EVOLUTION & DEVELOPMENT

3:6, 408414 (2001)

Conservation of the expression of Dll, en, and wg in the eye-antennal

imaginal disc of stalk-eyed flies
Imogen Hurley, Kevin Fowler, Andrew Pomiankowski, and Hazel Smith*
The Galton Laboratory, Department of Biology, University College London, 4 Stephenson Way, London, NW1 2HE, UK
The authors current address is The Galton Laboratory, Department of Biology, University College London, 4 Stephenson Way,
London, NW1 2HE, UK.
*Author for correspondence (email: ucbhhks@ucl.ac.uk)

SUMMARY
We studied the developmental basis of exaggerated eye span in two species of stalk-eyed flies (Cyrtodiopsis dalmanni and Sphyracephala beccarri). These flies have
eyes laterally displaced at the end of eyestalks, and males
have greatly exaggerated eye span, which they use as a sexual display. To investigate eye span development we have
compared eye-antennal disc morphology and the expression
of three key regulator genes of Drosophila head development,
Distal-less (Dll), engrailed (en), and wingless (wg), in the stalkeyed flies and Drosophila. We found great similarity in the basic division of the disc into anterior-antennal and posterior-eye
portions and in the general patterning of Dll, en, and wg. Unex-

pectedly, our results showed that although the eye and antenna are adjacent in adult stalk-eyed flies, their primordia are
physically separated by the presence of an intervening region
between the anterior and posterior portions of the disc. This region is absent from Drosophila eye-antennal discs. We chose
two stalk-eyed fly species that differed in the degree of eyestalk exaggeration but surprisingly we found no corresponding
difference in the size of the en-wg expression domains that
mark the boundaries of the dorsal head capsule primordia. In
summary, our expression data establish the regional identity of
the eye-antennal disc and provide a framework from which to
address the developmental genetics of hypercephaly.

INTRODUCTION

is far more extreme in C. dalmanni than S. beccarri. The ratio of eye span to body length in C. dalmanni males is about
2.5 times greater than that of S. beccarri males. The two
stalk-eyed fly species are also distantly related within the diopsid phylogeny (Baker et al. 2001), and so comparison between them is informative about general aspects of development when compared to the Drosophila outgroup.
The genetics and development of the head capsule are
well understood in Drosophila. The Drosophila head is
formed from the clypeolabral, labial, and eye-antennal discs.
The labrum and proboscis are derivatives of the clypeolabral
and labial discs, while the eye, antenna, head capsule, and
maxillary palps are formed by the fusion of a pair of eyeantennal discs (Gehring and Seippel 1967; Wildermuth and
Hadorn 1965). The regional identity of the eye-antennal disc
has been determined by classical fate mapping and using
molecular markers (Haynie and Bryant 1986; Royet and
Finkelstein 1996).
We examined the expression of three genes that show region-specific expression in Drosophila eye-antennal discs in
stalk-eyed flies. The first of these, Distal-less (Dll), is associated with outgrowths from the body in the proxiomodistal
axis and is expressed in the developing antennae (Panganiban et al. 1997). The other two, wingless (wg) and engrailed

Hypercephaly, in the form of lateral extensions of the head

capsule, is observed in at least eight families of Diptera
(Wilkinson and Dodson 1997). The most extreme examples
are seen in the family Diopsidae. All members of this family
show some degree of hypercephaly with both the eyes and
the antennae laterally displaced at the end of long eyestalks.
There is considerable variation between species within the
family both in eye span and the degree of sexual dimorphism
in eye span (Baker et al. 2001). In several sexually dimorphic
species there is evidence for sexual selection through strong
female mate preference for males with exaggerated eye span
(Burkhardt and de la Motte 1988; Hingle et al. 2001).
The identity and mode of action of the genes that underlie
exaggerated sexually selected traits in general, and hypercephaly in particular, are poorly understood. Here, we address the developmental and genetic mechanisms by which
the exaggerated morphology of stalk-eyed flies is achieved.
We used Drosophila melanogaster as a nonhypercephalic
outgroup against which to compare development in two species of stalk-eyed fly: Cyrtodiopsis dalmanni and Sphyracephala beccarri. Both species of stalk-eyed fly are sexually
dimorphic for eye span but the degree of male exaggeration
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(en), are expressed in restricted domains across the mediolateral axis of the adult Drosophila dorsal head capsule and in
the disc regions that give rise to these domains (Royet and
Finkelstein 1996). Wg is expressed in the orbital cuticle surrounding the compound eyes, while en expression is confined to the medial cuticle around the ocelli. It is believed
that these genes are involved in specifying head domain
identity: wg for lateral elements and en for medial structures
(Royet and Finkelstein 1996). Loss of wg causes the deletion
of lateral elements including the orbital cuticle, although ectopic wg expression causes an expansion of lateral pattern elements at the expense of medial structures such as the ocelli.
The en expression domain is eliminated in the imaginal discs
of ocelliless (oc) mutants and expanded in those of wg mutants, which develop an enlarged ocellar region.
We compared the expression of the medial head gene en
and the antennal-specific gene Dll in D. melanogaster, S.
beccarri, and C. dalmanni via immunohistochemistry using
pan-specific antibodies. We then cloned part of the C. dalmanni wg gene and examined its expression by in situ hybridization. We used the expression data that we generated
to define the region of the stalk-eyed fly eye-antennal disc,
which gives rise to the dorsal head capsule, including the exaggerated eyestalks, and compared it with the equivalent region in D. melanogaster.

MATERIALS AND METHODS

Fly stocks and rearing methods
Experimental samples of C. dalmanni were taken from laboratory
populations derived from field collections in 1993 near the Gombak
River in Malaysia. Stocks of S. beccarri were derived from collections in South Africa in 1993 (gift of G. Wilkinson). Both species
have been maintained in population cages containing at least 200
individuals, kept at 25C on a 12 h light:dark cycle and were fed
ground sweet corn. Samples of larvae for the experiments were collected as eggs and reared in uncrowded conditions (David et al.
1998; Baker et al. in press) until the late third-larval instar or early
pupal stages.
The sample of D. melanogaster came from a stock established
in 1972 from a collection in Dahomey (now Benin, West Africa).
Subsequently this has been maintained on standard sugar-yeast
food medium at large population size in cage culture at 25C and
exhibits high levels of genetic variation (Whitlock and Fowler
1999). Samples of late third-instar larvae were collected at the wandering stage from the culture bottles.
Scanning electron microscopy
The heads of flies were removed, fixed in 4% paraformaldehyde in
phosphate-buffered saline (PFA) overnight at 4C then postfixed in
OSO4dH2O. Following dehydration in an ethanol series, specimens
were incubated overnight in iso-amyl acetate at room temperature.
The heads were dried in a CO2 critical point drier and coated in

Dll, en, and wg in stalk-eyed flies

409

gold, and imaging was achieved in a Jeol 5410LV scanning electron

microscope (Oberkochen, Germany).
Immunohistochemistry
Larvae were dissected in phosphate-buffered saline (PBS) by inverting the heads so that the imaginal discs remained attached to the
cuticle and mouth hooks, and they were fixed in equal parts 4%
PFA and heptane for 20 min. The PFA layer was then replaced with
methanol for a brief wash followed by three quick washes in PBS
and two in PBS  0.1% Triton X-100 (PT). Samples were incubated overnight at 4C in primary antibody preabsorbed against
fixed larval heads and diluted in PT. All subsequent procedures
were performed at room temperature unless otherwise stated. Samples were washed in PT for 3  5 min and 3  30 min, blocked in
serum diluted 3:200 in PT for 1 h, then incubated for 2 h at 4C in
anti-mouse or rabbit secondary biotin-conjugated antibody, diluted
1:200 in blocking serum.
Antigen detection was performed using the Vectastain Avidin/
Biotin/Alkaline Phosphatase ABC System (Vector Laboratories;
Burlinghame, CA, USA) according to the manufacturers instructions. Prior to staining, endogenous alkaline phosphatase was removed with 3  5 min washes in NTMT (0.1 m NaCl, 0.05 m
MgCl2, 0.1 m Tris pH 9.5, 0.1% Tween-20, 0.001 m levamisole).
Staining was carried out in NTMT with 0.35% 5-bromo-4-chloro3-indolyl phosphate and 0.45% nitroblue tetrazolium, then discs
were dissected away from the cuticle and mounted. Eye-antennal
discs were photographed using a compound microscope. The primary antibodies were mouse anti-en mAb 4F11 (gift from N. Patel)
used at 1:20 dilution and rabbit anti-Dll (gift from G. Panganiban)
used at 1:500 dilution.
Isolation of diopsid-specific wingless sequence
Whole third-instar C. dalmanni larvae were frozen in liquid nitrogen and ground to a powder, and RNA was extracted using an
RNAqueous Total RNA Isolation Kit (Ambion; Huntingdon, UK)
as described in the manufacturers protocol. Samples were DNasetreated and reverse transcribed-polymerase chain reaction (RTPCR) was performed using diopsid-specific wg primers (Baker et
al. 2001): 5-GTT AGA ACW TGT TGG ATG CG-3 and 5 CTT
TCG ACG ACA ATC ACT TC-3 with an annealing temperature
of 55C. The resulting 581-bp product was cloned into a pGEM-T
vector (Promega; Southampton, UK) according to the manufacturers instructions. Plasmid DNA was prepared (Qiagen plasmid
mini-kit; Crawley, UK) and the identity of the insert was confirmed
via sequencing from the M13 reverse and forward primers in the
vector (Cambridge BioScience; Huntingdon, UK).
In situ hybridization
Detection of wg transcripts was performed by in situ hybridization as
described previously (Phillips et al. 1990) except that inverted larval
heads were digested for 2.5 min in 10 g ml1 proteinase K and hybridized in hybridization buffer (50% formamide, 5  sodium sodium
citrate (SSC), 0.05 mg ml1 tRNA, 0.05 mg ml1 heparin, 0.1% Tween
20). Hybridizations with denatured diopsid and D. melanogasterspecific wingless probes were performed at 56C overnight and detected via incubation at 4C overnight with sheep anti-digoxygenin
alkaline phosphatase Fab fragments (Boehringer Mannheim; Ingel-

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heim, Germany) diluted 1:1500. Staining and photography were performed as described for immunohistochemistry above.
All in situ hybridizations used digoxygenin 11-uridine tri-phosphate (UTP)-labeled RNA probes. Diopsid antisense probe (708
bp) was transcribed from the SP6 transcription start site in the vector following digestion with Aat II and sense probe (667 bp) from
the T7 start site of Pst I cut vector. Drosophila Dint-1 wg probes
were created from p wg C4 (gift from R. Phillips) in which mRNA
had been cloned into pNB40 (Brown and Kafatos 1988): sense (923
bp) and antisense (771 bp) probes were transcribed using T7 RNA
polymerase (on ClaI cut vector) and SP6 RNA polymerase (on PstI
cut vector) respectively.

RESULTS
Comparison of adult head morphology
In Drosophila the compound eyes occupy most of the head.
The antennae lie between the eyes and divide the head capsule into dorsal and ventral halves (Fig. 1A). We will focus

on the structure of the dorsal head capsule as this has been

best characterized in terms of gene expression and origins. In
Drosophila the most dorsal part of the head capsule can be
subdivided into three domains (Fig. 1B). The most medial
structures are the ocelli or simple eyes and the surrounding
cuticle and bristles. Next to this region, moving outward, is
the mediolateral domain consisting of the parallel ridged cuticle of the dorsal frons. Finally, the most lateral region,
which surrounds the compound eyes, is the orbital cuticle,
which is smooth but bears a number of large bristles at specified locations. Immediately ventral to the ocellar and frons
regions but still dorsal to the antennae is the ptilinum, a region of elastic cuticle. The antennae lie ventral to the ptilinum between the compound eyes. The ventral head capsule
(ventral to the antennae) includes the shingle cuticle and
gena (Fig. 1B).
In both species of stalk-eyed flies, the distance between
the medial ocelli and lateral orbital structures has been expanded dramatically, creating the cylindrical outgrowths or

Fig. 1. Dipteran head morphology. Hypercephaly is absent in D. melanogaster (AB), intermediate in S. beccarri (CE), and greatly exaggerated in C. dalmanni (FH), especially in males (G). The antennae in both species of stalk-eyed fly have moved laterally, compared
to D. melanogaster, and are located adjacent to the eye bulb at the distal end of the eyestalk (E and H). Cuticular markers of different
regions of the head capsule in D. melanogaster are illustrated in (B, adapted from Haynie and Bryant 1986). In the dorsal head ocellar
and orbital regions are characterized by a smooth cuticle bearing a number of large sensory bristles at specific locations. The frons region
between these consists of ridged cuticle with no bristles. Eyestalk cuticle in S. beccarri (E) is smooth but covered in sensory bristles; in
C. dalmanni bristles are widely spaced and arrayed in rows (H). Ant, antenna; oc, ocelli. Scale bars: 1 mm in B and E, 500 m in C, and
100 m in D and G.

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Hurley et al.

stalks from which their name is derived (Figs. 1CH).

Both the eyes and the antennae are displaced laterally at the
ends of these stalks. Cuticle patterning is not overtly conserved between these flies and Drosophila; for example,
there is no equivalent of the ridged frons cuticle (Figs. 1E
and 1H). In S. beccarri the stalks are short compared with
body length (Fig. 1C), and male flies have slightly longer
stalks than females. In C. dalmanni the degree of hypercephaly is much more marked. It is coupled to extreme sexual dimorphism, with female eye span typically being much
shorter than male eye span (Figs. 1F1G).
Comparison of eye-antennal disc morphology
The gross morphologies of stalk-eyed fly and Drosophila
eye-antennal discs are similar. Drosophila eye-antennal
discs can be divided into anterior and posterior portions. On
either side of the posterior portion are two lateral flaps (Fig.
2A). By late third-larval instar, the anterior portion of the
disc is marked by a series of concentric folds and gives rise
to the antennal segments in the adult (Fig. 2B). In the posterior portion of the disc under the lateral flaps, ommatidia
have begun to differentiate and form a regular array behind
the morphogenetic furrow. The dorsal lateral flap gives rise
to the dorsal head capsule, including the ocelli, frons, and orbital cuticle, while ventral head capsule features such as the
shingle cuticle are derived from the ventral lateral flap.
The stalk-eyed fly discs are similarly divided into anterior
and posterior portions. Morphologically the two portions resemble the halves seen in Drosophila discs (Figs. 2C2D).
As in Drosophila, the anterior portion is folded to form a series of concentric folds while the posterior portion is flanked
by two lateral flaps, and differentiating ommatidia and a
morphogenic furrow are visible (data not shown). However,
a third intervening section of disc tissue can be seen in stalkeyed fly discs, which is absent in Drosophila. This intervening tissue is similar in size in both species of stalk-eyed fly
(Figs. 2C2D).
Expression of Dll, en, and wg in eye-antennal discs
We investigated the distribution of DLL and EN protein in
the stalk-eyed fly eye-antennal disc using pan-specific antibodies to perform immunohistochemistry (Patel et al. 1989;
Panganiban et al. 1995). In Drosophila third-instar imaginal
discs, DLL was restricted to the center of the anterior portion
of the disc (Fig. 2B), which gives rise to the most distal second and third segments of the adult antenna (Panganiban et
al. 1997). Consistent with the hypothesis that the anterior
portion of the stalk-eyed fly disc corresponds to the antennal
primordia, central restriction of DLL expression was observed in the anterior portion of the disc in both species of
stalk-eyed fly (Figs. 2C2D).
In Drosophila third-instar discs, EN was detected in a
sector of the anterior portion. In the posterior portion, EN

411

was strongly expressed in a wedge-shaped patch in the dorsal lateral flap (arrow Fig. 3A), which gives rise to medial
head capsule including the ocelli (Royet and Finkelstein
1996). In addition, there was a faint band immediately behind the morphogenetic furrow in the posterior portion of the
disc (Fig. 3A). We examined the expression of EN in stalkeyed fly eye-antennal discs. No difference was observed in
the distribution of EN in the two stalk-eyed fly species discs.
EN protein expression was detected in a sector of the anterior
portion of the discs of both species and at two sites in the
posterior portion, one in the dorsal lateral flap and the other
behind the morphogenetic furrow (Figs. 3B3C). We propose that, as in Drosophila, the EN expression domain in the
dorsal lateral flap marks the medial head primordium.
A pan-specific antibody was not available for wg. To
study the expression of this gene we cloned a partial C. dalmanni wg cDNA by RT-PCR and compared wg expression in
this species and Drosophila via in situ hybridization. In late
third-instar Drosophila, wg transcripts were detected in a
wedge-shaped domain in the anterior portion. In the posterior
portion, wg was expressed along the edge of the ventral lateral flap in the shingle cuticle primordia and in two domains
in the dorsal lateral flap (Fig. 3D). These domains have been
shown to lie on either side of the en patch and to give rise to
the ptilinium and to the orbital cuticle in the lateral head capsule (Royet and Finkelstein 1996). Examination of wg transcripts in the C. dalmanni eye-antennal disc uncovered a similar pattern of wg expression in the anterior portion. Likewise,
the wg expression pattern was conserved along the edge of
the ventral lateral flap of the posterior portion (Figs. 3D3E).
In the dorsal lateral flap, also as in Drosophila, wg transcripts
were localized in two distinct domains separated by a slight
gap (arrows Figs. 3D3E). We propose that, as in Drosophila, one of these domains represents the primordium of the
orbital cuticle (arrowhead in Fig. 3E) and the other the primordium of the ptilinum (pt in Fig. 3E).

DISCUSSION
We compared the adult head morphology and the development of the eye-antennal disc in a pair of diopsid species
with that of Drosophila. The great differences in head morphology relate to an lateralward expansion of the head capsule causing a great increase in the distance between medial
structures such as the ocelli and the orbital cuticle around the
eyes. Cuticle patterning is not conserved between diopsids
and Drosophila. It was, therefore, not possible to determine
which of the domains within the mediolateral axis of the
Drosophila head defined by gene expression had increased
in size or whether all had done so to an equal extent.
There were no obvious morphological differences between
the eye-antennal discs of the two stalk-eyed fly species. Both

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Fig. 2. Eye-antennal imaginal discs
in Drosophila and stalk-eyed flies. In
each case, anterior is uppermost and
dorsal is to the right. (A) Structure
and fate map of D. melanogaster
eye-antennal disc. The disc (left) is
divided into anterior and posterior
portions; the sides of the posterior
portion folded over to form the dorsal and ventral lateral flaps (dotted
lines). The antenna is derived from
the anterior and the eye from the
posterior portion. Head capsule
structures are derived from the lateral flaps and are colored to match the
corresponding adult structure (see
key). In the diagram of the adult
head (right) dorsal is uppermost and
ventral below. (BD) Expression of
Dll in the third-instar eye-antennal
discs of D. melanogaster (B), C. dalmanni (C), and S. beccarri (D) is
shown using pan-specific antibodies
to perform immunohistochemistry.
Note the intervening tissue (brackets) separating the anterior and posterior portions of the discs in the two
stalk-eyed flies.

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413

Fig. 4. Proposed fate map of head region in C. dalmani based on marker gene expression. (A) Schematic diagram of the expression of
Dll (gray), wg (black), and en (pale gray) in the third-instar eye-antennal disc of D. melanogaster and C. dalmanni. Anterior is top and
dorsal to the right in both. In D. melanogaster imaginal discs the dorsal head primordium lies between the en and wg domains (bracketed). The equivalent domain in C. dalmani is marked and appears to be similar in size. (B) Predicted adult derivatives of Dll, wg, and en
expression domains in D. melanogaster and C. dalmani. In D. melanogaster the wg-expressing domain gives rise to orbital cuticle (black)
and the en-expressing domain to ocellar cuticle (pale gray). Note that the domain between the ocelli and orbital cuticle is much larger
(relative to other parts of the head) in C. dalmani (bracketed).

discs are extremely similar in their general structure to the

eye-antennal discs of Drosophila. However, the anterior and
posterior parts of the stalk-eyed fly discs were separated by an
intervening region not seen in Drosophila discs (Fig. 4).
To further probe this difference, we used gene expression
data of three key regulators of Drosophila head development
(Dll, en, and wg) to identify the antenna, eye, and head capsule
primordia within the stalk-eyed fly eye-antennal discs. In the
anterior portion of the stalk-eyed fly discs, expression of Dll in

the central region and en/wg in adjacent sectors matched the expression of the Drosophila homologs in the future antennal region (Fig. 4). En expression in the posterior portion, behind the
morphogenetic furrow, confirmed morphological evidence that
this part of the disc contained the presumptive eye region. Further distinctions within the posterior portion were possible using
Drosophila as a developmental model. The combined pattern of
wg and en in the dorsal lateral flap was consistent with this region giving rise to the dorsal head structures, and the expression

Fig. 3. Expression of en and wg in the third-instar eye-antennal disc of stalk-eyed flies. In each case the anterior portion of the disc is at
the top and dorsal is to the right. (AC) Immunohistochemistry against EN protein is shown in D. melanogaster (A), C. dalmanni (B),
and S. beccarri (C). Note EN expression in the dorsal lateral flap (arrows). In D. melanogaster this region gives rise to the ocellar cuticle
of the medial head capsule. EN is also expressed in a sector of the anterior portion of the disc (ant) and in the morphogenetic furrow
(mf). (DE) In situ hybridization with wg probes is shown in D. melanogaster (D) and C. dalmanni (E). Wg is expressed in three domains
in the dorsal and ventral lateral flaps associated with the posterior portions of both discs. Staining in the central part of the posterior
portion in the C. dalmanni disc (E) is artifactual and also seen with sense probes. In D. melanogaster, one domain (thin arrowheads) of
wg expression in the dorsal lateral flap gives rise to the orbital cuticle and one (pt) to the ptilinum. Expression in the ventral lateral flap
(s) is confined to the shingle cuticle primordium. Note that the intervening tissue in the C. dalmani disc (E) is twisted, such that wg expression in the anterior portion of this disc (ant) appears to occupy a broader domain than in the D. melanogaster disc (D).

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of wg along the edge of the ventral lateral flap suggested that

this part of the disc produces ventral head structures.
The striking similarities in expression of all three marker
genes in stalk-eyed flies and Drosophila was quite unexpected
given the differences in adult head. None of the genes that we
examined were expressed in the intervening tissue, which separates the anterior and posterior parts of stalk-eyed fly discs.
Given that this tissue is of similar size in species with very disparate degrees of hypercephaly, it is unclear what role it plays
in adult eyestalk development. Our results also show that although the eye and antenna are adjacent in the adult, their primordia are physically separated in the eye-antennal imaginal
disc. It is possible that the intervening diopsid-specific tissue
facilitates the bringing together of these two primordia during
metamorphosis, without contributing directly to the adult eyestalk. Fate mapping of the stalk-eyed fly eye-antennal disc is
currently being performed to resolve this issue.
In Drosophila, wg is expressed in the most lateral (orbital) and en in the most medial (ocelli) structures of the dorsal head. The expression of these two genes was therefore
expected to mark the lateral and medial boundaries of the
dorsal head capsule primordium in stalk-eyed flies. It is thus
quite surprising that despite the enormous variation in adult
head size between the three fly species, there is no corresponding difference in the size of the en-wg expression domain in the dorsal lateral flap. One explanation may be that
the growth that gives rise to the expanded head region in diopsids occurs after the stage at which we assayed gene expression (third-instar larvae). We have preliminary evidence
in favor of this hypothesis because development of the exaggerated eyestalks of male C. dalmanni is highly sensitive to
heat shocks applied during the later prepupal stage (Bjorksten et al. 2001). It is also possible that wg and/or en specify
different structures in stalk-eyed flies than they do in Drosophila. To address these issues and further characterize the
genetic basis of eyestalk development, we need to analyze a
wider range of markers during both larval and prepupal development. This work is underway in our laboratory.
Acknowledgments
We thank Gerald Wilkinson for providing S. beccarri, Rick Baker
for sharing his wg data, and Nipam Patel, Grace Panganiban, and
Roger Phillips for generously sharing their reagents. Scanning electron microscopy was performed in the Anatomy Department (UCL)

under the guidance of Mark Turmaine to whom we are indebted.

This work was funded by a NERC studentship awarded to I. H.

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