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Effects of heavy metals on insect immunocompetent cells
Journal of Insect Physiology 57 (2011) 760–770
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Effects of heavy metals on insect immunocompetent cells Joanna Borowska 1, Elz˙bieta Pyza * Department of Cytology and Histology, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Krako´w, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 July 2010 Received in revised form 10 February 2011 Accepted 21 February 2011
The influence of the following heavy metals, copper (Cu), zinc (Zn), cadmium (Cd) and lead (Pb), on haemocytes of the house fly Musca domestica L. was studied under laboratory conditions. House fly larvae were exposed to low or high, semi-lethal concentrations of metals. These particular metals were selected because they are present in polluted environments in Poland. In addition, we studied expression of the stress proteins HSP70 and HSP72 in haemocytes collected from larvae that had been exposed to heavy metal. The obtained results showed changes in haemocytes morphology and phagocytotic plasticity in the experimental flies in comparison to control. The number of prohaemocytes, regarded as stem cells, increased, while granulocytes, responsible for phagocytosis, decreased. However, we have not detected any clear changes in expression of HSP70 or HSP72 in flies treated with low or high concentrations of the heavy metals. ß 2011 Elsevier Ltd. All rights reserved.
Keywords: Biogenic metals Haemocytes HSP70 Musca domestica Xenobiotics
1. Introduction Haemolymph, the only extracellular liquid present in insects, is composed of plasma and cells called haemocytes. The classification of cells in the haemolymph is still controversial. It is also unclear whether specific categories of haemocytes make separate developing cell lines or if they are only morphological and physiological stages of the same line (Gupta, 1985; Lavine and Strand, 2002). Observations of living and fixed haemocytes do not bring univocal results concerning their types. The lack of clear results is caused by high polymorphism and sensitivity of these cells to changes in the environment. At present, the classification of haemocytes includes several types of cells. This classification is based on the function of these cells during metamorphosis and their involvement in cell defence. The classification of haemocytes includes prohaemocytes, granulocytes (granular cells) and plasmatocytes. It also includes those cells less often observed in the haemolymph: spherulocytes (spherule cells) and oenocytoids. Those types of cells have been described in many species of insects (Lavine and Strand, 2002; Ribeiro and Brehe´lin, 2006). In Drosophila melanogaster, a model species to study the mechanisms of innate immunity, only three types of haemocytes have been identified (Williams, 2007). These
* Corresponding author. Tel.: +48 12 663 2642; fax: +48 12 634 4951. E-mail addresses: [email protected], [email protected] (E. Pyza). 1 Present address: Department of Anatomy & Neurobiology, Sir Charles Tupper Medical Bldg, Dalhousie University, 5850 College Street, B3H 1X5 Halifax, NS, Canada. 0022-1910/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2011.02.012
include plasmatocytes, crystal cells and lamellocytes. Although haemocytes are responsible for cell-mediated immune defence they also play many other functions like storage and distribution of nutrients, regulation of development, synthesis and metabolism of various molecules and detoxification (Galloway and Depledge, 2001; Wood and Jacinto, 2007). The number and types of haemocytes within a species show high variability depending on the developmental and physiological stages. Among haemocytes, prohaemocytes are regarded as stem cells, differentiating into one or more types of haemocytes. The plasmatocytes seem to form capsules or nodules around foreign material while the main function of granular haemocytes is phagocytosis. In turn oenocytoids produce one of the phenoloxidases responsible for haemolymph darkening (Ribeiro and Brehe´lin, 2006). The functions of other haemocyte types, for example spherule cells, are unknown. In D. melanogaster the plasmatocytes, making up approximately 95% of circulating haemocytes, are responsible for phagocytosis, encapsulation of pathogens and production of antimicrobial peptides. The other 5% of haemocytes constitute crystal cells which are involved in melanization of invading pathogens and in wound healing. The lamellocytes are present in haemolymph only occasionally, for encapsulation of invading organisms which are too large for phagocytosis (Williams, 2007). In other arthropods and in molluscs haemocytes have similar functions as in insects and are responsible for phagocytosis, pathogen hydrolysis, production of reactive oxygen species and the phenoloxidase cascade (Jiravanichpaisal et al., 2006; Hooper et al., 2007). These functions are important in response to stress and can be affected by environmental pollutants, including heavy metals
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(Sauve´ et al., 2002; Gagnaire et al., 2004; Duchemin et al., 2008). Heavy metals may stimulate haemocyte activity or affect their functions and viability. In haemocytes of mussels, cytotoxic and genotoxic effects have been reported after exposure to low concentration of Cd (0.05–50 mM) (Dailianis, 2009; Banakou and Dailianis, 2010). Paradoxically, higher concentrations of Cd have been found to stimulate phagocytotic and lysosomal activities in haemocytes of the same species. This indicates that toxicity of heavy metals on immune cells depends on their concentration, route of administration and environmental factors (Olabarrieta et al., 2001). Mechanisms of heavy metal toxicity and detoxification are mostly unknown. Invertebrates are good models to study toxicity of heavy metals and are useful bioindicators of contamination of the environment. Moreover, some substances like metallothioneins and heat shock proteins (HSPs), the so-called biomarkers, increase in concentrations after heavy metal exposure (Piano et al., 2004; Said Ali et al., 2010). These substances can be used to evaluate the degree of heavy metal contamination in the environment (Bauman et al., 1993; Lewis et al., 1999; Kammenga et al., 2000) but also as indicators of stress in cells, including blood cells (Croute et al., 2000). HSPs, especially HSP70 and HSP60 have been suggested as sensitive biomarkers of environmental contamination (Ko¨hler et al., 1992; Nadeau et al., 2001; Karouna-Renier and Zehr, 2003) but their usefulness as biomarkers has also been questioned (Pyza et al., 1997; Warchałowska-S´liwa et al., 2005).
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The present study is a continuation of our earlier work (Borowska et al., 2004; Filipiak et al., 2010) on the effects of heavy metals on the immune system of Musca domestica used as an insect model organism. In addition to the house fly being a common insect species, it is also easy to breed, making it an excellent model to study effects of various toxins at population, individual and cellular levels. It is an especially good model to study basic cellular processes affected by toxic substances and mechanisms of detoxification (Szczerbina et al., 2008). In the house fly heavy metals (Zn, Cu, Cd, Pb), accumulate mostly in the abdomen tissues (Tylko et al., 2005). These metals affect development, fertility, life span, and survival (Raina et al., 2001; Borowska et al., 2004) possibly by inducing immunosuppression. We have already reported that heavy metals decrease the number of haemocytes and their adhesion (Borowska et al., 2004). The aim of the present study is to examine how haemocytes, their morphology and their functions are affected after intoxication of insects with heavy metals and to test if mortality increases as a result of the lack of immune defence. We also test if expression of constitutive (HSP70) and induced (HSP72) heat shock proteins in haemocytes are correlated with toxicity and concentration of heavy metals. Unlike other studies (Banakou and Dailianis, 2010; Filipiak et al., 2010) we did not expose haemocytes to heavy metals in tissue culture condition but collected them from insects reared on heavy metal polluted media.
Fig. 1. Types of haemocytes identified in the house fly. PR: prohaemocyte, PL: plasmatocyte, GR: granulocyte, I: intermediate cells between PL and GR. (a) May–Gru¨nwald– Giemsa staining and Light Microscopy. Scale bar: 10 mm. (b) Scanning Electron Microscopy (SEM), GR: granulocyte. Magnification: 3500. (c) PL: plasmatocyte. Magnification: 3500. (d) PR: prohaemocyte. Magnification: 3500.
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2. Materials and methods 2.1. Animals Insects were obtained from a laboratory culture maintained for many generations under laboratory conditions in a light/dark regime LD 12:12 (12 h of light and 12 h of darkness), at a constant temperature of 24 8C 1 8C. The humidity level was approximately 70%. The house fly larvae were reared from eggs until pupariation on one of two diets. One diet was composed of commercial rabbit food (200 g of pellets) and milk powder (10 g) dissolved in 300 ml of distilled H2O (control). The second diet consisted of an experimental solution, instead of distilled water, containing low or sublethal concentrations of heavy metals and of other compounds as in the first, control diet. The experimental insects were exposed to Cu2+ at final concentrations of 5 and 1000 mg/kg, respectively, to Zn2+ at final concentrations of 100 and 2000 mg/kg, respectively, to Cd2+ at final concentrations of 3 and 50 mg/ kg, respectively, or to Pb2+ at final concentrations of 20 and 10,000 mg/ kg, respectively. The larvae were reared on contaminated and control media after hatching from eggs until the end of the final (third) larval instar. As previously shown, heavy metals in the rearing media did not show a significant difference between deliberate, prepared concentrations and concentrations measured by atomic absorption spectrophotometry (AAS) (Borowska et al., 2004). Third instar larvae, just before pupariation, were used to examine haemocytes. 2.2. Reagents CuCl2H2O (POCH Gliwice); ZnCl2 (Merck); CdCl22.5H2O (POCH Gliwice); PbCl2 (Sigma–Aldrich); Giemsa stain (Aqua-Med); May– Gru¨nwald stain (Aqua-Med); HBSS (2 g NaCl, 0.1 g KCl, 0.25 g glucose, 0.015 g KH2PO4, 0.0758 g Na2HPO4 and 200 ml distilled H2O) with 1 mM EDTA; phosphate buffer (PBS) with 0.25% Triton X (Sigma–Aldrich) supplemented with 0.25% BSA (Sigma–Aldrich) and with 1% NGS – normal goat serum (Sigma–Aldrich); monoclonal mouse anti-HSP70 (HSC70) (Sigma) diluted with PBS 1:5000; polyclonal CyTM3-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories INC.) diluted with PBS 1:160 with 1% of NGS.
in wet condition at room temperature. The cells were fixed in 2.5% glutaraldehyde and 1% OsO4. Following washes with HBSS and dehydration in acetone, samples were dried in CO2-critical point and covered with gold. Haemocytes were observed using a scanning electron microscope (SEM) (JSM-5410, JEOL, Tokyo, Japan). 2.6. Heat shock protein detection The fixed cytospins of haemocytes were washed with PBS (pH 7.2) three times for 5 min. Then, preparations were incubated for 24 h with anti-HSP70 monoclonal mouse serum with 1% of NGS or with PBS as a negative control at 4 8C. After washing with PBS six times for 10 min, the preparations were incubated for 2 h at RT in the CyTM3-conjugated goat anti-mouse IgG with 1% of NGS. Next the preparations were washed six times for 10 min with PBS. Then, the preparations were dehydrated and mounted in a medium to preserve fluorescence and stored at 4 8C. This was all done prior to observation under a light microscope (Nikon Optiphot). 2.7. Haemocyte index Relative numbers of different types of haemocytes in control and in experimental groups exposed to each metal in low or high concentrations were analysed under a Nikon Optiphot light microscope (LM) with 100 and 40 oil immersion objectives in 50 randomly selected fields. For cell counting, a special measuring objective 40 1.25 6.3 with stereological net A10 (0.28 mm 0.28 mm) was used. The whole population of cells counted from 50 fields was taken as 100%. Cell images were taken with a Nikon DXM1200F digital camera.
2.3. Haemocyte retrieval Larvae were washed in distilled water and placed on ice for immobilization. Haemolymph was obtained by cutting the anterior part of the larva laterally with micro-dissecting scissors. Haemolymph obtained from 10 specimens (3 ml/larva) was dissolved in 300 ml of Hanks’ Balanced Salt Solution (HBSS, pH 7.4) with 1 mM of ethylenediaminetetraacetic acid (EDTA) and centrifuged for 10 min at 800 g at 21 8C (1K15, Sigma, Germany). Pellets were diluted with 350 ml of HBSS+ 1 mM of EDTA. 2.4. Haemocyte staining Cell samples were centrifuged for 10 min at 200 g in a cytospin centrifuge (Rotofix32, Hettich, Germany). Then, cytospins were fixed with a cold solution (1:1) of acetone and methanol for 5 min. After fixation cytospins were stained with May–Gru¨nwald dye for 3 min and then the same volume of distilled water was added for 1 min. After washing with distilled water, the slides were stained with Giemsa dye for 15 min. Next, the haemocytes were washed with distilled water, air-dried and finally dehydrated and mounted in Canada balsam. 2.5. Scanning electron microscopy of haemocytes Haemocytes were dissolved in 300 ml of HBSS with 1 mM of EDTA (pH 7.4), placed on a cover glass and incubated for 30 min
Fig. 2. (a and b) Relative numbers of four types of haemocytes (%) identified in the haemolymph of Musca domestica third instar larvae on the basis of May–Gru¨nwald– Giemsa staining – haemocyte index. Plotted are the means S.E.M., N = 50. (a) Pb 20 mg/kg, Pb 10,000 mg/kg; Cd 3 mg/kg, Cd 50 mg/kg; (b) Cu 5 mg/kg, 1000 mg/kg; Zn 100 mg/kg, Zn 2000 mg/kg. ANOVA and Bliss transformation; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).
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Fig. 3. Morphology of house fly haemocytes. (a and b) Control; (c) Pb 20 mg/kg; (d) Pb 10,000 mg/kg; (e) Cd 3 mg/kg; (f) Cd 50 mg/kg; (g) Cu 5 mg/kg; (h) Cu 1000 mg/kg; (i) Zn 100 mg/kg; (j) Zn 2000 mg/kg. (*) dots indicate granular cells with small, scattered granules in the cytoplasm; (*) stars indicate the cell with clustered granules in the cytoplasm; (&) squares indicate the small, irregular PR; (~) triangles indicate the impaired cells. Scale bar: 20 mm.
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2.8. Haemocyte sizes The measurements were carried out for 50 cells from the control group, and in 50 cells from each experimental group. The cell areas were measured using image analysis software (ImageJ., v. 1.27z). 2.8.1. Statistical analysis One way analysis of variance (ANOVA) was used to group the means of the control group and experimental groups for statistical analysis. The Shapiro–Wilk W-test for normality was performed. Next, the ANOVA or the non-parametric Kruskal–Wallis test (p 0.05) followed by post hoc Tukey test were used to determine statistically significant differences between multiple cell groups. For percentages, data Bliss transformation was applied. Application software (SigmaPlot 11.0, Systat Software Inc.) was used for data analysis and Microsoft Excel 2000 to prepare graphs. Data in graphs are represented as means standard error of the mean (SEM).
3.3.1. Lead The morphology of the cells of insects exposed to low concentration of Pb (20 mg/kg) was changed. In the nucleus chromatin was condensed. This was especially true of cells belonging to the PR type. The number of PR was increased by 71%. Only a few cells had the characteristic of GR. The majority of cells were identified as intermediate cells. In GR, granules were larger and sometimes formed clusters. The granules were less numerous than in the control group (Fig. 3c). In larvae exposed to Pb numerous bacteria were present in the haemolymph. In insects treated with high concentration of Pb (10,000 mg/kg) the PR number was increased by 20% compared to the control group. Many cells showed condensed chromatin in the nucleus or were fragmented. Only a few large GR were observed, but the majority of cells had the morphology of intermediate cells. In comparison with the control group and other experimental groups, the number of PL increased by 46%. The PL cytoplasm granules were considerably less numerous than in the control group but the granules were larger and very often concentrated in the cytoplasm (Fig. 3d).
3. Results 3.1. Haemocyte types and morphology Using May–Gru¨nwald–Giemsa staining methods, three main types of haemocytes were distinguished: prohaemocytes (PR), plasmatocytes (PL) and granulocytes (GR). In addition haemocytes with a feature of both plasmatocytes and granulocytes were identified as intermediate type (I) (Fig. 1a). These four categories of cells make up about 98% of all haemocytes of the house fly. Analysis with SEM confirmed the presence of three basic types of haemocytes (Fig. 1b–d). In addition, a few cells whose morphology resembled oenocytoids described by Gupta (1985) and some unidentified cells were observed. 3.2. Haemocyte index In larvae exposed to heavy metals changes were observed in the number of different cell types in their haemolymph. There was a significant increase of prohaemocytes. As many as twice the numbers of prohaemocytes were found in larvae exposed to heavy metals as in the control group. In addition to prohaemocytes, the number of granulocytes was also affected. In the control group about 1/3 of the haemocytes showed characteristic features of granulocytes while under the influence of heavy metals their percentage decreased, moving the balance towards intermediate cells (Fig. 2a and b).
3.3.2. Cadmium The exposure to Cd (3 mg/kg) increased the number of PRs by 74% but decreased GR by 92% compared to the control group. A single or a few scattered granules were observed in the cytoplasm of GR. The granules were larger than in the control group. Numerous cells showed intermediate features between PL and GR. In the haemolymph of experimental insects there was more bacteria than in the control group. In the case of larvae treated with 50 mg/kg Cd, the PR number was 104% higher than in the control group. Some cells had condensed chromatin in the nucleus. There was also lots of debris, originating probably from the impaired cells. There were only a small number of granular cells, 2.8% of the total number, and fewer (58% less) PL in haemolymph of experimental flies than in the control group. The granules of GR were larger than in the control group (Fig. 3e and f). 3.3.3. Copper In the blood of larvae reared on the medium contaminated with low (5 mg/kg) or high (1000 mg/kg) concentrations of Cu there was a large quantity of PR, 98% and 70% more, respectively. Numerous cells showed condensed chromatin in the nucleus in comparison to the control group. There was less GR and PL, by 96% and 38%, respectively after the low Cu concentration exposure and at
3.3. Effects of heavy metals on haemocyte type and morphology In the third instar larvae of the house fly, PR made up approximately 1/3 of the population of cells in haemolymph. Prohaemocytes have a homogeneous nucleus and very thin layer of cytoplasm (Figs. 1a and d and 3a and b). In the second type of cells, showing morphological features of PL, the nucleus occupies approximately 40 2% (N = 50) of the cell area. In the second type of cells a small number of granules was observed in the cytoplasm, or these organelles were not present (Figs. 1a and c and 3a and b). The third type of cells includes large cells having the features of GR. The nucleus of GR occupies approximately 15 2% (N = 50) of the cell area. These large cells have fine granules which are rather evenly scattered in the cytoplasm. The granules varied in colour from white, to greyish, to light violet, to blue after the May– Gru¨nwald–Giemsa staining (Figs. 1a and b and 3a and b). There were also cells having features intermediate between PL and GR (Fig. 1a).
Fig. 4. Area of the cytospined granulocytes of house fly larvae in control and in experimental groups treated with heavy metals in low and high concentrations. Plotted are the means S.E.M., N = 50; Kruskal–Wallis One Way ANOVA on Ranks; p < 0.001 (***).
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Fig. 5. Area of the cytospined plasmatocytes of house fly larvae in control and in experimental groups treated with heavy metals in low and high concentrations. Plotted are the mean S.E.M., N = 50; Kruskal–Wallis One Way ANOVA on Ranks; p < 0.006 (**).
Fig. 6. Area of the cytospined prohaemocytes of house fly larvae in control and experimental groups treated with heavy metals in low and high concentrations. Plotted are the means S.E.M., N = 50; Kruskal–Wallis One Way ANOVA on Ranks; p < 0.001 (***).
1000 mg/kg Cu, GR dropped to 85% and PL to 43%. Differences between GR, PL and intermediate cells were difficult to detect. In the cytoplasm of GR and PL, granules were larger than in the control group. In the haemolymph of the experimental flies there was also more bacteria than in the control group (Fig. 3g and h).
3.3.4. Zinc In the haemolymph cells of larvae treated with Zn (100 mg/kg or 2000 mg/kg) the number of PR increased (87% and 85%, respectively). Other cell types had a similar size with characteristics of intermediate cells. The intracellular granules were less numerous
Fig. 7. Expression of HSP70 in three types of haemocytes (PR, PL, GR) in the control and in experimental groups exposed to low (20 mg/kg) and semi-lethal (10,000 mg/kg) concentrations of Pb.
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than in the control group. These intracellular granules were larger and usually formed aggregates (Fig. 3i and j). 3.4. Size of haemocytes The area of granulocytes obtained from the larvae reared on the media contaminated with heavy metals showed a significant decrease in comparison with cells originating from the control group of larvae (Fig. 4). GR were noticeably reduced in size by 42% and 47% in larvae reared on the media containing 20 mg/kg and 10,000 mg/kg of Pb, respectively. A similar decrease in GR size was detected in larvae treated with low concentrations of Cu, Cd and high concentration of Zn. In larvae exposed to Cu and Cd in high concentrations and to Zn in low concentration, changes in the area of GR were smaller. GRs were smaller by 26%, 25% and 30%, respectively. Comparing with the control, plasmatocytes were smaller only after exposure of larvae to high concentration of Pb. This difference was statistically significant (p = 0.006) (Fig. 5). The analysis of the size of prohaemocytes showed a statistically significant increase, by 23% and 21%, of their area only in larvae treated with low and high Zn concentrations, respectively (Fig. 6). 3.5. Expression of HSPs The immunocytochemistry method revealed that HSP70 expression did not significantly change in haemocytes obtained
from experimental insects when compared with haemocytes of the control group. The expression of HSP70 was significantly increased, however, in larvae exposed to a heat shock (43 8C for 20 h) used as a positive control. Only after exposure to low concentrations of Pb, Cd, Cu and Zn some prohaemocytes and a few granular cells showed stronger intensity of fluorescence in the cytoplasm (Figs. 7–10). In all analysed preparations, including those of the control group, cells showing very strong fluorescence were observed (‘flash-type’ cells). In the case of HSP72 no differences were detected in the abundance of this protein between experimental and control groups. 4. Discussion The results obtained in the present study showed that haemocytes, insect immune cells, are strongly affected by intoxication of insects with heavy metals. They also confirmed our earlier results (Borowska et al., 2004) and the results of other authors, showing that heavy metals reduce immune responses of organisms (Victor, 1993; Marth et al., 2000; Galloway and Depledge, 2001; Matozzo et al., 2001), resulting in decrease of survival. As in vertebrates, the efficiency of the immune system in insects is correlated with the number of haemocytes and their function. In our previous study we showed that house flies, exposed to the same low and high concentrations of Cu, Zn, Pb and Cd as in the
Fig. 8. Expression of HSP70 in three types of haemocytes (PR, PL, GR) in the control and in experimental groups exposed to low (3 mg/kg) and semi-lethal (50 mg/kg) concentrations of Cd.
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Fig. 9. Expression of HSP70 in three types of haemocytes (PR, PL, GR) in the control and in experimental groups exposed to low (5 mg/kg) and semi-lethal (1000 mg/kg) concentrations of Cu.
present study, accumulate 0.9 and 4.5 times more of Cu, 0.8 and 1.5 times more Zn, 1.3 and 43.7 times more of Pb, and 4.9 and 33.8 times more of Cd, respectively than the control groups (Borowska et al., 2004). Since all tissues are penetrated by haemolymph, we assume that immune cells also accumulate heavy metals as has been found in molluscs (Victor, 1993; McIntosh and Robinson, 1999; Marigomez et al., 1990, 2002). In house fly larvae we determined three basic types of haemocytes: prohaemocytes (PR), plasmatocytes (PL) and granulocytes (GR), on the basis of haemocyte classification obtained in other insect species (Gupta, 1985; Franchini et al., 1996; Silva et al., 2002). We also determined cells similar in morphology to both PL and GR which were classified as intermediates (I). In the third instar, larvae haemocytes fluctuate in number, but their total number significantly decreased in heavy metal exposed insects (Borowska et al., 2004) although haemocytes of M. domestica seem to be more resistant to this type of stress than do the cells of D. melanogaster (Filipiak et al., 2010). In house flies exposed to high concentrations of Pb, Zn, Cu and Cd, the number of immune cells decreased by 26%, 18%, 17% and 16%, respectively (Borowska et al., 2004). Similar results have been observed in other organisms, such as shrimp and crabs (Victor, 1993; Lorenz et al., 2001). As found in Cd-treated haemocytes of molluscs the increase of cell death may result from oxidative damage within cells, such as lipid peroxidation, enhanced production of ROS and DNA damage (Banakou and Dailianis, 2010).
In addition to the previously reported decrease in the total number of haemocytes (Borowska et al., 2004), we found that the haemocyte index, the proportion of different cell types in a population of haemocytes, changed in flies exposed to heavy metals. The significant decrease was especially observed in the number of granulocytes and plasmatocytes. It may result from high mortality of granulocytes as trefocytes and/or from slowing down haemocyte differentiation and appearance of cells intermediate between PL and GR in the total pool of haemocytes. In turn, the observed higher proliferation of prohaemocytes and abnormal haemocyte morphology may indicate the occurrence of neoplastic transformation of haemopoietic organs or stimulation of the immune system under the influence of metals (Victor, 1993; Olabarrieta et al., 2001). The above changes were observed in haemocytes from all experimental groups except those originating from larvae exposed to high (10,000 mg/kg) concentration of Pb. In that case, the decrease in number of granulocytes, when compared with the control group, was more pronounced than in other groups. High concentrations of lead exposure did not show a significant increase in the PR number, as observed in all other experimental groups. Lead had the strongest negative effect on the total number of haemocytes (Borowska et al., 2004) and on the area of trefocytes, an effect possibly caused by high toxicity of accumulated Pb which induced mortality of proliferating PR and/or degeneration of haemopoietic organs.
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Fig. 10. Expression of HSP70 in three types of haemocytes (PR, PL, GR) in the control and in experimental groups exposed to low (100 mg/kg) and semi-lethal (2000 mg/kg) concentrations of Zn.
Exposure to heavy metals does not change the ratio of living cells to their total number but affects adhesion ability of haemocytes. The adhesion ability of haemocytes decreased by 56% and 59% in response to high concentrations of Pb and Cu, respectively (Borowska et al., 2004). Haemocytes also did not change from a round to an amoeboid shape, the shape characteristic of cells active in phagocytosis (Nieto-Fernandez et al., 2000). This indicates that phagocytosis, one of most important defence mechanisms, is disturbed in heavy metal intoxicated insects and molluscs, decreasing efficiency of their immune systems. Moreover, the observed decrease in the number of plasmatocytes and granulocytes which participate in phagocytosis leads to multiplication of pathogens which eventually kill the insect. A similar effect has been observed in other invertebrates (Brousseau et al., 2000; Matozzo et al., 2001; Olchawa et al., 2006). The analysis of changes in morphology of haemocytes under the influence of heavy metals also showed a significant decrease of the area of cells identified as granulocytes (Fig. 3), which participate in phagocytosis. They also store and excrete products of metabolism. The decrease in size may be induced by the toxic effects of the accumulated metals on cell metabolism and cytoskeleton structure (Fagotti et al., 1996). The decrease in the cell area was observed after treating the larvae with all the metals used in the study, with the strongest effect observed with lead exposure. The observed changes in haemocyte morphology and their functions may also result from the heavy metal induced stress on
the house fly hormonal system. The heavy metal induced stress could affect hormonal balance and homeostasis. It has been reported that metals and metallothioneins have an impact on cellular signal transduction induced, among other compounds, by steroid hormones (DeMoor and Koropatnick, 2000). In insects ecdysteroids regulate development and metamorphosis. Ecdysteroids also influence conversion of haemocytes into phagocytic cells as well as release of haemocytes from the haemopoietic organs to haemolymph. Since larval development and metamorphosis are delayed in metal-exposed flies (Borowska et al., 2004) this effect may result from the influence of heavy metals on haemocytes which take part in metamorphosis (Gupta, 1985; Raina et al., 2001; Kiger et al., 2001) and on the endocrine system. This system controls synthesis of haemolymph proteins in the fat body, and, in some species, protein synthesis in haemocytes, their accumulation and transition to haemolymph. The disruption of these processes may also significantly weaken the immune system (Go¨tz and Boman, 1985). The results obtained in the present study did not prove, however, a correlation between heat shock protein expression in haemocytes of M. domestica and heavy metal exposure. In some species a clear correlation between levels of HSPs, especially HSP70, and exposure to heavy metals has been reported (Ko¨hler et al., 1992, 1996; Bierkens et al., 1998; Steiner et al., 1998; Werner and Hinton, 1999; Wagner et al., 1999; Aı¨t-Aı¨ssa et al., 2000; Gibney et al., 2004). In other species, however, such a correlation
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has not been detected (Pyza et al., 1997; Ko¨hler et al., 2000; Werner and Hinton, 2000; Lewis et al., 2001). HSP70 was observed in all groups of haemocytes exposed to heavy metals and was generally similar to that of the control group. Low concentrations of some metals resulted in a higher abundance of HSP70, yet this was only observed in a small number of prohaemocytes and granulocytes. This confirms the observations of other authors (Pyza et al., 1997; Feder and Hoffman, 1999; Ko¨hler et al., 2000; Warchałowska-S´liwa et al., 2005) that high concentrations and/or long exposure to heavy metals often decrease HSP70 levels, compared to lower concentrations of the same metals. We also did not detect expression of the induced HSP72 after intoxication of larvae with heavy metals. It is possible that in vivo adaptation of cells to the presence of heavy metals decreased induction of HSP (Croute et al., 2000; Ko¨hler et al., 2000). 5. Conclusions The obtained results confirmed that heavy metals are immunotoxins for insects. They affect the morphology and haemocyte index. Haemocytes can be used as biomarkers of heavy metal intoxication. Disruption of haemocytes seems to be responsible for earlier observations of delayed development, metamorphosis and decreased survival of flies (Borowska et al., 2004) due to increased susceptibility to infections and parasitoids. Since M. domestica can be easily encountered in the natural environment and is easy to breed, its haemocytes could also be used as a cheap and easily available model to test toxicity of various substances in the polluted environment. Acknowledgement This study was supported by the Polish Ministry of Science and Higher Education. Grant nr. N304 066 32/2604. References Aı¨t-Aı¨ssa, S., Porcher, J., Arrigo, A., Lambre, C., 2000. Activation of the hsp70 promoter by environmental inorganic and organic chemicals: relationships with cytotoxicity and lipophilicity. Toxicology 145, 147–157. Banakou, E., Dailianis, S., 2010. Involvement of Na+/H+ exchanger and respiratory burst enzymes NDPH oxidase and NO synthase, in Cd-induced lipid peroxidation and DNA damage in haemocytes of mussels. Comparative Biochemistry and Physiology, Part C 152, 346–352. Bauman, J.W., Liu, J., Klassen, C.D., 1993. Production of metallothionein and heatshock proteins in response to metals. Fundamental and Applied Toxicology 21, 15–22. Bierkens, J., Maes, J., Vander, P.F., 1998. Dose-dependent of heat shock protein 70 synthesis in Rhapidocelis subcapita following exposure to different classes of environmental pollutants. Environmental Pollution 101, 91–97. Borowska, J., Sulima, B., Niklin´ska, M., Pyza, E., 2004. Heavy metal accumulation and its effects on development, survival and immuno-competent cells of the house fly Musca domestica from closed laboratory populations as model organism. Fresenius Environmental Bulletin 13, 1402–1409. Brousseau, P., Pellerin, J., Morin, Y., Cyr, D., Blakley, B., Boermans, H., Fournier, M., 2000. Flow cytometry as a tool to monitor the disturbance of phagocytosis in the clam Mya arenaria haemocytes following in vitro exposure to heavy metals. Toxicology 142, 145–156. Croute, F., Beau, B., Arrabit, C., Gaubin, Y., Delmas, F., Murat, J.C., Soleilhavoup, J.P., 2000. Pattern of stress protein expression in human lung cell-line A549 after short- or long-term exposure to cadmium. Environmental Health Perspective 108, 55–60. Dailianis, S., 2009. Production of superoxides and nitric oxide generation in haemocytes of mussel Mytilus galloprovincialis (Lmk.) after exposure to cadmium: a possible involvement of Na+/H+ exchanger in the induction of cadmium toxic effects. Fish & Shellfish Immunology 27, 446–453. DeMoor, J.M., Koropatnick, D.J., 2000. Metals and cellular signalling in mammalian cells. Cell and Molecular Biology 46, 367–381. Duchemin, M.B., Auffret, M., Wessel, N., Fortier, M., Morin, Y., Pellerin, J., Fournier, M., 2008. Multiple experimental approaches of immunotoxic effects of mercury chloride in the blue mussel, Mytilus edulis, through in vivo, in tubo and in vitro exposures. Environmental Pollution 153, 416–423. Fagotti, A., Di Rosa, I., Simoncelli, F., Pipe, R.K., Panara, F., Pascolini, R., 1996. The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes. Developmental and Comparative Immunology 20, 383–391.
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Heavy metals affect the circulating haemocyte number in the shrimp Palaemon elegans. Fish & Shellfish Immunology 11, 459–472. Marigomez, J.A., Cajaraville, M.P., Angulo, E., 1990. Cellular cadmium distribution in the common winkle, Littorina littorea (L.) determined by X-ray microprobe analysis and histochemistry. Histochemistry 94, 191–199. Marigomez, I., Soto, M., Cajaraville, M.P., Angulo, E., Giamberini, L., 2002. Cellular and subcellular distribution of metals in molluscs. Microscopic Research and Techniques 56, 358–392. Marth, E., Barth, S., Jelovcan, S., 2000. Influence of cadmium on the immune system. Description of stimulating reactions. Central European Journal of Public Health 8, 40–44. Matozzo, V., Ballarin, L., Pampanin, D.M., Marin, M.G., 2001. Effects of copper and cadmium exposure on functional responses of haemocytes in the clam, Tapes philippinarum. Archives of Environmental Contamination and Toxicology 41, 163–170. McIntosh, L.M., Robinson, W.E., 1999. 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Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Effects of heavy metals on insect immunocompetent cells Joanna Borowska 1, Elz˙bieta Pyza * Department of Cytology and Histology, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Krako´w, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 July 2010 Received in revised form 10 February 2011 Accepted 21 February 2011
The influence of the following heavy metals, copper (Cu), zinc (Zn), cadmium (Cd) and lead (Pb), on haemocytes of the house fly Musca domestica L. was studied under laboratory conditions. House fly larvae were exposed to low or high, semi-lethal concentrations of metals. These particular metals were selected because they are present in polluted environments in Poland. In addition, we studied expression of the stress proteins HSP70 and HSP72 in haemocytes collected from larvae that had been exposed to heavy metal. The obtained results showed changes in haemocytes morphology and phagocytotic plasticity in the experimental flies in comparison to control. The number of prohaemocytes, regarded as stem cells, increased, while granulocytes, responsible for phagocytosis, decreased. However, we have not detected any clear changes in expression of HSP70 or HSP72 in flies treated with low or high concentrations of the heavy metals. ß 2011 Elsevier Ltd. All rights reserved.
Keywords: Biogenic metals Haemocytes HSP70 Musca domestica Xenobiotics
1. Introduction Haemolymph, the only extracellular liquid present in insects, is composed of plasma and cells called haemocytes. The classification of cells in the haemolymph is still controversial. It is also unclear whether specific categories of haemocytes make separate developing cell lines or if they are only morphological and physiological stages of the same line (Gupta, 1985; Lavine and Strand, 2002). Observations of living and fixed haemocytes do not bring univocal results concerning their types. The lack of clear results is caused by high polymorphism and sensitivity of these cells to changes in the environment. At present, the classification of haemocytes includes several types of cells. This classification is based on the function of these cells during metamorphosis and their involvement in cell defence. The classification of haemocytes includes prohaemocytes, granulocytes (granular cells) and plasmatocytes. It also includes those cells less often observed in the haemolymph: spherulocytes (spherule cells) and oenocytoids. Those types of cells have been described in many species of insects (Lavine and Strand, 2002; Ribeiro and Brehe´lin, 2006). In Drosophila melanogaster, a model species to study the mechanisms of innate immunity, only three types of haemocytes have been identified (Williams, 2007). These
* Corresponding author. Tel.: +48 12 663 2642; fax: +48 12 634 4951. E-mail addresses: [email protected], [email protected] (E. Pyza). 1 Present address: Department of Anatomy & Neurobiology, Sir Charles Tupper Medical Bldg, Dalhousie University, 5850 College Street, B3H 1X5 Halifax, NS, Canada. 0022-1910/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2011.02.012
include plasmatocytes, crystal cells and lamellocytes. Although haemocytes are responsible for cell-mediated immune defence they also play many other functions like storage and distribution of nutrients, regulation of development, synthesis and metabolism of various molecules and detoxification (Galloway and Depledge, 2001; Wood and Jacinto, 2007). The number and types of haemocytes within a species show high variability depending on the developmental and physiological stages. Among haemocytes, prohaemocytes are regarded as stem cells, differentiating into one or more types of haemocytes. The plasmatocytes seem to form capsules or nodules around foreign material while the main function of granular haemocytes is phagocytosis. In turn oenocytoids produce one of the phenoloxidases responsible for haemolymph darkening (Ribeiro and Brehe´lin, 2006). The functions of other haemocyte types, for example spherule cells, are unknown. In D. melanogaster the plasmatocytes, making up approximately 95% of circulating haemocytes, are responsible for phagocytosis, encapsulation of pathogens and production of antimicrobial peptides. The other 5% of haemocytes constitute crystal cells which are involved in melanization of invading pathogens and in wound healing. The lamellocytes are present in haemolymph only occasionally, for encapsulation of invading organisms which are too large for phagocytosis (Williams, 2007). In other arthropods and in molluscs haemocytes have similar functions as in insects and are responsible for phagocytosis, pathogen hydrolysis, production of reactive oxygen species and the phenoloxidase cascade (Jiravanichpaisal et al., 2006; Hooper et al., 2007). These functions are important in response to stress and can be affected by environmental pollutants, including heavy metals
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(Sauve´ et al., 2002; Gagnaire et al., 2004; Duchemin et al., 2008). Heavy metals may stimulate haemocyte activity or affect their functions and viability. In haemocytes of mussels, cytotoxic and genotoxic effects have been reported after exposure to low concentration of Cd (0.05–50 mM) (Dailianis, 2009; Banakou and Dailianis, 2010). Paradoxically, higher concentrations of Cd have been found to stimulate phagocytotic and lysosomal activities in haemocytes of the same species. This indicates that toxicity of heavy metals on immune cells depends on their concentration, route of administration and environmental factors (Olabarrieta et al., 2001). Mechanisms of heavy metal toxicity and detoxification are mostly unknown. Invertebrates are good models to study toxicity of heavy metals and are useful bioindicators of contamination of the environment. Moreover, some substances like metallothioneins and heat shock proteins (HSPs), the so-called biomarkers, increase in concentrations after heavy metal exposure (Piano et al., 2004; Said Ali et al., 2010). These substances can be used to evaluate the degree of heavy metal contamination in the environment (Bauman et al., 1993; Lewis et al., 1999; Kammenga et al., 2000) but also as indicators of stress in cells, including blood cells (Croute et al., 2000). HSPs, especially HSP70 and HSP60 have been suggested as sensitive biomarkers of environmental contamination (Ko¨hler et al., 1992; Nadeau et al., 2001; Karouna-Renier and Zehr, 2003) but their usefulness as biomarkers has also been questioned (Pyza et al., 1997; Warchałowska-S´liwa et al., 2005).
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The present study is a continuation of our earlier work (Borowska et al., 2004; Filipiak et al., 2010) on the effects of heavy metals on the immune system of Musca domestica used as an insect model organism. In addition to the house fly being a common insect species, it is also easy to breed, making it an excellent model to study effects of various toxins at population, individual and cellular levels. It is an especially good model to study basic cellular processes affected by toxic substances and mechanisms of detoxification (Szczerbina et al., 2008). In the house fly heavy metals (Zn, Cu, Cd, Pb), accumulate mostly in the abdomen tissues (Tylko et al., 2005). These metals affect development, fertility, life span, and survival (Raina et al., 2001; Borowska et al., 2004) possibly by inducing immunosuppression. We have already reported that heavy metals decrease the number of haemocytes and their adhesion (Borowska et al., 2004). The aim of the present study is to examine how haemocytes, their morphology and their functions are affected after intoxication of insects with heavy metals and to test if mortality increases as a result of the lack of immune defence. We also test if expression of constitutive (HSP70) and induced (HSP72) heat shock proteins in haemocytes are correlated with toxicity and concentration of heavy metals. Unlike other studies (Banakou and Dailianis, 2010; Filipiak et al., 2010) we did not expose haemocytes to heavy metals in tissue culture condition but collected them from insects reared on heavy metal polluted media.
Fig. 1. Types of haemocytes identified in the house fly. PR: prohaemocyte, PL: plasmatocyte, GR: granulocyte, I: intermediate cells between PL and GR. (a) May–Gru¨nwald– Giemsa staining and Light Microscopy. Scale bar: 10 mm. (b) Scanning Electron Microscopy (SEM), GR: granulocyte. Magnification: 3500. (c) PL: plasmatocyte. Magnification: 3500. (d) PR: prohaemocyte. Magnification: 3500.
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2. Materials and methods 2.1. Animals Insects were obtained from a laboratory culture maintained for many generations under laboratory conditions in a light/dark regime LD 12:12 (12 h of light and 12 h of darkness), at a constant temperature of 24 8C 1 8C. The humidity level was approximately 70%. The house fly larvae were reared from eggs until pupariation on one of two diets. One diet was composed of commercial rabbit food (200 g of pellets) and milk powder (10 g) dissolved in 300 ml of distilled H2O (control). The second diet consisted of an experimental solution, instead of distilled water, containing low or sublethal concentrations of heavy metals and of other compounds as in the first, control diet. The experimental insects were exposed to Cu2+ at final concentrations of 5 and 1000 mg/kg, respectively, to Zn2+ at final concentrations of 100 and 2000 mg/kg, respectively, to Cd2+ at final concentrations of 3 and 50 mg/ kg, respectively, or to Pb2+ at final concentrations of 20 and 10,000 mg/ kg, respectively. The larvae were reared on contaminated and control media after hatching from eggs until the end of the final (third) larval instar. As previously shown, heavy metals in the rearing media did not show a significant difference between deliberate, prepared concentrations and concentrations measured by atomic absorption spectrophotometry (AAS) (Borowska et al., 2004). Third instar larvae, just before pupariation, were used to examine haemocytes. 2.2. Reagents CuCl2H2O (POCH Gliwice); ZnCl2 (Merck); CdCl22.5H2O (POCH Gliwice); PbCl2 (Sigma–Aldrich); Giemsa stain (Aqua-Med); May– Gru¨nwald stain (Aqua-Med); HBSS (2 g NaCl, 0.1 g KCl, 0.25 g glucose, 0.015 g KH2PO4, 0.0758 g Na2HPO4 and 200 ml distilled H2O) with 1 mM EDTA; phosphate buffer (PBS) with 0.25% Triton X (Sigma–Aldrich) supplemented with 0.25% BSA (Sigma–Aldrich) and with 1% NGS – normal goat serum (Sigma–Aldrich); monoclonal mouse anti-HSP70 (HSC70) (Sigma) diluted with PBS 1:5000; polyclonal CyTM3-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories INC.) diluted with PBS 1:160 with 1% of NGS.
in wet condition at room temperature. The cells were fixed in 2.5% glutaraldehyde and 1% OsO4. Following washes with HBSS and dehydration in acetone, samples were dried in CO2-critical point and covered with gold. Haemocytes were observed using a scanning electron microscope (SEM) (JSM-5410, JEOL, Tokyo, Japan). 2.6. Heat shock protein detection The fixed cytospins of haemocytes were washed with PBS (pH 7.2) three times for 5 min. Then, preparations were incubated for 24 h with anti-HSP70 monoclonal mouse serum with 1% of NGS or with PBS as a negative control at 4 8C. After washing with PBS six times for 10 min, the preparations were incubated for 2 h at RT in the CyTM3-conjugated goat anti-mouse IgG with 1% of NGS. Next the preparations were washed six times for 10 min with PBS. Then, the preparations were dehydrated and mounted in a medium to preserve fluorescence and stored at 4 8C. This was all done prior to observation under a light microscope (Nikon Optiphot). 2.7. Haemocyte index Relative numbers of different types of haemocytes in control and in experimental groups exposed to each metal in low or high concentrations were analysed under a Nikon Optiphot light microscope (LM) with 100 and 40 oil immersion objectives in 50 randomly selected fields. For cell counting, a special measuring objective 40 1.25 6.3 with stereological net A10 (0.28 mm 0.28 mm) was used. The whole population of cells counted from 50 fields was taken as 100%. Cell images were taken with a Nikon DXM1200F digital camera.
2.3. Haemocyte retrieval Larvae were washed in distilled water and placed on ice for immobilization. Haemolymph was obtained by cutting the anterior part of the larva laterally with micro-dissecting scissors. Haemolymph obtained from 10 specimens (3 ml/larva) was dissolved in 300 ml of Hanks’ Balanced Salt Solution (HBSS, pH 7.4) with 1 mM of ethylenediaminetetraacetic acid (EDTA) and centrifuged for 10 min at 800 g at 21 8C (1K15, Sigma, Germany). Pellets were diluted with 350 ml of HBSS+ 1 mM of EDTA. 2.4. Haemocyte staining Cell samples were centrifuged for 10 min at 200 g in a cytospin centrifuge (Rotofix32, Hettich, Germany). Then, cytospins were fixed with a cold solution (1:1) of acetone and methanol for 5 min. After fixation cytospins were stained with May–Gru¨nwald dye for 3 min and then the same volume of distilled water was added for 1 min. After washing with distilled water, the slides were stained with Giemsa dye for 15 min. Next, the haemocytes were washed with distilled water, air-dried and finally dehydrated and mounted in Canada balsam. 2.5. Scanning electron microscopy of haemocytes Haemocytes were dissolved in 300 ml of HBSS with 1 mM of EDTA (pH 7.4), placed on a cover glass and incubated for 30 min
Fig. 2. (a and b) Relative numbers of four types of haemocytes (%) identified in the haemolymph of Musca domestica third instar larvae on the basis of May–Gru¨nwald– Giemsa staining – haemocyte index. Plotted are the means S.E.M., N = 50. (a) Pb 20 mg/kg, Pb 10,000 mg/kg; Cd 3 mg/kg, Cd 50 mg/kg; (b) Cu 5 mg/kg, 1000 mg/kg; Zn 100 mg/kg, Zn 2000 mg/kg. ANOVA and Bliss transformation; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).
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Fig. 3. Morphology of house fly haemocytes. (a and b) Control; (c) Pb 20 mg/kg; (d) Pb 10,000 mg/kg; (e) Cd 3 mg/kg; (f) Cd 50 mg/kg; (g) Cu 5 mg/kg; (h) Cu 1000 mg/kg; (i) Zn 100 mg/kg; (j) Zn 2000 mg/kg. (*) dots indicate granular cells with small, scattered granules in the cytoplasm; (*) stars indicate the cell with clustered granules in the cytoplasm; (&) squares indicate the small, irregular PR; (~) triangles indicate the impaired cells. Scale bar: 20 mm.
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2.8. Haemocyte sizes The measurements were carried out for 50 cells from the control group, and in 50 cells from each experimental group. The cell areas were measured using image analysis software (ImageJ., v. 1.27z). 2.8.1. Statistical analysis One way analysis of variance (ANOVA) was used to group the means of the control group and experimental groups for statistical analysis. The Shapiro–Wilk W-test for normality was performed. Next, the ANOVA or the non-parametric Kruskal–Wallis test (p 0.05) followed by post hoc Tukey test were used to determine statistically significant differences between multiple cell groups. For percentages, data Bliss transformation was applied. Application software (SigmaPlot 11.0, Systat Software Inc.) was used for data analysis and Microsoft Excel 2000 to prepare graphs. Data in graphs are represented as means standard error of the mean (SEM).
3.3.1. Lead The morphology of the cells of insects exposed to low concentration of Pb (20 mg/kg) was changed. In the nucleus chromatin was condensed. This was especially true of cells belonging to the PR type. The number of PR was increased by 71%. Only a few cells had the characteristic of GR. The majority of cells were identified as intermediate cells. In GR, granules were larger and sometimes formed clusters. The granules were less numerous than in the control group (Fig. 3c). In larvae exposed to Pb numerous bacteria were present in the haemolymph. In insects treated with high concentration of Pb (10,000 mg/kg) the PR number was increased by 20% compared to the control group. Many cells showed condensed chromatin in the nucleus or were fragmented. Only a few large GR were observed, but the majority of cells had the morphology of intermediate cells. In comparison with the control group and other experimental groups, the number of PL increased by 46%. The PL cytoplasm granules were considerably less numerous than in the control group but the granules were larger and very often concentrated in the cytoplasm (Fig. 3d).
3. Results 3.1. Haemocyte types and morphology Using May–Gru¨nwald–Giemsa staining methods, three main types of haemocytes were distinguished: prohaemocytes (PR), plasmatocytes (PL) and granulocytes (GR). In addition haemocytes with a feature of both plasmatocytes and granulocytes were identified as intermediate type (I) (Fig. 1a). These four categories of cells make up about 98% of all haemocytes of the house fly. Analysis with SEM confirmed the presence of three basic types of haemocytes (Fig. 1b–d). In addition, a few cells whose morphology resembled oenocytoids described by Gupta (1985) and some unidentified cells were observed. 3.2. Haemocyte index In larvae exposed to heavy metals changes were observed in the number of different cell types in their haemolymph. There was a significant increase of prohaemocytes. As many as twice the numbers of prohaemocytes were found in larvae exposed to heavy metals as in the control group. In addition to prohaemocytes, the number of granulocytes was also affected. In the control group about 1/3 of the haemocytes showed characteristic features of granulocytes while under the influence of heavy metals their percentage decreased, moving the balance towards intermediate cells (Fig. 2a and b).
3.3.2. Cadmium The exposure to Cd (3 mg/kg) increased the number of PRs by 74% but decreased GR by 92% compared to the control group. A single or a few scattered granules were observed in the cytoplasm of GR. The granules were larger than in the control group. Numerous cells showed intermediate features between PL and GR. In the haemolymph of experimental insects there was more bacteria than in the control group. In the case of larvae treated with 50 mg/kg Cd, the PR number was 104% higher than in the control group. Some cells had condensed chromatin in the nucleus. There was also lots of debris, originating probably from the impaired cells. There were only a small number of granular cells, 2.8% of the total number, and fewer (58% less) PL in haemolymph of experimental flies than in the control group. The granules of GR were larger than in the control group (Fig. 3e and f). 3.3.3. Copper In the blood of larvae reared on the medium contaminated with low (5 mg/kg) or high (1000 mg/kg) concentrations of Cu there was a large quantity of PR, 98% and 70% more, respectively. Numerous cells showed condensed chromatin in the nucleus in comparison to the control group. There was less GR and PL, by 96% and 38%, respectively after the low Cu concentration exposure and at
3.3. Effects of heavy metals on haemocyte type and morphology In the third instar larvae of the house fly, PR made up approximately 1/3 of the population of cells in haemolymph. Prohaemocytes have a homogeneous nucleus and very thin layer of cytoplasm (Figs. 1a and d and 3a and b). In the second type of cells, showing morphological features of PL, the nucleus occupies approximately 40 2% (N = 50) of the cell area. In the second type of cells a small number of granules was observed in the cytoplasm, or these organelles were not present (Figs. 1a and c and 3a and b). The third type of cells includes large cells having the features of GR. The nucleus of GR occupies approximately 15 2% (N = 50) of the cell area. These large cells have fine granules which are rather evenly scattered in the cytoplasm. The granules varied in colour from white, to greyish, to light violet, to blue after the May– Gru¨nwald–Giemsa staining (Figs. 1a and b and 3a and b). There were also cells having features intermediate between PL and GR (Fig. 1a).
Fig. 4. Area of the cytospined granulocytes of house fly larvae in control and in experimental groups treated with heavy metals in low and high concentrations. Plotted are the means S.E.M., N = 50; Kruskal–Wallis One Way ANOVA on Ranks; p < 0.001 (***).
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Fig. 5. Area of the cytospined plasmatocytes of house fly larvae in control and in experimental groups treated with heavy metals in low and high concentrations. Plotted are the mean S.E.M., N = 50; Kruskal–Wallis One Way ANOVA on Ranks; p < 0.006 (**).
Fig. 6. Area of the cytospined prohaemocytes of house fly larvae in control and experimental groups treated with heavy metals in low and high concentrations. Plotted are the means S.E.M., N = 50; Kruskal–Wallis One Way ANOVA on Ranks; p < 0.001 (***).
1000 mg/kg Cu, GR dropped to 85% and PL to 43%. Differences between GR, PL and intermediate cells were difficult to detect. In the cytoplasm of GR and PL, granules were larger than in the control group. In the haemolymph of the experimental flies there was also more bacteria than in the control group (Fig. 3g and h).
3.3.4. Zinc In the haemolymph cells of larvae treated with Zn (100 mg/kg or 2000 mg/kg) the number of PR increased (87% and 85%, respectively). Other cell types had a similar size with characteristics of intermediate cells. The intracellular granules were less numerous
Fig. 7. Expression of HSP70 in three types of haemocytes (PR, PL, GR) in the control and in experimental groups exposed to low (20 mg/kg) and semi-lethal (10,000 mg/kg) concentrations of Pb.
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than in the control group. These intracellular granules were larger and usually formed aggregates (Fig. 3i and j). 3.4. Size of haemocytes The area of granulocytes obtained from the larvae reared on the media contaminated with heavy metals showed a significant decrease in comparison with cells originating from the control group of larvae (Fig. 4). GR were noticeably reduced in size by 42% and 47% in larvae reared on the media containing 20 mg/kg and 10,000 mg/kg of Pb, respectively. A similar decrease in GR size was detected in larvae treated with low concentrations of Cu, Cd and high concentration of Zn. In larvae exposed to Cu and Cd in high concentrations and to Zn in low concentration, changes in the area of GR were smaller. GRs were smaller by 26%, 25% and 30%, respectively. Comparing with the control, plasmatocytes were smaller only after exposure of larvae to high concentration of Pb. This difference was statistically significant (p = 0.006) (Fig. 5). The analysis of the size of prohaemocytes showed a statistically significant increase, by 23% and 21%, of their area only in larvae treated with low and high Zn concentrations, respectively (Fig. 6). 3.5. Expression of HSPs The immunocytochemistry method revealed that HSP70 expression did not significantly change in haemocytes obtained
from experimental insects when compared with haemocytes of the control group. The expression of HSP70 was significantly increased, however, in larvae exposed to a heat shock (43 8C for 20 h) used as a positive control. Only after exposure to low concentrations of Pb, Cd, Cu and Zn some prohaemocytes and a few granular cells showed stronger intensity of fluorescence in the cytoplasm (Figs. 7–10). In all analysed preparations, including those of the control group, cells showing very strong fluorescence were observed (‘flash-type’ cells). In the case of HSP72 no differences were detected in the abundance of this protein between experimental and control groups. 4. Discussion The results obtained in the present study showed that haemocytes, insect immune cells, are strongly affected by intoxication of insects with heavy metals. They also confirmed our earlier results (Borowska et al., 2004) and the results of other authors, showing that heavy metals reduce immune responses of organisms (Victor, 1993; Marth et al., 2000; Galloway and Depledge, 2001; Matozzo et al., 2001), resulting in decrease of survival. As in vertebrates, the efficiency of the immune system in insects is correlated with the number of haemocytes and their function. In our previous study we showed that house flies, exposed to the same low and high concentrations of Cu, Zn, Pb and Cd as in the
Fig. 8. Expression of HSP70 in three types of haemocytes (PR, PL, GR) in the control and in experimental groups exposed to low (3 mg/kg) and semi-lethal (50 mg/kg) concentrations of Cd.
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Fig. 9. Expression of HSP70 in three types of haemocytes (PR, PL, GR) in the control and in experimental groups exposed to low (5 mg/kg) and semi-lethal (1000 mg/kg) concentrations of Cu.
present study, accumulate 0.9 and 4.5 times more of Cu, 0.8 and 1.5 times more Zn, 1.3 and 43.7 times more of Pb, and 4.9 and 33.8 times more of Cd, respectively than the control groups (Borowska et al., 2004). Since all tissues are penetrated by haemolymph, we assume that immune cells also accumulate heavy metals as has been found in molluscs (Victor, 1993; McIntosh and Robinson, 1999; Marigomez et al., 1990, 2002). In house fly larvae we determined three basic types of haemocytes: prohaemocytes (PR), plasmatocytes (PL) and granulocytes (GR), on the basis of haemocyte classification obtained in other insect species (Gupta, 1985; Franchini et al., 1996; Silva et al., 2002). We also determined cells similar in morphology to both PL and GR which were classified as intermediates (I). In the third instar, larvae haemocytes fluctuate in number, but their total number significantly decreased in heavy metal exposed insects (Borowska et al., 2004) although haemocytes of M. domestica seem to be more resistant to this type of stress than do the cells of D. melanogaster (Filipiak et al., 2010). In house flies exposed to high concentrations of Pb, Zn, Cu and Cd, the number of immune cells decreased by 26%, 18%, 17% and 16%, respectively (Borowska et al., 2004). Similar results have been observed in other organisms, such as shrimp and crabs (Victor, 1993; Lorenz et al., 2001). As found in Cd-treated haemocytes of molluscs the increase of cell death may result from oxidative damage within cells, such as lipid peroxidation, enhanced production of ROS and DNA damage (Banakou and Dailianis, 2010).
In addition to the previously reported decrease in the total number of haemocytes (Borowska et al., 2004), we found that the haemocyte index, the proportion of different cell types in a population of haemocytes, changed in flies exposed to heavy metals. The significant decrease was especially observed in the number of granulocytes and plasmatocytes. It may result from high mortality of granulocytes as trefocytes and/or from slowing down haemocyte differentiation and appearance of cells intermediate between PL and GR in the total pool of haemocytes. In turn, the observed higher proliferation of prohaemocytes and abnormal haemocyte morphology may indicate the occurrence of neoplastic transformation of haemopoietic organs or stimulation of the immune system under the influence of metals (Victor, 1993; Olabarrieta et al., 2001). The above changes were observed in haemocytes from all experimental groups except those originating from larvae exposed to high (10,000 mg/kg) concentration of Pb. In that case, the decrease in number of granulocytes, when compared with the control group, was more pronounced than in other groups. High concentrations of lead exposure did not show a significant increase in the PR number, as observed in all other experimental groups. Lead had the strongest negative effect on the total number of haemocytes (Borowska et al., 2004) and on the area of trefocytes, an effect possibly caused by high toxicity of accumulated Pb which induced mortality of proliferating PR and/or degeneration of haemopoietic organs.
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Fig. 10. Expression of HSP70 in three types of haemocytes (PR, PL, GR) in the control and in experimental groups exposed to low (100 mg/kg) and semi-lethal (2000 mg/kg) concentrations of Zn.
Exposure to heavy metals does not change the ratio of living cells to their total number but affects adhesion ability of haemocytes. The adhesion ability of haemocytes decreased by 56% and 59% in response to high concentrations of Pb and Cu, respectively (Borowska et al., 2004). Haemocytes also did not change from a round to an amoeboid shape, the shape characteristic of cells active in phagocytosis (Nieto-Fernandez et al., 2000). This indicates that phagocytosis, one of most important defence mechanisms, is disturbed in heavy metal intoxicated insects and molluscs, decreasing efficiency of their immune systems. Moreover, the observed decrease in the number of plasmatocytes and granulocytes which participate in phagocytosis leads to multiplication of pathogens which eventually kill the insect. A similar effect has been observed in other invertebrates (Brousseau et al., 2000; Matozzo et al., 2001; Olchawa et al., 2006). The analysis of changes in morphology of haemocytes under the influence of heavy metals also showed a significant decrease of the area of cells identified as granulocytes (Fig. 3), which participate in phagocytosis. They also store and excrete products of metabolism. The decrease in size may be induced by the toxic effects of the accumulated metals on cell metabolism and cytoskeleton structure (Fagotti et al., 1996). The decrease in the cell area was observed after treating the larvae with all the metals used in the study, with the strongest effect observed with lead exposure. The observed changes in haemocyte morphology and their functions may also result from the heavy metal induced stress on
the house fly hormonal system. The heavy metal induced stress could affect hormonal balance and homeostasis. It has been reported that metals and metallothioneins have an impact on cellular signal transduction induced, among other compounds, by steroid hormones (DeMoor and Koropatnick, 2000). In insects ecdysteroids regulate development and metamorphosis. Ecdysteroids also influence conversion of haemocytes into phagocytic cells as well as release of haemocytes from the haemopoietic organs to haemolymph. Since larval development and metamorphosis are delayed in metal-exposed flies (Borowska et al., 2004) this effect may result from the influence of heavy metals on haemocytes which take part in metamorphosis (Gupta, 1985; Raina et al., 2001; Kiger et al., 2001) and on the endocrine system. This system controls synthesis of haemolymph proteins in the fat body, and, in some species, protein synthesis in haemocytes, their accumulation and transition to haemolymph. The disruption of these processes may also significantly weaken the immune system (Go¨tz and Boman, 1985). The results obtained in the present study did not prove, however, a correlation between heat shock protein expression in haemocytes of M. domestica and heavy metal exposure. In some species a clear correlation between levels of HSPs, especially HSP70, and exposure to heavy metals has been reported (Ko¨hler et al., 1992, 1996; Bierkens et al., 1998; Steiner et al., 1998; Werner and Hinton, 1999; Wagner et al., 1999; Aı¨t-Aı¨ssa et al., 2000; Gibney et al., 2004). In other species, however, such a correlation
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has not been detected (Pyza et al., 1997; Ko¨hler et al., 2000; Werner and Hinton, 2000; Lewis et al., 2001). HSP70 was observed in all groups of haemocytes exposed to heavy metals and was generally similar to that of the control group. Low concentrations of some metals resulted in a higher abundance of HSP70, yet this was only observed in a small number of prohaemocytes and granulocytes. This confirms the observations of other authors (Pyza et al., 1997; Feder and Hoffman, 1999; Ko¨hler et al., 2000; Warchałowska-S´liwa et al., 2005) that high concentrations and/or long exposure to heavy metals often decrease HSP70 levels, compared to lower concentrations of the same metals. We also did not detect expression of the induced HSP72 after intoxication of larvae with heavy metals. It is possible that in vivo adaptation of cells to the presence of heavy metals decreased induction of HSP (Croute et al., 2000; Ko¨hler et al., 2000). 5. Conclusions The obtained results confirmed that heavy metals are immunotoxins for insects. They affect the morphology and haemocyte index. Haemocytes can be used as biomarkers of heavy metal intoxication. Disruption of haemocytes seems to be responsible for earlier observations of delayed development, metamorphosis and decreased survival of flies (Borowska et al., 2004) due to increased susceptibility to infections and parasitoids. Since M. domestica can be easily encountered in the natural environment and is easy to breed, its haemocytes could also be used as a cheap and easily available model to test toxicity of various substances in the polluted environment. Acknowledgement This study was supported by the Polish Ministry of Science and Higher Education. Grant nr. N304 066 32/2604. References Aı¨t-Aı¨ssa, S., Porcher, J., Arrigo, A., Lambre, C., 2000. Activation of the hsp70 promoter by environmental inorganic and organic chemicals: relationships with cytotoxicity and lipophilicity. Toxicology 145, 147–157. Banakou, E., Dailianis, S., 2010. Involvement of Na+/H+ exchanger and respiratory burst enzymes NDPH oxidase and NO synthase, in Cd-induced lipid peroxidation and DNA damage in haemocytes of mussels. Comparative Biochemistry and Physiology, Part C 152, 346–352. Bauman, J.W., Liu, J., Klassen, C.D., 1993. Production of metallothionein and heatshock proteins in response to metals. Fundamental and Applied Toxicology 21, 15–22. Bierkens, J., Maes, J., Vander, P.F., 1998. Dose-dependent of heat shock protein 70 synthesis in Rhapidocelis subcapita following exposure to different classes of environmental pollutants. Environmental Pollution 101, 91–97. Borowska, J., Sulima, B., Niklin´ska, M., Pyza, E., 2004. Heavy metal accumulation and its effects on development, survival and immuno-competent cells of the house fly Musca domestica from closed laboratory populations as model organism. Fresenius Environmental Bulletin 13, 1402–1409. Brousseau, P., Pellerin, J., Morin, Y., Cyr, D., Blakley, B., Boermans, H., Fournier, M., 2000. Flow cytometry as a tool to monitor the disturbance of phagocytosis in the clam Mya arenaria haemocytes following in vitro exposure to heavy metals. Toxicology 142, 145–156. Croute, F., Beau, B., Arrabit, C., Gaubin, Y., Delmas, F., Murat, J.C., Soleilhavoup, J.P., 2000. Pattern of stress protein expression in human lung cell-line A549 after short- or long-term exposure to cadmium. Environmental Health Perspective 108, 55–60. Dailianis, S., 2009. Production of superoxides and nitric oxide generation in haemocytes of mussel Mytilus galloprovincialis (Lmk.) after exposure to cadmium: a possible involvement of Na+/H+ exchanger in the induction of cadmium toxic effects. Fish & Shellfish Immunology 27, 446–453. DeMoor, J.M., Koropatnick, D.J., 2000. Metals and cellular signalling in mammalian cells. Cell and Molecular Biology 46, 367–381. Duchemin, M.B., Auffret, M., Wessel, N., Fortier, M., Morin, Y., Pellerin, J., Fournier, M., 2008. Multiple experimental approaches of immunotoxic effects of mercury chloride in the blue mussel, Mytilus edulis, through in vivo, in tubo and in vitro exposures. Environmental Pollution 153, 416–423. Fagotti, A., Di Rosa, I., Simoncelli, F., Pipe, R.K., Panara, F., Pascolini, R., 1996. The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes. Developmental and Comparative Immunology 20, 383–391.
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