Phenoloxidase activity of acridid grasshoppers from the subfamilies melanoplinae and oedipodinae

Comp. Biochem. PhysioL Vol. 94B, No. 1, pp. 117-124, 1989 0305-0491/89 $3.00 + 0.00 Printed in Great Britain © 1989 Pergamon Press plc

PHENOLOXIDASE ACTIVITY OF ACRIDID GRASSHOPPERS FROM THE SUBFAMILIES

MELANOPLINAE AND OEDIPODINAE

MICHAEL J. BIDOCHKA, JEREMY P. GILLESPIE and GEORGE G. KHACHATOURIANS* Bioinsecticide Research Laboratory, Department of Applied Microbiology and Food Science,

University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0W0 (Tel: 306 966-5024)

(Received 23 January 1989)

Abstract--1. Phenoloxidase activity and wound melanization was studied in five species of grasshoppers representing the subfamilies Melanoplinae and Oedipodinae.

2. Most of the phenoloxidase activity was detected in the plasma fraction of grasshopper whole-body homogenates and supernatant fractions of the hemolymph. The species representing the Oedipodinae had 20-50% higher percentage of the total phenoloxidase activity associated with particulate matter from a whole-body homogenate when compared to the Melanoplinae.

3. Phenoloxidase activity could not be detected in sclerotized cuticle of adult grasshoppers. 4. The phenoloxidase existed as a zymogen which could be activated by chymotrypsin and inhibited

by KCN and NaCN while EDTA showed no effect. It had optimum activity at 37°C and pH 7.3. 5. These findings are discussed in relation to wound repair and immune responses to infection in

grasshopper species.

INTRODUCTION

Phenoloxidase (o-di-phenol:oxygen oxidoreduc- tase EC 1.10.3.1) is involved in melanization pro- cesses in insects. The enzyme oxidizes tyrosine derivatives to ortho-quinones which cross-link to terminal amines of proteins resulting in melanization (Andersen, 1985). This cross-linked product is an insoluble, protein-polyphenol matrix and is implicated in the encapsulation of pathogens (Gotz and Boman, 1985), cuticle wound repair and cuticle sclerotization (Andersen, 1985).

Phenoloxidase is present in most arthropods as the inactive precursor, prophenoloxidase. The enzyme can be activated by cuticular components, lysed hemocytes, or proteolytic enzymes such as chymotrypsin. Prophenoloxidase activation has been demonstrated in Bombyx mori (Ashida and Dohke, 1980), Manduca sexta (Aso et al., 1985), and Blaberus craniifer (Leonard et al., 1985). Reports on the localization of the prophenoloxidase and enzymes or compounds which activate it vary. Studies performed with B. mori (Ashida and Dohke, 1980) and M. sexta (Saul and Sugumaran, 1987) show phenoloxidase to be present in the hemolymph plasma, whereas Leonard et al. (1985) have suggested its location to be in the hemocytes. Schmit et al. (1977) have reported phenoloxidase activity in granulocytes and oenocytes of Galleria mellonella. A prophenoloxidase activating enzyme is present in the cuticle of M. sexta but the prophenoloxidase was not found in the cuticle (Saul and Sugumaran, 1986). The location of prophenoloxidase in insects is important when regarding its action against

*Author to whom correspondence should be addressed.

117

pathogens, response to cuticle damage and its role in cuticle sclerotization.

Insect prophenoloxidase was first recognized by Bodine and co-workers (Bodine et al., 1937; Bodine and Allen, 1938) in the eggs of the grasshopper, Melanoplus differentialis. Since then there have been no reports of phenoloxidase activity in the Melanoplinae or other grasshoppers. During our studies of hemocytic responses in another melanopline grasshopper, M. sanguinipes to the entomopathogenic fungus, Beauveria bassiana, we observed melanization of fungal conidia which had been encapsulated by hemocytes (Bidochka and Khacha- tourians, 1987). This observation suggested the presence of a phenoloxidase system in M. sanguinipes and necessitated a study of this topic. We have extended these previous observations by studying the localization and activation of prophenoloxidase in a laboratory non-diapause strain of the migratory grasshopper M. sanguinipes (Pickford and Randell, 1969), and field-collected acridid grasshoppers. M. sanguinipes, M. packardii, M. infantalis, Camnula pellucida and Spharagemon collare. The former three species are members of the Melanoplinae subfamily whilst the latter two species are members of the Oedipodinae subfamily.

MATERIALS AND METHODS

Non-diapausing adults of M. sanguinipes, 14 days after final ecdysis, were used in all experiments. Field specimens of adult M. sanguinipes, M. infantalis, M. packardii, C. pellucida and S. collare were collected at the university's fields during August 1988. The laboratory strain M. sanguinipes were kept in cages maintained at 30 _+ 2°C and illuminated with a 75 W floodlight and fed barley grass and

118 MICHAEL J. BIDOCHKA et al.

HOMOGENATE

Centrifuge

SUPERI IATANT

Microcentrifuge

I I SUPERNATANT PELLET

FRACTION [A] [B]

1 PELLET

i

Resuspend and recentfifuge I

I I SUPERNATANT PELLET

Micro- Resuspend and centr fuge microcentr fuge

I I SUPERNATANT PELLET SUPERNATANT PELLET

[C] [D] [El

Fig. 1. Procedure used for fractionating whole grasshopper homogenates. Fractions A to E were asssayed for phenoloxidase activity. Refer to Materials and Methods for details.

wheat bran ad libitum. The field-collected grasshoppers were brought in to the laboratory and kept in the same conditions as the laboratory strain grasshoppers for 1 week prior to experimentation.

Phenoloxidase activity was assayed from fractions of whole grasshopper homogenate. The scheme for fraction- ation of grasshopper homogenate is shown in Fig. 1. Four grasshoppers (2 male, 2 female) were frozen at -20°C for 30 min. The head, legs and wings of each insect were removed using sterile scissors so that only the thorax and abdomen remained. This was weighed and placed in a steel cup microhomogenizer (Sorvall Inc., Newtown, Conn.) with 0.01 M sodium phosphate buffer (pH 6.0; 3 ml buffer/2 g grasshopper) at 4°C. The preparation was homogenized in the steel cup microhomogenizer using an omnimixer (Sor- vall Inc., Newton, Conn.) at 30,000 rpm for 3 min at 4°C. All preparations were kept on ice to avoid possible auto- oxidation. The grasshopper homogenate was centrifuged in a RCSB centrifuge (Sorvall Inc.) using a SS-34 rotor at 5000g for 10 min. Fractions A and B are derived from the supernatant after centrifugation of the whole grasshopper homogenate. Fraction A represents the twice centrifuged supernatant from the original homogenate. Fraction B represents the pellet from the residual material left in the supernatant from centrifugation of the original homogenate. Fractions C, D and E are derived from the pellet after centrifugation of the whole grasshopper homogenate. Fraction C represents the supernatant from the pellet washing followed by two centrifugations. Fraction D represents the residual material after washing the pellet collecting the supernatant and recentrifuging. Fraction E represents the supernatant after two washings of the pellet derived from the whole grasshopper homogenate.

Hemolymph samples from the laboratory strain of M. sanguinipes were fractionated and assayed for phenoloxidase activity. Ten grasshoppers (5 male, 5 female) were incapaci- tated with CO 2 . The ventral thoracic plate of each insect was pierced with a sterile insect mounting pin (gauge 6) and l0/t 1 of hemolymph was withdrawn with an automatic pipettor (Pipetman P20, Mandel Scientific Co., Edmonton, Alberta) and stored on ice. The hemolymph samples were diluted with 1 ml phosphate buffer and centrifuged at setting no. 90 in a microcentrifuge (Microchemical Specialities Co., Berkley, CA) at 4°C for 5 min. The supernatant of each tube was removed and the remaining pellet resuspended in 1 ml phosphate buffer.

Phenoloxidase activity was monitored spectrophoto- metrically as the formation of dopachrome (Horowitz and Shen, 1952). The reaction mixture contained 3.9 ml 0.01 M phosphate buffer, l ml 0.02 M L-dihydroxyphenylalanine (L-DOPA; Sigma Chemical Co., St. Louis, MO) and 0.1 ml of sample. The reaction was initiated by the addition of the sample to the reaction mixture and measuring the increase

in absorbance at 490 nm for 10 min at 21°C. A standard curve was prepared using a reaction mixture containing 3.9 ml 0.01 M phosphate buffer, 1 ml L-DOPA and 0.1 ml of various concentrations of a commercial phenoloxidase derived from mushrooms (Sigma) with an activity of 5.2 x 104 units/mg. Grasshopper phenoloxidase activity was reported as equivalent units of mushroom-derived phenoloxidase activity.

Tyrosinase activity was also determined using the above protocol except that tyrosine was substituted for L-DOPA as the oxidative substrate. Tyrosinase activity was reported as equivalent units of mushroom-derived tyrosinase (Sigma) activity.

Prophenoloxidase activation was tested with a solution of bovine pancreas chymotrypsin (100/d of a 100/~g/ml solution; Sigma). The sample fractions were preincubated with the chymotrypsin solution for 15 min at 21°C then assayed for phenoloxidase activity as described above.

KCN, NaCN and EDTA were tested as inhibitors of phenoloxidase activity at concentrations from 10 -5 to 10-~ M. The pH and temperature activity profiles of phenoloxidase were determined using standard biochemical methods. The buffer used for generating the pH was a universal buffer solution (pH 2-12). However, above pH 10.4 the L-DOPA auto-oxidized and the pH profile was restricted to values below this. For the temperature activity profile the standard phenoloxidase assay solution was equilibrated over the range 0 to 90°C before the addition of the enzyme solution.

Non-denaturing polyacrylamide gel electrophoresis (PAGE) was carried out to assess the presence and activation of prophenoloxidase. The gel was polymerized in three layers. The top layer was 3.0% acrylamide, the middle layer 5.0% acrylamide and the bottom 7.5% acrylamide. To the acrylamide, 17.5ml of 1.5 M Tris-HCl (pH 8.8), 35 ml distilled water, 35 ~1 N,N,N',N'-tetramethylethylene- diamine (TEMED) and 0.70ml ammonium persulfate (75mg/ml) was added. From individual grasshoppers 30 #1 of hemolymph, 5 #1 bromophenol blue and 20/~1 of 0.25 M Tris-HC1 (pH6.8) with or without 100/~g chymotrypsin was placed into each well with a Hamilton syringe. The electrophoresis was performed at 10°C with 0.05 m Tris glycine buffer (pH 8.3). The electrophoresis was stopped when the bromophenol blue front reached the end of the gel. Phenoloxidase was located in the gel by staining with 40 mg L-DOPA in 100 ml of 0.01 M sodium phosphate buffer for I hr.

The effect of wounding grasshoppers and subsequent cuticle melanization was investigated. Grasshoppers were pierced with a sterile insect mounting pin (gauge 6) in the ventral abdominal sclerite. They were individually housed in Petri dishes kept in an environmental incubator (Hotpack Corp., Philadelphia, PA) along with the non-pierced

Phenoloxidase activity of grasshoppers 119

Fig. 2. Time course of male and female M. sanguinipes (lab strain) cuticle melanization after wounding. Grasshoppers were pierced at the first sclerite on the ventral abdominal side with a sterile insect mounting pin. (A) Male after I day; (13) male, 3 days; (C) male, 4 days; (D) male, 5 days; (E) female, 1 day; (F) female, 3 days; (G) female, 4 days; (H) female, 5 days. The wound position for all grasshoppers is indicated by

an arrow in (A).

controls at 27°C, in continuous light, and 27% relative humidity.

The site of wounding was photographed every 24 hr for 5 days.

RESULTS

Melanization, mediated by phenoloxidase, has been implicated in cuticular wound repair in insects. The effect of wounding on cuticle melanization was investigated in several grasshopper species. M. sanguinipes (lab strain), and wild M. sanguinipes, M. packardii, S. collare and C. pellucida were injured at

the first sclerite on the ventral surface of the abdomen using an insect mounting pin (gauge 6). Figure 2 shows a 5 day time course of melanization of the ventral abdominal first sclerite in laboratory-reared male and female M. sanguinipes. Within 2 days a dark spot was observed at and surrounding the point of injury, From 3 to 5 days females appeared to have a greater degree of melanization at the point of injury. Figure 3 shows the melanization response to wounding in grasshoppers representing four field-collected species. S. collare appeared to have the greatest amount of melanization at the point of injury. This individual also had natural melanization around the

120 MICHAEL J. BIDOCI-IKA et al.

Fig. 3. Melanization of cuticle in four grasshopper species 2 days after wounding. From left to right; S. collare, M. sanguinipes (wild), C. pellucida and M. packardii. Arrow on M. packardii indicates position

of wounding with a sterile insect mounting pin for all grasshoppers.

first, second and third sclerites which is common in the species.

Table 1 shows phenoloxidase activity (in the presence or absence of chymotrypsin) in the various fractions obtained from the grasshopper homogenates. Microscopic examination of fraction A revealed the absence of particulate material. In wild M. sanguinipes, M. packardii, and M. infantalis, fraction A contained the largest percentage of the total phenoloxidase activity in samples without chymotrypsin (denominator in Table 1). For all species phenoloxidase activity in fraction A ranged from 3 x 102 units/g body weight for M. packardii to 4.95 x 105units/g body weight for the laboratory strain of M. sanguinipes and represents 37.5-100% of the total phenoloxidase activity in the total homogenate without chymotrypsin. This suggested that a large proportion of the phenoloxidase is localized in the insect hemolymph. The pellet from the recentrifu- gation of the supernatant (fraction B) contained some particulate material. This fraction contained no

Table 1. Phenoloxidase activity in isolated fractions of grasshopper whole body homogenates

Activity of phenoloxidase ( × 103) in homogenate fraction*

Grasshopper species (source) A B C

964.5 147.3 994.9 Melanoplus sanguinipes (lab)

494.6 147.3 678.9

106.9 3.4 4.2 M. sanguinipes (wild)

13.1 3.4 0.5

1.0 0.1 0.0 M. packardii (wild) 0.3 0.0 0.0

29.2 0.0 0.4 M. infantalis (wild) 5.8 0.0 0.4

2601.7 0.0 37.8 Spharagemon collare (wild) 5.3 0.0 5.8

14.8 5.7 31.8 Camnula pellucida (wild)

0.6 0.0 0.7

*Phenoloxidase activity (units/g of grasshopper) before (denominator) and after (numerator) addition of chymotrypsin. Results are mean values from three determinations.

phenoloxidase activity in the absence of chymotrypsin in C. pellucida, S. collare and M. packardii but did contain 20.2% of the total phenoloxidase activity in wild M. sanguinipes. Fraction C consisted of the supernatant from the homogenate pellet that was resuspended in phosphate buffer. This fraction contained approximately 55% of the total phenoloxidase, without chymotrypsin addition, in C. pellucida and the laboratory-reared M. sanguinipes. Further- more, tyrosinase activity (1 × 103 units) was detected in fraction C from laboratory M. sanguinipes, both before and after the addition of chymotrypsin (data not shown). In the other grasshopper species tested the value for phenoloxidase, before chymotrypsin addition, in fraction C was below 7% of the total phenoloxidase activity. Fraction D contained lysed hemocytes but did not contain any phenoloxidase activity. Fraction E was the supernatant from the centrifugation of large particles from the homogenate such as cuticle and other insect tissues and showed no detectable phenoloxidase activity.

Chymotrypsin was added to the various grasshopper homogenate fractions shown in Table 1 (numerator). Activation of prophenoloxidase was found to be linear for chymotrypsin concentrations between 1 and 5 #g/ml. Maximum activation remained un- changed for concentrations between 8 and 15 #g/ml. When compared to the samples without chymotrypsin, the addition of 10 pg of chymotrypsin (50 units activity/mg) to fractions A resulted in a 25-fold increase in phenoloxidase activity in C. pellucida and a 5 x 102-fold increase in S. collare. The melanopline species showed a 2- to 8-fold increase in phenoloxidase activity in fraction A with the addition of chymotrypsin. These results indicate the presence of a prophenoloxidase in the hemolymph. An increase in phenoloxidase activity was also observed after pre- incubation of chymotrypsin with fractions B from C. pellucida. Other grasshopper species showed little or no change in phenoloxidase activity in fraction B with the addition of chymotrypsin. C. pellucida and S. collare showed a 45-fold and a 6.5-fold increase,


respectively, in fraction C. Little or no chymotrypsin- activated phenoloxidase was observed in fractions C in M. infantalis and M. packardii. On the other hand the wild M. sanguinipes showed an 8-fold increase in activity after activation by chymotrypsin. In all species tested no phenoloxidase activity, after chymotrypsin addition, was detected in fraction D or E.

The distribution of phenoloxidase activity in centrifuged hemolymph samples of the laboratory strain of male and female M. sanguinipes was examined (Table 2). There was no phenoloxidase activity in the centrifuged hemolymph supernatant or resuspended pellet before chymotrypsin activation. After chymotrypsin addition most of the total phenoloxidase activity occurred in the supernatant. Females had 1.6-fold more total phenoloxidase activity per 100 #1 hemolymph than males.

Chymotryptic activation of prophenoloxidase in- volves the hydrolysis of a peptide fragment from the proenzyme. Presumably the proenzyme is larger than the activated form. In order to assess the size difference between the activated (chymotrypsin treated) and non-activated prophenoioxidase we performed native PAGE on M. sanguinipes hemo-

Table 2. Phenoloxidase activities, after addition of chymotrypsin, in the supernatant and resuspended pellet of centrifuged hemolymph extracted from the

laboratory strain of M. sanguinipes Phenoloxidase activity*

(% total) in: Sex Supernatant Pellet Male 25.9 (96.2) 1.0 (3.8) Female 36.6 (87.8) 5.2 (12.3) *Phenoloxidase activity expressed as

units per 100/tl hemolymph after addition of chymotrypsin. Results are mean values from three determinations.

lymph samples. The results of staining the gel with DOPA are shown in Fig. 4. Chymotrypsin-activated hemolymph showed two bands of phenoloxidase activity (Fig. 4, lane A) as opposed to one band in the non-activated hemolymph (Fig. 4, lane B). The additional band in the chymotrypsin-activated sample migrated further in the native gel, suggesting cleavage of prophenoloxidase to an active, smaller phenoloxidase.

A 13

Fig. 4. Zymogram of M. sanguinipes phenoloxidase activity (A) after chymotrypsin activation, and (B) no chymotrypsin activation. Arrows indicate the two bands of phenoloxidase activity in (A).


IOO

.Q :E 80 E

6O

4O

o 2O

o. 0

+ l + i i

10-6 10 .5 10 .4 10 .3 10 -2 10 -1 Inhibitor concentration (M)

10

Fig. 5. Effect of KCN (1), NaCN (U]) and EDTA (&) on phenoloxidase activity in the laboratory strain of

M. sanguinipes.

Phenoloxidase had a pH optimum of 7.3 and temperature optimum of 37'C. The compounds KCN and NaCN inhibited phenoloxidase activity while EDTA showed no effect (Fig. 5).

DISCUSSION

Activation of insect prophenoloxidase was first reported in M. differentialis eggs (Bodine et al., 1937). Insect phenoloxidases occur as zymogens and are activated by serine proteases (Ashida et al., 1974), chymotrypsin (Ohnishi et al., 1970) /3-1,3 glucans (Gotz and Boman, 1985), peptidoglyeans (Yoshida and Ashida, 1986) and lipids (Heyneman and Ver- cauteren, 1964). Although prophenoloxidase has been shown to be activated by substances other than proteolytic enzymes, these substances most likely act as artificial intermediates for prophenoloxidase activation via proteolytic enzymes. For example, in Drosophila, activation of prophenoloxidase is the result of a cascade of reactions involving at least six proteins (Seybold et al., 1975). Chymotrypsin-treated M. sanguinipes hemolymph revealed two phenoloxidase bands, one of which migrated electrophoreti- cally further than in the non-treated hemolymph, suggesting proteolytic cleavage of the proenzyme. Furthermore, phenoloxidase activity in grasshopper species that we tested was increased with the addition of chymotrypsin. The prophenoloxidase of B. mori is activated by a specific proteolytic enzyme which removes a 5 kDa polypeptide from each subunit (Ashida and Dohke, 1980).

Several reports suggest that insect phenoloxidase is a cupric enzyme (Ashida, 1971; Heyneman and Vercauteren, 1964). However, the chelating agent EDTA did not inhibit phenoloxidase activity in M. sanguinipes while cyanide compounds did inhibit activity. Phenoloxidase activity in Spodoptera littoralis was inhibited by chelating agents and cyanide compounds (Ashaaya, 1972). pH and temperature activity profiles observed for M. sanguinipes phenoloxidase were similar to those observed for Spodo- ptera littoralis and Bombyx mori (Ashida and Dohke, 1980).

Our results with piercing of the grasshopper cuticle (Figs 2 and 3) show that melanization occurred at the point of injury. The melanization was localized and restricted to within 2 mm of the point of injury.

The darkening, or melanization of the cuticle, was assumed to be the result of the reaction of phenoloxidase on a cuticular or hemolymphal quinone precursor such as tyrosine, cresol, catechol or DOPA, the substrate used in our studies. Taylor (1969) has suggested that the insect phenoloxidizing system in the hemocyte plays a major role in sealing lesions in the integument. Wounding dramatically increases the number of hemocytes in circulation (Jones, 1967) and, in Calliphora, hemocytes containing phenoloxidizing enzymes accumulated at wound sites (Cross- ley, 1975). We have not examined whether hemocyte aggregation at the wound site for these grasshopper species takes place.

The activation of phenoloxidase for wound repair is a transient response. Therefore the insect must have a method by which the phenoloxidase is separated from its activator and substrate until needed. Several methods of phenoloxidase activation and separation from its substrate have been proposed. Our results suggest that acridid grasshopper phenoloxidase is not bound to the hemocytic membrane and probably exists in the hemocytic cytoplasm or hemolymph plasma. Mills and Whitehead (1970) suggest that phenoloxidase is located in the hemocyte and that a diuretic hormone may serve to increase the permeability of the hemocyte membrane to tyrosine and thus allowing the subsequent formation of N-acetyl- dopamine. Leonard et al. (1985) reported phenoloxidase of B. craniifer in the hemocytes but not in the plasma. There is evidence that G. mellonella prophenoloxidase is released from lysed granulocytes or oenocytoids (Schmit et al., 1977), and the activating substances are produced by granulocytes or cuticle basal cells (Hughes and Price, 1976). A lytic response in hemocytes, which would result in an activator-proenzyme complex, may be induced by wounding or by an invading microorganism (Gotz and Boman, 1985).

The significance of melanization, and phenoloxidase activity, in the insect defense reaction against internal parasites is well documented (Gotz and Boman, 1985). Aoki and Yanase (1970) noted that some silkworm larvae infected with the entomopathogenic fungus B. bassiana had blackened inte- gumentary spots. Granulocytes of M. sanguinipes lyse shortly after they come in contact with B. bassiana conidia with subsequent melanization of the encapsulated conidia (Bidochka and Khachatourians, 1987). The question as to whether the phenoloxidase is fungal- or insect-derived has recently been subject to investigation (St. Leger et al., 1988). Colonies of the fungi B. bassiana and Metarhizium anisopliae produced polyphenol pigments probably derived from phenoloxidase action (Zajic, 1963; St. Leger et al., 1988), but M. anisopliae does not produce phenoloxidase when grown on insect cuticle (St. Leger et al. 1988).

The laboratory strain of M. sanguinipes had an overall higher phenoloxidase activity than the wild M. sanguinipes. This difference may either be a difference in the genetic makeup of the strains or differences in the environmental conditions. The laboratory strain is a genetically monotypic non- diapausing strain that is kept in constant conditions of relative humidity, temperature and food supply


and type. On the other hand the field strain of M. sanguinipes may be of varied genotype (Chapco and Bidochka, 1986) and exposed to various environmental conditions. Therefore differences in phenoloxidase activity between the wild and laboratory strains of M. sanguinipes may be due to genetic differences or physiological differences which affect prophenoloxidase. A striking observation is the high overall phenoloxidase activity in the laboratory strain of M. sanguinipes, when compared to the other grasshoppers. Again, such high phenoloxidase activity values may be due to the favourable rearing conditions of laboratory grasshoppers, even though field-collected grasshoppers were housed for one week under laboratory conditions. We also observed differences between the wild Melanoplinae and the Oedopodinae. Specifically, fraction C in the Oedipo- dinae had a higher percent of the total phenoloxidase activity ( > 50%) when compared to the wild Melanoplinae before activation with chymotrypsin. After activation of fraction C with chymotrypsin the Oedipodinae showed up to 80-fold more phenoloxidase activity than the wild Melanoplinae. This fraction is a resuspension and centrifugation of particulate matter from the grasshopper homogenate. This suggests that in the Oedipodinae phenoloxidase may be associated with the hemocyte or other particulate material.

Since phenoloxidases have been suspected in the immune response of M. sanguinipes (Bidochka and Khachatourians, 1987) and other insects (Gotz and Boman, 1985), differences in phenoloxidase activity and localization in various grasshopper species may reflect differences in the grasshopper immune responses to various pathogens. For instance, the Melanoplinae were found to be more susceptible to infection by the protozoan, Nosema locustae than C. pellucida of the subfamily Oedipodinae (Ewen and Mukerji, 1980). On the other hand, C. pellucida was more susceptible to the entomopathogenic fungus, Entomophthora grylli pathotype I than the melanopline grasshoppers (Pickford and Reigert, 1964). These differences in resistance and suscepti- bility of grasshoppers to different entomopathogens may reflect the different methods of pathogen disease formation and the subsequent response of the grasshopper phenoloxidase system.

Acknowledgements--J. Chapman initially helped in these studies. Supported by an award of a Canadian Wheat Board Fellowship to M.J.B. and by the Saskatchewan Department of Agriculture through the Agriculture Devel- opment Fund.

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Phenoloxidase activity of acridid grasshoppers from the subfamilies melanoplinae and oedipodinae