting on mito-chondrial membrane transport. The chromatin clumping in nucleisuggests progressive inactivation of the nuclear component [34].

This ground beetle is a specialized snail predator in both theadult and larval stages. Since snails are metal concentrators [28],we can suppose that the rate of metals accumulated in the beetlebody is related to an increase of metal concentration in its diet.However, many studies have demonstrated the complexities ofpredicting the biotransfer of metals in terrestrial ecosystems. Theuptake and accumulation of metals depend not only on the natureof soil contamination but also on several soil- and organism-relatedvariables and on many biological factors (species, gender, age,weight, tissue). Our laboratory tests suggest that the metal contentsrecorded in the body of young imagines from the first laboratorygeneration depend on the route of uptake of the chemical elements.The significant differences in B, Cd and Cu concentrations recorded

in beetles reared from larval stages on polluted and control sub-strates and fed with snails from the same population show thatcontact with the substrate plays an important role in bio-accumulation of these metals. This is due to the high mobility oflarvae toward the substrate and the high surface area/body volumeratio. The biomagnification values recorded in emerging beetlesshow that ingestion influences the uptake of As, B, Be, Cr, Hg, Pb, Vand Zn and the preimaginal developmental phase is the mainexposure period for metal transfer along the food chain in thisspecialized predator. However, the ability of C. lefebvrei to accu-mulate metals is not related only to the uptake route and the bio-logical role of each metal; their doses must also be taken intoaccount. In general, the nutritionally essential elements seem to beregulated more efficiently than the non-essential ones [13,24].Moreover, metal kinetic studies in carabids indicate that differencesare probably due to the physiological capabilities of species fordetoxification by metal excretion [13,35]. Several authors havesuggested that species-specific differences in capability for metalassimilation and excretion are more important than the trophiclevel. A similar trend is evident for many investigated species ofcarabid beetles which are able to discharge assimilated metals byexcretion when their internal concentration exceeds a certainthreshold [9,10,17,36].

Studies on Diptera, Lepidoptera, Hymenoptera and Coleopterashowed that moulting and metamorphosis lowered the metalcontents in the investigated species [37e39]. The larval growth ofC. lefebvrei involves two moults and this process can help in theremoval of metals discharged into the midgut epithelium andcuticle. This detoxification process involves a low energy cost andremoves metals that can affect the survival and larval develop-mental time. As a result, the synergic effect of a combination ofmetals in both the food and soil exposure routes does not act onC. lefebvrei ontogeny; indeed, no significant differences wererecorded in the survival of animals reared on polluted and controlsoil. Nevertheless, in carabid beetles, different life traits such assurvival, growth and reproduction may be differently sensitive totoxicants [13,22,40e43].

Although C. lefebvrei has efficient detoxification mechanisms toneutralize the lethal effect of metal pollution in order to preservethe species, our study shows that metal contamination has suble-thal effects on immune function. The accumulation of metals caninteract with the immune capacity and affect the health of in-vertebrates [44e47]. We spectrophotometrically quantified theactive PO inside the cell-free hemolymph, measuring the conver-sion of the substrate DL-DOPA into dopachrome, and we comparedthe enzymatic activity among the life stages. The significantly lowerPO activity in larvae reared on polluted soil had negative conse-quences for C. lefebvrei fitness. Moreover, PO is a key chemical in thecascade of reactions ultimately leading to melanisation (cuticlepigmentation), moulting, tissue repair and defence against patho-gens [48e51]. Therefore, the response of PO to environmentalcontamination can be a reliable biomarker of exposure to physio-logical stress in organisms living in polluted habitats.

5. Conclusions

Environmental risk assessment requires more informative andefficient test systems including species with sensitive life stages.This study focused on the value of C. lefebvrei as a toxicologicalindicator according to the indications of Cortet [1]. This species iseasy to collect in the field and to maintain in the laboratory. Its roleas a specialized snail predator in both the adult and larval stagesmakes it suitable for the monitoring of pollution levels of somemetals in the food web of forest ecosystems. The results of thisstudy indicate that, in terms of accumulation rates and resistance to

F. Talarico et al. / European Journal of Soil Biology 61 (2014) 80e8988

metals, both adults and larvae of this species have efficient mech-anisms of detoxification to prevent negative effects on life traits andindividual fitness. Moreover, its immune response can be used as ahighly sensitive early warning parameter to assess the sublethaltoxic effects of heavy metal pollution when such effects are notevident in conventional bioassays such as food consumption,reproductive rate, survivorship and growth.

Acknowledgements

We thank the anonymous reviewers for their helpful commentsand suggestions, which significantly contributed to improve thequality of the paper. This research was supported by grantsassigned to A. Giglio from the Ministry of Education, University andResearch (MIUR).

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