1965 Taylor_Medici Amino Acid Grain Beetles
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Amino Acid Requirements of Grain Beetles 7<2 M. WIGHT TAYLOR ANDJOHN C. MEDICI Department of Biochemistry and Microbiology, Rutgers —The State University of New Jersey, New Brunswick, New Jersey ABSTRACT Development of a diet purified with respect to amino acids has led to the determination of amino acid requirements of several grain beetles. The con fused flour beetle, Tribolium confusum (Duval), required (in per cent of diet) arginine, 0.5; histidine, 0.2; isoleucine, 0.5; leucine, 0.8; lysine, 0.56; methionine, 0.3 (0.15 w ith excess cystine); phenylalanine, 0.7 (0.4 with excess tyrosine); threoni e, 0.4; tryptophan, 0.1; and valine 0.6. The red flour beetle, Tribol um castaneum (Herbst), required (in per cent) arginine, 0.4; histidine, 0.2; isoleucine, 0.3; and leucine, 0.7. T he saw-toothed grain beetle, Oryzaephilus surinamensis (L.), required (in per cent) arginine, 0.3; histidine, 0.2; isoleucine, 0.3; and leucine 0.6. Other amino acid requirements for the latter 2 insects were not determined. Requirements of these grain insects are compared with those of other organisms. The confused flour beetle was tested as an assay organism for amino acid content of protein but showed little promise. Due mainly to the difficulties involved in preparing diets adequately purified for the study of quantitative nutritional re quirements, most nutritional investigations of grain insects, and of insects in gen eral, have been of a qualitative nature (1-4). In the present study, emphasis was placed on the quantitativ amino acid re quirements of the confused flour beetle, Tribolium confusum (Duval), while the requirements o the red flour beetle, Tri bolium castaneum (Herbst), and the saw- toothed grain beetle, Oryzaephilus surina mensis (L.), fo 4 amino acids were also determined. The confused flour beetle re quires the same 10 amino a ids re uired by many vertebrates (5), and the relative order of protein quality for larvae of this insect is similar to that for the rat (3). Most other grain insects also require these 10 amino acids (2). The purposes of this study were to ob tain basic information on the nutrition of insects and to acquire enough information on the confused flour beetle to determine whether this insect might be used as an assay organism for the amino acid content of feedstuffs. EXPERIMENTAL The composition of the control diet which was used in all experiment is shown in table 1 This diet is nutritionally adequate, since it supports rapid growth of all insects studied for at least 3 genera tions. In each test, the amino acid being investigated was fed in a series of diets at different levels, the levels of the other die tary components being kept the same as those of the control diet. All insect colo nies were reared in a constant temperature room at 28°with a stock diet containing 95% unbleached wheat flour and 5% dried yeast. Eggs were sifted from this stoc diet 24 hours before the start of each ex periment. Three g oups of ten each, newly emerged larvae (average age 12 hours) per leve of nutrient tested were placed in small vials containing the experimental diets. Such replicates seldom varied m ore than 8% from the average of the 3 values and if so were discarded. The least differ ence between averages required for signifi cance at the 1% level was 0.123 mg/lar- vae. The flour beetle larvae were incubated at 28°and 75 ±5% relative humidity for 18 days, whereas the larvae of the saw- toothed grain beetle were incubated for only 14 days. At the end of the 14- or 18-day period, the surviving larvae were separated from the diet, counted and weighed. Usually all 10 larvae survived. A comparison of the final weights with the Received for publication July 23, 1965. ' This work was supported in part by the U. S. P ub lic H ea lt h T ra in in i; G ra nt n o. 2 -G -6 42 . 2 P aper of the Journal Series, New Jersey Agricul tu ra l E xp er im en t S ta tio n. 17 6 J. NUTRITION. 88; '66 b y o A u g u s t 1 8 , 2 0 1 0 j . u t r i t i o n . o r g D o w l o a d e d f r o m
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Amino Acid Requirements of Grain Beetles 7<2
M. W IGHT TAYLOR AND JOHN C. MEDICIDepartment of Biochemistry and Microbiology, Rutgers —The StateUniversity of New Jersey, New Brunswick, New Jersey
ABSTRACT Development of a diet purified with respect to amino acids has ledto the determination of amino acid requirements of several grain beetles. The confused flour beetle, Tribolium confusum (Duval), required (in per cent of diet)arginine, 0.5; histidine, 0.2; isoleucine, 0.5; leucine, 0.8; lysine, 0.56; m ethionine, 0.3(0.15 w ith excess cystine); phenylalanine, 0.7 (0.4 with excess tyrosine); threonine,0.4; tryptophan, 0.1; and valine 0.6. The red flour beetle, Tribolium castaneum(Herbst), required (in per cent) arginine, 0.4; histidine, 0.2; isoleucine, 0.3; andleucine, 0.7. T he saw -toothed grain beetle, O ryzaephilus surinam ensis (L.), required(in per cent) arginine, 0.3; histidine, 0.2; isoleucine, 0.3; and leucine 0.6. Otheram ino acid requirements for the latter 2 insects were not determ ined. Requirementsof these grain insects are compared with those of other organisms. The confusedflour beetle was tested as an assay organism for amino acid content of protein butsh ow ed little p ro mise.
Due m ainly to the difficulties involvedin preparing diets adequately purified forthe study of quantitative nutritional req ui remen ts , mo st n utritio na l in ve stig atio nsof grain insects, and of insects in general, have been of a qualitative nature(1-4).In the present study, emphasis was
placed on the quantitative amino acid requirements of the confused flour beetle,Tribolium confusum (Duval), while therequirements of the red flour beetle, Tribolium castaneum (Herbst), and the saw-
toothe d grain bee tle, O ryz aephilus surin amensis (L.), for 4 am ino acids were alsodeterm ined. The confused flour beetle requires the same 10 amino acids requiredby many vertebrates (5), and the relativeorder of protein quality for larvae of thisinsect is sim ilar to that for the rat (3).M ost other grain insects also require these10 amino acids (2).The purposes of this study were to ob
tain basic information on the nutrition ofinsects and to acquire enough inform ationon the confused flour beetle to determ inewhether this insect m ight be used as anassay organism for the am ino acid content
o f f eedstuf fs .EXPERIMENTAL
The composition of the control dietwhich was used in all experiments isshow n in table 1. T his diet is nutritionally
adequate, since it supports rapid growth ofall insects studied for at least 3 generations. In each test, the am ino acid beinginvestigated was fed in a series of diets atdifferent levels, the levels of the other dietary components being kept the same asthose of the control diet. A ll insect colonies w ere reared in a constant tem peratureroom at 28°with a stock diet containing95% unbleached wheat flour and 5% driedyeast. Eggs were sifted from this stockdiet 24 hours before the start of each experim ent. Three groups of ten each, newly
emerged larvae (average age 12 hours)per level of nutrient tested were placed insm all vials containing the experim entaldiets. Such replicates seldom varied m orethan 8% from the average of the 3 valuesand if so were discarded. The least difference betw een averages required for significance at the 1% level was 0.123 mg/lar-vae . T he flour beetle larvae w ere in cubatedat 28 °and 75 ±5% relative hum idity for18 days, whereas the larvae of the saw-toothed grain beetle were incubated foronly 14 days. At the end of the 14- or18-day period, the surviving larvae wereseparated from the diet, counted and
weighed. Usually all 10 larvae survived.A com parison of the final weights with the
Received for publication July 23, 1965.' This work was supported in part by the U. S.
P ub lic H ea lt h T ra in in i; G ra nt n o. 2 -G -6 42 .2 P aper of the Journal Series, New Jersey Agricul
tu ra l E xp er im en t S ta tio n.
176 J . NUTR IT ION .88; '66
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AM INO ACID REQUIREM ENTS OF GRAIN BEETLES 177
TA BL E 1Composition of control diet used in all
experiments
CornstarchAminoacids (all L -)Minerals2CornoilVitamin
mixtureCholesterol72.520.03.53.00.630.37
Com position of am ino acid m ixture: (as per cento f d iet) a rgin in e, 0 .9 ; histid in e, 0 .6 ; iso leu cin e, 1 .2 ;l eu cin e, 1 .4 ; ly si ne -HC l, 1 .3 ; m et hi on in e, 0 .6 ; p he ny l-a la nin e, 0 .8; th reo nin e, 0 .9; tryp to pha n, 0 .2 ; v alin e,1 .1 ; g lu tam ic a cid , 4 .5 ; g ly cin e, 4.5 ; a nd c ystin e, 2.0 .L evels of the essential am ino acids w ere based on thele ve ls p re se nt in g oo d q ua lity pro tein s.2Ash o f whol e whea t.3 V ita min s u sed w ere : (in m ic rp gram s/gra m o f d ie t)
choline chloride, 4000; nicotinic acid, 100; Ca D-p an to th en at e, 4 0; r ib of la vi n, 1 8; p yr id ox in e-HC l, 1 6;t hia rn in e-HC l, 1 2; fo lie a cid , 5 ; b io ti n, 0 .6 ; c ar ni ti ne ,10 ; inos ito l, 2000 ; } ) -aminobenzo icac id, 500; L-ascorbicacid, 10; m enadione, 1; and cobalam in, 0.5. T he last5 vitam ins are not am ong the know n requirem ents ofT . confusim i. C arnitine is required for pupal em erg
e nc e onl y.
experim ental diets and final weights withthe control diet (table 1) was used toevaluate the experim ental diets.Figure 1 is a plot of the data from the
isoleucine and valine experim ents. These2 am ino acids represent extrem es encountered with respect to slopes of the curves.The other 10 curves, including experiments on the sparing actions of cystineand tyrosine, ranged between these two.The point of maximal inflection in theupper portion of each curve was assumedto show the minimal requirement for the
am ino acid concerned. Larval grow th w iththe control diet was also considered ineach experiment as a check on the constancy of experimental conditions. Thenoticeably lower growth with valine wasbelow what is considered norm al and wasdue to excessive vibration of the flasksfrom a nearby shaking machine.Two other am ino acids, as well as the
control diet, run concurrently, gave sim ilarly low growth. W hen the incubator wasremoved to a quiescent spot for later assays, growth with the control and experim ental diets returned to the higher figures.It was not possible to weigh individual
larvae because of time lim itations and because the larvae dehydrated on exposureto air; it was not possible, therefore, to rundetailed statistical analyses. In a fewcases where this was tried there was onlym oderate scatter. There were occasional
very small individuals (runts), but theincidence was low. In over a thousandobservations the numbers ranged from1.4% with good diets to 6.3% with poor
diets.S tatistical analysis on responses withthe control diet, which was run with eachgroup of assays, indicated an undeterm ined source of variation occurring overa period of months or years. Therefore,weights obtained in experiments run atdifferent times may not reliably be compared with each other, but must be considered with respect to a standard such asthe control diet. In the present work eachexperim ent w as self-contained so that sucha com parison w as largely u nnecessary .
R ESUL TS AND D IS CU SS IO N
The results of the 12 experiments onthe confused flour beetle and the 4 experiments each on the red flour beetle and thesaw -toothed grain beetle are summarizedin table 2. For comparison this table alsocontains inform ation on the requirem entsof other organisms. The honey bee is theonly other insect that has been com pletelystudied in terms of amino acid requirements.In general, the requirements for the in
sects are higher for the larger insects(honey bee and confused flour beetle) and
0 .8 1 .0 1 .2
Am in o Ac id in D ie t {%}
Fig. 1 Weight of larvae of the confused flourbeetle at 18 days fed at graded levels of isoleucine and valine.
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17 8 M . W IG H T T A Y L O R A N D JO H N C . M E DIC I
T A B L E 2
A m in o acid req u ir em en ts of grain b eet les w ith com par ison of r eq u ir em en ts of cer tain oth er s pe cie s â €¢
Confused Red t S^w",j
Aminoácido fe ««» grain H«^
larva larva|„aArginine
0.5 0.4 0.3Histidine
0.2 0.2.2Isoleucine
0.5 0.3.3Leucine
0.8 0.7.6Lysine
0.56 ?Methionineno
cystine 0.3 ?excesscystine5
0.15 ?.14(11)Phenylalanineno
tyrosine 0.7 ?excesstyrosine
0.4 ?Threonine
0.4 ?Tryptophan
0.1 ?Valine
0.6 ?Glycine1
A ll requirem ents expressed as percentage of diet.2 A ll g rain in se ct re qu irem en ts sh ow n are fo r-amino3A llison, J. B . 1960 T he ideal am inogram .ifth4Davi s ( 8) r ep or ts a v al ue o f 0 .3 3% .0 .6 0. 31 .0 0. 90 .6 j0 .3 ?0 .5 0. 60 .2 0. 8â €” ac id s.Young
Adult
rat (7) rat7)0.20.3
0.070.5
0.430.8
0.250.9
0.140.6
0.230.3
0.110.9
?0.6
0.190.5
0.170.15
0.070.7
0.31—
—Young
chicken
(8)1.20.30.61.41.00.80.450.70.140.60.20.81.0International
Cong re ss o futrition,5E xcess cystine m eans an am ount w hich w e determ ined to be som ew hat above
with a minim um of methionine but not giving any indication of toxicity.thelevelYoung
human(9)3—J0.62.81.1y0.61.91.10.60.21
6.giving
aAdult
human(9)3——0.60.90.6?0.2
lower for the smaller insects (red flourbeetle and saw-toothed grain beetle). As
an hypothesis, it is suggested that thesedifferences in the requirements of threesuch sim ilar insects may be associatedwith the ad libitum feeding techniqueswhich must necessarily be used w ith suchsmall organisms. The smaller insects,having higher m etabolic rates because ofthe greater surface area per unit volumeand thus higher rates of feeding, m ight beexpected to eat more food per unit of bodyweight, and thus would show lower requirem ents in such feeding experim ents.Rate of growth should also be a factor.More data on a variety of other insectswould be needed to test this theory.
The principal amino acid which is required in markedly different amounts byinsects, as compared with higher organisms, is methionine. The higher requirements of mammals and the chicken maybe necessary for supplying sulfur amino
acids for the production of hair andfe ath ers. A rg in in e sh ow s c on sid era ble v ari
ation am ong the various species.It was found that the confused flourbeetle's grow th was slightly improved
when glycine was used as the supplier ofextra nitrogen in a diet containing minimal levels of the essential amino acids.This may be due to the method of nitrogenexcretion. Like the chicken, which requires glycine, insects excrete nitrogenlargely in the form of uric acid.W hen the essential amino acids were
fed to the confused flour beetle at their determ ined m inim al required levels in a dietcontaining adequate nonessential am inoacids, considerably lower growth was ob
served than that with the control diet.A dditional am ounts of ce rtain am ino acids,selected on the basis of some uncertainty as to the exact point of inflectionof the growth response curve, improvedgrowth somewhat, as shown in table 3, but
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AM INO ACID REQUIREM ENTS OF GRAIN BEETLES 17 9
TABLE 3Growth o f co nfused flo ur beetle la rva e fed diets co nta ining minima l levels
of the essential amino acids
Va ri ab le am ino a ci ds'2His%Minimal
l ev el s 0 .2 0. 30 .4 0. 40 .4 0. 40 .4 0. 4l ie u%0 .6 0. 60 .6 0. 70 .6 0. 60 .6 0. 6Leu%0 .8 0. 80 .8 0. 80 .8 0. 80 .9 0.9Phe%0. 70 .7 0. 70
growthmg1.761.751.901.972.152.192.172
Con tr ol d ie t 0. 6 1.2 1.4 0.8 0.20 2.50
1 Other dietary ingredients were the same as those in the control diet except for increases inglycine and glutam ic acid to m aintain a total of 20% am ino acids.2Leve ls i nc re as ed wher e i ta li ci ze d.3 Average of 30 larvae at the end of 18 days' incubation.
growth still failed to reach that obtainedwith the control diet. It appears that, foroptim al grow th, several of the am ino acidsshould be present at much more than them inimal requirement level, or that the requirements for various amino acids islower in the presence of an excess of theother essential amino acids. Thus the requirement for histidine was raised from0.2 to 0.4% when the low levels of theother acids were used.An assay procedure for am ino acids, us
in g co nfused flou r bee tle larvae, w as teste don sam ples of gelatin, casein hydrolysate,and sesam e m eal which had been analyzed
by automatic column Chromatographietechniques. Leucine and isoleucine werechosen for assay and the proteins wereadded to the control diet to furnish gradedlevels of the am ino acids, the amino aciditself being omitted. The glycine and glutamic acid content of the control diet wasreduced to 1% each to balance somewhatthe added nitrogen from protein.Results of the assays were unexpected.
Gelatin gave very poor growth at all levelsand hence was considered as toxic to theflour beetle. Casein hydrolysate gave opposite results; even the low levels, furnishing leucine and isoleucine m uch below
the m inimal requirement, gave growth of2.5 to 2.9 mg in 18 days.Sesame meal was the only test m aterial
which behaved in what m ight be called anormal manner. W hen it furnished 0.3%isoleucine, growth was the same as that
when 0.3% isoleucine was supplied as thefree acid. However, at 0.5% isoleucine,the requirement level, growth was muchsuperior with the sesame meal, and at0.6% isoleucine from sesame growth was3.34 mg, a value never reached with thecontrol diet. It appears from these 3 assaysthat there m ay be com plicated interrelations in the amino acid requirements ofthis insect. The casein and sesame mealmay have also contained non-amino acidgrowth factors of the type suggested byHorn and Warren (12) in their work withmicroorganisms.
ACKNOWLEDGMENTS
The authors are grateful to Dr. E. R. Ottand R. T. Amsden of the Rutgers Statistical Center for statistical analyses and toDr. R. W . W annamacher, Jr. of the Bureauof Biological Research for the amino acidanalyses.
L IT ERATURE C IT ED
1. Dadd, R. H. 1963 Feeding behaviour andnutrition in grasshoppers and locusts. Adv.Insect Physiol., 3: 47.
2. House, H . L. 1962 Insect nutrition. Ann.R ev. B iochem ., 33: 653.
3. Chirigos, M . A., A . N. M eiss, J. J. Pisano andM . W . Taylor 1960 Growth response of theconfused flour beetle, T ribolium confusum
(Duval) to six selected protein sources. J.N utrition, 72: 121.4. Fraenkel, G . S. 1959 A historical and com
parative survey of the dietary requirem entsof insects. Ann. N.Y. Acad. Sci., 77: 267.
5. Lemonde, A., and R. Bernard 1951 Nutrition des larves de Tribolium confusum,
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18 0 M. WIGHT TAYLOR AND JOHN C. MEDICI
Duval. II. Importance des acids amines.C aà ±ad. J. Z oology, 29: 80.
6. De Groot, A . P. 1953 Protein and aminoacid requirements of the honey bee (Apismellifica L.). W. Junk, The Hague, Netherlands.
7. National Research Council, Committee onAnimal Nutrition 1962 Nutrient requirements of laboratory animals, pub. 990. National Academ y of Sciences —National Research C ouncil, W ashington, D . C .
8. National Research Council, Committee onAnimal Nutrition 1960 Nutrient requirements of poultry, pub. 827. National Academ y of Sciences —N ational R esearch C ouncil, W ashington, D . C.
9. Block, R . J., and K. W . Weiss 1956 AminoAcid Handbook. Charles C Thomas, Springfield, Illinois, p. 161.
10. Davis, G . R. F. 1962 Quantitative L-argi-nine requirements of larvae of the saw-toothed grain beetle, Oryzaephilus surina-mensis (L.) (Coleoptera:Silvanidae'). J. In
sect P hysiol., 8; 377.
11. Davis, G . R. F. 1961 Sulfur-containingamino acids in the nutrition of saw-toothedgrain beetle, O ryzaephilus surinam ensis (L ).(C oleóptera: S ilvanidae~). J. N utrition, 75:
275.12. Horn, M . J., and H . W . Warren 1964
Availability of am ino acids to m icroorganisms. IV . Comparison of hydrolysates oflactalbum in, oatm eal and peanut butter w ithsimulated amino acid mixtures by growthresponse of m icroorganisms. J. Nutrition,83: 267.
