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Immune response in Porcellio scaber (Isopoda: Oniscidea): copper revisited
European Journal of Soil Biology 41 (2005) 77–83 http://france.elsevier.com/direct/ejsobi
Original article
Immune response in Porcellio scaber (Isopoda: Oniscidea): copper revisited Pinar Irmak a, Joachim Kurtz b, Martin Zimmer a,* a
b
Zoologisches Institut, Christian-Albrechts-Universität zu Kiel, Olshausenstr. 40, 24098 Kiel, Germany Department of Evolutionary Ecology, Max Planck Institute of Limnology, August-Thienemann Str. 2, 24306 Plön, Germany Available online 17 October 2005
Abstract Despite of numerous descriptive and experimental approaches, during the last 40 years, the function of vast copper stores in the midgut glands (hepatopancreas) of terrestrial isopods is still unknown. We tested the hypothesis that copper is involved in immune defence through the action of phenol-oxidising enzymes. We used Porcellio scaber fed on artificial diets with or without copper and with or without tyrosine for 2 months to reduce the copper store in the midgut glands and the tyrosine concentration in the hemolymph. To activate the immune response we implanted nylon filaments as artificial pathogens; we used phenoloxidase activity in hemolymph in vitro, and both cellular encapsulation and melanisation of filaments in vivo as measures of immune response. Isopods were capable of efficient immune reactions. Already 1 h after implantation of a nylon filament as an artificial pathogen, we observed heavy cellular encapsulation. At the same time, the implanted nylon filaments became partially covered with melanin that was likely due to hemolymph phenol oxidase (PO) activity. Hemolymph obtained from P. scaber also showed phenoloxidase activity in vitro, but to induce such activity, the addition of a denaturing detergent was necessary. Based on this, we propose that the hemocyanine of P. scaber can exert PO activity upon activation in vitro; whether, however, this process is of any significance in vivo is unclear. Our results on the effects of pre-experimental diets on immune response indicate that a deficiency of dietary copper might lead to the inability to raise and to regulate an efficient immune response, and thus, suggest an involvement of copper in immune response. © 2005 Elsevier SAS. All rights reserved. Keywords: Copper storage; Encapsulation; Hemocyanine; Hemocyte; Immune response; Melanisation; Pathogen infection; Phenol oxidation
1. Introduction During the last decade, the field of immunology has seen a dramatic shift of focus towards innate immunity, with a significant share of studies making use of invertebrates [15,20]. Yet, such work normally focuses on a limited number of model species, while the diversity of defence mechanisms that can be expected in the heterogeneous group of ‘invertebrates’ up to now remains largely unexplored * Corresponding author. Fax: +49 431 880 4368. E-mail address: [email protected] (M. Zimmer).
1164-5563/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.ejsobi.2005.09.011
[21,26]. Isopods may serve as particularly interesting examples for the study of immune defence under differing environmental conditions, since members of this taxon made the evolutionary step from marine to terrestrial habitats, with extant species still inhabiting these habitats, as well as intertidal zones and freshwater ecosystems. Not only parasite-mediated selection differs strongly between these habitats, also the environmental availability of resources needed for immune defence may deviate strongly. A considerable part of arthropod immune defence depends on phenol oxidases (POs), which are involved in the processes of encapsulation and melanisation of
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pathogens, wound healing, and sclerotisation of the cuticle [9,33]. These enzymes need phenolic compounds such as tyrosine as substrates. Enzymatic activity depends on copper, which is bound to evolutionary conserved binding sites. Here, we approach the potential roles of tyrosine and copper for immune defence of a terrestrial isopod species, the common woodlouse P. scaber. Terrestrial isopods store vast amounts of copper in specific organelles of S-cells in their digestive midgut glands (hepatopancreas [17,18,37]. Early studies tried to explain the phenomenon of copper storage in the light of land colonisation [14,35,36]. Owing to the difficult supply with copper in seawater, marine isopods may have developed mechanisms to extract copper effectively; if this ability has not been lost during the course of terrestrialisation, we will expect terrestrial isopods to effectively extract copper even in copper-rich environments [41]. More recently, we focused on considering the hepatopancreatic copper store an adaptation to the terrestrial life style (rather than a relict from marine ancestors). Thus, in previous studies we suspected a role of copper in digestive processes through copper-containing POs of endosymbiotic origin that act in the degradation of lignins and other phenolic compounds in phenol-rich food sources [39,40]. As mentioned above, another major function of POs is their involvement in immune defence, and copper storage may thus be relevant in this context. Within the immune system, POs are tightly regulated. In the course of a well-studied enzymatic cascade, a pro-PO is activated into PO that oxidises phenols (usually tyrosine) into quinones that, in turn, polymerise to form melanin to encapsulate pathogens [2,6,13,25]. However, in contrast to most other arthropods studied thus far, isopods may not exhibit a PO cascade upon immune stimulation [12,32]. As early as 1965, Wieser [35] suggested the use of hemocyanine (HC) as respiratory pigment as a reason for the storage of copper. Like PO, HC is a type-3 copper protein [4,5,16]. However, the storage capacity for copper in hepatopancreatic tissue surpasses the physiological needs for HC by orders of magnitude [7]. Interestingly, HC can be converted into a functional phenoloxidising enzyme upon conformation change in vitro [1, 8,11,28,42], although the significance of this process in vivo is as yet unknown. Since HC and PO probably evolved from a common ancestor enzyme, phenol-oxidising activity of HC might be an evolutionary relict [3]. With respect to immune defence, copper may therefore be relevant either through POs directly, or through
functional PO activity of HC. The present study is a first approach to evaluate the potential function of copper in the immune response of isopods in terms of phenol oxidation and pathogen encapsulation. In particular, we manipulated the nutritional availability of tyrosine and copper by feeding isopods with artificial diets, which were free of one or both of these substances. 2. Materials and methods 2.1. Animals Individuals of P. scaber were collected over a period of 4 months on the campus of the Christian-Albrechts University, Kiel, Germany. In the laboratory, isopods were maintained individually at 15 °C, 16 h L:8 h D, and were fed with an artificial diet [40] for 2 months. We distinguished four groups of isopods according to the diet provided: ● copper (central atom of both PO and HC) and tyrosine (substrate of PO in situ); ● copper, but no tyrosine; ● tyrosine, but no copper; ● neither copper nor tyrosine. According to Weißenburg and Zimmer [34], isopods that had fed on copper-free diet for 4 weeks were expected to be copper deficient. 2.2. PO assays For the in vitro PO assays, we added 5 mM MBTH (3-methyl-2-benzothiazoline hydrazone hydrochloride) [38] to a 40 mM HEPES buffer (N-2-Hydroxyethylpiperazine-N´-2-ethanosulfonic acid), pH 7.0. We used 4 mM dopamine as substrate, and 0.06% SDS (sodium-dodecyl-sulfate) was added according to Refs [11,19,22–24] to induce a conformation change of HC (see results and discussion for explanation). In pre-experiments with field-collected isopods, we added 0.5 μl of a saturated solution of phenylthiourea (PTU) in ethanol to specifically inhibit PO activity. This procedure served as a control for phenol oxidation through mechanisms other than PO activity. Each treatment was started with N = 15 replicates, but owing to death events, N was reduced to 5–8 per treatment. Hemolymph of field-collected isopods served as non-manipulated controls. For collection of hemolymph (2–10 μl) from isopods that had been fed with artificial diets mentioned above,
P. Irmak et al. / European Journal of Soil Biology 41 (2005) 77–83
animals were cooled on ice, before the cuticle was punctured dorsally on the thorax with a sterile injection needle. Hemolymph was immediately collected into a calibrated 5 μl glass microcapillary and added to 200 μl of the phenolic substrate in HEPES buffer in wells of a 96well ELISA plate, followed by 0.5 μl SDS. We recorded the change in absorption at 490 nm for 2 h on a BIOTEK® PowerWave × Select microplate scanning spectrophotometer (BIO-TEK Instruments, Inc., Winooski, Vermont, USA), using the BIO-TEK KC4 (© 2000) software. The activity was measured as the slope of product formation during 90 min of the linear phase. The concentration of total protein in the hemolymph was determined photometrically in 1 μl of hemolymph, using the test solution Roti® Nanoquant (K 880.1, Carl Roth GmbH & Co., Karlsruhe, Germany), following the protocol of the manufacturer. Measurements of OD590/ OD450 were taken in 96 well plates on the microplate spectrophotometer. Standard curves were obtained from serial dilutions of BSA (SIGMA, Taufkirchen, Austria) and included on each 96 well plate. 2.3. Encapsulation of nylon filaments as artificial pathogens In another experiment, we challenged individuals by implanting an artificial pathogen, a colourless nylon filament with a diameter of 0.12 mm [27,30,31] that was inserted dorsally between the fourth and fifth pereion segments. The length of the filament varied between 2 and 4 mm (we took this into account in the analyses, see below). Field-collected isopods served as control and reference for non-manipulated response to an artificial pathogen. Pathogen encapsulation is considered a two-step process. First, invertebrate blood cells (hemocytes) aggregate around the pathogen, thereafter dark-brown melanin is deposited in a PO-dependent reaction, called melanisation. We aimed at estimating the strength of both parameters, measuring first the total area of the capsule, and second the degree of darkening. To do so, we used the image analysis software on digital images obtained on a microscope (Zeiss standard 25) and digital camera (Olympus C-4040 Zoom). In a time series of isopod sampling, we removed the nylon filaments by dissection of field-collected isopods (N = 10 per sampling) after 1, 3, 24, and 48 h, and screened them under a microscope for both cellular and melanin encapsulation. Melanisation of the nylon filaments in terms of brown coloration was estimated on a relative scale (0– 5) by two test persons independently. Before applying
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their estimate to our experimental data, we tested for rank correlation (R = 0.645, P < 0.001, N = 341), and quantified melanisation of nylon filaments based on the mean personal estimates for each individual filament. We had observed maximum encapsulation 24 hours after insertion of the filaments in field-collected specimens. Isopods fed artificial diets (N = 5–8 per diet) were thus dissected 24 hours after implantation. To obtain simultaneous measurements of hemolymph protein and PO, we also took hemolymph samples from these individuals immediately before implantation and prior to dissection and handled them as described above. 2.4. Statistics Most of our data showed significant deviation from normal distribution. Thus, results are presented as median ± median absolute deviation, and non-parametric statistics were used for statistical comparison between species. Groups of data were analysed through Kruskal– Wallis H tests, pair-wise comparison was performed using Mann–Whitney U-tests (Bonferroni-corrected, where applicable). For the analysis of correlative relationships between measured parameters, we used Spearman’s rank correlation. 3. Results 3.1. Hemolymph PO activity Of the tested substrates, only dopamine revealed considerable PO activity in in vitro measurements of isopod hemolymph, while oxidation of other substrates was negligible (cf. Table 1). PO activity was not inhibited by PTU, but the addition of SDS was required to induce PO activity (Fig. 1). PO activity in isopods fed on artificial diets did not depend on the composition of the diet with respect to copper and tyrosine; treatments did not differ from each other (Fig. 2A; P > 0.1). By contrast, the hemolymph protein content of isopods fed on copper-containing diet was significantly higher than that of copper-deficient isopods (Fig. 2B; P < 0.05). One day after implantation of an artificial pathogen, PO activity was significantly reduced in all treatments (Fig. 3A; P < 0.01); however, treatments did not differ from each other in PO activity (P > 0.1). By contrast, the hemolymph protein content was significantly increased in copper-fed isopods (P < 0.01), but did not change in copper-deficient individuals (P > 0.4), in response to pathogen implantation (Fig. 3B).
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Table 1 In vitro PO activity (change in absorption over time: relative units) in hemolymph samples of P. scaber using different phenolic substrates and a substrate-free control (Blind), supplements and inhibitors (for explanation, see text). Data give the range (min–max) of three replicate measurements, each. Different lower-case letters indicate significant differences between treatments (α = 0.05); control, no supplement/inhibitor added; PTU, phenylthiourea (inhibitor) added; SDS, sodium dodecyl sulfate (denaturant) added Substrate Dopamine L-DOPA 4-methylcatechol Blind
control 0.7–1.2a 1.1–1.2a 0.02–0.03g
PTU 0.6–1.2a 0.1–0.8a,d 0.09–0.14d
SDS 7.6–8.2b 1.93–1.97e 0.89–0.95a
PTU/SDS 11.4–12.3c 1.40–1.45f 1.0–1.4f
0.11–0.16d
0.06–0.12d
0.09–0.22d
0.03–0.05g
Fig. 1. PO activity in hemolymph of P. scaber in vitro as tested with different supplements and inhibitors (for details see text). Activity is given as relative units, since extinction coefficients of phenol oxidation products cannot be provided, owing to random polymerisation processes; values are comparable, since each hemolymph sample was tested with each reaction mixture. control, no supplement/ inhibitor added; PTU, phenylthiourea (inhibitor) added; SDS, sodium dodecyl sulfate (denaturant) added. Bars indicate median ± median absolute deviation (N = 6). Different lower-case letters indicate significant differences between treatments (α = 0.05).
3.2. Encapsulation of nylon filaments as artificial pathogens
Fig. 2. PO activity in vitro (A) and protein content (B) in hemolypmph of P. scaber fed on artificial diets: diet without copper and without tyrosine (control), with copper and without tyrosine (Cu), diet without copper and with tyrosine (Tyr), diet with copper and with tyrosine (Cu/Tyr). Bars indicate median ± median absolute deviation (N = 5– 8). Different lower-case letters indicate significant differences between treatments (α = 0.05).
Diet-fed isopods exhibited cellular pathogen encapsulations that did not depend on the pre-experimental treatment (Fig. 5A; P > 0.9); values were in the same range of those observed in specimens freshly collected from the field. Isopods that had fed on a copper-containing diet achieved similar intensities of nylon filament melanisation as field specimens, but copper-deficient individuals exhibited significantly lower values (Fig. 5C; P < 0.05). 4. Discussion
In freshly field-collected isopods, implantation of nylon filaments induced both a cellular encapsulation and a melanisation of the artificial pathogens. The total area of cellular encapsulation increased during the first 24 h and then decreased again (Fig. 4A). However, when controlled for the length of the implant, the encapsulation area reached a maximum already after 1 h and remained stable thereafter (Fig. 4B). Similarly, melanisation of the nylon filament reached a maximum plateau value after 1 h (Fig. 4C).
According to our results, P. scaber is capable of efficient immune reactions: as early as 1 hour after implantation of a nylon filament as an artificial pathogen, we observed heavy cellular encapsulation that remained stable for at least 48 h. At the same time, the implanted nylon filaments became partially covered with melanin that was likely due to hemolymph PO activity. However, the processes of cellular encapsulation and melanisation were negatively correlated (RS = –0.55; P = 0.03;
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Fig. 3. Changes in PO activity in vitro (A) and protein content (B) in hemolymph of P. scaber in response to the implantation of nylon filaments; negative values indicate lower activity after implantation. Bars indicate median ± median absolute deviation (N = 5–8). Diet without copper and without tyrosine (control), with copper and without tyrosine (Cu), diet without copper and with tyrosine (Tyr), diet with copper and with tyrosine (Cu/Tyr). Different lower-case letters indicate significant differences between treatments (α = 0.05); asterisks indicate changes significant from zero (α = 0.05; ns, not significant).
N = 15), i.e. individuals with strong melanisation showed smaller areas of cellular capsules. Most likely, this indicates differences in the time course of the whole process, which starts with the aggregation of hemocytes and proceeds towards melanisation of the capsule. Since this comes along with degeneration of the cells, a reduction in size of the capsule during later stages of the process can be expected. Individuals with stronger melanisation (but smaller size of the capsule) would then show the faster and, thus, potentially superior immune response. Alternatively, the two parts of the process would also appear negatively correlated when individuals are constraint to optimise either one or the other part of the process. Hemolymph obtained from P. scaber also showed phenoloxidase (PO) activity in vitro. However, to induce such activity, the addition of the detergent SDS to the reaction mixture was necessary. SDS has previously been used to activate HC through a conformational
Fig. 4. Area of encapsulation (A), encapsulation area corrected for filament length (B), and relative melanisation (C) of nylon filaments used as artificial pathogen in P. scaber from the field. Data are median ± median absolute deviation (N = 12).
change to act as PO [11,19,22–24]. POs have been separated into two distinct groups. Tyrosinases (EC 1.14.18.1) catalyse the hydroxylation of monophenols and the oxidation of o-diphenols into o-quinones. Catechol oxidases (EC 1.10.3.1) are more specific and catalyse the oxidation of o-diphenols [4,10,29]. If HC serves as PO, it acts like a catechol oxidase [19]. Decker et al. [11] demonstrated that dopamine is a typical substrate for catechol oxidases, and dopamine yielded highest PO activity in our measurements. Thus, we propose that
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Fig. 5. Area of encapsulation corrected for filament length (A), and relative melanisation (B) of nylon filaments used as artificial pathogen in P. scaber fed on artificial diets: diet without copper and without tyrosine (control), with copper and without tyrosine (Cu), diet without copper and with tyrosine (Tyr), diet with copper and with tyrosine (Cu/Tyr). Data are median ± median absolute deviation (N = 5–8). Different lower-case letters indicate significant differences between treatments (α = 0.05).
the HC of P. scaber can exert PO activity upon activation in vitro; whether, however, this process takes place, or is of any significance, in vivo remains an open question that warrants further detailed investigation; we did not observe any correlation (RS = 0.11; P = 0.68; N = 15) between PO activity in vitro and melanisation of nylon filaments in vivo. Isopods fed on copper-free diet showed a lower hemolymph protein concentration and an absence of the normally observed increase in hemolymph protein after implantation. While we did not find any effects of tyrosine or copper deficiency on PO activity, we found reduced melanisation of a nylon implant in the absence of copper. This could indicate that the deficiency of dietary copper might lead to the inability to raise and to regulate an efficient immune response. We here assume that either the experimental hemolymph withdrawal or the implantation of the artificial pathogen induce the regeneration of hemolymph proteins under normal conditions, but not in copper-deficient isopods. This observation
suggests that a considerable proportion of the synthesised proteins are HC and/or POs, or that the regeneration of proteins in response to pathogen attack is somehow copper-dependent. Interestingly, the copperdependence of protein regeneration provides a (correlative) link between copper supply and the competence of cellular encapsulation. Correlation analyses revealed a positive correlation between the hemolymph protein content (prior to pathogen implantation) and the intensity of cellular encapsulation in response to pathogen implantation, but a negative correlation between the latter parameter and the hemolymph protein content after pathogen implantation. Thus, the ability to synthesise hemolymph proteins (in a copper-dependent process?) seems to indicate the capability of pathogen encapsulation. We were surprised to see reduced PO activity in vitro after the artificial pathogen attack, since we expected this procedure to induce an immune response and to result in a higher immune competence in terms of hemolymph PO activity. Possibly, the time interval (1 day) between pathogen implantation and subsequent PO determination was not long enough to allow for hemolymph protein regeneration, and we would have revealed a different picture had we given the isopods more time to elevate their weakened PO system. On the other hand, we found a positive correlation between PO activity before and after pathogen implantation (RS = 0.58; P = 0.02; N = 15). Thus, individuals with an efficient immune response (as indicated by in vitro PO activity) maintain a relatively high immune response after (artificial) pathogen attack, while individuals with a weaker immune response remain less immune competent than others. As to our knowledge, this is the first attempt to study the role of melanisation and encapsulation of pathogens in terrestrial isopods. Overall, our results present evidence for copper being essential for the immune response in P. scaber. Since the present study was designed as a first approach, however, numerous questions remain unanswered, warranting future detailed research on the immune competence of, and immune response in, terrestrial isopods. Acknowledgements We are grateful to Jens Rolff, University of Sheffield, for helpful hints on the implantation of nylon filaments, and to Elmar Jaenicke, Universität Mainz, for discussing PO and HC in arthropods.
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Original article
Immune response in Porcellio scaber (Isopoda: Oniscidea): copper revisited Pinar Irmak a, Joachim Kurtz b, Martin Zimmer a,* a
b
Zoologisches Institut, Christian-Albrechts-Universität zu Kiel, Olshausenstr. 40, 24098 Kiel, Germany Department of Evolutionary Ecology, Max Planck Institute of Limnology, August-Thienemann Str. 2, 24306 Plön, Germany Available online 17 October 2005
Abstract Despite of numerous descriptive and experimental approaches, during the last 40 years, the function of vast copper stores in the midgut glands (hepatopancreas) of terrestrial isopods is still unknown. We tested the hypothesis that copper is involved in immune defence through the action of phenol-oxidising enzymes. We used Porcellio scaber fed on artificial diets with or without copper and with or without tyrosine for 2 months to reduce the copper store in the midgut glands and the tyrosine concentration in the hemolymph. To activate the immune response we implanted nylon filaments as artificial pathogens; we used phenoloxidase activity in hemolymph in vitro, and both cellular encapsulation and melanisation of filaments in vivo as measures of immune response. Isopods were capable of efficient immune reactions. Already 1 h after implantation of a nylon filament as an artificial pathogen, we observed heavy cellular encapsulation. At the same time, the implanted nylon filaments became partially covered with melanin that was likely due to hemolymph phenol oxidase (PO) activity. Hemolymph obtained from P. scaber also showed phenoloxidase activity in vitro, but to induce such activity, the addition of a denaturing detergent was necessary. Based on this, we propose that the hemocyanine of P. scaber can exert PO activity upon activation in vitro; whether, however, this process is of any significance in vivo is unclear. Our results on the effects of pre-experimental diets on immune response indicate that a deficiency of dietary copper might lead to the inability to raise and to regulate an efficient immune response, and thus, suggest an involvement of copper in immune response. © 2005 Elsevier SAS. All rights reserved. Keywords: Copper storage; Encapsulation; Hemocyanine; Hemocyte; Immune response; Melanisation; Pathogen infection; Phenol oxidation
1. Introduction During the last decade, the field of immunology has seen a dramatic shift of focus towards innate immunity, with a significant share of studies making use of invertebrates [15,20]. Yet, such work normally focuses on a limited number of model species, while the diversity of defence mechanisms that can be expected in the heterogeneous group of ‘invertebrates’ up to now remains largely unexplored * Corresponding author. Fax: +49 431 880 4368. E-mail address: [email protected] (M. Zimmer).
1164-5563/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.ejsobi.2005.09.011
[21,26]. Isopods may serve as particularly interesting examples for the study of immune defence under differing environmental conditions, since members of this taxon made the evolutionary step from marine to terrestrial habitats, with extant species still inhabiting these habitats, as well as intertidal zones and freshwater ecosystems. Not only parasite-mediated selection differs strongly between these habitats, also the environmental availability of resources needed for immune defence may deviate strongly. A considerable part of arthropod immune defence depends on phenol oxidases (POs), which are involved in the processes of encapsulation and melanisation of
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pathogens, wound healing, and sclerotisation of the cuticle [9,33]. These enzymes need phenolic compounds such as tyrosine as substrates. Enzymatic activity depends on copper, which is bound to evolutionary conserved binding sites. Here, we approach the potential roles of tyrosine and copper for immune defence of a terrestrial isopod species, the common woodlouse P. scaber. Terrestrial isopods store vast amounts of copper in specific organelles of S-cells in their digestive midgut glands (hepatopancreas [17,18,37]. Early studies tried to explain the phenomenon of copper storage in the light of land colonisation [14,35,36]. Owing to the difficult supply with copper in seawater, marine isopods may have developed mechanisms to extract copper effectively; if this ability has not been lost during the course of terrestrialisation, we will expect terrestrial isopods to effectively extract copper even in copper-rich environments [41]. More recently, we focused on considering the hepatopancreatic copper store an adaptation to the terrestrial life style (rather than a relict from marine ancestors). Thus, in previous studies we suspected a role of copper in digestive processes through copper-containing POs of endosymbiotic origin that act in the degradation of lignins and other phenolic compounds in phenol-rich food sources [39,40]. As mentioned above, another major function of POs is their involvement in immune defence, and copper storage may thus be relevant in this context. Within the immune system, POs are tightly regulated. In the course of a well-studied enzymatic cascade, a pro-PO is activated into PO that oxidises phenols (usually tyrosine) into quinones that, in turn, polymerise to form melanin to encapsulate pathogens [2,6,13,25]. However, in contrast to most other arthropods studied thus far, isopods may not exhibit a PO cascade upon immune stimulation [12,32]. As early as 1965, Wieser [35] suggested the use of hemocyanine (HC) as respiratory pigment as a reason for the storage of copper. Like PO, HC is a type-3 copper protein [4,5,16]. However, the storage capacity for copper in hepatopancreatic tissue surpasses the physiological needs for HC by orders of magnitude [7]. Interestingly, HC can be converted into a functional phenoloxidising enzyme upon conformation change in vitro [1, 8,11,28,42], although the significance of this process in vivo is as yet unknown. Since HC and PO probably evolved from a common ancestor enzyme, phenol-oxidising activity of HC might be an evolutionary relict [3]. With respect to immune defence, copper may therefore be relevant either through POs directly, or through
functional PO activity of HC. The present study is a first approach to evaluate the potential function of copper in the immune response of isopods in terms of phenol oxidation and pathogen encapsulation. In particular, we manipulated the nutritional availability of tyrosine and copper by feeding isopods with artificial diets, which were free of one or both of these substances. 2. Materials and methods 2.1. Animals Individuals of P. scaber were collected over a period of 4 months on the campus of the Christian-Albrechts University, Kiel, Germany. In the laboratory, isopods were maintained individually at 15 °C, 16 h L:8 h D, and were fed with an artificial diet [40] for 2 months. We distinguished four groups of isopods according to the diet provided: ● copper (central atom of both PO and HC) and tyrosine (substrate of PO in situ); ● copper, but no tyrosine; ● tyrosine, but no copper; ● neither copper nor tyrosine. According to Weißenburg and Zimmer [34], isopods that had fed on copper-free diet for 4 weeks were expected to be copper deficient. 2.2. PO assays For the in vitro PO assays, we added 5 mM MBTH (3-methyl-2-benzothiazoline hydrazone hydrochloride) [38] to a 40 mM HEPES buffer (N-2-Hydroxyethylpiperazine-N´-2-ethanosulfonic acid), pH 7.0. We used 4 mM dopamine as substrate, and 0.06% SDS (sodium-dodecyl-sulfate) was added according to Refs [11,19,22–24] to induce a conformation change of HC (see results and discussion for explanation). In pre-experiments with field-collected isopods, we added 0.5 μl of a saturated solution of phenylthiourea (PTU) in ethanol to specifically inhibit PO activity. This procedure served as a control for phenol oxidation through mechanisms other than PO activity. Each treatment was started with N = 15 replicates, but owing to death events, N was reduced to 5–8 per treatment. Hemolymph of field-collected isopods served as non-manipulated controls. For collection of hemolymph (2–10 μl) from isopods that had been fed with artificial diets mentioned above,
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animals were cooled on ice, before the cuticle was punctured dorsally on the thorax with a sterile injection needle. Hemolymph was immediately collected into a calibrated 5 μl glass microcapillary and added to 200 μl of the phenolic substrate in HEPES buffer in wells of a 96well ELISA plate, followed by 0.5 μl SDS. We recorded the change in absorption at 490 nm for 2 h on a BIOTEK® PowerWave × Select microplate scanning spectrophotometer (BIO-TEK Instruments, Inc., Winooski, Vermont, USA), using the BIO-TEK KC4 (© 2000) software. The activity was measured as the slope of product formation during 90 min of the linear phase. The concentration of total protein in the hemolymph was determined photometrically in 1 μl of hemolymph, using the test solution Roti® Nanoquant (K 880.1, Carl Roth GmbH & Co., Karlsruhe, Germany), following the protocol of the manufacturer. Measurements of OD590/ OD450 were taken in 96 well plates on the microplate spectrophotometer. Standard curves were obtained from serial dilutions of BSA (SIGMA, Taufkirchen, Austria) and included on each 96 well plate. 2.3. Encapsulation of nylon filaments as artificial pathogens In another experiment, we challenged individuals by implanting an artificial pathogen, a colourless nylon filament with a diameter of 0.12 mm [27,30,31] that was inserted dorsally between the fourth and fifth pereion segments. The length of the filament varied between 2 and 4 mm (we took this into account in the analyses, see below). Field-collected isopods served as control and reference for non-manipulated response to an artificial pathogen. Pathogen encapsulation is considered a two-step process. First, invertebrate blood cells (hemocytes) aggregate around the pathogen, thereafter dark-brown melanin is deposited in a PO-dependent reaction, called melanisation. We aimed at estimating the strength of both parameters, measuring first the total area of the capsule, and second the degree of darkening. To do so, we used the image analysis software on digital images obtained on a microscope (Zeiss standard 25) and digital camera (Olympus C-4040 Zoom). In a time series of isopod sampling, we removed the nylon filaments by dissection of field-collected isopods (N = 10 per sampling) after 1, 3, 24, and 48 h, and screened them under a microscope for both cellular and melanin encapsulation. Melanisation of the nylon filaments in terms of brown coloration was estimated on a relative scale (0– 5) by two test persons independently. Before applying
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their estimate to our experimental data, we tested for rank correlation (R = 0.645, P < 0.001, N = 341), and quantified melanisation of nylon filaments based on the mean personal estimates for each individual filament. We had observed maximum encapsulation 24 hours after insertion of the filaments in field-collected specimens. Isopods fed artificial diets (N = 5–8 per diet) were thus dissected 24 hours after implantation. To obtain simultaneous measurements of hemolymph protein and PO, we also took hemolymph samples from these individuals immediately before implantation and prior to dissection and handled them as described above. 2.4. Statistics Most of our data showed significant deviation from normal distribution. Thus, results are presented as median ± median absolute deviation, and non-parametric statistics were used for statistical comparison between species. Groups of data were analysed through Kruskal– Wallis H tests, pair-wise comparison was performed using Mann–Whitney U-tests (Bonferroni-corrected, where applicable). For the analysis of correlative relationships between measured parameters, we used Spearman’s rank correlation. 3. Results 3.1. Hemolymph PO activity Of the tested substrates, only dopamine revealed considerable PO activity in in vitro measurements of isopod hemolymph, while oxidation of other substrates was negligible (cf. Table 1). PO activity was not inhibited by PTU, but the addition of SDS was required to induce PO activity (Fig. 1). PO activity in isopods fed on artificial diets did not depend on the composition of the diet with respect to copper and tyrosine; treatments did not differ from each other (Fig. 2A; P > 0.1). By contrast, the hemolymph protein content of isopods fed on copper-containing diet was significantly higher than that of copper-deficient isopods (Fig. 2B; P < 0.05). One day after implantation of an artificial pathogen, PO activity was significantly reduced in all treatments (Fig. 3A; P < 0.01); however, treatments did not differ from each other in PO activity (P > 0.1). By contrast, the hemolymph protein content was significantly increased in copper-fed isopods (P < 0.01), but did not change in copper-deficient individuals (P > 0.4), in response to pathogen implantation (Fig. 3B).
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Table 1 In vitro PO activity (change in absorption over time: relative units) in hemolymph samples of P. scaber using different phenolic substrates and a substrate-free control (Blind), supplements and inhibitors (for explanation, see text). Data give the range (min–max) of three replicate measurements, each. Different lower-case letters indicate significant differences between treatments (α = 0.05); control, no supplement/inhibitor added; PTU, phenylthiourea (inhibitor) added; SDS, sodium dodecyl sulfate (denaturant) added Substrate Dopamine L-DOPA 4-methylcatechol Blind
control 0.7–1.2a 1.1–1.2a 0.02–0.03g
PTU 0.6–1.2a 0.1–0.8a,d 0.09–0.14d
SDS 7.6–8.2b 1.93–1.97e 0.89–0.95a
PTU/SDS 11.4–12.3c 1.40–1.45f 1.0–1.4f
0.11–0.16d
0.06–0.12d
0.09–0.22d
0.03–0.05g
Fig. 1. PO activity in hemolymph of P. scaber in vitro as tested with different supplements and inhibitors (for details see text). Activity is given as relative units, since extinction coefficients of phenol oxidation products cannot be provided, owing to random polymerisation processes; values are comparable, since each hemolymph sample was tested with each reaction mixture. control, no supplement/ inhibitor added; PTU, phenylthiourea (inhibitor) added; SDS, sodium dodecyl sulfate (denaturant) added. Bars indicate median ± median absolute deviation (N = 6). Different lower-case letters indicate significant differences between treatments (α = 0.05).
3.2. Encapsulation of nylon filaments as artificial pathogens
Fig. 2. PO activity in vitro (A) and protein content (B) in hemolypmph of P. scaber fed on artificial diets: diet without copper and without tyrosine (control), with copper and without tyrosine (Cu), diet without copper and with tyrosine (Tyr), diet with copper and with tyrosine (Cu/Tyr). Bars indicate median ± median absolute deviation (N = 5– 8). Different lower-case letters indicate significant differences between treatments (α = 0.05).
Diet-fed isopods exhibited cellular pathogen encapsulations that did not depend on the pre-experimental treatment (Fig. 5A; P > 0.9); values were in the same range of those observed in specimens freshly collected from the field. Isopods that had fed on a copper-containing diet achieved similar intensities of nylon filament melanisation as field specimens, but copper-deficient individuals exhibited significantly lower values (Fig. 5C; P < 0.05). 4. Discussion
In freshly field-collected isopods, implantation of nylon filaments induced both a cellular encapsulation and a melanisation of the artificial pathogens. The total area of cellular encapsulation increased during the first 24 h and then decreased again (Fig. 4A). However, when controlled for the length of the implant, the encapsulation area reached a maximum already after 1 h and remained stable thereafter (Fig. 4B). Similarly, melanisation of the nylon filament reached a maximum plateau value after 1 h (Fig. 4C).
According to our results, P. scaber is capable of efficient immune reactions: as early as 1 hour after implantation of a nylon filament as an artificial pathogen, we observed heavy cellular encapsulation that remained stable for at least 48 h. At the same time, the implanted nylon filaments became partially covered with melanin that was likely due to hemolymph PO activity. However, the processes of cellular encapsulation and melanisation were negatively correlated (RS = –0.55; P = 0.03;
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Fig. 3. Changes in PO activity in vitro (A) and protein content (B) in hemolymph of P. scaber in response to the implantation of nylon filaments; negative values indicate lower activity after implantation. Bars indicate median ± median absolute deviation (N = 5–8). Diet without copper and without tyrosine (control), with copper and without tyrosine (Cu), diet without copper and with tyrosine (Tyr), diet with copper and with tyrosine (Cu/Tyr). Different lower-case letters indicate significant differences between treatments (α = 0.05); asterisks indicate changes significant from zero (α = 0.05; ns, not significant).
N = 15), i.e. individuals with strong melanisation showed smaller areas of cellular capsules. Most likely, this indicates differences in the time course of the whole process, which starts with the aggregation of hemocytes and proceeds towards melanisation of the capsule. Since this comes along with degeneration of the cells, a reduction in size of the capsule during later stages of the process can be expected. Individuals with stronger melanisation (but smaller size of the capsule) would then show the faster and, thus, potentially superior immune response. Alternatively, the two parts of the process would also appear negatively correlated when individuals are constraint to optimise either one or the other part of the process. Hemolymph obtained from P. scaber also showed phenoloxidase (PO) activity in vitro. However, to induce such activity, the addition of the detergent SDS to the reaction mixture was necessary. SDS has previously been used to activate HC through a conformational
Fig. 4. Area of encapsulation (A), encapsulation area corrected for filament length (B), and relative melanisation (C) of nylon filaments used as artificial pathogen in P. scaber from the field. Data are median ± median absolute deviation (N = 12).
change to act as PO [11,19,22–24]. POs have been separated into two distinct groups. Tyrosinases (EC 1.14.18.1) catalyse the hydroxylation of monophenols and the oxidation of o-diphenols into o-quinones. Catechol oxidases (EC 1.10.3.1) are more specific and catalyse the oxidation of o-diphenols [4,10,29]. If HC serves as PO, it acts like a catechol oxidase [19]. Decker et al. [11] demonstrated that dopamine is a typical substrate for catechol oxidases, and dopamine yielded highest PO activity in our measurements. Thus, we propose that
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Fig. 5. Area of encapsulation corrected for filament length (A), and relative melanisation (B) of nylon filaments used as artificial pathogen in P. scaber fed on artificial diets: diet without copper and without tyrosine (control), with copper and without tyrosine (Cu), diet without copper and with tyrosine (Tyr), diet with copper and with tyrosine (Cu/Tyr). Data are median ± median absolute deviation (N = 5–8). Different lower-case letters indicate significant differences between treatments (α = 0.05).
the HC of P. scaber can exert PO activity upon activation in vitro; whether, however, this process takes place, or is of any significance, in vivo remains an open question that warrants further detailed investigation; we did not observe any correlation (RS = 0.11; P = 0.68; N = 15) between PO activity in vitro and melanisation of nylon filaments in vivo. Isopods fed on copper-free diet showed a lower hemolymph protein concentration and an absence of the normally observed increase in hemolymph protein after implantation. While we did not find any effects of tyrosine or copper deficiency on PO activity, we found reduced melanisation of a nylon implant in the absence of copper. This could indicate that the deficiency of dietary copper might lead to the inability to raise and to regulate an efficient immune response. We here assume that either the experimental hemolymph withdrawal or the implantation of the artificial pathogen induce the regeneration of hemolymph proteins under normal conditions, but not in copper-deficient isopods. This observation
suggests that a considerable proportion of the synthesised proteins are HC and/or POs, or that the regeneration of proteins in response to pathogen attack is somehow copper-dependent. Interestingly, the copperdependence of protein regeneration provides a (correlative) link between copper supply and the competence of cellular encapsulation. Correlation analyses revealed a positive correlation between the hemolymph protein content (prior to pathogen implantation) and the intensity of cellular encapsulation in response to pathogen implantation, but a negative correlation between the latter parameter and the hemolymph protein content after pathogen implantation. Thus, the ability to synthesise hemolymph proteins (in a copper-dependent process?) seems to indicate the capability of pathogen encapsulation. We were surprised to see reduced PO activity in vitro after the artificial pathogen attack, since we expected this procedure to induce an immune response and to result in a higher immune competence in terms of hemolymph PO activity. Possibly, the time interval (1 day) between pathogen implantation and subsequent PO determination was not long enough to allow for hemolymph protein regeneration, and we would have revealed a different picture had we given the isopods more time to elevate their weakened PO system. On the other hand, we found a positive correlation between PO activity before and after pathogen implantation (RS = 0.58; P = 0.02; N = 15). Thus, individuals with an efficient immune response (as indicated by in vitro PO activity) maintain a relatively high immune response after (artificial) pathogen attack, while individuals with a weaker immune response remain less immune competent than others. As to our knowledge, this is the first attempt to study the role of melanisation and encapsulation of pathogens in terrestrial isopods. Overall, our results present evidence for copper being essential for the immune response in P. scaber. Since the present study was designed as a first approach, however, numerous questions remain unanswered, warranting future detailed research on the immune competence of, and immune response in, terrestrial isopods. Acknowledgements We are grateful to Jens Rolff, University of Sheffield, for helpful hints on the implantation of nylon filaments, and to Elmar Jaenicke, Universität Mainz, for discussing PO and HC in arthropods.
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