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Uric acid production in relation to protein metabolism in the silkworm, Bombyx mori, during pupal-adult development
Insect Biochem., x97I, I, z49-z63. [Scientechnica(Publishers)Ltd.]
z49
URIC ACID PRODUCTION IN RELATION TO PROTEIN METABOLISM IN THE SILKWORM, BOMB Y X MORI, DURING PUPAL-ADULT DEVELOPMENT SUMIO TOJO* Laboratory of Applied Entomology, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo, Japan
(Received 8 Aug., I97 o) ABSTRACT Accumulation and distribution of uric acid in the silkworm, Bombyx mori, were studied in relation to histolysis and histogenesis during the pupal period. In the male uric acid increased rapidly in the mid-pupal stage and again shortly before emergence, whereas in the female it was maintained at a constant level until the late pharate adult stage, when a rapid increase occurred. The rapid production of uric acid at these times was also shown by incorporation of ~'C into uric acid in pupae injected with 2-~C-glycine at various stages. The two sexes also differed in the changes of total protein content: in the male protein began to decrease from the mid-pupal stage, while in the female it changed little until shortly before emergence and then decreased rapidly. The sum of the nitrogen lost from protein and free amino-acids during the pupal period accounted for the nitrogen gained in uric acid. These results indicate that the lost protein is catabolized to uric acid via the amino-acid pool. When protein was labelled by injection of 2.x'C-glycine into male early pupae, ~4C was lost from protein and appeared in uric acid from the mid-pupal stage. In the female, 14C in both protein and uric acid changed only slightly. In the female silkworm a substantial amount of protein was transferred from the fat body to ovaries and other adult tissues in the mid-pupal stage, while total protein was essentially constant. The decrease of protein and the production of uric acid in the same stage of the male is believed to be due to the small development of the testes compared with the ovaries. Injected 2-~'C-uric acid was not metabolized any further, from which it is concluded that uric acid is really the end-product of nitrogen metabolism in silkworm pupae. Uric acid was distributed mainly in the fat body in the early half of the pupal period, but in the mid-pupa it was largely transferred to the rectal sac. After eclosion it was excreted with the meconium. THE occurrence of uric acid in the excreta of insects has been shown by m a n y investigators, as reviewed by Bursell (1967). As to uric acid formation in insects, however, knowledge is limited at present, except on the purine catabolic pathway (Gilmour, i96i ). T h a t nitrogen of uric acid originates f r o m amino-acids is supported b y the incorporation of labelled amino-acids into uric acid (McEnroe and Forgash, I957, i958; Brenner-Holzach and Leuthart, x96i , i965; Barrett and Friend, i97o), by nutritional experiments in which uric acid production was enhanced by rearing insects on diets of high protein content (Haydak, i953; Ito and Mukaiyama, 1964) , and also b y the *
Present address: Department of Biology, Yale University, New Haven, Connecticut, U.S.A.
250
TOJO
Insect Biochem.
correlation between nitrogen loss f r o m amino-acids and nitrogen gain in uric acid during the pupal period (Birt and Christian, r969). Endopterygote insects form a closed system with respect to nitrogen in the embryonic and pupal stages, and in m a n y cases during diapause. Such stages are very suitable for the study of nitrogen catabolism because metabolic relationships can be elucidated simply by comparing the accumulation of end-products with changes in other nitrogenous compounds. Moreover, analysis of when and how excretory nitrogenous compounds are produced during a closed stage is an important approach to elucidation of the mechanisms for conservation of reserves during differentiation and development. I n previous papers (Tojo and Hirano, x966 , i968), the accumulation of uric acid was shown to reflect the general metabolic activity of nitrogenous compounds in the pupae of Mamestra brassicae and the mature larvae of Chilo suppressalis. During diapause uric acid production was maintained at a low level, b u t during post-diapause development rapid accumulation occurred. I n this paper uric acid production is investigated in the silkworm, Bombyx morL during the pupal period,* mainly in relation to protein metabolism. Also, the incorporation of labelled amino-acids into protein and uric acid and the metabolism of 1*C-uric acid in vivo are examined. MATERIALS AND M E T H O D S ANIMALS
Silkworms of various strains were reared on mulberry leaves, and were selected at the time of pupation so as to give groups with not more than 6 hours' variation in age. When unavoidable, pupae were stored at 5° C. for not more than 24 hours in order to obtain animals of appropriate age. The grouped pupae were kept at 25 ° C. until use. The approximate pupal weight of experimental animals was x g. for males and I"5 g. for females. INJECTIONOF LABELLED COMPOUNDS For isotope incorporation studies, labelled materials dissolved in 2 l~l. of o'o67 M phosphate buffer, p H 7"o, were injected into the haemocoel of pupae under carbon dioxide anaesthesia through the interscgmental membrane of the lateral abdomen by means of a microsyringe. These treated pupae were kept at 25 ° C. until sacrifice. Labelled compounds used were as follows: 2-xIC-uric acid (specific activity 52"o me. per mmole), purchased from the Radiochemical Centre, Ameraham; and 2-x4C-glycine and U-x*Cleucine (specific activity 4'x2 and 6o.o me. per mmole respectively), prepared by the Dai-ichi Pure Chemical Co., Tokyo. DISSECTION OF TISSUES
Groups of 5 female pupae were used for each analysis. Blood was collected in a test-tube by applying gentle pressure on the abdomen after a puncture had been made by a needle at the prothorax region. The animals were then dissected along a ventral line, and remaining blood was washed out with ice-cold Ringer solution (2 per cent NaCI, o'2 per cent KC1, 0"04 per cent MgCI~, 0"08 per cent CaC10. The fat body, the ovary, the gut, and the rectal sac (with meconium) were each taken into Potter-Elvehjem glass homogenizers. The remaining bodies were put in a homogenizer and called the 'integument and muscle fraction'. This included the imaginal tissues such as wings, legs, muscles, etc., together with the integument. EXTRACTION OF URIC ACID
For extraction of uric acid from whole insects, groups of 5 pupae were homogenized with 5 volumes of water. The homogenate was heated at 9°0 C. for 3 minutes, and was centrifuged at * In this paper, for convenience, the entire period from the larval-pupal ecdysis to the pupaladult ecdysis is referred to as the 'pupal' stage; this includes both the pupa and the pharate adult.
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3000 r.p.m, for to minutes. After the supernatant was removed, the residue was re-extracted with water three times and the supernatants were combined and filtered. T h e filtrate was applied to a column for chromatography. For the respective tissues dissected as described above the extraction was repeated two or three times with 5 - I o ml. of water. For extraction from I ml. of blood, two portions of x ml. of water were used. Uric acid in the extracts was separated on an ion-exchange resin (Dowex i × io, formate type; I × 4-cm. column) and measured by ultra-violet absorption at z8o rag, essentially as described by Tojo and Hirano (I966). In tracer experiments, samples of each fraction from ion-exchange chromatography were placed in steel planchettes, dried, and counted in a gas-flow counter. SEPARATION OF FREE AMINO-ACIDS,URIC ACID, AND PROTEIN In the experiments to separate these three compounds, groups of pupae were homogenized with 5 volumes of 80 per cent ethanol and heated at 7 °o C. for 3 minutes, followed by centrifugation at 3ooo r.p.m, for io minutes. T h e supernatant was transferred to a flask, the residue was extracted twice more with 80 per cent ethanol, and the three extracts were combined. Aliquots were then used to determine total free amino-acids. If necessary, in order to separate uric acid from aminoacids, portions of the extract were added to a 0'5 x 3-cm. column of Dowex x × 1o (formate). Then amino-acids were eluted by 50 ml. of 80 per cent ethanol, the fractions being used for amino-acid determination and radioassay (by this procedure 2o-3o per cent of total free aminoacids were lost, but almost all the radioactivity in the amino-acid pool, derived from previously injected 2-1'C-glycine or U-l'C-leucine, was recovered). T h e next eluate by o.ox M formic acid was used to measure uric acid, based on absorption at z8o ml~. T h e residue from the extraction with 80 per cent ethanol was used for uric acid extraction by the procedure described above, using three portions of 30 ml. of water. Uric acid in the water extract was determined after chromatography on a I x 4-cm. column with Dowex x × Io. Total uric acid was obtained by addition of that in the ethanol extract and that in the water extract. T h e pellet which remained after the extraction with water was suspended in 2o ml. of 5 per cent perchloric acid and heated at 9 °0 C. for i o minutes, followed by centrifugation. After decanting the supernatant the residue was washed again with perchloric acid, heated as before. T h e n the pellet was successively washed at room temperature, twice with 3° ml. of ethanol/ether (3 : i) and then twice with 30 ml. of ether. From the residue thus obtained protein was extracted three times (3o ml. each) with o'25 M sodium hydroxide at 7 °° C. for xo minutes, and the extracts were combined. Portions were used for determination of total protein and for radioassay. In the experiment to investigate the changes of protein content in tissues, the extraction procedure for amino-acids was omitted. Uric acid was removed by hot water from the respective tissues separated from 5 animals. T h e remaining pellets were successively washed with hot 5 per cent perchloric acid, ethanol/ether, and then with ether as described above, and protein was extracted with o'25 M sodium hydroxide.
Amino-acids Amino-acids were analysed by the ninhydrin reaction (Yemm and Cocking, t954) using L-leucine as the standard.
Protein Protein was analysed by the method of Lowry, Rosenbrough, Farr, and Randall (x95 I), with albumin as the standard.
Radioactivity Radioactivity was measured in a windowless gas-flow counter, and was corrected for selfabsorption. RESULTS FATE OF INJECTED 2-14C-URIc ACID 2-14C-Uric a c i d w a s i n j e c t e d into p u p a e o f t h e 5"4 × 2"4 h y b r i d s i l k w o r m (a h y b r i d b e t w e e n C I 15 × N I 2 4 a n d N I 2 2 × C124) w i t h i n 12 h o u r s after p u p a t i o n , a n d g r o u p s o f t h e insects w e r e u s e d 2, 5, 8, a n d I i d a y s after t h e t r e a t m e n t . B y t h e e x t r a c t i o n p r o c e d u r e o f u r i c a c i d w i t h 5 v o l u m e s o f h o t w a t e r d e s c r i b e d above, 57"8, 3o'8, 7"9, 2.8, a n d
252
Insect Biochem.
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0.6 per cent of the injected radioactivity was recovered in the first, second, third, fourth, and fifth supernatant fluids respectively from female pupae 8 days after the injection. Extractions with hot 5 per cent perchloric acid, ethanol/ether (3 : i), ether, and 0"25 M sodium hydroxide were then performed in this order on the residue from the water extraction, but only traces of radioactivity were found in these extracts. It is therefore concluded that three or four extractions with water are sufficient for total recovery of uric acid. In Fig. I is shown an elution chromatogram of water extract from female pupae 8 days after 2-14C-uric acid injection. Peak D is the effluent position of uric acid. The elution
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Fraction number FIG. I.--Chromatogram of the hot-water extract of the female pharate adults of the silkworm injected with 2-14C-uric acid 8 days before, i.e., shortly after pupation. Peak D corresponds to uric acid. Silkworm: 5-4×2"4 . Resin: Dowex x×xo, formate type. Column: t ×4 cm. Solvent in stepwise elution: I, o'oo4 M formic acid; II, o.oi M formic acid; III, o'3 M formic acid. Fraction volume: 5 ml.
pattern of peak D by U.V. absorption coincides well with that by radioactivity, clearly indicating that U.V. absorption in this fraction is mainly due to uric acid. Peak C corresponds to the eluting position of ribosyluric acid as described in the previous paper (Tojo and Hirano, x968). Significant amounts of radioactivity were incorporated in peak C when Chilo suppressalis larvae were injected with 2-14C-uric acid (Tojo and Hirano, x97x), but none can be detected in this area from the silkworm pupa. Peak E is the elution area of an unknown metabolite of uric acid which characteristically exists in the wings of butterflies (Tojo and Yushima, z97t ), but only a trace .amount of label is detected in peak E. In Table I is given the distribution of radioactivity in the respective peaks on the chromatograms at various times after injection. In all cases almost all of the
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URIC ACID PRODUCTIONIN THE SILKWORM
253
radioactivity resides in peak D, that is, uric acid. It can be concluded that uric acid is a true end-product of nitrogen catabolism in silkworm pupae. DISTRIBUTION OF URIc ACID IN VARIOUSTISSUES As is shown in F~. z, in the female silkworm of 5"4 × 2"4 hybrid a large part of uric acid is distributed in the fat body and a small part in the integument and muscle fraction during the early half of pupal period. 2-1~C-Uric acid which has been injected into the I-day-old pupae disappeared rapidly from the blood and was concentrated mainly in the fat body. The accumulation pattern in the tissues changes strikingly from the mid-pupal
Table /.~DISTRIBUTION OF RADIOACTIVITYON THE COLUMNCHROMATOGRAMOF THE WATER EXTRACTOF THE SILKWORM,INJECTEDWITH2-taC-URIc ACIDSHORTLYAFTERPUPATION*
SEX
DAYSAFTER LARVAL-PUPAL ECDYSlS
Female II
(Adult)
Male
RECOVERY OF RADIOACTIVITY
(c.p.m.)t
TI~UE Peak A
Peak O
Peak E
Total
Whole Whole Whole
650 73° 82o
132,3OO 133,7OO 13o,2oo
115o 125o 950
I34,1oo • 135,68o
Body Wings Eggs Meconium
38 15 34 45°
314o 890 513o 116,11o
68 25 82 750
3246 930 5246 117,3Io
Total
537
I25,27o
925
126,732
Whole Whole Whole
4oo 860 67o
135,100
7oo 134o 950
136,2oo x34,6oo 131,220
132,4oo I29,6oo
131,020
* Race: 5"4 × 2"4.
The water extract was fractionated on a Dowex i activity distributed in each peak was measured.
×
IO column, as shown in Fig. I, and radio-
age. Uric acid decreases considerably from the fatbody and is transferred mainly to the rectal sac. Although it increases somewhat in the ovary and the integument and muscle fraction, it disappears from these tissues near emergence. In the final pharate adult almost all the uric acid exists in the rectal sac, and it is excreted with the meconium after emergence. Changes in the distribution of radioactivity from injected 2-14C-uric acid among tissues are quite similar to the changes of uric acid itself. It is noted that the concentration of z-14C-uric acid in blood is maintained at a nearly constant level, suggesting the existence of a regulatory mechanism. ACCUMULATIONOF URIC ACID IN THE WHOLE INSECT Uric acid contents in the silkworms of various strains or hybrids were investigated during the pupal period. In these experiments the uric acid concentrations in different stages were calculated on the basis of the weights just after pupation, in order to correct for weight-loss. The results are shown in Fig. 3, where stages are expressed as
254
Insect Biochem.
TOJO
percentages of pupal duration to make it easy to compare the patterns in different strains. The level of uric acid in the early pupa varies among strains, but the accumulation pattern is almost the same in the same sex of each strain. It is noteworthy that in the male, uric acid rapidly increases in the mid-pupal stage and increases further shortly before emergence, but in the female it does not change significantly until shortly before emergence, when rapid production occurs.
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Fla. 2.--Changes in the distributions of uric acid (A) and radioactivity of injected 2-14C-uric acid (B) among various tissues during pupal stage of the female silkworm. 2-z~C-Uric acid was injected into x-day-old pupae. Silkworm: 5.4×2.4 ('pupal' period, i x days). Integument and muscle fraction consists of the integument and the imaginal tissues such as wings, muscles, legs, etc. Values given for z4C in blood are on the basis of o'5 ml. of blood which was presumed to be contained in a pupa (z'5 g. wet weight). ®, Fat body; A, meconium; ×, integument and muscle; O, gut; , , ovary; O, blood. RELATIoNsHIP BETWEENURIC:ACID PRODUCTIONAND PROTEIN METABOLISM Fig. 4 shows the changes in the levels of free amino-acids and protein in relation to those of uric acid in the silkworms during metamorphosis. Also in these experiments the concentrations of these compounds were corrected for weight-loss. Although different strains were used for the two sexes, the comparison of patterns of nitrogenous compounds between sexes should be valid, since histolysis and histogenesis occur almost similarly in the same sex of both strains. In the male pupae the protein content is about 68 mg. per g. wet weight in the early half of the pupal period, then it decreases significantly from the mid-pupal age nearly at the time of the rapid uric acid production, reaching about 50 rag. per g. wet weight just before emergence. In the female pupae, on the other hand, the concentration of
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URIC ACID PRODUCTION
IN THE SILKWORM
255
protein remains almost unchanged around a level of 8o rag. per g. wet weight until 9 ° per cent of pupal duration, but it then decreases sharply to less than 7° nag. per g. at the same time that rapid production of uric acid is observed. As to amino-acids it may be said that the pool size does not significantly change, although it decreases to some degree in the latter half of the pupal period in the insects of both sexes. 20 20 Female
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FIG. 3.--Accumulation pattern of uric acid during pupal stage of the silkworm. O, Czo8 (xo days). A, Nz X Czo8 (8"5 days). 0 , Daizo X Oogusa (9 days). F1, 5"4 × 2"4 (z z days). ×, Daizo (z x days). Values in parentheses are days after larval-pupal eedysis to pupation. Based on the assumption that the nitrogen content is z6 per cent for protein and 33 per cent for uric acid, it is calculated that the nitrogen lost from protein and the nitrogen gained in uric acid during the pupal period are 3 mg. and 2"7 mg. for the male and z.8 mg. and 2"2 mg. for the female, respectively. These results strongly suggest that the lost protein is catabolized to uric acid. This view is supported by the data in Table II. T h e levels of uric acid and protein in three different hybrids were compared between the early pupa and the adult shortly after emergence. Although the levels differ with different hybrids, it can be said that the protein content in the male early pupa is lower (by 15-24 rag. per g. wet weight) than that in the female, and the decrease of protein during the pupal and pharate adult period is significantly greater in the male than in the female. It can also be pointed out that the nitrogen gained in uric acid is in each case equal to, or somewhat over, the nitrogen lost from protein. INCORPORATIONOF 2-14C-GLYCINE INTO PROTEIN AND URIC ACID To reveal the changes in metabofism of the amino-acid pool both in the synthetic pathway to protein and in the breakdown pathway to uric acid during metamorphosis,
256
Insect Biochem.
ToJo
2-14C-glycine was injected into pupae and pharate adults of different ages. Six hours after injection, they were used for extraction of free amino-acids, uric acid, and protein, and radioactivities in each fraction were measured. In this experiment Cio8 males and 5"4 × 2"4 females were used, as in the case of Fig. 4. Fig. 5 shows that the incorporation of z4C into protein increases conspicuously from the mid-pupal stage, reaching a maximum at 8o per cent pupal duration when about 35 per cent and 55 per cent of the total radioactivity given (after 6 hours) is distributed in protein for male and female, respectively. The recoveries of radioactivity in the amino-acid pool vary inversely with the incorporation into protein• A
Male
O
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stage (per cent) Pupal stage (per cent) Fxo. 4.--Variations in the concentrations of protein, free amino-acids, and uric acid during 'pupal' stage of the silkworm. Male: Cio8 ('pupal' period, io days); female: 5'4 × 2"4 ('pupal' period, Ii days)• O, Protein; O, uric acid; III, amino-acid. Pupal
The incorporation of I4C into uric acid increases markedly at those pupal stages when accumulation of uric acid has been noted (Figs. 4, 5)- The cumulative curve of the incorporation rate nearly coincides with the accumulation pattern of uric acid. In the female pupae the catabolism of glycine is maintained at a low level until 8o per cent pupal duration; it then increases rapidly• In contrast, in the male pupae it becomes active in the mid-pupal stage and again shortly before emergence. Furthermore, the distribution of 14C in these nitrogenous fractions of silkworms injected with 2-14C-glycine in early pupal stage was investigated after different lapses of time. As shown in Fig. 6, 14C increases in protein and in uric acid inversely with the loss from the amino-acid pool during the early period. In the male pupae of NI × Cio8, the quantity of label in protein declines from the mid-pupal stage, at the same time as a
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257
URIC ACIDPRODUCTIONIN THE SILKWORM
continuous increase of label in uric acid is observed. This agrees well with the decrease in total protein and rapid production of uric acid in the male from the mid-pupal stage (cf. Fig. 4). Even though protein synthesis becomes active during the latter half of pupal life, the disintegration of protein must be still greater in these stages. In the case of the female radioactivity in protein slightly increases, while the incorporation of x4C uric acid seems to stop almost completely during most of the pupal period. This agrees
Table //.--COMPARISON OF THE LABELSOF URIC ACID AND PROTEIN IN SILKWORMPUPAEJUST AFTERPUPATIONANDIN THE ADULTSJUSTAFTEREMERGENCE* AMOUNTPR~ENTt (mg. per g. wet weight) HYBRID
SEX
A--B
COMPOUND
Just after Pupation (A)
Just after Emergence (B)
rag. per g. Wet Weight
rag. Nitrogen per g. Wet Weight +*
Female Uric acid Protein
7"2 79"7
18"3 64"5
+I1"1 --15"2
+3"71 --2'54
Male
Uric acid Protein
8"7 54"6
25"9 33"8
+14"2 --20"3
+4'48 --3"56
Female Uric acid Protein
7"I
II'I
+ 4"O
+I"32
51"2
40"8
- - 1 o" 4
-- 1.63
Uric acid Protein
8"I 36"8
I9"9 19"8
+i1"8 --17"o
+ 3"94 --2"83
Female Uric acid Protein
8"4 63'5
I4"1 54"1
+ 5"7 - - 9"4
+ I
13.6
27'4 26'1
+13'8 --18"5
+4'60 --3.08
5'4×2"4
Kenko × Syungetsu
Syungetau× Housko
Male
Male
Uric acid Protein
44"6
"90 --1"57
* Values given are averages for two groups of each 5 insects. t Values for both stages are expressed on the basis of the wet weights previously measured soon after pupation. +*Calculated on the assumption that N = 16 per cent of protein and 33 per cent of uric acid. well with the maintenance of the protein and uric acid contents at almost constant levels in the female (cf. Fig. 4). DISTRIBUTION OF PROTEIN IN VARIOUSTissuEs OF FEMALEPUPAE To elucidate the mechanism of maintenance of protein at a constant level in the female pupa, changes in the distribution of protein in various tissues during metamorphosis were investigated, using Cxo8 strain. As shown in F/g. 7, in the first half of the pupal period a large part of the protein is distributed in the fat body, the remainder being distributed equally in the blood, the integument and muscle fraction, and the gut. In the mid-pupal stage, the pattern of distribution of protein among tissues changes markedly. It is lost rapidly from the fat body and slowly from the gut and the blood,
258
Insect Biochem.
TOJO
and increases simultaneously in the ovary and in the integument and muscle fraction (which includes imaginal tissues such as wings, legs, and muscles besides the integument). The sum total of protein in these tissues is almost constant during histolysis and histogenesis. In the late pupal stage (pharate adult), the major part of the protein becomes concentrated in the ovary and a smaller part in the integument and muscle fraction. Shortly before emergence the protein content decreases considerably from the integument and muscle fraction, which seems to cause the rapid decline of total protein and the production of uric acid in this stage (cf. Fig. 4).
Female
Male 6C
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FIG. 5.--Distribution of radioactivity in nitrogenous fractions of the silkworm in different pupal stages 6 hours after 2-'C-glycine injection. Male: C to8 (' pupal' period, to days); female: 5'4×2"4 ('pupal' period, zt days). I , In amino-acid; O, in protein; 0 , in uric acid. Also shown in Fig. 7 is the distribution of z4C in tissue proteins in the silkworms of the same strain injected with U-z4C-leucine on the second day after pupation (2o per cent pupal duration). The distribution of '4C in tissue proteins changes rapidly in the midpupal stage in nearly the same way as that of protein. In another experiment it was shown, after injection of U-z~C-leucine, that only trace amounts were incorporated into uric acid, and the quantities of label in the amino-acid pool and protein changed slightly. These results show that the nitrogenous compounds from the degraded tissues are efficiently re-utilized for histogenesis.
I97I,
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URIC ACID PRODUCTION
259
IN THE SILKWORM
DISCUSSION The occurrence of uric acid in pupae is known in many insects (Bursell, x967), but of the little work that has been done on its production during metamorphosis, most showed merely the increase of uric acid content during the pupal stage (Brown, I938; Patterson, i957; Taira and Nawa, x958). Only in Lucilia cuprina was the metabolic relationship between protein and uric acid suggested from comparison of changes in the concentrations of nitrogenous compounds during metamorphosis (Birt and Christian, I969).
t i
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3
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Days altar pupation
Days after pupation
A
B
8
10 Adult
FIG. 6.--Changes in the distribution of radioactivity among nitrogenous fractions of silkworm ' pupae' injected with z-'C-glycine. (A, male; B, female.) Stage of the silkworm at the time of the injection: male, I-day-old pupae of N1 × Cxo8 (' pupal' period, 8.5 days); female, 8-hour-old pupae of 5"4 × 2"4 (' pupal' period, xI days). O, Protein; 0 , uric acid; I , amino-acid. The silkworm seems to be the first example in which the pattern of uric acid accumulation during the pupal period has been elucidated. In the male silkworm rapid production of uric acid occurs in the mid-pupal stage and again shortly before emergence; in the female, on the other hand, uric acid is maintained at a constant level until shortly before emergence and then a rapid increase occurs. These changes were further demonstrated from the tracer study showing increased incorporation into uric acid during 6 hours after z-l~C-glycine injection in those stages when the rapid production of uric acid was observed (cf. Figs. 4, 5). It was also shown that the total protein content changes in a different manner in the two sexes. In male pupae it begins to decrease from the mid-pupal stage, while in female pupae it does not significantly change until 9° per cent pupal duration, when it declines rapidly.
260
Insect Biochem.
TOJO
As to the content of total free amino-acids, the pattern of change differs little between sexes. In both sexes, the size of the amino-acid pool decreases somewhat in the latter half of pupal stage, as has been shown in many species of insects (Chen, 1966). The decrease of total free amino-acids is calculated to be 0. 5 mg. nitrogen per g. wet weight at the most, on the basis of the analysis of free amino-acids (Kondo, x957) and the sensitivity of each amino-acid to ninhydrin reagent (Ishii, 1957). 80
6O
8 .o
.-./
.c 4c o
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E
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,'O
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,
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/
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aO 60 Pupal stage (per cent)
80
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Days after pupation B FIG. 7.--Changes in the distribution of protein among various tissues (A) and the incorporation of z4c of injected U-z4C-leucine into the tissue proteins (B) of the female pupae of the silkworm. Silkworm: CIo8 ( ' p u p a l ' period, Io days). The labelled leucine was injected into 2-day-old pupae. Identification of symbols as in Fig. 2. A
The nitrogen lost from protein (Table H) plus free amino-acids (above) during the pupal period accounts for more than four-fifths of the uric acid gained. T h e simultaneous occurrence of the protein decrease and the uric add accumulation supports the idea that the two processes are connected. This is confirmed by the finding that in the male silkworm injected with 2-z4C-glyeine, about equal amounts of 14C disappearing from protein and the amino-acid pool was recovered in uric acid, while in the female the ehanges in zaC in these fractions were much smaller (Fig. 6). According to Chinzei (1971) during adult development of the silkworm the nitrogen lost from total nucleic acids accounts for about 0.2 mg. per g. wet weight, and labelled purine and pyrimidine compounds are catabolized to uric acid in vivo. Therefore, the
i97x,
I
URIC ACID PRODUCTION IN THE SILKWORM
261
sum total of the nitrogen lost from amino-acids, protein, and nucleic acid is equal or dose to the uric acid accumulated during the pupal period. It has also been demonstrated that 2-14C-uric acid was not metabolized any further in the silkworm pupae in vivo. It is concluded that uric acid is really the end-product of nitrogen metabolism. In the female silkworm the distribution of protein among tissues changes greatly, mainly around the mid-pupal stage. The total protein content changes only slightly, since the sum of the protein lost from the fat body, gut, and blood is about equal to that gained in the ovary and other imaginal tissues. Thus, the protein from the degraded tissues is quite efficiently utilized for the formation of adult structures. As to the male pupae, such analyses were not performed, but it is possible to infer that a considerable part of the protein from lysing tissues is not utilized for histogenesis owing to the small size of the reproductive organs (testes), resulting in the decrease of total protein and the production of uric acid in the mid-pupal stage. The content of protein in the early male pupa is significantly lower than in the female, and the decrease during metamorphosis is far greater in the male than the female (Table H). This may be also understood by considering the small development of the testis in the male. It was found that protein in the integument and muscle fraction declined rapidly just before emergence. This probably comes from the pupal cuticle by the action of moulting fluid and accounts for the sharp increase of uric acid in both sexes at the same stage. The incorporation of radioactivity into protein of the whole insects during 6 hours after 2-1~C-glycine injection increases with development of the adult, reaching the maximum at 8o per cent pupal duration. The results for females agree well with those from the tracer study by Kawasaki, Sato, Suzuki, and Ojima (i969). They found the incorporation of 14C into ovarian protein, after 2-x4C-glycine injection, increased prominently with development of the adult, being maximum at about 8o per cent pupal duration when 4o per cent of the injected x4C was recovered in the ovarian protein. The two studies agree that the active incorporation of x4C from injected glycine during the latter half of pupal period in the female is mainly due to the formation of ovarian protein. In the early fifth-instar silkworm, uric acid is distributed mainly in the integument but in the mature larva it rapidly disappears from this site and accumulates in the fat body (Isaka, 1952, Yoshitake and Aruga, i952; Aruga, Yoshitake, and Ishihara, 1953; Hayashi, 1961). That the fat body becomes the site of uric acid storage in place of the integument after the mature larval stage is shown by the distribution of both endogenous uric acid and injected 2-1'C-uric acid in the present study. The transfer of uric acid to the rectal sac before emergence is known in many insects (Brown, 1938; Isaka, i952; Aruga and others, 1953; Wigglesworth, 1965). This is now dearly confirmed by quantitative estimations and tracer studies. In the female silkworm uric acid rapidly disappears from the fat body in the mid-pupal stage, which coincides with the time of the rapid loss of protein from this tissue (of. Figs. 2, 7), and almost all the uric acid is transferred to the rectal sac in the pharate adult, to be excreted with the meconium after emergence. Uric acid increases somewhat in the ovary and the integument and muscle fraction, but the increase in these tissues may be due to contamination with fat body, because the fat body becomes so dispersed in the mid-pupa that it is very difficult to separate completely from other tissues. These studies have shown that uric acid is produced from the surplus protein of disintegrating tissues via the amino-acid pool at certain stages in pupal and adult development. There remain the following questions to be elucidated in future: How is
262
ToJo
Insect Biochem.
the protein of the degraded tissues re-utilized for adult development ? W h a t kind of regulatory mechanism is operative in the rapid production of uric acid in a given period ? And further, I n what way is such insoluble material as uric acid transferred via the blood to the rectal sac ? ACKNOWLEDGEMENTS Thanks are due to Professor G. R. Wyatt of Yale University and Dr. Tadashi Kodama of the Institute for Plant Virus Research, Chiba, for reviewing the manuscript and offering suggestions for its improvement. I am grateful to Professor Teruo Yamasaki, Associate Professor Yoshiharu Matsumoto, and Mr. Yasuo Chinzei of the University of Tokyo for their invaluable discussion and advice during this study. REFERENCES
ARUGA,H., YOSHITAKE,N., and ISHIHARA,R. (I953), 'Studies on the mechanism of the expressions of the translucent and lemon genes in the silkworm. II. On some biochemical substances in the silkworm larva', ft. seric. Sci., Tokyo, 22, t x-I8. (In Japanese.) BARRETT, F. M., and FRmND, W. G. (I97O), 'Uric acid synthesis in Rhodnius prolix-us', ft. Insect Physiol., x6, I2X-x29. BIRT, L. M., and CHRISTIAN,B. (x969), 'Changes in nitrogenous compounds during the metamorphosis of the blowfly, Lucilia cuprina ', ~. Insect Physiol., I5, 7 xI-7 x9. BPmNNr:R-HOLZACH, O., and LEUTHARDT, F. ('96x), 'Untersuchung fiber die Biosynthese der Pterine bei Drosophila melanogaster', Helv. chim. .4cta, 44, x48o-I495. BRENNER-HOLZACH,O., and LEUTHARDT,F. (I965), ' l~ber die Herkunft des C-Atome 2 und 8 des Harnsiture bei Drosophila melanogaster', Helv. chim. Acta, 48, xx47-IxSI. BROWN,A. W. A. (x938), ' T h e nitrogen metabolism of an insect (Lucilia sericata Mg.). I. Uric acid, allantoin and uricase', Biochem. ft., 32, 895-9o2. BURfELL, E. (t967), ' T h e excretion of nitrogen in insects', Adv. Insect Physiol., 4, 33-67. CHI~, P. (x966), 'Amino acid and protein metabolism in insect development', Adv. Insect. Physiol., 3, 53-I32. CHINZEI, Y. (x97I), manuscript in preparation. GILMOUR, D. (I96x), The Biochemistry of Insects, p. 343. New York and London: Academic Press. HAYASHI,Y. (I96I), ' O n the uric acid formation in a few mutants and normal silkworm (Bombyx mori), with special reference to the xanthine dehydrogenase', ft. seric. Sci. Tokyo, $o, 89-94. (In Japanese.) HAYDAK,M. H. (I953), ' Influence of the protein level of the diet on the longevity of cockroaches ', Arm. ent. Soc. Am., 46, 547-560. ISAKA,S. (x952),' The stage of uric acid appearance in the adipose tissue of the silk-worm, Bombyx mori', Zool. Mag., Tokyo, 6x, 2x7-218. (In Japanese.) IsgII, S. (I957), 'Methods for systematic separation of amino acids', in Methods in Experimental Chemistry (ed. Japanese Chemical Society), vol. 23, pp. Io2-x3o. Tokyo: Maruzen. (In Japanese.) ITO, T., and MUKAIYAMA,F. (I964), 'Relationship between protein content of diets and xanthine oxidase activity in the silkworm, Bombyx mori L. ', ft. Insect Physiol., xo, 789-796. KAWASAKX,H., SATO, H., SUZUKI, M., and OJIMA, N. (I969), 'Conversion of serine to glycine during the formation of eggshells in the silkworm, Bombyx mori', y. Insect Physiol., I5, z5-32. KONDO, Y. (x957), 'Studies on the free amino acids and related compounds in the silkworm, Bombyx mori. III. On the free amino acids and related compounds in silkworm pupae and moths', ~. seric. Sd., Tokyo, 26, 341-344. (In Japanese.) LowRY, O. H., ROSm~BROUOH,N. J., F~mlL A. L., and RANDALL,R. J. (x95x), 'Protein measurement with the folin phenol reagent', )t. biol. Chem., I93, 265-275. McENRoE, W. D., and FORGASH,A. J. (I957), ' T h e in vivo incorporation of C 1~ formate in the ureide groups of uric acid by Periplaneta americana', .4nn. ent. Soc. Am., 50, 429-'43 L McENRoE, W. D., and FORQASH,A. J. (t958), 'Formate metabolism in the American cockroach, Periplaneta americana', Ann. ent. Soc. Am., 5I, x26-I29.
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PATTERSON, D. S. P. (I957), 'Qualitative and quantitative changes observed in the free a-amino nitrogen fraction of Tenebrio molitor pupal tissues during metamorphosis', Biochem..Z, 65, 729--735. TAIRA, T., and NAWA, S. (x9S8), ' N o direct metabolic relation between pterines and uric acid, ravines or folic acid in Drosophila melanogaster', .Tap. ~. Genet., 33, 42-45. To3o, S., and HmANO, C. (I966), ' An improved method for determination of uric acid in insects', .7. Insect Physiol., x2, i467-i47i. Tojo, S., and HIRANO, C. (I968), ' Uric acid production in Chilo suppressaEs larvae in relation to post-diapause development', ~. Insect Physiol., I4, xI2I-X 133. Tojo, S., and HmANO, C. (x97x), 'Nitrogen catabolism in two lepidopterous insects in relation to termination from diapause', Bull. natn. Inst. agric. Sci., Tokyo, C-2$, 47-96. (In Japanese.) Tojo, S., and YUSHIMA,T. (x97I), manuscript in preparation. WIGGLESWORTH,V. B. (1965), The Principles of Insect Physiology, 6th ed., p. 74 I. London: Methuen. YEMM, E. M., and COCKING, E. C. (I954), 'Determination of amino acids with ninhydrin', Analyst, Lond., 809 2o9-213. YOSmTAKE, N., and ARUGA,H. (I952), ' On the uric acid content in the integument and the blood of several mutants in the silkworm ', ~7. seric. Sci., Tokyo, 21, 7-x4.
Key Word Index: Uric acid, Bombyx mori, nitrogen catabolism.
z49
URIC ACID PRODUCTION IN RELATION TO PROTEIN METABOLISM IN THE SILKWORM, BOMB Y X MORI, DURING PUPAL-ADULT DEVELOPMENT SUMIO TOJO* Laboratory of Applied Entomology, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo, Japan
(Received 8 Aug., I97 o) ABSTRACT Accumulation and distribution of uric acid in the silkworm, Bombyx mori, were studied in relation to histolysis and histogenesis during the pupal period. In the male uric acid increased rapidly in the mid-pupal stage and again shortly before emergence, whereas in the female it was maintained at a constant level until the late pharate adult stage, when a rapid increase occurred. The rapid production of uric acid at these times was also shown by incorporation of ~'C into uric acid in pupae injected with 2-~C-glycine at various stages. The two sexes also differed in the changes of total protein content: in the male protein began to decrease from the mid-pupal stage, while in the female it changed little until shortly before emergence and then decreased rapidly. The sum of the nitrogen lost from protein and free amino-acids during the pupal period accounted for the nitrogen gained in uric acid. These results indicate that the lost protein is catabolized to uric acid via the amino-acid pool. When protein was labelled by injection of 2.x'C-glycine into male early pupae, ~4C was lost from protein and appeared in uric acid from the mid-pupal stage. In the female, 14C in both protein and uric acid changed only slightly. In the female silkworm a substantial amount of protein was transferred from the fat body to ovaries and other adult tissues in the mid-pupal stage, while total protein was essentially constant. The decrease of protein and the production of uric acid in the same stage of the male is believed to be due to the small development of the testes compared with the ovaries. Injected 2-~'C-uric acid was not metabolized any further, from which it is concluded that uric acid is really the end-product of nitrogen metabolism in silkworm pupae. Uric acid was distributed mainly in the fat body in the early half of the pupal period, but in the mid-pupa it was largely transferred to the rectal sac. After eclosion it was excreted with the meconium. THE occurrence of uric acid in the excreta of insects has been shown by m a n y investigators, as reviewed by Bursell (1967). As to uric acid formation in insects, however, knowledge is limited at present, except on the purine catabolic pathway (Gilmour, i96i ). T h a t nitrogen of uric acid originates f r o m amino-acids is supported b y the incorporation of labelled amino-acids into uric acid (McEnroe and Forgash, I957, i958; Brenner-Holzach and Leuthart, x96i , i965; Barrett and Friend, i97o), by nutritional experiments in which uric acid production was enhanced by rearing insects on diets of high protein content (Haydak, i953; Ito and Mukaiyama, 1964) , and also b y the *
Present address: Department of Biology, Yale University, New Haven, Connecticut, U.S.A.
250
TOJO
Insect Biochem.
correlation between nitrogen loss f r o m amino-acids and nitrogen gain in uric acid during the pupal period (Birt and Christian, r969). Endopterygote insects form a closed system with respect to nitrogen in the embryonic and pupal stages, and in m a n y cases during diapause. Such stages are very suitable for the study of nitrogen catabolism because metabolic relationships can be elucidated simply by comparing the accumulation of end-products with changes in other nitrogenous compounds. Moreover, analysis of when and how excretory nitrogenous compounds are produced during a closed stage is an important approach to elucidation of the mechanisms for conservation of reserves during differentiation and development. I n previous papers (Tojo and Hirano, x966 , i968), the accumulation of uric acid was shown to reflect the general metabolic activity of nitrogenous compounds in the pupae of Mamestra brassicae and the mature larvae of Chilo suppressalis. During diapause uric acid production was maintained at a low level, b u t during post-diapause development rapid accumulation occurred. I n this paper uric acid production is investigated in the silkworm, Bombyx morL during the pupal period,* mainly in relation to protein metabolism. Also, the incorporation of labelled amino-acids into protein and uric acid and the metabolism of 1*C-uric acid in vivo are examined. MATERIALS AND M E T H O D S ANIMALS
Silkworms of various strains were reared on mulberry leaves, and were selected at the time of pupation so as to give groups with not more than 6 hours' variation in age. When unavoidable, pupae were stored at 5° C. for not more than 24 hours in order to obtain animals of appropriate age. The grouped pupae were kept at 25 ° C. until use. The approximate pupal weight of experimental animals was x g. for males and I"5 g. for females. INJECTIONOF LABELLED COMPOUNDS For isotope incorporation studies, labelled materials dissolved in 2 l~l. of o'o67 M phosphate buffer, p H 7"o, were injected into the haemocoel of pupae under carbon dioxide anaesthesia through the interscgmental membrane of the lateral abdomen by means of a microsyringe. These treated pupae were kept at 25 ° C. until sacrifice. Labelled compounds used were as follows: 2-xIC-uric acid (specific activity 52"o me. per mmole), purchased from the Radiochemical Centre, Ameraham; and 2-x4C-glycine and U-x*Cleucine (specific activity 4'x2 and 6o.o me. per mmole respectively), prepared by the Dai-ichi Pure Chemical Co., Tokyo. DISSECTION OF TISSUES
Groups of 5 female pupae were used for each analysis. Blood was collected in a test-tube by applying gentle pressure on the abdomen after a puncture had been made by a needle at the prothorax region. The animals were then dissected along a ventral line, and remaining blood was washed out with ice-cold Ringer solution (2 per cent NaCI, o'2 per cent KC1, 0"04 per cent MgCI~, 0"08 per cent CaC10. The fat body, the ovary, the gut, and the rectal sac (with meconium) were each taken into Potter-Elvehjem glass homogenizers. The remaining bodies were put in a homogenizer and called the 'integument and muscle fraction'. This included the imaginal tissues such as wings, legs, muscles, etc., together with the integument. EXTRACTION OF URIC ACID
For extraction of uric acid from whole insects, groups of 5 pupae were homogenized with 5 volumes of water. The homogenate was heated at 9°0 C. for 3 minutes, and was centrifuged at * In this paper, for convenience, the entire period from the larval-pupal ecdysis to the pupaladult ecdysis is referred to as the 'pupal' stage; this includes both the pupa and the pharate adult.
I97I , I
URIC ACID PRODUCTION IN THE SILKWORM
25I
3000 r.p.m, for to minutes. After the supernatant was removed, the residue was re-extracted with water three times and the supernatants were combined and filtered. T h e filtrate was applied to a column for chromatography. For the respective tissues dissected as described above the extraction was repeated two or three times with 5 - I o ml. of water. For extraction from I ml. of blood, two portions of x ml. of water were used. Uric acid in the extracts was separated on an ion-exchange resin (Dowex i × io, formate type; I × 4-cm. column) and measured by ultra-violet absorption at z8o rag, essentially as described by Tojo and Hirano (I966). In tracer experiments, samples of each fraction from ion-exchange chromatography were placed in steel planchettes, dried, and counted in a gas-flow counter. SEPARATION OF FREE AMINO-ACIDS,URIC ACID, AND PROTEIN In the experiments to separate these three compounds, groups of pupae were homogenized with 5 volumes of 80 per cent ethanol and heated at 7 °o C. for 3 minutes, followed by centrifugation at 3ooo r.p.m, for io minutes. T h e supernatant was transferred to a flask, the residue was extracted twice more with 80 per cent ethanol, and the three extracts were combined. Aliquots were then used to determine total free amino-acids. If necessary, in order to separate uric acid from aminoacids, portions of the extract were added to a 0'5 x 3-cm. column of Dowex x × 1o (formate). Then amino-acids were eluted by 50 ml. of 80 per cent ethanol, the fractions being used for amino-acid determination and radioassay (by this procedure 2o-3o per cent of total free aminoacids were lost, but almost all the radioactivity in the amino-acid pool, derived from previously injected 2-1'C-glycine or U-l'C-leucine, was recovered). T h e next eluate by o.ox M formic acid was used to measure uric acid, based on absorption at z8o ml~. T h e residue from the extraction with 80 per cent ethanol was used for uric acid extraction by the procedure described above, using three portions of 30 ml. of water. Uric acid in the water extract was determined after chromatography on a I x 4-cm. column with Dowex x × Io. Total uric acid was obtained by addition of that in the ethanol extract and that in the water extract. T h e pellet which remained after the extraction with water was suspended in 2o ml. of 5 per cent perchloric acid and heated at 9 °0 C. for i o minutes, followed by centrifugation. After decanting the supernatant the residue was washed again with perchloric acid, heated as before. T h e n the pellet was successively washed at room temperature, twice with 3° ml. of ethanol/ether (3 : i) and then twice with 30 ml. of ether. From the residue thus obtained protein was extracted three times (3o ml. each) with o'25 M sodium hydroxide at 7 °° C. for xo minutes, and the extracts were combined. Portions were used for determination of total protein and for radioassay. In the experiment to investigate the changes of protein content in tissues, the extraction procedure for amino-acids was omitted. Uric acid was removed by hot water from the respective tissues separated from 5 animals. T h e remaining pellets were successively washed with hot 5 per cent perchloric acid, ethanol/ether, and then with ether as described above, and protein was extracted with o'25 M sodium hydroxide.
Amino-acids Amino-acids were analysed by the ninhydrin reaction (Yemm and Cocking, t954) using L-leucine as the standard.
Protein Protein was analysed by the method of Lowry, Rosenbrough, Farr, and Randall (x95 I), with albumin as the standard.
Radioactivity Radioactivity was measured in a windowless gas-flow counter, and was corrected for selfabsorption. RESULTS FATE OF INJECTED 2-14C-URIc ACID 2-14C-Uric a c i d w a s i n j e c t e d into p u p a e o f t h e 5"4 × 2"4 h y b r i d s i l k w o r m (a h y b r i d b e t w e e n C I 15 × N I 2 4 a n d N I 2 2 × C124) w i t h i n 12 h o u r s after p u p a t i o n , a n d g r o u p s o f t h e insects w e r e u s e d 2, 5, 8, a n d I i d a y s after t h e t r e a t m e n t . B y t h e e x t r a c t i o n p r o c e d u r e o f u r i c a c i d w i t h 5 v o l u m e s o f h o t w a t e r d e s c r i b e d above, 57"8, 3o'8, 7"9, 2.8, a n d
252
Insect Biochem.
'rojo
0.6 per cent of the injected radioactivity was recovered in the first, second, third, fourth, and fifth supernatant fluids respectively from female pupae 8 days after the injection. Extractions with hot 5 per cent perchloric acid, ethanol/ether (3 : i), ether, and 0"25 M sodium hydroxide were then performed in this order on the residue from the water extraction, but only traces of radioactivity were found in these extracts. It is therefore concluded that three or four extractions with water are sufficient for total recovery of uric acid. In Fig. I is shown an elution chromatogram of water extract from female pupae 8 days after 2-14C-uric acid injection. Peak D is the effluent position of uric acid. The elution
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Fraction number FIG. I.--Chromatogram of the hot-water extract of the female pharate adults of the silkworm injected with 2-14C-uric acid 8 days before, i.e., shortly after pupation. Peak D corresponds to uric acid. Silkworm: 5-4×2"4 . Resin: Dowex x×xo, formate type. Column: t ×4 cm. Solvent in stepwise elution: I, o'oo4 M formic acid; II, o.oi M formic acid; III, o'3 M formic acid. Fraction volume: 5 ml.
pattern of peak D by U.V. absorption coincides well with that by radioactivity, clearly indicating that U.V. absorption in this fraction is mainly due to uric acid. Peak C corresponds to the eluting position of ribosyluric acid as described in the previous paper (Tojo and Hirano, x968). Significant amounts of radioactivity were incorporated in peak C when Chilo suppressalis larvae were injected with 2-14C-uric acid (Tojo and Hirano, x97x), but none can be detected in this area from the silkworm pupa. Peak E is the elution area of an unknown metabolite of uric acid which characteristically exists in the wings of butterflies (Tojo and Yushima, z97t ), but only a trace .amount of label is detected in peak E. In Table I is given the distribution of radioactivity in the respective peaks on the chromatograms at various times after injection. In all cases almost all of the
1971, I
URIC ACID PRODUCTIONIN THE SILKWORM
253
radioactivity resides in peak D, that is, uric acid. It can be concluded that uric acid is a true end-product of nitrogen catabolism in silkworm pupae. DISTRIBUTION OF URIc ACID IN VARIOUSTISSUES As is shown in F~. z, in the female silkworm of 5"4 × 2"4 hybrid a large part of uric acid is distributed in the fat body and a small part in the integument and muscle fraction during the early half of pupal period. 2-1~C-Uric acid which has been injected into the I-day-old pupae disappeared rapidly from the blood and was concentrated mainly in the fat body. The accumulation pattern in the tissues changes strikingly from the mid-pupal
Table /.~DISTRIBUTION OF RADIOACTIVITYON THE COLUMNCHROMATOGRAMOF THE WATER EXTRACTOF THE SILKWORM,INJECTEDWITH2-taC-URIc ACIDSHORTLYAFTERPUPATION*
SEX
DAYSAFTER LARVAL-PUPAL ECDYSlS
Female II
(Adult)
Male
RECOVERY OF RADIOACTIVITY
(c.p.m.)t
TI~UE Peak A
Peak O
Peak E
Total
Whole Whole Whole
650 73° 82o
132,3OO 133,7OO 13o,2oo
115o 125o 950
I34,1oo • 135,68o
Body Wings Eggs Meconium
38 15 34 45°
314o 890 513o 116,11o
68 25 82 750
3246 930 5246 117,3Io
Total
537
I25,27o
925
126,732
Whole Whole Whole
4oo 860 67o
135,100
7oo 134o 950
136,2oo x34,6oo 131,220
132,4oo I29,6oo
131,020
* Race: 5"4 × 2"4.
The water extract was fractionated on a Dowex i activity distributed in each peak was measured.
×
IO column, as shown in Fig. I, and radio-
age. Uric acid decreases considerably from the fatbody and is transferred mainly to the rectal sac. Although it increases somewhat in the ovary and the integument and muscle fraction, it disappears from these tissues near emergence. In the final pharate adult almost all the uric acid exists in the rectal sac, and it is excreted with the meconium after emergence. Changes in the distribution of radioactivity from injected 2-14C-uric acid among tissues are quite similar to the changes of uric acid itself. It is noted that the concentration of z-14C-uric acid in blood is maintained at a nearly constant level, suggesting the existence of a regulatory mechanism. ACCUMULATIONOF URIC ACID IN THE WHOLE INSECT Uric acid contents in the silkworms of various strains or hybrids were investigated during the pupal period. In these experiments the uric acid concentrations in different stages were calculated on the basis of the weights just after pupation, in order to correct for weight-loss. The results are shown in Fig. 3, where stages are expressed as
254
Insect Biochem.
TOJO
percentages of pupal duration to make it easy to compare the patterns in different strains. The level of uric acid in the early pupa varies among strains, but the accumulation pattern is almost the same in the same sex of each strain. It is noteworthy that in the male, uric acid rapidly increases in the mid-pupal stage and increases further shortly before emergence, but in the female it does not change significantly until shortly before emergence, when rapid production occurs.
i ~=
i
",,3
o.
i
i
X 6.
0
20
40
60
80
Pupal stage (per cent)
A
I O0
!
3
5
l
9
Day:; dter pupation
B
Fla. 2.--Changes in the distributions of uric acid (A) and radioactivity of injected 2-14C-uric acid (B) among various tissues during pupal stage of the female silkworm. 2-z~C-Uric acid was injected into x-day-old pupae. Silkworm: 5.4×2.4 ('pupal' period, i x days). Integument and muscle fraction consists of the integument and the imaginal tissues such as wings, muscles, legs, etc. Values given for z4C in blood are on the basis of o'5 ml. of blood which was presumed to be contained in a pupa (z'5 g. wet weight). ®, Fat body; A, meconium; ×, integument and muscle; O, gut; , , ovary; O, blood. RELATIoNsHIP BETWEENURIC:ACID PRODUCTIONAND PROTEIN METABOLISM Fig. 4 shows the changes in the levels of free amino-acids and protein in relation to those of uric acid in the silkworms during metamorphosis. Also in these experiments the concentrations of these compounds were corrected for weight-loss. Although different strains were used for the two sexes, the comparison of patterns of nitrogenous compounds between sexes should be valid, since histolysis and histogenesis occur almost similarly in the same sex of both strains. In the male pupae the protein content is about 68 mg. per g. wet weight in the early half of the pupal period, then it decreases significantly from the mid-pupal age nearly at the time of the rapid uric acid production, reaching about 50 rag. per g. wet weight just before emergence. In the female pupae, on the other hand, the concentration of
I971 ,
I
URIC ACID PRODUCTION
IN THE SILKWORM
255
protein remains almost unchanged around a level of 8o rag. per g. wet weight until 9 ° per cent of pupal duration, but it then decreases sharply to less than 7° nag. per g. at the same time that rapid production of uric acid is observed. As to amino-acids it may be said that the pool size does not significantly change, although it decreases to some degree in the latter half of the pupal period in the insects of both sexes. 20 20 Female
Male
/
.~15 P ._~
15 la
II
•
,-10 O
o.
E
U
•
Q
0
0
_
rs
o
oB
~o
°
~" 5 8 "r"
u 'r" ::) J
~) 0
t
20
t
t
t
I
i
~
40 60 80 Pupal stage (per cent)
t
0
100
I
0
I
20
I
I
I
I
t
J
40 60 80 Pupal stage (per cent)
I
100
FIG. 3.--Accumulation pattern of uric acid during pupal stage of the silkworm. O, Czo8 (xo days). A, Nz X Czo8 (8"5 days). 0 , Daizo X Oogusa (9 days). F1, 5"4 × 2"4 (z z days). ×, Daizo (z x days). Values in parentheses are days after larval-pupal eedysis to pupation. Based on the assumption that the nitrogen content is z6 per cent for protein and 33 per cent for uric acid, it is calculated that the nitrogen lost from protein and the nitrogen gained in uric acid during the pupal period are 3 mg. and 2"7 mg. for the male and z.8 mg. and 2"2 mg. for the female, respectively. These results strongly suggest that the lost protein is catabolized to uric acid. This view is supported by the data in Table II. T h e levels of uric acid and protein in three different hybrids were compared between the early pupa and the adult shortly after emergence. Although the levels differ with different hybrids, it can be said that the protein content in the male early pupa is lower (by 15-24 rag. per g. wet weight) than that in the female, and the decrease of protein during the pupal and pharate adult period is significantly greater in the male than in the female. It can also be pointed out that the nitrogen gained in uric acid is in each case equal to, or somewhat over, the nitrogen lost from protein. INCORPORATIONOF 2-14C-GLYCINE INTO PROTEIN AND URIC ACID To reveal the changes in metabofism of the amino-acid pool both in the synthetic pathway to protein and in the breakdown pathway to uric acid during metamorphosis,
256
Insect Biochem.
ToJo
2-14C-glycine was injected into pupae and pharate adults of different ages. Six hours after injection, they were used for extraction of free amino-acids, uric acid, and protein, and radioactivities in each fraction were measured. In this experiment Cio8 males and 5"4 × 2"4 females were used, as in the case of Fig. 4. Fig. 5 shows that the incorporation of z4C into protein increases conspicuously from the mid-pupal stage, reaching a maximum at 8o per cent pupal duration when about 35 per cent and 55 per cent of the total radioactivity given (after 6 hours) is distributed in protein for male and female, respectively. The recoveries of radioactivity in the amino-acid pool vary inversely with the incorporation into protein• A
Male
O
o o
Female
~0
~0
o
e, o
E
i
O
E
<
"~
3
0 ~ 0 0 O0 0
m
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o
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60
~D "G u
,/
20c 10
,5
t~
t, 40 ~*
20C
40~,
,10
t,-
S
c
o ~k
,-| .J• --.a---I• --•-• • .. ai..,,o .
-o--I-o-I-T l® 5
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-
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stage (per cent) Pupal stage (per cent) Fxo. 4.--Variations in the concentrations of protein, free amino-acids, and uric acid during 'pupal' stage of the silkworm. Male: Cio8 ('pupal' period, io days); female: 5'4 × 2"4 ('pupal' period, Ii days)• O, Protein; O, uric acid; III, amino-acid. Pupal
The incorporation of I4C into uric acid increases markedly at those pupal stages when accumulation of uric acid has been noted (Figs. 4, 5)- The cumulative curve of the incorporation rate nearly coincides with the accumulation pattern of uric acid. In the female pupae the catabolism of glycine is maintained at a low level until 8o per cent pupal duration; it then increases rapidly• In contrast, in the male pupae it becomes active in the mid-pupal stage and again shortly before emergence. Furthermore, the distribution of 14C in these nitrogenous fractions of silkworms injected with 2-14C-glycine in early pupal stage was investigated after different lapses of time. As shown in Fig. 6, 14C increases in protein and in uric acid inversely with the loss from the amino-acid pool during the early period. In the male pupae of NI × Cio8, the quantity of label in protein declines from the mid-pupal stage, at the same time as a
I971, t
257
URIC ACIDPRODUCTIONIN THE SILKWORM
continuous increase of label in uric acid is observed. This agrees well with the decrease in total protein and rapid production of uric acid in the male from the mid-pupal stage (cf. Fig. 4). Even though protein synthesis becomes active during the latter half of pupal life, the disintegration of protein must be still greater in these stages. In the case of the female radioactivity in protein slightly increases, while the incorporation of x4C uric acid seems to stop almost completely during most of the pupal period. This agrees
Table //.--COMPARISON OF THE LABELSOF URIC ACID AND PROTEIN IN SILKWORMPUPAEJUST AFTERPUPATIONANDIN THE ADULTSJUSTAFTEREMERGENCE* AMOUNTPR~ENTt (mg. per g. wet weight) HYBRID
SEX
A--B
COMPOUND
Just after Pupation (A)
Just after Emergence (B)
rag. per g. Wet Weight
rag. Nitrogen per g. Wet Weight +*
Female Uric acid Protein
7"2 79"7
18"3 64"5
+I1"1 --15"2
+3"71 --2'54
Male
Uric acid Protein
8"7 54"6
25"9 33"8
+14"2 --20"3
+4'48 --3"56
Female Uric acid Protein
7"I
II'I
+ 4"O
+I"32
51"2
40"8
- - 1 o" 4
-- 1.63
Uric acid Protein
8"I 36"8
I9"9 19"8
+i1"8 --17"o
+ 3"94 --2"83
Female Uric acid Protein
8"4 63'5
I4"1 54"1
+ 5"7 - - 9"4
+ I
13.6
27'4 26'1
+13'8 --18"5
+4'60 --3.08
5'4×2"4
Kenko × Syungetsu
Syungetau× Housko
Male
Male
Uric acid Protein
44"6
"90 --1"57
* Values given are averages for two groups of each 5 insects. t Values for both stages are expressed on the basis of the wet weights previously measured soon after pupation. +*Calculated on the assumption that N = 16 per cent of protein and 33 per cent of uric acid. well with the maintenance of the protein and uric acid contents at almost constant levels in the female (cf. Fig. 4). DISTRIBUTION OF PROTEIN IN VARIOUSTissuEs OF FEMALEPUPAE To elucidate the mechanism of maintenance of protein at a constant level in the female pupa, changes in the distribution of protein in various tissues during metamorphosis were investigated, using Cxo8 strain. As shown in F/g. 7, in the first half of the pupal period a large part of the protein is distributed in the fat body, the remainder being distributed equally in the blood, the integument and muscle fraction, and the gut. In the mid-pupal stage, the pattern of distribution of protein among tissues changes markedly. It is lost rapidly from the fat body and slowly from the gut and the blood,
258
Insect Biochem.
TOJO
and increases simultaneously in the ovary and in the integument and muscle fraction (which includes imaginal tissues such as wings, legs, and muscles besides the integument). The sum total of protein in these tissues is almost constant during histolysis and histogenesis. In the late pupal stage (pharate adult), the major part of the protein becomes concentrated in the ovary and a smaller part in the integument and muscle fraction. Shortly before emergence the protein content decreases considerably from the integument and muscle fraction, which seems to cause the rapid decline of total protein and the production of uric acid in this stage (cf. Fig. 4).
Female
Male 6C
•
#
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% %
/
m\%
•
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%
=\ o
g
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00
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20
/
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• I
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5O
I t
Pupal stage (per cent)
I
I
100
0
I
t--'c-t
50
i, t',, 100
Pupal stage (per cent)
FIG. 5.--Distribution of radioactivity in nitrogenous fractions of the silkworm in different pupal stages 6 hours after 2-'C-glycine injection. Male: C to8 (' pupal' period, to days); female: 5'4×2"4 ('pupal' period, zt days). I , In amino-acid; O, in protein; 0 , in uric acid. Also shown in Fig. 7 is the distribution of z4C in tissue proteins in the silkworms of the same strain injected with U-z4C-leucine on the second day after pupation (2o per cent pupal duration). The distribution of '4C in tissue proteins changes rapidly in the midpupal stage in nearly the same way as that of protein. In another experiment it was shown, after injection of U-z~C-leucine, that only trace amounts were incorporated into uric acid, and the quantities of label in the amino-acid pool and protein changed slightly. These results show that the nitrogenous compounds from the degraded tissues are efficiently re-utilized for histogenesis.
I97I,
I
URIC ACID PRODUCTION
259
IN THE SILKWORM
DISCUSSION The occurrence of uric acid in pupae is known in many insects (Bursell, x967), but of the little work that has been done on its production during metamorphosis, most showed merely the increase of uric acid content during the pupal stage (Brown, I938; Patterson, i957; Taira and Nawa, x958). Only in Lucilia cuprina was the metabolic relationship between protein and uric acid suggested from comparison of changes in the concentrations of nitrogenous compounds during metamorphosis (Birt and Christian, I969).
t i
20 i
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4
/
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./
l
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l
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)".."
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3
5
7
2
4
6
Days altar pupation
Days after pupation
A
B
8
10 Adult
FIG. 6.--Changes in the distribution of radioactivity among nitrogenous fractions of silkworm ' pupae' injected with z-'C-glycine. (A, male; B, female.) Stage of the silkworm at the time of the injection: male, I-day-old pupae of N1 × Cxo8 (' pupal' period, 8.5 days); female, 8-hour-old pupae of 5"4 × 2"4 (' pupal' period, xI days). O, Protein; 0 , uric acid; I , amino-acid. The silkworm seems to be the first example in which the pattern of uric acid accumulation during the pupal period has been elucidated. In the male silkworm rapid production of uric acid occurs in the mid-pupal stage and again shortly before emergence; in the female, on the other hand, uric acid is maintained at a constant level until shortly before emergence and then a rapid increase occurs. These changes were further demonstrated from the tracer study showing increased incorporation into uric acid during 6 hours after z-l~C-glycine injection in those stages when the rapid production of uric acid was observed (cf. Figs. 4, 5). It was also shown that the total protein content changes in a different manner in the two sexes. In male pupae it begins to decrease from the mid-pupal stage, while in female pupae it does not significantly change until 9° per cent pupal duration, when it declines rapidly.
260
Insect Biochem.
TOJO
As to the content of total free amino-acids, the pattern of change differs little between sexes. In both sexes, the size of the amino-acid pool decreases somewhat in the latter half of pupal stage, as has been shown in many species of insects (Chen, 1966). The decrease of total free amino-acids is calculated to be 0. 5 mg. nitrogen per g. wet weight at the most, on the basis of the analysis of free amino-acids (Kondo, x957) and the sensitivity of each amino-acid to ninhydrin reagent (Ishii, 1957). 80
6O
8 .o
.-./
.c 4c o
"./:
Q.
E
l/
\
,'O
eL
.~a._ 2C x
"~---r---I:Z'.--. x,
0
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z
20
,
14
/
,
aO 60 Pupal stage (per cent)
80
I
I00
oL[2
.......
¶ "" "¢ "
I
t
10
Days after pupation B FIG. 7.--Changes in the distribution of protein among various tissues (A) and the incorporation of z4c of injected U-z4C-leucine into the tissue proteins (B) of the female pupae of the silkworm. Silkworm: CIo8 ( ' p u p a l ' period, Io days). The labelled leucine was injected into 2-day-old pupae. Identification of symbols as in Fig. 2. A
The nitrogen lost from protein (Table H) plus free amino-acids (above) during the pupal period accounts for more than four-fifths of the uric acid gained. T h e simultaneous occurrence of the protein decrease and the uric add accumulation supports the idea that the two processes are connected. This is confirmed by the finding that in the male silkworm injected with 2-z4C-glyeine, about equal amounts of 14C disappearing from protein and the amino-acid pool was recovered in uric acid, while in the female the ehanges in zaC in these fractions were much smaller (Fig. 6). According to Chinzei (1971) during adult development of the silkworm the nitrogen lost from total nucleic acids accounts for about 0.2 mg. per g. wet weight, and labelled purine and pyrimidine compounds are catabolized to uric acid in vivo. Therefore, the
i97x,
I
URIC ACID PRODUCTION IN THE SILKWORM
261
sum total of the nitrogen lost from amino-acids, protein, and nucleic acid is equal or dose to the uric acid accumulated during the pupal period. It has also been demonstrated that 2-14C-uric acid was not metabolized any further in the silkworm pupae in vivo. It is concluded that uric acid is really the end-product of nitrogen metabolism. In the female silkworm the distribution of protein among tissues changes greatly, mainly around the mid-pupal stage. The total protein content changes only slightly, since the sum of the protein lost from the fat body, gut, and blood is about equal to that gained in the ovary and other imaginal tissues. Thus, the protein from the degraded tissues is quite efficiently utilized for the formation of adult structures. As to the male pupae, such analyses were not performed, but it is possible to infer that a considerable part of the protein from lysing tissues is not utilized for histogenesis owing to the small size of the reproductive organs (testes), resulting in the decrease of total protein and the production of uric acid in the mid-pupal stage. The content of protein in the early male pupa is significantly lower than in the female, and the decrease during metamorphosis is far greater in the male than the female (Table H). This may be also understood by considering the small development of the testis in the male. It was found that protein in the integument and muscle fraction declined rapidly just before emergence. This probably comes from the pupal cuticle by the action of moulting fluid and accounts for the sharp increase of uric acid in both sexes at the same stage. The incorporation of radioactivity into protein of the whole insects during 6 hours after 2-1~C-glycine injection increases with development of the adult, reaching the maximum at 8o per cent pupal duration. The results for females agree well with those from the tracer study by Kawasaki, Sato, Suzuki, and Ojima (i969). They found the incorporation of 14C into ovarian protein, after 2-x4C-glycine injection, increased prominently with development of the adult, being maximum at about 8o per cent pupal duration when 4o per cent of the injected x4C was recovered in the ovarian protein. The two studies agree that the active incorporation of x4C from injected glycine during the latter half of pupal period in the female is mainly due to the formation of ovarian protein. In the early fifth-instar silkworm, uric acid is distributed mainly in the integument but in the mature larva it rapidly disappears from this site and accumulates in the fat body (Isaka, 1952, Yoshitake and Aruga, i952; Aruga, Yoshitake, and Ishihara, 1953; Hayashi, 1961). That the fat body becomes the site of uric acid storage in place of the integument after the mature larval stage is shown by the distribution of both endogenous uric acid and injected 2-1'C-uric acid in the present study. The transfer of uric acid to the rectal sac before emergence is known in many insects (Brown, 1938; Isaka, i952; Aruga and others, 1953; Wigglesworth, 1965). This is now dearly confirmed by quantitative estimations and tracer studies. In the female silkworm uric acid rapidly disappears from the fat body in the mid-pupal stage, which coincides with the time of the rapid loss of protein from this tissue (of. Figs. 2, 7), and almost all the uric acid is transferred to the rectal sac in the pharate adult, to be excreted with the meconium after emergence. Uric acid increases somewhat in the ovary and the integument and muscle fraction, but the increase in these tissues may be due to contamination with fat body, because the fat body becomes so dispersed in the mid-pupa that it is very difficult to separate completely from other tissues. These studies have shown that uric acid is produced from the surplus protein of disintegrating tissues via the amino-acid pool at certain stages in pupal and adult development. There remain the following questions to be elucidated in future: How is
262
ToJo
Insect Biochem.
the protein of the degraded tissues re-utilized for adult development ? W h a t kind of regulatory mechanism is operative in the rapid production of uric acid in a given period ? And further, I n what way is such insoluble material as uric acid transferred via the blood to the rectal sac ? ACKNOWLEDGEMENTS Thanks are due to Professor G. R. Wyatt of Yale University and Dr. Tadashi Kodama of the Institute for Plant Virus Research, Chiba, for reviewing the manuscript and offering suggestions for its improvement. I am grateful to Professor Teruo Yamasaki, Associate Professor Yoshiharu Matsumoto, and Mr. Yasuo Chinzei of the University of Tokyo for their invaluable discussion and advice during this study. REFERENCES
ARUGA,H., YOSHITAKE,N., and ISHIHARA,R. (I953), 'Studies on the mechanism of the expressions of the translucent and lemon genes in the silkworm. II. On some biochemical substances in the silkworm larva', ft. seric. Sci., Tokyo, 22, t x-I8. (In Japanese.) BARRETT, F. M., and FRmND, W. G. (I97O), 'Uric acid synthesis in Rhodnius prolix-us', ft. Insect Physiol., x6, I2X-x29. BIRT, L. M., and CHRISTIAN,B. (x969), 'Changes in nitrogenous compounds during the metamorphosis of the blowfly, Lucilia cuprina ', ~. Insect Physiol., I5, 7 xI-7 x9. BPmNNr:R-HOLZACH, O., and LEUTHARDT, F. ('96x), 'Untersuchung fiber die Biosynthese der Pterine bei Drosophila melanogaster', Helv. chim. .4cta, 44, x48o-I495. BRENNER-HOLZACH,O., and LEUTHARDT,F. (I965), ' l~ber die Herkunft des C-Atome 2 und 8 des Harnsiture bei Drosophila melanogaster', Helv. chim. Acta, 48, xx47-IxSI. BROWN,A. W. A. (x938), ' T h e nitrogen metabolism of an insect (Lucilia sericata Mg.). I. Uric acid, allantoin and uricase', Biochem. ft., 32, 895-9o2. BURfELL, E. (t967), ' T h e excretion of nitrogen in insects', Adv. Insect Physiol., 4, 33-67. CHI~, P. (x966), 'Amino acid and protein metabolism in insect development', Adv. Insect. Physiol., 3, 53-I32. CHINZEI, Y. (x97I), manuscript in preparation. GILMOUR, D. (I96x), The Biochemistry of Insects, p. 343. New York and London: Academic Press. HAYASHI,Y. (I96I), ' O n the uric acid formation in a few mutants and normal silkworm (Bombyx mori), with special reference to the xanthine dehydrogenase', ft. seric. Sci. Tokyo, $o, 89-94. (In Japanese.) HAYDAK,M. H. (I953), ' Influence of the protein level of the diet on the longevity of cockroaches ', Arm. ent. Soc. Am., 46, 547-560. ISAKA,S. (x952),' The stage of uric acid appearance in the adipose tissue of the silk-worm, Bombyx mori', Zool. Mag., Tokyo, 6x, 2x7-218. (In Japanese.) IsgII, S. (I957), 'Methods for systematic separation of amino acids', in Methods in Experimental Chemistry (ed. Japanese Chemical Society), vol. 23, pp. Io2-x3o. Tokyo: Maruzen. (In Japanese.) ITO, T., and MUKAIYAMA,F. (I964), 'Relationship between protein content of diets and xanthine oxidase activity in the silkworm, Bombyx mori L. ', ft. Insect Physiol., xo, 789-796. KAWASAKX,H., SATO, H., SUZUKI, M., and OJIMA, N. (I969), 'Conversion of serine to glycine during the formation of eggshells in the silkworm, Bombyx mori', y. Insect Physiol., I5, z5-32. KONDO, Y. (x957), 'Studies on the free amino acids and related compounds in the silkworm, Bombyx mori. III. On the free amino acids and related compounds in silkworm pupae and moths', ~. seric. Sd., Tokyo, 26, 341-344. (In Japanese.) LowRY, O. H., ROSm~BROUOH,N. J., F~mlL A. L., and RANDALL,R. J. (x95x), 'Protein measurement with the folin phenol reagent', )t. biol. Chem., I93, 265-275. McENRoE, W. D., and FORGASH,A. J. (I957), ' T h e in vivo incorporation of C 1~ formate in the ureide groups of uric acid by Periplaneta americana', .4nn. ent. Soc. Am., 50, 429-'43 L McENRoE, W. D., and FORQASH,A. J. (t958), 'Formate metabolism in the American cockroach, Periplaneta americana', Ann. ent. Soc. Am., 5I, x26-I29.
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PATTERSON, D. S. P. (I957), 'Qualitative and quantitative changes observed in the free a-amino nitrogen fraction of Tenebrio molitor pupal tissues during metamorphosis', Biochem..Z, 65, 729--735. TAIRA, T., and NAWA, S. (x9S8), ' N o direct metabolic relation between pterines and uric acid, ravines or folic acid in Drosophila melanogaster', .Tap. ~. Genet., 33, 42-45. To3o, S., and HmANO, C. (I966), ' An improved method for determination of uric acid in insects', .7. Insect Physiol., x2, i467-i47i. Tojo, S., and HIRANO, C. (I968), ' Uric acid production in Chilo suppressaEs larvae in relation to post-diapause development', ~. Insect Physiol., I4, xI2I-X 133. Tojo, S., and HmANO, C. (x97x), 'Nitrogen catabolism in two lepidopterous insects in relation to termination from diapause', Bull. natn. Inst. agric. Sci., Tokyo, C-2$, 47-96. (In Japanese.) Tojo, S., and YUSHIMA,T. (x97I), manuscript in preparation. WIGGLESWORTH,V. B. (1965), The Principles of Insect Physiology, 6th ed., p. 74 I. London: Methuen. YEMM, E. M., and COCKING, E. C. (I954), 'Determination of amino acids with ninhydrin', Analyst, Lond., 809 2o9-213. YOSmTAKE, N., and ARUGA,H. (I952), ' On the uric acid content in the integument and the blood of several mutants in the silkworm ', ~7. seric. Sci., Tokyo, 21, 7-x4.
Key Word Index: Uric acid, Bombyx mori, nitrogen catabolism.