Ecdysteroid titre and metabolism to novel apolar derivatives in adult female Boophilus microplus (Ixodidae).код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 2:39-54 (1985) Ecdysteroid Titre and Metabolism to Novel Apolar Derivatives in Adult Female Boophilus microplus (Ixodidae) Kim P. Wigglesworth, D a v i d Lewis, a n d Huw H. Rees Department of Biochemistry, University of Liverpool, Liverpool, England (K.P. W., H.H.R.), and Department of Animal Health Biology, Pfizer Central Research, Sandwich, Kent, England (0.L.) The free ecdysteroid titre determined by radioimmunoassay in adult female Boophilus microplus showed a peak just prior t o full engorgement and detachment of the ticks and decreased subsequently to a very low value. I n contrast, the titre of polar ecdysteroid conjugates was very low. Ecdysone was the major ecdysteroid at peak titre and was accompanied by much lower levels of 20-hydroxyecdysone. I n newly detached ticks, injected [3H]ecdysone was metabolized primarily (80%) into much less polar compounds, which could be resolved into at least three groups by reversed-phase h.p.1.c. These [3H] “apolar” metabolites were transferred to the newly laid eggs, where they accounted for the vast preponderance of ecdysteroids, the level of free hormone being low. Hydrolysis of the three groups of compounds with an esterase preparation from porcine liver yielding [3H]ecdysone, together with the release of [3H] ecdysteroid and fatty acids upon alkaline saponification of the compounds, suggests that they are of a fatty acyl ester nature. The chemical transformation of these “esters” into the corresponding acetonide derivatives indicates that the 2- and 3-hydroxyls of ecdysone remain unsubstituted in these compounds. Several tick tissues, including Malpighian tubules, ovaries, gut, and fat body, metabolized [3H]ecdysone in vitro forming the ”apolar esters” as major products. The maternal ecdysteroid “esters” may function as storage forms of hormone (presumably hormonally inactive), which could be hydrolysed enzymically during embryogenesis releasing free ecdysteroids. Such enzymic hydrolysis of [3H]ecdysone ”esters” by homogenates from developing eggs of B. microplus has been demonstrated. Key words: ecdysteroids, fatty acyl esters, ticks, Ixodidae, Soophilus micmphs, h.p.1.c. Acknowledgments: We thank the Science and Engineering Research Council and Pfizer Central Research for financial support and a C.A.S.E. Studentship (to K.P.W.), the Royal Society for an equipment grant, Miss P.A. Duxbury for investigating hydrolysis of the ecdysteroid esters in developing eggs, Dr. j.D. Gee and Dr. K.A.F. Gration for invaluable discussion, Mr. D. King and Mr. N. Leach-Bing for the supply of ticks, Professor K.-D. Spindler and J.D. O’Connor for generous gifts of antisera, and Dr. M.E. Rose and Mr. M. Prescott for the mass spectrometric analyses. Received April 18,1984; accepted June 20,1984. Address reprint requests to Dr. H.H. Rees, Department of Biochemistry, The University, P.O. Box 147, Liverpool, L69 3BX, U.K. 0 1985 Alan R. Liss,lnc. 40 Wigglesworth, Lewis, and Rees INTRODUCTION Moulting in immature stages of insects and other arthropods is controlled by the steroidal moulting hormones, the ecdysteroids. In many insect species investigated the major ecdysteroid in larvae and pupae is 20-hydroxyecdysone, which is generally accompanied by smaller quantities of ecdysone [1,2]. However, it has been demonstrated in many insect species that ecdysteroids occur not only in immature stages, but also in adult females, primarily in the ovaries . At least in the migratory locust, Locustu migrutoriu, ovarian ecdysteroids are synthesised in the follicle cells . In many insect species, it has been demonstrated that ovarian ecdysteroids are mainly present as polar conjugates which are largely passed into the eggs [3,5-81. In the locust, Schistocercu greguriu, the polar ecdysteroid conjugates in newly laid eggs have been identified as the 22-phosphate derivatives [9,10], whereas it has been reported that the major polar conjugates in newly laid eggs of the related species, L. migrutoriu, are 22-adenosine monophosphate derivatives [11,12]. Although the function of polar ecdysteroid conjugates in developing eggs is not known, the ecdysteroid 22-phosphates may represent inactive storage forms of hormone. In fact, embryonic tissues of S . gregariu can enzymically hydrolyse ecdysteroid 22-phosphates to liberate free ecdysteroids; other sources, such as de novo synthesis, of free ecdysteroids in embryos are not precluded . Studies on the developing eggs of a number of insect species indicate an apparent correlation between peaks of free ecdysteroids and the formation of embryonic membranelcuticle [for references see 101. The occurrence of ecdysteroids in nymphs of the hard tick, Amblyommu hebraeurn , and in the soft tick, Ornithodoros moubutu [El, has been reported. However, there have been no reports of the occurrence of ecdysteroids in adult female ticks. We now report the occurrence, titre, metabolism, and fate of ecdysteroids in adult females of the hard tick, Boophilus microplus. This work has been presented in preliminary form . MATERIALS AND METHODS Reagents H.p.1.c.* grade solvents were purchased from Rathburn Chemicals, Walkerburn, U.K.,Helix pornutiu arylsuphatase preparation (type H-1) and porcine esterase were from Sigma, and ecdysone was obtained from S h e s , Milan, Italy. [23,24-3H2]Ecdysone (80 Cilmmole) was purchased from New England Nuclear, Boston, U.S.A., and N-trimethylsilylimidazole was from Pierce and Warriner, Chester, U.K. Ticks One-host ticks, Boophilus microplus, were reared on calves at 20°C. Under the conditions used the nymphal-adult moult of B. microplus commenced on *Abbreviations: fast atom bombardment = FAB; gas chromatography/mass spectrometry (selected ion monitoring) = G U M S (SIM,); high-performance liquid chromatography = h.p.1.c.; 4-morpholine-ethanesulphonic acid = Mes; radioimmunoassay = RIA; relative retention time = RR,; thin layer chromatography = t.1.c.; N-trimethylsilylimidazole= TMSI. Ecdysteroids in Ticks 41 the 15th day from initial attachment to the host as larvae. Engorgement was completed around day 21, when the female ticks detached and began egg laying at day 24 postinfestation. Extraction Procedure Tick material (adult females and eggs) was extracted essentially according to the method of Dinan and Rees [S]. The adult ticks and eggs were macerated and extracted successively with methanollwater (7:3 vlv; 2 x), methanol ( 2 ~ ) ,and methanolldichloromethane (l:l, vlv; 2 X ) using Polytron and Potter-Elvehjem homogenisers, respectively. The volumes of each solvent used were: 2 x 15 mllg adult females; 2 x 1ml/lO mg eggs. After evaporation of solvent under vacuum, the extract was partitioned between methanollwater (7:3, vlv) and hexane (equal volumes) to remove apolar material, each phase being back-extracted with the respective counter phase. The residue from the aqueous methanol phase was evaporated on to Celite and applied to a silicic acid column. The column was developed sequentially with chloroform, 30% (vlv) methanol in chloroform* (which eluted free ecdysteroids), and 80% (vlv) methanol in chloroform (which eluted any highly polar ecdysteroids including conjugates [S]. During identification of free ecdysteroids at peak titre, further cleanup of the 30% methanol in chloroform silicic acid column fraction before analysis by h.p.l.c./RIA (Fig. 2) was effected on a reversed-phase Sep-Pak cartridge (Waters Associates). The sample dissolved in 10% (vlv) methanol in water (2 x 2 mi) was applied to the activated cartridge, which was eluted sequentially with 30% (vlv) methanol in water (4 ml), 60% (vlv) methanol in water (6 ml), and methanol (4 ml). Free ecdysteroids were eluted in the 60% methanol in water fraction. Radioimmunoassay Assay of 30% methanol in chloroform silicic acid column fractions was performed as described previously , using ecdysone as standard. Two antisera were used, ICT-1, a gift from Professor K. -D. Spindler, University of Dusseldorf 1181, and DHS-1-15 wk., a gift from Professor J.D. O’Connor, University of California . The cross-reaction factors of the antisera toward 20-hydroxyecdysone (ratio of the mass of 20-hydroxyecdysone required to displace 50% of [3H]ecdysone to the mass of ecdysone required) were: ICT-1 = 2.6; DHS-1-15 wk. = 2.8. Radiochemical Methods Radioactivity was assayed on a Beckman model LS 9800 liquid scintillation spectrometer, d.p.m. being computed using the H’ system. For assay of fractions from h.p.1.c. or t.l.c., Scintran Cocktail T (4 ml; BDH Chemicals *This fraction will be referred to as the “30% methanol in chloroform silicic acid column fraction” and was shown in this work to contain highly apolar derivatives of ecdysteroids in addition to free hormones. 42 Wigglesworth, Lewis, and Rees Ltd., U.K.) was used, whereas radioimmunoassay samples were assayed in Aquasol (4 ml; New England Nuclear). Hydrolysis of Highly Polar Ecdysteroid Conjugates During estimation of the highly polar ecdysteroid conjugate titre in adult females, a portion (three-quarters) of the 80% (vlv) methanol in chloroform silicic acid column fraction was evaporated to dryness, dissolved in 0.2 M Mes buffer pH 5.4 (1ml), and incubated with 0.5 ml of a crude arylsulphatase preparation from Helix pornutiu (125 units; 1 unit hydrolyses 1.0 pmole of nitrocatechol sulphate in 1 h at pH 5.0 and 37°C) for 18 h at 37°C. The reaction was terminated by the addition of ethanol (4 ml) and the protein precipitate was extracted four times with methanol (4 ml). Chromatography of the residue after evaporation of the combined alcoholic extracts on a silicic acid column as before allowed separation of a fraction containing ecdysteroids released from conjugation from any highly polar ecdysteroids refractory to enzymic hydrolysis. High-Performance Liquid Chromatography A high-performance liquid chromatograph (Waters Associates, Northwich, U.K.) incorporating two M6000A pumps in conjunction with a model 660 solvent programme controller, U6K injector, and a model 441 detector set at 254 nm, was used. Ecdysteroids were separated on the following systems: system 1, an Ultrasphere-ODS column (15 cm X 4.6 mm internal diameter; Anachem, Luton, U.K.) eluted at 1mllmin with methanollwater (9:11, vlv); system 2, a Partisil ODs-3 column (25 cm x 4.6 mm internal diameter; Whatman, Maidstone, U.K.) eluted at 2 mllmin with a linear gradient (20 min) of methanol in water changing from (a) 2:3 to 4:l (vlv), or (b) 2:3 to 1:O (vlv), or (c) 9:l to 1:O (vlv); system 3, an aminopropyl column (APS-Hypersil; 25 cm x 4.5 mm internal diameter; Shandon Southern Products, Runcorn, U.K.) eluted isocratically at 2 mllmin with either (a) dichloroethanelmethanol (19:1, vlv) or (b) dichloroethanelmethanol (47:3, vlv) [17 1. In the case of radioactive samples, fractions were collected at 0.5- or 1-min intervals for radioassay and scintillation cocktail (4 ml) added directly in the case of reversed-phase h.p.l.c., but after evaporation of the solvent when APS-Hypersil columns were used. Mass Spectrometry Fast atom bombardment mass spectra. Negative-ion FAB mass spectra were obtained as described previously . Gas chromatographylmass spectrometry (selected ion monitoring). Ecdysteroids were converted into their pertrimethylsilyl ether derivatives by heating with TMSI and subjected to GClMS (SIM) as described previously . Makisterone A was used as internal standard and fragment ions at mlz 567 and 561 were monitored. Administration of [3H]ecdysone [23,24-3H]ecdysonediluted to 40 Cilmmole was dissolved in insect Ringer solution and injected into day 22-23 adult female ticks through the dorsal Ecdysteroids in Ticks 43 wall (5 pl, generally containing 0.10-0.21 pCi 3H per tick). The animals were then kept at 27°C in a humid atmosphere and then either sacrificed by freezing 18 h after injection or allowed to lay eggs for 5-6 days, when the spent females were frozen; the eggs were frozen within 1day of oviposition. Thin-Layer Chromatography Samples were chromatographed on aluminium-backed precoated silica gel plates (0.2 mm thick Kieselgel 60Fm; E. Merck., A.G., Darmstadt, F.R.G.) developed as described in the appropriate points in the text. Authentic marker-compounds were located under UV light (253 nm) and the chromatograms were cut into 0.5-cm strips, which were radioassayed directly. Hydrolysis of Apolar Fractions With an Esterase Enzyme Fractions containing apolar derivatives of ecdysteroids were subjected to hydrolysis with an esterase preparation. Samples were dissolved in 0.1 M borate buffer (0.5 ml), pH 8.4, and incubated with a porcine esterase (10 units; 1unit will hydrolyse 1.0 pmole of ethylbutyrate per min at pH 8.0 and 25°C) for 24 h at 37°C. The reaction was terminated by the addition of ethanol (2 ml) and the protein precipitate was extracted four times with methanol (4 ml). Acetonide Derivative Formation Acetonide derivatives of ecdysteroids were prepared by the method of Galbraith and Horn . Incubation In Vitro of Tissues With [3H]ecdysone Malpighian tubules, ovaries, fat body, and gut were dissected from late day 20 females in tick saline (NaC1, 200 mh4; KC1, 10 mM; CaCl2, 2 mM; in 50 mh4 Tris-HC1buffer, pH 7.4) and each tissue incubated with [3H]ecdysone (1pCi; 40 Cilmmole) in 0.5 ml of tick saline pH 7.4 for 6 h at 27°C. The incubations were terminated by addition of ethanol (1ml) and the reaction mixtures homogenised and extracted as described earlier. Hydrolysis of Apolar Ecdysone "Esters" by a Homogenate of Developing Eggs of B. microplus Eggs of B. micropEus were collected within 24 h of oviposition and incubated for 15 days at 27°C and approximately 90% relative humidity. Under these conditions larvae hatch after 21 days. Day 15 developing eggs (500 mg) were homogenised at 0°C in 0.1 M potassium phosphate buffer pH 6.0 (6.7 ml) with a Potter Elvehjem homogenizer. Duplicate aliquots (2 ml) of the crude homogenate as well as 2 ml of boiled homogenate (control incubation) were preincubated in a reciprocating water bath at 50°C (approximate optimal temperature) for 10 min. The reaction was then initiated by addition of [23,24-3H]ecdysoneesters (58,000 d.p.m. 3H in 10 p1 methanol) purified by h.p.1.c. from newly laid eggs derived from female B microplus injected with [3H]ecdy~~ne. After 1 h, the incubations were terminated by addition of 44 Wigglesworth, Lewis, and Rees ethanol (5 ml), the mixture cooled, centrifuged (1,OOOg x 5 min), and the supernatant removed. The pellet was reextracted with ethanol (5 ml) followed by methanol (2 x 5 d)and the combined supernatants evaporated to dryness under reduced pressure. [3H]Ecdysone (Rf 0.33) produced by enzymic hydrolysis of the apolar [3H]ecdysone esters (Rf 0.56) was separated from the latter by t.1.c. of the extract (applied in methanol), developing with methano1:chloroform (1:4,vlv). The chromatograms were cut into 0.5-cm strips and radioassayed, and the 3H in the band corresponding to authentic ecdysone was expressed as a percentage of the total 3H recovered in ecdysone and apolar ecdysone esters. RESULTS Ecdysteroid Titre During Adult Female Development Batches of adult female B. microplus ticks (20-200 individuals) were collected at daily intervals from the time of the nymphal-adult moult (day 15) until commencement of egg laying (day 24) and stored at -20°C until they were extracted. The 30% methanol in chloroform silicic acid column fractions (which include the free ecdysteroids) and ecdysteroids released from conjugation with polar moieties by the Helix pomatia enzymes were subjected to radioimmunoassay using both the ICT-1 and DHS-1-15 antisera. The resulting titre curve (Fig. 1)shows the presence of one major peak of immunoreactive material in the 30% methanol in chloroform fraction at day 20 of development prior to complete engorgement; the levels of ecdysteroids released by hydrolysis of the polar ecdysteroid conjugate fractions with Helix enzymes were comparatively low. Similar titre curves were obtained in a separate experiment. ] antiserum erum Po,ar conjugated ecdysterolds T l m o f r o m i n l t i a l a t t a o h m o n t of t i c k s t o t h e h o a t (day.) Fig. 1 Titres of free and polar conjugated ecdysteroids (30% and hydrolysed 80% methanol in chloroform silicic acid column fractions, respectively) in adult female Boophilus microplus determined by RIA using two antisera. Ecdysteroids in Ticks 45 Identification of Free Ecdysteroids at Peak Titre The nature of the free ecdysteroids at the time of peak titre (day 20) was investigated initially by reversed-phase h.p.1.c. system 1collecting fractions every 30 s for radioimmunoassay (ICT-1 antiserum; Fig. 2). A major immunoreactive UV-absorbing peak, cochromatographing with authentic ecdysone, was observed, together with smaller peaks, one of which cochromatographed with 20-hydroxyecdysone. When the peak cochromatographing with ecdysone was collected and analysed on an APS-Hypersil column (system 3a), a single UV-absorbing peak (retention time, 13 min) cochromatographing with ecdysone was observed. Further evidence for the identity of the ecdysone was furnished by its negative-ion FAB mass spectrum, which showed a major peak at mlz 463 as expected. Analysis of another portion of the 30% methanol in chloroform silicic acid column fraction from day 20 ticks by GClMS [SIM] (Fig. 3) showed a peak in the chromatogram for the ion at mlz 567 having an identical retention time relative to the pertrimethylsilyl derivative of makisterone A (RR, 0.65) as that of silylated authentic ecdysone (RR, 0.65) [cf 211, as well as a smaller unidentified peak (RRt 0.84). The fact that the latter shows the ion at mlz 567 rather than at mlz 561 indicates that the compound is a 20-deoxyecdysteroid having an ecdysonelike nucleus. The chromatogram for the ion at mlz 561 suggested Ecd rone PO-Hydroxyecdyione Fig. 2 Reversed-phase h.p.1.c. fractionation (system I) of the 30% methanol in chloroform silicic acid column fraction from day 20 5. microplus females (equivalent to 2 ticks; 75 mg fresh weight) with collection of fractions every 30 sec for radioimrnunoassasy (ICI-I antiserum). The positions of authentic ecdysteroids are shown. 46 Wigglesworth, Lewis, and Rees ' O 0 . Total ion chromatogram lool I\ Intom8l Stmd8rd i m/z 567 3:20 1o:oo 20:oo Time (min:rec) Fig. 3 CC/MS(SIM) analysis of a silylated portion of the 30% methanol in chloroform silicic acid column fraction from day 20 B. microplus females. ions at mlz 561 and 567 were monitored. The positions of authentic ecdysteroids are shown. the presence of 20-hydroxyecdysone (RR, 0.49). Comparison of the levels (pglg tick) of free ecdysone and 20-hydroxyecdysone detected at peak titre by radioimmunoassay (2.8 and 0.08, respectively) and GUMS (SIM) (2.6 and 0.04, respectively) showed good agreement. Metabolism of [3H]ecdysone To obtain information on possible metabolic transformations responsible for the decrease in immunoreactive ecdysteroids during the period day 20-22 (see Fig. l), [3H]ecdysonewas injected into adult females at day 22-23 and its metabolism examined. Metabolism of ecdysone in adult females. Initially the fate of [3H]ecdysone was analysed in adult females sacrificed 18 h after injection of [3H] substrate. The females were extracted and the aqueous methanol phase of the methanollwater-hexane partition fractionated on a silicic acid column. Although the 80% methanol in chloroform fraction, which includes any highly polar ecdysteroids, contained some radioactivity, the majority (- 94% of the total recovered from the column) was associated with the 30% methanol in chlo- Ecdysteroids in Ticks 47 roform fraction. A portion of the latter fraction was analysed by reversedphase h.p.1.c. using a solvent gradient of 40-80% (vlv) methanol in water (system 2a), which should elute all known free ecdysteroids. The radiochromatogram observed comprised a major peak of residual ecdysone, a minute amount of radioactivity cochromatographing with 20-hydroxyecdysone, and a slightly more prominent peak chromatographing with ecdysone-3-acetate. In view of the very poor recovery of radioactivity from this chromatographic system, a portion of the original 30% methanol in chloroform silicic acid column was analysed by thin-layer chromatography with chloroformlmethan01 (4:l vlv) for development. The majority of the radioactivity (86%) chromatographed with ecdysone-3-acetate (Rf 0.56; cf Rf of ecdysone, 0.33), although a minor peak of 3H cochromatographed with this compound on the preceding reversed-phase h. p .l.c. system. Reanalysis of the original 30% methanol in chloroform silicic acid column fraction by reversed-phase h.p.1.c. eluting with a linear gradient of 40-100% (vlv) methanol in water (system 2b) revealed the presence of a major peak of radioactivity of an apolar nature as compared to ecdysone as well as small peaks cochromatographing with ecdysone and ecdysone-3-acetate, respectively (Fig. 4A). Because of the "apolar"* nature of the majority of this material, another portion of the original 30% methanol in chloroform silicic acid column fraction was reanalysed by reversed-phase h.p.l.c., but eluting with a linear gradient of 90-100% (vlv) methanol in water (system 2c). In this A B m,o Apolar ecdysteroids Apoler ecdysteroids m .-- m -Peak 2 I 0 b a c m 14 U a a a - I? P U 0 12 X c U 0 Tlmm(mln) tlmm (mln) Fig. 4 Reversed-phase h.p.1.c. (A, system 2b; B, system 2c) radiochromatograms of the 30% methanol in chloroform silicic acid column fraction from ticks following administration of [3H]ecdysoneto day 20 adult females. The positions of authentic ecdysteroids are shown. *In this paper the term "apolar" is used with reference to compounds that are appreciably less polar than ecdysone. 48 Wigglesworth, Lewis, and Rees system the large ”apolar” radioactive peak is resolved into three distinct peaks (Fig. 4B). Passage of [3H]ecdysteroidsinto the eggs. The possible passage of maternal ecdysteroids into the eggs was investigated by analysis of eggs derived from females injected with [TIlecdysone. In a typical experiment, ten adult females were injected with [3H]ecdysone (1.54 pCi total), the newly laid eggs (130 mg) were collected over the first 6 days of oviposition, the eggs extracted, and the aqueous methanol phase (0.70 pCi) of the methanoliwaterhexane partition fractionated on a silicic acid column. The 30% methanol in chloroform fraction (0.59 pCi) contained the majority of the radioactivity, with little (0.03 pCi) being present in the 80% methanol in chloroform (highly polar ecdysteroid) fraction. Analysis of the 30% methanol in chloroform fraction by reversed-phase h.p.1.c. using a linear gradient of 40-100% (vlv) methanol in water (system 2b) showed that it consisted almost entirely of apolar material, which was resolved using a 90-100% methanol in water gradient (system 2c) into three distinct peaks of the same polarity as the major radioactive metabolites observed in adult females (Fig. 5). Thus, it appears that the apolar compounds which are formed in females as major ecdysone metabolites are principal radioactive components in newly laid eggs and are probably formed maternally. Analysis of the Apolar Metabolites The apolar nature of the major [3H]ecdysone metabolites suggested that they could be fatty acyl ester derivatives. Therefore, each of the three [3H] Apolar ecdyateroida CI m 1 0 v- 01 0 .- X 0 -* E L Q Q 0 01 v U .-c > .-c > 0 a, m m 0 m .-0 L U ‘0 1 ID U c 2’0 15 10 5 0 Time (min) Fig. 5 Reversed-phase h.p.1.c. (system 2c) radiochromatogram of the 30% methanol in chloroform silicic acid column fraction from newly laid eggs of ticks which had been injected with [3H]ecdysone at day 20. Ecdysteroids in Ticks 49 apolar peaks were collected from reversed-phase h.p.1.c. (system 2c) and treated with a porcine liver esterase preparation; parallel control incubations without esterase were also carried out. Analysis of the products of the reactions by reversed-phase h.p.1.c. using a linear gradient of 40-100% methanol in water (system 2b) indicated that after enzymic hydrolysis in each case nearly all the radioactivity ( > 95%) cochromatographed with ecdysone, whereas control incubations yielded negligible radioactivity in that region (Fig. 6). Confirmation that the radioactivity was associated with ecdysone in each case was obtained by cochromatography with authentic material on an APS-Hypersil h.p.1.c. column (system 3b). These results are consistent with the notion that the apolar metabolites may be esters of ecdysone. The observed release of [3H]ecdysteroid in alkaline hydrolylsis (6% (wlv) sodium hydroxide in ethanollwater (lO:l, viv) at room temperature for 18 h) of the apolar compounds, as well as analysis by g.1.c. of the fatty acids released are also consistent with this view. To obtain some information on the position(s) substituted in the apolar ecdysone metabolites, acetonide derivative formation was attempted on two of these compounds (peaks 1and 2 ex. reversed-phase h.p.1.c.). Analysis of the products of the reaction by t.1.c. with chloroformlethanol (9:1, vlv) for development revealed the presence of a major peak of radioactivity (Rf 0.42) less polar than ecdysone-2,3-acetonide (Rf 0.20; cf Rf of underivatized compounds 1and 2 (0.03) and of ecdysone (0.03). These results suggest that C-2 Apolmr ecdymtmrold. 1 1 50 26 20 Tlma 16 10 6 0 (mln) Fig. 6 Reversed-phase h.p.1.c. (system 2b) radiochromatogram of the product of enzymic hydrolysis of apolar peak 2 (ex. Fig. 5) with a porcine liver esterase preparation. The position of authentic ecdysone is shown. Control incubation without esterase, - - -; esterasetreated, -. The radiochromatograms for the products of hydrolysis of apolar peaks 1 and 3 (ex. Fig 5) with the esterase preparation showed a similar pattern to that shown. 50 Wigglesworth, Lewis, and Rees and C-3 are free in the apolar metabolites 1 and 2, and that another position(s) is substituted in the latter compounds. Sites of formation of apolar ecdysone metabolites. Certain tissues from late day 20 females were incubated in vitro with [3H]ecdysone, the reaction mixture extracted, and the metabolites in the 30% methanol in chloroform silicic acid column fraction were analysed by reversed-phase h.p.1.c. (system 2b; Fig. 7). In the case of each tissue, when the peak corresponding to apolar ecdysteroids was collected and rechromatographed on reversed-phase h.p.l.c., eluting with a linear gradient of methanol in water changing from 9:l to 1 : O (v/v; system 2c; eg, Fig. 8), the radioactivity was resolved into three separate peaks cochromatographing with the apolar metabolites detected in females and eggs (Figs. 4,5). Thus, it is apparent that formation of the apolar ecdysone metabolites occurs, at least in vitro, in the Malpighian tubules, ovaries, fat body, and gut. 3 0 .. !! 1 Time (min) Fig. 7 Reversed-phase h.p.1.c. (system 2b) radiochromatograms of the 30% methanol in chloroform silicic actd column fractions derived from incubation of [3H)ecdysone with various tissues from late day 20 female B. microplus. The positions of authentic ecdysteroids are shown. Malpighian tubules, 0-0; ovaries 0-0; gut, 0--.-0. The results for fat body were similar to those for Malpighian tubules. Ecdysteroids in Ticks 51 T i m e (rnin) Fig. 8 Reversed-phase h.p.1.c. (system 2c) radiochromatograms of the 30% methanol in chloroform silicic acid column fractions derived from incubation of [3H]ecdysonewith ovaries (0-0) or gut (0--.-0)tissues from late day 20 female B. microplus. The relative proportions of apolar ecdysteroids formed in Malpighian tubules and fat body in vitro were similar to those observed in vivo. Hydrolysis of apolar ecdysone esters by a homogenate of developing eggs of B. microplus. When a homogenate (equivalent to 150 mg eggs) of B. microplus eggs, which had developed for 15 days, was incubated with apolar [3H]ecdysone esters under the conditions described, 20% hydrolysis of the substrate was observed. Negligible apparent hydrolysis of substrate was observed in a control incubation with boiled enzyme. In a separate incubation, the [3H]ecdysteroid product of the reaction cochromatographed with ecdysone on reversed-phases h.p.1.c. (system 2b). DISCUSSION The exact significance of the peak of immunoreactive ecdysteroids in the 30% methanol in chloroform silicic acid column fraction just before complete engorgement of adult female B. microplus is uncertain. This immunoreactivity reflects the titre of free ecdysteroids since the apolar ecdysteroid "esters," which also occur in the same silicic acid column fraction, are only marginally immunoreactive towards the two antisera employed. The apolar ecdysteroid "esters" have not been detected at peak titre, but apparently occur after day 22 of development. The location and source of the ecdysteroids have not yet been determined. The somewhat lower immunoreactivity detected with the ICT-1 antiserum than with the DHS-1-15 antiserum may reflect the possible presence of ecdysteroids having a modified ring structure, since, of the two 52 Wigglesworth, Lewis, and Rees antisera, the former shows greater specificity for that part of the ecdysteroid structure [18,19,23]. The occurrence predominantly of ecdysone with very much less 20hydroxyecdysone at peak titre in B. microplus adult females is reminiscent of the situation in the femalesleggs of most insect species investigated, where ecdysone, albeit often as a polar conjugate, predominates [see 31. In comparison with the titre of free ecdysteroids, that of polar conjugates hydrolysable with the Helix enzymes was much lower. The unlikely possibility still exists that polar conjugates which are not hydrolysed with such enzymes are present. Exogenous [3H]ecdysone was metabolised in adult female B. microplus primarily to appreciably less polar metabolites, which were readily resolved into three groups by reversed-phase h.p.1.c. Although these metabolites were hydrolysed upon treatment with an esterase enzyme preparation from porcine liver, it is always dangerous to draw conclusions regarding the structure of compounds solely from results of their hydrolyses with enzyme preparations which may not be pure. However, this observation taken together with the release of [3H]ecdysteroid and fatty acids upon alkaline saponification of the compounds is consistent with the notion that they are of an ester nature. It is noteworthy that ecdysone was the only ecdysteroid moiety in each of the three groups of apolar compounds resolved by reversed-phase h.p.1.c. The demonstration that the ecdysone "esters" (of peaks 1and 2; Fig. 4B)can be transformed chemically into the corresponding acetonide derivatives suggests that the 2- and 3-hydroxyls of ecdysone remain unsubstituted in the compounds. Of the remaining hydroxyls, the secondary 22-hydroxy group would probably be the most likely to be substituted. In fact, preliminary 'H n.m.r. evidence suggests that the C-22 hydroxyl group of ecdysone is substituted in at least two groups (peaks 1and 2; Fig. 4B)of these apolar derivatives. The possibility always exists that metabolism of exogenous [3H]ecdysone in adult female B. microplus may not accurately reflect that of endogenous hormone. The formation by several tissues of apolar ecdysone metabolites analogous to those formed in vivo suggests a wide distribution within the organism of the appropriate enzyme(s). However, the possible physiological signhcance of this is unclear at present. The observed metabolism of [3H]ecdysone into the "esters" in adult females, the passage of radioactivity into the eggs, together with the presence of a vast preponderance of apolar ecdysteroid "esters" compared with free hormone in the newly laid eggs (Fig. 5) suggest that the "esters" in the latter are of maternal origin. Although the ecdysteroids in newly laid eggs are apparently present predominantly as "esters," distinct peaks of free ecdysteroids have been detected during embryogenesis (K.P. Wigglesworth, unpublished results). The observed hydrolysis of the ecdysteroid esters by a homogenate of developing eggs of B. microplus suggests that such maternal "esters" may function as storage forms (presumably hormonally inactive), being enzymically hydrolysed at certain times during embryogenesis releasing free hormones. Of course, other sources of free ecdysteroids (eg, de novo synthesis) are also tenable. This enzymic hydrolysis of ecdysone "esters" by Ecdysteroids in licks 53 B microplus embryos is analogous to the release of free ecdysteroids from the corresponding 22-phosphates by a phosphatase enzyme preparation from developing embryos of the locust, Schistocercu greguriu . 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