Comparison of the myoanatomy of entoproct larval types
Sexual reproduction in entoprocts results in the formation of one of two different larval types: the more common, but most likely derived, planktotrophic swimming-type larva or the supposedly basal, lecithotrophic, creeping-type larva [1, 3].
The body wall musculature of the swimming-type larva of Loxosomella atkinsae consists of ring muscles which surround the entire larval body . The apical ring muscles and the ring muscles of the prototroch form the most prominent muscle sets of this larval type. Furthermore, about 40 prototroch longitudinal muscles originate from the most ventral prototroch ring muscle (“main prototroch constrictor” in ) and extend in direction of the body wall above the most dorsal prototroch ring muscle . In the swimming-type larva, the apical organ ring muscle is connected to several longitudinal muscles: one pair of main inner longitudinal retractor muscles, one abfrontal longitudinal muscle and a pair of median muscles . A pair of longitudinal muscles splits below the apical organ ring muscle, at the level of the frontal ganglion, and extends laterally to the digestive tract and establishes contact with the prototroch ring muscles. On the abfrontal side of the larva, a pair of diagonal muscles originates from the prototroch ring muscles and inserts below the apical organ ring muscles . An additional short abfronto-ventral pair of muscles arises close to the abfrontal diagonal muscle but seems to terminate already at the most ventral set of the episphere body wall ring muscles. A pair of frontal diagonal muscles inserts at the prototroch constrictor muscle and terminates below the apical organ ring muscle .
While this brief summary shows that a number of distinct larval muscle systems are present in this relative small and simple-looking entoproct swimming-type larva, the muscular architecture of the creeping-type larva appears far more complex. Similar to the swimming-type larva, the episphere of the larval body contains numerous outer ring muscles (Fig. 4b-d). Both larval types share a prominent apical organ muscle system, consisting of several densely packed ring muscles (cf.  and Figs. 4d, f; 5c herein).
Differences between the swimming- and creeping-type larva are found concerning the number of the prototroch longitudinal muscles, which is considerably higher in the swimming-type larva (40 instead of 28; cf.  and Figs. 4; 5a, d, e herein). Probably due to a much simpler architecture of the frontal organ in the swimming-type larva of Loxosomella atkinsae, the number of muscle bundles originating from the frontal organ is very low compared to the creeping-type larva of L. murmanica. Only one pair of muscles, the paired lateral longitudinal muscle, protrudes from the level of the frontal organ of the swimming larva and splits ventrally to form contact with the prototroch ring muscles . A similar muscle pair can be found in the creeping-type larva (the lateral longitudinal muscle), whereby small muscle fibers branch off to come in contact with the prototroch longitudinal muscles rather than with the prototroch ring muscles, as in the swimming larva (Fig. 5a, d, e). The dorsal end of this muscle seems to terminate not far from the frontal organ ring muscle, right below the prominent ring muscle set between apical and frontal organ (Fig. 5a). The position of the paired abfrontal diagonal muscles of the swimming-type larva between apical organ and prototroch is similar to the position of the abfrontal dorso-ventral muscles of the creeping-type larva (Fig. 5e). However, since the latter muscle pair does not contain fibers that intercross ventrally, homology of these two muscles between both larval types appears at least questionable.
Probably one of the most interesting muscle sets of the creeping-type larva, the horseshoe-shaped, posteriorly open enrolling muscle, is most likely a homolog of the main prototroch constrictor of the swimming-type larva (cf. ). We suggest the enrolling muscle/main prototroch constrictor muscle to be independent of the prototroch ring muscle system because (i) the position of this muscle changes by contraction of the prototroch longitudinal muscles from a ventral to dorsal position relative to the prototroch ring muscles and (ii) the shape of the enrolling muscle/main prototroch constrictor muscle is horseshoe-shaped (i.e., posteriorly open) in contrast to the prototroch muscle which forms a closed ring. The entoproct enrolling muscle may have a central function during metamorphosis, when the larva has already settled. During this process the hyposphere retracts and is enclosed through the contraction of a ring of cells above the prototroch [3, 4]. We suggest this ring of cells to be part of the enrolling muscle due to its position in the contracted larva.
Comparison of the musculature of the entoproct creeping-type larva with that of other lophotrochozoans
Due to the highly unique overall morphology of the entoproct creeping-type larva and the high complexity of its musculature, which undoubtedly contains numerous apomorphies, comparisons to other lophotrochozoans is somewhat difficult. To our surprise, however, we found some muscle systems that bear important differences as well as similarities to that of other taxa and we focus on these in the following.
Taxa with a so-called three-layered body wall musculature, consisting of an outer circular, inner longitudinal and intermediate oblique muscle layer, can be found in nearly all worm-shaped lophotrochozoan phyla, e.g., in various annelids including sipunculans and hirudineans [26–28], nemertines [29, 30], aculiferan molluscs [31–33] and in polyclad and rhabditophoran Platyhelminthes [34–36]. However, individual elements of the three-layered body wall pattern are often absent in the body wall of lophotrochozoan worms as, e.g., diagonal muscle fibers in many polychaetes [37–39]. The loss of specific muscle layers is therefore likely to be the result of secondary simplification.
Our data show that the episphere of the creeping-type larva is more or less covered with ring muscles (Figs. 3; 4), while longitudinal and oblique elements are completely missing. The adult muscle system of Loxosomella murmanica consists, among others, of longitudinal stalk musculature and the atrial ring muscles (Fig. 1). Muscle sets covering the complete adult body are lacking. In contrast, adult Loxosomella vivipara and L. parguerensis possess a fine outer ring muscle layer, covering parts of the body, as well as longitudinal and oblique muscle elements in the foot, stalk and calyx . A true body wall musculature could not be observed in any of the investigated entoproct species, and we suggest that a lophotrochozoan-like body wall is absent in entoprocts due to secondary loss. Whether or not the adult muscles develop from larval muscle sets is still uncertain, but rather than a complete loss of the complex larval musculature, incorporation of some larval elements into the adult muscular bodyplan by remodeling seems probable. A metamorphosing creeping-type larva already shows striking similarities to an adult entoproct: the larva attaches to the substratum with the contracted frontal organ, while a ring of cells above the prototroch contracts below the retracted hyposphere [3, 4]. The larval gut rotates by 90°, larval ciliary bands disintegrate and adult cilia are formed on tentacle buds, which are exposed when the atrium reopens [3, 4]. A transformation of the frontal organ ring musculature as foot musculature and/or of the prototroch ring muscles as atrial ring musculature (Fig. 2) appears highly likely, especially when considering the relative position of these prominent muscle sets in a settled larva. Further muscle units, such as the frontal organ retractors, may contribute to the formation of the longitudinal stalk musculature, but since details on the emergence of the adult entoproct body plan from the larval one are entirely lacking, these issues require reassessment of entoproct metamorphosis using modern methods.
Dorso-ventral muscle fibers are part of many lophotrochozoan body plans, e.g., polychaetes , platyhelminths  and mollusks [33, 41]. Thereby, diagnostic for mollusks and entoprocts alone is the medioventral intercrossing of parts of the dorso-ventral musculature [15–17]. Recent studies have shown that the larvae of polyplacophorans and the neomeniomorph Wirenia argentea share a transitory seven-fold seriality of dorso-ventral muscles in their ontogeny . The entoproct creeping-type larva possesses different sets of dorso-ventral muscle fibers (Figs. 5a, e, f; 6a, b). The most prominent set, the abfrontal dorso-ventral musculature, is located in the abfrontal part of the larvae, originating around the anus and running close to the hindgut, straight to the dorsal body wall (Figs. 5e; 6a, b).
Six pairs of rib-shaped muscles present another set of dorso-ventral musculature. These muscle fibers originate ventrally, surrounded by the prototroch longitudinal muscles, and bend laterally, probably inserting at the dorsal body wall (Figs. 5a; 6a, b). Additional dorso-ventral muscle pairs are found in the medio-frontal part of the larva and run towards the apical organ (Figs. 5a, b; 6a, b; frontal dorso-ventral muscles) and into the foot (Figs. 5a, d, f; 6a, b; pedal dorso-ventral muscles). These pedal dorso-ventral muscles are very strong muscle fibers, shaped like an inverted U, and seem to intercross ventrally in the posterior-third part of the animal, right above the pedal sole (Figs. 4b, f; 5a, d, f; 6a, b). These muscles are most likely the intercrossing dorso-ventral muscles previously recognized by transmission electron microscopy (see Fig. 6c in ).
Enrolling muscles are independent lateral muscle systems that occur in some larval and adult aculiferan mollusks [33, 41]. In both entoproct larval types, a muscle system is present which bears striking similarities to the enrolling muscle of polyplacophoran and larval neomeniomorph aplacophoran mollusks: in both phyla, the enrolling muscle is independent of the body wall and extends along the entire length of the pedal sole on the ventro-lateral side (Figs. 5f; 6a, b cf. [25, 33]). In the larva of the neomeniomorph Wirenia as well as in the swimming- and creeping-type entoproct larvae, this enrolling muscle is horseshoe-shaped, i.e., posteriorly open (Figs. 5f; 6a, b [25, 33]). Similar to (adult) polyplacophoran mollusks, the creeping-type larva is able to contract across the entire length of the body. Due to its position above the prototroch in the contracted creeping-type larva, the entoproct enrolling muscle might have an important role during metamorphosis. While this muscle system persists in adult polyplacophorans and is remodeled to become a part of the longitudinal body wall musculature in the neomeniomorph Wirenia, its postmetamorphic fate in entoprocts remains unknown.