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Tanned silksProc. R. Soc. Lond. B. 187, 133-170 (1974) Printed in Great Britain
Tanned silks
B y P. C. J . B r u n e t and B a rb a ra C. Coles
Department of Zoology, Oxford
(Communicated by N. A . M i t c h i s o n , - Received 23 May 1973 - Revised 8 April 1974)
The silks of the large cocoons of saturniid moths range in colour from pale to dark brown. They are invariably accompanied by phenolic compounds. The nature of these phenols and their interaction with silk form the subject of this paper.
Silk, though fluid when secreted, dries out almost instantly. I t is pure white; and if artificially kept dry remains white. I f the larva is allowed (as is in normal) to drench it with a watery secretion from the anus, or if artificially moistened, it darkens.
Saturniid silks are here shown to consist, not only of structural fibroin and sericin, but also of enzymes and phenols. The presence of moisture allows these to interact. The phenols in the silk gland are in the form of glucosides. They are cleaved by a glucosidase and the liberated phenols are then oxidized by an oxidase. The oxidation products act as tanning agents supposedly by introducing exogenous cross-links between protein chains.
Tanning brings about considerable physical and chemical changes in the properties of the silk. These are described.
Two of the tanning phenols have been identified. The O-glucoside of 3-hydroxyanthranilic acid has not been previously recorded as a natural metabolite of animals or plants (although it is said to appear in the excreta of Bombyx larvae following enforced administration of 3-hydroxy anthranilic acid). The 5-O-glucoside of gentisic acid has not been recorded in animals but has been found in boron-deficient plants. The identity of both has been confirmed by synthesis.
3-Hydroxyanthranilic acid glucoside is derived from dietary trypto phan, but gentisic acid does not arise from an aromatic amino acid. Our evidence is consistent with its being taken up from the food-plant as a conjugate, and with the phenolic moiety being used, without change, by the silk glands.
The biological and industrial implications of tanning of the cocoon silk are briefly considered.
1. I n t r o d u c t io n
Many insects - mainly moth larvae - secrete cocoons of silk and it is tacitly as sumed that these are ‘protective’. Yet, despite the fact th a t silk production draws heavily on an insect’s resources (about 50 % of the proteins of a larva may go into the construction of a cocoon), surprisingly few studies have been made as to their
134 P. C. J. Brunet and Barbara C. Coles
nature and purpose. If a biologist were to ask what purpose a cocoon serves - does it protect against radiation or humidity; or is it protective against a particular predator or pathogen; and, if it is protective, how is it suited for this? - he would find little guidance in the literature.
A typical silkworm’s cocoon is constructed from a continuous thread (of fibroin) cemented by so-called ‘gum ’ or sericin (Reaumur 1737). Both components are distinct proteins or belong to groups of related proteins. The fibroin, as it is being secreted, is coated with a wet film of sericin. This dries out and endows the other wise flexible fibroin feltwork with a measure of rigidity. The resulting cocoon is essentially an oval container showing a degree of resistance to deformation.
Commerce is interested in the fibroins, and an extensive literature on these exists. All fibroins share in common certain properties, such as a characteristic amino acid composition and crystallographic configuration. Far less is known about the sericins. However it is our contention tha t sericins are very relevant to an understanding of the physical and biological properties of cocoons for reasons th a t we shall put forward.
Cocoons show considerable superficial variation: a t one extreme, they are pale in colour, and the insect contained in them is able to escape by proteolytic digestion of the wall; a t the other extreme, cocoons are very dark, and escape is by way of a kind of mechanical trap-door.
In this paper we put forward the idea tha t this range of colours reflects funda mental differences in the nature of the silk, particularly in the sericin component. We offer evidence th a t in the paler cocoons the sericin is stabilized by, a t most, weak interactions while in the darker cocoons it is heavily and stably cross-linked.
We shall call the latter, ‘tanned silks’, purposely using a loose word to indicate th a t they have in some way been modified by one (or a variety of) cross-linking agents which endow them with physical and chemical stability. I t will be evident th a t a t the extremes (that is to say dark brown in contrast to pale silks) the differences are clear cut; one is tanned while the other is not. The situation is however somewhat more complicated in the case of some intermediates where evidently both kind and extent of tanning affect the properties of the silks.
Some of the so-called ‘wild silks’ of commerce, notably tussah silk, come into our category of tanned silks, and the isolation of the valuable fibroin from the cross-linked sericin in these cases presents manufacturing problems. An under standing of the underlying nature of this intractability could lead to improvements in their processing. From a biological point of view, an understanding of the nature of tanned silks should help to shed light on the purposes served by the cocoons th a t insects construct a t such considerable metabolic expense.
There has been no previous systematic treatm ent of the subject. In this pre liminary survey, several examples of tanned silks will be considered, and the properties of two cross-linking agents will be described.
Tanned silks 135
(a) Light and dark silks Silk is never dark when secreted. I t becomes dark only with the passage of
time. Early workers who studied this change (Dewitz 1921; Przibram 1922) spoke of this darkening as pigmentation, with the implication th a t perhaps no more was involved than a colour-change connected with camouflage; there was nothing to suggest th a t it carried with it a structural aspect.
Rudall (1962) first pointed out a resemblance between the manner in which the silks of the stalk of the cocoon of Antheraea (a tussah silk moth) and the cocoon of Nematus ribesii (a sawfly) darken and the colour-change undergone by insect cuticle as it hardens as a result of cross-linking (Pryor 19406); but no actual investigative work was carried out.
Another line of approach pointed to there being fundamental differences between silks of different species. Duspiva (1950) had shown th a t the moth Bornbyx makes use of a proteolytic enzyme with which it is able to make an escape hole in the cocoon; and later Kafatos & Williams (1964) investigated the protease used by Antheraeapernyito escape; they found th a t the enzyme is absent from Hyalophora cecropia, a moth th a t escapes from its brown cocoon by way of a preformed trap door.
Brunet (1967) took these findings together with the predictions of Rudall, to suggest th a t pale silks are untanned, relatively less stable proteins while dark silks are tanned and more stable. I t was found th a t the brown silk of Hyalophora is accompanied by substances of low molecular mass. One such substance, 3- hydroxyanthranilic (or 2-amino-3-hydroxybenzoic) acid, was detected in the silk gland in the form of a glucose conjugate (figure 9), and accompanying it were a glucosidase and a phenoloxidase. There were indications tha t the three components reacted to give first the free acid and then oxidation products, probably of a quinonoid nature, tha t would serve as polyfunctional links between the molecules of fibrous protein.
In Bombyx no such system is present. Here the silk most commonly is white, but there are races whose silk is coloured yellow with xanthophylls, and even others where the silk is greenish apparently owing to an unidentified glucoside th a t contains nitrogen (see, for example, Kikkawa 1953); however no phenoloxid ase has been reported. There is no evidence tha t these pigments affect the texture of the silk.
In this paper the above observations are extended. The unambiguous identity of two tanning phenols as 3-hydroxyanthranilic acid (figure 9) and gentisic (or 2,5-dihydroxybenzoic) acid (figure 10) are reported, and their metabolic origins are discussed. Further physical and chemical evidence is presented in support of our contention tha t brown silks, like sclerotins (Pryor 1940 a), are stabilized by primary valence bonds.
2. L iv e s t o c k , m e t h o d s and m a t e r ia l s
() Livestock The moths used in the experiments are members of the Bombycoidea.
Bombyxmori (L.), the mulberry silk-moth, the silk of which is not tanned and constitutes the greater part of commercially produced silk. Raised on mulberry ( M o r u s ). The Yellow European strain was used, supplied by Worldwide B utter flies Ltd, Over Compton, Sherborne, Dorset.
Antheraea pernyi Guerin-Meneville, the Chinese oak silk-moth, native of China, the silk of which is one of the ‘wild’ silks used in the production of tussah and shantung silks. The silk is tanned but fairly pale in colour. Raised mainly on hawthorn {Crataegus), but also on oak ( Qu). Supplied by Worldwide B utter flies Ltd.
Samia cynthia (Drury), the ailanthus silk-moth, a native of Asia but artificially transported and thriving elsewhere. Some varieties of this species are used in the production of ‘e ri’ silk; but not the variety used in this work, which produces light brown cocoons. Raised on privet {Lingustrum). Supplied by Mr W. Shibe, Moorestown, New Jersey, U.S.A., and Worldwide Butterflies Ltd, as Philosamia cynthia var. advena.
Hyalophora cecropia (L.), the cecropia moth or robin moth, a native of the U.S.A. The silk of this species is very dark and not used commercially. Raised on apple ( Malus). Obtained commercially from Mr W. Shibe, and as a gift from Dr G. P. Waldbauer and Dr Judith Willis of the University of Illinois.
Hyalophora gloveri (Strecker), Glover’s moth, a native of western U.S.A. The silk is dark and not used commercially. Raised on hawthorn {Crataegus). Supplied by Worldwide Butterflies Ltd.
Actias selene (Hiibner), the Indian moon moth, a native of Asia. The silk is dark and not used commercially. Supplied by the Butterfly Farm Ltd, Bilsington, Ashford, Kent.
There has been a restlessness among American taxonomists and generic names of Saturniidae have been changed and interchanged. We have adopted the te r minology of Ferguson (1972) for the American genera {Hyalophora and Samia). In his view the moth commonly known as Philosamia cynthia var. advena does not warrant subspecific distinction and should be known as Samia cynthia (Drury).
The nomenclature of the other genera {Bombyx, Antheraea and Actias) does not seem to be in dispute.
() Methods (i) Preparation of material for analysis
Silk glands. Larvae were anaesthetized with solid C0 2. For injections, a tuber culin syringe fitted with a 30-gauge needle was used, and inserted into the base of a dorsal tubercle of the seventh or eighth segment. Larvae were dissected under a saline (0.9 % aqueous sodium chloride containing 0.01 % calcium chloride). The extraction of phenols was carried out as described in the tex t (p. 139).
Silk. Direct extraction of soluble phenols was carried out as described in the tex t (p. 139).
(ii) Analytical methods Paper and thin layer chromatography. Paper chromatography was most commonly
carried out in the solvent mixture: w-butanol: acetic acid: water, 4 :1: 1.
Where no solvent is specified in the tex t this mixture will have been used. The following mixtures were on occasion used: acetic acid: water, 5 :100, methanol: n-butanol: benzene: water, 2 :1: 1: 1, ethanol:ammonia solution (relative density 0.88)-.water, 18: 1: 1, isopropanol:ammonia solution (relative density 0.88)-.water, 20:1:2, w-butanol:pyridine:water, 14:3 :3, benzene:acetic acid:water, 125:72:3, chloroform: toluene: acetic acid, 5:2:2. W hatman no. 1 paper or silica on plastic sheets (Macherey-Nagel MN-polygram
Sil N-HR/UV254) were used as supports. Electrophoresis. High voltage paper electrophoresis was carried out with a
vertically suspended sheet in an apparatus based on th a t of Michl (1951). I t was operated at about 70 V/cm for 30 min. 46 cm x 57 cm sheets of W hatman 3mm paper were used.
Separations were also carried out in a (horizontal) Camag HVE system operated at about 100 V/cm for 20 min with 40 cm x 20 cm sheets of Whatman no. 1 paper.
The buffers used were: pH 1.9 formic acid: acetic acid: water, 1:4 :45, pH 3.5 pyridine:acetic acid:water, 1: 10:89, pH 6.5 pyridine:acetic acid:water, 25: 1:225.
(c) Instruments used Ultraviolet absorption spectroscopy: Unicam SP800. Fluorescence spectroscopy: Aminco-Bowman. Electron spin resonance spectroscopy: carried out by Dr B. A. Coles on an instru
ment constructed by him in the Physical Chemistry Laboratory at Oxford. X-ray fluorescence spectroscopy: carried out by Mr C. R. Fagg in the Department
of Geology and Mineralogy on a Philips manual X-ray fluorescence spectrometer. Radioactivity: Packard radiochromatogram strip scanner; Tracerlab 4tt scanner;
Beckman liquid scintillation system. Extensometry: Instron model TM-M-L fitted with load cell type A.
(d) Materials 3-Hydroxyanthranilic acid: Mann Research Laboratories, and as a gift from Dr
Hegediis of Hoffmann-La Roche, Basle. fi-glucosidase ( emulsin): British Drug Houses and Koch-Light Laboratories. Palladium catalyst: Johnson-Matthey. All other chemicals: British Drug Houses.
Tanned silks 137
3. R e su l t s
(a) Soluble components associated with the silks of Antheraea pernyi, Hyalophora cecropia, Hyalophora gloveri and
Samia cynthia Extraction of brown silks with water or aqueous alcohols yields a number of
substances, separable by paper chromatography, variously giving positive reactions for phenols and with Ehrlich’s ^-dimethylaminobenzaldehyde reagent. Some are fluorescent. There was, however, a general smudginess following this procedure which suggested tha t not only was a mixture of phenols being detected but also their oxidation products.
This could be avoided by using either the silk glands or ‘white silk’ - freshly spun silk from animals kept over a desiccant (figured in Brunet 1967).
Glands of mature larvae tha t had just begun to spin were homogenized or left to steep in about twice their volume of methanol. Silk was steeped in 60- 80% aqueous methanol. Such solutions were examined by paper chromatography and high voltage electrophoresis and these techniques clearly showed the presence of several distinct compounds (figure 11), which could also be separated on a column of Sephadex G-25 by using 0.1 m acetic acid.
A quick scanning method was used tha t gives an indication of the presence of glucosides, and two were detected. Their identity and properties were then investigated.
(b) The presence of glucosides Silk glands or white silk were extracted in approximately 70 % aqueous meth
anol. Extracts were concentrated by standing in a desiccator over silica gel. Such dried extracts have been kept in a refrigerator for several years without deterioration.
Spots of the concentrate were scanned for glucosides by means of a simple two-dimensional method. Four sheets of W hatman no. 1 paper were spotted at one corner, 25 mm distant from the adjacent sides. The papers were formed into cylinders and developed in ^-butanol-acetic acid-water. After drying, two of the chromatograms were sprayed with crude (3-glucosidase (0.3 milligram emulsin per millilitre 0.01 m acetate buffer, pH 5.2). The remaining two sheets, which were to serve as controls, were sprayed with buffer alone. All four chromatograms were kept in a moist chamber (a polythene bag is suitable) for 4 h a t room temperature. After drying, each sheet was re-formed as a cylinder in such a way tha t the spots separated during the first run became the new starting line for chromatography in the second direction which was run in the same solvent. After completion of the run, the chromatograms were dried and examined under ultraviolet light (254 and 360 nm), and sprayed or dipped in ferric chloride-potassium ferricyanide (Barton, Evans & Gardner 1952) or diazotized sulphanilic acid (Cramer 1954) to locate phenols, or alkaline silver nitrate (Trevelyan, Procter & Harrison 1950) to detect reducing substances (including glucose).
Tanned silks 139
By using this method, substances tha t are not degraded by glucosidase lie on the diagonal th a t passes through the starting spot, having moved the same distance in both chromatographic separations. The aglycones of glucosides, after enzymic hydrolysis, lie elsewhere on the chromatograms (except in the unusual instances where glucoside and agly cone have the same mobility). The detection of glucose serves to confirm the existence of a glucoside. The method is obviously dependent on the specificity of the enzyme; critical confirmation is needed, but it serves as a valuable technique for preliminary survey.
-> second direction
Figure 1. Two dimensional ‘d iagonal’ chrom atogram s of an aqueous m ethanolic ex trac t of a silk gland of Sam ia cynthia, applied a t + . Only th e tw o m ajor ‘sp o ts’ are shown.
B oth chrom atogram s were run first in n-butanol-acetic acid-w ater. A fter drying, {a) was sprayed w ith buffer, (6) w ith (3-glucosidase solution. B oth (a) and (6), after drying, were rerun in the same chrom atographic solvent a t righ t angles to th e first direction.
(a) Shows glucosides (A and B) as do tted ‘ spots ’ on the diagonal. (b) Shows glucose (open ‘sp o ts’) and the aglycones (A and B, as black ‘sp o ts’) off the
diagonal.
Extracts of Antheraea pernyi showed the presence of one suspected glucoside (glucoside B). An additional glucoside (figure 1, glucoside A) was present in extracts of Hyalophora cecropia and gloveri and in Samia cynthia.
(c) The glucoside of 3-hydroxyanthranilic acid The more intensely fluorescent of the two glucosides showed a blue-violet
fluorescence and gave rise to an agly cone that fluoresced bright blue (figure 1, glucoside A and agly cone A).
The glucoside gave mostly negative results with commonly used colorigenic agents (table 1). Treatment of dried methanolic extracts of glands or white silk with (3-glucosidase (0.3 mg emulsin per ml 0.001 m acetate buffer a t pH 5.2) yielded the agly cone. Colour tests on this showed that it is phenolic and is a reducing agent, properties tha t are masked in the glucoside. Both glucoside and agly cone give a yellow colour with p-dimethylaminobenzaldehyde indicating perhaps an aromatic amine.
Table 1. Properties of glucoside A (3-h ydrox yanthr anilic acid glucoside) and aglycone A (3-hydroxyanthranilic acid)
glucoside A aglycone A
n-butanol-acetic acid-w aterf 0.45 0.85 m ethanol-w-butanol-benzene-waterf 0.48 — 5 % aqueous acetic acid f 0.80 0.65 n-butanol -pyridine -water f — 0.66
electrophoretic mobilities where serine = + 1 .0 (pH 1.9) or aspartic acid = - 1 .0 (pH 3.5, 6.5)
acetic acid-formic acid-w ater p H 1.9$ + 0.14 + 0.66 pyridine-acetic acid-w ater p H 3.5$ 0 0 pyridine-acetic acid-w ater p H 6.5$ -0 .5 0 -0 .6 7
reactions w ith colorigenic reagents
ninhydrin nil nil silver n itra te reagent § nil black diazotized sulphanilic acid§ nil orange-brown ferric chloride-potassium ferricyanide§ nil blue E hrlich’s reagent yellow yellow
spectroscopic properties
inspection under 360 nm light blue-violet pale blue fluorescence fluorescence
absorption Amax in 0.05 M phosphate, pH 7 315 315 absorption Amax in 0.1 M HC1 — 298 fluorescence Aexc in 0.05 M phosphate, pH 7 316 318 fluorescence Aem in 0.05 m phosphate, pH 7 405 415
f See p. 137. J See p. 137. § See p. 138.
Both glucoside and aglycone are uncharged a t pH 3.5, while a t pH 1.9 they behave as bases, and at pH 6.5 as acids.
The properties of potential acid and base (but not cc-amino acid), phenol, and reducing agent are consistent with those of 3-hydroxyanthranilic acid (figure 9). The presence of this acid has already been recorded from insects by Kikkawa (1953), and its immediate metabolic precursor, 3-hydroxykynurenin, has been regularly encountered (see, for example, Brunet 1965).
The aglycone and 3-hydroxyanthranilic acid show comparable mobilities when subjected to high voltage electrophoresis and paper chromatography in several solvents (table 1).
After purification by repeated chromatography and electrophoresis, both glucoside and aglycone show a characteristic absorption maximum at 315 nm (the spectrum of the glucoside is shown in figure 2). They have corresponding fluorescence excitation maxima at 316 and 318 nm respectively, and emission
Tanned silks 141
Figure 2. A bsorption spectrum of glueoside A (3-hydroxyanthranilic acid glucoside) in 0.05 M phosphate buffer, pH 7.0.
maxima a t 405 and 415 nm. This again is consistent with the aglycone being 3-hydroxyanthranilic acid.
The differences between the properties of glncoside and aglycone suggested tha t the glucoside is an O-glucoside, and this was accordingly synthesized from 3- aminobenzoic acid by way of 3-hydroxy-2-nitrobenzoic acid (Hegediis 1951). This compound was converted to the p-nitrophenyl ester and coupled to aceto- bromoglucose. Reduction of the nitro-group and de-esterification was accomplished with hydrogen with the aid of a palladium catalyst. Deacetylation was by sodium methoxide.
The properties of the synthetic and natural glucosides, 2-amino-3-0-(3-D- glucopyranosidobenzoic acid (figure 9), proved to be identical (table 1).
The glucoside is present in Hyalophom in H. and in Sarnia cynthia, and in small amounts in Actias selene, but is absent from Antheraea pernyi.
(d) The glucoside of gentisic acid The second suspected glucoside (figure 1, glucoside B), present in the silks of
Antheraea pernyi, Hyalophora cecropia and H. gloveri, and Samia cynthia (being particularly prevalent in extracts of Antheraea) was investigated with techniques similar to those described in the previous section. The properties of glucoside B and its aglycone are set down in table 2.
The aglycone is evidently a dihydroxybenzene derivative of an acidic nature, and its glucoside effectively a monohydroxybenzene (and thus an O-glucoside and not an ester). The blue fluorescence of the aglycone together with its electrophoretic mobilities were compatible with its being either 2,5-dihydroxyphenylacetic (homogentisic)acid (figure 10), or 2,5-dihydroxybenzoic (gentisic) acid (figure 10), or 2,3- dihydroxybenzoic acid. The last, being an orf^o-dihydroxybenzene, was ruled out as a possibility because the unknown did not form a chromatographically slow-
Table 2. Properties of glttcoside B (gentisic acid glucoside) AND AGLYCONE B (GENTISIC ACID)
glucoside B aglycone B
chrom atographic and electrophoretic mobilities
R t values w-butanol-acetic acid-w aterf 0.40 0.85 5 % aqueous acetic acid f 0.80 0.68 n-butanol-pyridine-w aterf 0.08 0.32 benzene-acetic acid-w aterf — 0.38 isopropanol-am m onia-w aterf — 0.39
electrophoretic mobilities where serine = + 1 .0 (pH 1.9) or aspartic acid = —1.0 (pH 3.5, 6.5)
acetic acid-formic acid-w ater p H 1.9$ -0 .1 5 -0 .1 6 pyridine-acetic acid-w ater p H 3.5$ -1 .1 7 -1 .6 4 pyridine-acetic acid-w ater pH 6.5$ -0 .5 8 -0 .8 2
reactions w ith colorigenic reagents
ninhydrin nil nil silver n itra te reagent § nil black diazotized sulphanilic acid§ weak pinkish-white
orange-brown ferric chloride-potassium ferricyanide § blue blue ferric chloride weak blue
blue-purple E hrlich ’s reagent nil nil
spectroscopic properties inspection under 360 nm light blue brigh t pale blue
fluorescence fluorescence inspection after fum ing w ith jSTH3 no change becomes yellower absorption Amax in w ater 311 324 absorption Amax in d ilute alkali 311 344 fluorescence Aexo in 0.05 m phosphate, p H 7 336 325 fluorescence Aem in 0.05 M phosphate, pH 7 445 454
f See p. 137. $ See p. 137. § See p. 138.
moving molybdate derivative (Pridham 1959). The aglycone, in fact, gives a positive cyanide test for para-dihydroxybenzenes (Feigl 1966). Of the two 2,5- dihydroxy acids, gentisic acid, which is far more rarely encountered in animals than homogentisic acid, seemed the more close to the aglycone, for example, homogentisic acid is unstable in the solvent mixture isopropanol-ammonia-water while the aglycone is not, and it has the same value as gentisic acid.
The further work required to verify the identity of the aglycone with gentisic acid and to establish the constitution of the natural glucoside involved the isola tion of quantities of glucoside from white silk or from silk glands by preparative
Tanned silks 143
paper chromatography with never less than two solvent systems (usually n- butanol-acetic acid-water followed by 5% aqueous acetic acid). The main fluores cent ‘bar5 was finally eluted and used in part for investigations on the nature of the glucoside itself, and in part for preparation of the aglycone, by using emulsin (as on p. 147) followed by two separations involving paper chromatography and/or electrophoresis.
The aglycone was dealt with first. Its ultraviolet absorption spectrum in distilled water shows a peak a t 324 nm and a shoulder a t about 234 nm (figure 3), which corresponds to tha t of gentisic acid. The fluorescence spectra of gentisic acid and aglycone B exhibit identical excitation and emission maxima, namely Aexc = 325 nm and Aem = 454 nm.
© 1.0
w avelength/nm
F igure 3. A bsorption spectrum of glucoside B (gentisic acid glucoside) in w ater, unchanged by alkali (full line).
Spectrum of synthetic gentisic acid in w ater (----------- ), and in very dilute alkali (-------------).
Ionization constants were obtained by measurement of the fluorescence intensity of gentisic acid and aglycone B solutions (1 p.g/ml) over a range of pH provided by citrate-phosphate buffer from pH 7.0 to 2.6, and by dilute hydrochloric acid for lower values. The pKa of gentisic acid was found to be 2.98 by this method; Weast (1971) gives 2.97. The value for the aglycone was found to be 3.0 (figure 4).
As a final comparison and identification, in collaboration with B. A. Coles, electron spin resonance spectra of gentisic acid and of the aglycone were obtained. Gentisic acid is a convenient compound for study by this method since it is readily converted to a free radical by oxidation with gaseous oxygen in alkaline solution, producing a semiquinone form which is stable for several minutes (see, for example, Ayscough 1967, p. 263).
The aglycone was prepared by electrophoresis a t pH 3.5 and chromatography in w-butanol-acetic acid-water on washed paper. 0.01 ml of 0.2 m sodium hydroxide was added to 0.01 ml of aglycone eluate (estimated to contain approximately
F igure 4. M easurements of fluorescence in tensity indicate a pK of 2.98 for synthetic gentisic acid (•) , and 3.0 for aglycone A (o).
10 pg/ml water) and shaken for a few seconds to allow oxidation to proceed. The resulting solution was transferred to a flat silica e.s.r. cell, degassed by bubbling oxygen-free nitrogen through the cell for about 15 s, then the cell was placed in a spectrometer and the spectrum scan was completed within 5 min. Comparison spectra of authentic gentisic acid were made at concentrations of 10 pg/ml and upwards, the higher concentrations being useful for improving the signal/noise ratio and hence the accuracy of the measurement of the hyperfine splittings. The spectrometer operated at a frequency of 9030 MHz.
0.32220.32200.32180.3214 0.3216 m agnetic flux density/T
F ig u r e 5. E lectron spin resonance spectra a t 9030 MHz of aglycone A (a), and gentisic acid (6).
Tanned silks 145
The spectrum obtained from the aglycone is shown in figure 5 a with the spec trum from a stronger sample of authentic gentisic acid in figure 5b for comparison. The three characteristic hyperfine splittings found for gentisic acid were 2.007, 2.187, and 2.577 x 10_4T (2.007, 2.187 and 2.577 G), and these are identical within experimental accuracy for the spectrum of the aglycone. This set of three hyper fine splittings may be regarded as a unique identification as it is extremely un likely th a t any other molecule could duplicate all three values. The additional weak lines visible in figure 5 a are due to an unidentified secondary free radical, formed when the primary free radical reacts with an impurity derived from the chromatography paper.
Regarding the nature of the natural glucosidic conjugate of gentisic acid, the anionic behaviour of the compound indicated by high voltage electrophoresis showed th a t the compound is not the glucose ester of the acid. The glucoside gives colour reactions of a mono-hydroxybenzene and cannot therefore be a diglucoside.
Several compounds of glucose and gentisic acid have been described in the liter ature from plants. Boron-deficient sunflowers contain a gentisic acid conjugate (Watanabe, Chorney, Skok & Wender 1964), which was identified as the 5-0-(3-d- glucoside of the acid by Zane & Wender (1964a), and its structure was verified by synthesis (Zane & Wender 19646). At the same time they also synthesized the other two monoglucosides, namely, the 2-O-glucoside and the 1-O-gentisoyl- glucose ester, and reported a number of characteristics of all three monoglucosides.
From their accounts the ester can easily be distinguished from the two 0 - glucosides, having characteristically different Rt values in several solvents. The O-glucosides, however, behave chromatographically in a very similar way and R t values are too close to be decisive for unambiguous identification. There was disagreement (Watanabe et al. 1964; Zane & Wender 1964a) as to the susceptibility of the glucosides towards emulsin; and both groups of authors recorded gentisic acid or its glucosides as giving no colour with diazotized sulphanilic acid, which was not in agreement with our findings.
In the circumstances it seemed essential to produce our own authentic compounds. For several reasons we thought tha t glucoside B would prove to be the 5-0- rather than the 2-O-glucoside and accordingly this was synthesized.
The method of Wagner (1958) was substantially used: gentisic acid was con verted to the diacetoxy derivative; the acetyl group was removed from the 5- position; the methyl ester was prepared. This was coupled with acetobromo- glucose by using silver oxide in quinoline. Deacetylation was brought about with sodium methoxide, and de-esterification with barium hydroxide (Zane & Wender 19646).
This synthetic 5-0 -(3-D-glucoside and glucoside B were found to have identical electrophoretic mobilities a t pH 1.9, 3.5 and 6.5. Both can be located by their blue fluorescence, which does not intensify on exposure to ammonia. Both give a dull blue-purple colour with 2 % aqueous ferric chloride. With diazotized sul phanilic acid they give a dull orange-brown colour tha t fades, which is contrary
to the report of Zane & Wender (1964a). They are both susceptible to hydrolysis by emulsin and by 1 m hydrochloric acid a t 100 °C for 30 min. They have the same Rt values in a range of solvents, and they exhibit identical fluorescence maxima, namely, Aexc = 336 nm and Aem = 445 nm in distilled water or 0.5 m phosphate buffer a t pH 7.0.
To make doubly certain of the identity of the insect glucoside, the two less likely mono-glucosides of gentisic acid were synthesized. The 2-O-glucoside and the glucoside ester were simultaneously prepared by a method based on K arrer’s (1917) synthesis of the corresponding salicylic acid glucosides. 5-Acetoxygentisic acid was made according to the method of Lesser & Gad (1926). The silver com plex of this (see Karrer, Nageli & Weidmann 1919) was coupled to acetobromo- glucose in toluene (Karrer 1917). The joint product was deacetylated with sodium methoxide. The yield of the ester far exceeded tha t of the O-glucoside.
The gentisic acid ester proved predictably different from either O-glucoside notably in its immobility during electrophoresis a t neutral or acidic pH. The 5-O-glucoside differs as follows from the 2-O-glucoside: the 5-O-glucoside fluoresces on paper a bright blue; it gives a dull orange-brown colour tha t fades with diazotized sulphanilic acid; its electrophoretic mobility a t pH 3.5 is 0.67 with respect to th a t of gentisic acid as unity; the 2-O-glucoside fluoresces a dull violet-blue th a t is intensified in ammonia; it gives a dark purple colour with diazotized sul phanilic acid; and its mobility a t pH 3.5 is 0.44 tha t of gentisic acid. Both O- glucosides are cleaved by emulsin.
I t was concluded tha t glucoside B, the predominant phenol present in the silk glands of Antheraea pernyi, and also present in Hyalophora and Sarnia species, is 5-0-(3-D-glucopyranosidogentisic acid (figure 10).
(e) The accumulation of tanning agents during the development of silk glands During the fourth instar of Sarnia, the silk glands are very small, thin tubes.
They remain so for the first 4 days of the fifth (last larval) instar. Quite suddenly, on the fifth day, silk production starts, and the glands rapidly increase in size until, a t the time of larval maturity, they occupy the major part of the body cavity (figure 6).
Further measurements were made of the rate of production of silk and 3- hydroxyanthranilic acid glucoside, and of their final concentrations in the glands; the former as it might give an indication whether synthesis of the two components was under joint or independent control; the latter as it would indicate the maxi mum of cross-linking tha t could subsequently occur.
In order to obtain the mass of glucoside present throughout the instar, silk glands of Sarnia were extracted three times in ten volumes of 80% aqueous methanol; the extracts were concentrated and aliquots separated by electrophoresis a t pH 1.9. Ten glands were used for study up to the fourth day of the last instar, and correspondingly fewer as the glands grew larger. Each fluorescent, 3-hydroxy - anthranilic acid glucoside, band was eluted with 0.01 m acetate buffer, pH 5.2,
Tanned silks 147
tim e after 4 th m oult/days
Figure 6. Growth of silk gland of Sam ia cynthia relative to body mass during final larval instar. The s tandard deviations are indicated by vertical lines.
and 1 mg of (3-glucosidase (emulsin) together with a trace of sodium metabisulphite (as antioxidant) were added. After 1 h the mixture was subjected to chromato graphy in w-butanol-acetic acid-water. The blue fluorescent bands representing 3-hydroxyanthranilic acid were located with a 360 nm ultraviolet lamp, and eluted with 0.05 m phosphate buffer, pH 7.0 The eluates were made up to a known volume with the same buffer, and the amount of 3-hydroxyanthranilic acid present was assessed fluorimetrically by comparison with authentic standards containing from 0.02 fig up to 1 fig per ml buffer. Settings used were Aexc = 318 nm and Aem = 415 nm.
The amount of acid (derived from its glucoside present in the gland) is shown in figure 7 a .None could be detected, even in combined extracts from ten larvae, during the first 4 days of the last instar. On the fifth day the fluorescent bar on chromatograms, representing the glucoside, became clearly visible and thereafter increased rapidly in intensity.
A final concentration of 2.48 fig of 3-hydroxyanthranilic acid per milligram of silk gland (dry mass) was reached; this corresponds to 5.09 fig glucoside per milligram gland (table 3).
Essentially the same method was applied in the study of gentisic acid in the silk glands of Samia and Antheraea, but with minor differences in technique. After methanolic extraction of the glucoside, electrophoresis was carried out a t pH 3.5 (instead of pH 1.9). After elution and treatm ent with glucosidase, the aglycone was separated by electrophoresis again a t pH 3.5. Elution was with phosphate buffer followed by fluorimetric assessment a t Aexc = 324 nm and Aem — 454 nm with solutions of authentic gentisic acid as standards.
9 •"
1 600 200 $
4 th moult
F igure 7. D ry mass of silk gland (broken line) during last larval instar. Mass of aglycone (derivable from its glucoside) present in a silk gland (solid line).
(a) 3-H ydroxyanthranilic acid in Sam ia (b) Gentisic acid in Antheraea pernyi.
Antheraea larvae spend an average of 10 days of the final larval instar in feeding (figure 76). Spinning starts 1 to 2 days later. Silk protein production begins on the fourth day of this instar, tha t is to say 1 day earlier than in Samia. Both species already contain detectable amounts of gentisic acid glucoside in their glands before silk protein production begins: the glucoside is even detectable in the glands of fourth instar larval Antheraea, and can also be detected in an extract prepared from ten, third day, fifth instar larvae of Samia, tha t is 2 days before any 3- hydroxyanthranilic acid glucoside can be found, despite the fact th a t the final concentration of gentisic acid glucoside reaches only one-tenth tha t of 3-hydroxyanthranilic acid in this species.
Table 3. Masses of mature larvae, silk glands, and of the GLUCOSIDES CONTAINED IN THE SILK GLANDS
3-hydr-
glucoside
glucoside per mg
fresh dry per silk silk per silk silk mass mass gland gland gland gland
g mg yg yg yg t*g Sam ia cynthia 6.7 183 928 5.09 96.1 0.52 Antheraea pernyi 19.6 489 nil nil 2660 5.38
Tanned silks 149
In Antheraea an average final concentration of gentisic acid per silk gland (dry mass 489 mg) was 1280 pig, tha t is to say 2.62 pig gentisic acid (corresponding to 5.38 (xg glucoside) per mg dry mass silk gland. had relatively far less with 0.25 pig gentisic acid (corresponding to 0.52 fxg glucoside) per mg dry mass silk gland.
A summary of these values is given in table 3. While the rate of accumulation of 3-hydroxyanthranilic acid glucoside in Samia is closely similar a t all times to the rate of accumulation of protein, the rate for gentisic acid glucoside in both Samia and Antheraea is, to begin with, in advance of th a t of protein accumulation. The relevance of this will be discussed in relation to the metabolic origin of the glucosides.
(/) Metabolic sources of the tanning agents (i) Z-Hydroxyanthranilic acid
Mammals and microorganisms are known to convert tryptophan, by way of kynurenin and 3-hydroxykynurenin, to 3-hydroxyanthranilic acid (Henderson, Gholson & Dalgliesh 1962). As kynurenin and 3-hydroxykynurenin are regularly encountered in insects it seemed reasonable to assume th a t such a pathway also exists in insects. To verify this, isotopically labelled tryptophan was injected into larvae shortly before they would begin to spin.
Samia larvae, whose age ranged from the fifth to thirteenth day of the final larval instar, were each injected with 5 pCi of DL-tryptophan [benzene C] in distilled water. They were allowed to feed for 10-12 h before their silk glands were dissected out, blotted and weighed. The glands were extracted in 80% aqueous methanol, and the extracts were concentrated. The concentrates were subjected to electrophoresis a t pH 1.9 on W hatman 3mm paper which serves to separate the original tryptophan from the glucoside. Strips cut from the electropherograms were scanned for radioactive zones with a strip counter.
The radioactivity on such an electropherogram (figure 8 a) appears as a very large peak th a t is evidently tryptophan, a second peak attributable to 3-hydroxy anthranilic acid glucoside, and a small peak a t the origin th a t is present when tryptophan is run by itself and which we consider to be a degradation product of tryptophan, presumably due to the acidity of the buffer.
The tryptophan and 3-hydroxyanthranilic acid glucoside areas were cut out and re-separated by paper chromatography. The presumed radioactive trypto phan region again corresponded precisely with the site of the authentic amino acid. The blue fluorescent region, considered to be 3-hydroxyanthranilic acid glucoside, was eluted and treated with emulsin (as on page 147). This product was separated by electrophoresis a t pH 1.9 followed by paper chromatography. The blue fluorescent region corresponded precisely with authentic 3-hydroxyanthranilic acid (figure 86).
I t was concluded th a t the 3-hydroxyanthranilic acid of the silk gland is (at least in part) derived from tryptophan, but the experiments gave no indication as to the intermediary metabolism. Were the 3-hydroxyanthranilic acid to arise by
12000
3-HAG
s ta r t electrophoretic m obility R t value
F igure 8. R adioactiv ity m easured on paper strip scanner or in scintillation counter (b), (c) and ( d)of electropherogram s and chrom atogram s of com ponents of silk glands following injection of 14C -tryptophan.
() E lectropherogram (acetate/form ate, pH 1.9) of an aqueous m ethanolic ex trac t of silk glands following injection of DL-tryptophan [benzene C]. The tw o larger peaks represent try p to p h an (Try) and 3-hydroxyanthranilic acid glucoside (3HAG).
() Chrom atogram (w-butanol-acetic acid-water) of 3-hydroxyanthranilic acid glucoside region shown in (a), after enzymic hydrolysis. The 3-hydroxyanthranilic acid peak (3HA) is visible.
(c) E lectropherogram (acetate/form ate, pH 1.9) of an aqueous m ethanolic ex trac t of silk glands following injection of l -tryp tophan C). Only the tryp tophan peak (Try) is visible.
(d) The alanine region of an electropherogram , re-separated by chrom atography (n-butanol-acetic acid-water), of a hydrolysate of the aqueous m ethanol insoluble com ponent of silk glands (effectively silk protein) following injection of L-tryptophan
[methylene-1*C), confirming the presence of radioactive alanine (Ala) in the silk.
way of kynurenin and 3-hydroxykynurenin by the recognized pathway (figure 9) we could expect that, if side-chain labelled tryptophan were injected, the label would appear, not in the 3-hydroxyanthranilic acid but in the by-product alanine.
Fifth instar larvae were injected with 5 p.Ci L-tryptophan C) and allowed to feed for about 10 h. Glands were extracted with 50 % aqueous methanol and subjected to electrophoresis a t pH 1.9. There was no indication of radioactivity associated with alanine; only with residual tryptophan and the supposedly arti ficial spot a t the start line (figure 8 c). Aqueous methanolic extracts of white silk likewise showed no labelled alanine.
Labelled alanine was then sought for in the protein. The methanol-insoluble fraction (in this case, of white silk) was subjected to hydrolysis with 6 m HC1 at 110 °C for 48 h in an evacuated, sealed tube. Following hydrolysis the HC1 was distilled off a t room temperature under vacuum and the brown residue was taken up in distilled water. After separation by electrophoresis a t pH 1.9 followed by chromatography in w-butanol-acetic acid-water or ethanol-ammonia-water,
Tanned silks 151
| H C — NH2
c h 2 |
c h 2
|
|
H C— N H 2
c h 3 c h 2i COOH 4-------- 1 ------- c=o1
f iY ™ * 2 A / N H 2
k ^ S ) H ^ ^ ^ O H
alanine and
3-hydroxyanthranilic 3-hydroxyanthranilic 3-hydroxykynurenin acid glucoside acid
F igure 9. Metabolic pathw ay from try p to p h an to 3-hydroxyanthranilic acid previously recorded in the literature , indicated by solid arrows. Evidence supporting its occurrence in certain silk-moths is given in th e tex t. The form ula of the glucoside found in the m oths is shown.
considerable radioactivity was detected in areas corresponding with authentic alanine (figure 8 d).Control hydrolysates of labelled tryptophan did not show any such activity.
The derivation of alanine from tryptophan is considered a very strong indication tha t the pathway from tryptophan to 3-hydroxyanthranilic acid is orthodox, resembling tha t encountered in mammals. More detailed work of a biochemical nature supporting this contention will be published elsewhere.
(ii) Gentisic acid The case of gentisic acid is different: while 3-hydroxyanthranilic acid is an
important intermediary metabolite, lying on the pathway to nicotinic acid, in, for example, mammals (though not in insects), gentisic acid serves no hitherto known function in metazoan animals; and when present in mammals it appears to be a detoxication product.
Four pathways to gentisic acid have been reported: (1) from tyrosine, (2) from salicylic acid, (3) from benzoic acid, and (4) from glucose.
The pathway from tyrosine to homogentisic acid (figure 10) is recognized in mammals and insects. Conversion of homogentisic to gentisic acid has been reported in rabbit and ra t liver (Ichihara, Ikedo & Sakamoto 1956; Watanabe 1964).
CH2—CH—COOH CH2—CO—COOH CH2—COOH
OH OH tyrosine p-hydroxyphenyl-
gentisic acid glucoside
F igure 10. Possible m etabolic routes to gentisic acid, the existence of which was shown to be unlikely by experim ents described in th e tex t. Gentisic acid appears to be supplied by th e diet.
The conversion of salicylates to gentisic acid has been reported by Kapp & Coburn (1942) and by Dumazert & El Ouachi (1954).
Benzoic acid (carboxyl-14C) is known to undergo hydroxylation to gentisic acid in plants (Kindi & Billek 1964).
Lastly Ehrensvard (1955) showed that Penicillium urticae raised on glucose medium will produce gentisic acid. However it is not usual for synthesis of an aromatic ring to take place in a multicellular animal, although Henry (1962) and Brunet (1963) have indeed shown that such synthesis takes place in some
insects and is attributable to intracellular symbionts. This route cannot altogether be ruled out.
Since tryptophan is the precursor of one tanning agent, the possibility tha t it also serves as a precursor of gentisic acid was considered. There is no precedent for this but a reasonable case could be pleaded.
In our attem pt to discover the pathway in silkworms, groups of larvae were injected with 5 [xCi of labelled presumptive metabolities on the eighth or ninth day of the final larval instar. Some larvae were also injected on the tenth day of the same instar.
As in the study of 3-hydroxyanthranilic acid metabolism, some larvae were allowed to feed for 10-12 h following injection. Their silk glands were then re moved and the contents were extracted in aqueous methanol. Other larvae were allowed to reach maturity and caused to spin their cocoons in a dry atmosphere (over calcium chloride). The silk was likewise extracted. After electrophoresis of the extracts, strips of the electropherograms were assayed for radioactivity.
The gentisic acid glucoside spot showed no radioactivity following injection of L-phenylalanine-14C(U), L-tyrosine-14C(U), DL-tryptophan [benzene ], sali cylic acid (carboxyl-1*C), or benzoic acid [C]. A clear peak, corresponding to gentisic acid glucoside was only obtained after injection of glucose-14C(U). Enzymic hydrolysis, however, showed that all the radioactivity resided in the glucose moiety.
This successful incorporation of radioactivity into gentisic acid glucoside in the case of animals injected with glucose, albeit only into the glucose moiety, was taken to indicate th a t the other negative results were meaningful and tha t the insects do not synthesize gentisic acid from any of the injected substances.
Attention was then paid to the diet of the insects. Antheraea had been mainly reared on hawthorn (Crataegus), as was H gloveri in which gentisic acid glucoside is also found. A few Antheraea had been raised on oak (Quercus). Samia had been raised on privet (Ligustrum).
Until 1958, gentisic acid was thought to occur only very rarely in higher plants (Karrer 1958), but Griffiths (1958, 1959) then showed that it was of widespread occurrence, not as the free acid but as a conjugate. This was detected in a very wide range of tropical plants, but none of these happened to be related closely to the food plants used in our work. A report on the substances contained in Crataegus leaves (Horhammer & Wagner 1963) does not include free gentisic acid or a conjugate.
In order to determine whether Griffith’s conjugate was present in our food plants 2 g (fresh mass) samples of hawthorn, oak and privet were shredded and digested with 10 ml 2 m hydrochloric acid. After cooling, the extracts were filtered and the filtrates were extracted with 2 ml diethyl ether. The ethereal extracts (0.2 ml) together with authentic gentisic acid were run chromatographically in benzene- acetic acid-water. Gentisic acid and a corresponding spot in all the extracts fluoresced pale blue and the fluorescence changed to yellowish on fuming with
Tanned silks 153
ammonia (a fairly specific test for gentisic acid). After re-chromatography in ^-butanol-acetic acid-water or electrophoresis the unknown again corresponded with gentisic acid.
Further samples of 2 g of air-dried oak and of hawthorn leaves were covered with absolute methanol and refluxed for 10 min. After cooling and filtration, the filtrates were extracted with light petroleum spirit (boiling range 40-60 °C) to remove chlorophyll. After concentration, the extracts were separated chromatographically in ^-butanol-acetic acid-water. There was no fluorescence at R t 0.85 (gentisic acid), nor a t R t 0.40 (gentisic acid-5-0 -(3-D-glucoside), but there was a slow-moving fluorescent spot apparently corresponding with Griffith’s (1959) methanol-soluble gentisic acid conjugate.
When subjected to ‘diagonal chromatography’ (as on page 139), the conjugate was found to have been cleaved by crude (3-glucosidase (emulsin) to gentisic acid and to a substance tha t reduced silver nitrate and had an value tha t corresponded with th a t of glucose. Emulsin is known to have other glycolytic activities, so th a t the analysis used was not precise enough uniquely to identify glucose. The conjugate is evidently a glucoside but its exact nature remains to be determined.
The investigation did, however, establish the presence of gentisic acid (in the form of a conjugate) in the food plants, and the measurements th a t follow show th a t there is a sufficiency to account for all the gentisic acid accumulated in the silk glands of the moths.
Estimates of the gentisic acid content made by visual comparison of the fluorescence of known amounts of leaf hydrolysates with known amounts of gentisic acid spotted on filter paper show that oak and hawthorn leaves contain about 100 [±g gentisic acid per gram fresh leaf mass. Privet yielded far less, having about 5-10 (xg g_1. Antheraea larvae eat on average about 4-5 g of fresh hawthorn leaves each day from the seventh to tenth day of the fifth instar; this is equivalent to 400-500 ;xg gentisic acid per day, and is sufficient to account for the amounts of gentisic acid glucoside in their silk glands (table 3), and the amounts of gentisic acid in privet are in good agreement with the amount of glucoside stored in the glands of 1Sarnia.
I t would seem then th a t the larvae ingest gentisic acid in the form of the plant conjugate. I t is presumably broken down in the gut and the free acid absorbed. Within the body, glucose is attached a t the 5-position of the ring, as was shown by the experiments with labelled glucose (p. 153).
I t was noted on page 149 that, while accumulation of 3-hydroxyanthranilic acid glucoside takes place in step with silk protein, the accumulation of gentisic acid glucoside does not: there is a slow intake into the silk glands before protein synthesis begins. This could be taken to indicate tha t in the case of 3-hydroxy anthranilic acid glucoside one method of control serves the two processes, by switching metabolism towards both silk protein and glucoside synthesis as in the case of the cockroach colleterial glands where a secretion of the corpora allata
Tanned silks 155
switches on both glucoside (Willis & Brunet 1966) and protein synthesis (Boden- stein & Sprague 1959). The precocious accumulation of gentisic acid glucoside, however, could indicate a separate controlling factor.
(g) Glucosides and $-glucosidase Insects are known to convert introduced phenols into the corresponding phenyl-
13-glucosides, this being one of their chief methods of detoxicating these reactive and potentially harmful substances (Smith 1955, 1962).
Most phytophagous insects, for example the mulberry silk worm ( ), (Ito & Tanaka 1959) and the cockroach, (Newcomer 1954), secrete a (3-glucosidase in their alimentary canal the function of which lies in the digestion of plant food. (3-Glucosidases also occur elsewhere in many insect tissues, for reasons not always convincingly explained.
Several instances are, however, known where (3-glucosidases have a definite and specific function. Thus, the defensive -quinones released from the tracheal glands of the cockroach, Diploptera punctata, are thought to be derived from phenyl glucosides; both a (3-glucosidase and a phenoloxidase have been demonstrated in these glands (Roth & Eisner 1962). Protocatechuic acid, which is involved in the tanning of the egg-capsule of Periplaneta is found as the 4-0 -(3-D-glucoside accompanying the protein to be tanned in the left colleterial gland, while a (3- glucosidase is separately present in the right gland (Brunet & Kent 1955). Simi larly 3,4-dihydroxybenzyl alcohol is also present as a glucoside in the cockroach, Blaberus (Pau & Acheson 1968). An O-glucoside of N -acetyldopamine is impli cated in cuticle tanning in the adult blowfly, (Sekeris 1964). More recently Brunet (1967) reported 3-hydroxyanthranilic acid glucoside in the silk gland of Hyalophora cecropia, and there was evidence tha t it was accompanied by a glucosidase.
Customary methods for the determination of glucosidase activity involve the liberation of a phenol (usually a nitrophenol) from a synthetic aryl-glucoside. Treatment of the incubate with alkali gives rise to the brightly coloured ionized species which is measured spectrophotometrically. In the case of Hyalophora silk gland, the large amount of indigenous 3-hydroxyanthranilic acid glucoside pre sented problems because on treatm ent with alkali the natural aglycone is rapidly oxidized to brown material tha t interferes with spectrophotometry of a nitrophenol.
To overcome this a simple qualitative test for glucosidase proved useful. Pieces of cocoons each weighing 60 mg were washed in several changes of distilled water to remove glucosides, dried, and transferred to small tubes containing 150 [jig of jp-nitrophenyl-[3-D-glucoside dissolved in 0.3 ml 0.1 m sodium phosphate buffer, pH 6.1. Following incubation at room temperature for 2 h, aliquots were removed from each tube, spotted on thin layer chromatographic foils. As controls, buffer alone, silk in buffer, glucoside in buffer, and 9-nitrophenol in buffer, all having been incubated for 2 h, were spotted on the foils which were then developed in chloroform-toluene-acetic acid.
IO-2
Both 39-nitroph.enol and its glucoside absorb strongly in 254 nm light and can easily be located against the fluorescent background of the foil. The i?f values found were: for ^-nitrophenyl glucoside 0.06, for p-nitrophenol 0.56.
The silks of Bombyx mori, Samia cynthia, gloveri, and Actias selene all showed glucosidase activity, and the glucoside spot was completely or all but completely absent after incubation.
Hyalophora gloveri b 0 - " O b v 0 3 Antheraea pernyi b £ > -
- bv
Actias selene O dkbv O b v O h Hyalophora cecropia b O C- ~ O b v O b v ~
Sam ia cynthia bQ c - - O b v 0 0 c o u c c bv pb b
3 -hydroxyanthranilic acid O b
+ 1 4 4 glucoside B s ta r t glucoside A
F igure 11. E lectropherogram (acetate/form ate, pH 1.9) showing the fluorescent substances (including glucosides A and B) present in aqueous m ethanolic ex tracts of silk glands of a varie ty of silk m oths.
F luorescent ‘sp o ts’ are indicated: ( ), trace fluorescence; b, blue; bv, blue-violet, dk, dark and p, pale.
The same species were examined for phenolic glucosides and aglycones. Silk glands, white silk and brown silk (but not the latter in the case of Bombyx) were extracted in aqueous methanol and the extracts were analysed by electrophoresis. Neither of the two glucosides was found in Bombyx. Gentisic acid glucoside is present alone in Antheraea, it accompanies 3-hydroxyanthranilie acid glucoside in Samia and Hyalophora. The latter glucoside seems to be present on its own in Actias (see figure 11). The significance of these specific differences is considered later (page 166).
(h) The oxidase-system of Antheraea pernyi and Samia cynthia As in the case of Hyalophora cecropia (Dewitz 1921), it was found th a t Antheraea
and Samia cocoons could be kept pure white by causing mature larvae to spin their coccons in the absence of moisture, over calcium chloride or silica gel. The fact th a t brown silks are, to begin with a t the time of spinning, white had already attracted the attention of several observers. I t was recorded for Antheraea by Levrat in 1899. Barwick (1912) considered tha t access to light was an essential for the darkening of Samia cynthia var. ricini silk to take place; Przibram (1922) disputed this, a t least for Hyalophora cecropia, and showed that humidity was the important factor. Brunet (1967) verified this and provided an explanation of the situation.
Tanned silks 157
Under normal circumstances the contents of the silk glands are spun into a coccon which is, from time to time, during late spinning and shortly after, drenched with a watery secretion tha t is according to Dewitz (1921) derived from the anus. The larva actively tramples the liquid into the cocoon which becomes sodden; and from this time onwards darkening proceeds. Although silk is spun as a fluid, the newly spun thread quickly dries out, and secondary wetting is needed to bring substrates into contact with enzymes, and products (the tanning agents) into contact with the silk protein. A cocoon will attain its normal brown colour in complete darkness.
Oxygen is also needed for browning to take place. White silk remains white indefinitely in oxygen-free distilled water. Parts of cocoons of Sarnia when dusted with sodium metabisulphite stay white even after the larva has moistened the whole cocoon.
Direct evidence for the existence of a phenoloxidase was obtained in the follow ing way. Two halves of the same white Antheraea cocoon, one of which had been kept a t 62-70 °C for 24 h, were moistened with distilled water and stored in a moist box for the subsequent 24 h. I t was found th a t only the unheated half had turned brown while the heat-treated part had remained white. Gentisic acid could be extracted with methanol from the heated part, which gave an indication tha t the glucosidase must have survived heat treatm ent but tha t a heat-labile oxidase had been inactivated.
Both halves of a cocoon of Samia, on the other hand, when treated as above, went dark in the moist chamber. We attribute this to the relative ease with which 3-hydroxyanthranilic acid is aerially oxidized, compared with the relative stability of gentisic acid. An aqueous solution of 3-hydroxyanthranilic acid rapidly darkens in air without the need for an oxidase; a solution of gentisic acid does not change colour for many hours.
Table 4. The oxidase of S a m i a silk
tube A tube B tube C tube D silk (oxidase) 36 m g silk 36 mg silk no silk 36 mg silk trea tm en t heat-treated u n trea ted — untrea ted 3 -hydroxyanthranilic
acid present or absent present present present absent
resulting colour brown tinge dark reddish-brown brown tinge colourless
A further experiment was carried out to clarify the situation. I t showed that while heat-treated and normal silk both bring about darkening of an aerated solution of 3-hydroxyanthranilic acid, the normal silk brings about darkening far more quickly (table 4). Absorption at 450 nm at pH 6.8 is characteristic of the oxidation products of 3-hydroxyanthranilic acid (Butenandt, Keck & Neubert 1957) and was used as a criterion of darkening; absorption spectra of the mixtures are shown in figure 12.
wavelength/nm
Figure 12. A bsorption spectra of reaction products derived from 3-hydroxyanthranilic acid; ---------------- , in the presence of inactivated s ilk ;----------------- , in the presence of un trea ted s i lk ; -------------------, in the absence of s i l k ; ---------------, spectrum of silk in buffer. (See also tab le 4.)
The specificity of the silk phenoloxidase is evidently not restricted. Antheraea silk, incubated as above, was found to oxidize the (unnatural) substrate, 3- hydroxyanthranilic acid at approximately the same rate as did the silk of Sarnia.
The enzyme showed the odd feature, characteristic of a wide variety of phenoloxidases (from potato tuber, pear fruit, cockroach colleterial gland), tha t of being inhibited by sodium chloride a t physiological concentrations. Most phenoloxidases are metalloenzymes: X-ray fluorescence measurements of both white and brown silks of Antheraea, Samia and Ilyalophora gloveri showed the presence of copper and iron.
These rather perfunctory investigations of the enzymes found in the secretion of the silk glands seem to suggest tha t a (3-glucosidase is customarily present. A phenoloxidase is secreted in the case of species the silks of which are tanned. These two enzymes act one after the other: the (3-glucosidase liberates the phenolic aglycone from its glucoside, and the oxidase then acts on the aglycone. The oxidation products, in some way not yet fully explained and perhaps involving polymerization, react with the silk protein which assumes a brown colour. Some consequences of this last reaction are considered in sections tha t follow.
(i) Gross-linking in silks The events attending the browning of silk resemble those tha t take place during
sclerotization of a cockroach egg-capsule (Pryor 1940 a). Here, changes in colour and hardness are accompanied by increased resistance to chemical and enzymic attack: the unhardened, white parts of an egg-capsule dissolve readily in cold, concentrated hydrochloric acid, in solutions of lithium salts, and during prolonged
Tanned silks 159
incubation in pepsin, while the brown parts withstand these treatments. Pryor attributed this stability to the existence of covalent cross-linking between protein chains. Precisely what happens is not known but the general consensus of opinion is th a t quinones or free radicals resulting from the action of an oxidase on a dihydroxybenzene serve to stabilize the proteins by acting as bi- or tri-functional links and perhaps, in addition, by becoming polymerized in the matrix. The model experiments of Hackman & Todd (1953) confirmed the reactivity of enzyme generated quinones towards amino groups (figure 13 a) and gave plausibility to Pryor’s supposition. Quinones are polyfunctional and can link more than one amino compound. There is also the possibility (figure 136) tha t tyrosyl groups in the proteins might link to aromatic tanning agents by way of biphenyl links such as have been described in resilin (Andersen 1954). The side-chain cross-linking proposed by Andersen (1971) could not apply in the case of these substituted benzoic acids.
COOH
OH
F igure 13. Possible modes of cross-linking, (a) Gentisic acid serving as a bifunctional link between amino acids of two protein chains. (6) Gentisic acid involved in a biphenyl link w ith a tyrosyl group. F u rth e r substitu tion , as in (a) or (6), could take place.
Despite the considerable amount of work tha t has been carried out on the pro cess of sclerotization, no definite figures exist relating the amount of tanning agent to protein. Tanned protein has a nitrogen value less than 15% and Hackman (1953) took the observed figure of 11.9 % to indicate tha t the protein was ‘diluted’ by 20.4% additional nitrogen-free material. Ideas have changed since then and many people would accept the view of Karlson & Sekeris (1962) th a t A-acetyldopamine (which is not nitrogen-free) is the universal cuticular tanning agent. A combined nitrogen value of 11.9% would in this case represent a mixture of 60 % protein and 40% tanning agent.
The simple tanning system in silk seemed to offer an opportunity for getting some measure of the extent of cross-linking in a sclerotin, and some preliminary investigations were carried out. Only stored material was available a t the time, and an extremely simple method of assay was used.
In the case of silk glands, samples of the aqueous methanol in which the glands had been stored were taken and, after suitable dilution with 0.5 m phosphate
buffer, pH 7.0, their relative fluorescence was measured at the peak value between 410 and 450 nm with excitation a t 320 nm. This would give a fair estimate of the glucosides of 3-hydroxyanthranilic acid and gentisic acid, which thin layer chro matography showed to be the only fluorescent substances present in significant amounts. The dry mass of the glands was measured, but no allowance was made for the fact th a t this weight included the gland as well as its contents.
In the case of silks, about 10 mg of white silk were extracted into 2 ml 0.5 m phosphate buffer, pH 7.0 a t 100 °C for 10 min. After appropriate dilution with buffer the relative fluorescence was read as above.
Our results suggested tha t no great reliability could be put on the figures ob tained, for example, silk, as compared with silk glands, from the same batch of larvae consistently gave far lower readings in the case of old material; evidently oxidation takes place slowly a t ambient humidity; and differences were also noted between batches, perhaps due to their having been raised on different food plants.
Despite these reservations, the fluorescence of the silks of Antheraea Samia cynthia and Actias selene on the one hand and Hyalophora cecropia on the other was so markedly different as to overcome any scepticism. The former group had a fluorescence equivalent to less than 10 micrograms of tanning glucoside per milligram silk while th a t of Hyalophora c exceeded 100 [xg. I f allowance is made for the molecular mass of the glucosides being about three times tha t of a typical amino acid, there is evidently about one molecule of tanning agent for every forty amino acids. Supposing th a t only the sericin component is involved (see p. 162) in the cross-linking, one molecule of tanning agent would be available for every fourteen or so amino acids. Such a high ratio of tanning agent to amino acid must exceed the number of particular amino acids tha t can participate in cross-linking and it offers a hint tha t the tanning agents are themselves involved in homopolymer formation with, presumably, peripheral linkages to protein as suggested by Pryor (1962). In the case of Actias , one molecule of tanning agent is present for every thousand amino acids in silk, or about three hundred if only sericin is cross-linked.
The established existence of tanning glucosides, glucosidase and phenoloxidase with silk proteins in those species the silks of which are naturally brown provides only circumstantial evidence and does not necessarily demonstrate tha t this is the cause of the browning of the silk tha t takes place nor that the properties of the silk are altered as a result of this association. In order to decide whether the oxid ized phenols represent no more than a pigmented product independent of the protein, or whether the oxidation products interact with the protein and serve to stabilize it, comparisons were made between white and brown silks with regard to (a) their chemical stability, ( b) their resistance to enzymic attack, and (c) their behaviour when subjected to mechanical strain.
Tanned silks 161
(j) Chemical changes on browning Concentrated aqueous solutions of lithium salts cause certain proteins, the
classical case being mulberry silk fibroin, to swell and eventually to dissolve completely. Since pieces of whole cocoon of Bombyx are likewise dispersed, both fibroin and sericin must be soluble. Lithium salts are said to produce this effect by disrupting the hydrogen bonds tha t are important in binding protein chain to protein chain. As a corollary, silks held together by more substantial cross-links might remain intact when treated with lithium salts.
Silks of various kinds (about 50 mg of each) were therefore put into tubes con taining 10 ml cold saturated aqueous lithium thiocyanate. The tubes were rotated a t 8 rev/min at room temperature for up to 3 days. Some silks dissolved; others resisted the treatment. In the cases where there were residues these were repeatedly washed in distilled water, dried in air and weighed.
Bombyx silk fibroin (table 5, tube 1) had completely dissolved within 20 min of treatment, as had white cocoon silk (fibroin together with sericin) of Antheraea and Sarnia tha t had been artificially prevented from darkening (table 5, tubes 2 and 7).
Table 5. Stability of silk towards lithium thiocyanate
mass before mass after trea tm en t trea tm en t
tube type mg mg
1 Bombyx mori, silk fibroin 50 0 2 Antheraea pernyi, white silk 50 0 3 norm al brow n silk 65 31.0 4 brow n silk (from white) 60 30.3 5 ‘degummed s ilk ’ 50 0.9 6 Actias selene, brown silk 49 10.0 7 Sam ia cynthia, w hite silk 49 0 8 norm al brown silk 60 34.0 9 ‘degum m ed’ silk 50 1.8
10 Hyalophora cecropia, brown silk 60 35.2
In contrast to this, about one-half by mass of normal brown cocoon silk (fibroin and sericin) of Antheraea, Sarnia and Hyaloph resisted the protracted treatment in the lithium solution (table 5, tubes 3, 8 and 10). A sample of white Antheraea silk (as in tube 2) th a t had been caused to darken by prior moistening showed resistance to dissolution (table 5, tube 4) comparable with normal, brown cocoon silk (table 5, tube 3).
This established tha t white silk, be it the normal condition of the silk {Bombyx), or as a result of artificial suppression of the normal darkening process {Antheraea, Sarnia and Hyalophora), undergoes disruption in lithium thiocyanate solution, while brown silks withstand the treatm ent to the extent of about 50 %.
In order to attribute the stability to the fibroin and/or the sericin component of
the silks, use was made of the alkaline ‘degumming’ which is considered by Lucas, Shaw & Smith (i960) to remove sericin while leaving the fibroin essentially intact. Cocoon silk was boiled first in a solution of soap (0.5 %) and sodium carbonate (1 %), followed by a solution of soap (0.5 %) for a total of 2 h. After this treatm ent brown cocoon silks of Antheraea and Samia (table 5, tubes 5 and 9) were rapidly dispersed in lithium solution, leaving only a small residue.
After degumming, the residual fibroins were not altogether white, and we conclude th a t the stability of brown silk resides chiefly in the sericin but tha t a small amount of cross-linking may take place as a result of some of the cross- linking agent diffusing into the non-crystalline parts of the fibroin.
When white and brown cocoon silks of Antheraea and Samia were immersed in cold concentrated hydrochloric acid, white Antheraea silk dissolved instantly, and tha t of Samia within 5 min. The respective brown silks, however, needed a t least 24 h: but neither showed anything like the resistance of cockroach egg- capsule.
Formamide is known to disrupt electrovalent linkages (Brown 1950). When the bright yellow cocoons of the yellow European strain of Bombyx which has caro tenoid pigment in the sericin layer, are treated with formamide, the yellow pig ment goes into solution, leaving the white fibroin. The colour of brown Antheraea and Samia cocoons is not removed by this treatment, indicating tha t stronger bonds are involved.
I t was concluded tha t with browning goes a resistance to chemical attack, indicative of strong primary valence links. Browning and resistance reside pri marily in the sericin layer of the silk.
(k) Extensometric tests Just as there are a t present no unambiguous chemical criteria for diagnosing a
sclerotin cross-link, so too there exist no reports of any distinctive, physical properties of sclerotins tha t could be used to support our claim that brown silks are a form of sclerotin. Nevertheless it seemed reasonable to assume that, if the differences between brown and white silks lay in tha t one was sclerotized and the other not, there would be differences in the physical properties of the silks. In addition, the availability of white silk (supposedly a pre-sclerotin) and brown silk (supposedly a sclerotin) in a convenient state, perhaps more readily accessible to physical measurement than, for example, insect cuticle, would offer a good opportunity of making a first diagnosis of the effects of sclerotization on the phy sical properties of a protein.
Extensometric properties of silks have been reported previously (Lucas, Shaw & Smith 1955), but these were all made on degummed cocoon silks, so tha t they refer only to fibroins; moreover the reported properties may to some extent reflect the effects of the alkali degumming procedure.
In the present work, measurements were made on single strands of untreated silk which had been detached from the loosely-spun outer parts of cocoons, and on
Tanned silks 163
percentage extension
F igure 14. Percentage extension in relation to tension of single strands of brown (solid lines) and w hite silks (broken lines).
( a ) Antheraea pernyi. ( b)Sam ia cynthia. The vertical lines indicate the breaking-points.
strands of white silk taken from cocoons spun over anhydrous calcium chloride. The results are shown in figure 14a for and in figure 146 for Samia cynthia, where the tension developed in the fibre is plotted against the percentage extension. (Tension rather than stress was plotted as no facilities were available for accurate measurements of the cross-sectional area.) Measurements were made at ordinary room temperature and humidity. The strand length varied from 30-40 mm and a constant rate of extension of 2 mm/min was applied.
The curve for the white Antheraea silk (figure 14 a) shows an initial steep elastic section, up to about 3 % extension, followed by a yield-point and a long region of flow. This may be interpreted in terms of unfolding of the amorphous regions of the molecular chains (owing to bulky side-chains) which are normally maintained in a folded form in the unstrained silk by weak bonding (Lucas, Shaw & Smith 1958). When sufficient stress is applied, these weak bonds are ruptured and the flow region is attained as the chains are progressively unfolded and slipping takes place; the stress required increases slightly with extension as the amorphous regions become oriented.
The behaviour of the silk which had been allowed to turn brown is strikingly different. An initial, very slightly steeper, elastic region is followed by a yield point a t a similar value of stress; but after a very short region of flow, the fibres become more resistant to extension presumably owing to their cross-linking.
I t is interesting to note tha t the curve given by Lucas et al. (1955) for Antheraea pernyi fibroin resembles the curve in figure 14a for whole brown silk, with a
difference tha t they observed a very much longer flow region. This would suggest th a t their samples were less strongly cross-linked, so tha t a greater degree of unfolding could occur before the covalent linkages began to resist further exten sion. As these fibres had been ‘ degummed ’ this may indicate tha t most of the cross-linking takes place in the sericin layer, a conclusion tha t is supported by the results described on page 162 with regard to resistance to lithium thiocyanate.
The behaviour of white Samia silk (figure 146) is similar to tha t of white aea silk: a short, elastic region, followed by a yield-point and a long region of flow; and this may be explained similarly in terms of the unfolding and slipping of amorphous regions of the protein molecule.
On tanning, however, the properties are completely altered: the stiffness of the fibre is greatly increased, giving a much steeper curve, scarcely any yield-point, and no trace of flow-behaviour. The tension in the brown silk at breaking-point is approximately five times tha t of white silk. These properties might be explained by assuming tha t a considerable amount of covalent cross-linking had occurred, so th a t much stronger bonds have to be ruptured before unfolding of the amor phous regions can take place.
(1) Cocoonase I t has been mentioned above (p. 134) th a t cocoons either present an oval form
with no preformed aperture, or one end is drawn out into a cone with the tip open (but guarded by loose strands of silk). Escape from the former type involves breaking down the wall of the cocoon with an enzyme secreted from the mouth region. Escape from the latter involves the newly emerged adult in thrusting its way out through the relatively unimpeded opening tha t already exists.
I t appeared at first tha t closed cocoons, susceptible to enzyme attack, would all prove to be lacking in primary valence cross-links and th a t the silks of open cocoons would be heavily cross-linked. These two extremes are exemplified by the mulberry silk worm, Bonibyx and by the robin moth, (Brunet 1967). However in the course of this work we have found th a t the situation is not as straightforward as this.
I f the tanning of silk involves the establishment of covalent cross-links, one might reasonably expect brown silk to show a considerable increase in stability, or even complete resistance, towards for instance the action of cocoonase, the proteinase obtained by Kafatos & Williams (1964) from emerging Antheraea. Indeed Pryor (1940 a) in his account of the properties of sclerotins speaks of their predictable resistance to enzymic attack. However our findings have indicated th a t Antheraea secretes gentisic acid glucoside, a potential cross-linking agent, in circumstances that would allow it to act as such; and chemical and physical tests (pp. 161 and 163) have indicated that the silk is cross-linked; yet even so it remains susceptible to cocoonase attack. In an attem pt to reconcile these apparently contradictory properties the experiments reported below were carried out.
Some cross-linked silks are indeed stable to cocoonase: Samia cynthia secretes a cocoon stabilized by the oxidation products of both 3-hydroxyanthranilic acid
Tanned silks 165
and gentisic acid (p. 139), yet curiously, this species still secretes a small amount of cocoonase. A number of cocoons were cut transversely and the pupae were re moved. Six pupae were replaced in the natural position, with their head ends a t the open end of the cocoon, and six were replaced in the inverted sense, with their heads abutting on the closed end. The split halves of the cocoons were then cemented together with Araldite AV100:HV100, 1:1.
All the normally oriented moths emerged and left the cocoons in the usual way. All the inverted pupae emerged from the pupal skins, but died inside the cocoons; and no signs of cocoonase attack were noticeable inside these cocoons. The same results were obtained when Bombyx (a species whose moths copiously secrete a cocoonase) were put in Sarnia cocoons.
When strips of Samia cocoon (10 mm x 4 mm) were moistened with a solution of cocoonase from Antheraea (obtained by placing pupae head downwards in glass funnels a t the time of emergence), or in 0.1 m potassium bicarbonate to serve as a control, the silk appeared unaffected by the treatm ent and showed no signs of attack after incubation for 20 min at 20 °C. From this, and from the inversion experiments, it is evident th a t some cross-linked silks resist the attack of cocoonase; but this is not always the case.
White and brown Samia silk, white and brown Antheraea silk and (white) Bombyx silk were all moistened with Antheraea cocoonase. Brown Samia silk survived the treatment, but all the other silks showed signs of attack, retaining little or no mechanical strength, and breaking readily when stretched. This is understandable in the case of the white silks, but less easy to explain in the case of brown Antheraea silk.
There is no reason why susceptibility to cocoonase necessarily conflicts with our hypothesis of cross-linking. The protein, resilin, which is undoubtedly a cross- linked protein (Andersen 1964; Andersen & Weis-Fogh 1964) is digested readily by a wide range of proteinases. I t will nevertheless be interesting to find out exactly what it is th a t endows one type of cross-linked silk with the ability to withstand cocoonase yet leaves another type susceptible.
I t seemed a t first tha t the resistant silks were those tha t incorporated 3- hydroxyanthranilic acid, and the less stable silks gentisic acid, but we found an exception to this in the case of Actias selene, which has a closed cocoon and apparently incorporates 3-hydroxyanthranilic acid into its silk (see table 6). Indeed there does seem to be a consistent relationship between susceptibility to cocoonase and the tanning acid concerned, 3-hydroxyanthranilic acid always being present in species that have a resistant cocoon. In the case of Actias which escapes by means of cocoonase from a cocoon tanned with 3-hydroxyanthranilic acid it must be the low content of tanning agent (p. 160) tha t permits this.
I t has been claimed tha t 3,4-dihydroxyphenylalanine is present in some cocoons (Przibram & Schmalfuss 1927). This substance could possibly act as a stabilizer; but when we made a search for it in Hyalojphora cecro'pia silk we came to the con clusion tha t it was not present.
T a b l e 6. D i s t r ib u t io n o f 3 -h y d r o x y a n t h r a n il ic a c id g l u c o s id e (3 H A G ) AND GENTISIC ACID GLUCOSIDE (GAG) IN RELATION TO COCOON COLOUR AND TYPE
silk gland or A
species cocoon colour cocoon type 3HAG GAG
Bombyx mori yellow closed - - Sam ia oynthia brown open + + + + Hyalophora gloveri grey-brown open + + + + + + Antheraea pernyi yellowish-brown closed - + + + Actias selene pale to dark brown closed + -
To sum up: all (naturally and artificially) white silks are susceptible to cocoonase attack, and so are some brown silks ( and Actias); the majority of brown silks, however, are very resistant ( and Sarnia). While inducing some definite changes in physical and chemical properties of a silk, exogenous aromatic cross-linking agents do not necessarily confer resistance to attack by cocoonase.
4. D i s c u s s i o n
This work emphasizes the versatility of the lepidopteran salivary gland, a gland th a t serves first to secrete digestive enzymes, switching, after feeding has ceased, to the production of silk and the ancillary substances described in this paper, and ultimately being reorganized to secrete the proteolytic enzyme, cocoonase, and the necessary adjuvants.
The, presumably primitive, digestive function of the gland may indeed explain why, a t a later stage when serving as a silk gland, it secretes a glucosidase tha t is found both in species where its presence can be explained, namely in those th a t produce tanned silk, and also in Bombyx where there is no obvious reason for its presence.
The other components of tanned silks are also interesting. 3-Hydroxyanthranilic acid proves to be the first tryptophan derivative unquestionably shown to be connected with tanning. Pryor (1955), finding 3-hydroxykynurenin in Calliphora and Lucilia, did suggest tha t it might be involved in tanning, but later (1962) showed less enthusiasm for the idea when no supporting evidence came to hand. One can suppose tha t the heavy commitments for tyrosine derivatives required to harden first the pupal case then the adult cuticle preclude their use in the tanning of silk. I f this is to take place an alternative source is needed. I t is probably not fortuitous th a t tryptophan metabolites are exploited since there is a tendency for these to build up with time for no obvious reason (see Brunet 1965) and, for example in the vanessids (Butenandt 1957), to be excreted in relatively huge quantities. I t is reasonable to speculate tha t the saturniids have exploited this source of aromatic compounds: instead of converting excess 3-hydroxykynurenin
Tanned silks 167
to phenoxazones which are excreted, saturniids appear to have switched 3-hydroxykynurenin metabolism to 3-hydroxyanthranilic acid, and to have made use of this in tanning. 3-Hydroxyanthranilic acid has been previously reported (Kikkawa 1953) from Bombyx eggs, but with no suggestion as to its function.
Neither gentisic acid nor its glucoside has been reported before as a normal metabolite of animals. In the absence of an alternative explanation it is considered tha t the gentisic acid in saturniids must originate from plant sources and be re used (after conversion to its glucoside). The re-use of plant materials by insects is not unusually encountered in other insects (Rothschild 1972).
The deactivation of aminophenol and dihydroxybenzene tanning agents in the form of (3-glucosides for the purpose of storage finds precedent in the aromatic glucosides reported from the cockroach colleterial gland (Brunet & Kent 1955; Pau & Acheson 1968).
I t seems justifiable to have put tanned silks into Pryor’s (1940 a) category of sclerotins. I f this is acceptable then the account of the properties of tanned as opposed to untanned silk is the first description of the differences between a sclerotin and its precursor, presclerotin. Differences in chemical properties between these two are much as would be expected; but the susceptibility of some tanned silks to attack by cocoonase is rather surprising.
Extension of this work could lead to the answering of a number of interesting biological questions. For example, the evolution of the habit of producing tanned silk as found in saturniids. There is also the question whether the secretion of white silk by Bombyx is primitive or arises as a result of breeding out the propensity to secrete tanning agents (as has occurred in the cultivated strains of Sarnia tha t produce white ‘eri’ silk). And, to revert to the question posed a t the beginning of this paper, it would be invaluable to have field observations tha t would shed light on the reasons why these moths have been driven to secrete such elaborate cocoons and tan them.
We owe thanks to Professor J. W. S. Pringle, F.R.S., and to Professor T. Weis-Fogh (in whose Department, in the University of Copenhagen, the work was started).
We gratefully acknowledge assistance from Fru Karen Krogh and Mrs Ailsa Massey.
Dr Hegediis, Dr Signe Nedegaard, Dr G. P. Waldbauer, Professor C. M. Williams and Dr Judith Willis kindly gave us chemicals or insects. Equipme

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