DIETARY AMINO ACID REQUIREMENTS OF THE ALMOND MOTH , CADRA CAUTELLA (WALKER) , BASED ON RADIOMETRIC AND CARCASS ANALYSIS TECHNIQUES By JACK MYRON HELLER A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1975 ACKNOWLEDGEMENTS I would like to thank the many individuals who assisted me throughout the study and preparation of this dissertation. My profound thanks go to Dr. R. E. Waites , who served as Chairman of the supervisory committee and whose help was invaluable throughout this study. Appreciation is also expressed to Dr. W. G. Eden, Chairman of the Department of Entomology, and Drs. D. L. Silhacek, B. J. Smittle, and D. S. Anthony, members of my supervising committee. Special appreciation goes to my wife, Barbara, for her patience and help during this period of graduate study, and for assisting in the preparation of this manuscript. TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii LIST OF TABLES V LIST OF FIGURES viii ABSTRACT X INTRODUCTION 1 LITERATURE REVIEW 4 Radioisotopes and the Determination of Amino Acid Requirements „ . 4 Carcass Analysis for the Estimation of Quantitative Amino Acid Requirements 11 General Insect Nutrition 12 Distribution and Metabolism of Amino Acids and Proteins 13 Methodology . 14 MATERIALS AND METHODS 22 Rearing 22 Radiochromatography and Autoradiography of U-1I+C-glucose 22 Radioactive Medium Preparation 24 Protein Extraction 25 Protein Hydrolysis 26 Thin-Layer Chromatography of Amino Acids 29 Radioactivity Measurements 30 Carcass Analysis 33 TABLE OF CONTENTS - Continued Paqe Gas Chromatography of Amino Acids for Carcass Analysis 35 Microbiological Study 40 RESULTS AND DISCUSSION 42 Qualitative Amino Acid Requirements by the Indirect Radioactive Method and Thin-Layer Chromatography 42 Carcass Analysis for the Determination of Quantitative Amino Acid Requirements Using Gas Chromatography 74 SUMMARY 87 APPENDIX 89 REFERENCES CITED 101 BIOGRAPHICAL SKETCH 110 LIST OF TABLES Table Page 1 Amino Acid Requirements of Some Insects Deter- mined by the Radioactivity Method 6 2 Experimental Parameters Used to Determine Amino Acid Requirements by the Indirect Radioactivity, Method 8 3 Rf and RLeucj.ne Values °f 22 Amino Acid Standards Separated by Two-Dimensional Thin-Layer Chromatography on Cellulose 45 4 Cpm/Carbon Atom of Amino Acids from Acid and Base Hydrolysates of Protein Extracted from Almond Moth Larvae Reared on Medium Containing ^C-Glucose 47 5 Radioactivity in Amino Acids from Acid and Base Hydrolysates of Proteins Extracted from Larval Rearing Media Containing 11+C-Glucose 53 6 Cpm/Carbon Atom of Free Amino Acids Extracted from Sterile Larval Rearing Medium Containing ^'C-Glucose and That Had Almond Moth Larvae Reared on It 58 7 Cpm/Carbon Atom of Free Amino Acids Extracted from Non-Sterile Larval Rearing Medium that was Incubated with ^C-Glucose for 12 Weeks and Had 'lo Larvae Reared on It 59 8 Cpm/Carbon Atom of Free Amino Acids Extracted from Sterile Larval Rearing Medium Containing 1 C-Glucose and on Which No Larvae were Reared 60 9 Result of Microbiological Study on Almond Moth Eggs and Larvae and 14C Media to Deter- mine the Source of Radioactive Free Amino Acid Synthesis 64 10 Specific Activity of Dried Supernatant Fractions from the Extraction of Fifth-Instar Almond Moth Larvae Reared on Medium Containing ll,C-Glucose 66 LIST OF TABLES - Continued Table Page 11 Specific Activity of Dried Supernatant Fractions from the Extraction of Larval Rearing Medium Containing ll+C-Glucose and That Had Almond Moth Larvae Reared on It 67 12 Specific Activity of Dried Supernatant Fractions from the Extraction of Larval Rearing Medium Containing ^C-Glucose and That Had No Larvae Reared on It 68 13 Weights of Dried Supernatant Fractions from the Extraction of Fifth-Instar Almond Moth Larvae Reared on Medium Containing 1 ^C-Glucose 69 14 Weights of Dried Supernatant Fractions from the Extraction of Larval Rearing Medium Containing 11+C-Glucose and That Had Almond Moth Larvae Reared on It 70 15 Weights of Dried Supernatant Fractions from the Extraction of Larval Rearing Medium Containing 11+C-Glucose and That Had 'To Larvae Reared on It „ 71 16 Pattern of Amino Acids in Fifth-Instar Almond Moth Larvae „ 75 17 Percent Composition of Amino Acids in Fifth- Instar Almond Moth Larvae 76 18 Retention Time, Relative Retention Time, and Sensitivity of 1*9 Trimethylsilyl Amino Acid Standards 79 19 _ Amino Acid Mixture Patterned After Carcass Analysis of Fifth-Instar Almond Moth Larvae ... 82 Table LIST OF TABLES - Continued A-l Effect of Thin-Layer Adsorbent and Thin-Layer Adsorbent and Minhydrin in Combination on Counting System Background 92 A-2 Quenching Properties of 20 Non-Visualized Amino Acid Standards Adsorbed on Cellulose Thin-Layers Using the Internal Standardization Method . 94 A-3 Quenching Properties of 20 Amino Acid Standards Adsorbed on Cellulose Thin-Layers and Visualized with 1/2 percent Ninhydrin in Acetone Spray Using the Internal Standardization Method . . . A-4 Quenching Properties of Visualized Amino Acids from Larval Protein Hydrolysates Separated by TLC and Using Automatic External Standardization 95 96 A-5 Quenching Properties of Five Supernatant Fractions from the Extraction of Larval Proteins Using the Internal Standardization Method g8 A-6 Quenching Properties of Supernatant Fractions from the Extraction of Larval Protein Using Automatic External Standardization 99 LIST OF FIGURES Principle of the radioactivity method for determining amino acid requirements Flow diagram for the extraction and clean-up of proteins used in this study 27 Separation of amino acids present in an acid hydrolysate of protein extracted from fifth-instar almond moth larvae reared on medium containing * ^C-glucose 43 Separation of amino acids present in a base hydrolysate of protein extracted from fifth-instar almond moth larvae reared on medium containing *■ ^C-glucose 44 Spot map of 22 amino acid standards separated on cellulose thin-layer plates 46 Separation of amino acids present in an acid hydrolysate of protein extracted from larval rearing medium which contained C-glucose and on which larvae were maintained 51 Separation of amino acids present in a base hydrolysate of protein extracted from larval rearing medium which contained C-glucose and on which larvae were maintained 52 Separation of free amino acids extracted from sterile larval rearing medium containing C-glucose and on which larvae were maintained 55 Separation of free amino acids extracted from non-sterile larval rearing medium containing 14C-glucose and that had no larvae reared on it 56 LIST OP FIGURES — Continued Figure Page 10 Separation of free amino acids extracted from sterile larval rearing medium containing 11+C-glucose and that had no larvae reared on it 57 11 Gas-liquid chromatogram of TMS amino acid derivatives from protein hydrolysate of fifth-instar almond moth larvae 77 12 Gas-liquid chromatogram of TMS-free amino acids extracted from fifth-instar almond moth larvae 78 13 Gas-liquid chromatogram of TMS protein amino acid standards 80 14 Gas-liquid chromatogram of a TMS derivatization of an alkaline acetone fraction from the extraction of protein from almond moth larvae 83 A-l One-dimensional co-chromatography of ll+C-Glucose with 1/2% glucose standard (in 3% aqueous ethanol) followed by auto- radiography of the thin-layer plate on Kodak No-Screen X-ray Film 91 Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DIETARY AMINO ACID REQUIREMENTS OF THE ALMOND MOTH , CADRA CAUTELLA (WALKER) , BASED ON RADIOMETRIC AND CARCASS ANALYSIS TECHNIQUES by Jack Myron Heller August, 1975 Chairman: Robert E. Waites Major Department: Entomology and Nematology The almond moth, Cadra cautella (Walker), synthesizes alanine, aspartic acid, glutamic acid, glycine, proline, and serine from U-14C-glucose. These amino acids are considered nutritionally non-essential. Amino acids that contained no radioactivity and are considered nutritionally essential include arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine. Cystine and cysteine contained an intermediate amount of activity and are still unclassified with respect to dietary need. Radioactive essential and non-essential free amino acids were isolated from larval rearing media. The specific activities were substantially higher in amino acids extracted from medium that had larvae reared on it than medium that had no larvae reared on it. There appears to be an insect-microorganism relationship at work here. However, the exact nature of this relationship and to what extent it contributes to the insects1 nutrition are at present undetermined. Since there was no detectable incorporation of radioactive essential free amino acids from the medium into the larval proteins (with the possible exception of cysteine) , the importance of this relationship seems questionable when the techniques I employed for this study are used. The study to isolate the source of the insect associated microorganisms is at present inconclusive. However, from the data available presently, it appears that the microorganisms come from within the almond moth eggs. Carcass analysis of fifth-instar almond moth larvae showed that proline, tyrosine, and glutamic acid made up almost 70 percent of the total free amino acids present in the larvae. At this stage in larval development prior to pupation, cuticular tanning and thickening are beginning and proline and tyrosine are major participants in these events. The protein amino acids, which contain a high percentage of glutamic acid and aspartic acid, in combination with the free amino acids are the basis for a dietary amino acid mixture at the 2 percent and 3 percent levels. By substituting this mixture for casein as the protein source in the almond moth diet, the requirements for vitamins and minerals can be determined more effectively. INTRODUCTION The study of insect nutrition, in particular the formulation of synthetic diets utilizing specific amino acids, vitamins, and minerals, has in recent years greatly expanded. When the specific nutrient requirements of an insect are known, many other facts about it become much clearer (i.e., physiological and biochemical). Once nutrition has been eliminated as a variable, many other aspects of the insect can be studied such as genetics, control (i.e., toxicant evaluation), biochemical relationships to higher or related organisms, etc. Indirect nutritional procedures have come into widespread use to study organisms that cannot be reared axenically or on defined media. They also support and enhance information gained through classic nutritional techniques in addition to adding information on the metabolic pathways of many nutrients. The indirect radioactivity method was first demonstrated in the work of Black, Kleiber, and Smith (1952) on the cow with radioactive carbonate and fatty acids being incorporated into non-essential amino acids. Shortly thereafter, Steele (1952) found that when U-ll+C-sucrose was ingested by the adult mouse, radioactivity appeared in the non-essential but- not in the essential amino acids extracted from the carcass proteins. Kasting and McGinnis (1958) confirmed this relationship for the blowfly, Phormia regina (Meig) , using U-^C-glucose. Their data were supported by the results from the classical deletion procedure which had been done earlier on this insect. The similarity in amino acid composition of the whole animal carcass and the pattern of amino acid requirements as determined by nutrition studies has been noted by Wu and Hogg (1952) for protozoa and Williams et al. (1954) for larger animals. The pattern of amino acids from carcass analysis studies has been used in several instances to formulate chemically defined diets with maximum larval growth being achieved (Auclair and Cartier 1963, Rock and King 1967b, and Rock and King 1967c). The almond moth, Cadra cautella (Walker) (Lepidoptera: Pyralidae) is a common pest of tobacco, cocoa beans, dried fruits, and nuts throughout much of the world (Bach 1930, Wadsworth 1933, Fraenkel and Blewett 1946a). Therefore, it was chosen as the subject of this study. The objective of the study was to determine the qualitative (i.e., indispensable) amino acid requirements of the almond moth. This was to be followed by determination of the amount of each protein amino acid present in the carcass, with the aim of determining the quantitative amino acid requirements (both dispensable and indispensable) of this insect. Once the qualitative and quantitative requirements had been established, a synthetic amino acid mixture could be developed. By substituting this mixture for casein as the protein source in the almond moth diet, the requirements for vitamins and minerals can be determined more effectively. This is because dietary compounds such as casein often contain contaminants or substances which confuse nutritional research (i.e., vitamins, minerals, etc.). Once a completely defined diet has been established, nutrition could be eliminated as a variable .factor in future studies on physiology, biochemistry, ecology, and ultimate control. These two nutritional techniques were applied to my study of the almond moth. LITERATURE REVIEW Radioisotopes and the Determination of Amino Acid Requirements Since the classic work of Steele (1952) , many investigators have used carbon-14 labeled substrates to determine the nutritionally essential amino acids of a variety of different insects. This procedure requires a compound that is normally present in the diet and readily metabolized as the carbon-14 substrate. Following administration by incorporation in the diet, injection, or some other suitable means, the organism is allowed time to metabolize the substrates. Metabolism of the substrate results in incorporation of label in the synthesized compounds (i.e., nutritionally non-essential) and no incorporation in the essential compounds that must be supplied in the diet. (See Figure 1.) The indirect radioactivity method has been applied to a number of phytophagus or other insects which cannot be reared on chemically defined media (Kasting and McGinnis 1958, 1960, 1962, 1964, Kasting et al. 1962, Strong and Sakamoto 1963, Rodriguez and Hampton 1966, Rock and King 1968, Rock and Hodgson 1971). A summary of the amino acid requirements of the above insects determined by this method- is shown in Table 1. RADIOACTIVE I*ADIOACTIVE GLUCOSE SUBSTRATE -X NUTRITIONALLY NON-ESSENTIAL C*0*C*C*C*C* C*u2" f INTERMEDIATES C*C*C* NUTRITIONALLY ESSENTIAL NATURAL FOOD SUPPLY NO - RADIO- ACTIVITY Figure 1. Principle of the radioactivity method for determining amino acid requirements.* S^'"-^,,=SLr,-jrsras.*- Table 1. Amino Acid Requirements of Some Insects Determined by the Radioactivity Method Amino Acid Blow Pale Wire- Green Wheat Two- Red- Boll Fly Western worm Peach Stem Spotted Banded Worm Cutworm Aphid Sawfly Spider Leaf Mite Roller Glutamic ac id _ _ + _ _ _ - - Aspartic ac id - - - - - - - - Alanine +? - - - - - - — Proline -I-? - - - - - - Serine - - - - - - - ~ Glycine - - - - - - - - Histidine + + + + + + + + Threonine + •(-? + + + - + + Leucine + + + + + + + + Isoleucine + + + + -!• + + + Valine + + + + + + + + Tyrosine + + + + + + + Phenylalanine + + + + + + + Lysine + + + + + + + + Arginine + + •i- + + + + + Methionine + + + + + + + Cystine + + - — Cysteine + = nutritionally essential - = nutritionally non-essential +?= some synthesis, possibly essential There are several important factors to consider when using the indirect radioactivity method to determine nutrient requirements. The first of these is the method by which the radioactive substrate is administered. The labeled compound may be administered as a single dose or the organism may be continuously exposed to the radioactive substrate. Organisms are often continuously exposed to a radioactive substrate by having it incorporated in their diet (Strong and Sakamoto 1963, Rodriguez and Hampton 1966, Rock and King 1968, Rock and Hodgson 1971). Most studies involve the administration of only a single dose of substrate injection. Kasting and McGinnis (1964) have devised a method of vacuum infiltration for organisms that are not easily injected. The radioactive substrate administered to the organism can affect both what compounds are labeled and the specific activity of these compounds. This was clearly shown in the study of Black et al. (1952) using bovine tissues. In this study, the labeling of amino acids varied depending on whether labeled acetate, propionate, butyrate, or bicarbonate was administered. Table 2 presents a summary of some of the substrates used, methods of administration, and metabolism periods for the determination of amino acid requirements using the indirect radioactivity method. Table 2. Experimental Parameters Used to Determine Amino Acid Requirements by the Indirect Radioactivity Method Organism Radioactive Method of Metabolism Source of Reference Substrate Adminis- Period Isolated tration (Hours) Amino Acid Cow Acetate- Single I-14C injection 3, 10, 22, Casein Black Acetate- 34 et al. II-^C 1957 Frog & Glucose- Single 4, 8, 12 Liver, Nakagawa Tadpole u-lt+c injection tail, muscle, carcass et al. 1964 Mouse Sucrose- Single 72 Whole Steele u-1(tc feeding mouse (minus intestines) 1952 Rat Glucose- Single 2.5 min.- Brain and Gaitonde U-^C injection 2 hr. liver et al. 1965 Blow fly Glutamic Single 48, 63 Whole Kasting & acid U-14C injection carcass McGinnis 1960 Wheat Glucose- Vacuum 24 Whole Kasting & Stem u-li+c infiltra- carcass McGinnis Sawfly tion 1964 Green Glucose- Continuous 24 Whole Strong & Peach U-^C feeding carcass Sakamoto Aphid 24 hr. 1963 Red- Glucose- Continuous 48 Whole Rock S Banded u-11+c feeding carcass King Leaf 48 hr. 1968 Roller Boll Glucose- Continuous 60-84 Whole Rock & Worm U-^C feeding 48-72 hr. carcass Hodgson 1970 The length of the metabolism period can affect the order of specific radioactivities among the protein and free amino acids. The level of specific radioactivity in free amino acids is not necessarily related to that of the protein amino acids. Nakagawa et al. (1964) showed that with a metabolism period of 4 hr. in the frog, the free amino acids that were labeled were not necessarily labeled in the protein amino acids from the same tissues. With a longer metabolism period following single administration of a radioactive substrate, there is generally greater incorporation of radioactivity into the protein amino acids, while radioactivity in the free amino acids may reach a peak and start to decline. The metabolism period often appears to be chosen by trial and error. Schaefer (1964) and Kasting et al. (1962) used the rate of production of 11+C02 and the total amount produced following administration of radioactive substrates as a guide to selecting the optimum metabolic period. To obtain reliable results using the radioactivity method, another factor that must be considered is the purity of isolated compounds (i.e., amino acids) before radioactivity measurement. This can be accomplished by simple two-dimensional thin-layer chromatography (TLC) with some organisms. However, with free amino acids from insect preparations, an ion exchange column followed by band paper chromatography in at least three solvent systems may be required to remove interfering materials (Kasting and McGinnis, 1960) . 10 The tissues from which amino acids are isolated also have a significant effect on the results obtained with the indirect radioactivity method. Specific radioactivities will depend on whether amino acids are isolated from the free or protein fractions (Nakagawa et al. , 1964) , or the whole carcass or specific organs (Nakagawa et al. 1964, Black et al. 1952, Gaitonde 1965). However, with most organisms, the different synthetic abilities of different organs are not a problem because the amino acids are isolated from the whole animal. A problem arises when amino acids with low or intermediate specific activities are isolated from whole insect larvae. The low level of radioactivity in certain amino acids may be due to dilution of these compounds synthesized in one organ by unlabeled amino acids from other tissues or organs. Studies such as those carried out by Lipke et al. (1965) on the biosynthetic capabilities of the different tissues and organs of the cockroach will aid in interpretation of results by demonstrating which organs and tissues are capable of synthesizing specific amino acids. The specific radioactivities of isolated amino acids may also vary depending on the concentration in which they are present. However, this has not been found with a number of insects (Kasting et al. 1962, Kasting and McGinnis 1962, 1964). In many instances, the classical deletion technique has shown tyrosine to be a non-essential amino acid while the indirect 11 radiometric technique has indicated that it is essential (i.e., lacks radioactivity) . Fukuda (1956) and Kasting and McGinnis (1962) have shown that tyrosine is synthesized from the essential amino acid phenylalanine and thus, even though it lacks radioactivity, it can still be classed as non-essential in many insects. Kasting and McGinnis (1966) have thoroughly reviewed the subject of radioisotopes and the determination of nutrient requirements . Carcass Analysis for the Estimation of Quantitative Amino Acid Requirements To determine quantitative amino acid requirements of an insect, feeding tests on graded levels of an amino acid mixture, such as one resembling a casein hydrolysate or some other protein, are evaluated on the basis of growth and development of the insect. A problem with this approach is that the initial balance of amino acids, both dispensable and indispensable, is probably far from optimum. Often, little consideration is given to the dispensable amino acid balance. Breuer et al. (1964) showed the importance of dispensable amino acid balance on total amino acid balance in the rat. Several workers have used new methods to gain a better starting point from which to develop an optimum amino acid mixture for various organisms. 1.2 Wu and Hogg (1952) using protozoa and Williams et al. (1954) using the rat, chick, and pig noted the similarity in amino acid composition of the whole animal carcass and the pattern of amino acid requirements determined by nutritional studies. Auclair and Cartier (1963) successfully reared the pea aphid Acyrbhosiphon pisum (Harris) on an amino acid diet based on the average concentration of these compounds in the blood and excreted honeydew. Rock and King (1967c) estimated the amino acid requirements for growth in the codling moth, Carpocapsa pomonella (Linneus) by carcass analysis. Rock and King (1967b) found that the quantitative pattern of amino acids in 1-day-old pupae of Argyro taenia velutinana (Walker) supported maximum larval growth when this insect was reared axenically on a chemically defined diet. Rock and King (1966) studied the amino acid composition in hydrolysates of the red-banded leaf roller during development. Their work indicated a shift in amino acid requirements during growth and development. This shift in requirements is probably mediated by the requirements of the particular tissue that is being formed in the rapidly developing and growing larvae. • General Insect Nutrition Several excellent reviews covering various phases of insect nutrition are currently available in the literature. Reviews concerning the general subject of insect nutrition include House (1961, 1962), Lipke and Fraenkel (1956), and Fraenkel (1959). 13 Friend (1962) discusses the nutritional requirements of phytophagus insects while House (1959) deals with the parasitoid Pseudosarco phaga affinis (Pall) and other insects. The current status of and future possibilities for research on the axenic culture of arthropods are discussed by Rodriguez (1966) . Richards and Brooks (1958) and Henry (1962) reviewed the significance of internal symbiosis and microorganisms in insect nutrition. Fraenkel and Blewett (1946b, 1946c) and Waites and Gothilf (1969) have studied the dietary requirements of the almond moth and several other closely related lepidopterous insects. Distribution and Metabolism of Amino Acids and Proteins Numerous articles and reviews on the distribution and metabolisms of proteins and amino acids in insect tissues and fluids are available in the literature. Florkin (1958) reviewed the subject of free amino acids in insect hemolymph and Chen (1962) the broader subject of free amino acids in insects. The free amino acids in Prodenia eridania, Culex pipiens, Glossina palpalis, and Drosophile melanogaster have been studied by Levenbook (1962), Chen (1963), Balogun (1969) and Mitchell and Simmons (1961), respectively. In the area of amino acid metabolism, Brunet (1965) reviews the subject of aromatic compounds while Bheemeswar (1958) 14 discusses general amino acid metabolism covering the subject of major biochemical reactions (i.e., deamination, transamination, decarboxylation, and peptide and protein synthesis). Chen (1966) has written a very extensive review covering the subject of amino acid and protein metabolism in insect development. He follows the changing patterns of amino acid composition throughout the life stages of various insects. Henry and Block (1961) report on the metabolism of sulfur-containing amino acids in the German cockroach, Blattella germanica (L. ) . Bursell (1963) follows the changing pattern of free amino acids in the thorax of the male tsetse flies during the hunger cycle and flight activity. Methodology Thin -Layer Chromatography Several two-dimensional solvent systems were investigated for the separation of complex amino acid mixtures before the one giving the best resolution was found. Heathcote and Jones (1965) devised a pair of solvent systems for ascending two-dimensional chromatography on cellulose thin- layers. This system produced unambiguous separation of 23 naturally occuring amino acids, including leucine and isoleucine, in 6 hr. This method required no tank saturation and using a ninhydrin staining solution could detect less than 1 \iq of amino acid. 1'5 The solvent systems were 2-propanol-formic acid-water (40:2:10 by volume) for development in the first dimension and tertiary butyl alcohol-methyl ethyl ketone-NH^OH-distilled water (50:30:10:10 by volume) for development in the second dimension. Jones and Heathcote (1966) used the same solvent system to separate the amino acids in protein hydrolysates. However, this time ninhydrin-collidine reagent was used to visualize the amino acids. This system made for easier identification of certain amino acids due to their characteristic color on staining. Haworth and Heathcote (1969) modified their previous method and accomplished the separation of up to 63 compounds. This new solvent system consisted of 2-propanol-methyl ethyl ketone-lN HC1 (60:15:25 v/v) for development in the first dimension and tertiary butyl alcohol-methyl ethyl ketone-acetone-methanol- NH^OH-distilled water (40:20:20:1:14:5 v/v) for development in the second dimension. With this new solvent system, a large spread was produced between the amino acid spots. In addition, highly reproducible results were obtained making it possible to use the Rf's of the various spots to identify the amino acids present in a protein hydrolysate. Using a ninhydrin-cadmium acetate dye system 5 X 10_1+ moles of amino acid can be detected following two-dimensional TLC. Heathcote and Haworth (1969a) again modified their solvent system to the following composition: tertiary pentyl alcohol-methyl ethyl ketone -ace tone -methanol- 16 NH^OH-distilled water (50:20 :10:5 :15 :5 v/v) . Heath cote et al. (1970) used selective staining to identify complex mixtures of amino acids and nitrogen containing metabolites separated by TLC. De Zeeuw (1968a) compared the use of saturated and unsaturated TLC chambers. He obtained better separation using an unsaturated chamber in addition to obtaining good reproducibility of R ' s and good spot shape. Other factors that affect separation and spot shape and should be kept constant are temperature, relative humidity, solvents, adsorbent, and geometry of the chamber. De Zeeuw (1968b) also studied the influence of humidity variations on the TLC of hypnotics. He showed that R 's changed considerably with variations in relative humidity of the TLC room. R ' s increased with increasing humidity and then fell sharply at higher humidities. Heathcote and Washington (1967) and Heathcote and Haworth (1969b) discussed the quantitation of small amounts of amino acids separated by thin-layer or paper chromatography using colorimetric or densitometric techniques respectively. Stahl (1968) stressed the need for standardization of terms in the literature so techniques could be repeated in other laboratories and results could be compared between laboratories. Several excellent texts on TLC include Smith (1960), Stahl (1969), and Pataki (1966). 17 Gas Chromatography A fairly recent advance in the quantitative analysis of amino acids was the adaptation of gas chromatography to these compounds. It is a very sensitive technique that requires only small amounts of the compound of interest to be injected into the instrument. Gehrke et al. (1969) did an extensive study of the trimethylsilyl (TMS) derivatives of protein amino acids examining such factors as chromatographic separation, precision and accuracy of the method, silylation as a function of reaction temperature and time, molar excess of reactants, stability of the TMS derivatives, quantitative analysis of a synthetic amino acid mixture, and application to biological samples. Gehrke and Leimer (1970b) studied the effect of solvents on derivatization of amino acids using bis (trimethylsilyl) trifluoroacetamide (i.e., BSTFA) . They found using polar solvents for the derivatization reaction produced two chromatographic peaks for glycine and one for arginine. In non-polar solvents, only the first chromatographic peak for glycine and no peaks for arginine were obtained. Gehrke and Leimer (1971) improved upon the previous work on trimethylsilylation of the 20 protein amino acids. Their major aim, which they accomplished, was to achieve a single derivatization, single injection method for the analysis of the 20 protein amino acids as the TMS derivatives in complex 18 biological substances. Amino acids were reproducibly converted in a single step, closed tube reaction to the TMS derivatives in 2.5 hr. at 150 C. Excellent separation of the 20 TMS protein amino acid derivatives was achieved on a single 6 m X 2 mm I.D. column packed with 10 percent OV-11 on 100/200 mesh Supelcoport in 60-80 min. Data from amino acid analysis of ribonuclease, B-casein, K-casein, soybean meal, and blood are in good agreement with values obtained by classical ion-exchange methods, and establishes the use of the TMS-/gas-liquid chromatographic (GLC) method for quantitative analysis of amino acids in biological materials. Several other derivatives are available for amino acid analysis by GLC. Vance and Feingold (1970) and Pisano and Bronzert (1972) studied the methylthiohydantoin derivatives of amino acids and Fu and Mak (1971) the N-acyl amino acid alkyl esters. Along with the TMS amino acids, the other major derivatives for GLC are the N-trifluoroacetyl (N-TFA) n-butyl ester and, in some cases, the methyl ester (Islam and Darbre 1969, Roach and Gehrke 1969a, 1969b, Casagrande 1970, Pellizzari et al. 1971, Gehrke et al. 1971) . Zumwalt et al. (1970) used the N-TFA n-butyl esters for quantitative analysis of amino acids in complex biological substances such as urine and blood plasma. He used ion-exchange 19 resins for sample clean-up and obtained quantitative recovery of amino acids from the exchange columns. Gehrke and Leimer (1970a) studied the effect of salts on the derivatization and chromatography of N-TFA n-butyl esters of amino acids. They found that inorganic salt at a ratio of 1:1, salt to total amino acids, was not a serious problem for qualitative work. However, for quantitative work, the following salts should be removed by ion-exchange chromatography: oxalate, manganese (II), cobalt (II), nickel, zinc, tin (II), lead (II), chromium (III), and iron (III) . Gehrke et al. (1971) used the N-TFA n-butyl ester derivatives to search for amino acids in hydrolysates of lunar fines from Apollo 11 and 12 missions. Zumwalt et al. (1971a, 1971b) refined the N-TFA n-butyl ester systems to the point where nanogram and picogram amounts of amino acids could be analyzed. Gehrke et al. (1971) have an excellent review article on the TMS and N-TFA n-butyl ester systems for the analysis of the 20 protein amino acids in biological samples. Burchfield and Storrs (1962) have a general text on the biochemical applications of gas chromatography. 20 Radioisotope Techniques The use of radioisotopes in metabolic studies has greatly increased the number of scientific questions that can be answered and the limits of detection that can be reached. Snyder (1965) reports on quantitative radioassay methods for TLC. He compares zonal versus autoradiographic scans and prefers zonal scans due to their greater resolving power and speed. Snyder (1966) also compares zonal versus strip scans of thin layer chromatograms and again prefers the zonal scans due to their greater sensitivity and resolving power. This is especially true with weak beta emitters such as H-labeled compounds in biological specimens. Three excellent reviews covering the subjects of TLC radioassay, instrumentation and procedures for lkC and 3H radioassay by TLC and liquid scintillation radioassay of thin layer chromatograms are presented by Snyder (1968, 1969a, 1969b). Bell and Hayes (1958) review many aspects of liquid scintillation counting. Protein and Amino Acid Extraction and Treatment Many texts are available covering the vast subject of protein and amino acid extraction, hydrolysis, clean-up, etc. Some of the more complete works include Block and Weiss (1956), Alexander and Block (1960a, 1960b), and Blackburn (.1968). 21 Roach and Gehrke (1970) developed a new rapid acid hydrolysis technique for proteins. Using aqueous 6N HC1 at a ratio of 1 mg. of protein to 1 ml. of acid, they heated the mixture in a sealed test tube containing a N2 atmosphere for 4 hr. at 145 C. +2 . Essentially, equivalent hydrolysis and yield were obtained when this method was compared with the standard 110 C. +1 for 26 hr. using ribonuclease as a protein source. MATERIALS AND METHODS Rearing A stock culture of the almond moth was maintained on a diet consisting of the following ingredients: 4 parts cornmeal, 4 parts whole wheat flour, 2 parts finely ground dog food, 1 part brewer's yeast, 1 part oatmeal, 1/2 part wheat germ, 1 part honey, and 1 part glycerine. These ingredients were mixed thoroughly and placed in 1/2 gal. wide-mouth mason jars. The medium was then innoculated with several hundred eggs and the jars covered with 12 cm. filter paper discs and sealed with metal jar rings. Following emergence of adult moths, the filter paper discs were replaced with screen wire discs. The jars were then inverted in a rack and eggs were collected in petri dishes as they dropped through the screen. These eggs were used to innoculate the next generation of the culture. Radiochromatography and Autoradiography of U-^C-Glucose" Uniformly labeled 14C-glucose was used throughout this study. The purity of the C-glucose was checked by TLC and autoradiography of the chroma tograms . 2 2 23 Twenty micrograms of unlabeled carrier glucose in a volume of 5 nl. was applied to an Eastman Chromagram Sheet of Silica Gel G. The spot was then dried in a cool air current. An aliquot of radio labeled glucose containing 0.01 uCi. of activity was then applied on top of the unlabeled carrier spot. In a second lane next to the lt+C-glucose, 10 ul. of an unlabeled 1/2 percent aqueous glucose solution was spotted. The solvent system used for development was n-butanol- isopropanol-water (5:3:1). The chroma togram was allowed to develop until the solvent front reached 1/2 in. from the top edge of the plate. Following development, the chromatogram was thoroughly dried to remove all traces of solvent. The plate was then sprayed with a solution consisting of 0.9 g. oxalic acid and 1.8 ml. aniline in 200 ml. of H20 and heated to 105°C. for 15 min. to visualize the glucose spot. The dried chromatogram was then wrapped in a single thickness of plastic film in order to protect the x-ray film from any substances on the thin-layer plate v/hich might cause fogging. Then, in a dark room with a safety light on, a sheet of unexposed x-ray film was placed in direct contact on top of the plastic wrapped chromatogram. A small notch was cut in the film and chromatogram. The separated film and plate could then be realigned after development by aligning the notches. The film and chromatogram were then placed 2 A between two pieces of wood cut approximately the same size as the x-ray film. The two pieces of wood were held together securely by placing several elastic bands around them. This procedure kept the film and chromatogram aligned correctly. The wood blocks were then wrapped in several layers of aluminum foil and placed in a drawer for 20 hr. Following this, the x-ray film was developed, washed, and air dried. R ' s for the radioactive and non-radioactive glucose spots were then determined and compared and the x-ray film examined for the 14C-glucose spot and any impurities or streaking. Radioactive Medium Preparation Radioactive medium was prepared by pipetting 160 uCi. of C-glucose* onto 30 g. of standard rearing medium. The medium was thoroughly mixed and placed in an 8 oz. baby food jar. It was then innoculated with approximately 200 eggs and the jar sealed with a filter paper disc and metal jar ring. Following development, mature larvae were removed from the radioactive medium for extraction of p"rotein. Supplied by Amersham/Searle. Specific Activity - 309 mCi./mM. 25 Protein Extraction A weighed amount of mature larvae that had been reared on medium containing C-glucose was placed in a tissue grinder and homogenized with 20 ml. of cold 10 percent trichloroacetic acid (TCA) . The grinder was rinsed with 10 ml. of cold 5 percent TCA and the liquid combined with the larval homogenate. The homogenate was then centrifuged for 5 min. and the supernatant decanted off for further analysis. The pellet containing the protein fraction plus other components (i.e., lipids, nucleic acids, etc.) was dried under a stream of N2. Twenty milliliters of acetone, made alkaline with NH^OH, was added to the centrifuge tube. The mixture was then placed in a 70 C. water bath and stirred gently for 5 min. Following this, it was again centrifuged for 5 min. and the supernatant poured off and saved. The pellet was then dried under- a stream of N2 . The acetone extraction, centrifugation, and drying steps were then repeated. The pellet was subjected to this treatment again, first using 95 percent ethanol and then using ether. It was repeated twice with each Solvent and all supernatant fractions were saved for weighing and scintillation counting. The pellet plus 10 ml. of 5 percent TCA were then placed in a 90 C. water bath for 15 min. with continuous stirring. The tube and its contents were then cooled under running water, centrifuged, the supernatant poured off and the pellet dried :>b under N2. The protein pellet was then washed with 3-5 ml. portions of 5 percent TCA centrifuged, dried, and weighed. The flow diagram in Figure 2 will help illustrate what components were extracted with the different solvents. As a control, protein was extracted from the radioactive media. Two types of samples were analyzed; medium that had larvae reared on it and medium that had no larvae reared on it. This protein was extracted and analyzed in the same way as the insect protein. Free amino acids from the media, which were contained in the first TCA supernatant fraction, were also analyzed. Before TLC could be used to separate the amino acids in this fraction, it first had to be cleaned up using the column method for carcass analysis of free amino acids. Protein and free amino acids were also extracted from mature larvae that had been reared on non-radioactive medium. These were used in the carcass analysis study. Protein Hydrolysis Proteins extracted from the larvae and media were subjected to acid and base hydrolysis so analysis of the amino acids could be accomplished by TLC and GLC. 27 Cold 10% TCA and larvae Homogenize Centrifuge Supernatant (glycogen, sugars, free amino acids, vitamins, nucleotides, etc.) TCA precipitate (lipids, nucleic acids, proteins) Alkaline acetone extraction Neutral lipids Phospholipids Nucleic acid * Residue 1 Ethanol extraction I Ether extraction Residue Hot TCA extraction Residue (protein) Figure 2. Flow diagram for the extraction and clean- proteins used in this study. •up of 28 Acid Hydrolysis: Ten milligrams of protein were placed in a 125 mm. screw top test tube with a Teflon-lined cap. The tube was flushed with a stream of filtered N2 and 10 ml. of 6N HC1 were added. The tube was again flushed with N2, sealed, and heated for 4 hr. at 145°C. The protein hydrolysate was then evaporated to dryness under vacuum in a 60 C. water bath. The residue was taken up in 2 ml. of 10 percent aqueous 2-propanol (v/v) and again evaporated to dryness. This step was repeated once again after which the residue was dissolved in 1/4 ml. of 10 percent aqueous 2-propanol for TLC. For GLC analysis, the amino acid residues were dissolved in 1 ml. of 0.05 N aqueous KC1. Base Hydrolysis; Base hydrolysis was used for the study of amino acids that were partially or completely destroyed by acid hydrolysis, such as tryptophan. Ten milligrams of protein, 65 mg. of Ba (OH) 2 '8^0 and 1 ml. of H2O were placed in a screw top test tube. The top of this tube had a small hole drilled in it and a silicone rubber septum from a gas chromatograph injection port was placed in the top. The top was screwed on and a hypodermic needle that had been attached by means of rubber tubing to a vacuum line was inserted into the tube through the hole in the top. The tube was then evacuated with the rubber septum keeping it air tight. 2 'J The tube was heated for 24 hr. at 125 -130 C. after which it was cooled. The protein hydrolysate was then adjusted to pH 6 with 2N H2SO4 and then heated to boiling. The tube and its contents were then centrifuged to separate the BaSO^. The BaSOit pellet v/as washed with a little water and the combined supernatant and washing were evaporated to dryness. The residue was then dissolved in 1/4 ml. of 10 percent aqueous 2-propanol for separation of the amino acids by TLC. Thin-Layer Chromatography of Amino Acids Amino acids from the protein hydrolysates were separated and identified by TLC. Five microliters of hydrolysate were spotted on a 20 X 20 cm. Eastman Chromagram sheet of cellulose without fluorescent indicator. The starting point was 1/2 in. from the edges of the plate at the bottom left hand corner. The spot was positioned by marking the edges of the plate with a soft lead pencil. The solvent front was also marked in this manner, care being taken so as not to disturb the thin-layer and cause distortion of the spots during chromatography. After application to the thin -layer plate, the spot was dried in a stream of warm air. Separation of 20 amino acids required the use of two-dimensional chromatography. The solvent systems used were 2-propanol -methyl ethyl ketone-lN HC1 (60:15:25 v/v) for 30 development in the first dimension and 2-methyl-2-butanol-methyl ethyl ketone-acetone-methanol-water-concentrated NH^oH (50:20:10:5:15:5 v/v) for development in the second dimension. Development in each phase was allowed to continue until the solvent front reached 1/2 in. from the top edge of the plate. Between development in the first and second dimensions, the plate was dried for 2 hr. in a fume hood. Following development in the second dimension, the plate was allowed to dry overnight. Visualization of the amino acids was accomplished by spraying the dried plates with 1/2 percent ninhydrin in acetone and then heating for 20 min. at 60 C. A standard plate (i.e., spot map) was prepared to facilitate identification of the amino acids. Standard solutions (0.025 M) of the 22 amino acids of interest were made up in 10 percent aqueous 2-propanol. These standards were chromatographed and their positions on the plate noted along with their R 's in both dimensions. Radioactivity Measurements The supernatant fractions from the larval and media protein extractions were placed in numbered scintillation vials that had been previously weighed. The liquid was then evaporated to dryness under a N2 stream and heat lamp and the vials again weighed. After the weight of each supernatant fraction was 31 known, 1 or 2 ml. of Soluene,* a sample solubilizer, was added to each vial. The samples were set aside for 48 hr. to allow the solubilizer to work. Fifteen milliliters of scintillation fluid consisting of 5 g. 2,5-diphenyloxazole (PPO) , 0.250 g. 1,4 bis-2- (4-methyl-5 phenylox-axolyl) -benzene (dimethyl POPOP) , and 1 L. of toluene were added to each vial. The vials were then placed in a Packard Tri-Carb Liquid Scintillation Spectrometer and allowed to temperature equilibrate (3-4 C.) for 1 hr. These samples were counted for only 10 min. due to the high activity present. The counts per minute (cpm) were corrected for any quenching with automatic external standardization (AES) . Following separation and visualization, the individual amino acids were scraped off the thin-layer plates into scintillation vials. The spots from 10 plates were pooled for each amino acid. For scintillation counting, a cocktail similar to the previous one was used except that Cab-O-Sil** (4 percent w/w) was added. Cab-O-Sil is a gelling agent which aided in suspension of the amino acids on the thin-layer adsorbent. The vials were then placed in the scintillation counter and allowed to temperature equilibrate for 1 hr. They were counted for 100 min. * Packard Instrument Company, Inc. ** Rohm and Haas. 32 AES was used to correct the counts for any quenching in the samples. Several vials containing different amounts of thin- layer adsorbent were also counted to determine if the adsorbent caused any increase in activity due to fluorescence. The quenching properties of 20 amino acids and the supernatant fractions from the larval protein extraction were studied using internal standardization. This study was undertaken to test the validity of the external standards method. Twenty microliters of a 1 mg./ml. standard of each of 20 amino acids were pipetted onto a cellulose thin-layer plate that had been divided into 20 sections. Each section of the plate contained a single amino acid standard. Twenty scintillation vials each containing 15 ml. of the same scintillation cocktail used in the counting of amino acids from the protein hydrolysate, 10 ul. of 1 C-glucose, and 2-3 drops of Bio-solv * solubilizer were counted in a Beckman LS-200 Liquid Scintillation Spectrometer prior to the addition of a single amino acid; each vial was recounted to see how much quenching resulted. This same study was repeated again with the exceptions that this time only 5 ul. of * C-glucose were used and the amino acids were visualized by the method noted previously before they were added to the scintillation vials. * Beckman Instrument Company. 33 The quenching properties of supematants from the protein extractions were studied in a similar manner. The supernatant fractions, from the extraction of protein from larvae reared on non-radioactive medium, were dried, weighed, and solubilized as previously discussed. Each one was then added to a separate vial which contained 15 ml. of the same scintillation fluid used previously for counting supernatant fractions. These vials also contained 10 pi. of ltfC-glucose and 1-2 drops of Bio-solv solubilizer. They had been counted before the addition of the supernatant fractions and were now counted a second time to determine the extent of quenching caused by the various fractions. Carcass Analysis Fifth-instar larvae were analyzed for the total amount of each amino acid they contained. The protein and free amino acids were analyzed separately and then combined later to arrive at a total for each individual amino acid. The carcass analysis was replicated twice using two separate groups of larvae. Protein extraction and subsequent hydrolysis for the liberation of amino acids were accomplished using the same method as described previously for the thin-layer work. The free amino acids were obtained from the supernatant of the larval-TCA homogenate following centrifugation. Before the free amino acids could be derivatized for GLC, they had to -be separated from any 34 interfering biological substances with an ion-exchange column. The resin, Amerlite CG-120 (100/200 mesh) , was prepared as follows: resin was placed in a 500 ml. beaker and covered with 3N NH4OH. It was then placed on a magnetic stirrer and swirled for 60 min. The resin was allowed to settle and the NH,f0H decanted off. This process was repeated twice more and the resin was then washed with double distilled water until it was approximately neutral. The resin was then regenerated by swirling for 30 min. three times with 3N HC1. It was then washed with double distilled water until it was approximately neutral. The columns, which consisted of 125 mm. test tubes with a 2 mm. hole in the bottom, were then filled to the 3/4 mark with wet resin. The level of liquid was never allowed to fall below the surface of the resin. The liquid in the column was then allowed to fall to approximately 3 mm. above the resin surface and the sample was added with a pasteur pipette. The entire 30 ml. of TCA supernatant fraction were passed through the column. Following this, 5-10 ml. portions of distilled water were used to wash the resin. The washes were discarded. The amino acids were then eluted from the column using five separate 2 ml. portions of 3N NHi+OH. This was followed by five, 5 ml. portions of distilled water. The flow rate through the column was approximately 1-2 ml . /min . 35 The effluent from the column was collected in a 125 ml. round bottom flask. It was evaporated to dryness on a rotary evaporator with the flask immersed in a 60 C. constant temperature water bath. The residue was dissolved in 1 ml. of aqueous 0.05N HC1 for derivatization. Free amino acids extracted from the rearing media were dissolved in 1/4 ml. of 10 percent aqueous 2-propanol for TLC. Gas chromatography of Amino Acids for Carcass Analysis Column Packing and Preparation: Twenty grams of Supelcoport* 100/200 mesh were placed in a round bottom flask and just covered with methylene chloride. The methylene chloride had been dried by running it through a silicic acid column and was then distilled into an all glass bottle to protect it from atmospheric moisture. OV-11 (2.22 g.) dissolved in a minimal amount of methylene chloride, was then added to the round bottom flask containing the Supelcoport. This gives a 10 percent loading of OV-11 on the solid phase. The flask was then placed on a rotary evaporator * Supelco Inc. 36 and the methylene chloride slowly evaporated at room temperature until the column packing was just damp. The flask was then immersed in a 60 C. water bath while under full vacuum on the rotary evaporator until no odor of methylene chloride remained. A 12 ft. by 2 mm. I.D. glass column was then silylated to prepare it for the column packing. The column was first filled with a 10 percent v/v solution of dimethyldichlorosilane in toluene and allowed to stand for 15 min. with the solution in it. The column was then flushed and filled with absolute methanol. After 5 min., the methanol was removed and the column was washed twice with acetone. It was then placed in an oven to dry. The packing of 10 percent OV-11 on 100/200 mesh Supelcoport was then added to the column. The column was then placed in the gas chrcmatograph oven and flushed with N2 carrier gas for 30 min. Following this, it was no-flow conditioned at 325 -330 C. for 12-15 hr. The oven was then cooled to room temperature. A flow of 10-15 ml. /min. of N2 carrier gas was used for the rest of the conditioning. The oven was then temperature -programmed to 300 C. at a rate of 1 C./min. and allowed to remain undisturbed at this temperature for at least 24 hr. Derivatization of Amino Acids: An aqueous aliquot of protein hydrolysate or free amino acid extract, containing from 0.5-6 mg. of total amino acids, was 37 added to a 65 mm. screw top culture tube with a Teflon-lined cap. The amino acid solution was just evaporated to dryness in a 70 C. sand bath while passing a stream of regulated, filtered N2 into the tube. Methylene chloride (0.5 ml.) was then added to the tube and evaporated just to dryness. This last step was repeated two more times. A known amount of internal standard, in this case decanoic acid in acetonitrile, was then added to the tube. The amount of internal standard should correspond to about the amount of each individual amino acid in the test tube and there should be 0.25 ml. of acetonitrile for each mg. of total amino acid. Therefore, in the standard tube which contained 0.1 mg. of each of 20 amino acids for a total of 2 mg. of amino acid, an internal standard of 0.2 mg./ml. acetonitrile would be used. By adding 0.5 ml. of this solution, the required 0.1 mg. of internal standard and 0.5 ml. of acetonitrile for the 2 mg, of total amino acid would be added. In derivatizing the protein hydrolysates, an aliquot corresponding to 4 mg. of total amino acids was used. Since the amount of each amino acid in the hydrolysate varied, 0.1 .mg. of internal standard was chosen as the arbitrary amount to use. An additional problem with the free amino acid extracts was that the amount of total amino acids was not known. Therefore, these several equal aliquots were derivatized using different total amounts of acetonitrile. 38 Following addition of the internal standard, 0.25 ml. of bis (trimethylsilyl) trif luoroacetamide (BSFTA) was added for each 1 mg. of total amino acids in the tube. Different amounts of BSFTA were also tried in the free amino acid derivatizations. The tubes were then securely closed and placed in an ultrasonic bath for 1 min. to insure complete mixing. The trimethylsilyl (TMS) derivatives of the amino acids were made by heating the tubes for 2.5 hr. at 150 C. in an oil bath. The tubes should be only 1/4 full and not immersed in the oil above the level of liquid. A reagent blank containing everything but amino acids was also run to check for extraneous peaks. In addition to the protein hydro lysates and free amino acid extracts which were used for carcass analysis, several other samples were studied. Supernatant fractions from the extraction of protein from non-radioactive larvae were studied to see if there was any loss of amino acids during the extraction procedure. The first of each of the following fractions were studied: alkaline acetone, ethanol, ether, and hot TCA. These fractions were cleaned up using the same procedure as that used for the larval free amino acids. A second larval protein extraction was then performed and the same fractions were analyzed. However, this time the fractions were evaporated to dryness and then hydro lyzed with 6H HCl. The hydrolysates were then cleaned up using the same column procedure 39 as that for the larval free amino acids. Both sets of fractions were then derivatized in the same manner as the protein hydrolysate samples. Gas Chromatography of Trimethylsilyl Amino Acids; The carcass analysis samples were chromatographed on a model 2100 Varian Aerograph using a flame ionization detector. Five microliters of sample were injected directly onto the column. The following parameters were used for the separation and detection of amino acids: injector temperature, 275 C. ; detector temperature, 300 C. ; N2 carrier gas flow rate, 17 ml./min. ; oven temperature, initial 100 C. ; 3-min. hold after start of solvent peak, 4°C./min. increase to 300 C. ; attenuator settings, 32 X lCf11. The samples prepared from the various protein extraction supernatants were analyzed on a Tracor MT 220 gas chromatograph using a flame ionization detector. The following chromatographic conditions were used with this machine: injector temperature, 270°C. ; detector temperature, 225 C. ; N2 carrier gas flow rate, 17 ml./min.; oven temperature, initial 100°C. ; 3-min. hold after start of solvent peak, 5°C./min. increased to 210 C. ; attenuator settings 8 X 10. 40 Microbiological Study When analysis of several free amino acid fractions from radioactive media, both with and without larvae on them, showed radioactive amino acids to be present, a study was undertaken to find the source of their, synthesis. The third replicate of the essential amino acid study was run under aseptic conditions. The larval rearing medium, containing C-glucose, was heat sterilized at 15 lb. pressure for 15 min. The medium was sterilized in the rearing jar, which was covered with aluminum foil. Almond moth eggs were surface sterilized by placing them in a 3 percent zephiran chloride solution, in sterile distilled water, for 15 min. The eggs were rinsed well with sterile distilled water and put on sterile filter paper in a sterile petri dish. All work was carried out in a sterile hood. The eggs were then placed on the sterile, radioactive medium and the rearing jar was placed in a closed TLC tank to minimize air movement and maintain a sterile environment. The free amino acids in this rearing medium were analyzed after the larvae were taken off for protein extraction. The free amino acids from both sterile and non-sterile medium, containing C-glucose but no larvae, were also extracted and analyzed as a control. The sterility of the almond moth eggs and radioactive medium was checked by incubating them separately in nutrient broth at 37 C. for 4 days and then streaking on nutrient agar plates. The 41 plates were then incubated at 37 C. for 4 days and read as + (i.e., growth) or - (i.e., no growth). Other materials studied in this way or simply by streaking on nutrient agar plates were: non-sterile eggs, non-sterile medium, sterile larvae, and sterile medium that had sterile larvae reared on it. Three replicates were run on each material studied. RESULTS AND DISCUSSION Qualitative Amino Acid Requirements by The Indirect Radioactive Method and Thin-Layer Chromatography Two-dimensional TLC permitted the identification of 19 amino acids (Figures 3 and 4) from acid and base hydrolysates of protein from fifth-instar almond moth larvae. The larvae had fed ad libitum on medium made radioactive with ltfC-glucose for approximately 3 weeks. Identification of amino acid spots was made with the aid of R,. and R, . values (Table 3) calculated f leucine from a spot map (Figure 5) of 22 amino acid standards. The cpm/carbon atom (Table 4) of amino acids isolated from larval protein shows that alanine, aspartic acid, glutamic acid, glycine, proline, serine, and an unknown ninhydrin positive compound from the acid hydrolysis fraction were highly labeled. Because these amino acids were synthesized from glucose by the almond moth, they are considered nutritionally non-essential. The cpm/carbon atom of arginine, histidine, isoleucine, leucine, lysine,' methionine, phenylalanine, threonine, tryptophan, valine, tyrosine, and two ninhydrin positive unknowns were not significantly above background. These amino acids were therefore not synthesized to any appreciable extent from glucose and must be considered nutritionally essential. Cystine and cysteine contained an intermediate amount of radioactivity. This 42 43 2 Dimension Figure 3. Separation of amino acids present in an acid hydrolysate of protein extracted from fifth-instar almond moth larvae reared on medium containing l^C-glucose. Unknown ninhydrin positive compounds. 44 2 ' Dinension Figure 4. Separation of amino acids present in a base hydrolysate of protein extracted from fifth-instar almond moth larvae reared on medium containing C-glucose. * Unknown ninhydrin positive compound. 45 Table 3. Rf and RLeucine Values of 22 Amino Acid Standards Separated by Two-Dimensional Thin-Layer Chromatography on Cellulose Amino acid First dimension* Second dimension** Rf Rleucine Rf Rleucine X 100 X 100 x ioo X 100 Alanine 55 » 63 12 25 Arginine 16 18 4 8 Asparagine 20 23 7 15 Aspartic acid 42 48 3 6 Cysteine 8 9 2 4 Cystine 4 5 2 4 Glutamic acid 52 60 3 6 Glutamine 26 30 8 17 Glycine 34 39 9 19 Histidine 9 10 13 27 Hydroxyproline 42 48 10 21 Isoleucine 36 99 44 92 Leucine 87 100 48 100 Lysine 15 17 8 17 Methionine 74 85 3 7 77 Phenylalanine 78 90 50 104 Proline 55 63 16 33 Serine 37 43 15 31 Threonine 46 53 40 33 Tryptophan 64 74 45 94 Tyrosine 69 79 31 65 Valine 76 87 29 60 * First dimension (60:15:25, v/v) . 2-propanol-methyl ethyl ketone-lN HCl ** Second dimension = 2-methyl-2-butanol-methyl ethyl ketone-acetone-methanol -water-ammonium hydroxide (50:20:10:5:15:5) . 46 lie val0(7ot 0 Try {^J o- Glu(NH2) Figure 5. Spot map of 22 amino acid standards separated on cellulose thin-layer plates. Solvent system: first dimension: 2-propanol -methyl ethyl ketone - IN HC1 (60:15:25, v/v) ; second dimension: 2-methyl-2- butanol-methyl ethyl ketone-acetone-methanol-water- concentrated ammonium hydroxide (50:20:10:5:15:5, v/v) 47 Table 4. Cpm/Carbon Atom of Amino Acids from Acid and Base Hydrolysates of Protein Extracted from Almond Moth Larvae Reared on Medium Containing li+C-Glucose Amino acid Carbon atoms/ Mg. of amino Cpm+/c arbon atom Rep. Rep. Rep. amino acid/ I II Ill acid 10 TLC plates Alanine 3 .125 112.5 532.5 2 76.0 Arginine 6 .075 1.0 0.5 0.5 Aspartic acid 4 .211 62.0 270.7 86.0 Cysteine 3 - 11.0 16.0 13.5 Cystine 6 - 5.0 10.8 5.8 Glutamic acid 5 .271 74.0 328.5 116.5 Glycine 2 .070 81.0 347.0 182.0 Histidine G - 0.0 0.0 0.0 Isoleucine 6 .102 0.0 0.0 0.0 Leucine 6 .154 0.0 0.0 0.0 Lysine 6 .128 0.0 0.3 0.3 Methionine 5 .030 1.2 2.6 2.6 Phenylalanine 9 .081 0.1 0.2 0.0 Proline 5 .078 15.5 63.8 30.8 Serine 3 .083 55.0 256.5 25.5 Threonine 4 .059 1.3 0.0 0.0 Tryptophan 11 - 0.7 0.9 0.9 Tyrosine 9 .103 0.2 0.0 0.0 Valine 5 .109 0.4 0.0 0.0 Unknown I - acid hydrolysate* 4 - 33.7 41.0 41.0 Unknown II - acid hydrolysate* 4 - 0.8 0.0 0.0 Unknown III - base hydrolysate* 4 - 2.2 3.1 2.6 + Cpm corrected for background and quenching. * Calculated on the basis of four carbon atoms per molecule. 48 situation could indicate one of several possibilities. These two amino acids may be synthesized to only a limited extent and still need to be supplied in the diet. Another possibility is that dietary cysteine or methionine may spare the need for biosynthesis. There may actually be no radioactivity in these compounds and the activity observed may be coming from labeled contaminants which have been detected at the origin of the thin-layer plate by liquid scintillation counting. Only cysteine was found in the acid hydrolysis fraction. The cystine, if present, may have been substantially destroyed by the hydrolysis procedure and/or converted to cysteine (Blackburn 1968) . Only cystine, or what appeared to be cystine, was found in the base hydrolysis fraction which is unusual since this procedure readily destroys both cysteine and cystine (Blackburn 1968) . These two compounds are often found to be among the lowest in concentration of any amino acids present in the insect which complicates their detection and quantitation (Strong and Sakamoto 1963, Rodriguez and Hampton 1966, Rock and King 1968, and Rock and Hodgson 1971) . Even though many insects do not require cysteine or cystine (Strong and Sakamoto 1963, Rodriguez and Hampton 1966, Rock and King 1968, and Rock and Hodgson 1971), I would supply these amino acids in the diet until further study could be done for the following reasons. The three carbon amino acids cysteine, serine, and alanine which are derived from pyruvate, should have a very high specific activity if they are indeed synthesized (Black etal. 1957, Rock and King 1968) . Dilution 49 with unlabeled carbon, and thus low specific activity, would be expected at the level or stage of metabolism where proline is synthesized. The synthetic route to the carbon chain of proline is indirect and multiple intermediates exist where dilution of labeled carbon would be expected from unlabeled dietary components. Also, these amino acids being present in such low concentrations, as was noted previously, precludes extensive dilution of labeled cysteine and cystine with unlabeled cysteine and cystine. Tyrosine, which contained no radioactivity, was not synthesized from the carbon chain of glucose. It must, therefore, be classified as nutritionally essential. Phenylalanine is known to be the principal precursor of tyrosine in the rat (Steele 1952). In insects, the synthesis of tyrosine from phenylalanine has been demonstrated in the silkworm larvae (Fukuda 1956) , the pale western cutworm (Kasting and McGinnis 1962) , and the prairie grain wireworm (Kasting et al. 1962) . In the above cases when phenylalanine is supplied in sufficient amounts, tyrosine can be classified as nutritionally non-essential . 50 As a control, protein was extracted from two types of radioactive media; one medium had larvae reared on it and one medium had no larvae reared on it. Figures 6 and 7 are spot maps of an acid and base hydrolysate of proteins extracted from larval rearing media. Table 5 presents the data from radiometric analysis of these proteins. There was no synthesis of any radioactive protein amino acids in the medium by any organism which might have contributed to the activity in the proteins of the insect. Several extractions of the larval rearing media for free amino acids were attempted. However, upon TLC, no satisfactory separation of amino acids was achieved. The amino acids tended to clump in three undistinguishable groups. These groups were pooled, counted by liquid scintillation, and found to contain activity. The ion exchange clean-up procedure which was used in the carcass analysis section of this paper was then tried prior to TLC of these free amino acids. This procedure resulted in a clean preparation, which upon TLC produced a high resolution, unambiguous separation of the amino acids present. Readable thin-layer plates were only obtained from the third replicate of this study which was carried out under aseptic conditions. The free amino acids from a sample of each of three radioactive media were studied. These were sterile medium that had larvae reared 51 2 Dimension Figure 6. Separation of amino acids present in an acid hydrolysate of protein extracted from larval rearing medium which contained ll+C-glucose and on which larvae were maintained. The same pattern and amino acids were present in an acid hydrolysate of protein from radioactive medium on which no larvae were reared. 52 "Y)f ~\ Leu / U 0 On- t /phe (X Ch A^.^N X)(P" V Jciy ""Orr X Origin Figure 7. Separation of amino acids present in a base hydrolysate of protein extracted from larval rearing medium which contained llfC-glucose and on which larvae were maintained. The same pattern and amino acids were present in a base hydrolysate of protein from radioactive medium on which no larvae were reared. 53 Table 5, Radioactivity in Amino Acids from Acid and Base Hydrolysates of Proteins Extracted from Larval Rearing Media Containing ^C-Glucose Amino acid Alanine Aspartic acid Cysteine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Medium containing larvae cpm/carbon atom* 0 0 0 0 0 0 0.1 0.3 0 0 0.3 0 0 0.3 0 0.3 0 Medium containing no larvae cpm/carbon atom* 0.2 0 0 0 0 0 0.1 0.2 0 0 0.4 0 0 0 0.2 0 0.3 * Corrected for background and quenching, replicates. Average of three 5-1 on it, non-sterile medium that had no larvae reared on it, and sterile medium that had no larvae reared on it. Figures 8, 9, and 10, respectively, show the amino acids present in these samples of media. Tables 6, 7, and 8 present the results of radiometric analysis of these amino acids. The amino acids extracted from sterile medium which had no larvae reared on it contained no radioactivity. This finding is self-explanatory and requires no further comment. The amino acids from non-sterile medium, which also had no larvae reared on it, contained substantial radioactivity. All 15 amino acids and the four ninhydrin positive unknown compounds contained activity to one degree or another. Most microorganisms are known to be capable of synthesizing all the protein amino acids. Even though the larval rearing medium is quite dry and this should preclude the need for aseptic conditions (Fraenkel 1959) , the synthesis of amino acids that is occurring is probably microbial in origin. The free amino acid extract from the sterile medium that had larvae reared on it contained 22 free amino acids and five ninhydrin positive unknowns, all but one substantially labeled. The activity of the amino acids in this extract ranged from 3 to 28 times the activity in the corresponding amino acids from the non-sterile extract. The presence of almond moth larvae from surface sterilized eggs in the sterile rearing medium somehow 55 Val o t> 0phe "•0 - So S ^i ■-oh .rt > • •■H (ii !M id a t id Tl .-H ■H O .C til P o 0 fi c •H TS h d () CO 33 :••; id H u IW id fii a o P -i-i 14-1 irl ■H h >W O V( >H .C 0 (1 111 TJ )J •H id ^ t/i >1 •H H H o 1 !■( (!) •M ill >i u ,C 78 79 Table 18. Retention Time, Relative Retention Time, and Sensitivity of 19 Trimethylsilyl Amino Acid Standards Amino Acid Retention Relative Sensitivity Time Retention (ng./mm. (sec.) Time of recorder deflection) Alanine .525 .432 11.6 Valine 725 .597 10.4 Leucine 825 .679 10.9 Isoleucine 870 .716 21.7 Glycine 895 .737 9.3 Proline 930 .765 16.1 Serine 1000 .823 8.5 Threonine 1035 .852 9.6 Internal Standard 1215 1.000 - (Decanoic acid) Hydroxyproline 1300 1.070 16.1 Aspartic acid 1320 1.086 9.4 Methionine 1370 1.128 17.2 Cysteine 1400 1.152 35.7 Glutamic acid 1475 1.214 18.5 Phenylalanine 1565 1.288 12.2 Arginine 1735 1.428 22.7 Lysine 1890 1.556 11.4 Tyrosine 2020 1.663 11.9 Tryptophan 2500 2.058 26.3 Cystine 2520 2.074 71.4 NOTE: Standard column and conditions - Column: 10 percent OV-11 on Supelcoport 100/120 mesh. 12 ft. x 2 mm. I.D. Conditions; injector temperature 275°C; detector temperature, 300°C. ; oven temperature, initial 100°C; 3-min. hold after start of solvent peak. 4°C./min. increased to 300°C; attenuator settings 32 x 10~ ,• N2 carrier gas flow, 17 ml./min.; flame ionization detector; internal standard, Decanoic acid. 80 o a u a . .0 G y ,-. r- ;■ 7 H -U 1 C s 0 n > a 4- H — c •n « 0^ Cj G 4- «-! V r» B (1. rH w oo a P a f • ■ ik % u ►• M K CM i a C a w -<• a< c ■n •n |k *H H M M M c +J C <$ rt f/: 1- •H £ CD T5 p; T1 a ., 0 c H 0) +J 1 ) -H ■H Ifl •a - s H ■n P H , ro SI •H u 0 (J 0 (J d) ( ) id •1-1 H C) ■» 0 ■H J CJ a u O D •H X) A , ; O o j-j rA H o U m CN M : trt o -H 0 •H p a) •: u ■ p !-l •H 0 +j •H (1) o IH 4-1 (1 to u !,'. ■H id Ut (S 0) X! 1 ) H ^J. • i.n oo Cj o >o ■ ■: rt O ") q -h H (u in • "■ •H O H si 0 (d IH rp B] 0 3. +J ']) •* c o id B • ■H -a G n C) R o a oo >S M < > <> (N ■ ) tji •H H ■•> o u o ■P \ no a) U • id o "« Q id t H < ! h >i H u - II 0 H 0 * • H 3 ■H P p ,9 Ifl u o o w u H 0 0) a, ■ (!) Oi J H -0 a a o 0) P •H H u rs c & 4-1 H 0) . il) > c ■H tn rfl tt H -H H 3 0) 3 ) 0 fi Ifl 00 a \ W O o iri (J (N U O H c r- >M r- m h •n o d 81 A small amount of hydroxyproline was detected among the protein amino acids. This is very unusual since hydroxyproline is not commonly found in proteins. It has, however, been found in fibrous proteins, such as collagen, and in plant proteins. Table 19 is a proposed mixture , at the 2 percent and 3 percent dietary level, of amino acids based on results from carcass analysis of fifth-instar almond moth larvae. Tryptophan is not included in this table since it is destroyed by acid hydrolysis and no base hydrolysates were analyzed. Histidine, cysteine, and cystine were not detected with the GLC method as they were with TLC. Cysteine and cystine are often in very low concentrations in late instar lepidoptera larvae (Rock and King 1966, 1967c) . A study was done to determine if there was any loss of amino acids during the protein extraction procedures. Figure 14 presents the results of this study. The derivatized extraction fractions contained no significant chromatographic peaks , aside from the internal standard, both before and after acid hydrolysis. This is not surprising since amino acids and ■ peptides are markedly hydrophilic and thus very insoluble in non-aqueous solvents. The organic solvents used for the extraction procedure (i.e., alkaline acetone, ether, and 95 percent ethanol) would have very little affinity for amino acids and not contribute to the loss of these compounds. The aqueous 82 Table 19. Amino Acid Mixture Patterned After Carcass Analysis of Fifth-Instar Almond Moth Larvae Amino Acid 2% Dietary 3% Dietary Dietary level level requirement (mg./lOO g. (mg ./100 g. from lltC of diet) o f diet) study Alanine , 146 219 _ Valine 126 189 + Leucine 178 267 + Isoleucine 118 177 + Glycine 82 123 - Proline 118 177 - Serine 98 147 - Threonine 68 102 + Aspartic acid 240 360 - Methionine 34 51 + Glutamic acid 324 486 - Phenylalanine 94 141 + Arginine 88 132 + Lysine 148 222 + Tyrosine 124 186 + Hydroxyprol ine 16 24 + = Not synthesized - nutritionally essential - = Synthesized - nutritionally non-essential. 83 O (J _°*'a gi u *« (> u 1(1 Eh Si -p P X 0 £ i ad O < > aj H N M Q M O ■H •-n M-l M +J Tl -a 01 a 1 nH >i > ■H N O X! ■H ii) >1 VI -P H S3 tJ O n () ■C -H T) u u -p (J a tj (U id (/I >1 :•' U-J X! ■P H H 0 a W 0 3 sh id m •H a> a -P m at) I) (» c Ol o in «J fB M C a\ M -M >< q -P +1 O U S S 01 oi ii t> o -P e o M rri !3 A nri m n u •H o Cr> ■> & G Hi o a 0 id -P 0 ■H ■H >