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Uric acid synthesis in Rhodnius prolixus
7. Insect Physiol.. 1970, Vol. 16, pp. 121 IO 129. PeTgamon Press. Printed in Great Britain
URIC ACID SYNTHESIS F. M. WRRETT* Zoology
IN RHODNIUS
PROLIXUS
and W. G. FRIEND
Department, university of Toronto, Toronto, Ontario, Canada (Received 7 July 1969)
Abstract-Glycine-l-iaC, glycine- 2-i&C, and sodium formate-*“C were used to investigate the origin of the carbon atoms of uric acid in Rhodnius prolixus. As in other uricotelic organisms, the carboxyl carbon of glycine is incorporated into position 4 of uric acid, the alpha carbon of glycine goes to position 5, and the carbon of sodium formate is incorporated into positions 2 and 8. The carboxyl carbon of glycine is converted to COz approximately twentyfour times more rapidly than is the alpha carbon, and much of this CO* is incorporated into carbon 6 of uric acid. The alpha carbon of glycine is used in the synthesis of formate. The use of isotopically labelled precursors to determine the origin of all five carbon atoms of uric acid has not previously been reported for any species of insect. INTRODUCTION
THE PRESENCEof the uricolytic pathway, by which nucleic acids are broken down to uric acid, has been well documented in insects (see BURSELL, 1967). However, the large amount of uric acid in the excreta of most insects cannot be accounted for solely on the basis of nucleic acid degradation. Consequently, a synthetic pathway has been sought by which uric acid could originate from other precursors. WEINER (1902) reported that birds synthesize uric acid by combining 2 molecules of urea with 1 molecule of the 3 carbon dicarboxylic acid, tartronic acid. Based on work with the Chinese Tussur moth, Antheraea pernyi, LEIFERT (1935) proposed a similar mechanism for uric acid synthesis in insects. This pathway was subsequently disproved both in birds (SCHULER and REINDEL, 1935) and in insects (ANDERSONand PATTON, 1955). The use of isotopically labelled precursors in pigeons has established the currently accepted pathway of uric acid synthesis in birds (BUCHANANet al., 1948; SONNE et al., 1948). BALDWIN (1963) has postulated a reaction sequence for uric acid synthesis in which glutamine, aspartate, glutamate, glycine, formate, carbon dioxide, and ribose-5-phosphate combine in the presence of ATP to give inosine. The conversion of inosine to hypoxanthine is followed by the action of xanthine dehydrogenase, which converts hypoxanthine to xanthine and xanthine to uric acid. Fig. 1 illustrates the origin of the individual carbon and nitrogen atoms of uric acid produced by this pathway. The reviews of GIL~MOUR(1961) and BURSELL (1967) indicate that insects and birds synthesize uric acid in the same way, although the complete pathway of uric * Present address : Zoology
Department,
Cambridge 121
University,
Cambridge,
England.
122
F. M. BARRETTand W. G. FRIEXD
acid synthesis in insects has not been fully documented. DESAI and KILBY (1958) reported that 4-amino-5-imidazole carboxamide increases the rate of uric acid production in fat body homogenates of the blowfly, CaZZiphoraerythrocephala, and the desert locust, Schistocerca gregaria. Since this compound could be converted to the corresponding ribotide, an intermediate on the uricotelic pathway (BALDWIN, 1963), this observation supports the hypothesis of a pathway in insects similar to that found in birds. In the Chinese Tussur moth, A. pernyi, the simultaneous addition of formate, ribose-S-phosphate, glutamate, and aspartate will stimulate uric acid synthesis (HELLER and JEZEWSKA,,1959). Using a more direct approach, MCENROE and FORGASH(1957) showed that sodium formate-14C injected into the American cockroach, Periplaneta americana, was incorporated almost exclusively into positions 2 and 8 of uric acid recovered after injection. This result also suggests a pathway similar to that found in birds, as does the work of TOJO and HIRANO (1968), who showed that glycine-1 -14C injected into the rice stem borer, Chile suppressalis, was incorporated into uric acid. The present study was undertaken to determine the origin of the carbon atoms of uric acid synthesized by the blood-sucking bug, Rhodnius pro&us (St%). Since fifth instar larvae of this insect ingest up to nine times their body weight in a single feeding of mammalian blood (FRIEND et al., 1965), they must eliminate considerable amounts of nitrogenous waste. Both the increased uric acid concentration in the haemolymph (BARRETT and FRIEND, 1966) and the rapid rate of uric acid excretion (WIGGLESWORTH, 1931) which follow feeding are indications of rapid The predominance of uric acid in the excreta of synthesis of this molecule. R. proZi,rms(WIGGLESWORTH, 1931) is an important practical consideration in the present investigation since appreciable amounts of purified radioactive acid are required for the chemical degradation procedure used to isolate the individual carbon atoms. In this investigation, 14C-labelled glycine and formate, known precursors of uric acid in the pigeon, were injected into fifth instar larvae of R. prolixus to examine the hypothesis that C, of uric acid comes from CO,, C, of uric acid comes from the carboxyl carbon of glycine, C5 of uric acid comes from the alpha carbon of glycine, and C, and C, of uric acid come from formate. The use of isotopically labelled precursors to determine the origin of all five carbon atoms of uric acid has not previously been reported for any species of insect. MATERIALS
AND METHODS
The animals used in this study were fifth instar larvae taken from a culture of R. prolixus reared at 25°C in a humid atmosphere. The stock animals were fed on rabbit blood once very 30 to 40 days. The fate of glycine and sodium formate in R. prolixus was followed by injecting known amounts of glycine-1-%Z, glycine-2-1%, or sodium formate-l”C into fifth instar larvae. The haemolymph, hindgut contents, and respiratory CO, were recovered 3 days after injection and counted to determine the percentage of the injected radioactivity present in each. In some instances radioactive CO4 was
URICACIDSYNrHESISIN RHODSIUS
123
PROLIXUS
The insects were held in Warburg collected on each of the 3 days after injection. vessels and the respired CO, was trapped in 1.0 N NaOH contained in the sidearm. Injections given 2 days after feeding were delivered from a 25 ,~l Hamilton
‘\
I
‘,
Glycine /’
!
/
Formote
\ Glutamate
Giutomme \ \ \
/
FIG. 1, Origin of the carbon and nitrogen uricotelic
pathway
(after
atoms of uric acid synthesized
by the
BURSELL,1967)
syringe held in a micromanipulator. The needle was inserted into a forelimb that had been amputated at the distal end of the femur; the junction between needle and forelimb was sealed with a mixture of beeswax and colophony (KROGH and WEIS-FOGH, 1951). After injection, the waxed junction was reheated slightly, the needle was withdrawn and the wax was allowed to harden and seal the wound. Leakage, which could have occurred during removal of the needle, was prevented by pinching the femur proximal to the tip of the needle just prior to withdrawal. The anus was then sealed with a small drop of the wax mixture so that uric acid synthesized after injection would accumulate in the hindgut. For the experiments summarized in Table 1 the’injected volumes and activities per insect were: 1 PC glycine-2-14C weighing 18 pg in 7.5 ~1 insect Ringer; 0.23 PC glycine-lJ4C weighing 3 ,ug in 7 ,ul of insect Ringer; 4.5.~~ sodium formate-14C weighing 66 pg in 7 ~1 insect Ringer. The insect Ringer solution was prepared by combining 11.0 g NaCl, 1.4 g KCl, and 1-O g CaCl, and diluting to 1 1. with distilled water., Radioactive samples were counted in a Packard Tri-Carb Liquid Scintillation Counter to a standard error, in net counting rate, of 5 per cent. The scintillation liquid was prepared by combining 6.0 g of 2,5-diphenyl-oxazole (PPO) and O-1 g
124
F. 31. BARRELSand 1%‘.G. FRIEND
of 1,4-bis-2-j-(5-phenyloxazolyl)-benzene (POPOP), diluting to 1 1. with sulphurfree toluene, and then mixing 600 ml of this solution with 386 ml of absolute alcohol. The hindgut contents were dissolved in hot, 0.1% LiCO, prior to counting while all other samples were added directly to the scintillation liquid. Corrections for quenching and background were applied in all cases to make comparisons between different types of samples valid. In the experiments summarized in Table 2, in which radioactive uric acid formed from labelled precursors was recovered for subsequent chemical degradation, 10 to 15 insects were injected as previously described, and then dissected 3 days later to obtain the hindgut and its contents. The contents were dissolved in 10 ml of hot 0.1% LiCO, in a 15 ml test tube, to which was added a large excess of unlabelled carrier uric acid to minimize losses of radioactive uric acid during purification. The purification procedure described by MCENROE and FORGASH (1957) was carried through three to five recrystallizations to remove radioactive impurities. Uric acid recovered in this manner was degraded chemically according to procedures previously described by other authors (BUCHAXANet al., 1948; SONNE et al., 1948). These procedures make possible the recovery of C, as CO,, C, plus Cs as glyoxylic acid semicarbazone, and C, plus C, as urea. Subsequent degradation of glyoxylic acid semicarbazone will release C, and C5 separately as CO, (SONNE et al., 1948). Carbon dioxide was trapped in 1 N NaOH and quantitated by titration with HCl. All degradations were carried out on composite samples weighing 80 to 103 mg and containing both radioactive uric acid recovered from injected insects and unlabelled carrier uric acid. RESULTS Three days after the injection of glycine-l-i% into fifth instar larvae, 47% of the injected activity was recovered in the hindgut contents and 29% was recovered in the respiratory CO,. In contrast, over the same period, glycine-2-r1C gave 89% of the injected activity in the hindgut contents and only 1.2% in the respiratory CO,. With sodium formate as precursor the hindgut contents had 68% of the injected activity while 3.7% was recovered in the respiratory CO,. These values, given in Table 1, will be used to predict the relative amounts of activity in the five carbon atoms of uric acid synthesized after the injection of each precursor. With glycine-lJ4C as precursor, over 90% of the radioactive CO, produced during the 3-day recovery period was collected in the first 24 hr after injection. There was also extensive incorporation of glycine into the hindgut contents in the first 24 hr. Little activity remained in the haemolymph by 3 days after injection, and even at 1 day the haemolymph contained less than 5% of the activity initially injected. The distribution of activity between the individual carbon atoms of uric acid recovered after the injection of each precursor is reported in Table 2. The carboxyl carbon of glycine was incorporated into C, of uric acid approximately twentyseven times more extensively than was the alpha carbon. The alpha carbon
LTRIC ACID
SEXTHESIS
RH0D.VIV.S
IN
125
PROLIXUS
contributed more than twice as heavily to the C, plus Cs portion of uric acid than did the carboxyl carbon of glycine. Both carbons of glycine were incorporated into positions 4 and 5 of uric acid but in greatly differing proportions (Table 2). was incorporated into Approximately 9776 of the injected sodium formateJ4C positions 2 and 8 ; only slight activity was recovered in the other 3 carbon atoms. TABLE CCz,
l-PERCEXTAGES AND HI&DGUT
OF THE IXJECTED ACTIVITY
FOUND
IN HAEMOLYMPH,
RESPIRATORY
COXTENTS AFTER THE IXJECTION OF SUSPECTED PRECURSORS OF URIC ACID ISTO FIFTH INSTAR LARVAE OF R. prolixus
Injected Suspected
precursor
Glycine-1-“C carbon
Haemolymph
(carboxyl (alpha
* Values
given
are averages
10 ~1 sample
haemolymph
TABLE
7
Carbon
1.2
carbon labelled) Sodium formate-ilC
FIFTH
(“/b) recorded
3 days after injection* Hindgut
dioxide
contents
29
47
labelled)
Glycine-2-W
7 A
activity
was
volume
1.4
1.2
89
-
3.7
68
for 3 or 4 insects.
counted
and
the
activity
recorded
was
corrected
for
a total
of 30 ~1.
~-DISTRIBUTION
OF ACTIVITY
INSTAR LARVAE OF R. prOkWS
IN THE CARBON ATOMS OF URIC ACID RECOVERED FROM INJECTED WITH
Initial
activity
SUSPECTED PRECURSORS OF URIC ACID
found
in purified
composite
samples
of uric acid (%) c, Precursors
‘*C-labelled Glycine-1
-r*C
plus
c,
as glyoxylic
used to obtain uric acid (carboxyl
C,
semicarbazone
C,
C5 3,7
8.0
74
70.3
carbon labelled) Glycine-2-r4C (alpha
0.3
56
161
carbon labelled) Sodium formate-‘%
0.s
* Values C4 plus
for CZ plus C, were
C5 and subtracting
t The
recovery
error
from
2.5
obtained
by adding
-
the percentages
c,
plus c,* 18.0
4oi
43.7
-
96.7
determined
for C, and
100%.
described
in the
text
has
been
arbitrarily
assigned
to the
C,
fraction.
The values given for C, plus C, in Table 2 were obtained by combining the percentages calculated for C, and C, plus Cs and subtracting from 100%. They presumably therefore contain any error involved in the determination of C,, C5,
126
F. Pd.
BARRETTand W. G. FRIEXQ
and C6. Replicate experiments using glycine-1 -r%Z as precursor, and measurement of activity in the unpurified Ca plus C? fraction, both indicated that this error is slight. Activities reported for C, and Cj in the experiment with glycine-2JJC as precursor should be viewed with caution. A recovery error during the degradation of glyoxylic acid semicarbazone made accurate individual quantitation of these carbon atoms impossible. For reasons described in the Discussion, the error has been attributed exclusively to C, and consequently the value reported may be much higher than was actually the case. DISCUSSION
WIGGLESWORTH (1931) reported that 64 to 84 per cent of the dry weight and approximately 90 per cent of the total nitrogen in the excreta of R. prolixus was in the form of uric acid. It is likely, therefore, that most of the radioactivity present in the hindgut contents after injection of l”C-labelled precursors (Table I) was contained in uric acid. The extensive accumulation of activity in the hindgut after these injections indicates that both sodium formate and glycine are used in the synthesis of uric acid in R. prolixus. Chemical degradation studies have revealed the positions in the uric acid molecule into which these precursor carbon atoms were incorporated. Origin of carbon 6 Injection of glycine-l- 14C gave rise to twenty-four times more radioactive CO, during the 3-day test period than did injections of glycine-Z-r4C (29%/1*2%, Table 1). This shows that R. prolixus converts the carboxyl carbon of glycine into CO, more readily than the alpha carbon. Consequently insects injected with glycine-1-r4C should have approximately twenty-four times more radioactive CO, available for metabolism than insects injected with glycine-2-r4C. Hence, if C, of uric acid does come from CO,, it should follow that C, in uric acid recovered from insects injected with glycine-l-r4C will be about twenty-four times more active than C, in uric acid recovered from insects injected with glycine-2-14C. The value of 26.7 actually obtained (80%/0.3%, Table 2) s h ows close agreement with this prediction and supports the hypothesis that C, of uric acid comes from CO,. A similar origin for C, has been demonstrated in the pigeon (BUCHANANet al., 1948; KARLSSON and BARKER, 1949) and in the snail, Otala Zactea (LEE and CA~IPBELL, 1965). Origin of carbons 2 and 8 There can be little doubt that sodium formate is used in the synthesis of carbons 2 and 8 of uric acid. After the injection of sodium formate-r4C 97% of the activity in recovered uric acid was contained in these two carbon atoms (Table 2). This value is in close agreement with those obtained in similar studies on other organisms: the pigeon, 98% (KARLSSON and BARKER, 1949) and 96% (E~~YN and SPRINSON, 1950); the snail, Otala Zactea, 94.4% (LEE and CAMPBELL, 1965); the cockroach, P. americana, 92% (MCENROE, 1957).,
CRIC
ACID
SYNTHESIS
IN
RHODNIUS
PROLIXUS
127
In this investigation, no attempt was made to determine the individual contributions of carbons 2 and 8 to the total activity in uric acid recovered after the injection of sodium formate. In the pigeon, equal amounts of activity were found in both carbon 2 and carbon 8 (1 : 1 ratio) (SONNE et al., 1948), while the cockroach had slightly more activity in carbon 8 (1 : 1*3 ratio) (MCEPU’ROE and FORGASH, 1957). If the cockroach were capable of reversible cleavage of the purine ring, and if the turnover of C, was slightly greater than that of C,, then the ratio of 1 : 1.3 would be the result of a dilution of the activity in C, (GILMOUR, 1961). Recently, HOPKINS and LOFGREN (1968) reported that CO? collected for 15 hr after the injection of r4C-8-adenine or W-2-adenine into the cockroach, Leucophaea maderae, contained 3-5 and 4.5% of the injected activity respectively. This observation suggests a slightly greater turnover of C, than C, and supports Gilmour’s earlier suggestion. Origins of carbons 4 and 5 If C, of uric acid comes exclusively from the carboxyl carbon of glycine, it should follow, assuming no exchange of the carboxyl carbon and the alpha carbon prior to incorporation, that all of the activity in the C, plus Cs fraction of uric acid recovered after the injection of glycine-l-i4C should be found in C, (Table 2). In fact, 95% of the activity of this fraction was present in C,, offering strong support to this hypothesis. The apparent presence of some activity in C, was also reported by other authors in a similar experiment on the pigeon (BUCHANANet al., 1948). They claimed that during the early stages of the chemical degradation of glyoxylic acid semicarbazone some C, may have been released along with the C, samples. In experiments where the activity of the C, fraction was great (i.e. after injection of glycine-1-r4C) such a contamination error would be noticeable. This observation explains the apparent activity in Cs and remains consistent with the hypothesis that C, of uric acid comes exclusively from the carboxyl carbon of glycine. If Cs of uric acid comes from the alpha carbon of glycine, it should also follow, again assuming no exchange of the carboxyl and alpha carbons of glycine prior to incorporation, that all of the activity in the C, plus Cs fraction of uric acid recovered after the injection of glycine-2-14C should be in Cs. In fact, 72% of the activity in the C, pIus C, fraction was attributed to C,, a result below the lOOo/opredicted (Table 2). However, it is possible to explain the activity in C, in a manner that is consistent with the hypothesis that C, comes exclusively from the alpha carbon of glycine. The recovery error mentioned earlier was arbitrarily assigned solely to C, and hence the value reported for C, (Table 2) may be much higher than was actuahy the case. Unfortunately the extent of the error could not be adequately determined. It is also possible that some exchange of the carboxyl and the alpha carbons of glycine did take place prior to incorporation. Such an exchange would transfer l”C atoms from the alpha position of the injected glycine-2J4C to the carboxyl group of freshly synthesized glycine and facilitate the incorporation of activity of the carboxyl carbon of glycine into C, of uric acid. Such a transfer of the alpha carbon of glycine to the carboxyl position could occur if the alpha carbon were converted to formate and the formate were subsequently used in the synthesis
128
of glycine.
F. M. BARRETTand W. G. FRIEXD BRICTEALX-GR~~GOIREand VERLY (1958)
reported that the silkworm,
Bombyx mori, can use formate for the synthesis of both carbon atoms of glycine and
results discussed below will show that R. prolixus can use- the alpha carbon of glycine, and probably the carboxyl carbon as well, for the synthesis of formate. When glycine-1 -W was metabolized by R. prolixus, 29% of the injected glycine was converted to respiratory CO, (Table 1). The loss of carboxyl carbon as CO, would presumably leave the alpha carbon as a l-carbon fragment which could be converted to formate and used in the synthesis of uric acid. If this were the case, then one would predict that injection of glycine-2-r4C would give more activity in the hindgut contents than would glycine-l-iaC, since both labelled glycine and labelled formate would be available for uric acid synthesis after injection of glycine-2-W. Results, given in Table 1, support this prediction; the hindgut contents contained 89% of the injected activity after the injection of glycine-2-14C but only 47% after the injection of glycine-l-i%. The higher percentage of activity resulting from injections of glycine-2-i% and the greater activity in the carbons 2 and 8 fraction of uric acid recovered after such injections (Table 2) clearly indicate that the alpha carbon of glycine is converted to formate. The fact that 89% of the injected activity was recovered in the hindgut contents 3 days after the injection of glycine-2J”C reflects the great extent to which glycine is used, either directly as glycine or indirectly as formate, in the synthesis of uric acid in R. prolixus. This observation suggests a possible reinterpretation of the findings of PICKETT and FRIEND (1965) concerning the essential nature of glycine in R. prolixus. When glucose-UJ4C was injected into fifth instar larvae no radioactivity could be found in the free glycine of the haemolymph 24 hr after injection, although serine was strongly labelled. Pickett and Friend therefore concluded that glycine was an essential amino acid in this insect. It now appears that the lack of radioactivity in glycine may be explained by the rapid conversion of this molecule to uric acid. More precise conversion times for glycine and the relative contributions it makes to its various metabolic pathways should now be determined. Uric acid, with its low solubility and consequently low toxicity, is an extremely efficient molecule to use as an end-product of nitrogen metabolism in animals where water conservation is a problem. Considering this, the similarities of uric acid metabolism found in such diverse groups as insects, birds, reptiles, and land snails become less surprising. Acknomledgements-The authors wish to thank the National supported this work through grants to W. G. F. and a scholarship
Research Council to F. R/I. B.
who
REFEREKCES ANDERSON A. D. and PATTON R. L. (1955) 1n vitro studies of uric acid synthesis in insects. J. exp. Biol. 128, 443151. B~ZLD‘CVIN E. (1963) Dynamic Aspects of Biochemistry. Cambridge University Press, London. B.%RRETT F. M. and FRIEND W. G. (1966) Studies on the uric acid concentration in the haemolymph of fifth instar larvae of Rhodnius prolixus (Stal) during growth and metamorphosis. J. Insect Physiol. 12, 1-7.
URIC ACIDSYXTHESIS IN RHOD,VKJS
PROLIXL’S
1ZY
BRICTEXJX-G~~GOIRE S. and VERLY W. G. (1958) Utilization of formate for the biosynthesis of glycine carbon-l and -2 in Bombyx mori. Nature, Lond. 182, 1515. BucH_~~ J. M., SONXE J. C., and DELLWA A. M. (1948) Biological precursors of uric acid--II. The role of lactate, acetate and carbon dioxide as precursors of the carbon chain and nitrogen atom 7 of uric acid. J. biol. Chem. 173, 81-98. BCX~ELL E. (1967) The excretion of nitrogen in insects. A&. Insect Physiol. 4, 33-67. DESX R. M. and KILBY B. A. (1958) Experiments on uric acid synthesis by insect fat body. Archs
int. Physiol.
Biochem.
66, 282-286.
ELWYN D. and SPRINSON D. B. (1950) The role of serine and acetate in uric acid formation. J. biol. Chem. 184, 465-474. FRIEND W. G., CHOY C. T. H., and CARTWRIGHTE. (1965) The effect of nutrient intake on the development and egg production of Rhodniusprolixus Stahl. Can.J. Zool. 43,891-904. GILMOUR D. (1961) The Biochemistry of Insects. Academic Press, New York. HELLER J. and JEZE~VSKAM. M. (1959) The synthesis of uric acid in the Chinese Tussur moth (Antheraea pernyi). Bull. Acad. pol. Sci. Cl. 11, Ser. Sci. Biol. 7, l-4. HOPKINS T. L. and LOFGRENP. A. (1968) Adenine metabolism in the cockroach, Leucophaea maderae. J. Insect Physiol. 14, 1803-1814. KARLSSONJ. L. and BARKER H. A. (1949) Biosynthesis of uric acid labelled with radioactive carbon. J. biol. Chem. 177, 597-599. KROGH A. and WEIS-FOGH (1951) The respiratory exchange of the desert locust, Schistocerca gregaria, before, during and after flight. J. exp. Biol. 28, 344-357. LEE T. W. and CAMPBELL J. W. (1965) Uric acid synthesis in the terrestrial snail, Otala Zactea.
Camp. Biochem.
Physiol.
15, 457-468.
LEIFERT H. (1935) Untersuchungen iiber den Exkretstoffwechsel bei Eiern, Raupen und Puppen von Antheraea pernyi. Zool. Jb. (Physiol) 55, 131-190. MCE~OE W. D. (1957) Uric acid metabolism in the American roach, Periplaneta americana (L.). Ph.D. Dissertation, Rutgers University, New Jersey. MCENROE W. D. and FORGASHA. S. (1957) The in viva incorporation of Cl”-formate in the ureide groups of uric acid by Periplaneta americana (L.). Ann. ent. Sot. Am. 50,429-431. PICKETT C. and FRIEND W. G. (1965) The nutritionally essential amino acids of Rhodnius prolixus (StHl) deterimned with glucose-4-C14. J. Insect Physiol. 11, 1617-1623. SCHULERW. and REINDEL W. (1935) Die Hamsluresynthese im Vogelorganismus. Hoppe SeyZer’s Z. physiol.
Chem. 234, 63-82.
SONNE J. C., BUCH~~ J. M., and DELLUVA A. M. (1948) Biological precursors of uric acid-I. The role of lactate, acetate and formate in the synthesis of the ureide groups of uric acid. J. biol. Chem. 173, 69-79. TOJO S. and HIRANO C. (1968) Uric acid production in larvae of the rice stem borer (Chile suppressalis) in relation to post-diapause development. J. Insect PhysioZ. 14, 1121-l 133. WEINER H. (1902) Die HamsPure. Ergebn. Physiol. 1, 555-650. WIGGLESWORTH V. B. (1931) The physiolo,v of excretion in a blood-sucking insect, Rhodnius prolixus (Hemiptera: Reduviidae). r. exp. Biol. 8, 41 l-451.
URIC ACID SYNTHESIS F. M. WRRETT* Zoology
IN RHODNIUS
PROLIXUS
and W. G. FRIEND
Department, university of Toronto, Toronto, Ontario, Canada (Received 7 July 1969)
Abstract-Glycine-l-iaC, glycine- 2-i&C, and sodium formate-*“C were used to investigate the origin of the carbon atoms of uric acid in Rhodnius prolixus. As in other uricotelic organisms, the carboxyl carbon of glycine is incorporated into position 4 of uric acid, the alpha carbon of glycine goes to position 5, and the carbon of sodium formate is incorporated into positions 2 and 8. The carboxyl carbon of glycine is converted to COz approximately twentyfour times more rapidly than is the alpha carbon, and much of this CO* is incorporated into carbon 6 of uric acid. The alpha carbon of glycine is used in the synthesis of formate. The use of isotopically labelled precursors to determine the origin of all five carbon atoms of uric acid has not previously been reported for any species of insect. INTRODUCTION
THE PRESENCEof the uricolytic pathway, by which nucleic acids are broken down to uric acid, has been well documented in insects (see BURSELL, 1967). However, the large amount of uric acid in the excreta of most insects cannot be accounted for solely on the basis of nucleic acid degradation. Consequently, a synthetic pathway has been sought by which uric acid could originate from other precursors. WEINER (1902) reported that birds synthesize uric acid by combining 2 molecules of urea with 1 molecule of the 3 carbon dicarboxylic acid, tartronic acid. Based on work with the Chinese Tussur moth, Antheraea pernyi, LEIFERT (1935) proposed a similar mechanism for uric acid synthesis in insects. This pathway was subsequently disproved both in birds (SCHULER and REINDEL, 1935) and in insects (ANDERSONand PATTON, 1955). The use of isotopically labelled precursors in pigeons has established the currently accepted pathway of uric acid synthesis in birds (BUCHANANet al., 1948; SONNE et al., 1948). BALDWIN (1963) has postulated a reaction sequence for uric acid synthesis in which glutamine, aspartate, glutamate, glycine, formate, carbon dioxide, and ribose-5-phosphate combine in the presence of ATP to give inosine. The conversion of inosine to hypoxanthine is followed by the action of xanthine dehydrogenase, which converts hypoxanthine to xanthine and xanthine to uric acid. Fig. 1 illustrates the origin of the individual carbon and nitrogen atoms of uric acid produced by this pathway. The reviews of GIL~MOUR(1961) and BURSELL (1967) indicate that insects and birds synthesize uric acid in the same way, although the complete pathway of uric * Present address : Zoology
Department,
Cambridge 121
University,
Cambridge,
England.
122
F. M. BARRETTand W. G. FRIEXD
acid synthesis in insects has not been fully documented. DESAI and KILBY (1958) reported that 4-amino-5-imidazole carboxamide increases the rate of uric acid production in fat body homogenates of the blowfly, CaZZiphoraerythrocephala, and the desert locust, Schistocerca gregaria. Since this compound could be converted to the corresponding ribotide, an intermediate on the uricotelic pathway (BALDWIN, 1963), this observation supports the hypothesis of a pathway in insects similar to that found in birds. In the Chinese Tussur moth, A. pernyi, the simultaneous addition of formate, ribose-S-phosphate, glutamate, and aspartate will stimulate uric acid synthesis (HELLER and JEZEWSKA,,1959). Using a more direct approach, MCENROE and FORGASH(1957) showed that sodium formate-14C injected into the American cockroach, Periplaneta americana, was incorporated almost exclusively into positions 2 and 8 of uric acid recovered after injection. This result also suggests a pathway similar to that found in birds, as does the work of TOJO and HIRANO (1968), who showed that glycine-1 -14C injected into the rice stem borer, Chile suppressalis, was incorporated into uric acid. The present study was undertaken to determine the origin of the carbon atoms of uric acid synthesized by the blood-sucking bug, Rhodnius pro&us (St%). Since fifth instar larvae of this insect ingest up to nine times their body weight in a single feeding of mammalian blood (FRIEND et al., 1965), they must eliminate considerable amounts of nitrogenous waste. Both the increased uric acid concentration in the haemolymph (BARRETT and FRIEND, 1966) and the rapid rate of uric acid excretion (WIGGLESWORTH, 1931) which follow feeding are indications of rapid The predominance of uric acid in the excreta of synthesis of this molecule. R. proZi,rms(WIGGLESWORTH, 1931) is an important practical consideration in the present investigation since appreciable amounts of purified radioactive acid are required for the chemical degradation procedure used to isolate the individual carbon atoms. In this investigation, 14C-labelled glycine and formate, known precursors of uric acid in the pigeon, were injected into fifth instar larvae of R. prolixus to examine the hypothesis that C, of uric acid comes from CO,, C, of uric acid comes from the carboxyl carbon of glycine, C5 of uric acid comes from the alpha carbon of glycine, and C, and C, of uric acid come from formate. The use of isotopically labelled precursors to determine the origin of all five carbon atoms of uric acid has not previously been reported for any species of insect. MATERIALS
AND METHODS
The animals used in this study were fifth instar larvae taken from a culture of R. prolixus reared at 25°C in a humid atmosphere. The stock animals were fed on rabbit blood once very 30 to 40 days. The fate of glycine and sodium formate in R. prolixus was followed by injecting known amounts of glycine-1-%Z, glycine-2-1%, or sodium formate-l”C into fifth instar larvae. The haemolymph, hindgut contents, and respiratory CO, were recovered 3 days after injection and counted to determine the percentage of the injected radioactivity present in each. In some instances radioactive CO4 was
URICACIDSYNrHESISIN RHODSIUS
123
PROLIXUS
The insects were held in Warburg collected on each of the 3 days after injection. vessels and the respired CO, was trapped in 1.0 N NaOH contained in the sidearm. Injections given 2 days after feeding were delivered from a 25 ,~l Hamilton
‘\
I
‘,
Glycine /’
!
/
Formote
\ Glutamate
Giutomme \ \ \
/
FIG. 1, Origin of the carbon and nitrogen uricotelic
pathway
(after
atoms of uric acid synthesized
by the
BURSELL,1967)
syringe held in a micromanipulator. The needle was inserted into a forelimb that had been amputated at the distal end of the femur; the junction between needle and forelimb was sealed with a mixture of beeswax and colophony (KROGH and WEIS-FOGH, 1951). After injection, the waxed junction was reheated slightly, the needle was withdrawn and the wax was allowed to harden and seal the wound. Leakage, which could have occurred during removal of the needle, was prevented by pinching the femur proximal to the tip of the needle just prior to withdrawal. The anus was then sealed with a small drop of the wax mixture so that uric acid synthesized after injection would accumulate in the hindgut. For the experiments summarized in Table 1 the’injected volumes and activities per insect were: 1 PC glycine-2-14C weighing 18 pg in 7.5 ~1 insect Ringer; 0.23 PC glycine-lJ4C weighing 3 ,ug in 7 ,ul of insect Ringer; 4.5.~~ sodium formate-14C weighing 66 pg in 7 ~1 insect Ringer. The insect Ringer solution was prepared by combining 11.0 g NaCl, 1.4 g KCl, and 1-O g CaCl, and diluting to 1 1. with distilled water., Radioactive samples were counted in a Packard Tri-Carb Liquid Scintillation Counter to a standard error, in net counting rate, of 5 per cent. The scintillation liquid was prepared by combining 6.0 g of 2,5-diphenyl-oxazole (PPO) and O-1 g
124
F. 31. BARRELSand 1%‘.G. FRIEND
of 1,4-bis-2-j-(5-phenyloxazolyl)-benzene (POPOP), diluting to 1 1. with sulphurfree toluene, and then mixing 600 ml of this solution with 386 ml of absolute alcohol. The hindgut contents were dissolved in hot, 0.1% LiCO, prior to counting while all other samples were added directly to the scintillation liquid. Corrections for quenching and background were applied in all cases to make comparisons between different types of samples valid. In the experiments summarized in Table 2, in which radioactive uric acid formed from labelled precursors was recovered for subsequent chemical degradation, 10 to 15 insects were injected as previously described, and then dissected 3 days later to obtain the hindgut and its contents. The contents were dissolved in 10 ml of hot 0.1% LiCO, in a 15 ml test tube, to which was added a large excess of unlabelled carrier uric acid to minimize losses of radioactive uric acid during purification. The purification procedure described by MCENROE and FORGASH (1957) was carried through three to five recrystallizations to remove radioactive impurities. Uric acid recovered in this manner was degraded chemically according to procedures previously described by other authors (BUCHAXANet al., 1948; SONNE et al., 1948). These procedures make possible the recovery of C, as CO,, C, plus Cs as glyoxylic acid semicarbazone, and C, plus C, as urea. Subsequent degradation of glyoxylic acid semicarbazone will release C, and C5 separately as CO, (SONNE et al., 1948). Carbon dioxide was trapped in 1 N NaOH and quantitated by titration with HCl. All degradations were carried out on composite samples weighing 80 to 103 mg and containing both radioactive uric acid recovered from injected insects and unlabelled carrier uric acid. RESULTS Three days after the injection of glycine-l-i% into fifth instar larvae, 47% of the injected activity was recovered in the hindgut contents and 29% was recovered in the respiratory CO,. In contrast, over the same period, glycine-2-r1C gave 89% of the injected activity in the hindgut contents and only 1.2% in the respiratory CO,. With sodium formate as precursor the hindgut contents had 68% of the injected activity while 3.7% was recovered in the respiratory CO,. These values, given in Table 1, will be used to predict the relative amounts of activity in the five carbon atoms of uric acid synthesized after the injection of each precursor. With glycine-lJ4C as precursor, over 90% of the radioactive CO, produced during the 3-day recovery period was collected in the first 24 hr after injection. There was also extensive incorporation of glycine into the hindgut contents in the first 24 hr. Little activity remained in the haemolymph by 3 days after injection, and even at 1 day the haemolymph contained less than 5% of the activity initially injected. The distribution of activity between the individual carbon atoms of uric acid recovered after the injection of each precursor is reported in Table 2. The carboxyl carbon of glycine was incorporated into C, of uric acid approximately twentyseven times more extensively than was the alpha carbon. The alpha carbon
LTRIC ACID
SEXTHESIS
RH0D.VIV.S
IN
125
PROLIXUS
contributed more than twice as heavily to the C, plus Cs portion of uric acid than did the carboxyl carbon of glycine. Both carbons of glycine were incorporated into positions 4 and 5 of uric acid but in greatly differing proportions (Table 2). was incorporated into Approximately 9776 of the injected sodium formateJ4C positions 2 and 8 ; only slight activity was recovered in the other 3 carbon atoms. TABLE CCz,
l-PERCEXTAGES AND HI&DGUT
OF THE IXJECTED ACTIVITY
FOUND
IN HAEMOLYMPH,
RESPIRATORY
COXTENTS AFTER THE IXJECTION OF SUSPECTED PRECURSORS OF URIC ACID ISTO FIFTH INSTAR LARVAE OF R. prolixus
Injected Suspected
precursor
Glycine-1-“C carbon
Haemolymph
(carboxyl (alpha
* Values
given
are averages
10 ~1 sample
haemolymph
TABLE
7
Carbon
1.2
carbon labelled) Sodium formate-ilC
FIFTH
(“/b) recorded
3 days after injection* Hindgut
dioxide
contents
29
47
labelled)
Glycine-2-W
7 A
activity
was
volume
1.4
1.2
89
-
3.7
68
for 3 or 4 insects.
counted
and
the
activity
recorded
was
corrected
for
a total
of 30 ~1.
~-DISTRIBUTION
OF ACTIVITY
INSTAR LARVAE OF R. prOkWS
IN THE CARBON ATOMS OF URIC ACID RECOVERED FROM INJECTED WITH
Initial
activity
SUSPECTED PRECURSORS OF URIC ACID
found
in purified
composite
samples
of uric acid (%) c, Precursors
‘*C-labelled Glycine-1
-r*C
plus
c,
as glyoxylic
used to obtain uric acid (carboxyl
C,
semicarbazone
C,
C5 3,7
8.0
74
70.3
carbon labelled) Glycine-2-r4C (alpha
0.3
56
161
carbon labelled) Sodium formate-‘%
0.s
* Values C4 plus
for CZ plus C, were
C5 and subtracting
t The
recovery
error
from
2.5
obtained
by adding
-
the percentages
c,
plus c,* 18.0
4oi
43.7
-
96.7
determined
for C, and
100%.
described
in the
text
has
been
arbitrarily
assigned
to the
C,
fraction.
The values given for C, plus C, in Table 2 were obtained by combining the percentages calculated for C, and C, plus Cs and subtracting from 100%. They presumably therefore contain any error involved in the determination of C,, C5,
126
F. Pd.
BARRETTand W. G. FRIEXQ
and C6. Replicate experiments using glycine-1 -r%Z as precursor, and measurement of activity in the unpurified Ca plus C? fraction, both indicated that this error is slight. Activities reported for C, and Cj in the experiment with glycine-2JJC as precursor should be viewed with caution. A recovery error during the degradation of glyoxylic acid semicarbazone made accurate individual quantitation of these carbon atoms impossible. For reasons described in the Discussion, the error has been attributed exclusively to C, and consequently the value reported may be much higher than was actually the case. DISCUSSION
WIGGLESWORTH (1931) reported that 64 to 84 per cent of the dry weight and approximately 90 per cent of the total nitrogen in the excreta of R. prolixus was in the form of uric acid. It is likely, therefore, that most of the radioactivity present in the hindgut contents after injection of l”C-labelled precursors (Table I) was contained in uric acid. The extensive accumulation of activity in the hindgut after these injections indicates that both sodium formate and glycine are used in the synthesis of uric acid in R. prolixus. Chemical degradation studies have revealed the positions in the uric acid molecule into which these precursor carbon atoms were incorporated. Origin of carbon 6 Injection of glycine-l- 14C gave rise to twenty-four times more radioactive CO, during the 3-day test period than did injections of glycine-Z-r4C (29%/1*2%, Table 1). This shows that R. prolixus converts the carboxyl carbon of glycine into CO, more readily than the alpha carbon. Consequently insects injected with glycine-1-r4C should have approximately twenty-four times more radioactive CO, available for metabolism than insects injected with glycine-2-r4C. Hence, if C, of uric acid does come from CO,, it should follow that C, in uric acid recovered from insects injected with glycine-l-r4C will be about twenty-four times more active than C, in uric acid recovered from insects injected with glycine-2-14C. The value of 26.7 actually obtained (80%/0.3%, Table 2) s h ows close agreement with this prediction and supports the hypothesis that C, of uric acid comes from CO,. A similar origin for C, has been demonstrated in the pigeon (BUCHANANet al., 1948; KARLSSON and BARKER, 1949) and in the snail, Otala Zactea (LEE and CA~IPBELL, 1965). Origin of carbons 2 and 8 There can be little doubt that sodium formate is used in the synthesis of carbons 2 and 8 of uric acid. After the injection of sodium formate-r4C 97% of the activity in recovered uric acid was contained in these two carbon atoms (Table 2). This value is in close agreement with those obtained in similar studies on other organisms: the pigeon, 98% (KARLSSON and BARKER, 1949) and 96% (E~~YN and SPRINSON, 1950); the snail, Otala Zactea, 94.4% (LEE and CAMPBELL, 1965); the cockroach, P. americana, 92% (MCENROE, 1957).,
CRIC
ACID
SYNTHESIS
IN
RHODNIUS
PROLIXUS
127
In this investigation, no attempt was made to determine the individual contributions of carbons 2 and 8 to the total activity in uric acid recovered after the injection of sodium formate. In the pigeon, equal amounts of activity were found in both carbon 2 and carbon 8 (1 : 1 ratio) (SONNE et al., 1948), while the cockroach had slightly more activity in carbon 8 (1 : 1*3 ratio) (MCEPU’ROE and FORGASH, 1957). If the cockroach were capable of reversible cleavage of the purine ring, and if the turnover of C, was slightly greater than that of C,, then the ratio of 1 : 1.3 would be the result of a dilution of the activity in C, (GILMOUR, 1961). Recently, HOPKINS and LOFGREN (1968) reported that CO? collected for 15 hr after the injection of r4C-8-adenine or W-2-adenine into the cockroach, Leucophaea maderae, contained 3-5 and 4.5% of the injected activity respectively. This observation suggests a slightly greater turnover of C, than C, and supports Gilmour’s earlier suggestion. Origins of carbons 4 and 5 If C, of uric acid comes exclusively from the carboxyl carbon of glycine, it should follow, assuming no exchange of the carboxyl carbon and the alpha carbon prior to incorporation, that all of the activity in the C, plus Cs fraction of uric acid recovered after the injection of glycine-l-i4C should be found in C, (Table 2). In fact, 95% of the activity of this fraction was present in C,, offering strong support to this hypothesis. The apparent presence of some activity in C, was also reported by other authors in a similar experiment on the pigeon (BUCHANANet al., 1948). They claimed that during the early stages of the chemical degradation of glyoxylic acid semicarbazone some C, may have been released along with the C, samples. In experiments where the activity of the C, fraction was great (i.e. after injection of glycine-1-r4C) such a contamination error would be noticeable. This observation explains the apparent activity in Cs and remains consistent with the hypothesis that C, of uric acid comes exclusively from the carboxyl carbon of glycine. If Cs of uric acid comes from the alpha carbon of glycine, it should also follow, again assuming no exchange of the carboxyl and alpha carbons of glycine prior to incorporation, that all of the activity in the C, plus Cs fraction of uric acid recovered after the injection of glycine-2-14C should be in Cs. In fact, 72% of the activity in the C, pIus C, fraction was attributed to C,, a result below the lOOo/opredicted (Table 2). However, it is possible to explain the activity in C, in a manner that is consistent with the hypothesis that C, comes exclusively from the alpha carbon of glycine. The recovery error mentioned earlier was arbitrarily assigned solely to C, and hence the value reported for C, (Table 2) may be much higher than was actuahy the case. Unfortunately the extent of the error could not be adequately determined. It is also possible that some exchange of the carboxyl and the alpha carbons of glycine did take place prior to incorporation. Such an exchange would transfer l”C atoms from the alpha position of the injected glycine-2J4C to the carboxyl group of freshly synthesized glycine and facilitate the incorporation of activity of the carboxyl carbon of glycine into C, of uric acid. Such a transfer of the alpha carbon of glycine to the carboxyl position could occur if the alpha carbon were converted to formate and the formate were subsequently used in the synthesis
128
of glycine.
F. M. BARRETTand W. G. FRIEXD BRICTEALX-GR~~GOIREand VERLY (1958)
reported that the silkworm,
Bombyx mori, can use formate for the synthesis of both carbon atoms of glycine and
results discussed below will show that R. prolixus can use- the alpha carbon of glycine, and probably the carboxyl carbon as well, for the synthesis of formate. When glycine-1 -W was metabolized by R. prolixus, 29% of the injected glycine was converted to respiratory CO, (Table 1). The loss of carboxyl carbon as CO, would presumably leave the alpha carbon as a l-carbon fragment which could be converted to formate and used in the synthesis of uric acid. If this were the case, then one would predict that injection of glycine-2-r4C would give more activity in the hindgut contents than would glycine-l-iaC, since both labelled glycine and labelled formate would be available for uric acid synthesis after injection of glycine-2-W. Results, given in Table 1, support this prediction; the hindgut contents contained 89% of the injected activity after the injection of glycine-2-14C but only 47% after the injection of glycine-l-i%. The higher percentage of activity resulting from injections of glycine-2-i% and the greater activity in the carbons 2 and 8 fraction of uric acid recovered after such injections (Table 2) clearly indicate that the alpha carbon of glycine is converted to formate. The fact that 89% of the injected activity was recovered in the hindgut contents 3 days after the injection of glycine-2J”C reflects the great extent to which glycine is used, either directly as glycine or indirectly as formate, in the synthesis of uric acid in R. prolixus. This observation suggests a possible reinterpretation of the findings of PICKETT and FRIEND (1965) concerning the essential nature of glycine in R. prolixus. When glucose-UJ4C was injected into fifth instar larvae no radioactivity could be found in the free glycine of the haemolymph 24 hr after injection, although serine was strongly labelled. Pickett and Friend therefore concluded that glycine was an essential amino acid in this insect. It now appears that the lack of radioactivity in glycine may be explained by the rapid conversion of this molecule to uric acid. More precise conversion times for glycine and the relative contributions it makes to its various metabolic pathways should now be determined. Uric acid, with its low solubility and consequently low toxicity, is an extremely efficient molecule to use as an end-product of nitrogen metabolism in animals where water conservation is a problem. Considering this, the similarities of uric acid metabolism found in such diverse groups as insects, birds, reptiles, and land snails become less surprising. Acknomledgements-The authors wish to thank the National supported this work through grants to W. G. F. and a scholarship
Research Council to F. R/I. B.
who
REFEREKCES ANDERSON A. D. and PATTON R. L. (1955) 1n vitro studies of uric acid synthesis in insects. J. exp. Biol. 128, 443151. B~ZLD‘CVIN E. (1963) Dynamic Aspects of Biochemistry. Cambridge University Press, London. B.%RRETT F. M. and FRIEND W. G. (1966) Studies on the uric acid concentration in the haemolymph of fifth instar larvae of Rhodnius prolixus (Stal) during growth and metamorphosis. J. Insect Physiol. 12, 1-7.
URIC ACIDSYXTHESIS IN RHOD,VKJS
PROLIXL’S
1ZY
BRICTEXJX-G~~GOIRE S. and VERLY W. G. (1958) Utilization of formate for the biosynthesis of glycine carbon-l and -2 in Bombyx mori. Nature, Lond. 182, 1515. BucH_~~ J. M., SONXE J. C., and DELLWA A. M. (1948) Biological precursors of uric acid--II. The role of lactate, acetate and carbon dioxide as precursors of the carbon chain and nitrogen atom 7 of uric acid. J. biol. Chem. 173, 81-98. BCX~ELL E. (1967) The excretion of nitrogen in insects. A&. Insect Physiol. 4, 33-67. DESX R. M. and KILBY B. A. (1958) Experiments on uric acid synthesis by insect fat body. Archs
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ELWYN D. and SPRINSON D. B. (1950) The role of serine and acetate in uric acid formation. J. biol. Chem. 184, 465-474. FRIEND W. G., CHOY C. T. H., and CARTWRIGHTE. (1965) The effect of nutrient intake on the development and egg production of Rhodniusprolixus Stahl. Can.J. Zool. 43,891-904. GILMOUR D. (1961) The Biochemistry of Insects. Academic Press, New York. HELLER J. and JEZE~VSKAM. M. (1959) The synthesis of uric acid in the Chinese Tussur moth (Antheraea pernyi). Bull. Acad. pol. Sci. Cl. 11, Ser. Sci. Biol. 7, l-4. HOPKINS T. L. and LOFGRENP. A. (1968) Adenine metabolism in the cockroach, Leucophaea maderae. J. Insect Physiol. 14, 1803-1814. KARLSSONJ. L. and BARKER H. A. (1949) Biosynthesis of uric acid labelled with radioactive carbon. J. biol. Chem. 177, 597-599. KROGH A. and WEIS-FOGH (1951) The respiratory exchange of the desert locust, Schistocerca gregaria, before, during and after flight. J. exp. Biol. 28, 344-357. LEE T. W. and CAMPBELL J. W. (1965) Uric acid synthesis in the terrestrial snail, Otala Zactea.
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LEIFERT H. (1935) Untersuchungen iiber den Exkretstoffwechsel bei Eiern, Raupen und Puppen von Antheraea pernyi. Zool. Jb. (Physiol) 55, 131-190. MCE~OE W. D. (1957) Uric acid metabolism in the American roach, Periplaneta americana (L.). Ph.D. Dissertation, Rutgers University, New Jersey. MCENROE W. D. and FORGASHA. S. (1957) The in viva incorporation of Cl”-formate in the ureide groups of uric acid by Periplaneta americana (L.). Ann. ent. Sot. Am. 50,429-431. PICKETT C. and FRIEND W. G. (1965) The nutritionally essential amino acids of Rhodnius prolixus (StHl) deterimned with glucose-4-C14. J. Insect Physiol. 11, 1617-1623. SCHULERW. and REINDEL W. (1935) Die Hamsluresynthese im Vogelorganismus. Hoppe SeyZer’s Z. physiol.
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SONNE J. C., BUCH~~ J. M., and DELLUVA A. M. (1948) Biological precursors of uric acid-I. The role of lactate, acetate and formate in the synthesis of the ureide groups of uric acid. J. biol. Chem. 173, 69-79. TOJO S. and HIRANO C. (1968) Uric acid production in larvae of the rice stem borer (Chile suppressalis) in relation to post-diapause development. J. Insect PhysioZ. 14, 1121-l 133. WEINER H. (1902) Die HamsPure. Ergebn. Physiol. 1, 555-650. WIGGLESWORTH V. B. (1931) The physiolo,v of excretion in a blood-sucking insect, Rhodnius prolixus (Hemiptera: Reduviidae). r. exp. Biol. 8, 41 l-451.