l ? i I I ‘ I MN I l H ,r 2! w * ‘ ”H h n. m “I 1‘ H (Ml H J: x l ‘ [ THE LIPOGENiC ACTIVITY OF FIVE AMINO ACIDS IN ASEPTICALLY REARED HOUSEFUES, MUSCA DOMESTICA L. Thesis for the Degree of M. S. MICHIGAN STATE UNWERSITY ROBERT THOMAS KON 1968 THESIS I, In I? {/1R 1, Michigim State University ABSTRACT THE LIPOGENIC ACTIVITY OF FIVE AMINO ACIDS IN ASEPTICALLY REARED HOUSEFLIES, MUSCA DOMESTICA L. By Robert Thomas Kon Five different free, radiolabelled amino acids com- prising five treatments were blended into a synthetic casein diet and fed to aseptically reared larvae of Musca domestica L. After four replications, analysis of pupae under 2A hours old showed that per mg of total lipid, leucine contributed 25.1% of the weight, glutamate 22.1%, alanine 4.3%, phenylalanine 4.2%, and methionine 0.3%. The values for fatty acids alone were 27.5%, 22.0%, A.2%, A.O% and O.A%, respectively. Except for leucine and methionine these values had good linearity with the cor- responding amino acid per cent values naturally occurring in casein. The fatty acids found by gas-liquid chromato- graphic analysis were, myristic (1.7%) palmitic (28.7%), palmitoleic (39.7%), stearic (trace), and oleic (29.9%). A treatment average of from 8A% to 96% of the radiolabel in the total lipids was found in the fatty acids, but it is believed that 95% more accurately represents actual biochemical occurrences. Among the unsaponifiable lipids, Robert Thomas Kon from 69% to 76.6% of the activity behaved as hydrocarbons, from 16.5% to 29.9% behaved as high molecular weight alco- hols, from 0% to 4.5% behaved as free sterols (column data, without additional analysis), and from 1.1% to 5.4% behaved as more polar compounds. The level of metabolites contrib- uted to the bio-fluids by each radiolabelled amino acid was found to be quite linear with the corresponding per cent value of each in casein and a hypothesis about this obser- vation was discussed. THE LIPOGENIC ACTIVITY OF FIVE AMINO ACIDS IN ASEPTICALLY REARED HOUSEFLIES, MUSCA DOMESTICA L. By Robert Thomas Kon A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1968 TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . iii LIST OF FIGURES . . . . . . . . . . . . . iv INTRODUCTION . . . . . . . . . . . . . . 1 LITERATURE REVIEW 2 MATERIALS AND METHODS 6 Diet Preparation . . . . . . 8 Rearing Procedure and Aseptic Culture . . . 8 Extraction and Chromatographic Fractionation .of Lipids . . . . . . 9 Gas- -Liquid Chromatography of Fatty Acids . . . . 13 RESULTS. . . . . . . . . . . . . . . . 15 DISCUSSION. . . . . . . . . . . . . . . 24 Pupae. . . . . . . . . . . . . . . . 24 Lipids . . . . . . . . . . . . . . . 24 Aqueous . . . . . . . . . . . . . . . 30 SUMMARY. . . . . . . . . . . . . . . . 38 REFERENCES. . . . . . . . . . . . . . . 40 ii Table LIST OF TABLES Gross data for housefly pupae aseptically reared on a synthetic diet containing free, radiolabelled amino acid Fresh body weight data of housefly pupae aseptically reared on a synthetic diet containing free, radiolabelled amino acid Radioactivity in total lipid fraction of housefly pupae aseptically reared on a synthetic diet containing free, radio- labelled amino acid. Radioactivity after saponification of total lipids of housefly pupae aseptically reared on a synthetic diet containing free, radiolabelled amino acid Relative distribution of radioactivity in unsaponifiable lipids from housefly pupae aseptically reared on a synthetic diet containing free, radiolabelled amino acid Gas-liquid chromatographic analysis of fatty acids of housefly pupae aseptically reared on a synthetic diet containing free, radiolabelled amino acids. Radioactivity in the aqueous fraction (bio- fluids) of housefly pupae aseptically reared on a synthetic diet containing free, radiolabelled amino acid Division of radioactivity between two fractions of bio-fluids of housefly pupae aseptically reared on a synthetic diet containing free, radiolabelled amino acid iii Page 15 16 17 19 2O 21 22 23 LIST OF FIGURES Figure 1. Relationship between casein composition and radioactivity in the total lipids of housefly pupae (under 24 hours old) aseptically reared on a casein diet con- taining free, radiolabelled amino acids 2. Relationship between casein composition and radioactivity in the bio-fluids of housefly pupae (under 24 hours old) aseptically reared on a casein diet containing free, radiolabelled amino acids. . . 3. Two curves relating dietary amino acid com- position to radioactivity in the bio—fluids of housefly pupae (under 24 hours old) aseptically reared on a synthetic diet with free, radiolabelled amino acids added. iv Page 25 31 34 INTRODUCTION A complete knowledge of insect biochemistry could possibly lead to the develOpment of a specific insecticide for any insect or small group of insects based upon slight metabolic variations. As a step toward further clarifica- tion of the biochemistry of the housefly, Musca domestica L., a study was made of the conversion of five radio- labelled amino acids into the fatty acids of aseptically reared housefly pupae. As the lipogenic activities of all the amino acids become known, the more important question of why they are different can be attacked. Because housefly larvae need only cholesterol supplemented protein as a carbon source (Brookes and Fraenkel, 1957; Monroe, 1962), they provide an excellent study subject by avoiding the variables of dietary carbohydrates and fats (assuming also that they possess no significant free CO fixation mechanism). 2 The amino acids chosen represent several structural classes. In this study the radiolabel was supplied as Alanine-U-Clu, Glutamate—U-Clu, Leucine-U-Clu, Methionine- G-H3, and Phenylalanine-U—Clu. LITERATURE REVIEW Studies have revealed that lipids serve as more than a reserve energy source for insects. Hobson (1935) discovered a dietary requirement for cholesterol by the blowfly, Lucilia sericata, which has since been accepted as a generality for all insects (Albritton, 1953). Clayton (1964) has reviewed the literature on cholesterol requirements by insects, and Monroe et a1. (1967) haveshown the benefit of cholesterol in houseflies for growth and reproduction. Ecdysone, the molting hormone of insects, was found to be a metabolite of cholesterol (Karlson and Hoffmeister, 1963). Another dietary requirement for a lipid was reported by Fraenkel and Blewett (1946). It was shown that the moth, Ephestia kuehniella, needed, but could not synthesize linoleic acid for successful emergence. Dadd (1961) found that Locusta migratoria did not mature on a linoleic deficient diet, while from 10% to 56% of Schistocerca gregaria did mature on the same diet, but with damaged wings. Fast (1964) reviewed the literature on insect lipids and found no consistent requirement for a lipid other than sterols. Structurally, lipoproteins were components of the nerve axons of insects studies by Richards (1944). Watanabe and Williams (1951) demonstrated that mito— chondria from the flight muscle of Phormia regina contained 29% lipid (dry weight). The insect cuticle was found to reduce water loss by using layers of hydrocarbons and waxes as waterproofing mechanisms (Ramsay, 1935; Gilby and Cox, 1963). Lipids have also been implicated in resistance to insecticides. Munson et al. (1954) presented numerous observations to show that temperature induced differences in saturation of lipids were related to insecticide resis— tance. The data were offered in support of the View that unsaturation increased the holding capacity of lipids for DDT and related compounds, rendering them non-toxic. Neri et a1. (1958) found a tendency for higher lipid content in DDT resistant strains of Anopheles atrgparvus compared to susceptible strains. Recently, Moore et al. (1967) found differences in fatty acid per cent composition between boll weevils, Anthonomus grandis, which died and those which survived exposure to insecticides. A higher percentage of palmitic and oleic acids were found in those which survived and a higher percentage of stearic, linoleic, and linolenic acids in those which died. These results contradicted those of Munson et a1. (1954).1 1In the two papers, different species were studied. Neither worker analyzed lipids by fractions to determine exactly where the differences in per cent composition occurred when such differences appeared related to insecti— cide resistance. The nutritional importance of amino acids, both essential (Mendel, 1914) and unessential (Snyderman, 1962) has been recognized. Now, elementary work is being done to characterize the mechanisms of insecticides by observing changes in free amino acid content of an insect. Lord and Solly (1964) studied DiazionR in concentra- tions which killed 50% and 90% of resistant and of sus- ceptible houseflies. They found a decrease in d- and B— alanine as well as proline in the dead flies. This effect was absent in surviving flies. A decrease in glycine proline, and glutamate was noticed in susceptible, malathion-poisoned roaches, Blatella germanica, by Mansingh (1965), while only a slight reduction occurred in poisoned, resistant roaches. Corrigan and Kearns (1963), monitored 14 amino acids and found only proline to decrease progres- sively with time in DDT poisoned roaches, Periplaneta americana, at 15°C. Despite the possible relationships of insecticide resistance with both amino acids and lipids, there is a lack of good information concerning the lipogenic activi- ties of amino acids in insects. Clements (1959) presented data on the incorporation of glycine and leucine into fats by whole tissue preparations of fat body from the desert locust, Schistocerca gregaria. Glycine was 1.6 times as lipogenic as leucine. However, in vitro studies do not consider total body needs for amino acids, or the varia- tion in composition of a total diet. In vivo studies of insect fatty acid synthesis from precursors included that by Louloudes et a1. (1961) from acetate-l-Clu injected into Periplaneta americana. Robbins et a1. (1960) performed a conversion study of injected acetate—l—Clu into lipids by adult houseflies. The synthesis of adult housefly lipids from injected mevalonate- 2-Clu was reported by Kaplanis et a1. (1961). Several studies have been performed to determine the amino acid requirements of insects (Vanderzant, 1957; Rock and King, 1967a, b; Earle et al., 1966). However, no reports were found describing from an in_vivg study, with insect materials, the relative tendencies of various amino acids to donate carbon to the body lipids. MATERIALS AND METHODS The parent flies for the pupae in this study were of a laboratory strain derived from the U.S.D.A., Insect Physiology Laboratory colony at Beltsville, Maryland. A synthetic housefly larval diet described by Monroe (1962) was modified to the following composition by omit- ting sodium oleate: Ingredient Parts Casein (vitamin free)1 72 Alphacel2 3 Wesson's salts2 4 RNA2 1 Agarl 20 An aqueous misture of B-vitamins was prepared to be added as the diet was used. Four to five drops of con- centrated ammonium hydroxide were added to keep the vitamins in suSpension. The composition per 10 m1 of vitamin mixture was: 1Supplied by Calbiochem, Los Angeles, California. 2From Nutritional Biochemicals Corp., Cleveland, 6 Ohio. Vitaminl Thiamine hydrochloride Riboflavin Nicotinic acid Pantothenic acid Pyridoxine hydrochloride Choline chloride Inositol Folic acid Biotin l 5 50 25 OO 50 25 1000 00 The vitamin-free dry mixture was ball milled 4 hours before adding 0.1% by weight of cholesterol in dichloro- methane which was then evaporated in a warm oven with fre- quent stirring. Radiolabelled amino acids2 unlabelled carrier amino acid 3 were mixed with and distilled water to form lmM solutions. Data for the amino acids were as follows: Quantity 12 12 12 120 12 yo no no pC pc 5 Radiochemical Purity 99% 96% 98% 96% 96% All vitamins were from Nutritional Biochemicals Amino Acid dpm/“gu Alanine-U-Clu 162 Glutamate-U-Clu 22 Leucine—U-Clu 53 Methionine-G-H3 1423 Phenylalanine—U-Clu 97 l Corp., Cleveland, Ohio. 2 From Nuclear Chicago, Des Plaines, Illinois. 3Nutritional Biochemicals Corp., Cleveland, Ohio. “Determined for each flask and based on the amino acid percentages for casein given by Hawk et a1. (13th ed. 1954). 5Added per flask. Radiochemical purity was determined by paper chromatography employing a system of n-butanol—acetic acid—HOH (125-30-125 v/v). Ten ml of modified Bray's fluor solution were used per vial for counting on a Nuclear Chicago Unilux I (model 6850) liquid scintillation counter. The formula for 2 liters of modified Bray's solution was: Ethylene glycol monomethyl ether 500 ml Toluene 1500 ml POPOP (1, 4-bis—[2-(5 phenyloxazoly)]- benzene) 100 mg PPO (2,5—Diphenyloxazole) 8 gm Diet Preparation Twenty synthetic larval diets were prepared in pyrex flasks (wide mouth, 250 ml) containing 7.5 g of dry diet, 26 ml of HOH, 24 m1 of 1mM amino acid solution and 0.75 ml of B-vitamin mixture. The flasks were sealed with a diSPoR plug (Scientific Products, Evanston, Illinois) and autoclaved at 15 pounds pressure (121°C) for 15 minutes. After sterilization, vigorous aggitation in ice water dispersed the diet as the agar gelled. Rearinngrocedure and Aseptic Culture For oviposition, a Petri dish holding a muslin bag wet with ammonium carbonate, was placed in cages contain- ing adult flies reared by the CSMA method (Anonomous, 1959). Six hours later, the collected eggs were loosely wrapped in moist paper toweling until used. The eggs were washed twice in water by shaking in a 50 ml Erlenmeyer flask and pouring off the eggs which floated. A 20 minute wash in 0.1% sodium hypochlorite (ChloroxR) was then given the eggs before aseptic transfer to the flasks with a pipette calibrated to deliver about 300 eggs. The innoculated flasks were incubated at 30°C for 2 days followed by 28°C until pupation began. A 600 m1 beaker under each flask insured that no migrating larvae would be lost from any sample. Pupating larvae were collected by breaking up the remaining diet with a Jet of water and washing it through a No. 20 standard screen. Larvae and pupae were then placed on Whatman filter paper in Petri dishes to set overnight at 38°C. The resulting pupae were counted, weighed,1 and frozen at -30°C until analyzed. Fluid thioglycollate medium was utilized to determine asepsis, and only proven sterile cultures were used in these studies. Extraction and Chromatographic Fractionation of Lipids The method of Kaplanis et a1. (1961) for extracting total lipids from adult houseflies was adopted for pupal lAll gravimetric determinations were made on a Mettler Gram—atic balance. lO extraction. The pupae were homogenized in a glass Ten— broeck homogenizer with water. The homogenate and rinses were collected in a 300 m1 round bottom flask and refluxed for 1.5 hours with acetone—ethanol (1:1 v/v) at 4 times the volume of water used. After reflux, the material was quantitatively1 vacuum filtered through a Bachner funnel. The acetone-ethanol were then removed in Xagug_and the total lipids extracted from the remaining aqueous (after acidification with HCl) with 3 equal washes of anhydrous ethyl ether. The ether was then dried over anhydrous sodium sulfate, collected in a tared flask, and removed in KEEEQ- Total lipids were weighed, radioassayed in 10 ml of a standard toluene— fluor mixture,2 and frozen in benzene under a nitrogen atmosphere. Radioactivity in the aqueous fraction was determined in modified Bray's solution. A 2.5 ml sample from each of the 5 pooled aqueous fractions was chromatographed on a cation exchange column (1.1 x 7.5 cm) of Dowex 50—Xl2 ground to pass a No. 100 standard sieve. The column was successively washed with 25 ml of water followed by 30 ml 1After every transfer of material throughout this study, the glassware yielding the material was rinsed at least 3 times into that which received it as an effort to be quantitative. 2Made as reported for modified Bray's solution, but replacing the ethylene glycol monomethyl ether with toluene. ll of 10% ammonium hydroxide and each fraction was assayed radiometrically. Saponification of the total lipids was accomplished by a slight modification of Kaplanis et a1. (1961). The total lipids were transferred into a 45 ml glass stoppered centrifuge tube with ethyl ether. A stream of nitrogen removed the ether and 5—6 ml of 10% (w/v) potassium hydroxide in 90% ethanol were added. The tube was flushed with nitrogen, stoppered and refluxed for 1.5 hours. A quantitative transfer was made from the centri- fuge tube to a separatory funnel with water and ethyl ether and the unsaponifiable lipids extracted with anhydrous ethyl ether. The extracts were placed into another separa— tory funnel, acidified to neutralize any potassium hydroxide, and then washed to neutrality with water. Anhydrous sodium sulfate was used to dry the ether as it was collected into a tared flask. After removing the solvent in vaggg, the weight of the unsaponifiable lipids were obtained and samples taken for radioassay. This fraction was then frozen in benzene in a nitrogen atmosphere for later analysis. The aqueous from the unsaponifiable fraction above was acidified to free the fatty acids from their potas— sium salts and then extracted with ethyl ether as described for the unsaponifiable fraction. The fatty acids were assayed radiometrically and gravimetrically before storage under nitrogen at -30°C. 12 The fatty acids were pooled by treatment and the solvent removed in zaggg. Transfer was made with ether to a 45 ml glass stOppered centrifuge tube and the ether removed by a stream of nitrogen. A boron trichloride— methanol solution1 was added and the tube was nitrogen flushed and refluxed 2 minutes (Metcalf and Schmitz, 1961). Transfer of the methyl esters to a separatory funnel was made with small aliquots of water (total 20 m1) and quantitatively extracted with anhydrous ethyl ether. The ether was then dried over anhydrous sodium sulfate and assayed gravimetrically in a nitrogen atmosphere. The fatty acid methyl esters were then stored under nitrogen at —30°C. A dual column analysis was made of the unsaponifiable lipids (Monroe et al., 1968). Woelm alumina (7.5g), neutral Grade I, deactivated with 1.5% water was packed in nghexane in a series of two 1.1 cm x 7.5 cm columns. About 1.5 g of anhydrous sodium sulfate was also added to the top of the column. The samples were evaporated in Eaggg and placed onto the first columns in small aliquots of benzene. The columns were then washed with 100 ml of benzene (eluting hydrocarbons and high molecular weight alcohols), anhydrous ethyl ether (eluting free sterols), lA prepared estrification kit from Applied Science Laboratories, Inc., State College, Pennsylvania. l3 and 50 m1 of methanol (eluting more polar molecules) (Kaplanis et al., 1961). The benzene fractions were evaporated in vagug and then placed onto the second columns in nfhexane. These columns were washed with 100 ml each of nfhexane (eluting hydrocarbons), benzene (eluting high molecular weight alcohols), and 50 m1 of methanol. The methanol fractions from both columns were pooled through a sintered glass funnel into one flask and thereafter considered as a single fraction. Weight and radiometric determinations were made on all fractions eluted. Gas-Liquid Chromatography of Fatty Acids The gas-liquid chromatograph used was a Research Specialities Model 600 Series equipped with a hydrogen- flame detector. The column used was 4.3 ft x 4 mm ID stainless steel with 12% HI-EFF 1B (Diethyleneglycol succinate)l filter coated onto Gas-Chrome Q.l Detailed operation procedures are presented under Results, and all peaks were identified by comparing the retention times of the unknown acids with those of authentic standards.1 The standards were: 1Applied Science Laboratories, Inc., State College, Pennsylvania. l4 K 108 N.I.H. Mix E Fatty acid Per cent Fatty acid Per cent C - 16 20 C - 8 6.28 C - 18 20 C - 10 9.26 C - l8-2H 20 C - 12 12.08 C - l8-4H 20 C - 14 23.29 C - 18-6H 20 C — 16 49.09 Relative per cent compositions were determined with a disc integrator which was checked against a stand- ard. Samples were adjusted to 4ug/ul and 201 injections were used. A 5u1 injection served as a template for trapping individual peaks for radio-assay. These peaks were trapped in n—hexane contained in glass tubes which were immersed in acetone and dry ice. The collecting tubes were rinsed into counting vials and the solvent evaporated by nitrogen before the addition of the fluor mixture and subsequent counting. RESULTS All rearing flasks proved to be sterile when the mature larvae were collected, and the gross data for the resulting pupae are presented in Table 1. The average weight per pupa was 16.7 mg compared to 20.5 mg reported by Monroe (1962), for the original version of this diet used with different rearing conditions. The high average weight per pupa from the alanine treatment was consistent through 4 replications.1 TABLE 1.--Gross data for housefly pupae aseptically reared on a synthetic diet containing fgee,radiolabelled amino acid. Labelled No. of Total Ave. wt./ Amino Acid Pupae** wt. (mg)** Pupa 14 Alanine-U-C 472 8,336 17.7 Glutamate—U—Clu 811 13,372 16.5 Leucine-U—Clu 815 13,530 16.6 Methionine-G-H3 607 10,020 16.5 Phenylalanine-U—Clu 688 11,506 16.7 * Pupae less than 24 hr. old. ** Total from 4 replications per amino acid. lSubsequent tests were made with varying concentra- tions of free alanine and glutamate to look again for weight differences, but the greatest concentration (2 mM alanine) evidently was not great enough to produce a 15 16 The average lipid content (Table 2) was 6.5% of live weight. The average residue fraction was 21.3%, and the aqueous fraction was 72.2% of the live weight. Aqueous fraction values were determined by subtracting the sum of residue and total lipid weight from total live weight. TABLE 2.--Fresh body weight data of housefly pupae aseptically reared on a synthetic diet containing free, radiolabelled amino acid.* Labelled Amino Acid Rgzidu°*; mgipi°*; A32°°us*;* Alanine-U-Clu 1,804 21.6 580 7.0 5,952 71.4 Glutamate-U-Clu 2,883 21.6 828 6.2 9.661 72.2 Leucine-U—cl” 2,824 20.9 901 6.6 9,805 72.5 Methionine-G—H3 2,159 21.6 642 6.4 7,219 72.1 Phenylalanine-U-Clu 2,399 20.9 720 6.3 8,387 72.8 * Pupae less than 24 hr. old. ** Total from 4 replications per amino acid. *** Determined by subtracting (residue + lipid) from total weight. The activity found in the total lipid fraction is presented as microgram equivalents (pg eq) of amino acid utilized by treatment in Table 3. Although glutamate was difference from the other treatments. However, it was shown that free amino acid is as effective as sodium oleate in dispersing the growth medium, thus the effect is physical rather than chemical. 17 TABLE 3.--Radioactivity in total lipid fraction of housefly pupae aseptically reared on a synthetic diet containing free, radiolabelled amino acid.* Specific Total Average Labelled Amino Acid Activity pg-eqi pg eq/mg lipid (dpm/pg)** l4 . Alanine-U-C 162 25,051 43.7 i 1.8 Glutamate-U—Clu 22 183,754 221.1 1 21.4 Leucine-U-Clu 53 225,973 250.8 i.29°2 Methionine—G H31“r 1,423 1,930 3.0 i 0.8 Phenylalanine-U-Clu 97 30,839 42.4 i 5.0 * Pupae less than 24 hr. old. ** Within each diet flask. fFrom 4 replications per amino acid. ++120 pC per flask, others with 12 p0 each. 2.4 times as abundant as leucine in each rearing flask, the leucine treatment produced more pg eq/mg total lipid than did glutamate (250.8 pg eq and 221.1 pg eq, respec— tively). Alanine and phenylalanine were approximately 1/6 as lipogenic as leucine. The methionine (3.0 pg eq) was converted to the total lipids at 1/84 the rate of leucine. Figure 1 shows the relationship between per cent composition and radioactivity incorporated into total lipids for the amino acids studied. Although alanine, phenylalanine and glutamate fall on the line, methionine 18 falls below and leucine above the linear curve. Table 4 shows that the fatty acids contained over 80% of total lipids' radioactivity. A comparison of activity determined after pooling fatty acid replications is shown in paren- theses. The pooled values represent a more accurately weighted estimate of the radioactivity per mg of fatty acid than the average obtained before pooling. Table 5 presents data on the distribution of radio- activity among the unsaponifiable lipids after column chromatography. From 69% to 76.6% of the carbon-l“ was found in the nfhexane fraction (hydrocarbons), 16.5% to 29.9% in the benzene fraction (high molecular weight alcohols), 0.0% to 4.5% in the ethyl ether fraction (free sterols), and 1.1% to 5.4% in the methanol fraction (more polar compounds). No detectable activity was recovered from the columns for the methionine treatment. Results of gas-liquid chromatographic analysis of the fatty acid methyl esters are presented in Table 6. Only myristic (C-l4), palmitic (C-l6), palmitoleic (C—l6zl), stearic (C-l8), and oleic (C-18:1) acids were found. By disc integration, the most abundant fatty acid was palmitoleic (39.7%), followed by oleic (29.9%), palmitic (28.7%), myristic (1.7%) and stearic acids (trace amount). 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