BackgroundSeveral independent meiofaunal lineages are suggested to have originated through progenesis, however, morphological support for this heterochronous process is still lacking. Progenesis is defined as an arrest of somatic development (synchronously in various organ systems) due to early maturation, resulting in adults resembling larvae or juveniles of the ancestors. Accordingly, we established a detailed neuromuscular developmental atlas of two closely related Dinophilidae using immunohistochemistry and CLSM. This allows us to test for progenesis, questioning whether i) the adult smaller, dimorphic Dinophilus gyrociliatus resembles a younger developmental stage of the larger, monomorphic D. taeniatus and whether ii) dwarf males of D. gyrociliatus resemble an early developmental stage of D. gyrociliatus females.ResultsBoth species form longitudinal muscle bundles first, followed by circular muscles, creating a grid of body wall musculature, which is the densest in adult D. taeniatus, while the architecture in adult female D. gyrociliatus resembles that of prehatching D. taeniatus. Both species display a subepidermal ganglionated nervous system with an anterior dorsal brain and five longitudinal ventral nerve bundles with six sets of segmental commissures (associated with paired ganglia). Neural differentiation of D. taeniatus and female D. gyrociliatus commissures occurs before hatching: both species start out forming one transverse neurite bundle per segment, which are thereafter joined by additional thin bundles. Whereas D. gyrociliatus arrests its development at this stage, adult D. taeniatus condenses the thin commissures again into one thick commissural bundle per segment. Generally, D. taeniatus adults demonstrate a seemingly more organized (= segmental) pattern of serotonin-like and FMRFamide-like immunoreactive elements. The dwarf male of D. gyrociliatus displays a highly aberrant neuromuscular system, showing no close resemblance to any early developmental stage of female Dinophilus, although the onset of muscular development mirrors the early myogenesis in females.ConclusionThe apparent synchronous arrest of nervous and muscular development in adult female D. gyrociliatus, resembling the prehatching stage of D. taeniatus, suggests that D. gyrociliatus have originated through progenesis. The synchrony in arrest of three organ systems, which show opposing reduction and addition of elements, presents one of the morphologically best-argued cases of progenesis within Spiralia.

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Dinophilus gyrociliatus is a typical representative of the synthetic group Archiannelida. Here we describe the structure of Dinophilus young adults ventral nerve cords. Five nerve cords, irregular commissures and intersegmental nerves are revealed by tubulin and FMRFamide immunostaining. 5-HT-stainning marks the complex varicose nerve plexus with numerous irregularly distributed small perikarya. Four nerve cords and solitary cell bodies occur after clyoxylic-induced reaction for catecholamines visualization. Altogether, our results indicated that morphology of Dinophilus ventral nerve cords differs from the Polychaetes archetype in many respects.

Despite the increasing data concerning the structure of the adult nervous system in various Lophotrochozoa groups, the early events during the neurogenesis of rare and unique groups need clarification. Annelida are a diverse clade of Lophotrochozoa, and their representatives demonstrate a variety of body plans, lifestyles, and life cycles. Comparative data about the early development are available for Errantia, Sedentaria, Sipuncula, and Palaeoannelida; however, our knowledge of Dinophiliformia is currently scarce. Representatives of Dinophiliformia are small interstitial worms combining unique morphological features of different Lophotrochozoan taxa and expressing paedomorphic traits. We describe in detail the early neurogenesis of two related species: Dimorphilus gyrociliatus and Dinophilus vorticoides, from the appearance of first nerve cells until the formation of an adult body plan. In both species, the first cells were detected at the anterior and posterior regions at the early trochophore stage and demonstrated positive reactions with pan-neuronal marker anti-acetylated tubulin only. Long fibers of early cells grow towards each other and form longitudinal bundles along which differentiating neurons later appear and send their processes. We propose that these early cells serve as pioneer neurons, forming a layout of the adult nervous system. The early anterior cell of D. vorticoides is transient and present during the short embryonic period, while early anterior and posterior cells in D. gyrociliatus are maintained throughout the whole lifespan of the species. During development, the growing processes of early cells form compact brain neuropile, paired ventral and lateral longitudinal bundles; unpaired medial longitudinal bundle; and commissures in the ventral hyposphere. Specific 5-HT- and FMRFa-immunopositive neurons differentiate adjacent to the ventral bundles and brain neuropile in the middle trochophore and late trochophore stages, i.e. after the main structures of the nervous system have already been established. Processes of 5-HT- and FMRFa-positive cells constitute a small proportion of the tubulin-immunopositive brain neuropile, ventral cords, and commissures in all developmental stages. No 5-HT- and FMRFa-positive cells similar to apical sensory cells of other Lophotrochozoa were detected. We conclude that: (i) like in Errantia and Sedentaria, Dinophiliformia neurogenesis starts from the peripheral cells, whose processes prefigure the forming adult nervous system, (ii) Dinophiliformia early cells are negative to 5-HT and FMRFa antibodies like Sedentaria pioneer cells.

Immunochemical detection of serotonin (5-HT) and tubulin in D. gyrociliatus and D. taeniatus revealed similar anatomical structures of ventral locomotory ciliary field and underlying 5-HT-immunoreactive nerve network in both young juvenile and adult worms. External application of 5-HT and serotonin biochemical precursor (5-HTP) resulted in increased level of 5-HT within nervous structures in both juveniles and adults. However, while 5-HT and 5-HTP increased speed of gliding locomotion in juvenile worms, the same concentration of drugs decreased significantly or did not affect locomotion of adults. Thus, 5-HT activates the gliding locomotion in juvenile Archiannelid worms similar to larvae of annelids, molluscs and flatworms which use cilia for locomotion. Altered mode of 5-HT action on the locomotion of adult Archiannelid worms indicates reorganization of the 5-HT cilia controlling system during Dinophilid maturation. How to cite this article: