Reduced cellular immune competence of a temperature-sensitive dopa decarboxylase mutant strain of Drosophila melanogaster against the parasite Leptopilina boulardi

Comp, Biochem. Physiol. Vol. 101B,No. 3, pp. 453--460,1992 Printed in GreatBritain

03054)491/92 $5.00+ 0.00 © 1992PergamonPressple

R E D U C E D C E L L U L A R I M M U N E C O M P E T E N C E OF A TEMPERATURE-SENSITIVE DOPA DECARBOXYLASE M U T A N T STRAIN OF D R O S O P H I L A M E L A N O G A S T E R A G A I N S T THE PARASITE L E P T O P I L I N A B O U L A R D I A, J. NAPPI,* Y. CARTON,'~J. LI* and E. VASS* *Department of Biology, Loyola University of Chicago, Chicago, IL 60626, USA (Tel: 312 508-3620); and ?Laboratoire de Biologie et Genetique Evolutives, CNRS, Gif-sur-Yvette, France (Received 28 June 1991) Abstraet--l. The melanotic encapsulation response made by larvae of a temperature-sensitive dopa decarboxylase (DDC) mutant strain of Drosophila against the parasitic wasp Leptopilina was severely compromised in hosts with reduced levels of DDC. 2. Dopa and 5,6-dihydroxyindole (DHI) were two hemolymph components identified in hosts exhibiting a melanotic encapsulation response. 3. This is the first study to implicate DDC in insect cellular immune responses, and to provide chemical evidence that the pigment formed during such responses is eumelanin derived from tyrosine.

INTRODUCTION When challenged by various pathogens and parasites, insects and other arthropods activate formidable humoral (Dunn, 1986, 1990; Rowley et al., 1986; Boman and Hultmark, 1987) and cellular defenses (Rizki and Rizki, 1984; Gotz and Boman, 1985; Ratcliffe et al., 1985; Gotz, 1986; Nappi and Carton, 1986; Nappi and Christensen, 1987; Christensen and Nappi, 1988; Christensen and Tracy, 1989; Sugumaran, 1990; Karp, 1990). Nonself components that are too large to be phagocytosed by individual cells are sequestered in multicellular capsules that harden and become pigmented. The pigment is believed to be eumelanin, a heteropolymer comprising various ohydroquinones and o-quinones derived from tyrosine or dopa (3,4-dihydroxyphenylalanine), but chemical evidence to support this idea is lacking. Implicated in the development of these pigmented capsules are certain catecholamine-metabolizing enzymes that function in various melanization and sclerotization reactions associated with the formation and repair of cuticle (Lai-Fook, 1966); because the phenol oxidase system is involved in the initial stages of the biosynthesis of melanin, considerable interest has focused on this enzyme system as a major component of the cellular immune responses of insects and other arthropods (see Sugumaran, 1990). This enzyme system hydroxylates the monophenol substrate tyrosine to dopa (monophenol oxidase activity), and oxidizes dopa and certain other diphenols (i.e. dopamine, N-acetyldopamine and N-fl-alanyldopamine) to their respective quinones (diphenol oxidase activity) which then are metabolized to melanin and/or sclerotin (Brown and Nestler, 1985; Andersen, 1985; Sugumaran, 1988). Various insect phenol oxidases have been reported to occur as proenzymes (prophenol oxidase) in the cellular and/or non-cellular compartments of the blood where they are activated in a series of steps by several endogenous proteins (Seybold

et al, 1975; Ashida et al., 1983, 1990; Pye, 1974; Rowley et al., 1986; Saul et al., 1987; Sugumaran, 1990). The participation of activated phenol oxidases in insect immune responses has been established

for some host-parasite associations (Nappi et al., 1987, 1991; Li et al., 1989, 1991), but the precise role the enzyme system plays in defense has not been ascertained. Surprisingly, no investigation has been made of the involvement of other catecholaminemetabolizing enzymes in insect immunity. Considering the key role played by dopa decarboxylase (DDC) (EC4.1.1.26; 3,4-dihydroxy-L-phenylalanine carboxylase) in converting dopa to dopamine, we were interested in knowing what effect this enzyme has on the immune capacity of an insect. This pathway in the biosynthesis of catecholamines is an important consideration not only because dopamine serves as a precursor for the N-acetyl and N-/~-alanyl catecholamines involved in the formation of dark sclerotin, but also because dopamine can be oxidized to form indole quinones which form melanin (Fig. 1) (Hori et al., 1984; Kramer et al., 1983, 1984; Hiruma et ai., 1985; Roseland et al., 1987). As an initial approach to studying the possible involvement of DDC in insect immunity we chose to evaluate the hemocytic encapsulation response of a DDCdeficient mutant of D. melanogaster when infected by the wasp parasitoid L. boulardi. A similar approach was taken recently by Rizki and Rizki (1990) in a detailed study using two phenol oxidase mutant strains of D. melanogaster. They showed that in the absence of phenol oxidase, some mutant hosts were able to encapsulate eggs of Leptopilina, but unable to produce pigmented capsules. We report here our investigations of a temperature-sensitive dopa decarboxylase mutant of D. raelanogaster (Ddc t'2) which was found to have a severely compromised immune capacity when reared at the restrictive temperature of 29°C.

453

454

A. J. NAPPIet al.

co2

MONOPHI~OL OXIDASE

COOH [~-CH2-CH-NH2

COOH HOHO""~,,~

.CH2.CH.NH 2

-

~

"

DECARBOXYLAS~

DOPA

TYROSZNE

0 II C-CH3 "O'[~'CHz-CHz-NH

_CH2.CH2.NH2 N-ACETYLTRANS]¢ERASE

DOPANZNE

N-AcEI"YLDOPN4XNE

,

o C-~ 3 ' -Oiz-CHz-NIt.

!] INDOI.E-S, 6-owr NONI[

MELANIN

N-ACETYLDOPAMZNE GUZNONE

SCLEROTZN

Fig. 1. Generalized diagram showing two major metabolic pathways of catabolism of dopa and dopamine that lead to the formation of melanin (i.e. eumelanin) and sclerotin.

MATERIALS AND METHODS The D. melanogaster used in this study were raised on a standard cornmeal and yeast medium. A Brazzaville strain of D. melanogaster was used as a host to rear and to maintain at 25°C a sympatric strain of the wasp parasite L. boulardi. Adult wasps were fed a 50% honey solution and kept at 20°C. Another strain of D. melanogaster not temperature-sensitive but highly immune-reactive (R = No. 445) against L. boulardi (Carton and Bouletreau, 1985; Carton and Nappi, 1991; Nappi et al., 1991) was used in parasite encapsulation studies and in comparative enzyme assays as controls with the temperature-sensitive dopa decarboxylase mutant, Ddc #:.

wound was collected with a microcapillary pipette. After extracting hemolymph each host larva was dissected and examined to verify the presence of the parasite, and to register the extent of the host immune response. For analysis of hemolymph catecholamines blood samples were placed into 50 pl 0.8 M citric acid stop buffer (pH 2.4) and frozen at -20°C. After each hemolymph sample was separately examined and a catecholamine profile obtained by highpressure liquid chromatography with electrochemical detection (HPLC-ED), blood samples from immune reactive larvae were then pooled to obtain sufficient volumes for multiple analyses that were required to identify endogenous eiectroactive components. Pooled hemolymph samples were also obtained from non-infected controls and from susceptible hosts that exhibited no immune response.

Infection o f Ddc #2 larvae

Chemicals

Parasitization of the temperature-sensitive dopa decarboxylase mutant, Ddc #2, was initiated by exposing fly larvae to several female wasps during a 4 hr period. Ddc #2 larvae reared at the permissive temperature of 20°C were parasitized at a host age of 72 + 3 hr, while Ddc *~2larvae reared at the restrictive temperature of 29°C were parasitized at 50 + 3 hr. Under these two different temperature conditions the Ddc #2 larvae were at a comparable stage in development when parasitized. However, at the restrictive temperature of 29°C Ddc #: mutants are reported to contain less than 3% of the normal dopa decarboxylase activity found in wild-type flies (Wright et aL, 1981; Tempel et al., 1984). Control experiments were conducted with wild-type larvae maintained at the same temperatures as mutant strains. In order to provide ample time for the complete development of an immune response, hosts were not dissected until at least 48 hr post-infection. At 48 hr post-infection melanotic encapsulation responses, if produced, were clearly manifested.

5,6-Dihydroxyindole (DHI) was obtained from Regis Chemical (Morton Grove, IL). All other reagents used were from Sigma Chemical Co. (St Louis, MO).

Insects

Hemolymph catecholamines

The term hemolymph used in this study refers to both the cellular (hemocytes) and non-cellular (plasma) components of the blood. Hemolymph samples were taken from nonparasitized and parasitized larvae at specified times postinfection when melanotic encapsulations were forming in the hosts. Larvae were first removed from the culture medium, washed in MOPS buffer, pH 6.5, and then dried on filter paper. Hemolymph was extracted from individual larvae by making a small incision in the cuticle just in front of the posterior spiracles. Hemolymph issuing from the

Enzyme assay

To determine by HPLC-ED the levels of dopa decarboxylase activity in Ddc t*2 larvae reared at restrictive and permissive temperatures, 12 third stage larvae maintained at these conditions were homogenized in 400 pl ice-cold MOPS buffer (pH 6.5), and the supernatant was used as the enzyme preparation. The reaction mixtures comprised 40 p l enzyme preparation, and 70nmol L-dopa, 0.56nmol pyridoxai phosphate, and 28 nmol PTU in a final volume of 140 pl. The components of the reaction mixture were prepared in 50 mM MOPS buffer (pH 6.5). Incubations were performed at 30°C. Enzyme reactions were terminated by removing 40 #1 aliquots of the reaction mixtures at 0, 15 and 30 min incubations and placing each aliquot into an equal volume of 0.8 M citric acid stop buffer (pH 2.4). Enzyme preparations were analyzed separately to establish base-line, endogenous levels of the substrate (dopa) and product (dopamine). Reaction velocities were calculated by measuring changes in the peak dimensions (height and/or area) of the product formed, and calculating concentrations based on data from standard curves. The latter were corrected for per cent recovery of external standards which were run prior to and following each test. Specific enzyme activity was expressed as rate of product formed per milligram protein. Protein concentrations were determined using the Bradford (1976) microassay with bovine serum albumin used as the standard.

Temperature-sensitive dopa decarboxylase mutant strain of Drosophila H P L C - E D analysis

Hemolymph samples for catecholamine determinations as well as the stopped reaction mixtures were analyzed by HPLC-ED. The HPLC-ED system consisted of a Biuanalytical Systems (West Lafayette, IN) LC-4B electrochemical detector equipped with a glassy carbon electrode maintained at a potential of +0.75mV vs an Ag/AgCI reference electrode and a sensitivity range of 2-20nA. Separations were achieved at 40°C by a BAS Phase-II, 3-#m ODS reverse-phase column (3.2 mm inner diameter × 10cm). A Gilson (Madison, WI) 712 HPLC System Controller was used to integrate peak dimensions. Two different chromatographic conditions were used to verify the identity of the electroactive components in the hemolymph that co-eluted with authentic standards. The first mobile system comprised 0.I M citrate buffer (pH 2.9) containing 5% acetonitrile, 0.5ram sodium octylsulfate, and 0.7mM Na2EDTA. The second mobile phase comprised 50raM citric acid (pH 3.2), 2.5% acetonitrile, 1 ram sodium octylsulfate and 0.7 mM Na2EDTA. The flow rate was maintained at 0.Smi/min. Attempts to identify unknown electroactive components in samples of hemolymph and in reaction mixtures were first made by comparing retention times of these components with those of authentic standards. Subsequently, the components were identified if they co-eluted with the authentic standards under two different chromatographic conditions, and if their peak heights were ampfified in proportion to the amount of standard added to the hemolymph sample. Standard curves were prepared by injecting increasing amounts of dopa and DHI. Peak dimensions (height and/or area) were measured and the data analyzed by linear regression. Statistical analysis

Results are presented as the means+SEM of the determinations specified. Differences between mean values were evaluated using the Student's t-test, and the difference between two means was considered significant when P <~0.05. RESULTS

At the permissive temperature of 20°C, larvae of the Ddc'~2 strain were highly immune-reactive against eggs of the parasitic wasp L. boulardi. The eggs of L. boulardi were found upon dissection of host larvae 48 hr after parasitization to be heavily melanized and encapsulated by hemocytes. The mean frequency of melanotic encapsulation in Ddc '~: hosts kept at the permissive temperature was approximately 80% (Table 1). However, Ddc '~2 hosts maintained at the restrictive temperature of 29°C showed a significant decrease in immune capacity, with the frequency of melanotic encapsulation averaging less than 9% (Table 1).

455

Of the 428 Ddc '~2 host larvae dissected and examined, none was found to contain a parasite egg enveloped solely by hemocytes. A pigmented capsule was a consistent feature of each cellular encapsulation response made by immune-reactive Ddc "2 hosts against eggs of Leptopilina. These observations were also made with the wild-type used as hosts. The restrictive temperature did not appear to adversely affect larvae of wild-type flies, at least in terms of their immune capabilities which were found to be unaltered. The encapsulation rate for R strain hosts was 73.5% (25/34) at the permissive temperature, and 69% (29/42) at the restrictive temperatures. The activity of dopa decarboxylase in non-parasitized third stage Ddc "2 larvae reared at the restrictive temperature of 29°C was assayed by measuring product formation with H P L C - E D (Fig. 2) Hemolymph samples were taken at a time that corresponded to the early development of melanotic capsules in Ddc t~2 hosts parasitized by Leptopilina. The enzyme assays were run at 5, 15, 30 and 45 min intervals and the rate of activity was found to be linear over this time range. The spec. act. of Ddc "2 dopa decarboxylase was calculated at 0.74nmol min-I rag protein -1 . The spec. act. of dopa decarboxylase in third stage larvae of the R strain of D. melanogaster that served as controls for these assays was 1.7 nmol r a i n -m mg protein -~ . Samples of hemolymph from Ddc "2 host larvae exhibiting melanotic encapsulations were analyzed by H P L C - E D and compared with samples of hemolymph from non-parasitized larvae to determine the catecholamines or catechol derivatives present during parasite encapsulation (Fig. 3). In immune-reactive Ddc 's2 larvae three electroactive components with retention times of 2.0, 3.02 and 3.57 rain were observed when solvent system 1 was used (Fig. 4). None of these components was found in hemolymph samples from non-parasitized control larvae. The first and the third components co-eluted with dopa and DHI, respectively. The identity of the remaining component could not be ascertained. When the same hemolymph samples were analyzed with solvent system 2, three components were observed, but with retention times of 2.55, 4.1 and 5.2 rain, respectively (Fig. 4). With the second solvent system the first and the second components co-eluted with dopa and DHI, respectively. The retention time for the unknown component was changed considerably (5.2rain) by the second solvent system so that it appeared after DHI (Fig. 4).

Table 1. Cellular immune responses of D. melanogaster larvae of the temperature-sensitive dopa decarboxylase mutant strain Ddc '~2 against the parasitoid L. boulardi at permissive and restrictive temperatures

Test No. l 2 3 4

Permissive temperature (20°C) % Melanized and Number encapsulated % Melanized hosts eggs larvae 62 75 52 27 216 Mean:

83.9 72.0 90.4 74.1

0 9.3 1.9 11.1

80.1

5.6

Restrictive temperature (29°C) % Melanized and Number encapsulated % Melanized hosts eggs larvae 71 64 50 27 212

0 9.4 18.0 I 1.1 8.49

32.4 21.9 4.0 40.7 24.8

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A. J. NApP]et al.

i '30 PMOL DOPAMINESTANDARD

0 MIN INCUBTION

I$ MIN INCUBATION

45 MIN INCUBATION

Fig. 2. Typical chromatograms obtained in assays of DDC activity from larvae reared at 29°C. Reaction mixtures were composed of 40/~I enzyme preparation, 70 nmol dopa, 0.56 nmol pyridoxal phosphate and 28 nmol PTU in a total volume of 140/~I. HPLC-ED conditions were +750 mV, 20nA full scale, and 5 #I of stopped reaction mixture. Rates of reaction were based on product (dopamine) formation calibrated against standard curves that were analyzed by linear regression. Using the same two solvent systems the peak heights of two endogenous components (dopa and DHI) increased in proportion to the amounts of authentic standards incorporated into the hemolymph samples (Fig. 4). These co-injection experiments complemented the co-elution data and confirmed the identifications of dopa and DHI in hemolymph from immune-reactive D r o s o p h i l a larvae. Five samples of hemolymph from immune-reactive D d c "2 larvae were combined and analyzed by HPLC-ED. Based on this single pooled sample, the amounts of dopa and DHI in the hemolymph of immune-reactive D d c t~2 larvae were calculated to

be 1.5 and 0.65pmol/#l hemolymph respectively. Neither dopa nor DHI was detected in the pooled samples of hemolymph from non-parasitized controls (not shown). DISCUSSION At the restrictive temperature of 29°C the immune capacity of the temperature-sensitive dopa decarboxylase mutant D d c 's2 was severely reduced. Under identical conditions the immune capabilities of wildtype larvae were unaltered. Since the restrictive temperature reduces the level of DDC activity in D d c '`z

HEMOLYMPHSAMPLES(I;~1) IMMUNEREACTIVE

1

HEMOLYMPHSAMPLES($ ~1) NONPARASrrIZED

Fig. 3. Representative samples of hemolymph from immune-reactive and non-parasitized control Ddc "2 larvae reared at 29°C and examined by HPLC-ED. Samples of hemolymph from parasitized larvae typically possessed varying amounts of three electroactive components not seen in hemolymph from control larvae. Two components co-eluted with the standards dopa and 5,6-dihydroxyindole (DHI). In 5/~l samples analyzed at +750 mV and 20 nA sensitivity a third component (arrow) was rarely evident in some samples, but it is more clearly resolved when larger volumes of sample were analyzed (see Fig. 4).

Temperature-sensitive dopa decarboxylase mutant strain of Drosophila

457

E .DOPA

-DOPA 4-

-ah SOLVENTSYSTEM#1

SOLVENTSYSTEM2

Fig. 4. Typical chromatograms produced when standards were co-injected with 20 #1 hemolymph samples from immune-reactive Ddct*2 larvae. Retention times for dopa and 5,6-dihydroxyindole (DHI) with solvent system 1 were 2.0 and 3.57 min, respectively, and with solvent system 2 these times were 2.55 and 4.1 min, respectively. The retention time for the unidentified component (arrow) was altered from 3.02 rain with solvent system 1 to 5.2 min with solvent system 2. Peak heights of DHI in each solvent system were increased in proportion to the amount of external standard that was added to the hemolymph sample. The above chromatograms show the proportional change when 2 nmol DHI was added. (Wright et al., 1981; Tempel et al., 1984), it is proposed that the decrease in immune competence that occurs in the mutant strain is at least partially associated with the genetic defect. This study is the first to implicate a catecholamine-metabolizing enzyme other than a phenol oxidase in the cellular immune response of an insect. The identification of elevated levels of dopa in the hemolymph of larvae exhibiting an immune response supports the recent observations of parasite-induced activation of insect phenol oxidases during melanotic encapsulation reactions (Nappiet al., 1987, 1991; Li et al., 1989, 1991). The identification of DHI in the hemolymph of immune-reactive larvae also establishes for the first time the involvement in insect immunity of a major precursor of eumelanin, and lends considerable support to the idea that the pigment comprising the hemocytic capsules is produced from the oxidation of o-hydroquinones and o-quinones derived from tyrosine. Pertinent questions raised by these observations include how does the enzyme function in immunereactive Ddc #2 larvae during melanotic encapsulations, and what regulatory processes are involved? It is difficult to explain why, when the dopa oxidation pathway is available for the synthesis of melanin (Fig. 1), Ddc'*2mutants lacking normal levels of DDC at the restrictive temperature are unable to form cellular melanotic capsules around eggs of Leptopilina. Apparently, DDC plays a significant role in the development of melanotic response against parasites. The synthesis of melanin is essentially an oxidation process that produces substances capable of destroying cells. It is well known that certain o-quinones derived from o-diphenols are cytotoxic,

inhibiting the activity of certain types of DNA polymerase (Graham et al., 1978a, b; Wick, 1980). The highly elevated levels of dopa we found in immunereactive Ddc #2 larvae most likely resulted from a marked increase in the activity of phenol oxidase. In the presence of activated tyrosinase, dopa itself is known to be cytotoxic and inhibits DNA polymerase in mammalian cells (Wick, 1989). Perhaps one of the functions of DDC is to modulate the levels of dopa and dopaquinone by diverting some of the dopa generated during parasite encapsulation to form dopamine. However, dopamine also can be oxidized to o-quinone and then converted to DHI and ultimately to melanin (Fig. 1). Previous studies have shown that in Drosophila, the oxidation of dopamine proceeds in vitro more rapidly than does the oxidation of dopa (Nappi and Seymour, 1991), but at present it is difficult to attribute any significance to this observation. It it possible that the o-quinones derived from the oxidations of dopa and dopamine have significantly different binding affinities for nucleophiles, and thus may function by different mechanisms to kill parasites. Although DDC from insects is believed to specifically decarboxylate dopa only (Sekeris and Fraguulis, 1989), it would be of interest to know if, under immune-reactive conditions, DDC decarboxylates other substances, including dopachrome and other intermediates in the synthesis of melanin (Fig. 5). It should be noted that dopamine is also an important precursor of components involved in cuticular sclerotization. Some of the major components involved in sclerotization include N-acetyl and N-p-alanyl dopamine and norepinephrine (Andsersen, 1985; Kramer and Hopkins, 1987;

A. J. NAPPIet al.

458

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LEUKODOPACHRONE L

DOPACHROME ~

DOPA C02

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HO-.~TCHx'CHz'I~8[z HO-",~,./ DOPAMZNE

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DOPAMZNEGUZNONE

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ZNDOLE-5,6-GUZNONE

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V MELANIN Fig. 5. Possible common reaction pathways involving the catabolism of dopa and dopamine to indole-5,6-quinone. The role of dopa decarboxylasein converting dopa to dopamine has been established, but the enzyme's participation in the other decarboxylation responses outlined here have not been confirmed. Wright, 1987; Sugumaran, 1988; Czapla et al., 1989). Acylation renders the amino nitrogen of these N-acyl quinone imines non-nucleophilic; thus they cannot cyclize to form melanin. Instead, nucleophilic groups of proteins and/or chitin are added to the N-acyl quinone imines which stabilize the interacting components. It is tempting to suggest that similar processes may be involved in the hardening of the hemocytic capsules that sequester parasites. Chemically suitable molecules for binding to non-self surfaces appear to be quinone methides and related molecules (Sugumaran, 1990). Our present knowledge of the mechanisms regulating the cellular and biochemical changes associated with insect immunity is less than adequate to do more than speculate on the nature of the interacting components, and on how the genetically encoded enzymes that are involved in catecholamine metabolism become activated in response to parasitic infection. Attempts to elucidate the components of the enzyme activation mechanism(s) and to show how activation is achieved during melanotic encapsulations of parasites have focused attention on the phenol oxidase system (Nappi et al., 1987; 1991; Li et al., 1989, 1991). Studies correlating enhanced immune-responsiveness with the concomitant activation in vitro of the prophenol oxidase system (Soderhall and Smith, 1983; Ratcliff et al., 1984) have led some investigators to ascribe to the proenzyme activating components an immune-recognition function (Soderhall and Smith, 1986a, b; Ashida and Soderhall, 1984; Ratcliffe et al., 1984; Leonard et al., 1985a, b; Johansson and Soderhall, 1989a). However, experimental evidence is required to clearly ascertain if activation of the phenol oxidase system is a direct result of an initial recognition response rather than its cause. This may be an arduous task, especially in Drosophila where at least 40 genes are said to be involved in catecholamine metabolism and cuticular sclerotization processes (Wright, 1987). Our present study inculpating DDC in the cellular immune response of Drosophila is promising in that it identifies an alternative path-

way and possibly new molecules that may be employed in immune-reactive insects. Equally intriguing for future studies are the possible cooperative interactions of catecholamine-metabolizing enzymes with the recently identified immune-like binding proteins (Soderhall et al., 1988; Johansson and Soderhali, 1989b; Reichhart et al., 1989; Duvic and Soderhall, 1990) and antibacterial immune peptides that are induced by bacterial infections (i.e. diptericins, cecropins, attacins, insect defensins, and the hemolins) (Reichhart et al., 1989; Duvic and Soderhall, 1990; Sun et al., 1990; Samakovlis et al., 1990; StanleySamuelson et al., 1991). Other enzymes involved in the synthesis of catecholamines that may be worth investigating in terms of their potential involvement in insect immune-reactions include dopamine fl-hydroxylase, which converts dopamine to norepinephrine, and N-methyltransferase which converts norepinephrine to epinephrine. Oxidative deamination by monoamine oxidase, O-methylation by catechol-O-methyltransferase and N-acetylation by N-acetyltransferase are but a few possible reaction pathways that degrade and inactivate catecholamines, and thus may constitute equally important mechanisms for regulating catecholamine metabolism in immune-reactive insects. SUMMARY Invertebrate immunity results from the integrative activities of several distinct cellular and humoral processes that cooperate to provide protection from invading pathogens and parasites. Drosophila larvae after parasitization by the parasitic wasp L. boulardi respond by forming melanized cellular capsules around the eggs of the parasite. Most authors recognize the role played by certain biogenic amines and their derivatives in the formation of melanotic capsules around parasites, but tittle is known of the enzyme activation mechanisms that are involved the process. In order to study the involvement of DDC in insect cellular immune-responses, we incorporated

Temperature-sensitive dopa decarboxylase mutant strain of Drosophila the use of a temperature-sensitive D D C m u t a n t strain of D. melanogaster (Ddct~Z). At the permissive temperature of 20°C larvae of Ddc t'2 were highly immune-reactive exhibiting melanotic encapsulation against the eggs of L. boulardi. However, at the restrictive temperature of 29°C when D D C activity is impaired, immune capacity was significantly reduced, decreasing the occurrence of melanotic capsules. Subsequent analysis of hemolymph samples by H P L C - E D from immune-reactive larvae identified dopa and 5,6-dihydroxyindole as two components involved during the melanotic encapsulation response, evidence that the pigment formed is eumelanin derived from tyrosine. Acknowledgements--We thank Drs T. Wright and E. Pentz, Department of Biological Sciences, University of Virginia for the Ddc ~'2 strain and for helpful discussions. Portions of this investigation were made possible by grant support from the National Institutes of Health (AO24199), the National Science Foundation and Centre National de la Recherche Scientifique Cooperative (INT-8815410), and the Franco-American Commission for Educational Exchange (Fullbright-Hays Grant to A.J.N.). We thank Ms Francoise Frey, Laboratoire de Biologie et Genetique Evolutives, C.N.R.S., (3if-sur-Yvette, France, for her technical assistance.

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