‘U 'a JJBRAR Y icHgan State 'Univcrsirv {2 Wrath ' N THES\$ ABSTRACT METABOLISM OF CHOLESTEROL-A-Clu IN RESISTANT AND SUSCEPTIBLE STRAINS OF ASEPTICALLY REARED HOUSE FLIES, MUSCA DOMESTICA L. By John William Bauer Six different resistant and susceptible strains of houseflies, Musca domestica L., were reared aseptically on a synthetic diet containing chol‘esterol-l-l-ClLl as the only sterol source. The uptake of cholesterol was different for all the strains, but there was no correlation of cholesterol content between the resistant and susceptible flies. The uptake of cholesterol by the females was higher than that for the males indicating a greater requirement for the females because of oogenesis. Esterification of the 3-8—. hydroxyl group accounted for an average of 28% with the free sterol fraction making up approximately 71% of the total sterols. Small amounts of radioactive polar steroids were recovered, indicating at least to a small extent, the metabolism of cholesterol to other more polar derivatives. As eXpected no radiolabelled hydrocarbons were found, indicating a lack of degradation of the steroid ring nucleus. Analyses of the free and esterified sterols by column chromatography, gas—liquid chromatography, ultra- violet Spectroscopy, and reverse isotOpe dilution demon- strated that unchanged cholesterol accounted for 96% of John William Bauer these fractions. Analyses by column chromatography and reverse isotope dilution showed that 0.9% behaved like 7—dehydrocholesterol. “1 METABOLISM OF CHOLESTEROL—A-Clu IN RESISTANT AND SUSCEPTIBLE STRAINS OF ASEPTICALLY REARED HOUSE FLIES, MUSCA DOMESTICA L. By John William Bauer A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1968 ACKNOWLEDGMENTS The author would like to eXpress his gratitude and appreciation to Dr. R. E. Monroe for his guidance and counsel throughout the course of this investigation and to the members of the graduate committee Drs. R. A. HOOpingarner, M. J. Zabik, and L. L. Bieber. Special thanks are expressed to my wife, Elizabeth, for her continually encouraging attitude and enthusiasm throughout the period of investigation. 11 TABLE OF CONTENTS ACKNOWLEDGMENTS . . . . . . . . ._ . . LIST OF TABLES . . . . . . . . . . .p . INTRODUCTION . . . . . . LITERATURE REVIEW . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . Experimental Insects . . . . . . . . . Diets and Rearing Methods . . . . Analytical Standards and Radiolabelled Cholesterol . . . . . Direct Extraction of Lipids . . . . Column Chromatography . . . Acetylation and Saponification of Sterols . . Separation of Sterols by Column Chromatography Sterol Purification . . . . p . Sterol Identification . . . . . . . . . RESULTS . . . . . . . . . . . . . . Direct Extraction of Lipids and Sterols . . . Composition of Radiolabelled Sterols . . . . Separation of Radiolabelled Sterols- . . . . Identification of Radiolabelled Sterols . DISCUSSION . . . . . . . . . . SUMMARY . . . . . . . . LITERATURE CITED . . . . . . . °. . . . iii Page 11 iv Table LIST OF TABLES Weights of resistant and susceptible strains of adult male and female houseflies fed cholesterol— A- CI“ in a larval aseptic synthetic diet . . . . . . . Total weight of lipids extracted from resistant and susceptible strains of adult male and female houseflies fed cholesterol— u-Cl“ in a larval aseptic synthetic diet . . Radiolabelled sterol content of resistant and susceptible strains of adult male and female houseflies fed cholesterol- A— C"+ in a larval aseptic synthetic diet . . . . . . . . Fractionation of total Cl“-compounds from males and females of resistant and suscepti- ble strains of houseflies reared on aseptic synthetic diets containing cholesterol- 4- C1“. Fractionation of the acetylated free and sterol ester fractions from males and females of resistant and susceptible strains of houseflies reared on an aseptic synthetic diet containing cholesterol— A- C . . ._ Results of gas-liquid chromatographic analyses of pooled steryl acetate fractions from resistant and susceptible strains of houseflies reared on an aseptic synthetic diet containing cholesterol- A— C . . . .\ Reverse isotope dilution and srecrystalliza- tion of the pooled A5 - and A5 ’7 -steryl acetate fractions from resistant and sus- ceptible strains of houseflies reared on an aseptic synthetic diet containing cholesterol- iv Page 23 25 26- 27 3O 31' 32 INTRODUCTION The development of resistance by insects to insecti- cides poses most serious problems in their control, and the occurrence of such resistance is not new, as this phenome- ft non has been known for more than 50 years. Despite the research of many investigators, the actual mechanism(s) of pesticide resistance is still not known.. Although many scientists have implicated potential mechanisms and have i? compared compounds and metabolites among different strains of insect species, they have not as yet found a sound physiological and biochemical basis for resistance. Because of the lipoidal-like nature of pesticides, the naturally occurring lipids of insects and other animals received much attention during early resistance studies. Unfortunately, such studies were usually gross in design and early instru- mentation greatly curtailed the scientist's ability to study the finer details of these investigations. One such comparison (Enan gt_al., 1964) made among several pesticide resistant strains of adult houseflies implicated different levels and/or absorption rates of cholesterol in these insects. Their results showed major differences between the resistant and susceptible strains, with the resistant strains containing far less cholesterol than the one strain of susceptible flies studied. These early investigations, however, were made without the use of aseptic synthetic methods and without modern instrumentation techniques, nor were the sterol levels studied in more than one susceptible strain of flies. This investigation, therefore, was con- cerned with the metabolism of cholesterol-A-Clu in differ- ent resistant and susceptible strains of aseptically reared houseflies, to determine if sterol metabolism or absorption rates actually enters into pesticide resistance per se or whether strains of flies differ widely even if they are resistant or not. LITERATURE REVIEW The importance of cholesterol and related steroids in insect nutrition was first demonstrated when Hobson (1935) showed that the larvae of the blowfly, Phaenicia sericata, required a dietary source of sterol. 'Lipke and Fraenkel (1959) further established that insects in general require a dietary sterol, and it is now recognized that insects do not possess the complete enzymatic system necessary for the synthesis of sterols from more simple compounds such as acetate and mevalonate. This character- istic is in direct contrast to most plants and animals which are able to synthesize their sterols from more simple compounds (Block, 1965). However, there are a few insects which have been reported to be capable of some degree of cholesterol biosynthesis, such as the silkworm' (Saito et al., 1963), the Silverfish, Ctenolepisma sp., (Clayton et a1., 1962), and the German cockroach, Blattella germanica L., (Clayton, 1960), but Clayton (196A) attributes these syntheses to the activity of symbionts. Though insects themselves are unable to synthesize sterols, these substances are vitally important for them, because as Clark and Block (1959) have shown, sterols serve a dual purpose. They have proposed that "sparing- fl sterols, which can replace up to 97% of the cholesterol 3 requirement of Dermestes maculatus, served in a structural capacity, and the remaining irreplaceable cholesterol served in a metabolic role possibly as the basic materials for some hormones that control essential life processes. Robbins (1963) has stated that every insect studied to date has been found to require a dietary or exogenous source of sterol for normal larval growth and metamorphosis. In addition to this, sterols have been reported to play a role in several other physiological processes in insects. Kobayashi, et a1. (1962)has demonstrated that certain sterols have "brain hormone" activity, and Gilbert (1963) has shown that some exhibit "Juvenile hormone" activity in insects. Ecdysone, from the silkworm, Bombyx mori L., has been shown to have a steroid structure (Karlson, g£_al., 1963). It has also been found that sterols are involved in the initiation of ovarian development (Robbins and Shortino, 1962) and are necessary for sustained viable egg production (Monroe, 1959, 1960). i For some functions the sterols may be utilized as such by the insect, but to satisfy other requirements they must first be converted into other sterols or steroids. Much work has been done recently on the mechanisms of these conversions as this may lead to the development of specific methods in insect control. According to Kaplanis et a1. (1963), the house fly, Musca domestica L., is unable to convert B—sitosterol into cholesterol or campesterol. This seems to be an exception because many, other insects such as the oriental housefly (Levinson, 1962) and the German cockroach (Robbins et a1” 1962) could convert B-sitosterol into cholesterol. Both Eurycotis floridana (Clayton and Edwards, 1962) and Blattella germanica L. (Louloudes et a1” 1962) were able to convert H1 cholestanol into A7-cholestenoL Also cholesterol could be dehydrogenated at the 7—position (Robbins et al., 1964) by B. germanica L. The conversion of cholesterol into 7- j dehydrocholesterol has also been observed in the housefly, J M. domestica, by Kaplanis et a1. (1960), Monroe (1964), and Monroe e£_a1. (1967). Several investigators have tried to synthesize cholesterol derivatives that could act as growth-inhibiting anti-metabolites to be used as possible insect controls. Clayton (1964) has come to the conclusion, however, that these cholesterol derivatives had no real inhibitory effects on the growth of insects. Some competitive inhi— bition was occasionally found, but this effect was usually completely reversed by cholesterol in normal concentrations. Earle et_al. (1967), however, recently has shown a partial inhibition of growth in the boll weevil by two different azasterols even in the presence of cholesterol. Also, inhibition of growth by another diazacholesterol was found in the tobacco hornworm (Svoboda et al., 1967). As mentioned above, Karlson (1963) found that the molting hormone ecdysone was a sterol, synthesized from cholesterol by the blowfly, Calliphora erythrocephala. Many investigators concluded that it was plausible that other hormones were also produced from cholesterol by insects. After administration of radioactive cholesterol . to insects, several investigators such as Kaplanis gt_al. {I} I (1960), Monroe (1964), Monroe et a1. (1967), Robbins gt El. (1961),IShdJ.et al (1963), and Lasser et a1. (1966) 4 have always found a certain percentage of the radioactivity in the so-called polar fractions of sometissues. So far, the nature of these radioactive components is completely unknown. MATERIALS AND METHODS Experimental Insects The houseflies (Musca domestica L.) used in these tests were obtained from the Entomology Research Division, U. S. Department of Agriculture, Corvallis, Oregon, and consisted of the following strains: 1.. DDT resistant. Orlando DDT strain immune to DDT with major genes for DDT dehydrochlorinase and for knockdown resistance (Kdr). 2. Malathion resistant. The "Grothe" strain resis— tant to malathion with about BOO-fold tolerance due to the altered ali-esterase factor for organophosphate resistance. 3. Parathion resistant. Parathion "classic wing" resistant to parathion with about 30—fold tolerance. A.. Sevin resistant. A strain immune to sevin due to a major 5th chromosomal factor for Sevin® tolerance and possibly additional factors. 5. Orlando susceptible. The Orlando regular strain completely susceptible to DDT, parathion, and- dieldrin. Another strain of flies employed in these studies was an insecticide-susceptible, maximum longevity strain obtained 7 from the Insect Physiology Laboratory, U. S. Department of- Agriculture, Beltsville, Maryland. Diets and Rearing Methods Pupae from each strain were placed in a large screen cage for eclosion and the adults fed a 1:1 mixture of non- fat dry milk and confectioners sugar. The larvae were routinely reared by the CSMA procedure (Anon., 1959) and kept at 2612° C. The test pupae were separated from the medium and 600 from each strain were placed into 50 ml beakers. Paper funnels with escape exits at the top were placed over the beakers to prevent the flies from having access to the puparial cases which have been found to con- tain sterols (Monroe, personal communication). The adults were allowed to emerge in large screen cages and were supplied with 10 g of a synthetic diet (Monroe, 1960) every third day. The synthetic diet, as modified by Monroe and Lamb (1968), was composed of the following constituents:l lThe casein was obtained from Calbiochem, Los Angeles, California; the sucrose was obtained locally as granulated cane sugar; the sodium oleate and nucleic acid were obtained from-Nutritional Biochemicals Corp., Cleveland, Ohio; and the cholesterol was a purified grade received from Fischer Scientific Co., Fair Lawn, New Jersey.* £2122: Casein (sodium salt)2 47.0 Sucrose A7.0 Sodium oleate 2.0 Wesson's salts A.0 RNA 0.1 Cholesterol (final concentration 0.1%) B—Vitamin mixture 3 The vitamin mixture used was as follows: Mg/lOOg diet Thiamine hydrochloride 50.0 Riboflavin 25.0 Nicotinic acid 100.0 Calcium pantothenate 50.0 Pyridoxine hydrochloride 25.0 Folic acid 5.0 Choline chloride 1,000.0 Inositol 500.0 Biotin 1.0 The RNA, salts, sodium oleate, sucrose, and casein were ball—milled for 3-5 hours. The cholesterol was added as 2500 g of casein was mixed with 500 g of 1% NaOH in distilled water, dried for 2—4 days, out into small pieces, and hammer-milled to pass a 0.009 mesh screen. 3All vitamins were obtained from Nutritional Biochemicals Corp., Cleveland, Ohio. 10 a dichloromethane solution and the mixture stirred occasionally until the solvent had evaporated. The vita- min mixture was dissolved in distilled water with the addition of 2—u drops of concentrated ammonium hydroxide. Ten m1 of the vitamin solution was added to 100 g of diet and ground in a mortar. The diet was then fan dried IMfi for approximately 20 minutes and subsequently reground until a fine homogeneous powder was obtained and the finished diet stored at —30° C until used. This adult synthetic diet was fed to the pretest flies in order to remove phytosterols from their tissues and thus allow finite studies with cholesterol only. Distilled water was provided in 100 m1 beakers with a thin piece of styrofoam floating on the surface to pre- vent drowning. Dead flies were removed from the cages daily to prevent cannibalism, which may serve as a source of nutrients (Ascher and Levinson, 1956). Petri dishes containing muslin bags soaked in ammonium carbonate solution were placed into the pretest fly cages for oviposition. After 6—7 hours the dishes were removed, and the eggs placed into 50 m1 Erlenmeyer flasks. The eggs were then surface sterilized for 20 minutes in 0.1% sodium hypochlorite. Using aseptic techniques, approximately 300 eggs were then transferred by a calibrated pipette to 250 ml Erlenmeyer flasks con- taining 7.5 g of a larval synthetic diet with 0.1% ll cholesterol -4dClu. This diet was similar to that reported by Monroe (1962) except that sodium oleate was omitted. The diet was composed of the following con- stituents: NutrientsLl Parts Casein 70.0 Alphacel 3.0 RNA 1.0 Wesson's salts 4.0 Agar 20.0 Cholesterol —4—Clu 0.1% B-Vitamins 0.75 m1/7.5 g of diet The casein, celluflour, ribose nucleic acid, Wesson's salts, and agar were ball—milled for 4-5 hours. The cholesterol —4—Clu was added to the dry diet as a dichloro- methane solution. The solvent was then allowed to evaporate from the diet with periodical stirring and heating in an oven at 45° C. To each of the flasks was added the vita— min solution and 50 ml of distilled water and the mixture autoclaved at 15 pounds (121° C) pressure for 20 minutes. After the eggs were transferred to the larval' synthetic diets, the flasks were placed in an incubator at 34° C for 2 days, at which time most of the larvae were “All nutrients from Nutritional Biochemicals Corp., Cleveland, Ohio, except the casein which was obtained from Calbiochem, Los Angeles, California and the cholesterol -4—Cl“ which was pUrchased from Nuclear Chicago, Des Plaines, Illinois. 12 in second or third instar. For the next four days the larvae were held at 26°:2° C, during which time they had pupated. Each flask was forcefully filled with distilled water, swirled, and the contents poured into a No. 20 standard sieve. Agar and other debris was washed through F“! the sieve by distilled water until only the pupae remained. 1‘ The pupae were then placed onto folded paper towels until dry. The dry pupae were put into 50 m1 beakers with A. J paper funnel escape assemblys and placed into quart Jars BJJ A fitted with screen lids, and the adults allowed_to emerge. The adults were supplied sucrose and distilled water by placing sugar cubes and cotton moistened with distilled water in inverted paper cups on the tops of the screen lids. After three days the flies were anesthetized with C02, sexed, weighed live, placed in vials, and kept at -30° C until analysed. All of these tests were conducted in triplicate. Analytical Standards and Radiolabelled Cholesterol The cholesterol (m.p. 149-150° C), cholesteryl acetate (m.p. 116-117° C), and cholestane (m.p. 80-81.5° C) were received from the Insect Physiology Laboratory, U. S. Department of Agriculture, Beltsville, Maryland. A Kofler block was used to determine melting points and the values recorded are uncorrected. 13 The 7-dehydrocholesterol (m.p. 143—144° C), (Nutritional Biochemicals Corp., Cleveland, Ohio) used was purified by successive recrystallizations from acetone, ethanol, methanol, acetone-ethanol (1:1), acetone-methanol (1:1), ethanol-methanol (1:1), and methanol. The 7- dehydrocholesteryl acetate (m.p. 130-13l° C) was made by acetylating the 7-dehydrocholesterol. Both were kept in yagug in the dark in a desiccator to minimize autodecompo— sition. The cholesterol—4—Clu was obtained from Nuclear- Chicago in two 50 uC lots. The radiolabelled cholesterol was diluted with authentic unlabelled cholesterol for a final observable specific activity of 917 counts per minute per pg. Radiochemical purity was found to be 99% as determined by paper chromatography using a distilled water- nfpropanol (2:3) system saturated with deobase (Kaplanis gtgal., 1960). Radioassays were made with a windowless_gas—flow counter attached to a Baird Atomic scaling unit.‘ The weightless samples were all prepared in triplicate and redistributed with 1»m1 of nyhexane before being counted, and all samples were counted for a sufficient time to give a maximum standard error of i5%. Direct Extration of Lipids The adult flies were homogenized with distilled water in an all—glass homogenizer. The homogenate was transferred 14 quantitatively into a 300 m1 round-bottom flask. A volume of acetone-ethanol (1:1) at 4 times the volume of aqueous used in the homogenization was then added to the reaction flask and the mixture refluxed for 90 minutes (Kaplanis et_al., 1960). The hot mixture was decanted into a Bachner funnel containing two circles of Whatman No. 1 filter paper and the supernatant was filtered into a vacuum filtering flask. Several rinses were made with small aliquots of acetone-ethanol (1:1) for the assurance of a complete and quantitative transfer. The residue from the filter paper was placed into a planchet and radioassayed as a quantitation check.‘ The solvent pair was then removed in vacuo from the aqueous extract, and the remaining aqueous quantitatively transferred to a separatory funnel to which was added approximately 0.5 ml of concentrated hydrochloric acid. The aqueous mixture was then extracted 3 times with equal volumescfi‘peroxide—free ethyl ether. The pooled ether fractions were back extracted with water until neutral and then-after drying over anhydrous sodium sulfate, the ether was removed in vacuo. The residue (total lipids) was weighed and then assayed radiometri— cally for the total sterol(s). The aqueous of the ether extracts was also radioassayed as a quantitative check. The extracts were then frozen in benzene in a nitrogen 9 atmOSphere at -300 C until further analysis. 15 Column Chromatography The total lipids were then fractionated by column chromatography on a 1.1 cm column of 7.5 g of neutral Woelm aluminum oxide, grade I, deactivated with 1.5% water (packed in nfhexane). One to 2 g of anhydrous sodium sulfate was also added to the top surface of the alumina. The total lipids were transferred quantitatively to the column in small aliquots of benzene, and then successively washed with 100 ml of benzene, ethyl ether, and methanol. The benzene fraction was evaporated in Em; vacuo and added to a second column which had been pre- pared in the same manner as the first column, but washed with nehexane, benzene, and methanol. After pooling the two methanol fractions, the 4 fractions consisting of nrhexane, benzene, ethyl ether, and methanol, which elute the hydrocarbons, sterol esters, free sterols, and more polar steroids, respectively,(Kap1anis et_a1., 1960) were assayed radiometrically. The free sterols and sterol ester fractions were kept at -30° C in benzene in a nitrogen atmosphere until further analysis. Acetylation and Saponification of Sterols The free sterol fractions were pooled and acetylated as reported by Johnston et a1. (1957) with some modifica— tions. The sterols were transferred to a glass stoppered graduated centrifuge tube with a Pasteur pipette and the, 16 solvent removed with a stream of dry nitrogen. Approxi- mately 1 m1 of acetic anhydride and pyridine (1:1) were added to the centrifuge tube. The tube was then flushed with nitrogen, stoppered tightly, and heated in a boiling water bath for 90 minutes. The tube was then removed from the water bath, and the solution quantitatively transferred to a separatory funnel with ice water (final volume 20 ml). The aqueous mixture was extracted 3 times with ethyl ether and the pooled ether fractions back extracted with 6.25% .0 hydrochloric acid-ice water (1:1), water until neutral, 5% sodium bicarbonate solution, and water until neutral. The ether fractions were then dried over anhydrous sodium sulfate, the ether removed in vacuo, and the residue assayed radiometrically. The sterol esters were subsequently saponified by transferring them quantitatively to a glass stoppered graduated centrifuge tube with a Pasteur pipette and the solvent removed with a stream of dry nitrogen. The volume of lipid was noted, and 0.5 ml of 10% potassium.hydroxide in 95% ethanol was added per 0.1 ml of lipid. The tube was flushed with nitrogen, stOppered tightly, and heated in a boiling water bath for 90 minutes. The solution was then quantitatively transferred to a separatory funnel containing 5 ml of ice water. The centrifuge tube was rinsed with small aliquots of ice water bringing the total volume of water in the separatory funnel to approximately 20 ml. The resulting aqueous was then extracted repeatedly 17 with ethyl ether, the pooled ether fractions back extracted with water until neutral, dried over anhydrous sodium sulfate, the ether removed in vacuo, and the resi- due assayed radiometrically. After radioassay, the sterol ester fractions were acetylated as described above. Separation of Sterols binolumn Chromatography The acetates of the free sterols and saponified steryl esters were subsequently analysed separately by column chromatography (Kaplanis gtéal., 1960). A 1.1 cm water Jacketed glass column was packed with 25 g of Woelm acid aluminum oxide, grade II, in nghexane. The steryl acetates were added to the column with small aliquots of nfhexane until a quantitative transfer had been made. The column was then wrapped with a heating coil so that the temperature of the column remained at 29° C to 31° C- The column was also wrapped in aluminum foil to prevent light from decomposing the A5’7 -conjugated dienes. The column was eluted with 3 L of nfhexane—benzene (95:5), and 20 m1 fractions were collected on an automatic frac-A tion-collector. After all of the nyhexane—benzene had passed through the column, 50 m1 of methanol was added and collected separately. The methanol was then reduced LE vagug_and the residue assayed radiometrically.' From each of the 20 m1 fractions, 200 pl aliquots were pipetted into liquid scintillation vials and radioassayed with a Nuclear- 18 Chicago Model 6850 "Unilux 1", an ambient temperature liquid scintillation spectrometer. Before the fluor mixture was added to the vials, the 200 pl aliquots were evaporated so that there would be no difference in quenching., The fluor mixture used (5 ml per vial) was- composed of 4 g of PPO (2, 5 diphenyloxazole) and 0.05 3 Of _h POPOP (p—bis [2-(5-phenyloxazolyl)] -benzene) per liter r5 of reagent grade toluene. Two peaks of radioactivity were observed that corresponded to two major groups of compounds which this column separates: AS—steryl acetate ”j ! 5’7-steryl acetate (as I (as cholesteryl acetate) and A 7—dehydrocholesteryl acetate). The fractions correspond-. ing to the two peaks were pooled separately, and then radioassayed on the windowless gas—flow counter to deter- mine total activity. These two major groups eluted from the column were frozen in benzene in a nitrogen atmosphere,. A5,? at —30° c and designated as A5- and —stery1 acetates. Sterol Purification The A5- and A5’7—stery1 acetate fractions were saponified separately using the method described above and then purified by digitonin precipitation (Sperry and Webb, 1950). The A5— and A5’7—steryl acetates were each trans-. ferred to centrifuge tubes, and the solvent removed with: a stream of dry nitrogen. To each tube, 1—2 ml of acetone— ethanol (1:1) was added and heated until the residue l9 dissolved. Sufficient digitonin5 to precipitate the sterols present was added as a 1% solution in 85% ethanol. The digitonin solution was added hot with a Pasteur pipette to insure mixing. The solution was stirred and then held for 3 hours in a 45° C water bath. Both tubes were removed from the bath, the sides were rinsed down with 85% ethanol, then centrifuged at 1200 rpm for 10 minutes. The supernatant of each tube was removed with a Pasteur pipette, and 3-4 ml of acetoneeethanol (1:1) was added and the mixture thoroughly stirred.. The tubes were again centrifuged for 10 minutes at 1200 rpm, the supernatant removed, and the washing process repeated with acetone—ether (1:1) and finally with ether. The washed digitonin—sterol precipitates were then suspended in 3-4 ml of ether and the ether removed with a fine stream of dry nitrogen in a water bath in order to coat the precipi- tate on the walls of the tubes. The sterol digitonides were then broken by dissolving the precipitate in 1-2 ml of pyridine and holding the mixture overnight at room temperature. The pyridine was removed with nitrogen to a volume of 0.2-0.3 ml. Both tubes were stirred thoroughly after the addition of 4 m1 of ether and centrifuged at 1200 rpm for 5 minutes. This step was repeated 3 times and each ether fraction was pooled in another centrifuge tube. The free purified sterols were then coated on the 5The digitonin required was 3.1 times the weight of sterols plus an excess of 20%. We 2O walls of the tubes by evaporating the ether with a stream of dry nitrogen. Both the AS- and A5’7-sterol fractions were then reacetylated using the same procedure as described above. Sterol Identification Analyses of the A5— and A5’7-steryl acetates were made by gas-liquid chromatography on a Research Specialties "600 Series" gas chromatograph equipped with a hydrogen flame detector and employing three different columns (VandenHeuvel et_al., 1961). Stainless steel columns, 4 ft x 7 mm (i.d.), were packed with a solid support of 100-120 mesh, silanized base-and acid-washed Gas Chrom P 6 coated with one of the following liquid phases: 3% SE-30, 1% QF-l, or 0.75% neopentyl glycol succinate. The SE-30 column was operated at 238° C, with the vaporizer at 214° C and a hydrogen flow rate of 60 ml per minute and air at 160 ml per minute. The flow rate for nitrogen, the carrier gas, was 125 ml per minute. The QF—l column was operated at 218° C, with the vaporizer at 272° C and a hydrogen flow rate of 60 ml per minute and the air at 160 ml per minute. The flow rate for nitrogen was 127.7 ml per minute. The neopentyl glycol succinate column was operated at 230° C, with the vaporizer at 280° C and a hydrogen and air flow rate of 60 and 160 ml per minute, respectively. The flow 6Gas Chrom P obtained from Applied Science Labora- tory, State College, Pennsylvania. 21 rate for nitrogen was 113.2 ml per minute. Both sterol fractions were diluted with benzene so that all samples injected were composed of 4 ug of steryl acetate in 1 ul of benzene. Three ul (12ug) of the samples were injected by a microliter syringe. All retention-times were calculated relative to the retention time of cholestane, and peaks were identified by comparison of these retention times with r1] those of authentic standards. 8 Further analyses of the steryl acetates were made by ultraviolet Spectroscopy and compared with spectra of 93 equal concentrations of cholesteryl acetate and 7- I? dehydrocholesteryl acetate standards. The samples were dissolved in absolute ethanol and the mixture placed in silica cuvettes. Analysis was made on a Beckman DB. recording spectrophotometer. The radiolabelled AS— and A5’7-stery1 acetates were admixed with 100 and 25 mg of authentic non-radioactive cholesteryl and 7—dehydrocholesteryl acetate, reSpectively, for reverse isotope dilution analysis. To each of the flasks, sufficient n—hexane was added to solubilize the mixtures and then evaporated to dryness. Tares were taken on two 5 ml volumetric flasks, and 10 mg of the labeled— unlabeled mixtures were added individually to the flasks. The flasks were then filled to the mark with benzene, and subsequently radioassayed. The mixtures were then returned to the flasks from which they came and the total mixtures now 22 transferred to small beakers with a Pasteur pipette. The benzene solvent was removed from the beakers with-a stream of dry nitrogen. The beakers were then filled half full with acetone and brought to a boil on a hot plate. After total solubilization had taken place, the beakers were removed from the hot plate and placed in the freezer. As the mixtures cooled, the beakers were occasionally swirled until a good crop of crystals had formed. The crystals were collected in a Sintered glass funnel under vacuum. When absolutely dry, 10 mg from each funnel were placed into the original 5 ml volumetric flasks, and the entire sequence was then repeated as described above. The mixtures were repeatedly recrystallized from ethanol (twice), methanol (twice), and ethanol-methanol (1:1, twice). After every recrystallization, radioassays were conducted for observable specific activity, and the results recorded as counts per minute per mg of steryl acetate. RESULTS Direct Extraction of Lipids and Sterols The average weight per fly for all strains investi- gated is shown in Table 1. TABLE 1.-—Weights of resistant and susceptible strains of adult male and female houseflies fed cholesterol—4-C1“ in a larval aseptic synthetic diet. Number of Average flies Weight (g) weight/fly (mg) Fly strain WWQQ a? 90 OWSQQ Beltsville susc. 277 296 2.9329 3.8846 10.6 13.1 Orlando susc. 37 69 0.4198 0.9047 11.3 13.1 MalathiOn resis. 172 210 1.8753 2.6124 10.9 12.4 Sevin resis. 158 193 1.8298 2.6592 11.6 13.8 DDT resis. 165 155 1.4610 1.8417 8.9 11.9 Parathion resis. 36 28 0~3828 0.4155 10.6 14.8 No significant difference in weight between the resistant and susceptible strains was observed. In all strains, the females were approximately 2.5 mg heavier than the males. The weight difference was lowest in the malathion resistant strain at 1.5 mg and highest in the parathion 23 24 resistant strain at 4.2 mg with the other two resistant and two susceptible strains falling between these two figures. Following direct extraction of lipids, the average weight of lipids per fly for each strain was calculated (Table 2). All strains showed approximately the same amount of lipid per mg of fly, ranging from 0.033 mg for the Sevin® resistant females to 0.057 mg for the parathion resistant males. The males in all the strains except for the malathion resistant flies contained slightly higher amounts of lipids per body weight than the corresponding females. The males of the malathion resistant strain con- tained 0.01 mg per mg of body weight less than the females of that strain. The sterol content of the flies as determined radio— metrically is shown in Table 3. The DDT resistant strain had the highest radio- labelled sterol content at 0.66 pg per mg of body weight for the males and 0.71 pg per mg of body weight for the females. The Orlando susceptible strain had the lowest sterol content at 0.40 ug and 0.46 ug for the males and females, respectively. The sterol content for all the other strains was between the DDT resistant and Orlando susceptible Strains. 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