Integrative and Comparative Biology

Integrative and Comparative Biology Advance Access originally published online on August 24, 2006
Integrative and Comparative Biology 2006 46(6):795-807; doi:10.1093/icb/icl033

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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

Correspondence: ¹E-mail: denizere{at}u.washington.edu

	Synopsis

Top Synopsis Introduction Imperfect eggs Oviform nymphs Embryonic metamorphoses The morphologists Endocrine-based theories Larval patterning The role of a... Future questions REFERENCES

 
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

Top Synopsis Introduction Imperfect eggs Oviform nymphs Embryonic metamorphoses The morphologists Endocrine-based theories Larval patterning The role of a... Future questions REFERENCES

 
Insect metamorphosis is one of the most common yet dramatic of biological phenomena, and problems associated 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 primordia 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.

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   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 = embryo; L = larva; N = nymph; P = pupa; and A = adult. The numbers associated with these letters indicate the instar (duration between molts) (for example, N1 is the first nymphal instar).

 
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 holometabolous 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 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

Top Synopsis Introduction Imperfect eggs Oviform nymphs Embryonic metamorphoses The morphologists Endocrine-based theories Larval patterning The role of a... Future questions REFERENCES

 
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 animals, 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 primordium, 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 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 development 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 theories that emerged in opposition to it.

	Oviform nymphs

Top Synopsis Introduction Imperfect eggs Oviform nymphs Embryonic metamorphoses The morphologists Endocrine-based theories Larval patterning The role of a... Future questions REFERENCES

 
"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 chrysalis, 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 superficial, 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 dimensions. Swammerdam furthermore considered the nymphal 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 metamorphosis, 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."

View larger version (146K): [in this window] [in a new window] [Download PowerPoint slide]   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 = the head; b = eyes; c = horns or antennae; d is "the lip;" e = "teeth or jaw bones;" f = first pair of joints on the proboscis; h = proboscis; i = the first pair of legs; k = "transparent stiff little parts;" l = the second pair of legs; m = the wings; n = "blade bones;" o = last pair of legs; p = "abdominal rings;" q = "the hinder parts of the body;" r = "2 little parts accompanying the sting;" s = 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.

 
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) considered the production of the adult to be more continuous:

The wings, horns and other parts which worms without 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 confined 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

Top Synopsis Introduction Imperfect eggs Oviform nymphs Embryonic metamorphoses The morphologists Endocrine-based theories Larval patterning The role of a... Future questions REFERENCES

 
"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 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 whenever 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.

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.

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Summary of various theories illustrating the homology of hemimetabolous stages and holometabolous stages. Adapted from Gillot (1995). hemi = hemimetabolous life history; holo = holometabolous life history; E = embryo; N = nymph; L = larva; P = pupa; A = 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 = the embryonic stage bearing the first embryonic cuticle. EPro-N = pronymphal stage, bearing the pronymphal cuticle EN = 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.

 
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 hemimetabolous 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 hemimetabolous 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 acquiring 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 development of the perfect animal. On the contrary, it is subject to the influence of natural selection and undergoes 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).

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   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 = cricket; 2 = earwig; 3 = Aphis; 4 = Scolytus; 5 = Anthrax; 6 = Balanius; 7 = Cynips; 8 = Ant; 9 = Wasp. Plate 2: ? = cricket nymph; 2 = Aphis nymph; 3 = earwig nymph; 4 = Scotylus larva; 5 = Anthrax larva; 6 = Balanius larva; 7 = larva of Cynips; 8 = ant larva; 9 = wasp larva.

 

	The morphologists

Top Synopsis Introduction Imperfect eggs Oviform nymphs Embryonic metamorphoses The morphologists Endocrine-based theories Larval patterning The role of a... Future questions REFERENCES

 
In the twentieth century, aspects of Harvey's second egg hypothesis were popularized by the work of Antonin Berlese (1913). Berlese was an insect morphologist, 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 differentiation, 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 appendages 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 of insect gut development. During embryonic development, the gut arises after 2 invaginations, the future stomodeum and proctodeum move posteriorly and anteriorly, respectively, towards the center of the blastula. 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 respectively, 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 during 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 developmental 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 nymphal 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 contained both polypodous and oligopodous postembryonic stages. That is, the ancestral insect would hatch to Holometabolous-like larval stages (polypodous) and then pass to hemimetabolous nymphal stages (oligopodous). 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 reinterpretation 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 tonofibrillae. 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 indirect 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 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 epidermis at molts. At the larval/pupal molt, the strands proliferate rapidly and divide to produce the entire assemblage of flight muscles (Hinton 1959). By showing 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

Top Synopsis Introduction Imperfect eggs Oviform nymphs Embryonic metamorphoses The morphologists Endocrine-based theories Larval patterning The role of a... Future questions REFERENCES

 
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 metamorphosis 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 hemimetabolous and holometabolous insects, and was found to regulate complete metamorphosis as well (Piepho 1942, 1946; Williams 1956). These experiments changed the debate on the origins of insect metamorphosis so that, instead of addressing the problem by comparing the development of a tissue or structure between hemimetabolous and holometabolous insects, the factors that regulate the process were now compared.

There is no "Wigglesworth theory" on the origin of insect metamorphosis. Instead, Wigglesworth downplayed the differences between hemimetabolous and holometabolous insects. Because he saw unity in the mechanisms that regulate hemimetabolous and holometabolous 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 theories that incorporated JH into Berlese's hypothesis emerged. The Czech insect physiologist, Novak proposed that earlier production of JH during embryonic development caused de-embryonization of the oligopod 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 promotes isometric growth. While JH is first produced during embryonic development in all insects, the corpora 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 progression 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 pronymph hypothesis. This idea is centered on the pronymph (the first postembryonic stage of basal insects and some other arthropods), which differs in morphology 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 development, creating the holometabolous larval stage (Truman and Riddiford 1999, 2002; Erezyilmaz and others 2004a, b).

Evidence for the homology of the larval and pronymphal stages comes from the degree of development 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 pronymph 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 formation (Ziese and Dorn 2003; Konopova and Zrzavy 2005). Whereas light microscopy had indicated only 2 embryonic "cuticles," transmission electron micrographs revealed a second layer that is produced at a time commensurate with pronymphal cuticle formation in hemimetabolous insects. Ziese and Dorn (2003) and Konopova and Zrzavy (2005) argued that the pronymphal 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 ecdysteroid 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

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Instead of simply focusing on the development of specific tissues during embryonic and metamorphic development, two developmental studies have compared the expression of genes that pattern the adult and larval organs to address the origin of metamorphic development. 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 produce 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 embryogenesis 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 morphogens 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 correspond 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 translocation 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 hawkmoth 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 hemimetabolous 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 conserved 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 morphologically 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 segment of the leg. The adult tarsus, however, is subdivided 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 programs 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 development, 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 expression. 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, production of the reduced larval eye, or stemmata of beetles, occurs in part through a premature arrest of the ancestral eye-patterning program. During metamorphosis in Drosophila, formation of ommatidial clusters occurs in a posterior to anterior wave of cell division and subsequent differentiation, called the morphogenetic furrow (Dickson and Hafen 1993). The extent of progression of the morphogenetic furrow is determined, in part, by Wg, which promotes head epidermis, 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 activated 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 differentiation initiating at the posterior margin, is conserved 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 development. 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 formation of the larval stemmata occurred through truncation, 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 development, 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

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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 holometabolous 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 br+ 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 imaginal 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 during 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 distinguishable 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 postembryonic 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

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The small body of current research supports the idea that pupal development is either a continuation or repetition of embryonic events. The mechanism attributed to the displacement or repetition of these events, however, has evolved with time, and has been influenced by available methods and innovations. 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 developmental genes that pattern either a worm-like larva or an adult-like nymph. Most comparative gene-expression studies have focused on the earliest embryonic 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 evolution 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 manuscript. 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.

	Footnotes

 
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.

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