Biological J o ~ r n a lofthe Linnean Society (1988), 33: 17-50. With 3 figures
Pupal polymorphism in the butterfly
Danaus chrysi’pus (L.): environmental,
seasonal and genetic influences
DAVID A. S. SMITH. F.L.S.
Department of Biology, Queen’s Schools, Eton College,
Windsor, Berkshire S L 4 6E W
EDDIE A. SHOESMITH
Department of L$e Sciences, Uniuersily of Buckingham,
Hunter Street, Buckingham MK18 1EG
AND
ALLISTER G. SMITH
Department of Biology, Oxford Potytechnic,
Headington, Oxford, O X 3 OBP
Received 15 December 1986, accepted f o r publication 13 February 1987
The pupae of the tropical butterfly Datraus chrysippus are either green or pink the switch being
operated by a ‘greening’ hormone produced in the larval head. Both environmental and genetic
cues are involved in controlling the endocrine mechanism.
T h e environmental factors identified are of two distinct kinds: proximate factors influence pupal
colour after the larva has selected its pupation site, whereas ultimate factors are effective at an
earlier stage, either prompting choice of pupation site by the larva or priming pupation physiology
in a particular direction. Genetic factors preadapt the larva to form a pupa which will be cryptic in
the normal or average conditions, climatic or biogeographical, anticipated in its environment.
T h e proximate factors demonstrated are background colour, darkness, light quality (wavelength)
and humidity. There is some evidence that substrate texture may also be relevant. Ultimate factors
are temperature, humidity and species of larval foodplant. Two closely linked gene loci which
govern the phenotype of adult morphs and races either have a pleiotropic effect on pupa colour or
are closely linked with other genes which do so. Moreover, the two loci interact epistatically with
respect to their pupation effects.
Factors producing predominantly green pupae are plant substrates, yellow background, darkness,
yellow light, high humidity, high temperature, the b allele at the B locus when homozygous and, on
non-plant substrates, the C allele at the C locus. High frequencies of pink pupae result on non-plant
substrates, red backgrounds, in blue light, low humidity, low temperatures and in B- and cc
genotypes. T h e C locus alleles, C and c, interact epistatically with the B alleles, their effect on choice
of pupation site being determined by linkage phase. Of the two foodplants tested, Calotrnpis
produced a high frequency of green pupae and Tylnphora of pinks. T h e seasonal cycling of rainfall,
temperature, availability or condition of foodplant, and gene frequencies are all correlated with
oscillations in the frequencies of green and pink pupae. Though genotype influences pupa colour,
.I/
0024-4066/88/010017 f 3 4 SOS.OO/O
0 1988 T h e Linnean
Society of London
D. A . S . S M I T H E T A L
18
,111 genotypes are capable of forming pupae of both colours. T h e variation can therefore be
attributed to an environmental polyphenism superimposed upon a genetic polymorphism .
The hormone producing green pupae emanates from the head during the prepupal period .
Denied hormonal influence, the pupa is pink . Pupal colour is judged to be aposematic at close
range and cryptic at distance .
K E Y t2'ORDS:
Aposematicism - apostatic selection - biogeography - crypsis - carotrnoids endocrine action - environmental cues - epistasis - larval behaviour .pleiotropy
.. polymorphism
polyphenism proximate factors .pupation ultimate factors.
Uanauj rhryrippu
~
~
~
CONTENTS
I ntroduc ti on . . . . . . . . . .
Description of the D . chrysippus pupa
. . . .
The gcnctics of D . chrysippus
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Alaterials and methods
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T h e experiments in Liverpool . . . . .
'The experiments in Dar es Salaam .
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Observations at Eton .
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Statistical methods
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Results
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The influence of plant and off-plant substrates .
Seasonal heterogeneity at Dar es Salaam . .
'l'he influence of background colour
. . .
Thc irilluence ofdarkness .
. . . . .
Thc influence of light quality (wavelength) . .
T h e influence of relative humidity (RH) . .
T h e seasonal effrct of variation in rainfall . .
T h e influenre of temperature: proximate effects
The influence of temperature: ultimate effects .
'The influence of larval foodplant .
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Genetic influences
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Ligation experiments .
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Discussion
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Classification of influences on pupation
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Proximate factors . . . . . . . .
U 1tima te factors . . . . . . . .
Larval feeding behaviour and pupation
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Genetic factors
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Biogeographical considerations
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Endocrine mechanisms
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Concluding remarks .
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Acknowledgements
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References
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INTRODU C T l O N
The pupae of many species of butterfly occur in two or more phenotypes.
most commonly green and brown although various other colours are
characteristic of particular species. Poulton ( 1887) showed that pupal coloration
in Aglais (Vanessa) urticae (L.), (Nymphalidae). Pieris brassicae L . and Artogeia
(Pieris)rupue (L.) (Pieridae) is considerably influenced by the colour of the
background on which pupation occurred . One of us (A. G . Smith. 1980) has
confirmed and extended Poulton's work on the two pierid species and also
Artogeia (Pieris)nupi (L.). The same phenomenon has been studied in at least ten
species of Papilionidae which exhibit the green-brown dimorphism
(Sevastopulo. 1948. 1974. 1975; Hidaka. 1956; Owen. 1971; Clarke &
Sheppard. 1972; Wiklund. 1972; A . G . Smith. 1976. 1978). The pupae of
Euploea core (Cramer) and Danaus chrysippus (L.) (Nymphalidae: Danainae) are
also polymorphic (A. G . Smith. 1976. 1978; Rothschild. Gardiner & Mummery.
PUPAL POLYMORPHISM IN D.CHRYSZPPUS
19
1978). Poulton's findings in respect of pupal response to background have been
extended since his time to include numerous species from several butterfly
families.
Many other factors in addition to background colour have been either
demonstrated or postulated as influences on pupal colour in particular species:
(1) Light quality (A. G. Smith, 1978, 1980).
(2) Size, shape or area of available substrate (Clarke & Sheppard, 1972; A.
G. Smith, 1978, 1980).
(3) Substrate texture (Sevastopulo, 1974, 1975; A. G. Smith, 1978, 1980).
(4)Relative humidity (Ishizaki & Kato, 1956; A. G. Smith, 1978, 1980).
(5) Temperature (Ishizaki & Kato, 1956; A. G. Smith, 1978, 1980).
(6) Photoperiod (Sheppard, 1958; West, Snellings & Herbeck, 1972; A. G.
Smith, 1978, 1980). (This factor is unlikely to be relevant to a predominantly
tropical species such as D.chrysippus.)
(7) Density of pupating larvae (Poulton, 1887) but apparently not
subsequently confirmed.
(8) Pupal diapausing behaviour (Ishizaki & Kato, 1956); diapause has not
been recorded in D. chrysippus.
(9) Season of the year (Owen, 1971) which could of course result from
variation in several of the above factors.
(10) The possibility that genetic factors might influence larval choice of
pupation site or response to environmental cues was pointed out by Owen
(1971) and has been demonstrated in Papilio polyxenes Fab. by Hazel (1977).
Although the existence of two or more distinct phenotypes in a population
suggests the possibility of direct genetic control, this appears not to be the case.
Segregation for alternative colours, governed by alleles, would produce
recognizable Mendelian ratios and this expectation does not fit the known facts
for either Papilio demodocus Esper (Owen, 1971) or D. chrysippus (this paper). A
common result is for almost all pupae within a brood to be of one colour and a
few of the other. Previous authors have therefore concluded that environmental
factors alone determine pupal colour and many such effects have indeed been
demonstrated (A. G. Smith, 1976, and references therein).
We describe here the influence, including the importance of their timing, of
several environmental factors on the determination of pupal colour in
D. chrysippus. We also investigate the possibility that there may be a genetic
component with the potential to produce a pupa of a particular colour. As the Dar
es Salaam population is polymorphic at three loci which are expressed in the
adult phenotype (D. A. S. Smith, 1975a, b, 1980), we have examined the Dar es
Salaam broods for correlations between pupation site, pupa colour and larval
genotype as revealed by the adults which enclosed successfully.
Our results come mainly from two series of experiments on pupation in
D.chrysippus carried out in Dar es Salaam (D.A.S.S.) and Liverpool (A.G.S.)
during 1973-1975. The data have been analysed statistically by E.A.S. In the
Dar es Salaam experiments we investigate the contribution of temperature,
rainfall (humidity), genetic factors and the selection of putation sites by larvae
to the marked seasonal variation in pupal polymorphism, which seems in some
respects to resemble the situation described for P . demodocus by Owen (1971).
The Liverpool results form a relatively small part of an extensive study (A. G.
20
D. A. S. SMITH E T A L
Smith, 1976) which included Papilio polytes L., P. demoleus L., P. polyxenes (A. G.
Smith, 1978), A . rapue, A . nupi and P. brassicae (A. G. Smith, 1980). T h e
papilionid and pierid species provide a convenient canvas against which to
compare our findings. O n the one hand, the D. chrysippus experiments in
Liverpool were specifically designed to discover and distinguish the influence on
pupa colour, immediately prior to pupation, of background colour, light
quality, relative humidity and temperature. T h e conditions were more precisely
controlled than was possible at Dar es Salaam. I n addition, some simple ligation
experiments were performed on prepupae which point to the physiological
mechanisms involved. O n the other hand, the continuity of the Dar es Salaam
observations, gathered over 14 successive months of variable weather, enable us
to study genetic and climatic variables and their influence over the whole period
of larval development. T h e two different approaches are thus complementary.
DESCRIPTION O F T H E PUPA OF D.CHRYSIPPUS
‘Two distinct phenotypes occur (Fig. 1) although both exhibit continuous
variation. The first is glossy green varying from exceedingly pale to deep jade.
‘The second is most commonly skin pink to dull white, “resembling a lump of
candle wax” (Rothschild el al., 1978), or occasionally pale peach or a yellowish
shade. Pupae of both types are adorned by a narrow gold band or diadem,
named the gddglanz (golden glance) by F. Muller (in titt. to E. B. Poulton,
1887), which consists of a series of tiny sparkling protuberances running along
the edge of the anterior keel. T h e potentially startling effect of the diadem,
especially on a pale ground colour, is highlighted by a thin line of black pigment
immediately below the keel. T h e pupa is further adorned by eight small
spangles of gold which are distributed according to a well defined pattern over
the body. Rothschild et al. (1978) have shown that carotenoids @-carotene and
lutein), derived from the larval foodplant, are a prerequisite for the
development of the golden diadem and flecks in the larger but otherwise similar
pupa of Danaus plexippus ( L . ) . If reared on an articifical diet lacking carotenoids,
the golden diadem is replaced by one of silver.
THE GENETICS OF D. CHRYSIPPUS
Adult D. chryszppus may vary at three gene loci which determine the colour
markers used in this study to identify genotypes (Fig. 1 ) : most Asian and
Australasian races are monomorphic with respect to these loci but populations
in East Africa are polymorphic €or all three, in West Africa at the B locus and in
Malaysia at the A locus (Table 1 ) Each locus has two identified alleles. The A
(ulcippus) locus determines presence or absence of a large patch of white on the
hindwing, which in genotype A A is orange or brown, the ua genotype having the
white patrh. T h e alleles at the B (brown) locus govern the ground colour of
both wings which may be orange or brown. T h e B allele gives brown while the
66 genotype is orange (Clarke, Sheppard & A. G. Smith, 1973). Bb
heterozygotes may be indistinguishable from BB but are often intermediate with
a brown forewing and a variable, sometimes largely orange, hindwing
(Fig. 1B). T h e C (chrysippus) locus has a dominant allele C, the CC genotype
having a forewing uniformly coloured (orange or brown) except for a black
PUPAL POLYMORPHISM IN D.CHRYSIPPUS
Figure 1. A-H, The phenotypes (genotypes in parenthesis) of Danaus chryszpjus from the
polymorphic population at Dar es Salaam (see Table 1). Top row, left to right: orange dorlf~pus(AA
bb C-); brown dorippus ( A A B b C-); orange albinus (aa bb C-). Second row, left to right: orange
aegyptius ( A A bb c c ) ; B brown aegyptius (AA B- cc); orange alcippus (aa bb cc). Third row, left to right:
orange semi-albinus ( A a bb C - ) ; orange alcippoides ( A a bb cc). Bottom left: the two phenotypes of
D.chrysipjus pupa, green (left) and pink (right). Note the diadem underlined in black with melanin
and the gold spangles on the body. Bottom right: a green pupa formed on the underside of a jackknifed (see p. 43) leaf of Asclepzas curussavica. Note the quality of the camouflage and the prominence
of the diadem.
21
22
D. A. S. SMITH E T AL.
TABLE
1. The genotypes, races, morphs and origins of D.chyippus used in this study
Genotype
na bb cc
Race
chrysippus
Morph
Origin
alcippus (orange)
Malaysia
aa 66 cc
en B- rr
aegyptius
alcippus (orange)
~k;Pp.r(brown)
Sierra Leone
A 3 bb
chrysippus
chrysippus (orange)
Sri Lanka
aegyptius
dorippus [brown)
dorippus (orange)
aeuptius (brown)
aepptius (orange)
albinus (brown)
albinus (orange)
alcippus (brown)
alc$)us (orange)
Uganda, Kenya, (Nairobi) and Tanzania
(Dar es Salaam)
cc
A- bh cc
aa B- Caa bb Can B- ci
aa bh cc
margin. The cc genotype has a large area of black at the apex of the forewing,
traversed by a row of white sub-apical spots. Many Cc individuals show traces of
the sub-apical spots, picked out in pale brown or orange, on the underside and
occasionally the upper side of the forewing. The extent to which heterozygotes
for any of the three loci are identifiable varies between populations, morphs
within populations, sexes and broods.
The B and C loci are linked fairly closely with a cross-over value ~ 4 y ;in
males (D. A. S. Smith, 1975a, b): crossing over has not been detected in females
and meiosis may be achiasmatic in this sex, as usual in Lepidoptera. Epistatic
interaction between the B and C loci has been demonstrated (D. A. S. Smith,
1980) and is tested for here as a possible influence on pupation. The A locus
probably assorts independently from the other two although the possibility of
loose linkage cannot yet be rejected.
For the purposes of this paper, we use only the frequency of the recessive
phenotypes for multiple regression analysis as distinguishing homozygous from
heterozygous dominant phenotypes by eye is not sufficiently reliable. For the
same reason, we have not used gene frequencies.
MATERIALS AND METHODS
The experiments in Liverpool
The experiments investigating the effect of background colour, of darkness
and of temperature on pupal colour determination were done with a stock
derived from Sierra Leone butterflies (genotypes aa B- cc and aa bb cc). The
humidity and ligaturing experiments used material of Ugandan origin
(genetically diverse); and Sri Lankan material (genotype AA bb cc) was used for
the light wavelength experiment.
Butterflies were free-flying in two heated greenhouses at the University of
Liverpool. Larvae were supplied with Asclepias curassauica L. and Gomphocarpus
PUPAL POLYMORPHISM IN D.CHRYSIPPUS
23
fruticosus (L.) Ait. as foodplants. Mature, fully-fed larvae preparing to pupate
often leave their foodplants and wander about in search of a pupation site.
Larvae have a conspicuous colour pattern of yellow, white and black bands and
possess three pairs of dorsal filaments. When preparing to pupate, the filaments
take on a withered appearance and their red bases become a lighter orange
colour. Also, t h h d y may become slightly contracted and develop a pink tinge.
These features are variable in expression and larvae were often selected for
experiments because of their large size rather than for any obvious indication of
approaching pupation.
The injuence of background colour
Fully-fed larvae were put into cylindrical tubes approximately 300 mm tall,
made from clear cellulose acetate sheeting, 0.25 mm thick, which had been lined
with a single layer of coloured tissue paper. One set of tubes was left unlined (see
Table 5). The cylinders were secured at the seam with paper clips and the
diameter was adjusted so that the ends fitted snugly into the top and bottom
sections of a 90-mm diameter ‘Sterilin’ plastic Petri dish, also lined with
appropriately coloured tissue paper. The cylinders were kept continuously
illuminated at approximately 25°C. No more than two larvae were put together
in any one cylinder.
The injuence of darkness
The importance of light cues to pupating larvae was tested using only red and
yellow lined cylinders (as described above), these colours having been found to
favour the occurrence of pink and green pupae respectively. Mature larvae were
randomly assigned to relevant cylinders and then left to pupate in conditions of
either continuous light or darkness at 25°C.
The injuence of light quality (wavelength)
The influence of light wavelength experienced by pupating larvae was
investigated by filtering light from coloured fluorescent light tubes (Thorn Atlas
Lighting Ltd.) through various Cinemoid filters (Strand Electric Ltd.). The
distribution of wavelengths obtained is shown in Table 8.
For this experiment, wooden compartments 0.25 x 0.50 x 0.40 m were
constructed. The front of each compartment was covered with the appropriate
Cinemoid filter(s) except that for white light, which was left open. T h e top of
each compartment could be removed to allow insertion and removal of
materials. The inside of each compartment was lined with reflective ‘Bacofoil’
aluminium foil.
Mature larvae were confined in clear acetate cylinders placed in each
compartment. The fluorescent light sources were fixed vertically to a supporting
framework approximately 0.3 m in front of each compartment. Black card
shields were used to screen each compartment from adjacent ones. Temperature
for this experiment was approximately 30°C and relative humidity was 15-23%.
The injuence of relative humidity ( R H )
Mature larvae were confined for pupation in large plastic boxes in each of
which were placed three small Petri dishes containing appropriate solutions to
regulate humidity. The three different solutions used were intended to control
24
D. A. S SMITH E T AL.
humidity as follows: saturated calcium chloride ( 3 5 % ) , saturated potassium
nitrate (757,) and water (lOO”/b) at 25°C (Weast, 1968). [In practice it
transpired that KNO, regulates RH at approximately 93% at this temperature
(Winston & Bates, 1960)]. Two replicates at each of the three humidity levels
were set up 48 h before the start of the experiment. T h e RH was checked with
cobalt thiocyanate paper, suspended on wire and inserted through a small hole
drilled in the lid of the box. After half a n hour, the papers were removed and
compared with a series of colour standards.
The experiment was performed in darkness at 25°C. T h e larvae were
introduced to the boxes through a small hole in the lid which was subsequently
plugged with cotton wool. T h e experimental design does not allow for possible
humidity gradients within the boxes, nor for any changes which might occur
while the experiment is running.
The proximate inluence o f temperature
Temperature influences at the time of pupation were studied by comparing
pupal colour frequences obtained from red and yellow-lined cylinders
maintained at either 14 or 25°C. These temperatures were thermostatically
controlled and the cylinders were illuminated continuously.
Iigation experiments
The involvement of endocrine mechanisms in pupal coloration was studied by
ligaturing larvae at different times in the prepupal stage and at different
locations on the body. Ligatures were tied using soft cotton thread and then
applied by slipping a loose knot over the head of a larva and drawing the thread
backwards until it reached the desired intersegmental position. The knot was
then slowly pulled tight. In the first experiment, larvae pupating on substrates
favourable for the production on either green or pink pupae were ligatured
midway along the body directly after they had assumed the suspended J-shaped
position in the prepupal period.
I n a second experiment the time at which hormonal influences act was
investigated. Larvae, having taken up the J-position on red or yellow tissue
paper, were ligatured between the thorax and abdomen during the first half
(early ligatured) of this period (about 6-7 h duration), or in the second half of
the period (late ligatured).
Finally, the source of the ‘greening’ hormone was sought by applying
ligatures to different parts of the body. Three positions were used, between the
head and first thoracic segment ( H / T I ) ; between first and second thoracic
segments (TI/T2); between second and third thoracic segments (T2/T3).
The experiments in Dar es Salaam
Eggs were obtained by sleeving individual females outdoors on a branch of
the principal local foodplant, Calotropus giganlea (L.) Ait. A few of the larger
broods were deposited on A . curassavica grown in a n outdoor insectary. The
larvae of each brood were raised together on one of five species of milkweed
Asclepiadaceae), the majority of C. gigantea and a few on each of C. procera
PUPAL POLYMORPHISM IN D.CHRYSIPPUS
25
(Ait.) Ait., A . curassavica, G. fruticosus and Tylophora stenoloba (K.Sch.) N.E.Br. In
retrospect, the diversity of foodplant used was unfortunate as it probably
introduced additional uncontrolled heterogeneity. By intention two broods were
split equally between C. gigantea and 1.stenoloba specifically to address the
question whether any aspect of pupation is influenced by foodplant.
All broods were reared separately in well ventilated cages measuring
1 .O x 0.5 x 0.5 m and constructed of a roughish wooden framework overlaid with
zinc gauze. The larvae were transferred daily to fresh shoots of foodplant, the
latter supported in a vertical position in jars of water. Broods larger than 20
were split between two or more cages. The cages were housed on a bench close
to a permanently open window, shaded by external steel louvres. The rearing
conditions approximated closely to those outdoors with respect to shade
temperature and humidity but the cages were always protected from direct
sunlight. When the fifth instar larvae wandered prior to pupation they were
free, either to remain on the foodplant, or to move to the lining or framework of
the cage, which was generally in contact with the plant shoots a t several points.
The pupae were left undisturbed until the winged adults had eclosed.
Meteorological data were obtained from the East African Meteorological
Department Station at Ubungo which is approximately 1 km from the
laboratory. The records used are monthly rainfall (as a n index of relative
humidity) and monthly mean temperature.
The pupal data were collected in the course of genetical studies on the colour
and pattern polymorphisms displayed on the wings of the adult butterflies
(D. A. S. Smith, 1975a, b, 1980). A potentially valuable consequence of this is
that the phenotype and some elements of the genotype of all successfully eclosed
adults could be identified. Thus, although adult genotypes cannot be matched
post facto to individual pupae, the phenotype frequency in segregations for up to
three pairs of alleles, is known for each brood which produced adults. We are
therefore able to investigate the influence of genotype on both the selection of
pupation sites by larvae and, with some caveats, the determination of pupa
colour after site selection. However, we emphasize that the experiments were not
specifically designed with the present paper in mind and certain controls were
lacking which might otherwise have been introduced. O n the other hand, the 51
broods available, of which 44 produced imagines, were raised over a period of
14 successive months and the continuity of the data renders them most suitable
for the detection by multiple regression analysis of seasonal influences on
pupation.
Two distinct hypotheses are tested, each using rainfall, temperature and
genotype as explanatory variates, with choice of pupation site and pupa colour
as response variates. I n Hypothesis I, the climatic and genetic factors are tested
for their influence in the month of pupation (explanatory variates with the suffix
‘I” below). Their effect is likely to be realized either during larval wandering
after feeding has ceased, in the course of which the larva chooses a pupation site,
or in the sensitive period, which lasts some 24 h, when the larva has stopped
wandering and is at rest on its selected pupation site, immediately prior to the
prepupal stage. I n Hypothesis 11, we investigate the possibility that the
environmental and/or genetic components are effective at an earlier (larval)
stage by applying explanatory data for the month in which larval development
below). As the
predominantly occurred (explanatory variates with the suffix ‘D’
26
D. A. S. SMITH ET A L
whole development from hatching to pupation often occurred within a single
calendar month, the two hypotheses are not mutually exclusive.
Observations at Eton
In the course of further genetical experiments on D. chrysz$pus at Eton during
1983---86,additional observations on pupation were recorded. The stocks from
which useful results were obtained had the following origins: (1) I n 1983,
butterflies of form alcippus (genotype aa bb cc), which originated from Malaysia,
were obtained from the Butterfly Farm, Bilsington, Kent and bred in Eton. The
distribution of green and pink pupae on plant and non-plant substrates was
recorded. (2) In 1986, the breeding programme involved three stocks: (a) form
chryszppus (genotype AA bb cc) from Sri Lanka, obtained from Worldwide
Butterflies, Sherborne, Dorset; (b) form dorippus (genotype AA bb C-) from
Mount Kenya and (c) a highly polymorphic stock, including forms dorippus,
aegyptius and alcippus, from Nairobi, Kenya, both supplied by Dr Ian Gordon.
The butterflies were allowed to fly free in a conservatory from late June to
mid September with occasional heat supplied only a t night. The conservatory
was planted with a variety of milkweed foodplants (Araujia sericiphora, Asclepias
syriaca, A . curassavica, Periploca laevigata and G.fruticosus). Additional plants were
also provided as nectar sources (Buddleia, Sedum, Lantana and Michaelmas
daisy) and for pyrrolizidine alkaloid sequestering (Senecio jacobea, Crotalaria
capensis and Heliotropiurn peruvianurn), the latter being essential for males to
produce the aphrodisiac pheromone used in successful courtship (Schneider et
al., 1975).
T o hasten development and provide controlled conditions, eggs were
harvested, transferred onto cut foodplant and reared in seed incubators in an
airing cupboard at a temperature which deviated little from 26°C. Five boxes of
larvae fed and pupated entirely in the dark without apparent ill effect. For
comparison, two boxes were given light from the sensitive period until pupation
was complete. The influence of light and darkness on the proportions of pink
and green pupae, all of which were suspended from the smooth transparent
plastic lids, is analysed.
Statistical methods
The following symbols are used with reference to the Dar es Salaam data:
brood identifier,
number of pupae in brood i,
number of pupae on plant in brood i,
probability of a pupa in brood i being on plant,
number of pupae on cage in brood i,
probability of a pupa in brood i being on cage,
number of green pupae on plant in brood i,
probability of a pupa on plant in brood i being green,
number of green pupae on cage in brood i,
probability of a pupa on cage in brood i being green,
rainfall during month of pupation of brood i,
PUPAL POLYMORPHISM IN D.CHRYSIPPUS
27
RD, rainfall during month of development of brood i,
TPi average temperature during month of pupation of brood i,
TDi average temperature during month of development of brood i,
pbbi proportion of surviving adults in brood i of bb genotype,
proportion of surviving adults in brood i of cc genotype.
pcc,
T h e focus of interest in the Dar es Salaam data was on relationships between
np, ngp and ngc on the one hand, and on the other hand the environmental
variates RP, RD, TP, T D and genetic variates pbb, pcc. Possible relationships
were investigated using generalized linear modelling (Nelder & Wedderburn,
1972), taking np, ngp, ngc in turn as response variate, and sub-sets of RP, RD,
T P , T D , pbb, pcc as explanatory variates. Models were fitted by the maximum
likelihood procedures in the G L I M 3.77 statistical computing program (Payne,
1985).
T h e models examined were of the form:
logi t (n** J = b’x;,
where n**, denotes one of np,, ngp,, ngc,; x1 is a column vector whose first
element is 1 and whose remaining elements are observations, for brood i, on a
subset of the environmental and genetic variates (and possibly interactions
between pairs of genetic variates); and b‘ is the transpose of a column vector of
linear parameters. If it is further assumed that individuals within broods are
stochastically independent of each other, the model implies that n**, (denoting
one of np,,ngp,,ngc,)has a binomial probability distribution with size parameter
a*, (respectively n,, np,,nc,) and probability parameter 1/ [1 exp( - b ’ x , ) ] .
A model of this form is fitted in GLIM, and estimates of the linear parameters
6 obtained, by declaring they-variate as n**, and the error term as binomial
with denominator n*. The default link function in GLIM for a binomial error
term is the logit function. Within this framework &), the estimate of the j t h
element of 6, is interpreted as the estimated increase in logit(n**) for a unit
increase in x b ) , the j t h explanatory variate, when the values of all other
explanatory variates are fixed. The model assumes bb) to be constant over all
values of n**. The corresponding estimated increase in n** for a unit increase
in xcI) is & ) n**( 1 -n**), which therefore varies with n**.
A number of transformations of the explanatory variates were investigated.
T h e genetic variates pbb and pcc were expressed as logits, or alternatively
untransformed. The estimated response surfaces were affected only marginally
by the alternative modes of expression. Power transformations of the
environmental variates were tried, but these did not in general improve the fit of
the model. The results reported below, with respect to the ‘logit’ model, have
the genetic variates expressed as logits and the environmental variates
untransformed. T h e logit expression of p b b and pcc implies that, if b3) is the
estimated linear coefficient for logit@--) (d
either pbb or pcc), the
estimated increase in n** for a ‘unit’ increase in p-- is b )n** ( 1 -n* *) /p--( 1 -p--).
Most of the fits revealed the data to be ‘over-dispersed’ relative to a binomial
distribution. I n these cases, estimated standard errors of the parameter estimates
were obtained using a heterogeneity or scale factor (Finney, 1971; McCullagh &
Nelder, 1983), calculated from the generalized Pearson x 2 goodness-of-fit
+
enotiv
D. A. S. SMITH E T A L
28
statistic (McCullagh & Nelder, 1983). Use of a scale factor is equivalent to a
quasi-likelihood model (Wedderburn, 1974; McCullagh & Nelder, 1983), in
which:
E[n**J =n*ln**l, u[n**,l =cpn*,n**,(l -n**,),
where E [ . ] denotes expectation, u[.] variance and cp the variance heterogeneity
factor. Model I1 of Williams (1982) was tried as an alternative to the use of a
straightforward heterogeneity factor, but residual plots revealed that the
mean:variance relationship embodied in Williams' model I1 was less suitable for
the data under examination than the one stated above. T h e plots in Fig. 2 ,
depicting the results of the fit for np, were constructed with the aid of the
Statgraphics program suite (S'rSC Inc., 1986).
The experimental data in Table 6 were also analysed using GLIM, in this
case declaring they-variate as the set of frequency counts in the body of the
table, the error term as Poisson, and using the three classifying characteristics
(substrate colour, light/darkness, colour of pupa) as factors.
RESULTS
The injuence of plant and off-plant substrates
The overall influence of substrate on pupal colour in the Dar es Salaam
butterflies is shown in Table 2. Averaged over the year, approximately 75y0 of
the larvae pupated on the sides or top of the cage compared with approximately
25y0 which remained on the plant. The distribution of pink and green pupae on
the two substrates is highly heterogeneous (P<O.OOl). T h e situation is similar to
that described by Poulton (1887) in pierids and vanessids and A. G. Smith
(1976) in papilionids: green pupae are much more frequent than expected on
plants and non-green ones on other surfaces.
A small brood ( n = 2 5 ) of Malaysian stock, race chrysippus f. alcippus (genotype
aa bb c c ) , reared in a greenhouse at Eton in 1983, gave a similar result:
x2(1)= 12.473; P <0.001 (with Yates' correction) which shows that the pupal
dimorphism is not confined to African race aegyptius.
Seasonal heterogeneity at Dar es Salaam
The overall effect described above was subject to considerable seasonal
variation at Dar es Salaam despite the fact that all the pupae were formed and
'I'ABLE2. T h e relation between pupation site and colour of
pupa in Danaus chrysippus a t Dar es Salaam. (Expected numbers
in parenthesis)
Pupation site
Colour
of pup"
Cage
Plant
Total
Pink
Gseen
Total
529 (430.8)
221 (319.2)
750
42 (140.2)
202 (103.8)
244
571
423
994
x',,,=211.9; P<0.001 (with Yates' correction).
PUPAL POLYMORPHISM I N D . CHRYSIPPUS
29
TABLE
3. Seasonal heterogeneity for p u p a t i o n site and p u p a l colour in D.chysippus a t D a r es S a l a a m
Pupation site
Pupal colour
.-
Montht
1974
August
September
Octobcr
November
December
1975
January
February
.March
April
May
June
July
August
September
'Iotals
Number
of
broods
Number
of
Plant
pupae
(%)
Green
Deviation§
(70)
(%I
x'
Deviation4
(46)
-
-
-
-
-
8
3
1
26
94
100
28
15.4
21.3
42.0
82.1
-11.9
-6.0
14.7
54.8
1.18
0.2 I
16.45** *
50.15***
30.8
38.3
41.0
60.7
-9.4
-1.9
0.8
20.5
2
5
1
89
149
50
43.8
30.9
16.0
16.5
3.6
-11.3
17.85***
3.22
1.97
67.4
37.6
20.0
27.2
-2.6
-20.2
10
11
4
171
150
88
~
~~
1
~
-
~
15.2
6.7
20.5
~
-12.1
-20.6
-6.8
-
-
5
49
6.1
-21.2
51
994
27.3:
~.
~
-
-
8.06**
25.89***
0.80
__
8.89**
134.75
51.5
48.7
29.5
.-
1.48
0.70
0.10
3.78
22.50***
1.51
10.41**
-
~
11.3
8.5
-10.7
5.55*
2.29
6.09*
-
-
-
16.3
-23.9
13.79***
68.19
49.2:
*P<0.05, **PcO.OI, ***P<O.OOl.
?Classified by month in which pupation occurred.
Tunweighted means.
§Difference between monthly value and unweighted overall mean.
TABLE
4. Meteorological and D.chrysipkus genetical d a t a for t h e period of study a t Dar es Salaam
Proportion of recessive
phenotypes (genotypes)
Weather
~~~~
.Man th *
1974
August
September
October
November
December
1975
January
February
March
April
May
June
July
August
September
Monthly
rainfall
(mm)
Mean
temperature
("C)
Per cent
orange
(bb)
Per cent
3
7
49
28
28
23.8
24.2
24.9
26.5
28.0
46.2
53.8
26
31
109
277
293
25
9
4
99
27.6
28.2
27.4
25.9
25.6
24.4
23.6
23.4
23.4
*Classified by month in which larval development mainly occurred.
aegvjtius
(6.1
-
-
30.3
0.0
56.8
74.3
100.0
0.0
0.0
31.0
20.8
18.1
10.0
31.6
30.4
16.7
55.6
26.2
31.3
29.8
20.0
36.8
26.1
52.4
-
-
D. A. S. SMITH E T A t .
30
remained under cover. A qualitative analysis, based on the raw data from the 5 1
broods, summarized in Tables 3 and 4,indicates that the seasonal variation had
the following aspects:
( 1 ) Pupation on plants (np) was more frequent in the hot months
(November- February) than the cooler ones (March-October) .
(2) Rainfall had no direct influence on choice of pupation site but it did
affect the frequency ‘green on cage’ (ngc).
( 3 ) Green pupae showed a bimodal frequency distribution, peaking in both
the cool and wet month of May, when they were common on off-plant as well as
plant substrates, and in the hot and drier months (November-February), when
they formed predominantly on plants.
(4)T h e only parameter showing no seasonal heterogeneity was the frequency
of ‘green on plant’ (ngp).
Similar seasonal variation was described by Own (1971) in Papilio demodocus in
Sierra Leone although his observations did not extend to choice of pupation site,
being confined to pupal colour (green/brown).
In the remainder of this section we examine the individual influence of several
environmental and genetic factors, on both choice of substrate and pupa colour,
as revealed by controlled experiments and multiple regression analysis.
The izjuence of background cotour
The influence of background colour on pupal coloration was tested in eight
treatments by allowing larvae to pupate in cylinders on either tissue paper
backgrounds of seven colours or colourless acetate sheet (Table 5). T h e
heterogeneity between backgrounds is highly significant ( x 2 ( 7=
) 7 1.1; P < 0.001).
Pink pupae were significantly more frequent than expected on green, white and
especially red backgrounds. Green pupae exceeded expectation very
significantly only on a yellow background on which they were twice as frequent
(65O;) as on any other type. It is clear that background colour exercises a
considerable influence on pupal colour. Poulton (1887) showed that in Pieris
brassicae and Artogeia rapae yellow and orange backgrounds have a strong
tendency to produce green pupae while substrates of other colours produced
TABLE
5. Thc influence of background colour on pupal coloration in L). ch~se‘ppus
Pupal colour
Cylinder
colour
~
Green
Pink
Red
Orange
Yellow
Green
Blue
BIown
White
Acetate
2
13
26
I
5
3
8
40
27
14
39
35
31
37
32
Totals
68
255
10
*P<0.05; **P<0.01;***P<0.001.
-~
~
Total
x2
4.8
32.5
65.0
2.5
12.5
24.4
7.5
25.0
42
40
40
40
40
41
40
40
6.706* *
3.154
46.482* * *
8.284**
1.760
0.275
4.420*
0.027
21.1
323
7 1.108***
Green
PUPAL POLYMORPHISM IN D. CHRYSIPPUS
31
mainly brown or non-green pupae. Danaus chrys$pus appears similar to the
pierid species in this respect.
The injuence of darkness
The data (Table 6) show a highly significant difference for pupa colour, in
the expected direction, between the two (control) illuminated samples on red
and yellow backgrounds ( P < O . O O l ) . However, in darkness the proportions of
the two colours did not differ significantly between substrates (P=0.7) and the
numbers of greens and pinks were more evenly balanced. Although the light
and dark treatments did not produce significantly different results on yellow
backgrounds (P=0.09), there was a significant difference on red backgrounds
( P < 0.001). Pupal coloration therefore results from an interaction between the
colour of the background on which pupation occurs and the presence or absence
=
P<O.OOl). As yellow and red backgrounds only produce
of light ( x ~ ( ~ ) 13.9;
significantly different results in the presence of light, reaction to the background
colour by the pupating larva is clearly an important factor in determining its
coloration. Danaus chrysippus always passes through the sensitive period during
the day, when light cues will operate. The pupal cuticle is often completed
during the night following the sensitive period though the timing is
temperature-dependent.
Butterflies of Sri Lanka stock, bred a t Eton,’ were also used to test the
influence of darkness. The larvae consisted of two first generation broods from
parents, which may also have been sibs, obtained from Worldwide Butterflies.
The genotype of all 84 offspring obtained was A A bb cc.
The larvae were raised in groups of 10-15, on cut foodplant, in seed
incubators, at a temperature of 26°C and 100% humidity. All pupae except five
(which are omitted) were suspended from the transparent plastic lid. The results
(Table 7) show that in darkness, when the sensitive larvae are unable to respond
to light quality, green pupae predominate, whereas in white light, on a
colourless background, most larvae are pink. The difference is very highly
significant (P<O.OOl).
I t should also be noted that the frequency of greens in darkness was higher
(75%) in the Eton experiment (Table 7) than in the two Liverpool experiments
involving both darkness and rough pupation substrates (57%) (combined result
from Tables 6 and 8); the difference approaches significance ( x * ( ~ ) =3.443 with
TABLE
6. T h e influence of light and darkness on pupal colour
determination in
D.chrysippus
Pupal colour
Conditions
for pupation
Green
Pink
Total
Yellow TP* in light
Yellow T P in darkness
Red T P in light
Red T P in darkness
15
9
4
I
11
9
15
7
19
18
16
18
Totals
36
35
71
*TP=Tissue paper.
D. A. S. SMITH E T A L .
32
‘I‘ABLE7. The influence of light and darkness on pupal rolour in
5ibling D.c h y i p p u s form chyszppus from Sri Lanka, when pupation
occurs on a clear, smooth plastic surface at 26°C. (Expected
numbers in parenthesis)
Pupal Lolour
~-
C:ondi tions
For pupation
Darkness
Light
.rotais
Grccn
Pink
Total
45 (35.7)
5 (14.3)
50
15 (24.3)
19 (9.7)
34
60
24
84
%’,,,=
18.689; P<0.001 (with Yates’ correction]
Yates’ correction; O.lO>P>O.O5). If this difference is real, it might result either
from the high humidity (see below) or the smooth substrate in the Eton
experiment (A. G. Smith, 1978). O n the other hand, if the Eton result is
compared with the Liverpool result (50”,; green) at high RH (93 100(’,1,
Table 9), also on a smooth plastic surface, the higher frequcncy of greens at
Eton is highly significant (x2(,)=8.771; P<O.Ol with Yates’ correction). The
latter comparison suggests that genetic factors might be responsible. HoweL cr,
comparing the Eton result in light with that in Liverpool on acetate (Table 5)
(also a smooth surface) gives x2(,,=0.058; 0.9>P>0.8: the rcsults arc
homogeneous.
+
The intuence o f light gualzty (wavelength)
The results of this Liverpool experiment (Table 8) show that light quality at
certain wavelengths is a powerful influence on pupa colour. I n particular,
yellow light (580-590 nm) produces a high frequency of green pupae, while blue
light (370 -500 nm) is uniquely effective in producing pinks. When the yellow
and blue samples are removed there is no significant heterogeneity remaining
= 3.056; 0.7>P>0.5): in other words, the other wavelengths used are
neutral insofar as pupal coloration is concerned.
(x’
I‘ABLE 8. The influence of light quality on pupal colour determination in D.chryJipjws
Pupal coloui
~-
LV<I\rlcngth
range (rim)
~
640-670
640-700
580-590
500-570
370-500
300-400
350-700
Pink
cl,)
~~
~-
-
14
14
15
I1
10
3
12
23
I1
13
10
94
93
14
II
3
14
9
-~
~
56 0
58 3
82 4
47 8
II 5
56 0
40 9
60 0
25
24
17t
23
26
25
22
25
50 3
~
P
’l’ot’ll
Grrcn
187
~
~~~
0.329
0.625
7.001**
0.055
1M O O * * *
0.329
0.i71
0.947
25,6j7***
~~
* * 1’ < 0.0 I : * * * P < 0 .OO 1 ,
t’l’lir sodium light w a s brokrn during thr exprrirnerit and could not be rrplacrd hefore thr o l d .
33
PUPAL POLYMORPHISM I N D. CHRYSIPPUS
The injuence of relative humidity ( R H )
No significant differences are observed between replicates at each RH level
(Table 9) and the data are pooled for comparison between levels. There is
= 7.4;
overall heterogeneity between the three humidity levels (x2(*)
0.05 > P > 0.02). Pairwise comparisons show significant differences between 35
and 93% R H (P=0.03) and between 35 and 1 0 0 ~ oRH (P=O.Ol) but not
between 93 and 100% (P=0.84).As the experiments were performed in
darkness, with a smooth transparent substrate for pupation, comparison with
the other experiment in which similar conditions obtained (Table 8) suggests
that, excepting R H , the other factors (darkness, substrate texture and
temperature) are unlikely to favour either pink or green. Therefore, we can
safely conclude that humidity exercises a role in determining pupal colour,
higher frequencies of pink pupae being produced at low than at high R H .
The seasonal efect o f variation in rainfall
The seasonal distribution of rainfall and, consequently R H , which was not
measured directly, is usually bimodal at Dar es Salaam, with ‘long rains’
centred on April and May and ‘small rains’ on December. However, the latter
are exceedingly variable in both timing and amplitude and in 1974-75 the
annual record is barely bimodal (Table4), September 1975 was
uncharacteristically wet.
The wettest month of May was associated with a significantly low proportion
of pupae formed on plant substrates (P<O.Ol) and yet the frequency of green
pupae was significantly high (P<0.05) (Table 3 ) . One result of the regression
analysis shows that ‘green on cage’ (ngc) is significantly and positively related to
rainfall at the time of pupation (RP)(P<0.05) and even more so (P%O.Ol)
It should
when matched against the whole period of larval development (RD).
be noted that the previous month (April) when much of the larval development
occurred, was also very wet. These results show that, despite the predominance of
off-plant pupation in May, a condition normally expected to produce a high
frequency of pinks (Table 2), green pupae predominate (51.5%). This is in
accord with the Liverpool experiment (Table 9) in suggesting a positive
proximate role for R H in producing green pupae.
O n the other hand, the highest frequency of green pupae occurred in the hot,
relatively dry month of January, associated in this case with pupation on plant
TABLE
9. The influence of relative humidity on pupal colour determination in D.
chrysippus
Replicate I
Relative
humidity
Replicatr I1
~
~~
Pupal colour
Total
.-
~~
Pupal colour
~
Total
~
(“0)
Green
Pink
4
Green
Pink
35 (CaCl,)
93 (KNO,)
100 (H,O)
19
12
11
23
23
21
7
11
14
11
10
13
I1
21
23
24
‘lotals
25
42
67
31
37
68
12
D. A. S. SMITH L'T .iL
34
' I ' A I ~ I X 10. Summary of' the results of multiple regression analysis on the Dar
Explanatory \ ariahlcs
- 12.3
0.44
0.30
-0.10
- 1.49
0.0037
-0.24
- 1.57
2.8
0.1 1
0.081
0.029
0.24
0.00 I4
0.084
0.18
eg
Salaam data
l)e\ ianw
.Icc(iiintcd
k1r
P-value*
Residual
0 0002
0 002
130
111
4h",,
40
I29
58
31",,*
'3 8
4.1
3.7
-3.5
d.f
0 005
2.6
-2.9
0.01
0.004
32
40
1 ' . =logit(. ; s.b.., e s t i m a t r d standard rrror; d L , drgrers offreedom.
*'l'hr significanrr levrls ( x ' ~ , ,a) r r derived from thc changc in residual deviance o n omission of thc rclcv,int
term from the equation. T h e y arr rrgarded as approximate and are therefore rounded up to only one
significant digit.
substrates. . I s most of the pupae formed on the underside of leabes, humidit)
from transpiration is expected to have been high when the larvae were i n the
sensitive period. Hobever, temperature (through choice of pupation site-p. 35), light quality and background colour were possibly all involved in this
response.
Significantly low frequencies of greens were recorded for the months of
hlarch, July and September 1975. Although March (as usual) and September
latypically) were wet while July was dry, the preceding months in each case
wcre relatively dry. Low RH, especially over the development period, probably
c d u w s the larvae to become physiologically preconditioned to form pink pupae.
Relatibe humidity is found not to be a significant factor affecting choice of
pupation site although its influence (high RH gives pupation off the plant) o\er
the whole dcvelopment period approaches significance (0.1 > P > 0.051.
The injuence
efects
of lemperalure: proximale
Given a choice of red and yellow background at temperatures of 14 and 25"C,
there was no effect ( P = 1.0) on either background (Table 1 1 i although
background colour influenced pupal colour in the manner expected ('lable 5 i.
'I'ABLF
I I . l ' h c influcnce of temperature at the time of pupation on
pupal colour determination i n D. chtysiflpus
Pupal colour
Tempcraturc
("C approx. 1
S u1xtr.i tr
Ycllow tissur
Yellow tissur
Kcd tissue
Red tissue
'I'U 181s
Pink
Green
~
~~~~~~~~
'lota 1
~~
14
"I 8
2
25
14
25
19
I
0
19
20
20
20
20
20
38
42
80
1
PUPAL POLYMORPHISM IN
D.CHRYSZPPUS
35
The injuence of temperature: ultimate efecls
The Dar es Salaam results agree with those from Liverpool in that the
regression ‘green on cage’ (ngc) against temperature is not a significant factor,
either at the time of pupation or over the period of development. O n the other
hand, the influence of temperature on ‘number on plant’ (np) is positive and
very significant (P=0.0002, Table 10). This influence is more pronounced for
the month in which pupation occurs than for the month of larval development,
though the difference is small. It is therefore probable that temperature
influences pupal colour late in larval life at the wandering stage. At high
temperatures, the larva is more likely to choose a plant substrate for pupation
which, in turn, raises the probability of its being green. I n other words,
temperature is important as a n ultimate factor although it has no influence as a
proximate factor.
Examination of the raw data (Tables 3 and 4) shows that pupation on plants
was indeed above average for four of the five hottest months
(November-March) and significantly so for three of them: two of these months
(December-January) also had much the highest frequencies of green pupae. O n
the other hand, off-plant pupation was above average in all the cooler months
(May-October). Pupal colour over this period varied between wide limits and
must be attributed to factors other than temperature.
The injuence of larval foodplant
Two Dar es Salaam broods (1 11, N = 30, 112, N = 15 pupae) were divided
equally as first instar larvae and fed on different foodplants, C. giganlea and
7.stenoloba (Table 12). Five of the 45 pupae, four of which were green, formed
on the plant and this analysis is restricted to the 40 pupae sited on the cage. T h e
higher frequency of green pupae from larvae which had fed on C. gigantea is very
significant ( P < O . O O l ) and there is no heterogeneity between the two broods
(x2(,!=0.307;0.7>P>0.5). As all the pupae formed away from the foodplant, a
proximate influence of the plants as pupation sites is ruled out.
Genetic injuences
Genotypes differ with respect both to choice of pupation site and the
frequency with which they form green pupae when pupating away from the
TABLE
12. The influence of larval foodplant on pupal colour in
D. chysippus (broods 11 1 & 112) from Dar es Salaam.
(Expected numbers in parenthesis)
Species
Pupal colour
or
Grem
Pink
Total
Calotropis gigantea
17 (10.9)
Tylophora stenoloba
6 (12.1)
2 (8.1)
15 (8.9)
19
21
17
40
roodplant
Totals
x’(,,=
23
12.750; P<O.OOl (with Yates’ correction).
D. A. S. SMITH E T AZ.
36
0
0.2
0.4
0.6
Proportion of bb
0.8
I
E 0.6
-a
0
c
-5-
0.4
L
0.
c;
0.2
0
Figure 2. Id! Contour plot showing the influence of the 6b and cc genotypes iexplanator) v;wiatc\m
on the proportion of larvae (np) choosing plants on which to pupatr (responsc variatr'i. 'l'he figurch
on the contours indicate the frequencies of np. (h) Response surfacr Tor rip illustrating the epistatic
interaction hrtween thr B and C loci as explanatory variates. The bulk of the data fall within the
trianglr delimited by 0,0 (bottom left), 0,l (top left) and 1,0 (bottom right). T h e positionb occupied
hy the grnotypcs are as follows: 0,0 is H-C- (brown dorippus), 0,l is B-cr (hrowm o e g y p i u ~I , 1.0 i\
b 6 G (orange dorippupus) and 1 , l is hhcc (orange n e y p t i u s ) . The calculations for both ( a ) and (bi
assume B ronstant temperature of 25.5"C which is the mean over the duration of the exprriments.
PUPAL POLYMORPHISM I N
D. CHRKSIPPUS
37
plant. As explained above, no attempt was made to distinguish between the
homozygous and heterozygous dominant phenotypes nor, consequently, to
estimate gene frequencies. Genotypes are scored simply as frequencies of the
recessive phenotype which is never in doubt. T h e raw data from the 44 broods
which produced adults are summarized in Table 4. It must be emphasized that
the monthly genotype frequencies for the reared broods do not reflect their
contemporary frequencies in the wild which are shown in Fig. 3.
Taking first the frequency of pupation on the plant (np), logit pbb has highly
significant positive coefficient ( P = 0.002, Table 10). The interaction term logit
pbbxlogit pcc has a negative coefficient (P=O.O05). For the Dar es Salaam
population, which shows substantial linkage disequilibrium between the B and
C loci (D. A. S. Smith, 1980), the meaning of this result in genetic terms is that
progeny with a high proportion of bC/bC and bC/bc (orange dorippus), on the one
hand, or Bc/Bc and Bclbc (brown aegyptius) on the other, produce a relatively
high frequency of pupation on plants, the influence of the former being greater
than the latter (Fig. 2A). Conversely, among the progeny of the common
mating Bc/bC x Bc/bC (brown dorippus), of which half is (Bc/Bc+ bC/bC) and the
other half BclcC, the majority will tend to pupate off the plant. I n practice, the
data points that are influencial in producing the negative coefficient for the
interaction term logit pbbxlogit pcc must be those that represent a high
frequency of the BcIbC genotype, as the bc/bc genotype is rare in the population
and turned u p in only small numbers in the reared broods.
The influence of genotype on ‘green on cage’ (ngc) is less complex than that
for np. The logit pcc term has a significant negative coefficient (P=0.004;
Table 10). I n other words, dorzppza (C-) forms a higher proportion of green offplant pupae than aegyptius (cc) .
No significant effect from the A locus is apparent but this is not surprising as
the frequency of the aa genotype (alcippus/albinus)was zero in most broods.
Ligation experiments
Three sets of ligation experiments were carried out in Liverpool. I n the first
(Table 13), small samples of larvae, ligatured early in the prepupal period, i.e.
before the pupal moult, were chosen from four different pupation sites, two of
which (green leaves and yellow tissue paper) were expected to produce mainly
TABLE
13. Pupal colour in ligatured D.chrysippus larvae
Cuticle colour (anterior/posterior)
~
~-
~
~~~
~
Greenigreen
Green/pink
Pink/green
Pinkipink
Green leaves
Brown twigs
and branches
Yellow tissue
Paper
Red tissue
paper
0
13
0
0
0
0
0
12
0
8
0
3
0
0
0
11
Totals
0
21
0
26
Pupation site
D. A. S. SMI'I'H E T .4L.
38
I'ABLE 14. The effect of delayed ligation on pupal colour determination in D.rhiysippu.r
Ycllo\\ tissuc
Kcd t i s u e
Ycllmv tissue
Kcd tissue
Totals
Early
Early
Latr
Late
0
0
7
0
7
0
0
0
0
0
0
0
3
10
7
7
0
26
10
3
green pupae and the other two (brown twigs and branches and red tissue paper)
to favour a majority of pinks. O n backgrounds favouring pink pupae, all were
pink both anterior and posterior to the ligature. However, on backgrounds
favouring greens, 2 1 of the 24 pupae were bicoloured being green anterior to the
ligature and pink posterior to it. None was entirely green. This result suggests
that a 'greening' hormone is produced in the head or thorax which is prevented
by the ligature from reaching the abdomen as the blood flow is cut off.
I n the second experiment, four groups of 10 larvae, selected in equal numbers
from red and yellow tissues on which they had suspended, were ligatured, either
early or late in the prepupal stage, (Table 14). Again, all larvae pupating on the
red background formed pink pupae. Those on a yellow background differed
according to the timing of ligation: if this was early, as in the previous
experiment, the majority was bicoloured, but, if late, entirely green with none
bicoloured. Therefore, if blood flow is uninterrupted until late in the prepupal
phase, the greening effect is by then established irreversibly.
The final experiment was an attempt to localize the source of the greening
hormone by applying ligatures in three different positions (Table 15). High
pupal mortality was a problem with this experiment despite which the result is
reasonably clear. As isolation of the larval head can produce a pupa with green
head cuticle but elsewhere pink, the greening hormone must be produced in the
head.
A brood ( 3 = 4 2 ) reared at Eton (1986), from a cross between a f. chrysippus
male from Sri Lanka and a f. dorippus female from Mount Kenya, displayed a
lethal defect which manifested itself at pupation in 41 of the 42 prepupae. The
sensitive period larvae appeared healthy and formed prepupac which were at
first normal but remained suspended perpendicularly instead of adopting the
characteristic J-shape with the head curled upwards. They eventually formed
TABLE
15. Positioned ligatures to localize the site of hormone secretion
PUPAL POLYMORPHISM IN D.CHRZSIPPW
39
truncated pupae with a constriction between the thorax and abdomen. Ten of
these pupae were bicoloured with a green head-thorax and pink abdomen, the
remaining 3 1 being pink. None was wholly green despite pupating in conditions
(darkness and high humidity) which generally favoured green. (None of the
truncated pupae eclosed successfully.) This result supports the Liverpool
experiments in showing that the greening hormone is secreted and targeted
early in the prepupal stage.
DISCUSSION
ClassiJication of inzuences on Pubation
The multifarious and disparate influences on pupa colour in D. chrysibbus are
most conveniently discussed under three heads, namely environmental factors,
both proximate and ultimate, and genetic factors. The various factors acting in
turn target an endocrine mechanism which is the final switch to pink or green.
First, proximate factors are those which influence pupation only during the
sensitive period, after the larva has ceased wandering and is at rest on its chosen
pupation site, immediately prior to formation of the prepupa.
Second, ultimate factors are of two kinds: either those which affect larval
choice of pupation site, hence indirectly influencing pupa colour; or those which
exert a direct influence on the physiology of colour determination which is
exercised at some stage earlier than the sensitive period. Both categories of
ultimate factor presumably act by canalizing, in the former case, larval
behaviour or, in the latter, the physiology of cuticular pigmentation, so as
normally to pre-empt the subsequent influence of proximate factors.
Alternatively, in some circumstances proximate factors might over-ride or reroute the outcome of ultimate conditioning, but our experiments provide no
confirmation for this possibility.
Third, those genes which influence pupation preadapt larvae to respond ab
initio, either physiologically or behaviourally, in the direction which is likely to
be in tune with the environment. In essence the genetic cues have been selected
to anticipate the norms of the environment and yet to be sufficiently flexible to
permit redirection or fine tuning of the hormonal mechanism by environmental
factors which are not infrequently quixotic and never entirely reliable. As
genotypes vary between both geographical races and morphs within races, the
latter often showing predictable seasonal variation in frequency, the genetic
influences on pupation are assumed to be the product of natural selection acting
on pleiotropic effects of genes which are also major determinants of the adult
phenotype.
Proximate factors
Our results for D . chtyszjjpus pupating on natural substrates in Dar es Salaam
support Wood's (1867) observation that pupal coloration is correlated with the
nature of the background on which the pupa is formed. The correlation may
result from larval response to any of a number of factors associated with green
and brown environments. We have shown that pupation site (on or away from
plants) is a highly significant factor in determining pupa colour, green pupae
$0
I).A.
S. SMITH E T A L .
being formed predominantly on plants and pink ones on other surfaces. As the
exact pupation sites on plants include the underside of leaves, fruits, buds,
occasionally flowers of various colours, or stems of different sizes and ages, the
precise cues involved must be diverse. Background colours, green in the case of
living leaves and young stems, or brown on old leaves and thick stems, substrate
texture, generally smooth though rough on old stems, or high humidity from
transpiration, are probably all common cues on plant substrates. Since the Dar
es Salaam larvae were given fresh food daily, background colour was
overwhelmingly green, substrate texture smooth and humidity from
transpiration high for larvae pupating on foodplants.
Clarke & Sheppard (1972) and A. G. Smith (1978) have demonstrated for
species of Papilionidae which show green/brown pupal dimorphism that the
diameter of shoots chosen as pupation sites influences the colour of pupae. Green
pupae are associated with twig diameters less than 10 mm whereas brown pupae
tend to form on twigs exceeding 10 mm. Although the latter have a higher
probability of being brown, they are often in fact green. We have not tested the
effect of twig diameter on pupa colour in D . chrysippus but it is certain that the
diameter of Calotropis twigs used often exceeded 10 mm and this may have
caused some pupae on plants to be pink. Clarke & Sheppard (1972) suggested
that some individual pupae on twigs which fail to match their background may
gain an advantage simply from being excentric or unexpected and thus
overlooked, always provided that they remain relatively rare and scattered
among a majority which is well camouflaged. T h e same might be true for pupae
which are occasionally found on unorthodox backgrounds where, although not
protected by crypsis, predators would not normally search for them. Both these
hypothetical cases would be examples of apostatic selection.
A. G. Smith (1978) has shown that substrate texture exercises a considerable
influence on pupa colour in Papilionidae, green predominating on smooth
surfaces and brown on rough ones. This is especially the case in P. polytes which
tends to pass through the sensitive period in darkness so that textural cues are
more functional than light. However, Sevastopulo (1974, 1975) found a higher
proportion of green pupae of P. demodocus on glass compared with sandpaper
substrates and this species passes the sensitive period largely in daylight. O u r
result (Tables 6 81 7) could imply that D.chrysippus is also able to respond to
textural cues in darkness as the smooth plastic surface used at Eton produced a
higher frequency of green pupae (75%) than the rough surface used in
Liverpool (57”/,). However, the two experiments may have been dissimilar in
other respects and, as the difference is slightly short of significance, the issue is
unresolved. Danaus chrysippus normally passes through the sensitive period during
the day and light cues are expected a priori to be more relevant than texture.
Humidity is a proximate factor of undoubted importance {Tableg and
below). It will generally be high for pupae on the underside of leaves, tending
to promote the frequency of green pupae. Morover, the absence of wind in the
experimental conditions at Dar es Salaam probably resulted in humidity
remaining high throughout development, at least during the day, an effect
which may have increased the frequency of green pupae compared with
conditions in the wild. However, during the dry seasons, humidity is expected to
fall at night when transpiration is much reduced. Moreover, pupae on stems are
always exposed to lower levels of transpiration and might be expected to show a
higher frequency of pink pupae irrespective of twig diameter or texture.
PUPAL POLYMORPHISM IN D. CHRYSfPPlJS
41
As the plant is a complex and varied pupation site, it is not possible to assess
the relative importance of background colour, texture and humidity from the
Dar es Salaam data but it is clear that its overall effect is to produce a high
proportion of green pupae, the only aspect of the data which was homogeneous
throughout the experimental period (Table 10).
The Liverpool experiments testing background colour and light quality as
proximate factors used artificial paper substrates of uniform colour and texture.
Yellow was found to be particularly effective, both as a background colour and
as incident light, in producing green pupae. Similar effects have been found in
papilionids, with the exception of P. polytes (A. G. Smith, 1978), and pierids
(A. G. Smith, 1980). For some of the species tested, though not D . chrysz$pus,
other coloured backgrounds (e.g. orange and brown) also produced high
frequencies of green pupae. Colourless acetate produced mainly green pupae in
the papilionids but overwhelmingly browns in pierids. I n this respect,
D. chys$pus when pupating on smooth, clear substrates in light (Tables 5 & 7)
gives results similar to the pierids. Red, blue, green and white backgrounds all
produced high frequencies of pink pupae in D. chrysippus as they do browns in
many other species. With regard to light quality, blue light alone was influential
in producing pink pupae.
Danaus chrysippus responds to light cues in a manner generally similar to many
other species which pass through the sensitive period during daylight.
Wavelengths towards the blue end of the spectrum enhance the production of
pink pupae while yellow light favours green pupae. Previous workers seem not
to have commented on the curious fact that green artificial backgrounds and
green incident light are generally associated with a majority of brown pupae in
experimental conditions. Pieris brassicae is an exception to this generalization
(A. G. Smith, 1980). However, various authors (for example, Moss & Loomis,
1952; Gates, 1962; Rackham, 1965) have shown that light transmitted through
green leaves contains an excess of yellow as well as green wavelengths. Gates
(1966) also showed that skylight, direct sunlight scattered by the atmosphere,
contains a high proportion of blue and ultraviolet wavelengths. Therefore, if the
period of larval sensitivity occurs in daylight, the use of light stimuli as
proximate cues is expected to be adaptive, producing a high frequency of green
pupae in the vicinity of foliage and of pinks away from it, as we have found.
Humidity is the only factor identified as having both ultimate and proximate
effects. The Liverpool experiment (Table 9) shows that high humidity has a
direct proximate influence, increasing the frequency of green pupae even when
plant substrates are not available. This result is supported by the Dar es Salaam
finding that ‘green on cage’ (ngc) is positively correlated with rainfall (RP &
RD) and peaks (62%) during the wettest and/or greenest months (May-June).
High humidity, whether encountered under a leaf in the form of transpiration or
away from the plant as rain, mist or cloud, probably acts as a cue for
environmental greeness. If background colour or light quality are actually ‘nongreen’, high humidity may reverse their influence.
Ultimate factors
It is evident from the Dar es Salaam data that R H (the larvae and pupae
were not exposed to the direct influence of rain) exercises an influence on pupa
colour which is realized at some stage of larval development earlier than the
12
U. A. S. SLII‘I H E T ‘41.
sensitive period. The partial regression for ‘green on cage’ (ngc) on ‘rainfall
during month of development’ ( R D ) attains a higher level of significance than
that for ngc on ‘rainfall during month of pupation’ ( R P ) . Although for many
broods the months of development and pupation were the same, the former
accounts for the variation found more satisfactorily. O n the other hand, the
negative regression of ‘number on plant’ ( n p ) on both RD and RP falls short of
significance (0.1 > P>O.O5 for R D ) : clearly RH has little impact on choice of
pupation site.
The roles of high RH as both an ultimate and a proximate factor can be
sumiiiarized as follows: as an ultimate factor it primes larval physiology, raising
thc probability of a grcen pupa 0 1 1 off-plant substrates (ngc); as a proximate
factor it raises the probability of green pupae on a colourless substrate and i t is
possible, though not demonstrated by us, that it may override other proximate
rues (e.g. blue light or red background) which would otherwise indicate
pinkness. The result of conflict between light cues (e.g. yellow) which produce
green pupae and low RH which stimulates pink was not tested. Further
experiments are needed to clarify the nature of the interaction betwecn
proximate R H on the one hand, and light and background cues on the other.
‘The varying morphology of recorded D.chrysippus foodplants with respect to
such features as size and shape of leaves, surface texture of leaves and stems and
diameter of twigs are expected to have a role as proximate factors in view of our
other findings. Howcver, the Dar es Salaam experiment (Table 12) does not test
for the proximate influence of different plant substrates as all the pupae
involved formed on the cage. T h e significantly higher proportion of green pupae
from larvae which had fed on C. giganlea compared with those reared on
7.slrnolohn i n the two split broods ( 1 1 1 & 112) must be explained by a n
ultimate factor. ‘Tjlophoria stenoloba is a xeromorphic climber with a thick leaf
cuticle and much smaller leaves than C.gigantea. It is probable that a higher
transpiration rate from the latter raises the humidity in the micro-environment
of the larvae throughout development and predisposes them to form green
pupae.
Although the majority of broods was raised on C. gigantea, excluding the two
split broods, other plants used were C.procera ( 3 ) , 7. stenoloba ( 6 ) , A . curassavica
( 2 ) and G. fruticosus ( 1 ) . T h e variety of foodplant used probably contributes to
the substantial amount of heterogeneity remaining unaccounted for after
multiple regression analysis on the Dar es Salaam data.
Temperature has no status as a proximate factor (Table 1 1 ) . Howcvcr, as a n
ultimate factor i t plays a key role. T h e positive regressions for ‘number on plant’
(np) against both ‘average temperature during months of pupation’ ( TP’i and
‘average temperature during month of development’ ( T D ) (Table 10) are
highly significant, the former accounting for the variation more fully than the
latter. This result implies that the impact of temperature is mainly on larval
choice of pupation site and occurs shortly before pupation, probably during the
wandering phase. At high temperatures, the larva is more likely to seek a plant
on which to pupate thus raising the probability of its being green.
There are two reasons why a larva might be expected to choose leafy
pupation sites during hot weather: ( 1 ) pupation beneath a leaf provides shade:
(2) transpiration promotes cooling. T h e Das es Salaam data show that the high
frequency of green pupae during the hottest months, when rainfall is moderatc
PUPAL POLYMORPHISM IN D. CHRYSIPPCS
43
or light, results rather from plant substrates being popular for pupation than
from high atmospheric humidity. T h e converse is that pupation in an open
sunny situation might be beneficial during the cooler months as the pupa will be
more exposed to radiant heat. Furthermore, non-plant substrates selected for
pupation are frequently those, such as rock, brick or concrete, which have good
heat retention and should assist the maintenance of high body temperature, by
conduction and convection, through the night.
Larval feeding behaviour and pupation
Danaus chrysz$pus and other danaid larvae have a curious habit of biting
through the petiole or main vein of the leaf on which they are feeding causing
part of the lamina to hang vertically downwards. The larva feeds, precisely
aligned with the midrib, on the ventral side of the leaf, which faces the plant’s
central axis: it is thus effectively hidden over a field of view well exceeding 180”.
Although the behaviour first appears in second instar larvae, often on flowers or
buds, it is especially developed in fifth instar larvae in their last 2-3 days of
feeding up before pupation. Larvae which remain on the foodplant to pupate
commonly d o so under the awning of a jack-knifed leaf which they must be
presumed to have prepared for their own benefit.
The value of this behaviour to a pupa could be any of the following: ( 1 ) to
improve camouflage as the pupa is suspended flush with its substrate and is
largely hidden from view (Fig. 1J); (2) to assist cooling by providing
continuous shade (although cooling from transpiration must be drastically
reduced as the leaf dries up); (3) to produce a progressively browner
background for improved crypsis when the pupa darkens at the approach of
eclosion; (4) by killing the leaf, to render it unattractive to later arriving larvae
which could threaten its anchorage or even its life (cannibalism of pupae by
larvae is a common occurrence in D. chrys$pus under conditions of crowding or
food shortage); (5) to prevent the plant from directing, as a defensive measure,
toxic or repellant substances to a damaged part. However, as danaids are
known to be highly adapted to subverting the plants’ defensive chemicals to
their own use (Rothschild, von Euw, Reichstein, Smith & Pierre, 1975), the last
is probably the least plausible explanation in this instance. Larvae which
choose, as is often the case, a plant other than the foodplant for pupation,
indulge in none of the behaviour described above.
Genetic factors
Possibly our most original finding is that genes a t two linked loci which
control major aspects of the adult phenotype also have a considerable influence
on pupal polymorphism. Thus Owen’s (1971) speculation for P. demodocus has
been shown to be correct but for a different species. The interest attaches mainly
to the following three results (Table 10):
(1) ‘Number of pupae on plant’ ( n p ) , expressed in logit form, has a highly
significant positive coefficient ( P= 0.002) when regressed on the proportion of
the 66 (orange) genotype, also expressed in logit form, in individual broods
(Fig. 2).
44
D. A S SMITH ET A L
( 2 ) The partial regression of np, expressed as logit, on the interaction term
phh X ~ C has
C
a very significant negative coefficient (P = 0.005).
(3) There is a significant negative coefficient (P=0.004) for 'number green
on cage' (ngc), expressed as logit, on proportion of the cc genotype k c c ) . This
result can be accepted despite the fact that approximately 250/, of larvae,
predominantly of bb genotype, pupated on the plant ( n p ) . The comparable test
for ngp on genotype shows no heterogeneity.
'The genes in question are two pairs of alleles, B, h and C, c at the closely
linked B and C loci, both pairs being subject to marked seasonal cycling at Dar
es Salaam (Fig. 3). The period January 1974 to September 1975, during which
the frequencies of genotypes at both loci were sampled in the field, embraces the
experimental period (August 1974 to September 1975). Cycling of the C locus
alleles was recorded over the 4 years 1972-1975 and the oscillation shown in
Fig. 3 was broadly replicated each year (see D. A. S. Smith, (1975b) for the
years 1972-1973). T h e census was carried out several times a week in grasdand
on the campus of the University of Dar es Salaam, the local wild population
being the exclusive source of all the breeding stock. All butterflies captured were
marked to avoid double counting. Monthly sample sizes varied between 79 and
339, only four being less than 100.
The frequency of the bb genotype varied between a low point of 23.8O6 in
*June and a maximum of 65.6% in January. Frequency was high during the
hotter months and low during the wet and/or cool months. This result suggests
that the propensity to pupate on plants during hot weather is not only promoted
PUPAL POLYMORPHISM IN D.CHRYSZPPUS
45
by temperature, the only environmental factor found to influence this
behaviour, but also by genotype. The cc genotype also varied in frequency from
a minimum of 5.8% in December up to 40.2% in June, its relationship with the
climatic cycle being almost the exact reverse of bb. Its low frequency in the hot
season implies that it contributes little to the high frequency of pupation on
plants a t this time. O n the other hand, a high proportion of cool season
pupation on plants must be attributed to the B-cc genotype with
B-C-predominating in off-plant sites (Fig. 2B).
Off-plant pupation peaks in the cool season and broadly coincides with
maxima of the interaction term p ( b b ) xp(cc) and the double dominant
phenotype (brown dorippus). Progeny tests have shown that most brown dor$pus
were double heterozygotes in repulsion phase (BbIbC), the males having a
significant mating advantage over the other phenotypes (D. A. S. Smith, 1981,
1984). Mating frequencies in 61 progeny test pairings were ranked as follows:
1 = . B c / b C x Bc/bC and Bc/cC male x Bc/Bc female (both .N = lo), 3. Bc/cC
male x Bc/bc female ( N = 3 ) , 4. all other pairings, ( N G 2 ) (D. A. S. Smith,
1980). The progenies of pairings 1-3 are predominantly brown and/or dorippus
phenotypes and the genetical explanation for the statistical interaction term is
probably that the high frequencies of B-C-genotypes precede by one or two
generations maxima for both pbb xpcc and off-plant pupation. I t is clear from
Fig. 2B that the B and C loci interact epistatically rather than additively: the
influence of the alleles C and c is reversed according to whether they are
associated with B-or bb; the influence of B and b is negligible in association with
cc but powerful with C-.
The contribution off. dorippus (C-) to a high frequency of green pupae occurs
by a route more direct than choice of pupation site. The finding that ngc has a
significant negative coefficient against pcc suggests that the main effect of the C
locus is to preadapt larvae to form pupae of a particular colour irrespective of
the substrate: C- pupae have a higher probability of being green and cc of being
pink when pupating off the plant. The B-C- genotype must be responsible for
the high frequency of green pupae in off-plant sites during the wet months of
April and May.
The overall impact of genetic factors on the determination of pupal colour is
considerable and they clearly exert influences a t least equivalent to those of the
ultimate environmental factors. The pleiotropic effects on the pupal phenotype
of genes governing the adult phenotype result in both their adult and pupal
manifestations being broadly synchronized with the seasonal cycling of
temperature and rain. A similar outcome could be produced by separate but
closely linked loci governing adult and pupal characters respectively although
this is less probable in view of the impressive pupal correlations with the B and
C adult effects. However, we emphasize that all genotypes are capable of
producing pupae of both colours and the pupal variation might best be
described as a polyphenism, determined environmentally, but subject to
influence by a genetic polymorphism. It is possible that other polyphenisms are
similarly controlled.
It is impossible on present evidence to be sure if the selective forces operating
locally at Dar es Salaam are alone sufficiently powerful to account for the steep
changes in gene frequency recorded in the field. Breeding is multivoltine and
continuous with approximately 13 overlapping generations a year: although
46
D. A. S.SXII'I'H E 7 A L
generation time (egg-egg), which is temperature-dependent, may be a s little a s
21 days and never exceeds one month, we suspect that seasonally oscillating,
north-south, migratory movements (D.A.S.S., personal observation] make a
major contribution to the observed changes. Otherwise, it is difficult t o
understand how the population could withstand the enormous segregation load
entailed in responding to rapid and often erratic environmental pressures.
Biogeographical consideralions
T h e geographical distribution of the morphs of D.chrysz&iuJ in Africa sug-gests
that the adult polymorphisms at the A, B and C loci in east Africa might have
arisen since the Pleistocene by the expansion and overlapping of three
previously distinct geographical races, dorippus in the northeast, aqjyptius in the
south and alcippus in the west (D. A. S. Smith, 1976, 1980). T h e species is
monomorphic for the orange dorippus morph (6CIbC) in the arid regions of
northern Kenya and Somalia and it is possible that the genetic cue to pupate
under leaves is appropriate in the hot, dry climate of the region. On the other
hand, the brown aeuptius morph (Bc/Bc)is the only form occurring throughout
the southern part of the continent where the climate is for the most part cooler
and less consistently dry. The climatic adaptations of the two morphs are clearly
reflected in their oscillating seasonal frequencies at Dar es Salaam (Fig. 3 ) . The
tendency for the BclBc genotype to form pink pupae in open sites away from
plant cover may be as advantageous in the cooler and more cloudy conditions of
the south as in the wet season in Tanzania. It would be interesting to know if
the colour frequencies of pupae differ between the brown and orange forms of
morph alcippus (genotypes aa Bcl c and aa bc/bc respectively) which apparently
occur together throughout West Africa. The contrast between wet and dry
seasons is more marked and predictable in coastal West Africa than in the east
and might be expected to exercise a seasonal influence on pupation similar t o
that described by Owen (1971) for P. demodocus in Sierra Leone.
Endocrine mechanisms
The ligation experiments, supplemented by observations on naturally
constricted pupae, lead to clear conclusions regarding the source, effect and
timing of release of a hormone which controls pupa colour. Ligation may
produce pupae which are green anteriorly but pink posterior to the ligature.
The reverse effect does not occur. Therefore, the hormone triggers a greening
response and, in the absence of the hormone, the pupa is pink. Bicoloured pupae
occur only if the ligature is applied at the start of the prepupal phase: if applied
late, the pupa may be either green or pink. T h e hormone presumably diffuscx
through the haemocoel or down neurosecretory tracts early in the prepupal
pcriod: if blood flow is cut off or the nerve cord constricted at a late stage, the
greening response has by then been determined irrevocably. Application of the
ligature at any point from the headlthorax boundary backwards can produce
bicoloured pupae which are invariably pink posterior to the ligature. 'This result
proves that the greening hormone is produced in the head, presumabl) from
neurosecretory tissue in a retrocerebral gland.
PUPAL POLYMORPHISM I N D.CHRYSIPPCS
47
The hormone apparently achieves its greening efrecc by routing yellow
carotenoids and the blue pigment pterobilin into the cuticle and superficial
tissues (Rothschild el al., 1978). I n pink pupae these pigments are diverted to
deeper levels, carotenoids being deposited in the cuticle only in the region of the
golden diadem and small gold spangles, where they are visiable as yellow specks
in the otherwise colourless exuviae.
Comparison with other species of Lepidoptera suggests that D.chrysippus
resembles the Pieridae in possessing a greening hormone (Ohtaki, 1960, 1963;
A. G. Smith, 1980) and differs from the Papilionidae which evidently have a
browning hormone, in the absence of which the pupa is green (Hidaka, 1960,
1961; A. G. Smith, 1978). Whether it is the same hormone which produces
opposite effects in the two families is unknown but seems unlikely. In many
pierids and some papilionids there is a correlation between pupal diapause and
usually brown (or non-green) pupal colour. However, no such relationship can
occur in D.chrysippus as diapause has not been recorded for any stage of the life
history.
CONCLUDING REMARKS
The high values for residual deviance in the Dar es Salaam data, after
extracting the independent effects of the various environmental and genetic
factors, indicate that much of the variation remains unaccounted for by
regression analysis (Table 10). There are a number of reasons why this is not
surprising. ( 1 ) Several different foodplant species were used, a factor which we
have shown to influence pupa colour but were unable to measure. (2) Both
plant and off-plant substrates were variable in colour, texture and form; ( 3 ) the
Dar es Salaam population is highly polymorphic and genes other than those
identified could affect pupation. Moreover, heterozygotes may differ from both
homozygotes a t individual loci or epistatic interactions between loci may occur,
especially as both effects have been demonstrated for other manifestations of the
B and C loci at Dar es Salaam (D. A. S. Smith, 1980). (4)The weather data
used were crude monthly averages and many broods may have pupated in
conditions very different from the mean values. (5) The Dar es Salaam data
could not be tested for the influence of the proximate factors revealed by the
Liverpool experiments. Despite these imperfections, the following factors have
been identified as pupation cues: background colour, light quality, relative
humidity, temperature, foodplant, alleles a t the B and C loci and interaction
between them. Hormonal control of pupa colour is governed by these cues and
emanates from a gland situated in the head.
T o the human eye the variable ground coloration of the D.chrysippus pupa
often confers crypsis upon it, at least when viewed from a distance against an
appropriate background. Green pupae are superbly camouflaged when
suspended among foliage. The pinks and whites can be cryptic on a variety of
backgrounds. The former blend well with dried leaves, brick or laterite walls
and the latter are beautifully camouflaged on concrete, a substance which is
popular and readily available in modern African towns and villages. Poulton
(1887) suggested that the gilded areas of danaines and vanessids had been
originally evolved to mimic the sparkle of mica and would be a cryptic feature
when the pupa was placed against a background of bare igneous rock.
48
D A. S. SMITH E T A L
Paradoxically, the lustrous metallic areas and irridescent sheen, which glint
and sparkle, especially in morning dew or sunlight after rain, are impressively
aposematic at close quarters. T h e paradox may perhaps be resolved by
supposing that a dual signalling system is involved (Rothschild et al., 1978)
camouflage at long distance backed by near range warning flashes which may
serve to startle a predator which stumbles unawares across the pupa. I n other
words, both aposematicism and crypsis may have survival value in different
circumstances (e.g. lighting conditions, distance, predator’s visual acuity). It is
mistaken to suppose that the two adaptations must necessarily conflict or that
either will reach perfection in all conditions (Dawkins, 1986). A combination of
crypsis at distance and aposematic or flash coloration at close quarters is
widespread in both adults and larvae of Lepidoptera (Owen, 1980) although
hitherto unremarked in pupae.
The D.chr_ysip@uswarning message is rarely bluff as the pupa, like the l a n a ,
adult and egg, is generally noxious and unpalatable, containing bitter tasting
cardiac glycosides (Rothschild et a/., 1975), sequestered by the larva from its
mildweed foodplant, and stored in the other stages. R . Trimen remarked (in litt.
to E. B. Poulton, 1887), “It is not improbable that this brilliant pupa stands in
no need of special protection, but, like the imago (and apparently the larva
also)) is avoided by insectivorous animals”. T h e strange glittering object is
probably well adapted to trigger a predator’s recall of any past unpleasant
encounter. However, we have shown that, in addition to being endowed with
proven deterrent and warning properties, which have acquired a welldocumentated chemical basis since Poulton’s day, both the larval behaviour of
D.chrysippus and the physiology of its pupation are intricately adapted to
providing the pupa with a high degree of crypsis giving long range protection in
spatially and temporally variable surroundings.
~
ACKNOWLEDGEMENTS
D.A.S.S. thanks the Royal Society and the Busk Fund, Eton, College, for
research grants enabling him to breed tropical butterflies in England. Also,
being the carrier of a well known X-linked gene inherited from his mother, he
gratefully acknowledges the assistance of his wife, daughters and colleagues who
have checked the colour identification of pupae. A.G.S. was financed by a
Science Research Studentship. H e is also most grateful to the late Professor
P. M. Sheppard, F.R.S. for much help and encouragement while supervising his
research and to Sir Cyril Clarke, F.R.S. for his interest and advice.
This year is the centenary of Edward Poulton’s classic paper “ A r t enquzry i ~ l o
the cause and extent of a special colour-relation between certain exposed LepidoplerouJ pupae
and the surfaces which immediate& surround them”. This splendid work is compulsive
reading for all who have subsequently worked on pupal polymorphism and we
dedicate our paper to the memory of its author.
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