Effect of Calcium on the Prophenoloxidase System Activation of the Brown Shrimp (Penaeus californiensis, Holmes)

Comp. Biochem. Physiol. Vol. 117A, No. 3, pp. 419–425, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00363-5

Effect of Calcium on the Prophenoloxidase System Activation of the Brown Shrimp (Penaeus californiensis, Holmes) Teresa Gollas-Galva´n,* Jorge Herna´ndez-Lo´pez,* and Francisco Vargas-Albores*† *CIBNOR, La Paz, BCS, 23000 Mexico, and †CIAD, Hermosillo, Son, 83000, Mexico ABSTRACT. The soluble prophenoloxidase (proPO) system of the brown shrimp (Panaeus californiensis) was obtained by centrifuging hemocytes (15,000 g) in low salt buffers. In these samples, proPO spontaneous activation was observed when calcium (.5 mM) was present in the buffers. Stable samples can be obtained in divalent cation-free buffer, and the sole addition of Ca21 resulted in the proPO activation. In contrast, Ca21 was not able to induce spontaneous activation in samples depleted of proPO activating enzyme (PPAE) obtained by passing the sample through a Blue Sepharose column. In addition, protease inhibitors like melittin and soybean trypsin inhibitor blocked the Ca21-induced spontaneous activation, indicating this cation is required for the proPO proteolytic activation. Although Ca21-induced spontaneous activation was not observed with intact hemocytes, this cation was necessary for the activation of proPO by β-glucans. Plasma Ca21 concentration of the brown shrimp is 8 mM, as determined by absorption spectroscopy. Thus, these results suggest Ca21 activates PPAE and then PPAE transforms proPO to an active form when both proteins are released from the cells after the stimulus. comp biochem physiol 117A;3:419–425, 1997.  1997 Elsevier Science Inc. KEY WORDS. Shrimp immunology, proPO system, crustacean, laminarin, hemocytes

INTRODUCTION The prophenoloxidase (proPO) system is an enzymatic cascade involved in invertebrate immune mechanisms (13,24,31). The major enzyme produced in proPO system activation is phenoloxidase (PO, o-diphenol-oxygen oxidoreductase; E.C. 1.10.3.1), which is necessary for the melanization process observed in response to foreign matter invading the hemocoele and during wound healing [for review, see (3,11,30,31)]. The proPO system can be specifically activated by β -1,3-glucans (1,8,14,27,33), bacteria cell walls (1,26) and lipopolysaccharide (32). Along with melanization, other cellular activities are stimulated during proPO system activation, including adhesion, encapsulation and phagocytosis (10,12,22,23,35). During the activation process, proPO activating enzyme (PPAE) has been identified as the proteinase responsible for the activation of the proPO and its conversion to active PO (4,30,31). The participation of Ca21 in the trigger of invertebrate defense processes has been demonstrated in several models. In crayfish, calcium is necessary for cell adhesion (9), and in the snail Helix pomatia, Ca 21 increases phagocytosis rate almost 15-fold (25). However, the role of Ca 21 on the

Address reprint requests to: F. Vargas-Albores; Centro de Investigacio´n en Alimentacio´ n y Desarrollo, Aptdo. Postal 1735, Hermosillo, Son, 83000 Mexico. Tel./Fax 152 (62) 80-00-58; E-mail: [email protected]. Received 6 February 1996; Accepted 19 August 1996.

proPO system activation is still unclear. Although some authors have noted Ca21 is necessary in the activating process of proPO in crustaceans (2) and insects (5,14), in other cases, Ca21 apparently is not needed for the proPO system activation (17,21). Furthermore, high concentrations of Ca21 apparently stabilize the proPO system and prevent the spontaneous activation that is observed at low concentrations of Ca21 (1,14,28). In crayfish, a proteolytic enzyme is involved in the activation of proPO. In turn, the proteinase activity is promoted by Ca21 (32), producing active PO. To the contrary, in the silkworm, proPO can be transformed by the PPAE in the absence of Ca21 (1). In addition to this apparently conflicting data, Ca 21 participation on the proPO system activation is assumed, and these differences may be associated with the source, extraction form, purity or other properties of the proPO preparation. In this study, we tested the effect of Ca21 on a crude preparation of shrimp proPO system to explain the physiological role of this cation in the shrimp defense system. MATERIALS AND METHODS All chemicals were purchased from Sigma Chemical Co. The glassware was washed with Toxa-Clean (Sigma Chem. Co.) to avoid any spontaneous activation of the proPO system by endotoxins. For the same reason, all solutions were prepared using pyrogen-free water.

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Solutions

proPO System Preparations

Shrimp anticoagulant solution was prepared by adding 10 mM EDTA–Na2 to shrimp salt solution (SSS), which corresponds to the ionic and osmotic values of shrimp hemolymph (34): 450 mM NaCl, 10 mM KCl, 10 mM HEPES, pH 7.3, 850 mOsm/kg. A cacodylate buffer (Cac, 10 mM sodium cacodylate, pH 7.0) was used for extraction of the proPO system and for determination of PO activity.

Soluble samples containing shrimp proPO system were obtained as previously (8) by inducing degranulation. In brief, hemolymph (200 µl) was centrifuged at 700 g and the cell pellet was washed by resuspending in 200 µl of Cac buffer and centrifuging at 15,900 g for 20 sec. Cell integrity was verified by microscopy, and hemocytic degranulation rather than citolysis was observed. The supernatant containing the granular material was separated and used as the proPO source.

Animals The shrimp, Penaeus californiensis, were grown in an experimental tidal pond (Center for Biological Research, La Paz, BCS, Mexico). The animals were maintained in an aquarium (24 6 4°C; salinity, 36 ppt) at least 2 days before the experiment. Only the hemolymph from intermolt apparently healthy shrimp was used. Extraction and Separation of the Hemolymph Hemolymph was extracted from the pleopod base of the first abdominal segment near the genital pore using a 0.5-ml syringe and a 27-gauge needle. Two volumes of precooled (10°C) anticoagulant solution (33) were preloaded per each volume of hemolymph. The diluted hemolymph was centrifuged at 560 g for 3 min and the supernatant separated.

Chemical Analysis The total protein content was measured according to Lowry et al. (18) using bovine serum albumin as standard. The calcium concentration of the shrimp plasma was measured using an absorption spectrophotometer (Buch Scientific, 200A) with 50% (v/v) acetylene-air as the combustion gas.

Enzymatic Activity Phenoloxidase activity was measured spectrophotometrically (in triplicate) by recording the formation of dopachrome from l-dihydroxyphenylalanine (l-DOPA). Fifty microliters of sample was placed in a microwell plate and 50 µl of l-DOPA (3 mg/ml) was added. After a 10-min incubation at 25°C, the optical density was read at 492 nm using an ELISA reader (Labsystems Uniskan II). The total amount of proPO available (PO 1 proPO) was determined after sample incubation with 50 µl of trypsin (100 µg/ml in Cac buffer), allowing the proPO activation reaction to proceed for 10 min at 25°C. The amount of residual inactive proPO in samples also containing PO activity was calculated as total available proPO minus the PO activity measured before the trypsin treatment. The specific activity was defined as the change of absorbance per min per mg of protein.

Role of Divalent Ions Effect of divalent ions (Ca21, Mg21, Mn21 ) on the proPO preparations was evaluated by obtaining samples in Cac buffer containing one of the cations (20 mM) and determining PO activity as described above. For other experiments, crude preparations of the shrimp proPO system were obtained in Ca 21-free Cac buffer (pH 7). Then, 50 µl of sample were incubated for 30 min at room temperature with 50 µl of Cac containing different C 21 concentrations (0, 5, 10, 20 and 100 mM). Effect of Calcium in Activation by Trypsin Fifty microliters of sample containing proPO was incubated for 30 min with different calcium concentrations (5, 50, 100 and 200 mM). Then, 50 µl of trypsin was added and incubated for 5 min at room temperature before determining PO activity. Separately, 50 µl of proPO sample was incubated with trypsin (50 µl) before addition of Ca 21 (5, 50, 100 and 200 mM) and the PO activity determination. For other experiments, the Ca21 effect was studied in shrimp PO obtained by incubating with trypsin (as described above), phenolase (EC 1.10.3.1) and tyrosinase (EC 1.14.18.1) separately. Phenolase and tyrosinase (Sigma Chem. Co.) concentrations were adjusted to obtain an absorbance equal to 0.1 after a 10-min incubation in Cac buffer (pH 7). Effect of Proteinase Inhibitors Melittin and soybean trypsin inhibitor (STI) were tested as inhibitors of the Ca21-dependent proPO activation effect. The samples were obtained in Cac with (50 µg/ml) or without melittin and incubated for 30 min at room temperature with Ca21 (10 mM), Ca21 1 STI (0.2 mg/ml), trypsin (0.1 mg/ml) or trypsin–STI before determining PO activity. Activation by b-Glucans Intact hemocytes were obtained by centrifuging shrimp plasma at 560 g for 3 min at 10°C and resuspending (106 cell/ml) in SSS. Granular material containing proPO was obtained as described above. In both cases, the samples were

Effect of Calcium on Shrimp proPO System

prepared in Ca21-free buffers. Fifty microliters of hemocyte suspension or granular content was incubated for 30 min at room temperature with 50 µl (1 mg/ml) of laminarin (βglucan) in the presence (20 mM) or absence of Ca21. Then, PO activity was recorded.

Stability One milliliter of hemolymph was obtained in 2 ml of precooled anticoagulant solution. The cell pellet was washed twice with SSS by centrifuging at 800 g. The cell pellet was resuspended in 2 ml of Cac and centrifuged for 20 sec at 15,900 g. The supernatant was separated into four aliquots and each was diluted (1 : 2) with Cac buffer containing 0, 5, 40 or 100 mM of CaCl2 . PO activity was recorded in each aliquot at 0, 30, 60, 90 and 120 min.

Preparation of PPAE-Free Sample A sample containing proPO was obtained from brown shrimp hemocytes in Ca21-free buffer as described above, and both PO activity and total available proPO were determined. One milliliter of the sample was passed through an 8-ml Blue-Sepharose column previously equilibrated with 10 mM cacodylate buffer (pH 8.0). The column was washed with 45 ml of the same buffer and the retained material was eluted by sequentially washing the column with 0.5, 1.0 and 1.5 M NaCl. Fractions containing proPO were detected by incubating the samples with trypsin or calcium and determining their PO activity.

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TABLE 1. Effect of divalent cations (20 mM) on the sponta-

neous activation of the brown shrimp proPO PO activity (%) Water Water 1 NaCl Water 1 Ca 21 Water 1 Mg 21 Water 1 Mn 21 Cacodylate buffer Cacodylate buffer 1 Ca 21

1.71 0.0 30.89 6.80 7.60 2.48 34.95

6 0.78 6 6 6 6 6

1.34 1.90 2.20 0.82 1.19

Values are means 6 SE (n 5 6) expressed as percent of PO activity detected, assuming the activation by trypsin as 100%.

served (Table 1). Therefore, an experiment was designed to study the effect of Ca21 concentration on proPO spontaneous activation. A preparation without proPO activation was obtained in Ca21-free Cac buffer, and then aliquots were incubated with different Ca21 concentrations (0–100 mM). ProPO spontaneous activation was observed from 10 to 100 mM of Ca 21 (P , 0.05) with maximum values from 50 to 100 mM (Fig. 1). Very little activation was measured when Mg21 instead Ca21 was assayed. In another series of experiments, shrimp hemocytes were resuspended in Cac containing different Ca21 concentra-

RESULTS AND DISCUSSION Previously, we reported the use of hemocyte centrifugation as an alternative method to extract proPO system (8). This method provides a crude proPO preparation with less contaminant unrelated proteins than the traditionally used hemocyte lysate supernatant (HLS). However, the addition of CaCl2 (20 mM) to the buffer resulted in spontaneous activation of proPO, detected as PO activity. This led us to a more careful examination of the reaction.

Effect of Cations Over proPO The effect of cations on the proPO activation was determined by addition of 20 mM Ca21, Mg21 or Mn21 to the buffer during the proPO extraction from shrimp hemocytes. Significant quantities (.30%) of active PO were detected when Ca 21 was present in the buffer (water or cacodylate), whereas the use of buffers containing 20 mM MgCl2 or MnCl2 gave only little activation. In contrast, when the proPO system from brown shrimp was obtained with divalent cation-free buffers, spontaneous activation was not ob-

FIG. 1. Spontaneous activation of the brown shrimp proPO

induced by Ca21. Samples obtained in cation-free buffer were incubated (n 5 5) with different Ca21 or Mg21 concentrations, and then their specific activity was determined (mean 6 SE).

422

FIG. 2. Influence of time on the Ca21-induced spontaneous

activation of the brown shrimp proPO. Percent (mean and SE) of the optical density at 492 nm for five samples are shown. The maximum value was considered as 100% for each sample.

tions (0, 5, 40 and 100 mM). The proPO system was extracted by centrifugation, and a time course of PO activity (0, 30, 60, 90 and 120 min) was determined for each supernatant. As shown in Fig. 2, all containing Ca21 had spontaneous activation, whereas the samples in Ca-free buffer were stable and modification of PO activity was not detected. A significant time influence was observed only in samples obtained with 5 mM Ca21 (Fig. 2), where 100% of activity was reached only after 90 min of incubation. The samples obtained with 40 or 100 mM of Ca21 showed high spontaneous activation at time zero (60%), and at 30 min proPO was not detected, indicating that 100% transformation had occurred. Although a Ca21-independent spontaneous activation of proPO has been reported in several insects (15,17,19–21), other authors have reported that low Ca21 concentrations (5–20 mM) produce proPO spontaneous activation in insects (1,6,14) and in crustaceans (2,28,32). The stability of the shrimp proPO system in the absence of Ca21 is similar to the results in the freshwater crayfish (2,28,32) where 5 mM of this cation stimulated the spontaneous activation. However, the spontaneous activation of the shrimp proPO system can not be inhibited by high Ca21 concentration, as recommended by some authors (2,14,28,32).

T. Gollas-Galva´n et al.

FIG. 3. Effect of calcium on the dopachrome formed by brown shrimp PO or trypsin-treated proPO. Mean (SE) of five determinations are shown.

Ca Effect Over Proteolytic Activation of proPO Because Ca21 has been shown to be necessary for proteolytic activation of proPO in Blaberus craniifer (14) and crayfish (2,32), we assayed the effect of different Ca21 concentrations on trypsin-induced proPO activation. High Ca21 concentration (100 mM) provoked a significant (P , 0.05) reduction of dopachrome formation, which can be interpreted as an inhibition of the proPO spontaneous activation. However, we did not observe an inhibition higher than 40% with 100 mM of Ca21, different from Leonard et al. (14), who reported total inhibition of activity with 100 mM of Ca21. Furthermore, a similar inhibitory pattern on the dopachrome formation was observed when Ca was added to proPO previously activated by trypsin (Fig. 3). This suggests high Ca21 concentrations (50–100 mM) affect the formation of dopachrome rather than the transformation of proPO to active PO. Thus, brown shrimp proPO can be activated by trypsin in the absence of Ca21, whereas the natural proteolytic proPO activation pathway apparently requires this cation. There is evidence indicating Ca21 is involved in the proPO activating system in insects and crustaceans, although the mechanism of action in spontaneous activation is still unclear. So¨derha¨ ll and Ha¨ll (32) demonstrated that Ca21 increases the protease activity in HLS of crayfish, and this can help to explain the role of Ca 21. Even though in some insects the action of PPAE apparently is Ca21 inde-

Effect of Calcium on Shrimp proPO System

423

TABLE 2. Effect of melittin on proPO activation produced

by trypsin or calcium

Trypsin Trypsin 1 STI Ca 21 (10 mM) Ca 21 (10 mM) 1 STI

Melittin

Melittin-free

100 46 4 10

100 4 25 7

Average values (n 5 5) are expressed as percent of proPO transformed in active PO.

pendent (1,15–17,19–21), it is possible, in crustaceans, the role of Ca21 on spontaneous activation is related to PPAE activation rather than directly affecting proPO transformation. Supporting this idea, we observed the spontaneous activation of the brown shrimp proPO system induced by Ca21 can be blocked by STI (Table 2). Additionally, when the shrimp proPO system was obtained in Cac buffer containing melittin (a noncompetitive proteinase inhibitor and blocker of proPO activation [29]), the spontaneous activation in the presence of Ca21 was inhibited. Thus, although melittin prevents proPO system activation by blocking the PPAE, Ca21 is required by this proteolytic enzyme and the subsequent proPO to PO transformation. Durrant et al. (7) separated the Blaberus discoidalis PPAE by passing HLS through a Blue Sepharose column. Using this method, we obtained a PPAE-free proPO preparation, which was not retained on the column. This PPAE-free preparation did not show spontaneous activation by incubation with Ca21 (10–50 mM) but could be activated by incubation with trypsin (Table 3). These results confirm Ca21 participation is associated with the activating system rather than directly with the proPO transformation to active PO.

Effect of Ca Over Dopachrome Formation To look for the effect of Ca 21 on dopachrome formation, we tested different Ca21 concentrations (0–100 mM) in trypsin-activated PO and two other phenol-oxidizing enzymes: phenolase (EC 1.10.3.1) and tyrosinase (EC 1.14.18.1). In all cases, the formation of dopachrome was significantly impaired (P , 0.05) by 10 mM or higher Ca21

TABLE 3. Effect of Ca 21 on proPO spontaneous activation in

PPAE-depleted samples obtained by passing them through a Blue Sepharose column

Original PPAE depleted

Trypsin

Cacodylate

Ca 21 (50 mM)

100 100

4 12

48 10

Average values (n 5 3) are expressed as percent of PO activity detected.

FIG. 4. Effect of Ca on the dopachrome formation reaction performed by oxidizing enzymes: phenolase, tyrosinase and brown shrimp phenoloxidase (n 5 6).

(Fig. 4). Only 60% of the original (without Ca21 ) shrimp PO activity was detected by using 100 mM Ca21. Thus, it is possible the apparent PO activity reduction, observed by using a high concentration of Ca21, reflects an effect on dopachrome formation rather than on the proPO activation or PO stability. Effect on b-Glucan-Induced Activation

β-Glucans are able to activate the crustacean proPO system probably by inducing protease activity as demonstrated on HLS of crayfish (2). In a similar manner, when the brown shrimp proPO system obtained by centrifugation was incubated with β-glucan in the presence of 20 mM Ca21, an additional activation was recorded. However, stimulation did not occur in Ca 21-free conditions (Fig. 5). Thus, Ca21 is necessary for glucan-induced proPO activation, and this result is compatible with physiological conditions where Ca21 is present. Ca21 participation during laminarin-induced stimulation was also determined in the intact hemocytes of brown shrimp. When circulating cells were stimulated with laminarin in a Ca21-free medium, PO activity was not observed, indicating an endogenous Ca21 requirement. When hemocytes were incubated with Ca21 alone, neither PO activity

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FIG. 5. Participation of calcium on the laminarin-induced

proPO activation. Means (SE) were compared by one-way ANOVA, and statistically significant differences (Tukey analysis) are denoted by different letters (n 5 9).

nor proPO were detected in the cell-free supernatant. However, when hemocytes were incubated with laminarin in a Ca21-containing medium, PO activity was detected (Table 4). In a cell-free proPO preparation (Table 4), a spontaneous activation (30%) was observed by incubating with Ca21, and an additional stimulation (up to 70%) was seen if laminarin was also added. Finally, laminarin alone (Ca21-free condition) was unable to produce PO activity in a cell-free proPO preparation, similar the results from intact hemocytes. proPO, Ca21 and PPAE The presence of Ca21 makes it difficult to explain why the proPO is not activated spontaneously in the hemolymph. Both PPAE and the inactive proPO are contained inside the hemocyte granules, and only under stimulus the proPO can be transformed in an active form by the PPAE. Consid-

ering our results and those obtained by other authors (2,5,14,30–32) demonstrating Ca21 stimulates the proteolytic activity that transforms the proPO, it is possible to suggest the manner of Ca21 participation. Either Ca21 is introduced to the hemocytic granule or, if already present, it is available only after stimulus. It is also possible Ca21 only participates in the process after PPAE and proPO have been released from the cells, which should occur after the stimulation. Our results suggest the proPO system is released from the cells under laminarin stimulation but in an inactive form. Outside the cell, proPO is activated by PPAE if Ca21 is present. We determined that in the brown shrimp plasma, the Ca21 concentration is nearly 8 mM. Thus, it is possible, in natural conditions, the activation of proPO system consists of two different steps. The first is related to cell degranulation induced by microbial stimulus. The second is the proPO transformation into its active form (PO), which is catalyzed by PPAE in presence of plasma Ca21. Although protein purification is necessary to continue the studies on shrimp proPO system, working with the granular content was helpful to determine the role of Ca 21 as the closest to in vivo conditions. In this study, only a PPAEdepleted preparation was sufficient to define that Ca21 is not directly required for PO activity. In addition, we found the conditions to obtain a stable proPO preparation necessary to purify the proteins in the future. This work was partially supported by FONSIMAC (Project: SIMAC/ 94/CM-04). We are grateful to F. Magallo´n and G. Portillo (Marine Aquaculture Program, CIBNOR, Mexico), who kindly supplied the experimental animals, and to Dr. G. Yepiz-Plascencia (CIAD, Mexico) for critical reading of the manuscript. Thanks also to Dr. Ellis Glazier, CIBNOR, for editing this English language text.

References 1. Ashida, M.; Ishizaki, Y.; Iwahana, H. Activation of prophenoloxidase by bacterial cell walls or β-1,3-glucans in plasma of the silkworm, Bombyx mori. Biochem. Biophys. Res. Commun. 113:562–568;1983. 2. Ashida, M.; So¨derha¨ll, K. The prophenoloxidase activating system in crayfish. Comp. Biochem. Physiol. 77B:21–26;1984. 3. Ashida, M.; Yamazaki, H. Biochemistry of the phenoloxidase system in insects: with special reference to its activation. In: Ohnishi, E.; Ishizaki, H. (eds). Molting and metamorphosis. Berlin: Springer-Verlag; 1990:239–265. 4. Aspa´n, A; Sturtevant, J.; Smith, V.J.; So¨derha¨ll, K. Purification and characterization of a prophenoloxidase activating en-

TABLE 4. Effect of Ca21 on laminarin-induced proPO activation

Hemocytes Cell-free centrifugate

Laminarin

Ca (20 mM)

Laminarin 1 Ca 21 (20 mM)

0.230 6 0.023 (1.0) 0.091 6 0.013 (1.0)

0.191 6 0.006 (0.8) 0.119 6 0.007 (1.3)

0.622 6 0.076 (2.7) 0.159 6 0.003 (1.7)

The role of Ca21 was tested in both cell-free proPO preparation and intact hemocytes. Values are means 6 SE (n 5 6) of optical density at 492 nm. The stimulation index setting of the laminarin treatment equal to 1 is indicated in parentheses.

Effect of Calcium on Shrimp proPO System

5. 6. 7.

8.

9. 10. 11. 12. 13. 14. 15. 16.

17.

18. 19.

20.

zyme from crayfish blood cells. Insect Biochem. 20:709–718; 1990. Brehe´lin, M.; Drif, L.; Baud, L.; Boemare, N. Insect haemolymph: cooperation between humoral and cellular factors in Locusta migratoria. Insect Biochem. 19:301–307;1989. Dularay, B.; Lackie, A.M. Haemocytic encapsulation and the prophenoloxidase-activation pathway in the locust Schistocerca gregaria forks. Insect Biochem. 15:827–834;1985. Durrant, H.J.; Ratcliffe, N.A.; Hipkin, C.R.; Asp N.A.; So¨derha¨ll, K. Purification of the pro-phenol oxidase enzyme from haemocytes of the cockroach Blaberus discoidalis. Biochem. J. 289:87–91;1993. Herna´ndez-Lo´pez, J.; Gollas-Galva´n, T.; Vargas-Albores, F. Activation of the prophenoloxidase system of the brown shrimp (Penaeus californiensis Holmes). Comp. Biochem. Physiol. 113C:61–66;1996. Johansson, M.W.; So¨derha¨ll, K. Isolation and purification of a cell adhesion factor from crayfish blood cells. J. Cell Biol. 106:1795–1803;1988. Johansson, M.W.; So¨derha¨ll, K. A cell adhesion factor from crayfish haemocytes has degranulating activity towards crayfish granular cells. Insect Biochem. 19:183–190;1989. Johansson, M.W.; So¨derha¨ll, K. Cellular immunity in crustaceans and the proPO system. Parasitol. Today 5:171–176; 1989. Johansson, M.W.; So¨derha¨ll, K. Cellular defence and cell adhesion in crustacean. Animal Biol. 1:97–107;1992. Leonard, C.; Ratcliffe, N.A.; Rowley, A.F. The role of prophenoloxidase activation in non-self recognition and phagocytosis by insect blood cells. J. Insect Physiol. 31:789–799;1985. Leonard, C.; So¨derha¨ll, K.; Ratcliffe, N.A. Studies on prophenoloxidase and protease activity of Blaberus craniifer haemocytes. Insect Biochem. 15:803–810;1985. Li, J.; Tracy, J.W.; Christensen, B.M. Hemocyte monophenol oxidase activity in mosquitoes exposed to microfilariae of Dirofilaria immitis. J. Parasitol. 75:1–5;1989. Li, J.; Tracy, J.W.; Christensen, B.M. Phenol oxidase activity in hemolymph compartments of Aedes aegypti during melatonic encapsulation reactions against microfilariae. Dev. Comp. Immunol. 16:41–48;1992. Lockey, T.D.; Ourth, D.D. Phenoloxidase activity independent of calmodulin and calcium in hemolymph of Heliothis virescens (Lepidoptera: Noctuidae) larvae. J. Econ. Entomol. 85:1069 –1071;1992. Lowry, O.H.; Rosebrough, N.L; Farr, A.L; Randall, R.J. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265 –275;1951. Nappi, A.J.; Carton, Y.; Frey, F. Parasite-induced enhancement of hemolymph tyrosinase activity in a selected immune reactive strain of Drosophila melanogaster. Arch. Insect Biochem. Physiol. 18:159–168;1991. Nappi, A.J.; Seymour, J. Hemolymph phenol oxidases in Drosophila melanogaster, Locusta migratoria, and Austropotamobius pallipes. Biochem. Biophys. Res. Commun. 180:748 –754; 1991.

425

21. Nayar, J.K.; Bradley, T.J. Comparative study of hemolymph phenoloxidase activity in Aedes aegypti and Anopheles quadrimaculatus and its role in encapsulation of Brugia malayi microfilariae. Comp. Biochem. Physiol. 109A:929–938;1994. 22. Rantamaki, J.; Durrant, H.; Liang, Z.; Ratcliffe, N.A.; Duvic, B.; So¨rderha¨ll, K. Isolation of a 90-kDa protein from haemocytes of Blaberus craniifer which has similar functional and immunological properties to the 76-kDa protein from crayfish haemocytes. J. Insect Physiol. 37:627–634;1991. 23. Ratcliffe, N.A.; Brookman, J.L.; Rowley, A.F. Activation of the prophenoloxidase cascade and initiation of nodule formation in locusts by bacterial lipopolysacharides. Dev. Comp. Immunol. 15:33–39;1991. 24. Ratcliffe, N.A.; Leonard, C.; Rowley, A.F. Prophenoloxidase activation: Non-self recognition and cell cooperation in insect immunity. Science 226:557–559;1984. 25. Richards, E.H.; Renwrantz, L.R. Two lectins on the surface of Helix pomatia haemocytes: a Ca21 -dependent, GalNAc-specific lectin and a Ca21-independent, mannose 6-phosphatespecific lectin which recognises activated homologous opsonins. J. Comp. Physiol. 161B:43–54;1991. 26. Rowley, A.F.; Rahmet-Alla, M. Prophenoloxidase activation in the blood of Leucophaea maderae by microbial product and different strains of Bacillus cereus. J. Insect Physiol. 36:931– 937;1990. 27. Smith, V.J.; So¨derha¨ll, K. β-1,3-Glucan activation of crustacean hemocytes in vitro and in vivo. Biol. Bull. 164:299–314; 1983. 28. So¨derha¨ll, K. Fungal cell wall β-1,3-glucans induce clotting and phenoloxidase attachment to foreign surface of crayfish hemocyte lysate. Dev. Comp. Immunol. 5:563–573;1981. 29. So¨derha¨ll, K. The bee venom melittin induces lysis of arthropod granular cells and inhibits activation of the prophenoloxidase-activating system. FEBS Lett. 192:109–112;1985. 30. So¨derha¨ll, K. Biochemical and molecular aspects of cellular communication in arthropods. Bull. Zool. 59:141–151;1992. 31. So¨derha¨ll, K.; Cerenius, L.; Johansson, M.W. The prophenoloxidase activating system and its role in invertebrate defense. Ann. N.Y. Acad. Sci. 712:155–161;1994. 32. So¨derha¨ll, K.; Ha¨ll, L. Lipopolysaccharide-induced activation of prophenoloxidase activating system in crayfish haemocyte lysate. Biochem. Biophys. Acta 797:99–104;1984. 33. Vargas-Albores, F.; Guzma´n-Murillo, M.A.; Ochoa, J.-L. An anticoagulant solution for haemolymph collection and prophenoloxidase studies of Penaeid shrimp (Penaeus californiensis). Comp. Biochem. Physiol. 106A:299–303;1993. 34. Vargas-Albores, F.; Ochoa, J.-L. Variations of pH, osmolality, sodium and potassium concentrations in the haemolymph of sub-adult shrimp (Penaeus stylirostris) according to size. Comp. Biochem. Physiol. 102A:1–5;1992. 35. Zahedi, M.; Denham, D.A.; Ham, P.J. Encapsulation and melanization responses of Armigeres subalbatus against inoculated Sephadex beads. J. Invertebr. Pathol. 59:258–263;1992.