795

Imperfect eggs and oviform nymphs: a history of ideas about

the origins of insect metamorphosis
Deniz F. Erezyilmaz1
Department of Biology, University of Washington, Box 351800, Seattle, WA 98195-1800, USA

Synopsis The problem of insect metamorphosis has inspired naturalists for centuries. One question that often arises is why
some insects, such as butterflies and bees, undergo a fairly radical metamorphosis while others, such as crickets and lice, do
not. Even before the concept of homology emerged scientists speculated which stage found in more direct-developing insects
would correspond with the pupal stage of metamorphosing insects. William Harvey (1651) considered the pupal stage to be a
continuation of embryonic events, calling it a “second egg.” Since then variations of this idea have emerged over the centuries
of scientific research and have been supported by a wide variety of methods and rationales. This review will follow those ideas
and the ideas that emerged in opposition to them to the present state of the field.

Introduction reduced or absent larval legs. The fate of the larval

Insect metamorphosis is one of the most common yet tissues differs among the holometabolous orders.

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dramatic of biological phenomena, and problems asso-
ciated with it have intrigued naturalists for centuries.
Specifically, many people have speculated about why
insects differ so extensively in the degree of change that
occurs between hatching and sexual maturity. These
different life history strategies are grouped according to
the extent of metatmorphosis into one of three classes:
ametaboly, hemimetaboly, or holometaboly (Fig. 1).
During ametabolous development, a miniature version
of the wingless adult emerges from the egg and simply
increases in size throughout the succeeding immature
(nymphal) stages. These wingless, more basal insects
often continue to molt after they reach sexual maturity.
Hemimetabolous development is also largely direct,
except that in these winged orders, external wing pri-
mordia grow throughout the nymphal stages, and the
wings and genitalia emerge at the final adult molt. In
contrast, the immature stages of holometabolous
(completely metamorphosing) insects are often widely
divergent from the adult form. The immature stages,
here called larvae, of the Holometabola undergo a
radical metamorphosis by passing from the larval to
the pupal stage (a process called pupal development),
and then from the pupal to the adult stage (adult
development). Larvae of holometabolous insects are
distinguished from the nymphs of hemimetabolous
insects by the presence of internally developing
wings, the presence of modified larval eyes, and
In some groups, larval tissues are degraded completely.
In others, the same tissue contributes to the larval,
pupal, and adult forms. The imaginal tissues destined
to give rise to the pupal and adult structures may be set
aside as early as embryogenesis, or created de novo at
metamorphosis.
Complete metamorphosis in insects arose once
from hemimetabolous ancestors, during the Permian
(Kukalova-Peck 1991; Labandeira and Phillips 1996).
In the ensuing
285 million years, the Holometabola
have radiated so extensively that holometabolous insect
species now account for 45–60% of all living organisms
(Hammond 1992). This explosion can be attributed to
the innovation whereby one insect is able to produce
more than one form. By decoupling larval and adult
development, larvae and adults were able to occupy
separate ecological niches (Kukalova-Peck 1991). In
addition, internalization of the developing wing
primordia allowed burrowing of the vermiform
(worm-shaped) larvae, and these juveniles were then
able to live inside ephemeral food sources, such as
fruits and detritus (Truman and Riddiford 1999).
Modern explanations for the evolution of holo-
metabolous metamorophosis from hemimetabolous
ancestors date back to the seventeenth century.
William Harvey (1651) considered the pupal stage to
be a continuation of embryonic events. Variations of
this argument of metamorphosis as a heterochrony

From the symposium “Metamorphosis: A Multikingdom Approach” presented at the annual meeting of the Society for Integrative and
Comparative Biology, January 4–8, 2006, Orlando, Florida.
1 E-mail: denizere@u.washington.edu

Integrative and Comparative Biology, volume 46, number 6, pp. 795–807

doi:10.1093/icb/icl033
Advance Access publication August 24, 2006
Ó 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecom-
mons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
796 D. F. Erezyilmaz

E E E
N1 L1
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A A
A

Ametaboly Hemimetaboly Hemimetaboly

(no metamorphosis) (partial metamorphosis) (complete metamorphosis)

Fig. 1 A figure adapted from a review by Sehnal and colleagues (1996). In ametabolous development, the immature stages
simply increase in size to produce the adult. Unlike other types of insect development, these insects, represented here
by a silverfish, continue to molt once they have become sexually mature (arrow). The immature stages (called nymphs)
of hemimetabolous insects resemble the ultimate adult, except that wings and genitalia are acquired at the final molt.
With holometaboly, or complete metamorphosis, the form of the larval stages often differs dramatically from that of the
adult. The group is represented here by a snake fly, with the pupal stage circled. E ¼ embryo; L ¼ larva; N ¼ nymph;
P ¼ pupa; and A ¼ adult. The numbers associated with these letters indicate the instar (duration between molts) (for
example, N1 is the first nymphal instar).

have arisen over the centuries of scientific research and
have been justified by a wide variety of methods. In this
essay, I will discuss the original ideas on the origins of
complete metamorphosis of insects, with the hope that
this will stimulate and focus future research. I will next
discuss current studies that use modern techniques to
address the origin of metamorphosis. Finally, I will briefly
suggest some future directions for studies of this topic.
Imperfect eggs
William Harvey is best known to modern science for
his 1628 work, An Anatomical Disquisition on the
Motion of the Heart and Blood in Animals, which
demonstrated, using observations and experiments,
that blood flows and that its movement is driven by
the heart. Harvey also completed a second book,
Disputations Touching the Generation of Animals,
which was published in 1651. The frontispiece of the
1651 edition depicts Zeus opening an egg to release a
spectrum of creatures. Significantly, 2 insects are
included, a grasshopper and a butterfly. In the 62nd
exercise, That an egg is the common original of all ani-
mals, Harvey disagreed with Aristotle by stating: “all
animals produce another animal either actually or
potentially. Those animals which produce another in
actuality are called viviparous, those which produce in
potentiality are oviparous.” To these 2 classes, however,
Aristotle counted a third group, the vermiparous,
which arise as worms spontaneously from putrescence
(Aristotle 322 BC; Harvey 1651). In defense of the
oviparous origins of caterpillars and maggots,
Harvey (1651) described Aristotle’s “worms” as the
product of imperfect eggs: “Imperfect eggs we call
those which are thrust out while they are immature
and have not yet reached their full size but continue to
grow outside the womb after they have been laid . . . in
this class should be included the primordia of insects,
which Aristotle calls worms.” Imperfect eggs produced
creatures that are intermediate between imperfect and
perfect creatures, Harvey (1651) argued:
Because in comparison with its own egg or primor-
dium, it is an animal endowed with sense and motion
that nourishes itself, but in comparison with the fly or
butterfly whose primordium exists in potentia, it is to
be accounted no more than a crawling egg, itself
Imperfect eggs and oviform nymphs
providing for its own growth. Such is a caterpillar
which, having acquired its proper size, is changed
into a chrysalis or a perfected egg, and ceasing to
move is, like an egg, an animal in potentia.
This idea that the larval state is a “crawling egg,” and
the pupal stage was a “second egg” was borrowed from
Aristotle (322 BC). In his argument, however, Harvey
articulated a new concept of insect life history and
development; larvae of metamorphosing insects were
thrust out of the egg “imperfect,” and were forced to
continue ontogenesis in a second period of develop-
ment that was separated from growth, the pupal stage.
This essay will follow the “second egg” idea through the
centuries of scientific discovery and examine the the-
ories that emerged in opposition to it.
Oviform nymphs
“His dissertation contains almost as many errors as
words,” wrote Jan Swammerdam (1669) of Harvey’s
“second egg” hypothesis, just 18 years after the
publication of The Generation of Animals. What
Swammerdam objected to was the concept of the chry-
salis, or pupa, as an egg that would produce one animal
from the matter of another. Instead, Swammerdam
considered the pupa to be a form of nymph. He wrote:
Those eggs, wherein the animalcules lie still without
food, in the figure of Nymphs, and which for that
reason, often have the form of the animalcules that are
to proceed from them, ought not to be called eggs, but
Nymphs in the form of eggs, or oviform Nymphs.
In this section, Swammerdam directly assaulted
Harvey’s conception of the pupa as an egg as super-
ficial, relying on the shape of the pupal case instead of
its contents, which he considered to be nymphal.
Swammerdam (1669) used the concept of the
nymph that was coined by Aristotle as the point
when insects “have received the outlines of shape
they are afterwards to wear.” That is, their adult dimen-
sions. Swammerdam furthermore considered the nym-
phal state to be the ground or foundation of insect
life. Insects, he argued, simply differed in the point
of development when the nymphal state was attained.
With this, he arranged insect life histories into 4 orders
that were based on the extent of metamorphosis,
roughly corresponding to ametaboly, hemimetaboly,
holometaboly (Fig. 1), and in the most extreme
order, a derived form of holometaboly found in
Dipterans. In the first order, exemplified by the
louse, the nymph is born perfect, in its final form,
and simply grows by “accretion” to produce the
adult. These nymphs that are born perfect, he called
797
“Nymph-Animals.” Insects that are born in the shape
of their parents, but are imperfect, or deficient in
regard to their wings belong to the second order,
which includes such insects as mayflies and crickets.
Swammerdam (1669) labeled the postembryonic
immature stages of this insect, “the Nymph-vermicle;
for the little creature, whilst it is and remains a real
Vermicle or Worm, has notwithstanding some of its
parts disposed and in an admirable manner beautifully
composed, just as they are in the Nymph state.”
In contrast, in insects that undergo complete metamor-
phosis, such as butterflies and bees, the nymphal state is
attained during pupal development, by the “Nymph-
Chrysalis” (Fig. 2). Finally, Swammerdam placed the
flies, which use their larval cuticle to form a pupal case,
or puparium, in the 4th order, where, like the Chrysalis
of the third order, the nymph is not created until the
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pupal stage. Because pupation occurs within the worm-
like larval case of the maggot, Swammerdam labeled
these immatures as “Vermiform Nymphs.”
Swammerdam’s equation of the pupal stage with the
nymph was based on his own extensive descriptions of
the imaginal tissues of the moth, which showed that the
primordia of the moth could be discerned as early as
the larval stages beneath the skin of the caterpillar
(Fig. 2). Unlike Harvey, who considered the pupal
stage to be an egg state during which one shape was
transformed to another, Swammerdam (1669) consid-
ered the production of the adult to be more continuous:
The wings, horns and other parts which worms with-
out legs seem to acquire about their chests, at the time
of their mutation, are not truly produced, during the
period of mutation, or, to speak more agreeably to
truth during the time of limbs shooting or budding
out; but that they have grown there by degrees under
the skin, and as the worm itself has grown by a kind of
accretion of parts, and will make their appearance in it
upon breaking the skin on its head or its back and
thereby give it the figure of a Nymph.
In revealing that imaginal development is not con-
fined to the immobile stage of the “second egg,”
Swammerdam discounted the notion that events that
occur in the pupal stage correspond to events that
occur in eggs.
Embryonic metamorphoses
“The metamorphoses of insects,” wrote the naturalist
Sir John Lubbock in 1883, “have always seemed to me
one of the greatest difficulties of the Darwinian
theory.” The problem for Lubbock was that, according
to accepted wisdom of the day, animals related by
common descent should resemble each other more
798 D. F. Erezyilmaz

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Fig. 2 Drawings of bee pupae from Swammerdam’s 1669 publication (1978 reprint), The Book of Nature or, the History of
Insects. (A) An intact pupa. a ¼ the head; b ¼ eyes; c ¼ horns or antennae; d is “the lip;” e ¼ “teeth or jaw bones;” f ¼
first pair of joints on the proboscis; h ¼ proboscis; i ¼ the first pair of legs; k ¼ “transparent stiff little parts;” l ¼ the
second pair of legs; m ¼ the wings; n ¼ “blade bones;” o ¼ last pair of legs; p ¼ “abdominal rings;” q ¼ “the hinder parts
of the body;” r ¼ “2 little parts accompanying the sting;” s ¼ the anus. (B) A ventral view of a bee pupa with the
thoracic pupal cuticle removed and the imaginal tissues pulled away. The original figure legend reads, “The worm of the
Bee, on the point of changing to a Nymph, and stripped of its skin, the better to show the infant parts of the future
Bee, which are here represented as they appear through the microscope, after extending them a little. aa: the antennae
or horns. b: the proboscis, with its parts. cc: the second pair of joints belonging to, or forming, the proboscis. dd: the
first pair. ee: the first pair of legs, lying against the breast. ff: the second pair of legs. gg: the third pair. hh: the greater
wings. ii: the smaller wings. k: the abdominal wings.” Reprinted from F Sehnal, P Svacha, and J Zrzavy, Evolution of insect
metamorphosis, in LI Gilbert, JR Tata, BG Atkinson (eds.), Metamorphosis: Postembryonic Reprogramming of Gene
Expression in Amphibian and Insect Cells. San Diego: Academic Press, p. 3–58, with permission from Elsevier.

closely in their immature stages. Only with further
ontogeny, the peculiarities of the species would appear
[Darwin (1859), however, did address exceptions to the
rule of embryonic resemblance in On the Origin of
Species. For instance: “The case, however, is different
when an animal during any part of its embryonic career
is active, and has to provide for itself. The period of
activity may come on earlier or later in life; but when-
ever it comes on, the adaptation of the larva to its
conditions of life is just as perfect and as beautiful
as in the adult animal. From such special adaptations,
the similarity of the larvae or active embryos of allied
animals is sometimes much obscured.”]. Insects defy
this, as Lubbock (1883) put it: “In most cases, the
development of the individual reproduces to a certain
extent that of the race; but the motionless, imbecile
pupa cannot represent a mature form.”
Exceptions to the doctrine of increasing disparity
accumulated. A corollary of this theory was that,
since traits proceeded from the general to the species-
specific, classification of organisms should agree in their
developmental history, with organisms that share an
increasing duration of developmental similarity
grouped together. Four years after Darwin published
the Origin of Species, the German naturalist, Fritz Muller
(1869) published a small book, Facts and Arguments
for Darwin. The premise of that book was to attempt
to apply Darwin’s theory to one particular group, the
Crustacea, and to form a picture of their ancestry. On
the classification of Crustacea, Muller summarily
rejected the notion that development of an individual
proceeds from the general type to the species-specific:
I cannot but think that we can scarcely speak of a
general plan, or typical mode of development of the
Crustacea, differentiated according to the separate
Sections, Orders, and Families, when for example,
among the Macrura, the River Crayfish leaves the
egg in its permanent form; the Lobster with
Schizopodal feet; Palemon, like the Crabs, as a Zoea;
and Peneus, like the Cirripedes, as a Nauplius,—and
when, still within this same sub-order Macrura,
Palinurus, Mysis and Euphausia again present
different young forms.
Imperfect eggs and oviform nymphs
Fig. 3 Summary of various theories illustrating the
homology of hemimetabolous stages and
holometabolous stages. Adapted from Gillot (1995).
hemi ¼ hemimetabolous life history; holo ¼
holometabolous life history; E ¼ embryo; N ¼ nymph; L
¼ larva; P ¼ pupa; A ¼ adult. (A–C) De-embryonization
theories. Harvey (1651) considered the pupal stage to
be a continuation of embryonic events. Berlese’s (1913)
theory subdivided the embryonic stages into 3 periods
of increasing development; the abdomen-less protopod
(Eproto), the differentiating polypod (Epoly), and the
terminally differentiating oligopod (Eoligo). Truman and
Riddiford (1999) subdivided embryonic development
according to embryonic molts. E1 ¼ the embryonic stage
bearing the first embryonic cuticle. EPro-N ¼ pronymphal
stage, bearing the pronymphal cuticle EN ¼ first nymphal
stage, produced just before hatching. (D) Poyarkoff
(1914a, b) suggested that the pupal stage arose through
subdivision of the imaginal stage. (E) Despite evidence
that the Holometabola evolved from hemimetabolous
insects, Heslop-Harrison (1958) believed that both
groups evolved from an ancestor containing both larval
and nymphal stages. (F) Hinton (1963) considered the
pupa to be homologous to the last stage nymph of
hemimetabolous insects.
The problem in grouping the Crustacea according to
the dogma of increasing diversity is that, like insects,
the greatest diversity is often seen in the immature
stages (Fig. 3). The issue of developmental history
and shared ancestry for Muller was whether variation
occurs by deviating from the ancestral form sooner, or
by passing through the ancestral forms and pass them
to a new morphology. When the latter case occurs, one
799
sees a record of historical transformations. However, as
Muller (1869) stated:
The historical record preserved in developmental
history is gradually EFFACED as the development
strikes into a constantly straighter course from the
egg to the perfect animal, and it is frequently
SOPHISTICATED by the struggle for existence
which the free-living larvae have to undergo.
Therefore, the effects of selection upon immature
stages will affect embryonic development, and thereby
obscure the developmental relationship of members of
the same group that encounter different circumstances
upon hatching.
By 1869, the fossil record had shown that hemime-
tabolous insects arose before metamorphosing insects,
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demonstrating that metamorphosis was acquired, not
inherited in the class (Muller 1869). Instead of focusing
on the pupal stage, Lubbock (1883) considered the
origin of the holometabolous larva. For Lubbock, the
problem insect metamorphosis posed to Darwinism
was that the immature stages of Holometabola, like
many Crustacea, bore less resemblance to their hemi-
metabolous ancestors than did the mature stages.
Lubbock’s resolution of this seeming paradox is derived
from Fritz Muller’s conclusions from the Crustacea. The
result was a rephrasing of William Harvey’s notion of
interrupted ontogeny couched in the terms of natural
selection. Lubbock (1883) stated: “there are, no doubt,
cases in which the earlier states are rapidly passed
through, or but obscurely indicated . . . either before
or after birth, animals undergo metamorphosis.”
Lubbock considered hemimetabolous insects, like
crickets, to pass through this “metamorphosis” in the
egg. Metamorphosing insects, however, would hatch
prior to the “embryonic metamorphosis.” Since acquir-
ing this mode of development, Lubbock reasoned, the
morphology of larvae have evolved so that “the larva
of an insect is by no means a mere stage in the devel-
opment of the perfect animal. On the contrary, it is
subject to the influence of natural selection and under-
goes changes which have reference entirely to its own
requirements and condition” (Fig. 4). Therefore,
Lubbock considered the holometabolous larva to be a
less-developed version of its ancestor that, once it
became free-living, was subject to intense selection
that would obscure its expected resemblance to the
hemimetabolous ancestor (Lubbock 1883).
The morphologists
In the twentieth century, aspects of Harvey’s second
egg hypothesis were popularized by the work of
800 D. F. Erezyilmaz

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Fig. 4 The hemimetabolous and holometabolous adults (Plate 1) matched with their immature stages in Plate 2. This
figure is originally from J. O. Westwood’s Introduction to the Modern Classification of Insects, but Lubbock (1883) used
these plates in a series of articles in Nature entitled, On the Origin and Metamorphoses of Insects, in which he discussed the
suitability of larval forms to their habitat. Plate 1: 1 ¼ cricket; 2 ¼ earwig; 3 ¼ Aphis; 4 ¼ Scolytus; 5 ¼ Anthrax;
6 ¼ Balanius; 7 ¼ Cynips; 8 ¼ Ant; 9 ¼ Wasp. Plate 2: ? ¼ cricket nymph; 2 ¼ Aphis nymph; 3 ¼ earwig nymph;
4 ¼ Scotylus larva; 5 ¼ Anthrax larva; 6 ¼ Balanius larva; 7 ¼ larva of Cynips; 8 ¼ ant larva; 9 ¼ wasp larva.

Antonin Berlese (1913). Berlese was an insect morphol-
ogist, and his ideas on the origin of the pupal stage
from direct developing ancestors are expressed in
English by Imms (1931). Berlese’s theory inspired
many opposing theories that focused on the homology
of the pupal stage to a corresponding stage of the
hemimetabolous ancestor (Fig. 3). These were often
derived from detailed studies of various tissues, as in
the case of Poyarkoff (1914a, b), Henson (1946), and
Hinton (1963).
Berlese (1913) proposed that the larval stage arose
after the developmental events of embryogenesis were
transposed to postembryonic life (Imms 1931). This
“de-embryonization” forced nymphal development to
occur later in life, compressing it into the pupal stage.
According to him, embryonic development progresses
through 3 phases: the protopod, polypod, and oligopod
stages. The protopod stage occurs prior to any differ-
entiation, when the head and thorax are segmented, but
the abdomen is still unsegmented. During the polypod
stage, the abdomen has completed segmentation, and
each “somite” bears a pair of rudimentary limbs. The
tracheal, nervous, and circulatory systems have also
completely formed. Further differentiation occurs
during the oligopod phase, and the abdominal appen-
dages undergo resorption (Imms 1931). In Berlese’s
view, the point in this ontogeny when embryos
hatch will determine their life history strategy.
Hemimetabolous insects pass through all 3 stages in
the embryo and hatch in the postoligopod stage. Most
holometabolous insects, however, hatch in the polypod
or oligopod stages, and therefore resume ontogenesis
during the pupal stage. For instance, the caterpillars
of Lepidoptera hatch in the polypod stage, Berlese’s
theory reasons, as the larvae possess a tracheal system,
but still retain abdominal appendages. The larvae of
Coleoptera and Neuroptera are examples of insects
that emerge from the egg during the oligopod stage.
Berlese (and Imms) considered dipteran larvae to be
derived oligopod larvae and so have secondarily lost
their limbs. As an example of a protopod larva,
Imms pointed to the endoparasitic larvae of some
Hymenoptera. These, he suggested, are little more
than a head and thorax with rudimentary abdomen,
forced to leave the egg before segmentation could be
completed (Imms 1931).
Harvey’s idea that the pupal stage is related to the
eggs of more “perfect” animals also appears in the
repetition theory of Henson (1946). Unlike the
Lubbock/Imms/Berlese concept, however, in which
the holometabolous pupal stage is considered to be a
continuation of interrupted embryonic events, Henson
considered pupal development to be a repetition of
embryonic development. Like Berlese, Lubbock, and
Imms, Henson was a morphologist, and his ideas on
embryonic repetition were derived from his knowledge
Imperfect eggs and oviform nymphs
of insect gut development. During embryonic devel-
opment, the gut arises after 2 invaginations, the future
stomodeum and proctodeum move posteriorly and
anteriorly, respectively, towards the center of the blas-
tula. After invaginating partway across the blastula, the
midgut is formed from mesenchymal cells that meet at
the center of the embryo. At the junctures of the mature
midgut with the foregut and with the hindgut respec-
tively, lie the anterior and posterior imaginal rings. At
metamorphosis, the anterior and posterior imaginal
rings proliferate rapidly and grow around the larval
midgut. In Henson’s (1946) view, activation of these
rings is a return to an embryonic condition, and the
anterior rings are interpreted as, “the resuscitated
embryonic blastopore’’ (Henson 1946). Therefore,
Henson regarded the process of midgut formation dur-
ing metamorphosis to be an embryonic process. In fact,
Henson considered each molt to be a continuation of
embryonic development in which morphogenesis was
suppressed in differing degrees. The metamorphic molt
was suppressed the least, allowing a complete repetition
of embryonic development.
A central feature of the Lubbock/Berlese/Imms view of
the evolution of the pupal stage is the idea that devel-
opmental stages can be compressed, or collapsed into
one stage, while others can be expanded or reiterated.
Another variation of this idea appeared in 1958. Heslop-
Harrison (1958), like Berlese, proposed that the pupal
stage arose through compression of the equivalent nym-
phal stages of the ancestral insect (Fig. 3E). Like Berlese,
Heslop-Harrison considered the holometabolous larval
stage to be an earlier stage of ontogeny, rather than one
equivalent to the hemimetabolous nymph. His concept
of the holometabolous ancestor, however, differed from
other theories. Heslop-Harrison believed that both
hemimetabolous and holometabolous strategies arose
by modification of a primitive insect life cycle that con-
tained both polypodous and oligopodous postembryo-
nic stages. That is, the ancestral insect would hatch to
Holometabolous-like larval stages (polypodous) and
then pass to hemimetabolous nymphal stages (oligopo-
dous). The hemimetabolous insects would arise by
embryonization of the larval stages, while the
Holometabola arose by compression of the nymphal
stages into the prepupal and pupal stadium (Heslop-
Harrison 1958; Sehnal and colleagues 1996).
In contrast to Berlese’s and Henson’s reinterpreta-
tion of Harvey’s second egg idea, the theory that gained
the most credence and has remained unchallenged
until recently was conceived by H. E. Hinton (1959,
1963). According to Hinton, the pupal stage is merely a
derived final stage nymph that bridges a developmental
gap between an increasingly divergent larval stage and a
relatively conserved adult morphology. It is a theory
801
written in opposition to 2 previous theories; first
Berlese’s and then Poyarkoff’s. Initially, Hinton was
a proponent of the Poyarkoff theory, and he claimed
responsibility for raising Poyarkoff’s 1914 papers on
the subject from obscurity. Poyarkoff’s ideas are also
in opposition to the “second egg” hypothesis, but in
this case, Poyarkoff held that the pupal stage arose after
a division of the imaginal (adult) stage into two instars
(Fig. 3D). As Hinton would later do, Poyarkoff came to
his conclusions by following the fate of skeletal muscles
through metamorphosis. In insects, the skeletal
muscles are attached to the cuticle by means of tonofi-
brillae. As Hinton wrote in his 1948 paper in support of
Poyarkoff’s theory, these tonofibrillae appear as the
cuticle is being formed. Because many muscles in
the adult are not present in the larval stages, Hinton
reasoned, these muscles, necessary for locomotion,
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must be formed de novo at the intervening molt. As
Hinton explained, however, the muscle growth must be
guided so that the correct spatial relations are attained.
Therefore, according to Hinton (1948) the Poyarkoff
theory states: “A mould of the same spatial relations as
in the adult is therefore required. This mould is the
pupa.” That is, the pupal cuticle is required to produce
the approximate dimensions of the adult to guide
muscle development. Once the muscle fibers reach
their approximate dimensions, they are then able to
attach to the newly forming adult cuticle. As evidence
for this ‘‘mould’’ constraint, Poyarkoff pointed to the
situation in Ephemeroptera, basal winged insects with
a subimaginal stage that is distinct in morphology
from either larval or adult forms. Poyarkoff cited a
paper by Durken (1907) who showed that some of
the muscles of the adult form are unstriated in the
subimago, but are functional during the adult stage.
From this Poyarkoff (through Hinton 1948) concluded
that the muscles become striated in the subimago, but
are not able to attach to the cuticle until the molt to the
imaginal stage.
Having endorsed the skeletal-muscle based view of
the Poyarkoff theory in which the pupal cuticle serves
as a “mould” for skeletal muscle development, Hinton
(1963) set out to demonstrate the “mould” constraint.
As he stated: “a study was made of the metamorphosis
of the skeletal muscles of several species. The result of
this work was unexpected; it showed that the theory
was untenable.” Essentially, what Hinton had to find
was that the imaginal skeletal muscles arise only during
pupal development. To do this he focused on the indir-
ect flight muscles. In general, these do arise during the
pupal stage; in most Diptera they appear at pupation
from myoblasts that adhere to muscle fibers, whereas in
Neuroptera and Coleoptera, larval muscle is directly
transformed into adult muscle during the early pharate
802
pupal stage. In the Simuliidae (Diptera), however,
Hinton discovered that the indirect flight muscles
arise very early in postembryonic development.
Instead of requiring a “mould” for development, the
muscles appear as thin strands that are attached to
the epidermis, rather than to tonofibrillae, and grow
independently of the larval muscles. The strands grow
with the larva, and appear to proliferate with the epi-
dermis at molts. At the larval/pupal molt, the strands
proliferate rapidly and divide to produce the entire
assemblage of flight muscles (Hinton 1959). By show-
ing that the flight muscles may arise during larval life,
Hinton felt this demonstrated that the pupal stage did
not arise out of an expansion of adult development, as
Poyarkoff had proposed (Hinton 1963).
Once he had shown that the skeletal muscles are able
to arise without a mould to guide development, Hinton
turned to the development of wings to shed light upon
the origin of the pupal stage. The internalization of
wings in the endopterygotes, argued Hinton (1963),
which allowed burrowing larvae to arise, created new
constraints upon the thorax. “The growth of the wing
anlage after the larval–pupal moult” he wrote, “is
enormous.” Moreover, after the larval–pupal molt,
“the volume of both the wings and the imaginal tissues
increases enormously. The indirect flight muscles are
destined to occupy almost the entire space in the
thorax, and there is simply no room in the thorax
for both developing wings and adult muscles.”
Therefore, Hinton reasoned, two molts are required
to produce a winged adult from a crawling larval
stage; one to evaginate the wings from the larval thorax,
and a second to free the wings from the pupal cuticle,
allowing further growth (Fig. 3F). As evidence for this,
he cited the situation in some larviform beetles in
which the female adults have become apterous. In
these females, the pupal stage is lost (Hinton 1963).
Endocrine-based theories
In 1934, V. B. Wigglesworth showed, by parabiosing a
5th stage nymph of the blood-sucking bug, Rhodnius to
a first-stage nymph, that a circulating factor regulated
the transition from nymph to the adult stage, as the 1st
instar molted to a miniature adult (Wigglesworth
1934). Then, in 1936 he showed that the source of
this factor were the corpora allata, as an allatum
taken from a 4th stage nymph could prevent meta-
morphosis when implanted into the abdomen of a
5th stage nymph, producing instead a supernumerary
6th stage nymph (Wigglesworth 1936). Subsequently,
the allatum factor, which was later named juvenile
hormone, or JH, was isolated from both hemimetabo-
lous and holometabolous insects, and was found to
D. F. Erezyilmaz
regulate complete metamorphosis as well (Piepho
1942, 1946; Williams 1956). These experiments chan-
ged the debate on the origins of insect metamorphosis
so that, instead of addressing the problem by compar-
ing the development of a tissue or structure between
hemimetabolous and holometabolous insects, the fac-
tors that regulate the process were now compared.
There is no “Wigglesworth theory” on the origin of
insect metamorphosis. Instead, Wigglesworth down-
played the differences between hemimetabolous and
holometabolous insects. Because he saw unity in the
mechanisms that regulate hemimetabolous and holo-
metabolous metamorphosis between the 2 strategies of
insects, Wigglesworth considered nymphs and larvae to
be polymorphisms of the same ontogenic stage, which
differed in appearance simply because they were
adapted to different environments. “The origin of
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metamorphosis is to be sought,” he stated “in the
divergent evolution of a polymorphic organism”
(Wigglesworth 1954). The mechanisms that were in
place in the hemimetabolous ancestors and regulated
their partial metamorphosis were simply exaggerated
in the Holometabola. Moreover, he considered
Lubbock’s conception of metamorphosis as a shift in
ontogeny to be an intellectual relic of the nineteenth
century, when the theory of recapitulation had great
influence. Instead, Wigglesworth believed, experiments
that showed that damage to holometabolous embryos
could affect either the larval or the imaginal stages,
depending upon the embryonic stage that was damaged
(Giegy 1931), demonstrated that both forms, larval and
imaginal, exist during embryogenesis. The role of JH
then was to suppress imaginal or adult differentiation
until metamorphosis (Wigglesworth 1954).
Despite Wigglesworth’s criticisms of the Harvey/
Lubbock/Berlese concept of metamorphosis, new the-
ories that incorporated JH into Berlese’s hypothesis
emerged. The Czech insect physiologist, Novak pro-
posed that earlier production of JH during embryonic
development caused de-embryonization of the oligo-
pod or polypod (the differentiating stages of embryonic
development) to create holometabolous larvae (Novak
1966). Novak distinguished 2 types of growth: gradient
growth, which occurs during embryonic development
and metamorphosis, and non-gradient or isometric
growth that occurs during the larval or nymphal stages.
JH, Novak argued, prevents gradient growth but pro-
motes isometric growth. While JH is first produced
during embryonic development in all insects, the cor-
pora allata appear earlier in holometabolous embryos.
Given the early appearance of the gland, JH production
could appear as early as the protopod stage. Once JH
levels had reached threshold levels, Novak proposed,
isometric growth would cause the polypod or oligopod
Imperfect eggs and oviform nymphs
form to be reiterated through postembryonic life until
JH levels declined at metamorphosis, and gradient
growth could resume. The difference between the
two groups, therefore, would be the amount of gradient
growth accomplished by the time of JH appearance
(Novak 1966).
In contrast to Berlese and Lubbock who saw the time
of hatching to be the event that interrupted the pro-
gression of embryonic development, Novak considered
the onset of JH production to be the event that halted
ontogenesis. This view was shared by Truman and
Riddiford (1999), and they expanded it into the pro-
nymph hypothesis. This idea is centered on the pro-
nymph (the first postembryonic stage of basal insects
and some other arthropods), which differs in morphol-
ogy from subsequent immature stages (Truman and
Riddiford 1999). In hemimetabolous insects, from
which the Holometabola are derived, the pronymphal
stage has become embryonized. In the Holometabola,
however, it was proposed that the pronymph has
become de-embryonized after JH production
encroached into earlier stages of embryonic develop-
ment, creating the holometabolous larval stage
(Truman and Riddiford 1999, 2002; Erezyilmaz and
others 2004a, b).
Evidence for the homology of the larval and pro-
nymphal stages comes from the degree of develop-
ment shared by the 2 forms. Truman and Riddiford
(1999, 2002) pointed to a set of early-born neurons in
the pronymph of grasshoppers that are contained
within each segment and appendage. A homologous
set is found in newly hatched larvae of the moth,
Manduca sexta, and the fly, Drosophila melanogaster.
In addition, they pointed out, the cuticle of the pro-
nymph is soft and lacks sclerites, like the cuticle of most
holometabolous larvae. Lastly, for both the pronymph
and the holometabous larvae, these cuticles are the
second embryonic cuticle produced and they form
just after dorsal closure, whereas the nymphal cuticle
is the third cuticle (Truman and Riddiford 1999). Data
that contradict this hypothesis have recently emerged
from electron microscopy of embryonic cuticle forma-
tion (Ziese and Dorn 2003; Konopova and Zrzavy
2005). Whereas light microscopy had indicated only
2 embryonic “cuticles,” transmission electron micro-
graphs revealed a second layer that is produced at a
time commensurate with pronymphal cuticle forma-
tion in hemimetabolous insects. Ziese and Dorn (2003)
and Konopova and Zrzavy (2005) argued that the pro-
nymphal cuticle was reduced, not de-embryonized as
Truman and Riddiford proposed. One way to determine
whether this layer is a vestigial cuticle or simply a
membrane would be to measure levels of the ecdyster-
oid hormones that trigger formation and lifting of
803
cuticles. If ecdysteroid levels rise at formation of the
second layer, this would strengthen the argument that
the layer is a remnant of the pronymphal cuticle.
Larval patterning
Instead of simply focusing on the development of spe-
cific tissues during embryonic and metamorphic devel-
opment, two developmental studies have compared the
expression of genes that pattern the adult and larval
organs to address the origin of metamorphic develop-
ment. To do this, both groups have focused on the
point in embryogenesis when hemimetabolous and
holometabolous development diverge. These authors
have analyzed the deployment of batteries of genes
that are required for patterning the adult eye
(Friedrich and Benzer 2000; Liu and Friedrich 2004)
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or leg (Tanaka and Truman 2004). What both groups
have found is that, although the ancestral patterning
programs are conserved during imaginal development,
progression of these programs are interrupted to pro-
duce the larval structure.
Patterning of the larval leg in the
tobacco hornworm
The specification of the proximal–distal (P–D) axis in
the insect leg was first described in genetic studies of
the fruit fly, Drosophila melanogaster (for review see
Kojima 2004). In this insect, the adult leg is formed
from imaginal cells that are set aside during embryo-
genesis and develop as an imaginal disc, a collapsed
epithelial sheet that develops inside the larva.
Patterning of the disc begins during the larval stages.
Briefly, the leg axis, which is composed of 5 segments,
(from proximal to distal), the coxa, trochanter, femur,
tibia, and tarsus, is determined by the activity of
2 morphogens, Wingless (Wg) and Decapentaplegic
(Dpp) (Lecuit and Cohen 1997; Wu and Cohen
1999). In the early leg disc, Wg is produced in the
ventral portion of the disc, while Dpp is expressed
in a dorsal stripe. The high, combined activity of
these two secreted factors activates expression of the
transcription factor Distal-less (Dll) in the center of the
disc (Cohen 1993; Diaz-Benjumea and others 1994).
Somewhat lower levels of the combined Wg/Dpp mor-
phogens activate the expression of dacshund (dac) in a
ring proximal to the Dll domain (Lecuit and Cohen
1997). Dll in turn activates expression of 2 subsequent
transcription factors: Arista-less (Al) in the most distal
domain, and Bric-a-brac (Bab) just proximal to the al
domain (Campbell and Tomlinson 1998). Correct
bab expression is required to subdivide the tarsus
into 5 segments, as bab mutants are missing tarsal
segments (Godt and others 1993). Initially, the protein
804
is found throughout the presumptive tarsus, but later
resolves into domains of higher expression that corre-
spond to tarsal segments (Godt and others 1993).
The most proximal region of the disc is determined
by two transcription factors: Homothorax (HTH) and
Extradenticle (EXD). Although Exd is ubiquitously
expressed, the presence of its cofactor, HTH in the
most proximal regions of the disc allows nuclear trans-
location and EXD function (Abu-Shaar and Mann
1998). The P–D expression pattern of these genes is
essentially conserved in the imaginal leg of the hawk-
moth Manduca, a more basal holometabolous insect
that produces the adult leg from a subset of the larval
leg epidermis (Tanaka and Truman 2004, 2006).
Studies of leg P–D axis specification in hemimeta-
bolous embryos has revealed that, although the axis
may not be established through a Wg/Dpp gradient,
limb development also converges upon a ground plan
whereby Exd, Dll, Dac, Bab, and Al are expressed and
function as they do in the Drosophila leg disc. (Jockusch
and others 2000; Inoue and others 2002; Niwa and
others 2000; Rogers and others 2002; Angelini and
Kaufman 2004; Erezyilmaz and others 2004a). For
instance, in the milkweed bug, loss of Wg signaling
through RNAi was unable to affect limb patterning,
but RNAi knockdown of Dll and Dac caused loss or
truncation of the tarsus and tibia, respectively
(Angelini and Kaufman 2004, 2005).
Given that a basic ground plan, with the P/D axis
patterned by Exd/Hth, Dll, Dac, Bab, and Al is con-
served between imaginal legs of holometabolous insects
and embryonic legs of hemimetabolous insects, Tanaka
and Truman (2006) asked whether the genetic ground
plan was altered, thereby producing the morphologi-
cally divergent larval leg of Manduca. As with other
holometabolous insects, the larval leg of Manduca has
only 1 segment within the tarsus, the distal-most seg-
ment of the leg. The adult tarsus, however, is subdi-
vided into 4 segments. Since most hemimetabolous
insects hatch with a full complement of tarsal segments,
the ground plan of the ancestral leg must have been
either (1) altered to produce novel patterning pro-
grams or (2) arrested to produce a truncated pattern.
Tanaka and Truman (2006) followed the expression of
Exd, Dac, Dll, Bab, and Al during larval leg develop-
ment, and did not detect significant deviations from
the ground plan of the leg during the initial subdivision
of the leg into segments. However, although the initial
uniform domain of Bab appeared in the tarsus, this
domain did not subdivide into rings of elevated expres-
sion. Because these rings correlate with tarsal segments,
and Bab expression is required for tarsal segmentation
in Drosophila, this suggests that the tarsal segments do
not form because Bab expression does not resolve
D. F. Erezyilmaz
into rings. From this, the authors concluded that the
larval leg arose, at least in part, from a truncation of the
ancestral program of patterning (Tanaka and Truman
2006).
Formation of the larval eye in Tribolium
Elements of truncation are also found by comparing
the expression of genes that pattern the adult eye
of holometabolous insects with their expression in
direct-developing embryos. In this instance, produc-
tion of the reduced larval eye, or stemmata of beetles,
occurs in part through a premature arrest of the ances-
tral eye-patterning program. During metamorphosis in
Drosophila, formation of ommatidial clusters occurs in
a posterior to anterior wave of cell division and sub-
sequent differentiation, called the morphogenetic fur-
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row (Dickson and Hafen 1993). The extent of
progression of the morphogenetic furrow is deter-
mined, in part, by Wg, which promotes head epider-
mis, but inhibits photoreceptor differentiation (Ma
and Moses 1995; Treisman and Rubin 1995; Hazelett
and others 1998). Such differentiation in Drosophila
requires expression of the glass (gl) gene, which is acti-
vated at the morphogenetic furrow and remains high in
the differentiating photoreceptors just posterior to the
furrow (Moses and Rubin 1991; Ellis and others 1993;
Treisman and Rubin 1996). The furrow, and therefore
gl expression, is initiated at the posterior edge of the
disc, opposing the wg expression domain (Ma and
Moses 1995; Triesman and Rubin 1996). This basic
configuration, with Wg expressed at the anterior of
the presumptive eye field and gl, or photoreceptor dif-
ferentiation initiating at the posterior margin, is con-
served in adult development of beetles and in
embryonic development of grasshoppers (Friedrich
and Benzer 2000; Liu and Friedrich 2004). However,
stemmatal development in the embryonic beetle
diverges from the ancestral program of eye develop-
ment. Instead of progressing across the eye field in a
morphogenetic furrow, gl expression was only present
in 2 clusters of cells, opposite the wg domain (Liu and
Friedrich 2004). Correspondingly, the patterning
mechanism produces only 2 fused stemmata composed
of
25 photoreceptors total, instead of producing rows
of rhabdomeres containing 8 photoreceptors each (Liu
and Friedrich 2004). These findings suggest that for-
mation of the larval stemmata occurred through trun-
cation, and subsequent modification of the ancestral
mechanism of eye patterning. In this scenario, the
first-born photoreceptors of the hemimetabolous eye
would be homologous to those that contribute to the
larval stemmata in the beetle (Liu and Friedrich 2004).
Therefore, for both the larval eye of Tribolium and the
Imperfect eggs and oviform nymphs
larval leg of Manduca, production of the larval form
occurs through an interruption of embryonic develop-
ment, consistent with the de-embryonization theories
inspired by Harvey (Fig. 3A–C). Metamorphosis of
these organs, however, does not occur simply by
resumption of the ancestral pattern from the point
where it was interrupted during embryogenesis. The
developmental program of the ancestral eye, for
instance, is re-deployed metamorphosis (Friedrich
and Benzer 2000).
The role of a pupal-specifier
in a hemimetabolous insect
Genetic support for the Lubbock/Berlese/Imms view of
the evolution of insect metamorphosis comes from
comparative studies of the pupal determinant, broad
(br). Epidermal expression of this transcription factor
is restricted to the larval–pupal transition in holome-
tabolous insects and its expression there is required for
progression to metamorphosis (Kiss and others 1978;
Karim and others 1993; Uhlirova and others 2003). For
instance, br null mutants fail to form a puparium but
continue to live as “over-aged larvae” (Kiss and others
1978). Moreover, larvae that were mosaics of br and
brþ tissue formed mosaics of larval and pupal cuticle at
metamorphosis (Kiss 1976). Loss of br in the silkmoth,
Bombyx, prevented growth and differentiation of the
adult eyes, legs, and wings, and prevented destruction
of the larval silk gland (Uhlirova and others 2003). The
role of br in regulating the larval–pupal transition is
probably conserved throughout the Holometabola,
since Konopova and Jindra (2006) have recently
reported that br is required for the pupal molt and ima-
ginal development in Chrysopa perla (Neuroptera), a
basal holometabolan.
To determine the role for this pupal-specifier in a
direct-developing insect, Erezyilmaz and colleagues
(2006) isolated a br ortholog from the milkweed
bug, Oncopeltus fasciatus. In contrast to its restricted
expression in metamorphosing insects, br is found dur-
ing embryonic development, is then expressed at each
nymphal molt, but is absent from the molt to the adult
stage. The nymphal stages of this insect are distinguish-
able by subtle differences in pigment pattern and by the
dimensions of the wing primordia. Loss of br by RNAi
knockdown prevented changes between the nymphal
stages; the nymphs were able to grow and molt to the
next stage, but they repeated the pigmentation pattern
and wing pad proportions of the stage at injection
(Erezyilmaz and others 2004b; Erezyilmaz and others
2006). In summary, br is required for all changes in
pattern and proportion that occur between the
nymphal stages. That this factor, which is required
805
for nymphal changes, has been restricted to one post-
embryonic stage in the Holometabola suggests that
metamorphosis arose as late embryonic and nymphal
expression of br became transposed to the penultimate
postembryonic instar.
Future questions
The small body of current research supports the idea
that pupal development is either a continuation or
repetition of embryonic events. The mechanism attri-
buted to the displacement or repetition of these
events, however, has evolved with time, and has
been influenced by available methods and innova-
tions. Today, the most likely explanations involve two
established regulators of insect life history, JH and its
effector br. What still remains to be learned, however,
is how these two stage-specifying factors interact with the
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developmental genes that pattern either a worm-like
larva or an adult-like nymph. Most comparative gene-
expression studies have focused on the earliest embryo-
nic events of hemimtabolous and holometabolous
insects, and have compared these events to imaginal
development of metamorphosing insects. However,
the two types of developmental trajectories diverge
later in embryonic development, after formation of
the phylotypic germ band. In order to determine which
developmental event brought about the departure from
hemimetabolous development that led to the evolu-
tion of complete metamorphosis, more studies should
focus on how global endocrine signals are integrated
with local patterning information during these later
stages of embryogenesis.
Acknowledgements
I thank Drs. J. Hodin, J. Edwards, K. Tanaka, and
J.W. Truman for helpful comments on this manu-
script. This work was supported by National Science
Foundation Grant IBN 9904959 to J.W.T. and L.M.R.,
and National Institute of Health Grant GM60122
to L.M.R.
Conflict of interest: None declared.
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