Comparison of neuromuscular development in two dinophilid species (Annelida) suggests progenetic origin of Dinophilus gyrociliatus

Alexandra Kerbl; Elizaveta G. Fofanova; Tatiana D. Mayorova; Elena E. Voronezhskaya; Katrine Worsaae

doi:10.1186/s12983-016-0181-x

Research
Open Access

Comparison of neuromuscular development in two dinophilid species (Annelida) suggests progenetic origin of Dinophilus gyrociliatus

Alexandra Kerbl†¹View ORCID ID profile,
Elizaveta G. Fofanova†²,
Tatiana D. Mayorova^{2, 3}Email author,
Elena E. Voronezhskaya² and
Katrine Worsaae¹Email author View ORCID ID profile

†Contributed equally

Frontiers in Zoology201613:49

https://doi.org/10.1186/s12983-016-0181-x

Received: 19 June 2016

Accepted: 20 October 2016

Published: 8 November 2016

Abstract

Background

Several 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.

Results

Both 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.

Conclusion

The 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.

Keywords

Meiofauna evolution
Paedomorphosis
Sexual dimorphism
Sister species
Nervous system
Musculature
Ciliation
Interstitial

Background

Meiofaunal life forms (specimens passing through a sieve with a mesh-size of 1 mm, while being retained on a sieve with 42 μm mesh-size, [1]) are represented in most extant macrofaunal bilaterian lineages as well as constituting numerous independent lineages (e.g., Acoela, Kinorhyncha, Gastrotricha, Gnathostomulida, etc. [2–4]). The meiofaunal lineages Gnathifera and Rouphozoa (with macroscopic forms of platyhelminths nested within) were recently shown to branch off first within Spiralia [3, 5]). As a consequence hereof, the ancestral spiralian condition might have been an acoelomate to pseudocoelomate, microscopic bodyplan with direct development (possibly inhabiting the interstitial realm) [3]. In Annelida, however, the most basally branching groups are macroscopic, therefore suggesting that meiofaunal groups such as the interstitial family Dinophilidae evolved by either gradual miniaturization or underdevelopment (paedomorphosis) [5, 6]. Paedomorphosis is caused by a change in developmental timing due to early offset (progenesis), late onset (post displacement) or slower developmental rate (neoteny). All these changes can be either local or global processes and result in the underdevelopment of either individual characters or sets of characters [7–21]. Global progenesis is considered a common pathway of the evolution of microscopic annelids from macroscopic juveniles, which grow up in the same interstitial environment between the sand grains. Progenesis hereby offers the possibility to become permanently small and colonize the favorable interstitial habitat through an inherited arrest of somatic growth in a larval or juvenile ancestor by a single speciation event, possibly initiated by an early maturation [5, 7–12, 14, 17, 22–29].

Dinophilidae has been discussed in early studies to represent ancestral features within Annelida, when it was considered an archiannelid lineage alongside other interstitial annelids, due to its’ members microscopic size and simple morphology [30–34]. It was later argued from morphological studies to have developed via progenesis from a primarily large ancestor because of its simple morphology and similarity to juveniles of macrofaunal families such as Dorvilleidae [10, 11, 14, 17, 22, 35–37]. The relationship of Dinophilidae to other annelids is still debated with a recent phylogenomic study [5], which is suggesting it to be part of the clade Orbiniida (with low support) together with the macrofaunal family Orbiniidae as well as the meiofaunal families Nerillidae, Parergodrilidae, Diurodrilidae and Apharyngtus. However, another study did not consider its position sufficiently supported [25], and none of the studies could determine the closest relative.

Dinophilids are 1 to 3 mm long, with all species counting 6 segments and lacking appendages, parapodia, and chaetae. They have externally indistinct segmentation, recognized only by the arrangement of transverse ciliary bands [32, 38, 39] and internal features such as lateral nerves, commissures, and nephridia [11, 40, 41]. The family Dinophilidae contains Trilobodrilus with six described species [30, 42–46] and Dinophilus, which is represented by approximately ten species [32, 38, 47–52], since the validity of several additional taxa is questioned due to ambiguous or insufficiently detailed morphological descriptions. Very few species have been barcoded, so further molecular sampling may reveal a higher cryptic diversity (Worsaae et al. unpublished). Two different morphotypes can be distinguished within Dinophilus: 1) monomorphic dinophilids with a long life cycle including an encystment stage for up to eight months [31, 53], 2) strongly dimorphic dinophilids with a rapid life cycle of only three weeks for adult females and less than a week for dwarf males [54, 55]. The dimorphic type has “normal-sized” females and miniature dwarf males [56–59], while in the monomorphic species the sexes cannot be distinguished from each other by outer morphological characters [56, 57]. Development in both morphotypes is direct, as found in most meiofaunal species, but different to the indirect life cycle of the annelid species used for developmental studies so far (e.g. Capitella teleta [60–62], Platynereis sp. [63, 64]). While the five to seven species of the monomorphic, bigger, orange type are limited to shallow colder waters of the arctic, subarctic and boreal coasts of e.g. Newfoundland, Greenland, Sweden, Denmark, Great Britain, and Russia [33, 38, 39, 48, 53], the hyaline, smaller, dimorphic type can be found in both boreal and temperate waters such as in Denmark [55, 58], France (pers. obs.), the Mediterranean [40], Brazil [65], North Carolina [66] and China [67]. Both monomorphic and dimorphic species of Dinophilus are found in the intertidal and subtidal region, where they are grazing on biofilm and small algae overgrowing macroalgae or in the interstices among sand grains in shallow waters [30, 34, 54, 68].

Despite several anatomical studies [11, 32, 40, 41, 56, 57, 69–72], little is known about the neuromuscular development in Dinophilus, which is thoroughly assessed in this study. However, previous studies did already assess the adult stages, stating that the musculature consists mainly of the pharyngeal [73] and body wall musculature, which is specified as layers of circular, diagonal, and longitudinal musculature [39]. The nervous system has likewise been investigated in mainly adults, assessing the relatively simple brain and a ventral nervous system consisting of five to seven longitudinal nerve cords [11, 40, 41, 71]. These are connected by one (in monomorphic) or three commissures (in dimorphic species) per segment, respectively. The neuromuscular system in D. gyrociliatus dwarf males is altered significantly from the pattern seen in females and also in other dinophilid males [56, 57]. Based on their diminutive size and ciliary pattern, they have been proposed to resemble a trochophore larva [56, 57], the resemblances however seem to be superficial.

Due to the size and morphological differences between the two morphotypes, it is proposed in this study that the smaller and simpler built D. gyrociliatus Schmidt, 1857 as representative of the dimorphic, fast developing morphotype has originated through a second progenetic process from the possibly already paedomorphic ancestor of Dinophilidae, which was most likely resembling the more complex and larger forms found in D. taeniatus. The evolutionary unravelling of D. gyrociliatus is further complicated by their possession of dwarf males, since males of D. gyrociliatus-ancestors probably have undergone a separate or ‘third’ progenesis relative to the females [58, 74], while D. taeniatus Harmer 1889 as representative of the monomorphic group with prolonged life cycle as well as the related dinophilid taxon, Trilobodrilus, have “normal-sized” males [31, 53, 75].

We hereby aim to establish a reference model for direct developing meiofaunal annelids by examining the neuromuscular system and its development in both sexes of D. gyrociliatus and D. taeniatus with immunohistochemistry and confocal laser scanning microscopy (CLSM), thereby also facilitating comparison across species and sexes. We will further examine whether the seemingly simpler morphology in female D. gyrociliatus reflects earlier developmental stages of D. taeniatus and whether the dwarf males resembles even earlier developmental stages of females, hereby seeking support for the hypotheses on a progenetic origin of the male and female D. gyrociliatus.

Methods

Specimens

Two different populations of Dinophilus gyrociliatus (originally from Xiamen, China and Naples, Italy) and two different populations of D. taeniatus (collected at the White Sea, Russia and in Quequertarsuaq, Disko Island, Greenland) were examined in the present study. No significant morphological intraspecific variations were detected between the populations. The presented illustrations are mainly based on D. gyrociliatus from lab cultures originally from China and D. taeniatus collected at the White Sea, Russia.

Dinophilus gyrociliatus

One culture of D. gyrociliatus was established by Bertil Åkesson at University of Gothenburg in the 1980’s from specimens sampled in Xiamen, China. A subsample of this culture is now kept at the Marine Biological Section, University of Copenhagen, Denmark, where the animals are maintained in seawater (salinity 28‰) at 18 °C and fed spinach twice a month after exchanging the water. Another culture of D. gyrociliatus (originally sampled in Naples, Italy) is kept in the institute of Developmental Biology RAS, Moscow, Russia. The worms are cultured in artificial seawater with 33‰ salinity at 20 °C and fed nettle once a week after exchanging the water.

For establishing the life cycle and stage-specific sampling, some females were separated from the main culture and checked on a daily basis. Newly laid cocoons were transferred to dishes, tracked and fixed after two days and subsequently every 12 h until hatching (after six days) for the establishment of the developmental series.

Dinophilus taeniatus

The Greenlandic specimens of Dinophilus taeniatus were obtained during a field trip to Disko Island, Southwest Greenland, from the shallow waters in the intertidal region in Quequertarsuaq harbour. The Russian specimens of D. taeniatus were obtained at the Pertsov White Sea Biological Station (White Sea, Russia). The worms were collected during low tide at the upper sublittoral zone. The culture of D. taeniatus was reared in the laboratory in natural filtered seawater at 10 °C and was checked twice a day for the presence of cocoons.

The cocoons were transferred to separate Petri dishes and kept in filtered seawater until fixation after four days and then every 24 h until hatching (approximately after 21 days). Juvenile and adult stages were also fixed similar to D. gyrociliatus.

Embryonic development is characterized by different duration of respective stages. We therefore use morphological markers (internal and external ciliary structures such as ciliary bands and ventral ciliary field, musculature and nervous system) and the sequence of their formation to compare the stages of the neuromuscular system in both morphotypes.

Staging of dinophilid development

We categorized Dinophilus development into 5 stages: early embryo (2.5–3 days after cocoon deposition in D. gyrociliatus and 5–6 days after cocoon deposition in D. taeniatus), late embryo (several ciliary bands and the ventral ciliary field developed, 4.5 days in D. gyrociliatus and 10–14 days in D. taeniatus,), prehatching/hatching (just before hatching from the fertilization envelope and – later on – the cocoon, 5.5–6 days in D. gyrociliatus and 14–21 days in D. taeniatus), juvenile (6.5–12 days in D. gyrociliatus and 21–40 days in D. taeniatus) and adult (12 and more days in D. gyrociliatus and 40 and more days in D. taeniatus,). The morphology is described in detail for D. gyrociliatus females and description of D. taeniatus is mainly focused on differences and similarities.

Immunohistochemistry and confocal laser scanning microscopy (CLSM)

Specimens (at least ten specimens per stage and used antibody) were anesthetized with isotonic MgCl₂ prior to fixation with 3.7 % paraformaldehyde in phosphate buffered saline (PBS, pH 7.4) at room temperature (RT); embryos were manually extracted from the cocoon and the fertilization envelope prior to fixation. Double as well as quadruple stainings were applied to investigate characters in the muscular, nervous, and ciliary system. These stainings included F-actin staining (Alexa Fluor 488-labelled phalloidin, A12379, INVITROGEN, Carlsbad, USA), DNA-staining (405 nm fluorescent DAPI, included in the embedding medium Vectashield) and immunostaining (monoclonal mouse anti-acetylated α-tubulin (T6793, SIGMA, St. Louis, USA), polyclonal anti-mouse anti-tyrosinated tubulin (T9028, SIGMA), polyclonal rabbit anti-serotonin (5-HT, S5545, SIGMA) and anti-FMRFamide (20091, IMMUNOSTAR, Hudson, USA)). Prior to adding the primary antibody-mix, the samples were preincubated with 1 % PBT (PBS + 1 % Triton-X, 0.05 % NaN₃, 0.25 % BSA, and 5 % sucrose). Afterwards, samples were incubated for up to 24 h at RT in the primary antibodies mixed 1:1 (in a final concentration of 1:400). Subsequently, following several rinses in PBS and 0.1 % PBT, specimens were incubated with the appropriate secondary antibodies conjugated with fluorophores (also mixed 1:1, in a final concentration of 1:400, goat anti-mouse labelled with CY5 (115-175-062, JACKSON IMMUNO-RESEARCH, West Grove, USA), goat anti-rabbit labelled with TRITC (T5268, SIGMA)) for up to 48 h at RT. This step was followed by incubation for 60 min in Alexa Fluor 488-labeled phalloidin solution (0.33 M phalloidin in 0.1 % PBT) after and prior to several rinses in PBS. Thereafter, specimens were mounted in Vectashield (including DAPI, VECTOR LABORATORIES, Burlingame, USA). The prepared slides were examined using an OLYMPUS IX 81 inverted microscope with a Fluoview FV-1000 confocal unit at the Marine Biology Section of the University of Copenhagen (property of K. Worsaae) and a Nikon A1 CLSM at the White Sea Biological Station. Acquired z-stacks were exported to the IMARIS 7.0 (BITPLANE SCIENTIFIC SOFTWARE, Zürich, Switzerland) software package to conduct further three-dimensional investigations and prepare representative images.

Image processing

Brightness, saturation, and contrast were adjusted in Adobe Photoshop CC 2015 (ADOBE Systems Inc., San Jose, USA) prior to assembling figure plates in Adobe Illustrator CC 2015, where also schematic drawings were created.

Results

Overall morphology and life cycle

Dinophilus gyrociliatus females and dwarf males

The adult female’s body is cigar-shaped, ranges in length between 1.0 to 1.5 mm and has a diameter of approximately 75–150 μm (Fig. 1a). The body is very hyaline and therefore internal organs such as the digestive system with the prominent pharyngeal bulb as well as developing eggs can be seen (Fig. 1a). One transverse ciliary band is found per segment in this species (cb, Fig. 1a). Adult dwarf males are about 50 μm in length and 20 μm in width with a roughly elongated ovoid shape (Fig. 1b). They do not form a digestive system, but the penile region with the muscular copulatory organ is prominently developed (co, Fig. 1b). The deposited cocoons contain several big female and small male eggs (dm labelling the male egg developing into a dwarf male, Fig. 1c), which are present in an average ratio of 1 male (dm, Fig. 1c, d) to 2–4 female eggs ([46], Fig. 1c, d), with the cocoons containing at least one male and one female egg. Male and female eggs retain their size difference throughout development.

Fig. 1
Light microscopic pictures of different life stages of *Dinophilus gyrociliatus* and *D. taeniatus*. Stages are indicated by silhouettes (*D. gyrociliatus* in white and *D. taeniatus* in orange), and the assignment to the respective stage next to them. Double-arrows indicate the antero-posterior axis (a-p) in the animals at prehatching stage. a-e *Dinophilus gyrociliatus*, a adult female, dorsal view, b dorsal view of an adult dwarf male, c cocoon with female embryos and dwarf males at 2 days after deposition, d cocoon with females and one dwarf male at 5 days after deposition (prehatching embryos), e early juvenile female, dorsolateral view, f-h *D. taeniatus*, f copulating male (on the left side) and female (on the right side), dorsal view, g encysted worm, h female next to a cocoon with eight eggs in dorso-lateral view, i-l embryogenesis, i two blastomere-stage with the apical pole up, j morula stage, k postgastrulation stage in ventral view, l prehatching embryo curling inside fertilization envelope with its anterior end up, m juvenile in dorsal view. Abbreviations: avcf – anteroventral ciliary field, bl – blastomere, c – cyst, cb - ciliary band, cch – compound cilia of the head, co – copulatory organ, coc – cocoon, dm – dwarf male, en – fertilization envelope, hg – hindgut, mam – macromere, mim – micromere, mo – mouth opening, np – neuropil, pcb – prostomial ciliary bands, phb – pharyngeal bulb, pro - prostomium, pyg – pygidium, s – sperm, sto – stomach, y – yolk

In contrast to the females, which were observed to hatch from the eggs after six days and mature afterwards, the dwarf males are already mature when hatching from the fertilization envelope (approximately five days after the cocoon has been deposited and before females hatch) and die one or two days later, after they fertilize the females inside the same cocoon and females passing in close proximity to the opened cocoon. Hereby the male has been observed to penetrate the body wall of the female and transfer sperm underneath the epidermis of the female in the posterior body region, where it is stored until the latter have developed eggs [76]. Hatching females and early juveniles are elongated and thin, with the individual ciliary bands located on broader body regions and thereby showing serial arranged structures (e.g. ciliary bands and intersegmental furrows) that suggest segmental arrangement of organ systems (Fig. 1e), while this pattern is continuously obscured when juveniles start feeding and extend their body circumference. The transition between juveniles and mature animals is fluent, with adults carrying eggs and dilating the posterior body region in the process (Fig. 1a).

The entire life cycle of the females from the time the cocoon is deposited to the time when the adult females lay their own cocoons takes a maximum of three weeks, including one week of embryonic development inside the cocoon (Fig. 1c-e).

Dinophilus taeniatus females and males

The external morphology in both males and females is similar to the one of D. gyrociliatus females described above, though the animals are larger (body length 2.3–3.1 mm, body width 100–300 μm, Fig. 1f). In contrast to D. gyrociliatus, the body of this species is strongly pigmented (animals are bright orange, Fig. 1f-m), differences between the sexes cannot be defined by outer morphology at any stage, except when females are carrying eggs. In contrast to D. gyrociliatus, where the dwarf male mainly fertilizes (pre-)hatching females of the same cocoon, copulation in D. taeniatus occurs in adults after hatching. When copulating (Fig. 1f), the male penetrates the body wall of the female with the penis. After a certain period of time, when encystment may take place (Fig. 1g), the female deposits a cocoon with eggs of both sexes, which cannot be distinguished by neither size, nor organization, nor colouration (Fig. 1h). Embryonic development takes two to three weeks; cleavage starts right after oviposition. Dinophilus is in general characterized by unequal, holoblastic spiral cleavage (Fig. 1i) resulting in a morula stage (Fig. 1j). The mouth opening is formed during gastrulation (mo, Fig. 1k). The embryos elongate and curl up inside their fertilization envelope at prehatching stage with their ventral side facing the fertilization envelope (Fig. 1l). The developmental sequence (i.e. the sequence of formation of musculature, ciliary structures and nerves) resembles that of female D. gyrociliatus, and thereby enables comparisons between specific stages. Similar to D. gyrociliatus (1A, E), the juvenile leaving the fertilization envelope resembles the adult (Fig. 1f, m).

Musculature

Dinophilus gyrociliatus females

Embryonic development

Body wall musculature. The first signs of muscular development can be detected after gastrulation (approximately 1.5–2 days after cocoon deposition), when a pair of ventrolateral longitudinal muscles (vllm) forms posterior to the mouth opening and then extends towards the anterior and the posterior end of the body (Fig. 2a, b). Subsequently, additional fibres join these, and a dorsolateral pair of longitudinal muscles (dllm, Fig. 2b) is formed, as well as a muscular ring around the mouth opening (mrmo, Fig. 2 b). Prior to elongation and curling of the animal inside the fertilization envelope, a pair of ventral longitudinal muscle bundles (vlm) is developed (Fig. 2c). They move medially and converged along the midline, embracing both the mouth opening (mo) and the ventral side of the developing pharyngeal bulb (phb, Fig. 2c). All longitudinal muscle bundles extend anteriorly into the prostomium, where they ramify towards the periphery, though their exact paths cannot be unravelled in early stages (Fig. 2c).

Fig. 2
Myogenesis in *Dinophilus gyrociliatus* females. Phalloidin-labelled actin-filaments shown in green, labelling of DNA with DAPI shown in blue, animals are oriented with the anterior end up (a-e, g, h) or to the left (f). Stages are indicated by silhouettes next to the figure capture, and the assignment to the respective stage next to them. The first signs of difference between the two species *D. gyrociliatus* and *D. taeniatus* are emphasized by a yellow dashed-lined frame around the picture. a Ventral view of the onset of myogenesis in the early embryo (3 days after the egg is deposited), b Ventral view of the female *D. gyrociliatus* with ventrolateral and dorsolateral longitudinal muscles developed in the early embryo (3.5–4 days after the egg is deposited), c ventral view of the exogastrically curled females in the late embryo (5–5.5 days after the egg is deposited), d prehatching females (left female still curled exogastrically inside the egg layer, right female free inside the cocoon with the dorsolateral side up, 5.5–6 days after the egg is deposited), e dorsal view of the head musculature in an early juvenile female, f dorsoventral view of the trunk musculature with longitudinal, circular and diagonal elements in an early juvenile female, g ventral view of the head musculature in an adult female, h lateral view of the posterior region of an adult female. Abbreviations: cb1-2 –ciliary band 1–2, cm – circular muscle, cmds – circular muscle of the digestive system, dlca – contralatero-anterior branch of the dorsolateral longitudinal muscle, dlcb – contralateral branch of the dorsolateral longitudinal muscle, dldb – dorsal branch of the dorsolateral longitudinal muscle, dlia – ipsilatero-anterior branch of the dorsolateral longitudinal muscle, dlib – ipsilateral branch of the dorsolateral longitudinal muscle, dllm – dorsolateral longitudinal muscle, dlvb – ventral branch of the dorsolateral longitudinal muscle, dm – diagonal muscle, lmds – longitudinal muscle of the digestive system, mo – mouth opening, mrmo – muscular ring around the mouth opening, np – neuropil, phb – pharyngeal bulb, phm – pharyngeal muscle, pyg – pygidium, sm – sigmoid muscle, vca - contralatero-anterior branch of the ventral longitudinal muscle, vcf – ventral ciliary field, vlcb – contralateral branch of the ventrolateral longitudinal muscle, vldb – contralatero-dorsal branch of the ventrolateral longitudinal muscle, vldb – dorsal branch of the ventrolateral longitudinal muscle, vlib – ipsilatero-anterior branch of the ventrolateral longitudinal muscle, vllm – ventrolateral longitudinal muscle, vlm – ventral longitudinal muscle, vlvb – ventral branch of the ventrolateral longitudinal muscle

First fragments of circular muscles (cm) start forming at the ventral side external to the longitudinal muscles at the same time as the ventrolateral and dorsolateral muscle bundles can be detected (Fig. 2a, b). Though several circular muscles are now added from anterior to posterior, they are incomplete in the earlier developmental stages (Fig. 2a-c), extending from the ventral towards the dorsal side, where they finally fuse at prehatching stage (Fig. 2d). In the late embryo stage, the circular muscles are forming an almost continuous sheath (Fig. 2c), which is not retained in later stages as the distance between the circular muscles increases (Fig. 2d-g).

Prostomial musculature. At the onset of muscular development, no muscles are formed anterior to the mouth opening. The developing brain and it neuropil, however, seem to be labelled by phalloidin, too, which is probably reacting to neuronal f-actin as was already shown previously in a wide range of animals such as molluscs [77, 78] and crustaceans [79] (Fig. 2a-c), and therby not related to musculature. Later on, the longitudinal muscle bundles of the posterior part of the body extend anteriorly (Fig. 2c), where they ramify and are joined by muscles emerging from the muscular ring around the mouth opening (mrmo, Fig. 2c). Supplementing these ramifications of the longitudinal muscles, three circular muscles are formed in the developing prostomium, which can be detected external to the attachment sites of the branching longitudinal muscles (cm1-3, Fig. 2c, d). During earlier developmental stages, the musculature is mainly dorsal to the neuropil (Fig. 2c), but also extends ventrally around the brain during subsequent stages (Fig. 2d, e, g, h). A more complete assessment of the pattern is possible in the hatching and juvenile stages (see below).

Musculature of the digestive system. The pharyngeal bulb (phb) is the most prominent and first developed part of the musculature of the digestive system emerging rather late in embryogenesis (approximately four days after cocoon deposition, Fig. 2c). The pharyngeal bulb itself consists of a tightly arranged stack of 27 plate-shaped muscle cells and dorsal and ventral longitudinal muscles [72], and is located posterior to the mouth opening (Figs. 2d).

The pharyngeal region differentiates in the developing embryo (Fig. 2b), and though cellular changes can be observed starting with the invagination of the mouth, muscular details can be detected much later. Similarly, the gut shows cellular differentiation of the adjacent cells prior to the formation of longitudinal and circular muscles, which can be detected after the formation of the pharyngeal bulb (4.5–5 days after the eggs have been deposited, Fig. 2d). However, the denser muscular layer of the body wall complicates the identification of the thin musculature of the alimentary channel. Compared to the longitudinal and circular muscles of the body wall, the respective elements in the digestive system were observed represented by one or two fibres only and spaced further apart.

Hatching & early juvenile stages

Body wall musculature. The layout of the longitudinal and circular muscles does not change significantly from the pattern detected during embryonic development, since only the dorsolateral longitudinal muscle bundles (dllm, Fig. 2d, f), which have been (ventro-)lateral in earlier stages, are shifted to the dorsal side. Internal to the longitudinal muscles, diagonal muscles (dm) are formed, which wind spiral-like around the body, starting at the level of the mouth opening and extending towards the posterior end of the animal (Fig. 2f). Their pattern does not seem to be fixed in development, since the muscles are arranged parallel to each other in some animals without any chiasmata, while the fibres are crossing each others’ paths more regularly in others.

Prostomial musculature. The musculature in the prostomium gets more defined, with additional fibres extending from the longitudinal muscle bundles on the ventral side of the body more dorsally, but also extending from the pharyngeal bulb to both the ventral and the dorsal side of the body (Figs. 2d, e, 3a, c). The circular muscles of the prostomium, in contrast to those of the body, consist of several fibres (two to seven, Figs. 2d, e, 3a, c). The ventrolateral longitudinal muscle bundles (vllm) extend ventrolaterally in a straight line to the level of the third circular muscular ring, where they then split into several branches of different thickness: The thinnest strand consists of one to a maximum of three fibres and extends ventrally to the prostomial epidermis anterior to the second circular muscle band (vlib, Figs. 2d, e, 3a). An additional muscle strand extends to the anterior tip ipsilateral to the midline (vlia, Fig. 3c). Furthermore, one strand extends contralaterally and connects to the epidermis at the level of the first circular muscle (vlca, Fig. 2e), and a short strand is directed more posterior and to the ventral side (vlvb, Fig. 2d, e).

Fig. 3
Musculature of the digestive system in juvenile *Dinophilus gyrociliatus* females. Phalloidin-labelled actin-filaments shown in green, labelling of DNA with DAPI shown in blue, animals are oriented with the anterior end up. Stages are indicated by silhouettes next to the figure capture, and the assignment to the respective stage next to them. a horizontal section through a juvenile female at the level of the sigmoid muscle, b detail of the pharyngeal bulb in dorsal view, c dorsal view of the head and pharyngeal musculature, d dorsal view of the posterior part of the body with sigmoid muscle and injected sperm lateral in an early juvenile female. Abbreviations: an – anus, cm – circular muscle, cmds – circular muscle of the digestive system, dlcb – contralateral branch of the dorsolateral longitudinal muscle, fmg – foregut-midgut transition, hg – hindgut, lmds – longitudinal muscle of the digestive system, mht – midgut-hindgut transition, mo – mouth opening, mrmo – muscular ring around the mouth opening, phb – pharyngeal bulb, phm – pharyngeal muscle, s – sperm, sm – sigmoid muscle, vcb – contralateral dorsal branch of the ventral longitudinal muscle, vlcb – contralateral branch of the ventrolateral longitudinal muscle, vlcb – contralatero-dorsal branch of the ventrolateral longitudinal muscle, vlia – anterior ipsilaterial branch of the ventrolateral longitudinal muscle, vlib – ipsilatero-anterior branch of the ventrolateral longitudinal muscle, vllm – ventrolateral longitudinal muscle, vlvb – ventral branch of the ventrolateral longitudinal muscle

The paths of the ventral and dorsolateral muscle bundles are less complex, but also show one to two splits: the dorsolateral muscle bundle bifurcates already anterior to the pharyngeal bulb into two strands of similar thickness, which are extending to the dorsolateral and ventrolateral side of the prostomium to the level of the first circular muscle. While one bundle is crossing the midline of the body and remains dorsolateral, extending contralaterally to the anterior tip (dlca, Fig. 2d, e), the lateral proportion extends ipsilateral to the first circular muscle (dlia, Fig. 2d, e). The third bundle is located most medial and extends contralaterally to the third circular muscle (dlcb, Figs. 2d, 3c). The ventral muscle bundles also split and traverse from the ventral to the dorsal side, also forming two furcations at this stage: a contralateral bundle extending to the epidermis at the level of the first circular muscle (vca, Fig. 3c) and another contralateral bundle extending to the level posterior to the third circular muscle (vcb, Fig. 3a). This pattern, once developed, can also be found in adult specimens of D. gyrociliatus female (Fig. 2g), though they get more refined and further splits are added.

Musculature of the digestive system. Additional circular fibres (cmds) form external to the longitudinal muscles of the gut musculature (lmds), extending from the mouth opening to the dorsal anus (Fig. 3a, c, d). Besides the muscular pharyngeal bulb, only a thin muscular ring is formed around the mouth opening (mrmo, Fig. 3a, c) and several thin circular fibres (spaced closer together than in the stomach (= midgut) and hindgut) are detected in the foregut (Fig. 3c). A thin muscular ring is formed around the anus (Fig. 2h). Additionally, an unpaired muscle traces the hindgut from the midgut-hindgut-transition to the ventral side anterior to the anus (sigmoid muscle – sm, Figs. 2h, 3d).

Adult

Body wall musculature. In contrast to D. taeniatus, where the multiple longitudinal muscle fibres can be seen spread along the entire body circumference (Fig. 5e, g, h), only six bundles are present in adult D. gyrociliatus females (one pair of dorsolateral, ventrolateral and ventral longitudinal muscles, Fig. 2h). All of them converge towards the posterior end of the body, where they seem to end blindly (Fig. 2h). Young adults can show a high number of diagonal muscles (dm, Fig. 2h), though this does not seem to be a fixed morphology.

Prostomial musculature. The muscles and their furcations as described in the juvenile stage get more defined (Fig. 2g).

Musculature of the digestive system. The gut musculature forms a thin layer of longitudinal and circular muscles (lmds, cmds, respectively, Fig. 2h). The latter are set further apart than the circular muscles of the body wall. The sigmoid muscle as described in females at hatching or juvenile stage extends ventrally in the hindgut, ending ventral close to the anus (sm, Fig. 2h).

The pharyngeal bulb is strongly connected to various muscles in the prostomial region, which are anchored in the epidermis of the prostomium (Fig. 2g). Additionally, the pharynx and the foregut are characterized by a series of circular muscle fibres positioned closely together, which cannot be observed in the posterior region of the digestive system.

Dinophilus gyrociliatus dwarf males

The onset of muscular development seems to be similar to the onset observed in females with longitudinal fibres emerging as two ventrolateral (vllm) pairs from the ventroanterior point of muscular origin (vpmo, Fig. 4a-c). In contrast to females, dwarf males do not develop a digestive system and a stomodeum could not be observed. We therefore used the formation of the anterior ciliary field (see below for a more detailed description) on the ventroanterior side of the animal as well as the pair of dorsomedian nephridia and the formation of the distinctive penile musculature as landmarks. During subsequent muscular growth and differentiation, the ventroanterior point of origin of muscles gets more refined and changes from an undefined mass (Fig. 4a, b) into the triangular form that can be observed in the adults (Fig. 4d, e). While the ventrolateral muscles line the lateral sides of the body, the dorsal fibres (dlm) extend dorsally as one bundle and bifurcate posterior to that (Fig. 4b, c). As they continue extending towards the posterior end of the body, where the copulatory organ is formed in subsequent steps, circular muscles (cm) are added in an anterior-to-posterior pattern (Fig. 4b, c). These structures consist of individual fibres, which emerge from the ventral side of the animal distal to the longitudinal muscle and extend further dorsally, until they fuse and form a ring (Fig. 4c-e), similar to the pattern seen in females (Figs. 2, 5).

Fig. 4
Muscular development in dwarf males of *Dinophilus gyrociliatus*. Phalloidin-labelled actin-filaments shown in green, labelling of DNA with DAPI shown in blue, animals are oriented with the anterior end up in dorso-ventral view (A-C), or to the left and in lateral view (D-I). a dorsal view of an one day old dwarf male with the anterior point of muscular origin formed; b dorsal region of the longitudinal and circular fibres forming in a two day old dwarf male in dorsal view; c ventral region of longitudinal and circular fibres in a two day old dwarf male, d musculature in a prehatching dwarf male; e-i section series with anterior to the left and posterior to the right through the copulatory organ in an adult dwarf male from the ventral (e) to the dorsal side (i), illustrating the penile cone (f, g) and the penile sheath (e-i). Abbreviations: alps – anterior loop of the outer sheath. cm – circular muscle, cmps – circular muscles of the penile sheath, dlm – dorsal longitudinal musculature, dlps – dorsal loop of the penile sheath, gpr – gonopore opening, lips – longitudinal inner muscles of the penile sheath, lops – longitudinal outer muscles of the penile sheath, pc – penile cone, pmr – posterior muscular ring, psm – penile sheath musculature, ros – ring around the gonopore formed by the outher sheath, test – testis, vllm – ventrolateral longitudinal muscle, vlps – ventral loop of the penile sheath, vpmo – ventroanterior point of muscular origin

Fig. 5
Myogenesis in *Dinophilus taeniatus*. Phalloidin-labelled actin-filaments shown in green, animals are oriented with the anterior end up (a-d, f-k) or to the left (e). Stages are indicated by silhouettes next to the figure capture, and the assignment to the respective stage next to them. The first signs of difference between the two species *D. gyrociliatus* and *D. taeniatus* are emphasized by a yellow dashed-lined frame around the picture. a-c embryonic development: a ventral view of an early embryo (5–6 days after eggs are deposited), b ventral view of a middle embryo (7–9 days after eggs are deposited), c ventral view of a prehatching embryo (10–14 days after eggs are deposited), d ventral view of an early juvenile animal, e-k adult worm: e ventrolateral view of the overall bodywall musculature, f detail of the head musculature in ventral view, g ventrolateral view of the body wall musculature in the trunk, h dorsolateral view of a virtually cropped stack of the caudal region of the body (arrowheads point at thin bundles of longitudinal muscles), i ventral view of the head (arrowhead points at basket-shaped musculature of the pharynx), j lateral view of the musculature in the digestive system in the posterior part of the body, k dorsal view of the copulatory organ and seminal receptacle. Abbreviations: alps – anterior loop of the penile sheath, an – anus, cm – circular muscle, cmds – circular muscle of the digestive system, cmps – circular muscles of the penile sheath, dlca – contralatero-anterior branch of the dorsolateral longitudinal muscle, dldb – dorsal branch of the dorsolateral longitudinal muscle, dlia – ipsilatero-anterior branch of the dorsolateral longitudinal muscle, dllm – dorsolateral longitudinal muscle, dlvb – ventral branch of the dorsolateral longitudinal muscle, dm – diagonal muscle, dsvp – muscular duct leading from the seminal receptacles to the penis, fmt – foregut-midgut transition, gpr – gonopore opening, lips – longitudinal inner muscles of the penile sheath, lm – longitudinal muscle, lmds – longitudinal muscle of the digestive system, lops – longitudinal muscles of the penile sheath, mo – mouth opening, mrmo – muscular ring around the mouth opening, np – neuropil, phb – pharyngeal bulb, phm – pharyngeal muscle, pyg – pygidium, sv – seminal vesicle, vca –contralatero-anterior branch of the ventral longitudinal muscle, vcb- – contralateral dorsal branch of the ventral longitudinal muscle, vlca – contralatero-anterior branch of the ventrolateral longitudinal muscle, vlcb – contralateral branch of the ventrolateral longitudinal muscle, vldb – dorsal branch of the ventrolateral longitudinal muscle, vlia – anterior ipsilaterial branch of the ventrolateral longitudinal muscle, vlib – ipsilatero-anterior branch of the ventrolateral longitudinal muscle, vllm – ventrolateral longitudinal muscle, vlm – ventral longitudinal muscle, vlvb – ventral branch of the ventrolateral longitudinal muscle

The penile region is the most prominent and complex muscular part in the dwarf males. It comprises a penile sheath (psm) with an organized inner and a meshwork-like outer layer (Fig. 4d-i). These components start to form after the ventrolateral longitudinal muscles have extended to the posterior end of the body and at least four circular muscles are formed. The penile sheath (psm) is joined by ventrolateral longitudinal muscles, which develop loop-like structures prior to additional details of the penile musculature (Fig. 4d). The dorsal longitudinal muscles join the structure prior to the development of the penile cone (pc), which forms independently of the penile sheath (Fig. 4f, g). Subsequently, the fibres forming the sheath start to get more defined and link the musculature of the copulatory organ to the ventrolateral and dorsal muscles of the body wall (Fig. 4d). At the same time, all six circular fibres have formed and the male starts moving (i.e. stretching and compressing) inside the egg layer, using motile cilia and muscular contractions (approximately half a day to a day before hatching, Fig. 4d).

Musculature of the copulatory organs in adults. The innermost layer of the penile sheath is dominated by ten to twelve fibres, which are arranged in a horizontal pattern extending from the anteriormost onset of the penile sheath to the most posterior point (lips, Fig. 4f, h). These muscles are weakly labelled with phalloidin, especially when compared to the strong labelling of the penile cone musculature and the outer layer and tightly enclose the penile cone (Fig. 4f, g). In the posterior part, muscle fibres form a ring, which is adjacent to the gonopore (gpr, Fig. 4g).

The outer layer of the sheath consists of circular (cmps) and longitudinal muscle fibres (lops), which are separate from the muscles of the body wall (Fig. 4e-h). In the most anterior part, several projections radiate into the body, but their ends could not be traced successfully in all specimens. In contrast to the organized pattern observed in the inner sheath, muscles in the outer sheath have a network-like appearance. Most obvious is a posterior ring formed by the fusion of ventrolateral longitudinal muscles with the penile sheath (ros, Fig. 4i). Furthermore, this ring encloses another ring-like structure, which is formed by the inner sheath.

Each of the ventrolateral longitudinal muscles of the body wall forms a bifurcation anterior to the penile sheath, so two smaller bundles can fuse with the sheath on each side. While the thinner part of this bifurcation seems to fuse with the posterior ring, the more prominent part terminates lateral at the penile sheath after forming a loop on each side of the animal (vlps, Fig. 4f). The dorsal longitudinal muscles contribute to the outer sheath by forming loops laterodorsal at the external surface. These loops join at the approximate body midline (dlps, Fig. 4i). Furthermore, the longitudinal muscular strands extend and form an additional loop anterior to the anterior loop of the outer sheath before bifurcating and merging with the sheath (alps, Fig. 4f). The pattern observed on the dorsal side of the sheath is more complex than the one found ventrally. Individual muscle fibres emerging from the posterior muscle ring and extending towards the anterior of the body form the main part of the network. They are thereby connecting to the loops, similar to fibres emerging from the ventral towards the anteriodorsal side of the sheath.

Dinophilus taeniatus (both sexes)

Embryonic development

The first muscle fibres differentiate soon after gastrulation during early embryogenesis. Similar to female D. gyrociliatus, the neuronal f-actin is labelled in the developing neuropil, which is why phalloidin-labelled materal can be detected in the prostomium (Fig. 5a-c). In the prostomial region, the first muscular element to develop is a muscular ring around the mouth opening (mrmo, Fig. 5a). The main structures of the body wall are established short thereafter in early D. taeniatus embryos: paired ventrolateral (vllm) and dorsolateral longitudinal muscles (dllm) can be distinguished, as well as fragments of future circular muscles (cm) on the lateral sides of the embryo (Fig. 5a).

The muscular system develops very quickly: a large number of muscles characteristic for adults is already present in late embryos. The body wall consists of multiple longitudinal muscles, the majority of which are grouped into ventral, ventrolateral, and dorsolateral longitudinal muscles (Fig. 5b). However, there are also thin separate longitudinal muscle fibres. Numerous circular muscles develop, forming complete rings tracing the body circumference. They occupy the entire length of the body. Longitudinal muscles bifurcate in the prostomial region organizing the prostomial musculature: both ventrolateral and dorsolateral muscles produce dorsal and ventral branches in the same manner (vldb, vlvb, dlvb, dldb, Fig. 5b). The pharyngeal bulb found posterior to the mouth opening is the first element of the musculature of the digestive system (phb, Fig. 5b).

Prehatching embryos demonstrate a regular organization of the body wall musculature and a complex prostomial musculature. Separate longitudinal muscles, seen earlier in the body, appear to be closer to the main muscle strands. Nevertheless, the main longitudinal muscles obviously consist of a group of up to ten muscle fibres (Fig. 5c). Diagonal muscles join the grid of circular and longitudinal muscles (dm, Fig. 5c). More muscle branches originating from the ventrolateral and dorsolateral longitudinal muscles are registered in the prostomial region. As the digestive system differentiates, a defined muscle layer emerges in the gut wall (Fig. 5c).

Hatching & early juvenile stages

Additional muscle fibres are detected in the prostomium and trunk of D. taeniatus juvenile worms. Main thick longitudinal muscles of the body wall are accompanied by several thin longitudinal muscles. The prostomial musculature adds branches (namely contralatero-anterior and contralateral dorsal branches) of a ventral longitudinal muscle (vca, vcb, Fig. 5d). In addition to the body circular muscles, several circular muscles differentiate in the prostomial region as well (cm1, cm2, Fig. 5d).

Adult

Body wall musculature. In adults, the number of longitudinal muscles increases dramatically compared with the previous stages. Additional multiple bundles branch out from ventral and dorsolateral longitudinal muscles (Fig. 5e, g, h). Ventrolateral muscles appear to be the most condensed among the other longitudinal muscles (vllm, Fig. 5e, g). Thus, longitudinal muscular bundles number up to 14 in an adult D. taeniatus. This is different in D. gyrociliatus, where the number of longitudinal muscles in the body wall is not altered during maturation and which therefore presents six longitudinal muscular bundles in juveniles and adults (Fig. 2f, h).

Prostomial musculature. The prostomial musculature of adult D. taeniatus is quite similar to that of a juvenile; however, several branches show more bifurcations than earlier (Fig. 5f).

Musculature of the digestive system. The pharyngeal bulb is very well seen posterior to the mouth opening (phb, Fig. 5e, f). Pharyngeal muscles connect the bulb with the other muscles in this region of the body (phm, Fig. 5f). The basket-like muscle sack surrounds the pharynx (Fig. 5i). The gut wall demonstrates a net of numerous longitudinal and circular muscles (lmds, cmds, Fig. 5j).

No sigmoid muscle as observed in females of D. gyrociliatus is detected around the gut of D. taeniatus at any developmental stage.

Musculature of the copulatory organs. The musculature of the male reproductive system in D. taeniatus males supports one pair of seminal receptacles and the penis (with the penile cone and the penile sheath, Fig. 5k). Regardless of the size difference between specimens of the two species, we found the arrangement of the inner longitudinal (lops) and outer circular muscles (cpms) in the penile sheath show close resemblance (compare Figs. 4d-i, 5k). Additionally, the connection between the copulatory organ and the longitudinal body wall musculature are similar in these two species with regards position and arrangement of muscle fibres.