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Ommochrome pigments of spiders
Comp. Biochem. Physiol., 1972, Vol. 42A, pp. 699 to 709. Pergamon Press. Printed in Great Britain
OMMOCHROME
PIGMENTS
OF SPIDERS*
V. L. SELIGY? Biochemistry
Laboratory, National Research Council of Canada, Drive, Ottawa, Ontario, Canada, KlA OR6
100 Sussex
(Received 25 October 1971) Abstract-l. Pigments with chemical properties characteristic to both ommatins and ommins were found to be present in the hypodermis and in some cases the intestinal diverticula of spiders from all families of the sub-order Labidognatha examined. 2. Based upon the natural physiological colours and the chemical redoxcolour changes the hypodermal pigments could be grouped into two major and one minor class. 3. Conventional ommochrome chemical techniques were used to show that (a) Class I pigments (orange to brown) consisted mostly, if not entirely, of an ommatin with properties nearly identical to xanthommatin. (b) Class II pigments (dark brown to black) contained both xanthommatin and an ommin, with the latter being the more predominant. (c) Class III pigments (yellow), which occur with the lowest frequency in nature, were variable in composition, consisting mostly of kynurenine and 3-hydroxykynurenine which are either precursors or degradation products of ommochromes.
INTRODUCTION is a wide range of coloration in spiders which may be distributed over the entire animal or localized in the prosoma, opisthosoma or legs as a series of bands or spots (Millot, 1926; Kaston, 1948; Levi et al., 1968). The colours are: white, yellow, green, various shades of red and brown, and black. While most colours are a result of pigments located within the epidermis (Millot, 1926), metallic or iridescent colours are compound: they are the resultant effect of light interference created by the tanned cuticle and the underlying epidermis. In some spiders, opisthosomal coloration and patterns are further complicated by the presence of a white layer made up of guanine storage cells located directly beneath the hypodermis and covering the intestinal diverticula (Millot, 1926). Although some studies have been made on changes in colour and colour pattern during development of spiderlings (McCook, 1888; Gabritschevsky, 1927; Moles, 1916; Millet, 1926; Rabaund, 1923) little is known about the biochemistry of the pigments contributing to the various patterns. This is in contrast to insect studies, where several classes of pigments occur (Cromartie, 1959; Fox & Vevers, THERE
* N.R.C.C. No. 12517. 7 Visiting Research Officer 1971. 699
700
V. L. SELIGY
1960; Ziegler & Harmsen, 1964). One of these pigment classes, the ommochromes, which have been extensively characterized (Becker, 1941; Butenandt, 1959; Linzen, 1967; Needham, 1970a, b), also occurs in the eyes of some spiders (Linzen, 1967; Vuillaume, 1969) and has been identified as the principal epidermal pigment of the theridid spider E. ovata (Cl.) (Seligy, 1969). This paper is a comparative study of the general properties of spider ommochromes and of the extent to which these pigments occur in the various spider families. MATERIALS
AND METHODS
Spiders of twenty-nine genera comprising fourteen families of the sub-class Labidognatha were collected in Carleton, Lincoln and York counties of Ontario and Summerland, British Columbia, Canada. Choice of species for study was based solely upon novelty of the colour patterns or interspecies colour similarities. In most cases the spiders that were chosen have been previously illustrated in colour (Levi et al., 1968). Hypodermal colour patterns were dissected out from various body parts of each spider and then peeled from their respective cuticles. The general presence of ommochromes was tested in situ by the ability of the hypodermal pigment to form the purple/violet halochrome when concentrated sulphuric acid is added (Becker, 1941). Ommatin-like redox colour changes were also examined in situ as previously described (Seligy, 1969) by alternate additions of O*OS-O*lml of saturated sodium dithionite and saturated (w/v) sodium nitrite or 5% (v/v) nitric acid by ~1. pipette. One experiment constituted four complete reduction and oxidation cycles on duplicate samples. For fractionation and partial purification of ommochromes, whole opisthosomal hypodermis was used. Each pigment pattern was dissected out, washed in distilled water (pH 7.0) and centrifuged at 20,000 g for 5 min. The resultant pellets were further extracted with 10 vol. of acetone, dried under vacuum at ambient temperature and stored at - 20°C until further use. To avoid the possible degradative effects of acid during extraction and analysis most operations, including short-term storage of extracts, were done at 4°C. Ommochromes were extracted from the acetone dried pigment pellets with either 5 N HCl, methanol-HCl(l4 : 1) or 90% formic acid. In most cases the procedure described by Needham (1970a) of total extraction with 90% formic acid, precipitation of ommatins and ommins by addition of diethylether and differential extraction of ommatins with water and ommins with 5 N HCl was found to be the most suitable for spider ommochromes. Absorption spectra of the extracts were obtained using a Cary-l4R spectrophotometer over the range of 700-200 nm. Whatman No. 1 paper (25 x 25 cm) was used for descending chromatography and formic acid-methanol-concentrated HCl-water (15 : 3 : 1 : 6) was used as the principal solvent system. nL-Rynurenine and L-3-hydroxykynurenine (A grade, Calbiochem) were used as chromatographic markers. RESULTS
AND DISCUSSION
In preliminary experiments it was established that pigment groups, such as pteridines (Stackhouse, 1966), carotenoids (Takeuchi, 1960) and melanins (Mason, 1959) were not present in detectable quantities in the hypodermal colour patterns of all spiders listed in Table 1. However, in addition to the hypodermal pigments being insoluble in neutral organic solvents all patterns were found to contain ommochromes as indicated by the formation of the violet-purple “halochrome” upon the addition of concentrated H&30, (Becker, 1941). Moreover, in almost all cases, chemical redox-colour changes were also observed. These results led to the
Agelenopti naevia (Walckenaer) Araneus diadematus (Clerck) Araneus triyolium (Hentz) Argiope aurantia (Lucas) Argiope t$zsc-iuta (Forskll) Chiracanthium mildei (L. Koch) Dictyna sp. Gnaphosa sp. Frontinella communis (Hentz) Pityophantes phrygiunw (C. L. Koch) Lycosa gulosa (Walckenaer) Dolomedes tenebrosus Metaphidippus gakzthea (Walckenaer) Phidippus clarus (Keyserling) SuZticus smicus (Clerck) Scytodes thoracica (Latrielle) Leuxsage wenusta (Walckenaer) Achaearanea tepiakriorum (C. L. Koch) Enoplognatha ovata (Clerck) Latrodktus variolus (Walckenaer) Steatoda borealis (Hentz) Theridion jkwuieum (Hentz) Theridion murarium (Emerton) Misumena walk (Clerck) Misumenops asperatus (Hentz) Philodromus rufhs (Walckenaer) Xisticus Zucttwsus (Blackwall) Tibellus obkmgus (Walckenaer)
Species
:(I) 4 (2) (2) 4 (4)
2 2
2 (1)
i(l) 2 (2) 2 3 3 (2) 15 (15) 6
(4) 17 (1) 12 (1) 10 (2) 3 3 1 2 2 2 1
1:
L, DO, VO, P L, DO, VO, P L, DO, VO, P DO, VO, P DO, VO, P L, DO L, DO, VO, P DO DO, VO DO L, DO, VO, P L, DO, VO, P DO, VO, P L, DO, VO, P DO, VO, P L, DO, VO, P DO, VO L, DO, VO DO, VO, P DO, VO, P L, DO, VO, P L, DO, VO L, DO, VO DO L, DO, P L, DO, VO, P L, DO, VO, P DO, P
Location? Medium to dark-brown ; black Light to dark-brown Light to medium-brown Yellow; black Yellow ; black Yellow; medium-brown Yellow-brown; dark-brown Dark brown Dark-brown to black Light to medium-brown Medium to dark-brown Medium to dark-brown Medium to dark-brown Red to dark-brown Medium to dark-brown Dark-brown to black Yellow; red to brown Medium to dark-brown Yellow; orange to red; black Black, red Dark brown to black Yellow; black Red; medium to dark-brown Yellow ; red Yellow; red-brown Red-brown to brown Red-brown to dark-brown Yellow brown; dark-brown
Physiological colour
Pigment
* Figures in parentheses are the number of males examined in addition to females. t Hypodermal pigments located in L, legs; DO, VO, dorsal and ventral opisthosoma; P, prosoma.
Thomisidae
Scytodidae Tetragnathae Theridiidae
Clubionidae Dictynidae Gnaphosidae Linyphiidae (Linyphiinae) Lycosidae Pisauridae Salticidae
Agelenidae Araneae
Family
Number * tested
TABLE ~-DISTRIBUTION OF OMMOCHROME PIGMENTSIN SPIDERSOF THE SUBORDER LABIDOGNATHA
I; II I; II I III; II III; II III; I I; II II II I I, II I, II I, II I, II I, II II III; I I, II III; I; II II, I II III; II I; I, II III; I III; I I I, II I; II
Classification
2 I
; g
!
8 q ij
B $ : 3
702
V. L. SELIGY
general observation that the natural epidermal colour variations of spiders could be grouped into two major and one minor class (Tables 1 and 2). The pigments of all three classes clearly differ in their natural or physiological colours and in the case of class I and II, they appear also to differ in their natural occurring redox-colour states as suggested by the redox-colour studies (Table 2). TABLE
2-SUM~IARY
OF REDOX COLOUR AND OF SPIDER HYPODERMAL
HALOCHROME-FORMING PIGMENTS
PROPERTIES
Pigment colour Classification I
Physiological
II
Orange, red, violet, red-brown, yellow-brown, light to medium-brown Dark-brown, black
III
Pale to bright yellow, green
Reduced *
Oxidized
Halochrome t
Red to red-brown
Yellow to Violet yellow-brown
Yellow to orangebrown
Dark-brown or black No change
Deep violet
Yellow to brown
* Dissected hypodermis was treated in situ with reducing and oxidizing reagents as described in Materials and Methods. t Dissected hypodermis was treated in situ with concentrated HsS04.
Since both redox and halochrome tests give only an indication that ommochromes are present, the general properties of spider pigments were further studied by conventional ommochrome extraction and fractionation procedures (see Materials and Methods). Although twenty-six species representing all three pigment classifications were studied in detail, the observations reported here are limited to only a few species. The species chosen to be most typical for class I pigment (red to brown) was A~uneus trifolium (Hentz) and for class II and III pigments (dark brown to black and bright yellow, respectively) Argiope uurantiu Lucas. A third species, the black widow spider, Latrodectus variolus Walckenaer, which contains a ventral red hour-glass pattern (class I) in addition to the overall black coloration (class II) was used for intraspecies pigment comparison. Extraction of opisthosomal epidermal pigments from A. trifolium (Hen&) and A. auruntiu Lucas with methanol-HCl (19 : 1) or HCl (5 N) resulted in extracts which were identical in colour, yellow to yellowish brown. However, differences were observed in the colours of the unextracted residual material as well as the precipitates recovered by centrifugation of the extracts after adjusting the pH to 5 and the sodium thiosulphate concentration to O*O5o/o. Class I precipitates were distinctly red, while class II precipitates were more variable in colour, red to dark brown. The visible absorption spectrum of either pigment extract in 1 N HCl resembled that of oxidized xanthommatin (Fig. 1). However, neither reagent was
OMMOCHROME
PIGMENTS
OF SPIDERS
703
effective in extracting all the colour from the epidermis of the two classes, particularly class II. Therefore it would appear that additions components such as ommins which are known to be only sparingly soluble in these reagents (Linzen, 1967) might be present. This assumption was further supported by the results
400
500 Wave
length,
600
nm
FIG. 1. Visible absorption spectra of opisthosomal hypodermal pigment extracts by 5 N HCl. ) and A. aurantiu Lucas (- -) from A. tnfolium (Hentz) (-
obtained using the formic acid extraction and ether fractionation procedure for ommatins and ommins as described by Needham (1970a) for isopod integumental ommochromes. This method proved to be most useful for not only achieving rapid and complete solubilization but greatly simplified comparative studies. Total colour extraction of A. trifoliunt (Hentz) hypodermal pigments (class I) was obtained at 4°C within 2 mm after brief homogenization in 90% formic acid, while pigments of Aruaw c&z&status Clerck, A. uurantia Lucas (class II) and L. oriole Walckenaer required longer exposures, S-30 min. The orange-brown coloured extract of A. trifoliunt was further fractionated into a bright yellow water soluble component and a faint reddish-violet acid soluble component. The spectral properties (Fig. 2) of the yellow extract closely resembled that of xanthommatin (Needham, 197Oa) while the reddish-violet pigment was similar to that described for ommin. Each formed the purple halochrome, (Fig. 3) upon redissolving in concentrated H,SO, after concentrating by flash evaporation at 15°C. The chromatographic properties of both extracts were similar to that of xanthommatin with only a faint trace of slow migrating component in the reddishviolet acid extract which corresponded to an ommin (Fig. 4). Consistent with the properties of ommatins and ommins was the observation that the yellow water soluble pigment was compfetely dialysable, whereas the reddish-violet pigment was only partially so. The ommin extract was also quite resistant to chemical oxidation. However, after 3 or more days’ exposure to 90% formic acid at room temperature (23°C) the colour changed from reddish violet to yellowish-brown. The two main products of this oxidation, when isolated after paper chromatography, had a similar
704
v. L.
I
400
.
I
500
.
I
600
Wave length, nm
.
SELIGY
I
400
.
.
.
500
a
.
600
Wavelength, nm
FIG. 2 FIG. 2. Visible absorption spectra of ommochromes from the hypodermis of A. trifolium (Hen@ initially extracted by 90% extracted by water from an ether-precipitate of the 90% from the residue and extracted by 5 N HCl (-*-) ( -) water extraction.
FIG. 3 dorsal opisthosomal formic acid (- - -), formic acid extract remaining after the
) and ommin (-a-) of FIG. 3. Visible absorption spectra of ommatin (A. trifolium in 100% sulphuric acid. Ommatin and ommin extracts were prepared from opisthosomal hypodermis as described in text and Fig. 2.
FIG. 4. Chromatogram of ommochrome extracts from class I hypodermal pigments. The extracted pigments were applied to Whatman No. 1 paper and developed by (v/v, descending chromatography using formic acid-methanol-HCl-water 15 : 4 : 5 : 1 : 5). A. trifolium, opisthosomal pigments: a, yellow water-soluble component extracted from ether-precipitate of 90% formic acid extract; b, reddishviolet 5 N HCl extract of water insoluble residue; c, kynurenine standard; d-i, 90% formic acid extracts of hypodermal pigments from d, A. hdematus; e, A. nuevia; f, A. saevw; g, L. variolus (ventral red hour glass); h, L. vem~sta;i, M. vatia. Abbreviations for U.V. spots: MB, medium blue; WR, whitish-blue; RB, reddish-brown; DV, dark-violet.
absorption spectrum as that of xanthommatin and reduced xanthommatin (dihydroxyxanthommatin). Since ommins, when exposed to formic acid for lengthy periods, can break down to yield smaller products including ommatin (Almedia,
OMMOCHROME
PIGMENTS
OF SPIDERS
70.5
1968) the xanthommatin and dihydroxyxanthommatin obtained from the slow oxidation of the reddish-violet ommin extract is probably a degradation product. When the same extraction procedure was used to study the class I pigments from other spiders, it was immediately apparent that variations in ommin but not ommatin content occurs (Fig. 4). In some cases, such as the orange-red hour glass pattern of L. variolus (Walckenaer) (Fig. 5), the red pattern of Enoplognatha ovata
I
a
1 400
.
1 500
9
1
1
600
Wave length, nm FIG. 5. Visible absorption spectra of ommochrome extracts from the opisthosomal
hypodermis of L. variolus Walckenaer. Formic acid (90%) (- -), water soluble component of the ether-precipitate of a 90% formic acid extract (), 5 N HCl extract of water insoluble residue (-.--). Inset figure: visible absorption spectra of the ventral red hour glass ommochromes. Extraction sequence and figure notations are the same as above.
Wave
length,
nrn
FIG. 6. Visible absorption spectra of ommochrome extracts from opisthosomal hypodermis of A. aurantia Lucas. Formic acid (90%) extract (- -), watersoluble component of the ether precipitate of a 90% formic acid extract (-), 5 N HCl extract of water insoluble residue (-*-). Inset figure : visible absorption spectra of 0.1 N HCl extract of yellow hypodermis pigment patch (-) and L-kynurenine (- -).
706
V. L.
SELIGY
(Clerck) and the red pattern of Misumena vatia (Clerck) (data not shown), only xanthommatin appears to be present. In others, the reddish-brown patterns of Agelenopsis naewia (Walckenaer), Leucusage venusta (Walckenaer), Araneus saevus (L. Koch) and Araneus dzizhnatus (Clerck), ommin is present in addition to ommatins. Upon closer examination of the pigment patterns in which ommins were also found it was noted that all the patterns were compound, consisting of additional fine sub-patterns involving class II pigments. A similar comparative study of the class II (black) pigments of L. variolus and A. aurantia indicated that these pigments differ significantly from class I in their marked increase in ommin content relative to xanthommatin (cf. Figs. 2, 5 and 6). The increase in ommin was also apparent in the initial formic acid extracts, since the orange-brown extracts of class I pigment of A. trifolium (Hentz) were much lighter in colour than the deep reddish-violet extracts of class II (Figs. 1 and 4a). Paper chromatography of the 5 N HCl extracts yield four characteristic spots using short wave U.V. light (2537 A) (Fig. 6). Whereas in class I pigment the characteristic slow migrating ommin spot was barely detectable by U.V. light, the ommin spots of class II were quite distinct. In addition to a slow migrating dark red ommin spot which is easily detected by visible light, a xanthommatin spot and a distinct bright whitish-blue fluorescent spot corresponding in R, to kynurenine, a fourth reddish-brown fluorescent spot with an R, between ommin and xanthommatin is present. The latter reddish-brown U.V. fluorescent spot is somewhat reminiscent to the violet U.V. fluorescence found in Ligia ommochrome extracts which Needham (1970a) has suggested may be due to a related metabolite of the indol-ommochrome pathway. In general, coloration of spiders is primarily due to pigments located within the hypodermis (Millot, 1926); however, differences in ground colour of the opisthosomal diverticular mass also occurs (McCook, 1888 ; Seligy, unpublished). In most cases, it was thought that the very dark colours of spiders were due only to the presence of class II pigments, but during the course of this study it was noted that both species A. aurantia Lucas and A. trifasciata (Forskil) also contained considerable amounts of xanthommatin in the gut diverticulae (Figs. 6 and 7). After dissection of this reddish-grey mass and exposure of it to the air for l-2 hr it slowly oxidized and became black in colour. In contrast, no such observation could be made for L. variolus Walckenaer or A. trifolium (Hentz). A further study showed that in addition to guanine, xanthommatin was also present in variable quantities in the gut of Agelenopis sp., L. venusta (Walckenaer) and a Lycosa sp. (class II pigmented hypodermis). It is conceivable that the presence of ommatins in the gut of some spiders and not in others could easily convey special differences in overall opisthosomal coloration. It should be noted that not all colour effects are the result of ommochromes or guanine cells. The pale to bright yellow patterns in the opisthosomal hypodermis of A. aurantia and A. trifasciata, for example, which are devoid of class I and II pigments, are a result of accumulated precursor or degradation products of the ommochrome pathway, since both kynurenine and 3-hydroxykynurenine have been identified as the major product in these patterns
OMMOCHROME PIGMENTS OF SPIDERS
707
(Figs. 6 and 7). Both of these products can also be detected in situ by the bright bluish-white fluorescence of the hypodermis during exposure to U.V. light at 2537 A. Since similar observations have been noted for one of the genetic colour variants of E. oaata (Clerck) (Seligy, 1969, and in preparation) it is likely that during the course of colour pattern evolution some of the hypodermal cells have lost the ability to complete the synthesis of ommochromes. 0.8 -
WE (J
(y
MB MB
WB o
WB WEWBWBWB Q;
qj””
MB MB DV
‘DV
z o’4 -
0
1
FIG. 7. Chromatogram of ommochrome extracts of class II hypodermal pigments. Chromatograms were developed descendingly on Whatman No. 1 paper as described in the text and Fig. 4. L. variolus pigments: a, yellow water soluble component extracted from the ether precipitate of a 90% formic acid extract; b, reddish-violet 5 N HCl extract of water insoluble residue; c, kynurenine standard. A. aurantia pigments: d, yellow water-soluble extract; e, reddish-violet 5 N HCl extract; f, 0.1 N HCl extract of dorsal opisthosomal yellow hypodermis pigment patch; g, 0.1 N HCI extract of A. aurantia opisthosomal mass after centrifugation for 10 min at 10,000 g; h and i, 0.1 N HCI extracts of L. variolus and A. trifolium opisthosomal mass after centrifugation as in g. Abbreviations for U.V. spots are the same as in Fig. 4.
In order of decreasing frequency, the colours associated with external coloration in spiders are: browns, black, red, green and yellow (cf. coloration of spiders, Levi et al., 1968). Recent electron microscope studies (Seligy et al., in preparation) indicate that these pigments are located in spherical ellipsoid granules, 04-1.0 p in dia. The basic morphology and dimensions of the pigment granules, whether from yellow, red, brown or black epidermal pigments, are the same. However, what is of major significance is that the two pigment granule classifications, class I and II, as distinguished by the redox-colour tests in this study, also exhibit marked differences in electron density (osmiophilia). Class I pigment granules are nonosmiophilic, whereas class II pigment granules are. From the experiments presented in this paper the only major difference between these two classes of pigment granules is in the relative amounts of ommatin and ommin present. But qualitative differences in proteins of the two classes of pigment granules and quantitative amounts of a metal have been noted (Seligy et al., in preparation). Recently it has been demonstrated (Ajami & Riddiford, 1971a, b) that insect ommin eye pigments are associated with proteins and that a metal may be also associated with this complex. In the present study the ubiquity of ommochrome pigments in 25
708
V. L.
SELIGY
spiders has been established. Although many of the variations in physiological colour expression appear to be a direct result of qualitative and quantitative differences in the ommochrome sub-groups present, it is most probable that additional components such as proteins and divalent cations will influence the permanent physiological redox-state of the chromogen. Acknowledgements-The author is grateful to Drs. J. R. Colvin, B. F. Johnson and J. M. Neelin of the National Research Council of Canada for use of equipment and chemicals. Thanks are also expressed to Dr. Robin Leech (Canada Department of Entomology, Ottawa) for helpful advice and Messrs. R. D. McMullen and C. V. G. Morgan (Canada Department of Agriculture, Summerland, B.C.) for providing spider material. REFERENCES AJAMI A. M. & RIDDIFORDL. M. (1971a) Identification of an ommochrome in the eyes and nervous system of saturniid moths. Biochemistry, N. Y 10, 1451-1454. AJAMI A. M. & RIDDIFORDL. M. (1971b) Purification and characterization of an ommochrome-protein from the eyes of saturniid moths. Biochemistry, N. Y. 10, 1455-1460. DE ALMEIDAF. F. (1968) Das ommochrom I als wahrschein lithe vorstufe des ommins in der ommochromsynthese von Plodia interpunctella und Ephestia kiihniella. Molec. gen. Genetics 102, 307-315. BECKERE. (1941) Die Pigmente der Ommin- und Ommatin-Gruppe: ein neue Klasse von Naturfarbstoffen. Naturwiss. 29, 237-238. BUTENANDT A. (1959) Wirkstoffe des Insektenreiches. Naturwiss. 46, 461-471. CROMARTIE R. I. T. (1959) Insect pigments. Ento. Reo. 4, 59-76. FORRESTH. S. (1959) The ommochromes. In Pigment Cell Biology (Edited by GORDONM.), pp. 619-628. Academic Press, New York. FOX H. M. & VEVERSG. (1960) The Nature of Animal Colours. Sidgwick& Jackson, London. GAEZRITSCHEVSKY E. (1927) Experiments on the colour changes and regeneration in the crab spider, Misumena vatia (Cl.). J. exp. 2001. 47, 251-267. KASTONB. J. (1948) Spiders of Connecticut. Connecticut State Geol. and Nut. Hist. Surwey, Bull. 70, l-874. LEVI H. W., LEVI L. R. & ZIM H. S. (1968) A Guide to Spiders and their Kin. Golden Press, New York. LINZEN B. (1967) Zur Biochemie der Ommochrome. Naturwiss. 54, 259-267. MCCOOKH. C. (1888) Notes on the relation of structure and function to colour changes in spiders. Acad. natn Sci. Phil. 40, 172-176. MASONH. S. (1959) Structure of melanins. In Pigment Cell BioZogy (Edited by GORDONM.) Academic Press, New York. MILLOT J. (1926) Contributions i l’histophysiologie des AranCides. Bull. biol. Fr. Belg. Sup@. VIII, l-238. MOLES M. L. (1916) The growth and colour patterns in spiders. J. Ent. 2001.8,129-158. NEEDHAMA. E. (1970a) The integumental pigments of some isopod Crustacea. Camp. Biochem. Physiol. 35, 509-534. NEEDHAMA. E. (1970b) Integumental pigments of the Amphipod, Jassa. Nature, Land. 228,1336-1337. RABAUNDE. (1923) Recherches sur la variation chromatique et l’homochromie des Arthropodes terrestres. Bull. biol. Fr. Belg. 57, 182-185. SELIGY V. L. (1969) Biochemical aspects of pigment variation in the spider Enoplognathu owata (Clerck) (Araneae: Theridiidae) Can. J. 2001. 47, 1103-1105. STACKHOU~E H. L. (1966) Some aspects of pteridine biosynthesis in amphibians. Camp. Biochem. Physiol. 17, 219-235.
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709
TAKEUCHIK. (1960) The behaviour of carotenoids and distribution of xanthophores during development of the Medaka (Oryzius Zatipes). Embryologica 5, 170-177. VUILLAUMEM. (1969) Les Ommochromes: Les Pigments des InvertLbbrb. Monographie 10,pp. 73-88. Masson et Cie, Paris. ZIEGLER I. & HARMSENR. (1969) The biology of pteridines in insects. Adv. Insect Physiol. 9, 139-203. Key Word Index-Spider matin; kynurenine.
ommochromes;
pigments; ommins; ommochromes;
xanthom-
OMMOCHROME
PIGMENTS
OF SPIDERS*
V. L. SELIGY? Biochemistry
Laboratory, National Research Council of Canada, Drive, Ottawa, Ontario, Canada, KlA OR6
100 Sussex
(Received 25 October 1971) Abstract-l. Pigments with chemical properties characteristic to both ommatins and ommins were found to be present in the hypodermis and in some cases the intestinal diverticula of spiders from all families of the sub-order Labidognatha examined. 2. Based upon the natural physiological colours and the chemical redoxcolour changes the hypodermal pigments could be grouped into two major and one minor class. 3. Conventional ommochrome chemical techniques were used to show that (a) Class I pigments (orange to brown) consisted mostly, if not entirely, of an ommatin with properties nearly identical to xanthommatin. (b) Class II pigments (dark brown to black) contained both xanthommatin and an ommin, with the latter being the more predominant. (c) Class III pigments (yellow), which occur with the lowest frequency in nature, were variable in composition, consisting mostly of kynurenine and 3-hydroxykynurenine which are either precursors or degradation products of ommochromes.
INTRODUCTION is a wide range of coloration in spiders which may be distributed over the entire animal or localized in the prosoma, opisthosoma or legs as a series of bands or spots (Millot, 1926; Kaston, 1948; Levi et al., 1968). The colours are: white, yellow, green, various shades of red and brown, and black. While most colours are a result of pigments located within the epidermis (Millot, 1926), metallic or iridescent colours are compound: they are the resultant effect of light interference created by the tanned cuticle and the underlying epidermis. In some spiders, opisthosomal coloration and patterns are further complicated by the presence of a white layer made up of guanine storage cells located directly beneath the hypodermis and covering the intestinal diverticula (Millot, 1926). Although some studies have been made on changes in colour and colour pattern during development of spiderlings (McCook, 1888; Gabritschevsky, 1927; Moles, 1916; Millet, 1926; Rabaund, 1923) little is known about the biochemistry of the pigments contributing to the various patterns. This is in contrast to insect studies, where several classes of pigments occur (Cromartie, 1959; Fox & Vevers, THERE
* N.R.C.C. No. 12517. 7 Visiting Research Officer 1971. 699
700
V. L. SELIGY
1960; Ziegler & Harmsen, 1964). One of these pigment classes, the ommochromes, which have been extensively characterized (Becker, 1941; Butenandt, 1959; Linzen, 1967; Needham, 1970a, b), also occurs in the eyes of some spiders (Linzen, 1967; Vuillaume, 1969) and has been identified as the principal epidermal pigment of the theridid spider E. ovata (Cl.) (Seligy, 1969). This paper is a comparative study of the general properties of spider ommochromes and of the extent to which these pigments occur in the various spider families. MATERIALS
AND METHODS
Spiders of twenty-nine genera comprising fourteen families of the sub-class Labidognatha were collected in Carleton, Lincoln and York counties of Ontario and Summerland, British Columbia, Canada. Choice of species for study was based solely upon novelty of the colour patterns or interspecies colour similarities. In most cases the spiders that were chosen have been previously illustrated in colour (Levi et al., 1968). Hypodermal colour patterns were dissected out from various body parts of each spider and then peeled from their respective cuticles. The general presence of ommochromes was tested in situ by the ability of the hypodermal pigment to form the purple/violet halochrome when concentrated sulphuric acid is added (Becker, 1941). Ommatin-like redox colour changes were also examined in situ as previously described (Seligy, 1969) by alternate additions of O*OS-O*lml of saturated sodium dithionite and saturated (w/v) sodium nitrite or 5% (v/v) nitric acid by ~1. pipette. One experiment constituted four complete reduction and oxidation cycles on duplicate samples. For fractionation and partial purification of ommochromes, whole opisthosomal hypodermis was used. Each pigment pattern was dissected out, washed in distilled water (pH 7.0) and centrifuged at 20,000 g for 5 min. The resultant pellets were further extracted with 10 vol. of acetone, dried under vacuum at ambient temperature and stored at - 20°C until further use. To avoid the possible degradative effects of acid during extraction and analysis most operations, including short-term storage of extracts, were done at 4°C. Ommochromes were extracted from the acetone dried pigment pellets with either 5 N HCl, methanol-HCl(l4 : 1) or 90% formic acid. In most cases the procedure described by Needham (1970a) of total extraction with 90% formic acid, precipitation of ommatins and ommins by addition of diethylether and differential extraction of ommatins with water and ommins with 5 N HCl was found to be the most suitable for spider ommochromes. Absorption spectra of the extracts were obtained using a Cary-l4R spectrophotometer over the range of 700-200 nm. Whatman No. 1 paper (25 x 25 cm) was used for descending chromatography and formic acid-methanol-concentrated HCl-water (15 : 3 : 1 : 6) was used as the principal solvent system. nL-Rynurenine and L-3-hydroxykynurenine (A grade, Calbiochem) were used as chromatographic markers. RESULTS
AND DISCUSSION
In preliminary experiments it was established that pigment groups, such as pteridines (Stackhouse, 1966), carotenoids (Takeuchi, 1960) and melanins (Mason, 1959) were not present in detectable quantities in the hypodermal colour patterns of all spiders listed in Table 1. However, in addition to the hypodermal pigments being insoluble in neutral organic solvents all patterns were found to contain ommochromes as indicated by the formation of the violet-purple “halochrome” upon the addition of concentrated H&30, (Becker, 1941). Moreover, in almost all cases, chemical redox-colour changes were also observed. These results led to the
Agelenopti naevia (Walckenaer) Araneus diadematus (Clerck) Araneus triyolium (Hentz) Argiope aurantia (Lucas) Argiope t$zsc-iuta (Forskll) Chiracanthium mildei (L. Koch) Dictyna sp. Gnaphosa sp. Frontinella communis (Hentz) Pityophantes phrygiunw (C. L. Koch) Lycosa gulosa (Walckenaer) Dolomedes tenebrosus Metaphidippus gakzthea (Walckenaer) Phidippus clarus (Keyserling) SuZticus smicus (Clerck) Scytodes thoracica (Latrielle) Leuxsage wenusta (Walckenaer) Achaearanea tepiakriorum (C. L. Koch) Enoplognatha ovata (Clerck) Latrodktus variolus (Walckenaer) Steatoda borealis (Hentz) Theridion jkwuieum (Hentz) Theridion murarium (Emerton) Misumena walk (Clerck) Misumenops asperatus (Hentz) Philodromus rufhs (Walckenaer) Xisticus Zucttwsus (Blackwall) Tibellus obkmgus (Walckenaer)
Species
:(I) 4 (2) (2) 4 (4)
2 2
2 (1)
i(l) 2 (2) 2 3 3 (2) 15 (15) 6
(4) 17 (1) 12 (1) 10 (2) 3 3 1 2 2 2 1
1:
L, DO, VO, P L, DO, VO, P L, DO, VO, P DO, VO, P DO, VO, P L, DO L, DO, VO, P DO DO, VO DO L, DO, VO, P L, DO, VO, P DO, VO, P L, DO, VO, P DO, VO, P L, DO, VO, P DO, VO L, DO, VO DO, VO, P DO, VO, P L, DO, VO, P L, DO, VO L, DO, VO DO L, DO, P L, DO, VO, P L, DO, VO, P DO, P
Location? Medium to dark-brown ; black Light to dark-brown Light to medium-brown Yellow; black Yellow ; black Yellow; medium-brown Yellow-brown; dark-brown Dark brown Dark-brown to black Light to medium-brown Medium to dark-brown Medium to dark-brown Medium to dark-brown Red to dark-brown Medium to dark-brown Dark-brown to black Yellow; red to brown Medium to dark-brown Yellow; orange to red; black Black, red Dark brown to black Yellow; black Red; medium to dark-brown Yellow ; red Yellow; red-brown Red-brown to brown Red-brown to dark-brown Yellow brown; dark-brown
Physiological colour
Pigment
* Figures in parentheses are the number of males examined in addition to females. t Hypodermal pigments located in L, legs; DO, VO, dorsal and ventral opisthosoma; P, prosoma.
Thomisidae
Scytodidae Tetragnathae Theridiidae
Clubionidae Dictynidae Gnaphosidae Linyphiidae (Linyphiinae) Lycosidae Pisauridae Salticidae
Agelenidae Araneae
Family
Number * tested
TABLE ~-DISTRIBUTION OF OMMOCHROME PIGMENTSIN SPIDERSOF THE SUBORDER LABIDOGNATHA
I; II I; II I III; II III; II III; I I; II II II I I, II I, II I, II I, II I, II II III; I I, II III; I; II II, I II III; II I; I, II III; I III; I I I, II I; II
Classification
2 I
; g
!
8 q ij
B $ : 3
702
V. L. SELIGY
general observation that the natural epidermal colour variations of spiders could be grouped into two major and one minor class (Tables 1 and 2). The pigments of all three classes clearly differ in their natural or physiological colours and in the case of class I and II, they appear also to differ in their natural occurring redox-colour states as suggested by the redox-colour studies (Table 2). TABLE
2-SUM~IARY
OF REDOX COLOUR AND OF SPIDER HYPODERMAL
HALOCHROME-FORMING PIGMENTS
PROPERTIES
Pigment colour Classification I
Physiological
II
Orange, red, violet, red-brown, yellow-brown, light to medium-brown Dark-brown, black
III
Pale to bright yellow, green
Reduced *
Oxidized
Halochrome t
Red to red-brown
Yellow to Violet yellow-brown
Yellow to orangebrown
Dark-brown or black No change
Deep violet
Yellow to brown
* Dissected hypodermis was treated in situ with reducing and oxidizing reagents as described in Materials and Methods. t Dissected hypodermis was treated in situ with concentrated HsS04.
Since both redox and halochrome tests give only an indication that ommochromes are present, the general properties of spider pigments were further studied by conventional ommochrome extraction and fractionation procedures (see Materials and Methods). Although twenty-six species representing all three pigment classifications were studied in detail, the observations reported here are limited to only a few species. The species chosen to be most typical for class I pigment (red to brown) was A~uneus trifolium (Hentz) and for class II and III pigments (dark brown to black and bright yellow, respectively) Argiope uurantiu Lucas. A third species, the black widow spider, Latrodectus variolus Walckenaer, which contains a ventral red hour-glass pattern (class I) in addition to the overall black coloration (class II) was used for intraspecies pigment comparison. Extraction of opisthosomal epidermal pigments from A. trifolium (Hen&) and A. auruntiu Lucas with methanol-HCl (19 : 1) or HCl (5 N) resulted in extracts which were identical in colour, yellow to yellowish brown. However, differences were observed in the colours of the unextracted residual material as well as the precipitates recovered by centrifugation of the extracts after adjusting the pH to 5 and the sodium thiosulphate concentration to O*O5o/o. Class I precipitates were distinctly red, while class II precipitates were more variable in colour, red to dark brown. The visible absorption spectrum of either pigment extract in 1 N HCl resembled that of oxidized xanthommatin (Fig. 1). However, neither reagent was
OMMOCHROME
PIGMENTS
OF SPIDERS
703
effective in extracting all the colour from the epidermis of the two classes, particularly class II. Therefore it would appear that additions components such as ommins which are known to be only sparingly soluble in these reagents (Linzen, 1967) might be present. This assumption was further supported by the results
400
500 Wave
length,
600
nm
FIG. 1. Visible absorption spectra of opisthosomal hypodermal pigment extracts by 5 N HCl. ) and A. aurantiu Lucas (- -) from A. tnfolium (Hentz) (-
obtained using the formic acid extraction and ether fractionation procedure for ommatins and ommins as described by Needham (1970a) for isopod integumental ommochromes. This method proved to be most useful for not only achieving rapid and complete solubilization but greatly simplified comparative studies. Total colour extraction of A. trifoliunt (Hentz) hypodermal pigments (class I) was obtained at 4°C within 2 mm after brief homogenization in 90% formic acid, while pigments of Aruaw c&z&status Clerck, A. uurantia Lucas (class II) and L. oriole Walckenaer required longer exposures, S-30 min. The orange-brown coloured extract of A. trifoliunt was further fractionated into a bright yellow water soluble component and a faint reddish-violet acid soluble component. The spectral properties (Fig. 2) of the yellow extract closely resembled that of xanthommatin (Needham, 197Oa) while the reddish-violet pigment was similar to that described for ommin. Each formed the purple halochrome, (Fig. 3) upon redissolving in concentrated H,SO, after concentrating by flash evaporation at 15°C. The chromatographic properties of both extracts were similar to that of xanthommatin with only a faint trace of slow migrating component in the reddishviolet acid extract which corresponded to an ommin (Fig. 4). Consistent with the properties of ommatins and ommins was the observation that the yellow water soluble pigment was compfetely dialysable, whereas the reddish-violet pigment was only partially so. The ommin extract was also quite resistant to chemical oxidation. However, after 3 or more days’ exposure to 90% formic acid at room temperature (23°C) the colour changed from reddish violet to yellowish-brown. The two main products of this oxidation, when isolated after paper chromatography, had a similar
704
v. L.
I
400
.
I
500
.
I
600
Wave length, nm
.
SELIGY
I
400
.
.
.
500
a
.
600
Wavelength, nm
FIG. 2 FIG. 2. Visible absorption spectra of ommochromes from the hypodermis of A. trifolium (Hen@ initially extracted by 90% extracted by water from an ether-precipitate of the 90% from the residue and extracted by 5 N HCl (-*-) ( -) water extraction.
FIG. 3 dorsal opisthosomal formic acid (- - -), formic acid extract remaining after the
) and ommin (-a-) of FIG. 3. Visible absorption spectra of ommatin (A. trifolium in 100% sulphuric acid. Ommatin and ommin extracts were prepared from opisthosomal hypodermis as described in text and Fig. 2.
FIG. 4. Chromatogram of ommochrome extracts from class I hypodermal pigments. The extracted pigments were applied to Whatman No. 1 paper and developed by (v/v, descending chromatography using formic acid-methanol-HCl-water 15 : 4 : 5 : 1 : 5). A. trifolium, opisthosomal pigments: a, yellow water-soluble component extracted from ether-precipitate of 90% formic acid extract; b, reddishviolet 5 N HCl extract of water insoluble residue; c, kynurenine standard; d-i, 90% formic acid extracts of hypodermal pigments from d, A. hdematus; e, A. nuevia; f, A. saevw; g, L. variolus (ventral red hour glass); h, L. vem~sta;i, M. vatia. Abbreviations for U.V. spots: MB, medium blue; WR, whitish-blue; RB, reddish-brown; DV, dark-violet.
absorption spectrum as that of xanthommatin and reduced xanthommatin (dihydroxyxanthommatin). Since ommins, when exposed to formic acid for lengthy periods, can break down to yield smaller products including ommatin (Almedia,
OMMOCHROME
PIGMENTS
OF SPIDERS
70.5
1968) the xanthommatin and dihydroxyxanthommatin obtained from the slow oxidation of the reddish-violet ommin extract is probably a degradation product. When the same extraction procedure was used to study the class I pigments from other spiders, it was immediately apparent that variations in ommin but not ommatin content occurs (Fig. 4). In some cases, such as the orange-red hour glass pattern of L. variolus (Walckenaer) (Fig. 5), the red pattern of Enoplognatha ovata
I
a
1 400
.
1 500
9
1
1
600
Wave length, nm FIG. 5. Visible absorption spectra of ommochrome extracts from the opisthosomal
hypodermis of L. variolus Walckenaer. Formic acid (90%) (- -), water soluble component of the ether-precipitate of a 90% formic acid extract (), 5 N HCl extract of water insoluble residue (-.--). Inset figure: visible absorption spectra of the ventral red hour glass ommochromes. Extraction sequence and figure notations are the same as above.
Wave
length,
nrn
FIG. 6. Visible absorption spectra of ommochrome extracts from opisthosomal hypodermis of A. aurantia Lucas. Formic acid (90%) extract (- -), watersoluble component of the ether precipitate of a 90% formic acid extract (-), 5 N HCl extract of water insoluble residue (-*-). Inset figure : visible absorption spectra of 0.1 N HCl extract of yellow hypodermis pigment patch (-) and L-kynurenine (- -).
706
V. L.
SELIGY
(Clerck) and the red pattern of Misumena vatia (Clerck) (data not shown), only xanthommatin appears to be present. In others, the reddish-brown patterns of Agelenopsis naewia (Walckenaer), Leucusage venusta (Walckenaer), Araneus saevus (L. Koch) and Araneus dzizhnatus (Clerck), ommin is present in addition to ommatins. Upon closer examination of the pigment patterns in which ommins were also found it was noted that all the patterns were compound, consisting of additional fine sub-patterns involving class II pigments. A similar comparative study of the class II (black) pigments of L. variolus and A. aurantia indicated that these pigments differ significantly from class I in their marked increase in ommin content relative to xanthommatin (cf. Figs. 2, 5 and 6). The increase in ommin was also apparent in the initial formic acid extracts, since the orange-brown extracts of class I pigment of A. trifolium (Hentz) were much lighter in colour than the deep reddish-violet extracts of class II (Figs. 1 and 4a). Paper chromatography of the 5 N HCl extracts yield four characteristic spots using short wave U.V. light (2537 A) (Fig. 6). Whereas in class I pigment the characteristic slow migrating ommin spot was barely detectable by U.V. light, the ommin spots of class II were quite distinct. In addition to a slow migrating dark red ommin spot which is easily detected by visible light, a xanthommatin spot and a distinct bright whitish-blue fluorescent spot corresponding in R, to kynurenine, a fourth reddish-brown fluorescent spot with an R, between ommin and xanthommatin is present. The latter reddish-brown U.V. fluorescent spot is somewhat reminiscent to the violet U.V. fluorescence found in Ligia ommochrome extracts which Needham (1970a) has suggested may be due to a related metabolite of the indol-ommochrome pathway. In general, coloration of spiders is primarily due to pigments located within the hypodermis (Millot, 1926); however, differences in ground colour of the opisthosomal diverticular mass also occurs (McCook, 1888 ; Seligy, unpublished). In most cases, it was thought that the very dark colours of spiders were due only to the presence of class II pigments, but during the course of this study it was noted that both species A. aurantia Lucas and A. trifasciata (Forskil) also contained considerable amounts of xanthommatin in the gut diverticulae (Figs. 6 and 7). After dissection of this reddish-grey mass and exposure of it to the air for l-2 hr it slowly oxidized and became black in colour. In contrast, no such observation could be made for L. variolus Walckenaer or A. trifolium (Hentz). A further study showed that in addition to guanine, xanthommatin was also present in variable quantities in the gut of Agelenopis sp., L. venusta (Walckenaer) and a Lycosa sp. (class II pigmented hypodermis). It is conceivable that the presence of ommatins in the gut of some spiders and not in others could easily convey special differences in overall opisthosomal coloration. It should be noted that not all colour effects are the result of ommochromes or guanine cells. The pale to bright yellow patterns in the opisthosomal hypodermis of A. aurantia and A. trifasciata, for example, which are devoid of class I and II pigments, are a result of accumulated precursor or degradation products of the ommochrome pathway, since both kynurenine and 3-hydroxykynurenine have been identified as the major product in these patterns
OMMOCHROME PIGMENTS OF SPIDERS
707
(Figs. 6 and 7). Both of these products can also be detected in situ by the bright bluish-white fluorescence of the hypodermis during exposure to U.V. light at 2537 A. Since similar observations have been noted for one of the genetic colour variants of E. oaata (Clerck) (Seligy, 1969, and in preparation) it is likely that during the course of colour pattern evolution some of the hypodermal cells have lost the ability to complete the synthesis of ommochromes. 0.8 -
WE (J
(y
MB MB
WB o
WB WEWBWBWB Q;
qj””
MB MB DV
‘DV
z o’4 -
0
1
FIG. 7. Chromatogram of ommochrome extracts of class II hypodermal pigments. Chromatograms were developed descendingly on Whatman No. 1 paper as described in the text and Fig. 4. L. variolus pigments: a, yellow water soluble component extracted from the ether precipitate of a 90% formic acid extract; b, reddish-violet 5 N HCl extract of water insoluble residue; c, kynurenine standard. A. aurantia pigments: d, yellow water-soluble extract; e, reddish-violet 5 N HCl extract; f, 0.1 N HCl extract of dorsal opisthosomal yellow hypodermis pigment patch; g, 0.1 N HCI extract of A. aurantia opisthosomal mass after centrifugation for 10 min at 10,000 g; h and i, 0.1 N HCI extracts of L. variolus and A. trifolium opisthosomal mass after centrifugation as in g. Abbreviations for U.V. spots are the same as in Fig. 4.
In order of decreasing frequency, the colours associated with external coloration in spiders are: browns, black, red, green and yellow (cf. coloration of spiders, Levi et al., 1968). Recent electron microscope studies (Seligy et al., in preparation) indicate that these pigments are located in spherical ellipsoid granules, 04-1.0 p in dia. The basic morphology and dimensions of the pigment granules, whether from yellow, red, brown or black epidermal pigments, are the same. However, what is of major significance is that the two pigment granule classifications, class I and II, as distinguished by the redox-colour tests in this study, also exhibit marked differences in electron density (osmiophilia). Class I pigment granules are nonosmiophilic, whereas class II pigment granules are. From the experiments presented in this paper the only major difference between these two classes of pigment granules is in the relative amounts of ommatin and ommin present. But qualitative differences in proteins of the two classes of pigment granules and quantitative amounts of a metal have been noted (Seligy et al., in preparation). Recently it has been demonstrated (Ajami & Riddiford, 1971a, b) that insect ommin eye pigments are associated with proteins and that a metal may be also associated with this complex. In the present study the ubiquity of ommochrome pigments in 25
708
V. L.
SELIGY
spiders has been established. Although many of the variations in physiological colour expression appear to be a direct result of qualitative and quantitative differences in the ommochrome sub-groups present, it is most probable that additional components such as proteins and divalent cations will influence the permanent physiological redox-state of the chromogen. Acknowledgements-The author is grateful to Drs. J. R. Colvin, B. F. Johnson and J. M. Neelin of the National Research Council of Canada for use of equipment and chemicals. Thanks are also expressed to Dr. Robin Leech (Canada Department of Entomology, Ottawa) for helpful advice and Messrs. R. D. McMullen and C. V. G. Morgan (Canada Department of Agriculture, Summerland, B.C.) for providing spider material. REFERENCES AJAMI A. M. & RIDDIFORDL. M. (1971a) Identification of an ommochrome in the eyes and nervous system of saturniid moths. Biochemistry, N. Y 10, 1451-1454. AJAMI A. M. & RIDDIFORDL. M. (1971b) Purification and characterization of an ommochrome-protein from the eyes of saturniid moths. Biochemistry, N. Y. 10, 1455-1460. DE ALMEIDAF. F. (1968) Das ommochrom I als wahrschein lithe vorstufe des ommins in der ommochromsynthese von Plodia interpunctella und Ephestia kiihniella. Molec. gen. Genetics 102, 307-315. BECKERE. (1941) Die Pigmente der Ommin- und Ommatin-Gruppe: ein neue Klasse von Naturfarbstoffen. Naturwiss. 29, 237-238. BUTENANDT A. (1959) Wirkstoffe des Insektenreiches. Naturwiss. 46, 461-471. CROMARTIE R. I. T. (1959) Insect pigments. Ento. Reo. 4, 59-76. FORRESTH. S. (1959) The ommochromes. In Pigment Cell Biology (Edited by GORDONM.), pp. 619-628. Academic Press, New York. FOX H. M. & VEVERSG. (1960) The Nature of Animal Colours. Sidgwick& Jackson, London. GAEZRITSCHEVSKY E. (1927) Experiments on the colour changes and regeneration in the crab spider, Misumena vatia (Cl.). J. exp. 2001. 47, 251-267. KASTONB. J. (1948) Spiders of Connecticut. Connecticut State Geol. and Nut. Hist. Surwey, Bull. 70, l-874. LEVI H. W., LEVI L. R. & ZIM H. S. (1968) A Guide to Spiders and their Kin. Golden Press, New York. LINZEN B. (1967) Zur Biochemie der Ommochrome. Naturwiss. 54, 259-267. MCCOOKH. C. (1888) Notes on the relation of structure and function to colour changes in spiders. Acad. natn Sci. Phil. 40, 172-176. MASONH. S. (1959) Structure of melanins. In Pigment Cell BioZogy (Edited by GORDONM.) Academic Press, New York. MILLOT J. (1926) Contributions i l’histophysiologie des AranCides. Bull. biol. Fr. Belg. Sup@. VIII, l-238. MOLES M. L. (1916) The growth and colour patterns in spiders. J. Ent. 2001.8,129-158. NEEDHAMA. E. (1970a) The integumental pigments of some isopod Crustacea. Camp. Biochem. Physiol. 35, 509-534. NEEDHAMA. E. (1970b) Integumental pigments of the Amphipod, Jassa. Nature, Land. 228,1336-1337. RABAUNDE. (1923) Recherches sur la variation chromatique et l’homochromie des Arthropodes terrestres. Bull. biol. Fr. Belg. 57, 182-185. SELIGY V. L. (1969) Biochemical aspects of pigment variation in the spider Enoplognathu owata (Clerck) (Araneae: Theridiidae) Can. J. 2001. 47, 1103-1105. STACKHOU~E H. L. (1966) Some aspects of pteridine biosynthesis in amphibians. Camp. Biochem. Physiol. 17, 219-235.
OMMOCHROME PIGMENTSOF SPIDERS
709
TAKEUCHIK. (1960) The behaviour of carotenoids and distribution of xanthophores during development of the Medaka (Oryzius Zatipes). Embryologica 5, 170-177. VUILLAUMEM. (1969) Les Ommochromes: Les Pigments des InvertLbbrb. Monographie 10,pp. 73-88. Masson et Cie, Paris. ZIEGLER I. & HARMSENR. (1969) The biology of pteridines in insects. Adv. Insect Physiol. 9, 139-203. Key Word Index-Spider matin; kynurenine.
ommochromes;
pigments; ommins; ommochromes;
xanthom-