Adult stemmata of the butterfly Vanessa cardui express UV and green opsin mRNAs.

Abstract

Adult stemmata are distinctive insect photoreceptors located on the posterior surfaces of the optic lobes. They originate as larval eyes that migrate inward during metamorphosis. We used a combination of light microscopy and in situ hybridization to examine their anatomical organization in the butterfly Vanessa cardui and to test for the presence of visual pigments, the light sensitive components of the visual transduction pathway. The bilateral cluster of six internal stemmata is located near the ventral edge of the lamina. They retain the dark screening pigment and overlying crystalline cones of the larval stemmata. We found two opsin mRNAs expressed in the stemmata that are also expressed, respectively, in UV-sensitive and green-sensitive photoreceptor cells in the compound eye. A third mRNA that is expressed in blue-sensitive photoreceptor cells of the compound eye was not expressed in the stemmata. Our results reinforce the idea that the adult stemmata are not merely developmental remnants of larval eyes, but remain functional, possibly as components of the circadian input channel.

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Cell Tissue Res (2005) 319: 175–179
DOI 10.1007/s00441-004-0994-3
SHORT COMMUNICATION
Adriana D. Briscoe.Richard H. White
Adult stemmata of the butterfly Vanessa cardui express UV
and green opsin mRNAs
Received: 27 June 2004 / Accepted: 6 September 2004 / Published online: 19 October 2004
# Springer-Verlag 2004
Abstract Adult stemmata are distinctive insect photo-
receptors located on the posterior surfaces of the optic
lobes. They originate as larval eyes that migrate inward
during metamorphosis. We used a combination of light
microscopy and in situ hybridization to examine their
anatomical organization in the butterfly Vanessa cardui
and to test for the presence of visual pigments, the light
sensitive components of the visual transduction pathway.
The bilateral cluster of six internal stemmata is located
near the ventral edge of the lamina. They retain the dark
screening pigment and overlying crystalline cones of the
larval stemmata. We found two opsin mRNAs expressed
in the stemmata that are also expressed, respectively, in
UV-sensitive and green-sensitive photoreceptor cells in the
compound eye. A third mRNA that is expressed in blue-
sensitive photoreceptor cells of the compound eye was not
expressed in the stemmata. Our results reinforce the idea
that the adult stemmata are not merely developmental
remnants of larval eyes, but remain functional, possibly as
components of the circadian input channel.
Keywords Extraretinal opsins.Photoreceptor.Visual
pigment.Vanessa cardui (Insecta)
Introduction
The larval photoreceptor organs of holometabolous
insects, known as stemmata, lose their attachment to the
head capsule at metamorphosis and migrate inward along
the developing optic nerve of the adult (e.g., Norlander
and Edwards 1969; Gilbert 1994). They end up on the
posterior surface of the adult optic lobe, readily distin-
guished by their dark screening pigment. Such adult
derivatives of larval stemmata have been reported for
lepidoptera (Ichikawa 1991), beetles (Schulz et al. 1984),
ants (Felisberti and Ventura 1996), flies (Seifert et al.
1987), caddisflies (Hagberg 1986), and other insects
(Fleissner and Fleissner 2003; Gilbert 1994). Originally
thought to be degenerate remnants in adults, accumulating
morphological and physiological evidence indicates that
the stemmata remain functional in adults (Ichikawa 1991;
Gilbert 1994).
The larval photoreceptor in Drosophila and other
muscomorphan flies is Bolwig’s organ (Strange 1961),
the homologue of the stemmata of other insects (Melzer
Von and Paulus 1989). Like stemmata, Bolwig’s photo-
receptors are retained in adults at the posterior margin of
the compound eye as the Hofbauer–Buchner eyelet
(Yasuyama and Meinertzhagen 1999). The green-sensitive
RH6 Drosophila rhodopsin (Salcedo et al. 1999; Yasuya-
ma and Meinertzhagen 1999) and blue-absorbing RH5
rhodopsin (Salcedo et al. 1999; Malpel et al. 2002) were
found to be expressed in eyelet cells, whereas known UV-
sensitive rhodopsins (RH3 and RH4) were not detected.
Little is known about the function of adult stemmata or
their photopigments in other insects.
Vanessa cardui has joined the swallowtail butterfly,
Papilio xuthus (Kitamoto et al. 1998, 2000), and the
sphingid moth, Manduca sexta (White et al. 2003), as a
lepidopteran species in which visual pigments have been
characterized (Briscoe et al. 2003). Three opsin-encoding
mRNAs are expressed in the compound eyes of Vanessa.
These encode a UV-sensitive rhodopsin with peak ab-
sorption at 360 nm, a blue-sensitive rhodopsin at 470 nm,
and a green-sensitive rhodopsin at 530 nm. The fact that
This work was supported by grants from the National Science
Foundation to A.D.B. (IBN-0346765) and R.H.W (IBN-9874493).
A. D. Briscoe (*)
Department of Ecology and Evolutionary Biology,
Comparative and Evolutionary Physiology Group, University
of California,
321 Steinhaus Hall,
Irvine, CA, 92697, USA
e-mail: abriscoe@uci.edu
Tel.: +1-949-8241118
Fax: +1-949-8242181
R. H. White
Department of Biology, University of Massachusetts–Boston,
100 Morrissey Blvd,
Boston, MA, 02125-3393, USA

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compound eye-specific opsins are also expressed in the
Hofbauer–Buchner eyelet of Drosophila makes it inter-
esting to see if this pattern of opsin expression is
conserved in other insects. To further our understanding
of the photoreceptor systems in day-flying lepidopterans,
we asked whether the opsins of the compound eye in
Vanessa are also expressed in the adult stemmata.
Materials and methods
Animals
Vanessa cardui pupae were purchased commercially
(Carolina Biological Supply Company) and allowed to
eclose in a humid chamber. The adults were hand-fed a
sucrose solution.
In situ hybridization
Riboprobes were made from three distinct opsin-encoding
3′RACE products isolated from a cDNA pool synthesized
from compound eye mRNA as previously described
(Briscoe et al. 2003). Approximately one third of the
coding region and all of the 3′UTR was included in the
synthesis reaction. The probes were 684 bp (UV opsin,
AF414074), 873 bp (blue opsin, AF414075), and 678 bp
(LW opsin, AF385333) in length. Briefly, M13 (−20) and
M13 forward primers were used in a 50-μl PCR reaction
with cloned plasmid DNA as a starting template. The PCR
product was separated on a 1% agarose gel, and the
appropriate band was cut out and purified using the
GeneClean kit (Qbiogene). The purified DNAwas used as
the template for a digoxigenin-labeling reaction using
either the SP6 or T7 RNA polymerase (DIG RNA labeling
kit, Roche). Probe yield was quantified using a dot blot.
The sections were incubated in hybridization buffer
(0.3 mM NaCl, 2.5 mM ethylene-tetraacetic acid,
20 mM TRIS-Cl, pH 8.0, 50% formamide, 10% dextran
sulfate, 200 μg/ml yeast tRNA, and ×1 Denhart’s medium)
for 30 min at 60°C. Then approximately 40 ng of probe
diluted in hybridization buffer was added to each slide.
The sections were coverslipped and incubated overnight at
60°C in a humid chamber. The sections were then washed
with ×2, ×1, and ×0.1 standard saline citrate (SSC) and
0.1% Tween, for 10 min to increase accessibility of the
probe. An alkaline-phosphatase conjugated anti-digoxi-
genin antibody, Fab fragment (1:1,000 in ×1 phosphate-
buffered saline, PBS) was added to the slides and
incubated 2 h at room temperature. Probe detection was
via 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt
(BCIP), nitro blue tetrazolium (NBT), and 0.1% Tween 20
colorimetric detection in alkaline phosphatase developing
solution (100 mM NaCl, 50 mM MgCl2, 100 mM TRIS,
pH 9.5).
Tissue preparation and light microscopy
For anatomy (Fig. 1a–c), opened head capsules were fixed
in cacodylate-buffered glutaraldehyde solution. After
aldehyde fixation for 1–2 h, the tissue was postfixed in
0.5% osmium tetroxide, dehydrated in ethanol and
propylene oxide, and embedded in Spurr’s resin (Poly-
sciences, Warrington, PA, USA). One-micrometer sections
were stained with methylene blue. Digital light micro-
graphs were obtained with an Olympus BX60 microscope
and Scionimage software, and processed in Adobe Photo-
shop with only magnification, contrast, and density
adjusted. For riboprobe detection (Fig. 1d–g), Vanessa
heads were fixed as in Briscoe et al. (2003). Cryostat
sections (14–16 μm) were cut (Microm, HM 500 OMV)
and photographed before and after in situ hybridization
using an Axioskop two plus microscope connected to an
AxioCam Hrc digital camera (Zeiss Goettingen, Ger-
many).
Results and discussion
Bilateral groups of six stemmata can be seen on the larval
head capsule. We found the six stemmata clustered near
the ventral edge of the lamina, similar to those described in
adult P. xuthus (Ichikawa 1991). The stemmatal cells
contained a dense concentration of screening pigment
granules surrounding rhabdoms. The stemmata were
surmounted by the crystalline cone cells that were present
in the larvae (Fig. 1a–d). In pierid and papilionid
caterpillars, each larval stemma contains seven photore-
ceptor cells (Ichikawa and Tateda 1982). Although larval
Vanessa has not yet been investigated, it is probably
similar. Thus, in principle there could be a total of 42
photoreceptors in the extraocular organ. It would be
interesting to investigate further whether all of these
photoreceptor cells are retained in the adult stemmata of
"
Fig. 1 Methylene-blue-stained light micrographs (a–c) of Vanessa
cardui retina and optic lobe showing the location of the adult
stemmata along the ventral margin of the lamina and (d–g) blue, UV,
and green opsin mRNA expression as indicated by detection of
digoxigenin-labeled riboprobes. c Arrowhead indicates location of
rhabdomeres. d Frozen section of retina and optic lobe photo-
graphed prior to performing the in situ hybridization. Arrow
indicates the presence of darkly pigmented stemmata. Arrowhead
indicates same ommatidia as in e. e Same section as in d, following
in situ hybridization using the blue opsin antisense riboprobe and
colorimetric detection using BCIP, NBT, and alkaline phosphatase.
Arrow indicates the location of the same stemmata shown in d,
showing the lack of screening pigments due to washing and the lack
of blue opsin mRNA expression. Arrowhead indicates the presence
of blue opsin mRNA in the retinular cells of the compound eye. f
Arrow indicates lobes of the stemmata that express UV opsin
mRNA, suggesting that some but not all photoreceptor cells of the
stemmata express the UV opsin transcript. Arrowhead indicates
crystalline cones. g Arrow indicates strong green opsin mRNA
expression in the stemmata. Dark staining of the photoreceptor cells
of the retina is also evident, as is lighter staining of the lamina.
Crystalline cone-like structures are also visible above the stemmata
(arrowhead). re Retina, la lamina, me medulla, 1. oc first optic
chiasma.
176

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Vanessa or whether (as in Drosophila) some are lost
during the course of development.
Green opsin riboprobe hybridization similar in intensity
to that observed in the compound eye was localized to the
stemmatal cells (Fig. 1g). Similar intensity was also
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observed between the retina and stemmata photoreceptors
with the UVopsin riboprobe (data not shown). Blue opsin
mRNA localized to the retinular cells of the compound
eye, in contrast, was not detected in the stemmata (Fig. 1d,
e). To our knowledge, this is the first report of specific
opsin transcripts localized to extraretinal photoreceptor
cells in a butterfly.
Our molecular results from Vanessa should be con-
sidered in light of the electrophysiological measurements
of Ichikawa (1991) on the adult stemmata of another
butterfly, P. xuthus. Whereas we found only UV- and
green-sensitive rhodopsins, Ichikawa’s spectral sensitivity
measurements identified three cell types—green-sensitive,
blue-sensitive, and UV-sensitive—the same three that are
present in the larval stemmata of Papilio and several other
lepidopteran species (Gilbert 1994). Thus it seems likely
to us that a blue-sensitive rhodopsin differing in sequence
from that of the compound eye is in fact expressed in the
adult (and perhaps larval) stemmata of Vanessa. However,
there are other possibilities: the larval stemmata of
Vanessa may lack blue-sensitive cells; blue-sensitive
cells present in the larva may degenerate at metamorpho-
sis; or blue-sensitive larval cells may cease to express their
blue-sensitive rhodopsin or switch to green or UV
expression. Clearly these alternatives can only be resolved
by more extensive morphological, electrophysiological,
and molecular studies with Vanessa.
Our results from Vanessa are also consistent with opsin
expression results from Drosophila. Several recent studies
indicate that the cells of its stemmatal homologue,
Bolwig’s organ, and its adult derivative, the Hofbauer–
Buchner eyelet, both express two rhodopsins: blue-sensi-
tive RH5 and green-sensitive RH6 (Yasuyama and
Meinertzhagen 1999; Malpel et al. 2002). We should
note that an early report (Pollock and Benzer 1988) on
opsin expression in Bolwig’s organ is incorrect (Malpel et
al. 2002; Pichaud and Desplan, personal communication).
Thus it is likely the case that larval and adult stemmata in
Vanessa (as in Drosophila) express the same opsins, and
except for the uncertainty discussed above, they are opsins
that are also expressed in the compound eye.
What might be the function of adult stemmata in
Vanessa and other insects? Stemmata, while immediately
useful to the larva for location of food (Harris et al. 1995)
and avoidance of predators, may also be important for
regulating life history traits such as the timing of pupation.
It has also been suggested that in adults they play a role in
entrainment of circadian rhythms (Ichikawa 1991; Fleiss-
ner 1982). In this regard, Drosophila is currently the most
intensely investigated species. Eyelet photoreceptors have
been shown to play a role in the entrainment of circadian
activity rhythms in Drosophila under normal 12-h
light / 12-h dark photoperiods in studies of mutant flies
that lack them (Regier et al. 2003). Since long-wavelength
receptors have been typically implicated in circadian
systems (e.g., Felisberti et al. 1997), it will be interesting
to see if all classes of adult stemmatal photoreceptors work
in concert to track changes in the spectral content of
ambient light.
Acknowledgements
Mangus, and Julia Stalleicken for technical assistance, and two
anonymous reviewers for their comments.
We thank Ivana Djuretic, Marilou Sison-
References
Briscoe AD, Bernard GD, Szeto AS, Nagy LM, White RH (2003)
Not all butterfly eyes are created equal: rhodopsin absorption
spectrum, molecular identification, and localization of ultravi-
olet-, blue- and green-sensitive rhodopsin-encoding mRNAs in
the retina of Vanessa cardui. J Comp Neurol 458:334–349
Felisberti F, Ventura DF (1996) Cerebral extraocular photoreceptors
in ants. Tissue Cell 28:25–30
Felisberti F, Ventura DF, Hertel H (1997) Cerebral extraocular
photoreceptors in beetles. Comp Biochem Physiol 118A:1353–
1357
Fleissner G (1982) Isolation of an insect circadian clock. J Comp
Physiol 149:311–316
Fleissner G, Fleissner G (2003) Nonvisual photoreceptors in
arthropods with emphasis on their putative role as receptors
of natural Zeitbeger stimuli. Chronobiol Int 20:593–616
Gilbert C (1994) Form and function of stemmata in larvae of
holometabolous insects. Annu Rev Entomol 39:323–349
Hagberg M (1986) Ultrastructure and central projections of
extraocular photoreceptors in caddisflies (Insecta Trichoptera).
Cell Tissue Res 245:643–648
Harris MO, Foster SP, Bittar T, Ekanayake K, Looij K, Howard A
(1995) Visual behaviour of neonate larvae of the light brown
apple moth. Entomol Exp Appl 77:323–334
Ichikawa T (1991) Brain photoreceptors in the pupal and adult
butterfly: fate of the lateral ocelli. Zoolog Sci (Tokyo) 8:471–
476
Ichikawa T, Tateda H (1982) Distribution of color receptors in the
larval eyes of four species of Lepidoptera. J Comp Physiol
149:317–324
Kitamoto J, Sakamoto K, Ozaki K, Mishina Y, Arikawa K (1998)
Two visual pigments in a single photoreceptor cell: identifica-
tion and histological localization of three mRNAs encoding
visual pigment opsins in the retina of the butterfly Papilio
xuthus. J Exp Biol 2001:1255–1261
Kitamoto J, Ozaki K, Arikawa K (2000) Ultraviolet and violet
receptors express identical mRNA encoding an ultraviolet-
absorbing opsin: identification and histological localization of
two mRNAs encoding short wavelength-absorbing opsins in
the retina of the butterfly Papilio xuthus. J Exp Biol 203:2887–
2894
Malpel S, Klarsfeld A, Rouyer F (2002) Larval optic nerve and adult
extra-retinal photoreceptors sequentially associate with clock
neurons during Drosophila brain development. Development
129:1443–1453
Melzer Von RR, Paulus HF (1989) Evolutionswege zum Larvalauge
der Insekten—Die Stemmata der hoeheren Dipteren und ihre
Abwandlung zum Bolwig-Organ. Z Zool Syst Evolut-Forsch
27:200–245
Norlander RH, Edwards JS (1969) Postembryonic brain develop-
ment in the Monarch butterfly, Danaus plexippus plexippus L,
II: the optic lobes. Wilhelm Roux Archiv 163:197–220
Pollock JA, Benzer S (1988) Transcript localization of four opsin
genes in the three visual organs of Drosophila: RH2 is ocellus
specific. Nature 333:779–782
Regier D, Stanewsky R, Helfrich-Forster C (2003) Cryptochrome,
compound eyes, Hofbauer–Buchner eyelets and ocelli play
different roles in the entrainment and masking pathway of the
locomotor activity rhythm in the fruit fly Drosophila melano-
gaster. J Biol Rhythms 18:377–391
Salcedo E, Huber A, Henrich S, Chadwell LV, Chou WH, Paulsen
R, Britt SG (1999) Blue- and green-absorbing visual pigments
of Drosophila: ectopic expression and physiological character-
ization of the R8 photoreceptor cell-specific Rh5 and Rh6
rhodopsin. J Neurosci 19:10716–10726
178

Page 5

Schulz W-G, Schluter U, Seifert G (1984) Extraocular photorecep-
tors in the brain of Epilachna varivestis (Coleoptera: Cocci-
nellidae). Cell Tissue Res 236:317–320
Seifert P, Smola U, Schinko I (1987) Internal extraocular
photoreceptors in a dipteran insect. Tissue Cell 19:111–118
Strange PH (1961) The spectral sensitivity of Calliphora maggots. J
Exp Biol 38:237–248
White RH, Xu H, Munch TA, Bennett RR, Grable EA (2003) The
retina of Manduca sexta: rhodopsin expression, the mosaic of
green-, blue- and UV-sensitive photoreceptors, and regional
specialization. J Exp Biol 206:3337–3348
Yasuyama K, Meinertzhagen IA (1999) Extraretinal photoreceptors
at the compound eye’s posterior margin in Drosophila melan-
ogaster. J Comp Neurol 412:193–202
179