W . . NUTRITION AND METABOLISM STUDIES OF ARTHROPODS Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY RICHARD H. ROSSJR.‘ 1970 L I B R A R Y . Michigan S Tate gee, Universi y f! ' 1'HES'S ABSTRACT NUTRITION AND METABOLISM STUDIES OF ARTHROPIDS By Richard H. Ross, Jr. A preliminary study to define a laboratory diet on which to rear the larvae of Rhyacionia buoliana (Schiff.) was made. The rearing vials were very important and gaseous exchange was essential for proper rearing and moisture levels. It was found that wheat germ appeared to contain essential components or balance of nutrients necessary for rearing this insect. Ascorbic acid also appeared to be an important component either for its nutritional value or as an antioxidant protecting other labile dietary components. It also appeared, because of the feeding habits of this insect and the length of its life cycle, that the larvae may have to be periodically transferred to new diet formulations to insure the pre- sence of changing essential nutrient(s). Aseptic rearing of the larvae seemed to be feasible. It was found that eggs, surface sterilized in 0.1% hypochlorite solu— tion for 10-15 minutes, could be aseptically introduced Richard H. Ross, Jr. into containers without appreciable decrease in egg hatch; however, sustained asepsis was difficult to maintain in the rearing vials employed. Female tarantulas, Aphonepelma sp., and female scorpions, Centruroides sculpturatus, were injected with luC. acetate-l- On analysis, both species expired over 30 percent as 1“C02. The tarantulas and scorpions had 6.3 and 3.6 times, respectively, the radioactivity in the fatty acids as in the unsaponifiable lipids, while they had 7.6 and 5.U Simes, respectively, the weight in the fatty acids as in the unsaponifiable lipids. Oleic acid was the predominant unsaturated fatty acid in both species with 65.9 percent in the scorpions and ”2.3% in the tarantulas. Palmitic acid was the predominant saturated fatty acid in both scorpions (13.3%) and tarantulas (19.8%). The tarantulas had nearly twice as much linoleic acid (22.7%) as the scorpions (12.1%), and the tarantulas had trace amounts of arachidonic acid. Cholesterol made up greater than 99% of the sterols in bOth species, but trace amounts of B—sitosterol and 7-dehydrocholesterol were also present. NUTRITION AND METABOLISM STUDIES OF ARTHROPODS By Richard H? Ross, Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1970 TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . LIST OF FIGURES . -. . . . . PART I: STUDIES ON TECHNIQUES FOR THE ZENIC AND ASEPTIC REARING OF THE EUROPEAN PINE SHOOT MOTH, RHYACIONIA BUOLIANA (SCHIFF.), (LEPIDOTERA:' OLETHREUTIDAE) Introduction . . . . . . . . . . . . Materials and Methods . . . . . . . . . Results . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . References . . . . . . . . . 1A PART II: UTILIZATION OF ACETATE-l- C BY THE TARANTULA, APHONEPELMA SP., AND THE SCORPION, CENTRUROIDES SCULPTURATUS, IN LIPID SYNTHESIS Introduction . . . . . . . . . . Materials and Methods . . . . . . . . . Results . . . . . . . . . . . . . . Discussion . . References . . . . APPENDIX I . . . . ‘. . . . . Literature Review for Part I . . °. . . . References . . . . . . . . . . . . . APPENDIX II 0 O O O O O O O O 0 O 0 O 0 Literature Review for Part II . . . . . . . References . . . . . . . . . . . . . ii Page 111 UWKOONN l8 19 23 32 35 37 38 A7 53 62 Table LIST OF TABLES Page Composition of the diets used in rearing European pine shoot moth larvae . . . . . A Growth of post diapausing European pine shoot moth larvae on different diet formulations at 25°C 0 o o o o o o 0 o o o o o 7 Growth of first instar European pine shoot 'moth larvae on different diet formulations at 25°C. 0 o o o o o o o o o o o 8 Effect of sodium hypochlorite on egg hatch and surface sterility in fluid thiogylcolate medium . . . . _. . . . . . . . . 10 Summary of the number, live weights, and treatment data of tarantulas and scorpions injected with acetate-l-luC . . . . . . 24 Recovery of radioactivity in the 1“002, residue, lipids, and aqueous fractions of tarantulas and scorpions injected with acetate-l-luc o o o o o o o o o o o 25 Weights of the total lipids and the relative percents of the saponifiable and unsaponifi- able lipids of tarantulas and scorpions injected with acetate-l-luc . . . . . . 27 Column chromatographic fractionation of the unsaponifiable lipids of tarantulas and scorpions injected with acetate-l-luC . . . 28 Gas-Liquid chromatographic analysis and relative % of the methyl esters of fatty acids of tarantulas and scorpions injected With acetate-I‘luc o o o o o o o o 0 30 iii Table 10 Gas-liquid chromatographic analysis and relative % of the free sterols of tarantulas and scorpions injected with acetate-l—luC iv Page 31 LIST OF FIGURES Figure Page 1 The respiration train used to analyze the l“€02 production of tarantulas and scorpions injected with acetate-l-luc . . . 21 PART I: STUDIES ON TECHNIQUES FOR THE ZENIC AND ASEPTIC REARING OF THE EUROPEAN PINE SHOOT MOTH, RHYACIONIA BUOLIANA (SCHIFF.), (LEPIDOPTERA: OLETHREUTIDAE) INTRODUCTION In order to more fully understand the European pine shoot moth, Rhyacionia buoliana_(Schiff.), physiologically, and for possible future host plant resistance studies, it was necessary to develop a defined diet on which to rear the larvae. Chawla and Harwood (1968) used a modifica— tion of Berger's (1963) diet which showed promise for further studies. A satisfactory mating technique has been developed by Daterman (1968, 1969). This paper concerns studies to define the synthetic diet both aseptically and nonaseptically to determine the nutritional requirements of the larvae. MATERIALS AND METHODS Diapausing larvae were collected from two Scotch pine plantations in the Cadillac-Traverse City area of Michigan. The infested buds were collected during the winter months and stored in a dark room maintained at A.S°C until ready to use. The larvae were allowed to emerge from the buds by placing the buds in screened cages at 29-33°C for "-8 days. The eggs used to provide the first instar larvae for the aseptic experiments were obtained by catching single and mating females in the plantations during the first week of July, 1969. They were caught and held in cylindri— cal one pint containers lined with Whatman No. 1 filter paper. The females were allowed to lay eggs on the filter paper which was then removed and kept moist in closed Petri dishes until the eggs were ready to hatch. The diets studied were based on Chawla and Harwood's (1968) modified Berger's diet. Table 1 lists the compo— nents of the diets examined. The vitamin mixture used was reported by Vanderzant and Reiser (1956) with the addition of choline (10 mg/ml) and inositol (5 mg/ml). This mixture was added to the diets as l ml/100 g solids except for diets l7 and 18 where it was added at 3 times that concen- tration. Conversion of casein to its sodium salt was accom- plished by wetting 1 kg of casein with l 1 of 1% aqueous sodium hydroxide. The casein-hydroxide mixture was Spread evenly in pans and the water was evaporated with the aid of a fan. The dried sodium caseinate chunks were then cut into small pieces and pulverized by passing them through a 0.008 screen of a hammer mill (Monroe & Lamb, 1968). The basic components of the diets were ball milled for 3-5 hours and stored dry. The cholesterol, ergosterol, .> .2 .Lounocoom .aamoaaocu cacamao casummm .m .w .z .cxmanaam ..oo owuaucoucm Lennfiu .m .caflao .noflowc< non .cozoofinamo .s .».z .xpo» 30: .nxpo: HaoHEozo unocxucaafimx .o .aHHmooH seaflmuno Lawam memo .m .o«zo .ccmao>oao ..Qnoo nHao«Eocoofim Hucofiuapuaz .2 .ucaawcm .cwsononcmsoq .uouaeaq naaufiuaouaecmcm seemam .m .wca>mH60u:n cophm cocoa cumunxzoucwo .N .HE C« nucoauoumc« um: mm Ca nucoaounwcd who .H .000 .oom cow com 000 com com cow com com com cow coo com com com com oow Lesa: m m e m un N.H N.H N.H N.H nu nn N.H N.H N.H N.H N.H N.H N.H N.H un ouauxds :«Emufi> nn nn m nn nn nn nn m m In un nn nn nn nu nn nn m 495:2. cofiuaoahaupog Casauu> nn In In nn nn m.N nu nn aHOLeLQOOOun a s~a.o nn nu haa.o In In nn NHH.o nn NHH.O In In In nn nn In In nn oHOLvunowLm saa.o nn nn NHH.o nn NHH.o NHH.o NHH.0 nn ~HH.0 nn nn NHH.o NHH.o BHH.o NHH.0 nn nn Hao poems“; FHH.o sam.o nn FHH.o han.o saa.o FHH.o NHH.o NHH.o safi.o NHH.o NHH.o sfla.o NHH.o smfi.o NHH.o NHH.0 nn mHOLopnoHocu ma ma ma wH ma 0H ma ma 0H ma ma 0H ma w” ma ea 0H mm :me< nn nn nn mm.o mm.o mm.o mw.o mm. mm.o mm.o mm.o mw.o mm.o mm.o mm.o mw.o mw.o mm.o vaom canpom nn nn nn H.H H.H H.a H.H H.H ~.H H.H H.H H.H H.H ~.fl H.H H.a H.H H.a ouhcuufiwELou n~.om nn nn nn m m m m m m m m m w m m m m m Hozooaa :« Uncencwnaxontmz nanahzuoe um“ nn nn a nu un z a nu nu nn nn 3 a a a nu nn a 10x um.NN m.N w.N m.N m.N m.N nn nn m.N m.N m.N w.N nn nn m.N m.N m.N m.N m.N k.3“; vapLOon< a: a: Na 2: a: a: a: a: a: a: a: a: a: a: a: a: as Na zavumcaa< nn nn un nn nn HN HN HN AN HN HN HN mN HN HN nn nn nn wonopuxoa HN flN HN AN AN nn nn nn nn nn un nu nn nn nn HN HN HN monOLuam N N N N N N N N N N N N N N N N N N anuamn conmoz II n! =N nn un nn nn nu nn nn un nn nu un nn nn nn 2N :Euew amen: mN oN nu mN mN un nu mN mN mN wN nu nn nn nu mN mN nn moua:«emmo a2 nn nu mN nn nn mN wN nn nn nn nu mN mN mN mN nn nu mN mewonao ma NH ma NmH Nafi ma NH HH 0H m m N w mm a m N g Hucocanoo upodu uduaau< .va>pw~ cues uoocm ocda camaouzm wcauaop c" vow: nuoao on» No cofiufinoaeoonn.fl mqmhma mo amass: co comma pcmohmm a .n nn 0.0 0.0: 00 cm ma n nn 0.0 >.HN 00 0m :H n nn 0.0 m.0 00 0m ma n nn 0.0 m.m 00 0m NH n 0.0 m.m n.0N 00 cm Ha n 0.0 N.H 0.mN 00 0m 0H n nn 0.0 0.0a 00 0m 0 n nn 0.0 m.ma 00 0m 0 n nn 0.0 -N.H 00 0: s n nn 0.0 m.N 00 0: 0 n nn 0.0 0.0H 00 0: m n nn 0.0 m.» 00 0: a n nn 0.0 m.s: 00 0: m n 0.0 m.mz 00 0: N N m.m a.» 0.Hm Hmm QHH H mahEmm mam: mCHMLmEm coapmasa wcfiooom om>pma mamfipp .oc mpaspm pcoopmm pcmonom .02 .02 code unmonmm Umwmem mpazpm Ooz .OomN pm mCOfipmHSEpom pmfiu pconmwmfin co mm>nma cpoE pooch mafia cmeOASM mcfimsmmmao umoa mo npzononn.N mqmmamn n J .mcoflpmofiaaoh m mo cmmza mm N NJ 00 Nm 0 0 0 0 0:. 0H.0 00H mm 00 :m mm 0 0 0m 0w 00H m0.0 00H no v: mm mm 0: 0m 00H 00H 00H H0.0 om mm mm as am In In In In nn Hohuzoo :Gz soaawpmwm .CHE mm .LWE 0N .:wE ma .zwu 0H .CNE m .gfl: 0N LN: 0N .:ME ma .CHE 0H .CNE m opHLoHLooaxc pcmogma Igopms pzoopom Kn cmpchEmpcco ucoogoa .Ezflwm: mpmfioohflmQNLp Uwsfim CH upwaflgmpn momaazu T? C Cd ,. >54 (\ I) n-J \_ r. I mac co muflgoficooamc Ezflnom no uommmmnn.: mamaa 11 the number of larvae noted at the first observation (after 15—25 days), while in this study it was calculated from the number of inoculated larvae. It was felt, because the larvae were allowed to crawl from the buds on their own and by handling them carefully with a brush, that all larvae inoculated were healthy. It was found, however, that many larvae never started feeding and died within a couple of weeks. The reason for this could possibly be attributed to some physical or chemical property of the diet rather that mechanical injury. House (1961) indicated that unnatural food and feeding conditions may not be conducive to optimum feeding. Heron (1965) working with spruce budworm larvae found that phagostimulants and at least one feeding deter- rent affected their feeding. Although Chawla and Harwood (1968) found that pine tissue was not a requisite for satisfactory growth, it was possible that there were phagostimulants present that would initiate feeding more rapidly. Of the pupae that were obtained in these diets few adults were able to emerge. When A2 male and 35 female pupae were taken out of buds collected in the field, brought into the laboratory, and placed in the growth chamber with the others, only 12% and 23% adults, respectively, were able to emerge. It was observed that most of them attempted to break the pupal case but were not able to complete eclosion. Pointing (1961) noted that the pupae moved 12 through the pupal chamber to the exit hole and protruded through the hole before emergence. Green (1956) also noted that emergence dropped sharply at temperatures exceeding 25°C. Therefore, in addition to having a nutritional deficiency in the laboratory diets, there may be some physi- cal aspect of movement through a tunnel that is conducive to adult emergence. It was also possible that a constant temperature of 25°C had some effect on the pupa so the initial mid-dorsal break in the pupal case was harder to make. Of the diets tested the modification of Berger's wheat germ diet (Chawla and Harwood, 1968) appeared to give the best results. The larvae also seemed to do quite well on the diets in which the carbohydrate was added after autoclaving. It appeared, however, that there was an essential unidentified component in the wheat germ or a critical balance of nutrients that must be present for larval growth and development. Vanderzant gt_al.(1962) working with three cotton insects noted that the diets deteriorated gradually, espec- ially with the loss of ascorbic acid by oxidation. This resulted in either death of the prepupae or incomplete emergence of the moths. In the tests conducted with the European pine shoot moth it was found that by omitting ascorbic acid the diets became very light colored in a period of a week. When a—tocopherol was added in place 13 of ascorbic acid the same light color was evident. If ascorbic acid was an antioxidant for some essential labile dietary component, it would be expected that a-tocopherol might also act the same way; however, the natural color was not restored when it was added. These experiments were inconclusive as to whether ascorbic acid was an essential nutritive component or a protecting additive to the diet or a combination of both. It may be necessary to transfer larvae to new diets periodically to insure that necessary dietary components have not deteriorated. Pointing (1963) reported that newly hatched larvae feed on needles before migrating to the buds where they feed during the late summer and fall. After overwintering in the buds he noted that they began their spring feeding in close correlation with shoot elongation. Because of these different feeding stages there may be a change in the dietary requirements with larval development. If this is so, it may be necessary to use several diets to insure the presence of necessary nutrient balances and components during the various stages of development. ' The aseptic rearing of these larvae appeared to be feasible. The problem in the contamination of these pre- liminary studies was shown to be in lid design. Because the mites were not original contaminants and were able to infest the vials, microorganisms could also enter. Care should also be taken to introduce as little hypochlorite 1A solution as possible with inoculation because the first instar larvae would drown. Experiments showed that the eggs could be surface sterilized with 0.1% hypochlorite for 10—30 minutes without an appreciable decrease in egg hatch. The development of the larvae within the egg was easy to follow and eggs ready to hatch within 2A hours could readily be detected. - Nutritional studies of these phytophagus insects were impeded because it was difficult to collect necessary quan- tities of infested buds, and the eggs could only be collected in the field for about two weeks while the adults were flying. The length of the life cycle and the diffi- culties in laboratory mating of the European pine shoot moth also made it a difficult insect to rear on artificial media. REFERENCES Berger, R. S. 1963. Laboratory techniques for rearing Heliothis species on artificial medium. USDA Agric. Res. Serv. ARS—33-8Azl-A. Chawla, S. S. and R. F. Harwood. 1968. Artificial diets for the European pine shoot moth, Rhyacionia buoliana (Schiffermuller) (Lepidoptera; Olethreutidae). Wash. Agric. Expt. Sta. Tech. Bull. No. 59:1-13. Wash. State Univ., Pullman. Daterman, G. E. 1968. Laboratory mating of the European pine shoot moth, Rhyacionia buoliana. Ann. Entomol. Soc. Amer. 61:920-923. Daterman, G. E. 1969. (personal communication). Green, G. W. 1965. The effect of physical factors on the emergence and subsequent behavior of adults of the European pine shoot moth, Rhyacionia buoliana (Schiff.). Canad. Entomol. 97:1077-1089.. Heron, R. J. 1965. The role of chemotactic stimuli in the feeding behavior of spruce budworm larvae on white spruce. Canad. J. Zool. A3z2A7-269. House, H. L. 1961. Insect nutrition. Ann. Rev. Entomol. 6:13-26. Monroe, R. E. and N. J. Lamb. 1968. Effect of commercial proteins on housefly reproduction. Ann. Entomol. Soc. Amer. 61:A56-A59. Pointing, P. J. 1961. The biology and behavior of the European pine shoot moth, Rhyacionia buoliana (Schiff.), in southern Ontario. 1. Adult. Canad. Entomol. 93:1098-1112. Pointing, P. J. 1963. The biology and behavior of the European pine shoot moth, Rhyacionia buoliana (Schiff.), in southern Ontario. II. Egg, larva, and pupa. Canad. Entomol. 95:8AA-863. 15 l6 Vandersant, E. S., M. C. Pool, and C. D. Richardson. 1962. The role of ascorbic acid in the nutrition of three cotton insects. J. Insect Physiol. 8:287-297. Vanderzant, E. S. and R. Reiser. 1956. Aseptic rearing of the pink bollworm on synthetic media. J. Econ. Entomol. A9:7-10. PART II. UTILIZATION OF ACETATE-l-luC BY THE TARANTULA, APHONEPELMA SP., AND THE SCORPION, CENTRUROIDES SCULPTURATUS, IN LIPID SYNTHESIS l7 INTRODUCTION Information on lipid metabolism in several insect species has been accumulated in recent years (Fast, 196A; Gilby, 1965). In addition Zandee (1967) has studied chol- esterol and fatty acid synthesis of crustacea, and he con- cluded that the absence of cholesterol biosynthesis may be characteristic of all arthropods. Zandee (1966b) working with the crayfish, Astacus astacus, Lamb and Monroe (1968) with the cereal leaf beetle, Oulema melanopus, Robbins g£_al. (1960) with the houSe fly, and Kodicek and Levinson (1960) with blowfly larvae found that these species util- ized at least twice the amount of acetate in the fatty acids as in the unsaponifiable lipids. Because many similarities as well as differences have been found in the lipid metabolism of the arthropods studied, it was felt that diverse groups of arthropods should be examined. Tarantulas and scorpions represent two groups of arthro- pods in which little is known of their metabolism. These studies were designed to examine the incorporation of ace- tate into tarantula and scorpion lipids and to determine possible similarities and differences from other arthropod groups that have been studied. 18 MATERIALS AND METHODS Experimental Animals. The animals used in these studies were female tarantulas, Aphonepelma sp., (obtained from the Southern Biological Supply Co., McKenzie, Tennessee) and female scorpions, Centruroides sculpturatus, (field collected by Lorin Honetschlager, Curator, Univer- sity of Arizona, Tempe). Prior to testing all of the animals were kept at 25—27°C and fed Tenebrio molitor larvae. Radiolabeled Acetate and Injection Techniques. The sodium acetate—l-luC (200 uCi, obtained from Amersham/ Searle Corp., Des Plaines, Illinois) was diluted to 10 ml with 0.13A M phosphate buffer (adjusted to pH 7.A7). Radioassays performed during these studies were made with 15 m1 of modified Bray's solutionl per vial and were counted in a Nuclear Chicago Unilux I (model 6850) liquid scintillation spectrometer. After dilution the acetate—l—luc solution had a specific activity of 1,595,892 dpm/US. The tarantulas, tested in 3 groups of 3 animals, were each injected with 10.8 pl acetate-l-luC through the costal membrane between the third and fourth pairs of legs. The scorpions, tested in 3 groups of 10 animals each, were injected with 0.A ul acetate-l-luC through the dorso- lateral intersegmental membrane of one of the posterior abdominal segments. 11000 ml ethylene glycol monomethyl ether, 2000 ml toluene, 12 g PPO, and 150 mg POPOP. l9 20 1“CO2 Analysis, Tissue Extraction,,and Combustion Analysis. The test animals were placed into a respiration train (Fig. 1) similar to that reported by Hopkins and Lofgren (1968). The 1“CO2 was collected for a 2A hour period in monoethanolamine and radioassayed. The groups of animals were then weighed live and frozen at -30°C until further analysis. The animals were homogenized in water, refluxed for 90 minutes in acetonezethanol (1:1) at A times the aqueous volume, and vacuum filtered (Kaplanis gt_§l., 1960). The residue was dried, weighed, and stored for combustion ana- lysis. The solvent pair was removed in vacuo, and the resulting aqueous transferred to a separatory funnel, acidified, and quantitatively extracted with ethyl ether to obtain the total lipids. The ether was dried over anhydrous sodium sulfate and removed in vacuo. The total lipids were analyzed gravimetrically and radiometrically and the aqueous fraction radioassayed. The residue was analyzed by placing 100 mg samples into bags prepared from 1 inch dialysis casing, and com- 1A busting them to CO in a combustion flask previously 2 reported by Hopkins and Lofgren (1968). The 14002 was trapped in 10 m1 monoethanolamine:methyl cellosolve (1:2) and radioassayed. Saponification and Column Chromatography. The total lipids were saponified under nitrogen in a glass-stoppered 21 Figure l.--The respiration train used to analyze the ”CO2 production of tarantulas and scorpions injected with acetate-l-luC. 22 tube with 10% potassium hydroxide in 95% ethanol for 90 minutes. The unsaponifiable lipids were extracted with ethyl ether, the aqueous acidified and the saponifiable lipids (fatty acids) extracted with ethyl ether. Both fractions were washed, dried over anhydrous sodium sulfate, and concentrated in vacuo. They were weighed and radio- assayed. The unsaponifiable lipids were fractionated by dual column chromatography on 1.1 X 7.5 cm columns each con- taining 7.5 g Woelm neutral aluminum oxide, grade 1, deactivated with 1.5% water (Kaplanis gt_al., 1960, as modified by Monroe §£_al., 1968). Each fraction was then weighed and radioassayed. Sterol Purification and Analysis. The ether fraction from the aluminum oxide column was purified by’2 successive digitonin precipitations (Louloudes §£_al., l96l),'and the purified sterols weighed and analyzed by a gas-liquid chromatograph equipped with a hydrogen flame detector (Research Specialities Corp., Series 600). Two stainless steel columns 3 ft X A mm ID were packed with 100-120 mesh Gas Chrom Q (Applied Science Laboratories, State College, Pennsylvania) coated with one of the following liquid phases: 1.0% QF-l or 0.75% neopentyl glycol succinate. Detailed column conditions are presented under results. Ultraviolet absorption spectroscopy was also used to aid in the identification of 7—dehydrocholesterol. 23 Fatty Acid Analysis. The saponifiable lipids (fatty acids) were methylated with borontrichloride-methanol (Applied Science Laboratories, State College, Pennsylvania) in a test tube in a boiling water bath for 2 minutes (Met- calf and Schmitz, 1961). The fatty acid methyl esters were analyzed by gas-liquid chromatography employing a 3 ft X A mm ID stainless stell column packed with 100—120 mesh Gas Chrom Q coated with 12% HI-EFF 1B. Detailed column con— 7 ditions are presented under results. The standards used were K 108 and N.I.H. Mix E fatty acid methyl esters, and mass spectroscopy was used to confirm the identities of the peaks when they deviated from the standards. RESULTS Table 5 summarizes the treatment data for the test animals. It presents the number of animals per test, the 1A live weights, ug of acetate-l- C injected, and total dpm injected. Although the tarantulas out-weighed the scor- pions considerably, an attempt was made to adjust the injected acetate-l-luC per unit of body weight so that approximately the same amounts were injected in both species. Table 6 presents the amounts of radioactivity recovered 1A in the CO residue, total lipids, and aqueous portions. 2 After 2A hours 31.2% and 3A.l% of the radioactivity were 1A recovered as CO2 from the tarantulas and scorpions, and U. 2 mm:.mm osH.Hmm 00HO.O o0:m.o 0000.0 smam.ma om coaaaccm 0NH.sm 00m.mms.: mmm0.0 stm.N 0NNN.:H mm00.0NH m mazpcmhme .pz Hmpoe .pz Hmuoe poaopnuhm Hmpoe pmmp poaopcpn< 2009 0009 now CH m nmm w pom .oz Umpomwcfi copooncfi Amv .03 m>HA ozananopmpmom ozfinfinmpmpoom mo EQU no N: . anopmpoom Spas Umpomncfi whoaapoom 0cm meSpcmpmp mo 0.: mpmw pCmEpmmsp 0:0 .mpnMH03 m>fiH .nmnesc one mo zpmeesmnn.m mqmfizwm Hmpop mpcmam>fljvm H0000 .03 nulmmr»flwpm H0000 .03 mpccam>flzcm H0000 n01 cc a n0; 00 0 fleece -0; no 1 H0000 .0; co 0 .L: 3N mSOTSUd mkuwfi H0009 :wamm: Lmuum NGOJH .vifinfinopmpmom 50H: popomncfi mcoflqpoom I 1 0:0 mwazpcmemp no wcoflpomam 0300300 0:0 .nVHSWH .msnwnop .oco . lfi 0:0 CH >0H>Hpomoficmh mo >Lm>000~nn.0 mqmae w 26 the residue, when combusted to CO and water, gave 13.9% 2 and 9.1% recovery, respectively. In both animals about 9% of the radioactivity was recovered in the aqueous fraction of the direct extraction while under 2% was recovered in the total lipid fraction. The total lipids comprised 2.0%' of the tarantula live weight and 5.8% of the scorpion live weight. After saponification of the total lipids, Table 7, it was found that the tarantulas had 7.6 times more fatty acids than unsaponifiable lipids while the scorpions had 5.A times more fatty acids. The tarantulas and scorpions had 6.3 and 3.6 times, respectively, more radioactivity in the fatty acids than in the unsaponifiable lipids. Table 8 shows that the tarantulas had more radio- activity, A2.l%, in the methanol fraction of the unsaponi- fiable lipids while most of the weight, 50.2%, was in the ether fraction (free sterols). The nfhexane fraction (hydrocarbons) had 20.5% of the weight but only 9.1% of the radioactivity. The scorpions had most of their radio- activity, 38.9%, in the benzene fraction with 23.2% in each of the ether and methanol fractions. .The n—hexane fraction had 1A.6% of the radioactivity. The ether frac- tion contained the most weight, 3A.0%, while the Q-hexane fraction had 30.8% of the weight. 27 m.HN o.ma. mwoa.0 , H.m0 0.2w :mwm.o m0m©.o COHQLoom 0.0H . 0.HH 0000.0 0.00 0.00 0000.H mamfi.m mascceeee spa>acce eaaaa A00 sca>apcm caaaq A00 A00 000000000 ncacem .03 ncaumm .03 0Haaa Hcpcc .03 H0000 00 0 H0000 00 0 man0fima:oa0m:0 man0fimfi:000m .ozananou0pmom :90: 0opomw:fi w:OHQLoom 0:0 m0azp:0h0p mo m0fiaaa mHQ0HmH:oa0m:s 0:0 00:0HMH:OQ00 m:p mo mp:mopma 0>Hp0amp c:p 0:0 00HQHH H0p0» 0:» Mo mpnwfimznn.0 mqmae 8 2 0.00 , 0.33 0.00 , 0.30 0.00 0.00 0.33 0.00 ccaacccm H.N: m.NH N.Hm N.0m m.0a 0.0a H.m 3.0: 003ps0p09 mufi>fipo0 mpfi>fipo0 mpH>Hpo0 mpfi>fipo0 0oaop:ph< I0000m .p3 30000003 nceccm .03 AwHOLmum mmpmv ncaemm .03 IOH00m .pz Am:onp0oopwmnv nmzpm Hmcpm 0:00:00 0:0xonn: m0HQfiH oHn0HNH:oa0m:s no 0 m>Hp0Hmm .ozananmp0pmo0 :00: 0opomn:fi 0:000poom 0:0 w0a§p:0m0p mo 00HQHH mHQ0HMHcoa0mcs 0:» 00 :0000:OHpo0pm 0H:Q0pw0p0Eo&:o :ESHoonn.m mqm<9 29 After analysis of the fatty acids, Table 9, it was found that arachidonic acid was present in trace amounts in the tarantulas, but was absent in the scorpion fatty acids. The C-l8 acids were predominant in both species, although both had only traces of linolenic acid. Oleic acid was most abundant in both species with 65.9% and U2.3%, respectively, in the scorpions and tarantulas. The tarantulas had relatively more linoleic acid, 22.7%, as compared to 12.1% in the scorpions. Palmitic and palmito- leic acids were present in both species with 19.8% and H.O%, respectively, in the tarantulas and 13.3% and a trace in the scorpions. Myristic acid was found in trace amounts in both species. Mass spectroscopy indicated that both of the species had trace amounts of two unidentified fatty acid methyl esters (MW of both was 28h) which represented two C-17 branched or straight chain fatty acids. In addi— tion two trace peaks with apparent molecular weights of 318 and 322 were present in the scorpion fatty acid methyl ester fraction. Table 10 presents the gas-liquid chromatographic analysis data of the sterols of the two species. Choles- terol was the predominant sterol with greater than 99% in both species. The NGS column indicated that small amounts of B—sitosterol and/or 7-dehydrocholesterol were present. The B—sitosterol was finally identified in trace amounts on the QF-l column and 7-dehydrocholesterol was determined 30 TABLE 9.--Gas-1iquid chromatographic analysis and relative % of the methyl esters of fatty acids of taran- tulas and scorpions injected with acetate—l-luC.l 2 Relative % Carbon no. Tarantula Scorpion C-luzo trace trace C—16:O 19.8 13.3 C-16:l H.O trace C—18:O 11.2 8.7 C-18:1 42.3 65.9 C-18:] 22.7 12.1 C-18:3 trace trace C-20:8 trace -- Unknowns traces“ traces5 1Column: 12% HI—EFF 15 on 100-120 mesh Gas-Chrom Q, nitrogen “9.6 ml/min., column 180°C (stainless steel 3 ft X H mm ID), hydrogen flame detector. 2Computed by disc integration. 3 5 The fatty acids were identified by comparison of the retention times with K 108 and N. I. H. Mix E standards of fatty acid methyl esters. Where the retentions times were significantly different, mass spectroscopy was used to confirm the identity. Mass spectroscopy indicated the presence of traces of two C-17 straight chain or branched fatty acid methyl esters (MW of both was 28”). Mass spectroscopy indicated the presence of traces of two C-17 straight chain or branched fatty acid methyl esters (MW of both was 28M) and two other peaks with apparent molecular weights of 318 and 322. 31 .00o00o000000 00HO0>I000H5 00 0000800000 0000a 0000000030 00scfizm .0O000000 0E0H0 :0wo0000 .AQH SE a x 00 m H0000 000Hcfi000v oowom CESHO0 ..:HE\HE m.:m 00wo00ac .0 Eo0001000 c005 omauooa 0o Hume uo.a "cadaoo .0O000000 0E0H0 00wo0000 .AQH as a x 00 m H0000 000HCH000V ooomm cesaoo «.005\.E m: 00wo0000 .0 Eo000|00w £008 omflsooa :o 002 0m0.o “meadow m H 00000 Ho00000aonoo0vhn0©|n 00000 o.m m.: m Ho000000fi0|m +mm o.m o.m 0.m m.m Ho00000aono "coam0oom 00000 H000000Hosoo0uzn0cun 00000 o.m w.: m Ho0000o0fi0lm +mm o.m o.m w.m m.m Ho00000ao00 "0H300000B 0ono0£00¢ 00000000 0oao0£00< 00000000 H0Eac0 0000 no UCSOQEoo manma Hmuz 0 0>fi00a0m 0:0000Ho00 o0 0>H00H00.00EH0 coa00000m .ozaual0000000 0003 00000n00 00OHQ0o00 0:0 00H0000000 0o 0Ho0000 0000 000 0o 0 0>fi00a00 000 0H0za000 0H0000wo00so0£0 00:00H100on|.oa 00009 32 by ultra violet spectroscopy even though it was not present in amounts great enough to compute a finite retention time. DISCUSSION After injection of the acetate—l-luC'into the tarantu- las and scorpions some of the compound should be eliminated 14C 0 These experiments showed that indeed greater than 2. 30% of the radioactivity was expired as 1”CO as 2, and some pre- liminary tests of the respiration train used in these studies showed that 50% or more may be expired. Zandee (1966a) recovered 25% of the radioactivity from crayfish and Lambremont and Stein (1965) were able to recover 50.7% l”C02 from the boll weevil after treatment with acetate-IMO. These experiments showed that both species utilized some of the radioactivity in the residue and aqueous fractions. It appearedthat the tarantulas utilized slightly more in the residue than did the scorpions while both had about the same amount in the aqueous portion of the direct extract. In both species only 1-2% of the total radioactivity was found in the total lipid fraction. These tests also showed that both species incor- porated more of the radioactivity into the fatty acid fraction than into the unsaponifiable lipids, and is, therefore, similar to results found for insects (Louloudes et al., 1961; Robbins et al., 1960; Lamb and Monroe, 1968), and in other arthropods and vertebrates (Zandee, 1962). 33 Analysis of the fatty acids demonstrated that the C-18 series was predominant in both species. In the two spotted spider mite (Walling et_al., 1968) and the cereal leaf beetle (Lamb and Monroe, 1968) the C-18 fatty acids were also predominant. In several insects and spiders, Barlow (196“) found that there were differences in the fatty acids, depending on the species studied. These stu- dies only showed differences in the relative amounts of the fatty acids except arachidonic acid, which was present in the tarantulas but not in the scorpions. Oleic acid was far more predominant in the scorpions than in the tarantulas while linoleic acid was more predominant in the tarantula fatty acids. Preliminary examination of the hydrocarbons by gas- liquid chromatography and mass spectroscopy demonstrated that the tarantulas had C-13, 1a, l6, 17, 18, 23, 3M, 25, and 26 straight chain saturated hydrocarbons. The scorpion had C-13, 1U, 15 and 25 saturated and C-27 unsaturated straight chain hydrocarbons. In addition both species had unknown peaks representing other unsaturated straight chain and branched hydrocarbons. Louloudes g£_§l. (1962) found that in house flies the odd numbered alkanes were' predominant but that even number chains, alkenes, branched, and cyclic hydrocarbons were also present. Both the tarantulas and the scorpions were found to have cholesterol as the predominant sterol. Although some 3H radioactivity was still present in the free sterols after two digitonin precipitations, it was possible that it represented higher molecular weight alcohols which precipi- tated with the sterols. This radioactivity was so low that it could not be attributed to sterol biosynthesis. Thus, the tarantula and scorpion, like other arthropods studied, cannot synthesize sterols from acetate. B-sitosterol and 7-dehydrocholestero1 were found as trace sterols in both species only after huge quantities of the sterol fraction were injected into the gas chromotograph. Both of these species demonstrated very similar patterns in the metabolism of acetate-l-luC. It was shown that both species metabolized acetate-l-luc into lipids in nearly the same proportions, although some differences did appear in the fatty acids and the various fractions of the unsaponifiable lipids. REFERENCES Barlow, J. S. 1964. Fatty acids in some insect and spider fats. Canad. J. Biochem. 42:1365—137A. Fast, P. G. 1964. Insect lipids: A review. Mem. Entomol. Soc. Canada 37:1-50. ~ Gilby, A. R. 1965. Lipids and their metabolism in insects. Ann. Rev. Entomol. 10:141-160. Hopkins, T. L. and P. A. Lofgren. 1968. Adenine metabolism and urate storage in the cockroach, Leucophaea maderae. J. Insect. Physiol. 1N:1803-1813. Kaplanis, J. N., w. E. Robbins, and L. A. Tabor. 1960. The utilization and metabolism of A-Clu-cholesterol by the adult house fly. Ann. Entomol. Soc. Amer. 53:260-26A. Kodicek, E. and Z. H. Levinson. 1960. Metabolism of B- sitosterol and other lipids in the presence of acetate-Z-luC by blowfly larvae. Nature. 188:1023-102A. Lamb, N. J. and R. E. Monroe. 1968. Lipid synthesis from acetate-l-Clu by the cereal leaf beetle, Oulema melanopus. Ann. Entomol. Soc. Amer. 61:1163-1166. Lambremont, E. N. and C. I. Stein. 1965. 'Clu02 production in the boll weevil, Anthonomus grandis, after injection of Clu—l—acetate. Ann. Entolmol. Soc. Amer. 58:765- 766. Louloudes, S. J., D. L. Chambers, D. B. Moyer, and J. H. Starkey, III. 1962. The hydrocarbons of adult house flies. Ann. Entomol. Soc. Amer. 55:442-A48. Louloudes, S. J., J. N. Kaplanis, W. E. Robbins, and R. E. Monroe. 1961. Lipogenesis from Ola-acetate by the American cockroach. Ann. Entomol. Soc. Amer. 5u:99— 103. 35 36 Metcalf, L. D. and A. A. Schmitz. 1961. The rapid prepara- tion of fatty acid esters for gas chromatographic analysis. Anal. Chem. 33:363-36“. Monroe, R. E., C. S. Polityka, and N. J. Lamb. 1968. Utilization of larval cholesterol—A-Clu for reproduc- tion in house flies fed unlabeled cholesterol in the adult diet. Ann. Entomol. Soc. Amer. 61:292-296. Robbins, W. E., J. N. Kaplanis, S. J. Louloudes, and R. E. Monroe. 1960. Utilization of l-Clu-acetate in lipid synthesis by adult house flies. Ann. Entomol. Soc. Amer. 53:128-129. Walling, M. U., D. C. White, and J. G. Rodriguez. 1968. Characterization, distribution, catabolism, and synthesis of the fatty acids of the two-spotted spider mite, Tetranychus urticae. J. Insect Physiol. 1&- lAAS-IASB. Zandee, D. I. 1962. Lipid metabolism in Astacus astacus (L.). Nature. 195:814-815. Zandee, D. I. 1966a. Metabolism in the crayfish Astacus astacus (L.). II. The energy-yielding metabolism. Archs. Int. Physiol. Biochim. 74:N5-57. Zandee, D. I. 1966b. Metabolism in the crayfish Astacus astacus (L.).- III. Absence of cholesterol synthesis. Archs. Int. Physiol. Biochim. 7U:A35-AA1. Zandee, D. I. 1967. Absence of cholesterol synthesis as contrasted with the presence of fatty acid synthesis in some arthropods. Comp. Biochem. Physiol. 20:811—822. APPENDIX I 37 LITERATURE REVIEW FOR PART I The European pine shoot moth, Rhyacionia buoliana (Schiff.) was a pest in Europe for more than a hundred years before it was introduced into America in 191A (Friend and West, 1933). Since then much work has been done on the biology and behavior of this insect (Green and Pointing, 1962; Green gt_a1., 1957, Pointing, 1961, 1963). The ecology of this insect has also been exten- sively studied (Green, 1962a, b; Haynes, 1961; Haynes and Butcher, 1961, 1962; Miller, 1967; Torgersen, 196A; Heik- kenen, 1960, 1963). Tests have been published on European pine shoot moth control with the use of insecti- cides (Haynes, 1959; Kulman and Dorsey, 1962) and with the practice of shearing (Rudolph and Lemmien, 1963). Para- sites and predators have also been studied by Watson and Arthur (1959), Juillet (1961), and Kulman (1965). Chawla and Harwood (1968) have reared the larvae of R. buoliana in the laboratory on artificial media. They used modifications of a wheat germ diet (Berger, 1963) and a bean diet (Shorey and Hale, 1965), and had fair results (per their methodology) but were unable to define the diets further. A satisfactory technique for mating the adults in the laboratory has been developed (Daterman, 1968, 1969). 38 39 It has been difficult to eliminate wheat germ and yeast from many diets. Charbonneau and Lemonde (1960) showed that there was an essential factor in Brewer's yeast required by Tribolium confusum, and Ephestia kuehniella required substances in the water soluble and water insoluble fractions of yeast (Fraenkel and Blewett, l9u3b). Adkisson g£_al. (1960) indicated that wheat germ contained sterols, fatty acids, tocopherols, protein, carbohydrates, vitamins, and minerals. The European corn borer, Pyrausta nubilalis, required an unidentified factor contained in corn leaves, grass juice concentrate, and other plant materials. The factor was not identical with the known B-vitamins, ascorbic acid, citrovorum factor, sodium nucleate, or carnitine. It was heat-stable, water soluble, and dialyzable (Beck, 1953). House (1965) showed that when insects were feeding on critical imbalances in the diet, they may have had metabolic difficulties result- ing in a decreased food consumption and a slower rate of weight increment. It may be possible that R. buoliana requires some chemotactic stimuli to initiate and maintain Optimal feeding behavior; because Heron (1965), working with spruce budworm larvae, showed that foliage and staminate flowers contained chemical substances which were phago—stimulants and at least one feeding deterrent. The phagostimulants in staminate flowers were mainly sugars and L-proline. Ho 40 stated that the accumulation of the deterrent glucoside, pungenin, was perhaps the reason for the limited consump— tion of mature needles. Leonard and Doane (1966) developed a wheat germ diet for the gypsy moth, Porthetria dispar, that gave larger specimens and more eggs that field collected ones. It was basically the diet reported by Vanderzant gt_a1. (1962) except that the wheat germ content was increased and linolenic acid was added. Water was a very essential component of insect diets (Fraenkel, 1943), and the amount needed varied tremendously (Trager, 19A7). The amount of water required in insect diets depended on the rate at which the water was lost by the body (Wigglesworth, 1965). With housefly larvae too much water was detrimental. Too little water reduced the availability of the medium and resulted in a longer period of larval development and undersized pupae and adults (Brookes and Fraenkel, 1958). Insects needed minerals and salts probably quite similar to those required by higher vertebrates (Trager, 19A7). The osmotic properties of the medium were impor— tant to the ability of the insect to utilize the food- stuffs. There was little evidence that pH had much effect and most diets had good buffering capacities (House, 1959). Consistency was very important (House, 1959; Brookes and Fraenkel, 1958) and unnatural food and feeding conditions 01 may not have been conducive to optimum nutrition (House, 1961). Many diets also required chemical attractants in addition to the nutritive components (House, 1959, 1961). The utilization of carbohydrates in the diet varied considerably among insects (House, 1961; Trager, 1947). Aedes aegypti larvae did not require a carbohydrate in their diet (Akov, 1962), and Vanderzant and Reiser (1956b) found that the pupation of the pink bollworm was acceler— ated when sucrose was reduced and/or Wesson salts increased. Brookes and Fraenkel (1958) stated that a few carbohydrates inhibited growth in housefly larvae. However, in spite of specific variations, all insects usually utilized glucose and fructose (House, 1961). Protein amino acids were vital nitrogen sources in the diet of an insect (Wigglesworth, 1965), and the require- ments were very complex (Trager, 1947; House, 1961). Singh and Brown (1957) determined that Aedes aegypti required arginine, isoleucine, leucine, lysine, phenylalanine, methionine, threonine, tryptophane, valine, and histidine in both larval and adult stages. These same ten essential amino acids appeared to be required by most insects (Wigglesworth, 1965). In many cases, however, these had to be supplemented with additional amino acids (House, 1961). Nucleic acids were not essential in the diets of most insects although their addition could have accelerated growth (Burnet and Sang, 1963). Aedes aegypti larvae A2 required RNA for optimal growth (Akov, 1962; Singh and Brown, 1957). Brookes and Fraenkel (1958) found that RNA or adenine and guanine increased the growth of housefly larvae, while uracil and cytosine had no effect. Vander- zant and Reiser (1956b) found that the addition of nucleic acid had no effect on the pink bollWorm. All insects studied so far required a sterol in the diet (Fraenkel, 19A3; Lipke and Fraenkel, 1956). Noland (195Aa) found after studying Al sterol derivatives with Blattella germanica that the no. 3 hydroxyl group, either free or esterified, was essential. The no. 5 double bond was not required for absorption and utilization. He con- cluded that the absorption of sterols in insects, as in vertebrates, involved the participation of a cholesterol esterase. After studying the effects of 31 nonutilizable cholesterol derivatives in German cockroaches in which the diets contained minimal optimal levels of cholesterol, Noland (1954b) concluded that there was a competitive inhibition of cholesterol by the other sterols. An excess of cholesterol in the diet of mosquito larvae inhibited pupation (Akov, 1962), and Sang (1956) reported that excess cholesterol had no effect on the mortality of Drosophila melanogaster but did affect the rate of develop- ment. In the nutrition of the pink bollworm Vanderzant and Reiser (1956b) found that cholesterol could be replaced by ergosterol, sitosterol, and stigmosterol. “3 Many insects have also required the addition of fatty acids to the diet. Ephestia kuehniella, E. elutella, E. cautella, and Plodia interpunctella required linoleic acid in the diet for wing development and adult emergence (Fraenkel and .Blewett, 19u6). Tamaki (1961) working with the smaller tea I tortrix, Adoxophyes orana, found that linolenic acid was required for emergence, and that olive oil, stearic, oleic, and linoleic acids had no effect. The salt marsh cater- pillar also required 1inolenic acid in the diet (Vanderzant, 1967). Rock g£_al. (1965) found that linolenic and linoleic acids gave better growth and adult emergence in Argyrotaenia velutinana. They found that oleic acid had a positive effect on growth but had no effect on adult emergence, while stearic and palmitic acids and methyl arachidonate had no effects on survival. Without linseed oil in the diet they found that larval growth was slow, mortality high, and that the adults were unable to emerge. They found that if the essential fatty acids were supplied as late as the fifth instar that normal adult emergence followed. Gordon (1959) noted that linoleic acid was required for the second generation of Blattella germanica. He found that linolenic and arachidonic acids were not effective, but possibly this was due to rapid deterioration in the diet. All insects required B-vitamins in their diets, although symbionts may have supplied certain of them 44 (Fraenkel, 1943). Singh and Brown (1957) found that vitamins were not required for adult nutrition in Aedes aegypti. The larvae, however, would not grow in the absence of the B-vitamins: thiamine, riboflavin, nico- tinic acid, pyridoxine, and patothenic acid. Folic acid or choline was required for pupation (Akov, 1962). Akov (1962) reported that riboflavin was the only vitamin that was detrimental in excess. Trager (1947) indicated that the insects tested thus far were all unable to utilize vitamin D. Vitamin B12 was not required by the pink boll- worm (Ouye and Vanderzant, 1964), but Gordon (1959) stated that it was required for rearing the German cockroach past Ithe first generation. The reason for this could have been that certain nitrients supplied in the egg were ample for growth and development of the progeny (House, 1959), but if not added to their diet, the next generation would be deficient. It was also found that choline and inositol were required in large enough quantities to be considered nutrients (Gordon, 1959). The omission of choline in the purified casein diet for pink bollworm prevented develop- ment (Vanderzant and Reiser, 1956b). Vanderzant and Reiser (1956a) presented a vitamin mixture for the pink bollworm. Gordon (1959) also stated that it was possible that a-tocopherol was either required or would improve growth in the German roach. Beck g£_a1. (1949) suggested that it was essential in the diet of Pyrausta nubilalis, but 45 Fraenkel and Blewett (1946) suggested that a-toc0pherol was an antioxidant preservative for the unsaturated fatty acids. Sang (1962) reported that the vitamin requirements for Drosophila varied considerably on axenic diets with different amounts of protein. He suggested that the requirements depended partially on the kind of nutrients present that were substrates in which the vitamins acted as coenzymes. Fraenkel and Blewett (1943a) found that the B— vitamin requirements of Lasioderma and Sitodrepa varied considerably from aseptic cultures to normally reared ones. The aseptic cultures were free of symbionts, thus suggesting that the symbionts were responsible for supplying accessory food substances. Using aseptic casein medium, Ouye and Vanderzant (1964) found that the pink bollworm required calcium pantothenate, folic acid, nicotinamide, pyridoxine, riboflavin, and thiamine. The vitamin requirements of aseptically reared onion maggots, Hylemya antigua, have been studied by Friend and Patton (1956). The diets for insects vary immensely and are extremely complex. Sang (1959), for example, found that there were a multiplicity of optimal diets for Drosophila melanogaster by partially substituting components. He also found that there appeared to be differences between various strains of Drosophila. Axenic techniques were also required to standardize test animals in order to separate specific metabolic needs from any host-symbiont relationships (House, 1961). 46 Another problem encountered while rearing some insects was that the diets gradually deteriorated, especially with the oxidative loss of ascorbic acid. This resulted in either death of the prepupae or incomplete emergence of moths with some cotton insects (Vanderzant g£_§1., 1962). There is still a need in nutritional studies to define diets further and to develop techniques by which large numbers of insect species can be aseptically reared. REFERENCES Adkisson, P. L., E. S. Vanderzant, D. L. Bull, and W. E. Allison. 1960. A wheat germ medium for rearing the pink bollworm. J. Econ. Entomol. 53:759-762. Akov, S. 1962. A qualitative and quantitative study of the nutritional requirements of Aedes aegypti L. larvae. J. Insect. Physiol. 8:319-335. Beck, S. D. 1953. Nutrition of the European corn borer, Pyrausta nubilalis (an.). III. An unidentified dietary factor required for larval growth. J. Gen. Physiol. 36:317-325. Beck, 3. 0., J. H. Lilly, and J. F. Stauffer. 1949. Nutrition of the European corn borer, Pyrausta nubilalis (an.). I. Development of a satisfactory purified diet for larval growth. Ann. Entomol. Soc. Amer. 42:483-496. Berger, R. S. 1963. Laboratory techniques for rearing Heliothis species on artificial medium. USDA Agric. Res. Serv. ARS 33-86:1-4. Brookes, V. J. and G. Fraenkel. 1958. The nutrition of the larva of the housefly, Musca domestica L. Physiol. Zool. 31:208-223. Burnet, B. and J. H. Sang. 1963. Dietary utilization of DNA and its derivatives by Drosophila melanogaster (Meig.) J. Insect Physiol. 9:553-562. Charbonneau, R. and A. Lemonde. 1960. Unidentified growth factors in Brewer's yeast. II. Some chemical and physical properties of these factors. Canad. J. Zool. 38:443-448. Chawla, S. S. and R. F. Harwood. 1968. Artificial diets for the European pine shoot moth, Rhyacionia buoliana (Schiffermuller) (Lepidoptera: Olethreutidae). Wash. Agric. Expt. Station Tech. Bull. No. 59:1-13. Wash. State Univ., Pullman. £17 48 Daterman, G. E. 1968. Laboratory mating of the European pine shoot moth, Rhyacionia buoliana. Ann. Entomol. Soc. Amer. 61:920-923. Daterman, G. E. 1969. (Personal communication) Fraenkel, G. 1943. Insect nutrition. Royal College Sci. J. 13:59-69. Fraenkel, G. and M. Blewett. 1943a. Intracellular sym- bionts of insects as a source of vitamins. Nature 152:506-507. Fraenkel, G. and M. Blewett. 1943b. The basic food requirements of several insects. J. Exptl. Biol. Fraenkel, G. and M. Blewett. 1946. Linoleic acid, vitamin E. and other fat-soluble substances in the nutrition of certain insects [Ephestia kuehniella, E. elutella, E. cautella and Plodia interpunctella (Lep.)] J. Exptl. Biol. 22:172-190. Friend, R. B. and A. S. West, Jr. 1933. The European pine shoot moth (Rhyacionia buoliana Schiff.). Yale Univ.: Sch. Forestry Bull. No. 37:1-66. New Haven, Conn.' Friend, W. G. and R. L. Patton. 1956. Studies on vitamin requirements of larvae of the onion maggot, Hylemya antigua (Mg.), under aseptic conditions. Canad. J. Zool. 34:152-162. Gordon, H. T. 1959. Minimal nutritional requirements of the German roach, Blattella germanica L. Ann. N. Y. Acad. Sciences. 77:290-351. Green, G. W. 1962a. Flight and dispersal of the European pine shoot moth, Rhyacionia buoliana (Schiff.). 1. Factors affecting flight, and the flight potential of females. Canad. Entomol. 94:282-299. Green, G. W. 1962b. Low winter temperatures and the European pine shoot moth, Rhyacionia buoliana (Schiff.), *in Ontario. Canad. Entomol. 94:314-336. Green, G. W., W. F. Baldwin, and C. R. Sullivan. 1957. The use of radioactive cobalt in studies of the dispersal of adult females of the European pine shoot moth, Rhyacionia buoliana (Schiff.). Canad. Entomol. 89:379-383. 49 Green, C. W. and P. J. Pointing. 1962. Flight and dis- gersal of the European pine shoot moth, Rhyacionia uoliana (Schiff. ). II. Natural dispersal of egg- laden females. Canad. Entomol. 94:299-314. Haynes, D. L. 1959. Dorsal contact toxicity of six insecticides to wintering larvae of the European pine shoot moth. J. Econ. Entomol. 52:588-590. Haynes, D. L. 1961. Studies on European pine shoot moth biology and interactions between the insect, its environment and Michigan host species. Dissertation Abst. 21:3216. Haynes, D. L. and J. W. Butcher. 1961. An evaluation of some larval growth criteria in European pine shoot moth larvae. Canad. Entomol. 93:561-563. Haynes, D. L. and J. W. Butcher. 1962. Studies on host preference and its influence on European pine shoot moth success and development. Canad. Entomol. 94:690-706. Heikkenen, H. J. 1960. The identification and dating of past attacks of the European pine shoot moth on red pine. J. Forestry 58:380-384. Heikkenen, H. J. 1963. Influence of site and other fac- tors on damage by the European pine shoot moth. Dissertation Abst. 25:9. Heron, R. J. 1965. The role of chemotactic stimuli in the feeding behaviour of spruce budworm larvae on white spruce. Canad. J. Zool. 43:247-269. House, H. L. 1959. Nutrition of the parasitoid Pseudosar- cophaga affinis (Fall.) and of other insects. Ann. N. Y. Acad. Sciences 77:394-405. House, H. L. 1961. Insect nutrition. Ann. Rev. Entomol. House, H. L. 1965. Effects of low levels of the nutrient content of a food and of nutrient imbalance on the feeding and nutrition of a phytophagus larva, Celerio euphorbiae (Linnaeus) (Lepidoptera: Sphingidae _ Canad. Entomol. 97:62-68. Juillet, J. A. 1961. Observations on arthrOpod predators of the European pine shoot moth, Rhyacionia buoliana (Schiff.) (Lepidoptera: Olethreutidae), in Ontario. Canad. Entomol. 93:195—198. 50 Kulman, H. M. 1965. Oviposition habits of Trichogramma minutum on artificial concentrations of eggs of the European pine shoot moth. Ann. Entomol. Soc. Amer. 58:241-243. Kulman, H. M. and C. K. Dorsey.’ 1962. Granular applica- tion of systemics for control of European pine shoot moth. J. Econ. Entomol. 55:304-305. Leonard, D. E. and C. C. Doane. 1966. An artificial diet for the gypsy moth, Porthetria dispar (Lepidoptera: Lymantriidae). Ann. Entomol. Soc. Amer. 59:462-464. Lipke, H. and G. Fraenkel. 1956. Insect nutrition. Ann. Rev. Entomol. 1:17-44. Miller, W. E. 1967. The European pine shoot moth-- Ecology and control in the lake states. Forest Sci. Monograph 14:1-72. Noland, J. L. 1954a. Sterol metabolism in insects. I. Utilization of cholesterol derivatives by the cock- roach, Blattella germanica L. Arch. Biochem. Biophys. 48:370-379- Noland, J. L. 1954b. Sterol metabolism in insects. II. Inhibition of cholesterol utilization by structural analogs. Arch. Biochem. Biophys. 52:323-330. ' Ouye, M. T. and E. S. Vanderzant. 1964. B-vitamin requirement of the pink bollworm. J. E0on. Entomol. 57:427-430. Pointing, P. J. 1961. The biology and behaviour of the European pine shoot moth, Rhyacionia buoliana (Schiff.), in southern Ontario. 1. Adult. Canad. Entomol. 93:1098—1112. Pointing, P. J. 1963. The biology and behaviour of the ' European pine shoot moth, Rhyacionia buoliana (Schiff.), in southern Ontario. II. Egg, larva, and pupa. Canad. Entomol. 95:844-863. Rock, G. C., R. L. Patton, and E. H. Glass. 1965. Studies of the fatty acid requirements of Argyrotaenia velu- tinana (Walker). J. Insect Physiol. 11:91-101. Rudolph, V. J. and W. A. Lemmien. 1963. Shearing scotch and red pine Christmas trees for control of the European pine shoot moth. Mich. Agric. EXpt. Station Quart. Bull. 46:186-205. 51 Sang, J. H. 1956. The quantitative nutritional requir- ments of Drosophila melanogaster. J. Exptl. Biol. 33:45-72. Sang, J. H. 1959. Circumstances affecting the nutritional requirements of Drosophila melanogaster. Ann. N. Y. Acad. Sciences 77:352-365. Sang, J. H. 1962. Relationships between protein supplies and B-vitamin requirements in axenically cultured Drosophila. J. Nutrition 77:355—368. Shorey, H. H. and R. L. Hale. 1965. Mass-rearing of the larvae of nine noctuid species on a simple artificial medium. J. Econ. Entomol. 58:522-524. Singh, K. R. P. and A. W. A. Brown. 1957. Nutritional requirements of Aedes aegypti L. J. Insect Physiol. 1:199-220. ‘ Tamaki, Y. 1961: Studies on nutrition and metabolism of the smaller tea tortrix, Adoxophyes orana (Fischer von Roslerstamm). II. An essential factor for adult emergence.- Japanese J. Appl. Entomol. Zool. 5:58-63. Torgersen, T. R. 1964. The bionomics of the European pine shoot moth, Rhyacionia buoliana (Schiffermuller) (Lepidoptera: Tortricidae), in Wisconsin. Disserta- tion Abst. 25:3768-3769. Trager, W. 1947. Insect nutrition. Biol. Rev. 22:148-177. Vanderzant, E. S. 1967. Wheat-germ diets for insects: Rearing the boll weevil and the salt-marsh caterpillar. Ann. Entomol. Soc. Amer. 60:1062-1066. Vanderzant, E. S., M. C. Pool, and C. D. Richardson. 1962. The role of ascorbic acid in the nutrition of three cotton insects. J. Insect Physiol. 8:287—297. Vanderzant, E. S. and R. Reiser. 1956a. Aseptic rearing of the pink bollworm on synthetic media. J. Econ. Entomol. 49:7-10. Vanderzant, E. S. and R. Reiser. 1956b. Studies of the nutrition of the pink bollworm using purified casein meda. J. Econ. Entomol. 49:454-458. Vanderzant, E. S., C. D. Richardson, and S. W. Fort. 1962. Rearing of the bollworm on artificial diet. J. Econ. Entomol. 55:140. 52 Watson, W. Y. and A. P. Arthur. 1959. Parasites of the European pine shoot moth, Rhyacionia buoliana (Schiff.), in Ontario. Canad. Entomol. 91:478-484. Wigglesworth, V. B. 1965. The principles of insect physiology, 6th ed. London: Methuen and Co., Ltd. 464-477. APPENDIX 11 53 LITERATURE REVIEW FOR PART II Acetate metabolism has been found to be highly complex in arthropods. Because acetate forms a central position among many avenues of anabolism and catabolism, acetate is a useful compound to use for intital general metabolism studies, especially lipid metabolism. After introducing acetate into a biosystem it would be expected thatsome of the compound would be eliminated as CO . Zandee (1966b) was able to recover 25% of the 2 injected radioactivity as 14 CO when he gave 4 injections 2 over a 4 day period in the crayfish, Astacus astacus. In work with the boll weevil, Lambremont and Stein (1965) were able to recover 25.3% of the radioactivity from 14 acetate-l-luC injection as CO within 3 hours, and 50.7% 2 after 12 hours. They found that the maximum luCO output 2 was 1 hour after injection, and by 2 hours it had decreased to about half the rate found at 1 hour. It has also been found that acetate-l-luC was incor- porated into the amino acids of the crayfish (Zandee, 1966a). Louloudes §£_al. (1961) reported that acetate-1N0 was incorporated into the unsaponifiable fraction of the 54 55 American cockroach at about the same rate as in the house fly, and most of the radioactivity was found to be in the hydrocarbon fraction. Robbins e£_al. (1960) found that with house flies, 80% of the unsaponifiable fraction's radioactivity from acetate-l-luC was in the hydrocarbon fraction. Zandee (1962) was able to recover radioactivity in hydrocarbons of the crayfish, and Lamb and Monroe (l968a,b) working with the cereal leaf beetle, Oulema melanopus, found that 1% of the total lipid radioactivity was in the unsaturated hydrocarbons and 13% was in the saturated ones. After analyzing the hydrocarbons of several adult fly species, Louloudes g£_al. (1962) found that the odd numbered compounds predominated over the even numbered ones. The alkanes were the major fraction, but alkenes were also present. All of the species had considerable branched chain compounds. The major distribution range was C-23 to C-29, but the relative distribution varied between species. Beament (1955) found the cuticular "grease" of cockroaches consisted of a hard wax and a "solvent," and that a series of paraffins and alcohols extended into the short, C-8 - C-l2, lengths to provide the "solvent." In analyzing the cuticular wax of the mormon cricket, Anabrus simplex, Baker gt_al. (1960) found that 48—58% of the wax was hydrocarbons of which 27-32% were C—30 and 0—31. PPGL 56 acids made up 15-18% with the C-18 acids being the major components. Esters made up 9-1l%, cholesterol 2-3%, polymers 12—14%, and 2-4% was unidentified. It has been found that acetate—lac was utilized for fatty acid synthesis in many insects to a much greater degree than for hydrocarbons. Kennedy (1957) stated that acetate was used in the synthesis of fatty acids and phospholipids. This was in agreement with that found for the intact rat in which acetate-l-luC was incorporated into fatty acids (Hutchens gt_a1., 1954). Louloudes g£_al. V (1961) found that acetate-lac incorporation in the American cockroach male was 14-17 times greater in the fatty acid fraction than in the unsaponifiable lipids. The male utilized more acetate for fatty acids than did the female. Kodicek (1960) found 8.8% of the radioactivity from dietary acetate-2-luC in the fatty acids of blowfly larvae as compared to 1.0% in the unsaponifiable fraction. Robbins §£_a1. (1960) found 2.5 times the acetate in the fatty acids of female house flies than in the unsaponifiable fraction, and they found that the females had 3.7-8 times the fatty acid synthesis as did the males. Lamb and Monroe (1968a) studying the cereal leaf beetle and Zandee (1962) studying the crayfish found that there were about 2 times the radioactivity in the fatty acids than in the unsaponifi- able fractions after injections of acetate-luC. This is in agreement with results obtained in vertebrates (Zandee, 57 1962). Lamb and Monroe (l968b) found radioactivity in all the fatty acids of the triglyceride fraction. Of these acids oleic and palmitic acids had the most radioactivity while linolenic acid had very little. Barlow (1964) found that there were fatty acid dif- ferences among the 30 insect and 2 arachnid species studied. He found that aphids had a high concentration of myristic acid while Diptera had more palmitoleic acid and he con— cluded that species living in colder environments had more highly unsaturated fatty acids. Fast (1966) also found a high concentration of palmitoleic acid in the neutral lipids of most Diptera. Walling g£_a1, (1968) working with the two-spotted spider mite, Tetranychus urticae, found that linolenic acid was the most abundant in both the neutral and phospholipids. They found other major acids to be palmitic, palmitoleic, stearic, oleic, and linoleic; and the C-18 series comprised 84% of the total fatty acids studied. Lamb and Monroe (l968a) found 10 fatty acids in the cereal leaf beetle. The radioactivity was found to be 25% in palmitic acid, 60% in oleic acid, and only 0.7% in linolenic acid even though it comprised 26% of the total fatty acids gravimetrically. Zandee (1966d) found large amounts of palmitic and oleic acids in crayfish, and he reported that palmitic acid had a central place in that animal's metabolism. He also found that the fatty acid composition varied from season to season. 58 The ability of animals to biosynthesize sterols varies throughout the animal kingdom. Holz gt_a1. (1961) found that it was likely that cholesterol was the princi- pal sterol or that it was the key intermediate to the formation of ciliate sterol in Tetrahymena corlissi Th-X. Squalene, mevalonic acid, and lanosterol could not replace cholesterol in the diet while certain other sterols as cholestanol, 22-dehydrocholesterol, B-sitosterol, and stigmosterol could partially replace the cholesterol; so they concluded that the impairment of the synthesis was between lanosterol and the ciliate sterol. Fagerlund and Idler (1960) found that a mussel and a clam could convert labeled squalene into sterols, and they also found that a clam could incorporate unsaturations at C-22 and C-25 (Fagerlund and Idler, 1961). In a carnivorous mollusc, Buccinum undatum, Voogt (1967b) found that it was unable to synthesize 3B—sterols from acetate. Howes and Whellock (1937) found that the snail, Helix pomatia, seemed to require cholesterol; however Voogt (1967a; l968a,b) showed that archeogastropod and pulmonate snails can synthesize 3B-stero1s from squalene and acetate. Wootton and Wright (1960; 1962) showed that Lumbricus terrestris could con- vert mevalonic acid to squalene but that there was a genetic block in the pathway of squalene to B-hydroxy and/or 5 0H sterols. 59 Van den Oord (1964) showed that in the crab, Cancer pagurus, neither acetate—l—luC nor mevalonic acid-2-luC was incorporated into cholesterol. Zandee (1966c; 1967) demonstrated that acetate was not utilized as a cholesterol precursor in Astacus astacus, Homarus gammarus, Avicularia avicularia (arachnid), or Graphidostreptus tumuliporus (myriapod). He suggested that the inability to synthesize cholesterol from acetate seemed to be characteristic of all arthropods. Clayton (1964) outlined the functions of sterols in insects. He mentioned that intestinal symbionts supplied sterols to many insects, and that in the cockroach a dietary source of sterol was necessary even though there was a high content of symbionts. Pant and Fraenkel (1950; 1954) showed that symbiotic yeasts within two insects species supplied most of the sterols and B-vitamins. Monroe (1959; 1960) and Kaplanis g£_al. (1960) showed that cholesterol was required exogenously and that it was efficiently used for viable egg production in the house fly. ' Clayton g£_§1. (1962) showed that acetate—l—luC was incorporated into cholesterol in a silver fish, Ctenole- pisma sp., but suggested that it could have been due to the presence of symbionts. Kaplanis g£_§l.(l963) working with a primitive insect, the firebrat, indicated that the incorporation of acetate-l—luC was so low that the insect 60 was probably unable to synthesize sterols. Levinson (1960) suggested that phytophagous insects could convert plant sterols to cholesterol while obligatory carnivores could not. The American cockroach may have been able to cleave the sterol side chain and resynthesize the isooctyl side chain of cholesterol (Casida §t_al., 1957). Clark and Bloch (l959b) indicated that the roach converted ergosterol to 22-dehydrocholesterol. Because cholesterol was not found in detectable quantities, they suggested that micro- organisms supplied the insect with cholesterol. Louloudes 'g§_al. (1961) found low levels of radioactivity in the sterol digitonides of the American roach. Since they pre— cipitated the sterols from the total unsaponifiable lipids, they suggested that possibly there were hydrocarbons present that were insoluble in the solvents used for digitonide formation so it resulted in decreased activity after repre- cipitation of digitonides. Bloch et a1. (1956) found that the larvae of Dermestis vulpinus incorporated acetate-l—luC into squalene but not into lanosterol or cholesterol; therefore, they concluded that cholesterol biosynthesis was interrupted at the squalene stage. Clark and Block (l959a,c) found that cholesterol could not be replaced by mevalonic acid, 8 squalene, lanosterol, or A , 4,4-dimethyl cholestenol, which indicated that cholesterol biosynthesis was multiply 61 blocked. Ishii and Hirano (1961) found that cholesterol was essential in the diet of the rice stem borer, and Happ and Meinwald (1966) working with an ant found that labeled acetate was incorporated into citronellal and citral but not into the digitonide fractions. Robbins g£_al. (1960) and Kaplanis g£_§1. (1961) found that there was no sterol synthesis in the house fly and that a sterol was required in the diet. Levinson and Bergmann (1957) stated that a sterol was the only lipid required by Musca vicina for growth and metamorphosis. Agarwal §§_al. (1961) found "mucasterol" in house flies and that CSMA reared house flies contained at least 4 sterols but lacked cholesterol. Thompson e£_§l.(1962) later identified this "house fly sterol" as campesterol. They found it originated from the CSMA medium and that the house fly showed a selective uptake of sterols that had the closest side chain to cholesterol. Kodicek and Levinson (1960) found that blowfly larvae were unable to synthesize sterols from acetate and that the cholesterol side chain was not formed by the re-synthesis of acetate. Sedee (1961) also found that blowfly larvae were unable to use squalene in place of cholesterol in their diet. REFERENCES Agarwal, H. C., J. E. Casida, and S. D. Beck. 1961. An unusual sterol from house flies. J. Insect. Physiol. 7:32—45. Baker, G., J. H. Pepper, L. H. Johnson, and E. Hastings. 1960. Estimation of the composition of the cuticular wax of the mormon cricket, Anabrus simplex. Hald. J. Insect Physiol. 5:47-60. Barlow, J. S. 1964. Fatty acids in some insect and spider fats. Canad. J. Biochem. 42:1365-1374. Beament, J. W. L. 1955. Wax secretion in the cockroach. J. Exptl. Biol. 32:514-538. Bloch, K., R. G. Langdon, A. J. Clark, and G. Fraenkel. 1956. Impaired steroid biogenesis in insect larvae. Biochem. Biophys. Acta. 21:176. Casida, J. E., S. D. Beck, and M. J. Cole. 1957. Sterol metabolism in the American cockroach. J. Biol. Chem. 224:365-371. Clark, A. J. and K. Bloch. 1959a. The absence of sterol synthesis in insects. J. Biol. Chem. 234:2578—2582. Clark, A. J. and K. Bloch., 1959b. Conversion of ergo- sterol to 22-dehydrocholesterol in Blattella germanica. J. Biol. Chem. 234:2589-2594. Clark, A. J. and K. Bloch. 1959c. Function of sterols in Dermestis vulpinus. J. Biol. Chem. 234:2583-2588. Clayton, R. B. 1964. The utilization of sterols by insects. J. Lipid Res. 5:3-19. Clayton, R. B., A. M. Edwards, and K. Bloch. 1962. Bio- synthesis of cholesterol in an insect, Silverfish (Ctenolepisma sp.). Nature 195:1125-1126. Fagerlund, U. H. M. and D. R. Idler, 1960. Marine sterols. VI. Sterol biosynthesis in molluscs and echinoderms. Canad. J. Biochem. Physiol. 38:997-1002. 62 63 Fagerlund, U. H. M. and D. R. Idler. 1961. Marine sterols. VIII. In vivo transformations of the sterol side chain by a clam. Canad. J. Biochem. Physiol. 39:505-509. Fast, P. G. 1966. A comparative study of the phospholipids and fatty acids of some insects. Lipids 1:209-215. Happ, G. M. and J. Meinwald. 1966. Biosynthesis of mono- terpenes in the ant (Acanthomyops claviger). Advan. Chem. Ser. 53:27-33. Holz, G. G., Jr., B. Wagner, and J. Erwin. 1961. Sterol requirements of the ciliate Tetrahymena corlissi Th-X. I. A nutritional analysis of the sterol requirements of T. corlissi Th-X. Comp. Biochem. Physiol. 2:202-213. ' Hutchens, T. T., J. T. VanBruggen, and E. S. West. 1954. Fatty acid and cholesterol synthesis rates in the intact rat. Archs. Biochem. Biophys. 52:261-268. Ishii, S. and C. Hirano. 1961. Absence of cholesterol biosynthesis in the rice stem borer, Chilo suppres- salis Walker. Botyi-Kagaku 26:71—74. Kaplanis, J. N., R. C. Dutky, and W. E. Robbins. 1961. The incorporation of 2-luC-mevalonate into house fly lipids. Ann. Entomol. Soc. Amer. 54:114-116. Kaplanis, J. N., W. E. Robbins, and L. A. Taylor. 1960. The utilization and metabolism of 4-Clu-cholesterol by the adult house fly. Ann. Entomol. Soc. Amer. 53:260-264. Kaplanis, J. N., W. E. Robbins, H. E. Vroman, and B. M. Bryce. 1963. The absence of cholesterol biosynthesis in a primitive insect — the firebrat, Thermobia domestica (Packard). Steroids 2:547-550. Kennedy, E. P. 1957. Metabolism of lipides. Ann. Rev. Biochem. 26:119-148. Kodicek, E. and Z. H. Levinson. 1960. Metabolism of B—sitosterol and other lipids in the presence of acetate-2-luC by blowfly larvae. Nature 188:1023- 1024. 64 Lamb, N. J. and R. E. Monroe. 1968a. Lipid synthesis from acetate-l-Clu by the cereal leaf beetle, Oulema melanopus. Ann. Entomol. Soc. Amer. 61:1164-1166. Lamb, N. J. and R. E. Monroe. 1968b. Studies of complex lipids synthesized from acetate-l-Clu by the cereal leaf beetle, Oulema melanopus. Ann. Entomol. Soc. Amer. 61:1167—1169. Lambremont, E. N. and C. I. Stein. 1965. C11402 production in the boll weevil, Anthonomusggrandis, after injec- tion of Clu-l-acetate. Ann. Entomol. Soc. Amer. 58:765-766. Levinson, Z. H. 1960. The function of dietary sterols in phytophagous insects. XI Int. Kongr. F. Entom. Wien 1960, Ver. H. B. III. (Verlog lst. Ent. Univ. Pavia, 1960):145-153. Levinson, Z. H. and E. D. Bergmann. 1957. Steroid utili- zation and fatty acid synthesis by the larva of the house fly, Musca vicina Macq. Biochem. J. 65:254- 260. ' . Louloudes, S. J., D. L. Chambers, D. B. Moyer, and J. H. Starkey III. 1962. The hydrocarbons of adult house flies. Ann. Entomol. Soc. Amer. 55:442-448. Louloudes, S. J., J. N. Kaplanis, W. E. Robbins, and R. E. Monroe. 1961. Lipogenesis from Clu-acetate by the American cockroach. Ann. Entomol. Soc. Amer. 54:99- 103. Monroe, R. E. 1959. Role of cholesterol in house fly reproduction. Nature 184:1513. Monroe, R. E. 1960. Effect of dietary cholesterol on house fly reproduction. Ann. Entomol. Soc. Amer. 53:821- 82“. ‘ Pant, N. C. and G. Fraenkel. 1950. The function of the symbiotic yeasts of two insect species, Lasioderma serricorne F. and Stegobium (Sitodrepa) paniceum L. Science 112:498-500. ' Pant, N. C. and G. Fraenkel. 1954. Studies on the symbio- tic yeasts of two insect species, Lasioderma serri- corne F. and Stegobium paniceum L. Biol. Bull. 107-420-431. 65 Robbins, W. E., J. N. Kaplanis, S. J. Louloudes, and R. E. Monroe. 1960. Utilization of 1-Clu-acetate in lipid synthesis by adult house flies. Ann. Entomol. Soc. Amer. 53:128-129. Sedee, J. W. 1961. Intermediary metabolism in aseptically reared blowfly larvae, Calliphora erythrocephala (Meig.). I. Biosynthesis of squalene and cholesterol. Archs. Int. Physiol. Biochim. 69:284-294. Thompson, M. J., S. J. Louloudes, W. E. Robbins, J. A. Waters, J. A. Steele, and E. Mosettig. 1962. Identity of the "house fly sterol." Biochem. Biophys. Res. Com. 9:113-119. Van den Oord, A. 1964. The absence of cholesterol syn- thesis in the crab, Cancer pagurus L. Comp. Biochem. Physiol. 13:461-467. Voogt, P. A. 1967a. Biosynthesis of 3B-sterols in a snail, Arion rufus L., from l—luC-acetate. Archs. Int. Physiol. Biochim. 75:492-500. Voogt, P. A. 1967b. Investigations on the capacity of synthesizing BB-sterols in Mollusca. I. Absence of 3B-sterol synthesis in a whelk, Buccinum undatum L. .Archs. Int. Physiol. Biochim. 75?809-815. Voogt, P. A. 1968a. Investigations of the capacity of, synthesizing 3B-stero1s in Mollusca. II. Study on the biosynthesis of 3B-sterols in some representatives of the order Basommatophora. Comp. Biochem. Physiol. 25:943-948. Voogt, P. A. 1968b. Investigations of the capacity of synthesizing 3B-sterols in Mollusca. III. The bio- synthesis of 3B-sterols in some archeogastropods. Archs. Int. Physiol. Biochim. 76:721-730. Walling, M. U., D. C. White, and J. G. Rodriguez. 1968. Characterization, distribution, catabolism, and synthesis of the fatty acids of the two-spotted spider mite, Tetranychus urticae. J. Insect Physiol. 14:1445-1458. Wootton, J. M. and L. D. Wright. 1960. Biosynthesis of squalene by the annelid, Lumbricus terrestris. Nature 187:1027-1028. Zandee, D. I. 1962. Lipid metabolism in Astacus astacus (L). Nature 195:814-815. 66 Zandee, D. I. l966a. Metabolism of the crayfish Astacus astacus (L). I. Biosynthesis of amino acids. Archs. Int. Physiol. Biochim. 74:35-44. Zandee, D. I. l966b. Metabolism in the crayfish Astacus astacus(L). II. The energy-yielding metabolism. Archs. Int. Physiol. Biochim. 74:45-57. Zandee, D. I. l966c. Metabolism in the crayfish Astacus astacus (L). III. Absence of cholesterol synthesis. Archs. Int. Physiol. Biochim. 74:435-441. Zandee, D. I. l966d. Metabolism in the crayfish Astacus astacus (L). IV. The fatty acid composition and the biosynthesis of the fatty acids. Archs. Int. Physiol. Biochim. 74:614—626. Zandee, D. I. 1967. Absence of cholesterol synthesis as contrasted with the presence of fatty acid synthesis in some arthropods. Comp. Biochem. Physiol. 20:811- 822. ~ . A ”71111111139111[[Efliijfflfltiifllulfllflll'fi