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On-line version ISSN 1678-8052
Neotrop. Entomol. vol.31 no.3 Londrina July/Sept. 2002
Activation of Prophenoloxidase and Removal of Bacillus subtilis from the Hemolymph of Acheta domesticus (L.) (Orthoptera: Gryllidae)
CLEONOR C.A. DA SILVA
Embrapa Recursos Genéticos e Biotecnologia, C. postal 02372, 70770-900, Brasilia, DF
Ativação da Profenoloxidase e Remoção de Bacillus subtilis da Hemolinfa de Acheta domesticus (L.) (Orthoptera: Gryllidae)
RESUMO - Fenoloxidase é considerada um importante mediador no sistema de defesa dos insetos contra patógenos e parasitóides. Essa enzima está presente na hemolinfa da maioria dos insetos estudados como uma proenzima inativa chamada profenoloxidase. Em Acheta domesticus (L.) a atividade da enzima foi estudada na presença de Bacillus subtilis. Injeções de bactéria na hemocele de ninfas desse inseto elevaram o nível de atividade da phenoloxidase. A taxa de aumento foi semelhante à observada nos insetos injetados com laminarin, um ativador da proenzima. A incubação de células de B. subtilis em soro com fenoloxidase ativada in vitro, e posterior injeção nos insetos, acelerou a remoção das bactérias da hemolinfa. A concentração das bactérias foi menor do que a observada nas ninfas injetadas apenas com bactéria em solução tampão. Por outro lado, ninfas injetadas com suspensão de bactéria incubada em soro com fenoloxidase inativada in vitro apresentaram alta concentração de bactéria na hemolinfa. Esses resultados indicam que B. subtilis é sensível à cascata de profenoloxidase de A. domesticus. Sugere-se que a fenoloxidase ativada adere à superfície das bactérias e aumenta a fixação dos hemócitos em torno das bactérias. Isso contribui para acelerar sua remoção da hemolinfa através da formação de nódulos.
PALAVRAS-CHAVE: Hemolinfa, fenoloxidase, hemócito, reação de defesa.
ABSTRACT - Insect phenoloxidase is considered an important mediator in defense reaction against pathogens and parasites. It is present in the hemolymph of most insects as an inactive proenzyme, called prophenoloxidase. In Acheta domesticus (L.) the phenoloxidase activity was studied in the presence of Bacillus subtilis. The bacteria induced the activation of the prophenoloxidase and increased the level of phenoloxidase activity when injected into the hemocoel of A. domesticus nymphs. The level of increase was comparable to those observed in nymphs injected with laminarin, an activator of the proenzyme. Incubating B. subtilis in serum with phenoloxidase activated in vitro and then injecting into the insects accelerated the removal of the bacteria from the hemolymph in vivo. The concentration of bacteria was lower than those observed in nymphs injected with Mes buffer alone. In control insects, injection of soybean trypsin inhibitor lowered phenoloxidase activity and the removal of bacteria from the hemolymph. These results indicate that B. subtilis is sensitive to the prophenoloxidase cascade of A. domesticus. It is suggested that activated phenoloxidase could bind to the surface of bacteria and increase the adhesion of hemocytes to bacteria, and thus accelerating their removal by nodule formation.
KEY WORS: Hemolynph, phenoloxidase, hemocyte, defense reaction.
It has become evident that insects possess an effective defense mechanism designed to resist the various microorganisms that use them as hosts. Certain events associated with these antimicrobial defenses are generally common to many insect species when exposed to different microbial attack and they include: recognition of nonself, changes in circulating hemocytes, antigen adhesion to hemocytes, enzyme activities and synthesis of antibacterial peptides in fat body (a functional equivalent of the vertebrate liver) (Brookman et al. 1989b, Gillespie et al. 1997). The change in the total and differential hemocyte counts, in parasitized or infected insects, is one of the first indication of a defense reaction (Wang et al. 1994, Gillespie et al. 2000, Silva et al. 2000b). Plasmatocytes and granular cells are the two types of hemocytes most often observed in hemocyte-mediated defenses such as phagocytosis, encapsulation and nodule formation (Gillespie et al 2000, Togo et al. 2000). Nodule formation consists of microaggregates of hemocyte entrapping large numbers of bacteria within a mucopolysaccharide matrix. The process ends with the melanization, which leaves darkened nodules attached to various organs of the insect. In orthopteran insects the immediate response to entomopathogenic fungus is nodule formation (Bidochka & Kachatourians 1987, Brookman et al. 1989a, Gillespie et al. 2000). In A. domesticus nodule formation around Xenorhabdus nematophilus, an intestinal symbiotic bacterium of the entomopathogenic nematode Steinernema carpocapsae, starts with a decrease in the total hemocyte counts during the first hour of exposure to the bacteria; the amount of bacteria in the hemolymph also declines during this time. Later the bacterium suppresses phenoloxidase activity (Silva et al. 2000a,b). Phenoloxidase participates in three important physiological processes: cuticular sclerotization, defense reaction and wound healing (Ratcliffe 1993, Sugumaran et al. 2000). The activation of prophenoloxidase through serine proteases produces phenoloxidase, the enzyme responsible for the synthesis of melanin (Sugumaran & Saul 1987, Brookman, et al. 1989b).
It has been demonstrated that insect parasitism and infection suppress phenoloxidase activity in vivo and in vitro (Richards & Edwards 2000, Silva et al. 2000b).
Pathogenic and non-pathogenic bacteria interact with the insect antimicrobial defenses in different ways. Studies in vitro and in vivo with the pathogenic X. nematophilus demonstrated that bacteria overcome the antimicrobial defenses of A. domesticus, by initially reducing the number of hemocytes, mainly granular cells, and later reducing phenoloxidase activity (Silva et al. 2000 a,b).
The interaction of Bacillus subtilis, a non-pathogenic Gram-positive bacterium, with the antibacterial system of insects is known for a few numbers of species. In Locusta migratoria (L.)and Galleria mellonella (L.) live and dead B. subtilis induced serum melanization and phenoloxidase activation in vitro, respectively (Brehélin et al. 1989, Dunphy & Bouchier 1992) and in locust Euprepocnemis shirakii (Bolivar),induced phagocytosis by both plasmatocytes and granular cells (Chang et al. 1992).
The objective of this study was the activation of the phenoloxidase of A. domesticus in vivo with B. subtilis and its mediation in removing the bacteria from the hemolymph. Fifth instar females and males of A. domesticus, weighing 200 ±10 mg, were used. The insects were reared on rabbit pellet chow at 30ºC and 55 ± 5% RH with a 14:10h light:dark photoperiod (McFarlane 1985). Gram-positive B. subtilis (ATCC no 6051) was maintained on tryptic soy agar. For experimental purposes B. subtilis was grown in 10 ml of tryptic soy broth in 50-ml Erlenmeyer flasks at 27ºC on a horizontal, gyratory shaker (150 rpm) for 12h. Upon achieving early stationary phase, (OD669 = 2.0), the bacteria were washed three times by centrifugation (12,000 g, 3 min., 27ºC) in N-morpholino-2-ethansulforic acid (Mes buffer, pH 6.5). Bacterial density was adjusted to 1.6 x 107 cells/ml for the experiments.
For phenoloxidase activation, twenty chilled (-20oC, 10 min.) nymphs were injected dorsolaterally between the third and fourth abdominal segments with 10 µl of bacterial suspension in Mes buffer. A positive control group was injected with 10µl of the phenoloxidase activator laminarin (1 mg/ml in Mes buffer), and the negative controls were injected with soybean trypsin inhibitor (500 mg/ml) and Mes buffer alone. At 5, 10, 20 and 30 min. post-injection the insects were bled by cutting ventral abdominal entersegmental membrane, the hemolymph was collected with micropipettes and centrifuged (12,000 g, 27ºC, 10 min.) to remove cell debris. Phenoloxidase activity was measured spectrophotometrically (OD490) 30 min. after the addition of 2 mg/ml of L-dihydroxyphenylalanine (L-DOPA; Sigma Chemical Co., St Louis, MO) in 10 ml of serum as the formation of dopachrome (Silva et al. 2000a). The use of serum more closely approximate the in vivo bacterial infection during which the hemocytes discharge cytoplasmic contents during the formation of nodules and eventually lyse releasing enzymes of the prophenoloxidase system into the plasma (Dunphy and Bourchier 1992). One enzyme unit (U) was defined as a change of 0.001 OD490 units/min. Phenoloxidase activity was expressed as units per mg total protein.
Phenoloxidase of A. domesticus bind to the surface of B. subtillis and increase the binding of hemocytes to the bacteria to in vitro (Silva et al. 2000a). To determine if components of the prophenoloxidase system accelerate the removal of the bacteria from the hemocoel, B. subtilis was incubated for 30 min. in: (a) serum with phenoloxidase activated by laminarin (1 mg/ml in Mes buffer); (b) serum with phenoloxidase inhibited with 500 mg of soybean trypsin inhibitor (Sigma) and (c) in Mes buffer alone. After the incubation time the bacteria were centrifuged and washed (12,000 g, 27ºC, 10 min.) three times in Mes buffer and 10 ml of each treatment was injected into the insects. At 5, 10, 20 and 30 min. post-injection the insects were bled, and the number of unattached bacteria was counted on a hemocytometer using phase contrast microscopy. Total serum protein was assayed by the Bradford method (1976) using bovine albumin as the standard.
Data are expressed as the mean ± standard error of the mean (n ³ 10 samples) and the percentage data are expressed as the decode mean of the arcsine ÖP transformed data (with 95% of confidence limits of the transformed data). The rate of bacterial removal was analyzed using one-way analyses of variance. Correlation coefficients were calculated for the relationship between phenoloxidase and activity bacterial removal.
B. subtilis activates the prophenolxidase of A. domesticus. Injection of B. subtilis into the hemocoel stimulated the activation of the proenzyme and increased the level of the phenoloxidase activity. By30 min. post-injection the enzyme activity reached 0.83 units and was higher than the concentration in control insects injected with Mes buffer (Fig. 1). The increase of phenoloxidase activity may be an indication of an immune response toward the bacteria. There was no larval death during the period of study. These results agree with Clarke and Dowds (1995) who found that injection of viable cells of B. subtilis into G. mellonela did not kill the larvae. However, injections of outer membrane fractions of the bacteria shown to be toxic and killed the larvae. Dead insects turned black into a few hours post-injection, probably due to melanization produced by phenoloxidase.
Injections of laminarin, a soluble b-1,3 glucan, into A. domesticus activated phenoloxidase throughout the 30-min. study when compared with the control. Phenoloxidase activity increased concomitant with a decrease in total hemocyte counts (5 min. post-injection, 1.5 ± 0.3 x 106 hemocytes/ml; 30 min. postinjection, 0.4 ± 0.2 x 106 hemocytes/ml). This decrease could be a result of hemocyte aggregation (nodule formation) induced by the presense of the bacteria. In a previous study using monolayers of A. domesticus hemocytes was found that B. subtilis adhere to more granular cells than plasmatocytes and serine proteases of the prophenoloxidase cascade had opsonic properties for B. subtilis. Also, phenoloxidase was present in greater quantities in the hemocytes than in the serum(Silva et al. 2000a).
Laminarin was also found to be a strong activator of prophenoloxidase in vivo in other orthopteran insects such as L. migratoria, Schistocerca gregaria (Forskal) and Poecilocerus pictus (F.) (Leonard et al. 1985, Bidochka et al. 1989, Rowley et al. 1990, Nellaiappan 1992) but it is failed to activate the proenzime of S. gregaria in vitro (Gillespie et al. 2000).
Phenoloxidase activity was greatly reduced by soybean trypsin inhibitor. This may suggest the presence of serine proteases in the prophenoloxidase cascade as reported for other insects (Brehelin et al. 1991, Boigegrain et al. 1992).
Incubating B. subtilis in vitro with phenoloxidase activated serum, and then injecting into the insects, accelerated its removal (6.4 x 0.7 x 106 bacteria/ml/min.) from the hemolymph when compared with control group injected with Mes buffer (8.3 x 0.6 x 106 bacteria/ml/min.) (Fig.2). The enhanced clearance of the bacteria may represents the binding of components of the prophenoloxidase cascade to the bacteria (Silva et al. 2000a) and thus removing bacteria by nodulation or bacterial surface modification by serine proteases (Dunphy & Webster 1991).
Inhibiting the serine proteases in phenoloxidase activated serum with soybean trypsin inhibitor lowered the bacterial removal from the hemocoel (10.1 x 0.6 x 106).
B. subtilis was sensitive to phenoloxidase of A. domesticus. It is known that in this insect granular cells/hemolymph are the source of prophenoloxidase prior to the formation of phenoloxidase active enzyme (Silva et al. 2000a). The insect eliminated most of the bacteria from the circulation. During the 30 min. of the study the phenoloxidase titer was correlated (r = 0.85 P<0.05) with the removal of bacteria from the hemolymph. As the level of PO activity increased the number of bacteria removed increased (Fig. 3).
Activation of the prophenoloxidase cascade is one of the early events of an immune response to bacteria. In A. domesticus the strategy of B. subtilis, whichactivates this proenzyme, differs for the pathogenic bacterium X. nematophilus that has lipopolysaccharide in the outer membrane, which damages hemocytes and reduce phenoloxidase activity and later kill the insect. The understanding of insect immune response to bacterial infection has potential to improve efforts at microbiological control of pest insect
The author are thankful to EMBRAPA for the financial suport and to Garry B. Dunphy for his valuable comments on this paper.
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Received 21/08/01. Accepted10/07/02.