Metabolism of hydroxybenzoic acids in Periplaneta americana

Comp. Biochem. Physiol. Vol. 77B, No. 4, pp. 791 798, 1984

0305-0491/84 $3.00 + 0.00 © 1984 Pergamon Press Ltd

Printed in Great Britain

METABOLISM OF HYDROXYBENZOIC ACIDS IN PERIPLANETA AMERICANA FRANK Y. T. SIN*, PETER C. J. BRUNET a n d IRIS L. SIN? Department of Zoology, South Parks Road, Oxford, UK

(Received 29 July 1983) Abstract--1. The metabolism of 2-hydroxybenzoic and 4-hydroxybenzoic acids have been compared in the cockroach, Periplaneta americana. 2. These phenolic acids did not play a direct role in the synthesis of tanning agents of the adult cuticle or of the egg capsule. 3. Most of the phenolic acids injected into the animals were excreted unchanged. 4. Degradation of these phenolic acids was mainly by decarboxylation. The resulting aromatic ring was used for the synthesis of tyrosine and phenylalanine. This interpretation was supported by the ability of the cockroach to utilize phenol for the synthesis of these aromatic amino acids.

INTRODUCTION Phenolic c o m p o u n d s are found in a b u n d a n c e in the animal a n d p l a n t kingdoms. In insects phenolic derivatives are involved in the f o r m a t i o n o f pigments (Linzen, 1974), in the sclerotization o f the cuticle ( K a r l s o n and Sekeris, 1962), in the t a n n i n g of the egg capsules of cockroaches (Brunet and Kent, 1955; Pau a n d Acheson, 1968) a n d in the t a n n i n g of silk (Brunet a n d Coles, 1974). Phenolic c o m p o u n d s are also used in defensive secretions of some insects (beetle: Tschinkel, 1969; grasshopper: Eisner et al., 1971; millipede: Blum et al., 1973; Duffey a n d Blum, 1977). In spite o f such a wealth o f knowledge on the metabolism a n d functions of some insect phenols, there is still a large n u m b e r o f phenolic c o m p o u n d s in insects awaiting identification, a n d whose metabolic role has yet to be clarified. T h e present p a p e r reports the isolation, identification a n d a metabolic study of the phenolic acids, salicylic acid (2-hydroxybenzoic acid) a n d 4-hydroxybenzoic acid in the cockroach, Periplaneta americana.

MATERIALS AND METHODS Insects Cockroaches, Periplaneta americana (L), were reared in an insectary at 26°C and approx. 60~ relative humidity with a daily photoperiod of 12 hr.

Radioactive chemicals [Ring UJ4C]2-hydroxybenzoic acid and [ring-U~4C]4-hydroxybenzoic acid were purchased as a mixture from the Radiochemical Centre, Amersham. These acids were separated by thin layer chromatography using silica gel G (Macherey-Nagel & Company, Duren) in solvent 1 (see below). The spots corresponding to 2-hydroxybenzoic acid and 4-hydroxybenzoic acid were scraped off the plastic backing sheet and the acids were extracted from the silica gel with 96~o ethanol. The extracts were concentrated by *Present address: Department of Zoology, University of Canterbury, Christchurch, 1, New Zealand. ?Present address: Department of Obstetrics and Gynaecology, Christchurch Womens Hospital, Christchurch, New Zealand.

evaporation under vacuum and rechromatographed in solvent 2 (see below). The spots were extracted as before, then concentrated and redissolved in 20~ ethanol. The u.v. spectrum was compared with the authentic acids (Sigma). The specific activities were 4.7 and 3.7mCi/mmole for 2-hydroxybenzoic acid and 4-hydroxybenzoic acid respectively. [Carboxyl-t4C]salicylic acid (2-hydroxybenzoic acid) (specific activity: 59 mCi/mmole); [UJ4C] phenol (35mCi/mmole); and L-lU-~4C]tyrosine (360mCi/mmole) were from the Radiochemical Centre, Amersham.

Extraction of phenolic compounds from cockroaches Cockroaches (about 30--150) were washed thoroughly with distilled water and then homogenized in 200-500 ml cold 80~ ethanol and 5 mM ascorbic acid in a Waring-type blender. The homogenate was stirred continuously for 30rain and then centrifuged for 15rain at 36,000g. The supernatant was kept for later use. The pellet was further extracted three times, each with 100-300ml cold ethanol-ascorbic acid as before. The pooled supernatants were concentrated by rotary evaporation at about 40°C to approximately 25 ml and centrifuged for 90 rain at 35,000 g. To remove the soluble protein an equal volume of chloroform was added to the supernatant which was shaken vigorously and then centrifuged at 35,000 g for 30 rain. The upper clear aqueous phase was evaporated to dryness in a rotary evaporator and redissolved in about 2-4 ml 0.1 M formic acid. This extract was ready for column chromatography.

Purification of 2-hydroxybenzoic acid and 4-hydroxybenzoic acid The phenolic compounds extracted from the cockroaches were fractionated by chromatography on a Sephadex G10 (particle size: 40-120 #m) column (1 cm 2 x 50 cm). The fractions were eluted with 0.1 M formic acid, Five ml fractions were collected. The fractions in which the acids were present were predetermined by running the authentic acids through the column. It was found that 2-hydroxybenzoic acid was present in fractions 78-85 and 4-hydroxybenzoic acid in fractions 98-105. In general, fractions 70-90 (compound A) and fractions 95-110 (compound B) were pooled, concentrated and redissolved in 96°/ethanol.

Injection of cockroaches To study the incorporation of radioactivity into the egg capsules and embryos, young female cockroaches after the adult moult were kept separately in plastic boxes with a 791

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FRANK Y. T. SIN et al.

mature male. After about 20 days, these females started laying an ootheca every 4-6 days. Only adult females which were about to complete the laying of an ootheca were chosen for the isotope experiments. A known amount of radioactive material was injected into slightly chilled animals between the sternites of the third and fourth abdominal segments. Any leakage of haemolymph during the injection was mopped up with a small piece of tissue paper and the radioactivity was determined by liquid scintillation counting. Thus the actual amount of labelled material injected could be corrected for. In general, the majority of the injected females laid the first ootheca within 4-6 days. To study the incorporation of radioactivity into cuticle, newly ecdysed animals and rnature animals were injected and allowed to develop for 14 and 5 days, respectively.

Analysis of radioactivity incorporated in oothecae and embryos The oothecae were washed thoroughly in water, then in 1 ml of a mixture containing equal volumes of 96% ethanol and ether. The cleaned ootheca was squashed lightly in the ethanol-ether mixture to separate the developing embryos. The soft tissue adhering to the inside surface of the egg capsule was removed by scraping with a blunt scalpel. The cleaned capsule was extracted six times with the ethanol-ether mixture and dried at 60":C. The dried capsule was weighed and digested in 0.4ml hydrogen peroxide: glacial acetic acid (1:1, v/v) at 60'C for 30 rain in a tightly sealed scintillation vial. After cooling, 15 ml of scintillant containing 5 g of 2,5-diphenyloxazole per litre of toluene: 2-ethoxyethanol (5:3, v/v) was added to the digested capsule for liquid scintillation counting (Bodnaryk and Brunet, 1974). The residual tissues and the squashed embryos were digested and assayed for radioactivity as described above. Adult cuticles were digested and the radioactivity was determined as described by Bodnaryk and Brunet (1974).

pH 6.5 as above or at ptt 1.9 (formic acid:glacial acetic acid:water, 78:148:2274, v/v/v), in a Camag HVE apparatus.

Gas" liquid chromatography Silylation was performed in conical vials tightly sealed with septum caps. Fifty microliters each of pyridine (GLC grade) and bis(trimethylsilyl)-trifluoracetamide containing 1,,~{,trimethylchlorosilane (Regis Chemical Co.) were added to the purified cockroach extract and heated at 60'C for 2hr. The mixture was cooled before being used for the analysis. Chromatographic analysis was pcrtormed in a Pyc series 104 Chromatograph. Chromatographs were temperalure programmed from 150 to 275 C at 4"C/min. The silanized glass column (5' x ~4"x 4 mm i.d.) was either packed with 3~00V-1 on Diatomite CLQ (100-200 mesh) or 3'!,,; OV-17 on Diatomite CLQ (100 120 mesh). The chromatographic behaviour of cockroach hydroxybenzoic acids, described in terms of methylene unit (MU) values as determined by the procedure of Dalgliesh et al. (1966), was compared with the authentic compounds using as standard even numbered straight-chain hydrocarbons from Cm to C:4.

Radiorespirometric analysis Radiorespirometric analysis was performed essentially as described by Murdock eta/. (1970a). Animals of similar weights were used.

Detection (ff'phenolic compounds Phenolic compounds after TLC or elcctrophoresis were detected by spraying with the diazotized sulphanilic acid reagent ("diazo" reagent) of Pauly (1904). RESUI.TS

Dissection o[" tissues

Isolation and idenafication

Various tissues were dissected in cold saline (0.9°0 NaC1 0.01°~i;CaCI~) and washed once in saline before use. In order to determine the distribution of radioactivity in different tissues, the tissues were extracted in 0.2 ml ethanol-ether at 60~'C for 15 30rain. The radioactivity was determined by liquid scintillation counting as for the digested capsule. In order to determine the amount of radioactivity in the intestinal tissue, the content of the intestine was washed out after the intestine was cut open. The intestinal tissue was then washed in two changes of cold saline and extracted as for the other tissues. Haemolymph was obtained by flushing the cut open body with 5 ml cold saline before the dissection of tissues.

Figure 1 shows the elution profile of the ethanol extract from the newly ecdysed and from the adult female cockroaches. The u.v. absorption spectrum o f c o m p o u n d A was identical to that of authentic 2-hydroxybenzoic acid. The spectrum of c o m p o u n d B was identical to that o f 4-hydroxybenzoic acid. The light absorption maxima o f c o m p o u n d s A and B under various conditions were comparable to 2-hydroxybenzoic acid and 4-hydroxybenzoic acid respectively (Table 1). The c h r o m a t o g r a p h i c properties o f c o m p o u n d A and c o m p o u n d B were congruent with authentic 2-hydroxybenzoic acid and 4-hydroxybenzoic acid respectively on TLC and G L C (Table 2). Low-voltage electrophoresis on cellulose thin layer at pH 6.5 revealed that these two unknown compounds were negatively charged, and A had identical R / t o that o f 2-hydroxybenzoic acid and B to that of 4-hydroxybenzoic acid.

Ultraviolet absorption spectra These were measured in a Unicam S.P. 500 Spectrophotometer.

Thin-layer chromatography Pre-coated sheets of cellulose thin layer (eel 300); polyamide-6; silica gel G were from Macherey-Nagel and Company, Duren. Chromograms were developed in the following solvents: (1) benzene:glacial acetic acid:water (6:7:1, v/v/v); (2) chloroform:glacial acetic acid:water (4:1:1, v/v/v); (3) butan- l-ol:glacial acetic acid:water (4:1:1, v/v/v); and (4) 0.1 M acetic acid.

Electrophoresis Thin layer electrophoresis was performed on cellulose thin layer (cel 300) at 500V for 20-40min at pH6.5 (pyridine:glacial acetic acid:water, 25:1:225, v/v/v) in a Camag apparatus (Switzerland). High voltage electrophoresis was performed on Whatman chromatography paper 3 mm at 3000 V for 30 rain, at

Occurrence C o m p o u n d A was extracted from adult animals as well as from the newly ecdysed animals, but it was found to be more a b u n d a n t in the newly ecdysed animals than in the adults (Table 3). F u r t h e r m o r e it was more a b u n d a n t in the females than in the males, The a m o u n t extracted was probably an underestimation as in a control experiment, in which radioactive 2-hydroxybenzoic acid was mixed with thirty animals and extracted as described above, only a b o u t 407,i', o f the added isotope was recovered.

Phenolic acid metabolism in cockroach

793

A o v

O

2~ <

A

110

310

5 '0

B

9 '0

7'0

". . . . . . . . . . . .

,~o

Fraction number

,%

Fig. 1. Elution profile of cockroach extract ( - - adult female; ... newly ecdysed female) obtained by fractionation on Sephadex GIO column, eluted with 0.1 M formic acid. However the extraction efficiency was very consistent from one experiment to the other. Compound B was much less abundant than A, and it was more abundant in the adults than in the newly ecdysed animals. Therefore there was no attempt made to make any quantitative estimation.

Metabolic fate of injected hydroxybenzoie acids (a) Tissue distribution of radioactivity. The distribution of radioactivity was determined in various tissues four to five days after injection ofisotopically labelled 4-hydroxybenzoic acid and 2-hydroxybenzoic acid respectively. Table 4 shows the radioactivity detected in fat body, ovary, sexual accessory gland, gut and haemolymph. Only a very small amount of radioactivity was detected in these tissues, but over 16~ of the injected radioactivity was recovered in the haemolymph. The nature of the radioactivity was later determined (see below).

(b) Radiorespirometric analysis. Radiorespirometric analysis of the CO2 exhaled over a period of 5 days after injection revealed that 15-36~ of the radioactivity was recovered when [carboxyl~4C]2-hydroxybenzoic acid was injected, but only about 5~o of the radioactivity was recovered when the ring-carbon atoms of 2-hydroxybenzoic acid were labelled. (c) Excretion. To analyse the nature of the excreta, animals were injected with uniformly [ring-labelled HC]2-hydroxybenzoic acid and 4-hydroxybenzoic acid respectively. The excreta was collected over a period of 1 month, and then extracted with ethanol. The ethanol extract was separated by high voltage electrophoresis at pH 6.5 followed by paper chromatography in solvent 3. The two hydroxylated benzoic acids were identified by co-running with the standard compounds. The "diazo" positive spots were cut out and the radioactivity was determined.

Table 1. Light absorption maxima of compounds A and B extracted from Periplaneta

americana Light

Compound

absorption

maxima

(nm)

Distilled water

0.1M formic acid

0.1M NaOH

297

302

297

nd

261

nd

2-hydroxybenzoic acid

4-hydroxybenzoic acid

Compound

A

297

302

297

Compound

B

nd

261

nd

ND: Not determined.

FRANKY. T. SIN et al.

794

Table 2. Chromatographic behaviour of compound A and compound B extracted from Periplaneta americana MU values of g.l.c.

Rf values of t.l.c.

Compound silica gel solvent 1

silica gel solvent 2

cellulose solvent 3

polyamide-6 solvent 4

OV-I

OV-17

2-hydroxybenzoic acid

0.67

0.84

0.88

0.08

14.97

16.17

4-hydroxybenzoic acid

0.45

0.58

0.85

0.12

16.21

17.22

Compound A

0.67

0.84

0.88

0.07

14.95

16.16

Compound B

0.45

0.58

0.84

0.ii

16.20

17.20

The two hydroxylated benzoic acids accounted for about 50~o of the radioactivity recovered from the excreta. (d) Incorporation of radioactivity into cuticle. Table 5 compares the amount of radioactivity incorporated into cuticles when 2-hydroxybenzoic acid labelled with ~4C at various carbon atom positions were injected into newly ecdysed animals. Higher radioactivity was recovered from the cuticle when the carbon atoms of the ring structure were labelled.

(e) Incorporation of radioactivity into egg capsules and embryos. Table 6 shows the amount of radioactivity incorporated into the egg capsules and the embryos. It can be seen that relatively higher radioactivity was recovered from the embryos when [ring U-'4C]4-hydroxybenzoic acid was injected. Approximately equal amounts of radioactivity were detected

in the egg capsule and in the embryos when 2-hydroxybenzoic acid was injected. However, when carboxyl-labelled 2-hydroxybenzoic acid was used, very low amounts of radioactivity were incorporated into both the egg capsules and embryos. In order to identify the radioactive substance incorporated into the egg capsule and embryo, newly formed egg capsules laid by females after receiving an injection of 2-hydroxybenzoic acid or 4-hydroxybenzoic acid, were extracted with an ether:ethanol (l :1, v/v) mixture. The extract was analysed by TLC in solvent 2. Two major radioactive spots were detected, having RI of 0.4 and 0.9 respectively. The slow spot corresponded exactly in position with 3,4-dihydroxybenzoic acid, the tanning agent of the egg capsule. The fast migrating spot has not yet been identified.

Table3. Amountof2-hydroxybenzoicacidextracted from Per~lanetaamericana Age of Animal

Sex

~g/g w e i g h t

of animal

Adult

female

1.56 + 0.15

Adult

male

0.28

+ 0.06

Newly

ecdysed

female

2.24

~ 0.23

Newly

ecdysed

male

0.86

~ 0.18

The newly ecdysed animals were collected and sacrificed within 15 min after the adult moult. The mean and standard error of mean are given. The concentration was determined, using authentic 2-hydroxybenzoic acid in 0.1 M formic acid as standard, at 300 nm.

Phenolic acid metabolism in cockroach

795

Table 4. Distribution of radioactivity in various tissues 5 days after injection of isotopically-labelled hydroxylated benzoic acids Tissue

Radioactivity recovered (%)

[Ring U-I~C]

[Ring U-I~C]

[Carboxyl-1"C]

2-hydroxybenzoic

4-hydroxybenzoic

2-hydroxybenzoic

acid

acid

acid

Sexual accessory gland

2.7

1

1

Ovary

0.5

0.5

0.3

Fat body

3.3

2.3

0.4

Gut content

nd

4-16

3-19

Gut

nd

2.0

2.0

Haemolymph

nd

13-19

16-18

Solid faeces

nd

nd

10-25

ND: Not determined. A second experiment was performed further to confirm the above observation. Six adult females, which were about to complete laying an egg capsule, were injected with uniformly ring-labelled 2-hydroxybenzoic acid (0.25#Ci). Four days after injection the animals were extracted with ethanol and the phenolic compounds were fractionated on a Sephadex G10 column. The radioactivity profile of the eluted fractions is shown in Fig. 2. Five distinct radioactive peaks were present. The identities of

peaks 3, 4 and 5 were determined by their u.v. absorption spectra and by TLC or by high voltage electrophoresis. The u.v. spectra are shown in Fig. 2. It is found that peak 5 had an identical spectrum to 3,4-dihydroxybenzoic acid. Peak 3 had an absorption spectrum identical to 4-o-fl-o-glucosido-3-hydroxybenzoic acid. The identity of peak 3 was further verified by incubating the peak fraction with /~-glucosidase in 1 ml distilled water at 37°C for 1 hr, followed by high voltage electrophoresis at pH 6.5.

Table 5. Recovery of radioactivity in the adult cuticle after injection of isotopicaUy-labelled hydroxylated benzoic acids Age of animal at time of injection

[Ring U-14C] 4-hydroxybenzoic acid

newly ecdysed

[Ring U-14C] 2-hydroxybenzoic acid

newly ecdysed

[Carboxyl-1~C] 2-hydroxybenzoic acid

Days after injection

Radioactivity recovered (%)

14

3.8

5

0.3

15

4.7

5

0.5

newly ecdysed

14

0.6

adult

14

0.6

adult

adult

FRANK Y. T. SIN et al.

796

Table 6. Recovery of radioactivity from egg-capsule and embryos after injection of 2-hydroxybenzoic acid and 4-hydroxybenzoic acid isotopically labelled with ~4C atoms at various carbon positions Compound

Days of development

[Ring U-14C]

embryos %

Total %

0.6+0.1(10)

6.1+0.9(10)

6.7

i0

0.9+0.1(5)

7.6+2.4(5)

8.5

i0

2.33+0.2(8)

3.2+0.3(8)

5.5

i0

0.6+0.1(6)

0.6+0.1(6)

1.2

i

egg capsule %

4-hydroxybenzoic acid

[Ring U-I~C] 2-hydroxybenzoic acid

[Carboxyl-14C] 2-hydroxybenzoic acid

Figure in parentheses is the number of egg capsules analysed. Each egg capsule had an average of 12 embryos. The mean and standard deviation is given.

After electrophoresis the electropherogram was sprayed with "diazo" reagent. The enzyme treated sample showed one brown spot which corresponded to 3,4-dihydroxybenzoic acid in colour and Rs value, the untreated sample had an orange colour and a slower mobility corresponding to 4-o-/3-glucosido-3-hydroxybenzoic acid. Peak 4 eluted from Sephadex GI0 in the position expected for 2-hydroxybenzoic acid. The radioactivity peaks in fractions 8-10 (peak 1) and in fraction 18 (peak 2) were not yet identified, but under such conditions, tyrosine and phenylalanine were eluted between fractions 8 10. From the comparison of incorporation of radioactivity into adult cuticle, egg capsule and embryo (see Tables 5 and 6), it became clear that the carboxyl groups of the phenolic acids were not incorporated significantly, but the ring structures were. This observation suggested that the phenolic acids might be decarboxylated, liberating ~4CO2and phenol, and the phenol be utilized for the synthesis of tyrosine, which could be used in the synthesis of the tanning agents of cuticle and egg capsule, as well as in protein synthesis. To test this hypothesis the following two experiments were performed.

Synthesis of aromatic amino acids ]'rom phenol [U-14C]Phenol was injected into 6-10 females. After two and a half hours the animals were homogenized and extracted with ethanol. The extract was pumped through a column (32 x 1.5 cm i.d.) containing a cation exchanger (Zerolit 225, BDH). The column was eluted with 400 ml 50~ ethanol, followed by 1 M

ammonia solution. The amino acids eluted with ammonia were analysed by a Locarte amino acid analyser. Tritiated tyrosine and histidine were added to the eluate as references. Fractions of 0.6 ml were collected and 200/~1 samples were taken for liquid scintillation counting. Figure 3 shows the radioactivity profile of free amino acids extracted from

o '~2.

2~5 v

l:ji

,

~5o

2zs

Wavelength

( nm

i ..,

30o

32~

i

, ,",

i

so

Fraction

ioo number

~so

Fig. 2. Radioactivity profile of cockroach extract tbur days after injection of [ring-UJ4C]2-hydroxybenzoic acid. The extract was fractionated on a Sephadex G 10 column, eluted with 0.1 M formic acid. Insert: u.v. spectra of peaks 3 and 5.

Phenolic acid metabolism in cockroach

797 DISCUSSION

Atkinson et al. (1973) reported the isolation of 2-hydroxybenzoic acid from the exuviae of a number of cockroaches. The present study confirms that 2-hydroxybenzoic acid and 4-hydroxybenzoic acid are present in the cockroach, Periplaneta americana (Fig. 1, Tables 1 and 2). Quantitative differences were found between the sexes and different ages (Table 3). The metabolic role of both these two hydroxylated \ ... ......... benzoic acids in the cockroach is a rather obscure ~ ,- ..... : : _ _ _ _I ........ _/",,_ : one. Lake and Mills (1975) proposed that A rr b to 4-hydroxybenzoic acid is an intermediate metabolite in the biosynthesis of the egg-capsule tanning agent, " - - - . .o.. "e e.o.o.e.e t O I O I O ! ! ~1 ! ~ O 3,4-dihydroxybenzoic acid. However their proposal 130 140 150 160 was based solely on in vitro analysis and there was no ., Fraction number direct demonstration of the incorporation of radioFig. 3. Radioactivity profile of amino acids separated by a activity into the egg capsule by injection of Locarte amino acid analyser. (a) Standards: L-[3H]tyrosine 4-hydroxybenzoic acid. From the present analysis, the low amount of and L-[3H]histidine; (b) amino acids extracted from cockroaches 2.5 hr after injection of [u-~aC]phenol. Fractions of radioactivity incorporated into the egg capsule after 0.6ml were collected and 200ml samples were taken for injection of [ring u J a c ] labelled 4-hydroxybenzoic liquid scintillation counting. acid strongly suggests that it is unlikely that 4-hydroxybenzoic acid is directly involved in the synthesis of 3,4-dihydrobenzoic acid (Table 6). cockroaches after injection of [U-UC]phenol. Peak A It is possible that these hydroxylated benzoic acids overlaps precisely with the tritiated tyrosine peak. have a fungicidal function in the cuticle, particularly Peak B was determined to be phenylalanine accord- during the post ecdysis period before the cuticles ing to the elution time relative to tyrosine and become fully tanned and resistant to fungal infection. histidine. Peak C was not identified. The metabolism of 4-hydroxybenzoic acid and 2-hydroxybenzoic acid occurs in a number of ways. Synthesis of egg capsule tanning agent from phenol Substantial amounts of the injected acids were decarReproductively mature females were injected with boxylated leading to the liberation of CO2 and pre[UJ4C]phenol. Four days later the colleterial glands sumably the formation of phenol; and significant were isolated and extracted with ethanol. A portion amounts were excreted unchanged. Degradation of of this colleterial extract was incubated with the phenolic acids through ring breakage was at an fl-glucosidase (10mg/ml) at 37"~C overnight. The extremely slow rate. This observation is consistent untreated and the enzyme treated extracts were anal- with that reported by Murdock et al. (1970). ysed chromatographically on cellulose thin layer in When the preferential uptake of radioactive carbon solvent 3. The result was compared with that ob- was compared using ring-labelled and carboxyltained when isotopically labelled tyrosine was injec- labelled 2-hydroxybenzoic acid, it became apparent ted. It was found that in the untreated sample a that the carbon atom of the carboxyl-group was not radioactive spot detected autoradiographically over- incorporated to the same extent as those of the lapped with the position of 3,4-dihydroxybenzoic aromatic ring (Table 6). acid glucoside and had an orange colour. After The reason for this difference is probably that some enzymatic cleavage a new radioactive brown spot of the hydroxybenzoic acid is decarboxylated to form corresponding to the position of 3,4-dihydroxy- CO2 and phenol. The insect is capable of utilizing benzoic acid was detected. An identical result was phenol to synthesize aromatic amino acids, e.g. tyobtained when labelled tyrosine was used. rosine, which are then used in the synthesis of the tanning agent and in protein synthesis. The incorporation of radioactivity into adult cuticle, egg case and Are symbionts involved in the utilization of phenol? embryos supports this hypothesis (Tables 5 and 6). In order to throw light on this question, adults Furthermore observations from experiments with were injected with 0.5 mg streptomycin (in saline). injection of [u-]aC]phenol and the subsequent isoTwenty-four hours later, further 0.05 mg was injec- lation of radioactive tyrosine and phenylalanine (Fig. ted. Twenty-four hours after the second injection of 3) and 3,4-dihydroxybenzoic acid glucoside are also streptomycin, the animals were injected with 1 yCi of strongly suggestive that this is the case. [U-HC]phenol. Two days after the injection, the fat The synthesis of tyrosine from phenol has been body, ovary and colleterial gland were dissected and demonstrated in millipedes, Oxidus gracilis (Duffey the soluble proteins were extracted with water. The and Blum, 1977). The enzyme involved in this process radioactivity from a known amount of protein (deter- has been isolated and identified as tyrosine phenol mined by the Folin method of Lowry et al., 1951) was lyase. The present observation in cockroaches sugcompared with that obtained from the control ani- gests that the tyrosine phenol lyase may exist in the mals. No significant difference was detected between cockroach. The results of the streptomycin experithe streptomycin treated animals and the control ment suggest that the conversion of phenol to tyanimals. rosine is unlikely to be due to bacterial symbionts.

/i .....

-

"

i

\,.;",..,:-.,

798

FRANK Y. T. Sly et al.

Acknowledgement--Financial support through the Science Research Council, Great Britain is gratefully acknowledged. REFERENCES

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