Table Of ContentJournaloftheLepidopterists'Society
55(2),2001,69-7.3
ONTOGENYOF DEFENSE AND ADAPTIVE COLORATION IN LARVAE OF THE COMMA
BUTTERFLY, POLYGON1A C-ALBUM (NYMPHALIDAE)
Soren Nylin1 Gabriella Gamberale-Stille, and Birgitta S. Tullberg
,
DepartmentofZoology,
StockholmUniversity,
SE-10691Stockholm,Sweden
ABSTRACT. Inmanybutterflyspeciesearlyandlatelarvalinstarsdifferincoloration.Thefirstthreelarval instarsinthecommabutter-
fly,Polygoniac-album,havecolorationthat,tothehumaneye,appearstobedisruptivelycryptic.Thelasttwoinstars,which aredefendedby
strongbranchingspines,areinsteadstrikinglycoloredinwhite,black,andorange. Ithasbeensuggestedthatthiscolorationisalsocryptic,mim-
ickingbirddroppings.Wetesttheideathatthiscolorationisinstead(orsimultaneously) aposematic,usingyoungchickensas modelsofbird
predators. Somechicksreadilyatethird-instarlarvae,bothinitiallyandafterhavingtastedthem,suggestingthatnochemicaldefenseispresent
incommalarvae.Thechicksalsoatedeadfifth-instarlarvaefromwhichthespineshadbeenremoved. Chicksinitiallyattackedfifth-instarlar-
vaewithintactspines,butlearnedtoavoidthemalreadyafterthefirstattack.Thissuggestsanaposematicfunctionofthecoloration,whichcan
haveevolvedbyindividualselectioninthisandmanyothernymphalidspecieswith spinylarvae,becauselarvaewerenotharmedduringthe
learningprocess.Theevolutionarycausesoftheontogeneticshiftindefensetacticsarediscussed.
Additionalkeywords: aposematism,mimicry,lifehistory,predation,adaptivefunction.
Adaptivecolorationinanimalshasbeenaveryactive The coloration ofthe first three larval instars in the
research field in evolutionary biology over the years comma butterfly Polygonia c-album L. (Nymphali-
(e.g., Poulton 1890, Cott 1940, Kettlewell 1973, Sillen- dae), appearstothehumaneyetobedisruptivelycryp-
Tullberg 1988, Malcolm 1990), and one in which the tic (Fig. 1A), atleastwhen seen againstanaturallyvar-
Lepidoptera have always featured prominently as iegated background oflight and shadow. The last two
model species. Adaptive coloration includes crypsis instars,whicharedefendedbystrongbranchingspines,
(helping to avoid detection by other animals), sig- are much more strikingly colored in white, black and
nalling, andthermoregulation. Crypsiscanbe achieved orange (Fig. IB).Thesametypeofcolorationispresent
byhavingcolors similarto the background, orbyhav- in the probable sisterspecies to P. c-album, the Nearc-
ing mosaic patterns ofspots or stripes that break up tic Pfaunus (Scudder 1889, Scott 1986). It has been
thecontourandsurface areaofthe animal (called"dis- suggestedthatthistype ofcolorationisalsocryptic,the
ruptivecrypsis";Cott 1940, Edmunds 1990). Signalscan largecontinuouswhiteareasupposedlymimickingbird
beofseveraltypes,including"aposematism,"definedas droppings (Thomas 1986). Resemblance to bird drop-
conspicuous colors advertisingunprofitabilitytopreda- pingsis seenin manybutterflylarvae, e.g., inLimenitis
tors (Guilford 1990a, b). A phenomenon that may in- and Papilio (Scott 1986), although to our knowledge
clude aspects ofboth signalling and crypsis is "mim- the function has neverbeen tested.
icry," a term used for organisms that adaptively Lateinstarlarvae ofotherPolygonia species (e.g., P.
resemble another species, as in the cases ofMullerian interrogationis, P. comma, P. satyrus, andP. c-aureum)
and Batesian mimicry (Malcolm 1990). In the former andthe relatedKaniskacanace aresimilarlycoloredin
case, unpalatable aposematic organisms mimic each conspicuouswhite, blackand orange, butwith nocon-
other. In the latter case, a palatable organism mimics tinuous white areas (Scott 1986, Teshirogi 1990). This
an aposematic one, capitalizingon its defenses. Some- suggests the possibility that the coloration is apose-
times the term mimicryis extended to cases when or- matic in these species, and thenperhaps in P. c-album
ganisms mimic the shape and colors ofobjects or im- and P.faunus as well. Here, we study the function of
mobile organisms for the purpose of crypsis, for the coloration oflate instar larvae ofP. c-album using
example as in stick insects (Edmunds 1990). Finally, young chickens as models ofbirdpredators. For com-
variation in coloration can be due to different require- parison and as a control for effects ofnovel food, we
ments for thermoregulation rather than predator alsotested the chicks with third-instarlarvae.
avoidance (e.g., Shapiro 1976, Kingsolver & Watt
Materials and Methods
1983). For all ofthese types of(presumably) adaptive
coloration, it is probablyfairto saythat the function is
often assumed, but seldom tested. Females of P. c-album were captured near Akers-
berga, north of Stockholm, Sweden. Eggs were ob-
tainedinflightcageswherethehostplant Urticadioica
1 Email: [email protected] Tel: +46-8-164044 Fax: (Urticaceae) was present. Larvae were reared on this
+46-8-167715. plant in the laboratory. Several asynchronous rearings
70 Journalofthe Lepidopterists' Society
Fig. 1. LarvaeofPohjgoniac-alhumonleavesofUrticadioica. A.Third-instarlarva. B. Fifth-instarlarva.
were made, so that third- and fifth-instar larvae were eral centimeters inlength (Figs. 1A and B are approx-
available fortrial simultaneously. imatelyto scale).
Third-instar larvae. In this instar, the larvae lack Chicks. Weuseddomesticchicks (Gallusgallusdo-
large areas of any color except for the background mesticus) aspredators. The chickshadnoteatenwhen
color, black. Smallspines arepresent, andtowards the theyarrivedfromthehatcheryatanageoflessthan20
end ofthis instarthe spines have ayellowish color on h. Batches of30-40 chicks were housed in cages (100
the foremost part ofthe body and awhitish color to- cm x 55 cm x 20 cm) with wooden sides, steel-net
wards the rear (as seen in Fig. 1A). The impression to floor, and a roof made partly of wood and partly of
W
the human eye is not that ofa striking coloration but chicken wire. The cages were heated with 60 car-
rather a pattern that could function disruptively bon light bulbs and the floorswere coveredwith saw-
against avariegatedbackground. dust. Chicks were fed chick startercrumbs andwater,
Fifth-instar larvae. In this instar, the foremost and at least from their second day on they were also
part ofthe body is colored orange, and the rear part fedlive mealworms (Tenebrio molitor).
is continuously colored white. The rest of the body Experiments. The experiments took place in an
is black with orange markings (Fig. IB). Large, arena ofthe same kind ofcage in which chicks were
chitinous, branching spines are present on the back housed. Part ofthe cage was screened off, leaving a
and sides of the body, and they are colored orange, testingfloorof30cmx55cm. Experiments tookplace
white or black, according to position. Larvae are sev- whenthechickswereaboutthree days old. Thechicks
Volume 55, Number 2 71
100 - -~~---~"--~~" or attacked third-instar larvae on all three occasions,
90 - . were tested a fourth time. All trial runs were per-
80 - \ formed consecutively on the same day within five
Co 70 - \ hours. We used Fisher's exact test for statistical com-
gm 6500-" 3dinstar parisons.
re 40 - 5thinstar Afollow-upexperimentwasperformednextseason,
5 30 - with new larval stock and a new batch ofchicks, this
\
20 - \ timetheywereoneweekold. Fivepairs ofchickswere
10 -
\ presented with a spineless fifth-instar larva together
- 1 I 2 I 3 with a mealworm in apetri dish. The larvae had been
Trial# killedbyfreezingandtheirspines hadbeencutoff.
Results
Fig. 2. Resultsfromtrialswithdomesticchickspresentedwith
third-instar(dashedline)orfifth-instar(solidline) larvaeofPolygo- General observations. The two mealworms pre-
niac-album.Thesamepairofchickswastestedonseveraloccasions
duringone experimentalday(subsequenttrials from leftto right). sentedatthestartofeachtrialwerealwayseatenrapidly,
Bothyoungandoldlarvaewereattackedininitialtrials,butfifth-in- demonstrating that the chicks were veryinterested in
tsrtiaarll#a1rvaanedw2erfeorabvootihdeldarvianltihnestnaersx,tNtri=al.7Npai=rs10ofpcahiirsckosficnhtircikals#i3n insect prey. In all trials the chicks were initially more
withyounglarvae(seetext). interested in attacking the mealworms than the
comma larvae when subsequently given achoice, not
weretestedinpairs,becausesinglechicksbecomedis- surprisinglysincethiswas afoodtypewithwhich they
tressed and do not feed normally (Gamberale & Tull- had previous experience. The third-instar P. c-album
berg 1996, and references therein). One ofthe chicks larvae might also have been somewhat hard to see
was fed as many mealworms as it would eat before a against the variegated background ofsawdust. How-
trial, which made it satiated and uninterestedin feed- ever, the experimental chicks were curious and in 19
ing duringthe trial. This chickwas used as acompan- out of 20 initial trials (both age classes combined)
iontotheexperimentalchick. We usedthe same com- eventuallypeckedatthe commalarvae.
panion chickin alltrials. Third-instar larvae. In the initial trials all 10 ex-
Preywas presented in a petri dishwith transparent perimentalchicks attackedthethird-instarcommalar-
bottom, i.e., chicks sawthe preyagainst abackground vae (Fig. 2). In five ofthese cases the larva was also
of sawdust. Before each trial, the chicks were pre- eaten (afteratime spanof, respectively, 19, 30, 35, 45,
sentedtwomealwormsinthepetridish. Thiswas done and 45 seconds). In the next trial the same chicks at-
to showthe place where preywas displayed, and as a tacked in nine out often cases (decrease in attackfre-
checkbefore each trial that the chicks were in factin- quencynot statistically significant: Fisher's exact test),
terested in insect prey. The chicks were then pre- and ate the larvain two cases (both after45 seconds).
sentedone mealworm andone larvaofP. c-album. We Both of these cases involved chicks which had also
collected data concerning chick attack behavior and eaten the commalarvainthe previous experiment.
the mortality ofthe attacked insects. Chicks were ex- Asexplainedin Materials and Methods onegroupof
posed to the preythroughout the trial and allowedto chickswastestedathirdtime,withattacksbyallseven
makeas manyattacks astheywished. Atrialwasended experimentalchicks (Fig. 2) andthelarvaebeingeaten
when the P. c-album larvahad been eaten, or after60 by the same two chicks (after 25 and 120 seconds).
seconds if the chick did not peck on the P. c-album When the two chicks that had eaten the larva on all
larva at all. Ifit did, wewaited another 60 seconds to three occasions were tested anew they did so again,
see ifthelarvawas eaten. this time within 20 seconds after exposure. When two
Twenty chicks (other than the companion chick) chicks thathadattackedbutnoteaten (onallthreeoc-
were used in total, originating from two separate casions)weretestedagaintheyrepeatedthis behavior.
batches. Halfofthemweretestedwiththird-instarlar- The last trials with this group took place 2 hours and
vae and the other halfwith fifth-instar larvae, using 40 minutes afterthe start ofthe experiment.
chicksfrombothbatches inbothtypes ofexperiments. In all cases when the larvawas not eaten itwas still
All chicks were tested twice with the same type of aliveaftertheexperiment (despitehavingbeenpecked
larva. In addition, to test forthe presence ofanyaver- at),butononeoccasionitwasvisiblydamaged, andfor
sion learning against third-instar larvae, seven chicks this reasonitwas killedbyus.
(ofthesamebatch)were alsotestedwithsuchlarvaea Fifth-instar larvae. The older larvae were never
thirdtime. Finally, fourofthesechicks, thathadeaten eaten by the chicks. In the initial trials attacks took
72 Journalofthe Lepidopterists' Society
placeinnineoutoftencases (Fig. 2). Theywereinthe matic function of the striking coloration. In the ab-
form ofpecks, in three cases followed by the chicks sence ofcolormanipulationswecannot, however, rule
liftingthe larvaanddroppingit. Inthenexttrial,which outthepossibilitythataversionwas in factto the sight
took place less than an hour after the first, the chicks ofthe spines themselves, independently ofcolor. In-
behavedverydifferently. Therewere no attacks in the terestingly enough, the spines are colored in such a
form of pecks (Fig. 2). The decrease in attack fre- way that might make them more conspicuous (Fig.
quency between trials is statistically significant IB), sothesetwopossibilities maybehardtoseparate.
(Fisher's exact test: two-tailedp < 0.05, one-tailedp < A dual function is also possible, so that the coloration
0.01). In all cases except one (N = 10) the experimen- indeedmimicsbirddroppings, aspreviouslysuggested
tal chick inspected the comma larvae closely some- (Thomas 1986) but has an aposematic function once
times for periods up to 15 seconds. In two cases at- the larva has been discovered. This would be useful if
tacks were initiated but terminated before contact. some predators are not deterred by the spines, or if
Larvaewere alive and apparentlyundamaged after all larvae are often damagedbynaive predators.
experiments, and they were returned to their host Inthiscontextitis,however, interestingtonotethat
plantswhere theysoon continuedto feed. the fifth-instarlarvaewere notdamagedbythe attacks
Fifth-instarlarvae without spines. In this exper- in the initial trials, when the chicks learned to avoid
imentlarvae fromwhichthe spines hadbeen removed them. This is in linewithprevious results from chemi-
were presented to the chicks. The first pair ofchicks callydefendedaposematicinsects (Boyden 1976, Jarvi
did not eat the larva, buttheyshowed no aversion and et al. 1981, Wiklund & Jarvi 1982, Wiklund & Sillen-
handled the larva throughout the experiment. This Tullberg 1985) but has not often been investigated in
larvawaspresentedalsotothe nextpairofchicks, and mechanically defended prey (but see Carrick (1936)
it was then eaten within 10 seconds. The three addi- for results from the related butterfly Aglais urticae).
tional pairs of chicks also ate the spineless larvae The importance of such observations is that they
within seconds. demonstrate that individual selection for aposematic
colorationispossible. Inotherwordsitisnotnecessary
Discussion
toinvoke kin selection orothertypes ofindirect selec-
Five out often chicks in initial trials ate third-instar tion, as wouldbe the case ifsome individuals must be
larvae, but only two chicks continued to eat them in sacrificed before predators can learn aversion (Fisher
subsequent trials. This suggests that they are not 1930, Benson 1971). Individual selection is a more
highlypalatabletobirds; possiblythe smallspines con- parsimonious explanation of aposematic coloration
fer them a limited defense. On the other hand, chicks when direct benefits to the individual are present, be-
showed no other signs ofbeginning to avoid third-in- cause for indirect selection additional assumptions of
star larvae after having experienced them. Theywere startingconditions arenecessaryregardingpreyfamily
almost always attacked (even though the previous ex- groupings and the movements and memoryofpreda-
periencewas lessthan anhourearlier), andtwochicks tors. Indeed, even aposematic butterflies often have
dideatthem in all fourtrialswithin atime span ofless crypticpupae, andthisisthe stageofthelifecyclethat
than three hours. Evidently no chemical defense ef- is mostlikelytobekilledbyinspectingpredators (Wik-
fective againstbirds is present in the commabutterfly. lund & Sillen-Tullberg 1985). Conversely, strong
This conclusion is also supported by the fact that the spines, which (as demonstrated here) should give di-
adults ofthe species are highly cryptic and also very rect benefits to the individual, are found very com-
palatable to great tits {Varus major) in laboratory ex- monly in nymphalid larvae, often togetiier with con-
periments (SN & BST unpubl.). However, this evi- trasting, bright colors or ajet-black color that should
dence is not conclusive, since butterfly larvae can be providelittlecrypsis againstgreenleaves (e.g., inother
unpalatableevenwhen adults arepalatable (Bowers & Nymphalini and in the Kallimini and Argynniti).
Farley 1990, Dyer& Bowers 1996). Spines are absentinsomechemicallydefendedgroups
The chicks showed no initial aversion to the older suchas the Danainae,with aposematiclarvae, andalso
commalarvae, butveryrapidlylearnedto avoid them. ingroups such as the Satyrinae andApaturinae,which
It seems highly improbable that a chemical defense have clearly cryptic larvae. In many cases, however,
shouldbepresentinoldlarvaebutnotinyounglarvae the patterns are far less clear and the function ofthe
or adults. More probably, the strong and sharp spines colorationuncertain (forinstance,Ladoga larvaeinthe
are what defend older larvae, as demonstrated bythe Limenitidini have spines butcryptic coloration; Teshi-
result of removing the spines. In any case, the rapid rogi 1990).
aversion learning suggests the possibilityofan apose- TheontogeneticshiftindefensetacticsinP. c-album,
Volume 55, Number 2 73
from cryptic coloration to aposematic coloration and tive mechanisms and strategies ofprey and predators. State
mechanical defense (seen also in A. urticae; Carrick UniversityofNewYorkPress,Albany.
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