Embryonic muscle development in direct and indirect developing

Embryonic muscle development in direct and indirect developing marine

flatworms (Platyhelminthes, Polycladida)

D. Marcela Bolanosa and Marian K. Litvaitisb,�

a,bDepartments of Zoology and Natural Resources, University of New Hampshire, Durham, NH 03824, USA�Author for correspondence (email: [email protected])

SUMMARY We compared embryonic myogenesis of thedirect-developing acotylean polyclad Melloplana ferrugineawith that of Maritigrella crozieri, a cotylean that develops via alarval stage. Fluorescently labeled F-actin was visualized withlaser confocal microscopy. Developmental times are reportedas percentages of the time from oviposition to hatching: 7 daysfor M. crozieri and 22 days for M. ferruginea. The epitheliumbegan to form at 30% development in M. crozieri and at 15%development in M. ferruginea. Random myoblasts appearedin peripheral areas of the embryo at 36% and 22--30%development in M. crozeri and M. ferruginea, respectively.Circular and longitudinal muscle bands formed synchronouslyat 37--44% development in M. crozieri; yolk obscuredobservations of early myogenesis in M. ferruginea. An orthog-

onal muscle grid was established by 45--50% development inboth species. Diagonal muscles developed in M. ferruginea at60--71% development. Hence, juveniles of this species hatchwith the same basic body-wall musculature as adults. Larvaeof M. crozieri did not hatch with diagonal muscles; thesemuscles are acquired postmetamorphosis. Additionally, aspecialized musculature developed in the larval lobes of M.crozieri. Oral musculature was complex and established by72% development in both species. Our results are comparableto the muscle differentiation reported for other indirect-developing polyclads and for direct-developing species ofmacrostomid flatworms. Furthermore, they provide additionalsupport that the orthogonal muscle pattern of circular andlongitudinal muscles is a symplesiomorphy of Spiralia.

INTRODUCTION

Rhabditophora comprise a diverse group of flatworms

characterized by highly variable body shapes that are mostly

due to the body-wall musculature. Functionally, their

body-wall musculature is not only linked to body shape but

also to locomotion, feeding, copulation, and egg-laying

(Hyman 1951). Fluorescent dye-conjugated phalloidin

has been used to study the muscle organization of free-

living rhabditophorans such as Macrostomum hystricinum

marinum Rieger 1977 and Hoploplana inquilina Wheeler

1894 (Rieger et al. 1994; Reiter et al. 1996), facultative

parasites, such as Urastoma cyprinae (Graff 1882) (Hooge

and Tyler 1999), and obligate endoparasites such as Fasciola

hepatica Linne 1758, Diplostomum pseudospathaceum

Niewiadomska 1984 and Schistosoma mansoni Sambon 1907

(Czubaj and Niewiadomska 1997; Mair et al. 1998, 2000,

2003).

Overall, the rhabditophoran body-wall musculature is

composed of layers of outer circular and inner longitudinal

fibers organized into an orthogonal, grid-like pattern (Hyman

1951; Prudhoe 1985). This organization is considered the

ground pattern for many flattened, vermiform invertebrates

(all lineages within Platyhelminthes, Acoelomorpha, Nemer-

tea) (Hyman 1951; Rieger et al. 1991, 1994; Hooge 2001).

Additionally, the rhabditophoran body wall may contain one

or several sheets of diagonally oriented muscle fibers located

between and below the circular and longitudinal layers (Ho-

oge 2001). However, these diagonal layers may be reduced or

even be completely absent (for a comprehensive review of

adult flatworm body-wall musculature, see Hooge and Tyler

2004). The orientation of the diagonal fibers is such that they

angle clockwise and counterclockwise around the animal (Ri-

eger et al. 1991), crossing over each other and forming a

second grid, offset by about 551 from the orthogonal pattern.

Finally, a parenchymal musculature formed by muscle fibers

traversing the central parenchyma and consisting of well-de-

veloped dorsoventral, transverse, and ventral longitudinal

muscles is present (Hyman 1951).

Although descriptions of flatworm muscle organization

have received considerable attention, studies of pattern for-

mation during developmental myogenesis remain scarce (Re-

iter et al. 1996; Younossi-Hartenstein and Hartenstein 2000;

Hartenstein and Jones 2003), and are mostly focused on the

EVOLUTION & DEVELOPMENT 11:3, 290 –301 (2009)

DOI: 10.1111/j.1525-142X.2009.00331.x

& 2009 Wiley Periodicals, Inc.290

emerging model species, M. h. marinum (Rieger et al. 1991,

1994; Reiter et al. 1996; Morris et al. 2004). In a comparative

study on the differentiation of body-wall musculature inM. h.

marinum and H. inquilina, Reiter et al. (1996) found that

muscle development varied depending on the life cycle that is

if worms hatched as miniature adults or as larvae.H. inquilina

is an acotylean polyclad with indirect development via a

Muller’s larva; M. h. marinum belongs to the direct-develop-

ing Macrostomida. Both Polycladida and Macrostomida rep-

resent early lineages within the Rhabditophora (Carranza et

al. 1997; Litvaitis and Rohde 1999; Baguna and Riutort

2004). Furthermore, they both belong to the Archoophora, a

grouping based on an organizational grade derived from the

homocellular arrangement of female gonads and the produc-

tion of entolecithal eggs (Hyman 1951). This organizational

grade contrasts with the Neoophora, a group characterized by

heterocellular gonads and ectolecithal eggs and include all

remaining rhabditophorans (Hyman 1951). The terms, how-

ever, carry no systematic value and are best used for devel-

opmental descriptions only.

In the larvae of H. inquilina muscle development was

strongly dependent on two founder muscles bands that were

laid down in a bilaterally symmetrical pattern along the lon-

gitudinal axis of the animal. Following this initial muscle

guide, two circular rings of muscles developed: one anteriorly,

demarcating the rostral end from the trunk region, the other

more posteriorly at the junction between trunk and tail end

(Reiter et al. 1996). This is in contrast to observations in the

direct-developing M. h. marinum, where several longitudinal

muscle bands could be seen to which many circular fibers

attached at right angles (Reiter et al. 1996). In this direct-

developing pattern, no initial bilateral symmetry or trunk de-

marcations are evident. It is tempting to speculate that such

observed differences in muscle development are related to

differences in developmental mode (direct vs. indirect). How-

ever, at present, there is no evidence to support or refute such

speculations.

In this study, we examined embryonic muscle differentia-

tion in Maritigrella crozieri (Hyman 1939) and Melloplana

ferruginea (Schmarda 1859), providing the first comparative

analysis on pattern formation during polyclad myogenesis.

M. crozieri is characterized by a Muller’s larva and is grouped

into the suborder Cotylea (polyclads with a ventral sucker,

Lang 1884), whereas M. ferruginea is a direct-developing

acotylean (polyclads without a ventral sucker, Lang 1884).

The main purpose of this study was to provide new devel-

opmental and morphological data that contribute to the un-

derstanding of body-wall muscle formation during

embryogenesis, and to determine whether observed differ-

ences in muscle development are related to differences in de-

velopmental mode. Additionally, we also compared our

findings to muscle development in other flatworms and other

Spiralia.

MATERIALS AND METHODS

CollectionAdult worms of M. crozieri were collected from submerged hang-

ing lines at Little Jim’s Marina, Fort Pierce, FL, USA (27128.420N,

80118.40W), where they were associated with their prey, the ascid-

ian Ecteinascidia turbinata Herdermann 1880. Adult specimens of

M. ferruginea were collected from under rocks in the intertidal and

shallow subtidal zones at Peanut Island (261 46.4280N; 801

82.6080W) on the Atlantic coast of Florida, USA. Animals were

lifted off the substrate using a soft paintbrush and were placed

individually into small plastic bags filled with seawater. Reproduc-

tive maturity of individuals was determined by inspecting the ven-

tral sides of the worms for eggs. When present, eggs are easily

visible in the oviducts. Egg-bearing specimens were acclimated at

221C for 2 days in individual plastic bags containing Millipore-

filtered seawater.

CultureAfter acclimatization, mature specimens were placed into Petri

dishes and their uteri were punctured with small tungsten needles.

This caused the release of zygotes without egg capsules (naked

embryos). The embryos were transferred into small gelatin-coated

Petri dishes of Millipore-filtered seawater that contained 200mg/ml

streptomycin and 60mg/ml penicillin to prevent microbial or fungal

growth and maintained at 221C. Embryonic development was fol-

lowed until the formation of larvae and juveniles. Embryos were

observed under a Leica DMLB microscope (Leica, Bannockburn,

IL, USA) equipped with a Nikon CoolPix 8700 (Nikon, Melville,

NY, USA).

Phalloidin staining and confocal microscopyEmbryos, larvae, and juveniles were fixed at different developmen-

tal stages at room temperature in 4% paraformaldehyde in 0.01M

phosphate buffer (PBS; pH 7.4) for 45min. Specimens were rinsed

(3 � ) for 15min with 0.01M PBS, permeabilized for 1h in 0.2%

Triton X-100 in PBS, stained for 2h or overnight with Alexa Fluor

488 phalloidin (Molecular Probes, Eugene, OR, USA), and rinsed

twice for 10min with PBS. Specimens were mounted on glass slides

in Gel/Mount (Biomeda Corp., Electron Microscopy Sciences,

Hatfield, PA, USA), and digital image acquisition and analysis

were performed using a Zeiss LSM 510 confocal microscope (Zeiss,

Thornwood, NY, USA). Samples were excited with a 488nm

multi-line argon-ion laser and emitted fluorescence was collected

using a 505–530nm bandpass filter. Measurements of the speci-

mens were taken with an ocular micrometer before fixation. For

each species, at least four specimens were examined for each de-

velopmental stage.

RESULTS

To conform to published studies on embryonic myogenesis in

archoophorans (Reiter et al. 1996; Ladurner and Rieger

2000), we define developmental stages as a percentage of total

Embryonic muscle development in marine £atworms 291Bolan� os and Litvaitis

developmental time, where 100% represents the time from

oviposition to hatching. However, we also include time since

oviposition in hours to allow for comparisons with studies of

other spiralians (Maslakova et al. 2004; Bergter and Paululat

2007). Total developmental time forM. crozieri was 6–7 days

and 18–22 days for M. ferruginea. These time frames are

comparable to other studies that also recorded much longer

developmental times for acotylean than cotylean polyclads

(Rawlinson et al. 2008; D. Bolanos & M. Litvaitis,

unpublished data).

Muscle differentiation in Maritigrella crozieri

Cleavage begins before oviposition. After oviposition (0%

development), zygotes (average diameter5134mm; n550)

underwent holoblastic, spiral cleavage, eventually dividing the

embryos into 64 cells. At about 17% of development (28h),

the embryos contained small amounts of yolk, which emitted

a dull autofluorescence. Gastrulation began at approximately

30% of development (50h). The surfaces of the embryos be-

came irregularly shaped, and a faintly staining, polygonal

pattern outlining the epithelial cells became visible (Fig. 1A).

Actin filaments inserting into the zonulae adhaerentes were

evident (Fig. 1B). Additional actin was distributed across the

embryo surface but was present in higher concentrations in

some regions (Fig. 1A). No muscle fibers were observed im-

mediately after epiboly. However, shortly after gastrulation

(approximately 36% development, 60h), a few small myo-

blasts were randomly dispersed throughout the embryo.

Once gastrulation was complete, the first cilia appeared

and minimal movement of the embryos was observed (37–

43% of development, 62–72h). A brightly staining, primary

circular muscle band was recognized (Fig. 1C), and unorga-

nized muscle fibers were distinguishable in the center of the

developing embryo. During this stage, many individual mus-

cle fibers appeared. At about 43–44% of development (72–

74h, day 4), the embryo still appeared spherical but muscle

fibers began to form concentrically around the apical organ

and in the oral region (Fig. 1D). Ciliary action caused the

embryos to slowly rotate within the egg capsules. Secondary

circular fibers derived by branching from the primary circular

band were detectable, although still no contractions were ob-

served at this point (Fig. 1E). Additionally, unorganized

weakly staining fibers could be distinguished deep within the

embryo. Also during this stage, a primary longitudinal muscle

formed and extended whip-like along the entire length of

the embryo, from anterior to posterior back again (Fig. 1E).

Presumably, this looped fiber extended into the area of a

future larval lobe.

At 45–52% of development (75–88h, day 4), a simple spi-

ral of circular muscle extending around the apical organ was

evident (Fig. 2A). During this time, additional longitudinal

muscles began to develop; they could be distinguished as

faintly staining branches (Fig. 2B). Thicker, circular fibers

encircled the periphery of the embryo, bifurcating and form-

Fig. 1. Confocal projections of the earlyembryonic development of Maritigrellacrozieri stained with phalloidin. (A) Em-bryo surface at about 30% of develop-ment, showing polygonal outlines ofepidermal cells. Scale bar550mm. (B)Higher magnification of embryo surface,arrowheads indicate actin in the zonulaeadhaerentes. Scale bar510mm. (C) Api-cal view of postgastrulation embryo,showing primary circular muscle band(pcm). Scale bar550mm. (D) Dorsolat-eral view of embryo at about 43%of development, showing concentric or-ganization of musculature surroundingapical organ (ao) and developing circularmusculature of mouth (m). (E) Lateralview of embryo showing primary longi-tudinal muscle (plm) extending whip-likealong the entire length, pcm delineatingextent of ao musculature, and orthogonalnetwork of circular (cm) and longitudinalmuscles (lm); asterisk indicates apexof embryo; ep, epidermis. Scale bars5

25mm.

292 EVOLUTION & DEVELOPMENT Vol. 11, No. 3, May--June 2009

ing additional muscle fibers (Fig. 2A). At this stage, first con-

tractions of the body were evident, and faster rotations of the

embryos were observed. On the ventral side of the embryo,

muscle fibers associated with the mouth were discernable (Fig.

2, A and C). The muscles of the mouth were seen as a separate

muscular field of concentrically distributed well-developed

muscle fibers. Unlike the circular muscle fibers of the apical

organ, oral muscle fibers did not run in a spiral fashion but

instead were densely packed, and formed a sphincter sur-

rounding an opening that extended deep into the embryo (Fig.

2, A and C). At 53–60% of development (89–101h, day 4),

the shape of the embryo was slightly ovoid, and the arrange-

ment of the circular muscle system became clearly evident

(Fig. 2, B and C). Apically, these circular rings of muscles

were more abundant and consisted of three to five individual

muscle fibers. At this stage, a few muscle bands completely

surrounded the embryo but toward the posterior end they

were less abundant and still incomplete (Fig. 2, B and C). In

the area of the equator, a paired set of circular fibers extended

along the periphery of the embryo, one from each side. How-

ever, instead of completely encircling the embryo, once they

reached about one half the circumference of the embryo, they

turned 1801 and doubled back, forming two highly flattened

ovals (Fig. 2B). It is possible that these fibers are associated

with the position of future larval lobes. At the very posterior

end, three areas of high muscle fiber concentrations were as-

sociated with the oral musculature. It is likely that these areas

will give rise to the dilator muscles of the mouth (Fig. 2C).

Furthermore, small, bright dots were localized around the

muscles of the mouth, which represented the insertion points

of future longitudinal muscles. Some of the circular muscle

bands bifurcated and gave rise to new small fibers.

During the next stage (61–71% of development, 102–120h,

day 5), the embryos elongated and their larval shapes became

clearly recognizable. At that time, eyes, larval lobes, an oral

hood, and ciliary bands were evident (Fig. 3, A and B). Ciliary

bands outlining the larval lobes could be recognized because

actin in the microvillar collars surrounding the sensory cells

(see Lacalli 1982) stained intensely. Furthermore, rhabdite

glands (actin staining in gland necks) became evident. This

change in morphology was associated with the growth of

many additional circular muscle bands and with the develop-

ment of a few longitudinal fibers. The circular muscles became

evenly spaced and completely encircled the embryo along its

entire body length (Fig. 3, A and C). The primary circular

muscle band was evident because of its prominent width and

extended along the rim of the oral hood (Fig. 3A). Contin-

uous rotations and strong uncoordinated contractions were

observed during this stage. With the exception of the oral

hood, which contained only circular fibers, all lobes contained

three or four longitudinal muscle fibers (Fig. 3A). Within each

lobe, these fibers originated from a single point located at the

distal end of each lobe. From there, they extended fan-like

through the lobes and eventually ran through the larval body

toward the apical end of the larva (Fig. 3).

The caudal region of the body contained five muscle fibers

(Figs. 3, C and 4, A and B), which connected dorsally with a

thick circular fiber and ventrally with the musculature of the

mouth (Fig. 4, A and B). These bands were partly responsible

for producing the extension and contraction of the larva.

The final stage ending with hatching, corresponded to 72–

100% of development (121–168h, days 6–7). A distinct

sphincter surrounded the ventrally located mouth, and

an interior view of the larva, revealed individual ventrolater-

al muscles that radiated from the rim of the mouth and

connected with the lateral body wall (Fig. 4, A and B). A

diffusely staining, tubular structure was observed in associa-

tion with the mouth, representing a rudimentary pharynx

Fig. 2. Confocal projections of the early embryonic development of Maritigrella crozieri stained with phalloidin. (A) Dorsolateral view ofembryo at 45–52% development, showing spiral organization of circular muscles around the apical organ (ao) and developing sphincter oforal musculature around the mouth (m). (B) Lateral view of circular muscle fiber distributed over the embryo at 53–60% development;arrowheads indicate a paired set of small circular fibers extending along the periphery and doubling back; longitudinal muscles (lm) arevisible. (C) Ventrolateral view showing organization of circular fiber bands (cm) surrounding the entire embryo and three sets of developingmouth dilators (md). Bright spots (small arrows) around the mouth represent insertion points of future longitudinal muscles. Asterisksindicate embryo apex. All scale bars550mm.


(Fig. 4, A and B). However, no specific pharyngeal muscu-

lature could be identified. Additionally, short, longitudinal

fibers, some crossing over each other, forming a distinct X

shape were located in the oral hood (Fig. 4, A and B).

At hatching, no diagonal muscle fibers were found and

the entire arrangement of the muscles consisted of sets of

circular and longitudinal fibers (Fig. 4, C and D). Two groups

of concentric muscles that overlapped each other and formed

a diagonally arranged, lattice-like pattern were observed (Fig.

4, C and D).

Muscle differentiation in Melloplana ferruginea

Embryogenesis in direct-developing polyclads generally takes

longer than that of indirect-developing species. Furthermore,

the embryos usually contain much larger amounts of yolk, a

phenomenon that can slow development in the early stages

(this study; Rawlinson et al. 2008). The embryos of M. fer-

ruginea were somewhat smaller than those of M. crozieri (av-

erage diameter5125mm; n550) and contained large

amounts of yolk. This yolk emitted a strong autofluores-

cence. The period from oviposition to the beginning of gas-

trulation corresponds to approximately 0–15% development

(0–79h). During early gastrulation (at about 14% develop-

ment, 74h), the fluorescence started to abate and at about

15% of development (79h), the polygonal outlines of epider-

mal cells were visible (Fig. 5A) and continued to be distinct

until about 16–21% of development (84–111h, day 4). The

first cilia appeared and some movement was noted at this

stage.

At 22–30% development (116–152h, days 5–6), ciliation

increased and slow rotational movements could be observed.

Additionally, the bright muscle anlagen visible on the surface

of the embryo increased in number and size (Fig. 5B). During

Fig. 3. Larval stages of Maritigrella cro-zieri at 61–71% of development. Confo-cal projection (A) and schematic diagram(B) of ventral view of Muller’s larvashowing oral hood (oh), primary circularmuscle (pcm), one lateral lobe (ll), andtwo ventrolateral lobes (vl), each contain-ing prominent longitudinal muscle fibers(arrowheads) converging at the distal endof the lobes. Small bright spots outliningthe lobes represent the ciliary band. Threeeyes (e) are visible at the apical end. Con-focal projection (C) and schematic dia-gram (D) of dorsal view of the larvashowing lateral (ll) and dorsolateral (dl)lobes, apical organ (ao) and caudal endcontaining distinct muscle fibers (arrows)that extend from the dorsal to the ventralsurface of the larva. Scale bars550mm.


the period of 31–44% of development (163–232h, days 7–9),

the embryo was still spherical in shape and a single eye was

present. The yolk cells migrated to the interior of the embryo

diminishing the ability to visualize individual muscle fibers.

Most myocytes appeared in an unorganized network. How-

ever, along the periphery of the embryo it was possible to

differentiate some circular fibers (Fig. 5B).

A second small eye appeared at 45–59% of development

(238–312h, days 10–13). In addition, faster rotations and first

contractions of the juveniles were observed. The body elon-

gated into a more ovoid shape, and the formation of a fine

orthogonal grid of circular and longitudinal muscles was ev-

ident (Fig. 5C). Unequally spaced circular fibers surrounded

the entire embryo, whereas a few longitudinal fibers extended

the length of the body, some of which bent diagonally at the

anterior end of the worm. Circular fibers were better devel-

oped than the longitudinal muscle fibers but a more dense

formation of longitudinal muscles was observed (Fig. 5C).

Well-developed, branched, dorsoventral muscles were visible

extending across the body of the embryo (Fig. 5D). These

muscles play an important role in locomotion and the main-

tenance of a flat body shape. Furthermore, the brain was

prominently visible in the anterior third of the embryo due to

actin staining in the neuropil (Figs. 5D and 6E). Also during

this period, the rudiments of the mouth on the ventral surface

was visible as an elongated shape consisting of aggregated and

unstructured muscle fibers (Fig. 5D).

At 60–71% of development (317–374h, days 14–16), the

growth of diagonal muscle bands on the dorsal (Fig. 6, A and

C) and ventral (Fig. 6, D and F) surfaces proceeded rapidly.

Another pair of eyes developed, and the mouth took on its

circular shape (Fig. 6, B and D–F). Dorsal, longitudinal

Fig. 4. Larval stages of Maritigrella cro-zieri at 72–100% of development. Con-focal projection (A) and schematicdiagram (B) of deep ventral view withventrolateral muscles (arrows) radiatingfrom the oral sphincter (os). Caudal mus-cle fibers (arrowheads) connect ventrallywith the mouth. Short, oral hood longi-tudinal muscles (ohl), some crossing toform an X are visible in the apical portionof the larva. Confocal projection (C) andschematic diagram (D) of lateral view ofa late-stage larva, showing two sets ofconcentric muscles arranged in a lattice-like pattern in the lateral lobes. clm, con-centric lobe musculature; ao, apical or-gan; e, eyes; dl, dorsolateral lobe; ll,lateral lobe; md, mouth dilators; oh, oralhood; ph, rudimentary tubular pharynx;rg, rhabdite glands. Scale bar550mm.


muscle fibers extended straight from the anterior to the pos-

terior end (Fig. 6, A, C and D). Dorsally, a paired set of

diagonal muscles covered the entire length of the worm,

crossing over each other at a 901 angle (Fig. 6, A–C). These

fibers were denser and closer at the anterior part of the body

becoming more spaced and still incomplete at the posterior

end (Fig. 6A). Ventrally, diagonal muscle bands covered the

anterior half of the body, crossing over each other at the body

midline and then extending in a more longitudinal orientation

along the lateral sides of the body (Fig. 6, D and F). All

diagonal muscle fibers were located between the longitudinal

and the circular muscles. Juveniles within the egg capsules

were observed to vigorously bend their anterior ends, a mo-

tion mediated by diagonal muscles. Ventral, longitudinal fi-

bers located along the body edges extended the entire length

of the juveniles, but at the posterior end behind the mouth

opening, they curved and formed U-shaped muscles (Fig. 6, D

and F).

At this stage, parenchymal muscles were present through-

out the body as disorganized, conspicuous fibers running in

varying directions (Fig. 6E). The concentric sphincter muscles

of the mouth became established as a smaller, inner and a

larger, outer ring of muscles (Fig. 7, A–C). At this stage, short

radial fibers seemed to connect the inner to the outer sphinc-

ter, however, they are most likely longitudinal muscles of the

future pharynx (Fig. 7, A–C). The ventral mouth was located

at the very posterior end. Elongation of the growing embryo

will cause the mouth to gradually move anterior to its final

midventral position characteristic of adult M. ferruginea.

Rhabdite glands could be distinguished and were more abun-

dant on the dorsal surface (Figs. 6, A and B and 7A).

The final stage corresponds to 72–100% of development

(380–528h, days 18–22). At approximately 78% of develop-

ment (411h, day 17), the juveniles had almost completed their

development but remained in their egg capsules for a few

additional days, continuously rotating and contracting until

hatching. Their bodies elongated and tapered posteriorly, ac-

quiring their typical worm shape. During that period, only a

few changes in the musculature were observed. The ventral

parenchymal muscles and the oral musculature became more

defined (Fig. 7). No changes were observed in the body-wall

musculature.

The oral musculature is complex and formed by distinct

sets of muscles (Fig. 7). A set of concentrically arranged

Fig. 5. Confocal projections of muscledifferentiation in Melloplana ferruginea.(A) Embryo at 15% of development withpolygonal outlines of epidermal cells. (B)Postgastrulation embryo at 22–30% de-velopment with bright spots of actin rep-resenting the anlagen of the body-wallmuscles. Arrows indicate short longitudi-nal muscle fibers in the periphery of theembryo. (C) Elongated embryo at 45–59% of development, showing a fine or-thogonal network of circular (cm) andlongitudinal muscles (lm). (D) Deep lat-eral view of embryo with anterior brainanlagen (b) and with branched, dorso-ventral muscles (arrowheads) traversingthe body. Aggregation of unstructuredmuscle fibers on the ventral side formingmusculature associated with the mouth(om). All scale bars525mm. rg, rhabditeglands.


muscles forms a larger sphincter at the ventral surface of the

worm and surrounds the mouth opening. The mouth

opening invaginates into the body wall, leading into a fun-

nel-like structure with a second, smaller sphincter located

where the funnel narrows to a tube. The second sphincter is

also composed of circularly arranged muscles. The funnel

and tube are lined by longitudinal muscles, which appear

to be arranged as radial muscles between the two sphincters

because they are splayed outwards (Fig. 7, A–C). Finally,

distinct dilator muscles extending from both sphincters

to the lateral body wall anchor the mouth (Fig. 7, A–C).

Random ventral parenchymal muscles that tended to extend

in a more longitudinal direction (Fig. 7B), eventually will at-

tach to the ventral and dorsal body wall and to organs.

Other parenchymal muscles remain suspended as individual

fibers throughout the parenchyma. During this stage, an in-

crease in the number of rhabdite glands was also observed

(Fig. 7A).

Fig. 6. Confocal projections of muscle differentiation in Melloplana ferruginea. (A) Projection showing network of longitudinal, circular,and diagonal muscles. (B) Deeper view showing oral musculature. (C) Schematic diagram of dorsal musculature. (D) Projection showingnetwork of longitudinal, circular, and diagonal muscles, and early oral musculature. (E) Deep view showing oral muscle sphincter andirregularly arranged parenchymal muscles. (F) Schematic diagram of ventral musculature. Arrowheads indicate longitudinal musclesbending in a U-shape at the posterior end. b, brain; cm, circular muscles; dm, diagonal muscles; e, eyes; lm, longitudinal muscles; om, oralmusculature; pm, parenchymal muscles; rg, rhabdite glands. All scale bars525mm.


DISCUSSION

Comparison of embryonic myogenesis inMaritigrella crozieri with other indirect developingpolyclads

In an ultrastructural and immunohistochemical study, You-

nossi-Hartenstein and Hartenstein (2000) describe muscle pre-

cursors in embryos of the polyclad Imogine mcgrathi Jennings

and Newman 1996 at about 84h (their stage 6), which develop

into circular and longitudinal fibers by the end of day 4 (their

stage 7). Considering that I. mcgrathi larvae hatched in about

7–8 days (Younossi-Hartenstein and Hartenstein 2000), these

events correspond to about 43–57% of development, a time

line that is comparable to our results for M. crozieri. Addi-

tionally, Younossi-Hartenstein and Hartenstein (2000) men-

tion the development of two distinct longitudinal muscle bands

in I. mcgrathi albeit without providing further description on

their position and fate. It is possible that these bands corre-

spond to the primary longitudinal muscle seen extending into

the area of the future larval lobes in M. crozieri.

A comparison of embryonic myogenesis of M. crozieri

with that of H. inquilina reveals similarities with respect to

primary muscle bands and the arrangement of the circular

musculature around the apical organ. According to Reiter et

al. (1996), a primary muscle grid appears at 80% of devel-

opment inH. inquilina. This orthogonal grid consists of a pair

of primary longitudinal fibers located laterally, an anteriorly

located primary circular muscle band, and a posterior pri-

mary circular muscle fiber (Reiter et al. 1996). In M. crozieri,

we also recognized the early formation of an anterior primary

circular muscle (43–44% of development); however, none of

the posterior circular muscle bands could be identified as a

primary muscle. We could, however, identify a major longi-

tudinal muscle that extended the length of the embryo and

then looped back to the anterior end. It is possible that this

muscle fiber corresponds to the primary longitudinal muscle

fiber described in H. inquilina (Reiter et al. 1996).

Also at 80% of development, the circularly arranged mus-

culature around the apical organ of H. inquilina is evident

(Reiter et al. 1996). Like in M. crozieri, it forms a simple

spiral. In both species, once the oral hood is established, this

apical muscular spiral terminates at the conspicuous primary

circular muscle band. In both species, the primary circular

muscle eventually extends along the rim of the oral hood.

A clear difference with respect to the timing of the ap-

pearance of major muscle fibers was found between the two

species. In H. inquilina, the first recognizable primary fibers

appeared only at 80% of development while the embryo was

still ovoid and had not yet acquired a typical larval form

(Reiter et al. 1996). In comparison, embryos of M. crozieri at

80% of development were clearly distinguishable as Muller’s

larva with well-developed larval lobes containing a complex

network of muscle fibers and a recognizable body-wall mus-

culature of circular and longitudinal muscles. The amount of

yolk and rearing temperatures are two factors that have been

shown to affect developmental time lines in polyclads (Raw-

linson et al. 2008; D. Bolanos & M. Litvaitis, unpublished

data). A case could be made that as an acotylean polyclad, the

embryos of H. inquilina contain large amounts of yolk, which

potentially could delay the growth of the myoblasts. How-

ever, I. mcgrathi also is an acotylean and its time line follows

the one observed in M. crozieri (Younossi-Hartenstein and

Hartenstein 2000). Although Reiter et al. (1996) do not pro-

vide culture temperature for their specimens, total develop-

mental time for H. inquilina was o4.5 days (compared with

6–7 days for M. crozieri at 221C), a fact that may invalidate a

claim of delayed development due to low temperature. Most

likely, these temporal differences are due to the fact that I.

mcgrathi and M. crozieri are subtropical species, whereas H.

inquilina is found in temperate regions.

Another difference between the two species involved the

presence of a few obliquely oriented muscle fibers in H. in-

quilina as early as 80% development (Reiter et al. 1996). We

did not observe such muscle fibers in M. crozieri, even at the

Fig. 7. Confocal projections of oralmuscle differentiation in Melloplana fe-rruginea. (A) Embryo with oral muscula-ture composed of concentric and splayedlongitudinal fibers and mouth dilators.(B) Late-stage embryo with complex oralmusculature forming a funnel-like struc-ture and with mouth dilators. (C) Sche-matic diagram of oral musculature. md,mouth dilators; om, oral musculature;pm, parenchymal muscles; rg, rhabditeglands. All scale bars525mm.


hatching stage. Hence, the diagonal muscles found in adult

M. crozieri are acquired at or after metamorphosis. This is

consistent with the function of such muscles. Contraction of

diagonal muscles causes lateral bending of the worms, a mo-

tion that is observed even in juveniles that are still in the egg

capsules (see section on M. ferruginea). Larvae of M. crozieri

do not require such motions and therefore do not have di-

agonal muscles.

The mouth of all polyclads is located ventrally as an inv-

agination of the body wall. The mouth opening ofM. crozieri

was surrounded by a well-developed circular muscle sphinc-

ter, consisting of at least three concentric fibers. From this

sphincter, paired ventrolateral longitudinal muscles extended

apically, and eventually connected with the lateral body wall.

Some of the ventrolateral muscles appear to connect with the

primary circular muscle fiber in the oral hood. In contrast, the

oral musculature of H. inquilina consists of an oral sphincter

surrounding the mouth from which an anteriorly located

mouth retractor muscle extends and also connects to the cir-

cular musculature of the apical organ (Reiter et al. 1996). It is

possible that this mouth retractor corresponds to one of the

ventrolateral muscles observed in M. crozieri.

Additionally, in M. crozieri an inner small muscular ring

was associated with a simple, straight pharynx outlined by

diffusely staining material. Pharyngeal muscles were not yet

developed at the larval stage. According to Ruppert (1978),

the development of a muscular pharynx occurs during larval

metamorphosis. Hence, the lack of a pharyngeal musculature

in M. crozieri reflects the planktonic life style of a larval sus-

pension feeder. The same has been observed in the larvae of I.

mcgrathi (Younossi-Hartenstein and Hartenstein 2000). This

is in contrast to the findings of Reiter et al. (1996), which

describe a complex pharyngeal musculature of radial, longi-

tudinal, and circular fibers in H. inquilina.

Finally, Reiter et al. (1996) mention changes in the orien-

tation of muscle fibers especially around the lobes and the

mouth as the larvae change in shape to a typical Muller’s

larva. However, they do not elaborate on these changes, and

no mention is made with regards to a unique double-set of

overlapping muscles as we found in the lobes of M. crozieri.

Comparison of embryonic myogenesis inMelloplana ferruginea with other direct developingRhabditophora

To date, no information is available on developmental myo-

genesis of direct-developing polyclads. Hence, we compare the

events in M. ferruginea to those of other rhabdiophorans,

especially to M. h. marinum. Time of development from ovi-

position to hatching of juvenile M. h. marinum is 4–5 days

(Reiter et al. 1996).

In addition to circular and longitudinal muscles,

rhabditophoran flatworms are characterized by a layer of di-

agonal muscles (Hyman 1951; Prudhoe 1985; Hooge 2001). In

M. ferruginea diagonal muscles appeared at about 60% de-

velopment and were well developed by 70% developmental

time. JuvenileM. ferruginea still within their egg capsules were

observed to laterally bend their anterior ends, a movement

possible because of diagonal muscles. An ultrastructural study

on muscle differentiation ofM. h. marinum times the presence

of circular, diagonal, and longitudinal muscle fibers between

50% and 64% of development (Rieger et al. 1991). Using

rhodamine–phalloidin staining, Reiter et al. (1996) show that

in M. h. marinum muscle fiber differentiation does not start

before 55% of development and that earlier stages of devel-

opment are obscured. Although we were able to identify

brightly staining actin muscle anlagen of M. ferruginea

around 21% of development, later differentiation events also

were obscured in our specimens. Comparable to M. h. mari-

num, a network of circular, longitudinal, and diagonal muscle

fibers was established between 45% and 70% of development.

We did not, however, observe ‘‘muscle lattices’’ as had been

described for M. h. marinum (Reiter et al. 1996).

Temporal congruence between the two species was also

found with respect to the development of dorsoventral muscle

fibers and of the oral musculature. In juveniles of M. fer-

ruginea the dorsoventral muscles are well developed by 60–

71% of development and function to maintain the flattened

shape of the worms. In comparison to M. crozieri, the mouth

of M. ferruginea is associated with a more developed but still

rudimentary pharynx. Two rings of circular muscles act as

sphincters and are connected to dilator muscles, which in turn

connect to the body wall. The mouth leads into a tubular

structure lined by longitudinal muscles. Because of a funnel-

shaped eversion of the mouth between the two circular

sphincters, these longitudinal muscles appear to be radially

arranged. However, this is the result of splaying out the oral

opening. In M. h. marinum on the other hand, Rieger et al.

(1994) describe distinct radial fibers associated with the phar-

ynx. In polyclads, pharyngeal musculature is important in

structuring the shape of the adult pharynx, (i.e., ruffled vs.

tubular pharynx), a differentiation that is of taxonomic im-

portance for familial distinctions (Faubel 1983, 1984; Prudhoe

1985). No gut musculature can be identified in embryos of

either M. ferruginea or M. h. marinum, a notion also con-

firmed for Macrostomum sp. (now described as M. lignano,

Ladurner et al. 2005) (Morris et al. 2004). Furthermore,

Morris et al. (2004) describe a muscle net in the anterior third

of Macrostomum sp. in which fibers follow the outer surface

of the brain as well as pass through the brain cortex and

neuropil. A similar arrangement of muscle fibers surrounding

and passing through the brain can be seen in M. ferruginea.

Muscle development of M. ferruginea can be compared

with that of free-living rhabditophorans other than macros-

tomids. However, these studies mention myogenesis only

within the context of development and organogenesis of the


entire embryo and do not involve specific muscle staining

approaches (Hartenstein and Ehlers 2000; Cardona et al.

2005). Hartenstein and Ehlers (2000) found that in the

rhabdocoel Mesostoma lingua (Abildgaard 1789) embryonic

myogenesis also results in a typical grid-like pattern of circular

and longitudinal muscles on day 7 (their stage 7; total devel-

opmental time 8 days with embryos maintained at 23–251C).

In the triclad, Schmidtea polychroa (Schmidt 1862) on the

other hand, layers other than the orthogon (second longitu-

dinal, diagonal) form during development and become part of

the body-wall musculature (Cardona et al. 2005). Because

polyclads and macrostomids are considered basal within

Rhabditophora (Carranza et al. 1997; Litvaitis and Rohde

1999; Baguna and Riutort 2004), the orthogonal muscle grid

of longitudinal and circular muscles appears to represent the

primitive arrangement within Platyhelminthes, with more

derived clades evolving additional muscle sets and/or

arrangements.

Comparisons with other Lophotrochozoa

Although Polycladida clearly belongs to the Spiralia based on

its cleavage pattern and dual origin of mesoderm (Boyer et al.

1996, 1998), a comparison of muscle development of larval

M. crozieri with that of other larvae in the clade is dependent

on the homology between polyclad larvae and trochophores.

Whereas homology among trochophores of mollusks, anne-

lids, and sipunculids has been established (Nielsen 2001; Ma-

slakova et al. 2004; Wanninger et al. 2005), homology of

polyclad larvae with lophotrochozoan larvae has not yet been

demonstrated. With this caveat in mind, we here compare the

larval musculature of M. crozieri to that of other spiralian

larvae.

Embryonic myogenesis of Capitella sp. 1 occurs sequen-

tially from anterior to posterior and results in the formation

of longitudinal, circular, and oblique body-wall muscles in

addition to specialized intrasegmental and setal sac muscle

fibers (Hill and Boyer 2001). According to Hill and Boyer

(2001), all larval muscles are retained postmetamorphosis and

develop into the adult musculature. In the polychaete Pom-

atoceros lamarckii (Quatrefages 1866) musculature associated

with the esophagus and intestine develop early and body-wall

musculature (which lacks circular muscles) is formed only

once the trochophore elongates. Again, the larval body-wall

musculature is retained in adult P. lamarckii (McDougall et

al. 2006). Unlike the weakly developed (or even absent) cir-

cular muscles bands in polychaetes where muscle formation

may be controlled by segmentation (Bergter et al. 2008), most

circular muscles in the larvae of M. crozieri appear synchro-

nously; diagonal body-wall muscles are acquired after meta-

morphosis. This is comparable to the process observed in the

sipunculid Phascolion strombus (Montagu 1804) in which cir-

cular muscles also appear synchronously, not sequentially

from anterior to posterior. Development of longitudinal mus-

cles, arranged in bands, follows during late larval develop-

ment (Wanninger et al. 2005).

Wanninger and Haszprunar (2002) describe the establish-

ment of an apical muscle grid during myogenesis in the larvae

of two species of chiton. Although the authors question un-

equivocal homology of the longitudinal fibers of the apical

grid with longitudinal fibers of the body-wall muscle grid,

they identify the apical grid as possible vestiges of the body-

wall musculature shared by the common ancestor of mollus-

can and vermiform spiralians.

From the above comparisons, it appears that in unseg-

mented spiralians the rudiments of circular muscles develop

synchronously. Our results in M. crozieri follow this pattern

supporting a close evolutionary relationship between unseg-

mented lophotrochozoans and polyclad flatworms based on

myogenesis. Furthermore, myogenetic events of body-wall

musculature in M. ferruginea also resemble the embryonic

muscle development observed in Acoela (Ladurner and Ri-

eger 2000; Semmler et al. 2008), a taxon occupying a basal

position in Bilateria.

With the present study, we have added information not

only from another indirect developing polyclad but also from

a direct developing species. Our results provide additional

support that the orthogonal muscle pattern of circular and

longitudinal muscle bands is a symplesiomorphy of Spiralia

and may already have been present in the stem species of

bilateral animals (Ladurner and Rieger 2000; Rieger and

Ladurner 2001, 2003; Semmler et al. 2008).

AcknowledgmentsThis work was supported by NSF grant DEB-0412932. A portion ofthis research was conducted at the Smithsonian Marine Station atFort Pierce, FL as part of a graduate student Link Fellowship. Weare grateful to Drs. Sigmer Quiroga and Kate Rawlinson for theirhelp in the field and for collecting embryos at different developmentalstages. Special thanks go to Dr. Mark Townley (InstrumentationCenter, UNH) for his technical assistance with the confocal micro-scope. We acknowledge NSF grant DBI-0618719 through which theconfocal microscope at UNH was acquired. This is Scientific Con-tribution No. 2378 from the New Hampshire Agricultural Experi-ment Station and Contribution No. 771 from the SmithsonianMarine Station at Fort Pierce, FL.

REFERENCES

Baguna, J., and Riutort, M. 2004. Molecular phylogeny of the Platy-helminthes. Can. J. Zool. 82: 168–193.

Bergter, A., Brubacher, J. L., and Paululat, A. 2008. Muscle formationduring embryogenesis of the polychaete Ophryotrocha diadema(Dorvilleidae)Fnew insights into annelid muscle patterns. Front. Zool.5: 1–18.

Bergter, A., and Paululat, A. 2007. Pattern of body-wall muscle differen-tiation during embryonic development of Enchytraeus coronatus (Annel-ida: Oligochaeta; Enchytraeidae). J. Morphol. 268: 537–549.


Boyer, B. C., Henry, J. J., and Martindale, M. Q. 1998. The cell lineage of apolyclad turbellarian embryo reveals close similarity to coelomate spi-ralians. Dev. Biol. 204: 111–123.

Boyer, B. C., Henry, J. Q., and Martindale, M. Q. 1996. Dual origins ofmesoderm in a basal spiralian: cell lineage analyses in the polyclad tur-bellarian Hoploplana inquilina. Dev. Biol. 179: 329–338.

Cardona, A., Hartenstein, V., and Romero, R. 2005. The embryonicdevelopment of the triclad Schmidtea polychroa. Dev. Genes Evol. 215:109–131.

Carranza, S., Baguna, J., and Riutort, M. 1997. Are the Platyhelminthes amonophyletic group? An assessment using 18S rRNA. Mol. Biol. Evol.14: 485–497.

Czubaj, A., and Niewiadomska, K. 1997. The muscular system of the cerc-aria of Diplostomum pseudospathaceum Niew., 1984 (Digenea): a phal-loidin-rhodamine fluorescence and TEM study. Acta Parasitol. 42: 199–218.

Faubel, A. 1983. The Polycladida, Turbellaria. Proposal and establishmentof a new system. Part I. The Acotylea. Mitt. Hamb. Zool. Mus. Inst. 80:17–121.

Faubel, A. 1984. The Polycladida, Turbellaria. Proposal and establishmentof a new system. Part II. The Cotylea. Mitt. Hamb. Zool. Mus. Inst. 81:189–259.

Hartenstein, V., and Ehlers, U. 2000. The embryonic development of therhabdocoel flatworm Mesostoma lingua (Abildgaard, 1789). Dev. GenesEvol. 210: 399–415.

Hartenstein, V., and Jones, M. 2003. The embryonic development of thebody wall and nervous system of the cestode flatworm Hymenolepsisdiminuta. Cell Tissue Res. 311: 427–435.

Hill, S. D., and Boyer, B. C. 2001. Phalloidin labeling of developingmuscle in embryos of the polychaete Capitella sp. I. Biol. Bull. 201:257–258.

Hooge, M. D. 2001. Evolution of body-wall musculature in the Platy-helminthes (Acoelomorpha, Catenulida, Rhabditophora). J. Morphol.249: 171–194.

Hooge, M. D., and Tyler, S. 1999. Musculature of the facultativeparasite Urastoma cyprinae (Platyhelminthes). J. Morphol. 241:207–216.

Hooge, M. D., and Tyler, S. 2004. Comparative morphology of the bodywall in flatworms (Platyhelminthes). Can. J. Zool. 82: 194–210.

Hyman, L. H. 1951. The Invertebrates. Platyhelminthes and Rhyn-chocoela: The Acoelomate Bilateria. Vol. II. McGraw-Hill, New York,vii1572pp.

Lacalli, T. C. 1982. The nervous system and ciliary band of Muller’s larva.Proc. R. Soc. Lond. B 217: 37–58.

Ladurner, P., and Rieger, R. 2000. Embryonic muscles development ofConvoluta pulchra (TurbellariaFAcoelomorpha, Platyhelminthes). Dev.Biol. 222: 359–375.

Ladurner, P., Scharer, L., Salvenmoser, W., and Rieger, R. M. 2005. Mac-rostomum lignano, n. sp. (Rhabditophora, Macrostomorpha): a newmodel organism among the lower Bilateria and the use of digital videomicroscopy in taxonomy of meiobenthic Platyhelminthes. J. Zool. Syst.Evol. Res. 43: 114–126.

Lang, A. 1884. Die Polycladen (Seeplanarien) des Golfes von Neapel underder angrenzenden Meeresabschnitte. Eine Monographie. Fauna FloraGolfes v. Neapel, Leipzig.

Litvaitis, M. K., and Rohde, K. 1999. A molecular test of platyhelminthphylogeny: inferences from partial 28S rDNA sequences. Invertebr. Biol.118: 42–56.

Mair, G. R., Maule, A. G., Day, T. A., and Halton, D. W. 2000. A confocalmicroscopical study of the musculature of adult Schistosoma mansoni.Parasitology 121: 163–170.

Mair, G. R., Maule, A. G., Fried, B., Day, T. A., and Halton, D. W. 2003.Organization of the musculature of schistosome cercariae. J. Parasitol.89: 623–625.

Mair, G. R., Maule, A. G., Shaw, C., Johnston, C. F., and Halton, D. W.1998. Gross anatomy of the muscle systems of Fasciola hepatica as vi-sualized by phalloidin-fluorescence and confocal microscopy. Parasito-logy 117: 75–82.

Maslakova, S. A., Martindale, M. Q., and Norenburg, J. L. 2004. Vestigialprotoroch in a basal nemertean, Carinoma tremaphoros (Nemertea; Pal-aeonemertea). Evol. Dev. 6: 219–226.

McDougall, C., Chen, W., Shimeld, S. M., and Ferrier, D. E. K. 2006. Thedevelopment of the larval nervous system, musculature and ciliary bandsof Pomatoceros lamarckii (Annelida): heterochrony in polychates. Front.Zool. 3: 16–29.

Morris, J., Nallur, R., Ladurner, P., Egger, B., Rieger, R., and Hartenstein,V. 2004. The embryonic development of the flatworm Macrostomum sp.Dev. Genes Evol. 214: 220–239.

Nielsen, C. 2001. Animal Evolution: Interrelationship of the Living Phyla.2nd Ed. Oxford University Press, Oxford, 563pp.

Prudhoe, S. 1985. A Monograph on Polyclad Turbellaria. Department ofZoology, British Museum (Natural History), Oxford University Press,Oxford, 259pp.

Rawlinson, K., Bolanos, D. M., Liana, M. K., and Litvaitis, M. K. 2008.Reproduction, development and parental care in two direct-developingflatworms (Platyhelminthes: Polycladida: Acotylea). J. Nat. Hist. 42:2173–2192.

Reiter, D., Boyer, B., Ladurner, P., Mair, G., Salvenmoser, W., and Rieger,R. 1996. Differentiation of the body wall musculature in Macrostomumhystricinum marinum and Hoploplana inquilina (Plathelminthes), as mod-els for muscle development in lower Spiralia. Roux’s Arch. Dev. Biol. 205:410–423.

Rieger, R., and Ladurner, P. 2001. Searching for the stem species of theBilateria. Belg. J. Zool. 131 (suppl. 1): 27–34.

Rieger, R., and Ladurner, P. 2003. The significance of muscle cells for theorigin of mesoderm in Bilateria. Integr. Comp. Biol. 43: 47–54.

Rieger, R., Salvenmoser, W., Legniti, A., and Tyler, S. 1994. Phalloidin-rhodamine preparations of Macrostomum hystricinum marinum(Plathelminthes): morphology and postembryonic development of themusculature. Zoomorphology 114: 133–147.

Rieger, R., et al. 1991. Organization and differentiation of the body-wallmusculature in Macrostomum (Turbellaria, Macrostomidae). Hydrobio-logia 227: 119–129.

Ruppert, E. E. 1978. A review of metamorphosis of turbellarian larvae. InF.-S. Chia and M. E. Rice (eds.). Settlement and Metamorphosis of Ma-rine Invertebrate Larvae. Elsevier, New York, pp. 65–81.

Semmler, H., Bailly, X., and Wanninger, A. 2008. Myogenesis in the basalbilaterial Symsagittifera roscoffensis (Acoela). Front. Zool. 5: 14–28.

Wanninger, A., and Haszprunar, G. 2002. Chiton myogenesis: perspectivesfor the development and evolution of larval and adult muscle systems inmollusks. J. Morphol. 251: 103–113.

Wanninger, A., Koop, D., Bromham, L., Noonan, E., and Degnan, B. M.2005. Nervous and muscle system development in Phascolion strombus(Sipuncula). Dev. Genes Evol. 215: 509–518.

Younossi-Hartenstein, A., and Hartenstein, V. 2000. The embryonic devel-opment of the polyclad Imogine mcgrathi.Dev. Genes Evol. 210: 383–398.


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Embryonic muscle development in direct and indirect developing