CADMIUM EFFECTS ON THE RATIO OF ACTIVITIES OF LYSOSOMAL AND TOTAL ACID PHOSPHATASES ( ACPLYS / ACPTOT ) IN lYMantRia DisPaR LARVAE

Searching for novel molecular biomarkers, we investigated cadmium effects on the ratio of specific activities of lysosomal and total acid phosphatases (ACPLys/ACPTot) in 4th instar gypsy moth larvae. After acute and chronic exposure to 10 and 30 μg Cd/g dry food, as well as after recovery from both concentrations, the trait values, plasticity, variability and genetic correlations were evaluated. The ACPLys/ACPTot ratio decreased during acute and chronic effects of both concentrations. Inhibition during long-term cadmium exposure was irreversible. Indices of phenotypic plasticity for ACPLys/ACPTot ratio were positive for all cadmium treatments. The variability of plasticity was higher after recovery from 10 μg Cd/g dry food, compared to recovery from 30 μg Cd/g dry food. A significant correlation coefficient was calculated between shortterm cadmium treatments. Significant changes in the ACPLys activity fraction during all treatments indicate the examined trait (ACPLys/ACPTot) could be used as a pollution exposure biomarker.


INTRODUCTION
Cadmium pollution has increased over the past decades as a result of anthropogenic activities.It is a non-degradable heavy metal that can be accumulated in animal tissues causing disruptions at all levels of organization.Cadmium was shown to reduce growth and longevity and to affect the development in various insect species, such as Epirrita autumnata (Van Oik et al. 2007), Lymantria dispar (Vlahović et al., 2009;Mirčić et al., 2010), Folsomia candida (Fountain and Hopkin, 2001), Oncopeltus fasciatus (Cervera et al., 2004).The effects of cadmium on detoxification and antioxidant systems in insects were noticed by Wang and Wang (2009) and Kafel et al. (2012).Planello et al. (2007Planello et al. ( , 2010) ) showed that cadmium exposure could alter the expression of ribosomal genes, as well as heat-shock and hormone receptor genes in Chironomus riparius.Ilijin et al. (2011) found the size of L2 type neurosecretory neurons was increased in cadmium-treated Lymantria dispar larvae.In addition, cadmium impedes cell cycle progression and cell proliferation (Templeton and Liu, 2010), DNA repair (Bertin and Averbeck, 2006) and cell-cell adhesion (Prozialeck et al., 2008).
After exposure, in Orchesella cincta approximately 90% of the cadmium is present in the gut (van Straalen and Roelofs, 2005).This study investigated cadmium effects on the midgut ratio of specific activities of lysosomal and total acid phosphatases (ACP Lys /ACP Tot ).Phosphatases are metalloenzymes that catalyze the hydrolysis of various phosphomonoesters and take part in transphosphorylation. (Calvo-Marzal et al., 2001).Acid phosphatases (ACP) are located mainly in the cytosol of midgut cells of Diptera and Lepidoptera, but can also be membranebound or present in the gut lumen (Terra and Ferreira, 1994).They participate in the final processes of digestion (Cheung and Low, 1975), excretion, water resorption and the mechanisms of active membrane transport, as well as in the cell replacement during apoptosis (Srivastava and Saxena, 1967).Lysosomal acid phosphatases take part in the hydrolysis of various macromolecules inside the lysosomal compartment, which is recognized as a center of heavy metal sequestration (Sterling et al., 2007).Cadmium is known to induce lysosomal damage (Fotakis et al., 2005), to increase lysosomal size and reduce its membrane stability (Lekube et al., 2000).Lysosomal enzyme release assay is generally considered as a biomarker of exposure to toxicants (Fotakis et al., 2005;Nazar et al., 2008).
The aims of this study were (i) to investigate the effects of cadmium exposure on the ratio of specific activities of lysosomal and total ACP (ACP Lys /ACP-Tot ) and to evaluate the possibility of using the trait as a biomarker of exposure; (ii) to study the variance and plasticity of the ACP Lys /ACP Tot ratio, and (iii) to evaluate the correlations of the ACP Lys /ACP Tot ratio across cadmium treatments.

Experimental animals and rearing conditions
Female gypsy moths lay a single egg mass that is the product of a single mating.All larvae hatched from a single egg mass represent full sibs.Twenty egg masses were collected from the poplar forest at Opovo (45°03´49"N and 20°27´26"E).Egg masses were kept at +4°C until they were allowed to hatch.Larvae were reared individually in plastic cups on a high wheat germ diet (HWG) (Bell et al., 1981;Odell et al., 1984) at 23°C with a 12 h light/dark photoperiod.They were fed daily.

Cadmium treatments
Ten larvae from each of the twenty egg masses were randomly selected and assigned to seven experimen-tal groups.These are as follows: 1 -C or control larvae that were not exposed to cadmium; 2 and 3 -Ac1 and Ac2 or larvae acutely exposed to two cadmium concentrations (10 and 30 µg Cd/g dry food, respectively); after molting into the 4 th instar they were fed the cadmium diet for three days; 4 and 5 -Chr1 and Chr2 or larvae chronically exposed to two cadmium concentrations (10 and 30 µg Cd/g dry food, respectively); these were provided a cadmium diet from hatching until sacrifice; 6 and 7 -Rec1 and Rec2 or larvae recovered after chronic exposure to two cadmium concentrations (10 and 30 µg Cd/g dry food, respectively); these were provided the cadmium diet from hatching until molting into the 4 th instar and then fed a control diet for three days until sacrifice.The cadmium test concentrations were based on the active component in Cd(NO 3 ) 2 •4H 2 O.

Preparation of midgut homogenates
Larvae were weighed and killed on ice by decapitation on the third day of the 4 th instar.The midguts were removed and kept at -20°C until homogenized.The homogenization was done in ice-cold saline 0.15M NaCl, followed by 10 min centrifugation at 10,000 x g.Supernatants were used for enzyme assays.For each pooled homogenate, five midguts from a single egg mass were used.

Enzyme assays
The activity of total acid phosphatases was measured spectrophotometrically by the modified method of Nemec and Socha (1988) based on the dephosphorylation of p-nitrophenyl phosphate (pNPP) which is a general substrate of phosphatases.The reaction was performed at 30°C in a mixture containing 100 mM citrate buffer pH 5.6, 5 mM MgCl 2 , homogenate and 5 mM pNPP.After 30 min, the reaction was stopped by the addition of 0.5 M NaOH, and the absorbance of the enzyme reaction product, p-nitrophenol, was measured at 405 nm.Lysosomal acid phosphatase activity was determined indirectly, by calculating the difference between the activities of total acid phosphatases and nonlysosomal phosphatases.The reaction mixture was the same as described above with the addition of 50 mM NaF, which is the specific inhibitor of lysosomal acid phosphatases.Protein concentrations were estimated according to the method of Bradford (1976).

Statistical methods
Mean values (MV) and standard errors of mean values (±SE) were calculated for the ratio of specific activities of lysosomal and total ACP.To evaluate the cadmium influence on the ACP Lys /ACP Tot ratio , oneway ANCOVA was used with the larval mass as covariate.The analysis of variance was carried out on the arc sin-transformed values of the trait.
Phenotypic plasticity (PP) for each egg mass was expressed by an index of PP, which was calculated according to the formula of Cheplick (1995): where Xc is the ACP Lys /ACP Tot ratio in the pooled homogenate from a single egg mass during control treatment, while Xt refers to cadmium treatment.Wilcoxon's test was used for comparing the PP of the trait between different cadmium treatments, and significance in index variability was estimated by the Ftest.Z-test was used for comparisons of correlation coefficients between different environments.Differences were considered significant when p<0.05 was achieved.

RESULTS
Fig. 1 shows the ratio of specific activities of lysosomal and total ACP (ACP Lys /ACP Tot ).During acute and chronic effects of both cadmium concentrations there were significant differences in relation to the control.Inhibition of the lysosomal fraction during long-term exposure to the effect of cadmium was irreversible considering that the activities did not return to the control level during a three-day recovery period.One-way ANOVA showed that the ACP Lys / ACP Tot ratio significantly depended on the cadmium concentrations during all treatments (Table 1).The indices of phenotypic plasticity for the ACP Lys /ACP-Tot ratio were positive for all cadmium concentration treatments (Table 2).The variability of plasticity was higher after recovery from 10 µg Cd/g dry food compared to recovery from 30 µg Cd/g dry food (Table 3).A statistically significant correlation coefficient was calculated between short-term cadmium treatments at 10 and 30 µg Cd/g dry food (Table 4).

DISCUSSION
The decrease in the ACP Lys /ACP Tot ratio during short-and long-term stress at both cadmium concentrations, as well as the failure of the lysosomal phosphatase fraction to recover from inhibition during chronic treatment, show that this enzyme is susceptible to cadmium toxicity.This finding is expected considering that lysosomes are known to respond intensely to heavy-metal stress.According to Fotakis et al. (2005), cadmium-induced lysosomal damage is one of the earliest events in the cell stress response, preceding mitochondria and DNA damage.It includes the permeabilization and disintegration of lysosomes (Messnera et al., 2012), leading to content leakage and pH changes.Consequently, the ACP Lys activity fraction might be reduced in such suboptimal conditions.Severe disruption of the lysosomal system could explain the irreversible nature of AC-P Lys inhibition that we have shown in this study.
Being accumulated in lysosomes, cadmium might inhibit ACP Lys directly, due to its high affinity to thiol groups and the forming of cadmium-thiol complexes with proteins (van Straalen and Donker, 1994).Since it is able to replace cations in the metalloproteins (Moulis and Thevenod, 2010), cadmium could target the ACP Lys active site, disabling enzyme function.In addition, it might prevent de novo synthesis of the enzyme.According to Planello and coworkers (2007), cadmium can inhibit the expression of ribosomal genes, consequently leading to the depletion of ribosomes.Stress may cause an energy redirection towards detoxification processes, which can also account for alterations in expression profiles in favor of proteins involved in stress adaptation response.Previously, we found that larval mass did not change during acute cadmium treatment, but de-creased during long-term exposure (Vlahović, 2009).These findings support the assumption of energetic shift during cadmium stress, as life history traits take longer to manifest changes compared to those at the molecular level.
Phenotypic plasticity represents a change of the phenotype of a specific genotype in response to environmental factors.The capacity of plasticity depends on genotype, hormones and environment, and does not have to be an adaptive trait (Scheiner, 1993).Cheplick's index of PP is used to explore plasticity when some covariates are expected to influence the target variable or trait showing the direction of fullsib plastic response (Valladares et al., 2006).The var-iability of plasticity was higher after recovery from 10 µg Cd/g dry food compared to recovery from 30 µg Cd/g dry food.
The significant positive correlation of the AC-P Lys /ACP Tot ratio between short-term treatments at 10 and 30 µg Cd/g dry food points to an overlapping in the genes that influence the ACP Lys /ACP Tot ratio at two different concentrations during acute treatment.Inter-environmental genetic correlations can considerably affect the rate and direction of evolution in traits related to the use of environmental resources (Via, 1984).Being significantly different from "one", the significant correlation we obtained is not a constraint for the evolution of plasticity.On the other Figure 1.Changes in ratio of specific activities of lysosomal and total ACP (ACPLys/ACPTot) (MV±SE) after acute (A) and chronic treatments (B) and after recoveries (C, D).Experimental groups were compared by one-way ANCOVA followed by LSD multiple range test.Bars marked with different letters differ significantly.C-control; Ac1 and Ac2 -acute exposure to 10 and 30 µg Cd/g dry food; Chr1 and Chr2 -chronic exposure to 10 and 30 µg Cd/g dry food; Rec1 and Rec2 -recovery from exposure to 10 and 30 µg Cd/g dry food   (2008) found that in the mussel Lamellidens marginalis cadmium affected the activity of lysosomal acid phosphatase (ACP L ) in an exposure duration-dependent manner, and Stubberud et al. (2000) evaluated ACP enzymes as a sensitive biomarker.
The significant influence of all cadmium treatments on the ACP Lys /ACP Tot ratio points to its great sensitivity, suggesting that ACP Lys could be considered as a bioindicator of dietary cadmium exposure.

Table 1 .
Mean square (x10 3 ) from the one-way ANCOVA of ratio of specific activities of lysosomal and total ACP (ACPLys/ACPTot) in gypsy moth larvae exposed to different cadmium treatments.Larval mass was used as the covariate.Bold values indicate P<0.05; df = degrees of freedom

Table 3 .
Significance of differences between means (Z, Wilcoxon's test) and standard deviations (F-test) of index of phenotypic plasticity for (ACPLys / ACPTot) ratio.Bold value indicates P<0.05

Table 4 .
Significance of correlations between cadmium treatments for percent of lysosomal acid phosphatase (ACPLys / ACPTot).marked absence of correlations between the treatment groups suggests that independent genetic mechanisms mediate the responses in different stress environments.Javakumar et al.