Experience-related changes in the brain of Agraulis vanillae (L.) (Nymphalidae)

6 Pages·2002·3.2 MB·

by  Vadim Kroutov

OURNAL
OF
J
The Lepidopterists' Society
Volume 56 2002 Number 4
JournaloftheLepidopterists'Society
56(4),2002, 193-198
EXPERIENCE-RELATED CHANGES IN THE BRAIN OF
AGRAULIS VANILLAE (NYMPHALIDAE)
(L.)
Vadim Kroutov
123 Bartram Hall, DepartmentofZoology, UniversityofFlorida, Gainesville, Florida32611, USA
Roger L. Reep
CollegeofVeterinaryMedicine, Health ScienceCenter, P.O. Box 100144Gainesville, Florida32610-0144, USA
AND
TOK FUKUDA
Centerfor Medical, AgriculturalandVeterinaryEntomology,Agricultural Research Service,
U.S. DepartmentofAgriculture, Gainesville, Florida32604, USA
ABSTRACT. In thebrain ofAgraulis vanillae, the size ofthe brain regions involvedin theprocessingofolfactoryinformationwas found
to dependon the butterfly's experience. Butterflies collectedin naturehave olfactoryglomeruli and mushroom bodycalyxes oflargerrelative
sizethan do butterflies reared and kept in the laboratoryin isolation from normal environmental stimuli. No size differencewas found in the
opticlobesorthecentralbodyin eithermalesorfemales.
Additional keywords: mushroom body, neuropil, olfactorylobes.
The brain of an insect is the principal associative nals to the mushroom bodies, cells that deliver signals
center of the body. It receives sensory information from the mushroom bodies to other parts ofthe ner-
from a variety of sense organs, processes it and con- vous system, and the intrinsic cells (Kenyon cells) that
trols all functions ofthe organism, including complex connect the first two types between themselves. The
forms ofbehavior. Several regions ofthe brain differ- Kenyon cells occupy the area around the mushroom
ingin morphologyand function are recognized and re- body neuropil.
ferred to as neuropils (Fig. 1). Neuropils are the cen- All information from the organs ofsmell (olfactory
ters of the regions and are formed by a complex of organs) is received in another brain region: antennal
denselypackednerve fibers. The neurons, which com- lobes, which are critically important in the delivery of
pose a region, lie at its periphery. On histological sec- olfactory information to the mushroom bodies. An-
tions of the brain neuropils appear as much denser, tennal lobes are composed of a series of neuropils-
darker than the rest ofthe brain areas. olfactory glomeruli, which receive and process olfac-
The neuropils ofparticular significance in the pro- tory signals from the antennae.
cessing of information in insect brains are the mush- Insect species with complex and flexible behavior
room bodies and antennal lobes. Mushroom bodies possess well-developed mushroom bodies and anten-
receive signals from different sense organs and experi- nal lobes, and larger insects have larger brains and
ments on Drosophila (Heisenberg et al. 1985, Han et more complex histological brain structure and gener-
al. 1992) and Apis (Erber et al. 1980, Menzel et al. ally exhibit greater complexity of behavior (Goossen
1974, Hammer & Menzel 1998) suggest that they are 1949, Bernstein & Bernstein 1969). The largest mush-
implicated in olfactory memory formation. They are room bodies (relative to the rest of the brain) are
composed ofthree types ofcells: cells that direct sig- found in social Hymenoptera. The morphological plas-
194 Journal of the Lepidopterists' Society
ant ments were collected duringthe 3-4 dayperiod ofthe
Opt.lbs
abundance peak ofthe species. Larvae were reared in
the laboratoryon their natural host-plant Passifiora in-
carnata (L.), picked in the same areawhere the larvae
were found. Laboratory reared adults spent 48 hours
aftereclosion in25 x 25 x 25 cm screen cages. The lab-
oratory conditions were 25°C, 65% relative humidity,
L:D 16:8 h. Butterflies were fed a 25% sugar solution.
For the preparation of the histological specimens
butterfly heads were removed and fixed in Bouin's fix-
ative, prepared 24 hours prior to usage, for 2 days.
Theywere then rinsed in 70% ethanol and embedded
in paraffin. Heads of 16 reared males, 10 reared fe-
Fig. 1. Diagram—ofthebutterflybrainsh—owingthemostimporta—nt males, 17 wild males and 22 wild females were sec-
neuropils. Opt.lbs. —optic lobes, Ant.lb—s. antenna] lobe—s, tnb tioned. The frontal microtome sections were 10 |lm
mushroombodi—es, Kc Kenyoncells,—cb central—body,be backof thick and were stained with hematoxylin-eosin.
theeye,ant.n. antennalnerve,ant. antenna,es esophagus.
Volumetric analysis was performed with an AIS/C
ticity ofthese brain structures has been demonstrated image analysis system (Imaging Research, Inc.) inter-
in bees (Withers et al. 1993, Wilmington et al. 1996, faced to a Zeiss Axiophot microscope via a Dage 72
Robinson 1998) and ants (Gronenberg et al. 1996). CCD camera. The following areas were measured in
Mushroom bodies increase in size when these insects selected spaced sections on both sides of the brain:
begin to perform complex and behaviorally more de- whole brain, antennal lobes, olfactory glomeruli, optic
manding tasks. Neuropil growth related to behavioral lobes, central body, mushroom body calyces, and the
changes has also been observed in non-social insects, regions occupied by Kenyon cells. When areas were
such as fruit flies and rove beetles (Bieber & Fuldner measured, this was done without awareness of the
1979, Technau 1984, Heisenberg et al. 1995). This group to which that individual belonged. The volume
growthwas foundto represent the further arborization ofa brain structure was calculated using the formula
and proliferation of existing brain cells, and not the ZAxtxN
Vol
production ofnew neurons. (object) i=i >
Flexibility of behavior and learning have been where n is die number ofsections on which measure-
demonstrated in different species of Lepidoptera ments were made, Ais die area ofa measured section,
&
(Swihart Swihart 1970, Papaj 1986, Weiss 1995, t is the distance between adjacent sections (e.g., sec-
1997, Hartlieb 1996, Fan et al. 1997). Butterflies and tion thickness), and N is the number ofsections repre-
moths have well-developed mushroom bodies (Ali sented by the section A.. Between 10 and 20 evenly
1974, Sivinsky 1989), and large antennal lobes (Mat- spaced sections were used to determine the volume of
sumoto & Hildebrand 1981). Both olfactory andvisual each region. This corresponded to 50-100% ofall the
learning have been described in Agraulis vanillae sections containingeach measured structure. The rela-
(Weiss 1995, Kroutov et al. 1999). tivevolume ofeach brain structure was calculated as a
Here we studied brain morphology in two groups of percentage ofthe volume ofthe whole brain.
Agraulis. One group comprisedbutterflies collectedin For statistical analysis ofthe data a fixed effects lin-
nature ("experienced" group) and the other groupwas ear model (ANOVA) was fit with PROC GLM (SAS
reared and maintained in the laboratory in isolation v.8). That is, size was modeled as a function of the
from normal environmental stimuli ("naive" group). fixed effects 'brain region', 'butterflygender' and 'but-
We
investigated the hypotheses that the sizes ofbrain terflygroup' ("experienced", "nai've" and "control"). All
structures involved in information processing and relevant assumptions such as constant variance and
learningvaryaccordingto the individual experience of normality were formally assessed. Due to the large
butterflies, and that such structures shouldbe largerin numberofmultiple Bonferroni comparisons we tested
butterflies exposed to various environmental stimuli at the 0.01 level ofsignificance throughout.
than in butterflies deprived ofthose. To exclude the possible effect ofage on the changes
in Agraulis brain, a control group of 10 males and 10
Materials and Methods
females, reared in the laboratorywas kept in cages for
Adults and larvae ofAgraulis vanillaewere collected 20-25 days aftereclosion underthe same conditions as
in Gainesville, Florida. All butterflies used in experi- described for the experimental group. The heads of
Volume 56, Number 4 195
&
Fig. 2. Sections ofthebrain ofAgraulisvanillae. a: mushroom body—calyx (ca) andKenyon cells (Kc);b: opticlobes (Opt.lbs); c: antennal
lobe wit—h glomeruli (Olf.gl.—); d: central body (cc); e: m—ushroom body calyx (ca), pedunculus (p), (3-lobe (B) and antennal lobe (Ant.lb.).
a,b,c,d frontalsections, e sagittal section. Scalebars 100(im.
controlbutterflies were sectioned, sections stained and mushroom bodies and olfactory glomeruli than did
brains measured as described above. "naive" or "control" individuals (Fig. 4; Table 1). The
relative volume ofmushroom body calyces in "experi-
Results
enced" butterflies was greater than in "naive" ones by
Figure 2 shows die sections ofthe measured brain 36% in males, and by 38% in females. Olfactory
structures in Agraulis vanillae. Most ofthe regions ex- glomeruli were larger in "experienced" Agraulis by
hibit clearly defined boundaries. Because of the ab- 48% in males, and 24% in females.
sence of a clear boundary between the mushroom The Kenyon cells region and antennal lobes showed
body's pedunculus andlobes, andthe surroundingneu- mixed outcomes. Within the Kenyon cells region,
ropil, attributable tothe staining method chosen fordie therewere no significant differences involume among
study, only mushroom bodycalyces were measured. the male groups, but "experienced" females exhibited
Whole brain volume ofAgraulis showed no signifi- smaller volumes than did "controls". For the antennal
cant variation according to group (Fig. 3). There was lobes, "experienced" males have larger volumes than
foundto be a significant interaction ofgender *group* do "naive" males. There were no differences among
brain region (p < 0.0001). Multiple pairwise compar- the female groups. The central bodyand optic lobe re-
isons revealed the following patterns: "experienced" gions exhibited no significant difference for any pair-
individuals ofboth sexes exhibited significantly larger wise comparison.
Journal of the Lepidopterists' Society
196
memory of the location of the host Passiflora patch,
because butterflies ofthis species utilize vast habitats
and linger at one spot for no longer than is necessary
to complete either feeding or egg-laying. Males, in
turn, need to locate the host-plant area to encounter
females and mate.
Detailed analysis of the captivity conditions and
their specific influence onAgraulis's experience, learn-
p 1 - ing and associated morphological changes in its brain
was not attempted. However, it seems evident that
captive laboratory-reared butterflies would have a
greatly reduced range of external stimuli, being de-
prived of space, visual stimuli, contacts with host-
—
Fig. 3. Wholebra—involumeinAgraulisva—nillae. EF "experi- plant, flowers and sex partners. It was also impossible
ence—d" females, NF "naive" f—emales, CF "control—" females, to determine precisely the age of "experienced" but-
EM "experienced" males, NM "naive" males, CM "control" terflies, collected in nature. But because we collected
males.
them in a period of 10-14 days after the beginning of
For most brain regions assessed, there was no sig- their abundance peak, we can estimate all collected
nificant difference between male and female volumes. butterflies to be ofapproximatelythe same age.
However, the antennal lobes exhibited the following Generally, measured brain structures were larger
pattern: "naive" and "control" females have larger an- (relative to the volume ofthe whole brain) in "experi-
tennal lobes than their male counterparts (p < 0.0001 enced" butterflies. But no difference in the relative
in each case), but "experienced" females do not differ volume ofoptic lobes and central body was recorded
significantly from "experienced" males. between two groups ofAgraulis. The most dramatic
For all brain regions, the "naive" and "control" increases in relative volume occurred in the mush-
groups exhibited no significant differences in volume, room bodies and olfactory glomeruli, whereas no size
within males or females. difference in optic lobes between "experienced" and
"naive" butterflies was observed. Therefore, olfactory
Discussion
stimuli may be of primary importance in driving the
The results of our research demonstrate that in structural changes indieAgraulis brain. Although but-
Agraulis differences in experience are correlated with terflies reputedlyrelyheavilyonvisual stimuli (Swihart
changes in thevolume ofseveral ofits brain regions in- 1970, Silberglied 1979, 1984), andAgraulis is capable
volved in sensoryinformation processing and memory of visual as well as olfactory learning (Weiss 1995),
formation. During their adult stage (2-4 weeks), their optic lobes only pass visual information to the
Agraulis vanillae butterflies must perform various ac- central brain, where processing and integration ofthis
tivities, the success of which can be enhanced by information takes place. Therefore such a resultis pre-
learning. Location offeeding sites with flowers thatof- dictable.
fer sufficient nectar reward, and recognition ofpoten- The relative decrease in volume ofthe Kenyon cells
tial danger are of importance to both sexes. Female region is rather hard to explain. However, because
Agraulis need to find suitable host-plants on which to there was no change in the whole brain volume, this
lay eggs. This involves not only recognition of the region's relative decrease could represent an actual
properplant amongst avarietyofotherplants, but also compression of the Kenyon cell clusters by the ex-
Table 1. Relativevolumes ofbrain regions as percentage ofthewhole brainvolumeinAgraulis vanillae. Within each box, differentsmall
case lettersindicate significantdifferences,whereasthesameletters indicatenodifference.
Mushroom Olfactory Kenyon cells Antennal Central Optic
bodycalyx glomeruli region lobes body Lobes
"Experienced" males 2.01 ± 0.08 a 1.45 ± 0.09a 0.69 ± 0.07a 3.74 ± 0.35b 0.61 ± 0.09a 64.4 ± 3.0a
"Naive"males 1.48 ± 0.05b 0.98 ± 0.05b 0.75 ± 0.05 a 3.41 ±0.10a 0.68 ± 0.04a 69.7 ±8.3a
"Control" males 1.38 ± 0.07b 0.98 ± 0.09 b 0.72 ± 0.04a 3.50 ± 0.10ab 0.66 ± 0.05 a 65.1 ±2.3a
"Experienced"females 2.18 ±0.10a 1.44 ± 0.08 a 0.56 ± 0.03a 3.90 ±0.10 a 0.63 ± 0.07a 61.8 ±2.6a
"Naive" females 1,58 ± 0.05 b 1.16 ± 0.05b 0.73 ± 0.05ab 4.00 ± 0.20 a 0.64 ± 0.03 a 62.4 ± 2.2a
"Control"females 1.58 ± 0.12b 1.11 ± 0.04b 0.84 ± 0.08b 4.00 ± 0.12 a 0.66 ± 0.02 a 61.1 ± 1.9a
—
Volume 56, Number 4 19"
Mushroom Body Olfactory glomeruli Kenyon cells
a b a 6
c
T
'ra
i_
JO
a
JL
o
j= JL JL
5
c
0o>
uk_
a.
i i i
EM NM CM EF NF CF EM NM CM EF NF CF EM NM CM EF NF CF
Antennal lobes Central body
Optic lobes
1.0
b a al1 a a a a a a a a a a a
= 4 .E 0.8
nIbaoS. 3 X9rOI1a 06 I T L . aaO 60 - T
.5c S3I
o ««-
£ 2 ° 04 f 40
c c
ao> 0o)
°- 1 8.0.2
i i
EM NM CM EF NF CF EM NM CM EF NF CF EM NM CM EF NF CF
—
EM NM
Fig. 4. Relativ—evolumesofbrain regi—ons aspercentageofthewhol—ebrainvolumeinAgrau—lis oanillae. "experienced"males,
"naive" males, CM "control" males. EF "experienced" females, NF "naive" females, CF "control" females. Different small case letters
indicatesignificantdifferences,whereas thesameletters indicatenodifferenceforeach sex.
panding mushroom body calyces. This problem could ference in olfactoryglomerulivolume between "naive"
be addressed by more detailed experimental analysis, and"experienced" males beingtwice as great as that in
for example assessment ofcell packing density. females, can perhaps be explained by the differences
The datapresentedhere are similarin manyways to in behavior ofmales and females. Females need to lo-
those ofstudies in other species ofinsects, which mea- cate host-plants and determine their suitability for
sured the size differences in brain regions caused by oviposition, and males need to search for females and
different experience and behavioral repertoire. As in recognize proper chemical cues from suitable part-
the present study an increase in relative volume of ners. Thus, each sex may rely on different environ-
mushroom bodies, and decrease in relative volume of mental stimuli. The change in the intensity of these
the Kenyon cells region were reported for ants (Gro- stimuli may effect butterflies ofdifferent sexes differ-
nenberg et al. 1996) and bees (Withers et al. 1993). ently, and cause the observed dissimilarityin the brain
Also, there was no increase in the relative volume of reconstruction. This dimorphism correspondswith our
the optic lobes in either of these insects. Olfactory earlier findings in Agraulis learning (Kroutov et al.
glomerularvolume was found to differbetween 1-day- 1999), where different learning capability was
old and nurse bees (larger in nurses), but the increase recorded for the two sexes.
was not maintained in foragers. For rove beetles Measurements in the control group show that mor-
mushroom body volume increase and no changes in phological changes in the brain ofAgraulis vanillae are
optic lobe volume were recorded (Bieber & Fuldner not age-related, but experience-related, since the rela-
1979). tive volumes of the studied brain structures in 2-day
The sexual dimorphism found in the reorganization ("naive") and 20-25-day ("control") butterflies were
of some brain structures in Agraulis, namely the dif- not significantlydifferent. These changes occurin only
198 Journal of the Lepidopterists' Society
a fewbrain compartments that are noted fortheir role tial expression ofthe Drosophila rutabaga gene in mushroom
bodies,neuralcentersforlearningininsects. Neuron9:619-627.
in information processing and learning in insects. This
Hartlieb, E. 1996. Olfactory conditioning in the moth Heliothis
further supports the hypothesis that growth of these virescens. Naturwissenschaffen83:87-88.
brain regions is related to learning experience and be- Heisenberg, M., A. Borst, S. Wagner & D. Byers. 1985.
Drosophila mushroom body mutants are deficient in olfactory
havioral complexity ofabutterfly.
learning. Nerogenet. 2:1-30.
Further experiments involving the manipulation of Heisenberg, JM.., M. Heusipp&Ch.Wanke. 1995. Structuralplas-
various elements of the environment may lead to a ticityintheDrosophila brain.J. Neurosci. 15 (3):1951-1960.
Kroutov,V, M. S.Mayer &T. C. Emmel. 1999. Olfactorycondi-
better understanding of the exact relation between
tioning of the butterfly Agraulis vanillae (L.) (Lepidoptera,
particular types of information, how they are pro- Nymphalidae)tofloralbutnothost-plantodors. InsectBehav.
J.
cessed, andchanges theycause indie brain ofAgraulis 12 (6):833-843.
vanillae. It would be especially interesting to analyze Matsumoto, S. G. & J. G. Hildebrand. 1981. Olfactory mecha-
nisms inthe mothManduca sexta: response characteristics and
the specific effects ofvarious environmental "depriva- morphologyofcentral neurons in the antenna! lobes. Proc. R.
tions" and, inverted, the effect ofadditional stimuli on Soc. Lond. B 213:249-277.
Menzel, R., Erber &T. Masuhr. 1974. Learning andmemory
the changes inAgraulis' brain structures. J.
inthehoneybee, pp. 195-218. In Browne, L. B. (ed.), Experi-
mental analysis ofinsectbehavior. Springer, Berlin.
Acknowledgments
Papaj, D. R. 1986. Conditioning of leaf-shape discrimination by
Wethank Dr. M. S. Mayerforhis helpful comments on ourpa- chemical cues in the butterfly, Battus philenor. Anim. Behav.
per, ScottWhittakerforhishelpwiththepreparationoftheillustra- 34:1281-1288.
tions and Galin Jones forhelping us with the statistical analysis of Robinson, G. E. 1998. From societ'ytogenes with the honeybee.
ourdata. Amer. Scientist86 (5):456-462.
Silberglied, R. E. 1979. Communication in the ultraviolet. A.
Literature Cited Rev. Ecol. Syst. 10:373^398.
. 1984. Visual communication and sexual selection among
Ali,F.A. 1974. Structureandmetamorphosisofthebrainandsub- butterflies, pp. 207-223. In Vane-Wright, R. I. & P. K. Ackery
oesophageal ganglion of Pieris brdssicae (L.) (Lepidoptera: (eds.). Thebiologyofbutterflies. Academic Press.
Pieridae).Trans. R. Ent. Soc. Lond. 125 (4):363^12. Sivinski, 1989. Mushroom bodydevelopmentin nymphalidbut-
Bernstein, S. & R. Bernstein. 1969. Relationships between for- terflieJ.s: acorrelate oflearning? Insect Behav. 2 (2):277-283.
agingefficiencyandthe size ofthe head andcomponentbrain Swihart, C. A. & S. L. Swihart.J. 1970. Colour selection and
and sensory structures in the red wood ant. Brain Research learned feedingpreferences in the butterfly, Heliconius chari-
16:85-104. tonius. Anim. Behav. 19:156-164.
Bieber, M. & D. Fuldner. 1979. Brain growth during the adult Swihart, S. L. 1970. The neural basis ofcolourvision in the but-
stageofaholometabolousinsect. Naturwissenschaffen66:426. terfly, Papiliotroilus. InsectPhysiol. 16:1623-1636.
Erber, J., T. Masuhr & R. Menzel. 1980. Localization ofshort- Technau,G. 1984. FiberJ.numberinthemushroombodiesofadult
term memory in the brain ofthe bee,Apis mellifera. Physiol. Drosophila melanogasterdependsonage,sexandexperience.
Entomol. 5:343-358. Neurogenet. 1:113-126. J.
Fan, R.-J., P.Anderson&B. S. Hansson. 1997. Behaviouralanaly- Weiss, M. R. 1995. Associativecolourlearninginanymphalidbut-
sis of olfactory conditioning in the moth Spodoptera littoralis terfly. Ecol. Entomol. 20:298^301.
(Bsdv.) (Lepidoptera: Noctuidae).J. Exp. Biol. 200:2969-2976. . 1997. Innate colourpreferences andflexible colourlearn-
Goossen, H. 1949. Untersuchungen an Gehirnen verschieden ingindiepipevineswallowtail. Anim. Behav. 53:1043-1052.
grosser,jeweilsverwandter Coleopteren- und Hymenopteren- Winnington, A. P., R. M. Napper & A. R. Mercer. 1996. Struc-
Arten. Zool. Jb. Abt.Allg. Zool. 62:1-64. turalplasticityofidentifiedglomeruliintheatennallobesofthe
Gronenberg, W., S. Heeren & B. Holldobler. 1996. Age- adultworkerhoneybee. Comp. Neurol. A365:479-490.
dependentandtask-relatedmorphologicalchangesinthebrain Withers, G., S. Fahrbach J&. G. E. Robinson. 1993. Selective
andthe mushroom bodiesoftheantCamponotusfloridanus. J. neuroanatomical plasticityand division oflabourin thehoney-
Exp. Biol. 199:2011-2019. bee. Nature364:238-240.
Hammer, M. & R. Menzel. 1998. Multiple sites of associative
odor learning as revealed bylocal brain microinjections ofoc- Receivedfor publication 6 March 2001; revised and accepted 21
topaminein honeybees. Learningand Memory5:146-156. February2002.
Han,P. L.,L. R. Levin, R. R. Reed&R. L. Davis. 1992. Preferen-