BiologicalJ oumal oflhe Linnean Society, 12:305-326.With 2 figures
December 1979
Pyrrolizidine alkaloids in arctiid moths (Lep.)
with a discussion on host plant relationships
and the role of these secondary plant
substances in the Arctiidae
,
MIRIAM ROTHSCHILD*, R. T. APLINt, P. A. COCKRUMS
J. A. EDGAR$, P. FAIRWEATHER7 ANDR.LEES?
Ashton, Peterborough, England
-t Dyson Perrins Laboratoly, University of Oxford,
South Parks Road, Oxford, England
$ CSIRO, Division ofAnima1 Health, Private Bag No. 1,
Parkwille, 3052 Australia
AccePted forpublication J anuary 1979
Pyrrolizidine alkaloids (PA) have been identified in six species of Arctiidae reared on Senecio and
Crotalaria. These include senecionine, seneciphylline, integerrimine, jacobine, jacozine, jacoline,
jaconine and a metabolite (C,,H,,NO,) from Senecio, and monocrotaline, trichodesmine and
crispatine from Crotalaria.
The all-red aberration of Tyriajacobaeae (var. conyij contained much less of the metabolite than
normal examples of this species. Female Spilosoma lutea reared on the same plants of S. Jhcobaea
contained markedly more jacobine and jacoline than the males.
Host plant relationships and secondaly plant substances are discussed. I t is suggested that the
Arctiid moths' own deterrent secretions, directed against vertebrate predators, pre-adapts them for
reeding on foliage likewise protected against large herbivores by toxic secondary plant substances
such as cardenolides and pyrrolizidine alkaloids. These latter substances are more toxic to vertebrate
than to insect herbivores, and their dual function of deterrent and insect aphrodisiac puts a premium
on their sequestration and storage once a species has achieved the initial steps, and occupied the plant
niche concerned. It is further suggested that the polyphagous habits of the Arctiidae result in a more
equitable distribution of the secondary plant substances within the Miillerian complex concerned,
thus providing a generalized warning message for the potential vertebrate predator.
KEY WORDS:- arctiid moths - host plant relationships
- pyrrolizidine alkaloid storage.
CONTENTS
Introduction
. . . . . . . . . . . . . . . . . .
Material and methods . . . . . . . . . . . . . . . .
Results
. . . . . . . . . . . . . . . . . . . .
Host plant relationship and secondary plant substances in the Arctiidae
The function of pyrrolizidine alkaloids (PA)in the Arctiidae
. .
The enigmatical character ofkctiid self-secreted toxins .
. . . .
Discussion
. . . . . . . . . . . . . . . . . . .
305
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. . . .
. . . .
. . . .
306
306
308
. . . .
313
. . . . .
317
319
321
. . . . .
. . . .
8 1979 The Linnean Society of London
306
M. ROTHSCHILD ET A L
I NTRO DUCT10 N
Senecio alkaloids were first identified in the Cinnabar moth (Tyriajacobaeae (L.1)
in 1968 by Aplin et al., and in the GardenTiger moth (Arctia caja (L.1)by Rothschild
8c Aplin ( 197 1). Subsequently their presence was reported (without the relevant
details) in Diaphora mendica (Clerck), Utetheisa bella (L.1. Utetheisa pulchelloides
Hampson, Amphicallia bellatrix Dalm and Argina cribraria Clerck (Rothschild, 1972a).
More detailed studies of Utetheisa pulchelloides and U . lotrix (Cram.) were made
simultaneously by Culvenor & Edgar (1972) and U. bella recently by Eisner
(personal communication).
In the present paper we provide further information concerning the species in
question, and the additional data relating to Spilosoma lutea (Huf.),S . lubricipeda (L.)
and the all-red aberration (var con# Watson) of Tyriajacobaeae. We were particularly
anxious to compare the relative quantities of pyrrolizidine alkaloids (PA)stored by
the two species of Spilosoma, in view of the suggestion (Rothschild, 1963) that the
Buff might be a mimic of the White Ermine.
MATERIAL A N D METHODS
The moths
Spilosoma lubricipeda and S . lutea were reared at Ashton, Peterborough (by MR)
from eggs laid by females captured in a mercury vapour trap. It proved very much
more difficult to breed adults of the latter species in the laboratory. The larvae
were fed on Senen'opcobaea L. and S. vulgaris L. The pupae (alive)and the imagines
(dried)were sent to Australia by air mail. Ninety living Amphicallia bellatrix reared
by G. R. Cunningham van Someren on Crotalaria lanata Beddome in Kenya were
sent to Oxford by air mail. Despite two weeks delay one or two specimens arrived
alive, after laying a number of bright yellow eggs en route, but unfortunately not
sufficient to test satisfactorily. Two additional dried specimens, locality unknown,
reared on Crotalaria capensis L.,from the Rothschild collection (British Museum
(Natural History))were also examined. Ten dried specimens (both sexes) ofArgtna
cribraria, reared by D. G. Sevastopulo in Mombasa on a Crotalaria tentatively
determined by the Nairobi Herbarium as C. agatiflora Schweinf. were sent air mail
to Oxford. (Judging from the alkaloids recovered from the host plant, this must
have been another species (Culvenor & Smith, 1972), possibly C. retusa L. o r C.
spectabilis Roth.).
The all-red variety of the Cinnabar moth (Tyriajacobaeae var. conyi) was reared by
Bernard Kettlewell in Oxford on three different species of Senecio, S.jacobuea, S.
vulgaris and S. squallidus L., and hatched at Ashton. They were dried after
ovipositing and sent to Australia by air mail.
The larvae of Diaphora mendica were collected in Germany and reared at Oxford
(by R. d'A. Ward) on local Senen'o vulgaris.
Theplants
The Senecio vulgaris on which the Spilosoma were reared was grown continuously
at Ashton under glass from seed obtained from the plant originally tested for us for
pyrrolizidine alkaloid content in 197 1 (Aplin & Rothschild, 1972; Tables 1, 2, 3).
The S.pcobaea were collected at Ashton from the garden, in the precise area from
PYRROLIZIDINE ALKALOIDS IN ARCTIID MOTHS
307
Table 1. Seasonal variation of alkaloids in the groundsel (Senecio vulguris)
(afterAplin8cRothschild, 1972)
Collecting date
14/6/68
(Garden, Elsfield,
Oxford)
19/7/68
71
33
Plant, dry
weight, (g)
Total 96of
alkaloid, dry
weight
0.23'
Senecionine (%)
Seneciphylline (%)
Integerriinine (%)
5/12/68
2018168
(Garden,Ashton, Peterborough)
0.20
0.28
0.19'
10
60
30
10
65
10
70
20
10
65
25
14
10
25
* Very young plants
Table 2. Seasonal variation of alkaloids in the ragwort (Seneciojacobueu)
(after Aplin 8c Rothschild, 1972)
20/5/68
~~
Collectingdate (atAshton)
19/7/68
19/7/68
20/8/68
2018168
4/10/68
5/12/68
5
36
35
14
0.25
T
40
0.22$
T
25
20
0.26
T
40
15
T
55
10
30
T
0.23.
T
20
10
45
~
Plant, dry weight, g
Total % of alkaloids
dry weight
Senecionine %
SeneciphyllineM
Integerrirnine X
Jacobine%
Jacozirie %
Jacoline M
~~~~~
18/6/68
~
21
70
55
0.33'
T
10
0.52
T
T
45
25
20
5
50
25
0.407
T
5
15
15
~~
10
60
5
10
~
~
~
~~~
44
0.79$
T
45
10
5
40
T
10
T
40
5
T
20
5
~
Very young plants
t Znreduction stageomitted
$ Different collecting site (water-garden, 100 yards distant)
T Trace
"'
Table 3. Site variation of alkaloids in the ragwort (Seneciojacobueu)
(afterAplin & Rothschild, 1972)
18/6/68
Ashton Terrace
Plant, dry weight, g
Total%ofalkaloids
dry,lweight
Senecionine
Seneciphylline%
Integerriinine %
Jacobine%
JacozineX
JacolineM
26/6/68
Weeting Heath
20/8/68
Ashton Terrace
20/8/68
Ashton
water-garden
70
51
35
14
0.52
Trace
10
5
50
25
0.24
Trace
Trace
5
0.25
Trace
40
0.23
Trace
25
20
Trace
55
Trace
10
40
20
30
10
Trace
40
5
308
M . ROTHSCHILD ET A L
which we obtained the plants tested previously, but more variation in PA content
could be expected compared with that of the isolated S. vulgaris.
The Crotalaria, the seeds of which were sent to us by G. R. C. van Someren, had
been tentatively identified as C. semperflorans Vint. (Rothschild & Aplin, 197 1).
However, when the flowers were obtained from the plants grown from the seeds at
the Dyson Perrins Laboratory (Oxford), R. M. Polhill (Royal Botanic Gardens,
Kew) was able to identify this species as C. lanata - a rare Crotalaria from the
Anamally Hills, Madras, now quite commonly grown in Nairobi gardens.
Unfortunately the seeds of the Crotalaria sent by D. G. Sevastopulo failed to
germinate so we were only able to extract the pods and seeds, not the foliage.
However, the green seed pods constitute the preferred diet of the larvae of
A . cri braria.
Methods
The moths and plants reared at Oxford were extracted by macerating three
times with methanol ( 10 ml/g) and twice with 80 % warm aqueous methanol ( 10
ml/g). The combined extracts after filtration were evaporated to dryness at 35OC in
a rotary evaporator.
The total alkaloids were obtained by partitioning the methanol soluble material
between chloroform and 2N aqueous sulphuric acid ( 1 :2; 25 ml/g) the organic
layer was washed twice with 2N acid ( 10 ml). The combined acidic aqueous extracts
were stirred overnight with zinc dust (c. 1 g) in order to convert alkaloid N-oxides to
the free base form. The resulting suspension was filtered, the aqueous filtrate made
alkaline with concentrated ammonia solution and extracted three times with
chloroform (10 ml). The chloroform layer was dried (Na,SO,) and evaporated to
dryness to afford the crude alkaloids.
A slight variation of this method was used for material worked up in Australia
(see Bernays, Edgar & Rothschild, 1977). The alkaloids were identified by comparison of their mass spectra and gas chromatographic retention times with those
of authentic samples using a combined gas chromatograph-mass spectrometer
(Varian MAT 111)equipped with a 1.5 m x 2 mm glass-lined stainless steel column
packed with 1% SE 30 on Chromosorb W,mesh size 80-100 and with temperature
programming from 150° or 180° to 230OC at Go/min and carrier gas (helium)
flowing at 15 ml/min. Quantitation was based on gas chromatographic peak areas.
RESULTS
Spilosoma lubricipeda (TheWhite Ermine)
Two samples of pu ae reared on Senecio vulgaris, one of six, the other of 13 1
specimens, were testeland found to contain the alkaloids from the food plant, i.e.
seneciphylline (I), senecionine (11)and a small amount of integerrimine (111).The
closely related alkaloids seneciphylline and senecionine were readily identified
from their mass spectra but were not resolved under the gas chromatographic
conditions employed. They were therefore quantitated together (Fig. 1A). The
pupae in the smaller sample each contained approximately 125 pg of
seneciphylline/senecioninewhile those in the larger sample contained 1 18 pg of
which about 75% was present as the N-oxides.
PYRROLIZIDINE ALKALOIDS I N ARCTIID MOTHS
Seneciphylline I
Senecionine
309
II
lntegerrirnine ID
S. lutea (The Buff Ermine)
Reared on the same sample of S. vulgaris (plants shared between both species of
caterpillar), the Buff (12 pupae (Fig. 1B)) stored similar but somewhat larger
amounts of seneciphylline/senecionine than the White Ermine, namely 170pg
per pupa, of which about 80% was present as N-oxide. Twenty-six pupae (Fig.
1C) reared on S. jacobaea stored approximately 90 pg per pupa of
seneciphylline/senecionine and 250 pg per pupa of jacobine (IV) (Fig. 2A) along
with small amounts of the other S . jacobaea alkaloids, jacoline (V),jacozine (VI),
jaconine (VII) and integerrimine. Only six adults (4 88 (Fig. lD), dry wt 330 mg,
and 2 99 (Fig. lE), dry wt 3 17 mg) of the Buff Ermine were available for testing
(also reared on S . ~acobaea).They contained mainly seneciphylline, senecionine,
jacobine and jacoline, with traces of jacozine, jaconine and integerrimine. The
seneciphylline/senecionine content ( 17 pg/8 and 27 pg/9) was approximately the
same for both sexes on a % dry wt basis, viz 0.02 1%for males and 0.01 7 % for the
females, but the jacobine content [ l 1 pg (0.013%)/8and 90 pg (0.57%)/91 and
jacoline content [11 pg (0.013%)/8and 57 pg (0.036%)/91were noticeably higher
in the females, This may have been due to additional sequestration of these two
alkaloids for the eggs. (PASare present in the eggs ofA. cuju and this genus is closer
to Spilosoma than to Tyrzajacobaeae which lack PASin their eggs (Aplin & Rothschild,
19721, or the females may be generally more efficient and selective storers than the
males, or the latter may metabolize jacobine and jacoline more rapidly.)
Tyrzujacobaeae (The Cinnabar)
We analysed approximately 40 pupae (Fig. 1F) reared on S.vulgaris and found
the alkaloids present in the food plant, together with metabolite C ~ ~ H ~ S N O ~
(already recorded by Aplin and Rothschild, 1972, mass spec. Fig. 1) (Table 4). It is
interesting that this metabolite is found in both A . cuju and T,Jucobueae, but is
lacking in the other related species reared on the same food plants. The all-red
18
M. ROTHSCHILD ET AL.
3 10
C
8
.-C
0
C
._
0
01
.-
C
mal
n
"
7
0
._
E
01
.0
.-
E
0
0
In
3
\
0
01
..-E
L
.-
0
0
c
2.
C
r
a
.-U
-\
C
01
dL
.-U
-
8
3
x
al
..-E
L
:
a
l
.-
a
C
8
0
0
-.
3
t
I
\
I
Figure I. Gas chromatograph of total alkaloids o f A, pupae ofS. lubricipeda reared on S.vulgaris; B, 12
pupae of's.lutea reared on S. vulgaris; C, 26 pupae of S. lutea reared on S. jacobaea; D, 4 male S.lutea
reared on S.jacobaea; E, 2 female S. lutca reared on S.jacobwa; F, pupae of T. jacobaeae reared on S.
vulgaris; G, the all red mutant T .jacobacac var con$ reared on three different species o n S m c i o .
PYRROLIZIDINE ALKALOIDS IN ARCTIID MOTHS
Jocobine Ip
Jocoline P
Jocozine Ill
Joconine Yll
JII
Table 4. Relative proportions of the alkaloids present in the pupae of Tyria
jacobaeae and in Seneciojucobaeu (after Aplin 8c Rothschild, 1972)
Pupae
T. jacobaear
(average of
2 trials)
Senecionine
I ntegerriinine
Seneciphylline
Jacobine
Jacozine
Jacolinr
Metabolite
i
i
Plant
S.jacobaea
(average of Sept.
Pupae
T.jacobaeae
Plant
S.vulganx
&A.caJa
& Oct. samples)
% 60-70
40-60
%
! 10-15
40-60
% 20-25
Absent
5
23-30
50-60
10
20-30
65-70
Absent
15-20
Absent
variety (var conyi) (Fig. 1G) had been reared on three species of Senecio (see above,
p. 310) and contained the alkaloids expected from this diet, namely
seneciphylline, senecionine, integerrimine, jacoline, jacobine, jacozine and
jaconine. The most striking difference between this aberration (var conyi) and the
‘normal’ species is the very small amount of the metabolite present. In the all-red
variety the metabolite represented only 1% of the total alkaloid content, whereas
the ‘normal’ form contained 15% of the metabolite (Fig. l G ) .
It is worth noting that if the females were offered a choice of plants of S. uulguris
and S. jucobaea in their breeding cages they selected the latter for oviposition
although occasionally eggs were also laid on S. vulgaris.
Diaphora mendica (The Muslin Moth)
Sixty pupae (3.5 g dry weight) from larvae reared on Senecio vulgaris in Oxford,
afforded 7 mg of total alkaloids (0.2%).Analysis by gas chromatography* (g.1.c.)
and mass spectrometry? (M.S.) demonstrated the presence of senecionine, its
* Pye 104 14 gas chromatograph; 1% SE 30 on 100-120 mesh “Gas Chrom Q”; column temperature 180°
with an argon carrier gas flow of35 rnlhnin.
t Direct insertion spectra run on an AEI MS9 mass spectrometer.
M . ROTHSCHILD ET AL.
312
geometrical isomer integerrimine and seneciphylline in a similar ratio to that
present in the food plant.
Argina cribraria
Ten adult moths (210 mg dry weight) reared in Mombasa on the seed pods of
Crotalaria ~ p (see
? below) afforded 5.6 mg of total alkaloids (2.6%).Analysis by
g.l.c., M.S., (Fig. 2B); nuclear magnetic resonance (n.m.r.*) (Table 5) confirmed
the presence of monocrotaline (VIII) (Bull et al., 1968).
Crotalaria spa
Seed pods: The dried seed pods (5 g) afforded 52 mg of total alkaloids (1.04%).
Crystallization from ethanol afforded monocrotaline as prisms m.p. 196- 198O
(report m.p. 197-198O, Leonard, 1950).
Seeds: Dried seeds (20g) afforded 662 mg of total alkaloid (3.31%). Crystallization from ethanol afforded monocrotaline as prisms m.p. 196-198O M.S., n.m.r.
(Table 5).
Amphicallia bellatrix
Eighty-four adult moths (7.4g dry weight) from larvae reared on Crotalaria fanata
in Nairobi afforded 84 mg of total alkaloids (1.13%).Analysis by g.1.c. and M.S.
(Fig. 2C); and n.m.r. (Table 5 ) confirmed the presence of trichodesmine (1x1(Atal
et al., 1966).
Crotalaria lanata
Dried leaves (57 g) from Nairobi afforded 75 mg of total alkaloids (0.13%).
Separation by preparative thin layer chromatography (20 x 20 cm, 1 mm, Merck,
Kieselgel 254/366 plate, run in chloroform methanol (3 :7)) afford trichodesmine
from the band with RF 0.2-0.4. A second minor constituent was tentatively
identified as crispatine (X).
* ' H n.1n.r.5peCtrd ofCDCI, rotations were run at 100 M/H, on a Perkin-Elmer R14 spectrometel
Table 5. Nuclear magnetic resonance spectra
T
4.0s
5.0m
5.3m
5.6m
6.0m
7.8m
8.6s
9.0d;
J=3.5cps
Nature of
signal
IX
Monocrotaline
VIII
H at C,
-CH,-O-CO-;C,
H, at C,
Hat C,
Hat C,
H, at C,
CH,-+OH
CH,-+H
X
d
8.96d
9.04d
J=6cps
s, Singlet;
Trichodesmine
d, doublet; m, multiplet.
X
PYRROLIZIDINE ALKALOIDS IN ARCTIID MOTHS
Monocrotaline
Pm
Trichodesmine
Crispatine
313
lX
X
HOST PLANT RELATIONSHIP A N D SECONDARY PLANT SUBSTANCES
IN THE ARCTIIDAE
Although the presence of toxic and distasteful substances in the aposematic
Lepidoptera, derived from their food plants, was deduced over a hundred years
ago (see Rothschild, 1972b1,chemical proof of this fact was not forthcoming until
relatively recent times. (Reichstein, 1967;Reichstein et al., 1968;Aplinet al., 1968).
The Arctiidae, a family which now includes the Aganaids (Hypsids)(Forbes, 1960;
Watson, 1975) and by some other authors also the Ctenuchids (Forbes, 1939)
present an unusually interesting example of this phenomenon. They form a
worldwide warningly coloured complex and, where species occur together, a
Mullerian mimicry complex (Seitz, 1930; Blest, 19641, and the species so far
examined pharmacologically have been shown to possess toxic secretions of their
own. There can be little doubt that these particular defences were first developed as
deterrents directed against nocturnal mammals, especially bats (Roeder, 1967 ;
Treat, 1955, 1963;etc.1, and to a lesser extent against crepuscular avian predators,
although many species (especially Pericopinae) can now rank as diurnal moths.
One may speculate that their bright colours were selected in reponse to bird
predation originally exerted during their daytime resting phase (Rothschild et al.,
1973).I t seems unlikely that any non-toxic moth would be able to bridge the dangerous gap between nocturnal and diurnal activity. Nevertheless the great majority
of aposernatic Tigers and Aganaids still pair and fly by night, for example Euplagta
quadripunctaria Pod., although this species is much in evidence feeding in
aggregations on ivy blossoms and Calluna in brilliant sunshine (Walker, 1966;
personal observation, M.R.). Arctiu caja does not feed as an adult and is therefore
not exposed to bird or wasp predation at nectar sources during daylight hours; it
is essentially nocturnal and the retention of its gaudy colouration and warning
displays is evidence of the degree of disturbance to which moths are subjected
while at rest. This suggests one route along which warning colour can be forced
upon a toxic species; we have often pointed out (Rothschild, 1972a,b; Rothschild
et al., 1979,in press) that the development of chemical defence mechanisms of the
Arctiid or Danaid type destines a species to adopt self-advertisement as a means of
protection. The fact that various present day Arctiids still appear somewhat
M . ROTHSCHILD ET A L
3 14
1007
-
A
-
50-
I36
I
lo0l
B
236
I
I36
3
LL
0
100
M+'
1
'
I40
150
C
2
!
M
+ -
353
o t -
1,1.11,
,
I,.
111
1111
111
I,. I
80
m/e
Figtiic 2 . Mars sprcrrutn o f : A,jac-obine; B, inonocrotaline;C, trichodesmine.
I
I
I.
I,
PYRROLIZIDINE ALKALOIDS IN ARCTIID MOTHS
315
ambivalent in this respect (Watson, 1975: 6) and adopt cryptic attitudes when at
rest“ is not surprising since crypsis-at-a-distance (Rothschild, 1975: 23) is a well
know strategy of many otherwise self-advertising and toxic species. Morton Jones
( 1932) drew attention to the fact that butterflies, on the whole, are less acceptable
to birds than moths. Thus in his first series of experiments he tested 78 species of
moths and 15 species of butterflies by exposing them to bird predation in a garden.
The former had a 78.5 overall acceptability rating and the latter only 37.3 The
majority of butterflies (Hesperidae excepted) are, broadly speaking, aposematic,
since even those groups like the tough and fast-flying Churuxes, which now largely
depend on speed and evasive behaviour for protection, are involved in a certain
amount of risky ‘public’ exposure during daylight hours. I t seems safe to assume
that the original day-flying Tiger, like the original butterfly, was a distasteful
insect. I t is therefore not surprising to find that many of their food plants can be
classified as toxic, for the association between toxic and aposematic insects and
poisonous plants is now well recognized (Rothschild, 1972a). But all such
associations do not necessarily arise in the same manner, nor at the same moment
in the insect’s evolutionary history. I t is likely that sequestering of toxic plant substances is generally a form of “stockpiling” of deterrents, increasing the insect’s unpalatability or even assuming a major role in the evolution of these characteristics,
but not necessarily providing the starting point of the specialized aposematic lifestyle. Thus, one might suppose that Danaids originally were no more or no less unpalatable than other ancestral Nymphalids, in which only the upper surface of the
wings were gaudily coloured but the undersides cryptic, and that the development
ofaposematic colouration to all wing surfaces and a characteristic slow flight, etc.,
etc., was associated with, or dependent upon, their conquest of the Asclepias plant
niche. Similarly the mimics which followed this new trend in their potential
models were also no more or no less toxic than the average Nymphalid-a
point supported by Jane Brower’s ( 1958) laboratory experiments with the
Monarch and the Viceroy.
The Arctiids are characterized by a general tendency towards polyphagy (see
Kostrowicki, 1969; Slansky, 1976) and species are known in both the old and new
worlds (Hayward, 1969; Forbes, 1960) which feed on many different families
including both ‘apparent’ and ‘unapparent’ species (Feeny, 19 761, and
sicotyledons as well as monocotyledons. Very few of the life-cycles of the 13,500
odd Arctiid species are known; a rather superficial survey of the recorded food
plants of 282 of them- (Table 6) shows they are distributed among 110 plant
families. This, however, is a considerable underestimate since twelve of the
species we noted were recorded as “feeding on low plants” and these cannot be
included in our table. In addition, even when specific food plants are mentioned,
the authors quite frequently add “etc., etc.” or “on various weeds”, or “low
plants including Listeru ouata (Orchidaceae)” o r similar phrases. It is evident,
however, that both in the old and new world the Compositae (82 records) are the
most favoured group. This suggests that the presence of PAS in the body tissues
of these moths could have played some part in their change from the nocturnal
to the diurnal aposematic life-style. On the other hand the number of species
feeding on the Boraginaceae, which are sometimes characterized by the presence
of PAS,are rather limited ( 14 records). The second most important family of host
* In aposematic moths it is the upper surface of the forewings which are cryptic in the resting position. In
nioderately distasteful butterHies it is the underside of the wings which are cryptic when at rest.
M. ROTHSCHILD E T AL..
316
Table 6 . Larval foodplants of 282 species' of Arctiidae (from varied sources in the
literature)
Plant family*
Compositae
Leguminosae
Papilionaceae
Graminae
Plantaginaceae
Rosaceae
Asclepiadaceae
Apocynaceae
Solanaceae
Salicaceae
Boraginaceae
Pol ygonaceae
Euphorbiaceae
Convolvulaceae
Moraceae
Ulmacaeae
Total number
of species
Old World
(Africa, India
Europe)
82
41
34
3
42
21
18
3
34
26
25
21
13
12
12
12
I3
23
7
15
18
15
14
13
12
11
11
11
1
8
I1
11
8
2
4
6
2
4
8
9
5
8
1
3
New World
(North,
South &
Central
America)
Australia
* Thereare 1-IOrecordsfrom96otherplant families.
plants are the Leguminosae (including the Papilionaceae) with 42 records. These
plants are rich in alkaloids (Harborne et al., 197 l),and Crotalaria, containing PAS,
is especially favoured, but Lupinus and Ulex (quinolizidine alkaloids), Cassia and
Erythrina (phenylalanine and tyrosine-derived alkaloids) are also eaten. Although
the Graminae feature as the third most favoured family of food plants, their
determination is so vague that it is virtually impossible to gauge the role of the
secondary plant substances in their selection. I t would not appear likely,
however, that the larvae feeding on Rosaceae-which is also well favouredobtain deterrents from these plants. Until recently the same comments might have
applied to the Plantaginaceae, but with the realization that the Iridoids play an
important role in insect defence mechanisms (Pavan, 1975) it is possible they
provide these substances for their Arctiid herbivores. Spilosoma lubricipeda and
Arctia caja are good examples of genera which although linked to Senecio,
nonetheless display the tendency to polyphagy. Over 50 species of plants,
including various trees, have been recorded as hosts of the latter, and larvae of
the former species have been found in greenhouses actually feeding on ripe
nectarines! ' (Lane, 1959). However, fairly closely related species may be
oligophagous, i.e. limited to a family of plants, or more rarely monophagous,
and apparently confined to a single genus or possible a species of food plant (f. e.
T.jacobaeae in the U.K.).Even so, the polyphagous larvae evince a predilection, or
at least the ability to feed and develop on toxic plants. Thus, for example, A . cuju
in the U.K. has been recorded from Aconitum, Smecio, Conualaria, Digitalis,
Solanurn, Mercurialis and Urtica, to mention only a few. In the laboratory it will
* Fruit eating may be less unusual than one might suppose. Half-grown A cqa larvae (previously reared on
of Asclcptar curmsautca, chose to feed on slices of ripe bananas which had been
provided for a tame bird.
Senecio), when offered leaves
PYRROLIZIDINE ALKALOIDS IN ARCTIID MOTHS
317
also feed on Cassia (personal observations, M.R.) and Cannabis (Rothschild et al.,
1977) and can also store various types of Secondary plant substances. This
tendency is seen in the new as well as the old world, and various Asclepias, Ficus,
Ricinus, Loranthus, Cestrum, Solanum, Chrysanthemum, Crotalaria, Zamia, etc., etc.,
figure in the list of Arctiid food plants (Table 6). As noted above, the Compositae,
especially Senecio, are favoured, and it is interesting that some species have
become monophagous on plants of isolated genera, from several different
families, which share in common the presence of pyrrolizidine alkaloids in their
tissues (Swain, 1963, 1966; Rothschild 8c Aplin, 1971). This suggests that, as in
the case of some Pierids, the secondary plant substances may become one of the
over-riding factors determining their food plant selection.
The Danaids have apparently evolved special adaptations which protect them
against the effects of the cardenolides in the foliage they consume (see below, p.
0001, but it is inconceivable that the Arctiids could develop specific methods for
dealing individually with the wide range of toxic secondary plant substances which
they can eat with impunity, and also sequester and store. One possible explanation
could be found in their own toxic secretions which may engender a generalized insensitivity to certain classes of other poisons (see also Rothschild & Kellett, 1972)
and thus pre-adapt them to the occupation ofvarious well protected plant niches,
not available to other species. Further refined adaptations would follow the initial
step.
For many aposematic insects, such as the Lygaeid plant-bugs, this must be the
path along which the stockpiling of deterrents has evolved, although in other cases
such as the Hadeninae (Lep. Noctuidae), their choice of food plant (bulbs of
Amaryllis and other lilies) could have initiated the aposematic life-style.
It is of interest that spiders, almost all of which are poisonous, are remarkably
insensitive to a wide range of toxins (Bettini & Brignole, 1978) in their arthropod
Prey*
THE FUNCTION OF PYRROLIZIDINE ALKALOIDS (PA)
IN THE ARCTIIDAE
White and Buff Ermines, as well as A . c q u , are only occasional storers of PAS
since they, too, are polyphagous and in nature other plants such as Taruxacum and
Rumex, which d o not contain these alkaloids, are often favoured. Since the
imagines do not feed, they cannot, like many Danaids, sequester these substances
after pupal emergence from appropriate wilting foliage or even nectar (Deinzer et
af., 1977; Dixon el af., 1978). It is therefore obvious that PASdo not play a crucial
role in their sexual behaviour patterns. It is worth noting that whereas the White
Ermine possesses well developed coremata these structures are lacking in both A .
c q u and S. lutea. However, no comparison was made by us of the relative fertility or
success of Ermines reared on Taraxucum and Senecio, it was merely noted that,
feeding on the former plants, females paired and laid many fertile eggs. It is not
impossible, however, that if these moths fortuitously sequester and store PASthey
could be metabolized and used to reinforce their aphrodisiacs-assuming
they have them (Eltringham, 1934). Culvenor & Edgar (1972) identified
dihydropyrrolizidines in the coremata of Utetheisa spp. It must be recalled that
various Arctiids, especially in the New World, which feed as adults, have been
recorded as regular visitors to PA sources (Pliske, 1975) such as wilting
M . ROTHSCHILD ET A L
318
Heliotropium, etc. But whether, like many Danaids, they are sequestering sex
pheromone precursors or adding to their defence repertoire, or both, is a matter
for conjecture.
Our original supposition (Aplin et al., 1968; Rothschild et al., 1973: 240) was
that PAS functioned as predator deterrents, a fact since demonstrated experimentally by Eisner and his collaborators (personal communication) and also
emphasized by Edgar et al. (1976) in connection with Danaids. We based this
hypothesis on the fact that those storers we tested were all aposematic and
relatively unacceptable to various predators (Frazer 8c Rothschild, 19611,while two
species of cryptic Noctuiids reared by us alongside T. jacobaeae on S. vulgaris
contained no PAS, and were also accepted by our tame birds (Lane, 1957).At the
time we drew attention to the rather slow-acting nature of these poisons, which,
considered as an aide mimoire for the naive bird predator, presented something of
an enigma. However, apart from their long range effects which can be disastrous
for domestic cattle, horses, poultry and man himself (Bull, Culvenor 8c Dick,
1968; Kay & Heath, 19691, Schoental (personal communication), from her own
observations, concluded that the ingestion of pyrrolizidine alkaloids can cause
immediate pain and discomfort to a mammal such as the laboratory rat. This is
apparent even if severe symptoms, prostration and death are delayed for weeks
or months. In several instances a single dose of seneciphylline (100-200 mg/kg),
the chief alkaloid stored by the Cinnabar and Garden Tiger moths, killed the six
experimental rats in one to ten days (Schoental 8c Magee, 1959).
I t would be extremely interesting to compare the relative acceptability to
predators of various Tiger moths, such as the Ermines and Arctia reared on
raraxacum or Senecio, especially with regard to the memory fixing qualities of the
PAS. Such a study might explain some of the contradictory results of many feeding
experiments with wild caught moths. It is quite possible that their main function is
a slow acting booster to the other toxic qualities of the imago. A very striking
observation made by Windecker (1939) and corroborated by us, is that for a period
of about three weeks following pupation, the pupa of Tyn'ajacobaeae is acceptable
to bird predators, despite the fact that it obviously contains its full complement
of larval-sequestered PAS from the food plant. We know from experiments with
A . caja that the toxic polypeptide, characteristic of the adult female abdomen
and eggs, is not developed in the early pupa (Rothschild et al., 1979), which at
this stage is innocuous if injected into a mouse or locust. These two observations
suggest that PAS alone are not very effective deterrents but probably complement
the pharate adult or adult insect's own unpalatable secretions.
I t is characteristic of the secondary plant substances/Lepidoptera interrelationships that there is a wide range in the apparent degree of dependence on
the chemicals concerned. On the whole, one gets the impression that even in the
Pieridae in which sinigrin lays such an important role as an oviposition cue,
larval feeding stimulant an in defensive stockpiling, the butterflies concerned are
not dependent upon it. We have found a field of apparently sinigrin-free kale
which nevertheless supported a large flourishing population of Pieris: on testing
for mustard oils, both plants and butterflies also proved negative (Aplin 8c
Rothschild, unpublished observation). I t is now very well known that Danaids
breed freely on plants which do not contain cardiac glycosides although
cardenolides must play a role in their defensive strategies.
In the Arctiidae we find both extremes of this type of association. Thus Arctia c q u
if.
PYRROLIZIDINE ALKALOIDS IN ARCTIID MOTltS
319
is an opportunist which stores PAS if they occur in the food plant encountered
during their larval perambulations. They are, however, in no way necessary either
for their reproductive success or for their defence mechanism. At the other end of
the scale, one finds Utetheisa which will lay only on plants containing PAS and
are therefore narrowly dependendent upon them-a striking fact when they occur in
unrelated families of plants such as Tournefrtia and Crotalaria. One is tempted to
assume that in such instances PAS can also act as oviposition cues - perhaps the
common esterifying acid part of the alkaloids can play this role in the case of the
Cinnabar. Yet, as we know, the Cinnabar does not lay indiscriminately on its U.K.
food plants Senecio vulgaris and S.jacobaea (Aplin & Rothschild, 1972; personal
observations), so that other factors can influence and determine the ovipositing
females’ final choice of food plant. Similarly, Bopprc ( 197 7 ) has demonstrated that
all PASare not equally effective in promoting copulation between specific Danaids.
The Arctiids, particularly A . cup, provide ideal material for investigating this
relationship in depth. Thus, for example, this latter species can be reared easily
on plants with or without PASand on artificial diets, and, if fed to predators, the
possible memory-fixing qualities of these alkaloids could be tested.
It would also be worth investigating their relative breeding success on S.jacobaea
and S. vulgaris, compared with other favoured food plants. In nature, even A . cqa,
an apparently haphazard ovipositor, may show a preference for laying on S.
vulgaris, although there is no evidence to suggest this at present. There is also an
excellent opportunity here for comparing the deterrent qualities of the eggs of A .
c q a which incorporate PAS, and the more warningly coloured eggs of Tyria
jacobaeae which, curiously enough, do not.
I t will also be interesting to compare the role of PASin the two sexes. In Danaids,
ofien only adult males contain them in their body tissues, and the male is generally
the principal sequesterer after emergence. However, both sexes ofDanausplexippus
and Euploea core (Cram.) obtain PAS as adults (Edgar et al., 1979 in press; Dixon
et al., 1978) from nectar and bruised plant tissues, and if these substances are
artificially introduced into their larval diet the body tissues of adult males contain
about twice as much PAS as the females (Rothschild & Edgar, 1978) suggesting
they are more effective storers. On the other hand we have seen (p. 309 above)
that female S. lutea store more of certain PAS than males reared on the same
plant.
T H E ENIGMATICAL CHARACTER OF ARCTIID SELF-SECRETED TOXINS
The multiplicity of Lepidopteran enemies, ranging from bacteria and moulds,
to spiders, mammals and birds, demands a plethora of defensive strategies.
Although some of these bear the stamp of classical defence mechanisms, for
example the presence of biogenic amines in the repugnatorial glands - a feature
shared by such varied organisms as snakes, whelks, hornets, octopuses and spiders,
etc., etc., - the function and purpose of many of these presumed protective devices
are unknown. Thus, when the resting A . cqa is disturbed, it stridulates, the sound
acco~npaniedby a sterotyped wing display, but this has never been observed in
nature directed towards a specific predator. One can only guess therefore at the
designated recipient of this elaborate visual and aural performance. We are even
more in the dark where the active proteins of polypeptides of the Lepidoptera
(Rothschild et af., 1979) are concerned. We assume they must function as ef’ective
320
M . ROTHSCHILD ET AL
deterrents against some range of predators, since they are lacking in cryptic
species, but often present in those aposematic types which rely for protection on
self-advertisement rather than concealment. These substances are not apparently
active via the oral route (Marsh 8c Rothschild, 1974; Bissett et al., 1960)but their
toxic nature can be demonstrated by injection into mice via the intraperitoneal
route. Thus a powerful toxin, cajin, is found in the female abdomen and eggs ofA.
cuja which is approximately equal in potency to bee venom. Yet this moth is not a
strikingly distasteful species compared with other Arctiids. Frazer and Rothschild
(1961) accorded A . cuju a rating of 4 (out of 7 ) compared with 7 for Tyriajacobaeae
and 6 for Callimorpha (= Panuxia) dominulu (L.) when tested on six species of
mammals, two reptiles and one amphibian predator. These authors, however, did
not record the sex of the prey offered. Lane (19571,who fed a variety of British
insects to a tama Shama (Kittucincla malabarica (Gm.))rated it “unpalatable but
eatable”, whileJanin (1972) recorded wild Great Tits (Parus mqor L.) attracted to a
feeding tray, eating it with evident relish. Some animals, of which the Bush Baby
(Galago) is an outstanding example, found the taste or feel of A . cuju in the mouth
intolerable, and one experience resulted in the avoidance of all adult Lepidoptera,
including those previously eaten with enthusiasm. The Garden Tiger also seemed
to exert a long-term deterrent effect on some bird predators, which ate it without
hesitation on the first occasion (several specimens in quick succession) and subsequently avoided it. This is in sharp contrast to the deterrent effects of T.jacobaeue,
which are generally immediate (Windecker, 1939 ; and personal observation) and
involve the hurried release of the captured insect. Curiously enough, an ant Lasius
nzger (L.) displayed the opposite reaction: T. jucobaeae was dragged into its nest
while eggs, pupa and adult of A . cqa, whole or dismembered, were carted off and
dumped some distance away (Parsons 8c Rothschild, 1964). The omnivorous
cockroach, however (Rothschild et al., 19791, will only eat A. caja eggs if very
hungry, although Pieris brussicue L. eggs were consumed with relish.
This type of insect deterrent is extraordinarily varied - a fact which is apparent
without any accurate knowledge of the chemistry of the substances in question.
Thus, for example, the macerated eggs of the Burnet Moth (Zygaena), when
injected intraperitoneally into the mouse, exert an astonishingly rapid response,
killing the animal in three minutes (Marsh 8c Rothschild, 1974), whereas
preserved in a refrigerator they rapidly lose their potency. However, frozen or
air-dried eggs of A . c q u are virtually as effective as fresh material even after long
periods of storage. Apart from such basic differences there is a wide range of
acceptability between one species of Arctiid and another, which suggests that the
chemical deterrents affecting taste, odour and acceptability by the naive bird are
at least as varied as these substances causing pain and discomfort after injection.
As we have indicated, the toxic substances we have found in T.jacobaeae and C.
dominulu are not so lethal to the injected laboratory mouse as those of A . caja, yet
both these moths are far more disagreeable to the taste of predators than the
Garden Tiger and are relatively rarely eaten. It is significant that if A . cuja is
reared on the same Senecio plants as T. jucobaeae it retains the same 4 : 7 rating of
unpalatability, and is far more readily accepted by predators than the latter
species. It is obvious, therefore, that PAS are not the primary cause of the striking
distastefulness of T . jacobaeae since both species are good storers of these
alkaloids.
Originally the toxic quality of A . cuja was noticed because, during handling, the
PYRROLIZIDINE ALKALOIDS IN ARCTIID MOTHS
32 1
moth (a male) inflicted what appeared to be a severe sting on the handler
(Rothschild, 1957). This isolated episode remains an unsolved mystery, but it
seems possible that the toxin can be - in some circumstances not yet elucidated introduced into the subcutaneous tissues, possibly via a broken spine, if the moth
is crushed in the mouth ofa predator.
The active polypeptide responsible for the lethal effect of abdominal extracts,
does not develop until the late pupal or pharate adult stage. A similar latent period
(in this case, of distastefulness) of four weeks (see above, p. 000) was noted by
Windecker ( 1939)for the early pupa of T,jacobaeae, a fact which demonstrated the
relatively unimportant deterrent role of the pyrrolizidine alkaloids present bequestered by the larvae) at this stage. It will be recalled that the emetic potency of
Danausplexippus (L.) increases during the pharate adult stage (Dixon et al., 19781,
but we do not know if this is due to a modification of the stored cardiac glycosides
(for instance by the suppression of an hydroxyl group) or by the secretion of some
substance which potentiates the effect on the predator’s nervous system and
increases the sensitivity of the smooth muscles of the stomach and crop. It is also of
considerable interest that in A . c q u the active toxins are concentrated in the ovaries
and eggs. It appears probable that the highly poisonous substance zygPnine
(Rocci, 1916; Marsh 8c Rothschild, 1974) is also mainly located in the eggs and
ovaries of the gravid female Burnet Moths (Zygaena), although Rocci, and Lane
( 1959),also found evidence for its presence in the haemolymph. The eggs of insects
are “sitting targets” and we should expect them to show varied chemical defence
mechanisms, especially those which are, like Zygaena and A . c q u , laid in large
batches. One of the most interesting examples is that of meloid beetles (Lytta), the
notorious ‘Spanish fly’, in which cantharadin secreted by the male beetle is passed
to the female during copulation (Sierra et al., 1976)and possibly directed onto her
eggs.
It is tempting to postulate that the evolution of the poisons associated with the
stinging apparatus of Hymenoptera originated as substances for the protection of
the eggs, especially those providing repellent extra-chorion material which, in
Lepidoptera, is also secreted by the accessory glands (Behan 8c Schoonhoven,
1978).
The distribution of the biogenic amines in non-nervous tissues within the family
appears as capricious as that of the polypeptides or toxic proteins, which suggests
they may perform different functions in the various organs concerned, but
probably also combine with toxic secondary plant substances to boost or
potentiate the latter’s deterrent effects. High concentrations of histamine have
been found in T.pcobaeae (also present in the larval hairs of A . c q a (Frazer, 196511,
ACh in the body of Spilosom lubricipeda in the abdomen of A . caja and A . villica, and
in the eggs of A . c q u . D/?-dimethylalcrylylcholine has been identified in the
defensive glands of A . c q u and Utetheisa bella. Unlike the polypeptides, the biogenic
amines are present in both sexes although their distribution in the body of males
and females may be different (Rothschild, 1972a, Table 2).
DISCUSSION
So far we have found that all aposematicTiger moths which feed on plants which
contain pyrrolizidine alkaloids store these substances. This is in marked contrast to
certain aposematic species feeding, for example, on plants containing cardiac
322
M. ROTHSCHILD ET A L
glycosides, several of which have proved negative for cardenolides (for example,
Digama aganais Felder and D. sinuosa Hampson feeding on Acokanthera (Rothschild,
19 72a, Table I), and Danaus chrysippus alcippus Cramer feeding on Gomphocarpus
(Rothschild et al., 1975)).It has been suggested that the Buff Ermine (S. lutea) is a
mimic of the White, but its ability to store PASeven more effectively than the latter
species renders this doubtful. Its relative acceptability to birds may be due to lack
of biogenic amines (Rothschild, 1972a), but it should probably be considered
merely as a slightly less toxic member of a large Miillerian complex. I t would be
interesting, however, to compare the feeding preferences in nature of these two
polyphagous larvae since their distastefulness could vary according to their diet
(seealsoMarshetal., 1977).
It should be emphasized that the polyphagy of species such as Spilosoma and
Arctia caju, which feed indiscriminately o n a wide variety of plants, and especially
on different species of the genus Senecio, some of which contain many PAS and
others only a few or none, results in an apparently capricious distribution of these
substances among the Miillerian mimics involved in the complex in question. I t
would thus seem to be a matter of pure chance which of the species is the more
toxic, at any one moment in time, depending on such factors as host plant
abundance, seasonal availability and variation (A lin & Rothschild, 1972). This
provides a stabilizing influence within the complex or it tends to distribute toxicity
among a certain proportion of all the insects involved - thus minimizing the
tendency to “lose ’ species from both ends of the spectrum. Brower et al. ( 1967)
have drawn attention to a somewhat similar situation within a single population of
the species Danaus plexippus. They designated those specimens which lacked
cardiac glycosides (owing to their absence from the food plant) as automimics of
those which stored them. In the case of the Arctiids which secrete various toxins
themselves in addition to “stockpiling” the secondary plant substances, the whole
situation must be seen as a fluid one, which engenders a generalized warning
message for potential vertebrate predators.
Species in which we have been able to assess the storage capacity of PAS by the
two sexes suggest that there are differences between males and females, as well as
selective storage of certain specific PAS. It is a pity that it is so difficult to obtain
sufficient females of Diaphoru mendica for testing, since their males are largely
cryptic in the U.K. race, through both sexes are white (aposematic)in Ireland. Are
the latter better storers than English males?
Of the two Crotalaria feeders, Amphicallia bellatrix is shown to be the most efficient
storer of PAS, if food plant and overall moth content are compared.
Trichodesmine has previously been isolated from the Himalayan species Crotalaria
rubiginosa Willd (Atal et al., 19661, while monocrotaline sequestered by A. cribraria is
more common, and has previously been extracted from a number of Crotalaria
species (Bull, Culvenor & Dick, 1968).
It should be noted, however, that even Arctiu caja is by no means a universal
storer of secondary plant substances, despite its ability to feed on so many toxic
species. Thus, reared on cabbage, it does not store mustard oils (Aplin &
Rothschild, unpublished observations) and although the larva fed on Cannabis
accumulates cannabinoids in its body tissues, these are stored mainly in the
cuticle and are shed at each moult. It effectively rids itself of both THC and CBD
before pupation (Rothschild et af., 1977).
Authors not infrequently (see Duffey, 1977) express surprise at the ability of
P
PYRROLIZIDINE ALKALOIDS IN ARCTIID MOTHS
323
insects to store PAS.But is this so suprising?- for the extremely toxic effect of these
substances is mainly due to one of the by-products of mammalian liver function,
which, in the process of “detoxification” of the PA, itself produces a harmful
metabolite. The “popularity” of Senecio alkaloids as a deterrent used by
aposematic insects may be explained by its deleterious effect on mammalian,
compared with insect herbivores. The fact that the Danaids appear to have
uncoupled Na+ and K + pumps (Jungreis 8c Vaughan, 19771, thus rendering the
toxicity of cardenolides somewhat irrelevant to these insects, is an analagous
situation. Unfortunately the Asclepiad-feeding Arctiids have not yet been
investigated from this point of view. Krieger et al. (19711 have also shown that
Arctiids feeding on more than eleven plant families have a higher epoxidase
activity in the midgut than species confined to 2-10 plant families (they did not
examine any monophagous Arctiidae) and suggest that this indicates an adaptive
strategy evolved to cope with a wide range of secondary plant substances.
Notwithstanding these various types of specializations which are now coming to
light, it is possible that in order to become “poison specialists” the ancestral
Arctiidae required the blanket protection afforded them by their own toxic
secretions. Once they had conquered the SenecidCrotalaria niche the dual purpose
of defence and sexual stimulation would immediately place a premium on the
sequestration and storage of PAS, and the trend to feed on plants containing
these substances would be reinforced. Gilbert (1979, in press), in his excellent
paper on insecdplant relationships, suggests that Tiger moths probably represent
an atypical group of toxic plant specialists. It seems likely that any groups of
insects which are examined in depth will present this degree of atypical features
and “special situations”, for the relationships between plants, insects and
predators are so infinitely complicated and the chemicals involved in defence so
diverse and complex (Zlotkin, 1973). Furthermore the Arctiids may well share
their type of specializations with several other groups which favour similar food
plants (Rothschild 8c Aplin, 197 1). For example the Pyrgomorphid grasshoppers
display a very similar life-style-possessing toxic secretions of their own
(Fishelson, 1960; Rothschild 8c Kellett, 1972) feeding on a wide range of rather
similar poisonous plants (Vuillaume, 1954; Bernays et al., 1975; Bernays et al.,
1977 ; Rothschild, 1972a1, and also possessing the ability to store cardenolides,
pyrrolizidine alkaloids and cannabinoids, etc. Their relationship with toxic
plants and that of various other “poison specialists” suggests they have evolved
along similar lines.
ACKNOWLEDGEMENTS
We are deeply grateful to Allan Watson for discussions, help with the literature
and for criticizing our manuscript. We would also like to thank D. G. Sevastopulo,
G. R. Cunningham van Someren and the late Bernard Kettlewell for sending us
material.
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