Pupa

A pupa (plural: pupae) is the third and immobile stage in the life cycle of holometabolous insects, which undergo complete metamorphosis, positioned between the active larval and reproductive adult phases; during this non-feeding period, the insect's internal tissues undergo histolysis and histogenesis to reorganize the body into its adult form, often encased in a protective structure.^[1] This stage is characteristic of the superorder Endopterygota, encompassing over 80% of insect species, including orders such as Lepidoptera (butterflies and moths), Coleoptera (beetles), Hymenoptera (bees, ants, wasps), and Diptera (flies).^[2] The pupal stage typically lasts from a few days to several months, depending on the species, environmental conditions like temperature and humidity, and whether the pupa is in diapause—a dormant state to overwinter or aestivate.^[3] Externally, the pupa appears quiescent and often forms a hardened exoskeleton or silk cocoon for protection against predators and desiccation, while internally, larval structures like mouthparts and musculature break down, and adult features such as wings, legs, and genitalia develop from imaginal discs.^[1] Upon completion, the adult ecloses by splitting the pupal case and expanding its wings, a process that can take hours as the exoskeleton hardens.^[1] Insect pupae are classified into three main morphological types based on the positioning of appendages and protective coverings: obtect pupae, where legs, wings, and antennae are appressed and glued to the body, forming a compact structure often called a chrysalis in butterflies; exarate pupae, with appendages free and extended from the body, typical in exposed or cocoon-protected forms like those of beetles and bees; and coarctate pupae, which resemble exarate but are enclosed within the hardened last larval skin (puparium), as seen in flies.^[4] These variations reflect adaptations to different habitats and lifestyles, with obtect forms common in Lepidoptera for aerial protection, and coarctate in soil-dwelling Diptera for durability.^[5] Notable examples include the chrysalis of monarch butterflies (Danaus plexippus), which hangs exposed and transforms over 8–15 days in temperate conditions,^[3] and the puparium of house flies (Musca domestica), buried in moist soil for 3–6 days before adult emergence.^[6] In social Hymenoptera like honeybees (Apis mellifera), pupae develop within wax cells, contributing to colony reproduction and taking about 12 days from larva to worker adult.^[7] The pupal stage's vulnerability underscores its ecological role, as it bridges larval herbivory and adult pollination or predation, influencing biodiversity in terrestrial ecosystems.^[8]

Overview and Role in Development

Definition and Characteristics

The pupa is the third developmental stage in the holometabolous metamorphosis of insects, succeeding the larval stage and preceding the emergence of the adult imago.^[9] This stage is characteristic of the Endopterygota superorder, encompassing orders such as Coleoptera (beetles), Lepidoptera (butterflies and moths), Hymenoptera (bees, ants, and wasps), and Diptera (flies).^[9] During pupation, the insect transitions from a feeding, motile larva to a non-feeding, largely immobile form focused on internal restructuring.^[10] Key features of the pupa include its quiescent nature, with minimal or no locomotion in most species, as larval muscles dissolve prior to the formation of adult musculature.^[10] The body is compact, with segments closely apposed and imaginal appendages—such as legs, wings, and antennae—folded tightly against the body or within protective pockets.^[9][10] Pupae typically cease feeding entirely, possessing reduced or non-functional mouthparts, and are often enclosed in a hardened exoskeleton or external case, such as a silk cocoon in some Lepidoptera, for protection during vulnerability.^[10] In contrast to hemimetabolous insects, which lack a pupal stage and exhibit gradual external changes from nymph to adult, the pupa represents a distinct resting phase enabling complete tissue reorganization in holometabolous forms.^[9] Basic anatomy includes the development of adult structures from imaginal discs—clusters of undifferentiated cells that form wings, limbs, and other features—and, in certain species, a fusion of the head and thorax into a cephalothorax.^[10]

Position in the Life Cycle

The pupa occupies a central position in the life cycle of insects that undergo complete metamorphosis, or holometaboly, serving as the quiescent stage that bridges the larval phase of feeding and growth with the adult phase of reproduction and dispersal. In this developmental sequence, the egg hatches into a larva specialized for nutrient acquisition and body mass increase, after which the larva enters the pupal stage for internal reorganization before eclosing as the imago, or adult, which prioritizes mating, oviposition, and environmental exploration. This four-stage progression—egg, larva, pupa, and adult—characterizes over 80% of insect species, underscoring the pupa's role as an obligatory intermediary in holometabolous development.^[1][11][12] The evolutionary advent of the pupal stage enabled radical shifts in insect morphology, allowing worm-like, feeding-focused larvae to transform into structurally diverse adults, such as those with wings or specialized sensory organs, thereby promoting ecological specialization and adaptive radiation. Fossil evidence indicates that holometaboly, including the pupa, first appeared approximately 310 million years ago during the Pennsylvanian subperiod (late Carboniferous).^[13][14] By comparison, insects with incomplete metamorphosis, termed hemimetaboly, bypass the pupal stage entirely, progressing through eggs that hatch into nymphs resembling miniature adults and undergoing gradual, incremental changes across multiple instars without a dedicated restructuring phase. This hemimetabolous pattern, seen in groups like orthopterans and hemipterans, contrasts sharply with holometaboly by limiting morphological discontinuity and emphasizing progressive development toward adulthood.^[15][11]

Physiological Processes

Formation and Hormonal Regulation

The formation of the pupa in holometabolous insects is initiated during the final larval instar through a precisely timed molting process known as pupation, which transforms the larval body into a non-feeding, immobile stage preparatory for adult emergence. This transition is triggered by a decline in juvenile hormone (JH) titers from the corpora allata glands, which normally maintain the larval state by preventing metamorphic gene expression. Concurrently, the brain secretes prothoracicotropic hormone (PTTH), stimulating the prothoracic glands to release ecdysone, which is rapidly converted to the active form 20-hydroxyecdysone (20E). The rise in 20E levels, in the absence of JH, commits epidermal and imaginal disc cells to pupal development, marking the onset of metamorphosis.^[16][17] Apolysis, the initial step in pupal formation, involves the separation of the old larval cuticle from the underlying epidermis, facilitated by 20E-induced secretion of molting enzymes such as chitinases and proteases. Following apolysis, the epidermis secretes a new, unsclerotized pupal cuticle beneath the detached larval one, which is later shed during ecdysis. This hormonal cascade is tightly regulated: low JH permits 20E to bind nuclear ecdysone receptors (EcR), activating early genes like Broad-Complex (BR-C), a transcription factor essential for pupal-specific gene expression and tissue remodeling. BR-C isoforms, such as Z1, are expressed in response to the first 20E pulse, suppressing larval traits while promoting pupal commitment without inducing adult differentiation.^[17][16][16] Recent studies using CRISPR-Cas9 genome editing have elucidated the role of ecdysone receptors in timing pupation, revealing that disruptions to EcR or downstream factors like E93 delay or arrest the process, leading to retention of larval tissues and incomplete metamorphosis. For instance, in the yellow fever mosquito (Aedes aegypti), CRISPR knockout of E93 results in pupae that eclose but retain larval tissues and die during the pupal stage, underscoring EcR's control over developmental timing via gene cascades.^[18] Additionally, environmental factors such as climate warming can disrupt this regulation; elevated temperatures in urban heat islands alter 20E titers during the prepupal stage, potentially advancing or desynchronizing pupation and affecting synchrony with host plants or predators in species like the Colorado potato beetle. These insights highlight the sensitivity of hormonal pathways to abiotic stressors, with implications for insect adaptation under global change.^[19][20]

Internal Metamorphosis

During the pupal stage, internal metamorphosis in holometabolous insects involves extensive histolysis, the programmed breakdown of larval tissues to recycle nutrients for adult development. Larval muscles, such as the dorsal external oblique muscles (DEOMs) in Drosophila, undergo apoptosis triggered by ecdysone signaling through the ecdysone receptor isoform B1, which activates cell-autonomous death pathways involving caspases.^[21] Similarly, the larval midgut histolyzes via a combination of apoptosis and autophagy, initiated by an early ecdysone pulse that upregulates genes like those encoding caspases and autophagic proteins.^[22] The nervous system also experiences selective histolysis, with many larval neurons pruned through caspase-mediated apoptosis to clear space for adult circuitry.^[23] Complementing histolysis, histogenesis drives the formation of adult structures from clusters of undifferentiated cells known as imaginal discs, which proliferate and differentiate during pupation. In Drosophila, wing imaginal discs evaginate and expand dramatically, transforming from folded sacs into flat epithelial sheets through oriented cell division and migration, regulated by signaling pathways such as Wingless (Wg, a Wnt homolog) for proliferation along disc margins and Decapentaplegic (Dpp, a BMP homolog) for patterning and growth control across the disc.^[24] These pathways interact dynamically; for instance, Dpp signaling represses Wg expression in central regions via transcription factors like Dorsocross, ensuring balanced disc expansion.^[25] Leg and eye discs follow analogous processes, with Wg and Dpp coordinating histoblast proliferation to form segmented appendages and ommatidia, respectively.^[26] Organ remodeling extends to vital systems, adapting them for adult function without external feeding, relying instead on larval reserves mobilized during histolysis. The brain undergoes profound rewiring, where larval circuits are partially dismantled via apoptosis of interneurons and Kenyon cells, while surviving neurons extend new processes to form adult-specific connections, such as in olfactory and memory circuits.^[27] This remodeling repurposes larval neurons for adult behaviors, with astrocytes aiding local neurite pruning to refine connectivity.^[28] Concurrently, Malpighian tubules—the insect renal organs—are remodeled by renal stem cells (RSCs) that partially activate during pupation to replace larval principal cells near the gut junction, with further remodeling in adults enhancing stone resistance and ion transport efficiency.^[29] The tubules shut down physiologically during early pupation, losing microvilli, before RSC-driven regeneration restores functionality.^[30] Recent research highlights additional layers of regulation in pupal metamorphosis. Post-2020 studies show that the pupal microbiome influences metamorphosis efficiency, with gut bacteria like Enterococcus species delaying pupation rates by influencing host immune responses and microbiota dynamics, while their reduction can accelerate the transition.^[31] In Drosophila and other insects, microbiota turnover during pupation reshapes community composition, supporting resource reallocation for histogenesis.^[32] Advances in live imaging have revealed the dynamic unfolding of imaginal discs; for example, in vivo time-lapse microscopy of Drosophila pupae captures wing disc evagination as a coordinated epithelial unfolding driven by actomyosin contractility, occurring over hours post-pupariation.^[33] Such techniques also document real-time cell rearrangements in notum regions, where crowding triggers delamination to limit proliferation.^[34]

Duration and Emergence

The duration of the pupal stage in insects varies widely across species and is heavily influenced by environmental conditions such as temperature, humidity, and photoperiod. In many dipterans like the housefly (Musca domestica), the pupal period typically lasts 3-6 days under optimal warm conditions, allowing rapid development to adulthood.^[35] In contrast, certain beetles, such as the warehouse beetle (Trogoderma variabile), can experience delayed pupation due to larval diapause extending up to 2 years.^[36] Higher temperatures generally accelerate pupal development and shorten the overall duration, while lower humidity or shorter photoperiods can extend it by inducing metabolic slowdowns.^[37][38] Diapause represents a key mechanism extending pupal duration, occurring as either obligatory (mandatory for the species, often one generation per year) or facultative (environmentally induced) dormancy to overwinter or avoid harsh conditions. It is commonly triggered by short day lengths (photoperiods) signaling seasonal change, leading to suppressed metabolic activity and halted histolysis or organogenesis. For instance, in the European corn borer (Ostrinia nubilalis), facultative pupal diapause can last several months under short-day conditions at moderate temperatures, enabling survival until spring.^[38][39] Warmer climates may shorten non-diapausing pupal stages but paradoxically increase diapause risks by altering photoperiod cues or inducing estival (summer) diapause in some species, as observed in the oriental tobacco budworm (Helicoverpa assulta), where high temperatures prolong the pupal phase.^[40] Emergence, or eclosion, marks the end of the pupal stage, involving enzymatic degradation of the pupal cuticle followed by adult expansion. Chitinases, such as those in the insect chitinase family (e.g., CHT5 in beetles), are secreted to soften and break down the chitinous pupal exoskeleton, facilitating the adult's exit.^[41] Once free, the adult insect pumps hemolymph into its wings and body under increased pressure generated by abdominal contractions, expanding and hardening the new cuticle within minutes to hours.^[42] Eclosion timing is often synchronized to minimize predation, with many butterflies emerging at dawn when visibility is low and predators are less active.^[43]

Behaviors and Defenses

Defensive Adaptations

Pupae, being immobile and vulnerable during metamorphosis, employ a range of physical defenses to evade detection by predators. Cryptic coloration is prevalent, allowing pupae to blend seamlessly with their environment; for instance, swallowtail butterfly (Papilio spp.) pupae exhibit green or brown hues that mimic twigs or leaves, enhancing survival against visually hunting birds.^[44][45] Immobility further reinforces this camouflage, as pupae remain stationary to resemble inert plant parts.^[45] Chemical defenses in pupae often build on larval strategies, involving both sequestration of hostplant toxins and endogenous synthesis. Monarch butterfly (Danaus plexippus) pupae sequester cardenolides acquired during the larval stage from milkweed (Asclepias spp.), rendering them toxic to avian predators and thereby deterring attacks.^[46][47] In contrast, some beetle pupae synthesize alkaloids de novo.^[45] These compounds are stored in hemolymph and exuded as a reflexive defense, highlighting the pupal stage's reliance on pre-formed chemical armaments.^[45] Behavioral and social adaptations compensate for pupal immobility, particularly in species with symbiotic interactions. Myrmecophilous lycaenid butterfly pupae benefit from ant tending, where attendant ants (Crematogaster spp.) aggressively defend the pupae against predators and parasitoids, improving survival rates in ant-colonized habitats.^[48] Acoustic signaling serves as another mechanism; pupae of some Hymenoptera and lycaenids produce substrate-borne vibrations via stridulation to alert tending ants or startle potential predators, modulating ant attendance and deterring attacks.^[49][50] Recent research post-2020 has illuminated how environmental stressors impact these defenses. In tobacco hornworm (Manduca sexta) pupae, mechanical disturbance via vibrations triggers sex-specific behavioral responses such as twitching and pulsating, with males exhibiting more frequent and prolonged reactions.^[51] These findings emphasize the need for integrated approaches to understand pupal resilience amid global environmental shifts.^[52]

Reproductive Behaviors

In certain species of Lepidoptera, such as butterflies in the genus Heliconius, pupal mating occurs where males copulate with females immediately upon or during their emergence from the pupal case, a behavior first documented in the late 19th century.^[53] This facultative strategy is observed sporadically; for instance, in Heliconius erato, field studies recorded only two instances among 11 emerging females, with males patrolling host plants like Passiflora species to locate pupae using chemical cues from the plants and potentially from the pupae themselves.^[54] Females in these species store sperm received during pupal mating in a spermatophore, allowing them to fertilize eggs post-emergence without further copulation, as evidenced by high mating success rates (19 out of 20 recaptured virgin females were found mated).^[53] Pupal mating is facilitated by sex pheromones emitted from female pupae in some moths, which arrest and attract patrolling males prior to adult eclosion. In the codling moth Cydia pomonella, female pupae release pheromones that draw males to the site, enabling pre-emergence copulation and reducing post-eclosion mate-searching risks. Calling behaviors during the pupal stage are less common but include vibrational signals in select species; for example, some lepidopteran pupae produce substrate-borne vibrations to signal readiness or deter rivals, though these are primarily documented in adult courtship contexts and extended to pre-emergence interactions in low-mobility pupae. Silk pheromones, deposited on pupal cocoons by some moths, may also aid in mate location by providing persistent chemical trails that males detect near emergence sites. Parthenogenesis in the pupal stage, known as pupal pedogenesis, is a rare asexual reproductive mode observed in certain insects, particularly gall midges of the family Cecidomyiidae (Diptera), where unfertilized eggs develop within the pupa into larvae that emerge viviparously. This process allows reproduction without fertilization, often in isolated or low-density conditions, and involves endosomatic development of reproductive structures inside the pupal body, leading to the mother's death upon offspring emergence. The evolutionary advantages of pupal mating include securing mates in sparse populations by enabling males to monopolize emerging females on predictable host plants, thereby increasing reproductive success amid high larval mortality and asynchronous eclosion.

Classification and Variations

General Morphological Types

Pupae are primarily classified into three morphological types based on the configuration of appendages and protective structures: exarate, obtect, and coarctate. In exarate pupae, the appendages—such as legs, wings, and antennae—are free and not fused to the body wall, allowing for a more adult-like appearance and limited voluntary movement.^[55] This type is prevalent in orders like Coleoptera (beetles) and Hymenoptera (such as bees and wasps).^[56] In obtect pupae, the appendages are appressed and glued to the body, forming a compact structure often seen in exposed forms. This type is common in Lepidoptera (butterflies and moths) and some Diptera (flies).^[56] In contrast, coarctate pupae are enclosed within the hardened larval exoskeleton (puparium), which conceals the typically exarate appendages of the internal pupa and provides enhanced protection during vulnerable stages.^[55] A secondary classification distinguishes pupae by mouthpart structure: decticous and adecticous. Decticous pupae possess functional, articulated mandibles that enable the pupa to bite or chew its way out of enclosures, facilitating escape and reflecting a more active phase.^[55] These are always exarate and occur in basal Hymenoptera, such as sawflies (e.g., Arge pagana in Argidae).^[57] Adecticous pupae, conversely, have reduced or non-functional mandibles, relying instead on other mechanisms like silk-dissolving enzymes or external aids for emergence, and are typically immobile.^[55] This type is widespread in advanced Lepidoptera, including moths.^[56] These morphological types carry functional implications tied to their ecology and development. Exarate pupae's free appendages permit slight repositioning or defensive responses, aiding survival in exposed environments, while obtect forms prioritize compactness for aerial or silk-protected contexts, and coarctate forms emphasize fortification for protection in concealed or soil-dwelling habitats.^[55] Distributionally, exarate pupae dominate in Coleoptera and most Hymenoptera, coarctate pupae are characteristic of Diptera (higher flies), and obtect forms prevail in Lepidoptera.^[56] Evolutionarily, decticous pupae represent the primitive condition, with adecticous forms derived secondarily, though direct fossil evidence for transitional hybrids remains limited.^[55] Variations in pupal development include the prepupa, an interpolated stage in insects exhibiting hypermetamorphosis, such as certain beetles in the family Meloidae (oil beetles), where it precedes the true pupa between the active larva and pupation.^[58][59] In contrast, some insects with incomplete metamorphosis, such as thrips and whiteflies, feature a pseudopupa or false pupa, which is not a true pupal stage but a quiescent pre-adult phase lacking the full metamorphic remodeling seen in holometabolous insects.^[60]

Specific Enclosing Structures

In the pupal stage of many Lepidoptera, particularly butterflies, the chrysalis serves as a distinctive enclosing structure, consisting of a naked, obtect pupa that is externally hardened without silk. This form arises from the sclerotization process of the larval cuticle, where the outermost layers tan and rigidify following apolysis, providing a protective shell for metamorphosis. The chrysalis is secured to a substrate, often a plant stem or leaf, via the cremaster—a specialized, hook-like appendage at the posterior abdominal end that anchors it firmly. Many butterflies also produce a silk girdle, a simple thread spun around the body just prior to pupation, which provides additional support by holding the pupa in place against the substrate.^[61][62].pdf)^[63] Emergence of the adult occurs when the butterfly splits the chrysalis along a weakened dorsal seam, allowing the insect to extricate itself and expand its wings.^[64] Cocoons represent another specialized pupal enclosure, primarily constructed by larvae of various moths in the Lepidoptera and certain beetles in the Coleoptera, through the extrusion and weaving of silk glands into a multilayered protective case. These structures incorporate calcium oxalate crystals embedded within the silk matrix, which significantly bolster tensile strength and puncture resistance, adapting the cocoon to withstand predation and environmental pressures. Adult moths typically escape the cocoon by exploiting pre-formed exit valves in the silk or by secreting proteolytic enzymes, such as cocoonase, that selectively degrade the sericin protein binding the fibroin fibers.^[65][66] In Diptera, such as houseflies (Musca domestica), the puparium forms a robust, barrel-shaped case from the tanned and contracted final larval exoskeleton, encasing the coarctate pupa and recycling the cuticle for protection. Prominent respiratory horns project from the anterior end through specialized slits in the puparium wall, enabling tracheal gas exchange while minimizing exposure. Post-2020 biomechanical analyses have highlighted the puparium's capacity to endure compressive forces, with its layered, sclerotized integument distributing stress to safeguard internal tissues during burial or handling.^[67][68][69] These enclosing structures exhibit notable variations in construction and function: chrysalides depend on direct sclerotization of the pupal integument for rigidity without extrinsic materials, contrasting with the externally fabricated silk of cocoons; puparia, meanwhile, repurpose the larval skin into a seamless, hardened barrel. Not all pupae require such enclosures, as seen in exarate pupae of many beetles (Coleoptera), which are often placed in earthen cells or remain exposed in soil habitats without additional casings.^[70][62]