Biological Journal oJthe Linnean Sociely (1991),42: 457-465. With 1 figure

Carotenoids and colouration of poplar hawkmoth caterpillars ( populi) Downloaded from https://academic.oup.com/biolinnean/article/42/4/457/2654249 by guest on 01 October 2021 JOY GRAYSON, MALCOLM EDMUNDS, E. HILARY EVANS

Departmenl of Applied Biology, Lancashire Polytechnic, Preston PRl 2TQ

AND

GEORGE BRITTON

Department of Biochemistry, Universip of Liverpool, P.O. Box 147, Liverpool L69 3BX

Receiued 9 NoNovember 1989, accepted for publication 7 March 1990

Carotenoids and chlorophylls a and b were extracted from final instar caterpillars of the poplar hawkmoth () and the eyed hawkmoth (Smm'nihus occllafu), as well as from their food plants. Both species of caterpillar absorb the two chlorophylls and the carotenoids lutein, cis-lutein and B-carotene in the gut and deposit lutein and cis-lutein in the integument. It is the lutein, together with pterobilin, that is largely responsible for the colour of the : yellow-green poplar hawkmoth caterpillars have more lutein in the integument than dull green ones which in turn have more than white ones. Yellow-green and dull green caterpillars both sequester lutein and cis-lutein in the gut wall, but the yellow-greens translocate more of these pigments to the integument than the dull greens. The white caterpillars absorb very little lutein and cis-lutein into the gut, and so they have much less also in the integument. The mechanism by which the reflected light perceived by the caterpillar is translated into differential absorption of pigment by the gut and deposition in the integument is not known.

KEY WORDS:-Carotenoids - chlorophyll - lutein - colouration - Laofhoe populi - hawkmoth caterpillars - plant pigment sequestration.

CONTENTS Introduction ...... 458 Material ...... 458 Method of analysis ...... 459 Results ...... 460 Carotenoids and chlorophyll in food plants ...... 460 Carotenoids and chlorophyll in the integument of poplar hawkmoth caterpillars . 460 Fate of carotenoids and chlorophyll ingested by caterpillars ...... 462 Pigments in the integument of the eyed hawkmoth ...... 463 Discussion ...... 463 Acknowledgements ...... 464 References ...... 464 457 0224-4066/91/040457+09 $03.00/0 0 1991 The Linnean Society of London 458 J. GRAYSON ET AL.

INTRODUCTION Carotenoids are polyene pigments usually containing 40 carbon atoms and built up of eight isoprene residues. Over 400 carotenoids are known (Weedon, 1980), and modern analytical techniques are revealing more. Carotenoids are widely distributed in both plants and . There is no convincing report, however, of any de nouo formation of carotenoids in animals, and it is believed that they are derived either directly or indirectly from plants or microorganisms (Britton et al., 1977). After ingestion by an , carotenoids may be stored Downloaded from https://academic.oup.com/biolinnean/article/42/4/457/2654249 by guest on 01 October 2021 unchanged (either selectively or indiscriminately), structurally modified, or totally rejected. Thus, knowledge of an insect’s food is a prerequisite to consideration of the origin of its carotenoids (Kayser, 1982). Carotenoids are one of the commonest types of pigment found in the (Feltwell, 1978). Although the chemical techniques for extraction and identification of pigments were very primitive by modern standards, as long ago as 1885 Poulton identified green and yellow pigments from the haemolymph of eyed hawkmoth larvae (Smerinthus ocellata) as being derived directly from the chlorophyll and xanthophyll found in their foodplant. (‘Xanthophyll’ was used at that time to include the hydrocarbon carotenes as well as true xanthophylls.) Work on identification of carotenoids in Lepidoptera has continued in the century since Poulton’s work. Feltwell & Rothschild (1974) identified the carotenoids present in 38 species of Lepidoptera. The large white (Pieris brassicae) sequesters 14 different carotenoids from its food, some of which are present in all stages of the life history. Carotenoids are often sequestered selectively, rather than in proportion to their occurrence in the food: for example, P. brassicae pupae have more carotenoids, especially b-carotene, than do Pieris rapae pupae when larvae are fed on the same food (Rothschild, Valadon & Mummery, 1977). In silkmoth larvae (Hyalophora cecropia) comparable differences occur in different individuals of a single species: Mummery, Valadon & Rothschild (1976) found that normal green caterpillars have 157.6 pg g-I carotenoid per individual while a recessive blue mutant has only 2.4 pg g-l. The possible functions of these carotenoids, particularly in animal signalling, have been discussed by Feltwell & Rothschild (1974), Rothschild (1975, 1978) and Rothschild et al. (1978, 1986). However, since the work of Poulton (1886), very little has been published on carotenoids in the integument contributing to the camouflaging greens of caterpillars. In 1978 a study was begun on the colouration of eyed and poplar hawkmoth caterpillars in Lancashire. The colour varies on different food plants in the field (Edmunds & Grayson, 1991), and under artificial conditions with different lighting (Grayson & Edmunds, 1989). In this paper we report on carotenoids that are sequestered from the ’ food and deposited in the integument, and on the patterns of pigment deposition in different colour forms of the poplar hawkmoth caterpillar.

MATERIAL Samples of foodplant for pigment analysis were collected during the summer of 1982 near Preston. The species used were Salix fragilis L. (crack ), S. cinerea L. (sallow), S. uiminalis L. (osier) and alba L. (white poplar). CAROTENOIDS IN POPLAR HAWKMOTH CATERPILLARS 459 Leaves were removed from the stems, and the petioles and median veins were excised. About 1.5 g wet weight of leaves of each species was then divided into two halves, one half being analysed fresh, the other after storage in deep freeze (- 15°C) for 12 months to detemine if the pigments decay with time. Full grown final instar caterpillars of the poplar and eyed hawkmoths (Laothoe populi L. and Smerinthus ocellata L.) were also analysed. In initial experiments caterpillars were starved for 24 h, killed by immersion in liquid nitrogen, and then stored for between 2 and 6 months at - 15°C. However, it was found that

24 hours is not a long enough period of starvation to clear the gut of plant Downloaded from https://academic.oup.com/biolinnean/article/42/4/457/2654249 by guest on 01 October 2021 material, freezing and thawing caterpillars made it difficult to separate the integument and gut sac from the rest of the body, and both carotenoids and chlorophylls deteriorate with time even in a freezer. In later experiments analysis was therefore carried out on fresh larvae which had been starved for 48 h, and then killed with ethyl acetate vapour. The dead caterpillars were opened up by means of a dorsal incision, the integument was dissected away from adhering connective tissue, and the gut sac was also removed after first ligaturing it at both ends. Both integument and gut sacs were washed thoroughly with tap water before freezing at -15°C for a few days before analysis. Frass from final instar caterpillars was also collected, frozen and analysed.

METHOD OF ANALYSIS Chlorophylls and carotenoids were exhaustively extracted in cold acetone by the method of Britton & Goodwin (1971). The lipid free residue was dried at 110°C for dry weight determination. Chlorophyll was estimated on the total extract in ether by measuring absorbance at 660 nm and 642.5 nm using the following formulae (Strain, Cope & Svec, 1971): Chlorophyll a (pg ml-’) = 9.93 (Asm)-0.777 (A642.5) Chlorophyll b (pg ml-’) = 17.6 (A642,5)-2.81 (Aa,,) The carotenoids were separated by thin layer chromatography under alkaline conditions to prevent isomerization of violaxanthin and neoxanthin to auroxanthin and neochrome respectively (Britton & Goodwin, 197 1). Carotenoids were identified on the basis of co-chromatography, absorption spectrum (Davies, 1976) and mass spectrum. Violaxanthin and neoxanthin were characterized by a modified HC1-ether test Uungalwala & Cama, 1962). Quantitative estimation of carotenoids used the absorbance coefficients of Davies (1976). Cis-lutein was identified by its light absorption spectrum, which had A,,, at 418, 442 and 468 nm with a weak ‘cis-peak’ at 330 nm; its mass spectrum, which was identical to that of (all-trans-)lutein; and by iodine-catalysed photoisomerization, which yielded (all-trans-)lutein as the main product. Some 2 to 3 years after completion of this work, a stored sample was compared with a well-characterized set of lutein isomers (available from other work) by adsorption phase high performance liquid chromatography on a silica column (Barry, 1988). It had an identical retention time and absorption spectrum to (9<+ 9’3-lutein. This mixture appears to be present universally in small amounts in all green leaves. 460 J. GRAYSON ET AL.

RESULTS Carotenoids and chlorophyll in food plants Table 1 shows the carotenoid and chlorophyll content of fresh food plants. Lutein, p-carotene, violaxanthin and neoxanthin are present in all four plants. Cis-lutein was only distinguished from normal (all-trans-)lutein in S. fragilis and S. viminalis; it probably occurs in the other two plants as well, but confirmatory

tests were not carried out. No obvious differences in either the concentrations or Downloaded from https://academic.oup.com/biolinnean/article/42/4/457/2654249 by guest on 01 October 2021 the percentage occurrence of the different carotenoids were found in the four plants. Lutein and /3-carotene are the most abundant carotenoids and cis-lutein is the scarcest. Carotenoids comprise only about 13% of the total pigment extracted, the remainder of which was chlorophylls a and b in the ratio of about 3 : 1. Analysis of plants that had been frozen and stored for 12 months showed that carotenoids decline by 40-52% and chlorophylls by 21-40y0 on a dry weight basis compared with fresh material. There is also differential breakdown of pigments with time: violaxanthin is the least stable pigment and cis-lutein and lutein are the most stable. Chlorophyll a also appears to be less stable than chlorophyll b so that the chlorophyll a : b ratio is decreased to 2 : 1 or less. These results underline the importance of using fresh material.

Carotenoids and chlorophyll in the integument of poplar hawkmoth caterpillars Table 2 presents the results of the analysis of pigments in the integument of differently coloured caterpillars fed on three different foods. The main pigments in the integument are lutein and cis-lutein with just traces of p-carotene and chlorophylls a and b. Because of the minute quantities of these pigments positive identification of the pigment with a peak absorption at 670 nm was not possible: it is probably chlorophyll but it could be some other blue-green pigment. Cis-lutein runs as a broad band on thin layer chromatography plates just below lutein, but it is not always possible to draw a clear line between the two. This may account for some of the variation in the cis-lutein: lutein ratio. The

TABLE1. The pigments extracted from fresh food plants (pg g dry weight-’), together with percentages of the principal carotenoids

Carotenoids Chlorophylls Carotenoid: Lutein & /3-caro- Viola- Neo- a : b chlorophyll Plant cis-lutein tene xanthin xanthin Total Ch1.a Ch1.b Total ratio ratio

S. fragdzs 413.0 258.3 83.3 115.2 869.8 4216 1382 5598 3.1 0. I5 47.5% 29.7% 9.So/, 13.2”/, S. cinerea 555.9 363.5 128.2 149.8 1197.4 4798 1771 6568 2.7 0.18 46.494 30.4% 10.7T0 12.5% S. vtmtnalis 614.9 507.7 78.1 156.2 1356.9 5750 2164 7915 2.7 0 17 45.30% 37.4% 5.e0 11.5°/0 P. alba 449.7 317.1 127.5 113.9 1008.2 6752 2338 9090 2.9 0.11 44.S0/, 31.5% 12.7% 11.2% CAROTENOIDS IN POPLAR HAWKMOTH CATERPILLARS 461 TABLE2. The pigments in the integument of 48 hour starved poplar hawkmoth larvae (pg g dry weight - ' )

Larval Larval Cis-lutein: rolour food plant Lutein Cis-lutein /?-carotene lutein ratio

Yellow-green S. fragilis 72.7 5.7 * 0.08 S. fragilis 146.4 9.7 * 0.07 S. fragilis 75.4 t * n.d.

S. fragilis 46.9 7.7 * 0.16 Downloaded from https://academic.oup.com/biolinnean/article/42/4/457/2654249 by guest on 01 October 2021 S. fragilis 80.0 6.1 * 0.08 S. fragilis 82.2 4.1 * 0.05 S. fragilis 57.4 4.4 * 0.08 S. cinerea 70.9 3.3 * 0.08 P. alba 29.4 8.7 * 0.30 P. alba 32.3 2.2 * 0.07 P. alba 37.8 4.0 * 0.1 I Dull green S. fragilis 5.0 I .6 * 0.32 S. fragilis 12.5 t * n.d. S. fragilis 21.1 I .3 * 0.06 S. fragilis 13.2 I .4 * 0.11 S. fragilis 21.6 I .2 0.06 S. fragilis 27.8 1.6 0.06 S. fragilis 37.7 t * n.d. S. cinerea 27.4 9.8 * 0.36 White S. fragilis 4.13 n.d. n.d. n.d. P. alba 2.77 n.d. n.d. n.d. P. alba 3.43 n.d. n.d. n.d. P. alba 0.9 - - n.d.

*, Trace present; t, the lutein figure includes cis-lutein; -, absent; n.d. not determined. comparison of pigments in different coloured caterpillars shown in Table 2 is summarized in Fig. 1. Yellow-green caterpillars on either S. fragilis or S. cinereu have 70-85 pg g-' ofcarotenoid (lutein plus cis-lutein), dull green caterpillars on the same plants have 20-30 pg g-I, while white caterpillars on white poplar have only 2-3 pg g-'. Thus differences in colour of caterpillar can be explained in terms of different quantities of carotenoid in the integument. Very occasionally caterpillars fed on S.frugilis become white while some fed on P. alba become yellow-green (Grayson & Edmunds, 1989). Table 2 also gives the

White Dull green lorvoe lorvoe Yellow-green Iorvoe

J 0 20 40 60 80 100 120 Carotenoid content (pg g-I

Figure 1. The carotcnoid content (lutein and cis-lutein) of the integument of three colour forms of final instar poplar hawkmoth caterpillan: means and standard deviations (pg g dry weight-'). 462 J. GRAYSON ElAL. carotenoid content of these larvae: white larvae on S. fragilis have just slightly more carotenoid than normal white larvae, while yellow-green larvae on P. alba have much less carotenoid than normal yellow-green caterpillars, but ten times as much as white caterpillars reared on this plant. Quite remarkable is the selectivity by which p-carotene is eliminated, presumably in the gut, allowing lutein and cis-lutein to be sequestered in the integument.

Fate of carotenoids and chlorophyll ingested by caterpillars Downloaded from https://academic.oup.com/biolinnean/article/42/4/457/2654249 by guest on 01 October 2021 Table 3 gives the carotenoid and chlorophyll content of frass from yellow- green and from white caterpillars that had all recently fed on P. alba. It shows that considerable quantities of all the carotenoids and chlorophylls found in leaves pass through the body unchanged. Frass from white caterpillars has much more chlorophyll than frass from yellow-green caterpillars, and the chlorophyll a : b ratio is much higher than it is in plant leaves. Table 3 also gives the amounts of carotenoid and chlorophyll in the gut sac. It shows that lutein, cis-lutein and B-carotene are all taken up from the food by the gut, but there is rather little violaxanthin and no neoxanthin. Chlorophylls a and b are also taken up by the gut, but in very small quantities relative to their occurrence in the food. Much of the lutien and cis-lutein is transported through the haemocoel and deposited in the integument (see Table 2), but some lutein also enters the reproductive system since eggs were observed to have 16.6 pg g-' of lutein. Yellow-green and dull green caterpillars have about five times as much lutein and cis-lutein in the gut sac as do white caterpillars. This suggests that white caterpillars have little pigment in the integument because they fail to absorb it in the gut. Dull green caterpillars absorb it in the gut but fail to deposit as much in the integument as do yellow-green caterpillars. Dull green caterpillars have much more chlorophyll in the gut sac than yellow-green larvae, while white ones have only a trace of chlorophyll. The

TABLE3. Pigments in the frass and in the gut sacs of final instar poplar hawkmoth caterpillars (pg g dry weight-')

~~~~ ~~~ Carotenoids (pg g-') Chlorophylls (pg g-') Carotenoid: Larval Qs- /3- Viola- Neo- a : b Chlorophyll colour Lutein lutein carotene xanthin xanthin Total a b Total ratio ratio

Frass of caterpillars Yellow- green 209.6 36.4 86 25.7 48.6 406 2299 197 2496 11.7' 0.16 White 237.4 55.3 100 23.7 20.6 437 6494 552 7046 11.8 0.06 Gut sacs (five specimens per extraction) Yellow- green 25.9 2.5 16.2 2.9 - 47.5 6.5 4.1 10.6 1.6 4.5 Dull green 24.7 7.8 34.0 - - 66.5 23.2 28.8 52.0 0.8 1.3

White 5. I * 5.0 - - 10.1 * * *- ~

*, Trace present; -, absent. CAROTENOIDS IN POPLAR HAWKMOTH CATERPILLARS 463 TABLE4. Pigments in the integument of eyed hawkmoth caterpillars (pg g dry weight-’)

Carotenoids Chlorophylls

Larval Food Cis- a:b colour plant Lutein lutein a b ratio

Yellow-green S. fragilis 69.2 t - ~ - Grey -green S. cinerea 42.9 t 19.5 31.5 0.6 Grey-green S. cinerea 54.5 4.1 - - -

Whitish green S. fragilis 27.6 * 8.2 9.1 0.9 Downloaded from https://academic.oup.com/biolinnean/article/42/4/457/2654249 by guest on 01 October 2021 Whitish green S. uiminalis 19.2 1.3 19.5 5.0 3.9

*, Trace present; t, the lutein figure includes cis-lutein; -, absent. reason for this difference is not known, though it is possible that the same mechanism determines how much carotenoid and chlorophyll is absorbed.

Pigments in the integument of the eyed hawkmoth The eyed hawkmoth has two colour forms of caterpillar in Lancashire, yellow-green and grey-green, but in addition a whitish green form was produced by rearing caterpillars in the field but enclosed in a white muslin sleeve (Grayson & Edmunds, 1989). Only five caterpillars were available for analysis, but the procedure used was the same as for the poplar hawk. The results of this analysis are presented in Table 4. The main carotenoids in the integument of this species are again lutein and cis-lutein, but there appeared to be more chlorophyll than in the poplar hawk. However, the amounts of chlorophyll varied widely and may perhaps be an artefact (for example, chlorophyll may occur just below the integument but adhere to it so that it is not always completely washed off). Although the sample size is very small, the results suggest that yellow-green caterpillars have the most lutein and whitish green ones the least, which is similar to the situation reported here for the poplar hawkmoth.

DISCUSSION Several workers have examined the carotenoids present in the bodies of Lepidoptera (e.g. Mummery et al., 1976), but very few have concentrated on the carotenoids in the larval integument. Dahlman ( 1969) extracted pigments from the integument of Manduca sexta, but he referred to them simply as ‘xanthophylls’. Clark ( 197 l), however, showed that the normal development of colour in Hyalophora cecropia larvae is dependent on the presence of lutein in the diet, and Kayser (1974) found that lutein is the predominant pigment in the integument of Pieris brassicae larvae. We have here shown that in two species of hawkmoth caterpillar the main carotenoid pigments in the integument are (all- trans-)lutein and cis-lutein, and these are also present in the gut wall, presumably having been sequestered from the food. Why caterpillars use lutein instead of any other dietary carotenoid for integument colour is not clear, but it is possible that a lutein-specific binding protein or lipoprotein is present in the integument or in the haemolymph. Although our work has concentrated on carotenoids, it must 464 J. GRAYSON El,4L. be remembered that the colour of a green caterpillar is partly due to lutein and partly to the bile pigment pterobilin (Rothschild, 1978). p-Carotene is also taken up from food by the gut wall, but it is not then moved to the integument, and its fate thereafter is unknown. It is a precursor of vitamin A which is important in insect vision, but it is not clear why dull green caterpillars should have the most j-carotene and white ones the least. Some of it may be translocated to the eyes since retinal, which is a degradation product of @-carotene, is the light absorbing prosthetic group of insect visual pigments (see

Kayser, 1982). More detailed study would be required to determine the relative Downloaded from https://academic.oup.com/biolinnean/article/42/4/457/2654249 by guest on 01 October 2021 proportions of the different carotenoids voided as frass, digested in the gut, or absorbed by the gut and translocated to various parts of the body. Kayser (1982) has recently classified Lepidoptera into two groups on the basis of their carotenoid composition: some like Pieris brassicae absorb lutein and p-carotene in equal amounts, while others like Aglais urticae L. absorb lutein in preference to @-carotene. Laothoe populi clearly belongs to the first group (see Table 3), but when we analysed the entire body of large caterpillars we found that they have four to eight times as much lutein as p-carotene. Although much of the lutein is deposited in the integument, some passes into the eggs. Feltwell & Rothschild (1974) have suggested that the lutein in the eggs of P. brassicae may protect the developing embryo from solar radiation, but this is unlikely to be true for L. populi because females tend to lay eggs on the undersides of leaves where insolation is low. Grayson & Edmunds (1989) have shown that whether a caterpillar becomes white or yellow-green depends on visual cues perceived by the young larva: when second and third instars are kept on white surfaces they become white while when they are reared on green, grey or black surfaces they tend to become yellow-green. There must therefore be a system by which the visual cue perceived by the eye causes the gut wall either to take up lutein and produce a yellow-green caterpillar, or to reject lutein and so to produce a white caterpillar. By contrast the colour of dull green caterpillars is under genetic control (Grayson & Edmunds, 1989). Dull green larvae take up lutein in the gut wall but deposit much less of it in the integument than do yellow-green caterpillars. The hormonal or other control system that mediates these processes is completely unknown.

ACKNOWLEDGEMENTS This work was supported by the Natural Environment Research Council (Grant no. GR/4365). We also thank Dr Janet Edmunds for critically reading the manuscript, and an anonymous referee for helpful comments.

REFERENCES BARRY, P., 1988. Faclors aJec1ing carolenoid content and composition of higher plan6 tissues. Unpublished Ph.D. thesis, University of Liverpool. BRITTON, G. & GOODWIN, T. W., 1971. Biosynthesis ofcarotenoids. Melhods in Enumology, IN,':654-701. BRITTON, G., GOODWIN, T. W., HARRIMAN, G. E. & LOCKLEY, W. J. S., 1977. Carotenoids of the ladybird beetle, Coccinella septempunclala. Insect Biochemistry, 7: 337-345. CLARK, R. M., 1971. Pigmentation of Hyalophora cecropia larvae fed artificial diets containing carotenoid additives. Journal of Insect Physiology, 17: 1593-1598. CAROTENOIDS IN POPLAR HAWKMOTH CATERPILLARS 465

DAHLMAN, D. L., 1969. Cuticular pigments of the tobacco hornworm (Manduca sexta) larvae: effects of diet and genetic differences. Journal of Insect Physiology, 15: 807-814. DAVIES, B. H., 1976. Carotenoids. In T. W. Goodwin (Ed.), Chemistry and Biochemistry of Plant Pigments, Vol. 2: 38-165, 2nd edition. London: Academic Press. EDMUNDS, M. & GRAYSON, J., 1991. Camouflage and selective predation in caterpillars ofthe poplar and eyed hawkmoths (Laothoe populi and Smerinthus ocellatn). Biologirnl Journal of the Linnean Soriep, 42: 467-480. FELTWELL, J., 1978. The distribution ofcarotenoids in insects. In J. B. Harborne (Ed.), Biochemical Aspects of Plant and Animal Coeuolution: 277-307. London: Academic Press. FELTWELL, J. & ROTHSCHILD, M., 1974. Carotenoids in thirty-eight species of Lepidoptera. Journal of