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Vitamin B12 levels in selected insects
Insect Biochem. Vol. 14, No. 2, pp. 175-179, 1984 Printed in Great Britain.All rightsreserved
0020-1790/84$3.00+ 0.00 Copyright © 1984PergamonPress Ltd
VITAMIN B12 LEVELS IN SELECTED INSECTS EDGAR J. WAKAYAMA,*t JACK W. DILLWITH,*§ RALPH W. HOWARD~ and GARY J. BLOMQUIST*II *Departments of Biochemistry and tMedical Technology, University of Nevada, Reno, NV 89557 and .+Southern Forest Experiment Station, Forest Service, U.S. Department of Agriculture, Gulfport, MS 39503, U.S.A. (Received 3 May 1983) Abstract--Vitamin B~2 concentrations were determined by radioassay in the housefly, five species of termites, and 17 other phylogeneticallydiverse insect species. Vitamin B~2was not detected in the housefly Mtisca domestica, which apparently cannot interconvert propionate and succinate. In contrast, the termite Zootermopsis angusticollis readily interconverts succinate and methylmalonate, and contains high amounts of vitamin B~2(940 pg/mg dry tissue), as do four other species of termites. Experiments involving selective elimination of either gut tract protozoa or bacteria in Coptotermes formosanus indicate that intestinal bacteria are the major source of vitamin B~2 in this termite. The other insect species examined have undetectable to moderate amounts of vitamin B~z. Key Word Index: Vitamin B~2, termites, cobalt, Musca domestica
INTRODUCTION
proach the question of natural levels of vitamin B~2 in insects. This report presents our analyses of housefly and termite tissues for vitamin Bt2, and our attempt to correlate the levels found with the biochemistry of these insects. We also report the levels of vitamin B~2 in 17 other insect species representing seven phylogenetically diverse orders.
A requirement for vitamin B~2 in insects is not well documented. Most of the evidence comes from classical dietary deletion experiments which are difficult to perform and interpret. The literature suggests that vitamin B~2 is required as a dietary supplement for some insects, but that it is not required for others (House, 1974; Dadd, 1977). MATERIALS AND METHODS Studies on the nutritional requirements of insect cells cultured in vitro have shown that some may Insects utilize vitamin B~2. For example, fibroblastic cells Insects were obtained from Biology Section, S. C. Johnfrom Periplaneta americana require vitamin B12 for son and Son, Racine, Wisconsin: Blatta orientalis (L.), normal growth (Landureau and Steinbuch, 1969). Periplaneta americana (L.), Lepisma saccharina (L.), ,4tBecker (1975) found that uridine, in the presence of tagenus megatoma (F.), Sitophilus oryzae (L.), Tribolium vitamin Bt2, restored growth in cultured Drosophila castaneum (Herbst), Tineola bisseliella (Hummel), Plodia melanogaster cells whose growth was inhibited by interpunctella (Hiibner) and Musca domestica (L.). Flucker's Cricket Farm, Baton Rouge, Louisiana: Acheta domesticus added adenosine. He suggested that vitamin B~2 (L.). Carolina Biological Supply Co., Burlingame, North functions as a coenzyme for vitamin B~2 dependent Carolina: Sareophaga bullata (Parker), Tenebrio sp. and ribonucleotide reductase. Manduca sexta (Johannson). Uniform Services: Glossina m. The housefly Musca domestica directly converts morsitans (Westwood). Musca domestica were maintained as propionate to acetate by a pathway which does not described by Dillwith et al. (1982) and the other insects were involve conversion to methylmalonyl-CoA and used immediately upon arrival. Colonies maintained at succinyl-CoA (Diliwith et al., 1982). The termite UNR: ,4pis mellifera (L.) (obtained locally), Acyrthosiphon Zootermopsis angusticollis, however, readily converts pisum (Harris) (reared as described by Ryan et al., 1982), succinate to methytmalonate (Chu and Blomquist, Trichoplusia ni (Hiibner) (cultured as described by de Renobales and Blomquist, 1983) and Drosophila melanogaster 1980; Blomquist et al., 1980), suggesting that these (Meigen) (Oregon R wild strain) (cultured on Formula 4-24 two species may differ in their requirements for, or Drosophila food, Carolina Biological Supply Company). their ability to utilize, vitamin B~2. Coptotermes formosanus (Shiraki) was collected from The availability of a sensitive and specific assay for standing Cypress snags in lake Charles, Louisiana. Navitamin B~2 offers the opportunity to directly ap- sutitermes corniger (Motschulsky) was collected on Barro Colorado Island, Republic of Panama. Reticulitermes §Present address: Department of Entomology, University of flavipes (Kollar) and R. virginicus (Banks) were collected from logs on the Harrison Experimental Forest about 30 km Missouri, Columbia, MO 65211, U.S.A. north of Gulfport, Mississippi. Zootermopsis angusticollis IIPlease address correspondence to: Gary J. Blomquist, (Hagen) was obtained from logs in the vicinity of Chico, Department of Biochemistry, University of Nevada, California. Reno, NV 89557, U.S.A. Mention of company or trade source is solely for Defaunation experiments identification of material used and does not imply Groups of approx. 200 workers of C. formosanus were endorsement by the U.S. Department of Agriculture. used in six treatment combinations: (1) whole termites, 175
EDGAR J. WAKAYAMA et al.
176
normally faunated, containing all normal protozoa, bacteria and spirochaetes; (2) whole termites minus the hindgut containing the protozoa, bacteria and spirochaetes; (3) hindguts from group 2 containing the protozoa, bacteria and spirochaetes; (4) whole termites, lacking hindgut protozoa, but containing hindgut bacteria and spirochaetes; (5) whole termites lacking hindgut bacteria and spirochaetes, but containing hindgut protozoa; and (6) whole termites lacking all hindgut micro-organisms. All treatments were prepared using the methods of Mauldin et al. (1972, 1978).
Extraction of vitamin B~2 Between 200 and 1500 (100-700mg dry weight) of the smaller insects (aphids, termites, etc.) and 6 and 100 (300-4000 mg dry weight) of the large insects (cockroaches, tobacco hornworm, etc.) were used in each experiment. Insects were dried in an oven at 70°C for 12hr and then weighed. Homogenation was performed in a minimal volume of borate-KCl buffer (pH 9.4) containing 0.3 mM KCN. Samples were then centrifuged at 2000g for 10 min to remove cell debris and the supernatant was stored at -20°C in the dark until assayed.
Radioassay Standard vitamin BI2 radioassay kits supplied with [57Co]cyanocobalamin, borate-KCI-KCN buffer, intrinsic factor, dithiothreitol and vitamin B~2 standards were obtained from Diagnostic Products Corp., Los Angeles, California. This radioassay is a modification of the methods of Liu and Sullivan (1972) and Mollin et al. (1976). It involves mixing 200 #1 of samples or vitamin Bu standards with 1 ml of borate (0.1 M)-KCI (0.1 M) buffer (pH 9.4) containing 0.3 mM KCN, approx. 10.2 nCi of [57Co]cyanocobalamin, and 1.0 mg of dithiothreitol. The tubes capped with loose-fitting caps were placed in a covered boiling water bath (100°C) for 1 hr to denature proteins and to release and convert vitamin B~2to the stable cyanocobalamin derivatives. After cooling the tubes in an ambient water bath for 5 to 10min, 100#1 of "purified" intrinsic factor (IF) was added and incubated at ambient temperature for 2 hr during which [SVCo]cyanocobalamin and the sample Bu derivatives compete for IF binding. After equilibrium was reached, dextran-coated charcoal (400/~1) was added to each tube and incubated for 10 rain before separating the free (unbound) vitamin Biz from the bound (IF-B~2 complex) by adsorption on the dextran-coated charcoal. Separation of the free vitamin B~2 derivative from the bound was performed by centrifuging at 3000g for 10 min in a refrigerated (4°C) centrifuge. The supernatant containing the IF-B~2 complex was completely transferred into a tube for counting. The radioactivity (cpm) of the IF-B,2 complex was determined in a Beckman gamma scintillation counter.
Cobalt Cobalt concentrations in insects were determined using an IL 251 Atomic Absorption Spectrophotometer with flameless Atomizer IL 555, Instrumentation Laboratory, Inc., Wilmington, Massachusetts. The supernatant of homogenized insects in borate-KCl-KCN buffer, as described in extraction of vitamin B~z, was further diluted to 1:100 with the borate-KC1 buffer and aspirated into the flameless atomizer. The standard curve was obtained by analyzing 0-25 ng/ml cobalt chloride solutions, and unknowns determined from this curve. RESULTS An analysis of the vitamin B~2 content of adult mixed sex and larval houseflies showed no detectable amounts (Table 1). Both the larval growth media (1067pg/mg) and the adult diet (1.5pg/mg) contained vitamin B~2 suggesting that the absence of
vitamin B,2 in this insect was not due to a dietary deficiency. Consistent with the absence of detectable amounts of vitamin B12 in the housefly was the absence of detectable amounts of cobalt. In contrast to the housefly, five species of termites representing three diverse families contain large amounts of vitamin B~2 (Table 1). Values range from 258 + 2 6 p g / i n s e c t for C. formosanus to 9703 _ 372 pg/insect for Z. angusticollis (Table 1). When converted to a dry weight basis, the values range from 4 5 5 p g / m g in C. formosanus to 3211 pg/mg in N. corniger (Table 1). All of these termites also had relatively high cobalt levels: 13 _ 2 ng/insect (mean _ SD, n = 3) in N. corniger, 74 ___ 1 ng/insect in C. formosanus, 1500 ___200 ng/ insect in R. flavipes and R. virginicus, and 25,600 + 3300 ng/insect in Z. angusticollis. A comparison of vitamin B~2 levels in gut tissue versus all other tissues in both Z. angusticollis and C. formosanus showed that gut tissue contained the majority of the vitamin B12 (Table 2). This indicated that the bacteria, spirochaetes and/or protozoa in the gut tract were the most likely source of the vitamin. To determine which was the source, groups of C. formosanus were treated to remove either bacteria plus spirochaetes, protozoa or all three. Elimination of bacteria and spirochaetes reduced vitamin B,2 levels from 258 ___26 pg/insect to 87 + 3 pg/insect within 10 days after streptomycin treatment was initiated. This value is in the same range as that of termites whose hindgut had been removed (64 ___ 16 pg/insect). In contrast, elimination of protozoa by CO2 and 02 under pressure did not significantly affect vitamin Bt2 levels (Table 2), suggesting that the protozoa do not contribute to vitamin BI2 production. When all micro-organisms are eliminated from the gut, vitamin BI2 values decrease to 68 _ 3 pg/insect. The residual values of vitamin Bt2 that remain after the removal of bacteria are similar to those present in control insects without gut tracts. This suggests that the amounts of vitamin B~2 in streptomycin treated insects reflect vitamin B~2 present in insect tissues, and that this did not decrease significantly in the 10-day treatment period. Termites contain anaerobic bacteria in their hindgut (Breznak, 1982) and based on the above data, these are the most likely site of vitamin B~2 production. The marked differences in vitamin B~2 concentrations found in houseflies and termites prompted us to examine insects from several phylogenetically diverse orders to determine if any trends in the presence or absence of vitamin Bu were apparent. In general, all taxa, except termites, contain considerably lower vitamin B~2 levels than is usually observed in vertebrates. M o s t of the data in Table 1 were obtained from whole insects, and it is possible that certain tissues, particularly gut tissue, may contain higher concentrations. The housefly and fruitfly D. melanogaster (Table 1) have undetectable levels of vitamin B~2, but other Diptera, including the blowfly S. bullata and the Tse-tse fly, G. m. morsitans, contain low but measurable amounts of vitamin B12 (Table 1). Analysis of the Drosophila diet (formula 4-24, Carolina Biological, Burlingame, North Carolina) showed no detectable vitamin B~2.
Vitamin B~2 in insects
177
Table 1. Concentrations of vitamin B~2 in selected insects
Species
Vitamin B,2 pg/insect pg/mg't (mean + SD, n = 3)
Life stage*
Diptera Musca domestica
a
1
ND ND 225 ± 11 66 ± 6.9 ND ND
ND~ ND 23.4 ± 1.1 1.5 .+ 0.2 ND ND
m m m m m
9703 ± 372 258 + 26 281 ± 5 721 _+59 1455 ± 95
940 + 35 455 __.48 563 ± 9 2131 ± 177 3211 -+ 203
a a a a I
6.4 ± 1.4 7.4 ± 0.4 ND ND 37.+4
2.8 ± 0.6 3.8 .+ 0.2 ND ND 0.5+0.1
Homoptera Acyrthosiphon pisum
1
ND
ND
Hymenoptera Apis mellifera
a
ND
ND
Lepidoptera Manduca sexta Trichoplusia ni
1 a
p 1 a 1 a 1
283 + 60 ND ND 25±6 13±0.9 28+4 2.5+0.4 15.+4.2
0.3 ± 0.1 ND ND 0.3_+0.1 7.0+0.5 7.7± hi 1.1 _+0.2 3.5±0.9
Orthoptera Periplaneta americana Blatta orientalis Acheta domesticus
a a a
390 ± 21 478 ± 84 ND
4.6 ± 0.1 0.4 ___0.1 ND
Thysanura Lepisma saccharina
a
53 + 6.4
17 ± 2.1
1
Gloss±ha m. morsitans Sarcophaga bullata Drosophila melanogaster
a p a
Isoptera Zootermopsis angusticollis Coptotermes formosanus Reticulitermes flavipes Reticulitermes virginicus Nasutitermes corn±get Coleoptera
Attagenus megatoma Tribolium castaneum Sitophilus oryzae Tenebrio species
Plodia interpunctella Tineola bisseliella
*a--adults, mixed ages and mixed sex; l--larvae; p~pupae; m--mixed castes. "['pg/mgdry weight of tissue. :[:NI)---not detectable; sensitivity = 0.04 pg/mg. T w o o f the Coleoptera c o n t a i n e d significant levels o f v i t a m i n B n , w h e r e a s S. oryzae h a d u n d e t e c t a b l e a m o u n t s ( T a b l e 1). O n e species o f Orthoptera (the cricket A. domesticus) h a d u n d e t e c t a b l e levels o f v i t a m i n B~2 while t w o c o c k r o a c h e s (P. americana a n d B. oriental±s) h a d m e a s u r a b l e a m o u n t s o f vitam i n B,2. L a r g e a m o u n t s o f v i t a m i n B,2 were p r e s e n t
in the h i n d g u t o f P. americana ( T a b l e 2) s u g g e s t i n g t h a t a n a e r o b i c b a c t e r i a ( B r e z n a k , 1982) in the h i n d gut are the s o u r c e o f the v i t a m i n B,2. N e i t h e r the p e a a p h i d , A . p i s u m ( H o m o p t e r a ) n o r the h o n e y b e e , A. mellifera ( H y m e n o p t e r a ) c o n t a i n detectable a m o u n t s o f v i t a m i n B12 ( T a b l e 1). T h e p e a a p h i d w a s g r o w n o n b o t h alfalfa a n d f a v a b e a n
Table 2. Vitamin B,2 concentrations in whole insects, insects minus gut tissue, hindguts, and in selectively defaunated termites Species
Zootermopsis angusticollis Coptotermes formosanus
Periplaneta americana
Tissue Whole insects Insects minus gut tissue Gut tissue Whole insects Insects minus gut tissue Gut tissue Insects minus bacteria and spirochaetes~ Insects minus protozoa Insects minus bacteria, spirochaetes and protozoa Whole insects Insects minus hindgut Hindgut
Vitamin B~2* pg/insect 9703 + 372f 3824 _+250 6741 + 40 258 ± 26 64 + 16 197 _+ 10 87 ± 3 248 _ 23 68 + 3 390 + 21 130 + 10 230 __. 17
*pg/mg dry weight, tMean +SD, n = 3. ~Elimination of gut organisms is described in the text.
EDGAR J. WAKAYAMAel aL
178
plants, and, as expected, neither of these contain detectable amounts of vitamin Bt2. Within the Lepidoptera, both adult and larval forms of P. interpunctella and T. bisseliella contain significant amounts of vitamin B~2.Larvae of M. sexta also contained appreciable amounts of vitamin B~2. L. saceharina, a member of the Thysanura and one of the most primitive insects, contains appreciable levels of vitamin B~2 (Table 1). Differences in the vitamin Bt2 content of insects in different life stages were noted in two cases. Larvae of Trichoplusia ni and Tenebrio species contain appreciable amounts of vitamin Bt2 while adults of these species do not contain detectable levels. In addition, P. interpunctella larvae contain larger amounts of vitamin B~2 than does the adult insect. DISCUSSION Anaerobic bacteria utilize vitamin Bt2 as a cofactor in many enzymatic reactions, but higher animals have been shown to utilize only a few B12 requiring enzymes (Baker, 1972). The best studied of these enzymes is the methylmalonyl-CoA mutase which interconverts succinyl-CoA and methylmalonyl-CoA and is an important reaction in the metabolism of odd chain fatty acids and branched chain amino acids (Reitz, 1982). Vitamin B~2 requiring L-leucine-2,3-aminomutase, which catalyzes the conversion of L-leucine into fl-leucine, has been shown to be present in animal tissues and may function in the metabolism of branched chain amino acids (Baker and Stadtman, 1982). Vitamin B~2 has also been shown in animals to function in B~2-dependent methionine biosynthesis (Taylor, 1982). There is little evidence for the synthesis of vitamin B~2 in animals and higher plants (Wuest and Perlman, 1968), and in general, vertebrate animals require dietary vitamin B~2. Many aerobic and anaerobic micro-organisms can synthesize vitamin B,2, and they are a major source of this vitamin (Coates, 1968). The lack of vitamin B,2 in the housefly is consistent with the metabolism of propionate in this insect. Studies with propionate isotopically labelled in the 1, 2 or 3 position with carbon-13 indicated that propionate was directly converted to acetate and was not metabolized via methylmalonyl-CoA to succinylCoA. Carbon 3 of propionate became the carboxyl carbon of acetate and carbon 2 of propionate became the methyl carbon of acetate (Dillwith et aL, 1982). Thus, it appears that the housefly metabolizes propionate by a unique pathway (for animals). Plants, many of which do not contain appreciable amounts of vitamin B12, metabolize propionate by a similar pathway as that suggested for the housefly (Hatch and Stumpf, 1962; Giovanelli and Stumpf, 1958). The lack of vitamin B12 in the housefly along with its presence in the diet suggests that it is not utilized by this insect. The high concentration of vitamin B~2 in Z. angusticollis is consistent with its ability to readily convert succinate to the methylmalonyl derivative used in branched alkane synthesis (Chu and Blomquist, 1980; Blomquist et al., 1980). Isolated epidermal tissue from this insect is able to incorporate
[1,4J4C]succinate into branched alkanes (Chu and Blomquist, unpublished), suggesting that the termite tissue itself utilizes vitamin B12. The concentration of vitamin B~2 in these termites is in the same general range as that found in mammalian tissues. For example, the normal vitamin B~2 range for human liver is 800-3,000 pg/mg. Rat liver contains 60-300 pg/mg dry weight (Baker and Frank, 1968). In performing the assays of vitamin B12 isolated from C.formosanus, it was noted that the vitamin B~2 appeared to be in tightly bound complexes, which required longer heat treatment (1 hr at 100°C) to release the free cyanocobalamin. Some microorganisms have been shown to tightly bind vitamin B~2; both Lactobacillus bulgaricus and L. thermophilus in saline suspension show a cyanocobalamin-binding capacity which is reduced, although not completely destroyed, by heating at 90°C (Skeggs, 1969). Also, extracts of Ochromonas cells can preferentially bind cyanocobalamin (Ford et al., 1953). The analysis of the vitamin B~2 content of insects from several orders revealed no general trends within orders, between orders or between life stages of individual species. However, the results of our analysis are very consistent with the available literature on vitamin B~2 in insects. The lack of vitamin B,2 in the housefly is consistent with the reports that vitamin B~2 is not required for normal growth (Brookes and Fraenkel, 1958; House and Barlow, 1958). Also, the presence of vitamin B12 in T. castaneum is consistent with the reported dietary requirement for this vitamin (Armstrong, 1978). The presence of vitamin B,2 in P. americana is consistent with the observation that cells from this insect grown in culture require vitamin B~2 for normal growth (Landureau and Steinbuch, 1969). The absence of vitamin B~2 in D. melanogaster was somewhat surprising, in view of the observation that the addition of vitamin B,2 and uridine restored growth to cultured cells whose growth was inhibited by added adenosine (Becker, 1975). Our results suggest that under normal physiological conditions, vitamin B~2 does not play a significant role in this insect. This observation is supported by the finding that the normal rearing media for Drosophila contained no vitamin Bt2. Some general statements can be made regarding the presence of vitamin B~: in insects: (1) the occurrence of vitamin B~2 correlates with the observed metabolism of propionate and succinate in the housefly and a termite; (2) termites contain large amounts of vitamin B~2, apparently synthesized by their symbiotic gut bacteria; (3) no general trends are apparent in the presence or absence of vitamin B~2 within members of the orders Coleoptera, Diptera, Lepidoptera and Orthoptera which were examined; (4) the apparent lack of vitamin B~2 in some species poses a number of intriguing questions including how these organisms perform metabolic reactions which in many animals require vitamin Bt2 as a cofactor. REFERENCES
Armstrong E. (1978) The effects of vitamin deficiencies on the growth and mortality of Tribolium castaneum infected with Noserna whitei. J. Invert. Path. 31, 303-306.
Vitamin B~2 in insects Baker H. A. (1972) Corrinoid-dependent enzymic reactions. A. Rev. Biochem. 41, 55-90. Baker H. A. and Frank O. (1968) Vitamin Bl2. In Clinical Vitaminology: Methods and Interpretation, p. 136. Wiley, New York. Baker J. J. and Stadtman T. C. (1982) Amino mutases. In B12, Vol. 2, Biochemistry and Medicine (Edited by Dolphin D.), pp. 203-232. Wiley, New York. Becker J. L. (1975) Role of vitamin Bi2 in the reduction of ribonucleotides into deoxyribonucleotides in Drosophila cells grown in vitro. Biochimie 58, 427-430. Blomquist G. J., Chu A. J., Nelson J. H. and Pomonis J. G. (1980) Incorporation of [2,3J3C]succinate into methylbranched alkanes in a termite. Archs Biochem. Biophys. 204, 648-650. Breznak J. A. (1982) Intestinal microbiota of termites and other xylophagous insects. A. Rev. Microbiol. 36, 323-343. Brookes V. J. and Fraenkel G. (1958) The nutrition of the larvae of the housefly Musca domestica L. Physiol. Zool. 31, 208-223. Chu A. J. and Blomquist G. J. (1980) Biosynthesis of hydrocarbons in insects: succinate is a precursor of the methyl branched alkanes. Archs Biochem. Biophys. 201, 304-312. Coates M. E. (1968). Vitamin B12: deficiency effects in animals. In The Vitamins (Edited by Sebrell W. H., Jr and Harris R. S.), pp. 212-220. Academic Press, New York. Dadd R. H. (1977) Qualitative requirements and utilization of nutrients: insects. In CRC Handbook Series in Nutrition and Food (Editor-in-chief, Rechcigl M., Jr), Section D: Nutritional Requirements, Vol. 1, Comp. and Qualitative Requirements. de Renobales M. and Blomquist G. J. (1983). A developmental study of the composition and biosynthesis of the cuticular hydrocarbons of Trichoplusia ni. Insect Biochem. 13, 493-502. Dillwith J. W., Nelson J. H., Pomonis J. G., Nelson D. R. and Blomquist G. J. (1982) A 13C NMR study of methylbranched hydrocarbon biosynthesis in the housefly. J. biol. Chem. 257, 11305-11314. Ford J. E., Holdsworth E. S., Kon S. K. and Porter J. W. G. (1953) Occurrence of the various vitamin B~2 active compounds. Nature 171, 150-151. Giovanelli J. and Stumpf P. K. (1958) Fat metabolism in plants. X. Modified fl-oxidation of propionate by peanut mitochondria. J. biol. Chem. 231, 411-426. Hatch M. D. and Stumpf P. K. (1962) Fat metabolism in
179
higher plants. XVIII. Propionate metabolism by plant tissues. Archs Biochem. Biophys. 96, 193-198. House H. L. (1974) Nutrition. In The Physiology oflnsecta (Edited by Rockstein M.), Vol. V, pp. 1~52. Academic Press, New York. House H. L. and Barlow J. S. (1958) Vitamin requirements of the housefly Musca domestica L. (Diptera: Muscidae). A. Ent. Soc. Am. 51, 299-302. Kubasik N. P., Ricotta M. and Sine H. E. (1980) Commercially-supplied binders for plasma cobalamin (Vitamin B~2), analysis-purified intrinsic factor, "cobinamide"-blocked R-protein binder, and non-purified intrinsic factor-R-protein binder compared to microbiological assay. Clin. Chem. 26, 598-600. Landureau J. C. and Steinbuch M. (1969) Cyanocobalamine as a support of the in vitro cell growth-promoting activity of serum proteins. Experientia 25, 1078-1079. Liu Y. K. and Sullivan L. W. (1972) An improved radioisotope dilution assay for serum vitamin B~2 using hemoglobin-coated charcoal. Blood 39, 426-429. Mauldin J. K., Rich N. M. and Cook D. W. (1978) Amino acid synthesis from ~4C-acetate by normally and abnormally faunated termites, Coptotermes formosanus. Insect Biochem. 8, 105-109. Mauldin J. D., Smythe R. V. and Baxter C. C. (1972) Cellulose catabolism and lipid synthesis by the subterranean termite, Coptotermes formosanus. Insect Biochem. 2, 209-217. Mollin D. L., Anderson B. C. and Burman J. F. (1976) The serum vitamin B~2 level: Its assay and significance. Clin. Haemat. 3, 521-546. Reitz J. (1982) Methylmalonyl-CoA mutase. In Bl2 Vol. 2 Biochemistry and Medicine (Edited by Dolphin D.), pp. 357-379. Wiley, New York. Ryan R. O., de Renobales M., Dillwith J. W., Heisler C. R. and Blomquist G. J. (1982) Biosynthesis of myristate in an aphid: Involvement of a specific acylthioesterase. Archs Biochem. Biophys. 213, 26-36. Skeggs H. R. (1969) Vitamin Bl~. In The Vitamins (Edited by Gyorgy P. and Pearson W. N.), Vol. VII, 2nd edn, pp. 277-302. Academic Press, New York. Taylor R. T. (1982) Bt2-dependent methionine biosynthesis. In Bt2 Vol. 2 Biochemistry and Medicine (Edited by Dolphin D.), pp. 37-355. Wiley, New York. Wuest H. M. and Perlman D. (1968) Vitamin Bl2: Industrial preparations. In The Vitamins (Edited by Sebrell W. H., Jr and Harris R. S.), pp. 139-144. Academic Press, New York.
0020-1790/84$3.00+ 0.00 Copyright © 1984PergamonPress Ltd
VITAMIN B12 LEVELS IN SELECTED INSECTS EDGAR J. WAKAYAMA,*t JACK W. DILLWITH,*§ RALPH W. HOWARD~ and GARY J. BLOMQUIST*II *Departments of Biochemistry and tMedical Technology, University of Nevada, Reno, NV 89557 and .+Southern Forest Experiment Station, Forest Service, U.S. Department of Agriculture, Gulfport, MS 39503, U.S.A. (Received 3 May 1983) Abstract--Vitamin B~2 concentrations were determined by radioassay in the housefly, five species of termites, and 17 other phylogeneticallydiverse insect species. Vitamin B~2was not detected in the housefly Mtisca domestica, which apparently cannot interconvert propionate and succinate. In contrast, the termite Zootermopsis angusticollis readily interconverts succinate and methylmalonate, and contains high amounts of vitamin B~2(940 pg/mg dry tissue), as do four other species of termites. Experiments involving selective elimination of either gut tract protozoa or bacteria in Coptotermes formosanus indicate that intestinal bacteria are the major source of vitamin B~2 in this termite. The other insect species examined have undetectable to moderate amounts of vitamin B~z. Key Word Index: Vitamin B~2, termites, cobalt, Musca domestica
INTRODUCTION
proach the question of natural levels of vitamin B~2 in insects. This report presents our analyses of housefly and termite tissues for vitamin Bt2, and our attempt to correlate the levels found with the biochemistry of these insects. We also report the levels of vitamin B~2 in 17 other insect species representing seven phylogenetically diverse orders.
A requirement for vitamin B~2 in insects is not well documented. Most of the evidence comes from classical dietary deletion experiments which are difficult to perform and interpret. The literature suggests that vitamin B~2 is required as a dietary supplement for some insects, but that it is not required for others (House, 1974; Dadd, 1977). MATERIALS AND METHODS Studies on the nutritional requirements of insect cells cultured in vitro have shown that some may Insects utilize vitamin B~2. For example, fibroblastic cells Insects were obtained from Biology Section, S. C. Johnfrom Periplaneta americana require vitamin B12 for son and Son, Racine, Wisconsin: Blatta orientalis (L.), normal growth (Landureau and Steinbuch, 1969). Periplaneta americana (L.), Lepisma saccharina (L.), ,4tBecker (1975) found that uridine, in the presence of tagenus megatoma (F.), Sitophilus oryzae (L.), Tribolium vitamin Bt2, restored growth in cultured Drosophila castaneum (Herbst), Tineola bisseliella (Hummel), Plodia melanogaster cells whose growth was inhibited by interpunctella (Hiibner) and Musca domestica (L.). Flucker's Cricket Farm, Baton Rouge, Louisiana: Acheta domesticus added adenosine. He suggested that vitamin B~2 (L.). Carolina Biological Supply Co., Burlingame, North functions as a coenzyme for vitamin B~2 dependent Carolina: Sareophaga bullata (Parker), Tenebrio sp. and ribonucleotide reductase. Manduca sexta (Johannson). Uniform Services: Glossina m. The housefly Musca domestica directly converts morsitans (Westwood). Musca domestica were maintained as propionate to acetate by a pathway which does not described by Dillwith et al. (1982) and the other insects were involve conversion to methylmalonyl-CoA and used immediately upon arrival. Colonies maintained at succinyl-CoA (Diliwith et al., 1982). The termite UNR: ,4pis mellifera (L.) (obtained locally), Acyrthosiphon Zootermopsis angusticollis, however, readily converts pisum (Harris) (reared as described by Ryan et al., 1982), succinate to methytmalonate (Chu and Blomquist, Trichoplusia ni (Hiibner) (cultured as described by de Renobales and Blomquist, 1983) and Drosophila melanogaster 1980; Blomquist et al., 1980), suggesting that these (Meigen) (Oregon R wild strain) (cultured on Formula 4-24 two species may differ in their requirements for, or Drosophila food, Carolina Biological Supply Company). their ability to utilize, vitamin B~2. Coptotermes formosanus (Shiraki) was collected from The availability of a sensitive and specific assay for standing Cypress snags in lake Charles, Louisiana. Navitamin B~2 offers the opportunity to directly ap- sutitermes corniger (Motschulsky) was collected on Barro Colorado Island, Republic of Panama. Reticulitermes §Present address: Department of Entomology, University of flavipes (Kollar) and R. virginicus (Banks) were collected from logs on the Harrison Experimental Forest about 30 km Missouri, Columbia, MO 65211, U.S.A. north of Gulfport, Mississippi. Zootermopsis angusticollis IIPlease address correspondence to: Gary J. Blomquist, (Hagen) was obtained from logs in the vicinity of Chico, Department of Biochemistry, University of Nevada, California. Reno, NV 89557, U.S.A. Mention of company or trade source is solely for Defaunation experiments identification of material used and does not imply Groups of approx. 200 workers of C. formosanus were endorsement by the U.S. Department of Agriculture. used in six treatment combinations: (1) whole termites, 175
EDGAR J. WAKAYAMA et al.
176
normally faunated, containing all normal protozoa, bacteria and spirochaetes; (2) whole termites minus the hindgut containing the protozoa, bacteria and spirochaetes; (3) hindguts from group 2 containing the protozoa, bacteria and spirochaetes; (4) whole termites, lacking hindgut protozoa, but containing hindgut bacteria and spirochaetes; (5) whole termites lacking hindgut bacteria and spirochaetes, but containing hindgut protozoa; and (6) whole termites lacking all hindgut micro-organisms. All treatments were prepared using the methods of Mauldin et al. (1972, 1978).
Extraction of vitamin B~2 Between 200 and 1500 (100-700mg dry weight) of the smaller insects (aphids, termites, etc.) and 6 and 100 (300-4000 mg dry weight) of the large insects (cockroaches, tobacco hornworm, etc.) were used in each experiment. Insects were dried in an oven at 70°C for 12hr and then weighed. Homogenation was performed in a minimal volume of borate-KCl buffer (pH 9.4) containing 0.3 mM KCN. Samples were then centrifuged at 2000g for 10 min to remove cell debris and the supernatant was stored at -20°C in the dark until assayed.
Radioassay Standard vitamin BI2 radioassay kits supplied with [57Co]cyanocobalamin, borate-KCI-KCN buffer, intrinsic factor, dithiothreitol and vitamin B~2 standards were obtained from Diagnostic Products Corp., Los Angeles, California. This radioassay is a modification of the methods of Liu and Sullivan (1972) and Mollin et al. (1976). It involves mixing 200 #1 of samples or vitamin Bu standards with 1 ml of borate (0.1 M)-KCI (0.1 M) buffer (pH 9.4) containing 0.3 mM KCN, approx. 10.2 nCi of [57Co]cyanocobalamin, and 1.0 mg of dithiothreitol. The tubes capped with loose-fitting caps were placed in a covered boiling water bath (100°C) for 1 hr to denature proteins and to release and convert vitamin B~2to the stable cyanocobalamin derivatives. After cooling the tubes in an ambient water bath for 5 to 10min, 100#1 of "purified" intrinsic factor (IF) was added and incubated at ambient temperature for 2 hr during which [SVCo]cyanocobalamin and the sample Bu derivatives compete for IF binding. After equilibrium was reached, dextran-coated charcoal (400/~1) was added to each tube and incubated for 10 rain before separating the free (unbound) vitamin Biz from the bound (IF-B~2 complex) by adsorption on the dextran-coated charcoal. Separation of the free vitamin B~2 derivative from the bound was performed by centrifuging at 3000g for 10 min in a refrigerated (4°C) centrifuge. The supernatant containing the IF-B~2 complex was completely transferred into a tube for counting. The radioactivity (cpm) of the IF-B,2 complex was determined in a Beckman gamma scintillation counter.
Cobalt Cobalt concentrations in insects were determined using an IL 251 Atomic Absorption Spectrophotometer with flameless Atomizer IL 555, Instrumentation Laboratory, Inc., Wilmington, Massachusetts. The supernatant of homogenized insects in borate-KCl-KCN buffer, as described in extraction of vitamin B~z, was further diluted to 1:100 with the borate-KC1 buffer and aspirated into the flameless atomizer. The standard curve was obtained by analyzing 0-25 ng/ml cobalt chloride solutions, and unknowns determined from this curve. RESULTS An analysis of the vitamin B~2 content of adult mixed sex and larval houseflies showed no detectable amounts (Table 1). Both the larval growth media (1067pg/mg) and the adult diet (1.5pg/mg) contained vitamin B~2 suggesting that the absence of
vitamin B,2 in this insect was not due to a dietary deficiency. Consistent with the absence of detectable amounts of vitamin B12 in the housefly was the absence of detectable amounts of cobalt. In contrast to the housefly, five species of termites representing three diverse families contain large amounts of vitamin B~2 (Table 1). Values range from 258 + 2 6 p g / i n s e c t for C. formosanus to 9703 _ 372 pg/insect for Z. angusticollis (Table 1). When converted to a dry weight basis, the values range from 4 5 5 p g / m g in C. formosanus to 3211 pg/mg in N. corniger (Table 1). All of these termites also had relatively high cobalt levels: 13 _ 2 ng/insect (mean _ SD, n = 3) in N. corniger, 74 ___ 1 ng/insect in C. formosanus, 1500 ___200 ng/ insect in R. flavipes and R. virginicus, and 25,600 + 3300 ng/insect in Z. angusticollis. A comparison of vitamin B~2 levels in gut tissue versus all other tissues in both Z. angusticollis and C. formosanus showed that gut tissue contained the majority of the vitamin B12 (Table 2). This indicated that the bacteria, spirochaetes and/or protozoa in the gut tract were the most likely source of the vitamin. To determine which was the source, groups of C. formosanus were treated to remove either bacteria plus spirochaetes, protozoa or all three. Elimination of bacteria and spirochaetes reduced vitamin B,2 levels from 258 ___26 pg/insect to 87 + 3 pg/insect within 10 days after streptomycin treatment was initiated. This value is in the same range as that of termites whose hindgut had been removed (64 ___ 16 pg/insect). In contrast, elimination of protozoa by CO2 and 02 under pressure did not significantly affect vitamin Bt2 levels (Table 2), suggesting that the protozoa do not contribute to vitamin BI2 production. When all micro-organisms are eliminated from the gut, vitamin BI2 values decrease to 68 _ 3 pg/insect. The residual values of vitamin Bt2 that remain after the removal of bacteria are similar to those present in control insects without gut tracts. This suggests that the amounts of vitamin B~2 in streptomycin treated insects reflect vitamin B~2 present in insect tissues, and that this did not decrease significantly in the 10-day treatment period. Termites contain anaerobic bacteria in their hindgut (Breznak, 1982) and based on the above data, these are the most likely site of vitamin B~2 production. The marked differences in vitamin B~2 concentrations found in houseflies and termites prompted us to examine insects from several phylogenetically diverse orders to determine if any trends in the presence or absence of vitamin Bu were apparent. In general, all taxa, except termites, contain considerably lower vitamin B~2 levels than is usually observed in vertebrates. M o s t of the data in Table 1 were obtained from whole insects, and it is possible that certain tissues, particularly gut tissue, may contain higher concentrations. The housefly and fruitfly D. melanogaster (Table 1) have undetectable levels of vitamin B~2, but other Diptera, including the blowfly S. bullata and the Tse-tse fly, G. m. morsitans, contain low but measurable amounts of vitamin B12 (Table 1). Analysis of the Drosophila diet (formula 4-24, Carolina Biological, Burlingame, North Carolina) showed no detectable vitamin B~2.
Vitamin B~2 in insects
177
Table 1. Concentrations of vitamin B~2 in selected insects
Species
Vitamin B,2 pg/insect pg/mg't (mean + SD, n = 3)
Life stage*
Diptera Musca domestica
a
1
ND ND 225 ± 11 66 ± 6.9 ND ND
ND~ ND 23.4 ± 1.1 1.5 .+ 0.2 ND ND
m m m m m
9703 ± 372 258 + 26 281 ± 5 721 _+59 1455 ± 95
940 + 35 455 __.48 563 ± 9 2131 ± 177 3211 -+ 203
a a a a I
6.4 ± 1.4 7.4 ± 0.4 ND ND 37.+4
2.8 ± 0.6 3.8 .+ 0.2 ND ND 0.5+0.1
Homoptera Acyrthosiphon pisum
1
ND
ND
Hymenoptera Apis mellifera
a
ND
ND
Lepidoptera Manduca sexta Trichoplusia ni
1 a
p 1 a 1 a 1
283 + 60 ND ND 25±6 13±0.9 28+4 2.5+0.4 15.+4.2
0.3 ± 0.1 ND ND 0.3_+0.1 7.0+0.5 7.7± hi 1.1 _+0.2 3.5±0.9
Orthoptera Periplaneta americana Blatta orientalis Acheta domesticus
a a a
390 ± 21 478 ± 84 ND
4.6 ± 0.1 0.4 ___0.1 ND
Thysanura Lepisma saccharina
a
53 + 6.4
17 ± 2.1
1
Gloss±ha m. morsitans Sarcophaga bullata Drosophila melanogaster
a p a
Isoptera Zootermopsis angusticollis Coptotermes formosanus Reticulitermes flavipes Reticulitermes virginicus Nasutitermes corn±get Coleoptera
Attagenus megatoma Tribolium castaneum Sitophilus oryzae Tenebrio species
Plodia interpunctella Tineola bisseliella
*a--adults, mixed ages and mixed sex; l--larvae; p~pupae; m--mixed castes. "['pg/mgdry weight of tissue. :[:NI)---not detectable; sensitivity = 0.04 pg/mg. T w o o f the Coleoptera c o n t a i n e d significant levels o f v i t a m i n B n , w h e r e a s S. oryzae h a d u n d e t e c t a b l e a m o u n t s ( T a b l e 1). O n e species o f Orthoptera (the cricket A. domesticus) h a d u n d e t e c t a b l e levels o f v i t a m i n B~2 while t w o c o c k r o a c h e s (P. americana a n d B. oriental±s) h a d m e a s u r a b l e a m o u n t s o f vitam i n B,2. L a r g e a m o u n t s o f v i t a m i n B,2 were p r e s e n t
in the h i n d g u t o f P. americana ( T a b l e 2) s u g g e s t i n g t h a t a n a e r o b i c b a c t e r i a ( B r e z n a k , 1982) in the h i n d gut are the s o u r c e o f the v i t a m i n B,2. N e i t h e r the p e a a p h i d , A . p i s u m ( H o m o p t e r a ) n o r the h o n e y b e e , A. mellifera ( H y m e n o p t e r a ) c o n t a i n detectable a m o u n t s o f v i t a m i n B12 ( T a b l e 1). T h e p e a a p h i d w a s g r o w n o n b o t h alfalfa a n d f a v a b e a n
Table 2. Vitamin B,2 concentrations in whole insects, insects minus gut tissue, hindguts, and in selectively defaunated termites Species
Zootermopsis angusticollis Coptotermes formosanus
Periplaneta americana
Tissue Whole insects Insects minus gut tissue Gut tissue Whole insects Insects minus gut tissue Gut tissue Insects minus bacteria and spirochaetes~ Insects minus protozoa Insects minus bacteria, spirochaetes and protozoa Whole insects Insects minus hindgut Hindgut
Vitamin B~2* pg/insect 9703 + 372f 3824 _+250 6741 + 40 258 ± 26 64 + 16 197 _+ 10 87 ± 3 248 _ 23 68 + 3 390 + 21 130 + 10 230 __. 17
*pg/mg dry weight, tMean +SD, n = 3. ~Elimination of gut organisms is described in the text.
EDGAR J. WAKAYAMAel aL
178
plants, and, as expected, neither of these contain detectable amounts of vitamin Bt2. Within the Lepidoptera, both adult and larval forms of P. interpunctella and T. bisseliella contain significant amounts of vitamin B~2.Larvae of M. sexta also contained appreciable amounts of vitamin B~2. L. saceharina, a member of the Thysanura and one of the most primitive insects, contains appreciable levels of vitamin B~2 (Table 1). Differences in the vitamin Bt2 content of insects in different life stages were noted in two cases. Larvae of Trichoplusia ni and Tenebrio species contain appreciable amounts of vitamin Bt2 while adults of these species do not contain detectable levels. In addition, P. interpunctella larvae contain larger amounts of vitamin B~2 than does the adult insect. DISCUSSION Anaerobic bacteria utilize vitamin Bt2 as a cofactor in many enzymatic reactions, but higher animals have been shown to utilize only a few B12 requiring enzymes (Baker, 1972). The best studied of these enzymes is the methylmalonyl-CoA mutase which interconverts succinyl-CoA and methylmalonyl-CoA and is an important reaction in the metabolism of odd chain fatty acids and branched chain amino acids (Reitz, 1982). Vitamin B~2 requiring L-leucine-2,3-aminomutase, which catalyzes the conversion of L-leucine into fl-leucine, has been shown to be present in animal tissues and may function in the metabolism of branched chain amino acids (Baker and Stadtman, 1982). Vitamin B~2 has also been shown in animals to function in B~2-dependent methionine biosynthesis (Taylor, 1982). There is little evidence for the synthesis of vitamin B~2 in animals and higher plants (Wuest and Perlman, 1968), and in general, vertebrate animals require dietary vitamin B~2. Many aerobic and anaerobic micro-organisms can synthesize vitamin B,2, and they are a major source of this vitamin (Coates, 1968). The lack of vitamin B,2 in the housefly is consistent with the metabolism of propionate in this insect. Studies with propionate isotopically labelled in the 1, 2 or 3 position with carbon-13 indicated that propionate was directly converted to acetate and was not metabolized via methylmalonyl-CoA to succinylCoA. Carbon 3 of propionate became the carboxyl carbon of acetate and carbon 2 of propionate became the methyl carbon of acetate (Dillwith et aL, 1982). Thus, it appears that the housefly metabolizes propionate by a unique pathway (for animals). Plants, many of which do not contain appreciable amounts of vitamin B12, metabolize propionate by a similar pathway as that suggested for the housefly (Hatch and Stumpf, 1962; Giovanelli and Stumpf, 1958). The lack of vitamin B12 in the housefly along with its presence in the diet suggests that it is not utilized by this insect. The high concentration of vitamin B~2 in Z. angusticollis is consistent with its ability to readily convert succinate to the methylmalonyl derivative used in branched alkane synthesis (Chu and Blomquist, 1980; Blomquist et al., 1980). Isolated epidermal tissue from this insect is able to incorporate
[1,4J4C]succinate into branched alkanes (Chu and Blomquist, unpublished), suggesting that the termite tissue itself utilizes vitamin B12. The concentration of vitamin B~2 in these termites is in the same general range as that found in mammalian tissues. For example, the normal vitamin B~2 range for human liver is 800-3,000 pg/mg. Rat liver contains 60-300 pg/mg dry weight (Baker and Frank, 1968). In performing the assays of vitamin B12 isolated from C.formosanus, it was noted that the vitamin B~2 appeared to be in tightly bound complexes, which required longer heat treatment (1 hr at 100°C) to release the free cyanocobalamin. Some microorganisms have been shown to tightly bind vitamin B~2; both Lactobacillus bulgaricus and L. thermophilus in saline suspension show a cyanocobalamin-binding capacity which is reduced, although not completely destroyed, by heating at 90°C (Skeggs, 1969). Also, extracts of Ochromonas cells can preferentially bind cyanocobalamin (Ford et al., 1953). The analysis of the vitamin B~2 content of insects from several orders revealed no general trends within orders, between orders or between life stages of individual species. However, the results of our analysis are very consistent with the available literature on vitamin B~2 in insects. The lack of vitamin B,2 in the housefly is consistent with the reports that vitamin B~2 is not required for normal growth (Brookes and Fraenkel, 1958; House and Barlow, 1958). Also, the presence of vitamin B12 in T. castaneum is consistent with the reported dietary requirement for this vitamin (Armstrong, 1978). The presence of vitamin B,2 in P. americana is consistent with the observation that cells from this insect grown in culture require vitamin B~2 for normal growth (Landureau and Steinbuch, 1969). The absence of vitamin B~2 in D. melanogaster was somewhat surprising, in view of the observation that the addition of vitamin B,2 and uridine restored growth to cultured cells whose growth was inhibited by added adenosine (Becker, 1975). Our results suggest that under normal physiological conditions, vitamin B~2 does not play a significant role in this insect. This observation is supported by the finding that the normal rearing media for Drosophila contained no vitamin Bt2. Some general statements can be made regarding the presence of vitamin B~: in insects: (1) the occurrence of vitamin B~2 correlates with the observed metabolism of propionate and succinate in the housefly and a termite; (2) termites contain large amounts of vitamin B~2, apparently synthesized by their symbiotic gut bacteria; (3) no general trends are apparent in the presence or absence of vitamin B~2 within members of the orders Coleoptera, Diptera, Lepidoptera and Orthoptera which were examined; (4) the apparent lack of vitamin B~2 in some species poses a number of intriguing questions including how these organisms perform metabolic reactions which in many animals require vitamin Bt2 as a cofactor. REFERENCES
Armstrong E. (1978) The effects of vitamin deficiencies on the growth and mortality of Tribolium castaneum infected with Noserna whitei. J. Invert. Path. 31, 303-306.
Vitamin B~2 in insects Baker H. A. (1972) Corrinoid-dependent enzymic reactions. A. Rev. Biochem. 41, 55-90. Baker H. A. and Frank O. (1968) Vitamin Bl2. In Clinical Vitaminology: Methods and Interpretation, p. 136. Wiley, New York. Baker J. J. and Stadtman T. C. (1982) Amino mutases. In B12, Vol. 2, Biochemistry and Medicine (Edited by Dolphin D.), pp. 203-232. Wiley, New York. Becker J. L. (1975) Role of vitamin Bi2 in the reduction of ribonucleotides into deoxyribonucleotides in Drosophila cells grown in vitro. Biochimie 58, 427-430. Blomquist G. J., Chu A. J., Nelson J. H. and Pomonis J. G. (1980) Incorporation of [2,3J3C]succinate into methylbranched alkanes in a termite. Archs Biochem. Biophys. 204, 648-650. Breznak J. A. (1982) Intestinal microbiota of termites and other xylophagous insects. A. Rev. Microbiol. 36, 323-343. Brookes V. J. and Fraenkel G. (1958) The nutrition of the larvae of the housefly Musca domestica L. Physiol. Zool. 31, 208-223. Chu A. J. and Blomquist G. J. (1980) Biosynthesis of hydrocarbons in insects: succinate is a precursor of the methyl branched alkanes. Archs Biochem. Biophys. 201, 304-312. Coates M. E. (1968). Vitamin B12: deficiency effects in animals. In The Vitamins (Edited by Sebrell W. H., Jr and Harris R. S.), pp. 212-220. Academic Press, New York. Dadd R. H. (1977) Qualitative requirements and utilization of nutrients: insects. In CRC Handbook Series in Nutrition and Food (Editor-in-chief, Rechcigl M., Jr), Section D: Nutritional Requirements, Vol. 1, Comp. and Qualitative Requirements. de Renobales M. and Blomquist G. J. (1983). A developmental study of the composition and biosynthesis of the cuticular hydrocarbons of Trichoplusia ni. Insect Biochem. 13, 493-502. Dillwith J. W., Nelson J. H., Pomonis J. G., Nelson D. R. and Blomquist G. J. (1982) A 13C NMR study of methylbranched hydrocarbon biosynthesis in the housefly. J. biol. Chem. 257, 11305-11314. Ford J. E., Holdsworth E. S., Kon S. K. and Porter J. W. G. (1953) Occurrence of the various vitamin B~2 active compounds. Nature 171, 150-151. Giovanelli J. and Stumpf P. K. (1958) Fat metabolism in plants. X. Modified fl-oxidation of propionate by peanut mitochondria. J. biol. Chem. 231, 411-426. Hatch M. D. and Stumpf P. K. (1962) Fat metabolism in
179
higher plants. XVIII. Propionate metabolism by plant tissues. Archs Biochem. Biophys. 96, 193-198. House H. L. (1974) Nutrition. In The Physiology oflnsecta (Edited by Rockstein M.), Vol. V, pp. 1~52. Academic Press, New York. House H. L. and Barlow J. S. (1958) Vitamin requirements of the housefly Musca domestica L. (Diptera: Muscidae). A. Ent. Soc. Am. 51, 299-302. Kubasik N. P., Ricotta M. and Sine H. E. (1980) Commercially-supplied binders for plasma cobalamin (Vitamin B~2), analysis-purified intrinsic factor, "cobinamide"-blocked R-protein binder, and non-purified intrinsic factor-R-protein binder compared to microbiological assay. Clin. Chem. 26, 598-600. Landureau J. C. and Steinbuch M. (1969) Cyanocobalamine as a support of the in vitro cell growth-promoting activity of serum proteins. Experientia 25, 1078-1079. Liu Y. K. and Sullivan L. W. (1972) An improved radioisotope dilution assay for serum vitamin B~2 using hemoglobin-coated charcoal. Blood 39, 426-429. Mauldin J. K., Rich N. M. and Cook D. W. (1978) Amino acid synthesis from ~4C-acetate by normally and abnormally faunated termites, Coptotermes formosanus. Insect Biochem. 8, 105-109. Mauldin J. D., Smythe R. V. and Baxter C. C. (1972) Cellulose catabolism and lipid synthesis by the subterranean termite, Coptotermes formosanus. Insect Biochem. 2, 209-217. Mollin D. L., Anderson B. C. and Burman J. F. (1976) The serum vitamin B~2 level: Its assay and significance. Clin. Haemat. 3, 521-546. Reitz J. (1982) Methylmalonyl-CoA mutase. In Bl2 Vol. 2 Biochemistry and Medicine (Edited by Dolphin D.), pp. 357-379. Wiley, New York. Ryan R. O., de Renobales M., Dillwith J. W., Heisler C. R. and Blomquist G. J. (1982) Biosynthesis of myristate in an aphid: Involvement of a specific acylthioesterase. Archs Biochem. Biophys. 213, 26-36. Skeggs H. R. (1969) Vitamin Bl~. In The Vitamins (Edited by Gyorgy P. and Pearson W. N.), Vol. VII, 2nd edn, pp. 277-302. Academic Press, New York. Taylor R. T. (1982) Bt2-dependent methionine biosynthesis. In Bt2 Vol. 2 Biochemistry and Medicine (Edited by Dolphin D.), pp. 37-355. Wiley, New York. Wuest H. M. and Perlman D. (1968) Vitamin Bl2: Industrial preparations. In The Vitamins (Edited by Sebrell W. H., Jr and Harris R. S.), pp. 139-144. Academic Press, New York.