Phenological fate of plant-acquired pyrrolizidine alkaloids in the polyphagous arctiidEstigmene acrea

Chemoecology 14:207–216 (2004)0937–7409/04/040207–10© Birkhäuser Verlag, Basel, 2004DOI 10.1007/s00049-004-0276-2

CHEMOECOLOGY

Phenological fate of plant-acquired pyrrolizidine alkaloidsin the polyphagous arctiid Estigmene acreaThomas Hartmann1, Claudine Theuring1, Till Beuerle1 and Elizabeth A. Bernays2

1Institut für Pharmazeutische Biologie der Technischen Universität Braunschweig, Mendelssohnstrasse 1, D-38106 Braunschweig, Germany2Department of Entomology of the University of Arizona, P.O. Box 210088, Tucson, AZ 85721-0088, USA

Summary. The alkaloid profiles of the life history stagesof the highly polyphagous arctiid Estigmene acrea wereestablished. As larvae individuals had free choice betweena plain diet (alkaloid-free) and a diet that was supplementedwith Crotalaria-pumila powder with a known content andcomposition of pyrrolizidine alkaloids (PAs). Idiosyncraticretronecine esters (insect PAs) accounted for approximatelyhalf of the PAs recovered from the larvae. These alkaloidswere synthesized by the larvae through esterification of dietarysupinidine yielding the estigmines, and esterification ofretronecine yielding the creatonotines. The retronecine isderived from insect-mediated degradation of the sequesteredpumilines (macrocyclic PAs of the monocrotaline type).With one exception, the PA profiles established for larvaewere found almost unaltered in all life-stages as well aslarval exuviae and pupal cocoons. The exception is themales, which in comparison to pupae and adult females,showed a significantly decreased quantity of the cre-atonotines and pumilines. These data support the idea thatthe creatonotines are direct precursors of the PA-derived malecourtship pheromone, hydroxydanaidal. Crosses of PA-freemales with PA-containing females and vice versa confirmedan efficient trans-mission of PAs from males to females andthen from females to eggs. In single cases a male bestowedalmost his total PA load to the female, and a female her total loadto the eggs. The results are discussed with respect to phero-mone formation, PA transmission between life-stages, and thedefensive role of PAs against predators and parasitoids.

Key words. Alkaloid sequestration – pyrrolizidine alkaloids –insect alkaloids – alkaloid transmission – male courtshippheromone – chemical defense – Arctiidae – Estigmene acrea

Introduction

Plant-acquired pyrrolizidine alkaloids (PAs) are utilized asdefensive chemicals in a number of arctiids (Hartmann 1999;Weller et al. 1999; Hartmann & Ober 2000; Eisner et al.2002). PA-sequestering arctiids incorporate PAs as pro-toxicfrees bases that are efficiently converted into their non-toxicN-oxides by means of a specific soluble NADPH-dependentflavin monooxygenase in their hemolymph (Lindigkeit et al.

1997; Naumann et al. 2002). This enzyme ensures that PAsingested with the larval food are maintained in the safe stateof their N-oxides at all times and through all life-stages.Since the N-oxides are easily reduced in the gut of potentialpredators and thus converted into the pro-toxic state, bothN-oxides and tertiary PAs possess the same potential toxicityfor any predator. PAs are toxic for insects and vertebratesbecause cytochrome P450 enzymes of xenobiotic metabolismconvert (bioactivate) the free base to highly reactive pyrrolicintermediates that react with biological nucleophiles (Brattsten1992; Cheeke 1994; 1998). The primary consequences are celltoxicity and mutagenicity. PA-N-oxides are not bioactivated.In insects mutagenic effects of PAs have been proved in theDrosophila wing test (Frei et al. 1992) and direct toxicity ofPAs on growth and survival was demonstrated withPhilosamia ricini (Saturniidae), an exemplary generalist her-bivore: larvae showed reduced growth and all died as pupae(Narberhaus et al. 2004). Detrimental effects of PAs onpredators are probably rare in nature since PAs are stronglydeterrent and thus a PA-protected prey is generally not eaten.

The role of PAs as host-derived defensive chemicals inthe performance of arctiid moths has been most completelyelucidated in the monophagous Utetheisa ornatrix (Eisneret al. 2002). Larvae of this moth obtain their PAs fromCrotalaria spp. and retain the alkaloids through metamor-phosis. At mating the male advertises to the female his loadof PAs, via the male courtship pheromone, hydroxydanaidal.This is emitted from a pair of scent brushes (coremata) whichhe everts during close-range precopulatory interactionwith the female. High hydroxydanaidal levels in the malecorrelates not only with high systemic PA load (Dussourdet al. 1991) but also with large body size (Conner et al.1990; Iyengar & Eisner 1999) – a trait that is heritable.Females are able to differentiate between males that containunequal quantities of hydroxydanaidal and appear to favormales having higher corematal levels (Conner et al. 1990).During precopulatory assessment of males hydroxydanaidalappears to be the only criterion of choice (Iyengar et al.2001). At mating, the male transmits a portion of his PAs tothe female during insemination (Dussourd et al. 1988). Inthe female the male-derived PAs are immediately distributedthroughout her body including the wings (Rossini et al.2001). At oviposition these PAs are deposited in the ovariesand a large portion of the female’s PA load is transmitted tothe eggs. Correspondence to: Thomas Hartmann, e-mail: [email protected]

Compared to monophagous arctiids like Utetheisa thatalways stays and feeds on its alkaloidal host plant, there are arc-tiids that are extreme generalists and include different PA-containing plants in their diets. Among these is Estigmeneacrea for which 69 larval host plant species are listed(Robinson et al. 2002) among them Senecio longilobus(Asteraceae) and Crotalaria pumila (Fabaceae) known tocontain PAs. In the insects’ habitat, in southern Arizona(e.g. grassland and savanna habitats), these two species areoften relatively uncommon and must be located by thewandering larvae.

E. acrea is well adapted to sequester, metabolize andtransmit PAs (Hartmann et al. 2004). Males are known topossess coremata and emit hydroxydanaidal (Krasnoff &Roelofs 1989; Davenport & Conner 2003). As in manyother arctiids (Boppré 1986; Schneider 1987) PAs arestrong phagostimulants for caterpillars of E. acrea(Bernays et al. 2002b) and they possess two PA-sensitivegustatory neurons in the galeal styloconic sensilla, oneof which is highly specific to PAs, and the other respond-ing to PA concentrations as low as 10−12 mol l−1 (Bernayset al. 2002a, b). This great gustatory sensitivity correlateswith the strong feeding stimulation causing the caterpil-lar to feed actively on a suitable PA source. Moreover, ithas been shown that the stimulating gustatory sensitivitycan be modulated in two directions. Firstly, the respon-siveness of the PA sensitive cells in the two sensilla con-tinuously declined if the caterpillars were reared on analkaloid-free diet but the decline could be reversed byexperience with PAs in the diet (Chapman et al. 2003).Secondly, extensive feeding on plants rich in PAs causeda loss in response of the PA-sensitive cells; the sensiti-vity loss lasts for approximately two hours (Bernayset al. 2003). This reduction of responsiveness could becorroborated by behavioral experiments and field obser-vations, indicating some specific mechanisms that, inspite of the strong feeding stimulation, allow the cater-pillars to leave their PA source and search for alternativeand potentially better nutrient sources.

In this paper, we examined the sequestration andprocessing of PAs by larvae of E. acrea reared under condi-tions where they always had the choice between PA-containing and PA-free artificial diets and thus wereunlikely to have had doses of PAs that reduced gustatorysensitivity. The following aspects were studied: (1) thequantitative distribution of plant-sequestered PAs and idio-syncratic retronecine esters (insect PAs) in the differentlife history stages; (2) both sexes of pupae and adults wereanalyzed to confirm the previously observed sexual dimor-phism of the creatonotines, which most likely function asprecursors of hydroxydanaidal biosynthesis in males(Hartmann et al. 2003); (3) the transmission of PAs to eggs,directly from females, and from males via females.

Methods

Insects

Laboratory cultures of Estigmene acrea were obtained fromcaterpillars collected at Gardner Canyon and Box Canyon in southernArizona. Caterpillar cultures were reared on a wheat-germ-based

artificial diet (Yamamoto 1969). Insects were raised individually in200-ml plastic cups containing a small cube of diet that wasreplaced daily. Two kinds of diet were used: (a) plain diet withoutaddition, (b) PA diet, the same diet mixed with Crotalaria pumilapowder at 10 % of dry weight. The C. pumila powder containedPAs at a level of 0.18 %, giving a final concentration in the diet of0.018 %; the alkaloid composition is given in Table 1. Larvae wereraised either on plain diet without any access to PAs, or had accessto both foods. Larvae feeding on PA diet chosen for PA analysiswere always confined with plain diet for 14 h to ensure that PAs inthe gut were evacuated.

Larvae were allowed to complete development either on theplain diet or the choice of the two diets. Pupae for chemical analy-sis were first sexed and then frozen. Pupae retained for obtainingadults were sexed and one of each sex placed in a plastic container(8 liter) with a Petri dish containing wet cotton. Sheets of papertowel and wax paper provided substrates for mating and oviposi-tion respectively. Crosses were made between males and femaleswhich had both had access to PAs as larvae, between PA-free malesand PA-containing females, and between PA-containing males andPA-free females. Eclosion, copulation and oviposition was moni-tored twice each day. Copulation was observed in some cages butnot others, and because this appeared to be a lengthy process, it ispossible that eggs were unfertilized in cages where it was notobserved. After oviposition in a cage, a process that took one tofour days, males and females were frozen and eggs were collectedfrom the paper. Clutches from each female were separately frozenin Eppendorf tubes prior to analysis for PAs.

Females vary greatly in the number of eggs deposited and theway in which they are laid. In general, unfertilized females layscattered eggs or small clusters of eggs (not usually more thatabout 24 per cluster). In contrast, fertilized females tend to laylarge clusters of up to several hundred. In this work, “clutch” isused to define the combined small or large egg clusters from asingle female. Clutches were arbitrarily defined as large or small ifthey weighted 12.8 to 36.6 mg and 1.5 to 3.4 mg, respectively.

PA analysis

All insect materials, i.e. larvae, exuviae, cocoons, sexed pupae,male and female adults, and eggs from the different experimentswere preserved at −20 °C. All samples allotted to PA analysis werelyophilized and kept in closed vials until analysis. Alkaloid extrac-tion, separation and quantitative PA analysis by capillary GC andassignment of structures by GC-MS were performed as describedpreviously (Witte et al. 1993; Hartmann et al. 2004).

Results

PA profiles of life history stages

All data presented below refer to laboratory cultures ofEstigmene acrea reared as larvae under identical conditions.Larvae had always the choice between a PA-containing diet(0.018 % alkaloids on dry weight basis) and alkaloid-freeplain diet. Larvae appeared to prefer the PA diet. The alka-loid composition was determined for larvae, sexed pupae,and adult males and females as well as exuviae and cocoons(Table 1). With the exception of the simple necine epoxide,subulacine, all PAs present in the diet were sequestered bythe larvae. These alkaloids (Fig. 1) have previously been iso-lated and identified from Crotalaria pumila (Hartmann et al.2004). PA-X is a still unknown alkaloid of the monocrotalinetype, structurally related to the pumilines. The estigmines,creatonotines and isocreatonotines (Fig. 1) are idiosyncraticnecine esters (insect PAs) synthesized by the insect fromplant PAs. Their structures have previously been elucidatedfrom E. acrea (Hartmann et al. 2004) and leaf beetles(Hartmann et al. 2001; Pasteels et al. 2001).

208 T. Hartmann et al. CHEMOECOLOGY

Vol. 14, 2004 Pyrrolizidine alkaloids in Estigmene acrea 209

Tabl

e 1

Pyrr

oliz

idin

e al

kalo

ids

prof

iles

of la

rvae

,exu

viae

and

coc

oons

,and

mal

e an

d fe

mal

e pu

pae

and

adul

ts o

f Est

igm

ene

acre

are

ared

on

artif

icia

l die

t sup

plem

ente

d w

ith C

rota

lari

apu

mil

aPA

s (f

inal

PA

con

cent

ratio

n 0.

018

%,d

ry w

eigh

t bas

is).

All

larv

ae h

ad th

e ch

oice

bet

wee

n pl

ain

diet

and

die

t+PA

s

RI

[M+ ]

Rel

ativ

e ab

unda

nce

(%)

Lar

val

Lar

vaea

Lar

vaeb

Mal

e pu

pae

Fem

ale

pupa

eM

ale

adul

tsFe

mal

e ad

ults

Alk

aloi

dD

B-1

m/z

diet

4thin

star

fina

l ins

tar

Exu

viae

cC

ocoo

nsd

n=

12n

=9

n=

8n

=8

Pla

nt a

cqui

red

Supi

nidi

ne12

4913

919

42

−2

25.3

±2.

419

.6±

2.1

13.3

±2.

52.

6±

1.1

Subu

laci

ne12

8215

513

−−

−−

−−

−−

Ret

rone

cine

1425

155

−3

1−

17.

3±

1.3

7.9

±2.

41.

7±

1.2

1.3

±0.

5Pu

mili

ne B

2159

323

3123

1738

166.

5±

0.8

10.3

±1.

511

.6±

1.2

15.8

±2.

2PA

-X22

1633

52

11

32

0.8

±0.

30.

6±

0.3

2.4

±0.

51.

3±

0.4

Pum

iline

C22

5133

713

128

2012

3.8

±0.

22.

3±

0.8

7.5

±1.

47.

9±

1.7

Pum

iline

A23

5935

122

1513

2817

13.3

±1.

116

.7±

2.7

24.9

±2.

518

.8±

2.1

Pla

nt P

As

(% o

f to

tal)

100

5842

8950

57.0

±±3.

857

.2±±

3.1

62.3

±±5.

947

.7±±

6.7

Inse

ct s

ynth

esiz

edE

stig

min

e A

1730

239

14

24

1.3

±0.

41.

2±

0.5

2.8

±0.

70.

9±

0.5

Est

igm

ine

B18

3025

36

186

2111

.3±

1.6

14.0

±2.

120

.4±

3.4

9.4

±2.

8Is

ocre

aton

otin

e A

1865

255

−−

−1

0.7

±0.

30.

7±

0.3

0.4

±0.

21.

0±

0.4

Cre

aton

otin

e A

1874

255

54

−3

4.8

±0.

65.

1±

1.0

1.2

±0.

43.

6±

1.0

Isoc

reat

onot

ine

B19

5726

99

7−

14.

3±

0.5

4.4

±1.

23.

3±

142

8.8

±0.

9C

reat

onot

ine

B19

7226

920

223

1419

.8±

2.5

17.4

±2.

69.

6±

4.0

28.5

±4.

0

Inse

cts

PAs

(% o

f to

tal)

4155

1144

42.9

±±3.

943

.0±±

3.1

37.6

±±5.

952

.1±±

6.8

Tota

l PA

s pe

r in

divi

dual

(µg

):16

4.3

±16

.713

7.3

±30

.473

.2±

14.3

151.

2±

30.1

PA c

onc.

(m

g·g

−1dr

y w

t):

0.69

±0.

070.

46±

0.10

0.64

±0.

131.

14±

0.23

a 4thIn

star

(14

indi

vidu

als)

; b fina

l ins

tar

(5 p

oole

d in

divi

dual

s); c ex

uvia

e of

4th

to f

inal

inst

ars

(fro

m 2

4 po

oled

indi

vidu

als)

; d coco

ons

(fro

m 1

4 po

oled

indi

vidu

als)

A comparison of the quantitative PA profiles estab-lished for male and female pupae and adult males andfemales (Table 1) shows almost identical profiles for thesexes in pupae but obvious differences between the profilesof adult males and females. An analysis of variance of totalPAs per individual revealed that overall the sexes and lifestages were not different but males had less alkaloid in theadult stage but not in the pupal stage (ANOVA: sex, F 37,11.179, P = 0.286; life stage F37,1 2.679, P = 0.111; sex x lifestage, F37,1 4.959, P = 0.032). For a better comparison totalalkaloids recovered from the life stages were considered infour fractions: (1) creatonotines, (2) estigmines, (3) totalinsect PAs [sum of (1) plus (2)], and (4) plant acquired PAs(pumilines, supinidine and retronecine). Retronecine whichdoes not occur in the diet must be formed by hydrolysis ofthe pumilines; it was included in the fraction of plantacquired PAs. From Figure 2 it can be seen that the quanti-ties of the creatonotines are lower in male adults than infemale adults or pupae of either sex. A detailed ANOVA(Table 2) revealed that for creatonotine the differences are

significant (sex x life stage interaction term, P = 0.015).Using t-tests to compare pupae and adults also indicate thatin males, creatonotines, total insect PAs, and plant derivedPAs all drop from pupa to adult, while the estigmines do notchange (see legend of Fig. 2). By contrast, there are no sig-nificant changes among females (see Fig. 2). Thus, in malesthe transition from pupal to adult stage is associated with asignificant disappearance of the creatonotines and the plantpumilines.

PA concentrations in exuviae and cocoons

The data in Table 1 show that exuviae and cocoons containPAs. To evaluate the PA concentrations associated with exu-viae and cocoons an experiment was performed in whichfinal instar exuviae, cocoons, and pupae of eight individualswere quantitatively analyzed for their PA contents and con-centrations. Although only four of the eight cocoons couldbe recovered, the results clearly indicate that Estigmeneacrea lose a considerable proportion of their PAs with thelarval exuviae and incorporate a large proportion into thepupal cocoons (Fig. 3). Exuviae of the final larval stagecontain about 30 % of the PA load found in the respectivepupae, and the pupal cocoons contain almost the same alka-loid load as pupae. Exuviae and cocoons exhibit equal PAconcentration that were about tenfold higher than the con-centrations found in pupae.

Transmission of larval PAs via malesand females to eggs

A preliminary experiment was performed to show whetherEstigmene acrea endow their eggs with PAs. For thisfemales and males which both as larvae had access to a PA-containing diet were crossed and the egg clutches analyzedfor PAs. The egg clusters from six pairs all contained highlevels of PAs (Table 3). The number of eggs per clutch variedconsiderably. Small egg clutches appeared to contain ahigher PA concentration (ca. 4 mg/g) than large ones (ca, 1.7mg/g). The total concentrations were up to 4-times higherthan those measured for female adults (Table 1).

To examine transmission of PAs from females to eggsand from males to females in a more quantitative manner thefollowing two experiments were performed: Experiment I,females that as larvae have had access to PAs were crossedwith PA-free males; experiment II, PA-free females werecrossed with males reared as larvae on a PA-containing diet.After oviposition the adults and egg clutches were analyzedfor PAs. The results of two experiments are summarized inFigures 4 and 5. In experiment I eggs were obtained fromeight pairs. PA analysis of the females and eggs revealedgreat differences between the individual pairs (Fig. 4).These differences concern both the total amounts of PAsrecovered from the females and their eggs as well as theallocation between the life stages. However, in all cases PAswere found in eggs. The relative amounts of total PAs asso-ciated with the eggs vary between about 10 % of availablePAs (e.g. pairs 2 and 8) and 80 to 100 % (e.g. pairs 1, 4 and 5).In pair 5 the female was devoid of PAs and thus must havedonated all of her PAs to the eggs. The numbers of eggs per


N

OO

O

O

H

HO

1

27

N

OO

O

O

H

HOO

Pumiline A

N

OO

O

O

H

HO

9

N

OH

HHO

7

(P + I)

(P + I)

Pumiline B(P + I)

Pumiline C(P + I)

N

OH

H

N

OH

HO

R = HR = CH3

N

O

HO

OH

R 7

N

OH

HO

O

HO

R

7

9

N

O

HO

HO

OH

R

7

N

CHOHO

Supinidine

Estigmine AEstigmine B

R = HR = CH3

Isocreatonotine AIsocreatonotine B

R = HR = CH3

Creatonotine ACreatonotine B

(7R)-Hydroxydanaidal(male courtship pheromone)

(I)

(I) (I)(I)

Retronecine

(P)

Subulacine

Fig. 1 Pyrrolizidine alkaloids (PAs) of Crotalaria pumila fed tolarvae of Estigmene acrea and idiosyncratic PAs (insect PAs)recovered from E. acrea. The arrows indicate the assumed originof the necine base moiety of the insect PAs. The letters in paren-thesis indicate the occurrence of the respective alkaloid in the larvalhost plant (P), the sequestering arctiid (I) or both (P + I). Thealkaloids are illustrated as tertiary amines, in plants and insects allare maintained as their N-oxides

egg clutch varied also considerably, i.e. between 62 (pair 2)and 2,335 (pair 4) (Fig. 4, numbers at columns). The totalamount of PAs associated with a single egg ranged from 33 ngto 240 ng. It should be noted that the pairs with fewesteggs (2, 3, 7 and 8) were those with smaller amounts oftransferred alkaloid and it is quite likely that some wereunfertilized, since no copulation was observed.


µg alkaloid per individual

0 20 40 60 80 100 120

Plant acquired PAs

Total insect PAs

Estigmines

Creatonotines

Relative abundance (%)

20 40 60 80 100

Relative abundance (%)

20 40 60 80 100

µg alkaloid per individual

0 20 40 60 80 100 120

male

female

Plant acquired PAs

Total insect PAs

Estigmines

Creatonotines

Pupae

Adults

male

female

Fig. 2 Amount (left) andrelative proportion (right) ofthe fractions of “insect PAs”and “plant acquired PAs”recovered from female andmale pupae and adults ofEstigmene acrea (mean ± stan-dard error). Creatonotines andestigmines together make upthe “insect PAs”; “plantacquired PAs” contain thepumilines plus supinidine andfree retronecine. Analysesusing t-tests to compare pupaeand adults indicate that inmales creatonotines, totalinsect PAs, and plant derivedPAs all drop from pupa toadult, while estigmines do notchange (creatonotines, t 3.328,p 0.004; estigmines t 0.749,p 0.463; total insect PAs, t 2.941,p 0.009; plant-derived PAs,t 3.006, p 0.0076 − df = 18).There are no significant changesamong females (creatonotines, t1.117, p 0.282; estigminest 1.402, p 0.181; total insectPAs, t 0.63, p 0.538; plant-derived PAs, t 0.136, p 0.894 −df = 15). See also text foranalysis of variance

Table 2 Analysis of variance of pyrrolizidine alkaloid contents:absolute contents (µg per individual) of four alkaloid fractions(creatonotines + estigmines represent total insect PAs; plant PAsrepresent plant acquired PAs + retronecine)

PA group Factor F P

Creatonotines Sex 3.875 0.057Life stage 0.196 0.661S × L 6.525 0.015**

Estigmines Sex 0.884 0.354Life stage 2.316 0.138S × L 0.228 0.636

Insect PAs (total) Sex 1.895 0.178Life stage 0.738 0.396S × L 4.065 0.050

Plant PAs Sex 0.172 0.681Life stage 3.636 0.065S × L 2.829 0.102

df = 37, 1

PA

con

cent

rati

on (

mg/

g dr

y w

eigh

t)

0.0

0.5

1.0

1.5

2.0

2.5

3.0Exuviae CocoonsPupae

Tot

al P

As

(µg

per

indi

vidu

al)

0

20

40

60

80Exuviae CocoonsPupae

Fig. 3 Concentrations and amounts of total PAs associated withexuviae (final instar) (n = 8), cocoons (n = 4) and pupae (n = 8) larvaeof Estigmene acrea (mean ± standard error)

Table 3 Endowment of eggs of Estigmene acrea with PAs. Aslarvae both males and females were reared on an artificial diet andhad free choice between the plain diet and the diet containing0.018 mg Crotalaria-PAs/g diet (dry weight basis)

Small Large Totalegg clutch egg clutch egg clutch

Parameter n = 3 n = 3 n = 6

Egg mass (mg) 2.8 ± 0.6a 20.9 ± 7.8 11.9 ± 5.4Total PAs (µg/egg mass) 10.3 ± 1.5 33.7 ± 8.8 22.0 ± 6.6PA concentration (mg/g) 4.0 ± 0.7 1.7 ± 0.2 2.9 ± 0.6

amean ± standard error

In experiment II, eggs were obtained from 8 pairs.Alkaloid analysis revealed the presence of PAs in females,eggs or both in three pairs (Fig. 5), while females and eggsof the remaining five pairs were devoid of PAs. In all pairs,as expected, males contained PAs. At least pairs 1 and 2(Fig. 5) indicate a transfer of PAs from males throughfemales to the eggs. In pair 1 the male as the primary PAsource retained only traces of alkaloid. Pair 3 was the onlyone in which no PAs could be detected in the eggs thoughthe female had received somewhat of the male’s alkaloidload.

The PA profiles established for adults and eggs in thetwo experiments clearly reflect to origin of the PA load.In experiment I the profiles of donating females andaccepting eggs are almost identical (Fig. 6); similarly forexperiment II, where females and their eggs reflect the

males’ patterns (Fig. 7). However, the patterns were differ entbetween the two experiments. Actually, the creatonotineswere not even detectable in remaining PA profiles ofmales, which corroborates the discussed (see Fig. 4) loss ofcreatonotines in males, but appeared to increase fromfemales to eggs.

Discussion

Evidence for the role ofcreatonotine as direct pheromone precursor

Caterpillars of Estigmene acrea not only sequester plant PAsoffered in an artificial diet enriched with Crotalaria pumilapowder but also convert almost half of these plant acquired


% of total PAs per pair

0 20 40 60 80 100 120

Pai

rs

1

2

3

4

5

6

7

8

µg PAs per individual or egg cluster

0 25 50 75 100 125 150 175 200

Pai

rs

1

2

3

4

5

6

7

8

800 (157 ng)

62 (98 ng)

152 (142 ng)

2335 (33 ng)

900 (198 ng)

760 (204 ng)

200 (240 ng)

123 (171 ng)

*

*

*

FemaleEggs

FemaleEggs

Fig. 4 PAs recovered fromfemales and eggs of eight pairsof Estigmene acrea. Femalesthat as larvae had access toPA-containing diet werecrossed with males reared aslarvae on PA-free diet.Numbers on the columns givethe quantity of eggs per clutchand in parenthesis, ng PAsper single egg. *Pairs wheremating was directly observed

PAs / individual or egg cluster

0 20 40 60 80 100 120

Pai

rs

1

2

3

% of total PAs per individual

0 20 40 60 80 100

Pai

rs

1

2

3

956 (8 ng)

1158 (37 ng)

190

EggsFemalesMales

EggsFemalesMales

*

Fig. 5 PAs recovered frommales, females and eggs ofthree pairs of Estigmene acrea.Males that as larvae had access toPA-containing diet were matedwith females reared as larvaeon a PA-free diet. Numbers onthe columns give the quantityof eggs per clutch and in paren-thesis, ng PAs per single egg.*Pairs where mating wasdirectly observed

alkaloids into idiosyncratic PAs (insect PAs), the estigminesand creatonotines. The estigmines A and B are the esters ofsupinidine, supplied with the plant diet (see Table 1), with 2-hydroxy-3-methylbutyric acid (2-hydroxy analogue ofvaline) and 2-hydroxy-3-methylpentanoic acid (2-hydroxyanalogue of isoleucine). The respective O2 esters ofretronecine are the creatonotines A and B, which are foundtogether with their O2-esters, isocreatonotines A and B(Hartmann et al. 2004). The 2-hydroxy acids are insectmetabolites (Ehmke et al. 1990; Hartmann et al. 1990;Schulz et al. 1993).

Both plant-acquired and idiosyncratic PAs stored bylarvae are retained through pupation to adulthood. The sexualdimorphism found in adults confirms a previous observation(Hartmann et al. 2003). In contrast to adult females andmale and female pupae, adult males showed a decreasedlevel of total PAs primarily caused by a significant decreasein the amount of the creatonotines and a substantial loss ofthe pumilines, while the level of the estigmines remainedunaltered. (see Fig. 2). It appears reasonable to relate theloss of creatonotines and pumilines to the formation ofhydroxydanaidal, the male courtship pheromone (Hartmannet al. 2003). The most plausible route for the formationhydroxydanaidal in Estigmene acrea that as larva fed onCrotalaria pumila PAs is outlined in Figure 1. As suggested inthe scheme the first step would be degradation of a portionof the pumilines to retronecine, which is always detectableas free necine base in insect extracts, but not present in thePA-containing diet. Retronecine provides the necine basefor the formation of the creatonotines which are suggestedto act as direct precursors of hydroxydanaidal. This view isindirectly supported by the behavior of the estigmines thatpersist stably during the development from pupa to adult.The estigmines lack the hydroxyl group at C-7, and there-fore cannot act as a pheromone precursor.

Evidence favoring the role of creatonotine as an inter-mediate in the formation of hydroxydanaidal comes fromprevious studies with the Asian arctiid Creatonotos transienswhich as larva was reared on a diet containing heliotrine, aplant derived O9-monoester like creatonotine. In Creatonotos,the formation of the pheromone occurs within the first hoursafter eclosion of the males (Nickisch-Rosenegk et al. 1990)most likely from heliotrine which was found to accumulatein the coremata of 8-days-old male pupae just one daybefore emergence of the moth (Egelhaaf et al. 1990). Thistiming of pheromone production corresponds with the tim-ing of decrease of the creatonotine in Estigmene, i.e. in adultmales but not yet male pupae. Biosynthetic studies withheliotrine revealed that the aromatization of the retronecinemoiety yielding dehydro-retronecine occurs prior to theester cleavage at C-9 (Schulz et al. 1993). The same authorsprovided evidence that free retronecine is not a direct pre-cursor of hydroxydanaidal. Interestingly they observed theformation of creatonotine (now named creatonotine B) as aside product and simultaneously pheromone formation (seeSchulz 1998). It is still not known whether heliotrine isdirectly converted into the pheromone or whether thisoccurs via free retronecine and creatonotine. Altogether thevarious lines of evidence strengthen the assumption that thenecine moiety of macrocyclic PAs of the monocrotaline typeis incorporated into hydroxydanaidal through freeretronecine following its esterification to creatonotine.

Transfer of larval PAs through the life-stages

We showed that larval PAs were retained to adulthood andthat there is an efficient transfer of PAs from males tofemales and from females to eggs. Among the individualpairs analyzed there were single cases where a female sacri-ficed her total PA load to the eggs and the same wasobserved with a male that provided his alkaloids to thefemale. These examples indicate highly specific and efficient


Developmental stage

Rela

tive a

bu

nd

an

ce

(%

)

0

20

40

60

Supinidine

Pumilines

Creatonotines

Estigmines

Females Eggs

Fig. 6 PA profiles (mean ± standard error) of females and eggs ofthe seven pairs illustrated in Fig. 4. Females that as larvae hadaccess to PA-containing diet were mated with males reared as larvaeon a PA-free diet

Developmental stage

Rel

ativ

e ab

unda

nce

(%)

0

20

40

60

80SupinidinePumilinesCreatonotinesEstigmines

Males Females Eggs

Fig. 7 PA profiles (mean ± standard error) of males, females andeggs of the three pairs illustrated in Fig. 5. Males that as larvae hadaccess to PA-containing diet were mated with females reared aslarvae on a PA-free diet

mechanisms channeling the PAs via the various life stages tofinally provide the eggs as the most vulnerable and endan-gered life stage with the best possible chemical protection.Some pairs showed less or no transfer of PAs to eggs and itis likely that, in such cases, copulation had not occurred. It isalso possible that there is natural variability in PA transfer,but further studies are required. During the maintenanceof this E. acrea culture, it has often proved difficult to getsuccessful mating (Singer, unpubl. observations).

Chemical protection of eggs is, of course, a well knownphenomenon. There are numerous examples of autogenouslyproduced or plant-acquired chemicals used by insects toprotect their eggs and offspring (for review see Blum &Hilker 2002). This defense can be provided maternally,paternally or by both parents (for review see Eisner et al.2002). Of the plant-acquired defense chemicals the PAs,particularly in the Lepidoptera, provide the most impressiveexamples. In the ithomiine butterflies visitation of PA-containing plant material is strongly male-biased and adultmales are presumed to be the collectors of most PA (Pliske1975). Experimental evidence indicates an efficient trans-mission of PAs from males to females and subsequently tothe eggs (Brown 1984; 1987). A comparable paternal allo-cation of sequestered PAs to females and eggs has beendemonstrated for the danaine butterfly Danaus gillipus(Dussourd et al. 1989). In the arctiids where larvae sequesterthe PAs, a biparental endowment of the eggs appears to bethe rule, as illustrated in this study. This has also been demon-strated for Creatonots transiens (Nickisch-Rosenegk et al.1990) and most completely studied for Utetheisa ornatrixalready referred to in the introduction.

Basically Estigmene acrea behaves in its allocation ofPAs like Creatonotos and Utetheisa. However, in contrast toUtetheisa whose larvae monophagously stay on the hostplant, Crotalaria, and where both sexes have unlimitedaccess to PAs, the highly polyphagous larvae of Estigmenemay encounter PA-containing plants rarely or irregularlyduring their wanderings. (Bernays et al. 2004) PA plantsencountered by larvae in the field are highly acceptable, buton many occasions, larvae leave the PA plant after somehours (Bernays et al. 2003) so that continued access to PAsfor sequestration is unlikely. Even for oviposition, femalesappear to regularly use non-PA-containing plants such asHelianthus annua (Asteraceae) (Singer, unpubl. observations).There is some evidence that Estigmene is capable of utiliz-ing almost any PA-containing plant as PA source and able toallocate and process the sequestered PAs properly and formales to advertise their PA load through the pheromone.Such versatility would be of value in nature where PA plantsare only found opportunistically or not at all. Some individ-uals do not find PA plants, as judged by the absence of PAsin field-collected larvae ready to pupate (Hartmann &Singer, unpubl. data). In such cases adult females devoid ofPAs may be inseminated by males rich in PAs. In this situa-tion the pheromone-directed mating behavior would beextremely valuable and may illicit a paternal investment inegg protection.

Protective functions of pyrrolizidine alkaloids

PAs are present in all life stages of E. acrea. The observedhigh PA concentrations in the larval exuviae and pupal

cocoons indicate a preferred peripheral localization of thePAs. Although a preferential localization of PAs in theintegument is well documented for arctiid species (Egelhaafet al. 1990; Ehmke et al. 1990; Hartmann et al. 1990;Nickisch-Rosenegk et al. 1990; Nickisch-Rosenegk & Wink1993), no PAs could be detected in larval exuviae and pupalcocoons of Creatonotos transiens (Nickisch-Rosenegk et al.1990) or larval exuviae of Arctia caja (Nickisch-Rosenegk &Wink 1993). The latter, however, contained substantialamounts of alkaloid in the cocoon. The presence of PAs inE. acrea at high levels in peripheral tissues of larvae andadults, in eggs or extracellular materials like cocoons appearsreasonable, and strongly suggest a deterrent function of PAsagainst predators.

Utetheisa ornatrix reared as larvae on Crotalaria spp., areprotected against spiders, both as larvae and adults (Eisner &Meinwald 1987; Eisner & Eisner 1991; Gonzalez et al.1999). The PA endowment has been shown to protect theeggs against coccinellid beetles (Dussourd et al. 1988), ants(Hare & Eisner 1993) and chrysopid larvae (Eisner et al.2000). An impressive example provides PA sequesteringithomiine butterflies that sequester PAs only as adults. Theorb-weaving spider Nephila clavipes, one of the mostimportant predators of butterflies, accepts ithomiine butter-flies as prey only immediately after emergence from the pupawhen they still do not have PAs. The spider treats PA-ladenithomiine (Brown 1984; Trigo et al. 1996) and arctiids(Eisner 1982; Trigo et al. 1993) by cutting them from theweb and allowing them to escape unharmed. The prompt-ness with which the spider rejects its prey could beaccounted for by the presence of PAs on the moth’s surfaceparticularly scale coating (Rossini et al. 2003). A compari-son of the deterrent activity of various PAs on N. clavipesrevealed similar effects with macrocyclic diesters (e.g.senecionine) and monoesters like O9-senecioylretronecineand their N-oxides while simple necines like retronecine andits N-oxide were ineffective (Silva & Trigo 2002). Thus,Estigmene acrea in its various life stages appears to be wellprotected against a variety of predators.

In southern Arizona a major cause of mortality in arctiidsis attack by parasitoids, especially tachinid flies (Stireman &Singer 2003) A recent study with Grammia geneura, a relatedarctiid living in the same environment, revealed a higher resis-tance of caterpillars that had access to a PA-containing plantsto tachinid parasitoids. The resistance of individual caterpil-lars is associated with the relative amount of PA-plant eatenas well as with the amount of sequestered PAs from this plant,Senecio longilobus (Singer, Carriere, Theuring & Hartmann,submitted). In both E. acrea and G. geneura substantial con-centrations of PA were found associated with hemolymph(Hartmann, Bernays & Singer, unpublished results). The roleof PAs in the protection of larvae against parasitoids is thefocus of continuing work.

Acknowledgements

This work was supported by grants from the DeutscheForschungsgemeinschaft and Fonds der ChemischenIndustrie to T. H. We thank M. S. Singer for help with thecaterpillar culture, and R. F. Chapman for help with rearinginsects on the specific diets.


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Received 11 March 2004; accepted 14 April 2004.

Documents

Phenological fate of plant-acquired pyrrolizidine alkaloids in the polyphagous arctiidEstigmene acrea