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Origin of arachidonic acid in the salivary glands of the lone star tick, Amblyomma americanum
Insect Biochem. Molec. Biol. Vol.25, No. 2, pp. 225-233,1995
Pergamon
09~17,18(94)00059-X
Copyright© 1995ElsevierScienceLtd Printed in GreatBritain.All rightsreserved 0965-1748/95$9.50+ 0.00
Origin of Arachidonic Acid in the Salivary Glands of the Lone Star Tick, Amblyomma
americanum ALAN S. BOWMAN,t:~ JOHN R. SAUER,t PAUL A. NEESE,t JACK W. DILLWITH~" Received 9 May 1994; revised and accepted 23 July 1994
The contribution of synthesis and dietary sequestration to the high arachidonate content of the lone star tick, Amblyomma americanum, salivary glands was investigated by assessing the salivary metabulites of various radioinheled fatty acid substrates administered to partially fed females. A portion of each of the fatty acids studied was incorporated into the fatty acid moiety of the phosplmlipid fraction. [14C]acetate was metabolized only into myristic, palmitic, palmitoleic, stearic, and olcic acids. [~-l]oleic acid, p4C]linoleic acid, [14C]~-Iinolenic acid and [14C]cicosatrienoic acids were incorporated into salivary gland phospholipids but underwent Httle change including elongation and/or desuturation to arachidonate. Ingested [~H]arachidonic acid was readily taken up by the salivary gland and distributed among the lipid classes in a pattern markedly different from that of the other fatty acids tested. We conclude that ticks are unable to synthesize arnchidonic acid for incorporation into the salivary glands, but rather sequester it from the host bloodmeal. Fatty acids Biosynthesis Phospholipids Eicosatrienoic acid Arachidonic acid
Acetate
Oleicacid
Linoleicacid
Linolenicacid
Prostaglandins of the 2-series are synthesized from the precursor arachidonic acid via the cyclooxygenase Ticks surpass all other arthropods in the number and pathway (Holtzman, 1991). The salivary glands of variety of diseases that they transmit to domestic aniA m b l y o m m a americanum contain an unusually high conmals, and rank second only to mosquitoes as vectors of centration of arachidonic acid (8% of the total fatty human disease (Sonenshine, 1991). The effectiveness of acids) which is present only in the phospholipid ticks as pathogen vectors is enhanced by their prolonged fraction (Shipley et al., 1993a). Animals, including most host attachment and the immunosuppressive properties arthropods that have been studied to date, synthesize of tick salivary gland products (Ramachandra and arachidonic acid (20: 4, n-6) by elongation/ Wikel, 1992). Problems associated with prolonged desaturation of dietary linoleic acid (18:2, n-6) attachment are ameliorated by compounds in tick saliva (Stanley-Samuelson et al., 1988). Because nearly all exhibiting anti-inflammatory; analgesic; anti-coagulant; animals lack a A n desaturase, linoleic acid is considered anti-hemostatic; vasodilatorary; and/or immunosupan essential fatty acid. Blomquist and co-workers have pressive properties (Ribeiro, 1987; Sauer et al., 1995). shown that some insect species are capable of de novo Prostaglandins possess many of these properties and linoleic acid synthesis from acetate; e.g. a termite, cockhave been found in the saliva of several tick species roach, cricket (Blomquist et al., 1982) and pea aphid (Higgs et al., 1976; Dickenson et al., 1976; Ribeiro et al., (de Renobales et al., 1986). Conversely, dietary studies 1985, 1992). Prostaglandins are believed to play a major on the mosquito, Culex pipiens, have demonstrated that role in tick feeding (Sauer et aL, 1993) and, together with complete adult emergence requires a dietary source of their immunosuppressive activity, would appear to play both linoleic and arachidonic acid (Dadd, 1983). This a major role in tick-borne disease transmission and indicates that C. pipiens is incapable of converting pathogenesis. linoleie to arachidonic acid, and, therefore, is dependent upon the host bloodmeal for both of these essential fatty tDcpartrnent of Entomology, Oklahoma State University,Stillwater, acids. The origin of arachidonic acid in salivary glands is of OK 74078, U.S.A. :[:Author for correspondence. importance to an appreciation of the role of eicosanoids INTRODUCTION
225
226
A L A N S. B O W M A N e t al.
in tick feeding. In this study, we assessed the relative contributions of de novo synthesis, conversion of dietary fatty acid precursors, and direct sequestration from the host to the high salivary gland arachidonate content. In addition, the fate of ingested fatty acids in the salivary gland was investigated.
[3H]oleic acid (2 #Ci/tick), [~4C]linoleic acid (0.75#Ci/tick) or [3H]arachidonic acid (1 #Ci/tick). Treatments with [~4C]7-1inolenic acid and [14C]di-homo7-1inolenic acid were restricted to ingestion (0.65/~Ci/tick) and injection (0.65/~Ci/tick) studies in mated females only. For ingestion studies, the solvent carrier was evaporated under nitrogen and the label resuspended in a 0.5ml microcentrifuge tube in 15#1 MATERIALS AND METHODS 68 mM NaCI, 0.25 mg/ml bovine serum albumin diluent with incubation at 37°C and vigorous vortexing. Label Materials [l-laC]Sodium acetate (54 mCi/mmol); [9,10-3H(N)]9 - was fed via glass capillary tubes placed over the tick's octadecenoic (oleic) acid (7.2Ci/mmol); [1-14C]9,12- mouthparts. The microcentrifuge tube was rinsed twice octadecadienoic (linoleic) acid (50mCi/mmol); and with a further 15/~1 diluent and the rinse fed to the ticks. [5,6,8,9,11,12,14,15-3H(N)] 5,8,11,14-eicosatetraenoic By this method, more than 85% of the label was ingested (arachidonic) acid (80 Ci/mmol) were purchased from by the ticks as assessed by the amount of radioactivity DuPont-New England Nuclear, Wilmington, Del. remaining in the microcentrifuge and capillary tubes. [ 1-~4C]6,9,12-Octadecatrienoic (7-1inolenic) acid For the injection studies, the labelled compound was (55mCi/mmol) and [1-~4C]8,11,14-eicosatrienoic (di- resuspended, as above, in 20/~1 diluent and 2 #1 carefully homo-7-1inolenic) acid (55 mCi/mmol) were obtained injected into the hemocoel of each tick with a 10#1 from American Radiolabeled Chemicals Inc., St Louis, syringe fitted with a 22 gauge needle. For the topical Mo. and [1-t4C]methanol (42mCi/mmol) from ICN, application studies, the label was resuspended in 15 pl Irvine, Penn. Thin-layer chromatography plates were ethanol and 1.5#1 applied to the dorsal cuticle and normal phase silica gel without fluorescent indicator allowed to evaporate. The ticks were maintained in a acquired from Analtech, Newark, Del. Silicic acid humid environment at room temperature. The salivary (BioSil A; 100-200 mesh) was purchased from BioRad, glands were dissected from ticks injected and fed with label after 48 and 72 h, respectively. Ticks undergoing Richmond, Calif. BondElut solid-phase extraction (SPE) columns topical application were so treated daily for up to 5 days, (500 mg) were obtained from P. J. Cobert Associates and their glands removed 24 h after the last application. Inc., St Louis, Mo. Lipid standards and other chemicals Mated females were administered the labelled precursors were acquired from Sigma Chemical Company, St Louis, by injection and feeding only, and their glands removed Mo. Liquid scintillation cocktail (BioCount) was ob- after 24 and 48 h, respectively. tained from Research Products International Corp., Lipid analysis Mount Prospect, I11. Reagent grade diethyl ether and Lipids were extracted from the salivary glands imdichloromethane were purchased from EM Science, mediately after collection by the method of Bligh and Gibbstown, N.J. High performance-liquid chromatogDyer (1959) and stored in chloroform with butylated raphy grade acetone, 2-propanol and acetonitrile were hydroxytoluene (5mg/100ml) at - 2 0 ° C for further supplied by Fisher Scientific, Pittsburgh, Penn. Hexane, analysis. Phospholipids, free fatty acids, and neutral chloroform and methanol (Fisher Scientific) were glass lipids were isolated from the lipid extract using 500 mg redistilled before use. amino-propyl SPE columns as described by Shipley et al. (1994). Following base hydrolysis of the phospholipid Collection of tissue fraction, fatty acid methyl esters (FAMEs) were preAdult female lone star ticks, Amblyomma americanum pared and purified as described by Shipley et al. (1993a). (L.), were reared on sheep according to the methods of Aliquots of the salivary gland phospholipid FAMEs Patrick and Hair (1976) and removed during the "slow- were fractionated according to their degree of unfeeding" phase (7-14 days on sheep). Adult virgin female saturation on silver impregnated 500 mg benzene sulA. americanum were reared in a similar manner in the fonic acid-SPE (Ag+-SPE) columns, as described by absence of males. Salivary glands were dissected while Christie (1989). The radioactivity in each fatty acid ticks were submerged in ice cold 0.1 M MOPS buffer, class was determined by liquid scintillation counting pH6.8, containing 0.02M ethylene glycol bis-(fl- (Beckman LS6000SC, Fullerton, Calif.) employing an aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). automatic quench correction curve. The separation Ticks which had undergone topical treatment with efficiency using the Ag+-SPE columns was determined radiolabelled substrate were rinsed by three 20 s rinses in using FAMEs prepared from salivary gland phosphohexane prior to salivary gland removal. lipid containing authentic labelled fatty acids. Radio-HPLC was performed on salivary gland phosFatty acid biosynthesis pholipid FAMEs with a C-8 reverse-phase column Groups of 10 partially fed adult virgin female (Supelcosil LC-8, particle size 5/~m, 25cm x 4.6mm A. americanum were fed, topically applied or injected i.d., Supelco, Bellafonte, Penn.) using a Waters Model into the hemocoel with either [~4C]acetate (1/~Ci/tick), 510 solvent delivery system. Authentic FAMEs were
TICK SALIVARYGLAND ARACHIDONATE
227
mixed with salivary gland phospholipid FAMEs mined by radio-scanning. Phospholipids were identified and introduced into the system dissolved in aceto- by comparing the chromatographic pattern of the salinitrile:chloroform (1:1, v/v) and eluted isocratically vary gland and phospholipid standards (Sigma, St Louis, with acetonitrile:water (85:15, v/v) at a flow rate of Mo.) under iodine vapor. 1 ml/min. Elution of FAMEs was monitored at 205 nm and radioactivity by an on-line Radiomatic Model CT Fit-one/Beta radioactivity flow detector (Radiomatic RESULTS Instruments and Chem. Co., Tampa, Fla.) Utilizing Scinti Verse LC (Fisher) as the liquid scintillation Fatty acid biosynthesis A portion of each of the fatty acid substrates tested cocktail. Counting efficiencies with this system were 64 and 46% for ~4C and 3H, respectively. Retention times was incorporated into the fatty acid moiety-of the of radioactive peaks were compared with those of phospholipid fraction in the salivary glands. Radiounlabelled authentic standards and a similar mixture HPLC of the salivary gland phospholipid FAMEs from of standards labelled by transmethylation with ticks after administration of [~4C]acetate (Fig. la) [14C]methanol. showed de novo biosynthesis of palmitate, palmitoleate, The tick salivary gland phospholipid FAMEs were stearate, and oleate. No evidence for the conversion of also subjected to gas chromatography (HP5890II, oleate to linoleate (Fig. lb) or any other product followHewlett-Packard, Sunnydale, Calif.) on a DB-225, ing [3H]oleic acid administration was observed. Neither 15 m x 0.53 mm, 1/~m film thickness megabore capillary elongation or desaturation products of administered column (J&W Scientific, Folsom, Calif.). The column [14C]linoleic acid, namely ~-linolenic, di-homo-~-linotemperature program was 120°C for 2 min, 5°C/min to lenic, or arachidonic acids, were detected (Fig. lc). No 200°C, 2°C/min to 225°C, hold for 2 min. This program further metabolism of [~4C]~-linolenic acid was detected gave complete baseline resolution for all the major (Fig. ld). Neither injected nor ingested [~4C]di-homo-vFAMEs. Authentic standards were added to the samples linolenic acid was converted to arachidonic acid and introduced via an on-column injector employing an (Fig. le), but there was evidence of fl-oxidation and oven-track program operating at 3°C above the oven subsequent synthesis of palmitate, palmitoleate, stearate temperature. Elution of FAMEs was monitored with a and oleate from ingested [~4C]eicosatrienoic acid thermal conductivity detector operating at 225°C. The (Fig. le). Attempts to isolate and concentrate possible eluting FAMEs were collected, at the appropriate times, desaturation products from each of the labelled prein chilled glass pasteur pipettes plugged with chloro- cursors studied using Ag2+-SPE prior to radio-HPLC form-dampened glass wool and then rinsed into vials resulted in essentially no radioactivity in the desired with 2 ml methanol. The methanol was evaporated and • fractions. Ticks fed [3H]arachidonic acid incorporated the radioactivity determine by liquid scintillation count- the label into the salivary gland phospholipid where it ing. Retention times of salivary gland FAMEs were was not converted into other fatty acids (Fig. If). The analytical results of the radio-HPLC determinations compared to those of authentic standards. were confirmed by radio-GC (data not shown) which Fate of ingested fatty acids were essentially the same except that radio-GC detected Virgin and mated A. americanum were fed [14C]acetate, a very small amount of myristate in ticks administered [3H]oleic acid, [14C]linoleic acid or [3H]arachidonic acid, [~4C]acetate. Virgin female ticks were topically applied [14C]linoleic as before. [laC]~-Linolenic and [14C]di-homo-~-linolenic acid were only fed to mated females. Salivary glands acid daily for up to 5 days and the salivary gland were removed 24 and 48 h later from the mated and phospholipid FAMEs fractionated on Ag÷-SPE virgin ticks, respectively, and the lipids extracted im- columns according to the degree of fatty acid unsatumediately (Bligh and Dyer, 1959). An aliquot of the ration (Table 1). The amount of radioactivity eluted in extracted lipid was separated on 20 x 20 cm silica gel G the triene and tetraene fractions were similar using plates, 250/~m thickness (Analtech, Neward, Del.) in FAMEs prepared from authentic [~4C]linoleic acid added hexane/diethyl ether/acetic acid (75: 25 : 1, v/v/v) and the to unlabelled salivary gland phospholipid. No further distribution of the label determined by radio-scanning desaturation of the diene fraction was observed over the (Bioscan 2000, BioScan, Washington, D.C.). Lipid experimental period as evidenced by the lack of increasclasses were identified by comparison to known lipid ing amounts of radioactivity eluted in the triene and standards (Sigma, St Louis, Mo.) under iodine vapor. tetraene fraction. That the diene fraction of the salivary The phospholipid fraction was isolated from the total gland phospholipid FAMEs is metabolically dynamic lipid extract using amino-propyl SPE columns, as above was shown by the time-dependent decrease in the diene (Shipley et al., 1994). Individual phospholipid classes fraction (r = 0.983, P < 0.01) and the concomitant inwere resolved on 20 x 20 cm, 250 #m thickness silica gel crease in the monoene (r = 0.955, P < 0.05) and the G channelled plates with pre-absorbent zone (Analtech, saturated fractions (r = 0.983, P < 0.02). Little difference was observed between virgin and Newark, Del.) in chloroform/hexane/methanol/acetic acid/boric acid (4:30:20:10:1.8, v/v/v/v/w) (Gilfillan mated ticks regarding biosynthesis and incorporation of et al., 1983) and the distribution of radioactivity deter- fatty acids. The route of radiolabel administration also
228
ALAN S. BOWMAN et al.
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FIGURE 1. Radio-HPLC chromatograms of fatty acid methyl esters from the phospholipid fraction of salivary glands from partially fed female A. americanum 24-48 h after administration of radiolabelled precursors. (a) females injected with [14C]acetate; (b) females fed [3H]oleicacid; (c) females fed with [14C]linoleicacid; (d) females fed with [t4C]~,-linolenicacid; (e) females fed and injected (inset) with [14C]di-homo-~-linolenicacid and (f) females fed with [3H]arachidonic acid. had no effect on the subsequent fatty acid biosynthesis in ticks, except for [t4C]di-homo-y-linolenic acid. The American cockroach, Periplaneta americana, has been shown to synthesize linoleic acid and arachidonic TABLE 1. Distribution of radioactivity among the fatty acid classes of salivary gland phospholipid from groups of 10 virgin female A. amerieanum topically applied daily with [[4C]linoleic acid (0.75#Ci/tick). Glands were dissected 24h after the last topical apphcation and the phospholipid FAMES fractionated according to degree of unsaturation on silver-impregnated benzene sulfonic acid solid-phase extraction columns, as described in the Materials and Methods Fatty acid class
Radioactivity distribution (% age) Day 2 Day 3 Day 4 Day 5
Saturated Monoene Diene Triene Tetraene Pentaene Hexaene
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1.42 4.69 93.53 0.25 0.06 0.04 0.0!
2.02 5.09 92.66 0.15 0.05 0.02 0.01
"Note: Triene and tetraene fractions re-chromatographed as diene.
acid de novo from [14C]acetate (Jurenka et al., 1987) and was employed in the present study as a positive control. In our hands, linoleic acid (18:2, n-6), eicosanoic acid (20: 0), eicosadienoic acid (20: 2, n-6), eicosatrienoic acid (20:3, n-6), and arachidonic acid (20:4, n-6) were synthesized when adult female P. americana were injected with [14C]acetate, [3H]oleic acid, or [14C]linoleic acid and the whole b o d y lipids analysed as for the tick salivary glands (data not shown).
Fate o f ingested f a t t y acids Distribution o f radioactivity a m o n g the different lipid classes in salivary glands fed various fatty acid precursors was assessed by radio-TLC. Radioactivity was not incorporated into the sterol fraction with any o f the labelled precursors (Fig. 2). The phospholipid fraction contained the majority o f the radioactivity when [~4C]acetate was ingested, sterol esters contained only 2% with the remainder present equally in the free fatty acid and triglyceride pools (Fig. 2a). Following ingestion o f [3H]oleic acid (Fig. 2b), [~4C]linoleic acid (Fig. 2c),
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F I G U R E 2. Radio-chromatogram of TLC-separated lipid extracts of salivary glands from partially fed female A. americanum 24 h after ingesting: (a) [14qacetate; (b) [3H]oleic acid; (c) [t4qlinoleic acid; (d) [14C]y-linolenic acid; (e) [14qdi-homo-7-1inolenic acid; and (f) [3H]araehidonic acid. Arrows indicate position of the lipid standards: PL, phospholipid; S, sterol; FFA, free fatty acid; TG, triglyceride; and SE, sterol ester.
[~4C]7-1inolenic acid (Fig. 2d) and [14C]di-homo-7-1inolenic acid (Fig. 2e) radioactivity was largely present in the phospholipid fraction with lesser amounts in the triglyceride pool, and small but detectable radioactivity in the free fatty acid and sterol ,ester fractions. In contrast, radioactivity was incorporated solely in the phospholipid fraction of salivary glands when ticks were fed [3H]arachidonic acid (Fig. 20. Phospholipid was isolated by solid-phase extraction from tick salivary glands fed radiolabelled fatty acid precursors and the distribution of the radioactivity among the phospholipid classes assessed by radio-TLC (Fig. 3). An unidentified phospholipid migrating near the sphingomyelin standard, as reported by Shipley et al. (1993a), incorporated radioactivity only when [14C]acetate was the ingested precursor (Fig. 3a). Label was distributed between phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in a ratio approximating 70:30 when ticks had been fed [~4qacetate, [3I-I]oleic acid, and [14qlinoleic acid (Fig. 3a-c) and 60:40 with [t4C]~-linolenic acid and [~4qdi-homo-7-1inolenic acid (Fig. 3d-e). Conversely, [3H]arachidonic acid was incorporated into PC and PE in the ratio 25:75 (Fig. 30. No difference in the distribution of radioactivity among the
salivary gland lipid classes and phospholipid subclasses was observed between virgin and mated ticks. DISCUSSION In most organisms, even-numbered fatty acids of 16 (palmitate) and 18 (stearate) carbons long are synthesized de novo from acetate by the fatty acid synthase multi-enzyme complex and then interconverted to the required fatty acids by desaturation and elongation, as appropriate. Ticks are also capable of synthesizing palmitate (16:0) and stearate (18:0) de novo. In addition, these fatty acids are desaturated to form palmitoleate (16:1, n-7) and oleate (18:1, n-9). Recently a gene exhibiting high sequence homology to stearoyl CoA desaturase was identified in a cDNA library prepared from A. americanum salivary glands (Luo, 1993). It is accepted that linoleic acid is a dietary requisite as animals are unable to convert oleate to linoleate due to the absence of A~2 desaturase (Whitehead, 1984). However, the American cockroach, Periplaneta americana; the termite, Zooterrnopsis angusticollis; the house cricket, Acheta domesticus (Blomquist et al., 1982); and the Australian field cricket, Teleogryllus commodus
230
A L A N S. B O W M A N et al.
tJurenka et al., 1988) have been shown to be capable of synthesizing linoleic acid de novo. Linoleic acid biosynthesis in insects has been further studied to include 15 insect species representing four orders (see de Renobales et al., 1987) and has also been demonstrated in the land slug and garden snail (Weinert et al., 1993). Earlier reports suggested that the pea aphid, Acyrthosiphon pisum could not synthesize linoleate de novo (Blomquist et al., 1982), but later experiments showed that this insect is capable of synthesizing linoleic acid but required an extended incubation period, i.e. > 2 h (de Renobales et al., 1986). The tick does not synthesize linoleic acid from acetate nor oleic acid, despite incubation periods of up to 5 days. Rather it appears that ,4. americanum is dependent upon the host bloodmeal for its linoleate requirements. The majority of animals, including arthropods, cannot synthesize linoleic acid but can readily convert this dietary necessity into arachidonic acid by a series of desaturation and elongation steps (Stanley-Samuelson et al., 1987). In the present study, `4. americanum did not synthesize detectable amounts of arachidonic acid from any of the fatty acid precursors tested. Incubation periods of up to 5 days with linoleic acid failed to
produce any arachidonic acid even though the phospholipid linoleate was metabolically active as evidenced by increasing proportions of labelled saturated and unsaturated fatty acids being formed, presumably by fl-oxidation and subsequent de novo biosynthesis. Similarly, ticks seemed incapable of converting 7-1inolenic and di-homo-7-1inolenic acids to arachidonic acid. Indeed, apart from being able to synthesize palmitate and stearate, presumably by the action of the fatty acid synthase enzyme complex, and the subsequent conversion to palmitoleate and oleate by Agdesaturase, the tick was demonstrated to exhibit very limited fatty acid biosynthetic capability (Fig. 4). Therefore, it would appear that any fatty acid found in the tick salivary gland containing more than one double bond must originate from the host blood (Fig. 4). Interpretation of previous fatty acid biosynthetic studies on arthropods has been confounded by the problem of attributing the synthetic capability to insect tissues and dismissing contributions from symbiotic microorganisms (Borgeson et al., 1991; Borgeson and Blomquist, 1993). In the present study the fatty acid precursors were fed to ,4. americanum, thus, permitting an assessment of any contributions gut microflora may
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F I G U R E 3. R a d i o - c h r o m a t o g r a m o f T L C - s e p a r a t e d p h o s p h o l i p i d s f r o m salivary g l a n d s from p a r t i a l l y fed female A. americanum 24 h after ingesting: (a) ["C]acetate; (b) [3H]oleic acid; (c) []4C]linoleic acid; (d) [14C]~,-linolenic acid; (e) [14C]di-homo-y-linolenic acid; a n d (f) [3H]arachidonic acid. U N K = u n k n o w n p h o s p h o l i p i d ; P C = p h o s p h a t i d y l c h o l i n e ; PE = phosphatidylethanolamine.
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AS.~sa~rMe Diet ~
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FIGURE 4. Apparent fatty acid biosynthetic capability of A. americanum and origin of salivary gland fatty acids. Abbreviations: 2: 0, acetate; 16:0, palmitate; 18:0, stearate; 16:1, palmitoleate; 18:1, oleate; 18:2, linoleate; 20:2, eicosadienoate; 18:3, 7-1inoleneate,20:3, di-homo-7-1inolenic;20:4, arachidonate; and FAS, fatty acid synthaseenzymecomplex. provide to the fatty acid requirement of the tick. No effect of the route of precursor administration on subsequent fatty acid production in ticks was noted, except that di-homo-7-1inolenic acid underwent fl-oxidation only when ingested, presumably within the gut or possibly by gut microflora. The inability of the tick to convert linoleate to arachidonate is highly unusual, only the domestic cat (Kendall, 1984), rainbow trout (Yu and Sinnhuber, 1976) and the mosquito, Culex pipiens (Dadd, 1983) have been shown incapable of converting linoleate to arachidonate as inferred from nutrition studies, though the cat is reported capable of synthesizing arachidonate acid from y-linoleic acid (Kendall, 1984). Whilst salivary prostaglandins are believed to be important in successful tick feeding (Sauer et al., 1993), the necessity of the tick to be able to synthesize the prostaglandin precursor araehidonic acid appears to be unimportant. This is due to the tick's ability to sequester arachidonate from the diet and accumulate it in the salivary glands. Further evidence that tick arachidonate originates from the diet is seen in the dramatic increase in salivary gland arachidonate content in A. americanum females following attachment to the host (Shipley et al., 1993b). Previously, we demonstrated that, at least for sheep (Bowman et al., 1993) and dogs (A. S. Bowman, unpublished observations), the host blood and the components within would provide A. americanum with an adequate supply of arachidonate. Since both the mosquito and tick appear to lack the ability to synthesize araehidonic acid, it is tempting to postulate that this may be a common characteristic of blood-feeding arthropods. Fatty acid precursors were incorporated into various lipid classes in A. americanum salivary glands including phospholipid, free fatty acid, triglyceride and sterol ester, but not the sterol fraction. Thus, it appears that A. americanum, like other arthropods, lacks the ability
to synthesize sterols de novo. Acetate, oleate, linoleate, 7-1inoleneate, and di-homo-7-1inolenic were incorporated primarily into the phospholipid fraction, but also appeared in the triglyceride pool as suggested by endogenous fatty acid analysis of salivary gland lipids (Shipley et al., 1993a). Labelled arachidonic acid was recovered only in the phospholipid fraction, reflecting the distribution of endogenous arachidonate (Shipley et al., 1993a). That arachidonate is preferentially esterified in the phospholipid pool is common is eicosanoid-producing tissues (MacDonald and Sprecher, 1991). The specific distribution of the salivary gland lipids is likely due to the substrate affinities of lysophosphatide acyltransferase and acyl transferase involved in the phospholipid and triglyceride synthesis, respectively. Within the phospholipid fraction, label was incorporated mainly into PC and PE. Lesser amounts of acetate were also incorporated into an unknown phospholipid that migrates near sphingomyelin by TLC. This phospholipid contains predominantly (>70%) saturated fatty acids (Shipley et al., 1993a) and, is thus, less likely to be labelled by the unsaturated fatty acid treatments. Acetate, oleate, and linoleate were incorporated into PC and PE in a ratio approximating 70: 30, respectively, and 7-1inoleate and di-homo-y-linolenic acid in a ratio 60: 40. Arachidonate was recovered in PC and PE in the ratio 27:75, respectively. This specific distribution is thought to be due to the remodelling process involving transacylase, which transfers arachidonate from PC and PE, and is a common feature in eicosanoid-producing tissues (MacDonald and Sprecher, 1991). Shipley et al. (1993a, b) noted the endogenous arachidonate distribution between PC and PE was highly variable. Based on studies with A. americanum salivary glands in vitro (Bowman et al., 1995), we believe that ingested arachidonate is incorporated initially primarily into PC and then later transacylated in PE, eventually PE containing
232
ALAN S. BOWMAN et al.
the majority of arachidonate. Thus, the variable results of Shipley et al. (1993a, b) could be due to the feeding status of the tick at the time of detachment from the host and the length of time before the glands were dissected. The data obtained in the present study suggest that the tick has evolved to its parasitic life style with regards to eiconsanoid precursors in the salivary gland. The high dietary arachidonate intake has negated the necessity for its synthesis, whilst that arachidonate which is sequestered into the salivary gland is processed in a highly preferential manner. Current studies on the incorporation, remodelling and release of arachidonic acid in tick salivary glands will further our understanding of the regulation of eiconsanoid production in arthropods.
REFERENCES Bligh E. G. and Dyer W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917. Blomquist G. J., Dwyer L. A., Chu A. J., Ryan R. O. and de Renobales M. (1982) Biosynthesis of linoleic acid in a termite, cockroach and cricket. Insect Biochem. 12, 349-353. Borgeson C. E. and Blomquist G. J. (1993) Subcellular location of the A 12 desaturase rules out bacteriocyte contribution to linoleate biosynthesis in the house cricket and the American cockroach. Insect Biochem. Molec. BioL 23, 297-302. Borgeson C. E., Kurtti T. J., Munderloh U. G. and Blomquist G. J. (1991) Insect tissues, not microorganisms, produce linoleic acid in the house cricket and the American cockroach. Experientia 47, 238-241. Bowman A. S., Sauer J. R., Shipley M. M., Gengler C. L., Surdick M. R. and DiUwith J. W. (1993) Tick salivary prostaglandins: their precursors and biosynthesis. In Host Regulated Developmental Mechanisms in Vector Arthropods (Edited by Borovsky D. and Speilman A.), Vol. 3, pp. 169-177. University of Florida-IFAS, Vero Beach, Florida. Bowman A. S., Dillwith J. W., Madden R. D. and Sauer J. R. (1995) Uptake, incorporation and remodelling of arachidonic acid in isolated salivary glands of the lone star tick. Insect Biochem. Molec. Biol. (in press). Christie W. W. (1989) Silver ion chromatography using solid-phase extraction columns packed with a bonded-sulfonic acid phase. J. Lipid Res. 30, 1471-1473. Dadd R. H. (1983) Essential fatty acids: insects and vertebrates compared. In Metabolic Aspects o f Lipid Nutrition in Insects (Edited by Mettler T. E. and Dadd R. H.), pp. 107-142. Westview Press, Boulder, Colorado. de Renobales M., Ryan R. O., Heisler C. R., McLean D. L. and Blomquist G. J. (1986) Linoleic acid biosynthesis in the pea aphid, Acyrthosiphon pisum (Harris). Arch. Insect Biochem. Physiol. 3, 193-203. de Renobales M., Cripps C., Stanley-Samuelson D. W., Jurenka R. A. and Blomquist G. J. (1987) Biosynthesis of linoleic acid in insects. TIBS 12, 364-366. Dickenson R. G., O'Hagan J. E., Schotz M., Binnington K. C. and Hegarty M. P. (1976) Prostaglandin in the saliva of the cattle tick Boophilus microplus. Aust. J. Exp. Biol. Med. Sci. 54, 475-486. GilfiUan A. M., Chu A. J., Smart D. A. and Rooney S. A. (1983) Single plate separation of lung phospholipids including disaturated phosphatidylcholine. J. Lipid Res. 24, 1651-1656.
Higgs G. A., Vane J. R., Hart R. J., Potter C. and Wilson R. G. (1976) Prostaglandins in the saliva of the cattle tick, Boophilus microplus (Canestrini) (Acarina, Ixodidae). Bull. Entomol. Res. 66, 665-670. Holtzman M. J. (1991) Arachidonic acid metabolism. Am. Rev. Respir. Dis. 143, 188-203. Jurenka R. A., de Renobales M. and Blomquist G. J. (1987) De novo biosynthesis of polyunsaturated fatty acids in the cockroach Periplaneta americana. Arch. Biochem. Biophys. 255, 184-193. Jurenka R. A., Stanley-Samuelson D. W., Loher W. and Blomquist G. J. (1988) De novo biosynthesis of arachidonic acid and 5, I 1,14-eicosatrienoic acid in the cricket Teleogryllus commodus. Biochim. Biophys. Acta 963, 21-27. Kendall P. T. (1984) The use of fat in dog and cat diets. In Fats in Animal Nutrition (Edited by Wiseman J.), pp. 383-404. Butterworths, London. Luo C. (1993) Molecular biology approach to investigate enzymes in the tick salivary glands which are important in tick feeding. M.S. thesis, Oklahoma State University. MacDonald J. I. S. and Sprecher H. (1991) Phospholipid fatty acid remodeling in mammalian cells. Biochim. Biophys. Acta 1084, 105-121. Patrick C. D. and Hair J. A. (1976) Laboratory rearing procedures and equipment for multihost ticks (Acarina: Ixodidae). J. Med. Entomol. 12, 389-390. Ramachandra R. N. and Wikel S. K. (1992) Modulation of hostimmune responses by ticks (Acari: Ixodidae): effect of salivary gland extracts on host macrophages and lymphocyte cytokine function. J. Med. EntomoL 29, 818-826. Ribeiro J. M. C. 0987) Role of saliva in blood-feeding by arthropods. A. Rev. Entomol. 32, 463-478. Ribeiro J. M. C., Makoul G. T., Levine J., Robinson D. R. and Speilman A. (1985) Antihemostatic, anti-inflammatory, and immunosuppressive properties of the saliva of a tick, Ixodes dammini. J. Exp. Med. 161, 332-344. Ribeiro J. M. C., Evans P. M., McSwain J. L. and Sauer J. R. (1992) Amblyomma americanum: characterization of salivary prostaglandins E 2 and F2~ by RP-HPLC/bioassay and gas chromatographymass spectrometry. Exp. parasitoL 74, 112-116. Sauer J. R., Bowman A. S., Shipley M. M., Gengler C. L., Surdick M. R., McSwain J. L., Luo C., Essenberg R. C. and Dillwith J. W. (1993) Arachidonate metabolism in tick salivary glands. In Insect Lipids: Chemistry, Biochemistry and Biology (Edited by StanleySamuelson D. W. and Nelson D. R.), pp. 99-138. University of Nebraska Press, Lincoln. Sauer J. R., McSwain J. L., Bowman A. S. and Essenberg R. C. (1995) Tick salivary gland physiology. A. Rev. Entomol. 411 (in press). Shipley M. M., Dillwith J. W., Essenberg R. C., Howard R. W. and Sauer J. R. (1993a) Analysis of lipids in the salivary glands of Amblyomma americanum (L.): detection of a high level of arachidonic acid. Arch. Insect Biochem. Physiol. 23, 37-52. Shipley M. M., Dillwith J. W., Bowman A. S., Essenberg R. C. and Sauer J. R. (1993b) Changes in lipids of the salivary glands of the lone star tick, Amblyomma americanum, during feeding. J. Parasitol. 79, 834-842. Shipley M. M., Dillwith J. W., Bowman A. S., Essenberg R. C. and Sauer J. R. (1994) Distribution of arachidonic acid among phospholipid subclasses of lone star tick salivary glands. Insect Biochem. Molec. Biol. 2,4, 663-670. Sonenshine D. E. (1991) Biology of Ticks, Vol. 1, 447 pp. Oxford University Press, New York. Stauley-Samuelson D. W., Jurenka R. A., Loher W. and BIomquist G. J. (1987) Metabolism of polyunsaturated fatty acids by larvae of the waxmoth, Galleria mellonella. Insect Biochem. Physiol. 6, 141-149. Stauley-Samuelson D. W., Jurenka R. A., Cripps C., Blomquist G. J. and de Renobales M. 0988) Fatty acids in insects: composition, metabolism, and biological significance. Arch. Insect Biochem. Physiol. 9, 1-33. Weinert J., Blomquist G. J. and Borgeson C. E. (1993) De novo
TICK SALIVARY GLAND ARACHIDONATE biosynthesis of linoleic acid in two non-insect invertebrates: the land slug and the garden snail. Experientia 49, 919-921. Whitehead C. C. (1984) Essential fatty acids in poultry nutrition. In Fats in Animal Nutrition (Edited by Wiseman J.), pp. 153-167. Butterworths, London. Yu T. C. and Sinnbuber R. O. (1976) Growth responses of rainbow trout (Saline gairdneri) to dietary w 3 and w6 fatty acid. Aquaculture 8, 309-317.
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Acknowledgements--The authors thank Jerry Bowman and Mike Doss for supplying the ticks and cockroaches, respectively. We acknowledge Sharon White's secretarial skills and Robin Madden's help with figures. Approved for publication by the Director, Oklahoma Agricultural Experiment Station. This research was supported by NIH Grant AI-31460.
Pergamon
09~17,18(94)00059-X
Copyright© 1995ElsevierScienceLtd Printed in GreatBritain.All rightsreserved 0965-1748/95$9.50+ 0.00
Origin of Arachidonic Acid in the Salivary Glands of the Lone Star Tick, Amblyomma
americanum ALAN S. BOWMAN,t:~ JOHN R. SAUER,t PAUL A. NEESE,t JACK W. DILLWITH~" Received 9 May 1994; revised and accepted 23 July 1994
The contribution of synthesis and dietary sequestration to the high arachidonate content of the lone star tick, Amblyomma americanum, salivary glands was investigated by assessing the salivary metabulites of various radioinheled fatty acid substrates administered to partially fed females. A portion of each of the fatty acids studied was incorporated into the fatty acid moiety of the phosplmlipid fraction. [14C]acetate was metabolized only into myristic, palmitic, palmitoleic, stearic, and olcic acids. [~-l]oleic acid, p4C]linoleic acid, [14C]~-Iinolenic acid and [14C]cicosatrienoic acids were incorporated into salivary gland phospholipids but underwent Httle change including elongation and/or desuturation to arachidonate. Ingested [~H]arachidonic acid was readily taken up by the salivary gland and distributed among the lipid classes in a pattern markedly different from that of the other fatty acids tested. We conclude that ticks are unable to synthesize arnchidonic acid for incorporation into the salivary glands, but rather sequester it from the host bloodmeal. Fatty acids Biosynthesis Phospholipids Eicosatrienoic acid Arachidonic acid
Acetate
Oleicacid
Linoleicacid
Linolenicacid
Prostaglandins of the 2-series are synthesized from the precursor arachidonic acid via the cyclooxygenase Ticks surpass all other arthropods in the number and pathway (Holtzman, 1991). The salivary glands of variety of diseases that they transmit to domestic aniA m b l y o m m a americanum contain an unusually high conmals, and rank second only to mosquitoes as vectors of centration of arachidonic acid (8% of the total fatty human disease (Sonenshine, 1991). The effectiveness of acids) which is present only in the phospholipid ticks as pathogen vectors is enhanced by their prolonged fraction (Shipley et al., 1993a). Animals, including most host attachment and the immunosuppressive properties arthropods that have been studied to date, synthesize of tick salivary gland products (Ramachandra and arachidonic acid (20: 4, n-6) by elongation/ Wikel, 1992). Problems associated with prolonged desaturation of dietary linoleic acid (18:2, n-6) attachment are ameliorated by compounds in tick saliva (Stanley-Samuelson et al., 1988). Because nearly all exhibiting anti-inflammatory; analgesic; anti-coagulant; animals lack a A n desaturase, linoleic acid is considered anti-hemostatic; vasodilatorary; and/or immunosupan essential fatty acid. Blomquist and co-workers have pressive properties (Ribeiro, 1987; Sauer et al., 1995). shown that some insect species are capable of de novo Prostaglandins possess many of these properties and linoleic acid synthesis from acetate; e.g. a termite, cockhave been found in the saliva of several tick species roach, cricket (Blomquist et al., 1982) and pea aphid (Higgs et al., 1976; Dickenson et al., 1976; Ribeiro et al., (de Renobales et al., 1986). Conversely, dietary studies 1985, 1992). Prostaglandins are believed to play a major on the mosquito, Culex pipiens, have demonstrated that role in tick feeding (Sauer et aL, 1993) and, together with complete adult emergence requires a dietary source of their immunosuppressive activity, would appear to play both linoleic and arachidonic acid (Dadd, 1983). This a major role in tick-borne disease transmission and indicates that C. pipiens is incapable of converting pathogenesis. linoleie to arachidonic acid, and, therefore, is dependent upon the host bloodmeal for both of these essential fatty tDcpartrnent of Entomology, Oklahoma State University,Stillwater, acids. The origin of arachidonic acid in salivary glands is of OK 74078, U.S.A. :[:Author for correspondence. importance to an appreciation of the role of eicosanoids INTRODUCTION
225
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A L A N S. B O W M A N e t al.
in tick feeding. In this study, we assessed the relative contributions of de novo synthesis, conversion of dietary fatty acid precursors, and direct sequestration from the host to the high salivary gland arachidonate content. In addition, the fate of ingested fatty acids in the salivary gland was investigated.
[3H]oleic acid (2 #Ci/tick), [~4C]linoleic acid (0.75#Ci/tick) or [3H]arachidonic acid (1 #Ci/tick). Treatments with [~4C]7-1inolenic acid and [14C]di-homo7-1inolenic acid were restricted to ingestion (0.65/~Ci/tick) and injection (0.65/~Ci/tick) studies in mated females only. For ingestion studies, the solvent carrier was evaporated under nitrogen and the label resuspended in a 0.5ml microcentrifuge tube in 15#1 MATERIALS AND METHODS 68 mM NaCI, 0.25 mg/ml bovine serum albumin diluent with incubation at 37°C and vigorous vortexing. Label Materials [l-laC]Sodium acetate (54 mCi/mmol); [9,10-3H(N)]9 - was fed via glass capillary tubes placed over the tick's octadecenoic (oleic) acid (7.2Ci/mmol); [1-14C]9,12- mouthparts. The microcentrifuge tube was rinsed twice octadecadienoic (linoleic) acid (50mCi/mmol); and with a further 15/~1 diluent and the rinse fed to the ticks. [5,6,8,9,11,12,14,15-3H(N)] 5,8,11,14-eicosatetraenoic By this method, more than 85% of the label was ingested (arachidonic) acid (80 Ci/mmol) were purchased from by the ticks as assessed by the amount of radioactivity DuPont-New England Nuclear, Wilmington, Del. remaining in the microcentrifuge and capillary tubes. [ 1-~4C]6,9,12-Octadecatrienoic (7-1inolenic) acid For the injection studies, the labelled compound was (55mCi/mmol) and [1-~4C]8,11,14-eicosatrienoic (di- resuspended, as above, in 20/~1 diluent and 2 #1 carefully homo-7-1inolenic) acid (55 mCi/mmol) were obtained injected into the hemocoel of each tick with a 10#1 from American Radiolabeled Chemicals Inc., St Louis, syringe fitted with a 22 gauge needle. For the topical Mo. and [1-t4C]methanol (42mCi/mmol) from ICN, application studies, the label was resuspended in 15 pl Irvine, Penn. Thin-layer chromatography plates were ethanol and 1.5#1 applied to the dorsal cuticle and normal phase silica gel without fluorescent indicator allowed to evaporate. The ticks were maintained in a acquired from Analtech, Newark, Del. Silicic acid humid environment at room temperature. The salivary (BioSil A; 100-200 mesh) was purchased from BioRad, glands were dissected from ticks injected and fed with label after 48 and 72 h, respectively. Ticks undergoing Richmond, Calif. BondElut solid-phase extraction (SPE) columns topical application were so treated daily for up to 5 days, (500 mg) were obtained from P. J. Cobert Associates and their glands removed 24 h after the last application. Inc., St Louis, Mo. Lipid standards and other chemicals Mated females were administered the labelled precursors were acquired from Sigma Chemical Company, St Louis, by injection and feeding only, and their glands removed Mo. Liquid scintillation cocktail (BioCount) was ob- after 24 and 48 h, respectively. tained from Research Products International Corp., Lipid analysis Mount Prospect, I11. Reagent grade diethyl ether and Lipids were extracted from the salivary glands imdichloromethane were purchased from EM Science, mediately after collection by the method of Bligh and Gibbstown, N.J. High performance-liquid chromatogDyer (1959) and stored in chloroform with butylated raphy grade acetone, 2-propanol and acetonitrile were hydroxytoluene (5mg/100ml) at - 2 0 ° C for further supplied by Fisher Scientific, Pittsburgh, Penn. Hexane, analysis. Phospholipids, free fatty acids, and neutral chloroform and methanol (Fisher Scientific) were glass lipids were isolated from the lipid extract using 500 mg redistilled before use. amino-propyl SPE columns as described by Shipley et al. (1994). Following base hydrolysis of the phospholipid Collection of tissue fraction, fatty acid methyl esters (FAMEs) were preAdult female lone star ticks, Amblyomma americanum pared and purified as described by Shipley et al. (1993a). (L.), were reared on sheep according to the methods of Aliquots of the salivary gland phospholipid FAMEs Patrick and Hair (1976) and removed during the "slow- were fractionated according to their degree of unfeeding" phase (7-14 days on sheep). Adult virgin female saturation on silver impregnated 500 mg benzene sulA. americanum were reared in a similar manner in the fonic acid-SPE (Ag+-SPE) columns, as described by absence of males. Salivary glands were dissected while Christie (1989). The radioactivity in each fatty acid ticks were submerged in ice cold 0.1 M MOPS buffer, class was determined by liquid scintillation counting pH6.8, containing 0.02M ethylene glycol bis-(fl- (Beckman LS6000SC, Fullerton, Calif.) employing an aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). automatic quench correction curve. The separation Ticks which had undergone topical treatment with efficiency using the Ag+-SPE columns was determined radiolabelled substrate were rinsed by three 20 s rinses in using FAMEs prepared from salivary gland phosphohexane prior to salivary gland removal. lipid containing authentic labelled fatty acids. Radio-HPLC was performed on salivary gland phosFatty acid biosynthesis pholipid FAMEs with a C-8 reverse-phase column Groups of 10 partially fed adult virgin female (Supelcosil LC-8, particle size 5/~m, 25cm x 4.6mm A. americanum were fed, topically applied or injected i.d., Supelco, Bellafonte, Penn.) using a Waters Model into the hemocoel with either [~4C]acetate (1/~Ci/tick), 510 solvent delivery system. Authentic FAMEs were
TICK SALIVARYGLAND ARACHIDONATE
227
mixed with salivary gland phospholipid FAMEs mined by radio-scanning. Phospholipids were identified and introduced into the system dissolved in aceto- by comparing the chromatographic pattern of the salinitrile:chloroform (1:1, v/v) and eluted isocratically vary gland and phospholipid standards (Sigma, St Louis, with acetonitrile:water (85:15, v/v) at a flow rate of Mo.) under iodine vapor. 1 ml/min. Elution of FAMEs was monitored at 205 nm and radioactivity by an on-line Radiomatic Model CT Fit-one/Beta radioactivity flow detector (Radiomatic RESULTS Instruments and Chem. Co., Tampa, Fla.) Utilizing Scinti Verse LC (Fisher) as the liquid scintillation Fatty acid biosynthesis A portion of each of the fatty acid substrates tested cocktail. Counting efficiencies with this system were 64 and 46% for ~4C and 3H, respectively. Retention times was incorporated into the fatty acid moiety-of the of radioactive peaks were compared with those of phospholipid fraction in the salivary glands. Radiounlabelled authentic standards and a similar mixture HPLC of the salivary gland phospholipid FAMEs from of standards labelled by transmethylation with ticks after administration of [~4C]acetate (Fig. la) [14C]methanol. showed de novo biosynthesis of palmitate, palmitoleate, The tick salivary gland phospholipid FAMEs were stearate, and oleate. No evidence for the conversion of also subjected to gas chromatography (HP5890II, oleate to linoleate (Fig. lb) or any other product followHewlett-Packard, Sunnydale, Calif.) on a DB-225, ing [3H]oleic acid administration was observed. Neither 15 m x 0.53 mm, 1/~m film thickness megabore capillary elongation or desaturation products of administered column (J&W Scientific, Folsom, Calif.). The column [14C]linoleic acid, namely ~-linolenic, di-homo-~-linotemperature program was 120°C for 2 min, 5°C/min to lenic, or arachidonic acids, were detected (Fig. lc). No 200°C, 2°C/min to 225°C, hold for 2 min. This program further metabolism of [~4C]~-linolenic acid was detected gave complete baseline resolution for all the major (Fig. ld). Neither injected nor ingested [~4C]di-homo-vFAMEs. Authentic standards were added to the samples linolenic acid was converted to arachidonic acid and introduced via an on-column injector employing an (Fig. le), but there was evidence of fl-oxidation and oven-track program operating at 3°C above the oven subsequent synthesis of palmitate, palmitoleate, stearate temperature. Elution of FAMEs was monitored with a and oleate from ingested [~4C]eicosatrienoic acid thermal conductivity detector operating at 225°C. The (Fig. le). Attempts to isolate and concentrate possible eluting FAMEs were collected, at the appropriate times, desaturation products from each of the labelled prein chilled glass pasteur pipettes plugged with chloro- cursors studied using Ag2+-SPE prior to radio-HPLC form-dampened glass wool and then rinsed into vials resulted in essentially no radioactivity in the desired with 2 ml methanol. The methanol was evaporated and • fractions. Ticks fed [3H]arachidonic acid incorporated the radioactivity determine by liquid scintillation count- the label into the salivary gland phospholipid where it ing. Retention times of salivary gland FAMEs were was not converted into other fatty acids (Fig. If). The analytical results of the radio-HPLC determinations compared to those of authentic standards. were confirmed by radio-GC (data not shown) which Fate of ingested fatty acids were essentially the same except that radio-GC detected Virgin and mated A. americanum were fed [14C]acetate, a very small amount of myristate in ticks administered [3H]oleic acid, [14C]linoleic acid or [3H]arachidonic acid, [~4C]acetate. Virgin female ticks were topically applied [14C]linoleic as before. [laC]~-Linolenic and [14C]di-homo-~-linolenic acid were only fed to mated females. Salivary glands acid daily for up to 5 days and the salivary gland were removed 24 and 48 h later from the mated and phospholipid FAMEs fractionated on Ag÷-SPE virgin ticks, respectively, and the lipids extracted im- columns according to the degree of fatty acid unsatumediately (Bligh and Dyer, 1959). An aliquot of the ration (Table 1). The amount of radioactivity eluted in extracted lipid was separated on 20 x 20 cm silica gel G the triene and tetraene fractions were similar using plates, 250/~m thickness (Analtech, Neward, Del.) in FAMEs prepared from authentic [~4C]linoleic acid added hexane/diethyl ether/acetic acid (75: 25 : 1, v/v/v) and the to unlabelled salivary gland phospholipid. No further distribution of the label determined by radio-scanning desaturation of the diene fraction was observed over the (Bioscan 2000, BioScan, Washington, D.C.). Lipid experimental period as evidenced by the lack of increasclasses were identified by comparison to known lipid ing amounts of radioactivity eluted in the triene and standards (Sigma, St Louis, Mo.) under iodine vapor. tetraene fraction. That the diene fraction of the salivary The phospholipid fraction was isolated from the total gland phospholipid FAMEs is metabolically dynamic lipid extract using amino-propyl SPE columns, as above was shown by the time-dependent decrease in the diene (Shipley et al., 1994). Individual phospholipid classes fraction (r = 0.983, P < 0.01) and the concomitant inwere resolved on 20 x 20 cm, 250 #m thickness silica gel crease in the monoene (r = 0.955, P < 0.05) and the G channelled plates with pre-absorbent zone (Analtech, saturated fractions (r = 0.983, P < 0.02). Little difference was observed between virgin and Newark, Del.) in chloroform/hexane/methanol/acetic acid/boric acid (4:30:20:10:1.8, v/v/v/v/w) (Gilfillan mated ticks regarding biosynthesis and incorporation of et al., 1983) and the distribution of radioactivity deter- fatty acids. The route of radiolabel administration also
228
ALAN S. BOWMAN et al.
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FIGURE 1. Radio-HPLC chromatograms of fatty acid methyl esters from the phospholipid fraction of salivary glands from partially fed female A. americanum 24-48 h after administration of radiolabelled precursors. (a) females injected with [14C]acetate; (b) females fed [3H]oleicacid; (c) females fed with [14C]linoleicacid; (d) females fed with [t4C]~,-linolenicacid; (e) females fed and injected (inset) with [14C]di-homo-~-linolenicacid and (f) females fed with [3H]arachidonic acid. had no effect on the subsequent fatty acid biosynthesis in ticks, except for [t4C]di-homo-y-linolenic acid. The American cockroach, Periplaneta americana, has been shown to synthesize linoleic acid and arachidonic TABLE 1. Distribution of radioactivity among the fatty acid classes of salivary gland phospholipid from groups of 10 virgin female A. amerieanum topically applied daily with [[4C]linoleic acid (0.75#Ci/tick). Glands were dissected 24h after the last topical apphcation and the phospholipid FAMES fractionated according to degree of unsaturation on silver-impregnated benzene sulfonic acid solid-phase extraction columns, as described in the Materials and Methods Fatty acid class
Radioactivity distribution (% age) Day 2 Day 3 Day 4 Day 5
Saturated Monoene Diene Triene Tetraene Pentaene Hexaene
0.57 2.80 96.43 0.14a 0.04" 0.01 0.01
0.80 4.17 94.81 0.13 0.08 0.01 0.00
1.42 4.69 93.53 0.25 0.06 0.04 0.0!
2.02 5.09 92.66 0.15 0.05 0.02 0.01
"Note: Triene and tetraene fractions re-chromatographed as diene.
acid de novo from [14C]acetate (Jurenka et al., 1987) and was employed in the present study as a positive control. In our hands, linoleic acid (18:2, n-6), eicosanoic acid (20: 0), eicosadienoic acid (20: 2, n-6), eicosatrienoic acid (20:3, n-6), and arachidonic acid (20:4, n-6) were synthesized when adult female P. americana were injected with [14C]acetate, [3H]oleic acid, or [14C]linoleic acid and the whole b o d y lipids analysed as for the tick salivary glands (data not shown).
Fate o f ingested f a t t y acids Distribution o f radioactivity a m o n g the different lipid classes in salivary glands fed various fatty acid precursors was assessed by radio-TLC. Radioactivity was not incorporated into the sterol fraction with any o f the labelled precursors (Fig. 2). The phospholipid fraction contained the majority o f the radioactivity when [~4C]acetate was ingested, sterol esters contained only 2% with the remainder present equally in the free fatty acid and triglyceride pools (Fig. 2a). Following ingestion o f [3H]oleic acid (Fig. 2b), [~4C]linoleic acid (Fig. 2c),
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F I G U R E 2. Radio-chromatogram of TLC-separated lipid extracts of salivary glands from partially fed female A. americanum 24 h after ingesting: (a) [14qacetate; (b) [3H]oleic acid; (c) [t4qlinoleic acid; (d) [14C]y-linolenic acid; (e) [14qdi-homo-7-1inolenic acid; and (f) [3H]araehidonic acid. Arrows indicate position of the lipid standards: PL, phospholipid; S, sterol; FFA, free fatty acid; TG, triglyceride; and SE, sterol ester.
[~4C]7-1inolenic acid (Fig. 2d) and [14C]di-homo-7-1inolenic acid (Fig. 2e) radioactivity was largely present in the phospholipid fraction with lesser amounts in the triglyceride pool, and small but detectable radioactivity in the free fatty acid and sterol ,ester fractions. In contrast, radioactivity was incorporated solely in the phospholipid fraction of salivary glands when ticks were fed [3H]arachidonic acid (Fig. 20. Phospholipid was isolated by solid-phase extraction from tick salivary glands fed radiolabelled fatty acid precursors and the distribution of the radioactivity among the phospholipid classes assessed by radio-TLC (Fig. 3). An unidentified phospholipid migrating near the sphingomyelin standard, as reported by Shipley et al. (1993a), incorporated radioactivity only when [14C]acetate was the ingested precursor (Fig. 3a). Label was distributed between phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in a ratio approximating 70:30 when ticks had been fed [~4qacetate, [3I-I]oleic acid, and [14qlinoleic acid (Fig. 3a-c) and 60:40 with [t4C]~-linolenic acid and [~4qdi-homo-7-1inolenic acid (Fig. 3d-e). Conversely, [3H]arachidonic acid was incorporated into PC and PE in the ratio 25:75 (Fig. 30. No difference in the distribution of radioactivity among the
salivary gland lipid classes and phospholipid subclasses was observed between virgin and mated ticks. DISCUSSION In most organisms, even-numbered fatty acids of 16 (palmitate) and 18 (stearate) carbons long are synthesized de novo from acetate by the fatty acid synthase multi-enzyme complex and then interconverted to the required fatty acids by desaturation and elongation, as appropriate. Ticks are also capable of synthesizing palmitate (16:0) and stearate (18:0) de novo. In addition, these fatty acids are desaturated to form palmitoleate (16:1, n-7) and oleate (18:1, n-9). Recently a gene exhibiting high sequence homology to stearoyl CoA desaturase was identified in a cDNA library prepared from A. americanum salivary glands (Luo, 1993). It is accepted that linoleic acid is a dietary requisite as animals are unable to convert oleate to linoleate due to the absence of A~2 desaturase (Whitehead, 1984). However, the American cockroach, Periplaneta americana; the termite, Zooterrnopsis angusticollis; the house cricket, Acheta domesticus (Blomquist et al., 1982); and the Australian field cricket, Teleogryllus commodus
230
A L A N S. B O W M A N et al.
tJurenka et al., 1988) have been shown to be capable of synthesizing linoleic acid de novo. Linoleic acid biosynthesis in insects has been further studied to include 15 insect species representing four orders (see de Renobales et al., 1987) and has also been demonstrated in the land slug and garden snail (Weinert et al., 1993). Earlier reports suggested that the pea aphid, Acyrthosiphon pisum could not synthesize linoleate de novo (Blomquist et al., 1982), but later experiments showed that this insect is capable of synthesizing linoleic acid but required an extended incubation period, i.e. > 2 h (de Renobales et al., 1986). The tick does not synthesize linoleic acid from acetate nor oleic acid, despite incubation periods of up to 5 days. Rather it appears that ,4. americanum is dependent upon the host bloodmeal for its linoleate requirements. The majority of animals, including arthropods, cannot synthesize linoleic acid but can readily convert this dietary necessity into arachidonic acid by a series of desaturation and elongation steps (Stanley-Samuelson et al., 1987). In the present study, `4. americanum did not synthesize detectable amounts of arachidonic acid from any of the fatty acid precursors tested. Incubation periods of up to 5 days with linoleic acid failed to
produce any arachidonic acid even though the phospholipid linoleate was metabolically active as evidenced by increasing proportions of labelled saturated and unsaturated fatty acids being formed, presumably by fl-oxidation and subsequent de novo biosynthesis. Similarly, ticks seemed incapable of converting 7-1inolenic and di-homo-7-1inolenic acids to arachidonic acid. Indeed, apart from being able to synthesize palmitate and stearate, presumably by the action of the fatty acid synthase enzyme complex, and the subsequent conversion to palmitoleate and oleate by Agdesaturase, the tick was demonstrated to exhibit very limited fatty acid biosynthetic capability (Fig. 4). Therefore, it would appear that any fatty acid found in the tick salivary gland containing more than one double bond must originate from the host blood (Fig. 4). Interpretation of previous fatty acid biosynthetic studies on arthropods has been confounded by the problem of attributing the synthetic capability to insect tissues and dismissing contributions from symbiotic microorganisms (Borgeson et al., 1991; Borgeson and Blomquist, 1993). In the present study the fatty acid precursors were fed to ,4. americanum, thus, permitting an assessment of any contributions gut microflora may
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F I G U R E 3. R a d i o - c h r o m a t o g r a m o f T L C - s e p a r a t e d p h o s p h o l i p i d s f r o m salivary g l a n d s from p a r t i a l l y fed female A. americanum 24 h after ingesting: (a) ["C]acetate; (b) [3H]oleic acid; (c) []4C]linoleic acid; (d) [14C]~,-linolenic acid; (e) [14C]di-homo-y-linolenic acid; a n d (f) [3H]arachidonic acid. U N K = u n k n o w n p h o s p h o l i p i d ; P C = p h o s p h a t i d y l c h o l i n e ; PE = phosphatidylethanolamine.
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231
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FIGURE 4. Apparent fatty acid biosynthetic capability of A. americanum and origin of salivary gland fatty acids. Abbreviations: 2: 0, acetate; 16:0, palmitate; 18:0, stearate; 16:1, palmitoleate; 18:1, oleate; 18:2, linoleate; 20:2, eicosadienoate; 18:3, 7-1inoleneate,20:3, di-homo-7-1inolenic;20:4, arachidonate; and FAS, fatty acid synthaseenzymecomplex. provide to the fatty acid requirement of the tick. No effect of the route of precursor administration on subsequent fatty acid production in ticks was noted, except that di-homo-7-1inolenic acid underwent fl-oxidation only when ingested, presumably within the gut or possibly by gut microflora. The inability of the tick to convert linoleate to arachidonate is highly unusual, only the domestic cat (Kendall, 1984), rainbow trout (Yu and Sinnhuber, 1976) and the mosquito, Culex pipiens (Dadd, 1983) have been shown incapable of converting linoleate to arachidonate as inferred from nutrition studies, though the cat is reported capable of synthesizing arachidonate acid from y-linoleic acid (Kendall, 1984). Whilst salivary prostaglandins are believed to be important in successful tick feeding (Sauer et al., 1993), the necessity of the tick to be able to synthesize the prostaglandin precursor araehidonic acid appears to be unimportant. This is due to the tick's ability to sequester arachidonate from the diet and accumulate it in the salivary glands. Further evidence that tick arachidonate originates from the diet is seen in the dramatic increase in salivary gland arachidonate content in A. americanum females following attachment to the host (Shipley et al., 1993b). Previously, we demonstrated that, at least for sheep (Bowman et al., 1993) and dogs (A. S. Bowman, unpublished observations), the host blood and the components within would provide A. americanum with an adequate supply of arachidonate. Since both the mosquito and tick appear to lack the ability to synthesize araehidonic acid, it is tempting to postulate that this may be a common characteristic of blood-feeding arthropods. Fatty acid precursors were incorporated into various lipid classes in A. americanum salivary glands including phospholipid, free fatty acid, triglyceride and sterol ester, but not the sterol fraction. Thus, it appears that A. americanum, like other arthropods, lacks the ability
to synthesize sterols de novo. Acetate, oleate, linoleate, 7-1inoleneate, and di-homo-7-1inolenic were incorporated primarily into the phospholipid fraction, but also appeared in the triglyceride pool as suggested by endogenous fatty acid analysis of salivary gland lipids (Shipley et al., 1993a). Labelled arachidonic acid was recovered only in the phospholipid fraction, reflecting the distribution of endogenous arachidonate (Shipley et al., 1993a). That arachidonate is preferentially esterified in the phospholipid pool is common is eicosanoid-producing tissues (MacDonald and Sprecher, 1991). The specific distribution of the salivary gland lipids is likely due to the substrate affinities of lysophosphatide acyltransferase and acyl transferase involved in the phospholipid and triglyceride synthesis, respectively. Within the phospholipid fraction, label was incorporated mainly into PC and PE. Lesser amounts of acetate were also incorporated into an unknown phospholipid that migrates near sphingomyelin by TLC. This phospholipid contains predominantly (>70%) saturated fatty acids (Shipley et al., 1993a) and, is thus, less likely to be labelled by the unsaturated fatty acid treatments. Acetate, oleate, and linoleate were incorporated into PC and PE in a ratio approximating 70: 30, respectively, and 7-1inoleate and di-homo-y-linolenic acid in a ratio 60: 40. Arachidonate was recovered in PC and PE in the ratio 27:75, respectively. This specific distribution is thought to be due to the remodelling process involving transacylase, which transfers arachidonate from PC and PE, and is a common feature in eicosanoid-producing tissues (MacDonald and Sprecher, 1991). Shipley et al. (1993a, b) noted the endogenous arachidonate distribution between PC and PE was highly variable. Based on studies with A. americanum salivary glands in vitro (Bowman et al., 1995), we believe that ingested arachidonate is incorporated initially primarily into PC and then later transacylated in PE, eventually PE containing
232
ALAN S. BOWMAN et al.
the majority of arachidonate. Thus, the variable results of Shipley et al. (1993a, b) could be due to the feeding status of the tick at the time of detachment from the host and the length of time before the glands were dissected. The data obtained in the present study suggest that the tick has evolved to its parasitic life style with regards to eiconsanoid precursors in the salivary gland. The high dietary arachidonate intake has negated the necessity for its synthesis, whilst that arachidonate which is sequestered into the salivary gland is processed in a highly preferential manner. Current studies on the incorporation, remodelling and release of arachidonic acid in tick salivary glands will further our understanding of the regulation of eiconsanoid production in arthropods.
REFERENCES Bligh E. G. and Dyer W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917. Blomquist G. J., Dwyer L. A., Chu A. J., Ryan R. O. and de Renobales M. (1982) Biosynthesis of linoleic acid in a termite, cockroach and cricket. Insect Biochem. 12, 349-353. Borgeson C. E. and Blomquist G. J. (1993) Subcellular location of the A 12 desaturase rules out bacteriocyte contribution to linoleate biosynthesis in the house cricket and the American cockroach. Insect Biochem. Molec. BioL 23, 297-302. Borgeson C. E., Kurtti T. J., Munderloh U. G. and Blomquist G. J. (1991) Insect tissues, not microorganisms, produce linoleic acid in the house cricket and the American cockroach. Experientia 47, 238-241. Bowman A. S., Sauer J. R., Shipley M. M., Gengler C. L., Surdick M. R. and DiUwith J. W. (1993) Tick salivary prostaglandins: their precursors and biosynthesis. In Host Regulated Developmental Mechanisms in Vector Arthropods (Edited by Borovsky D. and Speilman A.), Vol. 3, pp. 169-177. University of Florida-IFAS, Vero Beach, Florida. Bowman A. S., Dillwith J. W., Madden R. D. and Sauer J. R. (1995) Uptake, incorporation and remodelling of arachidonic acid in isolated salivary glands of the lone star tick. Insect Biochem. Molec. Biol. (in press). Christie W. W. (1989) Silver ion chromatography using solid-phase extraction columns packed with a bonded-sulfonic acid phase. J. Lipid Res. 30, 1471-1473. Dadd R. H. (1983) Essential fatty acids: insects and vertebrates compared. In Metabolic Aspects o f Lipid Nutrition in Insects (Edited by Mettler T. E. and Dadd R. H.), pp. 107-142. Westview Press, Boulder, Colorado. de Renobales M., Ryan R. O., Heisler C. R., McLean D. L. and Blomquist G. J. (1986) Linoleic acid biosynthesis in the pea aphid, Acyrthosiphon pisum (Harris). Arch. Insect Biochem. Physiol. 3, 193-203. de Renobales M., Cripps C., Stanley-Samuelson D. W., Jurenka R. A. and Blomquist G. J. (1987) Biosynthesis of linoleic acid in insects. TIBS 12, 364-366. Dickenson R. G., O'Hagan J. E., Schotz M., Binnington K. C. and Hegarty M. P. (1976) Prostaglandin in the saliva of the cattle tick Boophilus microplus. Aust. J. Exp. Biol. Med. Sci. 54, 475-486. GilfiUan A. M., Chu A. J., Smart D. A. and Rooney S. A. (1983) Single plate separation of lung phospholipids including disaturated phosphatidylcholine. J. Lipid Res. 24, 1651-1656.
Higgs G. A., Vane J. R., Hart R. J., Potter C. and Wilson R. G. (1976) Prostaglandins in the saliva of the cattle tick, Boophilus microplus (Canestrini) (Acarina, Ixodidae). Bull. Entomol. Res. 66, 665-670. Holtzman M. J. (1991) Arachidonic acid metabolism. Am. Rev. Respir. Dis. 143, 188-203. Jurenka R. A., de Renobales M. and Blomquist G. J. (1987) De novo biosynthesis of polyunsaturated fatty acids in the cockroach Periplaneta americana. Arch. Biochem. Biophys. 255, 184-193. Jurenka R. A., Stanley-Samuelson D. W., Loher W. and Blomquist G. J. (1988) De novo biosynthesis of arachidonic acid and 5, I 1,14-eicosatrienoic acid in the cricket Teleogryllus commodus. Biochim. Biophys. Acta 963, 21-27. Kendall P. T. (1984) The use of fat in dog and cat diets. In Fats in Animal Nutrition (Edited by Wiseman J.), pp. 383-404. Butterworths, London. Luo C. (1993) Molecular biology approach to investigate enzymes in the tick salivary glands which are important in tick feeding. M.S. thesis, Oklahoma State University. MacDonald J. I. S. and Sprecher H. (1991) Phospholipid fatty acid remodeling in mammalian cells. Biochim. Biophys. Acta 1084, 105-121. Patrick C. D. and Hair J. A. (1976) Laboratory rearing procedures and equipment for multihost ticks (Acarina: Ixodidae). J. Med. Entomol. 12, 389-390. Ramachandra R. N. and Wikel S. K. (1992) Modulation of hostimmune responses by ticks (Acari: Ixodidae): effect of salivary gland extracts on host macrophages and lymphocyte cytokine function. J. Med. EntomoL 29, 818-826. Ribeiro J. M. C. 0987) Role of saliva in blood-feeding by arthropods. A. Rev. Entomol. 32, 463-478. Ribeiro J. M. C., Makoul G. T., Levine J., Robinson D. R. and Speilman A. (1985) Antihemostatic, anti-inflammatory, and immunosuppressive properties of the saliva of a tick, Ixodes dammini. J. Exp. Med. 161, 332-344. Ribeiro J. M. C., Evans P. M., McSwain J. L. and Sauer J. R. (1992) Amblyomma americanum: characterization of salivary prostaglandins E 2 and F2~ by RP-HPLC/bioassay and gas chromatographymass spectrometry. Exp. parasitoL 74, 112-116. Sauer J. R., Bowman A. S., Shipley M. M., Gengler C. L., Surdick M. R., McSwain J. L., Luo C., Essenberg R. C. and Dillwith J. W. (1993) Arachidonate metabolism in tick salivary glands. In Insect Lipids: Chemistry, Biochemistry and Biology (Edited by StanleySamuelson D. W. and Nelson D. R.), pp. 99-138. University of Nebraska Press, Lincoln. Sauer J. R., McSwain J. L., Bowman A. S. and Essenberg R. C. (1995) Tick salivary gland physiology. A. Rev. Entomol. 411 (in press). Shipley M. M., Dillwith J. W., Essenberg R. C., Howard R. W. and Sauer J. R. (1993a) Analysis of lipids in the salivary glands of Amblyomma americanum (L.): detection of a high level of arachidonic acid. Arch. Insect Biochem. Physiol. 23, 37-52. Shipley M. M., Dillwith J. W., Bowman A. S., Essenberg R. C. and Sauer J. R. (1993b) Changes in lipids of the salivary glands of the lone star tick, Amblyomma americanum, during feeding. J. Parasitol. 79, 834-842. Shipley M. M., Dillwith J. W., Bowman A. S., Essenberg R. C. and Sauer J. R. (1994) Distribution of arachidonic acid among phospholipid subclasses of lone star tick salivary glands. Insect Biochem. Molec. Biol. 2,4, 663-670. Sonenshine D. E. (1991) Biology of Ticks, Vol. 1, 447 pp. Oxford University Press, New York. Stauley-Samuelson D. W., Jurenka R. A., Loher W. and BIomquist G. J. (1987) Metabolism of polyunsaturated fatty acids by larvae of the waxmoth, Galleria mellonella. Insect Biochem. Physiol. 6, 141-149. Stauley-Samuelson D. W., Jurenka R. A., Cripps C., Blomquist G. J. and de Renobales M. 0988) Fatty acids in insects: composition, metabolism, and biological significance. Arch. Insect Biochem. Physiol. 9, 1-33. Weinert J., Blomquist G. J. and Borgeson C. E. (1993) De novo
TICK SALIVARY GLAND ARACHIDONATE biosynthesis of linoleic acid in two non-insect invertebrates: the land slug and the garden snail. Experientia 49, 919-921. Whitehead C. C. (1984) Essential fatty acids in poultry nutrition. In Fats in Animal Nutrition (Edited by Wiseman J.), pp. 153-167. Butterworths, London. Yu T. C. and Sinnbuber R. O. (1976) Growth responses of rainbow trout (Saline gairdneri) to dietary w 3 and w6 fatty acid. Aquaculture 8, 309-317.
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Acknowledgements--The authors thank Jerry Bowman and Mike Doss for supplying the ticks and cockroaches, respectively. We acknowledge Sharon White's secretarial skills and Robin Madden's help with figures. Approved for publication by the Director, Oklahoma Agricultural Experiment Station. This research was supported by NIH Grant AI-31460.