Table Of ContentJournaloftheLepidopterists'Society
57(4),2003,279-283
MALE-SPECIFIC STRUCTURES ON THEWINGS OFTHE GULF FRITILLARY BUTTERFLY,
AGRAULIS VANILLAE (NYMPHALIDAE)
Casandra L. Rauser1 and Ronald L. Rutowski2
DepartmentofBiology,ArizonaStateUniversity,Tempe,Arizona85287-1501, USA
ABSTRACT. MalesoftheGulfFritillarybutterfly,AgraulisvanillaeLinnaeus(Nymphalidae),havedistinctivestructuresoncertainveins
ofthe dorsalwingsurfacethat appeartobeinvolvedinpheromoneproduction. Hereweconfirm andextendan earlierdescriptionofthese
structures.Observationsusinglightandscanningelectronmicroscopyindicatethatinthesestructures,patchesofseveralscaletypesalternate
withscalelessareasalongtheveins. Someoftheseopenareashaveporesthatwesuggestmightbetheroutebywhichthepheromonemoves
fromcellsinthewingintegumentontobrush-bearingscalesfromwhichitisdisseminatedduringcourtship.Wehavealsofoundthatalthough
thedimensionsofthebasicunitsofthesestructuresontheveinsarenotcorrelatedwithbodysize,largermalesdohavegreatertotalveinlength
devotedtothesestructures.Thesefindingsarediscussedinlightofthecourtshipofthisspeciesandthepotentialforthesestructurestobein-
volvedinmatechoiceandtobeaproductofsexualselection.
Additionalkeywords: pheromones,androconia,morphology, matingbehavior,SEM.
In many butterflies and moths, males have struc- servations could suggest how the scent was produced
tures on their wings or body that produce chemical and disseminated in A. vanillae. The second objective
signals, orpheromones, used by males when courting wastodeterminehowmuch,ifany,inter-individualvari-
females (Myers & Brower 1969, Pliske &Eisner 1969, ationthereisintheoverallsize ofthese structures. This
Vane-Wright & Boppre 1993, Kan & Hidaka 1997, could suggest die extent to which males might vary in
Iyengaret al. 2001). In some cases, behavioral experi- uheir abilityto produce critical chemical signals during
mentshaveconfirmedthefunctionofthesestructures, courtship. Lastly,wewantedtorelate Midler's andour
but more often aphermonal function is inferred from observationstopublisheddescriptions ofthecourtship
the high surface area ofall parts ofthem, and the ob- behaviorofA. vanillae (Rutowski & Schaefer 1984).
servation that they are often brought close to the an-
tennae ofthe female during courtship (Lundgren & Methods
Bergstrom 1975, Rutowski 1977, Boppre 1984). Al- We examineddie FWs ofmale andfemaleA. vanil-
though much is known ofthe morphology and opera- lae usingboth lightandscanningelectron microscopy.
tion ofsuch structures in danaine butterflies, the full Specimens were either laboratory-reared from field-
diversityoftheirstructureandfunctioninbutterfliesis collected larvae andeggs oncuttings ofPassiflora spp.
generallynotwell documented. orfield-caught as adults inTempe,Arizona. Forexam-
Miiller (1877) first discovered and described pre- ination ofscale morphology, we chose butterflies with
sumptive scent-producing structures on the wings of little or no wing wear. In some cases, we removed
males of the Gulf Fritillary, Agraulis vanillae Lin- scales with a small paintbrush to expose the cuticle
naeus (Nymphalidae), acommonheliconiinebutterfly coveringtheveins underlyingthe scales. Forlight mi-
in the American tropics and subtropics. In particular, croscopy we mounted FWs onto slides and pho-
he reportedthat along six ofthe forewing (FW) veins tographed specialized structures and scales. For scan-
there were patches oflongthin scales each bearing a ningelectron microscopy(SEM),weattachedwingsto
brush at the distal end, which alternatedwith rows of stubs with conducting paint and no coating, and ob-
normal scales crossing the vein. Our preliminaryob- served them with a Philips/FEI XL 20 scanning elec-
servations ofthese structures using light microscopy tron microscope.
suggested that the arrangement ofscales along these To determine whether larger males also have a
veins while serially repeated, as Miiller (1877) re- largerandroconialareaondieirFWs, anddius greater
ported, was more complex than indicated by his de- pheromone-disseminating ability, we measured die
scription and sowe undertook a more detailed study length ofthe portion ofeach ofthe veins containing
oftheir morphologyusinglight andscanningelectron specialized structures onthe male FW. Foreach spec-
microscopy. imen (n = 20), we then totaled these lengths andplot-
FW
Ourfirstobjectivewastorepeatandextend Miillers tedthistotalagainst length andsubjectedthe data
observations because we believed more detailed ob- tocorrelationanalysis.Wealsodeterminedthe density
ofspecialized structures along the veins to investigate
1Correspondingauthor'scurrentaddress:DepartmentofEcology whetheritalsovarieswith FWsize. To dothisweesti-
andEvolutionaryBiology,321 Steinhaus Hall,UniversityofCalifor- mated the number ofstructures per mm on each of
nia,2EImrvaiinle:,[email protected],USA,email:crauser®uci.edu the six veins on a male FW We then calculated the
280 Journalofthe Lepidopterists' Society
ca. 10mm
FlG. 1. Asketchofthedorsalsideofthe FWofthemaleGulf Fig. 2. An SEM ofasegmentofthevein containingthe spe-
Fritillary butterfly with the veins containing the presumptive cializedstructureswiththescalesintact(250x).Thebrush-likescent
pheromone-disseminating structures labeled accordingto Scoble's scalesareattachedtotheveinatSS-2andthebroadflatscalesareat-
(1992) notation. The segments oftheveins containingthe special- tachedatSS-1.
izedstructuresarehighlighted.
One type is broad at the top, narrow at the base and
mean numberofstructurespermm forall6veins and shinyblack, whereas the other type is equallyas long,
compared the mean number ofstructures per mm to but broad at the base and tip and translucent, shiny
the FWlength, alsousingcorrelationanalysis (n = 20). gold. Both types ofscales are flat and the black scales
fit snuglyoverthe gold, scales as shownin Fig. 5. IVS-
Results 2 (mean length = 0.01 mm) is the area containing no
As reported by Miiller (1877), male-specific scales scale attachmentsbetween SS-1 and SS-2. SS-2 (mean
andotherstructures arefoundonthe dorsal FWofthe length = 0.04 mm) is a region with the scale attach-
male on veins M,, M„ M CuA CuA and 1A 2A mentsbetween IVS-2andPS. Thescalesarisingin SS-2
(Fig. 1;wingveinnotation,3,Scoblre 1992)2., Onvein2sthat are denselypacked, long(meanlength = 0.4mm), nar-
had not been disturbed we observed that, along at row, and have brush-like ends with high surface area.
least part ofeach ofthe six veins, rows ofbrush-like In addition, the bases of these scales are black, fol-
scales alternated with rows of scales with broad, lowed byathin transparent area, thenbyablack, nar-
straightedges (Fig. 2). The averagelengthofeachvein row section which contracts into another thin trans-
segment that contains the male-specific structures is parent area, followed bya black brush-like apex (Fig.
1m11.m.577om±nm1C.4uo3nAmMam3n,d1o3n1.25M.9±6v1+.1639.14.m73+mm1.om5n1ComnumA1Apon1+4.M27~A2±., 11T3.h7e±4 t6o)vh.eerTlthireeassnesPpSbarraeusnssthh-bobawesanarliinnargFeisagc.a(l6m.eesPaSanre(lmepenoagstnihtlie=onng0et.dh0s4=om0tm.h0a)6t
FWlengthof2the20specimensweexamineLdaveraged
33.7 ± 2.6 mm.
With the scales removed from the veins, we found
mm
thatasetoffive distinctareas makeup aunit0.24
in length that is serially repeated along each ofthe six
veins (Fig. 3). Inorderfrom thewingbase tothewing
edge, the five distinct areas are the inter-scale vein-
section 1 (IVS-1), the scale-section 1 (SS-1), the inter-
scale section 2 (IVS-2), the scale-section 2 (SS-2) and
the porous section (PS) (Figs. 3, 4).
As shown in Fig. 4, the IVS-1 (mean length = 0.12
mm) occurs between the repetitive structures and
does not contain any scale attachment sites. Every
third row ofscales occuring on the inter-vein part of
the wing runs uninterrupted across the veins. SS-1 theFviegi.n3o.faTmhaele'rsepFetWitiivsesnhaotwunrewiotfhthtehespseccailaeslirzeedmostvreudct(u2r0exs).alTohneg
(mean length = 0.02 mm) is the scale attachment row fivedistinctsectionsofthespecializedstructuresarelabeledaccord-
after IVS-1 and contains two different types ofscales. ingtotheirfunction: Scale Section 1 and2: SS-1 and SS-2; Inter-
scaleVeinSection 1and2:IVS-1andIVS-2;andPorousSection:PS.
Volume 57, Number4 281
Fig.5. AnSEMofthetwotypesolscalesandtheirattachment
pointsatSS-1 (1500x). Bothscaletypesareflatandonefitssnuggly
overtheother.Thescaletypethatliesontopisbroadatdietop,nar-
rowatthebase andshinyblack. The otherscaletype is equallyas
long,broadatthebaseandtipandtranslucent,shinygoldincolor.
vanillae. Inaddition,we foundthatdiese male-specific
structures consist of serially repeated units of scale
arrangements along the veins. However, each unit
consists of five rather than two distinct elements, as
describedby Midler. One ofthese elements is apatch
ofbrush-bearing scales and rows of scales typical of
those found crossing the vein elsewhere on the wing
(Magnus 1950). We also found other scale types and
Fig. 4. An SEM of the male Gulf Fritillary pheromone- three regionswithoutscales thatincludedan areadot-
dPiSssaenmdinSaSt-i2ngarsetrfuucrttuhreerwmiatghntihfeiefidveindiosrtdiencrttsoecctlieoanrlsylsaebeeltehde(d7i5f0fxe)r.- tedwith large pores openingontothewingsurface.
ence between the two areas (1500x). SS-2 is the scale attachment The specific elements ofthe structures found along
point for the scent scales and PS is the hypothesized area of these veins mayinteract in the followingways to dis-
pheromonesecretion. seminate a pheromone. The brush-bearing scales lo-
cated in SS-2 lie directly over the porous area (PS)
mm) istheporous areacontainingnoscaleattachment (Fig. 6). These pores could be the openings through
sites adjacentto SS-2. which the product of pheromone-producing cells in
The total length ofthe vein segments that contain
the presumptive pheromone-disseminating structures
FW
ispositivelycorrelatedwith length (r2 = 0.858, p <
FW
0.0001) (Fig. 7). This means thatthelongerthe of
the male, the longerthe length oftheveins containing
the pheromone-disseminating structures. However,
there is no relationship between the mean number of
mm FW
structuresper anddielength ofthe (p > 0.40)
mm
(Fig. 8). Therefore, the number ofstructures per
alongtheveins remains constant regardless ofthe size
oftheFW. Collectively,theseresultsindicatedratmales
with longer FWs have more total scent-disseminating
structures than males with shorter FWs and thus
greaterscentdisseminatingability.
Discussion ontFihge.ma6.leFAWphwoittohgtrhaephscoefntaspchaelersominotnaec-tdaitsSsSe-m2in(2a0txi)n.gTshterutcrtaunrse-
Our observations confirm and extend Midler's parentportionofthescentscalesclosesttothepointofattachment
liesdirectlyovertheporousarea(PS). PSishypothesizedtosecrete
(1877) description of male-specificFsWtructures found pheromonesthatdisseminateontothehighsurfaceareabrush-like
along certain veins on the dorsal surface of A. scales.
282 Journalofthe Lepidopterists' Society
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Forewing length (mm) Forewing length (mm)
Fig. 7. The positive relationship between the FWlength and Fig.8. Norelationshipexistsbetweenthenumberofstructures
the total length of the vein segments containing pheromone- permmperindividualandtheFWlength.Inotherwords,theden-
disseminatingstructures(r2=0.858,p<0.001). sityofandroconiaalongtheveinsremainsconstantregardlessofFW
size(p >0.40).
the wing integument is secreted. This arrangement
would allowthe compounds to be directlytransferred disseminating structures and the quantity of phero-
ontothe denselypacked, high surface areabrushes on mone secretedarepositivelycorrelatedwithmalesize.
the overlying scales. The black and gold scales de- A similar correlationwas found in the moth Utetheisa
scribed in SS-1 lie over the narrow stalk of delicate ornatrix and was determined to be a heritable trait
scent-scales (Fig. 2). These SS-1 scales, which were (Iyengar& Eisner 1999). Ifthe amountofpheromone
difficulttoremove, appeartoform aprotectivebarrier disseminatedbythe male duringcourtshipiscriticalto
over the relatively fragile and easily removable SS-2 female mate choice inA. vanillae, largermales should
brush-bearingscales. This protective barrier mayhelp be more successful at matingthan smaller males. This
prevent the loss ofbrush-bearing scales and excessive is the case in U. ornatrix where the amount ofphero-
evaporation ofpheromone secretions. mone disseminated by the male is the only criterion
Rutowski and Schaefer (1984) described the usedbythefemalewhenchoosingamate(Iyengaretal.
courtship behavior ofA. vanillae and reported a male 2001). Similarly, malesinDrosophilagrimshawideposit
display, which they called the wing clap. During apheromonetoattractfemalestoamatingsitebyrub-
courtship the male positions himself head-to-head bing their abdomen on the substrate. Males who de-
alongsidethefemalewithhisbodyatabouta45° angle posit the greatest amount ofpheromone at their site
relative to the body ofthe female. Initially his wings are the most successful at attracting females and mat-
are slightlyspread and the female's ipsilateral antenna ing (Droney& Hock 1998).
comes to laybackbetween the male's wings. He then The evidence discussed above indicates that the
quicklycloses and reopens hiswings repeatedlycatch- morphologyandlocation ofthe male androconiainA.
ing the female's antenna between them and bringing vanillae maybe a product ofsexual selection (Fisher
the female antennainto briefcontactwith the distinc- 1958, Baker&Carde 1979, Eisner& Meinwald 1995).
tive structures on the dorsal FW veins. Hence, as in Thequantityofthepheromonesecretedmaybeanin-
other species, we find a male courtship behavior that dicator of male quality that females evaluate when
stronglysuggeststhatthese male-specific, highsurface choosing a mate (Dussourd et al. 1991, LaMunyon
area structures are producing a pheromone that may 1997, Iyengar & Eisner 1999). Quality here could
be important in mate choice byfemales (Tinbergen et mean speciesidentityorabilitytoprovideeithermate-
al. 1942, Magnus 1950, Broweret al. 1965, Conner et rial (i.e., defensive secretions ) orgeneticbenefits (i.e.,
al. 1980, Pivnicket al. 1992). genes forlarge size). Pheromone quantitymayalsoin-
Our data show that males with longer FWs have a dicateamale's ageand/ormatingstatusbecausescent-
greater total length of veins containing pheromone- scales are likelytobe lostwith age andwith each mat-
disseminating structures (Fig. 7). Because the density ing or courtship. In this way a female maybe able to
of androconia per individual is not affected by FW assess the physiological state ofa malewhen choosing
FW
length (Fig. 8), and length is a good measure of amate.
male size, larger males should have a greater total However, sometimes the GulfFritillary female will
number ofpheromone-disseminating structures than mate with a male even when he does not perform the
smaller males. Thus, the number of pheromone- wing-clap display (Rutowski & Schaefer 1984). Per-
Volume 57, Number 4 283
hapspheromones arenottheonlyindicatorofamale's Eisner, T. &J. Meinwald. 1995. The chemistryofsexual selec-
quality, female choice is not based on this character, Fishetiro,n.R.PrAo.c.1N9a5t8l..ATchaed.geSncie.tiUc.aSl.At.he9o2r:y50o-f5n5a.tural selection. 2nd
some females are less discriminating than others, or reviseded. DoverPublications,NewYork.
some males have a strong enough odor to elicit a re- Iyengar,V. K.&TEisner. 1999. Heritabilityofbodymass,asex-
ceptive response from the female without using the uallyselectedtrait,inanarctiidmoth(Utetheisaornatrix).Proc.
Nad.Acad. Sci. U.S.A.96:9169-9171.
display. To better understand the functions of diese Iyengar,V. K., C. Rossini & T. Eisner. 2001. Precopulatoryas-
pheromone-disseminatingstructures, furtherinvestiga- sessmentofmalequalityinanarctiidmoth(Utetheisaornatrix):
tionneedstobeundertakentoidentifydiepheromones, hydroxydanaidalistheonlycriterionofchoice.Behav.Ecol.So-
ciobiol.49:283-288.
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pheromone amountaffects female mate choice. Ethol. 15:8W7-93.
LaMunyon,C. 1997. Increasedfecundity,asafunctionofmul-
tiplemating,inanarctiidmoth, Utetheisaornatrix.Ecol.Ento-
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