Three-dimensional reconstruction of the naupliar musculature and a scanning electron microscopy atlas of nauplius development of Balanus improvisus (Crustacea: Cirripedia: Thoracica)

Arthropod Structure & Development 38 (2009) 135–145

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Three-dimensional reconstruction of the naupliar musculature and a scanning electron microscopy atlas of nauplius development of Balanus improvisus (Crustacea: Cirripedia: Thoracica) Henrike Semmler a, b, *, Jens T. Høeg b, Gerhard Scholtz a, Andreas Wanninger b a b

¨t zu Berlin, Institut fu ¨ r Biologie, Vergleichende Zoologie, Philippstr. 13, D-10115 Berlin, Germany Humboldt-Universita University of Copenhagen, Department of Biology, Research Group for Comparative Zoology, Universitetsparken 15, DK-2100 Copenhagen Ø, Denmark

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 April 2008 Accepted 29 September 2008

An atlas of the naupliar development of the cirripede Balanus improvisus Darwin, 1854 using scanning electron microscopy (SEM) is provided. Existing spikes on the hindbody increase in number with each moult and are an applicable character for identification of the different nauplius stages, as is the setation pattern of the first antennae. The naupliar musculature of B. improvisus was stained with phalloidin to visualise F-actin, followed by analysis using confocal laser scanning microscopy (CLSM) with subsequent application of 3D imaging software. The larval musculature is already fully established in the first nauplius stage and remains largely unchanged during all the six nauplius stages. The musculature associated with the feeding apparatus is highly elaborated and the labrum possesses lateral muscles and distal F-actin-positive structures. The alimentary tract is entirely surrounded by circular muscles. The extrinsic limb musculature comprises muscles originating from the dorsal and the ventral sides of the head shield, respectively. The hindbody shows very prominent postero-lateral muscles that insert on the dorso-lateral side of the head shield and bend towards ventro-posterior. We conclude that the key features of the naupliar gross anatomy and muscular architecture of B. improvisus are important characters for phylogenetic inferences if analysed in a comparative evolutionary framework. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Nauplius Phalloidin Development SEM 3D reconstruction Confocal microscopy

1. Introduction The Cirripedia constitute a clade of sessile crustaceans with a very distinct adult body anatomy, whose members are mostly filter feeders, while others are commensals or parasites. The position of cirripedes within the Animal Kingdom has been widely debated for almost two centuries, until Thompson (1830) demonstrated that the settlement of Balanus is preceded by a swimming crustacean-type larval stage (Scholtz, 2008). After Mu¨nter and Buchholz (1870) had described the first two nauplius stages of Balanus improvisus, later nauplius stages of this species were found but could not be assigned to specific stages within the life cycle (Filatowa, 1902; Hoek, 1909; Tengstrand, 1931; Lucks, 1940). It was not until Buchholz (1951) and Jones and Crisp (1954) that all six nauplius stages of B. improvisus were described in detail, resulting in semi-schematic drawings of the first antenna and ventral views of the posterior end of all developmental stages. In addition,

* Corresponding author. University of Copenhagen, Department of Biology, Research Group for Comparative Zoology, Universitetsparken 15, DK-2100 Copenhagen Ø, Denmark. Fax: þ45 35 32 12 00. E-mail address: [email protected] (H. Semmler). 1467-8039/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2008.09.003

illustrations of all six nauplii and the lecithotrophic cypris larva of B. improvisus are available, but the detailed morphological descriptions are given in Russian only (Murina and Grintsov, 1995). Since absolute size of a specimen alone does not suffice to discern between cirripede nauplius stages due to a relative high plasticity of this feature even within a single species, the aforementioned studies used the so-called ‘‘setation formula’’ (sensu Bassindale, 1936) for this purpose. In addition, the gross morphology of the hindbody has proven to yield important characters for staging cirripede nauplii (Buchholz, 1951). Accordingly, we reinvestigated in detail the gross anatomy of B. improvisus and provide herein one of the first SEM-based accounts of the entire series of the nauplius stages in a cirripede crustacean (cf. Rybakov et al., 2002; Chan, 2003). Such data may serve as a basis for future comparative developmental analyses of this phylogenetically important crustacean taxon. While a wealth of information exists concerning the anatomy of adult cirripedes, surprisingly few recent studies have focused on the inner anatomy of the nauplius, e.g., the larval central nervous system (Semmler et al., 2008). The muscular systems of the larvae of the balanomorph Semibalanus balanoides (formerly B. balanoides) and the pedunculate barnacle Ibla quadrivalvis were described in some detail using light microscopy (Walley, 1969; Anderson, 1987).

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In addition, the embryonic and larval musculature of the goosebarnacle Calantica spinosa (syn. Pollicipes spinosus) and the musculature of the cypris larva of Lernaeodiscus porcellanae had been depicted earlier (Batham, 1946; Høeg, 1985). More recently, data on the ultrastructure of muscle fibre cells and the first antennae musculature of the cyprid of B. amphitrite as well as the overall muscular arrangement of B. improvisus nauplii have become available employing transmission electron microscopy and/or F-actin labelling in conjunction with confocal laser scanning microscopy (CLSM) (Lagersson, 2002; Lagersson and Høeg, 2002; Semmler et al., 2006). The scarcity of larval myoanatomical data not only applies to cirripedes but to Crustacea as a whole. Earlier studies described the naupliar trunk anatomy of the anacostracan branchiopod Artemia salina and Branchinecta ferox by using semi-thin sections (Benesch, 1969; Fryer, 1983), while the development of the limbs and the limb musculature of the branchiopod Triops longicaudatus were investigated by means of phalloidin staining (Williams and Mu¨ller, 1996). In addition, myogenesis of the dendrobranchiate decapod shrimp Sicyonia ingentis and of the branchiopod A. salina were described recently (Kiernan and Hertzler, 2006). Furthermore, myogenesis of the first antennae of the ostracod Heterocypris incongruens was investigated by means of light microscopy (Smith and Tsukagoshi, 2005) and muscle development of the two isopods, Porcellio scaber and Idotea baltica, was studied by immunocytochemistry (Kreissl et al., 2008). In order to broaden the database for our understanding of the functional anatomy and the evolution of cirripede organ systems, the present study shows a detailed three-dimensional reconstruction of the musculature of the nauplius larva of B. improvisus. In combination with the description of the ontogenetic sequence of the larval development of this species we also contribute to questions concerning naupliar development, identification, and morphology of Cirripedia and Crustacea as a whole. 2. Material and methods 2.1. Animal collection and fixation Adult B. improvisus were collected from buoys and wooden planks in the Koster-Fjord and the Ide-Fjord at the Northwest coast of Sweden (Skagerrak). The specimens were maintained in running seawater tanks at the Tja¨rno¨ Marine Biological Laboratory (TMBL) and were fed with Artemia larvae. The nauplii were captured by filtering the water through 60 mm nets and were kept in 0.2 mm millipore-filtered seawater. Antibiotics (Streptomycin: 36.5 mg/l and Penicillin G: 21.9 mg/l) were added to prevent bacterial and fungal growth. The animals were fed with the microalgae Thalassiosira pseudonana (200 cells/ml) and Skeletonema costatum (100 cells/ml), respectively. The larvae were relaxed in 7.4% MgCl2 and fixed twice a day in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) for 1–2 h at room temperature. Subsequently, they were washed several times in 0.1 M PB and stored in 0.1 M PB þ 0.1% NaN3 at 4  C. Since the free-living first nauplius stage is only existent for up to 2 h, gravid adults were dissected in order to obtain the nauplius I stage. 2.2. Fluorescence staining, confocal microscopy, and imaging procedures In order to overcome permeabilization problems, the larval carapace was punctured or cut with insect needles and the larvae were subsequently sonicated in a Branson 2210 sonicator (Branson Ultrasonics, Danbury, CT, USA) at 42 kHz for 20 s to up to 1 min. Then, the specimens were washed three times in PB and incubated for 1 h in 0.4% Triton-X 100 in 0.1 M PB (PBT). The following steps

were carried out in the dark. The specimens were incubated in 1:40 diluted Oregon Green 514 phalloidin (Molecular Probes, Eugene, OR, USA) in 0.1 M PBT over night. The specimens were washed for 3  15 min in 0.1 M PB and embedded in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). To assess the degree of tissue autofluorescence, the incubation step in Oregon Green 514 phalloidin was skipped in the negative probes. In addition, photo-bleaching experiments, in which phalloidin-stained specimens were exposed to 405–488 nm laser light until the signal of the fluorochrome faded, were carried out. These procedures demonstrated high specificity of the phalloidin staining for the musculature of the study specimens with little unspecific or autofluorescent signal. A total of five specimens of nauplius I larvae, 17 nauplii II, eight nauplii III, eight nauplii IV, six nauplii V, and 10 nauplius VI larvae were analysed with a Leica DM RXE 6 TL fluorescence microscope with a TCS SP2 AOBS laser scanning device (Leica Microsystems, Wetzlar, Germany), using a HCX PL APO CS 40.0 1.25 oil UV objective lens and 1.0–1.35 confocal zoom settings. Images were edited with the Leica confocal software and Adobe Photoshop CS2 and Adobe Illustrator CS2 imaging software, respectively (Adobe Systems, San Jose, CA, USA). For three-dimensional computer reconstruction, the CLSM image stacks, which each comprised between 90 and 150 optical sections with a 0.3 mm step-size, were processed with the IMARIS 5.7.1 software (Bitplane AG, Zu¨rich, Switzerland). 2.3. Scanning electron microscopy The specimens were fixed in 4% PFA in PB and stored in 0.1 M PB þ 0.1% NaN3 at 4  C. They were washed twice in distilled water and then dehydrated in a graded acetone series. After two additional washes in 100% acetone the specimens were critical point dried with a Baltec CPD 030 critical point dryer (BAL-TEC AG, Balzers, Liechtenstein) and sputter-coated with platinum–palladium for 140 s in a JEOL JFC 2300HR sputter coater (Jeol Ltd., Tokyo, Japan). Subsequently, the specimens were analysed with a JEOL JSM-6335F scanning electron microscope. 3. Results 3.1. Gross morphology of B. improvisus naupliar stages Crustacean nauplii are characterized by three pairs of appendages, i.e. the first and second antennae and the mandibles (e.g., Williams, 1994; Scholtz, 2000; Dahms et al., 2006). In addition, all cirripede nauplii have a pair of characteristic frontolateral horns (Fig. 1). Naupliar development involves both an increase in size (Table 1) and an increase in the number of setae on the appendages (Figs. 1 and 2) and the number of spines on the posterior process (Figs. 1 and 3). Nauplius I is about 160 mm long, whereas the nauplius VI is about 600 mm long. According to Bassindale (1936) the nauplius stages differ in the number of setae on the first antenna. Along the four segments of the first antennae, both the nauplius I and II exhibit four terminal setae and four ventral setae but lack dorsal setae (Fig. 2A, B). Dorsal setae appear incrementally with one in nauplius III, (Fig. 2C), two in nauplius IV, and three in nauplius V. Nauplius V bears an additional ventral seta. The final nauplius stage (VI) possesses three dorsal and six ventral setae (Fig. 2E). The posterior process proves to be the most distinct character to distinguish the different nauplius stages from each other, whereas the absolute size of the individuals may vary in different populations and some stages do show overlaps (Table 1). Nauplius I exists for only a few hours after hatching and carries neither setae nor spines on the posterior process. In nauplius II a pair of spines

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Fig. 1. SEM micrographs of the six nauplius stages of B. improvisus. (A) Nauplius I shows no setation on the labrum (la). The first antennae (a1), the second antennae (a2) and the mandibles (md) possess only few setae. (B–F) Setation increases in the successive stages. (B þ C) Nauplii II (B) and III (C) hardly differ in morphology and size. (D–G) The head shields of the nauplii IV (D), V (E) and VI (G) stage show posterior spines (arrows). The best characters for separating the naupliar stages are size and differences of the posterior process. ff, frontal filament; fh, frontolateral horn. Scale bars: A: 50 mm, B–G: 100 mm.

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Table 1 Parameters characterizing the larval stages of Balanus improvisus. Number of specimens investigated per stage was 10 except for stage V where there were only three specimens available. Parameter

Period of occurrence after release from mother animal

Nauplius stage I Hour 0–2 II Day 1–2 III Day 2–3 IV Day 3–4 V Day 4–5 VI Day 5–6

Body length (head–furca), mm

Carapace width, mm

Distance between the frontolateral horns, mm

137–161 250–281 312–343 344–406 453 587–609

80–98 135–150 165–187 203–234 213–296 343–375

80–95 217–250 249–265 313–328 310–375 406–438

‘‘s1’’ (sensu Norris and Crisp, 1953) and anterior to these a semicircle of outgrowths on the ventral surface of the posterior process appears, while in the nauplius III a line of outgrowths is present anterior to the spines (Fig. 3B, C). In addition to the pair of ‘‘s1’’spines found in II–VI, nauplius IV adds a second pair of spines (‘‘s2’’) and a median spine (Fig. 3D). In the anterior region of the posterior process, rows of outgrowths are present and the head shield possesses a pair of spines on its posterior margin (Figs. 1 and 3). Nauplius V exhibits an additional pair of spines (‘‘s3’’) close to the ‘‘s1’’ pair (Fig. 3E). In the region anterior to the ‘‘s3’’ spines the last (VI) nauplius stage features six pairs of ventral spines which mark the developing thoracic segments of the ensuing cypris larva (Fig. 3F).

3.2. Musculature 3.2.1. General The various striated muscles differ in diameter in the six nauplius stages, but the relative arrangement to each other remains constant from nauplius I through nauplius VI. The naupliar musculature consists of several distinct functional units: the musculature associated with the feeding apparatus, dorsal and ventral extrinsic limb muscles, and muscle fibres for the moving of the hindbody. 3.2.2. Musculature of the feeding apparatus The musculature associated with the feeding apparatus is very complex (Figs. 4 and 5A, G, H). The labrum bears on the lateral sides two pairs of muscles (Figs. 4C–E and 5G, H) of which the proximal pair consists of two fibres and the distal muscle pair of three fibres. Between the lateral muscles four pairs of F-actin-positive protuberances are present (Fig. 5G, arrows). They are connected to each other by myofibres (Fig. 5G). The posterior-most interconnection is distally extended, forming a V-shaped structure (Figs. 4C, E, arrow, and 5G, H). The tip of the labrum holds three distal pairs of F-actin-positive structures. Close to the more distal lateral muscles (l2), a transversal fibre (tv) is present on the dorsal side of the labrum (Figs. 4F, G and 5C, H). The oesophagus and the intestine are contractible by circular muscles (Figs. 4A, I and 5A, H). The hindgut possesses strong circular muscles around the intestine, while the midgut shows only very fine circular muscles (Figs. 4A and 5A, B, E). Close to the oesophageal ring muscles (oc), two ventral and two dorsal fibres

Fig. 2. SEM images of the right first antennae (a1). (A) Nauplius I lacks dorsal setae. (B) In nauplius II the first antenna is divided into four segments. (C) Nauplius III exhibits one dorsal seta (marked green) on the first antennae. (D) In nauplius IV one additional dorsal seta is present on the first antennae (marked blue). (E) In nauplius V the first antennae feature one additional dorsal (marked pink) and ventral seta (marked yellow). (F) Nauplius VI features one additional ventral (marked light green) seta. ts, terminal setae; v1, ventral seta 1; v2, ventral seta 2; v3 þ 4, ventral setae 3 and 4. Scale bars: A: 10 mm, B–F: 25 mm.

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Fig. 3. Semi-schematic representation of the six nauplius stages focusing on the body outline and the posterior process. (A) Nauplius I is much smaller than the later stages and shows only a few setae and spines. (B) The hindbody of nauplius II is elongated (pp). A caudal spine (cs) and a pair of spines (s1) are present at the base of the posterior process. The small outgrowths (arrow) on the hindbody resemble a semi-circle. (C) In nauplius III a line of outgrowths is present on the hindbody (double arrow). (D) In nauplius IV there are additional spines present on the posterior process, viz., a pair of ‘‘s2’’ spines and a median spine (ms). Lines of small outgrowths (double arrowheads) are present on the hindbody anterior to the median spine. The head shield carries one pair of spines (hs). (E) Nauplius V shows a pair of ‘‘s3’’ spines in addition to spines already present on the posterior process in previous stages. (F) The hindbody of nauplius VI carries six pairs of small spines (arrowheads). la, labrum.

run into the direction of the nauplius eye (Figs. 4D, H, I and 5G, H; od, ov). At the base of the frontal filaments two fibres (ffm) are found (Figs. 4F–H and 5 in purple). There are diverse fibres which connect the muscles of the hindbody to each other. These ‘‘reinforcement muscles’’ are mainly located in the region of the intestine (Figs. 4G, H, arrowheads and 5A, B in brown). The musculature of the second antennae and the mandibles, which are also involved in the food intake, are discussed below. 3.2.3. Dorsal extrinsic muscles Four dorsal muscle pairs are involved in the movement of the first antennae (Fig. 5I), three of which arise from the dorsal midline (a1d1, a1d3, a1d4), whereas the fourth pair originates from a more dorso-

lateral region (a1d2). Three pairs of second antenna muscles have their origin at the dorsal midline (Fig. 5, a2d1, a2d3, a2d7), whereas two pairs of fibres insert slightly lateral to the midline (a2d2, a2d8). From the vicinity of the a2d2-muscle three additional muscle pairs proceed to the second antennae (a2d4–6). Only one pair of the dorsal mandibular muscles inserts at the midline (Fig. 5I, mdd3), whereas two other muscle pairs insert laterally to the dorsal midline (mdd1, mdd2). Furthermore, two mandible fibre pairs originate from a dorso-lateral region of the head shield (mdd4, mdd5). 3.2.4. Ventral extrinsic muscles One muscle pair, originating close to the oesophageal base, bends from the ventral side of the animal and reaches into the first

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Fig. 4. CLSM images of the musculature of the third nauplius stage of B. improvisus. (A) In the projection image the musculature associated with the feeding apparatus (boxed area), and the postero-lateral muscles (plm) show strong signal. The intestine is surrounded by circular muscles (im). (B) Overlay of the transmission light micrograph and the respective projection image shows the location of the appendages and the frontolateral horns (fh). (C–J) Different focus planes of the CLSM stack are depicted from ventral to dorsal. (C–E) The lateral muscles (l1, l2) are situated on the lateral sides of the labrum. In the distal part of the labrum, three paired structures are located (black asterisks). The arrows point to the ‘‘Vshaped’’ structure in the tip of the labrum. In the proximal region of the labrum, the ventral oesophageal muscles (ov) are located. (F) Posterior to the stomodeum (white asterisk) the transversal muscle (tv) and the third ventral muscle of the second antennae (a2v3) are present. (G–I) The extrinsic muscles of the second antennae (a2v3–5), the mandibles (mdv1–2), and a muscle interconnecting the latter one with the postero-lateral muscle (mdpl), insert on the ventral side. ‘‘Reinforcement’’ muscles interconnect several fibres (arrowheads). (I) Oesophageal circular muscles (oc) surround the mouth opening. (J) Dorsal extrinsic muscles insert in the first antennae (a1d1, a1d3–4), the second antennae (a2d1, a2d7–8), and the mandibles (mdd3). a2, antenna 2; cs, caudal spine; ffm, frontal filament muscle; md, mandible; od, dorsal oesophageal muscle.

antennae (Fig. 5C, a1v1). There are five ventral pairs of muscles which extend into the second antennae (Figs. 4G–J and 5C, a2v1–5). One muscle pair leads to the mandible from the ventral midline, whereas an additional fibre inserts at a ventro-lateral position (Figs.

4G, H, J and 5C, mdv1, mdv2). A third very subtle extrinsic muscle fibre arises at the muscle which interconnects the mandibular ventral midline muscle and the postero-lateral fibres (Supplementary data, Movie clip 1). These three mandibular ventral

Fig. 5. Three-dimensional reconstruction of the nauplius II musculature. The musculature compromises several units: the muscles of the first antennae (white), the second antennae (yellow), the mandibles (green), postero-lateral muscles (blue), the intestinal muscles (orange) and the feeding apparatus (pink). (A) Ventral view. (B) Dorsal view. (C) The ventral muscles are shown from the dorsal side by omitting the dorsal-most part. (D) Dorsal view of the right body half. The dorsal (white, yellow, green) and ventral (light blue, orange, dark green) extrinsic muscles are shown in different colours. (E) Musculature of the nauplius in posterior view. (F) Overlay of a three-dimensional reconstruction of the musculature and a light micrograph of the specimen shown in A, indicating the relative location of the musculature within the specimen. (G þ H) The musculature of the feeding apparatus features lateral muscles (l1, l2), a pair of ventral (ov) and dorsal (od) oesophageal muscles, oesophageal circular muscles (oc), a transversal muscle (tv) and small F-actin-positive protuberances (arrows); viewed from the ventral (G) and the dorsal (H) sides, respectively. (I) The dorsal musculature is schematically redrawn. a1, antenna 1; a1d1–4, dorsal muscles of the first antennae 1–4; a1v1, ventral muscle of the first antennae; a2, antenna 2, a2d1–8, dorsal muscles of the second antennae 1–8; a2v1–5, ventral muscle of the second antennae 1–5; ffm, frontal filament muscle; md, mandible; mdv1–2, ventral mandible muscles 1–2; mdd1–5, dorsal mandible muscles 1–5; mdpl, mandibular postero-lateral muscle; omd1, oblique mandilbe muscle 1; omd2, oblique mandible muscle 2; brown, ‘‘reinforcement’’ muscles; purple, frontal filament muscles. Scale bars: A–F, I: 50 mm, G, H: 25 mm.

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muscles and the ‘‘mdpl’’-muscle are interconnected by two oblique fibres (Fig. 5C, omd1, omd2). 3.2.5. Muscles of the hindbody The naupliar hindbody bears only one pair of strong posterolateral muscles which insert at the dorsal side of the animal in lateral position and extend posteriorly and further into ventroposterior direction (Figs. 4 and 5, plm). At the anterior part of the postero-lateral muscle a fibre inserts which extends towards the mandibular ventral midline muscle (Fig. 5B, C, Supplementary data, Movie clip 1, mdpl). 3.2.6. Musculature of the nauplius I stage The musculature of nauplius I (while still in the eggshell) is very delicate and consists of very thin fibres (Fig. 6). Therefore, posterolateral muscles (plm) and distinct extrinsic dorsal muscles of the first antennae (a1d3, a1d4) and ventral muscles of the first antennae (a1v1), second antennae (a2v2) and the mandibles (mdv1) are weakly stained. No connection between foregut and midgut was detectable, suggesting that the gut musculature is not fully developed in the non-feeding nauplius I stage. 4. Discussion 4.1. Gross morphological details for identification and discrimination of larval stages In general, a nauplius (orthonauplius) is characterized by the presence of three pairs of appendages (first antennae, second antennae, and mandibles), a posterior growth zone, a posteriorly directed labrum, and a median eye (Sanders, 1963). In addition, it is an autapomorphy for the Cirripedia that their nauplii possess frontolateral horns on the head shield (Høeg, 1992; Anderson, 1994; Watanabe et al., 2008). Size, shape of the head shield, relative lengths of dorsal thoracic spines and abdominal process, and the shape of the labrum have been used to develop keys to assign the larval stages of barnacles to a respective species (Lang, 1979). Due to

their relative small size, B. improvisus nauplii are easily distinguishable from those of a number of other cirripede species (Jones and Crisp, 1954). However, the nauplii of B. amphitrite and Elminius modestus resemble B. improvisus larvae in a number of details and may thus be easily confused with the latter (Jones and Crisp, 1954). The relative length of the middle lobe of the labrum with respect to both lateral lobes in E. modestus is greater than in any other known Balanus species (Jones and Crisp, 1954; Ross et al., 2003). B. amphitrite is distributed in warm and temperate waters, but not in areas where the other two species occur, which in addition differ in their breeding time. We are of course aware that the fine details in our SEM micrographs lend themselves to additional comparative purposes, but here we limit ourselves to the discrimination of stages.

4.2. Musculature 4.2.1. Postero-lateral muscles Muscles in the hindbody of nauplii have hitherto been described by light microscopy for some cirripede species including B. perforatus, Chthamalus stellatus, Lepas anatifera, L. pectinata, Couchodernia virgata, Dichelaspis darwinii, and B. improvisus (Mu¨nter and Buchholz, 1870; Groom, 1894), but recent data on such posterior muscles are lacking. The pedunculate barnacle C. spinosa (syn. P. spinosus) features a lateral pair of muscles in the posterior region of the nauplius (Batham, 1946). Observations of the swimming habits of nauplii may reveal the function of these muscles. Swimming of B. perforatus is regularly interrupted by periods of apparent inactivity in which the long exopodite setae are folded backwards and close over the posterior process, which is drawn forwards and backwards over the interior surfaces of the appendages (Norris and Crisp, 1953). Thus, material accumulated on the limbs may be combed and, aided by movements of the endopodites, subsequently pushed forward towards the labrum (Norris and Crisp, 1953). Food might also be broken up by rasping on the posterior process. A cleaning function for the movement of the hindbody has been discussed for B. perforatus (Lochhead, 1936), although the back-and-forth

Fig. 6. Musculature of the nauplius I. (A) Overlay of a light micrograph and the respective confocal projection image. The nauplius I (n I), still within the eggshell (e), already possesses extrinsic muscle fibres in the first and second antennae (a2) and in the mandibles (md). (B) Distinct extrinsic dorsal muscles of the first antennae (a1d3, a1d4) and ventral muscles of the first antennae (a1v1), the second antennae (a2v2) and the mandibles (mdv1) are present. Postero-lateral muscles (plm) are located in the hindbody. (C) Boxed area in ventral view. ffm, frontal filament muscle; la, labrum. Scale bar: A, B: 25 mm.

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movement is also found in cirripede larvae that swim in very clean water (e.g., Verruca and Chthamalus). The architecture of the naupliar musculature of S. balanoides and B. improvisus shows a large number of similarities. Surprisingly, however, no muscles resembling the postero-lateral muscles in B. improvisus were found in S. balanoides (Walley, 1969). Instead, Walley (1969) found in S. balanoides one pair of muscles (‘‘no. 22’’) inserting at the dorsal side of the nauplius and extending posteroventrally into the hindbody. The pedunculate barnacle I. quadrivalvis features three muscles in the hindbody. The pair of ‘‘middorsal dorsal shield to anterior edge caudal papilla’’ (¼dva)muscles was observed through transversal sections and the ‘‘transversal muscle to caudal papilla’’ (¼dam)-muscle by longitudinal sectioning and light microscopy (Anderson, 1987). If considered an entity, these muscles resemble the postero-lateral muscle in B. improvisus, which first extends towards an antero-posterior direction and then bends ventro-posteriorly. 4.2.2. Muscles in the region of the labrum The three pairs of distal labral structures in B. improvisus may be associated with glands which are known to be located in the tip of the labrum in Balanus and in S. balanoides (Groom, 1894; Walley, 1969). The secretion produced by these glands may glue the food together thus assisting the feeding process, but interestingly we have also observed these glands, although in a reduced state, in cirripede nauplii that are entirely lecithotrophic (unpublished). Unfortunately, there are no detailed studies on the feeding process in cirripede nauplii beyond the SEM-based account of Rainbow and Walker (1976). A single transverse muscle passes behind the oesophagus of S. balanoides nauplii and connects the anterior sternites to each other (Walley, 1969). The transverse muscle of B. improvisus is located in a similar position and may thus be considered homologous. Radial dilator muscles are attached to the cuticle of the oesophagus of S. balanoides (Walley, 1969). B. improvisus also possesses (in addition to the oesophageal circular muscles) a number of muscles which are involved in the contraction of the oesophagus, namely the dorsal and the ventral oesophageal muscles and the lateral muscles of the labrum. A. salina nauplii possess oesophageal circumferential muscles and a suspensor dilator which originates at the posterior base of the second antennae and inserts at the oesophagus (Kiernan and Hertzler, 2006). Such oesophageal muscles seem to be lacking altogether in nauplii of S. ingentis (Kiernan and Hertzler, 2006). 4.2.3. Frontal filament muscles By applying three-dimensional reconstruction software a muscle pair in the oesophageal region could be clearly identified as muscles that are situated at the base of the frontal filaments rather than being connected to the oesophagus (cf. fig. 4G herein and Semmler et al., 2006: fig. 1G). A pair of fibres close to the base of

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the frontal filaments is also present in S. balanoides (‘‘no. 1’’; see Walley, 1969). This muscle pair joins the ventral and dorsal regions of the head shield and passes between the nauplius eye and the protocerebrum (Walley, 1969). Video observations of nauplii of Verruca stroemia and B. improvisus revealed occasional rapid and independent movements of each individual frontal filament and we, therefore, suggest that these structures are indeed connected to muscle fibres (unpublished observation). If, as appears likely, the frontal filaments are chemoreceptors (Walker, 1974) such rapid movements might assist the sensory function in a low Reynolds number environment just as is the case for antennular flicking in decapod crustaceans (Schmitt and Ache, 1979). 4.2.4. Dorsal extrinsic muscles The number of dorsal muscles in B. improvisus is higher than previously assumed (Semmler et al., 2006). The nauplii of B. improvisus possess 17 pairs of dorsal extrinsic muscles (Table 2). The dorsal region of the head shield of S. balanoides bears 19 pairs of muscles of which four pairs insert in the first antennae, 10 pairs in the second antennae and five pairs in the mandibles (Walley, 1969). The nauplii of the pedunculate I. quadrivalvis possess only 18 pairs of dorsal muscles with the second antennae exhibiting nine muscle pairs and the mandible bearing five muscle pairs (Anderson, 1987). However I. quadrivalvis nauplii are lecithotrophic and could, therefore, be prone to reductions in muscular complexity due to more simplified limb functions. S. ingentis and A. salina also show extrinsic muscles of dorsal and ventral origin (Benesch, 1969; Kiernan and Hertzler, 2006). The first antennae of B. improvisus nauplii are moved by the use of four muscle pairs which have dorsal origin. Similar numbers and arrangement of the first antennae muscles are found in S. balanoides (Walley, 1969), while only two pairs of muscles insert at the first antennae of Sicyonia (Kiernan and Hertzler, 2006) and no dorsal muscles at all extend into the first antennae in the branchiopod species B. ferox and A. salina (Fryer, 1983; Benesch, 1969; Kiernan and Hertzler, 2006). The second antennae of B. improvisus nauplii are moved by eight pairs of dorsal muscles. S. balanoides possesses 10 pairs and I. quadrivalvis nine pairs of dorsal muscle pairs in the second antenna (Walley, 1969; Anderson, 1987). Eight pairs of dorsal extrinsic muscles of the second antennae are present in S. ingentis and nine pairs in the branchiopods (Fryer, 1983; Kiernan and Hertzler, 2006). In A. salina, all of these muscles attach to the dorsal midline, whereas in S. ingentis most of them originate in a more lateral position. Four pairs of dorsal mandibular muscles are present in the dendrobranchiate nauplius S. ingentis and in the branchiopod nauplii A. salina and B. ferox (Fryer, 1983; Kiernan and Hertzler, 2006). In contrast to that the cirripede nauplii of B. improvisus, S. balanoides (Walley, 1969) and I. quadrivalvis (Anderson, 1987) possess five pairs of dorsal mandibular extrinsic muscles.

Table 2 Number of muscles associated with the naupliar appendages of crustacean species studied so far. Clade

Cirripedia

Species (reference)

B. improvisus (this study)

Branchiopoda S. balanoides (Walley, 1969)

Malacostraca

I. quadrivalvis (Anderson, 1987)

B. ferox (Fryer, 1983)

A. salina (Benesch, 1969; Kiernan and Hertzler, 2006)

S. ingentis (Kiernan and Hertzler, 2006)

Appendage First antenna Ventral Dorsal

1 4

1 4

1 4

3 0

4 0

4 2

Second antenna Ventral Dorsal

5 8

6 10

4 9

8 9

8 9

3 8

Mandible Ventral Dorsal

3 5

3 5

5 5

5 4

5 4

4 4

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H. Semmler et al. / Arthropod Structure & Development 38 (2009) 135–145

4.2.5. Ventral extrinsic muscles One pair of ventral muscles extends into the first antennae of B. improvisus nauplii. The first antennae of both S. balanoides and I. quadrivalvis also bears one muscle which inserts on the ventral side (Walley, 1969; Anderson, 1987). In contrast to this, four pairs of ventral antennule muscles were found in S. ingentis and in A. salina (Kiernan and Hertzler, 2006). B. improvisus nauplii possess five pairs of ventral muscles inserting at the second antennae. In S. balanoides nauplii, six pairs pass through the second antennae (Walley, 1969) while only four pairs of ventral muscles are present in the second antennae of I. quadrivalvis (Anderson, 1987). Three pairs of ventral muscle fibres in S. ingentis and eight pairs of ventral muscle fibres in A. salina lead towards the second antennae of the nauplii (Kiernan and Hertzler, 2006). B. improvisus and S. balanoides only possess three pairs which all insert at the mandibles on the ventral side (Walley, 1969). I. quadrivalvis nauplii bear five pairs of ventral mandible fibres (Anderson, 1987). Four pairs of ventral muscles insert at the mandible of nauplii of S. ingentis and three pairs in nauplii of A. salina (Kiernan and Hertzler, 2006). 4.2.6. Phylogenetic perspectives Muscles similar in position and structure could be identified in the cirripede nauplii of B. improvisus and S. balanoides (Table 3).

Table 3 Homologous muscles in Balanus improvisus (data from this study) and Semibalanus balanoides (data from Walley, 1969). The numbers refer to the labelling of the muscles used by Walley (1969). Dorsal side

Ventral side

Species

B. improvisus

Appendage First antenna

Dorsal muscle 1 (a1d1)

5

Dorsal muscle 2 (a1d2) Dorsal muscle 3 (a1d3) Dorsal muscle 4 (a1d4)

12 7 8

Dorsal muscle 1 (a2d1)

3

Dorsal muscle 3 (a2d3)

6

Dorsal muscle 4 (a2d4)

13

Dorsal muscle 5 (a2d5)

14

Dorsal muscle 6 (a2d6)

15

Dorsal muscle 7 (a2d7) Dorsal muscle 8 (a2d8)

9 16

Dorsal muscle 1(mddl)

10

Second antenna

S. balanoides

B. improvisus

S. balanoides

Ventral muscle 1 (a1v1)

25

Ventral muscle 1 (a2v1) Ventral muscle 2 (a2v2) Ventral muscle 3 (a2v3) Ventral muscle 4 (a2v4) Ventral muscle 5 (a2v5)

31 (?)

Ventral muscle 1 (mdv1) Ventral muscle 2 (mdv2)

36

32 (?)

28

27

34

Homologous muscles were also found for the two branchiopod species A. salina and B. ferox based on the criteria for assigning muscle homologies such as (1) similar position including segment location, anterior–posterior, dorsal–ventral, or proximal–distal extension, (2) similar structural details including origin and insertion sites and intermediate attachments, and (3) similar transitional forms throughout subsequent larval stages (see Remane, 1963; Kiernan and Hertzler, 2006). The numbers of extrinsic muscles in the first antennae are identical in all three cirripede nauplii and the numbers of the other appendage muscles are relatively similar (Table 2). The number of appendage muscles is also relatively similar in the two branchiopod and the malacostracan nauplii (Fryer, 1983; Kiernan and Hertzler, 2006). Whether this general correspondence of muscle numbers between nonmalacostracan and malacostracan nauplii speaks against the independent evolution of malacostracan nauplii, as suggested by Scholtz (2000), remains to be tested by more detailed analyses of the position and attachment patterns of the corresponding muscles and by the investigation of muscle patterns in embryonised eggnauplii commonly found in malacostracans (Scholtz, 2000). The loss or gain of muscles over evolutionary time may be explained by the acquisition of different functions of the limbs for swimming or feeding. To complicate matters further, locomotory modes are diverse even among closely related crustacean species (Williams, 1994). The second antenna, where the number of muscles differs most among the species studied so far, is the main locomotory organ in free-swimming crustacean nauplii (except in copepods [Williams, 1994]; see Table 2). Changes in amplitude, limb beat frequency, size and shape lead to changes in swimming behaviour (Williams, 1994) and this may involve different muscles in the appendages. Although our survey of the crustacean nauplius appendage musculature is far from complete, some tentative conclusions are possible. It appears that the most dramatic reduction of muscle numbers is seen in the dorsal muscles of the first antennae in branchiopod nauplii. In addition, the number of ventral muscles in the second antennae of branchiopods is higher than in the other taxa. Both phenomena may be correlated with the specific shape and function of the reduced first antennae and, in particular, the large second antennae apomorphic of branchiopod nauplii (Olesen, 2004). For cirripede nauplii, the low number of dorsal and ventral extrinsic muscles entering the first antennae (compared to the second antennae and mandibles) dovetails with these appendages not participating actively in naupliar swimming but remaining more or less statically extended forward from the body, but next to no data exist on musculature and swimming movements in nauplii of the two other thecostracan taxa, the Facetotecta and the Ascothoracida (Høeg and Kolbasov, 2002; Pe´rez-Losada et al., 2002). We conclude that data on the architecture of the naupliar musculature for additional species are necessary for any sound conclusions concerning the muscular ground pattern of larval Cirripedia and Crustacea in general. Acknowledgements

Mandible

Others

Dorsal muscle 2 (mdd2)

11

Dorsal muscle 3 (mdd3) Dorsal muscle 5 (mdd5) Dorsal muscle 4 (mdd4)

19 20 23

Transversal muscle (tv) Frontal filament muscle (ffm) Postero-lateral muscle (plm)

24 1 22 þ 21 (?)

39

The authors are grateful to Kent Berntsson (Tja¨rno¨) for help with specimen collection and for providing access to an established B. improvisus culture. We extend our thanks to Alexander Schulz and Michael Hansen (Copenhagen) for technical assistance with the CLSM and we thank Bjarne Bisballe (Copenhagen) for SEM assistance. Ekaterina Ponomarenko (Berlin) is gratefully acknowledged for providing the CLSM stack used for the three-dimensional reconstruction presented in Fig. 5 and Movie clip 1 of the electronic supplement to this article and Diego Maruzzo (Copenhagen) for comments on the manuscript. AW is grateful for financial support by the Danish Research Council (grants nos. 21-04-0356 and 27105-0174) and the Carlsberg Foundation (grant no. 2005-1-249) and

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