Pleonal muscle development in the shrimp Penaeus (Litopenaeus) vannamei (Crustacea: Malacostraca: Decapoda: Dendrobranchiata)

Arthropod Structure & Development 38 (2009) 235–246

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Pleonal muscle development in the shrimp Penaeus (Litopenaeus) vannamei (Crustacea: Malacostraca: Decapoda: Dendrobranchiata) Philip L. Hertzler*, William R. Freas Department of Biology, Brooks 217, Central Michigan University, Mount Pleasant, MI 48859, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 August 2008 Accepted 10 December 2008

Penaeoidean shrimp pleonal muscle is a valuable economic resource worldwide, but little is known of its development during larval stages. The development of pleonal muscle in Penaeus (Litopenaeus) vannamei was studied by rhodamine-phalloidin staining and laser-scanning confocal microscopy. Dorsal pleonal muscle was first evident at the protozoea I stage while ventral pleonal muscle was present by the protozoea II stage. Identifiable ventral pleonal muscles were evident by the protozoea III stage and all ventral muscle types were present in the mysis I. The tail flex response began at the mysis stage and growth of existing pleonal muscles continued. The pleopods formed during the mysis stages, with coxal and basis muscles developed by mysis III. The pleopods became functional beginning with the first postlarval stage. We conclude that the pleonal muscle pattern of P. vannamei larvae is similar to that of adult Penaeus setiferus, and that homologous muscles are present. The major formation of dorsal pleonal muscles occurs during the protozoea II stage, while significant development of ventral pleonal muscles occurs during the protozoea III stage. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Penaeoidean Larvae Pleon Phalloidin Confocal microscopy

1. Introduction The modern studies of muscle development in crustaceans are limited to a handful of studies in penaeoidean shrimp embryos and nauplii (Kiernan and Hertzler, 2006); barnacle nauplii (Semmler et al., 2006), and isopod (Kreissl et al., 2008) and amphipod embryos (Price and Patel, 2008). Penaeoidean shrimp, of the suborder Dendrobranchiata, are the only decapod crustaceans to freely spawn rather than brood their eggs, undergo complete rather than meroblastic cleavage, and hatch as a non-feeding nauplius larva rather than a later larval stage. They develop through a series of naupliar, protozoeal, and mysis larval stages before assuming the body plan of the adult as a postlarva (Fig. 1A–C). Between molts, growth occurs in the posterior region. The muscle progenitor cells have at least two embryonic origins: (1) the naupliar mesoderm gives rise to muscle of the head and first three appendages of the nauplius, and (2) the primordial mesoteloblast gives rise to posterior mesoderm as the larva grows and adds posterior segments (Anderson, 1973; Zilch, 1978, 1979; Hertzler and Clark, 1992; Hertzler, 2002; Gerberding and Patel, 2004; Hertzler, 2005). Naupliar mesoderm progenitor cells form during gastrulation and migrate into the limbs. During the limb bud stage, foci of muscle cells in the limbs and trunk grow and connect to form complete * Corresponding author. Tel.: þ1 989 774 2393; fax: þ1 989 774 3462. E-mail address: [email protected] (P.L. Hertzler). 1467-8039/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2008.12.003

muscle fibers (Kiernan and Hertzler, 2006). Further growth of muscle occurs by enlargement and splitting during the naupliar stages. The posterior mesoderm forms by teloblastic cell divisions of the primordial mesoteloblast. While this process has not been examined in penaeoidean shrimp, it has been described in detail for several malacostracans, the higher crustaceans to which penaeoidean shrimp belong (reviewed by Dohle and Scholtz (1997). In a pattern conserved among the malacostracan species examined, the primordial mesoteloblast gives rise to four pairs of mesoteloblasts; these stem cells and their progeny form the post-naupliar mesoderm which gives rise to most of the subsequent muscle tissue. As dendrobranchiate larvae progress to later stages, more posterior appendages are used for locomotion (Dall et al., 1991; Chu et al., 1996). Naupliar and protozoeal stages use cephalic propulsion: nauplii use the first and second antennae and mandibles, and protozoea swim with the two pairs of antennae and three pairs of maxillipeds. Mysis stages swim using the pereopods, while postlarvae use the pleopods. In the present study, the terms ‘‘pereon’’ and ‘‘pleon’’ are substituted for ‘‘thorax’’ and ‘‘abdomen’’ in the older studies, as these terms are preferred by modern workers in malacostracan crustaceans (see Schram and Koenemann, 2004). In addition to limb locomotion, starting with the mysis stages, shrimp can move rapidly backwards by flexing the powerful pleonal trunk muscles.

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Fig. 1. Larval stages and pleonal muscles of dendrobranchiate shrimp. (A) Naupliar stages I and V of Penaeus (Farfantepenaeus) duorarum. The naupliar stages (of which there are five or six) use cephalic appendages (antennules, antennae, mandibles) for swimming. First and second maxillae and maxillipeds appear in later naupliar stages. (B) Protozoea I, II, III of Penaeus esculentus. Antennules and antennae are used for swimming while mandibles are used for feeding. Some pereonal appendages are functional by PZ III. (C) Mysis I, II, III, and early postlarva of P. duorarum. Pereonal appendages (pereopods) are used for swimming while pleonal appendages (pleopods) develop during mysis stages and function in swimming in post-larval stages. Abbreviations: antennules (an1), antennae (an2), mandibles (md), endopod (en), exopod (ex), first and second maxillae (mx1, mx2), first, second, and third maxillipeds (mxp1, mxp2, mxp3), pereopods (per), pleopods (ple). (D) Diagram of pleonal muscles of Penaeus (Litopenaeus) setiferus, anterior left, dorsal top. Yellow: dorsal pleonal muscles, cyan: external (superficial) arms of anterior oblique muscles, blue: (deep) anterior oblique muscles, green: posterior oblique muscles, red: central muscles, black: transverse muscles. T1–T6: transverse muscles of pleonal somites 1–6. A and C from Dobkin (1961), D modified from Young (1959) with permission from NOAA, Seattle, USA; B from Fielder et al. (1975), with permission from CSIRO, Collingwood, Australia. Scale bars in mm.

No studies exist of the development of pleonal muscle in the larval stages of dendrobranchiate shrimp. Muscle development in the closest relatives of zoea and mysis larval stages of caridean shrimp was described by Daniel (1930). In a comparative analysis, he was able to discern a generalized pattern of ventral muscles in the malacostracan pleon (1931b). This pattern appears to hold for adult dendrobranchiate shrimp muscles as well, as described by Young (1959) for Penaeus (Litopenaeus) setiferus. The pleonal trunk muscles are arranged in a complex, interwoven pattern for rapid and powerful flexion of the tail (Fig. 1D). The main (deep) muscles are (1) the dorsal pleonal muscles, which function as extensors to oppose the massive ventral pleonal muscles, (2) the anterior and (3) posterior oblique pleonal muscles, which provide the primary flexion, (4) the central muscles, which support the flexion of the oblique muscles, and (5) the transverse (stator) muscles, which function as fulcral support for the central muscles and in lateral compression of the pleon (Young, 1959). Additional minor muscle groups were also identified: the posterior loop of anterior oblique,

the external arm of the anterior oblique, the posterior oblique, and oblique transverse muscles (Young, 1959). Crustacean muscles originate from specialized regions on the cuticle and insert onto apodemes, internal projections of exoskeleton functionally equivalent to vertebrate tendons (Pringle, 1972). To follow the development of pleonal muscles in penaeoidean larva to the adult body plan, we stained larval stages of Penaeus vannamei with rhodamine-phalloidin and examined them using laser-scanning confocal microscopy. A recent phylogenetic analysis (Lavery et al., 2004) confirmed that P. (Litopenaeus) vannamei is closely related to P. (Litopenaeus) setiferus, meaning that the muscle pattern is likely to be similar in the two species. We found that the dorsal pleonal muscles had formed by the protozoea I stage, while the major ventral pleonal muscles formed during the protozoea III stage. By mysis I, all of the major pleonal trunk muscle groups of the adult were present, while the pleopod muscles formed during the mysis III stage. The pleonal muscle pattern of P. vannamei larvae was similar to that of adult P. setiferus, and homologous muscles could

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be identified based on the extensive description of overall pattern, origings, and insertions by Young (1959).

analyzed in three dimensions using an evaluation version of IMARIS 6.3.1 (BitPlane, Inc.).

2. Materials and methods

3. Results

2.1. P. vannamei broodstock and larvae

3.1. Overview of naupliar and protozoeal development

P. vannamei larvae were obtained from Hawaii Oahu Suisan, Inc. (Kahuku, Oahu, Hawaii), the Oceanic Institute (Makapuu Point, Honolulu, Hawaii), and SyAqua/Genus PLC (Franklin, Kentucky). Broodstock were maintained and induced to spawn as described in Wyban and Sweeney (1991). Larvae were cultured in either filtered natural seawater or artificial seawater at 28  C by the hatchery staff. Nauplius V through protozoea II stages were provided with an algal diet of Chaetoceros gracilis, and subsequent larval stages were provided with Artemia larvae of the appropriate size. L. vannamei protozoea stages each last 30–40 h, while mysis stages last about 24 h each at 27  C (Wyban and Sweeney, 1991). No effort was made in this study to obtain younger or older samples within each larval stage.

Penaeoidean shrimp develop through several nauplius larval stages, characterized by a planktonic (free-swimming) habit, the absence of pereonal somites (body divisions), locomotion by appendages on the first three somites (first and second antennae and mandibles), and a single medial eye (Anderson, 1971; Dall et al., 1991; Anger, 2001). The early naupliar stages bear unsegmented appendages, becoming segmented in the later stages, and swim by an intermittent rowing motion (Fig. 1A; Dall et al., 1991). Fine setae extend from the appendages and are added with each successive molt. Between molts, growth occurs in the posterior region; the next appendages (1st and 2nd maxillae and 1st and 2nd maxillipeds) appear by the nauplius IV stage. P. vannamei develops through six naupliar stages, distinguished by the setation on the naupliar appendages (Kitani, 1986). In contrast, five naupliar stages are described for most penaeoidean species. Lateral body muscles extend into the posterior of the nauplius (Kiernan and Hertzler, 2006), but as the naupliar stages lack pleonal somites, no pleonal muscles are present. The last nauplius stage metamorphoses into the protozoea I (PZ I), with pereonal somites, unsegmented pleon, and growth zone anterior to the terminal telson and uropods (Fig. 1B; Anderson, 1971; Dall et al., 1991; Anger, 2001). The 3rd maxillipeds and five pairs of pereopods develop from the pereonal somites, and protozoea swim using their antennae and first two maxillipeds (Dall et al., 1991). Muscle nomenclature in the following description is taken from Young (1959). There are five major pleonal muscle groups which appear in the somites of the adult pleon (Fig. 1D). These are the dorsal pleonal muscles (DA) and four groups of ventral muscles: anterior oblique (AO) muscles, posterior oblique (PO) muscles, transverse (T) muscles, and central (C) muscles. Details of the origins and insertions of these muscles observed in the present study and by Young (1959) are summarized in Table 1.

2.2. Larval staining and imaging Larval stages were sampled from rearing tanks and fixed with 4% formaldehyde in artificial seawater (ASW) at 22  C for 1.5–3 h. Larvae were rinsed with ASW and sonicated in a bath sonicator for 3–5 s to permeabilize the exoskeleton. Larvae were then incubated with gentle rocking for 18–24 h with 2 mg/ml rhodamine-phalloidin (Sigma, Inc.), 0.1% Tween-20 in ASW. After the incubation with stain, samples were rinsed in ASW, dehydrated in four changes of 100% ethanol, and cleared and mounted in methyl salicylate as described in Kiernan and Hertzler (2006). Some larvae were oriented ventrally by adhering them to slides with silicon vacuum grease (Dow Corning, Inc.). Samples were viewed using 10 0.4 NA Planapo, 20 0.75 NA Planapo or 40 0.75 NA Plan fluor objectives on an Olympus Fluoview 300 laser-scanning confocal microscope at Central Michigan University. Z-series were collected at a step size of 2– 3 mm. Extended focus and stereo 3D views were constructed using the Fluoview software. Image plates were assembled using Adobe Photoshop CS2. Some images were rotated or flipped horizontally or vertically for more favorable orientation, but no further image processing was performed. XY images were stitched together into composites by manually aligning landmark features of the images. Drawings were made in Adobe Photoshop using an Intuos 2 graphics tablet (Wacom Co., Ltd.). Some confocal data sets were

3.2. Pleonal muscle development in protozoea P. vannamei protozoea I (PZ I) contained dorsal pereonal–pleonal longitudinal muscles which extended through the pereonal somites to the pleonal growth zone (Fig. 2A, B). No major ventral muscles were present in the pleon at this time. The dorsal pereonal–pleonal

Table 1 Origins and insertions of pleonal muscle groups in P. vannamei, P. setiferus. Muscle

#1

#2

#3

#4

#5

#6

#7

Dorsal medial Dorsal lateral

ori: a a1 ins: p a1 ori: a a1 ins: p a1

ori: a a2 ins: p a2 ori: a a2 ins: p a2

ori: a a3 ins: p a3 ori: a a3 ins: p a3

ori: a a4 ins: p a4 ori: a a4 ins: p a4

ori: a a5 ins: p a5 ori: a a5 ins: p a5

–

–

ori: a a6 ins: p a6

–

Transverse (T)

ori: dl a1 ins: m a1 ori: t5 ins: d to T1

ori: dl a2 ins: m a2 ori: d to T1 ins: d to T2

ori: dl a3 ins: m a3 ori: d to T2 ins: d to T3

ori: dl a4 ins: m a4 ori: d to T3 ins: d to T4

ori: dl a5 ins: m a5 ori: d to T4 ins: d to T5

ori: a a6 ins: a, m a6 ori: d to T5 ins: d to T6

–

ori: pd t5 ins: pv a1 –

ori: ad a1 ins: pv a2 ori: pv t5 ins: AO2 ori ori: d to T1 ins: pv a2

ori: ad a2 ins: pv a3 ori: avl a1 ins: AO3 ori ori: d to T2 ins: pv a3

ori: ad a3 ins: pv a4 ori: avl a2 ins: AO4 ori ori: d to T3 ins: pv a4

ori: ad a4 ins: pv a5 ori: avl a3 ins: AO5 ori ori: d to T4 ins: pv a5

ori: ad a5 ins: pv a6 ori: avl a4 ins: AO6 ori –

–

Central (C) Anterior oblique (AO) External arm of AO (X) Posterior oblique (PO)

ori: pd t5 ins: pv a1

–

ori: avl a5 ins: AO7 ori –

Abbreviations: ori, origin; ins, insertion; a, anterior; p, posterior; a1–a6, pleonal somites 1–6; t5, pereonal somite 5; dl, dorsal-lateral; m, medial; d, dorsal; pd, posterior-dorsal; pv, posterior-ventral; ad, anterior-dorsal; avl, anterior-ventral-lateral. Data from the present study and text of Young (1959).

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Fig. 2. P. vannamei protozoea I, II, and III pleonal trunk muscles. Images are composites of multiple optical sections. (A, B) PZ I, frontal sections, anterior left, right side top, dorsal view. (A) The cephalothorax (cth) extends anterior and pleon (ple) extends posterior of the vertical line. Compound eyes (ce), first and second antennae (an1, an2), mandibles (md), maxillipeds (mxp1, mxp2) are labeled. (B) Higher magnification of the unsegmented pleonal region of protozoea in A. Medial (MDA) and lateral (LDA) dorsal pereonal–pleonal longitudinal muscles are present. Posterior to the growth zone (gz), the gut (g) bulges as it passes through the pleon to the anus (an), flanked by the two lobes of the telson (tel). (C, D) PZ II, longitudinal sections, anterior left, dorsal top. Pereonal and pleonal segments are labeled as pe1–5 and 1–6, respectively. Dorsal pereonal (MDT, LDT) and pleonal muscles (MDA, LDA) are prominent and ventral muscles are beginning to form. Note the segmentation in the dorsal pleonal muscle and traces of ventral muscle (VM) in D. (E) PZ III, anterior left, dorsal top. Ventral muscles (VM) are more developed. Scale bar in A, C, D, E ¼ 200 mm, B ¼ 50 mm.

muscles were composed of dorsomedial longitudinal muscles and dorsolateral longitudinal muscles on each side (Fig. 2B). The dorsal pereonal–pleonal muscles were segmented in the pereon but not in the pleon at this stage. The medial pereonal–pleonal muscles inserted on the anterior, dorsal part of the pleon, while the lateral pereonal–pleonal muscles extended further posterior, inserting

onto the dorsal surface of the telson. A complex network of muscle fibers supported the hindgut and anus in the telson and extended into the furcal spines (Fig. 2B). The pleonal somites had formed by the PZ II stage (Fig. 2C, D). Ventral exoskeletal projections (latero-tergal plates) were wider in the pleonal somites than in the pereonal somites. Developing

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ventral muscle was evident in the pereon and pleon. Segmentation was also evident within the dorsal pleonal muscle (Fig. 2D). The pleon had lengthened considerably at the PZ III stage (Fig. 2E). Spines were present on the dorsal side and the telson was clearly separate from a6 at this stage. 3.3. Protozoea III pleonal muscles Analysis of individual optical sections and comparison to later stages and the adult (Young, 1959) allowed the identification of the developing ventral muscles. The external arms of the anterior oblique muscles were found in superficial longitudinal sections (Fig. 3A). The bulk of the oblique muscles were found in deeper longitudinal sections (Fig. 3B). The ventral interior of the trunk had expanded and space was evident between the gut and the developing ventral muscles. The transverse muscles and central muscles were difficult to identify in longitudinal sections at this stage. In frontal sections of PZ III, the external arms of the anterior oblique, transverse, and main anterior oblique muscles could be identified (Fig. 3C). 3.4. Mysis pleonal muscles Mysis stages swim backwards and upside down using their pereonal appendages and rapid tail flicks, and the locomotory function of the antennae is lost (Dall et al., 1991). Pleopods, the pleonal limbs, develop during this time (Fig. 1C). Three superficial

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pleonal muscle groups – ventral, lateral, and dorsal – were described in adult P. setiferus (Young, 1959), but these thin muscles were not observed in P. vannamei larvae. In the mysis I (M I) all of the major adult trunk muscles could be identified according to the descriptions by Young (1959). These muscles are illustrated in longitudinal (Fig. 4) and frontal sections (Fig. 5) of the M I larva. Three-dimensional analysis of the confocal data set shown in Fig. 5 was performed to compare with longitudinal views and confirm the identity of the muscles (see electronic supplement). The ventral muscles in pleonal somites 2 and 3 showed the most significant serial repetition of pattern, while somites 1, 4, 5, 6 showed more variation, as first recognized for malacostracan crustaceans generally by Daniel (1931b). The pleonal muscles had grown to fill the space which was present in the PZ III (Fig. 4A, C). Superficial longitudinal sections revealed the external arms of the anterior oblique muscles (Fig. 4B), while deeper sections revealed the main anterior and posterior oblique (Fig. 4C), central, and transverse muscles (Fig. 4D). The main dorsal pleonal muscles, which function as extensor muscles to oppose the massive ventral pleonal flexor muscles (Young, 1959), were found in M I larval stages. In frontal sections they could be seen in two bilateral groups, the lateral dorsal pleonal muscles and the medial dorsal pleonal muscles (Fig. 5A). The dorsal pleonal muscles were segmented according to the pattern of the pleon, and originated and inserted on dorsal apodemes dividing the segments. They ran beneath the dorsal cuticle and above the gut, linked endto-end from the anterior of pleonal somite 1 to the posterior end of

Fig. 3. P. vannamei protozoea III pleonal trunk muscles. (A, B) Single longitudinal optical sections of pleonal segments 2–5 from same sample as in Fig. 2E. Anterior left, left side sections shown. (A) The external arms of the anterior oblique muscles (X) are present. (B) Deep muscles. A muscle-free space (*), lateral to the gut, is present between the dorsal pleonal muscles (DA) and the nascent ventral anterior oblique (AO) muscles. (C) Composite of frontal optical sections through pereonal segments 3–5 (pe5) and pleonal segments 1– 6. Image is a mosaic of two separate scans. Anterior left, ventral view. External arms of the anterior oblique (X), transverse (T), and anterior oblique (AO) muscles are present. Scale bars in A, B ¼ 50 mm, C ¼ 100 mm.

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Fig. 4. P. vannamei mysis I pleonal trunk muscles. (A) Composite of longitudinal optical sections. Four Z series were used to make the mosaic image, which was then flipped horizontally so anterior is at the bottom and dorsal is to the left. Pereopods (per3–5) and pleonal somites (1–6) are indicated. Note that the pleonal muscles are more massive than in the PZ III. (B–D) Extended focus images through right side of pleonal somites 2–4. (B) External arms of anterior oblique muscles (X), in right superficial sections. (C) Deeper muscles of right side, showing dorsolateral pleonals (LDA), anterior oblique (AO) and posterior oblique (PO) muscles. (D) Deeper sections to the left of midline, showing dorsomedial pleonals (MDA), central muscles (C2, C3, C4), transverse muscles (T3, T4), and gut (g). Dorsal (d) and ventral (v) posterior branches of C4 are indicated. Scale bar ¼ 50 mm for all panels.

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Fig. 5. P. vannamei mysis I pleonal muscles, frontal optical sections. Anterior at bottom, posterior at top, ventral views. (A) Composite of dorsal sections, showing dorsal pereonal– pleonal muscle (DTA), medial (MDA) and lateral (LDA) dorsal pleonal muscles. (B) Composite of mid-frontal sections. The central muscles (C1–C6) and transverse muscles (T1–T6) are evident. External arms of anterior oblique muscle (X) flank the gut (g) in somite 6. (C) Composite of ventral sections. The anterior oblique muscles (AO1–6) originate dorsally at the midline and run ventral-laterally to insert on each side. The external arms of the anterior obliques (X4–X7) are also evident. Scale bar ¼ 50 mm for all panels. See electronic supplementary file for animation of optical sections through this sample.

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pleonal somite 5. A small amount of dorsal pleonal muscle was found in the sixth pleonal somite at the M I stage. 3.5. Mysis central pleonal muscles The central muscles of the pleon support the oblique muscles during their contractions (Young, 1959). Six pairs of central muscles in P. vannamei mysis larvae, labeled C1–C6, were located deep inside the pleonal muscle mass, best seen in longitudinal sections (Figs. 4D and 6B). They were arranged end-to-end in a wavy line beginning in the last pereonal segment and ending in the anterior of the 6th pleonal segment, as described by Young (1959). The central muscles were linked to one another on the dorsal side of the transverse muscles, with each muscle originating on a dorsal branch insertion of the previous one, and with ventral branches running medially in association with the transverse muscles (Figs. 4D and 5B), as described for the adult (Young, 1959). 3.6. Mysis transverse pleonal muscles The transverse muscles function as fulcral supports of the central muscles and in lateral compression of the pleon (Young, 1959). Six transverse pleonal muscle pairs were present in mysis larvae (Figs. 4D, 5B and 6C; compare Young, 1959, Figs. 61, 64). The paired transverse muscles ran from dorsolateral attachments on the tergum (the arched dorsal part of each pleonal somite; Pe´rezFarfante and Kensley, 1997) to meet at the midline (Fig. 5B). The thickness of the transverse muscles decreased from anterior to posterior. T5 and T6 ran in different patterns from T1 to T4, as seen in the adult (Young, 1959). 3.7. Mysis oblique pleonal muscles The oblique muscles provide the main force for tail flexion. Frontal sections revealed different aspects of the M I pleonal oblique muscles (Fig. 5; compare Young (1959), Figs. 62, 64). There were seven left–right pairs of anterior oblique (AO) muscles , which formed a series of V-shaped series in ventral view (Fig. 5C), and ran anterior to the transverse muscles in mid-longitudinal sections (see Fig. 6C). They originated near the anterior-dorsal midline below the gut on one somite and inserted ventrolaterally on the posterior of the next somite (Table 1). The first anterior oblique (AO1) muscle originated in the last pereonal somite and inserted ventrolaterally into the posterior of the first pleonal somite (Fig. 5C). AO2–6 showed a similar pattern, spanning two segments as described for adult muscle (Young, 1959). In longitudinal sections of the mysis III, the anterior, medial origin of the anterior obliques below the gut was evident in deep sections (Fig. 6C), and their posterior, ventrolateral insertions were present in more lateral section planes (Fig. 6B). The AO6 was more slender than AO2–5, as observed in the adult (Young, 1959), and ran into the long 6th pleonal somite to insertion sites at the posterior in association with the tail fan. The external arms of the anterior obliques (X) formed an interlocking V with the anterior obliques. The external arms of the anterior oblique muscles were thicker in the mysis III (Fig. 6A) compared to the mysis I (Fig. 4B). The external arms 2 and 3 were shorter than the others and inserted on the ventrolateral posterior of a1 rather than in association with a main anterior oblique muscle (Fig. 5C, 6A). External arms X4–X6 ran as extensions from AO4 to 6, respectively. They originated on the posterior, ventrolateral part of each somite, ran up around the deeper muscles, and inserted beneath the gut at the dorsal midline, in association with the origin of their conjugate anterior oblique muscles (Fig. 5C). Two branches of external arms 4–6 were observed (Figs. 4B and 6A). The dorsal branches inserted on the central muscle origins, while the ventral

branches inserted near the origins of the main anterior oblique muscles. What Young (1959) identified as external arm muscle extended into the long 6th pleonal somite to insert dorsolaterally to the main AO6 muscles (Figs. 5B, C and 6B). As observed by Young (1959), the AO muscles and associated external arms functionally span 3 segments, from the ventral–lateral origin of one external arm to its insertion on the dorsal, medial AO origin to the AO ventral–lateral insertion. This pattern also holds for caridean shrimp (Daniel, 1930). The posterior oblique (PO) muscles were best observed in longitudinal sections of the mysis III (Fig. 6C; compare Young, 1959, Fig. 61). Each PO muscle originated dorsal to the transverse muscle, closely apposed to the anterior end of the central muscles. The PO ran posterior to the each transverse muscle and inserted ventrolaterally with its associated anterior oblique muscle. No PO muscle was found in the sixth pleonal somite, as for the adult (Young, 1959). 3.8. Pleopod muscle development Postlarvae adopt a benthic lifestyle and swim forward by rhythmic beating of the fully functional pleonal pleopods. The pleopods develop during the mysis stages. In the M I, the coxal regions of the pleopod buds are present but no muscles are found within them (Fig. 7A). By the M II stage, the pleopod buds had elongated to the basis region but muscle was still not present (Fig. 7B). By the M III stage the major muscles of the coxa, basis and exopod had formed (Fig. 7C, C0 ). Pleopod muscles were larger in the functional limbs of the postlarva (Fig. 7D, D0 ). Promotor, remotor, and adductor muscles were found in the coxa and basis, and rotators, extensors, and flexors were found for the exopodite, as in the adult pleopod (Young, 1959). 4. Discussion 4.1. Homology of larval and adult muscle of related penaeoidean species The complex pleonal muscle pattern of mysis stage P. vannamei larvae is very similar to the adult P. setiferus. Since these are also closely related species within subgenus Litopenaeus (Lavery et al., 2004), the muscles are considered to be homologous. A careful study of the text and figures of Young (1959) enabled muscle identification in mysis stages, which were then traced backward in developmental time through earlier larval stages. Young (1959), in turn, based his nomenclature on the comparative work on other Malacostracan crustaceans by Daniel (1930, 1931a,b). While the trunk pleonal muscles were present by the mysis I, the pleopod muscles were not formed until the mysis III. Some muscles described by Young (1959) in the adult were not found in P. vannamei larvae. The labeling technique used provided high resolution details of the main muscles but did not reveal the thinner superficial muscles described for the adult. These include the dorsal, lateral and ventral superficial muscles shown in Figs. 60, 62, 63 of Young (1959). Either these superficial muscles were not yet present in larvae or present but not detected by the current method. Of the main muscles, two branches of the anterior oblique muscles of the adult were not identified in larvae: the posterior loop of the anterior oblique muscles and the oblique transverse muscles (Young, 1959). Further investigation may reveal the presence of these muscles. The muscle attachment sites were difficult to visualize using the present technique, even though some background staining of the exoskeleton was evident. Preliminary work suggests that the internal attachment sites (apodemes) are present in the

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Fig. 6. P. vannamei mysis III pleonal muscles, composite longitudinal optical sections. (A) Left lateral sections, with lateral dorsal pleonal muscle (LDA), and external arms of anterior obliques shown (X3–X7). Dorsal (d) and ventral (v) insertions of X5, and coxa promoter (CP) and remoter (CR) of the 3rd pleopod are indicated. (B) Deeper sections, showing dorsal pereonal–pleonal muscle (DTA) and central muscles (C1–6). External arm muscle extending into somite 6 (X) is also shown in these sections. (C) Medial sections, showing medial dorsal pleonal muscle (MDA), pleonal anterior obliques (AO1–6), posterior obliques (PO1–5), and transverse muscles (T1–6). Abbreviations: pereopod 5 (per5), pleopods (ple1–5), gut (g). Scale bar ¼ 100 mm for all panels.

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Fig. 7. Development of pleopod muscles in P. vannamei. (A–D) Extended focus views of longitudinal confocal sections of left side, anterior left, dorsal top. (C0 , D0 ) Interpretive drawings based on C and D respectively. (A) Mysis I, pleonal somites 2–4 (2–4). (B) Mysis II, pleonal somites 2–4. (C, C0 ) Mysis III, pleopod 3. (D, D0 ) Postlarva 2, 3rd pleopod. Abbreviations: cox, coxa; CP, coxa promotor; CAD, coxa adductor; CR, coxa remotor; bas, basis; BAD, basis adductor; BP, basis promotor; BR, basis remotor; exo, exopod; XRO, exopod rotator; XEX, exopod extensors; XF, exopod flexor. Scale bar ¼ 50 mm for all panels.

data when analyzed with 3D visualization software, due to the background fluorescence of the cuticle. It became clear in the course of this study that the anterior and posterior oblique muscles are so-named by their relationship to the transverse muscles. For example, AO2 ran anterior to T1 while PO2 ran posterior to T1. While the origin of each PO muscle is proximally associated with the next AO muscle (for example, PO2 lies anterior to and follows AO3), each PO inserts in association with its anterior partner (for example, PO2 inserts with AO2). There appeared to be a conflict between the numbering of the oblique muscles described in the text and those illustrated in the figures of Young (1959). We based our homologies on the text descriptions of Young (1959),

which agreed with the numbering of the oblique muscles illustrated in Crangon (Daniel, 1930). Thus, AO1 and PO1 ran anterior to the pleonal transverse muscles, AO2 and PO2 ran anterior and posterior to T1, and so forth. 4.2. Development of dorsal pleonal muscles The major formation of dorsal pleonal muscles occurs during the protozoeal II stage. These muscles are extensions of the dorsal pereonal muscles present in the PZ I stage and possibly the longitudinal muscle in naupliar stages (Kiernan and Hertzler, 2006). Studies are in progress to follow the development of the pleonal–pereonal

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(thoraco-abdominal) muscles from the nauplius onwards, which should determine whether the naupliar longitudinal muscle becomes dorsal or lateral muscle. The dorsal pleonal muscles are segmental, as seen in the adult. At the mysis I they do not yet cover the gut on the dorsal side, as they do in the adult. In addition, the fibers of the medial dorsal pleonal muscle in larvae run longitudinally, rather than obliquely, as is seen in the adult. Therefore, growth and patterning of dorsal pleonal muscles must continue through later larval and post-larval stages. 4.3. Development of ventral pleonal muscles While the ventrolateral portions of the ventral pleonal muscles are present in the PZ II, the classes of ventral muscles first become identifiable at the PZ III stage. Both main oblique muscles and external arms of the anterior obliques could be identified in longitudinal and frontal sections of the PZ III. Considerable space was present within the PZ III pleon which is filled with ventral muscles by the M I stage. Therefore, significant growth of ventral pleonal muscles occurs during the PZ III stage. It would be interesting to focus higher spatial and temporal resolution studies on early and later PZ III stages to gain further understanding of the development of the ventral pleonal muscles at both the tissue and cellular levels. In isopods, the use of myosin heavy chain antibodies revealed the formation of muscle from single progenitor cells and subsequent division and differentiation into muscle fibers in directdeveloping embryos (Kreissl et al., 2008). We saw no evidence of this pioneer muscle cell mode in P. vannamei larvae, but a higher spatial resolution study could be done to determine the mechanism of myogenesis, whether by pioneer cells or splitting of existing muscles. Development of isopod trunk (primarily pereonal) muscles is described in the electronic supplementary material of Kreissl et al. (2008); the ventral longitudinal muscles develop first in early embryos, while dorsal and transverse muscles form later. In pre-hatching embryos, the dorsal longitudinal muscles are segmentally iterated, as in P. vannamei dorsal pleonal muscles. The intersegmental extensor muscles traverse multiple segments in the same way as the oblique muscles do in penaeoideans. An anterior– posterior gradient of muscle development was observed in the thoracic somites of this direct-developing species (Kreissl et al., 2008). However, at the time resolution of the present study, the formation of both trunk and limb muscles in P. vannamei appeared to occur simultaneously within pleonal somites 1–6. A more frequent sampling interval within each larval stage would be necessary to detect an anterior–posterior gradient of muscle development within the pleon. Since each larval stage lasts 1–2 days at typical culture temperatures, there exists the potential for significant muscle growth during each stage. Comparisons can also be made between penaeoidean ventral pleonal muscles and those of other decapod crustaceans. A similar if not homologous pattern of central muscles, transverse muscles, anterior and posterior oblique muscles was found in the caridean prawns Crangon, Praunus (Daniel, 1930) and Pandalus (Berkeley, 1928; Young, 1959), and (with the possible exception of the central muscles) the crayfish Astacus (Young, 1959). The complex pattern of ventral pleonal muscles therefore predates the divergence of the Dendrobranchiata (penaeoidean shrimp) and the Pleocyemata (caridean shrimps, crayfish, lobsters, crabs) from their last common ancestor. While subtle differences in pleonal muscle pattern could be found between two caridean species (Daniel, 1930), the muscle pattern appears to be identical in the two penaeoidean species examined to date. A closer analysis may reveal subtle differences in penaeoidean muscle patterning that could be analyzed from a phylogenetic perspective. Finally, the generalization for malacostracan crustaceans made by Daniel (1931b) that only pleonal

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somites 2 and 3 show close serial repetition of muscle pattern, while somites 1, 4, 5, and especially 6 show variations, also holds for dendrobranchiate shrimp. In P. vannamei, the development of the trunk pleonal muscles after the M I occurs by the growth of existing muscles rather than addition of new muscle groups. In contrast, the pleonal limb muscles are formed along with the development of the pleopods during M I–III. While the details of limb muscle formation were not the focus of this study, it was clear that the adult pleopod muscle homologs in P. setiferus could be identified in P. vannamei larvae. Limb muscle development in the Dendrobranchiata merits further investigation. In summary, the homologs of adult pleonal muscles could be found in penaeoidean larvae. Segmented dorsal pleonal muscle was evident in PZ II, while the four ventral muscle groups were present by the M I stage. This study provides a foundation for more detailed investigations of myogenesis in this important group of crustaceans. Acknowledgements We thank Jessica Lapp, Phil Oshel, and two anonymous reviewers for editorial comments on the manuscript, and Dr. Athula H. Wikramanayake for hosting PLH at the University of Hawaii Manoa, where this study was initiated. Thanks also to Komarey Moss, Kathy Rasher (now at the Oceanic Institute, Oahu, HI), John Ho at Hawaii Oahu Suisan, Inc. and Dr. Alok Deoraj at SyAqua/Genus PLC for providing larval shrimp. This work was made possible by Research Professorship and President’s Research Incentive Funds awards from Central Michigan University and support from the CMU Biology Department Microscopy Facility. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi: 10.1016/j.asd.2008.12.003. References Anderson, D.T., 1973. Embryology and Phylogeny in Annelids and Arthropods. Pergamon Press, Oxford. Anger, K., 2001. The Biology of Decapod Crustacean Larvae. Crustacean Issues 14. A.A. Balkema Publishers, Lisse. Berkeley, A.A., 1928. The musculature of Pandalus danae Stimpson. Transactions of the Royal Canadian Institute, Toronto 16, 181–321. Chu, K.H., Sze, C.C., Wong, C.K., 1996. Swimming behavior during the larval development of the shrimp Metapenaeus ensis (De Haan, 1844) (Decapoda, Penaeidae). Crustaceana 69, 368–378. Dall, W., Hill, B.J., Rothlisberg, P.C., Sharples, D.J., 1991. Biology of the Penaeidae. Advances in Marine Biology 27, 7–54. Daniel, R.J., 1930. The abdominal muscular systems of the zoe¨a and mysis stages of the shrimp (Crangon vulgaris Fabr.) and their bearing on phylogeny. Proceedings and Transactions of the Liverpool Biological Society 44, 95–109. Daniel, R.J., 1931a. The abdominal muscles of the shore crab (Carcinus maenas) and of the zoea and megalopa stages. Proceedings and Transactions of the Liverpool Biological Society 45, 50–56. Daniel, R.J., 1931b. Comparative study of the abdominal musculature in Malacostraca. Part I. The main ventral muscles of the typical abdominal segments. Proceedings and Transactions of the Liverpool Biological Society 45, 57–71. Dobkin, S., 1961. Early developmental stages of pink shrimp, Penaeus duorarum, from Florida waters. Fishery Bulletin U.S. 61, 321–349. Dohle, W., Scholtz, G., 1997. How far does cell lineage influence cell fate specification in crustacean embryos? Seminars in Cell and Developmental Biology 8, 379–390. Fielder, D.R., Greenwood, J.G., Ryall, J.C., 1975. Larval development of the tiger prawn, Penaeus esculentus Haswell, 1879 (Decapoda, Penaeidae) reared in the laboratory. Australian Journal of Marine and Freshwater Research 26, 155–175. Gerberding, M., Patel, N.H., 2004. Gastrulation in crustaceans: germ layers and cell lineages. In: Stern, C.D. (Ed.), Gastrulation: From Cells to Embryo. Cold Spring Harbor Press, Cold Spring Harbor, pp. 79–89. Hertzler, P.L., 2002. Development of the mesendoderm in the dendrobranchiate shrimp Sicyonia ingentis. Arthropod Structure and Development 31, 33–49.

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