FATE OF THE CYPRIS AND ADULT ADDUCTOR MUSCLES DURING
METAMORPHOSIS OF BALANUS AMPHITRITE (CIRRIPEDIA: THORACICA)
Henrik Glenner a n d Jens T. Høeg
A B S T R A C T
The ontogeny of the cypris adductor muscle (CAM) and the adult adductor muscle (musculus
adductor scutorum, M A S ) of the acorn barnacle Balanus amphitrite was examined during the
cypris/juvenile metamorphosis. By using section series of epoxy-resin embedded material, we found
that the MAS of the adult barnacle develops de novo in a preoral position, while the postoral C A M
degenerates but remains present. Eventually the C A M of the cypris larva disintegrates and the remaining MAS serves to close the opercular plates of the juvenile and adult barnacle. The C A M
and the MAS accordingly do not represent homologous structures and should be encoded as separate characters in a phylogenetic analysis. The character states of the CAM and M A S in the taxa
comprising the Thecostraca are reviewed.
In all cirripede cypris larvae, the cypris adductor muscle, or CAM, connects the two
sides o f the carapace, which derive directly
from downfolding of the naupliar headshield
(Walossek et al., 1996). The CAM is situated
postorally and ventral to the gut, but dorsal
to the postoral parts of the central nervous
system.
All juvenile and adult Thoracica except
Ibla have a preorally situated musculus adductor scutorum (MAS). This adductor muscle connects the two scuta and effects closure o f all four opercular plates, the scuta by
direct action and the terga by indirect action.
Despite a lack of data, the literature has often referred to the MAS as originating from
the translocated cypris adductor (CAM)
(Doochin, 1951; Daniel, 1958). However, as
pointed out by Hessler (1964), this interpretation entails a topological transition which is
very difficult to envision. Either the median
part o f the cypris muscle (CAM) must disintegrate, thereby allowing the remaining two
lateral parts to pass by the alimentary tract
and reunite preorally, or the alimentary tract
must break apart and allow the intact adductor muscle to move to the preoral position, after which a continuous alimentary tract is
reestablished. Both of these scenarios imply
that the CAM develops into the MAS of the
adult, and, therefore, that the CAM and MAS
are homologous structures. Opposed to this,
Walley (1969) in the benchmark study on cirripede metamorphosis stated very briefly that
the larval CAM and adult MAS are unrelated
in Semibalanus balanoides (L.). Although her
study was commendably detailed for many
other organ systems, she did not provide morphological details on this problem.
The present study was carried out to investigate the ontogeny of the adductor muscle during cirripede metamorphosis and to clarify
whether the larval CAM and the adult MAS
represent the same homologous structure or
have a fundamentally different origin. As discussed by Klepal (1985) and Glenner et al.
(1995), the answer to this question is also important for cirripede phylogeny in general.
We chose to investigate adductor muscle
ontogeny in Balanus amphitrite Darwin because this species has been reported to have
a chaotic stage during metamorphosis (Bernard and Lane, 1962).
MATERIALS AND METHODS
Adult Balanu.s amphitrite from Beaufort, North Carolina. U.S.A., were induced to release nauplii in the laboratory. The larvae were kept at 28°C from release as nauplius I to the cypris stage (Rittschof et al., 1984).
Cypris larvae were observed continuously through a
dissection microscope both before and after settlement.
The timing of events in settlement and metamorphosis became known within an accuracy of about 5 min.
Cypris larvae and metamorphosing specimens were
fixed in Bouin's fluid at intervals of I h after settlement
until the commencement of cirral beat. After fixation,
the specimens were embedded in TAAB 812 epoxy resin,
cut serially in 2-pm sections on an ultramicrotome, and
examined with a Leica DM RXA light microscope.
RESULTS
The Pelagic Cypris
Our results on Balanus amphitrite and our
observations on cypris larvae from pedunculate
Thoracica, Acrothoracica, and Rhizocephala
Fig. 1. A, Unsettled cypris, horizontal section, anterior end left, section plane slightly tilted with micrograph showing only left part of C A M (unlabeled arrow indicating position of right part), no developing MAS present on this
section or anywhere else in section series; B, Cypris, early metamorphosis 3 0 - 9 0 min after settlement; section oriented as in A; two still unconnected parts of MAS have appeared, widely separated from disintegrating CAM, two
parts of M A S not meeting anywhere in section series. Legends: AN = antennuic; CAM = cypris adductor muscle;
M A S = musculus adductor scutorum; PMC = posterior mantle cavity; T H O = thorax.
agree on a similar position and morphology
of the C A M (Høeg, 1985; unpublished data).
The CAM consists of two cone-shaped arrays
of distinctly cross-striated muscle fibers interconnected by a median tendon. In Balanus
amphitrite, the cypris is 5 4 0 - 5 9 0 p m long
with the CAM located 275 p m from the anterior end (CAM, Figs. 1A, 4A). On either
side of the cypris body, the bases of the coneshaped muscular arrays attach to the inner
surface of the carapace in an approximately
6 5 - g m wide circular zone (CAM, Fig 3A).
From each side of the carapace, the cross-striated fibers converge medially from these attachments and connect with the median acellular tendon. No muscular elements extend
from one side of the body to the other (Fig.
3A).
When contracting, the C A M moves the two
halves of the carapace toward each other. This
results in closure of the ventral aperture that
leads into the anterior and posterior mantle
cavities, which house the antennules and the
thorax with its natatory appendages (Walley,
1969; Hoeg, 1985; Walossek et al., 1996).
The Metamorphosing Cypris
Soon after attachment the cypris initiates
the metamorphic molt. This eventually results
in the shedding o f the cypris carapace, developed from the naupliar headshield, and liberation of the first juvenile instar, which immediately commences setose feeding with the
thoracopods (Walley, 1969; Glenner and
Hoeg, 1993). The CAM remains in place during the first part of metamorphosis (CAM,
Fig. 2. Metamorphosing cypris 2 - 3 h after settlement, two sections (A, B) from horizontal section series of same
specimen, anterior end left, MAS forming one continuous muscle attaching on carapace (A) and extending across
body with no intervening tendon (B). Legends and scale as in Fig. 1.
Fig. 3B). It does not show signs of reconfiguration or anterior migration. Instead, it gradually degenerates and finally disappears altogether before metamorphosis is completed
(CAM, Fig. IB).
Preorally, and in the same metamorphosing
specimens that still had a postorally situated
CAM, we observed the development of the
incipient MAS (Figs. 1B, 3B, C). Serial sections confirmed that the developing MAS was
at all times far separated from the CAM. The
MAS first appears as two separate muscular
bundles, each attached to the carapace cuticle on its respective side of the body. Both
bundles bend toward the midline, but initially
fail to contact (again confirmed by serial sections: MAS in Figs. 1B, 3C). Slightly later,
they contact so that the final MAS forms a
continuous band of muscular tissue connecting the two sides of the carapace (MAS, Figs.
2A, B, 3D). In addition to the differences in
ontogeny and position, the C A M and MAS
also differ distinctly in two morphological
features. Unlike the CAM, the MAS does not
have a median tendon at any stage of development (Figs. 2B, 3D). Moreover, the C A M
is always distinctly cross-striated, while we
could not detect any cross-striation in the
MAS at the light microscopical level. Matsuno
and Hirota (1989) found that the MAS of the
balanomorph Tetraclita squamosa Bruguiere
is cross-striated at the TEM level, but in a disordered fashion and with incomplete Z-discs.
Thus, the MAS has the characteristics of slow
muscle capable of sustained contraction (see review of cirripede muscles in Anderson, 1993).
Shedding of the cypris carapace and initial beating of the thoracopods (= cirri) can
start as early as 10 h after settlement (Glenner and H0eg, 1993). At this stage, the juvenile barnacle can already close the newly
formed opercular plates, a function vital to its
survival.
DISCUSSION
Homology of the CAM and the MAS
The development from a pelagic and mobile cypris to a permanently attached juvenile
barnacle is a classic example of an elaborate
invertebrate metamorphosis (Walley, 1969).
Our study revealed that during the metamorphosis of Balanus amphitrite the postorally
sited cypris adductor muscle (CAM) degenerates, while a new and ontogenetically separate muscle, musculus adductor scutorum,
(MAS) develops preorally (Fig. 4). We observed both muscles, spatially widely separated, within the same metamorphosing specimens. We therefore conclude that the cypris
and adult adductor muscles represent different, nonhomologous organs with no ontogenetic or phylogenetic relation. This solves a
long-standing discussion and has important
phylogenetic implications (Klepal, 1985;
Glenner et al., 1995). The impression gained
from earlier literature on the adductor muscle
is that the cypris CAM develops directly into
the adult MAS (Doochin, 1951; Daniel,
1958). However, in a benchmark paper, Hessler (1964) expressed serious doubt about this,
since a translocation of the postoral CAM into
a preoral MAS would be "topographically impossible." Our results prove Hessler right. Finally, our sections, those of Takenaka et al.
(1993), and the SEM observations of Glenner
and H0eg (1993) all agree that Bernard and
Lane (1962) were in error when describing a
"chaotic phase" in the metamorphosis of Balanus amphitrite.
Implications for Cirripede Phylogeny
The Cirripedia is a taxon within the Thecostraca (Maxillopoda), which also comprises
the Ascothoracida and the Facetotecta (Table
1), and with the Tantulocarida as the likely extant sister group. In all Thecostraca, the terminal larval stage is a cypris-like form which
accomplishes settlement (Grygier, 1987;
Walker, 1992; Anderson, 1993; Moyse et a l ,
1995; Newman, 1996).
The Cirripedia comprises three taxa, the
boring barnacles (Acrothoracica), the parasitic
barnacles (Rhizocephala), and the stalked and
acorn barnacles (Thoracica). Recent evidence
from rRNA sequencing and from larval ultrastructure suggests that the Acrothoracica is the
sister group to a taxon comprising the Rhizocephala and the Thoracica (Jensen et al., 1994;
Spears et al., 1994).
Except for the Iblidae, all adult Thoracica
have an MAS situated preorally just as in Balanus amphitrite. The monotypic Iblidae stand
apart, in that the adult has the adductor situated postorally, i.e., coinciding with the position of the CAM in the cypris. A similar situation occurs in the Acrothoracica and in the
Ascothoracida, suggesting that this is the plesiomorphic condition. As discussed by Klepal
(1985), this is one reason why the Iblidae are
often considered to be the sister group of all
remaining Thoracica. The importance of following the fate of the adductor muscle during the metamorphosis of Ibla is now evident.
It is necessary to establish whether the adult
muscle in Ibla derives directly from the larval one, or whether the adult MAS develops
de novo as shown for other cirripedes (Walley, 1969, for Semibalanus balanoides; present study for Balanus amphitrite). Although
the metamorphosis was not observed, Batham's
(1945) study indicates that a de novo development also takes place in the Iblidae. She
observed that the postoral adult adductor in
Ibla idiotica Batham consists of "unstriated
�
—
Fig. 3. A, Unsettled cypris; transverse section showing two separate cone-shaped parts of C A M attached to carapace and converging on median tendon; B, Cypris, early metamorphosis 3 0 - 9 0 min after settlement, section oriented as in A, C A M unchanged compared to A; C, Same specimen as in B, but 30 u m apart in section series as indicated in Fig. 4B, two lateral halves of developing MAS not meeting here or anywhere else in section series; D,
Metamorphosing cypris 2 - 3 h after settlement, section oriented as in C, but two halves of developing MAS now
connected medially to form continuous adductor (as in specimen in Fig. 2); E, Early juvenile 8 h after settlement,
transverse section, cypris carapace shed and MAS extending across body between areas of future scutal plates (MAS
shortened compared with condition in 2D). Scale as in Fig. 1. Legends: C A M = cypris adductor muscle; M A S =
musculus adductor scutorum; MC = mantle cavity; T H O = thorax.
Table 1.
The adductor muscle in the Crustacea Thecostraca.
(1) For these taxa there is no certain information whether the larval adductor is conserved in (he adult.
(2) Known only as larvae. The cypris-y of the Facetotecta may lack an adductor, since this structure was not seen by Grygier (1987, and personal c o m m u n i cation).
(3) T h e adult Rhizocephala are so specialized that they have lost almost all traces of cirripede morphology.
fibers," just as is the case for the preoral adductor, MAS, in B. amphitrite, when viewed
with the light microscope. In contrast, the
cypris adductor muscle, CAM, consists of
distinctly striated fibers in all cirripedes, also
when viewed only with the light microscope
(Walley, 1969; Hoeg, 1985; this study).
In the first cladistic analysis of the Thoracica, Glenner et al. (1995) used the position
o f the adult adductor muscle as one of their
soft-tissue characters. With some reservation,
they coded the character states into a single
binary character, namely, (0) adult adductor
muscle postoral, (1) adult adductor muscle
preoral. However, our results herein show that
the postoral cypris adductor muscle and the
preoral adult adductor muscle represent nonhomologous organs. This suggests that the
adult adductor in the chosen outgroup Ascothoracida is not homologous to the adult adductor in the cirripede ingroup taxa and, that,
therefore, a revised coding scheme o f the
Glenner et al. (1995) matrix is called for.
Such a recoding, incorporating all existing information on larval and adult adductor muscles and their fate in metamorphosis, must
await studies of metamorphosis in both the
Ascothoracida and the Iblidae. In addition,
the Facetotecta warrants close attention, since
it is unknown whether the cypris-y larva of
this thecostracan taxon has a carapace adductor (Grygier, 1987, and personal communication). The answer to these questions in
the Thecostraca will be valuable when discussing the homology and phylogenetic significance of "carapaces" and their adductor
muscles in Crustacea at large (see reviews in
Hessler, 1964; Walossek, 1993; Fryer, 1996).
The intricate nature of adductor muscle
morphology and ontogeny in the Thecostraca
once again illustrates the importance in phylogenetic systematics of a detailed morphological analysis followed by a careful consideration of alternative character-coding
schemes.
ACKNOWLEDGEMENTS
We are indebted to Prof. R. R. Hessler, Scripps Institution of Oceanography, for inducing us to complete this
study and for many inspiring discussions and intriguing
lectures, and also to Prof. D. Rittschof, Duke University
Marine Laboratory, for unfailing hospitality to HG.
Dr. M. J. Grygier helped us with information on the
fFig. 4. Metamorphosis in Balanus amphitrite showing topological relations of cypris adductor muscle (CAM) and
adult musculus adductor scutorum (MAS), diagrammatic representation of median sagittal sections. A, Unsettled cypris
with postoral CAM, horizontal arrows showing section plane of Fig. 1 A; B, Newly settled cyprid retaining CAM,
but with clearly separate MAS developing preorally, thorax rotated approximately 90° compared to original cypris
long axis making preoral MAS appear "posterior" to C A M in some section planes, anterior mantle cavity (AMC)
with antennules (AN) almost obliterated, horizontal arrows showing section plane of Fig. 2B, near vertical arrows
showing section planes of Figs. 3B and 3C; C, Early juvenile after shedding of cypris carapace and 180' rotation of
thorax completed, with CAM completely disappeared and M A S functioning to operate opercular valves, scutum and
tergum not appearing in truly median sagittal section, but included here for clarity, attachment organs of cypris antennules (AN) retained revealing anteriormost end o f body, vertical arrows showing section plane of Fig. 3E, adapted
from Walley (1969), Walker (1992), and Takenaka et al. (1993). Legends: AMC = anterior mantle cavity; AN = antennule; C A M = cypris adductor muscle; CIR = cirri; MAS = musculus adductor scutorum; MC = mantle cavity; NE
= nauplius eye; NS = nervous system; P M C = posterior mantle cavity; SC = scutum; STO = stomach; T E = tergum;
T H P = thoracopods.
Ascothoracida and Facetotecta. Finally, we gratefully
a c k n o w l e d g e the financial support of the University
of C o p e n h a g e n to H G and the Danish Natural Science
Foundation (grant number 9 4 - 0 1 1 6 3 6 for general support to JH and grant number 9 6 - 0 1 4 0 5 for a Leica photomicroscope).
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RECEIVED: 1 August 1997.
ACCEPTED: 8 December 1997.
Address: Department of Cell Biology and Anatomy,
Institute of Zoology, University o f Copenhagen, D K 2 1 0 0 Copenhagen, Denmark (e-mail: [email protected])