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Comparative studies with Tribolium (Coleoptera, Tenebrionidae) — II: Productivity of T. castaneum (herbst) and T. confusum Duv. on natural, semi-synthetic and synthetic diets
f.
Stored Prod,
1966, Vol. 1, pp. 313-324.
Res.,
Pergamon Press Ltd.
Printed in Great Britain.
Comparative Studies with Tribolium (Coleoptera, Tenebrionidae) - II: Productivity of T. castaneum (Herbst) and T. confusum Duv. on Natural, Semi-synthetic and Synthetic Diets ALEXANDER
SOKOLOFF,
IAN R. FRANKLIN
and RAJ K. LAKHANPAL*
Departments of Genetics and Nutritional Science, University of California, Berkeley, California, U.S.A. (First receiued 15 September,
1965 and infinal form
16 December,
1965)
Abstract-Productivity of T. cnrtaneum and T. confwm has been determined in the following diets: Naylor’s synthetic medium; corn supplemented with the amino acids cystine, glutathione, proline and tryptophan in amounts necessary to bring them to the level found in wheat; corn and whole wheat flour without any supplements, and corn and whole wheat flour supplemented with brewer’s yeast and vitamins. Relative developmental rates of the two species in these media were also determined by counting the numbers of adults emerging in 14-day periods. Significant differences in productivity (the number of eggs surviving to the adult stage) have been detected within a species in the different media and between species within a given medium. The Naylor medium is unfavorable for both species, and judging from relative developmental rates less favorable to T. confwm than to 1. castaneum. ‘f: castaneum
performs poorest in non-supplemented media, better in media supplemented with vitamins, and the best in media supplemented with yeast. While T. confwum also shows an increased productivity in vitamin- and yeast-supplemented media, this increase is not as marked as in 1. castanezm. Considering both the productivity and the developmental rates, it appears that the addition of the four amino acids to corn had a beneficial effect to T. castaneum but not to T. conftcsum. The two species were most productive in corn plus yeast, but this productivity was not significantly higher than in whole wheat flour plus yeast. Drawing upon the evidence available in the literature, and the present data, it is concluded that 1. confusum is better adapted to utilize a wider variety of foods than T. castaneum and in order to satisfy its greater quantitative requirements for certain nutrients, this species engages in cannibalism more often than 1. confusum. The result is that, when the two species are introduced into the same container, i.e. compete for food and space, T. confuum is more often eliminated than 1. castaneum. *Present address: Department Colorado, U.S.A.
of Home Economics,
313
Colorado State University,
Fort Collins,
314
ALEXANDER SOKOLOFF, IAN R. FRANKLIN and RAJ K.
LAKHANPAL
1. INTRODUCTION IN THE first paper of this series (SOKOLOFFet al., 1966) the pertinent literature was reviewed and data on productivity and relative developmental rates were obtained for Tribolium castaneum (henceforth abbreviated CS) and T. confmum (CF) in several commercially available diets with and without yeast. In the present paper the same criteria for performance of C’S and CF are used to assess natural, semisynthetic and synthetic diets in order to substantiate further that nutritional differences exist in these two species of flour beetles. 2. MATERIALS AND METHODS The 1. castaneum (C’S) and T. confusum (CF) beetles used in these experiments as in the previous ones were derived from the stocks synthesized from a number of wild type strains in 1958 and maintained in large populations at the Department of Genetics, University of California, Berkeley, California. Details of the methods of synthesis and maintenance of these stocks are given by LERNER and Ho (1961). For any given species, freshly formed pupae were removed from the cultures, sexed, and fifteen males introduced into each of one series of eight creamers each containing a different test diet; fifteen females were introduced into each ofanother series of eight creamers containing these diets. The two series of creamers were returned to the incubator (maintained in darkness at 29°C and 70 per cent r.h.) Two weeks later, when the imagoes were about lo-days old, five female and five male beetles from each diet were introduced into each of two creamers containing 4g of this same diet. Each group of adults was transferred to a new creamer containing the same diet twice a week for a total of 3 weeks. Each group remained in one set of creamers for 3 days and in the next for 4 days. At each transfer the sex of every dead beetle was determined, but none was replaced. The creamers containing eggs were returned to the incubator to allow development of the progeny. Census of adults was begun in each medium at 42 days, and subsequent counts were made, ifjuvenile stages were still present, at 2-week intervals until all the prosupplies of synthetic or geny had died or reached the adult stage. Uninoculated semisynthetic media were kept under refrigeration until needed. No attempts were made to “acclimate” the medium to the conditions prevailing in the incubator, but the creamers were left at room temperature for a few hours before introducing the test beetles. A preliminary experiment was undertaken with three “synthetic” diets which have been developed for feeding mammals but was unsuccessful. These diets, GPD-2, MO-l and R-l are normally used for guinea pigs, mice and rats, respectively. Since flour beetles require a sterol in addition to the unsupplemented mammalian diets, a parallel experiment was performed to test these diets supplemented with 1% cholesterol. Neither species produced any progeny on any of these diets, and the adults introduced died during the 3-week experimental period. It was observed that the diets that contained large amounts of sucrose (MO-l and R-l) turned yellowish to brown and became lumpy. Rancidity developed in all the diets, possibly because of exposure of the oil to the high temperature and humidity in the incubator.
Comparative Studies with Tribolium (Coleoptera, Tenebrionidae)-II
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successful experiments were performed with the following diets: “natural’‘-fine stoneground whole wheat flour on yellow cornmeal sifted through No. 5 silk bolting cloth (56 meshes/in.) to remove the large particles of bran or corn and to make the particle size uniform, with no additives; c‘semi-synthetic”-the same whole wheat flour (W) and yellow cornmeal (C) as above, but with the additives described below and summarized in Table 1; a synthetic medium (NM) reported by NAYLOR (1964) consisting of the various ingredients shown in Table 2 and supplemented with vitamins and zinc chloride at the levels indicated in Table 3. TABLE 1. COMPOSITION OF TESTDIETS(EXPRESSED IN PERCENTAGES) Diet number
Ingredient
1. 2. 3. 4. 5. 6.
Wheat flour Corn flour Brewer’s yeast Naylor’s diet* Naylor’s B-vitamin mix. Amino acid mix.t
Total
1
2
3
4
5
100 -
100
95
99
100
5
-
-
-
-
100
100
7
8
1
100 -
95 5 -
99 1
95.4 _ 4.6
100
100
100
100
-
*See Tables 2 and 3. TContained: cyst., 0.37g; gluta., 3.298; proline, 0.82g; and tryp., 0.12g. TABLE 2. COMPOSITION OFNAYLOR’SMEDIUM Ingredient Casein Wesson’s salts Corn starch Sucrose Cholesterol
Percentage 15.00 4.00 64.00 16.00 1.00
TABLE 3. AMOUNTOF VITAMINS ANDZINCCHLORIDE IN NAYLOR’SMEDIUM (EXPRESSED INpg/g) Thiamine HCl Riboflavin Folic acid Pyridoxin HCl Nicotinic acid Ca pantothenate Choline chloride Biotin Insitol Dr.-carnitine Zinc chloride
6
25.0 12.5 2.5 12.5 50.0 25.0 500.0 250.0 0.5 6.0 200.0
100
ALEXANDER SOKOLOFF,IAN R. FRANKLIN and RAJ K. LAKHANPAL
316
The corn and whole wheat flour were supplemented with brewer’s yeast in a weight ratio of 19 : 1 to make up two of the “semisynthetic” diets (WY and CY). The same flours were supplemented with the vitamin mixture given in Table 3 to make up two additional semisynthetic diets (WV and CV). The protein contents of whole wheat flour (13.3 per cent) and cornmeal (7.8 per cent) differ considerably and the amino-acid compositions of their proteins also differ. Among the larger differences are those shown in Table 4 (derived from BLOCK and WEISS 1956, and expressed as mg per 16g protein nitrogen content of the diet) : TABLE
4.
DIFFERENCES IN CONTENT OF FOUR AMINO ACIDS IN TWO “NATURAL”
Amino acid Tryptophan Cystine Glutamic acid Proline *Derived of the diet.
from BLOCK and WEISS (1956)
Whole wheat
Cornmeal
1.2 3.3 31.4 10.3
0.7 1.6 18.4 7.2
and expressed
as mg per 16g protein
DIETS*
nitrogen
content
To bring up the level of these amino acids to the level found in whole wheat, corn flour obtained from the cornmeal was enriched with a mixture consisting of 0.1 lg tryptophan, 0.36g cystine, 3.28g glutamic acid and 0.81g proline, or 4.56g of this mixture for every 95.4g of corn medium (this diet will be symbolized CAA). All chemical additives were obtained from Nutritional Biochemical Corporation Cleveland, Ohio; brewer’s yeast was from Standard Brands Inc., Peekskill, New York; cornmeal and whole wheat flour from the Food Mill, Oakland, California. 2.1. Productivity The number of adult Fl that developed in each of the eight media was determined from two replicates, each containing five pairs of beetles. The latter were transferred to fresh containers twice a week for a period of 3 weeks to minimize cannibalism of eggs by the adults, and of larvae on smaller larvae or larvae on pupae. Each of The transfers were not of equal these periods will be referred to as a “transfer”. length: the odd-numbered transfers were 3 days and the even-numbered transfers 4 days in length. The yield for each replicate, transfer and the total for each diet is given in Table 5. The sex and date on which a parental beetle was found dead was recorded, and the data on productivity have been corrected if the dead beetle was a female. This procedure is somewhat different from that followed in the first paper of this series (SOKOLOFF et al., 1966), and it was adopted because the length of transfers was not equal. However, this procedure does not materially alter the conclusions, and the data from the two experiments can be reduced to a common denominator, as will be seen later.
Comparative
Studies with Tribolium (Coleoptera,
317
Tenebrionidae)-II
TABLE 5. PRODUCTIVITYOF 1. castaneum AND T. confuum (INEACHOFTWOREPLICATES,SIXTRANSPERS AND THE TOTAL) IN THE NAYLOR MEDIUM (NM), IN CORN (C) AND WHOLE WHEAT FLOUR (W) WITHAND wlTHOUTYEAsT(Y),VITAMINS (V)ORAMMO ACIDS(fbi)
Medium Transfer
Replicate
1 : 2 E 3 ; 4 z 5 Fl 6 : Total
a+b
NM
W
28* 9 28* 13 ll* 2 11* 7 4* 1 IO* 8*
81 31 79 63 78 47 71 59 82 34 83 46 -
-
132
754
2325
103 55 105 70 95 63 124 84 82 63 77 74 -
136 117 163 164 118 140 135 148 101 121 162 150 -
995
1655
16 11* 8* 6* 1* 2* 2* 13f 2*
;: 2 ; 3 1: 4 E 5 ;: 6
:: 127
: -
*Corrected tCorrected
atb
166 160 200 197 174 187 194 180 186 188 249 244
-
1
Total
WY
84
WV
C
T. castaneum 101 116 37 121 69 127 115 123 66 156 88* 146* 37 167 89* 162* 80 160 112* 141* 105 206 141* 189* 1040 1814 T. confusum 108 87 126 91 150 107 144 101 116 64 90 124 130 80 121 105 105 80 112 112 171 124 132 19 _ 1505 1094
CY
cv
CAA
87 90 133 135 125 137 110 188 144 144 164 184
99 66 77 79 109 68 91 67 125 125 132 145
2590
1641
1183
155 118 202 197 163 146 200 173 186 140 219 187
145 141 173 178 136 126 131 160 114 153 149 169
134 114 137 150 115 118 125 125 129 130 136 164
2086
1775
1577
163 170 167 187 251* 171 251* 190 272* 211 302* 255
for loss of one female. for loss of two females.
IS we define productivity as the number of eggs laid by a certain number of females in a given period and surviving to the adult stage, then it is immediately evident that the productivity of the two species of Tribolium in the Naylor medium (NM) is very low: only 132 T. castaneum and 84 T. confusum adult Fl were obtained for the whole 3-week period (six transfers) in the two replicates. Many factors could be invoked to explain such results, but the most likely explanation is that the NM is not suitable for the growth of the larvae or for survival of the adults (and yet, with the addition of brewer’s yeast the productivity of Tribolium exceeds that in whole wheat flour enriched with brewer’s yeast, as some unpublished experiments have shown).
318
ALEXANDER SOKOLOFF, IAN R. FRANKLIN and RAJ K. LAKHANPAL
The evidence that NM is not suitable for Tribolium is derived from these experiments and NAYLOR’S (1964) own data. In NAYLOR’S (1964) experiments, fifty-two to sixty out of sixty larvae survived to metamorphosis which would indicate the medium is adequate for development. However, the developmental periods were 35 per cent longer in NM than in the casein yeast controls, which would mean that the medium is nutritionally poor. In the present experiments the low productivity may be attributed to the following factors: (1) the medium may not be adequate to bring about normal development of the gonads, particularly in the female; (2) the medium may not provide the necessary requirements for the survival of the adults-as indicated in Table 5, 25 per cent of the parental females died within 3 weeks of the duration of the experiment; (3) adults may lay as many eggs as in other media, but cannibalism is intense; (4) the eggs laid may be numerous and the parents may not cannibalize them to a large extent, but the medium is poor for the development of the larvaethis last possibility is supported by NAYLOR’S (1964) and our data on developmental rates. Regardless of which factors may be in operation, it is evident that productivity as above defined is very low in Naylor’s medium. An analysis of variance of productivity in the Naylor medium showed that there was no significant difference between the two species. The numbers were, however, small. The analysis of variance for the seven remaining media is given in Table 6. While there are no significant differences between species summed over the whole experiment, there are significant differences in their performance on different media and productivity at different transfers as illustrated by the significant species x media and species ?: transfers interaction mean squares. 6. ANALYSIS OFVARIANCE
TABLE
Source
d.f.
Species (S) Media (M) SxM Groups (within S x M) (G) Transfers(T) SxT MxT MxSxT GxT Total *Significant tsignificant SSignificant
6 6 14
5 5 30 30 70 167
Sum
of squares
2592.857 234,491.060 66,941.060 30,160.OOO 31,678.619 16,826.072 17,671.298 9004.011 11,881.OoO 42 1,245.977
Mean squares 2592.86 39,081.84 11,156.84 2154.29 6335.72 3365.21 589.04 300.13 169.73
v.r. 1.20 n.s. 18.14: 5.18t (12.69t) 37.33$ 19.831 3.471 1.77* -
at the 0.05 level. at the 0.01level. at the 0 .OOllevel.
The productivity of these two species on the above media were further investigated The pairwise comparisons using SCHEFF~‘S (1953) multiple comparison test.
Comparative
Studies with Triboliun
(Coleoptera,
Tenebrionidae)-II
319
pooled over the two species and significant at the 10 per cent level, are: CY > CV, C, W; and CV>W (see Fig. 1). WV, CAA, C, W; WY>CAA,
c?
w
WY
CF
,
CY
C
WV
MEDIUM
FIG. I. Productivity of T. castawum (CS) and T. confusum (CF) expressed as a mean number of progeny averaged over six transfers in W=wheat flour; WY= wheat flout-f yeast; WV=wheat CV=corn+vitamins; CAA=corn+amino acids. flour-tvitamins; C=con; CY=corn+yeast;
The productivity on the corn and this difference was almost performance on corn was more shows the highest productivity than in wheat + yeast (WY).
media was larger than for wheat in both species, significant at the 10 per cent level. The higher striking for CF than for CA’. Corn + yeast (CY) in both species, but was not significantly higher
L 1! 3
4
5
6
TRANSFERS
FIG. 2. Productivity of 1. castaneum (CS) and 1. confurum (CF) expressed as mean number of progeny per five females in all the media at given transfer. Odd number of transfers are 3 days and even numbers are 4 days in length.
ALEXANDER SOKOLOFF,IAN R. FRANKLIN and RAJ K. LAKHANPAL
320
It is also apparent from these data that the increase in productivity as the result of yeast or vitamin supplements was greater in C’S than in CF, i.e. CS shows greater response to enrichment of the media than CF. With respect to transfer the two species react quite differently: productivity at transfer 2 is higher in CF, but at transfer 5 and 6 productivity is higher in C’S (see Fig. 2). There is a significant medium x transfer mean square, but contrasts were not calculated as no pattern is evident. 2.2.
Relative developmentalrates
The data summarized in Table 7, show the following. (1) Judging by the distribution of adults eclosing during the four periods observed, the Naylor medium is less favorable to CF than to CS. About 45 per cent of the C’S beetles had hatched by day 56, while only 13 per cent of CF had hatched by the same date. Three quarters of the CF beetles emerged in the 47-70-day period, while for CS in the corresponding interval 35 These differences are significant at the 0.1 per cent per cent emerged. level (x2, 1 d.f., = 24.28). (2) For the other media it is evident that nearly all the CF adults had hatched by day 42 in W, WY, WV, and CAA, and all had emerged by this day in CY and CV. A similar picture was obtained for CS in WY, WV and CY, while in CV there were sixty-one beetles (about 4 per cent) that hatched later. The most striking differences between CS and CF are in the W, C, and CAA. In W and C about 20 per cent of the CS beetles were delayed in reaching the imago stage by day 42, while the developmental rate in CAA was intermediate between that in the non-supplemented and the yeastThe differences in the two species are illustrated supplemented media. in Fig. 3.
NM
bv
WI
WV *IDI”Y
c
CI
C”
CIA
HY
w
Vf
WV
c
CI
C”
CAA
I,DI”I
FIG. 3. Relative developmental rates of T. castaneumand 1. confusum in eight media (NM=Naylor’s synthetic medium; for explanation of other symbols see caption to Fig. 1).
Studies with Tribolium (Coleoptera,
Comparative TABLE
7.
RELATIVE
RATES OF T. castaneum
DEVELOPMENTAL
IN THE VARIOUS
Medium
n
%
NM W WY WV C CY CV CAA
14 596 2324 1681 766 2373 1580 1059
NM W WY WV C CY cv CAA
0 991 1654 1093 1503 2086 1775 1575
12.61 79.05 99.96 99.64 80.29 99.87 96.28 89.52
%
36 145 1 6 159 3 60 116
32.43 19.23 0.04 0.36 16.67 0.13 3.66 9.81
8 4 1 1 2 0 0 2
12.90 0.40 0.06 0.09 0.13 0.13
AND
T. confurum
TEST MEDIA
Adults observed 56 _______ n
42
321
Tenebrionidae)-II
by day 70 %
n l-. castaneum 39 10 0 0 24 0 1 8
84 n
35.14 1.33 2.52 0.06 0.67
Total
%
22 3 0 0 5 0 0 0
19.82 0.40 0.52
111* 754 2325 1687” 954* 2376* 1641 1183
-
T. confusum
99.60 99.94 99.91 99.86 100 100 99.87
47 0 0 0 0 0 0 0
*These values differ from the total in Table 5 because account for shortages resulting from death of Fl females.
2.3.
Evaluation
11.29 -
75.81 -
-
in the latter
of productivity as a criterion for performance
62* 995 1655 1094 1505 2086 1775 1577
corrections
were made
to
in nutritional studies
Since the criterion of performance in this study and the previous one in this series (SOKOLOFF et al., 1966) has been chiefly productivity, it is of interest to examine how the data in the two experiments compare. Since the two experiments were of unequal length, the total productivity in equivalent media has been divided by 180 in the first and by 210 in the second experiment to convert the data to a per female-day basis. The results are shown in Table 8. TABLET.
PRODUCTIVITYOF T. castaneum (CS) AND 1. confurum (CF)
EXPERIMENTS(DATAAREPER
FEMALE-DAYBASIST~MAKETHE
Experiment
INFOURMEDIAINTWOSEPARATE
PERIOD
1
OFOBSERVATIONEQUAL)
Experiment
2
Medium CF
cs (ti f
c CY W WY *Standard
6.04 10.16 4.55 11.43
S.E.*)
(* (* (5 (*
errors, computed
0.86) 0.86) 0.86) 0.86)
(6 f
6.63 9.43 7.66 9.49
from analysis
(* (& (A (*
S.E.*)
0.86) 0.86) 0.86) 0.86)
of variance.
cs (6i f
4.95 12.33 3.59 11.07
CF
S.E.*)
(5 (5 (* (&
0.77) 0.77) 0.77) 0.77)
(5 f
7.17 9.93 4.74 7.88
S.E.*)
(* (& (3 (-&
0.77) 0.77) 0.77) 0.77)
322
ALEXA~VDER SoKotaFe, IAN R. FRANKLINand RAJ K. LAKHANPAL
The figures show remarkable agreement especially if it is remembered that only ten females were used for each medium. The greatest discrepancy is for CF in W and WY where the results in the second experiment were lower than in the first. Because of the large differences between replicates the sensitivity of future experiments could be considerably improved by increasing the number of adults per replicate and possibly by transferring the beetles more frequently to fresh creamers to minimize cannibalism. Nevertheless, the results are clearly promising enough and the labor involved is sufficiently small, to justify the use of this criterion for testing the performance of different species in various nutritional diets. The relative developmental rates of the beetles at arbitrary intervals in the test diets provide useful adjunct information as will be shown below. However, these intervals should perhaps be decreased so that adults are counted once or twice a week (instead of once every two weeks) and the counts begun on the 35th day instead of on the 42nd day after the parents are introduced. 3. DISCUSSION
The present study, like the first in this series (SOKOLOFF et al., 1966) establishes that the two species of flour beetles behave differently in the media used. CS produces fewer progeny than CF in corn flour (C) and whole wheat flour (W) when yeast is absent, but produces more than CF when yeast is added, The addition of vitamins to either kind of flour is a greater stimulus to productivity for CS than for CF. The addition of amino acids to corn flour had no appreciable effect on the productivity ofCF and caused an increase of productivity for C’S that was not significant. However, taking productivity and relative developmental rates as criteria for performance, the developmental rates in this it is clear that CS benefited from this supplement: medium were intermediate between those in corn without yeast and corn with yeast. No differences were detected for CF in these media. The previous study and the present one have shown that CS is a species which requires certain components of brewer’s yeast to a greater extent than CF. It was shown in the previous paper (SOKOLOFF et al., 1966) that white rice and brown rice with and without yeast differ in productivity, and it was inferred, from the known vitamin requirements for CF and the reported content of these vitamins and certain minerals, that these two media (particularly white rice) are deficient in thiamine, riboflavin and nicotinic acid. The productivity data indicate that CS requires more riboflavin and nicotinic acid than CF. PANT and GABRANI (1963) have shown that for CS. although CF requires thiamine, this vitamin is of secondary importance White and brown rice are also deficient in potassium for CF. If CS requires this cation, it probably needs it at a higher concentration than CF. The present study has demonstrated that the addition of 0.37g cystine, 3.29 glutathione, 0.82g proline and 0.12g tryptophan to corn flour did not benefit CF, but did benefit CS, since it improved productivity and the relative developmental rates of this species. A number of correlated studies are now available to help explain why CS is usually the winner in competition between strains of CS and CF. DAWSON (1964 and 1965) on the basis of selection experiments for fast and slow developmental rates in C’S and CF and the response of the two species following
Comparative Studies with Tribolium (Coleoptera, Tenebrionidae)-II
323
relaxation of selection has concluded that “the CF synthetic strain possesses relatively greater homeostatic properties than the corresponding CS strain”. CRENSHAW and LERNER (1964) have assayed productivity of strains of CS and CF inbred by brother-sister mating. Significant differences in means and variances in length of oviposition period were found for CS, but not for CF. For total productivity in 120 days, significant intraspecies, between strain variation in means and variances was found for both species. Daily productivity over a 40-day period revealed significant strain differences for CF, but interstrain differences in variance could not be established. On the other hand, in CS significant interstrain variation in means and variances was present. The previous study in this series (SOKOLOFF et al., 1966) and the present one indicate that the dietary requirements of CF are met better than those of CS in a wide variety of foods. SOKOLOFF et al. (1965) have shown that when eggs of the other species are supplied to CF or to CS adults, the latter species consumes nearly all the eggs of CF, but CF consumes fewer CS eggs. The result is that CS eventually eliminates CF. INOUYE and LERNER (1965) h ave shown that the elimination of CF proceeds at a slower rate in media supplemented with tryptophan than in unsupplemented media, apparently because CS obtains enough of this ingredient from the food and it does not have to eat eggs and pupae of CF to get it to the extent that it does in unsupplemented media. STANLEY (1938) sh owed that CF is more fecund when eggs are added in large numbers. Ho and DAWSON (1966) have shown that CS larvae are more cannibalistic than CF larvae, and that CS becomes more fecund than CF following egg cannibalism. The interest in this and the preceding study (SOKOLOFF et al., 1966) has centered on whether nutritional differences could be detected by the performance of the two species, by their productivity primarily in chemically undefined media. Such differences have now been demonstrated for a number of components of the diet, chiefly vitamins of the B-group and in certain amino acids. Considering that these are fairly closely related species, it is indeed surprising that the simple assays used in these experiments were sufficient to demonstrate these differences. On the other hand, that nutritional differences in the requirements of the two species of Tribolium exist is not surprising. SANG (1959) has shown that inbred strains of Drosophila melanogaster differ in their minimal vitamin and amino-acid requirements. In future studies it will be necessary to test various amino acids in varying quantities as additives in a synthetic medium such as that devised by NAYLOR (1964). Whether Ttibolium will be more amenable than Drosophila to reveal the taxonomic differences at the level of requirements rather than at the level of metabolism remains to be determined. assistance of Mesdames BARBARAB. DALY, MARJORIEA. HOY and Miss SYDNEYSMITH and the advice of Drs. HARLLEY MCKEAN and R. W. Howe is gratefully acknowleged. This investigation was supported by USPHS grant GM-08942. Acknowledgements-The
REFERENCES BLOCK, R. J. and WEISS, K. W. (1956) Methods and results of protein analysis. In Amino Acid Handbook. 386 pp. Thomas, Springfield, Ill. CRENSHAW,J. W. and LERNER, I. M. (1964) Productivity of inbred strains of Tribolium confmum and Tt+bolium castaneum. Ecology 45,697-705.
324
ALEXANDERSOKOLOFP,IAN R. FRANKLINand RAJ K. LAKHANPAL
DAW~ONP. S. (1964) The Genetics of Developmental Rate and its Relationship to Competitive Ability in Tri6uZium. 167 pp. Ph.D. Dissertation, University of California at Berkeley. DAWSON,P. S. (1965) Genetic homeostasis and developmental rate in Tribolium. Genetics 51, 873-885. Ho, F. K. and DAWSON,P. S. (1966) Egg cannibalism by Tribolium larvae. Ecology 47, (In press.) INOUYE,N. and LERNER,I. M. (1965) Competition between Tribolium species (Coleoptera, Tenebrionidae) on several diets. J. stored Prod. Res. 1, 185-191. LERNER,I. M. and Ho, F. K. (1961) Genotype and competitive ability of Tribolium species. Am. Nat. 95, 329-343. NAYLOR,A. F. (1964) Possible value of casein, gluten, egg albumin or fibrin as whole proteins in the diet of two strains of the flour beetle Tribolium confwum (Tenebrionidae). Can. 3. ZooI.42, l-9. PANT, N. C. and GABRANI, K. (1963) Qualitative vitamin B requirements of Tribolium castaneum Herbst. Indian 3. Ent. 25, 110-l 15. SANG,J. H. (1959) Circumstances affecting the nutritional requirements of Drosophila melanogaster. Ann. N. Y. Acad. Sci. 77, 352-365. SCHEFF~, H. (1953) A method for judging all contrasts in the analysis of variance. Biometrika 40, 87-104. SOKOLOFF, A., FRANKLIN, I. R. OVERTON, L. F. and Ho, F. K. (1966) Comparative studies with Tribolium-I: Productivity of T. castaneum and T. confusum in several commercially-available diets. 3. stored Prod. Res. 1, 295-311. SOKOLOFF, A., LERNER, I. M. and Ho, F. K. (1965) Self-elimination of T. castaneum following xenocide of 1. confusum. Am. Nat. 99, 399S404. STANLEY, J. (1938) The egg-producing capacity of populations of Tribolium confusum Duv. as affected by intensive cannibalistic egg-consumption. Can. 3. Res. 16, 300-306.
Stored Prod,
1966, Vol. 1, pp. 313-324.
Res.,
Pergamon Press Ltd.
Printed in Great Britain.
Comparative Studies with Tribolium (Coleoptera, Tenebrionidae) - II: Productivity of T. castaneum (Herbst) and T. confusum Duv. on Natural, Semi-synthetic and Synthetic Diets ALEXANDER
SOKOLOFF,
IAN R. FRANKLIN
and RAJ K. LAKHANPAL*
Departments of Genetics and Nutritional Science, University of California, Berkeley, California, U.S.A. (First receiued 15 September,
1965 and infinal form
16 December,
1965)
Abstract-Productivity of T. cnrtaneum and T. confwm has been determined in the following diets: Naylor’s synthetic medium; corn supplemented with the amino acids cystine, glutathione, proline and tryptophan in amounts necessary to bring them to the level found in wheat; corn and whole wheat flour without any supplements, and corn and whole wheat flour supplemented with brewer’s yeast and vitamins. Relative developmental rates of the two species in these media were also determined by counting the numbers of adults emerging in 14-day periods. Significant differences in productivity (the number of eggs surviving to the adult stage) have been detected within a species in the different media and between species within a given medium. The Naylor medium is unfavorable for both species, and judging from relative developmental rates less favorable to T. confwm than to 1. castaneum. ‘f: castaneum
performs poorest in non-supplemented media, better in media supplemented with vitamins, and the best in media supplemented with yeast. While T. confwum also shows an increased productivity in vitamin- and yeast-supplemented media, this increase is not as marked as in 1. castanezm. Considering both the productivity and the developmental rates, it appears that the addition of the four amino acids to corn had a beneficial effect to T. castaneum but not to T. conftcsum. The two species were most productive in corn plus yeast, but this productivity was not significantly higher than in whole wheat flour plus yeast. Drawing upon the evidence available in the literature, and the present data, it is concluded that 1. confusum is better adapted to utilize a wider variety of foods than T. castaneum and in order to satisfy its greater quantitative requirements for certain nutrients, this species engages in cannibalism more often than 1. confusum. The result is that, when the two species are introduced into the same container, i.e. compete for food and space, T. confuum is more often eliminated than 1. castaneum. *Present address: Department Colorado, U.S.A.
of Home Economics,
313
Colorado State University,
Fort Collins,
314
ALEXANDER SOKOLOFF, IAN R. FRANKLIN and RAJ K.
LAKHANPAL
1. INTRODUCTION IN THE first paper of this series (SOKOLOFFet al., 1966) the pertinent literature was reviewed and data on productivity and relative developmental rates were obtained for Tribolium castaneum (henceforth abbreviated CS) and T. confmum (CF) in several commercially available diets with and without yeast. In the present paper the same criteria for performance of C’S and CF are used to assess natural, semisynthetic and synthetic diets in order to substantiate further that nutritional differences exist in these two species of flour beetles. 2. MATERIALS AND METHODS The 1. castaneum (C’S) and T. confusum (CF) beetles used in these experiments as in the previous ones were derived from the stocks synthesized from a number of wild type strains in 1958 and maintained in large populations at the Department of Genetics, University of California, Berkeley, California. Details of the methods of synthesis and maintenance of these stocks are given by LERNER and Ho (1961). For any given species, freshly formed pupae were removed from the cultures, sexed, and fifteen males introduced into each of one series of eight creamers each containing a different test diet; fifteen females were introduced into each ofanother series of eight creamers containing these diets. The two series of creamers were returned to the incubator (maintained in darkness at 29°C and 70 per cent r.h.) Two weeks later, when the imagoes were about lo-days old, five female and five male beetles from each diet were introduced into each of two creamers containing 4g of this same diet. Each group of adults was transferred to a new creamer containing the same diet twice a week for a total of 3 weeks. Each group remained in one set of creamers for 3 days and in the next for 4 days. At each transfer the sex of every dead beetle was determined, but none was replaced. The creamers containing eggs were returned to the incubator to allow development of the progeny. Census of adults was begun in each medium at 42 days, and subsequent counts were made, ifjuvenile stages were still present, at 2-week intervals until all the prosupplies of synthetic or geny had died or reached the adult stage. Uninoculated semisynthetic media were kept under refrigeration until needed. No attempts were made to “acclimate” the medium to the conditions prevailing in the incubator, but the creamers were left at room temperature for a few hours before introducing the test beetles. A preliminary experiment was undertaken with three “synthetic” diets which have been developed for feeding mammals but was unsuccessful. These diets, GPD-2, MO-l and R-l are normally used for guinea pigs, mice and rats, respectively. Since flour beetles require a sterol in addition to the unsupplemented mammalian diets, a parallel experiment was performed to test these diets supplemented with 1% cholesterol. Neither species produced any progeny on any of these diets, and the adults introduced died during the 3-week experimental period. It was observed that the diets that contained large amounts of sucrose (MO-l and R-l) turned yellowish to brown and became lumpy. Rancidity developed in all the diets, possibly because of exposure of the oil to the high temperature and humidity in the incubator.
Comparative Studies with Tribolium (Coleoptera, Tenebrionidae)-II
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(2)
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successful experiments were performed with the following diets: “natural’‘-fine stoneground whole wheat flour on yellow cornmeal sifted through No. 5 silk bolting cloth (56 meshes/in.) to remove the large particles of bran or corn and to make the particle size uniform, with no additives; c‘semi-synthetic”-the same whole wheat flour (W) and yellow cornmeal (C) as above, but with the additives described below and summarized in Table 1; a synthetic medium (NM) reported by NAYLOR (1964) consisting of the various ingredients shown in Table 2 and supplemented with vitamins and zinc chloride at the levels indicated in Table 3. TABLE 1. COMPOSITION OF TESTDIETS(EXPRESSED IN PERCENTAGES) Diet number
Ingredient
1. 2. 3. 4. 5. 6.
Wheat flour Corn flour Brewer’s yeast Naylor’s diet* Naylor’s B-vitamin mix. Amino acid mix.t
Total
1
2
3
4
5
100 -
100
95
99
100
5
-
-
-
-
100
100
7
8
1
100 -
95 5 -
99 1
95.4 _ 4.6
100
100
100
100
-
*See Tables 2 and 3. TContained: cyst., 0.37g; gluta., 3.298; proline, 0.82g; and tryp., 0.12g. TABLE 2. COMPOSITION OFNAYLOR’SMEDIUM Ingredient Casein Wesson’s salts Corn starch Sucrose Cholesterol
Percentage 15.00 4.00 64.00 16.00 1.00
TABLE 3. AMOUNTOF VITAMINS ANDZINCCHLORIDE IN NAYLOR’SMEDIUM (EXPRESSED INpg/g) Thiamine HCl Riboflavin Folic acid Pyridoxin HCl Nicotinic acid Ca pantothenate Choline chloride Biotin Insitol Dr.-carnitine Zinc chloride
6
25.0 12.5 2.5 12.5 50.0 25.0 500.0 250.0 0.5 6.0 200.0
100
ALEXANDER SOKOLOFF,IAN R. FRANKLIN and RAJ K. LAKHANPAL
316
The corn and whole wheat flour were supplemented with brewer’s yeast in a weight ratio of 19 : 1 to make up two of the “semisynthetic” diets (WY and CY). The same flours were supplemented with the vitamin mixture given in Table 3 to make up two additional semisynthetic diets (WV and CV). The protein contents of whole wheat flour (13.3 per cent) and cornmeal (7.8 per cent) differ considerably and the amino-acid compositions of their proteins also differ. Among the larger differences are those shown in Table 4 (derived from BLOCK and WEISS 1956, and expressed as mg per 16g protein nitrogen content of the diet) : TABLE
4.
DIFFERENCES IN CONTENT OF FOUR AMINO ACIDS IN TWO “NATURAL”
Amino acid Tryptophan Cystine Glutamic acid Proline *Derived of the diet.
from BLOCK and WEISS (1956)
Whole wheat
Cornmeal
1.2 3.3 31.4 10.3
0.7 1.6 18.4 7.2
and expressed
as mg per 16g protein
DIETS*
nitrogen
content
To bring up the level of these amino acids to the level found in whole wheat, corn flour obtained from the cornmeal was enriched with a mixture consisting of 0.1 lg tryptophan, 0.36g cystine, 3.28g glutamic acid and 0.81g proline, or 4.56g of this mixture for every 95.4g of corn medium (this diet will be symbolized CAA). All chemical additives were obtained from Nutritional Biochemical Corporation Cleveland, Ohio; brewer’s yeast was from Standard Brands Inc., Peekskill, New York; cornmeal and whole wheat flour from the Food Mill, Oakland, California. 2.1. Productivity The number of adult Fl that developed in each of the eight media was determined from two replicates, each containing five pairs of beetles. The latter were transferred to fresh containers twice a week for a period of 3 weeks to minimize cannibalism of eggs by the adults, and of larvae on smaller larvae or larvae on pupae. Each of The transfers were not of equal these periods will be referred to as a “transfer”. length: the odd-numbered transfers were 3 days and the even-numbered transfers 4 days in length. The yield for each replicate, transfer and the total for each diet is given in Table 5. The sex and date on which a parental beetle was found dead was recorded, and the data on productivity have been corrected if the dead beetle was a female. This procedure is somewhat different from that followed in the first paper of this series (SOKOLOFF et al., 1966), and it was adopted because the length of transfers was not equal. However, this procedure does not materially alter the conclusions, and the data from the two experiments can be reduced to a common denominator, as will be seen later.
Comparative
Studies with Tribolium (Coleoptera,
317
Tenebrionidae)-II
TABLE 5. PRODUCTIVITYOF 1. castaneum AND T. confuum (INEACHOFTWOREPLICATES,SIXTRANSPERS AND THE TOTAL) IN THE NAYLOR MEDIUM (NM), IN CORN (C) AND WHOLE WHEAT FLOUR (W) WITHAND wlTHOUTYEAsT(Y),VITAMINS (V)ORAMMO ACIDS(fbi)
Medium Transfer
Replicate
1 : 2 E 3 ; 4 z 5 Fl 6 : Total
a+b
NM
W
28* 9 28* 13 ll* 2 11* 7 4* 1 IO* 8*
81 31 79 63 78 47 71 59 82 34 83 46 -
-
132
754
2325
103 55 105 70 95 63 124 84 82 63 77 74 -
136 117 163 164 118 140 135 148 101 121 162 150 -
995
1655
16 11* 8* 6* 1* 2* 2* 13f 2*
;: 2 ; 3 1: 4 E 5 ;: 6
:: 127
: -
*Corrected tCorrected
atb
166 160 200 197 174 187 194 180 186 188 249 244
-
1
Total
WY
84
WV
C
T. castaneum 101 116 37 121 69 127 115 123 66 156 88* 146* 37 167 89* 162* 80 160 112* 141* 105 206 141* 189* 1040 1814 T. confusum 108 87 126 91 150 107 144 101 116 64 90 124 130 80 121 105 105 80 112 112 171 124 132 19 _ 1505 1094
CY
cv
CAA
87 90 133 135 125 137 110 188 144 144 164 184
99 66 77 79 109 68 91 67 125 125 132 145
2590
1641
1183
155 118 202 197 163 146 200 173 186 140 219 187
145 141 173 178 136 126 131 160 114 153 149 169
134 114 137 150 115 118 125 125 129 130 136 164
2086
1775
1577
163 170 167 187 251* 171 251* 190 272* 211 302* 255
for loss of one female. for loss of two females.
IS we define productivity as the number of eggs laid by a certain number of females in a given period and surviving to the adult stage, then it is immediately evident that the productivity of the two species of Tribolium in the Naylor medium (NM) is very low: only 132 T. castaneum and 84 T. confusum adult Fl were obtained for the whole 3-week period (six transfers) in the two replicates. Many factors could be invoked to explain such results, but the most likely explanation is that the NM is not suitable for the growth of the larvae or for survival of the adults (and yet, with the addition of brewer’s yeast the productivity of Tribolium exceeds that in whole wheat flour enriched with brewer’s yeast, as some unpublished experiments have shown).
318
ALEXANDER SOKOLOFF, IAN R. FRANKLIN and RAJ K. LAKHANPAL
The evidence that NM is not suitable for Tribolium is derived from these experiments and NAYLOR’S (1964) own data. In NAYLOR’S (1964) experiments, fifty-two to sixty out of sixty larvae survived to metamorphosis which would indicate the medium is adequate for development. However, the developmental periods were 35 per cent longer in NM than in the casein yeast controls, which would mean that the medium is nutritionally poor. In the present experiments the low productivity may be attributed to the following factors: (1) the medium may not be adequate to bring about normal development of the gonads, particularly in the female; (2) the medium may not provide the necessary requirements for the survival of the adults-as indicated in Table 5, 25 per cent of the parental females died within 3 weeks of the duration of the experiment; (3) adults may lay as many eggs as in other media, but cannibalism is intense; (4) the eggs laid may be numerous and the parents may not cannibalize them to a large extent, but the medium is poor for the development of the larvaethis last possibility is supported by NAYLOR’S (1964) and our data on developmental rates. Regardless of which factors may be in operation, it is evident that productivity as above defined is very low in Naylor’s medium. An analysis of variance of productivity in the Naylor medium showed that there was no significant difference between the two species. The numbers were, however, small. The analysis of variance for the seven remaining media is given in Table 6. While there are no significant differences between species summed over the whole experiment, there are significant differences in their performance on different media and productivity at different transfers as illustrated by the significant species x media and species ?: transfers interaction mean squares. 6. ANALYSIS OFVARIANCE
TABLE
Source
d.f.
Species (S) Media (M) SxM Groups (within S x M) (G) Transfers(T) SxT MxT MxSxT GxT Total *Significant tsignificant SSignificant
6 6 14
5 5 30 30 70 167
Sum
of squares
2592.857 234,491.060 66,941.060 30,160.OOO 31,678.619 16,826.072 17,671.298 9004.011 11,881.OoO 42 1,245.977
Mean squares 2592.86 39,081.84 11,156.84 2154.29 6335.72 3365.21 589.04 300.13 169.73
v.r. 1.20 n.s. 18.14: 5.18t (12.69t) 37.33$ 19.831 3.471 1.77* -
at the 0.05 level. at the 0.01level. at the 0 .OOllevel.
The productivity of these two species on the above media were further investigated The pairwise comparisons using SCHEFF~‘S (1953) multiple comparison test.
Comparative
Studies with Triboliun
(Coleoptera,
Tenebrionidae)-II
319
pooled over the two species and significant at the 10 per cent level, are: CY > CV, C, W; and CV>W (see Fig. 1). WV, CAA, C, W; WY>CAA,
c?
w
WY
CF
,
CY
C
WV
MEDIUM
FIG. I. Productivity of T. castawum (CS) and T. confusum (CF) expressed as a mean number of progeny averaged over six transfers in W=wheat flour; WY= wheat flout-f yeast; WV=wheat CV=corn+vitamins; CAA=corn+amino acids. flour-tvitamins; C=con; CY=corn+yeast;
The productivity on the corn and this difference was almost performance on corn was more shows the highest productivity than in wheat + yeast (WY).
media was larger than for wheat in both species, significant at the 10 per cent level. The higher striking for CF than for CA’. Corn + yeast (CY) in both species, but was not significantly higher
L 1! 3
4
5
6
TRANSFERS
FIG. 2. Productivity of 1. castaneum (CS) and 1. confurum (CF) expressed as mean number of progeny per five females in all the media at given transfer. Odd number of transfers are 3 days and even numbers are 4 days in length.
ALEXANDER SOKOLOFF,IAN R. FRANKLIN and RAJ K. LAKHANPAL
320
It is also apparent from these data that the increase in productivity as the result of yeast or vitamin supplements was greater in C’S than in CF, i.e. CS shows greater response to enrichment of the media than CF. With respect to transfer the two species react quite differently: productivity at transfer 2 is higher in CF, but at transfer 5 and 6 productivity is higher in C’S (see Fig. 2). There is a significant medium x transfer mean square, but contrasts were not calculated as no pattern is evident. 2.2.
Relative developmentalrates
The data summarized in Table 7, show the following. (1) Judging by the distribution of adults eclosing during the four periods observed, the Naylor medium is less favorable to CF than to CS. About 45 per cent of the C’S beetles had hatched by day 56, while only 13 per cent of CF had hatched by the same date. Three quarters of the CF beetles emerged in the 47-70-day period, while for CS in the corresponding interval 35 These differences are significant at the 0.1 per cent per cent emerged. level (x2, 1 d.f., = 24.28). (2) For the other media it is evident that nearly all the CF adults had hatched by day 42 in W, WY, WV, and CAA, and all had emerged by this day in CY and CV. A similar picture was obtained for CS in WY, WV and CY, while in CV there were sixty-one beetles (about 4 per cent) that hatched later. The most striking differences between CS and CF are in the W, C, and CAA. In W and C about 20 per cent of the CS beetles were delayed in reaching the imago stage by day 42, while the developmental rate in CAA was intermediate between that in the non-supplemented and the yeastThe differences in the two species are illustrated supplemented media. in Fig. 3.
NM
bv
WI
WV *IDI”Y
c
CI
C”
CIA
HY
w
Vf
WV
c
CI
C”
CAA
I,DI”I
FIG. 3. Relative developmental rates of T. castaneumand 1. confusum in eight media (NM=Naylor’s synthetic medium; for explanation of other symbols see caption to Fig. 1).
Studies with Tribolium (Coleoptera,
Comparative TABLE
7.
RELATIVE
RATES OF T. castaneum
DEVELOPMENTAL
IN THE VARIOUS
Medium
n
%
NM W WY WV C CY CV CAA
14 596 2324 1681 766 2373 1580 1059
NM W WY WV C CY cv CAA
0 991 1654 1093 1503 2086 1775 1575
12.61 79.05 99.96 99.64 80.29 99.87 96.28 89.52
%
36 145 1 6 159 3 60 116
32.43 19.23 0.04 0.36 16.67 0.13 3.66 9.81
8 4 1 1 2 0 0 2
12.90 0.40 0.06 0.09 0.13 0.13
AND
T. confurum
TEST MEDIA
Adults observed 56 _______ n
42
321
Tenebrionidae)-II
by day 70 %
n l-. castaneum 39 10 0 0 24 0 1 8
84 n
35.14 1.33 2.52 0.06 0.67
Total
%
22 3 0 0 5 0 0 0
19.82 0.40 0.52
111* 754 2325 1687” 954* 2376* 1641 1183
-
T. confusum
99.60 99.94 99.91 99.86 100 100 99.87
47 0 0 0 0 0 0 0
*These values differ from the total in Table 5 because account for shortages resulting from death of Fl females.
2.3.
Evaluation
11.29 -
75.81 -
-
in the latter
of productivity as a criterion for performance
62* 995 1655 1094 1505 2086 1775 1577
corrections
were made
to
in nutritional studies
Since the criterion of performance in this study and the previous one in this series (SOKOLOFF et al., 1966) has been chiefly productivity, it is of interest to examine how the data in the two experiments compare. Since the two experiments were of unequal length, the total productivity in equivalent media has been divided by 180 in the first and by 210 in the second experiment to convert the data to a per female-day basis. The results are shown in Table 8. TABLET.
PRODUCTIVITYOF T. castaneum (CS) AND 1. confurum (CF)
EXPERIMENTS(DATAAREPER
FEMALE-DAYBASIST~MAKETHE
Experiment
INFOURMEDIAINTWOSEPARATE
PERIOD
1
OFOBSERVATIONEQUAL)
Experiment
2
Medium CF
cs (ti f
c CY W WY *Standard
6.04 10.16 4.55 11.43
S.E.*)
(* (* (5 (*
errors, computed
0.86) 0.86) 0.86) 0.86)
(6 f
6.63 9.43 7.66 9.49
from analysis
(* (& (A (*
S.E.*)
0.86) 0.86) 0.86) 0.86)
of variance.
cs (6i f
4.95 12.33 3.59 11.07
CF
S.E.*)
(5 (5 (* (&
0.77) 0.77) 0.77) 0.77)
(5 f
7.17 9.93 4.74 7.88
S.E.*)
(* (& (3 (-&
0.77) 0.77) 0.77) 0.77)
322
ALEXA~VDER SoKotaFe, IAN R. FRANKLINand RAJ K. LAKHANPAL
The figures show remarkable agreement especially if it is remembered that only ten females were used for each medium. The greatest discrepancy is for CF in W and WY where the results in the second experiment were lower than in the first. Because of the large differences between replicates the sensitivity of future experiments could be considerably improved by increasing the number of adults per replicate and possibly by transferring the beetles more frequently to fresh creamers to minimize cannibalism. Nevertheless, the results are clearly promising enough and the labor involved is sufficiently small, to justify the use of this criterion for testing the performance of different species in various nutritional diets. The relative developmental rates of the beetles at arbitrary intervals in the test diets provide useful adjunct information as will be shown below. However, these intervals should perhaps be decreased so that adults are counted once or twice a week (instead of once every two weeks) and the counts begun on the 35th day instead of on the 42nd day after the parents are introduced. 3. DISCUSSION
The present study, like the first in this series (SOKOLOFF et al., 1966) establishes that the two species of flour beetles behave differently in the media used. CS produces fewer progeny than CF in corn flour (C) and whole wheat flour (W) when yeast is absent, but produces more than CF when yeast is added, The addition of vitamins to either kind of flour is a greater stimulus to productivity for CS than for CF. The addition of amino acids to corn flour had no appreciable effect on the productivity ofCF and caused an increase of productivity for C’S that was not significant. However, taking productivity and relative developmental rates as criteria for performance, the developmental rates in this it is clear that CS benefited from this supplement: medium were intermediate between those in corn without yeast and corn with yeast. No differences were detected for CF in these media. The previous study and the present one have shown that CS is a species which requires certain components of brewer’s yeast to a greater extent than CF. It was shown in the previous paper (SOKOLOFF et al., 1966) that white rice and brown rice with and without yeast differ in productivity, and it was inferred, from the known vitamin requirements for CF and the reported content of these vitamins and certain minerals, that these two media (particularly white rice) are deficient in thiamine, riboflavin and nicotinic acid. The productivity data indicate that CS requires more riboflavin and nicotinic acid than CF. PANT and GABRANI (1963) have shown that for CS. although CF requires thiamine, this vitamin is of secondary importance White and brown rice are also deficient in potassium for CF. If CS requires this cation, it probably needs it at a higher concentration than CF. The present study has demonstrated that the addition of 0.37g cystine, 3.29 glutathione, 0.82g proline and 0.12g tryptophan to corn flour did not benefit CF, but did benefit CS, since it improved productivity and the relative developmental rates of this species. A number of correlated studies are now available to help explain why CS is usually the winner in competition between strains of CS and CF. DAWSON (1964 and 1965) on the basis of selection experiments for fast and slow developmental rates in C’S and CF and the response of the two species following
Comparative Studies with Tribolium (Coleoptera, Tenebrionidae)-II
323
relaxation of selection has concluded that “the CF synthetic strain possesses relatively greater homeostatic properties than the corresponding CS strain”. CRENSHAW and LERNER (1964) have assayed productivity of strains of CS and CF inbred by brother-sister mating. Significant differences in means and variances in length of oviposition period were found for CS, but not for CF. For total productivity in 120 days, significant intraspecies, between strain variation in means and variances was found for both species. Daily productivity over a 40-day period revealed significant strain differences for CF, but interstrain differences in variance could not be established. On the other hand, in CS significant interstrain variation in means and variances was present. The previous study in this series (SOKOLOFF et al., 1966) and the present one indicate that the dietary requirements of CF are met better than those of CS in a wide variety of foods. SOKOLOFF et al. (1965) have shown that when eggs of the other species are supplied to CF or to CS adults, the latter species consumes nearly all the eggs of CF, but CF consumes fewer CS eggs. The result is that CS eventually eliminates CF. INOUYE and LERNER (1965) h ave shown that the elimination of CF proceeds at a slower rate in media supplemented with tryptophan than in unsupplemented media, apparently because CS obtains enough of this ingredient from the food and it does not have to eat eggs and pupae of CF to get it to the extent that it does in unsupplemented media. STANLEY (1938) sh owed that CF is more fecund when eggs are added in large numbers. Ho and DAWSON (1966) have shown that CS larvae are more cannibalistic than CF larvae, and that CS becomes more fecund than CF following egg cannibalism. The interest in this and the preceding study (SOKOLOFF et al., 1966) has centered on whether nutritional differences could be detected by the performance of the two species, by their productivity primarily in chemically undefined media. Such differences have now been demonstrated for a number of components of the diet, chiefly vitamins of the B-group and in certain amino acids. Considering that these are fairly closely related species, it is indeed surprising that the simple assays used in these experiments were sufficient to demonstrate these differences. On the other hand, that nutritional differences in the requirements of the two species of Tribolium exist is not surprising. SANG (1959) has shown that inbred strains of Drosophila melanogaster differ in their minimal vitamin and amino-acid requirements. In future studies it will be necessary to test various amino acids in varying quantities as additives in a synthetic medium such as that devised by NAYLOR (1964). Whether Ttibolium will be more amenable than Drosophila to reveal the taxonomic differences at the level of requirements rather than at the level of metabolism remains to be determined. assistance of Mesdames BARBARAB. DALY, MARJORIEA. HOY and Miss SYDNEYSMITH and the advice of Drs. HARLLEY MCKEAN and R. W. Howe is gratefully acknowleged. This investigation was supported by USPHS grant GM-08942. Acknowledgements-The
REFERENCES BLOCK, R. J. and WEISS, K. W. (1956) Methods and results of protein analysis. In Amino Acid Handbook. 386 pp. Thomas, Springfield, Ill. CRENSHAW,J. W. and LERNER, I. M. (1964) Productivity of inbred strains of Tribolium confmum and Tt+bolium castaneum. Ecology 45,697-705.
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ALEXANDERSOKOLOFP,IAN R. FRANKLINand RAJ K. LAKHANPAL
DAW~ONP. S. (1964) The Genetics of Developmental Rate and its Relationship to Competitive Ability in Tri6uZium. 167 pp. Ph.D. Dissertation, University of California at Berkeley. DAWSON,P. S. (1965) Genetic homeostasis and developmental rate in Tribolium. Genetics 51, 873-885. Ho, F. K. and DAWSON,P. S. (1966) Egg cannibalism by Tribolium larvae. Ecology 47, (In press.) INOUYE,N. and LERNER,I. M. (1965) Competition between Tribolium species (Coleoptera, Tenebrionidae) on several diets. J. stored Prod. Res. 1, 185-191. LERNER,I. M. and Ho, F. K. (1961) Genotype and competitive ability of Tribolium species. Am. Nat. 95, 329-343. NAYLOR,A. F. (1964) Possible value of casein, gluten, egg albumin or fibrin as whole proteins in the diet of two strains of the flour beetle Tribolium confwum (Tenebrionidae). Can. 3. ZooI.42, l-9. PANT, N. C. and GABRANI, K. (1963) Qualitative vitamin B requirements of Tribolium castaneum Herbst. Indian 3. Ent. 25, 110-l 15. SANG,J. H. (1959) Circumstances affecting the nutritional requirements of Drosophila melanogaster. Ann. N. Y. Acad. Sci. 77, 352-365. SCHEFF~, H. (1953) A method for judging all contrasts in the analysis of variance. Biometrika 40, 87-104. SOKOLOFF, A., FRANKLIN, I. R. OVERTON, L. F. and Ho, F. K. (1966) Comparative studies with Tribolium-I: Productivity of T. castaneum and T. confusum in several commercially-available diets. 3. stored Prod. Res. 1, 295-311. SOKOLOFF, A., LERNER, I. M. and Ho, F. K. (1965) Self-elimination of T. castaneum following xenocide of 1. confusum. Am. Nat. 99, 399S404. STANLEY, J. (1938) The egg-producing capacity of populations of Tribolium confusum Duv. as affected by intensive cannibalistic egg-consumption. Can. 3. Res. 16, 300-306.