Imperfect eggs and oviform nymphs.pdf

Imperfect eggs and oviform nymphs: a history of ideas aboutthe 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.

IntroductionInsect metamorphosis is one of the most common yet

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

reduced or absent larval legs. The fate of the larval

tissues differs among the holometabolous orders.

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: [email protected]

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

doi:10.1093/icb/icl033

Advance Access publication August 24, 2006

� 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

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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 ofmetamorphosis. 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 . . . inthis 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

E E

P

EN1 N1

L1L2

L3

L4

L5

L6

L7

L8

N2N2

N3

N3N4

N4

N5

N5

N6

N6

N7

N8

A

Ametaboly(no metamorphosis)

Hemimetaboly(partial metamorphosis)

Hemimetaboly(complete metamorphosis)

A

A

Fig. 1 A figure adapted from a review by Sehnal and colleagues (1996). In ametabolous development, the immature stagessimply increase in size to produce the adult. Unlike other types of insect development, these insects, represented hereby 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 theadult. 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) (forexample, N1 is the first nymphal instar).

796 D. F. Erezyilmaz

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

“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

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 bemore 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

Imperfect eggs and oviform nymphs 797

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

theOrigin of Species, the German naturalist, FritzMuller

(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.

Fig. 2 Drawings of bee pupae from Swammerdam’s 1669 publication (1978 reprint), The Book of Nature or, the History ofInsects. (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 ¼first pair of joints on the proboscis; h ¼ proboscis; i ¼ the first pair of legs; k ¼ “transparent stiff little parts;” l ¼ thesecond 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 partsof the body;” r ¼ “2 little parts accompanying the sting;” s ¼ the anus. (B) A ventral view of a bee pupa with thethoracic pupal cuticle removed and the imaginal tissues pulled away. The original figure legend reads, “The worm of theBee, on the point of changing to a Nymph, and stripped of its skin, the better to show the infant parts of the futureBee, which are here represented as they appear through the microscope, after extending them a little. aa: the antennaeor horns. b: the proboscis, with its parts. cc: the second pair of joints belonging to, or forming, the proboscis. dd: thefirst pair. ee: the first pair of legs, lying against the breast. ff: the second pair of legs. gg: the third pair. hh: the greaterwings. ii: the smaller wings. k: the abdominal wings.” Reprinted from F Sehnal, P Svacha, and J Zrzavy, Evolution of insectmetamorphosis, in LI Gilbert, JR Tata, BG Atkinson (eds.), Metamorphosis: Postembryonic Reprogramming of GeneExpression in Amphibian and Insect Cells. San Diego: Academic Press, p. 3–58, with permission from Elsevier.


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

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,

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

fromFritzMuller’s conclusions from theCrustacea. 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 beforeor 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

Fig. 3 Summary of various theories illustrating thehomology of hemimetabolous stages andholometabolous stages. Adapted from Gillot (1995).hemi ¼ hemimetabolous life history; holo ¼holometabolous life history; E ¼ embryo; N ¼ nymph; L¼ larva; P ¼ pupa; A ¼ adult. (A–C) De-embryonizationtheories. Harvey (1651) considered the pupal stage tobe a continuation of embryonic events. Berlese’s (1913)theory subdivided the embryonic stages into 3 periodsof increasing development; the abdomen-less protopod(Eproto), the differentiating polypod (Epoly), and theterminally differentiating oligopod (Eoligo). Truman andRiddiford (1999) subdivided embryonic developmentaccording to embryonic molts. E1 ¼ the embryonic stagebearing the first embryonic cuticle. EPro-N ¼ pronymphalstage, bearing the pronymphal cuticle EN ¼ first nymphalstage, produced just before hatching. (D) Poyarkoff(1914a, b) suggested that the pupal stage arose throughsubdivision of the imaginal stage. (E) Despite evidencethat the Holometabola evolved from hemimetabolousinsects, Heslop-Harrison (1958) believed that bothgroups evolved from an ancestor containing both larvaland nymphal stages. (F) Hinton (1963) considered thepupa to be homologous to the last stage nymph ofhemimetabolous insects.


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

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Fig. 4 The hemimetabolous and holometabolous adults (Plate 1) matched with their immature stages in Plate 2. Thisfigure is originally from J. O. Westwood’s Introduction to the Modern Classification of Insects, but Lubbock (1883) usedthese plates in a series of articles in Nature entitled, On the Origin and Metamorphoses of Insects, in which he discussed thesuitability 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.


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.

Acentral featureof theLubbock/Berlese/Immsviewof

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

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,

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

frommyoblasts that adhere to muscle fibers, whereas in

Neuroptera and Coleoptera, larval muscle is directly

transformed into adult muscle during the early pharate


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

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

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


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

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)

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 thetobacco 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


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 theDrosophila 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

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-

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


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-specifierin 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 formedmosaics 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

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,

ishowthese twostage-specifying factors interactwith the

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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