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Developmental studies in Pieris brassicae (Lepidoptera)—II. A study of nitrogenous excretion during the last larval instar
Comp. Biochem. Physiol., 1975, Vol. 51B, pp. 445 to 449. Pergamon Press. Printed in Great Britain
DEVELOPMENTAL STUDIES IN PIEIUS BRASSICAE (LEPIDOPTERA) II. A STUDY OF NITROGENOUS EXCRETION DURING THE LAST LARVAL INSTAR B. MAUCHAMPz AND R. LAFONT~ qnstitut National Agronomique, Centre Griguon, 78850 Thiverval Grignon; and 2Ecole Normale Sup6rieure, Laboratoire de Zoologie, 46 rue d'Ulm, 75230 Paris Cedex 05, France (Received 10 M a y 1974)
Abstract--1. Nitrogenous excretion products (uric acid and its main metabolite allantoic acid) have been studied during the last larval instar. 2. These compounds are either excreted by the animal or stored in the body (essentially the fat body). 3. Enzyme activities related to uric acid biosynthesis (guanine deaminase and xanthine dehydrogenase) or degradation (uricase) have been examined in several tissues in order to determine accurately their respective roles in the nitrogenous excretion processes. INTRODUCTION DURING the last few years, the nitrogenous metabolism of insects has been extensively studied (reviews by Razet, 1966; Bursell, 1967; Corrigan, 1970). The larva of Pieris brassicae has been investigated in an attempt to define the nature of the excreted nitrogenous compounds. The animals used by Razet (1961) were not specifically fed under constant conditions nor on the same host plant. This last aspect is very important, because excretion is dependent on the food composition; for example, the percentage of protein in the diet modifies enzyme activities. Ito & Mukayama (1964) have shown that the xanthine dehydrogenase (a key enzyme in uric acid synthesis) level in B o m b y x is dependent on food composition, so that we have followed a more detailed study including: (i) the analysis of the main excretory compounds (uric acid and allantoic acid) and the modification of their relative abundance in fecal pellets; (ii) the determination of uric acid in various tissues of the larva, which is known to accumulate uric acid before pupation into its fat body (Wigglesworth, 1925; Harmsen, 1966); (iii) the measurement in several organs of enzymatic activities related to uric acid metabolism, anabolism (guanine deaminase and xanthine dehydrogenase) or catabolism (uricase). The cabbage used in our experiments did not contain any noticeable amount of uric acid, so that we can conclude that this compound results from metabolic activities of Pieris larva.
immediately lyophilized and stored at -27°C until they were used. Two series of animals were used. 2. Analysis o f animals and excreta
Variations of uric acid were studied throughout the instar in the fat body, carcass and gut; imaginal discs were not studied because of the low amount present. All these organs were extracted with cold 0.2 N perchloric acid, but this method was not applied to excreta. Low pH's may break allantoic acid into glyoxylic acid and urea (a method used for allantoic acid measurements), so that excreta were extracted by hot water (Tojo & Yushima, 1972). Uric acid was then determined either by a colorimetric reaction (Carroll et al., 1971) or by the uricase method of Kageyama (1971). Allantoic acid was measured by the Schryver-Fosse method (described in Razet, 1961). 3. Enzyme determinations
All enzyme determinations were performed with the use of radioactive substrates (as previously described in mg
/
1. Animals
The animals were fed as previously described (Lafont et al., 1975). Excreta, such as dissected organs, were
® /~-'b9
50
MATERIALS AND M E T H O D S
mg
®
i00
D 50
i00
Fig. 1. Variations of uric acid content of animals. A. Total uric acid. B. Variations into fat body ( - e - - e - ) and "carcass" (o . . . . o). The time scale (abscissa) of all figures is expressed as a percentage of instar duration.
445
446
B. MAUCHAMP AND R. LAFONT
mg~
'mg
® 300'
//
s'o
5O
Fig. 2. Estimation of excreta during the last larval instar (dry weight). A. Rate of excretion, in mg/hr and animal. B. Sum of excreta.
' mg/h
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Fig. 3. Excretion of uric acid into fecal pellets. A. Rate of uric acid excretion (mg/hr and animal). B. Total uric acid excretion. C. Fraction of excreta represented by uric acid. Two series of experiments are presented here.
Developmental studies in Pieris brassicae-II
447
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Fig. 4. Excretion of allantoic acid into fecal pellets (same legend as Fig. 3). the case of xanthine dehydrogenase, see Lafont & Papillon, 1972). Guanine deaminase and xanthine dehydrogenase were studied together, whereas uricase, which was less active, needed longer incubation periods of several hours. (a) Guanine deaminase (GD) and xanthine dehydrogenase (XDH). Whole animals or tissues were ground in distilled water containing a trace of phenylthiourea to prevent GD inactivation by tyrosinase (Hodge & Glassman, 1967). This crude extract was centrifuged (10min at 25,000 g) and ammonium sulphate added up to 40% saturation, in order to precipitate XDH; GD precipitates in the 40-60 % saturation fraction. The XDH containing sediment was resuspended in a 0"1 M Tris-HC1 buffer, pH 8.5, and treated with charcoal. Charcoal was then removed by two successive centrifugations at 40,000 g. GD was resuspended in a phosphate buffer (0.05 M, pH 7"5). 2-14C guanine and xanthine (CEA) were used as substrates and kinetics were performed at 30°C. (b) Uricase. Organs were extracted with water and ammonium sulphate added to 50% saturation; the sediment containing uricase was redissolved in borate buffer
(0'I M, pH 9.2) and 8-14C uric acid added; this method proved the only satisfactory one with regard to specificity and sensibility.
RESULTS 1. Uric acid in the larva Our experiments (Fig. 1) show that: (i) the content of the whole animal rises a great deal during the instar (fourfold); (ii) parts of the animals show various patterns during development (Fig. 1B). Most of the uric acid of the young larva lies in the integument, associated with protein granules (Tsujita & Sakurai, 1964, 1966, 1967; Barbier, 1972). The integument also contains ommochromes that are excreted before the pupal moult into red fecal pellets also rich in uric acid; both compounds arise, at least partially from the larval integument. During the second half of the instar, uric acid rises in the
448
B. MAUCrIAMVANt) R. LArONT
fat body, which plays a fundamental role in nitrogen metabolism (Kilby, 1963).
®
®
2. Analysis of excreta
t
The dried excreta were weighed and the levels of total excretion are reported in Fig. 2. The occurrence of uric acid and allantoic acid has been reported in Figs. 3 and 4, uric acid is generally more abundant than its metabolite, except early in the instar. The last excreta are poor in allantoic acid, while they are rich in uric acid (up to 14 per cent of dry weight in the red fecal pellets). As the gut does not contain noticeable amounts of uric acid, while the animal lays fecal pellets containing this compound, uric acid must be excreted only in the posterior part of the gut.
t
2
/
/ /1
lOO
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3. Enzymatic studies (a) Uric acid synthesizing activities. Figure 5 shows the varying activities of G D and XDH. These enzymes are not very active in the young larva, but we note a rapid increase and a maximum at the middle of the instar. They then decrease to a lower level at pupation. Their patterns are quite different: G D is essentially found in the fat body, while X D H exists in several organs, including the fat body, epidermis and digestive tract. (b) Uric acid breakdown. P. brassicae larva is known to contain enzymes for uric acid breakdown to allantoic acid, e.g. active uricase and allantoinase (Razet, 1961). We have only studied uricase, the limiting factor in this degradative pathway (Fig. 6). There is little enzyme activity in the gut wall and Malpighian tubules, as expressed by one animal (this fact is not necessary in conflict with Razet's results, because this author gives enzyme values per mg of protein, and the mass of Malpighian tubules, where he finds a large amount of uricase, is very
~oo
Fig. 5. Activities of enzymes related to uric acid synthesis. A. Total XDH variations. B. Activity of XDH into fat body (t2--z~) and gut ( . . . . ). C. Total guanine deaminase; arbitrary units. low). Much more uricase was detected in the fat body and body wall. Uricase falls to a low level in the prepupa, as it does in Calliphora (Desai & Kilby, 1958), but reappears in the pupa, with a different pattern. DISCUSSION Our brief study has shown that uric acid and allantoic acid undergo important changes during
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II
. 100
-
~
\
,
\
~
/
X ~t,
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100
Fig. 6. Activity of uricase. A. Total activity. B. Variations into fat body (z2 - - - t3), gut wall ( .... ) and carcass ( 4 ~ - - - . - ) ; arbitrary units.
Developmental studies in Pieris brassicae-II the last larval instar. The complementary variations of uric acid content observed for fat body and carcass (Fig. 1) have been further investigated by the means of radioisotopic experiments. After a 2-14C guanine injection at mid-instar, nearly all the radioactivity is excreted; after a similar injection in the pharate pupa, the main part of the radioactivity is found in the fat body as uric acid. We may thus conclude that, in the prepupa, fat body accumulates guanine and transforms it into uric acid, according to our enzymatic studies. When integumental uric acid disappears, uricase is not very active, so that this compound is not markedly metabolized; the larva does not excrete fecal pellets and the gut contains only a small amount of uric acid (about 0.1 rag)--uric acid from the integument (for 0.5 mg) must be accumulated in the fat body. This organ has a double property: it may both accumulate and synthesize uric acid (similar data have been previously found with pupal wings in the case of pteridines, Lafont, 1972); it may also store proteins arising from the haemolymph (Chippendale & Kilby, 1969; Chippendale, 1971). Excreta are characterized by a ratio of allantoic acid to uric acid lower than 1, according to the low values of uricase found. As most of the excreta are laid down at the fifth instar, we may suggest that during the larval stage, P. brassicae excretes about twice as much uric acid than it does allantoic acid, a report in conflict with the experiments of Razet (1961). However, the breeding of larvae on another variety of cabbage gave us very different results, so that the influence of food composition seems to be very important. Acknowledgements The authors thank the C.E.A. for the partial supply of radiolabeled products. REFERENCES BARBmRR. (1972) Origine et formation des granules pigmentaires de la cuticule larvaire de Tyriajacobeae (L6pidopt6re, Arctiide). C. R. hebd. S~anc. Acad. Sci., Paris 274, 1839-1842. BURSELL E. (1967) The excretion of nitrogen in insects. Adv. Insect Physiol. 4, 33-67. CARROLLJ. J., COBURNH., DOUGLASSR. & BAn,SONA. L. (1971) A simplified alkaline phosphotungstate assay for uric acid in serum. Clin. Chem. 17, 158-160. CHIPPENDALE G. M. (1971) Selective protein storage by the fat body of the Angoumois grain moth Sitotroga cerealla. Insect Biochem. 1, 122-124. CHn'PENDALEG, M. & KtLnYB. A. (1969) Relationship between the proteins of the haemolymph and fat body duringdevelopmentof Pieris brassicae. J. Insect Physiol. 15, 905-926.
449
CORRIGAN J. J. (1970) Nitrogen metabolism in insects. In Comparative Biochemistry of Nitrogen Metabolism. Vol. 1, The Invertebrates (Edited by CAMPaELLJ. W.), pp. 388-488. Academic Press, New York. D~AI R. M. & KILBYl~. A. (1958) Experiments on uric acid synthesis by insect fat body. Archs int. Physiol. Biochim. 66, 282-286, HAgMSEN R. (1966) The excretory role of pteridines in insects. J. exp. Biol. 45, 1-13. HODGE L. D. & GLASSMANE. (1967) Purine catabolism in Drosophila melanogaster--II. Guanine deaminase, inosine phosphorylase and adenosine deaminase activities in mutants with altered xanthine dehydrogenase activities. Genetics 57, 571-577. I1"o T. & MUKAIYAMAF. (1964) Relationship between protein content of diets and xanthine oxidase activity in the silkworm, Bombyx mori. J. Insect Physiol. 10, 789-796. KAGEYAMAN. (1971) A direct colorimetric determination of uric acid in serum and urine with uricase--catalase system. Clin. chim. Acta 31, 421-426. IOLBY B. A. (1963) The biochemistry of insect fat body. Adv. Insect Physiol. 1, 111-174. LAFONTR. (1972) Les pt6rines des Pieridae (Lepidoptera) et leur biosynth~se--II. Synth6se et transport de l'isoxanthopt6rine au cours de la vie nymphale et adulte chez Pieris brassicae. Biochimie 54, 73-82. LAFONT R., MAUCHAMPB., BOULAYG. • TARROUXP. (1975) Developmental studies in Pieris brassicae (Lepidoptera)--I. Growth of various tissues during the last larval instar. Comp. Biochem. Physiol. 51B, 439--444. LAFONTR. & PAPILLONJ. (1972) Les pt6rines des Pieridae (Lepidoptera) et leur biosynth6se--III. Etude de l'activit6 xanthine d6shydrog6nase (E.C. 1.2.3.2) au cours du d6veloppement. Biochimie 54, 365-370. RAZET P. (1961) Rccherches sur l'uricolyse chez les insectes. Th6se, Imprimerie bretonne, Rennes. RAZET P. (1966) Les 616ments terminaux du catabolisme azot6 chez les insectes. Ann. Biol. 5, 43-73. ToJo S. & YUSHIMAT. (1972) Uric acid and its metabolites in butterfly wings. J. Insect Physiol. 18, 404422. TSUJITA M. & SAKURAIS. (1964) Relationship between chromogranules and uric acid in hypodermal ceUs of silkworm larvae. Proc. Jap. Acad. 40, 561-565. TSUJITA M. & SAKURAIS. (1966) Chemical composition of chromogranules produced in the hypodermal cells of silkworm larvae. Proc. Jap. Acad. 42, 956-959. TSUJITA M. & SAKURAIS. (1967) Pteridine granules in hypodermal cells of the silkworm larva causing nontransparency of larval skin. Proc. Jap. Acad. 43, 991996. WIGGLESWORTHV. B. (1925) Uric acid in Pieridae: a quantitative study. Proc. R. Soc. Load. B 97, 149-155. Key Word Index--Nitrogen excretion; Pieris brassicae ; insect development; larval; fat body; uric acid; guanine deaminase; xanthine dehydrogenase; uricase.
DEVELOPMENTAL STUDIES IN PIEIUS BRASSICAE (LEPIDOPTERA) II. A STUDY OF NITROGENOUS EXCRETION DURING THE LAST LARVAL INSTAR B. MAUCHAMPz AND R. LAFONT~ qnstitut National Agronomique, Centre Griguon, 78850 Thiverval Grignon; and 2Ecole Normale Sup6rieure, Laboratoire de Zoologie, 46 rue d'Ulm, 75230 Paris Cedex 05, France (Received 10 M a y 1974)
Abstract--1. Nitrogenous excretion products (uric acid and its main metabolite allantoic acid) have been studied during the last larval instar. 2. These compounds are either excreted by the animal or stored in the body (essentially the fat body). 3. Enzyme activities related to uric acid biosynthesis (guanine deaminase and xanthine dehydrogenase) or degradation (uricase) have been examined in several tissues in order to determine accurately their respective roles in the nitrogenous excretion processes. INTRODUCTION DURING the last few years, the nitrogenous metabolism of insects has been extensively studied (reviews by Razet, 1966; Bursell, 1967; Corrigan, 1970). The larva of Pieris brassicae has been investigated in an attempt to define the nature of the excreted nitrogenous compounds. The animals used by Razet (1961) were not specifically fed under constant conditions nor on the same host plant. This last aspect is very important, because excretion is dependent on the food composition; for example, the percentage of protein in the diet modifies enzyme activities. Ito & Mukayama (1964) have shown that the xanthine dehydrogenase (a key enzyme in uric acid synthesis) level in B o m b y x is dependent on food composition, so that we have followed a more detailed study including: (i) the analysis of the main excretory compounds (uric acid and allantoic acid) and the modification of their relative abundance in fecal pellets; (ii) the determination of uric acid in various tissues of the larva, which is known to accumulate uric acid before pupation into its fat body (Wigglesworth, 1925; Harmsen, 1966); (iii) the measurement in several organs of enzymatic activities related to uric acid metabolism, anabolism (guanine deaminase and xanthine dehydrogenase) or catabolism (uricase). The cabbage used in our experiments did not contain any noticeable amount of uric acid, so that we can conclude that this compound results from metabolic activities of Pieris larva.
immediately lyophilized and stored at -27°C until they were used. Two series of animals were used. 2. Analysis o f animals and excreta
Variations of uric acid were studied throughout the instar in the fat body, carcass and gut; imaginal discs were not studied because of the low amount present. All these organs were extracted with cold 0.2 N perchloric acid, but this method was not applied to excreta. Low pH's may break allantoic acid into glyoxylic acid and urea (a method used for allantoic acid measurements), so that excreta were extracted by hot water (Tojo & Yushima, 1972). Uric acid was then determined either by a colorimetric reaction (Carroll et al., 1971) or by the uricase method of Kageyama (1971). Allantoic acid was measured by the Schryver-Fosse method (described in Razet, 1961). 3. Enzyme determinations
All enzyme determinations were performed with the use of radioactive substrates (as previously described in mg
/
1. Animals
The animals were fed as previously described (Lafont et al., 1975). Excreta, such as dissected organs, were
® /~-'b9
50
MATERIALS AND M E T H O D S
mg
®
i00
D 50
i00
Fig. 1. Variations of uric acid content of animals. A. Total uric acid. B. Variations into fat body ( - e - - e - ) and "carcass" (o . . . . o). The time scale (abscissa) of all figures is expressed as a percentage of instar duration.
445
446
B. MAUCHAMP AND R. LAFONT
mg~
'mg
® 300'
//
s'o
5O
Fig. 2. Estimation of excreta during the last larval instar (dry weight). A. Rate of excretion, in mg/hr and animal. B. Sum of excreta.
' mg/h
® //
\
//
0.1.
\
i
mgT, 5O
(~
'g
5
I
~ /
4
.,0
l
!
l© 10
/
JI//~
t
/
/ 2i
I
50
S
.50
Fig. 3. Excretion of uric acid into fecal pellets. A. Rate of uric acid excretion (mg/hr and animal). B. Total uric acid excretion. C. Fraction of excreta represented by uric acid. Two series of experiments are presented here.
Developmental studies in Pieris brassicae-II
447
~'mg/h 0.1,
II II
Ai I
I
J
'mg~o
/\,
so
/
mg
,
1
©
//
2
io
/
\
I
D
I
i
so
Fig. 4. Excretion of allantoic acid into fecal pellets (same legend as Fig. 3). the case of xanthine dehydrogenase, see Lafont & Papillon, 1972). Guanine deaminase and xanthine dehydrogenase were studied together, whereas uricase, which was less active, needed longer incubation periods of several hours. (a) Guanine deaminase (GD) and xanthine dehydrogenase (XDH). Whole animals or tissues were ground in distilled water containing a trace of phenylthiourea to prevent GD inactivation by tyrosinase (Hodge & Glassman, 1967). This crude extract was centrifuged (10min at 25,000 g) and ammonium sulphate added up to 40% saturation, in order to precipitate XDH; GD precipitates in the 40-60 % saturation fraction. The XDH containing sediment was resuspended in a 0"1 M Tris-HC1 buffer, pH 8.5, and treated with charcoal. Charcoal was then removed by two successive centrifugations at 40,000 g. GD was resuspended in a phosphate buffer (0.05 M, pH 7"5). 2-14C guanine and xanthine (CEA) were used as substrates and kinetics were performed at 30°C. (b) Uricase. Organs were extracted with water and ammonium sulphate added to 50% saturation; the sediment containing uricase was redissolved in borate buffer
(0'I M, pH 9.2) and 8-14C uric acid added; this method proved the only satisfactory one with regard to specificity and sensibility.
RESULTS 1. Uric acid in the larva Our experiments (Fig. 1) show that: (i) the content of the whole animal rises a great deal during the instar (fourfold); (ii) parts of the animals show various patterns during development (Fig. 1B). Most of the uric acid of the young larva lies in the integument, associated with protein granules (Tsujita & Sakurai, 1964, 1966, 1967; Barbier, 1972). The integument also contains ommochromes that are excreted before the pupal moult into red fecal pellets also rich in uric acid; both compounds arise, at least partially from the larval integument. During the second half of the instar, uric acid rises in the
448
B. MAUCrIAMVANt) R. LArONT
fat body, which plays a fundamental role in nitrogen metabolism (Kilby, 1963).
®
®
2. Analysis of excreta
t
The dried excreta were weighed and the levels of total excretion are reported in Fig. 2. The occurrence of uric acid and allantoic acid has been reported in Figs. 3 and 4, uric acid is generally more abundant than its metabolite, except early in the instar. The last excreta are poor in allantoic acid, while they are rich in uric acid (up to 14 per cent of dry weight in the red fecal pellets). As the gut does not contain noticeable amounts of uric acid, while the animal lays fecal pellets containing this compound, uric acid must be excreted only in the posterior part of the gut.
t
2
/
/ /1
lOO
©
3. Enzymatic studies (a) Uric acid synthesizing activities. Figure 5 shows the varying activities of G D and XDH. These enzymes are not very active in the young larva, but we note a rapid increase and a maximum at the middle of the instar. They then decrease to a lower level at pupation. Their patterns are quite different: G D is essentially found in the fat body, while X D H exists in several organs, including the fat body, epidermis and digestive tract. (b) Uric acid breakdown. P. brassicae larva is known to contain enzymes for uric acid breakdown to allantoic acid, e.g. active uricase and allantoinase (Razet, 1961). We have only studied uricase, the limiting factor in this degradative pathway (Fig. 6). There is little enzyme activity in the gut wall and Malpighian tubules, as expressed by one animal (this fact is not necessary in conflict with Razet's results, because this author gives enzyme values per mg of protein, and the mass of Malpighian tubules, where he finds a large amount of uricase, is very
~oo
Fig. 5. Activities of enzymes related to uric acid synthesis. A. Total XDH variations. B. Activity of XDH into fat body (t2--z~) and gut ( . . . . ). C. Total guanine deaminase; arbitrary units. low). Much more uricase was detected in the fat body and body wall. Uricase falls to a low level in the prepupa, as it does in Calliphora (Desai & Kilby, 1958), but reappears in the pupa, with a different pattern. DISCUSSION Our brief study has shown that uric acid and allantoic acid undergo important changes during
®
®
/ • 5t)
II
. 100
-
~
\
,
\
~
/
X ~t,
L 50
100
Fig. 6. Activity of uricase. A. Total activity. B. Variations into fat body (z2 - - - t3), gut wall ( .... ) and carcass ( 4 ~ - - - . - ) ; arbitrary units.
Developmental studies in Pieris brassicae-II the last larval instar. The complementary variations of uric acid content observed for fat body and carcass (Fig. 1) have been further investigated by the means of radioisotopic experiments. After a 2-14C guanine injection at mid-instar, nearly all the radioactivity is excreted; after a similar injection in the pharate pupa, the main part of the radioactivity is found in the fat body as uric acid. We may thus conclude that, in the prepupa, fat body accumulates guanine and transforms it into uric acid, according to our enzymatic studies. When integumental uric acid disappears, uricase is not very active, so that this compound is not markedly metabolized; the larva does not excrete fecal pellets and the gut contains only a small amount of uric acid (about 0.1 rag)--uric acid from the integument (for 0.5 mg) must be accumulated in the fat body. This organ has a double property: it may both accumulate and synthesize uric acid (similar data have been previously found with pupal wings in the case of pteridines, Lafont, 1972); it may also store proteins arising from the haemolymph (Chippendale & Kilby, 1969; Chippendale, 1971). Excreta are characterized by a ratio of allantoic acid to uric acid lower than 1, according to the low values of uricase found. As most of the excreta are laid down at the fifth instar, we may suggest that during the larval stage, P. brassicae excretes about twice as much uric acid than it does allantoic acid, a report in conflict with the experiments of Razet (1961). However, the breeding of larvae on another variety of cabbage gave us very different results, so that the influence of food composition seems to be very important. Acknowledgements The authors thank the C.E.A. for the partial supply of radiolabeled products. REFERENCES BARBmRR. (1972) Origine et formation des granules pigmentaires de la cuticule larvaire de Tyriajacobeae (L6pidopt6re, Arctiide). C. R. hebd. S~anc. Acad. Sci., Paris 274, 1839-1842. BURSELL E. (1967) The excretion of nitrogen in insects. Adv. Insect Physiol. 4, 33-67. CARROLLJ. J., COBURNH., DOUGLASSR. & BAn,SONA. L. (1971) A simplified alkaline phosphotungstate assay for uric acid in serum. Clin. Chem. 17, 158-160. CHIPPENDALE G. M. (1971) Selective protein storage by the fat body of the Angoumois grain moth Sitotroga cerealla. Insect Biochem. 1, 122-124. CHn'PENDALEG, M. & KtLnYB. A. (1969) Relationship between the proteins of the haemolymph and fat body duringdevelopmentof Pieris brassicae. J. Insect Physiol. 15, 905-926.
449
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