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De novo biosynthesis of prostaglandins by the australian field cricket, Teleogryllus commodus
0306-4492/86$3.00 + 0.00 Pergamon Journals Ltd
Camp. Biochem. Physiol. Vol. 85C, No. 2, pp. 303-307, 1986 Printed in Great Britain
DE NO VO BIOSYNTHESIS OF PROSTAGLANDINS BY THE AUSTRALIAN FIELD CRICKET, TELEOGRYLLUS DAVID
STANLEY-SAMUELSON*,
and *Department
of Entomological Sciences, tDepartment of Biochemistry,
COMMODUS
RUSSELL A. WERNER
University University
JURENKA?,
GARY
J. BLoMQu1s-rt
LOHER*
of California, Berkeley, CA 94720, of Nevada, Reno, NV 89557, USA
USA
and
(Received 3 February 1986) Abstract-l. The accumulation and metabolism of certain polyunsaturated fatty acids by testes from the Australian field cricket, Teleogryllus comtnodus,are described. Testes accumulated a substantial proportion (about 16%) of label from radioactive C20: 3n6 that was injected into the haemocoel. Fifty percent of the label accumulated by testes was associated with the phospholipid fraction, whereas in the remainder of the body 30% was incorporated into the phospholipid fraction. 2. Prostaglandins (PG) E,, E, and F,, were quantified in extracts of the testes of adult insects by radioimmunoassay. 3. Label from injected radioactive Cl8: 2n6, C20: 3n6 and C20:4n6 was recovered as prostaglandins PGE and PGF. The radioactivity from C18:2n6 that was recovered as PGE, and PGF,, indicated elongation/desaturation to C20:3n6 followed by conversion to PG. 4. Since C18: 2n6 is readily formed from acetate in T. commodus, these findings indicate the de nova biosynthesis of C20 polyunsaturated fatty acids and prostaglandins by this species.
INTRODUCTION
(PGs), derivatives of certain C20 polyunsaturated fatty acids (PUFAs), or PGsynthesizing activity have been detected in reproductive tracts of the crickets A&eta domesticus (Destephano and Brady, 1977) and Teleogryllus corn modus (Loher et al., 1981), the moths Bombyx mori (Yamaja Setty and Ramaiah, 1979) and Trichopfusia ni (Hagen and Brady, 1982), the locust Locusta migratoria (Lange, 1984) and the housefly Musca domestica (Wakayama et al., 1980). In addition to reproductive tissues, PGs were detected in heads, thoraces and muscles in a number of species (Murtaugh and Denlinger, 1982) and in heads and ventral nerve cords of T. commodus (StanleySamuelson et al., 1983). So far, knowledge of biological activities of PGs in insects is limited. PGs are implicated in the release of egg-laying behavior in the crickets A. domesticus (Destephano and Brady, 1977) and T. commodus (Loher, 1979; Loher et al., 1981) and, to a lesser extent, in the moth Bombyx mori (Yamaja Setty and Ramaiah, 1980). In light of the wide distribution of PGs among insect tissues and species, we adopt the working hypothesis that other biological roles, possibly analogous to known cellular and tissue-level modulation in mammals (Horrobin, 1978), will eventually be discerned. Even less is known about the biochemistry of the C20 PUFAs, C20: 3n6 and C20:4n6, that are direct precursors to the l- and 2-series PGs, respectively. While C20 PUFAs may be obtained directly from the diets of carnivorous and omnivorous insects, strictly phytophagous insects may further elongate and desaturate dietarily supplied Cl8 PUFAs to C20, as suggested for the waxmoth, G. Mellonella (Dadd, 1983a; Stanley-Samuelson and Dadd, 1984). Still Prostaglandins
303
other insects are able to biosynthesize at least one Cl8 PUFA, linoleic acid (Blomquist et al., 1982), which, in concert with further desaturation and elongation, provides the basis for de nouo biosynthesis of C20:3n6 in males of T. commodus (Stanley-Samuelson et al., 1986). If, as observed in other insects (Destephano et al., 1974; Yamaja Setty and Ramaiah, 1979), PGE, is formed in tissues of T. commodus, the biosynthesis of C20: 3n6 would suggest the complete synthesis of a PG. Here it is reported that C20: 3n6 is efficiently taken up from the circulating hemolymph and incorporated into testicular phospholipids. Label associated with radioactive C20: 3n6 and C20:4n6 injected into the haemocoel of males was recovered as, respectively, l-series and 2-series PGs. Since C18: 2n6 and C20:3n6 are formed from injected acetate (Stanley-Samuelson et aI., 1986), de nouo synthesis of PGE, and PGF,, is suggested. MATERIALS
AND METHODS
field cricket, Teleogryllus comcolony maintained at 27 & 1°C and an LD 12: 12 hr cycle. Details are described by Loher (1981). [1-14C]C20:3n6 (54.9mCi/mmole), [5,6,8,9,11,12,14, 15-3H]C20:4n6 (83.8 Ci/mmole) and [U-14C]C18:2n6 (0.5 mCi/mmole) were purchased from New England Nuclear. Various amounts of substrate, specified in context of individual results, were injected into the abdomens of adult males by inserting the needle of a 10 ~1 Hamilton 701 syringe between the seventh and eighth abdominal sclerites, pushing the needle along the lateral body wall about two sclerites anteriorly, then depressing the plunger. After specified incubation periods, the testes and the rest of the bodies were separately extracted. To determine the distribution of label between the testes and bodies and the incorporation of labelled fatty acid into lipid fractions, lipids were Males of the Australian
modus, were taken from a laboratory
304
DAVID STANLEY-SAMUELSONet al.
according to Bligh and Dyer (1959). The lipid extract was separated into phospholipids (PLs), triacylglycerols (TGs), monoacylglycerols, diacylglycerols and free fatty acids (MG, DG, FFA) and waxes by developing on Kieselgel thin layer chromatography (TLC) plates in hexanediethyl ether-formic acid (90: 10: 1, v/v/v). Material was visualized under U.V. light after spraying with Rhodamine 6G. Each fraction was scraped into a test tube. The PL were extracted according to the method of Arvidson (1967) and the other lipid fractions were extracted with diethyl ether. Radioactivity of lipid extracts and fractions was estimated by liquid scintillation counting (LSC) of aliquots in 0.4% diphenyloxazole in toluene at 4547% efficiency for tritium and f&88% for carbon-14. For studies of PG biosynthesis, testes and bodies were separately extracted three times in acidified ethyl acetate. PGs were separated by chromatography on Biosil A columns (Wakayama et al., 1986). The PGE and PGF fractions were collected and either counted by LSC (above) or further separated into l- and 2-series PGs by chromatography on 10% AgNO, impregnated Silica Gel Type H (w/w) TLC plates in chloroform-methanol-acetic acid (80: 10: 10, v/v/v). Label associated with PGE,, PGE,, PGF,, and PGF,, was identified by co-chromatography with authentic standards. Individual fractions were extracted from the Silica gel three times with ethyl acetate and counted by LSC. Quantities of PGE, , PGE, and PGF, were estimated in extracts of testes taken from 2- and ldday old adults by commercial radioimmunoassay (RIA) kits (Seragen, Inc., Boston, Ma.). Given values reflect correction for extraction efficiency, monitored in separate tests by estimating recoveries of [‘HIPG. extracted
RESULTS
Incorporation of labelled C20: 3n6 into tissue lipid fractions The differential incorporation of C20 : 3n6 by testes with respect to the rest of the body and distribution
of radioactivity among various lipid fractions were investigated. Radiolabelled C20 : 3n6 (0.2 PCi) was injected into adult males; after 2 hr the testes and the remaining insect body were separately extracted, the total extract was separated into four lipid fractions and radioactivity assayed by liquid scintillation counting. The testes accumulated 16.4 (SD = lO.S%, N = 5) of the radioactivity injected as C20: 3n6, the other 83.6% (SD = 10.5%) being recovered from the rest of the body (data not shown). Table 1 shows the distribution of radioactivity recovered in various lipid fractions after 2 hr following injection of C20:3n6 into abdomens of adult males. Of the label recovered from testes, 50% was in PL, about 14% in TG, 35% in MG, DG, FFA, and Table 1. Distribution of [1-‘4C]20: 3 by lipid fractions in various bodv Darts of male TeleowvNus commodus*
Table 2. Quantities of prostaglandins, as RIA-equivalents, in extracts of testes taken from 2- and 16-day old adult crickets. Given values represent PG RIA-equivalents + I SE (number of individual aairs of testes) PGE, PGE, PGP,,
Day 2 adults
Day 16 adults
635.0 + 66.2 (4) 1032.3 + 178.7 (7) 163.7 + 18.7 (12)
452.0 + 103.2 (5) 1770.0 + 135.4(5) 368.3 k 87.9 (6)
0.5% in waxes. The pattern differed for label recovered from the rest of the body, with lower proportions in PL and higher in TG and MG, DG, FFA. This pattern is also similar to results on distribution of arachidonic acid with respect to lipid fractions (Stanley-Samuelson et al., 1986); C20 PUFAs accumulated by testes thus appear to be efficiently incorporated into PLs. Determination of prostaglandins in extracts of testes
PGs have been detected in spermathecae, ovaries and nervous tissues from females of T. commodus (Loher et al., 1981; Stanley-Samuelson et al., 1983, and one aspect of the significance of C20: 3n6 and C20 :4n6 in these tissues (references above) is thought to be their roles as PG precursors. Table 2 shows the PGE,, PGEl and PGF,, levels that have been determined as RIA-equivalents in extracts of testes from 2- and 16-day old adults. The occurrence of the analogous parental fatty acids has been documented elsewhere (Stanley-Samuelson et al., 1986). Prostaglandin biosynthesis in males of T. commodus
Following determination of 1- and 2-series PGs and their analogous precursor C20 PUFAs in extracts of testes, the possibility was tested that male crickets would be able to convert radiolabelled fatty acid substrate into PGs in vivo. [‘4C]C20: 3n6 (0.6 PCi) and t3H]C20:4n6 (0.75 PCi) were separately injected into the abdomens of adult males and after various incubation times the testes and the remainder of the body were separately extracted and analyzed for PGs. Table 3 presents the proportions of injected radiolabel associated with C20 PUFAs that were recovered as PGs at 2, 10 and 120 min after injection. The proportion of radioactivity associated with C20: 3n6 recovered as PG in the testes declined from 1.1 to 0.1% over the 2 min to 2 hr incubation range. Similar declines were observed with C20:4n6 injections. These findings are consistent with the prod-
Table 3. Incorporation of ‘%20:3 and ‘H20:4 into prostglandins after injection into male Teleogryllus commodus for various time Percentage of injected radiolabel recovered in PG fraction 20:4 20:3
Percent of recovered [I-“C]20: 37 Lipid fraction PL MG, DG, FFA TG Waxes
Testes 50.3 + 34.9 + 14.4 k 0.5 f
20.0 25.0 12.8 0.4
Rest of insect 29.4 5 49.4 + 19.8 k I .5 f
7.9 12.7 5.8 0.4
*[l-“C]20:3 was injected into adult males and after 2 hr. the insects were killed. The testes and remainder of the insect were extracted, lipid fractions separated and radioactivity assayed, as described in Materials and Methods. tMean f SD (N = 4).
Incubation
_...._
time
(min)
Testes
Rest of insect
Testes
2 10 120
I.1 0.4 0.1
1.2 0.7 0.4
0.8 0.4 0.1
Rest ~~ of insect 0.8 0.6 0.4
l“C20: 3 and ‘H20:4 were injected into adult male crickets and after various incubation periods the testes and remainder of the insect were extracted and analyzed for prostaglandins (see Materials and Methods).
Synthesis of PG by cricket uction, followed by uptake and excretion, of radioactivity associated with PGs by the Malpighian tubule/hindgut complex, as described for females of T. commodus (Stanley-Samuelson and Loher, 1985) and for the housefly (Wakayama et al., 1986). The proportions of radioactivity recovered as PGs were approximately equal in testes and the rest of the body (Table 3). Given that the testes represent about 10 times less tissue than the whole animal (about 35 vs over 300mg), these data suggest that either the testes convert 20 : 3 to PG very efficiently or that they accumulate PGs produced elsewhere in the body. The idea that PGs may be taken up from the circulation by individual tissues is supported by the uptake of PG-related label by ovaries of T. commodus (StanleySamuelson and Loher, 1985). Table 3 shows that 2 min after injection of radiolabelled fatty acids, approximately similar proportions of the injected radiolabel recovered in the PG fraction came from C20 : 3n6 and C20: 4n6, indicating substantial potential for production of l- and 2-series PGs. Based on previous work which demonstrated the biosynthesis of C20:3, but not C20:4, from acetate and from linoleic acid (Stanley-Samuelson et al., 1986), it was reasoned that any PGs formed from these latter substrates would be derived from C20: 3n6 and yield l-series rather than 2-series PGs. This idea was tested by injecting radiolabelled Cl8 :2n6 into males daily for three days. Testes and the remainder of the bodies were separately extracted for PGs, followed by separation into E- and F-series PGs by column chromatography. The PGE and PGF fractions were further separated into 1- and 2-series PGs by AgNO,-TLC. This procedure separates the PGs according to the number of double bonds, with the l-series PGs ascending slightly ahead of the 2-series. This order of separation necessitates correction for label associated with l-series PGs tailing with the 2-series, done by spotting radioactive PGE, and PGF, standards on the TLC plates, followed by calculating the proportions of counts recovered in the l-series location. Table 4 shows the amount of radioactivity recovered as 1- and 2-series PGs from the testes and the rest of the body. Allowing for the 25-30% of the l-series label tailing with the 2-series (Table 4), it appears that all of the label recovered from the whole body (less testes) occurs as l-series PG. Respectively, 55 and 65% of the label recovered from testes occurred as PGE, and PGF,,. Subtracting l-series Table 4. Separation of one and two series prostaglandins after in uiuo synthesis from “C-18 :2 in the male cricket Teleogryllus commodus * DPM recovered source of prostaglandin Testes Rest of insect PGE, standard PGE,, standard
PGE,t 3871 (54.7) 27,766 (72.8) 1373 (71.7)
in each fraction
PGE,
PGF,,
3201 10,380 542
5364 (65.5) 31,419 (70.7)
2830 13,000
1900 (76.0)
600
PGF,,
*%-IS:2 was injected into adult male crickets for three days and testes and remainder of the insect were extracted for prostaglandins. E and F series prostaglandins were separated on Biosil A columns and these were separated further into the one and two series by AgNO, TLC (see Materials and Methods). tNumbers in parentheses refer to the percent of label recovered in the one series. C.B.P.85,2C--D
305
trailing, it would appear that about 15% of the PGE, and about 10% of PGF, radioactivity elutes with the 2-series PG. DISCUSSION
The data presented here show that testes of T. commodus differentially incorporate injected C20: 3n6 into PLs. Including results from previous work on testicular bioaccumulation of injected fatty acids in this species (Stanley-Samuelson et al., 1986), this efficient incorporation is probably specific to PUFAs: Cl 8 : 2n6, C20 : 3n6 and C20 : 4n6 are all rapidly taken up relative to the saturated fatty acid, stearate. Similarly, the housefly, M. domestica, efficiently accumulates labelled C20 : 3n6 and C20 : 4n6 into the PL fraction, specifically into the 2-position, compared to a saturated fatty acid (Wakayama et al., 1985). This work also reports the detection of PGE,, PGE, and PGF,, as RIA-equivalents in extracts of testes, as well as in vivo biosynthesis of PG from injected substrate. The biological significance of PGs in the testes of T. commodus remains unclear. Following mating and transfer of PG-synthetase activity, PGs formed in the spermathecae release egg-laying behavior in the female; but the PGs per se are not transferred in the spermatophore (Loher et al., 1981). On the other hand, PGs have been detected in many tissues of this cricket (Stanley-Samuelson et al., 1983; Stanely-Samuelson and Loher, unpublished observations), including Malpighian tubules and certain glands. If tissue and cellular activities are in part regulated by local turn-over of PGs, as thought for mammals, the detection of PGs and of PG-synthesis activity in testes may be considered under the mantle of local modulation. In vivo biosynthesis of PGs from three substrates, C20 : 4n6, C20 : 3n6 and C 18 : 2n6, was observed. The data from experiments with C20: 3n6 and C20:4n6 indicates substantial capacity for biosynthesis of land 2-series PGs from immediate precursors by males, and extends the known locations of PG biosynthesis in T. commodus. The formation of PGs from linoleic acid is to be interpreted with respect to PUFA metabolism. In mammals, Cl 8 : 2n6 undergoes desaturation to Cl 8 : 3n6, elongation to C20 : 3n6, and another desaturation to C20:4n6 (Sprecher, 1977). Formation of two l-series PGs from Cl8: 2n6 in T. commodus is consistent with desaturation and elongation to C20 : 3n6, followed by formation of l-series PGs. Table 4 shows that in the testes about 25 and lo%, respectively, of the PGE and PGF radioactivity chromatographed as 2-series PGs after correcting for l-series tailing on AgN03-TLC. It would appear from this finding that a small proportion of C20 : 3n6 may undergo an additional desaturation to C20:4n6, followed by derivatization to 2-series PG. Since no evidence for the desaturation to C20 : 4n6 emerged in earlier work on this question (Stanley-Samuelson et al., 1986), F-series PGs, prepared after incubation with injected radioactive C18:2, were chromatographed on a radio-high pressure liquid chromatograph (radio-HPLC) optimized for separation of l- and 2-series PGs. This yielded a single peak of radioactivity corresponding with l-series PG. In metabolism of injected separate experiments,
DAVID STANLEY-SAMUELSON et al.
306
Cl8 : 2n6 to C20 PUFAs was reexamined by radioHPLC. This also supported the earlier findings of synthesis of C20: 3n6 with no radioactivity associated with C20:4n6. Summarizing literature and analytical evidence, Stanley-Samuelson and Dadd (1983) concluded that long-chain PUFAs were probably a general feature of insect lipids, most readily observed in PLs and extracts of more membraneous tissues. One implication of this idea, especially for strictly phytophagous insects, but also for all insects whose dietary PUFA complement differs from local tissue and cellular requirements, would be a greater or lesser capability for metabolism of long-chain PUFAs. T. commodus, now shown capable of de novo biosynthesis of at least two PUFAs and the 1-series PGs, represents a condition that differs fundamentally from the commonly appreciated mammalian pattern and from some other insect groups. The de novo biosynthesis of linoleic acid, the parental unit of all n6 PUFAs, is now known in many insects representing several orders (Blomquist et al., 1982; Cripps et al., 1986). If these linoleate biosynthesizers can perform further elongations and desaturations, the biosynthesis of C20 PUFAs, and possibly PGs, may eventually be perceived as wide-spread among insects. Just as we do not yet known the distribution of biosynthetic capability with respect to PUFAs and PGs among insect species and orders, the range of fatty acid metabolic patterns is also unclear. So far, we can identify three patterns. The waxmoth pattern features the elongation and desaturation of dietary Cl8 : 3n3 to C20: 5n3 without a comparable elongation and desaturation of the linoleic acid family members (Dadd, 1983a; Stanley-Samuelson and Dadd, 1984). The mosquito pattern, most thoroughly analyzed in Culex pipiens, is characterized by dietary requirement for vitamin-level quantities of arachidonic or certain structurally related fatty acids (Dadd, 1983b). Since linoleic acid cannot fulfill this need, an inability to introduce more unsaturation into the parental PUFA is implied. T. commodus represents a third, cricket, pattern. Future work will uncover still other patterns and help clarify how many insect species are represented by the known patterns; as work proceeds, metabolism of PUFAs may come to be of increasing significance in insect physiology and biochemistry. Acknowledgements-We thank Dr R. H. Dadd for reading and suggesting improvements to the manuscript. Supported by NIH Grant ROl HDO3619 to W.L., by NSF Grant DCB-8309146 and the Nevada Agricultural Experiment Station funds to G.J.B.
REFERENCES Arvidson G. A. E. (1967) Reversed-phase partition thinlayer chromatography of rat liver lecithens to yield eight simple phosphatidyl cholines. J. Lipid Res. 8, 155-158. Bligh E. G. and Dyer W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 91 l-917. Blomquist G. J., Dwyer L. A., Chu A. J., Ryan R. 0. and de Renobales M. (1982) Biosynthesis of linoleic acid in a termite, cockroach and cricket. Insect B&hem. 12, 349-353.
Cripps C., de Renobales M. and Blomquist G. J. (1986) De nova biosynthesis of linoleic acid in insects. Biochim. biophys. Acia 876, 572-580. Dadd R. H. (1983a) Long-chain polyenoics and the essential dietary fatty acid requirement of the waxmoth, Galleria mellonella. J. Insect Physiol. 29, 7799786. Dadd R. H. (1983b) Esserftial fatty acids-insects and vertebrates compared. In Metabolic Aspects of Lipid Nutrition in Insects (Edited by Mittler T. E. and Dadd R. H.), pp. 107-147. Westview Press, Boulder, Colorado. Destephano D. B. and Brady V. E. (1977) Prostaglandin and prostaglandin synthetase in the cricket, Acheta domesticus. J. Insect Physiol. 23, 905-911, Destephano D. B., Brady U. E. and Lovins R. E. (1974) Synthesis of prostaglandin by reproductive tissue of the male house cricket, Acheta domesticus. Prostaglandins 6, 71-79. Hagen D. V. and Brady V. E. (1982) Prostaglandins in the cabbage looper, Trichoplusia ni. J. Insect Physiol. 28, 761-766. Horrobin D. F. (1978) Prostaglandins. Physiology, Pharmacology and Clinical Sigmjkance. The Book Press, Brattleboro, Vt. Lange A. B. (1984) The transfer of prostaglandinsynthesizing activity during mating in Locusta migratoria. Insect Biochem. 14, 551-556. Loher W. (1979) The influence of prostaglandin E2 on oviposition in Teleogryllus commodus. Entomologia exp. appl. 25, 107-109. Loher W., Ganjian I., Kubo I., Stanley-Samuelson D. and Tobe S. (198 1) Prostaglandins: their role in egg-laying of the cricket Teleogryllus commodus. Proc. Nat1 Acad. Sci., U.S.A. 78, 783557838. Murtaugh M. P. and Denlinger D. L. (1982) Prostaglandins E and F, in the house cricket and other insects. Insecf Biochem. 12, 599603. Sprecher H. (1977) Biosynthesis of polyunsaturated fatty acid and its regulation. In Polyunsalurared Fatly Acids (Edited by Kanau W.-H. and Holman R. T.). American Oil Chemists’ Society, Champaign, IL, USA. Stanley-Samuelson D. W. and Dadd R. H. (1983) Longchain polyunsaturated fatty acids: patterns of occurrence in insects. Insect Eiochem. 13, 5499558. Stanley-Samuelson D. W. and Dadd R. H. (1984) Polyunsaturated fatty acids in the lipids from adult Galleria mellonella reared on diets to which only one unsaturated fatty acid had been added. Insect Biochem. 14, 321-327. Stanley-Samuelson D. W. and Loher W. (1985) The disappearance of injected prostaglandins from the circulation of adult female Australian field crickets, Teleogryllus commodus. Archs Insect Biochem. Physiol. 2, 367-374. Stanley-Samuelson D. W., Klocke J. A., Kubo I. and Loher W. (1983) Prostaglandins and arachidonic acid in nervous and reproductive tissue from virgin and mated female crickets Teleogryllus commodus. Entomologia exp. appl. 34, 35-39. Stanley-Samuelson D. W., Loher W. and Blomquist G. J. (1986) Biosynthesis of polyunsaturated fatty acids by the Australian field cricket, Teleogryiius commodus. Insect Biochem. 16, 387-393. Wakayama E. J., Dillwith J. W. and Blomquist G. J. (1980) In vitro biosynthesis of prostaglandins in the reproductive tissues of the male house fly, Musca domestica (L.). Am. 2001. Abstract No. 1010. Wakayama E. J., Dillwith J. W. and Blomquist G. J. (1985) Occurrence and metabolism of arachidonic acid in the housefly, Musca domestica (L.). Insect Biochem. 15, 367-374. Wakayama E. J., Dillwith J. W. and Blomquist G. J. (1986) Occurrences and metabolism of prostaglandins in the housefly, Musca domestica (L.). Insect Biochem. (in Press).
Synthesis of PG by cricket Yamaja Setty B. N. and Ramaiah T. R. (1979) Isolation and identification of prostaglandins from the reproductive organs of male silkmoth, Bombyx mori. Insect Biochem. 9, 613417.
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Yamaja Setty B. N. and Ramaiah T. R. (1980) Effect of prostaglandins and inhibitors of prostaglandin biosynthesis of oviposition in the silkmoth Bombyx mori. Znd. J. exp. Biol. 18, 539-541.
Camp. Biochem. Physiol. Vol. 85C, No. 2, pp. 303-307, 1986 Printed in Great Britain
DE NO VO BIOSYNTHESIS OF PROSTAGLANDINS BY THE AUSTRALIAN FIELD CRICKET, TELEOGRYLLUS DAVID
STANLEY-SAMUELSON*,
and *Department
of Entomological Sciences, tDepartment of Biochemistry,
COMMODUS
RUSSELL A. WERNER
University University
JURENKA?,
GARY
J. BLoMQu1s-rt
LOHER*
of California, Berkeley, CA 94720, of Nevada, Reno, NV 89557, USA
USA
and
(Received 3 February 1986) Abstract-l. The accumulation and metabolism of certain polyunsaturated fatty acids by testes from the Australian field cricket, Teleogryllus comtnodus,are described. Testes accumulated a substantial proportion (about 16%) of label from radioactive C20: 3n6 that was injected into the haemocoel. Fifty percent of the label accumulated by testes was associated with the phospholipid fraction, whereas in the remainder of the body 30% was incorporated into the phospholipid fraction. 2. Prostaglandins (PG) E,, E, and F,, were quantified in extracts of the testes of adult insects by radioimmunoassay. 3. Label from injected radioactive Cl8: 2n6, C20: 3n6 and C20:4n6 was recovered as prostaglandins PGE and PGF. The radioactivity from C18:2n6 that was recovered as PGE, and PGF,, indicated elongation/desaturation to C20:3n6 followed by conversion to PG. 4. Since C18: 2n6 is readily formed from acetate in T. commodus, these findings indicate the de nova biosynthesis of C20 polyunsaturated fatty acids and prostaglandins by this species.
INTRODUCTION
(PGs), derivatives of certain C20 polyunsaturated fatty acids (PUFAs), or PGsynthesizing activity have been detected in reproductive tracts of the crickets A&eta domesticus (Destephano and Brady, 1977) and Teleogryllus corn modus (Loher et al., 1981), the moths Bombyx mori (Yamaja Setty and Ramaiah, 1979) and Trichopfusia ni (Hagen and Brady, 1982), the locust Locusta migratoria (Lange, 1984) and the housefly Musca domestica (Wakayama et al., 1980). In addition to reproductive tissues, PGs were detected in heads, thoraces and muscles in a number of species (Murtaugh and Denlinger, 1982) and in heads and ventral nerve cords of T. commodus (StanleySamuelson et al., 1983). So far, knowledge of biological activities of PGs in insects is limited. PGs are implicated in the release of egg-laying behavior in the crickets A. domesticus (Destephano and Brady, 1977) and T. commodus (Loher, 1979; Loher et al., 1981) and, to a lesser extent, in the moth Bombyx mori (Yamaja Setty and Ramaiah, 1980). In light of the wide distribution of PGs among insect tissues and species, we adopt the working hypothesis that other biological roles, possibly analogous to known cellular and tissue-level modulation in mammals (Horrobin, 1978), will eventually be discerned. Even less is known about the biochemistry of the C20 PUFAs, C20: 3n6 and C20:4n6, that are direct precursors to the l- and 2-series PGs, respectively. While C20 PUFAs may be obtained directly from the diets of carnivorous and omnivorous insects, strictly phytophagous insects may further elongate and desaturate dietarily supplied Cl8 PUFAs to C20, as suggested for the waxmoth, G. Mellonella (Dadd, 1983a; Stanley-Samuelson and Dadd, 1984). Still Prostaglandins
303
other insects are able to biosynthesize at least one Cl8 PUFA, linoleic acid (Blomquist et al., 1982), which, in concert with further desaturation and elongation, provides the basis for de nouo biosynthesis of C20:3n6 in males of T. commodus (Stanley-Samuelson et al., 1986). If, as observed in other insects (Destephano et al., 1974; Yamaja Setty and Ramaiah, 1979), PGE, is formed in tissues of T. commodus, the biosynthesis of C20: 3n6 would suggest the complete synthesis of a PG. Here it is reported that C20: 3n6 is efficiently taken up from the circulating hemolymph and incorporated into testicular phospholipids. Label associated with radioactive C20: 3n6 and C20:4n6 injected into the haemocoel of males was recovered as, respectively, l-series and 2-series PGs. Since C18: 2n6 and C20:3n6 are formed from injected acetate (Stanley-Samuelson et aI., 1986), de nouo synthesis of PGE, and PGF,, is suggested. MATERIALS
AND METHODS
field cricket, Teleogryllus comcolony maintained at 27 & 1°C and an LD 12: 12 hr cycle. Details are described by Loher (1981). [1-14C]C20:3n6 (54.9mCi/mmole), [5,6,8,9,11,12,14, 15-3H]C20:4n6 (83.8 Ci/mmole) and [U-14C]C18:2n6 (0.5 mCi/mmole) were purchased from New England Nuclear. Various amounts of substrate, specified in context of individual results, were injected into the abdomens of adult males by inserting the needle of a 10 ~1 Hamilton 701 syringe between the seventh and eighth abdominal sclerites, pushing the needle along the lateral body wall about two sclerites anteriorly, then depressing the plunger. After specified incubation periods, the testes and the rest of the bodies were separately extracted. To determine the distribution of label between the testes and bodies and the incorporation of labelled fatty acid into lipid fractions, lipids were Males of the Australian
modus, were taken from a laboratory
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DAVID STANLEY-SAMUELSONet al.
according to Bligh and Dyer (1959). The lipid extract was separated into phospholipids (PLs), triacylglycerols (TGs), monoacylglycerols, diacylglycerols and free fatty acids (MG, DG, FFA) and waxes by developing on Kieselgel thin layer chromatography (TLC) plates in hexanediethyl ether-formic acid (90: 10: 1, v/v/v). Material was visualized under U.V. light after spraying with Rhodamine 6G. Each fraction was scraped into a test tube. The PL were extracted according to the method of Arvidson (1967) and the other lipid fractions were extracted with diethyl ether. Radioactivity of lipid extracts and fractions was estimated by liquid scintillation counting (LSC) of aliquots in 0.4% diphenyloxazole in toluene at 4547% efficiency for tritium and f&88% for carbon-14. For studies of PG biosynthesis, testes and bodies were separately extracted three times in acidified ethyl acetate. PGs were separated by chromatography on Biosil A columns (Wakayama et al., 1986). The PGE and PGF fractions were collected and either counted by LSC (above) or further separated into l- and 2-series PGs by chromatography on 10% AgNO, impregnated Silica Gel Type H (w/w) TLC plates in chloroform-methanol-acetic acid (80: 10: 10, v/v/v). Label associated with PGE,, PGE,, PGF,, and PGF,, was identified by co-chromatography with authentic standards. Individual fractions were extracted from the Silica gel three times with ethyl acetate and counted by LSC. Quantities of PGE, , PGE, and PGF, were estimated in extracts of testes taken from 2- and ldday old adults by commercial radioimmunoassay (RIA) kits (Seragen, Inc., Boston, Ma.). Given values reflect correction for extraction efficiency, monitored in separate tests by estimating recoveries of [‘HIPG. extracted
RESULTS
Incorporation of labelled C20: 3n6 into tissue lipid fractions The differential incorporation of C20 : 3n6 by testes with respect to the rest of the body and distribution
of radioactivity among various lipid fractions were investigated. Radiolabelled C20 : 3n6 (0.2 PCi) was injected into adult males; after 2 hr the testes and the remaining insect body were separately extracted, the total extract was separated into four lipid fractions and radioactivity assayed by liquid scintillation counting. The testes accumulated 16.4 (SD = lO.S%, N = 5) of the radioactivity injected as C20: 3n6, the other 83.6% (SD = 10.5%) being recovered from the rest of the body (data not shown). Table 1 shows the distribution of radioactivity recovered in various lipid fractions after 2 hr following injection of C20:3n6 into abdomens of adult males. Of the label recovered from testes, 50% was in PL, about 14% in TG, 35% in MG, DG, FFA, and Table 1. Distribution of [1-‘4C]20: 3 by lipid fractions in various bodv Darts of male TeleowvNus commodus*
Table 2. Quantities of prostaglandins, as RIA-equivalents, in extracts of testes taken from 2- and 16-day old adult crickets. Given values represent PG RIA-equivalents + I SE (number of individual aairs of testes) PGE, PGE, PGP,,
Day 2 adults
Day 16 adults
635.0 + 66.2 (4) 1032.3 + 178.7 (7) 163.7 + 18.7 (12)
452.0 + 103.2 (5) 1770.0 + 135.4(5) 368.3 k 87.9 (6)
0.5% in waxes. The pattern differed for label recovered from the rest of the body, with lower proportions in PL and higher in TG and MG, DG, FFA. This pattern is also similar to results on distribution of arachidonic acid with respect to lipid fractions (Stanley-Samuelson et al., 1986); C20 PUFAs accumulated by testes thus appear to be efficiently incorporated into PLs. Determination of prostaglandins in extracts of testes
PGs have been detected in spermathecae, ovaries and nervous tissues from females of T. commodus (Loher et al., 1981; Stanley-Samuelson et al., 1983, and one aspect of the significance of C20: 3n6 and C20 :4n6 in these tissues (references above) is thought to be their roles as PG precursors. Table 2 shows the PGE,, PGEl and PGF,, levels that have been determined as RIA-equivalents in extracts of testes from 2- and 16-day old adults. The occurrence of the analogous parental fatty acids has been documented elsewhere (Stanley-Samuelson et al., 1986). Prostaglandin biosynthesis in males of T. commodus
Following determination of 1- and 2-series PGs and their analogous precursor C20 PUFAs in extracts of testes, the possibility was tested that male crickets would be able to convert radiolabelled fatty acid substrate into PGs in vivo. [‘4C]C20: 3n6 (0.6 PCi) and t3H]C20:4n6 (0.75 PCi) were separately injected into the abdomens of adult males and after various incubation times the testes and the remainder of the body were separately extracted and analyzed for PGs. Table 3 presents the proportions of injected radiolabel associated with C20 PUFAs that were recovered as PGs at 2, 10 and 120 min after injection. The proportion of radioactivity associated with C20: 3n6 recovered as PG in the testes declined from 1.1 to 0.1% over the 2 min to 2 hr incubation range. Similar declines were observed with C20:4n6 injections. These findings are consistent with the prod-
Table 3. Incorporation of ‘%20:3 and ‘H20:4 into prostglandins after injection into male Teleogryllus commodus for various time Percentage of injected radiolabel recovered in PG fraction 20:4 20:3
Percent of recovered [I-“C]20: 37 Lipid fraction PL MG, DG, FFA TG Waxes
Testes 50.3 + 34.9 + 14.4 k 0.5 f
20.0 25.0 12.8 0.4
Rest of insect 29.4 5 49.4 + 19.8 k I .5 f
7.9 12.7 5.8 0.4
*[l-“C]20:3 was injected into adult males and after 2 hr. the insects were killed. The testes and remainder of the insect were extracted, lipid fractions separated and radioactivity assayed, as described in Materials and Methods. tMean f SD (N = 4).
Incubation
_...._
time
(min)
Testes
Rest of insect
Testes
2 10 120
I.1 0.4 0.1
1.2 0.7 0.4
0.8 0.4 0.1
Rest ~~ of insect 0.8 0.6 0.4
l“C20: 3 and ‘H20:4 were injected into adult male crickets and after various incubation periods the testes and remainder of the insect were extracted and analyzed for prostaglandins (see Materials and Methods).
Synthesis of PG by cricket uction, followed by uptake and excretion, of radioactivity associated with PGs by the Malpighian tubule/hindgut complex, as described for females of T. commodus (Stanley-Samuelson and Loher, 1985) and for the housefly (Wakayama et al., 1986). The proportions of radioactivity recovered as PGs were approximately equal in testes and the rest of the body (Table 3). Given that the testes represent about 10 times less tissue than the whole animal (about 35 vs over 300mg), these data suggest that either the testes convert 20 : 3 to PG very efficiently or that they accumulate PGs produced elsewhere in the body. The idea that PGs may be taken up from the circulation by individual tissues is supported by the uptake of PG-related label by ovaries of T. commodus (StanleySamuelson and Loher, 1985). Table 3 shows that 2 min after injection of radiolabelled fatty acids, approximately similar proportions of the injected radiolabel recovered in the PG fraction came from C20 : 3n6 and C20: 4n6, indicating substantial potential for production of l- and 2-series PGs. Based on previous work which demonstrated the biosynthesis of C20:3, but not C20:4, from acetate and from linoleic acid (Stanley-Samuelson et al., 1986), it was reasoned that any PGs formed from these latter substrates would be derived from C20: 3n6 and yield l-series rather than 2-series PGs. This idea was tested by injecting radiolabelled Cl8 :2n6 into males daily for three days. Testes and the remainder of the bodies were separately extracted for PGs, followed by separation into E- and F-series PGs by column chromatography. The PGE and PGF fractions were further separated into 1- and 2-series PGs by AgNO,-TLC. This procedure separates the PGs according to the number of double bonds, with the l-series PGs ascending slightly ahead of the 2-series. This order of separation necessitates correction for label associated with l-series PGs tailing with the 2-series, done by spotting radioactive PGE, and PGF, standards on the TLC plates, followed by calculating the proportions of counts recovered in the l-series location. Table 4 shows the amount of radioactivity recovered as 1- and 2-series PGs from the testes and the rest of the body. Allowing for the 25-30% of the l-series label tailing with the 2-series (Table 4), it appears that all of the label recovered from the whole body (less testes) occurs as l-series PG. Respectively, 55 and 65% of the label recovered from testes occurred as PGE, and PGF,,. Subtracting l-series Table 4. Separation of one and two series prostaglandins after in uiuo synthesis from “C-18 :2 in the male cricket Teleogryllus commodus * DPM recovered source of prostaglandin Testes Rest of insect PGE, standard PGE,, standard
PGE,t 3871 (54.7) 27,766 (72.8) 1373 (71.7)
in each fraction
PGE,
PGF,,
3201 10,380 542
5364 (65.5) 31,419 (70.7)
2830 13,000
1900 (76.0)
600
PGF,,
*%-IS:2 was injected into adult male crickets for three days and testes and remainder of the insect were extracted for prostaglandins. E and F series prostaglandins were separated on Biosil A columns and these were separated further into the one and two series by AgNO, TLC (see Materials and Methods). tNumbers in parentheses refer to the percent of label recovered in the one series. C.B.P.85,2C--D
305
trailing, it would appear that about 15% of the PGE, and about 10% of PGF, radioactivity elutes with the 2-series PG. DISCUSSION
The data presented here show that testes of T. commodus differentially incorporate injected C20: 3n6 into PLs. Including results from previous work on testicular bioaccumulation of injected fatty acids in this species (Stanley-Samuelson et al., 1986), this efficient incorporation is probably specific to PUFAs: Cl 8 : 2n6, C20 : 3n6 and C20 : 4n6 are all rapidly taken up relative to the saturated fatty acid, stearate. Similarly, the housefly, M. domestica, efficiently accumulates labelled C20 : 3n6 and C20 : 4n6 into the PL fraction, specifically into the 2-position, compared to a saturated fatty acid (Wakayama et al., 1985). This work also reports the detection of PGE,, PGE, and PGF,, as RIA-equivalents in extracts of testes, as well as in vivo biosynthesis of PG from injected substrate. The biological significance of PGs in the testes of T. commodus remains unclear. Following mating and transfer of PG-synthetase activity, PGs formed in the spermathecae release egg-laying behavior in the female; but the PGs per se are not transferred in the spermatophore (Loher et al., 1981). On the other hand, PGs have been detected in many tissues of this cricket (Stanley-Samuelson et al., 1983; Stanely-Samuelson and Loher, unpublished observations), including Malpighian tubules and certain glands. If tissue and cellular activities are in part regulated by local turn-over of PGs, as thought for mammals, the detection of PGs and of PG-synthesis activity in testes may be considered under the mantle of local modulation. In vivo biosynthesis of PGs from three substrates, C20 : 4n6, C20 : 3n6 and C 18 : 2n6, was observed. The data from experiments with C20: 3n6 and C20:4n6 indicates substantial capacity for biosynthesis of land 2-series PGs from immediate precursors by males, and extends the known locations of PG biosynthesis in T. commodus. The formation of PGs from linoleic acid is to be interpreted with respect to PUFA metabolism. In mammals, Cl 8 : 2n6 undergoes desaturation to Cl 8 : 3n6, elongation to C20 : 3n6, and another desaturation to C20:4n6 (Sprecher, 1977). Formation of two l-series PGs from Cl8: 2n6 in T. commodus is consistent with desaturation and elongation to C20 : 3n6, followed by formation of l-series PGs. Table 4 shows that in the testes about 25 and lo%, respectively, of the PGE and PGF radioactivity chromatographed as 2-series PGs after correcting for l-series tailing on AgN03-TLC. It would appear from this finding that a small proportion of C20 : 3n6 may undergo an additional desaturation to C20:4n6, followed by derivatization to 2-series PG. Since no evidence for the desaturation to C20 : 4n6 emerged in earlier work on this question (Stanley-Samuelson et al., 1986), F-series PGs, prepared after incubation with injected radioactive C18:2, were chromatographed on a radio-high pressure liquid chromatograph (radio-HPLC) optimized for separation of l- and 2-series PGs. This yielded a single peak of radioactivity corresponding with l-series PG. In metabolism of injected separate experiments,
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Cl8 : 2n6 to C20 PUFAs was reexamined by radioHPLC. This also supported the earlier findings of synthesis of C20: 3n6 with no radioactivity associated with C20:4n6. Summarizing literature and analytical evidence, Stanley-Samuelson and Dadd (1983) concluded that long-chain PUFAs were probably a general feature of insect lipids, most readily observed in PLs and extracts of more membraneous tissues. One implication of this idea, especially for strictly phytophagous insects, but also for all insects whose dietary PUFA complement differs from local tissue and cellular requirements, would be a greater or lesser capability for metabolism of long-chain PUFAs. T. commodus, now shown capable of de novo biosynthesis of at least two PUFAs and the 1-series PGs, represents a condition that differs fundamentally from the commonly appreciated mammalian pattern and from some other insect groups. The de novo biosynthesis of linoleic acid, the parental unit of all n6 PUFAs, is now known in many insects representing several orders (Blomquist et al., 1982; Cripps et al., 1986). If these linoleate biosynthesizers can perform further elongations and desaturations, the biosynthesis of C20 PUFAs, and possibly PGs, may eventually be perceived as wide-spread among insects. Just as we do not yet known the distribution of biosynthetic capability with respect to PUFAs and PGs among insect species and orders, the range of fatty acid metabolic patterns is also unclear. So far, we can identify three patterns. The waxmoth pattern features the elongation and desaturation of dietary Cl8 : 3n3 to C20: 5n3 without a comparable elongation and desaturation of the linoleic acid family members (Dadd, 1983a; Stanley-Samuelson and Dadd, 1984). The mosquito pattern, most thoroughly analyzed in Culex pipiens, is characterized by dietary requirement for vitamin-level quantities of arachidonic or certain structurally related fatty acids (Dadd, 1983b). Since linoleic acid cannot fulfill this need, an inability to introduce more unsaturation into the parental PUFA is implied. T. commodus represents a third, cricket, pattern. Future work will uncover still other patterns and help clarify how many insect species are represented by the known patterns; as work proceeds, metabolism of PUFAs may come to be of increasing significance in insect physiology and biochemistry. Acknowledgements-We thank Dr R. H. Dadd for reading and suggesting improvements to the manuscript. Supported by NIH Grant ROl HDO3619 to W.L., by NSF Grant DCB-8309146 and the Nevada Agricultural Experiment Station funds to G.J.B.
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Yamaja Setty B. N. and Ramaiah T. R. (1980) Effect of prostaglandins and inhibitors of prostaglandin biosynthesis of oviposition in the silkmoth Bombyx mori. Znd. J. exp. Biol. 18, 539-541.