Essential Dietary Amino Acids for Growth of Larvae of the Yellow Mealworm, Tenebrie molitor L.1 G. R. F. DAVIS Research Station, Research Branch, Agriculture Canada, University Campus, Saskatoon, Saskatchewan, Canada S7N 0X2 ABSTRACT Larvae of the yellow mealworm, Tenebrìamolitor L., have been used to evaluate nutritional quality of proteins and protein isolates. However, such investigations have been complicated by lack of knowledge of dietary requirements of the larvae. To determine essential dietary amino acids for growth of Tenebria molitor, single amino acids were deleted from the amino acid mixture of the diet. Diets were maintained isonitrogenous with supplementary glycine and, in the case of deleted glycine, with glutamic acid. Growth, as measured by gain in weight, and survival were observed over a 4-week period at 27 ±0.25°and 65 ±5% relative humidity. The results indicate that larvae of Tenebria molitor require a dietary source of the same 10 amino acids essential for growth in rats, other vertebrates, and some protozoa. They also showed that serine, tyrosine, glutamic acid, and possibly glycine were dis pensable for growth in this insect. Alanine, cystine, proline, and aspartic acid appeared semidispensable. Survival over the 4-week experimental period was unaffected by deleting amino acids from the diet. The results are discussed in relation to amino acid requirements of other insects and to suggested improvement of the diet of the present investigation. J. Nutr. 105: 1071-1075, 1975. INDEXING KEY WORDS insect nutrition •amino acids •Tenebria molitor •growth
The rationale for using larvae of Tenebria molitor L. to compare the nutritional value of proteins was published several years ago (1). Recently, these larvae have been used successfully to determine the nutri tive value of oilseed proteins (2, 3) and oilseed protein isolates (4). Interpretation of results of such nutritional studies has, however, been complicated by the failure of larvae of T. molitor fed amino acid mix tures to develop (5). The assumption has to be made that T. molitor require the same ten essential amino acids as most other insects (5). Previously, larvae of T. molitor fed a diet containing an amino acid mixture as the sole protein source developed as quickly as when fed a casein diet (6). Although growth was not as rapid as when larvae werethefeddieta diet wheat brewer's yeast, couldof be usedplus to determine essential amino acid require ments by the classical deletion method. 1071
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The present paper reports the effect of deleting single amino acids from the amino acid mixture of diet on growth and sur vival of larvae of T. molitor, Gembloux strain, race F. MATERIALS AND METHODS
Experimental diets. The optimal con centration of dietary protein for larvae of T. molitor has been shown to be in the broad range of 2 to 32% (7). Because com parative nutritional investigations are in progress using these larvae and mice or rats, and because mammalian diets regu larly contain 10% protein, this level of di etary protein was chosen for the diets of the present investigation. The experimental diets were similar to that used previously in an investigation of the nutritional value of oilseed protein isolates with this insect Received for publication February 24, 1975. 1 Contribution no. 596, Agriculture Canada search Station, Saskatoon, Saskatchewan.
Re
1072
G. R. F. DAVIS
( 4 ). However, an amino acid mixture based on the composition of larval tissues of this insect (4) was substituted for the protein portion of the diet, and guanine and cytosine were added to the diet. Details of the complete diet are outlined in table 1. Deletions of single amino acids were made from this diet to provide 18 deficient diets. Additional amounts of glycine were used to keep the experimental diets isonitrogenous and additional glutamic acid, in the case of the glycine-free diet. The diet containing the complete amino acid mix ture served as the reference diet; however, a natural of 90brewer's parts whole ground wheat and diet 10 parts yeast was in cluded in each test to determine the uni formity of response of test larvae from one test to another. The latter diet is used routinely in stock rearing of T. molitor. Mealworm experiments. Larvae of T. molitor, Gembloux strain, race F, were se lected from stock rearing and were starved for 48 hours. For each test diet, 30 larvae were selected within the weight range 8.4
to 12.5 mg and placed in individual glass vials containing l g of diet. The average group weights for 30 larvae were main tained at 10 mg. The vials containing larvae and diet were kept at 27 ±0.25° and 65 ±5% relative humidity for 4 weeks. The larvae were then reweighed, and changes in weight during the experimental period were calculated. Because of the large numbers of larvae required for the full test of 18 diets de ficient in a single amino acid, 5 tests were made. Each first plus four brewer's consisted yeast of a complete diet,of athe wheat diet, and four deficient diets. The fifth test consisted yeast of a complete diet,remaining a wheat plus brewer's diet, and the two deficient diets. Analysis of variance showed no difference ( P > 0.05 ) among gains in weight of groups of larvae fed the wheat plus brewer's yeast diet or the complete diet. Therefore, the data were grouped for further statistical treatment. Data on gains in fresh weight were ana lyzed using analysis of variance and Dun-
TABLE 1 Composition of the diet used to determine dietary amino acid requirements of larvae of Tenebrie molitor L.1 Amino acid mixture8
Other components
(resolved)Arginine Alanine base)Aspartic (free pureCystineGlutamic acid, chemically
bacteriologicalMcCollum-Davis 185sCholesterolGuanine salt mixture, no.
acidGlycineHistidine
baseCytosine free (H2O)Linoleic %)Vitamin-B acid (95 + solutionVitamin-B
base)Isoleucine (free free)Leucine(allo ml/g free)Lysine (methionine solutione29.8921.2790.3360.0140.0140.2620.10 mono HC1a0.3040.1610.2970.0590.3670.1920.0980.1540.2660.224Dextrin,
ml±500.0050.0025.0025.0012.5012.506.002.500.2540 /O.I
MethioninePhenylalanineProline
chlorideNicotinic acidThiamin-HClD-Calcium
free)SerineThreonine (hydroxyproline
pantothenateRiboflavinPyridoxine-HClDL-Carnitine free)TryptophanTyrosineValine0.0940.2310.2590.1430.1330.0490.2380.224Choline (allo HC1Folie • acidD-BiotinZinc chloride4IV 1 All dietary components obtained from ICN Nutritional Biochemicals Corporation, Cleveland, Ohio, except as otherwise noted. 1 All L isomers. »McCollum, E. V. & Davis, M. (1914) Observations on the isolation of the substance in butterfct which eierts a stimulating influence on growth. J. Biol. Chem. 19, 245. Composition as follows (in %): calcium lactate-5H2Q,. 35.19; calcium biphosphate • HjO, 14.60; potassium monophosphate, 25.78; sodi um monophosphate, 9.38; sodium chloride, 4.67; uagnçajum suJSate anhydrous, 7.19; ferrie citrate, 3.19. « Fisher Scientific Co., Fair Lawn, N.J.
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ESSENTIAL AMINO ACIDS FOR T. molitor
1073
TABLE 2 Effect of deleting single amino acids from the diet on growth and survival of larvae of Tenebrio molitor L. reared for 4 weeks at 27 ±0.25°and 66 ±5% relative humidity1 freshInitialmg10.1 deletedNone Amino acid
0.8°26.3 ± ±0.110.1 1.9°26.4 ± 0.210.0 ± 1.5"26.1 ± 0.210.0 ± 1.6°23.4 ± ± acidGlycineAlanineCystineProlineAspartic 0.210.0 1.8°"22.1 ± 0.210.0 ± 1.7»20.9 ± 0.210.0 ± 1.8»20.9 ± 0.210.0 ± 1.4620.9 ± 0.210.0 ± 1.7»4.4 ± 0.210.0 ± acidHistidineLysineArginineIsoleucineTryptophanMethionineLeucinePhenylalanineValineThreonineWheat 0.3«3.8 ± 0.210.0 ± 0.2"3.0 ± 0.210.0 ± 0.2'2.9 ± 0.210.0 ± 0.4«2.7 ± 0.210.0 ± 0.2°2.5 ± 0.210.0 ± 0.2C2.3 ± 0.210.0 ± 0.2'2.3 ± 0.210.0 ± ±0.1C2.4 0.29.9 ± 0.1"2.1 ± 0.210.0 ± 0.2«55.4 ± 0.210.0 ± plus brewer's yeast4Avg ±0.1weight3Gain«26.2 ±1.2SurvivalNo.146/15028/3030/3027/3029/3029/3029/3030/3029/30
(complete)SerineTyrosineGlutamic
1 Values are the mean of 30 larvae per group, except for complete and wheat plus brewer's yeast diets. ! Values are mean ±SEM. >Values followed by the same letter superscript do not differ from one another at P *=0.05. *Control diet included in each test but not in the statistical analyses.
can's multiple range test (8). Differences at the 5% level of confidence were con sidered significant. RESULTS Larvae of T. molitor, fed a diet contain ing amino acids in the proportions occur ring in larval tissues of this insect as the sole protein source, gained 2.6 times their initial weight during the 4-week experi mental period (table 2). This gain in weight was about half the potential gain as observed with larvae fed a diet of ground whole wheat plus brewer's yeast. Deletion of serine, tyrosine, glutamic acid, or glycine from the amino acid mix ture did not affect the growth of larvae of T. molitor (table 2). However, a slight growth depression did occur when alanine, cystine, proline, or aspartic acid were re moved from the diet. In contrast, growth of larvae was negligible when they were fed diets lacking histidine, lysine, arginine, isoleucine, tryptophan, methionine, leucine, phenylalanine, valine, or threonine. Survival of larvae of T. molitor in this investigation ranged from 90 to 100% and
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was apparently unrelated to the adequacy of the diet for larval growth. DISCUSSION
The present results show that larvae of T. molitor require a dietary source of the same 10 amino acids essential for growth in rats, other vertebrates, and some pro tozoa ( table 2 ) ( 5 ). The data also indicate that serine, tyrosine, glutamic acid, and possibly glycine can be deleted from the diet of T. molitor without adversely af fecting the rate of growth. The intermediate position of alanine, cystine, proline, and aspartic acid suggests that the complete diet may contain suboptimal concentrations of other compo nents. Both dietary alanine and dietary proline have been shown to accelerate growth of larvae of the saw-toothed grain beetle, Oryzaephilus surinamensis (L.), but only when they were fed diets con taining suboptimal concentrations of argi nine (9). Because dietary arginine is es sential for growth of T. molitor (table 2), and because the nutritional value of lactalbumin for these larvae can be increased
1074
G. R. F. DAVIS
by addition of supplemental arginine (10), the present results suggest that the concentration of arginine in the amino acid mixture of this study may be suboptimal. This would require some biosynthesis of the amino acid from alanine or proline, or both. The beneficial effect of supplement ing lactalbumin with proline on larvae of T. molitor in a previous study (10) may also be explained on this basis. The role of aspartic acid is difficult to assess because of the involvement of this amino acid in the synthesis of purines and pyrimidines and because of possible in hibition of its metabolism by glutamic acid. However, larval growth of Tribolium confusum (Duval) (11) and of Bombyx mori L. ( 12) was accelerated by the inclusion of aspartic acid in the diet. Under certain conditions, aspartic acid has also been con sidered essential in the diet of larvae of T. molitor ( 13) and of O. surinamensis (14). Nevertheless, contrary to observations with O. surinamensis (14), dietary aspartic acid did not influence the survival of larvae of T. molitor in the present investigation (table 2). The slight growth depression in larvae of T. molitor in the absence of di etary aspartic acid may indicate suboptimal concentrations of guanine or cytosine in the experimental diet of this study. Cystine is generally considered a dis pensable dietary amino acid. However, it accelerated growth in larvae of O. surina mensis fed a diet containing methionine ( 15 ) and could not be replaced by concen trations of methionine greater than optimal for that amino acid. The present results suggest that, like larvae of O. surinamensis, larvae of T. molitor probably require a small amount of dietary cystine, irreplace able by additional methionine, for optimal growth. In the present investigation, these four amino acids, which have intermediate di etary importance, may be required to meet the metabolic demands of the insect tis sues, to facilitate transaminations, or to function in the synthesis of such nitrogencontaining compounds as purines and pyrimidines. The slight growth depression observed with deletion of any one from the diet ( table 2 ) is probably an expression of a requirement for such metabolic pro cesses. As for vertebrates, dietary mixtures
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containing only essential amino acids are inadequate or useless for insect growth, even at optimal nitrogen levels, and must be supplemented with individual amino acids that are individually dispensable (15). Both alanine and aspartic acid, as individual supplements of mixtures of es sential amino acids, have been shown to improve the growth of some insects (16), as in the present investigation, probably for the reasons indicated above. Phenylalanine is converted to tyrosine by many insects, but the reverse does not occur (17). The present results (table 2) are in agreement with this observation. Larval growth was unaffected by a lack of tyrosine in this study but was retarded by the absence of dietary phenylalanine. The beneficial effect of supplemental tyrosine for this insect, reported in a previous paper (10), must therefore have been through a sparing effect on phenylalanine metabolism. Growth of larvae of T. molitor fed the diet containing the complete amino acid mixture was of the same order as that ob tained previously (4) when vitamin-free casein was used as the protein source. However, this growth was only about half the potential growth as indicated by growth of the larvae fed a diet of wheat plus brewer's yeast (table 2). These re sults suggest that improved growth of this insect may be obtained by increasing the dietary concentration of arginine, by in cluding a low concentration of cystine in the diet and by modifying the concentra tions of aspartic acid, guanine, and cytosine in the diet. The present study does pro vide a preliminary basis for interpreting results of investigations being pursued in this laboratory using larvae of T. molitor concerning the nutritional quality of pro teins. It also indicates possible useful amino acids to be used in experiments of supple mentation of deficient proteins with indi vidual amino acids. ACKNOWLEDGMENTS
The conscientious technical assistance of Mr. F. T. Eves is gratefully acknowledged. LITERATURE CITED 1. Leclercq, J. Si De Bast, D. (1965) Projet d'utilisation des larves de Tenebria molitor pour comparer la valeur nutritive des pro téines.Ann. Nutr. Aliment. 19, 19-25.
ESSENTIAL AMINO ACIDS FOR T. molitor 2. Davis, G. R. F. & Sosulski, F. W. (1972) Use of larvae of Tenebria molitor L. to deter mine nutritional value of proteins in six de fatted oilseed meals. Arch. Int. Physiol. Biochem. 80, 501-509. 3. Davis, G. R. F. (1974) Evaluation of the nutritional value of proteins of rape, turnip rape and yellow mustard seed by larvae of the yellow mealworm, Tenebria molitor L. Arch. Int. Physiol. Biochem. 82, 141-148. 4. Davis, G. R. F. & Sosulski, F. W. (1974) Nutritional quality of oilseed protein isolates as determined with larvae of the yellow meal worm, Tenebria molitor L. J. Nutr. 204, 1172-1177. 5. Dadd, R. H. (1970) Arthropod nutrition. In: Chemical Zoology, vol. 5, part A ( Florkin, N. & Scheer, B. T., eds.), pp. 35-95, Aca demic Press, New York. 6. Davis, G. R. F. (1974) Protein nutrition of Tenebria molitor L. XVII. Improved amino acid mixture and interaction with dietary carbohydrate. Arch. Int. Physiol. Biochem. 82, 631-637. 7. Davis, G. R. F. & Sosulski, F. W. (1973) Protein nutrition of Tenebria molitor L. XVI. Effects of dietary protein concentration on larvae of race F. Arch. Int. Physiol. Bio chem. 81, 661-665. 8. Robinson, P. (1959) Tests of significance for use in comparison of several with particular reference to Duncan'smeans, multiple range test. Can. Dept. Agr. Processed Pubi, no. 4. 9. Davis, G. R. F. (1968) Dietary alanine and proline requirements of the beetle,
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10.
11.
12.
13.
14. 15.
16. 17.
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Oryzaephilus surinamensis. J. Insect Physiol. 14, 1247-1250. Davis, G. R. F. (1969) Protein nutrition of Tenebria molitor L. X. Improvement of the nutritional value of lactalbumin by sup plementation with amino acids. Arch. Int. Physiol. Biochem. 77, 741-748. Naylor, A. F. (1963) Glutamic and aspartÃ-c acids and sucrose in the diet of the flour beetle, Tribolium conjusum ( Tenebrionidae). Can. J. Zool. 41, 1127-1132. Ito, T. & Arai, N. (1965) Nutrition of the silkworm, Bombyx mori. Vili. Amino acid requirements and nutritive effects of various proteins. Bull. Serie. Exp. Stn., Japan 19, 345-373. Leclercq, J. & Lopez-Francos, L. (1964) Nutrition protidique chez Tenebria molitor L. VI. de remplacementd'acides de la aminés. caséine par desEssais mélangesartificiels Arch. Int. Physiol. Biochem. 72, 276-296. Davis, G. R. F. (1967) Nutritional rela tionships in larvae of Oryzaephilus surina mensis. J. Insect Physiol. 13, 1737-1743. Davis, G. R. F. (1961) Sulfur-containing amino acids in the nutrition of the sawtoothed grain beetle, Oryzaephilus surinamen sis (L.) (Coleóptera:Silvanidae). J. Nutr. 75, 275-278. Dadd, R. H. (1973) Insect nutrition: cur rent developments and metabolic implications. Annu. Rev. Entomol. 18, 381-420. Chefurka, W. (1965) Intermediary metab olism of nitrogenous and lipid compounds in insects. In: The Physiology of the Insecta, vol. 2 (Rockstein, M., ed.), pp. 670-678, Academic Press, New York.
The rationale for using larvae of Tenebria molitor L. to compare the nutritional value of proteins was published several years ago (1). Recently, these larvae have been used successfully to determine the nutri tive value of oilseed proteins (2, 3) and oilseed protein isolates (4). Interpretation of results of such nutritional studies has, however, been complicated by the failure of larvae of T. molitor fed amino acid mix tures to develop (5). The assumption has to be made that T. molitor require the same ten essential amino acids as most other insects (5). Previously, larvae of T. molitor fed a diet containing an amino acid mixture as the sole protein source developed as quickly as when fed a casein diet (6). Although growth was not as rapid as when larvae werethefeddieta diet wheat brewer's yeast, couldof be usedplus to determine essential amino acid require ments by the classical deletion method. 1071
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The present paper reports the effect of deleting single amino acids from the amino acid mixture of diet on growth and sur vival of larvae of T. molitor, Gembloux strain, race F. MATERIALS AND METHODS
Experimental diets. The optimal con centration of dietary protein for larvae of T. molitor has been shown to be in the broad range of 2 to 32% (7). Because com parative nutritional investigations are in progress using these larvae and mice or rats, and because mammalian diets regu larly contain 10% protein, this level of di etary protein was chosen for the diets of the present investigation. The experimental diets were similar to that used previously in an investigation of the nutritional value of oilseed protein isolates with this insect Received for publication February 24, 1975. 1 Contribution no. 596, Agriculture Canada search Station, Saskatoon, Saskatchewan.
Re
1072
G. R. F. DAVIS
( 4 ). However, an amino acid mixture based on the composition of larval tissues of this insect (4) was substituted for the protein portion of the diet, and guanine and cytosine were added to the diet. Details of the complete diet are outlined in table 1. Deletions of single amino acids were made from this diet to provide 18 deficient diets. Additional amounts of glycine were used to keep the experimental diets isonitrogenous and additional glutamic acid, in the case of the glycine-free diet. The diet containing the complete amino acid mix ture served as the reference diet; however, a natural of 90brewer's parts whole ground wheat and diet 10 parts yeast was in cluded in each test to determine the uni formity of response of test larvae from one test to another. The latter diet is used routinely in stock rearing of T. molitor. Mealworm experiments. Larvae of T. molitor, Gembloux strain, race F, were se lected from stock rearing and were starved for 48 hours. For each test diet, 30 larvae were selected within the weight range 8.4
to 12.5 mg and placed in individual glass vials containing l g of diet. The average group weights for 30 larvae were main tained at 10 mg. The vials containing larvae and diet were kept at 27 ±0.25° and 65 ±5% relative humidity for 4 weeks. The larvae were then reweighed, and changes in weight during the experimental period were calculated. Because of the large numbers of larvae required for the full test of 18 diets de ficient in a single amino acid, 5 tests were made. Each first plus four brewer's consisted yeast of a complete diet,of athe wheat diet, and four deficient diets. The fifth test consisted yeast of a complete diet,remaining a wheat plus brewer's diet, and the two deficient diets. Analysis of variance showed no difference ( P > 0.05 ) among gains in weight of groups of larvae fed the wheat plus brewer's yeast diet or the complete diet. Therefore, the data were grouped for further statistical treatment. Data on gains in fresh weight were ana lyzed using analysis of variance and Dun-
TABLE 1 Composition of the diet used to determine dietary amino acid requirements of larvae of Tenebrie molitor L.1 Amino acid mixture8
Other components
(resolved)Arginine Alanine base)Aspartic (free pureCystineGlutamic acid, chemically
bacteriologicalMcCollum-Davis 185sCholesterolGuanine salt mixture, no.
acidGlycineHistidine
baseCytosine free (H2O)Linoleic %)Vitamin-B acid (95 + solutionVitamin-B
base)Isoleucine (free free)Leucine(allo ml/g free)Lysine (methionine solutione29.8921.2790.3360.0140.0140.2620.10 mono HC1a0.3040.1610.2970.0590.3670.1920.0980.1540.2660.224Dextrin,
ml±500.0050.0025.0025.0012.5012.506.002.500.2540 /O.I
MethioninePhenylalanineProline
chlorideNicotinic acidThiamin-HClD-Calcium
free)SerineThreonine (hydroxyproline
pantothenateRiboflavinPyridoxine-HClDL-Carnitine free)TryptophanTyrosineValine0.0940.2310.2590.1430.1330.0490.2380.224Choline (allo HC1Folie • acidD-BiotinZinc chloride4IV 1 All dietary components obtained from ICN Nutritional Biochemicals Corporation, Cleveland, Ohio, except as otherwise noted. 1 All L isomers. »McCollum, E. V. & Davis, M. (1914) Observations on the isolation of the substance in butterfct which eierts a stimulating influence on growth. J. Biol. Chem. 19, 245. Composition as follows (in %): calcium lactate-5H2Q,. 35.19; calcium biphosphate • HjO, 14.60; potassium monophosphate, 25.78; sodi um monophosphate, 9.38; sodium chloride, 4.67; uagnçajum suJSate anhydrous, 7.19; ferrie citrate, 3.19. « Fisher Scientific Co., Fair Lawn, N.J.
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ESSENTIAL AMINO ACIDS FOR T. molitor
1073
TABLE 2 Effect of deleting single amino acids from the diet on growth and survival of larvae of Tenebrio molitor L. reared for 4 weeks at 27 ±0.25°and 66 ±5% relative humidity1 freshInitialmg10.1 deletedNone Amino acid
0.8°26.3 ± ±0.110.1 1.9°26.4 ± 0.210.0 ± 1.5"26.1 ± 0.210.0 ± 1.6°23.4 ± ± acidGlycineAlanineCystineProlineAspartic 0.210.0 1.8°"22.1 ± 0.210.0 ± 1.7»20.9 ± 0.210.0 ± 1.8»20.9 ± 0.210.0 ± 1.4620.9 ± 0.210.0 ± 1.7»4.4 ± 0.210.0 ± acidHistidineLysineArginineIsoleucineTryptophanMethionineLeucinePhenylalanineValineThreonineWheat 0.3«3.8 ± 0.210.0 ± 0.2"3.0 ± 0.210.0 ± 0.2'2.9 ± 0.210.0 ± 0.4«2.7 ± 0.210.0 ± 0.2°2.5 ± 0.210.0 ± 0.2C2.3 ± 0.210.0 ± 0.2'2.3 ± 0.210.0 ± ±0.1C2.4 0.29.9 ± 0.1"2.1 ± 0.210.0 ± 0.2«55.4 ± 0.210.0 ± plus brewer's yeast4Avg ±0.1weight3Gain«26.2 ±1.2SurvivalNo.146/15028/3030/3027/3029/3029/3029/3030/3029/30
(complete)SerineTyrosineGlutamic
1 Values are the mean of 30 larvae per group, except for complete and wheat plus brewer's yeast diets. ! Values are mean ±SEM. >Values followed by the same letter superscript do not differ from one another at P *=0.05. *Control diet included in each test but not in the statistical analyses.
can's multiple range test (8). Differences at the 5% level of confidence were con sidered significant. RESULTS Larvae of T. molitor, fed a diet contain ing amino acids in the proportions occur ring in larval tissues of this insect as the sole protein source, gained 2.6 times their initial weight during the 4-week experi mental period (table 2). This gain in weight was about half the potential gain as observed with larvae fed a diet of ground whole wheat plus brewer's yeast. Deletion of serine, tyrosine, glutamic acid, or glycine from the amino acid mix ture did not affect the growth of larvae of T. molitor (table 2). However, a slight growth depression did occur when alanine, cystine, proline, or aspartic acid were re moved from the diet. In contrast, growth of larvae was negligible when they were fed diets lacking histidine, lysine, arginine, isoleucine, tryptophan, methionine, leucine, phenylalanine, valine, or threonine. Survival of larvae of T. molitor in this investigation ranged from 90 to 100% and
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was apparently unrelated to the adequacy of the diet for larval growth. DISCUSSION
The present results show that larvae of T. molitor require a dietary source of the same 10 amino acids essential for growth in rats, other vertebrates, and some pro tozoa ( table 2 ) ( 5 ). The data also indicate that serine, tyrosine, glutamic acid, and possibly glycine can be deleted from the diet of T. molitor without adversely af fecting the rate of growth. The intermediate position of alanine, cystine, proline, and aspartic acid suggests that the complete diet may contain suboptimal concentrations of other compo nents. Both dietary alanine and dietary proline have been shown to accelerate growth of larvae of the saw-toothed grain beetle, Oryzaephilus surinamensis (L.), but only when they were fed diets con taining suboptimal concentrations of argi nine (9). Because dietary arginine is es sential for growth of T. molitor (table 2), and because the nutritional value of lactalbumin for these larvae can be increased
1074
G. R. F. DAVIS
by addition of supplemental arginine (10), the present results suggest that the concentration of arginine in the amino acid mixture of this study may be suboptimal. This would require some biosynthesis of the amino acid from alanine or proline, or both. The beneficial effect of supplement ing lactalbumin with proline on larvae of T. molitor in a previous study (10) may also be explained on this basis. The role of aspartic acid is difficult to assess because of the involvement of this amino acid in the synthesis of purines and pyrimidines and because of possible in hibition of its metabolism by glutamic acid. However, larval growth of Tribolium confusum (Duval) (11) and of Bombyx mori L. ( 12) was accelerated by the inclusion of aspartic acid in the diet. Under certain conditions, aspartic acid has also been con sidered essential in the diet of larvae of T. molitor ( 13) and of O. surinamensis (14). Nevertheless, contrary to observations with O. surinamensis (14), dietary aspartic acid did not influence the survival of larvae of T. molitor in the present investigation (table 2). The slight growth depression in larvae of T. molitor in the absence of di etary aspartic acid may indicate suboptimal concentrations of guanine or cytosine in the experimental diet of this study. Cystine is generally considered a dis pensable dietary amino acid. However, it accelerated growth in larvae of O. surina mensis fed a diet containing methionine ( 15 ) and could not be replaced by concen trations of methionine greater than optimal for that amino acid. The present results suggest that, like larvae of O. surinamensis, larvae of T. molitor probably require a small amount of dietary cystine, irreplace able by additional methionine, for optimal growth. In the present investigation, these four amino acids, which have intermediate di etary importance, may be required to meet the metabolic demands of the insect tis sues, to facilitate transaminations, or to function in the synthesis of such nitrogencontaining compounds as purines and pyrimidines. The slight growth depression observed with deletion of any one from the diet ( table 2 ) is probably an expression of a requirement for such metabolic pro cesses. As for vertebrates, dietary mixtures
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containing only essential amino acids are inadequate or useless for insect growth, even at optimal nitrogen levels, and must be supplemented with individual amino acids that are individually dispensable (15). Both alanine and aspartic acid, as individual supplements of mixtures of es sential amino acids, have been shown to improve the growth of some insects (16), as in the present investigation, probably for the reasons indicated above. Phenylalanine is converted to tyrosine by many insects, but the reverse does not occur (17). The present results (table 2) are in agreement with this observation. Larval growth was unaffected by a lack of tyrosine in this study but was retarded by the absence of dietary phenylalanine. The beneficial effect of supplemental tyrosine for this insect, reported in a previous paper (10), must therefore have been through a sparing effect on phenylalanine metabolism. Growth of larvae of T. molitor fed the diet containing the complete amino acid mixture was of the same order as that ob tained previously (4) when vitamin-free casein was used as the protein source. However, this growth was only about half the potential growth as indicated by growth of the larvae fed a diet of wheat plus brewer's yeast (table 2). These re sults suggest that improved growth of this insect may be obtained by increasing the dietary concentration of arginine, by in cluding a low concentration of cystine in the diet and by modifying the concentra tions of aspartic acid, guanine, and cytosine in the diet. The present study does pro vide a preliminary basis for interpreting results of investigations being pursued in this laboratory using larvae of T. molitor concerning the nutritional quality of pro teins. It also indicates possible useful amino acids to be used in experiments of supple mentation of deficient proteins with indi vidual amino acids. ACKNOWLEDGMENTS
The conscientious technical assistance of Mr. F. T. Eves is gratefully acknowledged. LITERATURE CITED 1. Leclercq, J. Si De Bast, D. (1965) Projet d'utilisation des larves de Tenebria molitor pour comparer la valeur nutritive des pro téines.Ann. Nutr. Aliment. 19, 19-25.
ESSENTIAL AMINO ACIDS FOR T. molitor 2. Davis, G. R. F. & Sosulski, F. W. (1972) Use of larvae of Tenebria molitor L. to deter mine nutritional value of proteins in six de fatted oilseed meals. Arch. Int. Physiol. Biochem. 80, 501-509. 3. Davis, G. R. F. (1974) Evaluation of the nutritional value of proteins of rape, turnip rape and yellow mustard seed by larvae of the yellow mealworm, Tenebria molitor L. Arch. Int. Physiol. Biochem. 82, 141-148. 4. Davis, G. R. F. & Sosulski, F. W. (1974) Nutritional quality of oilseed protein isolates as determined with larvae of the yellow meal worm, Tenebria molitor L. J. Nutr. 204, 1172-1177. 5. Dadd, R. H. (1970) Arthropod nutrition. In: Chemical Zoology, vol. 5, part A ( Florkin, N. & Scheer, B. T., eds.), pp. 35-95, Aca demic Press, New York. 6. Davis, G. R. F. (1974) Protein nutrition of Tenebria molitor L. XVII. Improved amino acid mixture and interaction with dietary carbohydrate. Arch. Int. Physiol. Biochem. 82, 631-637. 7. Davis, G. R. F. & Sosulski, F. W. (1973) Protein nutrition of Tenebria molitor L. XVI. Effects of dietary protein concentration on larvae of race F. Arch. Int. Physiol. Bio chem. 81, 661-665. 8. Robinson, P. (1959) Tests of significance for use in comparison of several with particular reference to Duncan'smeans, multiple range test. Can. Dept. Agr. Processed Pubi, no. 4. 9. Davis, G. R. F. (1968) Dietary alanine and proline requirements of the beetle,
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Oryzaephilus surinamensis. J. Insect Physiol. 14, 1247-1250. Davis, G. R. F. (1969) Protein nutrition of Tenebria molitor L. X. Improvement of the nutritional value of lactalbumin by sup plementation with amino acids. Arch. Int. Physiol. Biochem. 77, 741-748. Naylor, A. F. (1963) Glutamic and aspartÃ-c acids and sucrose in the diet of the flour beetle, Tribolium conjusum ( Tenebrionidae). Can. J. Zool. 41, 1127-1132. Ito, T. & Arai, N. (1965) Nutrition of the silkworm, Bombyx mori. Vili. Amino acid requirements and nutritive effects of various proteins. Bull. Serie. Exp. Stn., Japan 19, 345-373. Leclercq, J. & Lopez-Francos, L. (1964) Nutrition protidique chez Tenebria molitor L. VI. de remplacementd'acides de la aminés. caséine par desEssais mélangesartificiels Arch. Int. Physiol. Biochem. 72, 276-296. Davis, G. R. F. (1967) Nutritional rela tionships in larvae of Oryzaephilus surina mensis. J. Insect Physiol. 13, 1737-1743. Davis, G. R. F. (1961) Sulfur-containing amino acids in the nutrition of the sawtoothed grain beetle, Oryzaephilus surinamen sis (L.) (Coleóptera:Silvanidae). J. Nutr. 75, 275-278. Dadd, R. H. (1973) Insect nutrition: cur rent developments and metabolic implications. Annu. Rev. Entomol. 18, 381-420. Chefurka, W. (1965) Intermediary metab olism of nitrogenous and lipid compounds in insects. In: The Physiology of the Insecta, vol. 2 (Rockstein, M., ed.), pp. 670-678, Academic Press, New York.
