Behaviour in vitro of separated fractions of haemocytes of the locust Schistocerca gregaria

Summary The haemocytes of locusts (Schistocerca gregaria) have been partially separated, by centrifugation on discontinuous Percoll gradients, into 3 subpopulations of plasmatocytes of which the most dense (band 5) displays more than 95% morphological homogeneity. The cells of band 5 are very granular, adhere and spread on glass, and contain nearly all the phenoloxidase activity of the total haemocyte population, as shown by 1,3-glucan (laminarin)-stimulation of both haemocyte lysate supernatant and monolayers of living cells. Cells from band 5 show negligible endocytosis of fluorescent latex beads, whereas those of band 4, which are less granular, plasmatocyte-like cells, are actively endocytic in vitro. The majority of the haemocytes are found in band 3, which is a mixture of coagulocytes and agranular plasmatocytes, with negligible phenoloxidase activity. The in vitro locomotory behaviour of adherent cells from bands 3 and 5 was compared, and addition of laminarin-activated haemocyte lysate supernatant, in which the prophenoloxidase activation sequence had been initiated, stimulated chemokinesis in cells of band 5 but not band 3. The separated fractions thus show marked behavioural and biochemical differences.     

Insect haemolymph: Cooperation between humoral and cellular factors in Locusta migratoria

In Locusta migratoria, prophenoloxidase is present in the plasma and serum, but in reduced amounts relative to the haemocytes. This phenoloxidase activity cannot be induced by either heating or freezing and thawing and it is lost by heating at 70°C for 30 min. Both lipopolysaccharides and laminarin can elicit the prophenoloxidase activating system. These elicitors of prophenoloxidase activation are active in haemocyte lysate and in serum but never induce any phenoloxidase activity in plasma. In haemocyte lysate, the activating system is not heat resistant, and heating at 56°C for 30 min prior to incubation with laminarin or lipopolysaccharide precludes any phenoloxidase activity. Plasma contains a strong inhibitor of the prophenoloxidase activating system but serum does not. This inhibitor does not affect the phenoloxidase enzyme itself. The possible role of the activating system in immune recognition and the strategies evolved by parasites or pathogens to escape being recognized by their host are discussed.     

Studies on prophenoloxidase and protease activity of Blaberus craniifer haemocytes

Using a citrate-EDTA buffer as an anticoagulant it was possible to isolate intact haemocytes from the insect, Blaberus craniifer, without causing extensive degranulation and subsequent clotting. A haemocyte lysate from this insect contained prophenoloxidase (proPO), which could be activated by β 1,3-glucans. The activation process was dependent upon Ca²⁺ ions and seemed to occur by a limited proteolysis, since several serine protease inhibitors such as soybean trypsin inhibitor, benzamidine and $\text{p-}\text{nitrophenyl}\text{-p′-}\text{guanidobenzoate}$ blocked convertion of proPO to the active enzyme. Treatment of proPO with urea or heat also caused proPO activation but probably without the intervention of serine proteases, since the protease inhibitors used failed to block the activation. Within the haemocyte lysate, several endopeptidases were present, which were enhanced in activity by prior treatment with β 1,3-glucans. These endopeptidases were inhibited in activity when the haemocyte lysate was incubated with benzamidine prior to the addition of β 1,3-glucan. This provides further indications that the activation of proPO involves a limited proteolytic attack. The active phenoloxidase enzyme became strongly bound to foreign surfaces and this phenomenon may assist in providing opsonic properties for the proPO cascade.     

Exocytosis and uptake of bacteria by isolated haemocyte populations of two crustaceans: evidence for cellular co-operation in the defence reactions of arthropods

Summary The role of exocytosis in the cellular defence reactions of arthropods was investigated using in vitro cultures of isolated haemocytes (blood cells) from the freshwater crayfish Pacifastacus leniusculus, and the shore crab Carcinus maenas. In both species, activated lysates of those cell types that contain the prophenoloxidase activating system (granular cells of crab and crayfish and semigranular cells of crayfish) were found to induce degranulation (exocytosis) of semigranular and granular cells. A cell lysate, in which the prophenoloxidase system was kept inactive, did not have this effect. Limited degranulation of granular cells of crab was also induced by lipopolysaccharides as has earlier been shown for crayfish semigranular cells. The phagocytic capability of semigranular cells from crayfish was lost after exocytosis induced by the Ca²⁺ ionophore A23187, and under no conditions were the granular cells of crabs or crayfish seen to ingest bacteria in vitro. An opsonic function for the attaching proteins of a 1,3-glucan-activated haemocyte lysate was demonstrated using the phagocytic hyaline cells from crabs. Phenoloxidase appeared to lack opsonic properties.We suggest that, in crustaceans, opsonization takes place through hierarchically stimulated exocytotic release, and biochemical activation of the prophenoloxidase activating system: first from lipopolysaccharide-sensitive cells (semigranular cells of crayfish or granular cells of crabs) and then from granular cells, triggered by the initially released and activated prophenoloxidase system. Finally, sticky proteins of the activated prophenoloxidase system coat the invader, rendering it susceptible to the phagocytes (hyaline cells in both crab and crayfish and, to a lesser extent, semigranular cells of crayfish). These processes would, together, constitute a cellular communication pathway not previously demonstrated for invertebrates.Abbreviations DMSO dimethyl sulfoxide - L-DOPA L-dihydroxy-phenylalanine - GLS granular cell lysate supernatant - HLS haemocyte lysate supernatant - HyLS hyaline cell lysate supernatant - LPS lipopolysaccharide - proPO prophenoloxidase - SGLS semigranular cell lysate supernatant - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid disodium salt     

The role of prophenoloxidase activation in non-self recognition and phagocytosis by insect blood cells

Experiments indicate that the prophenoloxidase activating system, which is responsible for melanin production, is also involved in immunorecognition in insects. Using haemocyte monolayer preparations of Blaberus craniifer, Galleria mellonella and Leucophae maderae, it was shown that laminarin, a β 1,3-glucan extracted from fungal cell walls and an activator of the prophenoloxidase system, enhanced the phagocytosis of test bacteria.Scanning electron microscopy of haemocyte monolayers showed that incubation of test bacteria with laminarin significantly increased the number of microorganisms attached to both the plasmatocytes and the granular cells. Furthermore with the granular cells, these bacteria became entrapped in an amorphous matrix. This material probably consists of the “sticky” proteins previously reported to be produced by crustacean haemocytes following prophenoloxidase activation. Pretreatment of haemocytes with laminarin abolished the stimulatory effect on ingestion, indicating that these “sticky” proteins are opsonic, since they would have been discharged from the haemocytes onto the glass monolayer leaving few molecules available for subsequent coating of the test particles.Preliminary biochemical studies on the G. mellonella prophenoloxidase system demonstrated that it was activated by trypsin, laminarin and laminarin G, a highly purified β 1,3-glucan, but not by dextran. Serine protease activities were also enhanced by adding laminarin to a haemocyte lysate supernatant, suggesting that the stimulatory mechanism may involve the proteolytic activity of such enzymes.     

Isolation and Partial Purification of Prophenoloxidase from Daucus carota L. Cell Cultures

The enzyme, phenoloxidase, was isolated and partially purified as an inactive enzyme, a proenzyme, from plant cell cultures of Daucus carota, Nicotiana tabacum, and Haplopappus gracilis. The prophenoloxidase was found to be specifically activated by Ca²⁺ or Mn²⁺ ions in concentrations above 1 millimolar. Calmodulin was not involved in this activation. Concentrations of Ca²⁺ or Mn²⁺ below 1 millimolar could not induce activation of the prophenoloxidase, but if trypsin was added simultaneously with Ca²⁺ or Mn²⁺ at a concentration of 1 millimolar or below, the proenzyme was converted to its active form. The inactive form of phenoloxidase was found to be a soluble enzyme, whereas after activation the enzyme aggregated, and a significant amount of the enzyme activity could become pelleted.     

In the spiny lobster (Panulirus interruptus) the prophenoloxidase is located in plasma not in haemocytes

In the spiny lobster (Panulirus interruptus), unlike other crustaceans most of the prophenoloxidase (proPO) was detected in cell-free plasma (86.3%). In spite of its location, lobster proPO activating system has a similar activation mechanism to other crustacean proPO systems. Haemocyte lysate was able to activate the plasma proPO indicating location of the prophenoloxidase activating enzyme (PPAE) in haemocytes. Lobster haemocyte PPAE was isolated by affinity chromatography and its participation as activating enzyme was demonstrated. This enzyme is a serine-proteinase that transforms the inactive form (proPO) to an active one (phenoloxidase). The PPAE was also present in the cell-free supernatant of haemocytes previously incubated with Vibrio alginolyticus.     

Studies on the activation of the prophenoloxidase system of insects by bacterial cell wall components

The ability of bacterial cell walls to activate the prophenoloxidase cascade was tested using Blaberus craniifer, Clitumnus extradentatus, Locusta migratoria and Schistocerca gregaria. Effects of modifying components of the cell wall on the activation of prophenoloxidase in a haemocyte lysate supernatant preparation were examined. Peptidoglycan was found to be an important factor for the activating ability of Gram-positive bacteria. Lysozyme treatment of Micrococcus luteus cell wall showed that the soluble peptidoglycan was the active component. Teichoic acid isolated from Staphylococcus aureus did not activate the prophenoloxidase cascade. However, removal of teichoic acid from the cell wall enhanced activation, probably by exposure of peptidoglycan. Several Escherichia coli K-12 strains, with differing lipopolysaccharide compositions, were also tested for activation of prophenoloxidase. Differences in the ability of the various strains to activate the prophenoloxidase cascade were apparent although no clear conclusions could be made. The role of capsular polysaccharides was investigated too, using two Klebsiella pneumoniae strains, a noncapsulate mutant and its capsulate parent strain. The capsular polysaccharide conferred an increased activating potential. This difference in activation was lost by removal of the capsule from the parent strain. These results are interpreted in terms of the nonself recognition process in insect haemolymph.     

Purification of prophenoloxidase from crayfish blood cells,and its activation by an endogenous serine proteinase

A prophenoloxidase was purified from blood cells of the crayfish Pacifastacus leniusculus. The purified proenzyme was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and had a molecular mass of 76 kDa under both non-reducing and reducing conditions. The crayfish prophenoloxidase was a glycoprotein, with an isoelectric point of about 5.4.A 36 kDa serine proteinase, isolated and purified from crayfish blood cells (Aspán et al., 1990b, Insect Biochem.20, 709–718), could convert the 76 kDa prophenoloxidase to phenoloxidase by an apparent proteolytic cleavage, since the molecular masses of two active enzymes, phenoloxidases, were 60 and 62 kDa. A commercial serine proteinase, trypsin, activated prophenoloxidase to phenoloxidase, and as a result a 60 kDa protein was produced.In the blood cells of crayfish four serine proteinases or ³H-DFP binding proteins are present, with masses of 36, 38, 50 and 67 kDa. However, ³H-DFP labelling of proteins in blood cells lysate, prepared in its inactive form, only yielded labelled bands of 50 and 67 kDa, whereas addition of an elicitor to prophenoloxidase system activation, a β-1,3-glucan, resulted in the appearance of four ³H-DFP labelled proteins, with molecular masses of 67, 50, 38 and 36 kDa, respectively. Thus, the 36 kDa endogenous serine proteinase, the prophenoloxidase activating enzyme, ppA, may be present as an inactive precursor in crayfish blood cells. The 38 and 36 kDa proteinases could both cleave the chromogenic peptide S-2337 [Bz-Ile-Glu-(γ-O-Piperidyl)-Gly-Arg-p-nitroaniline], and specifically bind prophenoloxidase.These results show that crayfish prophenoloxidase, the terminal enzyme of the prophenoloxidase activating cascade, a proposed defence pathway in arthropod blood, can be converted to active enzyme by an apparent proteolytic cleavage, not only by a commercial proteinase, but also by an endogenous serine type proteinase.     

Comparative study of hemolymph phenoloxidase activity in Aedes aegypti and Anopheles quadrimaculatus and its role in encapsulation of Brugia malayi microfilariae

Hemolymph phenoloxidase activity of sugar-fed and blood-fed females of Anopheles quadrimaculatus and Aedes aegypti showed similar characteristics. Phenoloxidase was present as an inactive proenzyme in both mosquito species and was partially activated during collection of the hemolymph. In both mosquito species, phenoloxidase activity was modulated by different buffers and activated phenoloxidase did not need Ca²⁺. Enzymatic activity was higher in the hemocytes than in the plasma in both mosquito species. Trypsin, laminarin, and blood-feeding on uninfected and Brugia malayi-infected jirds enhanced hemolymph phenoloxidase activity in both mosquito species. The appearance of hemolymph phenoloxidase activity was inhibited by p-nitrophenyl p′-guanidinobenzoate HCl, soybean trypsin inhibitor, ethylenediaminetetraacetic acid, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea and reduced glutathione, but not by benzamidine in A. quadrimaculatus. The appearance of hemolymph phenoloxidase activity was inhibited by benzamidine, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea, reduced glutathione, β-nitrophenyl p′-guanidinobenzoate and soybean trypsin inhibitor, but not by ethylenediaminetetraacetic acid in A. aegypti. It is suggested that in both mosquito species, blood-feeding and migration of sheathed microfilariae in the homocoel activated the prophenoloxidase in the hemolymph and caused the encapsulation and melanization of microfilarial sheaths and microfilariae of B. malayi.     

The 76 kD cell-adhesion factor from crayfish haemocytes promotes encapsulation in vitro

Summary Semigranular cells from the crayfish, Pacifastacus leniusculus, were separated by Percoll gradient centrifugation and were used to study the encapsulation of foreign particles. The semigranular cells were found strongly to encapsulate glass beads coated with haemocyte lysate in which the prophenoloxidase-activating system had been activated with laminarin or with a low concentration of calcium ions. The granular cells only weakly encapsulated these particles. The encapsulationpromoting factor was purified from haemocyte lysates and found to be a 76 kD protein which was recognized by an antiserum to the previously described 76 kD cell-adhesion factor. After the last step in purification (Con A-Sepharose chromatography), the flowthrough consisted of several proteins, which had some, but less, encapsulation-promoting activity and contained a 30 kD band that was also recognized by the antiserum to the 76 kD cell-adhesion factor. If the haemocyte lysate prepared in low [Ca²⁺] was incubated with a -1,3-glucan prior to purification, no 76 kD protein could be isolated but only a 30 kD protein. The 30 kD protein thus seems to be a degradation product of the 76 kD cell-adhesion factor. We conclude that the 76 kD protein which is released from degranulating haemocytes, and to a lesser extent its 30 kD fragment, can promote encapsulation. Phenoloxidase did not have any encapsulation-promoting activity.     

Adipokinetic hormone enhances laminarin and bacterial lipopolysaccharide-induced activation of the prophenoloxidase cascade in the African migratory locust,Locusta migratoria

Lom-AKH-I enhances the activation in vivo of prophenoloxidase in the haemolymph of the African migratory locust, Locusta migratoria, in response to challenge with laminarin. AKH does not influence the speed or initial magnitude of the phenoloxidase response to laminarin, but prolongs the period of activation of the enzyme in a dose-dependent manner. Injections of preparations of bacterial lipopolysaccharide (LPS) do not activate prophenoloxidase in vivo, but co-injection of Lom-AKH-I with commercial preparations of LPS from Klebsiella pneumoniae, Escherichia coli, or Shigella flexneri (but not one from Pseudomonas aeroginosa) results in dose-dependent increases in the levels of phenoloxidase that persist in the haemolymph for several hours. It is argued that the effects of AKH on phenoloxidase activation in locusts described here are, at least in part, related directly to changes in lipid metabolism brought about by the hormone.     

绿僵菌侵染对椰心叶甲酚氧化酶活性的影响

对椰心叶甲Brontispa longissima(Gestro)成虫血淋巴中酚氧化酶的特性进行分析,并研究绿僵菌(Metarhizium anisopliae)侵染对血浆甲酚氧化酶活性的影响。结果显示,椰心叶甲成虫的血浆及血细胞裂解液中均检测到酚氧化酶活性,且昆布多糖及胰蛋白酶可显著提高其活性。绿僵菌MA-4侵染组在侵染后第1至第5d的血浆酚氧化酶活性高于未侵染组(P<0.05),但是椰心叶甲成虫体内注射10μg昆布多糖后,侵染组的酚氧化酶活性显著低于未侵染组(P<0.05),表明绿僵菌一方面对可激活椰心叶甲的酚氧化酶原激活系统,另一方面又可抑制昆布多糖对椰心叶甲酚氧化酶原激活系统的诱导作用。     

Phenoloxidase activity in the hemolymph of the spiny lobster Panulirus argus

The prophenoloxidase activating system plays a major role in the defense mechanism of arthropods. In the present study, the phenoloxidase activity and its location in the hemolymph of the spiny lobster Panulirus argus is presented. Phenoloxidase activity was observed in the hemocyte lysate supernatant (HLS) and plasma after their incubation with trypsin. Higher amounts of trypsin were required to activate the HLS prophenoloxidase, due to the presence of a trypsin inhibitor in this fraction. Activation of prophenoloxidase was found when HLS was incubated with calcium, with an optimal pH between 7.5 and 8. This spontaneous activity is due to the prophenoloxidase activating enzyme, a serine proteinase that activates the prophenoloxidase once calcium ions were available. SDS was able to induce phenoloxidase activity in plasma and hemocyte fractions. Prophenoloxidase from HLS occurs as an aggregate of 300kDa. Electrophoretic studies combining SDS-PAGE and native PAGE indicate that different proteins produced the phenoloxidase activity found in HLS and plasma. Thus, as in most crustaceans, Panulirus argus contains a prophenoloxidase activating system in its hemocyte, comprising at least the prophenoloxidase activating enzyme and the prophenoloxidase. Finally, it is suggested that phenoloxidase activity found in plasma is produced by hemocyanin.     

A cell adhesion factor from crayfish haemocytes has degranulating activity towards crayfish granular cells

A factor able to mediate cell adhesion of semigranular and granular haemocytes of the crayfish Pacifastacus leniusculus was recently purified from crayfish haemocyte lysate (Johansson and Söderhäll, J. Cell Biol.106, 1795–1803, 1988). It is a protein with a mass of 76 kDa, and its activity seems to be generated concomitantly with the activation of the prophenoloxidase (proPO) activating system. In this paper, we present evidence that this same protein is also responsible for the previously reported degranulating activity of a crayfish haemocyte lysate, in which the proPO system has been activated. First, the 76 kDa band in SDS-polyacrylamide electrophoresis seems to be a single protein, since in isoelectric focusing the purified cell adhesion factor fraction migrated as one band with an isoelectric point of 7.2. Second, this fraction was also able to degranulate crayfish granular cells in vitro, and third, antibodies to this 76 kDa protein, which are known to block cell adhesion, could also inhibit degranulation in vitro.     

Induction of degranulation and lysis of haemocytes in the freshwater crayfish,Astacus astacus by components of the prophenoloxidase activating system in vitro

To study the role of the prophenoloxidase activating system, an enzyme cascade located in the haemocytes of crustaceans, in the cellular defences of the freshwater crayfish, Astacus astacus in vitro, monolayer cultures of mixed or separated haemocyte populations, isolated by density gradient centrifugation, were challenged with the bacterium, Moraxella sp. pre-coated with phenoloxidase and the other attaching proteins in crayfish haemocyte lysate (HLS), or in the case of controls, with saline or Moraxella sp. pre-incubated in saline alone. Examination of the coverslips 1 h after inoculation revealed that, in the mixed haemocyte cultures, most of the cells had undergone profound degranulation and lysis following exposure to the HLS-coated bacteria. Cell lysis was also evident in the experimental semigranular cell monolayers, but not in the controls, although in those controls treated with the saline-incubated bacteria, the semigranular haemocytes had undergone degranulation without lysis. In contrast, the granular cells appeared to be unaffected by the saline-incubated Moraxella sp., and with the HLS-coated bacteria displayed only marked degranulation. Greater numbers of bacteria were always associated with the cells or cell remnants in the experimental cultures compared to the controls. We suggest that the attaching proteins of the prophenoloxidase cascade are strong nonself signals for the haemocytes, causing them to degranulate and release previously cell-bound recognition factors into the haemolymph, where they are free to trigger activation of adjacent haemocytes.     

The Trypsin Inhibitor Panulirin Regulates the Prophenoloxidase-activating System in the Spiny Lobster Panulirus argus

The melanization reaction promoted by the prophenoloxidase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms that are still not fully understood. We report here the finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined by combining Edman degradation/N-terminal sequencing and electrospray ionization-MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin (K_i = 8.6 nm) with a notable specificity because it does not inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin, and plasmin. The removal of panulirin from the lobster hemocyte lysate leads to an increase in phenoloxidase response to LPS. Likewise, the addition of increasing concentrations of panulirin to a lobster hemocyte lysate, previously depleted of trypsin-inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme.     

Signal transduction of the prophenoloxidase activating system of prawn haemocytes triggered by CpG oligodeoxynucleotides

Intracellular phenoloxidase (PO) activity in haemocyte lysate supernatant (HLS) of giant freshwater prawn (Macrobrachium rosenbergii) was shown to be enhanced by CpG oligodeoxynucleotide (ODN) 2006, but not by so-ODN13. When haemocytes were treated in vitro with 50 microg/ml of ODN2006 for 30 min, the increases in both intra- and extracellular stimulated PO activity (POS) and extracellular total PO activity (POT) and the reduction of POT suggest that the PO activity of haemocytes is enhanced by ODN2006 stimulation, but new prophenoloxidase (proPO) is not synthesised. In an attempt to determine which signal transduction pathway is involved in the activation of the proPO system, haemocytes were separately treated with activators or inhibitors of specific signalling components. The results show that there was an increase in both intra- and extracellular POT of haemocytes treated with sodium fluoride (a G-protein activator); the addition of phosphokinase A (PKA)-activating 8-bromo-cAMP to haemocytes only increased intracellular POT, and the addition of either phorbol-12-myristate-13-acetate (PMA; a phosphokinase C (PKC) activator) or caffeine (a phosphodiesterase inhibitor) only increased extracellular POT. When PMA-stimulated haemocytes were treated with chelerythrine (a PKC inhibitor), the induced extracellular POT was significantly reduced. Furthermore, the study of ODN2006-stimulated haemocytes treated with chelerythrine or palmitoyl-DL-carnitine (a PKC inhibitor) showed that the enhancement effects of ODN2006 on the intra- and extracellular POS and extracellular POT were significantly decreased. ODN-stimulated haemocytes treated with genistein (an inhibitor of protein tyrosine kinase) showed a further increase in extracellular POT, but the other PO activities remained the same as those of the ODN-stimulated group. These results suggest that the activation of the proPO system of prawn haemocytes, including degranulation and PO activity, is induced by ODN2006 via a PKC-activating signalling pathway, but negatively regulated via the tyrosine kinase pathway.     

The immune response of the desert locust Schistocerca gregaria during mycosis of the entomopathogenic fungus,Metarhizium anisopliae var acridum

Topical application of Metarhizium anisopliae var acridum to the desert locust Schistocerca gregaria resulted in changes in the biochemistry and antimicrobial defenses of the haemolymph. M. anisopliae var acridum colonized the host haemolymph from day two post application. The haemocytes did not attach to, phagocytose or nodulate elements of the fungus. However, the presence of the fungus appeared to stimulate hemocyte aggregation over the first few days of mycosis though the number of aggregates declined subsequently. The total hemocyte count increased two days after application, indicating an overall stimulation of the immune system, but declined to a value below that for uninoculated controls by day four. The differential haemocyte count showed that the initial increase in total haemocyte count was primarily due to a larger number of coagulocytes. After day two consistent declines in cell number were observed for all haemocyte classes in mycosed insects. The activity of the enzyme, phenoloxidase, decreased during the course of infection. However, the converse was true for prophenoloxidase. Lysozyme levels were significantly smaller in infected than control locusts. There was a significant correlation between lysozyme and PO activities when data from mycosed and control insects were combined. The total protein content of the haemolymph decreased during the course of infection.     

Cresolase, catecholase and laccase activities in haemocytes of the red swamp crayfish

Phenoloxidase activity in crayfish haemocyte lysates and extracts of haemocyte membranes were studied using native PAGE and SDS-PAGE gels and staining for cresolase, catecholase and laccase activities. The activation of the proenzyme, prophenoloxidase to phenoloxidase, in native PAGE was demonstrated following exposure to SDS. By staining samples separated in SDS-PAGE followed by renaturation, a high molecular mass phenoloxidase activity was identified in both the soluble and membrane fractions of haemocyte preparations. The membrane-associated activity appeared at only relatively high molecular mass (> 300 kDa), and could easily be eluted from membranes using detergents or NaCl. Further, this membrane-associated activity has a catecholase activity but not the cresolase activity seen in the soluble preparations. In addition, several other phenoloxidase enzymes were identified with different relative mobilities (250, 80, 72 and 10 kDa). Crayfish haemocytes also contained laccase activity, thought to be restricted to cuticle sclerotisation in the integument. Laccase activity in haemocytes might aid in the formation of capsule used to contain pathogens.