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Identification, estimation, and localization of catecholamines in eggs of the house cricket, Acheta domesticus (L.)
7. Ins. Physiol., 1965, Vol. 11, pp. 591 to 600. Pergamon Press Ltd.
Printed in Great Britain
IDENTIFICATION, ESTIMATION, AND LOCALIZATION OF CATECHOLAMINES IN EGGS OF THE HOUSE CRICKET, ACHETA DOMESTICUS (L.)” P. J. S. FURNEAUX”f
and J. E. McFARLANE
Department of Entomology, Macdonald College, McGill Macdonald College P.O., P.Q., Canada (Received
9 October
University,
1964)
Abstract-DOPA, DOPamine, and N-acetyl DOPamine have been identified as the only polyphenols to occur during embryogenesis of Acheta dome&us. The quantitative variation and histochemical localization of these substances indicate that they are involved in the development of the serosal cuticle. In view of the absence of melanogenesis during normal development and the probability that a tanning reaction is involved in the termination of the first phase of water absorption, it is suggested that N-acetyl DOPamine is the precursor of a tanning quinone. The other phenols are thought to be intermediates in its biogenesis. A relatively large amount of free DOPamine has been found in the extra-embryonic fluid of post-catatrepsis eggs. This is thought to originate from the digestion of the serosa and serosal cuticle, and by de-acetylation of N-acetyl DOPamine through the action of the hatching enzymes. INTRODUCTION
THE eggs of the house cricket are laid containing complete
development.
phases which
Water
are separated
is absorbed
less water than they require
from the surrounding
by a brief period
of water loss shortly
medium
to
in two
after catatrepsis
(MCFARLANE and FURNEAUX, 1964). At the onset of the first phase of water absorption a change which has been called fragmentation takes place in the maternal epicuticle
(MCFARLANE, 1960,
(1962) is used here to describe
1963).
The
terminology
proposed
the layers of the cricket egg shell.
by MCFARLANE The first phase of
water absorption ends at the time that the embryo undergoes catatrepsis. The maternal and serosal epicuticles each possess an active tyrosinase, and it has been suggested that the first phase of water absorption is initiated and terminated by the differential tanning of these two layers of the shell (MCFARLANE, 1960). The fragmentation
of the maternal
epicuticle
of this layer at the onset of water absorption. been reported
indicates a change in the properties However, no observable change has
to take place in the serosal epicuticle
at the end of the first phase of
* Ths work has been supported by a grant from the National Research Council of Canada. 7 Present address: Milstead Laboratory of Chemical Enzymology, Broad Oak Road, Sittingboume, Kent, England. 591
P. J. S. FURNEAUX AND J. E. MCFARLANE
592
water absorption. The evidence for tanning of the serosal epicuticle is twofold. An unidentified, potentially melanogenic phenol is present in the serosal cuticle together with the active serosal epicuticular tyrosinase at this stage (MCFARLANE, 1960). It is also known that the serosal epicuticle and a thin layer of the serosal endocuticle bordering the epicuticle are the only parts of the serosal cuticle to remain undigested at eclosion (MCFARLANE, 1962). The remainder of the serosal endocuticle is digested between catatrepsis and eclosion, probably by the action of hatching enzymes secreted by the pleuropodia. The serosal endocuticle of Melanoplus diferentialis is known to be digested in this way (SLIFER, 1937, 1938). Polyphenols have not previously been identified in insect eggs. This paper is a report of the identification of polyphenols in house-cricket eggs and of the results of investigations which suggest that their occurrence is related to the development of the serosal cuticle. MATERIAL A culture of the house cricket is maintained in this laboratory according to the directions of GHOURI and MCFARLANE (1958). Eggs were collected from this culture in paper cups (14 oz, Lily Paper Cups Ltd., Toronto) partially filled with moist sand. After 24 hr the cups were removed; the eggs were separated from faeces and sand and incubated on moist filter paper at 34°C. Since the eggs are fertilized immediately before oviposition, the age of eggs in any sample was known to vary by up to 24 hr from the experimental time, which was arbitrarily fixed at zero at the start of incubation. Under these conditions house-cricket eggs hatch after 9 days ; the beginning and end of the first phase of water absorption occur at 36 and 84 hr respectively. METHODS Preparation and chromatography of egg homogenates Eggs from which the maternal cuticle had been removed (MCFARLANE, 1960) were thoroughly washed in distilled water and ground with a glass grinder in 2 ml of 5% HCl in 95% ethanol. The homogenate was centrifuged and the supernatant concentrated to about 0.02 ml. The concentrate was applied to the origin of a piece of Whatman No. 1 chromatography paper and developed by ascending flow. The following solvent mixtures were used for development: (1) (2) (3) (4) (5)
1-butanol-acetic acid-water 1-butanol-acetic acid-water 1-butanol-acetic acid-water Ethyl acetate-acetic acid-water 1-butanol-acetate buffer, pH 4.4
4-l-5 7-l-2 4-l-l 9-2-2 2-l
(Acetate buffer: 63 ml O-1 N acetic acid+ 37 ml 0.1 M sodium acetate) The reagents used for the detection of phenolic substances are listed in Table 1. Pauly’s diazotized sulphanilic acid, p-anisidene, silver nitrate, ferric chloride, and 1-nitroso-2naphthol were used according to the directions of SMITH (1960).
CATECHOLAMINES IN EGGSOF THE HOUSECRICKET,ACHETA
DOMESTICUS
(L.)
593
Sodium molybdate was used as a O-1 M aqueous solution and was sprayed onto the chromatograms (PRIDHAM, 1959). An ammoniacal solution of ethylenediamine was used according to the directions of SOURKES et al. (1963) ; the fluorescent spots were observed by ultraviolet irradiation. Ninhydrin was sprayed onto the papers as a 0.1% solution in acetone and the papers were subsequently heated at 110°C for 3 min. The other ninhydrin test used is according to SMITH (1960). Fluorometric estimation of catecholamines DOPA (3,4_dihydroxyphenylalanine), DOPamine (3,4_dihydroxyphenylethylamine), and N-acetyl DOPamine (N-acetyl-3,4-dihydroxy-fi-phenylethylamine) were estimated by a modification of the fluorometric technique of SOURKES et al. (1963). This technique is based on the formation of a water-soluble, fluorescent condensation product when catechols are treated with ethylenediamine under alkaline conditions. The specificity of the method depends on prior chromatographic separation of the substances to be estimated. Samples of 200 eggs from which the maternal cuticle had been removed were homogenized and the concentrate subjected to paper partition chromatography in solvent system (2) as described above. The papers were dried, dipped through a mixture of ethylenediamine and 10% ammonia (1 : 4), blotted between sheets of clean chromatography paper, and then dried in a cold-air draught. The areas corresponding to DOPA, DOPamine, and N-acetyl DOPamine were cut out, and the condensation product was leached into 10 ml of distilled water in tubes held at 60°C for 40 min. Frequent shaking with a Vortex mixer was found necessary to ensure maximum leaching. The fluorescence of the extracts was measured with a Coleman filter fluorometer (PC6 as primary filter and PC2 as secondary filter) and compared to graded amounts of authentic catecholamines” treated in the same way. A blank was prepared by leaching an equivalent area of the treated chromatogram into 10 ml of distilled water. Histochemical techniques The iodate methods and other techniques described by PEARSE (1960) for the localization of tissue phenols were applied to whole eggs and both paraffin and frozen sections. Only the iodate reagent was found to be satisfactory for this material. Paraffin sections were cut at 7 p and frozen sections at 15 p. The serosal shell and serosa were removed from fixed eggs by dissection with iridectomy scissors and watchmakers’ forceps. RESULTS
AND DISCUSSION
IdentiJcation of catecholamines in house-cricket eggs Three catecholamines were identified as DOPA, DOPamine, and N-acetyl DOPamine in samples of 200 eggs from which the maternal cuticle had been * DOPA was obtained from British Drug Houses Ltd., Poole, Dorset, England. DOPamine was obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio. A sample of N-acetyl DOPamine was generously provided by Dr. C. E. SEKERIS,Physiologisch-chemisches Institut der Universitat Miinchen, Germany. 38
594
P. J. S. FURNEAUXANDJ. E. MCFARLANE anbruqaa) awuoqma saddoa-uppKqufN u!IP-%u!N
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CATRCHOLAMINES
IN EGGS OF THE HOUSE CRICKET, ACBETA
DOMESTICUS
(L.)
595
removed. They were present in all samples taken between 60 hr and eclosion. Identification was based on paper partition chromatography and the use of a variety of general and specific reagents for phenols (Table 1). In every case the phenol under investigation behaved in a similar way to the authentic substance. These three catecholamines can be distinguished by their reaction with the last three detecting reagents as well as by their R, values. The ammoniacal ethylenediamine reagent produces fluorescent condensation products with most o-dihydroxyphenols. (However, in our experience, fluorescent products are not formed with catechol and protocatechuic acid.) The reagent does not seem to have been used previously for the detection of o-dihydroxyphenols in insect material. Of these three catecholamines, N-acetyl DOPamine is ninhydrin-negative because the amino group is substituted. DOPA and DOPamine can be distinguished by the last test : the neutralized sample is applied to the origin of a piece of chromatography paper previously rubbed with cupric carbonate with which a-amino acids form a Cu-complex. After development of the chromatogram, DOPamine remains ninhydrin-positive while the amino acid, DOPA, is ninhydrin-negative (SMITH, 1960). No other polyphenols were found in homogenates of cricket eggs at any stage of development. These three substances were further characterized as catecholamines by adsorption onto alumina at about pH 8 and elution with 0.5 M acetic acid (SOUR= et al., 1963). The identity of N-acetyl DOPamine was further established by hydrolysis. Two similar samples of 150 pre-catatrepsis eggs were homogenized and the homogenates purified by alumina adsorption. The acetic acid eluates were concentrated and the concentrate chromatographed in solvent system (3). The chromatogram of one sample was treated with ammoniacal ethylenediamine, and the area corresponding to N-acetyl DOPamine on the other chromatogram was cut out and shaken with 2 ml of 2 N HCl. The acid solution was then sealed into an ampoule and heated at 110°C for 6 hr. Paper chromatography of the hydrolysate revealed a single spot corresponding to DOPamine after application of the ammoniacal ethylenediamine reagent. Variation of catecholamines throughout embryonic development Visual estimation of the fluorescent condensation products formed between the catecholamines described above and the ammoniacal ethylenediamine reagent indicated that DOPamine was present at a higher concentration than either DOPA or N-acetyl DOPamine, especially after catatrepsis. A modification of the technique of SOURKESet al. (1963) was applied to estimate each catecholamine at different stages of embryonic development. The results of duplicate estimations on samples of 200 eggs are shown in Fig. 1. The origin of each catecholamine was fixed at 12 hr before the first readings were obtained. This was justified because fluorescent spots of weak intensity were observed with the naked eye at this time. However, when leached into water, they were beyond the sensitivity of the instrument.
P. J. S. FUFWEAUX AND J. E. MCFARLANE
596
Variation between samples is attributed to some stage in the estimation procedure prior to separation by chromatography because variation between standards of the same concentration was slight. Loss probably occurred when the concentrated supernatant was transferred to chromatography paper. This could have
I1
o-8_0_8/-~-~-~ 0
0
I1
24
I
11
49
(1
72
’
96
1
‘1 120
” 144
‘1’ 166
192
”
216
Age,hr FIG.
1.
Variation of DOPamine (top), N-acetyl DOPamine (middle), and DOPA (bottom) throughout embryonic development of Acheta domesticus.
been lessened by taking an aliquot from the supernatant of a larger quantity of eggs, but the large size of any such sample would have allowed only a few samples to be taken from any one egg collection. To avoid errors which might arise if the sampling were split into series;the sample size was limited so as to allow all samples to be taken from the same egg collection. The results are therefore a compromise between precision and practical considerations. The concentration of DOPA rose to 0.7 pg/lOO eggs at 36 hr. It fell off from then until 108 hr, and then rose slowly until eclosion. During the second day of incubation this appeared to be the only catecholamine present in the egg. During the third day of incubation N-acetyl DOPamine rose to about the same level as DOPA, while DOPamine reached a level of about O-3 pg/lOO eggs. Catatrepsis occurs at 84 hr and samples taken at this time revealed a marked rise in the level of The concentration of N-acetyl DOPamine DOPamine and N-acetyl DOPamine. then fell off to a lower fluctuating level. On the other hand, the level of DOPamine continued to rise until it reached a maximum of nearly 6 pg/lOO eggs at 156 hr. From then until eclosion the level of DOPamine fell off, but there still remained about 4.5 pg/lOO eggs in samples taken just before eclosion. The concentration of DOPamine at 84 hr was estimated in two sets of sampleseggs which had undergone catatrepsis and those which had not. It is clear from Fig. 1 that there is a marked rise of detectable DOPamine at catatrepsis. In order
CATECHOLAMINES
IN EGGS OF THE HOUSE CRICKET, ACHETA
DOMESTICUS
(L.)
597
to determine whether some of the DOPamine was present in the pre-catatrepsis egg in a form which was not freely extractable, the residue after centrifugation was washed, centrifuged again, and then subjected to acid hydrolysis. The washed residues from several samples of 200 eggs were hydrolysed under a variety of conditions ; the optimum conditions were found to be hydrolysis in 0.5 N HCl for 20 hr at 60°C when about 1-O pg DOPamine per 100 eggs could be detected. This would account for the difference in the level of DOPamine between eggs sampled immediately before and after catatrepsis. These hydrolytic conditions were less severe than those required to hydrolyse the glucoside of N-acetyl DOPamine in Drosophila (OKUBO, 19.58) or the glucosides of m-aminophenol and hydroquinone in the locust (MYERS and SMITH, 1954). This indicates that the mechanism by which DOPamine is bound in pre-catatrepsis eggs does not involve the formation of a glucoside. DOPamine in the extra-embryonic jluid After catatrepsis, the house-cricket embryo is surrounded by a fluid-filled space bounded externally by the serosal endocuticle. Some of the properties of this extra-embryonic fluid in the egg of Locusta have been described by JONES (1958). It contains a protein rich in phenolic groups and a soluble tyrosinase. When exposed to the air or released from the egg under liquid paraffin it forms a tanned protein membrane at the air-liquid or liquid-liquid interface. Jones claimed that the phenolic precursor of the tanning quinone was an integral part of the protein which was tanned. When post-catatrepsis eggs of the house cricket were punctured in a drop of 0.1 M sodium molybdate, so that only the extra-embyronic fluid escaped, the drop immediately became orange. This indicated the presence of an ene diol (PRIDHAM,1959). The fluid from ten post-catatrepsis eggs was applied directly to chromatography paper which had been previously treated with 0.1 M sodium molybdate. After development in solvent system (1) the molybdate complex remained as a single spot (Rf 0.05). When the extra-embryonic fluid of ten eggs was applied to untreated paper, chromatographed, and subsequently sprayed with sodium molybdate, a single spot was revealed which corresponded with authentic DOPamine. The absence of any coloured complex at or near the origin suggested that none of the phenolic component of the extra-embryonic fluid of the housecricket egg is attached to protein. No other phenolic substances were detected in the extra-embryonic fluid after application of any of the reagents listed in Table 1. The identity of this free DOPamine was further established by ultraviolet spectrophotometry. The fluid of 1500 eggs aged 156 hr was chromatographed in solvent system (l), and the area corresponding to DOPamine was cut out. The substance was eluted with methanol and the eluate examined with a Leitz U.V. Spectrophotometer. An absorption peak at 280 rnp with a shoulder at 288 rnp was found in a solution of authentic DOPamine, DOPamine isolated from the extraembryonic fluid, and a mixture of the two solutions. A peak at 225 rnp in the
598
P. J. S. FUJXNEAUX ANDJ. E. MCFARLANE
solution of authentic DOPamine was not apparent in the eluate, probably because of insufficient purification. Localization of catecholamines in house-cricket eggs A 10% aqueous solution of potassium iodate (PEARSE,1960) was found to be satisfactory for localization of catecholamines in the house-cricket egg. In most other histochemical techniques for the localization of tissue phenols, fixation precedes the application of the reagent. Poor results were encountered with these other techniques, and it was considered that these were due to leaching of catecholamines into the fixative. Eggs at various stages of development were punctured in the iodate solution and examined 24 hr later with a binocular microscope. The brown oxidation product could be seen clearly; dissection revealed that the brown coloration was confined to the shell and serosa of pre-catatrepsis eggs and to the shell and extra-embryonic fluid of post-catatrepsis eggs. Other iodate-treated eggs were fixed in Baker’s formalin-Ca (PANTIN, 1946). From this material paraffin and frozen sections were prepared. In sections of pre-catatrepsis eggs the brown colour was restricted to the serosal cuticle and serosal cells, and in sections of post-catatrepsis eggs it was found in the serosal cuticle and the space occupied by the extra-embryonic fluid of the intact egg. The maternal cuticle, yolk system, and embryo were colourless in all sections examined. The serosal cuticle of all iodate-treated eggs was at all stages of uniform colour. However, the intensity of coloration of the serosal cells varied. Prolongation of the iodate treatment did not increase the intensity of coloration of the serosal cells. This seemed to exclude the possibility that the intensity of reaction depended on the access of the reagent. It suggested that the serosal cells differed in their catecholamine content. This is consistent with the suggestion that insect epidermal cells are out-of-phase in the performance of a sequence of metabolic syntheses leading to the production of the cuticle (WIGGLESWORTH,1957). GENERAL DISCUSSION The biogenesis of N-acetyl DOPamine in late third-instar larvae of Calliphora erythrocephala has been shown to involve DOPA and DOPamine as intermediates (SEKERISand KARLSON,1962). The occurrence of these three metabolites suggests that a similar pathway of tyrosine metabolism exists in the house-cricket egg. The localization of these catecholamines in pre-catatrepsis eggs indicates that the pathway is one of the metabolic activities of the serosal cells, and that it is related to the development of the serosal cuticle. N-acetyl DOPamine has been reported to function in insects only as a sclerotizing agent (KARLSONet al., 1962; KARLSON and SEKERIS,1962). It is possible that this substance may have a similar role in the serosal epicuticle of the house-cricket egg, and its occurrence supports the mechanism of termination of the first phase of water absorption proposed by MCFARLANE (1960). It is noteworthy that melanogenesis does not normally occur in the
CATECHOLAMINES IN EGGSOF THE HOUSECRICKET,ACHETA
DOMESTICUS
(L.)
599
house-cricket egg. The localization of these catecholamines in the serosa, therefore, strongly implies the existence of a tanning reaction in the serosal cuticle. The extra-embryonic fluid of the eggs of Melanoplus diferentialis is known to contain proteolytic enzymes secreted by the pleuropodia (SLIFER, 1937, 1938; SHUTTS, 1952). These enzymes digest the serosal endocuticle prior to eclosion. The serosal endocuticle of the house-cricket egg is also digested between catatrepsis and eclosion (MCFARLANE, 1960) and it is probable that a similar mechanism is involved. The possibility that DOPamine is secreted into the extra-embryonic fluid by the pleuropodia was excluded because these structures were at all times negative to the iodate reagent. However, the proteolytic enzymes secreted into the extra-embryonic fluid by the pleuropodia may be able to de-acetylate ‘N-acetyl DOPamine. This would explain the fall in level of N-acetyl DOPamine after the house-cricket embryo has undergone catatrepsis. Further DOPamine may be added to the pool of DOPamine in the extra-embryonic fluid as the serosal cells disintegrate because a portion of the DOPamine of pre-catatrepsis eggs required mild acid hydrolysis for its release. DOPamine occurs at a considerably higher concentration in house-cricket eggs than has been reported for post-embryonic stages of insects (OSTLUND, 1954). The maximum concentration of DOPamine in the house-cricket egg occurs at 156 hr. Eggs of this age weigh about 0.642 mg. The concentration of DOPamine in these eggs would be about 935 pg/g live eggs. REFERENCES GHOURIA. S. K. and MCFARLANEJ. E. (1958) Observations on the development of crickets. Canad. Ent. 90, 158-165. JONESB. M. (1958) Enzymatic oxidation of protein as a rate-determining step in the formation of highly stable surface membranes. Proc. roy. Sot. (B) 148, 263-277. KARLSONP. and SEKERISC. E. (1962) N-acetyl-dopamine as sclerotising agent of the insect cuticle. Nature, Land. 195, 183-184. KAFZSON P., SEKERISC. E., and SEKERIK. E. (1962) Zum Tyrosinstoffwechsel der Insekten. VI. Identifizierung von N-acetyl-3,4-dihydroxy-/I-phenathylamin (N-Acetyl-dopamin) als Tyrosinmetabolit. Hoppe-Seyl. Z. 327, 86-94. MCFARLANEJ. E. (1960) Structure and function of the egg shell as related to water absorption by the eggs of Acheta domesticus (L.). Canad.J. Zool. 38, 231-241. MCFARLANEJ. E. (1962) The cuticles of the egg of the house cricket. Canad. -7. Zool. 40, 13-21. MCFARLANE J. E. (1963) Induction in vitro by buffer solutions of a structural change occurring normally during the embryonic development of the house cricket. Canad. J. Zool. 41, 23-28. MCFARLANEJ. E. and FURNEAUXP. J. S. (1964) Revised curves for water absorption by the eggs of the house cricket, Acheta dome&us (L.). Canad.2. Zool. 42, 239-243. MYERS C. M. and SMITH J. N. (1954) Comparative detoxication. 2. Glucoside formation from phenols in locusts. Biochem. J. 56, 498-503. OKUBO S. (1958) Occurrence of N-acetylhydroxytyramine glucoside in Drosophila mekmogas&. Med.?. Osaka Univ. 9, 327-337. &TLUHD E. (1954) The distribution of catecholamines in lower animals and their effect on the heart. Acta physiol. Stand. 31 (suppl.), 112, l-67.
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P. J. S. FURNEAUXAND J. E. MCFARLANE
PANTIN C. F. A. (1946) Notes on Microscopical Techniqueforzoologists. Cambridge University Press. PEARCEA. G. E. (1960) Histochemistry, Theoretical andApplied. 2nd Ed. Churchill, London. PRIDHAM J. B. (1959) Paper electrophoresis and paper chromatography of phenolic compounds. r. Chromatog. 2, 605-611. SEKERISC. E. and KARLSON P. (1962) Zum Tyrosinstoffwechsel der Insekten. VII. Der katabolische Abbau des Tyrosins und die Biogenese der Sklerotisierungssubstanz, N-acetyl-dopamin. Biochim. biophys. Acta 62, 103-I 13. SHUTTSJ. H. (1952) Some characteristics of the hatching enzyme in the eggs of MeZanopZus differentialis (Thomas). PYOC. S. Dak. Acad. Sci. 31, 158-163. SLIFERE. H. (1937) The origin and fate of the membranes surrounding the grasshopper egg; together with some experiments on the source of the hatching enzyme. Quart. J. micr. Sci. 79, 493-506. SLIFER E. H. (1938) A cytological study of the pleuropodia of MetanopEus differentialis (Orthoptera, Acrididae) which furnishes new evidence that they produce the hatching enzyme. J. Morph. 63, 181-205. SMITH I. (1960) Chromatographic and Electrophoretic Techniques Vol. I. Heinemann, London. SOURKEST. L., DENTON R. L., MURPHY G. F’., CHAVEZB., and SAINT CYR S. (1963) The excretion of dihydroxyphenylalanine, dopamine, and dihydroxyphenylacetic acid in neuroblastoma. Pediatrics 31, 660-668. WIGGLESWORTHV. B. (1957) The physiology of insect cuticle. Annu. Rev. Ent. 2, 37-54.
Printed in Great Britain
IDENTIFICATION, ESTIMATION, AND LOCALIZATION OF CATECHOLAMINES IN EGGS OF THE HOUSE CRICKET, ACHETA DOMESTICUS (L.)” P. J. S. FURNEAUX”f
and J. E. McFARLANE
Department of Entomology, Macdonald College, McGill Macdonald College P.O., P.Q., Canada (Received
9 October
University,
1964)
Abstract-DOPA, DOPamine, and N-acetyl DOPamine have been identified as the only polyphenols to occur during embryogenesis of Acheta dome&us. The quantitative variation and histochemical localization of these substances indicate that they are involved in the development of the serosal cuticle. In view of the absence of melanogenesis during normal development and the probability that a tanning reaction is involved in the termination of the first phase of water absorption, it is suggested that N-acetyl DOPamine is the precursor of a tanning quinone. The other phenols are thought to be intermediates in its biogenesis. A relatively large amount of free DOPamine has been found in the extra-embryonic fluid of post-catatrepsis eggs. This is thought to originate from the digestion of the serosa and serosal cuticle, and by de-acetylation of N-acetyl DOPamine through the action of the hatching enzymes. INTRODUCTION
THE eggs of the house cricket are laid containing complete
development.
phases which
Water
are separated
is absorbed
less water than they require
from the surrounding
by a brief period
of water loss shortly
medium
to
in two
after catatrepsis
(MCFARLANE and FURNEAUX, 1964). At the onset of the first phase of water absorption a change which has been called fragmentation takes place in the maternal epicuticle
(MCFARLANE, 1960,
(1962) is used here to describe
1963).
The
terminology
proposed
the layers of the cricket egg shell.
by MCFARLANE The first phase of
water absorption ends at the time that the embryo undergoes catatrepsis. The maternal and serosal epicuticles each possess an active tyrosinase, and it has been suggested that the first phase of water absorption is initiated and terminated by the differential tanning of these two layers of the shell (MCFARLANE, 1960). The fragmentation
of the maternal
epicuticle
of this layer at the onset of water absorption. been reported
indicates a change in the properties However, no observable change has
to take place in the serosal epicuticle
at the end of the first phase of
* Ths work has been supported by a grant from the National Research Council of Canada. 7 Present address: Milstead Laboratory of Chemical Enzymology, Broad Oak Road, Sittingboume, Kent, England. 591
P. J. S. FURNEAUX AND J. E. MCFARLANE
592
water absorption. The evidence for tanning of the serosal epicuticle is twofold. An unidentified, potentially melanogenic phenol is present in the serosal cuticle together with the active serosal epicuticular tyrosinase at this stage (MCFARLANE, 1960). It is also known that the serosal epicuticle and a thin layer of the serosal endocuticle bordering the epicuticle are the only parts of the serosal cuticle to remain undigested at eclosion (MCFARLANE, 1962). The remainder of the serosal endocuticle is digested between catatrepsis and eclosion, probably by the action of hatching enzymes secreted by the pleuropodia. The serosal endocuticle of Melanoplus diferentialis is known to be digested in this way (SLIFER, 1937, 1938). Polyphenols have not previously been identified in insect eggs. This paper is a report of the identification of polyphenols in house-cricket eggs and of the results of investigations which suggest that their occurrence is related to the development of the serosal cuticle. MATERIAL A culture of the house cricket is maintained in this laboratory according to the directions of GHOURI and MCFARLANE (1958). Eggs were collected from this culture in paper cups (14 oz, Lily Paper Cups Ltd., Toronto) partially filled with moist sand. After 24 hr the cups were removed; the eggs were separated from faeces and sand and incubated on moist filter paper at 34°C. Since the eggs are fertilized immediately before oviposition, the age of eggs in any sample was known to vary by up to 24 hr from the experimental time, which was arbitrarily fixed at zero at the start of incubation. Under these conditions house-cricket eggs hatch after 9 days ; the beginning and end of the first phase of water absorption occur at 36 and 84 hr respectively. METHODS Preparation and chromatography of egg homogenates Eggs from which the maternal cuticle had been removed (MCFARLANE, 1960) were thoroughly washed in distilled water and ground with a glass grinder in 2 ml of 5% HCl in 95% ethanol. The homogenate was centrifuged and the supernatant concentrated to about 0.02 ml. The concentrate was applied to the origin of a piece of Whatman No. 1 chromatography paper and developed by ascending flow. The following solvent mixtures were used for development: (1) (2) (3) (4) (5)
1-butanol-acetic acid-water 1-butanol-acetic acid-water 1-butanol-acetic acid-water Ethyl acetate-acetic acid-water 1-butanol-acetate buffer, pH 4.4
4-l-5 7-l-2 4-l-l 9-2-2 2-l
(Acetate buffer: 63 ml O-1 N acetic acid+ 37 ml 0.1 M sodium acetate) The reagents used for the detection of phenolic substances are listed in Table 1. Pauly’s diazotized sulphanilic acid, p-anisidene, silver nitrate, ferric chloride, and 1-nitroso-2naphthol were used according to the directions of SMITH (1960).
CATECHOLAMINES IN EGGSOF THE HOUSECRICKET,ACHETA
DOMESTICUS
(L.)
593
Sodium molybdate was used as a O-1 M aqueous solution and was sprayed onto the chromatograms (PRIDHAM, 1959). An ammoniacal solution of ethylenediamine was used according to the directions of SOURKES et al. (1963) ; the fluorescent spots were observed by ultraviolet irradiation. Ninhydrin was sprayed onto the papers as a 0.1% solution in acetone and the papers were subsequently heated at 110°C for 3 min. The other ninhydrin test used is according to SMITH (1960). Fluorometric estimation of catecholamines DOPA (3,4_dihydroxyphenylalanine), DOPamine (3,4_dihydroxyphenylethylamine), and N-acetyl DOPamine (N-acetyl-3,4-dihydroxy-fi-phenylethylamine) were estimated by a modification of the fluorometric technique of SOURKES et al. (1963). This technique is based on the formation of a water-soluble, fluorescent condensation product when catechols are treated with ethylenediamine under alkaline conditions. The specificity of the method depends on prior chromatographic separation of the substances to be estimated. Samples of 200 eggs from which the maternal cuticle had been removed were homogenized and the concentrate subjected to paper partition chromatography in solvent system (2) as described above. The papers were dried, dipped through a mixture of ethylenediamine and 10% ammonia (1 : 4), blotted between sheets of clean chromatography paper, and then dried in a cold-air draught. The areas corresponding to DOPA, DOPamine, and N-acetyl DOPamine were cut out, and the condensation product was leached into 10 ml of distilled water in tubes held at 60°C for 40 min. Frequent shaking with a Vortex mixer was found necessary to ensure maximum leaching. The fluorescence of the extracts was measured with a Coleman filter fluorometer (PC6 as primary filter and PC2 as secondary filter) and compared to graded amounts of authentic catecholamines” treated in the same way. A blank was prepared by leaching an equivalent area of the treated chromatogram into 10 ml of distilled water. Histochemical techniques The iodate methods and other techniques described by PEARSE (1960) for the localization of tissue phenols were applied to whole eggs and both paraffin and frozen sections. Only the iodate reagent was found to be satisfactory for this material. Paraffin sections were cut at 7 p and frozen sections at 15 p. The serosal shell and serosa were removed from fixed eggs by dissection with iridectomy scissors and watchmakers’ forceps. RESULTS
AND DISCUSSION
IdentiJcation of catecholamines in house-cricket eggs Three catecholamines were identified as DOPA, DOPamine, and N-acetyl DOPamine in samples of 200 eggs from which the maternal cuticle had been * DOPA was obtained from British Drug Houses Ltd., Poole, Dorset, England. DOPamine was obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio. A sample of N-acetyl DOPamine was generously provided by Dr. C. E. SEKERIS,Physiologisch-chemisches Institut der Universitat Miinchen, Germany. 38
594
P. J. S. FURNEAUXANDJ. E. MCFARLANE anbruqaa) awuoqma saddoa-uppKqufN u!IP-%u!N
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CATRCHOLAMINES
IN EGGS OF THE HOUSE CRICKET, ACBETA
DOMESTICUS
(L.)
595
removed. They were present in all samples taken between 60 hr and eclosion. Identification was based on paper partition chromatography and the use of a variety of general and specific reagents for phenols (Table 1). In every case the phenol under investigation behaved in a similar way to the authentic substance. These three catecholamines can be distinguished by their reaction with the last three detecting reagents as well as by their R, values. The ammoniacal ethylenediamine reagent produces fluorescent condensation products with most o-dihydroxyphenols. (However, in our experience, fluorescent products are not formed with catechol and protocatechuic acid.) The reagent does not seem to have been used previously for the detection of o-dihydroxyphenols in insect material. Of these three catecholamines, N-acetyl DOPamine is ninhydrin-negative because the amino group is substituted. DOPA and DOPamine can be distinguished by the last test : the neutralized sample is applied to the origin of a piece of chromatography paper previously rubbed with cupric carbonate with which a-amino acids form a Cu-complex. After development of the chromatogram, DOPamine remains ninhydrin-positive while the amino acid, DOPA, is ninhydrin-negative (SMITH, 1960). No other polyphenols were found in homogenates of cricket eggs at any stage of development. These three substances were further characterized as catecholamines by adsorption onto alumina at about pH 8 and elution with 0.5 M acetic acid (SOUR= et al., 1963). The identity of N-acetyl DOPamine was further established by hydrolysis. Two similar samples of 150 pre-catatrepsis eggs were homogenized and the homogenates purified by alumina adsorption. The acetic acid eluates were concentrated and the concentrate chromatographed in solvent system (3). The chromatogram of one sample was treated with ammoniacal ethylenediamine, and the area corresponding to N-acetyl DOPamine on the other chromatogram was cut out and shaken with 2 ml of 2 N HCl. The acid solution was then sealed into an ampoule and heated at 110°C for 6 hr. Paper chromatography of the hydrolysate revealed a single spot corresponding to DOPamine after application of the ammoniacal ethylenediamine reagent. Variation of catecholamines throughout embryonic development Visual estimation of the fluorescent condensation products formed between the catecholamines described above and the ammoniacal ethylenediamine reagent indicated that DOPamine was present at a higher concentration than either DOPA or N-acetyl DOPamine, especially after catatrepsis. A modification of the technique of SOURKESet al. (1963) was applied to estimate each catecholamine at different stages of embryonic development. The results of duplicate estimations on samples of 200 eggs are shown in Fig. 1. The origin of each catecholamine was fixed at 12 hr before the first readings were obtained. This was justified because fluorescent spots of weak intensity were observed with the naked eye at this time. However, when leached into water, they were beyond the sensitivity of the instrument.
P. J. S. FUFWEAUX AND J. E. MCFARLANE
596
Variation between samples is attributed to some stage in the estimation procedure prior to separation by chromatography because variation between standards of the same concentration was slight. Loss probably occurred when the concentrated supernatant was transferred to chromatography paper. This could have
I1
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I1
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I
11
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(1
72
’
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1
‘1 120
” 144
‘1’ 166
192
”
216
Age,hr FIG.
1.
Variation of DOPamine (top), N-acetyl DOPamine (middle), and DOPA (bottom) throughout embryonic development of Acheta domesticus.
been lessened by taking an aliquot from the supernatant of a larger quantity of eggs, but the large size of any such sample would have allowed only a few samples to be taken from any one egg collection. To avoid errors which might arise if the sampling were split into series;the sample size was limited so as to allow all samples to be taken from the same egg collection. The results are therefore a compromise between precision and practical considerations. The concentration of DOPA rose to 0.7 pg/lOO eggs at 36 hr. It fell off from then until 108 hr, and then rose slowly until eclosion. During the second day of incubation this appeared to be the only catecholamine present in the egg. During the third day of incubation N-acetyl DOPamine rose to about the same level as DOPA, while DOPamine reached a level of about O-3 pg/lOO eggs. Catatrepsis occurs at 84 hr and samples taken at this time revealed a marked rise in the level of The concentration of N-acetyl DOPamine DOPamine and N-acetyl DOPamine. then fell off to a lower fluctuating level. On the other hand, the level of DOPamine continued to rise until it reached a maximum of nearly 6 pg/lOO eggs at 156 hr. From then until eclosion the level of DOPamine fell off, but there still remained about 4.5 pg/lOO eggs in samples taken just before eclosion. The concentration of DOPamine at 84 hr was estimated in two sets of sampleseggs which had undergone catatrepsis and those which had not. It is clear from Fig. 1 that there is a marked rise of detectable DOPamine at catatrepsis. In order
CATECHOLAMINES
IN EGGS OF THE HOUSE CRICKET, ACHETA
DOMESTICUS
(L.)
597
to determine whether some of the DOPamine was present in the pre-catatrepsis egg in a form which was not freely extractable, the residue after centrifugation was washed, centrifuged again, and then subjected to acid hydrolysis. The washed residues from several samples of 200 eggs were hydrolysed under a variety of conditions ; the optimum conditions were found to be hydrolysis in 0.5 N HCl for 20 hr at 60°C when about 1-O pg DOPamine per 100 eggs could be detected. This would account for the difference in the level of DOPamine between eggs sampled immediately before and after catatrepsis. These hydrolytic conditions were less severe than those required to hydrolyse the glucoside of N-acetyl DOPamine in Drosophila (OKUBO, 19.58) or the glucosides of m-aminophenol and hydroquinone in the locust (MYERS and SMITH, 1954). This indicates that the mechanism by which DOPamine is bound in pre-catatrepsis eggs does not involve the formation of a glucoside. DOPamine in the extra-embryonic jluid After catatrepsis, the house-cricket embryo is surrounded by a fluid-filled space bounded externally by the serosal endocuticle. Some of the properties of this extra-embryonic fluid in the egg of Locusta have been described by JONES (1958). It contains a protein rich in phenolic groups and a soluble tyrosinase. When exposed to the air or released from the egg under liquid paraffin it forms a tanned protein membrane at the air-liquid or liquid-liquid interface. Jones claimed that the phenolic precursor of the tanning quinone was an integral part of the protein which was tanned. When post-catatrepsis eggs of the house cricket were punctured in a drop of 0.1 M sodium molybdate, so that only the extra-embyronic fluid escaped, the drop immediately became orange. This indicated the presence of an ene diol (PRIDHAM,1959). The fluid from ten post-catatrepsis eggs was applied directly to chromatography paper which had been previously treated with 0.1 M sodium molybdate. After development in solvent system (1) the molybdate complex remained as a single spot (Rf 0.05). When the extra-embryonic fluid of ten eggs was applied to untreated paper, chromatographed, and subsequently sprayed with sodium molybdate, a single spot was revealed which corresponded with authentic DOPamine. The absence of any coloured complex at or near the origin suggested that none of the phenolic component of the extra-embryonic fluid of the housecricket egg is attached to protein. No other phenolic substances were detected in the extra-embryonic fluid after application of any of the reagents listed in Table 1. The identity of this free DOPamine was further established by ultraviolet spectrophotometry. The fluid of 1500 eggs aged 156 hr was chromatographed in solvent system (l), and the area corresponding to DOPamine was cut out. The substance was eluted with methanol and the eluate examined with a Leitz U.V. Spectrophotometer. An absorption peak at 280 rnp with a shoulder at 288 rnp was found in a solution of authentic DOPamine, DOPamine isolated from the extraembryonic fluid, and a mixture of the two solutions. A peak at 225 rnp in the
598
P. J. S. FUJXNEAUX ANDJ. E. MCFARLANE
solution of authentic DOPamine was not apparent in the eluate, probably because of insufficient purification. Localization of catecholamines in house-cricket eggs A 10% aqueous solution of potassium iodate (PEARSE,1960) was found to be satisfactory for localization of catecholamines in the house-cricket egg. In most other histochemical techniques for the localization of tissue phenols, fixation precedes the application of the reagent. Poor results were encountered with these other techniques, and it was considered that these were due to leaching of catecholamines into the fixative. Eggs at various stages of development were punctured in the iodate solution and examined 24 hr later with a binocular microscope. The brown oxidation product could be seen clearly; dissection revealed that the brown coloration was confined to the shell and serosa of pre-catatrepsis eggs and to the shell and extra-embryonic fluid of post-catatrepsis eggs. Other iodate-treated eggs were fixed in Baker’s formalin-Ca (PANTIN, 1946). From this material paraffin and frozen sections were prepared. In sections of pre-catatrepsis eggs the brown colour was restricted to the serosal cuticle and serosal cells, and in sections of post-catatrepsis eggs it was found in the serosal cuticle and the space occupied by the extra-embryonic fluid of the intact egg. The maternal cuticle, yolk system, and embryo were colourless in all sections examined. The serosal cuticle of all iodate-treated eggs was at all stages of uniform colour. However, the intensity of coloration of the serosal cells varied. Prolongation of the iodate treatment did not increase the intensity of coloration of the serosal cells. This seemed to exclude the possibility that the intensity of reaction depended on the access of the reagent. It suggested that the serosal cells differed in their catecholamine content. This is consistent with the suggestion that insect epidermal cells are out-of-phase in the performance of a sequence of metabolic syntheses leading to the production of the cuticle (WIGGLESWORTH,1957). GENERAL DISCUSSION The biogenesis of N-acetyl DOPamine in late third-instar larvae of Calliphora erythrocephala has been shown to involve DOPA and DOPamine as intermediates (SEKERISand KARLSON,1962). The occurrence of these three metabolites suggests that a similar pathway of tyrosine metabolism exists in the house-cricket egg. The localization of these catecholamines in pre-catatrepsis eggs indicates that the pathway is one of the metabolic activities of the serosal cells, and that it is related to the development of the serosal cuticle. N-acetyl DOPamine has been reported to function in insects only as a sclerotizing agent (KARLSONet al., 1962; KARLSON and SEKERIS,1962). It is possible that this substance may have a similar role in the serosal epicuticle of the house-cricket egg, and its occurrence supports the mechanism of termination of the first phase of water absorption proposed by MCFARLANE (1960). It is noteworthy that melanogenesis does not normally occur in the
CATECHOLAMINES IN EGGSOF THE HOUSECRICKET,ACHETA
DOMESTICUS
(L.)
599
house-cricket egg. The localization of these catecholamines in the serosa, therefore, strongly implies the existence of a tanning reaction in the serosal cuticle. The extra-embryonic fluid of the eggs of Melanoplus diferentialis is known to contain proteolytic enzymes secreted by the pleuropodia (SLIFER, 1937, 1938; SHUTTS, 1952). These enzymes digest the serosal endocuticle prior to eclosion. The serosal endocuticle of the house-cricket egg is also digested between catatrepsis and eclosion (MCFARLANE, 1960) and it is probable that a similar mechanism is involved. The possibility that DOPamine is secreted into the extra-embryonic fluid by the pleuropodia was excluded because these structures were at all times negative to the iodate reagent. However, the proteolytic enzymes secreted into the extra-embryonic fluid by the pleuropodia may be able to de-acetylate ‘N-acetyl DOPamine. This would explain the fall in level of N-acetyl DOPamine after the house-cricket embryo has undergone catatrepsis. Further DOPamine may be added to the pool of DOPamine in the extra-embryonic fluid as the serosal cells disintegrate because a portion of the DOPamine of pre-catatrepsis eggs required mild acid hydrolysis for its release. DOPamine occurs at a considerably higher concentration in house-cricket eggs than has been reported for post-embryonic stages of insects (OSTLUND, 1954). The maximum concentration of DOPamine in the house-cricket egg occurs at 156 hr. Eggs of this age weigh about 0.642 mg. The concentration of DOPamine in these eggs would be about 935 pg/g live eggs. REFERENCES GHOURIA. S. K. and MCFARLANEJ. E. (1958) Observations on the development of crickets. Canad. Ent. 90, 158-165. JONESB. M. (1958) Enzymatic oxidation of protein as a rate-determining step in the formation of highly stable surface membranes. Proc. roy. Sot. (B) 148, 263-277. KARLSONP. and SEKERISC. E. (1962) N-acetyl-dopamine as sclerotising agent of the insect cuticle. Nature, Land. 195, 183-184. KAFZSON P., SEKERISC. E., and SEKERIK. E. (1962) Zum Tyrosinstoffwechsel der Insekten. VI. Identifizierung von N-acetyl-3,4-dihydroxy-/I-phenathylamin (N-Acetyl-dopamin) als Tyrosinmetabolit. Hoppe-Seyl. Z. 327, 86-94. MCFARLANEJ. E. (1960) Structure and function of the egg shell as related to water absorption by the eggs of Acheta domesticus (L.). Canad.J. Zool. 38, 231-241. MCFARLANEJ. E. (1962) The cuticles of the egg of the house cricket. Canad. -7. Zool. 40, 13-21. MCFARLANE J. E. (1963) Induction in vitro by buffer solutions of a structural change occurring normally during the embryonic development of the house cricket. Canad. J. Zool. 41, 23-28. MCFARLANEJ. E. and FURNEAUXP. J. S. (1964) Revised curves for water absorption by the eggs of the house cricket, Acheta dome&us (L.). Canad.2. Zool. 42, 239-243. MYERS C. M. and SMITH J. N. (1954) Comparative detoxication. 2. Glucoside formation from phenols in locusts. Biochem. J. 56, 498-503. OKUBO S. (1958) Occurrence of N-acetylhydroxytyramine glucoside in Drosophila mekmogas&. Med.?. Osaka Univ. 9, 327-337. &TLUHD E. (1954) The distribution of catecholamines in lower animals and their effect on the heart. Acta physiol. Stand. 31 (suppl.), 112, l-67.
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PANTIN C. F. A. (1946) Notes on Microscopical Techniqueforzoologists. Cambridge University Press. PEARCEA. G. E. (1960) Histochemistry, Theoretical andApplied. 2nd Ed. Churchill, London. PRIDHAM J. B. (1959) Paper electrophoresis and paper chromatography of phenolic compounds. r. Chromatog. 2, 605-611. SEKERISC. E. and KARLSON P. (1962) Zum Tyrosinstoffwechsel der Insekten. VII. Der katabolische Abbau des Tyrosins und die Biogenese der Sklerotisierungssubstanz, N-acetyl-dopamin. Biochim. biophys. Acta 62, 103-I 13. SHUTTSJ. H. (1952) Some characteristics of the hatching enzyme in the eggs of MeZanopZus differentialis (Thomas). PYOC. S. Dak. Acad. Sci. 31, 158-163. SLIFERE. H. (1937) The origin and fate of the membranes surrounding the grasshopper egg; together with some experiments on the source of the hatching enzyme. Quart. J. micr. Sci. 79, 493-506. SLIFER E. H. (1938) A cytological study of the pleuropodia of MetanopEus differentialis (Orthoptera, Acrididae) which furnishes new evidence that they produce the hatching enzyme. J. Morph. 63, 181-205. SMITH I. (1960) Chromatographic and Electrophoretic Techniques Vol. I. Heinemann, London. SOURKEST. L., DENTON R. L., MURPHY G. F’., CHAVEZB., and SAINT CYR S. (1963) The excretion of dihydroxyphenylalanine, dopamine, and dihydroxyphenylacetic acid in neuroblastoma. Pediatrics 31, 660-668. WIGGLESWORTHV. B. (1957) The physiology of insect cuticle. Annu. Rev. Ent. 2, 37-54.