The effects of dietary nitrogen levels on glycine, formate, and xanthine incorporation into urates in the German cockroach, Blattella germanica L. (Dictyoptera: Blattellidae)

Comp. Biochem. Physiol. Vol. 75B, No. 2, pp. 293 to 300, 1983

0305-0491/83/060293-08503.00/0 © 1983 Pergamon Press Ltd

Printed in Great Britain

THE EFFECTS OF DIETARY NITROGEN LEVELS ON GLYCINE, FORMATE, AND XANTHINE INCORPORATION INTO URATES IN THE GERMAN COCKROACH, B L A T T E L L A G E R M A N I C A L. (DICTYOPTERA: BLATTELLIDAE) Jo A. ENGEBRETSON* and DONALD E. MULLINS Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA (Received 29 September 1982) Abstract--1. German cockroaches were injected with either [14C]glycine ' [~ 4C]formate, or [14C]xan-

thine and maintained on different dietary nitrogen levels. They were analyzed for whole body radiolabel retention and incorporation of radiolabel into the urate fraction after feeding on various diets for one to two weeks. Groups of cockroaches were injected with labelled urate precursors and the 1'*CO2 released was collected. Release of 14COz was examined in relation to dietary nitrogen levels. 2. Radiolabelled glycine was metabolized rapidly, some of it being released as ~4CO2. There was a direct relationship between x4C incorporation into body urates and maintenance on different dietary nitrogen levels. 3. Radiolabelled formate was rapidly partitioned into body urates by cockroaches maintained on specific diets. Comparatively low levels of ~4CO2 were released. 4. Incorporation of 14C-xanthine into urates was found to increase with elevated dietary nitrogen levels. Dietary effects on its metabolism were mirrored by ~4CO2 released and other metabolites excreted in the feces.

may be used by developing embryos to supplement their nitrogen requirements. Cockroach urate storage is directly related to dietary nitrogen level and age (Mullins & Cochran, 1975a; Cochran, 1979; Cochran et al., 1979; Valovage & Brooks, 1979). The large build up of urates is most likely due to de novo synthesis. Both glycine and formate are precursors in de novo urate synthesis in birds (Henderson, 1972) and insects (Barrett & Friend, 1970; Cochran, 1975). These materials are incorporated into the purine skeleton early in the uricotelic pathway. On the other hand, conversion of xanthine to uric acid represents the last step of urate production in both the nucleicolytic and uricotelic pathways (Cochran, 1975). Although information on urate build up in cockroaches is available, little is known about the influence of diet on the incorporation of specific precursors or purine metabolites of uric acid. The purpose of this study was to examine the effects of dietary nitrogen on uric acid synthesis using radiolabelled glycine, formate and xanthine.

INTRODUCTION

Three general pathways have been described in insect uric acid (urate) metabolism. They are: (1) de novo urate synthesis or the uricotelic pathway involving the incorporation of excess (dietary) nitrogen into this purine, (2) the nucleic acid to uric acid degradation (nucleicolytic) pathway and (3) uric acid degradative or uricolytic pathway (Cochran, 1975). The uricotelic pathway appears to be the most important in urate production, with the nucleicolytic pathway contributing only small amounts (Bursell, 1967; Cochran, 1975). All of the steps involved in the uricotelic pathway have not been demonstrated in insects, but the pathway appears to be similar to that which occurs in birds (Barrett & Friend, 1970; Cochran, 1975). Invertebrates from several taxonomic groups, including insects, store urates internally (Mullins & Cochran, 1974). Among insects, cockroaches can store quite large amounts (Mullins & Cochran, 1975a, 1976; Valovage & Brooks, 1979). The adaptive value and functions of these urate stores have not been clearly established, but they may serve as a nitrogen reserve under certain dietary conditions (Nolfi, 1970; Mullins & Cochran, 1976) or they may function as an "ion sink" in facilitating hemolymph homeostasis (Mullins & Cochran, 1974; Hyatt & Marshall, 1977; Tucker, 1977a,b,c). Mullins & Keil (1980) reported that significant amounts of urates from male B. 9ermanica are presented to females during mating and

* Present address: Department of Environmental Toxicology, University of California, Davis, CA 95616, USA.

METHODS AND MATERIALS Adult virgin male and female German cockroaches were used in this study. Stock cultures were held at ca. 20°C and uncontrolled humidity in 11.41. glass aquaria with dog food and water provided. The sexes were separated as older nymphs and were held until the adult molt in I 1. glass jars also provided with dog food and water. All insects used were 3-7-day old adults. They were maintained singly in 10 × 2 cm plastic petri dishes which were fastened to the top of a 20-dram plastic vial containing distilled water. A glass tube (0.3 × 7 cm) packed with a

293

294

.Io A. ENGIiBRETSON and DONALD E. MULLINS

cotton wick was inserted through the top of the vial and the bottom of the petri dish cage. allowing cockroaches easy access to water. Diets used included those with t), 5.25, 42 or 66"o casein protein formulated as described by Mullins (1974L Dog food served as the control diet. They were dispensed into stainless steel planchets, packed, steamed (10rain) and oven dried (80'C) prior to presentation to the insects. A motor-driven microapplicator fitted with 0.5 ml syringe and 26 gauge needle was used to inject I,,1 volumes of radiolabelled materials into the cockroaches. Chilled insects (4 CI were moved onto the needle so that it penetrated between the tirst and second abdominal sternites to a depth of (1.5 cm. Upon completion of an experiment, cockroaches were frozen ( 20 C) and later analyzed b) whole body extraction. This extraction included homogenization of each insect with I ml 0.6",, g i 2 c o 3 ill a glass homogenizer, heating (80C for lOmin) and centrifugation (10000 for 10 mini. Total bod) urates were determined from whole body extract aliquots using an enzymatic spectrophotometric assay modified from Dubbs e t a / . {1956), Whole body radioactivity was determined by placing 0.5 ml of the supernatant in 20 ml scintillation vials with S ml xylene and PCS (Amershaln Scarle)cocktail (1:1). Samples were counted on a Beckman LS 3150 scintillation counter and results tabulated after correction for counting elficiency, backgronnd and dihltion. Radiolabelled uric acid and xanthme were isolated by thin-laycr chromatography (TLC). Whole body extract (25 Itll was spotted on 2 mm thick cellulose TLC plates (20 x 20 cm), developed in tl-bntanol:methanol: H 2 0 : N H 4 O H (60:20:20:11 and visualized using u.v. light. Cellulose containing marked spots was removed from the plates, extracted with 2 ml 0.6". l_i,('O_~ t12 ml centrifuge tubc: 5 10 min at 60 C), centrifuged (5 rain at 1000.qL and u 0.5 ml aliquot of the supernutant was removed for scintillation counting.

Radiolahelled :/Iveme uml lormate Whoh" hodv stl,liev Groups of 10 males and females were placed on one of the following dietary levels: 0. 5, 25 or 42". casein protein or dog food. After one week they were injected with either U-["~C]glycine (Amersham Searle: SA. 107 mCi/mM) or ['aC]formate (ICN Pharmaceuticals. Inc.: SA. 56 mCi/mMI. One week after injection (two weeks maintenance on the respective diets), the insects were frozen prior to analysis. Radiolahelled CO2 studies. Glvcine and formate metabolized to and released as *'*COz after injection into males or females maintained on dog food was studied. Four replicates consisting of 4 adults (2 males and 2 femalesl were injected with either radiolabelled glycine or formate and placed into glass cages (Mullins & Eaton, 1977) provided with diet and drinking water (12/12:L/D: 20 + 3 CI. The ~4CO_, released was measured at 1, 3. 6, 8, 12, 24, 48 and 72 hr intervals by removal and analysis of KOH scrubbing solution (apparatus and technique described by Mullins & Eaton, 1977t. Adjustments for mortality were made in the final tabulations. To determine radiolabel incorporation into ufates, additional cockroaches were injected and fi'ozen im replicate groups of four insects each) at the time intervals corresponding to those times when ~*C'02 samples were obtained. They were analyzed for both whole body and urate radiolabel content.

Radiolahelled wmthim, 14"hoh, ho,h' .studies. Females were placed on diets containing either 0, 25 or 66"i, casein protein or dog food for 1 week prior to injection with [~'*C]xanthine IICN Pharmaceuticals, lnc. SA. 54mCi/mMI. Two females from each diet group were frozen 1 day and five females from each diet group were frozen I week post injection. All insects

were extracted and whole body, uratc and xanthme fudiolabel were determined by TLC and scintillation counting. Radiolabelled C O 2 studies. Xanthine metabolized to and released as ~'*CO2 after injection into females maiutaincd on either a 0, 25 or 66'~,, casein protein or dog Ik}od diel was studied. After maintenance on the respective diets. groups of 4 females each were injected wdh [~ ~C ]xanthinc and placed in the ~'~CO,-collection apparatus for one week. Since all four dietary groups were examined at one time. it was not possible to use replicates in this portion of lhc study. The insects were frozen one week post injeclion and radiolabelled whole body urates and xanthine were determined by TLC and scintillation counting. Radiolabc[ excreted in the feces was determmcd bx extraction (10 mg/ml 0.6'!~; LiaCO 3 10 min at 6(I (' with mixing, asmg a vortex mixer) and centrifugation I1000,q h~l It) mini. Total fecal radiolabel was determined on I).5 ml aliquots ot supernatant placed in 8 ml of the scintillation cocktail.

RESULTS

Radiolaheled glycine and/ormate stmlie,~ The data for radiolabel retention in thc wholc insect a n d that i n c o r p o r a t e d into b o d y urates from [14C]glycine injected c o c k r o a c h e s m a i n t a i n e d on various diets are p r e s e n t e d in Table 1. Tolal radiolabel retained in the w h o l e insect r a n g e d froln 20.228 (0!Ii, p r o t e i n diet) to 27,576 d p m (42",, protein dietl. r e p r e s e n t i n g 34~46% o f the initial injected a m o u n l . T h e r e was a general trend of increased radiolabel retention in insects m a i n t a i n e d on the higher nitrogen diets. However, significant differences were found onl~ w h e n the e x t r e m e s of dietary protein levels [0 and 42'~;t were c o m p a r e d . T h o s e insects m a i n t a i n e d on higher dietary nitrogen levels c o n t a i n e d greater radiolabel m their stored urates. Radiolabel i n c o r p o r a t e d into urates ranged from 5460 (0!I:~, protein dietl to 11.071 d p m (42",, protein diet), r e p r e s e n t i n g 9 18". of the initial injected [~'~C]glycine. T h e r e were greater differences o b s e r v e d b e t w e e n the m e a n s of radiolabelled urates than in whole b o d y radiolabel retention. A l t h o u g h radiolabel i n c o r p o r a t i o n into whole b o d y and urates was the same in males a n d females, total urate c o n t e n t was higher in females than in males. U r a t e c o n t e n t in males increased from 0.99 to 2.22 mg. U r a t e c o n t e n t in females increased from 1.31 to 3.76 m g with a c o r r e s p o n d i n g increase of 0 42",, casein protein in the diet. Total urate c o n t e n t increased as available dietary nitrogen increased. Table 2 p r e s e n t s data o b t a i n e d when insects were m a i n t a i n e d on diets c o n t a i n i n g difi'erent levels of nitrogen a n d injected with radiolabeled formate. W h o l e b o d y retention of radiolabel ranged from 48,494 d p m (00; p r o t e i n diet) to 79,670 d p m (42'!;, protein diet), r e p r e s e n t i n g 42-70'!; of the initial injected radiolabel, respectively. R e t e n t i o n of total radiolabel in the [~4C]formate injected insects was greater in individuals m a i n t a i n e d on the higher protein diets. This trend was similar to that o b s e r v e d in insects injected with [14C]glycine. Radiolabel i n c o r p o r a t e d into b o d y urates ranged from 16,404 d p m (0°{i p r o t e i n diet} to 34.569 d p m (dog food d i e t ) i n insects injected with [ l * C ] f o r m a t e . H o w ever, the p a t t e r n of [~'*C]formate i n c o r p o r a t i o n into the b o d y urates differed from that observed for [14C]glycine. T h o s e individuals m a i n t a i n e d on the

295

Dietary nitrogen levels and urates in the German cockroach Table 1. Radiolabel retained in the whole insect and in the whole body urates plus the total urates present one week after [~4C]glycine injection in cockroaches maintained on various diets for two weeks* Diet (% protein)t 0 5 25 Dog food (25) 42

Radiolabel content:~§ Whole insect Whole body urates Total urates (dpm) (%) (dpm) (%) (mg/Female) (mg/Male) 20,228 a 22,435 ab 21,962 ab 23,653ab 27,576 b

34 37 37 39 46

5460 a 7470 ab 9603 bc 9737 bc 11,071c

9 12 16 16 18

1.31 ab 0.97 a 1.84 b 2.56 bc 3.76c

0.99 ab 0.68 a 1.44 b 1.65 b 2.22b

* Initial injected dose = 60,031 dpm [14C]glycine/insect. ? Dog food was included as a control diet. Data from males and females were combined since there was no significant difference between sexes within dietary groups (Student's t-test, P < 0.05, n = 20). (%) = Percent of injected dose. § Similar letters within columns indicate means that are statistically the same according to Duncan's Multiple Range Test (P < 0.05).

42~0 protein diet incorporated approximately the same amount of [~4C]formate (16~0 of initial injected dose) as those which fed on the 0~o protein diet (140/o of initial injected dose). There was a general trend of increased total body urates in males and females corresponding with increased dietary nitrogen available (Table 2). In males, urates increased from 0.87 to 2.74mg. In females, urates increased from 0.79 mg at the 0 ~ protein level to 2.63 mg at the 25~ protein level, which was not significantly different from 2.45 mg at the 4 2 ~ protein level. Thus, total urate content increased with increased dietary nitrogen regimes. Results obtained on respired 14CO2 after injection of [a4C]glycine in dog food fed insects are shown in Fig. 1. There was a large ~4CO2 release (22,000 dpm) during the 12 hr interval after injection, which corresponded with a decrease in whole body radiolabel retention from 74,000 to 50,000dpm (24,000dpm change). Whole body radiolabel losses coincided with 14CO2 released. The cumulative 14CO2 release increased steadily and after 72 hr, represented 74~, of

the initial injected amount. Radiolabel incorporated into the urate fraction was rapid during the first 6 hr, but then appears to have stabilized, representing c a . 16,000 dpm incorporated (21~0 of the initial injected dose) during the 72 hr interval. Results of 14CO2 respired from [14C]formate injected dog food fed insects are shown in Fig. 2. At 6 hr, cumulative 14CO2 release was c a . 17,000dpm and increased slowly over 72hr to a total of 19,000 dpm, representing 12~o of the initial injected amount. Whole body radiolabel decreased rapidly from 161,000 to 100,000dpm in the first 6hr, then stabilized at approx 100,000 dpm. However, there was no corresponding large increase in t4CO2 release during this 6 hr interval. Radiolabel incorporation into body urates increased rapidly during the first 12 hr, from 22,000 to c a . 72,000 dpm. After 72 hr, it represented 52~0 of the initial injected dose. A total of 64~0 of the initial dose was recovered in ~4CO2 released and in [~4C]urates. After 72 hr, [14C]formate incorporated into [~4C]urates represented 86~0 of the whole body radiolabel retained.

Table 2. Radiolabel retained in the whole insect and in the whole body urates plus the total urates present one week after [-14C]formate injection in cockroaches maintained on various diets for two weeks* Diet ('~ protein)? 0 5 25 Dog food ( 2 5 ) 42

Radiolabel content:~§ Whole insect Whole body urates Total urates (dpm) (%) (dpm) (%) (mg/Female) (mg/Male) 48,494 a 49,682 a 61,600 b 64,649b 79,670 c

42 43 54 56 70

16,404a 22,047ab 26,844bc 34,569c 18,164a

14 19 23 30 16

0.79 a 1.05 a 1.90 b 2.63 b 2.45b

0.87 a 0.90 a 1.64 ab 1.30 ab 2.74b

* Initial injected dose = 114,588 dpm [t4C]formate/insect. t Dog food was included as a control diet. Data from males and females were combined since there was no significant difference between sexes within dietary groups (Student's t-test, P < 0.05, n = 20). (%) = Percent of injected dose. § Similar letters within columns indicate means that are statistically the same according to Duncan's Multiple Range Test (P < 0.05).

296

Jo A. ENGEBR} ISON ~:lnd [)()NAI.I) I!. m[ll LINS 90 WHOLE

8O - - - -E}

INSECT

URATES

70

0 0 ,~

60

>~--

40

~

3O

/

.I / /

0 cl 2O n-I0

, 0

5

' IO

' 15

2'0

25'

3

TIME

5

. 40

.

. . 45

50

55

0

' 65

' 70

75'

(hrs)

Fig. 1. Radiolabel retention ill the whole insect, in body urates a n d respired us (-'02 from cockroaches feeding on dog food after injection with [14C]glycine {74,00(1 dpm), measured for 72 hr. Vertical lines represent 95
Radiolabelled .vanthine studies Radiolabel retention in whole body, body urates and the total body urate content of females maintained on various diets 1 day (Day 1) and 1 week (Day 7) after injection with [l'*C]xanthine is shown in Table 3. There was a general trend of increased incorporation of xanthine in insects maintained on higher dietary nitrogen levels. One day after injection (Day l), whole body retention ranged from 68,881 (0'!~, protein diet) to 292,580 dpm (dog food diet), representing 27-100% of the initial injected amount, respectively.

After I week, whole body retention ranged from 78.079 ((Y',, protein diel} to 221.790 dpm (66
200

180 160

-

-

O* WHOLE INSECTS . . . . -Q URATES - - ~ CUMULATIVE CO2 RELEASE

~

0 0 _o ~40 X

'~

120

o >-

.±

60

o

9

40

0

0

,

i

5

I0

15

20

25

30 TIME

35

40

i

i

i

I

i

i

i

45

50

55

60

65

70

75

(hrs)

Fig. 2. RadJolabel retention m the w h o l e insect, in b o d y urates and respired as (-'02 f r o m cockroaches

feeding on dog food after injection with [l 4.C]format e {161,000 dpml. measured Ibr 42 hr. Vertical lines represent 95";, confidence inter~als.

Dietary nitrogen levels and urates in the German cockroach

297

Table 3. Radiolabel retained in the whole insect and the whole body urates plus the total urates present one day and one week after ["*C]xanthine injection in female cockroaches maintained on various diets for two weeks*

Diet (% protein)f 0 25 Dog food (25) 66

Radiolabel content + Whole insect Whole body urates Day 1 Day 7 Day 1 Day 7 (dpm) (%) (dpm) (%) (dpm) (%) (dpm) (%) 68,881 a 215,010 b 292,580b 243,403 b

27 84 115 95

78,079a 176,724ab 178,344ab 221,790 b

31 69 70 87

63,316a 209,892b 177,118b 232,817b

25 82 69 91

71,966a 174,709b 164,065b 223,914b

28 69 64 88

Total urates Day 1 Day 7 (rag/insect) (mg/insect) 0.52a 0.96 b 0.65a 0.83 ab

0.51 a 0.69 a 0.61a 0.86 b

* Initial injected dose = 255,000 dpm [~4C]xanthine/insect. f Dog food was included as a control diet. ++Similar letters within columns indicate means that are statistically the same according to Duncan's Multiple Range Test (P < 0.05), n = 2 at Day 1 and n = 5 at Day 7. (%) = Percent of initial injected dose.

porated isotope into body urates at a rate of 7380 + 2258dpm/hr for a total of 69.4% of the injected amount. Total body urate content ranged from 0.52 (0~/;, protein diet) to 0.96 mg (25% protein diet) at Day 1 and from 0.51 (0% protein diet) to 0.86 mg (66% protein diet) at Day 7. Urate content remained approximately the same from Day 1 to 7 in females, with the exception of a decrease from 0.96 to 0.69 mg in females maintained on 25% protein diet. At Day 7, the total urate content in the 66% protein fed females was significantly higher than the other dietary nitrogen regimes. Release of 14CO2 from [14C]xanthine injected females maintained on various dietary nitrogen regimes is shown in Fig. 3. At the 0% protein level, '4CO2 was released at a fairly steady rate, representing a total of 25% of the initial injected amount over 1 week. Dog food and 25% protein fed females released 18% of the initial injected amount over one week. Females fed 66% protein diet released low

amounts of t4CO2 after the first 48 hr, totaling 12°~ of the initial injected amount over 1 week. Table 4 contains a tabulation of radiolabel recovered in CO 2, urates, xanthine and feces from [14C]xanthine injected females. Cockroaches fed 0% protein diet retained less radiolabel in the body urates (42%) and released more 14CO2 than females fed either the 25 of the 66% protein diets. Females maintained on 0% protein diet also excreted more radiolabel in the feces (52,364 dpm) than 25% (11,799 dpml or 66% (7391 dpm)--protein fed females. A range of 1.3 2.7% (3215-6955, respectively) of the injected radiolabelled xanthine was detectable in the females 1 week after injection. Recovery of initial injected dose in the analyzed components ranged from 88 to 100%. DISCUSSION The results obtained after injection of radiolabelled glycine and formate indicate that both of these precursors are incorporated into uric acid in Blattella

7O O 0..

cJ'~NO0 60 (I) 0'~

..... 0 ---o ---~ .... ~

0% PROTEIN 25% PROTEIN DOG FOOD 66%PROTEIN

/

_

50

LLIj LU 40

/

//

. ~"

//

>-~: I-

"''"

/ ///" /

. . . . . . .

u
o

. . . . . . . . . .

o

8~---:...~---°- ~ 5 5

o

,~ n-

o.-~ 20

J

0

24

~

48

i 72

i 96

I 120

I

t

144

168

TIME (hrs) Fig. 3. Cumulative '4C02 released from females maintained on different dietary nitrogen levels after injection of ['4C]xanthine (255,000 dpmt, measured over a 1 week interval.

298

.lo A. ENGHIRFISONand DONALDE. MULLINS Table 4. Radiolabel recovered in CO2, urates, xanthine and feces from lemales maintained on various diets one week after injection with [~'*C]xanthine* fit; protein (dpm) C,,)

Radiolabel content+ 25",, protein 66°, protein (dpm) (",3 (dpml (',3

CO 2 Body urates Body xanthine Feces

63,133 106,559 6955 52,364

25 42 3 21

46.485 191,641 4688 11,799

18 75 2 ~

31,322 182.916 3215 7391

12 72 1 3

Total

229,01 I

91

254.613

100

224,844

88

Radiolabeled components

Dog food+ Idpm) (<',,I 46.444 146,419 4229

I~ 57 2

* Initial injected dose - 255,000 dpm [l'*C]xanthine/insect. n - 4. + ('~;D= Percent of initial injected dose. + Dog food was included as a control diet: containing 25<',i crude protein. Data is not reported for fecal radiolabel since feces could not be reliably collected and separated from ground dog food diet scattered on the bottom of the glass cages while insects were feeding.

germanica in a manner similar to that reported in Rhodnius prolixus (Barrett & Friend, 1970) and Periplaneta americana (McEnroe & Forgash, 1957). There were differences found in whole body [14C]urate retention of the initial injected dosage for glycine (16%) and formate (30'~;,) on a dog food diet (Tables 1 and 2). This represents a 1 : 1.9 ratio of glycine to formate incorporation into the uric acid molecule. Similar results were obtained in the ~'*CO2 experiments. Over a 72hr interval, glycine incorporation into urates (representing 39°~i of the whole body radiolabel retained) can be compared to formate incorporation into urates (68% of the whole body radiolabeL Figs 1 and 2). These retention rates represent a 1:1.7 ratio of glycine to formate incorporation into urates in cockroaches maintained on dog food. Ratios of 1:1.9 and 1:1.7 glycine to formate compares favorably to 1 glycine:2 formate molecules incorporated into uric acid in uricotelic organisms. This observation supports the accepted origins of carbon and nitrogen in insect de novo uric acid synthesis. Other observations can be made regarding glycme and formate metabolism in B. germanica. Whole insect retention of [14C]glycine not appearing in the body urates may represent incorporation into proteins (i.e. into vitellogenins or hemolymph proteins) or other metabolic pools (Hill, 1965; Chefurka, 1965). The pattern of incorporation of [~4C]glycine into the body urates probably reflects the status of urate synthesis and storage as it relates to the insect's dietary nitrogen regime (Table 1). For example, at 07, protein, some mobilization of urates could be expected to occur because the insect would presumably be on a negative nitrogen balance regime (Mullins & Cochran, 1975b). Cockroaches on a high nitrogen diet (42°,i~ protein) are synthesizing and storing more urates. Indeed, the large amount of [14C]glycine found in the urate fraction reflects high rates of body urate storage. Whole insect radiolabel retention after rejection with [14C]formate also appears to be related to the insect's dietary nitrogen status. However, the pattern of radiolabel incorporation into body urates is somewhat different than that observed for [14C]glycine. Cockroaches fed a high protein diet (42,~,.} incorporated a similar amount of label into urates as those fed

a 0", protein diet (16 and 14'!,, respectively, see Table 2). In this study, radiolabel retention in the body urates was 16<:;; in insects fed a 42"i, protein diet. Similarly, McEnroe & Forgash 11957) reported a low rate of labelled formate retention in urates {3 5";;i in American cockroaches fed a high {60°.g) protein dict. They also reported rapid incorporation of labelled formate into amino acids such as proline, glutamine, with the most significant of these being serine. They suggested that free amino acids may serve as a depot for functional one carbon groups such as formate. Formate is important in intermediary metabolism of amino acids (Chefurka. 1965: Corrigan, 1970i. Since formate represents a readily available carbon source, and insects on a high protein diet would probably have abundant amino acids available, B. ,qermanica may metabolize formate through this amino acid pool rather than converting it to urate. Formate is also important in pyrimidine formation and other purine pathways. For example, Johnson et al. (19801 showed that incorporation of large quantities of labelled formate into inosinate and adenylsuccinate occurred with very little incorporation into uric acid in Dro,sophila larvae. Similar kinds of alternate metabolic pathways for [l"~C]formate metabolism may be occurring in cockroaches maintained on high levels of dietary nitrogen. Radiolabel retention m the whole body and in body urates was not significantly different for males and females in either the glycine or the formate experiments. This indicates that both sexes are metabolizing these compounds at similar rates. It was hypothesized that radiolabel incorporated into urates might serve as an index to total urate content. Even though total urate content was significantly higher in females, rates of radiolabel incorporation into urates were similar to those of males. Radiolabel incorporation into urates and total urate content are both influenced by dietary nitrogen level, but the amount of radiolabel found in urates was not a direct reflection of total uratc content. In the overall metabolism portion of this stud3 (b*CO2 release), cockroaches fed dog food diet (25
299

Dietary nitrogen levels and urates in the German cockroach incorporated into urates was readily metabolized through other pools in the body. In addition, glycine may have been incorporated into urates with 14CO2 subsequently being released if these [14C]urates were mobilized. Glycine is required in insects for porphyrin rings necessary for the synthesis of cytochromes and enzymes such as catalase, peroxidase and tryptophan pyrrolase (Corrigan, 1970). As mentioned earlier, glycine interconverts with serine and one carbon units. Mansingh (1965) found glycine to be incorporated into amino acids, primarily serine, proline and glutamine. Thus, glycine not found in urates could be metabolized through these other pathways, perhaps resulting in 14CO2release. The 14C02 release pattern from German cockroaches maintained on dog food and injected with [14C]formate showed a similar slow release to that observed in P. americana (McEnroe & Forgash, 1957) (Fig. 3). These cockroaches released less 1'~CO2 than cockroaches injected with [lgC]glycine. As suggested previously, formate not incorporated into the urate fraction may be useful in formation of other substances. There was an initial drop in 14C label in the whole insect without a corresponding increase in 14CO2 release. It is possible that [~4C]formate (or its metabolites) was eliminated through the gut. However, this could not be established since feces were not collected in this experiment. Results obtained from these studies clearly show that the dietary nitrogen status of German cockroaches may directly affect incorporation rates of glycine and formate into the whole insect, particularly into the urate fraction. They also indicate that there are differences in utilization of these two purine precursors. Radiolabeled xanthine incorporation into urates was rapid at all dietary nitrogen levels (Table 3). The amount of radiolabel found in urates at Day 1 and 7 was very similar. Females maintained on the higher dietary nitrogen levels had increased amounts of whole body radiolabel contained in the body urates. When these results are compared with 14CO2 release (Fig. 3), the effect of dietary nitrogen level on xanthine metabolism is clearer. All females from dietary groups produced an initial burst of 14CO2 release, which may have resulted from increased urate metabolism due to trauma associated with the anesthesia and injection procedures. After the initial 24 hr period, the patterns of 14CO2 release were related to the dietary nitrogen level on which the cockroaches were maintained. Those on the 0% protein diet (negative nitrogen balance diet) were probably utilizing pathways mobilizing newly synthesized or stored urates (Mullins & Cochran, 1975b), releasing relatively higher levels of ~4CO2. Those cockroaches on the 25% protein diet or dog food appear to have been at an intermediate dietary nitrogen balance. They initially released a'*CO2 at rates comparable to the 0% protein fed insects, but over time, the ~'~CO2 release rate decreased. Urate metabolism in these insects appears to be in a more modulating state between synthesis and mobilization. They may be utilizing a portion of the injected purine through the urate pool (including storage and subsequent mobilization) or by direct degradation (bypassing the storage and mobilization steps). At some point, a metabolic equilibrium may be estab-

lished in the radiolabeled urate pool such that synthesis and storage prevail. Presumably, urate synthesis and storage predominate in females maintained on 66~o protein diet (a highly positive nitrogen balance regime). Thus, after the initial burst of 14CO2 release, little additional 14CO2 was released. The recovery of radiolabel from [~4C]xanthine injected cockroaches in various components reflects the dietary nitrogen level on which the insect was maintained (Table 4). Since the largest amount of 14CO2 was released by females on the 0!'~; protein diet, it appears that they (or their internal symbiontst are capable of metabolizing xanthine directly or through the urate pool. The large amounts of label found in feces from 0% protein fed cockroaches would also suggest catabolism of [~4C]xanthine or [14C]ur" ate, or their metabolites that are eliminated through the gut. The identity of these materials was not determined. Because those females maintained on higher protein diets are in an intermediate (250~i) or positive (66%) nitrogen balance, their urate metabolism is oriented more towards urate storage. Radiolabel found in the body urates represented 75 and 72% of the initial injected amount retained in 25 and 66% protein fed insects, respectively (Table 4). Examination of excretion of radiolabel in 14COz and in feces revealed that 25% protein fed females excreted more label in these fractions than 66°)i protein fed insects. These results indicate greater metabolism of [l'*C]xanthine or [~4C]urates which are not incorporated into stored urates in insects fed on an intermediate dietary nitrogen level or which are metabolized rapidly through the urate pool.

Acknowledgements--Appreciation is extended to Drs Donald G. Cochran and John L. Eaton for critically reviewing the manuscript. We also thank Douglas Howell for his assistance in computer analysis and graphics.

REFERENCES

BARRETTF. M. & FRIENDW. G. (1970) Uric acid synthesis in Rhodnius prolixus. J. Insect Physiol. 16, 121-129. BURSELLE. (1967) The excretion of nitrogen in insects. In Advances in Insect Physiology. (Edited by BEAMENTJ. W. L., TREHERNEJ. E. • W1GGLESWORTHV. B.), Vol. 4, pp. 33-67. Academic Press, London. CHEFURKAW. (1965) Intermediary metabolism of nitrogenous and lipid compounds in insects. In The Physiology c?l" lnsecta. (Edited by ROCKS~]N M.), Vol. 2, pp. 669-768. Academic Press, New York. COCHRAND. G. (1975l Excretion in insects. In Insect Biochemistry and Function (Edited by CANDYD. J. & KILBV B. A.), pp. 177-281. Chapman and Hall, London. COCHRAN D. G. (19791 Comparative analysis of excreta and fat body from various cockroach species. Comp. Biochem. Physiol. 64A, 1-4. COCHRAN D. G., MULLINSD. E. 8¢. MULLINS K. J. (1979) Cytological changes in the fat body of the American cockroach, Periplaneta americana, in relation to dietary nitrogen levels. Ann. ent. Soc. Am. 72, 197-205. CORR~GANJ. J. (1970) Nitrogen metabolism in insects. In Comparative

Biochemistry

Of Nitroyen

Metabolism

(Edited by CAMPBELLJ. W.), Vol. 1, pp. 387-488. Academic Press, New York.

300

Jo A. [Z.NGEBRIH'SON and DONALD F.. MUI.IlNS

DUBBS C. A., DAVIS F. W. & ADAMS W. S. (1955) Simple microdetermination of uric acid. ,I. biol. Chem. 218, 497 504. HI~NI)ERSON J. F. (1972) Regulation of Purine Biosynthesis. ACS Monograph 170. American Chemical Society, Washington, D.C. HllA. g. (1965)The incorporation o f [ l~Cjglycine into tim proteins of the fat body of the desert locust during ovarian development. J. Insect Physiol. I I, 1605 1615. HYA'II A. D. and MARSHALL A. T. (1977) Sequestration of hemolymph sodium and potassium by fat body in the water stressed cockroach, Periphmeta americcma. J. h l w e t Physiol. 23, 1437 1441. JOHNSON M. M.. NAStt D. & HI!NI)t!RSON J. F. (1980) Purine metabolism in larvae of Drosophila mehmooaster fed radioactive hypoxanthine, inosine, or formate. Comp. Biochem. Physiol. 66B, 555 561. MANSlX(}H A. (1965) Glycine catabolism in Blattella ~lermauica L. J. Insect Phv.siol. l l , 1031 1037. McENRot z W. & FO~G,XSH A. 11957) The m ciro incorporation of [ ~ C ] f o r m a t e in the ureide groups of uric acid by Periphmeta americamt (L.~..tml. ent. Soc, Am. 50, 429 431. MUI,IANS D. E. (1974) Nitrogen metabolism in the American cockroach: An examination of whole body ammonium and other cations excreted in relation to water requirements. J. exp. Biol. 61, 541 556. Mt~LLINS D. E. & CO('HRAN D. G. (1974) Nitrogen metabolism in the American cockroach: An examination of whole body and fat body regulation of cations in response to nitrogen balance. ,l. exp. Biol. 61, 557 570. m u I I,INS D. E. & COCItRAN D. G. (1975a) Nitrogen metabolism in the American cockroach l. An examination of

positive nitrogen balance with respect to uric acid stores. Comp. Bioehem. Physiol. 50A, 489 500. MUI.LINS D. E. & COCttRAN D. G. 11975bj Nitrogen metabolism in the American cockroach ll. An examination of negative nitrogen balance with respect to mobilization of uric acid stores. Comp. Biochem. Plm'~iol. 50A, 501 51i). MULLINS I). E. & CO(II~.:A", D. (i. [1976) A comparatixe study of nitrogen excretion in 23 cockroach ,~pccics. ('omp. Bioehem. Phl'siol. 53A, 393 399. MULLINS 1). [_:. & EAI()'-, J. k. 11977) Labeled ts(-()z production by moths injected with { ~aC ]glucose in responsc 1o lighl stimuli: A method and a preliminary investigalion. Conlp. Bioehem. Phl'siol. 58B, 3{~5 375. m I I 3 INS l). E. & KIHI (" I ( l {}8()) Paternal invcstmclH of urates in cockroaches. Noture, Lond. 283, 567 569. NoLlq J. R. (19701 Bioswlthesis of uric acid in the tunicate Mol#ula manhatten.si* with general scheme for the funclion of stored purincs in animals. (',rap. Biochem. Phlsiol. 35, 827 842. TL('KI!R L. (1977a1 Effect of dehydration and rchydration on the water content and Na + and K t balance in aduh male Periphmeta amcricaml. J. evp. Biol. 71, 49 6(~. TIXKI-:P, L. (1977b) The influence of diet, agc, and state of hydration of Na +, K ' and urate balance ill the fat body of the cockroach Periphmelaamericana..l evp. Biol. 71, 67 79. "FtI('KIR L. (1977c1 The inlluence of age. diet, and lipid content on survival water balance, and Na ~ and K" regulation m dehydrating cockroaches..I, evp. Biol 71, 81 93. VAI,O\.A(;I W. D. & BROOKS M. A. (19791 Uric acid quantities in the fat body of normal and aposymbiotic German cockroaches, Blattella !termani('a. Ann. ent. Soe. Am. 72, 687 689.