Millipede Study Shows Epidermal Invagination Drives Postembryonic Segment Addition

A detailed anatomical investigation of the millipede Niponia nodulosa has revealed that postembryonic segment addition proceeds through a coordinated process of local epidermal invagination and muscular reorganization prior to molting. While most research on arthropod body segmentation has centered on embryonic patterning, postembryonic segment addition, termed anamorphosis, represents a widespread developmental mode across many lineages including millipedes, centipedes, and crustaceans. In millipedes belonging to the class Diplopoda, trunk growth occurs through the insertion of newly formed segmental units, known as rings, situated anterior to the terminal telson. Despite historical documentation of this developmental phenomenon under the classical law of anamorphosis, the precise three-dimensional tissue dynamics and cellular architecture executing this morphological insertion have remained largely unresolved. By combining high-resolution X-ray micro-computed tomography with serial resin histological sectioning and confocal fluorescence microscopy, a research team examined how epidermal folding and muscle repositioning generate new functional trunk units within a confined posterior anatomical region.

Arthropods exhibit diverse modes of body tagmosis and segmentation, with developmental pathways broadly categorized into epimorphosis, where the complete complement of segments is finalized during embryonic life, and anamorphosis, where additional segments appear sequentially during juvenile molts. Anamorphosis is widely considered the ancestral condition of arthropod development, from which epimorphosis evolved independently in several specialized groups such as geophilomorph and scolopendromorph centipedes. Within anamorphic systems, biologists distinguish between hemianamorphosis, in which segment addition halts at a specific developmental stage while molting continues, euanamorphosis, where new segments emerge at every molt throughout life, and teloanamorphosis, where segment addition coincides with molting until sexual maturity is attained. The millipede Niponia nodulosa, classified within the order Polydesmida and family Cryptodesmidae, develops through teloanamorphosis, sequentially appending rings across juvenile instars until reaching sexual maturity at stadium VIII with a fixed count of twenty rings including the telson. Each millipede ring developmentally represents two fused true segments bearing two pairs of walking legs, rendering the structural integration of new units uniquely complex.

To capture the internal tissue remodeling that precedes cuticle shedding, researchers collected juvenile specimens of Niponia nodulosa from Iwadono, Higashimatsuyama City, Saitama Prefecture, Japan, and maintained them under controlled laboratory conditions at twenty-five degrees Celsius in darkness on a moist humus substrate. The developmental investigation focused primarily on the transition between stadium VI and stadium VII, a phase when juveniles are sufficiently large for intricate microstructural imaging and undergo the transition of ring seventeen from an apodous to a podous state while inserting a new ring eighteen between ring seventeen and the telson. Individual monitoring of stadium VI juveniles showed that the intermolt duration lasted an average of 69.3 plus or minus 34.7 days, with considerable variation ranging from 35 to 128 days across eight tracked specimens. Because external pigmentation or general activity provided unreliable temporal indicators of approaching ecdysis, the researchers utilized stereotypic chamber construction behaviors to establish precise staging criteria for experimental sampling.

Juveniles of Niponia nodulosa construct dome-shaped protective molting chambers using compacted fecal pellets, dividing the pre-molt phase into a distinct preparatory period and a subsequent rigidation period. During the early preparatory period, individuals gather fecal matter to assemble the outer chamber shell. Over the next forty-eight hours, termed the late preparatory period, the juvenile remains active inside the sealed chamber, depositing a fine lining of silk-like threads over the inner fecal walls, an architectural feature documented here for the first time within the family Cryptodesmidae. Following this lining activity, the juvenile enters the rigidation period, curling completely with its ventral side directed inward and entering an immobile state refractory to external mechanical stimuli. This behavioral shift correlates with apolysis, the physiological separation of the living epidermis from the overlying cuticular exoskeleton destined to be shed during ecdysis, establishing reliable temporal markers for sampling sequential developmental stages.

To visualize the internal morphological progression in three dimensions, the authors fixed anesthetized specimens in Bouin solution, stained them with one percent iodine in absolute ethanol, freeze-dried them using liquid tertiary-butyl alcohol, and scanned the posterior trunk with an inspeXio SMX-100CT micro-computed tomography system at thirty-five to forty kilovolts and ninety to one hundred microamperes. These volumetric scans yielded reconstructions with a voxel resolution of one to two micrometers, which were analyzed in Amira software alongside conventional histological sections. For light microscopy, specimens embedded in Technovit 8100 resin were cut into five to ten micrometer sections and stained with hematoxylin and eosin. Additionally, F-actin filaments and cell nuclei were visualized by dissecting posterior tissues, embedding them in optimal cutting temperature compound, sectioning along the sagittal plane, and staining with rhodamine-phalloidin and 4′,6-diamidino-2-phenylindole for confocal laser scanning microscopy.

The anatomical reconstructions demonstrated that the ring primordium initiates development during the early preparatory period, well before any external cuticular changes or body elongation become visible. The earliest histological sign of segmentation is a localized inward invagination of the single-layered epidermis situated ventrally between the telson and ring seventeen, flanking the posterior terminus of the ventral nerve cord. As the animal enters the late preparatory period, a complementary invagination appears in the dorsal epidermis. These invaginating epithelial folds deepen and coalesce laterally, producing a circumscribed ring-shaped constriction. In specimens undergoing earlier instars, specifically stadia III, IV, and V, which add three, two, and one ring per molt respectively, histological sections revealed three, two, and one distinct epithelial folds within the invaginated pocket, confirming that the number of invaginated primordia matches the exact quantity of rings added during that developmental molt.

As the animal transitions into the rigidation period, systemic apolysis detaches the epidermis from the old exoskeleton in an anterior-to-posterior progression. Concurrently, the juvenile trunk elongates along its anteroposterior axis, causing the previously invaginated ring primordium to expand outward from between ring seventeen and the telson as if being drawn from an internal fold. The lateral margins of the primordium develop prominent bilateral projections that establish the paranota, which are lateral extensions of the pleurotergite plate. Simultaneously, paired leg primordia emerge from ring seventeen beneath the old cuticle, and the primordium develops a raised transverse boundary demarcating the anterior prozonite from the posterior metazonite. By the late rigidation period, apolysis is completed, and the primordium displays a textured surface. Upon ecdysis to stadium VII, the fully expanded ring eighteen possesses a diameter approximately four times greater than the compacted primordium observed in late rigidation.

Alongside epidermal folding, the research detailed the complex muscular rearrangements required to insert a functional ring between existing trunk structures. In stadium VI intermolt juveniles, adjacent rings are linked dorsally by two distinct skeletal muscle groups: long dorsal internal muscles, which bridge the anterior margin of one ring to the anterior margin of the next ring or telson, and short dorsal retractor muscles, which originate at the prozonite-metazonite boundary of a ring and insert onto the anterior margin of the subsequent ring or telson. Histological and phalloidin staining demonstrated that the dorsal retractor muscle of ring seventeen initially anchors to the anterior edge of the preanal ring of the telson, immediately adjacent to the origin of anal valve muscle one. During the preparatory period, the epidermal invagination begins immediately between these two muscle attachment points, allowing both muscles to maintain their skeletal anchors as tissue folding commences.

When the juvenile enters the rigidation period and apolysis detaches the epidermal sheet from the cuticle, the dorsal retractor muscle of ring seventeen and the companion dorsal internal muscle sever their original attachments to the telson. Rather than degenerating, these existing muscle bundles shift their posterior insertion points and reattach directly to the anterior margin of the newly formed ring eighteen primordium. Simultaneously, two novel longitudinal muscle groups differentiate de novo inside the dorsal territory of the ring primordium, extending posteriorly to form fresh structural linkages with the anterior margin of the telson. One of these newly differentiated bundles corresponds to the dorsal internal muscle of ring eighteen, running from its anterior margin to the telson, while the second represents the dorsal retractor muscle of ring eighteen, originating at its prozonite-metazonite boundary. This coordinated process preserves anterior-posterior muscular continuity while accommodating the intercalated segment.

In contrast to the dynamic reorganization of dorsal trunk musculature, the muscular architecture situated within the telson remained stable throughout ring addition. Muscle av1 connects the dorsal preanal ring to the posterior margin of the anal valves, while av2 connects the anterolateral preanal wall to their anterior borders; the eversible anal sac muscles link the preanal ring laterally to the rectal cavity. These telson muscles persisted without positional alteration or degradation across all observed preparatory and rigidation stages, exhibiting only temporary relaxation during late rigidation when the anal valves open laterally and the rectal sac everts. Preserving these contractile tissues intact enables juveniles to defecate and manipulate fecal pellets for molting chamber assembly during the preparatory phase.

Histological analyses further uncovered a specialized, strongly eosinophilic tissue that develops transiently within the telson strictly during the preparatory period. Composed of hypertrophied cells possessing expanded cytoplasm filled with eosinophilic material and scattered nuclei, this tissue occupies the internal space between the epidermis and hindgut, surrounding anal valve muscle one without disrupting its structure. The tissue extends distally toward two pairs of setae-like spinnerets located on the preanal ring but displays no anatomical connection to the digestive tract. The transient proliferation of this tissue coincides temporally with the behavioral deposition of silk-like lining threads inside the fecal chamber, leading researchers to hypothesize that it functions as a temporary spinning gland. Because this gland regresses and becomes undetectable upon entering the rigidation period, and because no permanent cuticular ducts or dedicated extrusion muscles were detected, the physiological mechanism governing thread emission remains an open question requiring further biochemical and ultrastructural analysis.

The morphogenetic mechanisms documented in Niponia nodulosa illustrate an evolutionary strategy for postembryonic growth wherein segmentation is integrated with molting under spatial constraints. While segment addition in branchiopod crustaceans such as Thamnocephalus platyurus involves continuous posterior body elongation without molting or epidermal invagination, millipedes achieve segment proliferation inside an intact, rigid exoskeleton by folding the epidermis internally before shedding the cuticle. By providing a three-dimensional morphological framework for anamorphic ring formation, this study establishes a structural foundation for future genetic and cellular analyses of postembryonic development across arthropods.

Subject of Research: Morphogenetic and muscular mechanisms of postembryonic segment addition during anamorphosis in millipedes

Article Title: Epidermal invagination and muscle rearrangement associated with the segment addition during anamorphosis in a millipede, Niponia nodulosa

Article References: Chiyoda, S., Oguchi, K., Aonuma, H., & Miura, T. (2026). Epidermal invagination and muscle rearrangement associated with the segment addition during anamorphosis in a millipede, Niponia nodulosa. Developmental Biology Advances, 17(1), Article 1. https://doi.org/10.1186/s13227-025-00257-3

Image Credits: AI Generated

DOI: 10.1186/s13227-025-00257-3

Keywords: Niponia nodulosa, anamorphosis, millipede development, Diplopoda, arthropod segmentation, epidermal invagination, muscle rearrangement, ring primordium, X-ray micro-computed tomography, apolysis, telson musculature, molting chamber, Cryptodesmidae, postembryonic development, teloanamorphosis, spinning gland