Biological Journal oJthe Linnean Sociely (1991),42: 457-465.With 1 figure
Carotenoids and colouration of poplar
hawkmoth caterpillars (Laothoe populi)
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 3 B X
Receiued 9 NoNovember 1989, accepted f o r publication 7 March 1990
Carotenoids and chlorophylls a and b were extracted from final instar caterpillars of the poplar
hawkmoth (Laothoe populi) 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 insect: 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
caterpillars - plant pigment sequestration.
-
lutein - colouration
-
Laofhoe populi
-
hawkmoth
CONTENTS
Introduction . . . . . . . . . . . . . . . . . . .
Material . . . . . . . . . . . . . . . . . . . .
Method of analysis
. . . . . . . . . . . . . . . . .
Results
. . . . . . . . . . . . . . . . . . . .
Carotenoids and chlorophyll in food plants
. . . . . . . . . .
.
Carotenoids and chlorophyll in the integument of poplar hawkmoth caterpillars
Fate of carotenoids and chlorophyll ingested by caterpillars
. . . . . .
Pigments in the integument of the eyed hawkmoth
. . . . . . . .
Discussion
. . . . . . . . . . . . . . . . . . .
Acknowledgements
. . . . . . . . . . . . . . . . .
References
. . . . . . . . . . . . . . . . . . .
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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 animals. 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 animal, carotenoids may be stored
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
Lepidoptera (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 butterfly (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 insects’ 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 willow),
S. cinerea L. (sallow), S. uiminalis L. (osier) and Populus 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. I n 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
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, 1971).
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). I t 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.
J. GRAYSON ET AL.
460
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
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
Plant
Lutein &
cis-lutein
/3-caro- Violatene
xanthin
S. fragdzs
413.0
258.3
47.5%
S.cinerea
S. vtmtnalis
555.9
46.494
614.9
45.30%
P. alba
449.7
44.S0/,
29.7%
363.5
30.4%
507.7
37.4%
317.1
31.5%
Chlorophylls
Neoxanthin
Carotenoid:
a : b chlorophyll
Total Ch1.a Ch1.b Total ratio
ratio
115.2
869.8 4216 1382 5598
3.1
0.I5
6568
2.7
0.18
78.1
156.2
1356.9 5750 2164 7915
5.e0 11.5°/0
2.7
0 17
2.9
0.11
83.3
9.So/,
128.2
10.7T0
127.5
12.7%
13.2”/,
149.8
1197.4 4798 1771
12.5%
113.9
11.2%
1008.2 6752 2338 9090
CAROTENOIDS IN POPLAR HAWKMOTH CATERPILLARS
461
TABLE
2. The pigments in the integument of 48 hour starved poplar hawkmoth larvae (pg g dry
weight - )
'
Larval
Larval
food plant
rolour
Lutein
Cis-lutein
/?-carotene
72.7
146.4
75.4
46.9
80.0
82.2
57.4
5.7
9.7
*
*
*
Yellow-green
S. fragilis
S. fragilis
S. fragilis
S. fragilis
S. fragilis
S. fragilis
S. fragilis
t
70.9
3.3
P. alba
P. alba
P. alba
29.4
32.3
37.8
8.7
2.2
4.0
S. fragilis
S. fragilis
S. fragilis
S. fragilis
S. fragilis
S.fragilis
S. fragilis
5.0
12.5
21.1
13.2
21.6
27.8
37.7
I .6
S. cinerea
27.4
S.fragilis
P. alba
P. alba
P. alba
0.08
0.07
n.d.
0.16
0.08
0.05
0.08
*
7.7
6.1
4.1
4.4
S. cinerea
Cis-lutein:
lutein ratio
*
*
*
*
*
*
0.08
0.30
0.07
0.1 I
*
Dull green
*
t
*
t
*
0.32
n.d.
0.06
0.11
0.06
0.06
n.d.
9.8
*
0.36
4.13
n.d.
n.d.
n.d.
2.77
3.43
0.9
n.d.
n.d.
n.d.
n.d.
-
-
n.d.
n.d.
n.d.
*
*
I .3
I .4
I .2
1.6
White
*, 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
lorvoe
Dull green
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-').
J. GRAYSON E l AL.
462
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
Table 3 gives the carotenoid and chlorophyll content of frass from yellowgreen and from white caterpillars that had all recently fed on P. alba. I t 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
TABLE
3. Pigments in the frass and in the gut sacs of final instar poplar hawkmoth caterpillars
(pg g dry weight-')
~~~~
Carotenoids (pg g-')
Larval
colour
Qs-
Lutein
lutein
/3ViolaNeocarotene xanthin xanthin Total
Frass of caterpillars
Yellow209.6
36.4
green
White
237.4
55.3
White
48.6
406
2299
197
2496
11.7'
0.16
100
23.7
20.6
437
6494
552
7046
11.8
0.06
1.6
4.5
0.8
1.3
7.8
34.0
5. I
*
5.0
absent.
b
25.7
24.7
*, Trace present; -,
a
Carotenoid:
a : b Chlorophyll
Total ratio
ratio
86
Gut sacs (five specimens per extraction)
Yellow25.9
2.5
16.2
2.9
green
Dull
green
~~~
Chlorophylls (pg g - ' )
-
47.5
-
-
-
-
66.5
10.1
6.5
4.1
10.6
23.2 28.8
52.0
*
*
*
-
~
463
CAROTENOIDS IN POPLAR HAWKMOTH CATERPILLARS
TABLE
4. Pigments in the integument of eyed hawkmoth caterpillars (pg g dry weight-’)
Carotenoids
Larval
colour
Yellow-green
Grey-green
Grey-green
Whitish green
Whitish green
Food
plant
S.fragilis
S. cinerea
S. cinerea
S.fragilis
S. uiminalis
Lutein
69.2
42.9
54.5
27.6
19.2
Cislutein
t
t
4.1
*
1.3
Chlorophylls
b
a:b
ratio
19.5
31.5
0.6
-
-
-
8.2
19.5
9.1
5.0
0.9
3.9
a
-
-
~
*, 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 ( 197l ) , 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 (alltrans-)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 E l ,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
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 u p 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.
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