JOURNAL

OF INVERTEBRATE

PATHOLOGY

56, 261-279 (199@

High-Pressure Liquid Chromatographic Analysis of Hemolymph Plasma Catecholamines in Immune-Reactive Aedes aegypti DIMITRI

D. MUNKIRS,

BRUCE M. CHIUSTENSEN,~

AND JAMES

W. TRACY*

Department of Veterinary Science and *Departments of Comparative Biosciences and Pharmacology, University of Wisconsin, Madison, Wisconsin 53706 Received December 21, 1989; accepted February 9, 1990 Tyrosine and catecholamines have been implicated as substrates for the encapsulation reactions involved in the immune response of mosquitoes to microftiae (mff). Identification and quantitation of tyrosine and catecholamines present in Aedes aegypti hemolymph plasma were accomplished by ion-pair high-pressure liquid chromatography with electrochemical detection at either + 650 or + 850 mV vs Ag/AgCl. Tyrosine, dopamine, and N-+&nyldopamine were detected in the hemolymph plasma of naive A. aegypti. Although no differences in these compounds were observed in hemolymph plasma from A. aegypti inoculated with DirojZaria immitis ti, the chromatogram showed a single major peak (PI) (65 PM, expressed as dopamine equivalents) that was not present in naive hemolymph plasma. Saline-inoculated controls contained only 5% of the PI in immune reactive hemolymph plasma. A high concentration of PI (127 f 39 ~.LM)was also detected after treatment of hemolymph plasma with mild alkaline conditions (PH 9.01, indicating that it is normally present as an electrochemically inert form in naive mosquitoes. High concentrations of PI were also detected in the naive hemolymph plasma from three other mosquito species, but no PI was found in A. trivittatus under any conditions. PI did not cochromatograph with any of the catecholamines commonly thought to be involved in immune responses of dipterans against metazoan parasites, suggesting that it may be a unique substrate for these reactions. The biological relevance of PI was evidenced by its appearance in the hemolymph plasma of two strains of D. immitis-inoculated A. aegypti. o 1990Academic press, IIIC.

INTRODUCTION The ability of insects to recognize foreign and/or abiotic substances and their capacity to mount immune responses against them is well established (Lackie, 1981). The immune response against multicellular parasites, which are too large to be phagocytosed, is termed melanotic encapsulation and generally involves the aggregation of hemocytes to the surface of the parasite and the formation of a contiguous, multilayered, darkly pigmented capsule (Nappi, 1975). The formation of this melanotic capsule likely involves the production of stable protein-polyphenol complexes via crosslinking and polymerization reactions of catecholamines and quinones derived from the catabolism of tyrosine (Christensen and Tracy, 1989). Many of the enzymes involved in these reactions, phenol oxidases, L-aromatic amino acid decarboxylases, and i To whom correspondence

should be addressed.

ZV-acetyltransferases are present in the hemolymph of insects (Brunet, 1980), and phenol oxidase activity has been quantitated in plasma and hemocytes from immune reactive mosquitoes (Nappi et al., 1987; Li et al., 1989). It is well established that hemocytes actively participate in the melanotic encapsulation response against microfilariae (Christensen et al., 1989; Li et al., 1989), but the humoral factors (with the exception of phenol oxidase) that may be involved have yet to be identified. Because of the significance placed on the hypothesis that the encapsulation reaction employs the production of melanin and sclerotin compounds, biochemical analysis of the hemolymph to identify and localize the substrates, intermediates, and enzymes involved in their synthesis is required to delineate specific details of the immune capabilities of mosquitoes and other insects (Christensen and Tracy, 1989). 267 0022-2011190 $1.50 Copyri&t 8 1990 by Academic Press, Inc. All rights of reproduction in any form resewed.

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Tyrosine is the initial substrate in the biosynthesis of catecholamines and quinones produced during the formation of melanin/ sclerotin. Tyrosine is sequestered during the larval feeding stages in dipterans and is not known to be synthesized de novo. Large reservoirs of tyrosine and/or its derivatives are needed for normal cuticle production, and the formation of inert storage forms is one means of accumulating sufficient amounts for this purpose. Inert conjugates of tyrosine and/or its derivatives may provide a protective factor by preventing the premature oxidation of free metabolites by phenol oxidases present in the hemolymph. Also, the addition of phosphate or glucosidic esters to tyrosine increases it solubility, thus making it easier to amass large reservoirs. Storage forms of tyrosine have been found in numerous insects. Lu et al. (1982) reported that tyrosine+-0-glucoside was the predominant storage form in the larvae of all lepidopteran species examined and that it seemed to provide a supply of both tyrosine and glucose for use in cuticular sclerotization. Mitchell and Lunan (1964) and Lunan and Mitchell (1969) identified tyrosine-O-phosphate in Drosophila melanogaster. Subsequent studies of 10 other Drosophila species revealed that tyrosineO-phosphate was also the reservoir of this amino acid in these insects, although in D. busckii the tyrosine storage form was a B-0-glucoside (Chen et al., 1978). Other forms of inert conjugates of tyrosine and its derivatives have been identified. Some are B-alanyl-L-tyrosine in Sarcophaga bullata (Levenbook et al., 1969), dopamine3-O-sulfate in Periplaneta americana (Bodnaryk et al., 1974), 3-O-phosphate and sulfate esters of N-acetyldopamine in P. americana (Bodnaryk and Brunet, 1974), Ltyrosyl-0-acetyldopamine in Celerio euphorbiae (Sienkiewich and Piechowska, 1973), and y+glutamyl-L-phenylalanine in Musca domestica (Bodnaryk, 1970). Insects that have inert conjugates of tyrosine require a greater number of enzymatic

AND

TRACY

steps to synthesize melanin and/or sclerotin than do insects that have inert forms of catecholamines. Thus, insects that possess storage forms of compounds which occur later in these pathways may be able to respond more rapidly to parasitic infection. To date, no studies have been conducted to determine if any of these compounds actively participate in the immune response of mosquitoes. What, if any, tyrosine, catecholamines, and inert storage forms of these compounds are present in the cellfree plasma of adult mosquitoes is not known. One of the difficulties in ascertaining what factors are synthesized, stimulated, or repressed in mosquito immune responses is the minute quantities of hemolymph available for biochemical studies. This necessitates the need for a highly sensitive method of tyrosine and catecholamine analysis. High-pressure liquid chromatography with electrochemical detection (HPLC-EC) is routinely used for the separation and quantitation of tyrosine and catecholamines at the picomole level in mammalian tissues (Kissinger et al., 1981). This sensitivity makes possible the analysis of microliter samples of hemolymph plasma. The first objective of this study was to survey the cell-free plasma of Aedes aegypti for the presence of tyrosine and catecholamines via HPLC-EC analysis. Second, we tested the hypothesis that plasma tyrosine and/or catecholamines are substrates/intermediates in the immune response of these mosquitoes against microfilariae. Third, we determined whether storage forms of tyrosine and/or catecholamines were present in the A. aegypti plasma. We report here that although tyrosine and catecholamines are present in the plasma of A. aegypti mosquitoes, there is no evidence for a storage form of tyrosine. Significantly, we did observe the presence of a catecholamine-like compound that seems to be involved in the immune response. We have designated this compound Peak I (PI) and discuss the possible significance it may have in the immune response.

HPLC

MATERIALS

ANALYSIS

AND METHODS

OF

HEMOLYMPH

PLASMA

269

and the cysteinyl conjugates of dopa and dopamine were synthesized by previously Mosquitoes described methods (Andersen, 1971; A. aegypti black-eyed Liverpool strain Kramer et al., 1983; Morgan et al., 1987; Ito (LVP) and A. aegypti Rockefeller strain et al., 1986). All other chemicals were of the (RKF) were obtained from laboratory col- best commercially available grade. Water onies at the University of London (1977) used was purified through a Milli-Q filtraand the University of Notre Dame (1983), tion system (Millipore Corp., El Paso, respectively. Rearing and maintenance fol- Texas). lowed previously described methods (Christensen and Sutherland, 1984). Adult Hemolymph Plasma Collection A. trivittatus were reared from the eggs of Three- to 5-day-old female mosquitoes field-collected adults (Christensen and were anesthetized by a slow stream of CO2 Rowley, 1978). Armigeres subalbatus were gas and immediately placed on a cold table obtained from the University of Notre at 4°C. Two incisions were made, one Dame (1986) and were reared as described through the tip of the proboscis and one in Beemtsen et al. (1989). Anopheles gamthrough the last two abdominal segments. biae were obtained from the Center for DisFifty incised mosquitoes were placed in a ease Control (Atlanta, Georgia) in 1987and chilled Pasteur pipette with a small glass were reared according to their protocols. wool trap inserted to separate the mosquitoes from the hemolymph collected. The Chemicals tubes were centrifuged at 600g for 10min at Dopa, dopamine, L-tyrosine, N-acetyl- 4°C. Hemolymph volume was quantitated dopamine, epinephrine, norepinephrine, with a chilled SO-p1Hamilton syringe. Hecu-methyldopa, 6-hydroxydopa, dihydroxy- molymph was kept on ice and was analyzed benzylamine, metanephrine, normetaneph- on the same rine, 3,4-dihydroxyphenylacetic acid, wise noted. day of collection unless other3,4-dihydroxyphenylglycol, 3,4-dihydroxymandelate, deoxyepinephrine, N-acetyl- Inoculations tyrosine, L-cysteine, arylsulfatase (abalone entrails, type VIII), alkaline phosphatase Dirofilaria immitis mff used for inocula(calf intestine, type VIII), acid phosphatase tions were obtained from the blood of an (sweet potato, type X), a-D-glucosidase infected beagle (provided by R. B. Grieve, (yeast, type III), p-D-glucosidase (al- University of Wisconsin, School of Veterimonds), p-nitrophenylphosphate, p-nitro- nary Medicine). Isolation of mff from the catecholsulfate, p-nitrophenyl p-D-&COblood was accomplished as described in side, p-nitrophenyl o-D-glucoside, and O- Nappi et al. (1987). Isolated mff were phospho-L-tyrosine were purchased from placed on a slide and heat killed by passing Sigma Chemical Co. (St. Louis, Missouri). a flame under the slide until no further 6-Hydroxydopamine, DL-threo-3,4-dihymovement of the mff was observed. Ten to droxyphenylserine, L-4-hydroxy 3-me- 15 mff were aspirated into a micropipette thoxyphenylalanine, and L-3,4-dihyand inoculated intrathoracically into 3- to droxyphenylalanine methyl ester were pur- 5-day-old female mosquitoes as previously chased from the Chemical Dynamics Corp. described (Christensen, 1981). Mosquitoes (South Plainfield, New Jersey). N-B-Ala- inoculated with Aedes saline (Hayes, 1953) nyldopamine was donated by A. J. Nappi in the same manner served as controls. (Department of Biology, Loyola University Mosquitoes were maintained on 0.3 M suof Chicago, Chicago, Illinois). N-Acetyl- crose until the hemolymph was collected at 18-21 hr postinoculation. norepinephrine, iV-p-alanylnorepinephrine,

270

MUNKIRS,

CHRISTENSEN.

HPLC-EC Analysis of Tyrosine, Catecholamines, and Hemolymph Plasma

Catecholamine standards were prepared as stock solutions of 200 pglml in 0.1 M perchloric acid containing 0.1% L-cysteine (HClO,/cys) as an antioxidant. Hemolymph samples were diluted 1: 10 (v/v) in ice-cold 0.1 M HClO,/cys and centrifuged at 11,750g for 5 min at 4°C. The acid-soluble portion was analyzed. Chromatography

A liquid chromatographic system consisting of a Beckman 114M pump and Rheodyne 7125 injector port was used with a Bioanalytical Systems Model LC-4B amperometric detector equipped with a glassy carbon electrode. The detector was set at either +650 mV for the detection of catecholamines or + 850 mV for the detection of tyrosine vs an Ag/AgCl reference electrode. Isocratic separation was achieved with a C,s reverse-phase column (Altex Ultrasphere ODS, 5-Km particle size, 4.6 mm id. x 25 cm) equilibrated with a mobile phase consisting of 0.1 M citrate buffer containing 0.5 mM sodium octylsulfate (HPLC grade, Pierce Chem. Co., Rockford, Illinois), 0.5 mM Na,EDTA, and 5% acetonitrile (HPLC grade, J. T. Baker Chem. Co., Phillipsburg, New Jersey) at 40°C. The standard pH of the mobile phase was 2.9, although in some cases it was varied to give better resolution of some peaks. The flow rate was 1.0 ml/min. Enzymatic Treatment of Hemolymph Plasma

Hemolymph plasma collected from naive A. aegypti (LVP) was treated with a variety of enzymes in order to determine if storage forms of tyrosine or catecholamines were present. The enzymes used were chosen because of the types of inert storage forms which have been reported in insects to date. (1) Alkaline phosphatase. Enzyme activ-

AND

TRACY

ity was confirmed spectrophotometricahy using p-nitrophenylphosphate as a substrate in 0.1 M glycine buffer, pH 9.0, containing 1.0 mM ZnCl, and 1 .O rnM MgCl* (Sigma Chemical Co.). The specific activity was found to be 400 units/mg protein. Incubation of tyrosine-O-phosphate with the enzyme and subsequent analysis of HPLCEC for the liberation of free tyrosine served as proof that the enzyme were active with this substrate. Twenty microliters of A. aegypti hemolymph plasma were incubated with 8 x IOH units enzyme in 70 ~10.1 M glycine buffer, pH 9.0, containing 1.0 mM ZnCl, and 1 .O mM MgCl, at 30°C for 5-min intervals up to 40 min. Samples were then diluted 1:2 (v/v) in ice-cold 0.1 M HC104/ cys. All samples were centrifuged for 5 min at 4°C and the acid-soluble fraction was analyzed by HPLC-EC at +850 mV vs Agl AgCl. Controls for all enzyme assays consisted of hemolymph plasma diluted (l:lO, v/v) directly into 0.1 M HClO&ys, plasma incubated with buffer in the absence of enzyme (1: 10, v/v), and enzyme incubated in buffer without plasma (l:lO, v/v). (2) Acid phosphatase. Enzyme activity was confirmed spectrophotometrically as described (Moss, 1984) except that the reaction was carried out in 46 mM sodium citrate, pH 5.0. Specific activity was 30 units/ mg protein. Enzyme activity with the substrate tyrosine-O-phosphate also was confirmed as mentioned above. Ten microliters of A. aegypti plasma were incubated with 1 x lo-’ units enzyme in 46 mr+i sodium citrate, pH 5.0 (1:5, v/v), at 37°C for 15 and 30 min. The samples were then diluted 1:2 (v/v) in 0.1 M HClO&ys, centrifuged, and analyzed at +850 mV vs Ag/ A&l. (3) Sulfatase. Enzyme activity was confirmed spectrophotometrically using pnitrocatecholsulfate as a substrate, and specific activity was 20 units/mg protein (Breslow and Sloan, 1972). TyrosineO-sulfate was unavailable for use as a substrate. Twenty microliters of A. aegypti plasma were incubated with 2.3 units en-

HPLC

ANALYSIS

OF HEMOLYMPH

zyme in 0.5 mM sodium acetate, pH 5.0, containing 0.5 mM Na,P20, and 1.71 M NaCl (1:5, v/v), at 37°C for 15, 30, and 60 min. Samples were diluted 1:2 (v/v) in 0.1 M HClOJcys, centrifuged, and analyzed at +850 mV vs Ag/AgCl. (4) cx+Glucosiduse. Enzyme activity was confiied spectrophotometrically using p-nitrophenyl a-D-glucoside as a substrate in 1 M sodium acetate buffer, pH 6.8 (Hers and von Hoof, 1966). Specific activity was found to be 77 units/mg protein. Thirty microliters of A. aegypti plasma and 0.77 units of enzyme were incubated in M sodium acetate, pH 6.8 (1:5, v/v), at 37°C for 15, 30, and 60 min. The samples were then diluted I:2 (v/v) in 0.1 M HClO&ys, centrifuged, and analyzed at +850 mV vs AglAgCl. (5) @&Glucosidase. Enzyme activity with the substrate p-nitrophenyl p-D-glucoside in 1.0 M sodium acetate, pH 5.0, was measured spectrophotometrically (Gatt, 1969)and was found to be 13 units/mg protein. Thirty microliters of A. aegypti plasma and 1.3 units of enzyme were incubated in 0.1 M sodium acetate, pH 5.0 (1:5, v/v), at 37°C for 15,30, and 60 min. Samples then were treated exactly as described for the a-glucosidase assay. Protein

Precipitation

Experiments

In order to determine if a plasma protein is required for the production of PI during alkaline treatment, the following experiments were done. (1) Hemolymph plasma was added directly to 10vol of 80% methanol or acetone at - 20°C. Precipitated proteins were eliminated by centrifugation at 11,750g for 5 min at 4°C. The organic soluble fraction was then evaporated to dryness in a SpeedVat Concentrator (Savant Instruments Inc., Farmingdale, New York). Residues were dissolved in 0.1 M glycine, pH 9.0, and incubated at 30°C for 15 min. HClO&ys was added and samples were analyzed by HPLC-EC at +650 mV vs Ag/AgCl. (2) Hemolymph plasma was placed in a

PLASMA

271

l-ml Reacti-Vial (Pierce, Rockford, Illinois), sealed, and heated at 100°Cfor 5 min. Half of the hemolymph was diluted 1:10 (v/ v) in ice-cold HClOJcys. The other half was diluted 1:5 (v/v) in 0.1 M glycine, pH 9.0, and incubated at 30°C for 15 min. HClOJcys was added to give a final dilution of 1:10 (v/v), and both samples were analyzed by HPLC-EC at + 650 mV vs Ag/ AgCl. A second experiment was done in which equal volumes of heated and unheated hemolymph were incubated together in 0.1 M glycine,pH 9.0 (1:5, v/v), at 30°C for 15 min. HClO&ys was added to give a final dilution of 1:lO (v/v) and this sample was analyzed under the same conditions . (3) Twenty-five microliters of hemolymph plasma was applied to a Sephadex G-25 fine column (3 mm i.d. x 6.5 cm) equilibrated with 50 mM ammonium acetate, pH 6.0. The column was calibrated with Blue Dextran (M, > 2 x 106), cyanocobalamin (M, - 1200)) and buffer-treated hemolymph plasma containing PI. Two fractions were collected, with the elution volume of cyanocobalamin as the separation point for each fraction. Fractions 1and 2 were both divided in half and evaporated to dryness in a Speed-Vat. One half of each fraction was diluted directly into 0.1 M HClO,/cys and the other half was incubated in 0.1 M glycine, pH 9.0, at 30°C for 15min. Another sample consisted of equal volumes of each fraction incubated together with 0.1 M glycine buffer, pH 9.0, at 30°C for 15 min. Each of the glycine buffer-treated samples was then diluted (v/v) into HClOJcys to give an equivalent volume to the control. All samples were analyzed by HPLC-EC at +650 mV vs Ag/AgCl. Aflnity

Chromatography

Buffer-treated hemolymph plasma was applied to a small disposable chromatography column packed with 200 ~1 of boronyl agarose and equilibrated with 0.3% NH40H. The column plus sample was centrifuged for approximately 20 set in a table-

272

MUNKIRS,

CHRISTENSEN,

top clinical centrifuge, then washed twice with l-ml aliquots of water. Subsequent washes were in order, 1 ml of methanol, 1 ml of 5050 acetonitrile:O.Ol M (NH&SO,, and 1 ml of water. Bound catechols were eluted with 0.5 ml of 0.1 M HCl and the eluate was evaluated by HPLC-EC (Dimson, 1984). Treatment of catecholamine standards (dopamine and epinephrine) in the same manner served as controls.

+850

AND

TRACY

RESULTS Aedes aegypti (LVP) hemolymph plasma was shown to contain tyrosine and dopamine by HPLC-EC via coinjections with authentic standards (Fig. 1). Quantitation of these two components was accomplished from peak height data by linear extrapolation from standard curves. Dopamine concentration was about 2.6 PM and tyrosine

a Ir

mV

C

ColnJoctlon with tyroslne

Hemolymph

Colnjoctlon wlth dopamlne

k

I.-

12

8

0

Time

12

0

,

I

4

a

(minutes)

FIG. 1. Tyrosine and catecholamine analysis of Aedes aegypti (LVP) hemolymph plasma from 3- to 5-day-old females. The first injection shows the chromatogram of hemolymph plasma diluted 1: 10 (v/v) in 0.1 M HClO, containing 0.1% cysteine immediately after collection. The total volume of the injection represents 0.5 pl of hemolymph plasma from approximately five mosquitoes. The second injection was a mixture of 50 ng of tyrosine and 0.5 pl of plasma. The third injection shows a coinjection of hemolymph plasma with 1.O ng of dopamine. In all cases the final dilution of the hemolymph was 1: 10 (v/v). Sensitivity was 10 n4 full scale, the flow rate was 1.O ml/min, and detection was at + 850 mV vs Ag/AgCl.

J

HPLC

ANALYSIS

OF

about 110 PM. N-B-Alanyldopamine was also detected in the plasma by coinjection with a synthetic standard. The concentration was not calculated but was clearly less than that of either tyrosine or dopamine. Analysis of tyrosine levels in naive hemolymph plasma compared with hemolymph plasma from immune reactive mosquitoes showed that no change in the quantity of this amino acid is seen during an immune response. To test for the presence of tyrosine storage forms, A. aegypti hemolymph plasma was treated with several hydrolytic enzymes as described under Materials and Methods. Such treatment failed to produce an increase in the concentration of tyrosine, indicating that phosphate, glucoside, and sulfate storage forms of this amino acid are not present. No changes in the chromatographic profiles of hemolymph plasma treated with acid phosphatase, arylsulfatase, or B-glucosidase were seen. The mild alkaline conditions required for alkaline phosphatase treatment of hemolymph resulted in the appearance of a distinct catecholamine-like peak in high concentrations, which we designate peak I. PI was not detectable by HPLC-EC in naive hemolymph plasma without prior exposure to alkaline buffer. Table 1 lists some of the conditions that result in the appearance of PI in hemolymph plasma. PI was formed when hemolymph plasma was incubated with three different buffers at pH 9.0, but was not detected when plasma was incubated in buffers of near neutral or acidic pH. Incubation of plasma with alkaline buffer at 4°C resulted in a progressive increase of PI over time with maximal peak heights detected at 30 min. PI is the major component of alkaline-treated hemolymph plasma by HPLC-EC analysis (Fig. 2). Coinjection of PI with catechols commonly isolated from insects is shown in Figure 3. Although PI does not cochromatograph with these compounds, its chromatographic behavior is similar. By varying the pH of

HEMOLYMPH

273

PLASMA

TABLE 1 pH DEPENDENCE IN PEAK I FORMATION IN NAIVE HEMOLYMPH PLASMA OF Aedes aegypti (LVP) Temperature CC)

Buffer

PH

0.1 hi glycine 0.1 h4 glycine 0.1 ht carbonatebicarbonate 0.1 M carbonatebicarbonate 0.1 h3 ammonium bicarbonate 1 M sodium acetate 2.5 M sodium acetate 46 mst citrate

9.0 9.0

30 4

+a +

9.0

30

+

9.0

4

+

9.0 6.8 5.0 5.0

30 31 31 31

+ -

PI

Note. Aedes aegypfi (LVP) hemolymph plasma was diluted 1:5 (v/v) in the buffers listed under the described conditions. After incubation, the solutions were diluted 1:2 (v/v) in 0.1 M HC104 containing 0.1% cysteine. The sample were then centrifuged to remove particulates and the acid soluble fraction was analyzed by HPLC-EC at +650 mV vs Ag/AgCI. (+) denotes samples that were positive for PI and (-) indicates that PI was not detected under those conditions. a Typical (+) values are in the range of 125 PM dopamine equivalents.

the mobile phase to shift retention times, PI was shown to elute similarly with dopamine. Because of this similarity, we have quantified PI in terms of dopamine equivalents. Calculations of four separate samples show that PI is present in 127 + 39 PM dopamine equivalents. One difference that was noted for PI in terms of the other catechols was its greater susceptibility to pH change of the mobile phase. A shift in retention time occurred for all catecholamines tested, but the effect was most dramatic on PI. As the pH of the mobile phase was increased, the retention time for PI decreased. In an effort to determine the identity of PI, coinjections of many commercially available standards and several synthesized standards were done. Table 2 lists the catechols that were evaluated. None of these compounds cochromatographed with PI. Synthesis of cysteinyl dopas and dopamines resulted in several peaks by HPLC-EC. Identification and purification of these peaks were not done because none of the peaks cochromatographed with PI. Preliminary data suggest that the appearance of PI is dependent on one or more

274

MUNKIRS, t660

mV

CHRISTENSEN, +sSO

3POOk

AND TRACY

mt

I:r I i-650

0

I.

I

.

.

‘4

6

12

16

mV

3

FIG. 2. Comparison of naive vs alkaline-treated Aedes aegypti (LVP) hemolymph plasma at + 650 mV vs Ag/AgCl. Sensitivity was 10 nA full scale and the flow rate was 1.0 ml/mm. The first panel represents 0.5 tr.1of hemolymph plasma diluted 1: 10 (v/v) in 0.1 M HClO, containing 0.1% cysteine. The second panel represents 0.5 pl of hemolymph plasma that was fust diluted 1:5 (v/v) in 0.1 M glycine buffer, pH 9.0, and incubated at 30°C for 15 min. After incubation, the sample was diluted 1:2 (v/v) in HClOJcys. Both samples were centrifuged prior to injection and all manipulations were carried out at 4°C unless otherwise stated.

hemolymph plasma proteins. Precipitation of hemolymph plasma proteins with 80% methanol or acetone at -20°C resulted in the failure of alkaline treatment to produce PI in the cytosolic fraction. Hemolymph plasma samples that were alkaline treated prior to methanol or acetone precipitation were positive for PI. PI was stable for several months in plasma treated in this manner and stored at -20°C. Boiling of the plasma to inactivate proteins prior to alkaline treatment also resulted in samples that were negative for PI. However, when equal volumes of boiled and fresh hemolymph were mixed and then alkaline treated, PI was detected, but in about 50% of the amount seen in buffer-treated hemolymph that was not boiled. This was expected because any active proteins necessary for the

=z0 0 : 0

I

8

4 Time

12

1

16

(mid

FIG. 3. Coinjection of catecholamine standards with alkaline-treated Aedes aegypti hemolymph plasma at +650 mV vs Ag/AgCl. Sensitivity was 0.5 nA full scale and the flow rate was 1.O ml/mm. Ten microliters of hemolymph plasma were diluted 1:5 (v/v) in 0.1 M glycine buffer, pH 9.0, and incubated at 30°C for 15 mm. This solution was then diluted 1:2 (v/v) with 0.1 M HClO&ys containing 0.4 ng/ul dopa, dopamine (DA), epinephrine (Epi), norepinephrine (Norepi), and 1.5 ng/pl N-acetyldopamine (NADA) and N-p alanyldopamine (NBAD).

formation of PI would also be diminished by 50% due to the boiling of half of the sample. Since incubation times were kept constant for all samples, the amount of PI formed would be a function of the amount of required protein(s) in the sample. Samples of plasma from which the highmolecular-weight proteins were removed by gel filtration chromatography also failed to elicit PI after alkaline treatment. PI is also present in the alkaline-treated

HPLC ANALYSIS

OF HEMOLYMPH

TABLE 2 CATECHOLS EXAMINED THAT Do NOT COELUTE WITH PEAK I BY HPLC-EC ANALYSIS a-Methyldopa 6Hydroxydopa Metaaephiine Normetaaephrine 3,4-Dihydroxyphenylaetic acid 3,4Diiydroxyphenylglycel 3,4-Dihydroxymandelate

Deoxyepinepkrioe N-Acetyltyrosine CHydroxydopamine oL-Threo-3,4-dihydroxyphenylserine r+Hydroxy-3-methoxyphenylakmine t.-3.4~Diydroxyphenyl-alanine methyl ester r,-Tyrosine”

Note. Catechols were prepared as stock solutions of 200 p&al in 0. I M HClO, containing 0.1% cysteine as an antioxidant. Five microliter injections of l-10 ng were analyzed at + 650 mV vs Ag/Agcl. L?L-Tyrosine was analyzed at + 850 mV vs Ag/AgCl.

hemolymph plasma of A. aegypti (RKF), An. gambiae, and Ar. subalbatus; however, it was not detected in A. trivittutus. In A. aegypti undergoing an immune response to D. immitis mff, PI is the major component of the plasma as compared with saline-

II

+660

mv

mif

I:c

+660

In”

I:r

1:t :; 21 i-0 4 8 1216

275

PLASMA

inoculated controls (Fig. 4). PI in mffinoculated hemolymph plasma was present in approximately 65 PM, expressed as dopamine equivalents, while saline-inoculated hemolymph plasma contained only 3.3 PM PI in dopamine equivalents. The major peak in the immune reactive hemolymph plasma was positively identified as PI based on the chromatographic behavior of both peaks at four different mobile phase pH values. In all cases, PI generated by alkaline conditions and the major peak present in hemolymph plasma of immunereactive mosquitoes cochromatographed. PI was also the major component of the plasma of n&inoculated A. aegypti (RKF), but it was not detected in m&inoculated A. trivittatus. PI is tentatively classified as a catecholamine based on its pH-dependent chromatographic behavior and electrochemical reactivity. The voltammagram of PI in Figure 5 is consistent with the electrochemical properties of many catecholamines. Further evidence for PI having a catechol moiety is the fact that it binds to boronyl agarose, a matrix with high affinity for compounds containing coplanar vicinal hydroxyl groups (Dimson, 1984). Unfortunately, re16-

saline

inoculated

2

10-

E F !! 5 0

5-

nutosl nut.*)

FIG. 4. Presence of Peak I in the immune-reactive hemolymph plasma of Aedes aegypti. Three- to 5 day-old females were intrathoracically inoculated with l&15 Dirc$iluria immitis mff. Hemolymph plasma was collected at 1g21 hr postinoculation and immediately diluted 1:lO (v/v) into 0.1 M HClOJcys. As a control, the same number of mosquitoes were inoculated with Aedes saline, and hemolymph plasma was collected at the same time intervals and treated exactly as was described for the parasite-inoculated mosquitoes. HPLC-EC analysis was done at + 650 mV vs Ag/AgCl, 10 nA full scale, and the flow rate was 1.0 mllmin.

200

400

600

600

1000

E CmV)

FIG. 5. Hydrodynamic voltammagram of Peak I from the hemolymph plasma of Aedes aegypti. Repetitive injections of plasma containing PI were done in which the amount injected and the sensitivity remained constant, while the potential of the working electrode was varied in 50 mV increments over the range of +35@-950 mV vs AgfAgCl.

276

MUNKIRS,

CHRISTENSEN.

coveries were poor for both the standards and the PI. Only 71% dopamine and epinephrine were recovered as compared to the 96% values recorded in Dimson (1984). Recovery was only 20% for PI as compared to buffer-treated hemolymph prior to chromatography . DISCUSSION The presence of tyrosine, dopamine, and ZV-B-alanyldopamine in the plasma of A. aegypti (LVP) mosquitoes was not surprising since it is currently hypothesized that tyrosine and catecholamines are important constituents of insect immune mechanisms (Soderhall, 1982; Nappi and Christensen, 1987). However, there was no significant change in the concentrations of these molecules in naive vs immune-reactive plasma. Plasma from mosquitoes inoculated with heat-killed mff was collected at 18-21 hr postinoculation. This time was used because of the report that A. aegypti (LVP) inoculated with heat-killed mff elicit a rapid and complete melanotic encapsulation reaction within 24 hr (Christensen et al., 1984). It is possible that tyrosine or catecholamine concentrations could be affected earlier, and a time course study of the changes in immune reactive plasma would be helpful to explore this possibility. Unfortunately, the paucity of material available for analysis makes this difficult. Results from the enzymatic treatment of hemolymph plasma show that tyrosine is not present as a glucoside, phosphate, or sulfate storage form. This, in conjunction with the absence of any change in tyrosine levels in immune-reactive plasma, strongly implies that tyrosine is not directly involved in the immediate response to parasitic infection. Likewise, dopamine and N-B-alanyldopamine storage forms were also not detected. This was somewhat surprising since all three compounds have been implicated as substrates for the melanotic capsule formation in the immune response (Nappi and Christensen, 1987). If the dark pigment associated with encapsulation reactions is a melanotic compound,

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some form of catechol would be required for its synthesis. We report here the presence of a catecholamine-like compound, PI, that occurs in relatively high concentration in the naive plasma of A. aegypti (LVP). The formation of PI is a pH-dependent phenomenon. Since detection is possible only after naive plasma is treated under mild alkaline conditions, the results suggest that PI is normally present as an electrochemically inert storage form. Possession of inert storage forms of these compounds could be advantageous to the mosquito. They may provide a protective factor by preventing premature oxidation by enzymes involved in the synthesis of the melanotic capsule. Similarly, storage forms may simply aid in the prevention of the autooxidation of catechols that occur later in the biosynthetic pathway leading to melanotic products. Many of these later intermediates are converted to melanin compounds nonenzymatically (Brunet, 1980). Our results have shown that PI is not stored as a glucoside, phosphate, or sulfate conjugate. At this time, no work has been done to try to identify the precursor of PI. HPLC-EC chromatograms show that PI is the major electrochemically active component of alkaline-treated plasma. The biological significance of this peak is suggested by the observation that it is also the major component of hemolymph plasma from two strains of A. aegypti inoculated with D. immitis. Although PI was detected in salineinoculated controls, it was about 5% of the amount in mff-inoculated samples. This may be attributed to the wound-healing process initiated by the inoculation procedure. Further evidence of the significance of PI is the fact that it is the major peak of alkaline-treated hemolymph plasma from two other mosquito species, Anopheles gambiae and Armigeres subalbatus. The presence of PI in these mosquitoes, which are quite different in their habitats, physiologies, and vector capacities, suggests that it may be a frequently occurring element in mosquito immune mechanisms. However,

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PI was not detected in either alkalinetreated or D. immitis-inoculated A. trivittatus. The absence of PI in this mosquito species was not altogether surprising, given the work that has been done recently comparing the immune responses of A. aegypti and A. trivittatus. A. trivittatus is a highly immunocompetent host, capable of encapsulating mff in a much more rapid and effective manner than A. aegypti (LVP) (Christensen et al., 1984). The reasons for this difference in immune competence are not completely known. Naive A. trivittatus have been reported to have a significantly greater number of immune-competent hemocytes than naive A. aegypti (Nappi and Christensen, 1986; Li and Christensen, 1990). In response to infection, A. aegypti circulating hemocyte populations increase, while no increase is noted in A. trivittatus (Christensen et al., 1989; Li et al., 1989). It is possible that A. trivittatus can react faster than A. aegypti because more immunoreactive hemocytes are readily available to respond initially. The active proliferation of hemocytes in A. aegypti may be a means in which this mosquito attempts to increase its subpopulation of immunoreactive hemocytes, thus explaining the slower response. Naive and immune-reactive A. trivittatus hemocytes have been found to contain three- to fourfold higher MPO activity than A. aegypti hemocytes (Li et al., 1989). MPO catalyzes the oxidation of tyrosine to dopa, and dopa can then be oxidized in a number of steps to form melanin compounds. Further contrasts are seen in the microscopic examination of encapsulated mff from these two species. A. trivittatus produce black capsules, typical of what would be expected if a melanin pigment were present, whereas A. aegypti produce golden brown capsules (unpubl.), a pigmentation more commonly associated with sclerotin compounds in insects (Brunet, 1980). At present, only preliminary observations have been made on the role that tyrosine or catecholamines in the hemolymph may play in A. trivittatus immune re-

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sponses. A higher concentration of tyrosine was noted in naive plasma of A. trivittatus as compared to A. aegypti; however, no change in tyrosine concentration was detected in naive vs immune-reactive A. trivittatus plasma, leaving the role of this amino acid in the immune response unclear (unpubl.). It is evident that the two species respond differently on the biochemical level to mff, but further investigation is needed to clarify these differences. It is possible that A. trivittatus immune mechanisms operate in a manner similar to what commonly has been hypothesized, i.e., the hydroxylation of tyrosine to dopa with subsequent oxidation to melanin. Elevated levels of MPO and tyrosine, in addition to the coloration of the capsules, makes creditable the idea that this could be a true melanization response. More studies are required to show that A. trivittatus possess other enzymes and substrates involved in melanin synthesis. Our results indicate that A. aegypti utilize different biochemical means to respond to mff than A. trivittatus. In A. aegypti, PI seems to be intimately involved in the immune response. Although the structure of PI is still not known, our results show that it has the characteristics of a catecholamine. The formation of PI seems to require the presence of one or more plasma proteins, although the nature of this protein is presently not known. Since it is presumed that PI is present in an inert storage form in naive hemolymph plasma, it is likely that some form of cleavage is necessary to liberate it during the alkaline treatment. Whether enzymatic cleavage occurs is not clear. Another possibility is that PI could be a rearrangement product caused by the change in pH. Significantly, we have shown that PI is not one of the catechols commonly associated with melanin synthesis in insects. We have also eliminated numerous other catechols as well as some synthesized cysteinyl conjugates of dopa and dopamine. That PI is not identical with any of the compounds commonly associated with melanin production suggests that

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mosquitoes which synthesize PI may not produce a melanotic capsule, but instead have capsules composed of a sclerotinic compound. It also is possible that PI could be a conjugate of a catecholamine(s) involved in melanin or sclerotin production that we have not investigated. In summary, our results did not support our original hypothesis that A. aegypti hemolymph plasma has an inert storage form of tyrosine or that tyrosine is involved in the immediate response to parasitic infection. However, our data do show that A. aegypti hemolymph plasma contains PI, a catecholamine-like compound, in high concentration. We have shown that PI is normally present as an inert storage form, but that the storage form is not a phosphate, sulfate, or glucoside. More significantly, we have shown that PI is a biologically relevant compound as evidenced by its appearance in A. aegypti undergoing an immune response against inoculated D. immitis mff. The identification of PI and its immediate precursor are now necessary to further clarify biochemical aspects of A. aegypti immune mechanisms. ACKNOWLEDGMENTS We thank Linda A. Christensen for technical support in rearing and maintaining the mosquitoes required for this project. This study was supported by the National Institutes of Health, Grant AI 19769.

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