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The crayfish pathogen Psorospermium haeckeli activates the prophenoloxidase activating system of freshwater crayfish in vitro
Aquaculture, 99 ( I991 ) 225-233 Elsevier Science Publishers B.V., Amsterdam
225
The crayfish pathogerrPls~mq~~~ium haeckeli activates the prophenoloxidase,activating system of freshwater crayfish in vitro Lage Cereniusa, Paula Menttonenb, Ossi V. Lindqvistb and Kenneth SijderhZlla ‘Department of PhysiologicalBotany, Universityof Uppsala,Box 540, S- 75 i 21 clppsala,Sweden bDepartment of Applied Zoology, Uniwrs~$~qfKuopnio, P.0. Box 6, SF- 7021 I Kuopio, Finland
(Accepted 28 March 1991)
L., Henttonen, P., Lindyvist, O.V. and Siiderhiill, K., I99 1. The crayfish pathogen Psorospermium haeckeli activates the prophenoloxidase activating system of freshwater crayfish in vitro. Aquaculture, 99: 225-233.
Cerenk,
Preparations of the crayfish pathogen Psorospermium haeckeli were found to activate Ihe prophenoloxidase activating (proPO) system in haemocyte lysates of two crayfish species Pacifastacusleniusculus and Ask~cusastacus. This is the tilrstreport showing that this parasite can elicit the activation of a defence system in crayfish and that the activation appears to be similar to the activation of the prop0 system triggered by different microbial polysaccharides.
INTRODUCTION
Psorospermiumhaeckeli has been described as a parasite on freshwater crayfish from the mid nineteenth century (Haeckel, I 857 ), but its taxonomic status is obscure and it has not yet been cultured in vitro. It has been observed in several species of crayfish, e.g. Astacusastacus (Haeckel, 1857; Ljungberg and Monne, 1968; Nylund and Westman, 1978 ), A. leptodactylus(Haeckel, 1857; Vey, 1978; Vranckx and Durliat, 198 1; Fiirst and SiiderhQll, 1987), Pacifastacusleniusculus (Unestam, 1973; Vey, 1978 ) and Cambarusaffinis (Krucinska and Simon, 1968) . The presence of this parasite is correlated with mortality in wild populations, and during culture of crayfish this parasite can cause high mortalities (S5derh%ll, 1988). The immune system of freshwater crayfish has been described in great detail at the molecular level (for recent reviews see Siiderhtill and Smith, 1986; Johansson and SiiderhHll, 1989; Siiderhill et al,, 1990). Parasites which try to gain entry into the body cavity of crayfish will trigger the activation of the prophenoloxidase ( proPQ j activating system, which functions as a non-self 0044-8486/91/$03.50
Q 1991 Elsevier Science Publishers B.V. All rights reserved.
226
L. CERENIUS ET AL.
recognition and defence system in crustaceans (Siiderhtill, 1982). Factors produced by activation of this system such as opsonic, cytotoxic, fungicidal, cell encapsulation promoting activities will aid in the elimination of foreign intruders (for reviews see Siiderhiill et al., 1988, 1990) e The prop0 system is specifically triggered by minute amounts of certain microbial polysaccharides such as j?-1,3-glucans from fungal cell walls (Sederhal, 1982; Duvic and Siiderhall, 1990) or by lipopolysaccharides (LPS) from Gram negative bacteria (Siiderhtill and H%ll, 1984). This type of defence reaction can be surveyed in the laboratory, for example, by monitoring different enzymic activities of the prop0 system in lysates of crayfish blood cells. In this way the potential to trigger a defence against a pathogen can be provisionally assessed. The final outcome of an infection by a particular pathogen, however, will of course be dependent upon a multitude of factors including environmental conditions. It has earlier been stated in the literature (e.g. Unestam, 1973) that crayfish do not mount any visible defence against the parasite P. haeckeli.However, Vranckx and Durliant ( 198 1) showed that this organism sometimes was found encapsulated with a thin layer of haemocytes in the crayfish A. l’e’entodactylus. Recently, Kobuyashi and SiiderhIll ( 1990) provided data indicating that haemocytes from P. haeckeli-infected A. astacusare affected by the presence of the parasite. This was done by examining the degree of capping, i.e. the non-uniform binding of the lectin concanat alin A to the surface of the blood cells. Animals infected by P. haeckeli exhibited a higher degree of capping, indicative of haemocyte activation: than non-infected animals (Kobayashi and Siiderhgll, 1990). This was confirmed by the frequent observations of encapsulated and melanized P. haeckeliin the tissues of A. astacus ( Kobayashi and Siiderhtill, 1990). In this paper we demonstrate that P. haeckeli does indeed trigger the activation of the crayfish prop0 system in vitro similar to the microbial poiysaccharides p- 1,3&cans or LPS. MATERIAL AND METHODS
Cra.$sh. Astacusastacusand Pacifastacuslenrusculuswere kept in separate aquaria with tap water at 12-14°C. Only intermoult animals were used. Preparationof Psorospermiumhaeckelifor in vitroexperiments. The soft tissues of A. astaciwere dissected from individuals originating from stocks known to be heavily infected by this parasite. The dissected material was cut into smaller pieces and to 1 g of the fragmented tissues was added to 10 ml 0.0 1% (w/u) pepsin in 0.0 1 M HCl and incubated with stirring at 37°C for 1 h. The preparation was then filtered through cheese-cloth and washed four times with 30 ml 0.0 1 Mcacodylate buffer, pH 7.0, and 100 mM CaCl, by centrifugation at 2000Xg for 2 min. This material still contains a considerable amount of
ACTIVATION OF A DEFI’NCE SYSTEM IN CRAYFISH %Y PSOROSPERMIUM
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crayfish tissue fragments, but preparations made in the same way from noninfected crayfish resulted in material without the capacity to trigger the activation of the prop0 system (see results section). No bacteria or other organisms apart from P. haeckeli could be observed in the P. haeckeli preparations during careful examination under the microscope. Preparation of haemocyte lysatesupernatants(HLS). HLS were prepared from P. leniuscuius or A. astacus according to SklerhHll and Hill ( 1984). These
preparations contain a prop0 system in its inactive form, which can be rrctivated by microbial polysaccharides. The HLS preparations were tisel as a source of enzymes for the experiments described below. Enzyme assaysof the prophenoloxidase activatingenyme (ppA) andphenoloxidase. The prophenoloxidase activating enzyme, a serine proteinase, was as-
sayed as described in Siiderhtill ( 1983) using the chromogenic substrate S23 37 ( Bz-ile-Glu- ( y-O-pipridyl )-Gly-Arg-paranitroanilide ) from Kabi Vitrum, Stockholm, Sweden. Phenoioxidase was monitored by using L-dihydroxy-phenylalanine (DOPA) as a substrate as detailed in Siiderhill ( 198 1) . The complete reaction mixture contained 50 ~1 P. haeckeli preparation in a total reaction volume of 400 ~1. The number of P. hrieckeli in each mixture was ca. 1000 if not otherwise indicated. Electron microscopy. The isolated P. haeckeli particles were fixed in 2.5% glu-
taraldehyde in 0.05 Mcacodylate buffer, pH 7.2; postfixed in 2% OsOs, and, after dehydration, the material was embedded in TAAB 8 12. The thin sections were contrasted with uranyl acetate and lead citrate and viewed in a Jeol 1OOBmicroscope. Protein determination. Protein was determined according to Bradford ( 1976 )
using bovine serum albumin as a standard. Chemicals. Lipopolysaccharide (E. coli 0 127:B8 ), pepsin, benzamidine, and
soybean trypsin inhibitor were all obtained from Sigma. L-dihydroxy-phenylalanine was acquired from Ajinmoto Co. Inc., Japan, and Polymyxin B was provided from Burroughs Wellcome, NC, USA. The laminarin was purchased from Calbiochem Corp., La Jolla, CA, USA. RESULTS
Prompted by our observations through the microscope that P. haeckefi was sometimes melanized within the crayfish tissues (Fig. 1) we set out to determine whether isolated preparations of the pathogen would be able to activate the prop0 system in vitro. To haemocyte lysates containing a prop0 system
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Fig. 1. .Psorospermium hue&e/i from muscle tissue of As&w ustacus. One parasite is heavily melanized. kale bar = 40 pm.
in its inactive, not yet activated form, different amounts of isolated P. haeckefi were added. As shown in Fig. 2, about 100 P. haeckeli particles were required to achieve a measurable increase in phenoloxidase activity. Activation of the prop0 system in lysates made from either A. astacusor P. leniusculus haemocytes was triggered to about the same degree (Fig. 2). The effect of P. haeckefion crayfish prop0 system was then compared with the activation induced by /3-1,3-gllucans or lipopolysaccharides (Siiderhiill and Smith, 1986). For these experiments P. leniusculuswas used since the prop0 system is known in greatest detail in this species ( SiiderhHl et al., 1990). The degree of activation of the prop0 system was studied by the addition of either isolated P. haeckelior the different microbial polysaccharides to a prop0 system in its inactive, not yet activated form. Table 1 demonstrates that P. haeckeli could induce activation of the prophenoloxidase activating enzyme (ppA), a serine proteinase, as well as prophenoloxidase. The activation caused by this parasite was about as high as that induced by either a-l ,3glucans or LPS (Table 1). No detectable ppA or phenoloxidase activities were recorded in the P. haeckelipreparation. To test whether an inactive prop0 was present in the P. haeckelipreparation, laminarin, LPS or trypsin were added to such preparations. No ppA or phenoloxidase activities were detected after the additions of these proPO-system triggers to a P. haeckeli preparation, showing that the parasite itself did not have these enzymes and no contamination of these crayfish enzymes occurred during preparation of the parasite. The activation
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0.6 -
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Number of P. haeckeli
Fig. 2. Phenoloxidase activity after addition of P. haeckefi to haemocyte lysate supernatants. Different amounts of the parasite were added to haemocyte lysate supematants from A. astacus (0 ) or P. leniusculus(0). Phenoloxidase activities are expressed as rlA490/min/mgproteinMean values k standard deviations from three experiments.
TABLE I
Enzyme activities of the prophenoloxidase activating enzyme (ppA) and phenoloxidase in crayfish haemocyte lysate supematant (HLS) after addition of P. haeckeh, laminarin or lipopolysaccharides (LPS) Addition to HLS
None P. haeckeli P. haeckeh + ST1 P. haeckeli+ benzamidine P. haeckeli+ polymyxin B Laminarin Laminarin+ STI Laminarin +benzamidine LPS LPS + ST1 L?S + benzamidine LPS f polymyxin B
Enzyme activities Proteinase ( ppA ) &05/min/mg~rot.
Phenoloxidase AA490/min/mgprot.
0.0 1 f 0.00 0.31 f0.08 0.01 +o.oo 0.01 +o.oo NT 0.44f0.13 0.01~0.00 0.01 fO.OO 0.48 f 0.09 0.01 fO.OO
0.03 + 0.00 0.45kO.13 0.02 + 0.0 1 0.03 + 0.00 0.02 f 0.0 1 0.20 f 0.07 0.03 f 0.01 0.03 2 0.02 0.23 f. 0.10 0.02 + 0.01 0.02 f 0.00
. .+ 0*0° :01!
0.01 fO.OO
The concentrations of soybean trypsin inhibitor (STI) and benzamidine were 1 mg/ml and 10 w, respectively. The amount of polymyxin B used was 10 000 units/ml ( Wderhlll and Hill, 1984). NT; not tested. Mean values f s.d. of three experiments.
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of the prop0 system elicited by j?-1,3-glucans or lipopolysaccharides is known to be inhibited by serine proteinase inhibitors (SiiderhZll, 198 1, Asp&net al., 1990; Siiderhall et al., 1990). Therefore, commercial serine proteinase inhibitors were tested for their ability to inhibit the activation induced by P. haeckeli. This was done by incubating the prop0 preparations with either benzamidine or soybean trypsin inhibitor, which are known inhibitors of prop0 system activation (Aspan et al., 1990), together with an activator of the proPa cascade. Table 1 shows that these proteinase iahibitors blocked the P. haeckeli-induced prop0 activation as efficiently as the activation caused by p1,3glucan or LPS. Polymyxin B which prevents LPS-triggered prop0 activation in crayfish (Siiderhal and HU, 1984) as well as LPS-mediated coagulation in Limuluspdyghemus (SiiderhZill et al., 1985 ) and endogenous pyrogen production in mammals (Duff and Atkins, 1982) could not prevent the activation caused by the addition of P. haeckelipreparation to the haemocyte lysate (Table 1). This shows that the P. haeckeZipreparation was not contaminated with Gram negative bacteria. DISCUSSlON
The data presented in this report show that P. haeckeliis capable of triggering activation of the prop0 system in vitro. The means by which this is achieved are obviously similar to the immune activation induced by different microbial polysaccharides, since the P. haeckeli-mediated activation also seems to be accomplished by limited proteolysis (Aspan et al., 1990; Aspan and Siiderhtill, 199 1). It remains to be established which component of the thick capsule wall of P. haeckeli (Fig. 3) will trigger this activation or whether other elements such as adhered bacterial cell wall remnants on the shell of P. haeckeliare responsible for the prop0 activation. Such remnants might easily have been deposited if this parasite had been taken up through the alimentary tract. It is, however, not known which way this parasite infects its host and sxperiments with fractionated P. haeckeli capsule components will probably be needed to fully understand how this organism achieves the activation of the prop0 systeill. However, the following observations strongly suggest that the activation of the prop0 system was brought #about by the parasite itself and not by any contaminating bacteria: Polymyxin B did not inhibit the P. haeckeli-triggered activation of the prop0 system demonstrating that endotoxin (e.g. LPS) of Gram negative bacteria is not responsible for this activation. Furthermore, no bacteria were visible on the P. haeckeli particles when examined under the light microscope or by electron microscopy (Fig. 3 ). Thus, it appears likely that, contrary to previous opinions, this pathogen has the potential to induce defence reactions within the host. Further support for this conclusion is provided by the observations by Kobayashi and S6derhgll ( 1990) that a P. haeckeli infection in A. astacusis manifested by a change in
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Fig. 3. Electron micrograph of an isolated P. haeckefi showing the capsule wall surrounding the parasite. Scale bar=&2 pm.
haemocyte behaviour an-d by Vranckx and Durliant ( 198 1) who found a thin haemocyte capsule around some P. haeckeliwithin A. feptodactylustissues. It is therefore likely that this pathogen affects the immune system of the host. This is further corroborated by the findings of Ftirst and Siiderh311 ( 1987) that P. haeckeli infected A. leptodactyluscrayfish were more prone to succumb to an Aphanomycesastaci (crayfish plague) infection than crayfish free of P. haeckeli. This might suggest tha.t even if a 1G.haeckeli infection itself is not lethal to the host, the reduced immune capatity of the animal will make it more sensitive to other pathogens. Thus, P. hawkeli should be considered
232
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as a serious threat to crayfish during farming where different pathogens always constitute a danger. ACKNOWLEDGEMENTS
This work was supported by Carl Tryggers Foundation, the Fishery Board of Sweden and the Swedish Research Council of Forestry and Agricultural Research.
REFERENCES A. and Siiderhlll, K., 199 1. Purification of prophenoloxidase fromcrayfish bloodcells, andits activation by an endogenous serine proteinase. Insect. Biochem., 21: 363-373.
Aspan,
Aspln, A., Sturtevant, J., Smith, V.J. and Siiderhill, K., 1990. Purification and characterization of a prophenoloxidase activating enzyme from crayfish blood cells. Insect Biochem., 20: 709718. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254. Duff, G.W. and Atkins, E., 1982. The inhibitory effect of polymyxin B on endotoxin-induced endogenous pyrogcn production. J. Immunol. Methods, 52: 333-340. Duvic, B. and Stiderhlll, K., 1990. Purification and characterization of a p-1,3-glucan binding protein from plasma of the crayfish Pucifas~acus leniusculus J. Biol. Chem., 265: 9327-9332. Fiirst, M. and Sijderhiill, K., 1987. The crayfish Aslacus feptoductylus in Turkey. Diseases and present distribution of the crayfish plague fungus, Aphanomyces astaci. FAO Report, Rome, 26 PP. Haeckel. E., 1857. Ober die Gewebe des Flusskrebses. Arch. Anat. Physiol. Med., 24: 561-562. Johansson, M.W. and Siiderhtill, K., 1989. Cellular immunity in crustaceans and the prop0 system. Parasitol. Today, 5: 17 l-1 76. Kobayashi, M. and Siiderhlll, K., 1990. Comparison of concanavalin A reactive determinants on isolated haemocytes of parasite-infected and non-infected freshwater crayfish. Dis. Aquatic Org., 9: 141-147. Krucinska, J. and Simon, E., 1968. On the parasites and epibionts of the branchial cavity in crayfish at Wrocklaw and vicinity. Przegl. Zool., 12: 288-290. Ljungberg, 0. and Monne; L., 1968. On the eggs of an enigmatic nematode parasite encapsulated in the connective tissue of the European crayfish, Astucus astucus, in Sweden. Bull. OIY. Int. Epizooties, 69: 1231-1235. Nylund, V. and Westman, K., 1978. Psorospermium haeckeli, a parasite on the European crayfish Astucus atucus, found in Finland. In*. P.J. Laurant (Editor), Freshwater Crayfish, IV. Papers from the Fourth International Symposium on Freshwater Crayfish, Thonon-les-Bains, France, pp. 385-390. Soderhall, K., 198 1. Fungal cell wall & 1,3-glucans induce clotting and phenoloxidase attachment to foreign surfaces of crayfish hemocyte lysate. Dev. Comp. Immunol., 5: 565-573. S~dcrhall, K., 1982. Prophenoloxidase activating system and melanization - a recognition mechanism of arthropods? A review. Dev. Comp. Immunol., 6: 60 1-6 11, Stiderhlll, K., 1983. Beta- I ,Iglucan enhancement of protease activity in crayfish hemocyte lysate. Comp. Biochem. Physiol. B, 74: 22 l-224. Soderhill, K., 1988. Fungal parasites and other diseases on freshwater crayfish. In: J. Kovonen
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and R. Lappalainen (Editors), Rapautlous 2000, Keski-Suomen Kalastuspiirin Kalastustoimisto Tiedotus nro 5, Jyvtiskyll, Finland, pp. 23-46. SSderhBll, K. and Hill, L., 1984. Lipopolysaccharide-induced activation of prophenoloxidase activating system in crayfish haemocyte iysate. Biochim. Biophys. Acta, 797: 99-104. SiiderhBll, K. and Smith, V.J., 1986. The prophenoloxidase activating system: the biochemistry of its activation and role in arthropod cellular immunity, with special reference to crustaceans. In: M. Brehelin (Editor), Immunity in Invertebrates. Springer, Berlin, pp. 208-223. SGderhiill, K., Levin, J. and Armstrong, P.B., 1985. The effects of j&l ,f-glucans on blood coagulation and amebocyte release in the horse-shoe crab Limufus pofyphemus. Biol. Bull., 169: 66 l-674. Siiderhill, K., Johansson, M.W. and Smith, V.J., 1988. Internal defence mechanisms. In: D.M. Holdich and R.S. Lower-y (Editors), Freshwater Crayfish: Biology, Management and Exploitation. Croom Held, London, pp. 2 13-235. Siiderhiill, K., Aspan, A. and Duvic, B., 1990. The proPO-systems and associated proteins; role in cellular communication in arthropods. Res. Immunol., 141: 896-907. Unestam, T., 1973. Significance of diseases on freshwater crayfish. In: S. Abrahamsson (Editor), Freshwater Crayfish. Papers from the First International Symposium on Freshwater Crayfish, Austria 1972. Studentlitteratur, Lund, pp. 135-l 50. Vey, A., 1978. Recherches sur une maladie des echervisses due au parasite Psorospermiumhaeckeli Hilgendorf. In: P.-J. Laurant (Editor j, Freshwater Crayfish, IV. Papers from the Fourth International Symposium on Freshwater Crayfish, Thonon-les-Bains, France, pp. 4 19-426. Vranckx, R. and Durliat, M., 198 1. Encapsulation of Psorospermiumhaeckeli by the haemocytes of Astacuslepotodactylus.Experientia, 37: 40-42.
225
The crayfish pathogerrPls~mq~~~ium haeckeli activates the prophenoloxidase,activating system of freshwater crayfish in vitro Lage Cereniusa, Paula Menttonenb, Ossi V. Lindqvistb and Kenneth SijderhZlla ‘Department of PhysiologicalBotany, Universityof Uppsala,Box 540, S- 75 i 21 clppsala,Sweden bDepartment of Applied Zoology, Uniwrs~$~qfKuopnio, P.0. Box 6, SF- 7021 I Kuopio, Finland
(Accepted 28 March 1991)
L., Henttonen, P., Lindyvist, O.V. and Siiderhiill, K., I99 1. The crayfish pathogen Psorospermium haeckeli activates the prophenoloxidase activating system of freshwater crayfish in vitro. Aquaculture, 99: 225-233.
Cerenk,
Preparations of the crayfish pathogen Psorospermium haeckeli were found to activate Ihe prophenoloxidase activating (proPO) system in haemocyte lysates of two crayfish species Pacifastacusleniusculus and Ask~cusastacus. This is the tilrstreport showing that this parasite can elicit the activation of a defence system in crayfish and that the activation appears to be similar to the activation of the prop0 system triggered by different microbial polysaccharides.
INTRODUCTION
Psorospermiumhaeckeli has been described as a parasite on freshwater crayfish from the mid nineteenth century (Haeckel, I 857 ), but its taxonomic status is obscure and it has not yet been cultured in vitro. It has been observed in several species of crayfish, e.g. Astacusastacus (Haeckel, 1857; Ljungberg and Monne, 1968; Nylund and Westman, 1978 ), A. leptodactylus(Haeckel, 1857; Vey, 1978; Vranckx and Durliat, 198 1; Fiirst and SiiderhQll, 1987), Pacifastacusleniusculus (Unestam, 1973; Vey, 1978 ) and Cambarusaffinis (Krucinska and Simon, 1968) . The presence of this parasite is correlated with mortality in wild populations, and during culture of crayfish this parasite can cause high mortalities (S5derh%ll, 1988). The immune system of freshwater crayfish has been described in great detail at the molecular level (for recent reviews see Siiderhtill and Smith, 1986; Johansson and SiiderhHll, 1989; Siiderhill et al,, 1990). Parasites which try to gain entry into the body cavity of crayfish will trigger the activation of the prophenoloxidase ( proPQ j activating system, which functions as a non-self 0044-8486/91/$03.50
Q 1991 Elsevier Science Publishers B.V. All rights reserved.
226
L. CERENIUS ET AL.
recognition and defence system in crustaceans (Siiderhtill, 1982). Factors produced by activation of this system such as opsonic, cytotoxic, fungicidal, cell encapsulation promoting activities will aid in the elimination of foreign intruders (for reviews see Siiderhiill et al., 1988, 1990) e The prop0 system is specifically triggered by minute amounts of certain microbial polysaccharides such as j?-1,3-glucans from fungal cell walls (Sederhal, 1982; Duvic and Siiderhall, 1990) or by lipopolysaccharides (LPS) from Gram negative bacteria (Siiderhtill and H%ll, 1984). This type of defence reaction can be surveyed in the laboratory, for example, by monitoring different enzymic activities of the prop0 system in lysates of crayfish blood cells. In this way the potential to trigger a defence against a pathogen can be provisionally assessed. The final outcome of an infection by a particular pathogen, however, will of course be dependent upon a multitude of factors including environmental conditions. It has earlier been stated in the literature (e.g. Unestam, 1973) that crayfish do not mount any visible defence against the parasite P. haeckeli.However, Vranckx and Durliant ( 198 1) showed that this organism sometimes was found encapsulated with a thin layer of haemocytes in the crayfish A. l’e’entodactylus. Recently, Kobuyashi and SiiderhIll ( 1990) provided data indicating that haemocytes from P. haeckeli-infected A. astacusare affected by the presence of the parasite. This was done by examining the degree of capping, i.e. the non-uniform binding of the lectin concanat alin A to the surface of the blood cells. Animals infected by P. haeckeli exhibited a higher degree of capping, indicative of haemocyte activation: than non-infected animals (Kobayashi and Siiderhgll, 1990). This was confirmed by the frequent observations of encapsulated and melanized P. haeckeliin the tissues of A. astacus ( Kobayashi and Siiderhtill, 1990). In this paper we demonstrate that P. haeckeli does indeed trigger the activation of the crayfish prop0 system in vitro similar to the microbial poiysaccharides p- 1,3&cans or LPS. MATERIAL AND METHODS
Cra.$sh. Astacusastacusand Pacifastacuslenrusculuswere kept in separate aquaria with tap water at 12-14°C. Only intermoult animals were used. Preparationof Psorospermiumhaeckelifor in vitroexperiments. The soft tissues of A. astaciwere dissected from individuals originating from stocks known to be heavily infected by this parasite. The dissected material was cut into smaller pieces and to 1 g of the fragmented tissues was added to 10 ml 0.0 1% (w/u) pepsin in 0.0 1 M HCl and incubated with stirring at 37°C for 1 h. The preparation was then filtered through cheese-cloth and washed four times with 30 ml 0.0 1 Mcacodylate buffer, pH 7.0, and 100 mM CaCl, by centrifugation at 2000Xg for 2 min. This material still contains a considerable amount of
ACTIVATION OF A DEFI’NCE SYSTEM IN CRAYFISH %Y PSOROSPERMIUM
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227
crayfish tissue fragments, but preparations made in the same way from noninfected crayfish resulted in material without the capacity to trigger the activation of the prop0 system (see results section). No bacteria or other organisms apart from P. haeckeli could be observed in the P. haeckeli preparations during careful examination under the microscope. Preparation of haemocyte lysatesupernatants(HLS). HLS were prepared from P. leniuscuius or A. astacus according to SklerhHll and Hill ( 1984). These
preparations contain a prop0 system in its inactive form, which can be rrctivated by microbial polysaccharides. The HLS preparations were tisel as a source of enzymes for the experiments described below. Enzyme assaysof the prophenoloxidase activatingenyme (ppA) andphenoloxidase. The prophenoloxidase activating enzyme, a serine proteinase, was as-
sayed as described in Siiderhtill ( 1983) using the chromogenic substrate S23 37 ( Bz-ile-Glu- ( y-O-pipridyl )-Gly-Arg-paranitroanilide ) from Kabi Vitrum, Stockholm, Sweden. Phenoioxidase was monitored by using L-dihydroxy-phenylalanine (DOPA) as a substrate as detailed in Siiderhill ( 198 1) . The complete reaction mixture contained 50 ~1 P. haeckeli preparation in a total reaction volume of 400 ~1. The number of P. hrieckeli in each mixture was ca. 1000 if not otherwise indicated. Electron microscopy. The isolated P. haeckeli particles were fixed in 2.5% glu-
taraldehyde in 0.05 Mcacodylate buffer, pH 7.2; postfixed in 2% OsOs, and, after dehydration, the material was embedded in TAAB 8 12. The thin sections were contrasted with uranyl acetate and lead citrate and viewed in a Jeol 1OOBmicroscope. Protein determination. Protein was determined according to Bradford ( 1976 )
using bovine serum albumin as a standard. Chemicals. Lipopolysaccharide (E. coli 0 127:B8 ), pepsin, benzamidine, and
soybean trypsin inhibitor were all obtained from Sigma. L-dihydroxy-phenylalanine was acquired from Ajinmoto Co. Inc., Japan, and Polymyxin B was provided from Burroughs Wellcome, NC, USA. The laminarin was purchased from Calbiochem Corp., La Jolla, CA, USA. RESULTS
Prompted by our observations through the microscope that P. haeckefi was sometimes melanized within the crayfish tissues (Fig. 1) we set out to determine whether isolated preparations of the pathogen would be able to activate the prop0 system in vitro. To haemocyte lysates containing a prop0 system
228
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Fig. 1. .Psorospermium hue&e/i from muscle tissue of As&w ustacus. One parasite is heavily melanized. kale bar = 40 pm.
in its inactive, not yet activated form, different amounts of isolated P. haeckefi were added. As shown in Fig. 2, about 100 P. haeckeli particles were required to achieve a measurable increase in phenoloxidase activity. Activation of the prop0 system in lysates made from either A. astacusor P. leniusculus haemocytes was triggered to about the same degree (Fig. 2). The effect of P. haeckefion crayfish prop0 system was then compared with the activation induced by /3-1,3-gllucans or lipopolysaccharides (Siiderhiill and Smith, 1986). For these experiments P. leniusculuswas used since the prop0 system is known in greatest detail in this species ( SiiderhHl et al., 1990). The degree of activation of the prop0 system was studied by the addition of either isolated P. haeckelior the different microbial polysaccharides to a prop0 system in its inactive, not yet activated form. Table 1 demonstrates that P. haeckeli could induce activation of the prophenoloxidase activating enzyme (ppA), a serine proteinase, as well as prophenoloxidase. The activation caused by this parasite was about as high as that induced by either a-l ,3glucans or LPS (Table 1). No detectable ppA or phenoloxidase activities were recorded in the P. haeckelipreparation. To test whether an inactive prop0 was present in the P. haeckelipreparation, laminarin, LPS or trypsin were added to such preparations. No ppA or phenoloxidase activities were detected after the additions of these proPO-system triggers to a P. haeckeli preparation, showing that the parasite itself did not have these enzymes and no contamination of these crayfish enzymes occurred during preparation of the parasite. The activation
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0.6 -
1 o4
Number of P. haeckeli
Fig. 2. Phenoloxidase activity after addition of P. haeckefi to haemocyte lysate supernatants. Different amounts of the parasite were added to haemocyte lysate supematants from A. astacus (0 ) or P. leniusculus(0). Phenoloxidase activities are expressed as rlA490/min/mgproteinMean values k standard deviations from three experiments.
TABLE I
Enzyme activities of the prophenoloxidase activating enzyme (ppA) and phenoloxidase in crayfish haemocyte lysate supematant (HLS) after addition of P. haeckeh, laminarin or lipopolysaccharides (LPS) Addition to HLS
None P. haeckeli P. haeckeh + ST1 P. haeckeli+ benzamidine P. haeckeli+ polymyxin B Laminarin Laminarin+ STI Laminarin +benzamidine LPS LPS + ST1 L?S + benzamidine LPS f polymyxin B
Enzyme activities Proteinase ( ppA ) &05/min/mg~rot.
Phenoloxidase AA490/min/mgprot.
0.0 1 f 0.00 0.31 f0.08 0.01 +o.oo 0.01 +o.oo NT 0.44f0.13 0.01~0.00 0.01 fO.OO 0.48 f 0.09 0.01 fO.OO
0.03 + 0.00 0.45kO.13 0.02 + 0.0 1 0.03 + 0.00 0.02 f 0.0 1 0.20 f 0.07 0.03 f 0.01 0.03 2 0.02 0.23 f. 0.10 0.02 + 0.01 0.02 f 0.00
. .+ 0*0° :01!
0.01 fO.OO
The concentrations of soybean trypsin inhibitor (STI) and benzamidine were 1 mg/ml and 10 w, respectively. The amount of polymyxin B used was 10 000 units/ml ( Wderhlll and Hill, 1984). NT; not tested. Mean values f s.d. of three experiments.
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of the prop0 system elicited by j?-1,3-glucans or lipopolysaccharides is known to be inhibited by serine proteinase inhibitors (SiiderhZll, 198 1, Asp&net al., 1990; Siiderhall et al., 1990). Therefore, commercial serine proteinase inhibitors were tested for their ability to inhibit the activation induced by P. haeckeli. This was done by incubating the prop0 preparations with either benzamidine or soybean trypsin inhibitor, which are known inhibitors of prop0 system activation (Aspan et al., 1990), together with an activator of the proPa cascade. Table 1 shows that these proteinase iahibitors blocked the P. haeckeli-induced prop0 activation as efficiently as the activation caused by p1,3glucan or LPS. Polymyxin B which prevents LPS-triggered prop0 activation in crayfish (Siiderhal and HU, 1984) as well as LPS-mediated coagulation in Limuluspdyghemus (SiiderhZill et al., 1985 ) and endogenous pyrogen production in mammals (Duff and Atkins, 1982) could not prevent the activation caused by the addition of P. haeckelipreparation to the haemocyte lysate (Table 1). This shows that the P. haeckeZipreparation was not contaminated with Gram negative bacteria. DISCUSSlON
The data presented in this report show that P. haeckeliis capable of triggering activation of the prop0 system in vitro. The means by which this is achieved are obviously similar to the immune activation induced by different microbial polysaccharides, since the P. haeckeli-mediated activation also seems to be accomplished by limited proteolysis (Aspan et al., 1990; Aspan and Siiderhtill, 199 1). It remains to be established which component of the thick capsule wall of P. haeckeli (Fig. 3) will trigger this activation or whether other elements such as adhered bacterial cell wall remnants on the shell of P. haeckeliare responsible for the prop0 activation. Such remnants might easily have been deposited if this parasite had been taken up through the alimentary tract. It is, however, not known which way this parasite infects its host and sxperiments with fractionated P. haeckeli capsule components will probably be needed to fully understand how this organism achieves the activation of the prop0 systeill. However, the following observations strongly suggest that the activation of the prop0 system was brought #about by the parasite itself and not by any contaminating bacteria: Polymyxin B did not inhibit the P. haeckeli-triggered activation of the prop0 system demonstrating that endotoxin (e.g. LPS) of Gram negative bacteria is not responsible for this activation. Furthermore, no bacteria were visible on the P. haeckeli particles when examined under the light microscope or by electron microscopy (Fig. 3 ). Thus, it appears likely that, contrary to previous opinions, this pathogen has the potential to induce defence reactions within the host. Further support for this conclusion is provided by the observations by Kobayashi and S6derhgll ( 1990) that a P. haeckeli infection in A. astacusis manifested by a change in
ACTIVATION
OF A DEFENCE SYSTEM IN CRAYFISH BY PSOROSPERMUM
HAECKELI
231
Fig. 3. Electron micrograph of an isolated P. haeckefi showing the capsule wall surrounding the parasite. Scale bar=&2 pm.
haemocyte behaviour an-d by Vranckx and Durliant ( 198 1) who found a thin haemocyte capsule around some P. haeckeliwithin A. feptodactylustissues. It is therefore likely that this pathogen affects the immune system of the host. This is further corroborated by the findings of Ftirst and Siiderh311 ( 1987) that P. haeckeli infected A. leptodactyluscrayfish were more prone to succumb to an Aphanomycesastaci (crayfish plague) infection than crayfish free of P. haeckeli. This might suggest tha.t even if a 1G.haeckeli infection itself is not lethal to the host, the reduced immune capatity of the animal will make it more sensitive to other pathogens. Thus, P. hawkeli should be considered
232
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as a serious threat to crayfish during farming where different pathogens always constitute a danger. ACKNOWLEDGEMENTS
This work was supported by Carl Tryggers Foundation, the Fishery Board of Sweden and the Swedish Research Council of Forestry and Agricultural Research.
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