Journal of

Anatomy

J. Anat. (2015) 227, pp325--340

doi: 10.1111/joa.12335

Musculoskeletal anatomy and feeding performance of pre-feeding engyodontic larvae of the European eel (Anguilla anguilla) Mathias Bouilliart,1 Jonna Tomkiewicz,2 Peter Lauesen,3 Barbara De Kegel1 and Dominique Adriaens1 1

Research Group Evolutionary Morphology of Vertebrates, Ghent University, Ghent, Belgium National Institute of Aquatic Resources – Section for Marine Ecology and Oceanography, Technical University of Denmark, Charlottenlund, Denmark 3 Billund Aquaculture Service, Billund, Denmark 2

Abstract Being part of the elopomorph group of fishes, Anguillidae species show a leptocephalus larval stage. However, due to largely unknown spawning locations and habitats of their earliest life stages, as well as their transparency, these Anguilla larvae are rarely encountered in nature. Therefore, information regarding the early life history of these larvae, including their exogenous feeding strategy and feeding performance, is rather scarce. To better understand the structural basis and functional performance of larval feeding in captivity, the functional morphology of the cranial musculoskeletal system in pre- and first-feeding engyodontic leptocephali of the European eel (Anguilla anguilla) was studied. A 3D reconstruction of the feeding apparatus (head of the leptocephali < 1 mm) was used to visualize and describe the musculoskeletal changes throughout these stages. To analyze the ontogenetic changes in the functionality of the feeding apparatus towards the active feeding phase, 3D data of joints, levers and muscles derived from the reconstructions were used to estimate bite and joint reaction forces (JRFs). Observing a maximum estimated bite force of about 65 lN (and corresponding JRFs of 260 lN), it can be hypothesized that leptocephalus larvae are functionally constrained to feed only on soft food particles. Additionally, potential prey items are size delimited, based on the theoretically estimated average gape of these larvae of about 100 lm. This hypothesis appears to be in line with recent observations of a diet consisting of small and/or gelatinous prey items (Hydrozoa, Thaliacea, Ctenophora, Polycystenia) found in the guts of euryodontic leptocephalus larvae. Key words: Anguilla anguilla; bite force; engyodontic leptocephali; feeding performance; functional morphology.

Introduction The family Anguillidae comprises 16 eel species (and three subspecies) that are widely distributed in tropical and subtropical areas (Minegishi et al. 2005; Teng et al. 2009; Inoue et al. 2010). Being catadromous species, these elongated fishes spend most of their juvenile and adult life in estuarine and freshwater areas, before migrating over several hundred kilometers to their spawning grounds in tropical ocean waters (van Ginneken & Maes, 2005; Correspondence Mathias Bouilliart, Research Group Evolutionary Morphology of Vertebrates, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium. E: [email protected] Accepted for publication 6 May 2015 © 2015 Anatomical Society

Tsukamoto, 2006; Righton et al. 2012; Schabetsberger et al. 2013). After hatching, so-called engyodontic leptocephalus larvae are formed that are completely transparent and characterized by the presence of yolk and a few needle-like teeth (Leiby, 1989; Miller, 2009). Later on, these engyodontic larvae become euryodontic larvae when yolk is depleted and multiple short, broad-based teeth start to appear (Leiby, 1989). Due to the largely unknown deep-water marine birthplaces, engyodontic leptocephalus larvae are rarely encountered in nature. Current information regarding the anatomical, physiological and behavioral characteristics of leptocephali is therefore limited to observations on euryodontic larvae (Norman, 1926; Miller, 2009). As engyodontic larvae usually undergo the transition from endogenous to exogenous feeding, information on the

326 Bite performance of pre-feeding leptocephali, M. Bouilliart et al.

feeding strategy of first-feeding leptocephali is thus also scarce in the literature. One larval feeding strategy suggests a nutritional intake through the integument of the leaf-like body of the older, euryodontic larvae (Otake et al. 1993). However, being more ribbon-like than leaf-like in shape, the benefits of the increased surface area are limited in engyodontic larvae. Additionally, discoveries of detritus remnants and digested material in the guts of euryodontic larvae also suggest the presence of a more active feeding mechanism (Mochioka & Iwamizu, 1996; Lecomte-Finiger et al. 2004). Although several hypotheses exist on the identity of these recovered particles (going from whole zooplanktonic organisms to marine snow), none of the suggestions can currently be verified by observations of larval feeding in nature or in captivity (Mochioka & Iwamizu, 1996; Riemann et al. 2010; Miller et al. 2013). To better understand the structural basis for active larval feeding, the functional morphology of the cranial musculoskeletal system was examined in pre- and first-feeding engyodontic leptocephali of the European eel (Anguilla anguilla Linnaeus 1758). Being unable to obtain pre-feeding larvae from nature, captively bred leptocephali were acquired as part of a European research project (PRO-EEL). Since 2010, this project strives for a self-sustaining breeding program of the European eel under controlled conditions. The motivation for this project is the observed decline in the natural populations of the Anguilla species over the past 40 years. At present, this decline is attributed to a combination of biological phenomena and anthropogenic influences (Knights, 2003; van Ginneken & Maes, 2005; Friedland et al. 2007; Belpaire et al. 2009; Crook, 2010; Munk et al. 2010; Pacariz et al. 2014). Dealing with rather small heads (< 1 mm long), computergenerated 3D reconstructions of the musculoskeletal anatomy of the feeding apparatus in pre- and first-feeding larvae were used. This allowed us to: (i) visualize and describe the musculoskeletal changes in the feeding apparatus throughout the transition from endogenous to exogenous feeding; and (ii) analyze the ontogenetic changes in the functional performance (bite and joint reaction forces (JRFs)) of the developing feeding apparatus towards the active feeding phase. These forces were calculated using a static state equilibrium model based on quantitative and qualitative data of the larva’s musculoskeletal topography (Herrel et al. 1998a,b). Also, in vivo video-recordings of larvae in captivity were used to qualitatively study the kinematics of the feeding apparatus during mouth opening and closing. This video-footage allowed not only a preliminary estimation of jaw gape angle in each investigated age, but also the identification of: (i) the major contributors; (ii) the sequence of operation; and (iii) the duration, speed and acceleration of skeletal elements during mouth opening and closing.

Materials and methods Specimens Engyodontic European eel (Anguilla anguilla) larvae from captively bred parents (Butts et al. 2014) were cultured (~20 °C, ~36 ppt) at the Lyksvad research facility of the Technical University of Denmark (Ødis, Denmark). For as long as larvae were available, daily samplings from two batches were performed (hatched 10/04/2011 and 29/01/2012, respectively). Larvae were kept in total darkness at a constant water temperature in order to simulate natural conditions (Knights, 2003). From the first batch of specimens (exact number not known), eight specimens were fixed in glutaraldehyde 2.5% in phosphate-buffered saline and transported to Ghent University for further functional morphological analysis. From the second batch of larvae (exact number not known), about 60 specimens were used for video recording to obtain kinematic data on jaw mobility.

Specimen preparation After fixation, the larvae of the first batch were rinsed in a 0.2 M cacodylate-buffer (pH 7.4) before being post-fixed in osmium tetroxide (1% OsO4). After gradual dehydration with 100% ethanol and propylene oxide, larvae were embedded in epon (Fluka; Sigma; epoxyembedding medium (45345), DDSA hardener (45346), MNA hardener (45347) and accelerator (45348)). Using a Microm HM360 microtome provided with a diamond knife (Diatome, histo, 8 mm), series of 1-lm transverse sections of the head region of each embedded specimen were obtained (a total of three specimens was used for histological sectioning). All sections were stained with toluidin blue (pH 9). In addition, non-embedded reference specimens were stained for bone and cartilage following Walker & Kimmel (2007).

3D reconstructions The histological sections were photographed using a digital camera € nster, Germany) (ColorView 1; Soft Imaging Solutions, GmbH Mu mounted on a light microscope (Olympus BX41, 40 9 magnification), using ANALYSIS 5.0 software (Soft Imaging System). Images were cropped to the same dimensions in Adobe Photoshop CS4 before being loaded into AMIRA 5.4.1 (64-bit version; Computer Systems Mercury), where they were visually and manually aligned. The correct alignment was checked using pictures of the original (intact) specimens. All recognizable muscles, cartilaginous and bony structures, ligaments and tendons were reconstructed and identified using the works of Norman (1926), Winterbottom (1974) and Hulet (1978) for nomenclatural guidance. Morphometric data on the muscles were gathered by calculating the fiber lengths and volumes of these muscles using the Measurement and SurfaceArea modules in AMIRA, respectively. To obtain the mean fiber length for an individual muscle, the lengths of five separate muscle fibers were measured and averaged in the 3D space. The reported muscle volumes were obtained by averaging the derived muscle volumes over both sides of the head, as the larvae are considered to be bilaterally symmetrical.

Calculating the output forces Theoretical maximal output forces that these larvae can generate were calculated using a static state equilibrium model following the work of Cleuren et al. (1995) and Herrel et al. (1998a,b). Two types © 2015 Anatomical Society

Bite performance of pre-feeding leptocephali, M. Bouilliart et al. 327

of data were implemented in this model. First, input forces (in N) of jaw-closing muscles acting across the jaw joint were estimated. All force estimations were based on the physiological cross-sectional area (PCSA (cm2)) of the muscle. This PCSA represents the ratio of the volume of the muscle and the average muscle fiber length, which was multiplied by a maximal force per unit of surface of 25 N . cm-2 (Aerts et al. 1987) to obtain maximal muscle force. Second, 3D coordinates of: (i) the required points to define the lower jaw (including the tip of Meckel’s cartilage and the lower jaw joint); (ii) the origin and insertion of the adductor mandibulae; and (iii) the tip of the first, second and third dentary tooth (bite points) were derived from the reconstructions using the PointProbe module in Amira. Due to the larva’s bilateral symmetry, 3D coordinates implemented in the model were obtained from one body side only. Total larval bite forces were then obtained by doubling the values of the results obtained from modeling one body side. Making use of the moment exerted by the musculus adductor mandibulae at the level of the jaw joint, the magnitude of the output force, hereafter referred to as food reaction force (FRF; Fig. 1a), at the predefined bite points along the jaw was determined. In order to make comparison throughout the ontogenetic series possible, the FRF force vector perpendicular to the lower jaw (90 °) was estimated in every investigated larval stage. More important, as each FRF exerts, in turn, a moment onto the jaw joint, JRFs that keep the lever system in a static equilibrium state (Fig. 1a) were also determined. To ensure comparability between all investigated larval stages, forces were calculated over a predefined range of lower jaw depression angles, going from a closed to a fully opened mouth. The fully closed mouth represented the minimum gape angle (model depression angle: 0 °), which was determined by artificially rotating the lower jaw in the 3D model until the teeth in both jaws contacted each other (performed in RHINOCEROS 4.0, NURBS modeling for Windows, Robert McNeel, USA). The maximum gape angle was obtained by depressing the larva’s lower jaw until the processus retroarticularis of Meckel’s cartilages contacted the quadrata.

a

Theoretically defining a minimum and maximum gape angle was required as no information is currently available on the actual angles over which these larvae depress their jaws in nature. As these force calculations were based on the relative position of the involved skeletal elements in the feeding apparatus in the way the larvae were embedded for sectioning, a sensitivity analysis was performed. Again, RHINOCEROS 4.0 was used to change the reconstructed 3D configuration of the musculoskeletal system, after which FRFs were recalculated using the new coordinates. As a result, two additional bite models were run for every investigated ontogenetic stage, using: (i) a configuration with an abducted hyosymplecticum so that the most ventral point of the hyosymplecticum was located perpendicular underneath the structure’s articulation point with the neurocranium; and (ii) a configuration with an adducted hyosymplecticum so that the most ventral points of both hyosymplectica met at the mid-sagittal plane. However, no abduction or adduction was added to the hyosymplectica of the 9 days post-fertilization (dpf) stage, as the configuration of the mandibular arch did not allow an unambiguous medial or lateral displacement of the ventral tip of the hyosymplectica. Adding both configurations, it is assumed to have covered the natural range of hyosymplecticum positions possible in living larvae.

Video-recordings Of the second batch, 9 dpf larvae were screened for a pulsing heart and the absence of musculoskeletal deformations in the head region. Daily recordings were made for a period of 1 week, until larval mortality was absolute. In the end, 18 leptocephalus larvae were filmed of which five depressed their jaws while being filmed (Table 1). Because larvae did not respond to the presence of provided food particles, recorded lower jaw movements were limited to moments of larval breathing and seemingly random depressions of the lower jaw (Table 1). A digital camera [Nikon DS-Fi1, Nikon Instruments, USA, 1280 9 960 pixels (max. 12 fps)] mounted on an

b

Fig. 1 (a) Representation of the feeding apparatus of a 15-days-old leptocephalus larva with indication of the action and reaction forces at: (i) the jaw joint (blue arrows); and (ii) a selected bite point (black arrows). All forces and angles are estimated with respect to the lower jaw (red arrow). (b) Lateral view on a 12-days-old larva of the European eel (Anguilla anguilla; ID A_anguilla_Lepto12) with indication of the digitized points (Table 2), and the translated and rotated coordinate system (x- and y-axis). © 2015 Anatomical Society

328 Bite performance of pre-feeding leptocephali, M. Bouilliart et al.

Table 1 List of recorded in vivo (pre)leptocephalus larvae with indication of: (i) the age of the larvae (dpf); (ii) the presence of lower and/or upper jaw deformations; and (iii) the presence of jaw movements in the absence of food.

Table 2 Description of the digitized landmarks represented in Fig. 1b. LM 1

Deformations Age (dpf) ID A_anguilla_Lepto01 A_anguilla_Lepto02 A_anguilla_Lepto03 A_anguilla_Lepto04 A_anguilla_Lepto05 A_anguilla_Lepto06 A_anguilla_Lepto07 A_anguilla_Lepto08 A_anguilla_Lepto09 A_anguilla_Lepto10 A_anguilla_Lepto11 A_anguilla_Lepto12 A_anguilla_Lepto13 A_anguilla_Lepto14 A_anguilla_Lepto15 A_anguilla_Lepto16 A_anguilla_Lepto17 A_anguilla_Lepto18

9 9 10 11 11 11 11 11 11 11 12 12 14 14 14 15 15 15

Upper jaw

– – – – – – x – x – – – – – x – x x

Lower jaw

x x x – – – – – – – – – – – – – – –

Jaw depression

– – – – – – – – – x x x – x – – x –

dpf, days post-fertilization. optical microscope (Nikon Eclipse 55i, 10 9 magnification) and driven by NIS-Elements D(ocumentation) (Imaging Software) was used for these recordings. During filming, all specimens were placed at the center of a droplet of seawater and manipulated so that they were oriented in a plane parallel to the camera with maximal overlap of paired anatomical structures as to minimize orientation errors.

Kinematic analysis To identify the major contributors to the jaw-opening and -closing systems, as well as the sequence of operation and the jaw depression angles, the video-files were converted to separate frame-files using MIDASPLAYER (Xcitex). In each frame, 11 kinematically relevant landmarks (Fig. 1b; Table 2) were digitized using DIDGE (Image Digitizing Software). To obtain relative movements of all elements with respect to the neurocranium, the 2D-coordinates of these landmarks were translated and rotated for every frame in MICROSOFT EXCEL (Microsoft Office Professional Plus 2010, Version 14.0.7147.5001) using two points on the neurocranium (indicated by an asterisk in Table 2) that define a reference axis. From these coordinates, movements of skeletal elements could be quantified, calculating distances and rotation angles. Calculations included: (i) the maximum distance between the anterior tip of the neurocranium and the lower jaw (lm); (ii) the maximum distance between the tip of the anterior teeth in upper and lower jaw (lm); (iii) the maximum gape angle between the upper and lower jaw (°); and (iv) the caudoventral rotation angle of the hyoid arch (°). Unfortunately, due to variable time intervals between consecutive frames, the obtained recordings could not be used to perform a quantitative

*2 *3 4 5 6 7 8 9 10 11

Description The anterior tip of the rostrum at the level of the ventral attachment with the praemaxillary tooth The most dorsal point of the rostrum The intersection between the posterior part of the otic vesicle and the dorsal part of the notochord The anterior end of the interhyal The anterior end of the ceratohyal The posterior end of the ceratohyal The lower jaw joint The attachment site of the second maxillary tooth The anterior tip of the lower jaw The anterior tip of the praemaxillary tooth The anterior tip of the first dentary tooth

LM, digitized landmarks.

kinematic analysis on the duration, speed and acceleration of the skeletal elements during jaw movements.

Results Three larval stages during the transition from endogenous to active feeding were reconstructed in this study. The first stage (9 dpf; Fig. 2) was characterized by a significant amount of yolk and therefore represented an endogenously feeding leptocephalus larva. The second stage specimen (12 dpf; Fig. 3) also represented endogenous feeding, but several anatomical observations (including a reorientation of the head and an eruption of the most anterior teeth) suggested that the onset of exogenous feeding was near. The exogenously feeding larva, with complete absence of an oil-droplet, was represented by the last stage (15 dpf; Fig. 4). A detailed description of the changing musculoskeletal anatomy over these three stages is given below and illustrated in Figs 2–4. Nomenclature of the chondrocranial elements in European eel follows Norman (1926). Morphometric data on the muscular changes are summarized in Table 3.

Stage 1: endogenous feeder (9 dpf) – TL 6.6 mm Chondrocranium All skeletal elements present at this stage are composed of hyaline cartilage. The neurocranium (Fig. 2e) is formed by a single piece of cartilage. In the ethmoid region, the anteriorly located round rostrum continues dorsally in the mesethmoid cartilage, which forms a connection with a pair of supraorbital bars or taenia marginales (Adriaens & Verraes, 1997). These round bars bridge part of the orbital region by running along the dorso-anterior margin of the eyes, without connecting to the cartilaginous supports, or cartilagines oticales, of the auditory capsule. Ventrally, the rostrum continues in the ethmoid plate, which is connected © 2015 Anatomical Society

Bite performance of pre-feeding leptocephali, M. Bouilliart et al. 329

a

Fig. 2 Lateral view of the reconstruction of a 9 dpf larva of (a) the European eel, and (b) lateral view, (c) caudolateral view and (d) ventral view of the reconstruction of the feeding apparatus of the 9 dpf larva, and (e) dorsolateral view on the reconstructed neurocranium of the larva. Elements in blue are composed of hyaline cartilage, bony elements are represented in yellow, and elements in red/pink represent muscles and ligaments. a.c., auditory capsule; cb., ceratobranchial; ch., ceratohyal; dent., dentary tooth; eh., epihyal; ethm., ethmoid plate; fen.hypo., fenestra hypophysea; for.tr.h., foramen truncus hyomandibularis VII; ih., interhyal; m.a.a.p., musculus adductor arcus palatini; m.a.m., musculus adductor mandibulae complex; m.ax., axial muscles; M.c., Meckel’s cartilage; m.im., musculus intermandibularis; m.l.a.p., musculus levator arcus palatini; m.p.h., musculus protractor hyoidei; m.sh., musculus sternohyoideus; max., maxillary tooth; mesethm., mesethmoid cartilage; n., neurocranium; not., notochord; oes., oesophagus; o.v., otic vesicle; os dent., os dentale; os max., os maxillare; p.f., pectoral fin; p.g., pectoral girdle; p.p., parachordal plate; p-otic., pro-otic cartilage; prmax., praemaxillary tooth; q., quadratum; r., rostrum; s.b., supraorbital bar; t.c., trabecula communis.

b

c

d

e

to the trabecula communis. This cartilaginous bar is round in cross-section and runs along the orbital region in a straight line between both eyes towards the pro-otic cartilage of the otical region. At the level of the connection between these cartilaginous masses, a fenestra hypophysea can be observed. Posteriorly, the pro-otic cartilage makes contact with a pair of parachordal plates, located on both sides of the notochord in the otical region. The lateral side of each parachordal plate is connected to the cartilagines oticales by means of a commissura basicapsularis. Additional processes, located anterior to the commissura basicapsularis, are protruding laterally from the pro-otic cartilage towards the cartilagines oticales. Occipital elements are not present yet. The mandibular arch usually comprises two pairs of structures: Meckel’s cartilages and the palatoquadrata (Adriaens & Verraes, 1997; Fig. 2c,d). In this developmental stage, only the quadratum part of the palatoquadratum has developed. This structure is triangular and is vertically © 2015 Anatomical Society

directed downwards. Meckel’s cartilages are curved, elongated pieces of cartilage that anteriorly touch at the mid-sagittal plane. These elements lie in the same direction as the quadrata. Of the hyoid arch (Fig. 2c), three pairs of elements are present: the epihyals, the interhyals and the ceratohyals. The epihyals or hyosymplectic cartilages are flat and triangular, and are located between the quadrata and the cartilagines oticales of the neurocranium. A small foramen (foramen truncus hyomandibularis VII) is located in the center of these hyosymplectica. The interhyals are small rod-like elements located between the ventral margin of the hyosymplectica and the dorsal part of the ceratohyals. The latter are elongated structures that are directed ventrally and inclining towards the mid-sagittal plane. In the branchial region (Fig. 2b,d), three pairs of elongated cartilaginous masses are located behind the ceratohyals. These masses represent the onset of the ceratobranchials of the first three branchial arches.

330 Bite performance of pre-feeding leptocephali, M. Bouilliart et al.

a

b

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d

e

Caudal to the neurocranium, the scapulocoracoid cartilage of the pectoral girdle (Fig. 2d) is supporting the cartilaginous base of the pectoral fin. Osteocranium Two pairs of dermal bones have already formed (Fig. 2a–d). At the level of the upper jaw, a pair of ossa maxillaria articulates anteriorly with the lateral edges of the ethmoid plate and curves posteriorly underneath the eyes without connecting to any other cartilaginous or bony element. In the lower jaw, three pairs of fragments of ossa dentalia are located on top of the anterolateral parts of the Meckel’s cartilages. Although all of the observed bony elements bear elongated and curved teeth that are still surrounded by epithelial tissue, not all of the observed teeth are supported by bone. An anterior pair of teeth in both upper and lower jaw lack any kind of bony support, and appear to be inserted directly upon the dorsal part of the rostrum of the

Fig. 3 Lateral view of the reconstruction of a 12 dpf larva of (a) the European eel, and (b) lateral view, (c) caudolateral view and (d) ventral view of the reconstruction of the feeding apparatus of the 12 dpf larva, and (e) dorsolateral view on the reconstructed neurocranium of the larva. Elements in blue are composed of hyaline cartilage, bony elements are represented in yellow, and elements in red/pink represent muscles and ligaments. a.c., auditory capsule; bb., basibranchial; cb., ceratobranchial; ch., ceratohyal; dent., dentary tooth; eh., epihyal; ethm., ethmoid plate; fen.hypo., fenestra hypophysea; for.tr.h., foramen truncus hyomandibularis VII; hb., hypobranchial; ih., interhyal; m.a.a.p., musculus adductor arcus palatini; m.a.m., musculus adductor mandibulae complex; m.ax., axial muscles; M.c., Meckel’s cartilage; m.im., musculus intermandibularis; m.l.a.p., musculus levator arcus palatini; m.p.h., musculus protractor hyoidei; m.sh., musculus sternohyoideus; max., maxillary tooth; mesethm., mesethmoid cartilage; mnd-h. lig., mandibulo-hyoid ligament; n., neurocranium; not., notochord; oes., oesophagus; o.v., otic vesicle; os dent., os dentale; os max., os maxillare; p.f., pectoral fin; p.g., pectoral girdle; p.p., parachordal plate; p-otic., pro-otic cartilage; prmax., praemaxillary tooth; proc. retroart., processus retroarticularis; q., quadratum; r., rostrum; s.b., supraorbital bar; t.c., trabecula communis.

neurocranium and the ventral side of the anterior tips of the Meckel’s cartilages, respectively. Myology Individual muscle fibers of six pairs of muscles can already be identified in this larval stage (Fig. 2b–d; Table 3). First, a single precursor of the musculus adductor mandibulae complex, which originates at the dorsal margin of the quadratum, runs along the anterior side of the quadratum before bending posteriorly over the tip of this cartilaginous element. This muscle then inserts onto the dorsal edge of Meckel’s cartilage at a 2/3 distance from the tip of this structure. Second, a conically shaped musculus levator arcus palatini extends laterally from the dorsal to the ventral edge over the anterior part of the hyosymplectica. Medially to each hyosymplecticum, a conically shaped musculus adductor arcus palatini is located between the dorsal and the ventral edge over the posterior part of this cartilaginous element. Between the anterior tip of the ceratohyal and the © 2015 Anatomical Society

Bite performance of pre-feeding leptocephali, M. Bouilliart et al. 331

a

Fig. 4 Lateral view of the reconstruction of a 15 dpf larva of (a) the European eel, and (b) lateral view, (c) caudolateral view and (d) ventral view of the reconstruction of the feeding apparatus of the 15 dpf larva, and (e) dorsolateral view on the reconstructed neurocranium of the larva. Elements in blue are composed of hyaline cartilage, bony elements are represented in yellow, and elements in red/pink represent muscles and ligaments. a.c., auditory capsule; bb., basibranchial; bh., basihyal; cb., ceratobranchial; ch., ceratohyal; dent., dentary tooth; eh., epihyal; ethm., ethmoid plate; fen.hypo., fenestra hypophysea; for.tr.h., foramen truncus hyomandibularis VII; hb., hypobranchial; ih., interhyal; m.a.a.p., musculus adductor arcus palatini; m.a.m., musculus adductor mandibulae complex; m.ax., axial muscles; M.c., Meckel’s cartilage; m.im., musculus intermandibularis; m.l.a.p., musculus levator arcus palatini; m.p.h., musculus protractor hyoidei; m.sh., musculus sternohyoideus; max., maxillary tooth; mesethm., mesethmoid cartilage; mndh. lig., mandibulo-hyoid ligament; n., neurocranium; not., notochord; oes., oesophagus; o.v., otic vesicle; os dent., os dentale; os max., os maxillare; p.f., pectoral fin; p.g., pectoral girdle; p.p., parachordal plate; p-otic., pro-otic cartilage; prmax., praemaxillary tooth; proc. retroart., processus retroarticularis; q., quadratum; r., rostrum; s.b., supraorbital bar; t.c., trabecula communis.

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anterior tip of Meckel’s cartilage, a long and slender musculus protractor hyoidei is located. Anteriorly, at the level of the attachment with Meckel’s cartilage, the muscle fibers of the protractor hyoidei become intertwined with the muscle fibers of the musculus intermandibularis. The latter is a small and flat muscle connecting both halves of the lower jaw anteriorly. Finally, the musculus sternohyoideus is a rather long, broad and flat muscle that originates at the most anterior part of the pectoral girdle, runs forward along the lateral side of the heart and passes the branchial region to insert on the ventromedial edge of the ceratohyal.

Stage 2: transition phase from endogenous to exogenous feeding (12 dpf) – TL 7.0 mm Chondrocranium The neurocranium (Fig. 3e), still comprising a single piece of hyaline cartilage, has undergone several small modifications compared with the previous stage. In the ethmoid © 2015 Anatomical Society

region, three modifications are observed. First, relatively to the posterior part of the skull, all elements of the ethmoid region have moved towards a more dorsal position. Second, the rostrum has become more pronounced, creating a short and blunt anterior tip to the neurocranium. Third, the median part of the mesethmoid cartilage has become thinner, while the lateral parts now form a thicker connection with the pair of laterally compressed taenia marginales. In the orbital region, the dorsal displacement of the ethmoid region is accompanied by a dorsal curving of the anterior part of the trabecula communis. In the otical region, the cartilagines oticales have undergone both a rostrodorsal and caudal growth. In addition, the small processes protruding laterally from the pro-otic cartilage now extend towards the cartilagines oticales, creating the two facial foramens. Caudally oriented cartilaginous growth is also observed within the parachordal plates. As a result, these plates now extend into the occipital region of the skull (Fig. 3).

332 Bite performance of pre-feeding leptocephali, M. Bouilliart et al.

Table 3 Morphometric data (volume, fiber length and PCSA) of the observed head muscles in the three reconstructed stages of larval development of the European eel. Muscle Volume (lm³) (mean  SD) (df = 1) Musculus adductor arcus palatini Musculus adductor mandibulae Musculus levator arcus palatini Musculus protractor hyoidei Musculus intermandibularis Musculus sternohyoideus Fiber length (lm) (mean  SD) (df = 4) Musculus adductor arcus palatini Musculus adductor mandibulae Musculus levator arcus palatini Musculus protractor hyoidei Musculus intermandibularis Musculus sternohyoideus PCSA (lm²) (mean  SD) (df = 3) Musculus adductor arcus palatini Musculus adductor mandibulae Musculus levator arcus palatini Musculus protractor hyoidei Musculus intermandibularis Musculus sternohyoideus

9 dpf

12 dpf

15 dpf

10 109.50 82 192.00 11 488.00 12 306.50 7024.00 99 846.50

     

976.11 6028.79 101.82 19.09 0.00 8889.04

20 079.50 146 157.00 20 532.50 19 969.50 7534.00 120 663.00

 17.68  10035.26  2312.95  37.48  0.00  9192.39

35.42 153.71 41.30 140.56 94.96 414.49

     

6.53 13.27 1.17 6.29 3.24 9.34

40.40 325.69 73.17 356.76 83.59 422.26

     

3.69 7.90 8.02 0.65 5.05 7.48

43.97 351.20 73.94 364.81 85.08 337.79

     

1.38 4.81 4.62 5.98 0.24 10.43

295.51 538.76 279.40 87.73 85.62 241.01

     

69.75 70.60 9.58 4.53 0.23 25.56

501.24 449.03 284.03 55.97 90.46 285.84

     

52.93 37.77 51.70 0.17 6.31 25.82

563.87 527.01 413.81 108.89 186.77 313.43

     

127.93 12.84 41.49 6.42 0.61 37.08

24 770.50 185 052.00 30 476.00 39 712.00 15 890.00 105 770.00

 4801.96  2968.43  1835.65  1921.92  0.00  10329.42

df, degrees of freedom; dpf, days post-fertilization; PCSA, physiological cross-sectional area.

The two pairs of structures forming the mandibular arch (Fig. 3c,d) have grown along their longitudinal axes. The quadrata are rather flat and elongated cartilaginous masses, with their broad, convex, ventral margins articulating with concave surfaces at the posterior end of the Meckel’s cartilages. Caudal to these concave surfaces, two distinct processus retroarticulares have developed at the Meckel’s cartilages. Both the quadratum and Meckel’s cartilage have become tilted upwards, taking in a horizontal position now. In the hyoid arch (Fig. 3c), the hyosymplectica extend ventrally and are now making contact with the dorsal margin of the quadrata. At the level of this connection, a single processus extends from both of the hyosymplectica to form an articulation point with the interhyals. Instead of its prior rod-like shape, the interhyals are now broad and flat elements. The elongated ceratohyals have also become tilted from a vertical to a horizontal orientation. In the branchial region (Fig. 3b,d), four pairs of slender, cylindrical ceratobranchials are present. Medial to the anterior ends of the first and second ceratobranchials, two pairs of small hypobranchials have developed. In between the first pair of hypobranchials, a small basibranchial can be observed. Despite the presence of elements of four branchial arches, no signs of gill development are present on any of the arches. The pectoral girdle (Fig. 3d) still only consists of the scapulocoracoid connected to the cartilaginous base of the pectoral fin.

Osteocranium No other bones have formed besides the ossa maxillaria and the ossa dentalia (Fig. 3c). The ossa maxillaria have straightened out compared with their configuration in the previous stage. These bones now each bear three long, needle-like teeth. The different fragments of the ossa dentalia have joined into two flat dermal bones. Each of them now bears three elongated teeth and is located alongside the outer edge of the Meckel’s cartilages. Myology No additional muscles have developed compared with the previous stage, but all increased in volume and mean fiber length (Fig. 3b–d; Table 3). Additionally, small changes are observed in the point of origin and/or insertion of some of the muscles present. The musculus adductor mandibulae has nearly doubled not only its muscle fiber length but also its volume. Moreover, the muscle now originates laterally below the foramen truncus hyomandibularis VII, runs along the lateral side of the quadratum and inserts on the dorsal edge of Meckel’s cartilage at a 3/5 distance from the tip of the latter. The musculus levator arcus palatini, as well as the adductor arcus palatini have doubled in volume. Although the former has also doubled its mean fiber length, the latter only displays a limited increase in fiber length. The levator muscle now inserts on the dorsolateral face of the hyosymplecticum (anterior to the foramen), while its dorsal apex is located anterior to the base of the auditory capsule. The adductor palatini muscle inserts on the dorsomedial © 2015 Anatomical Society

Bite performance of pre-feeding leptocephali, M. Bouilliart et al. 333

face of the hyosymplecticum, dorsal to the foramen. The muscle fiber length of the musculus protractor hyoidei increased two and a half times, while volume increased one and a half times. Both the musculus sternohyoideus and the musculus intermandibularis slightly increased in volume, while the mean muscle fiber length of both muscles did not change. Additionally, one pair of ligaments is observed in this larval stage. This mandibulo-hyoid ligament attaches anteriorly to the most posterior point of the processus retroarticularis, while it is posteriorly attached to the posterior end of the ceratohyal.

Stage 3: exogenous feeder (15 dpf) – TL 7.1 mm Chondrocranium All modifications to the neurocranium (Fig. 4e) compared with stage 2 are concentrated around the otical region of the skull. The cartilagines oticales have extended both rostrodorsally as well as caudally, while the free posterior ends of the taenia marginales have grown towards the anterior margins of the cartilagines oticales, without connecting to the latter. Both the quadrata and the Meckel’s cartilages (Fig. 4c,d) have grown along their longitudinal axes, causing the anterior tip of the lower jaw to exceed the anterior point of the rostrum. Apart from the now cylindrical interhyals and the appearance of a small round basihyal at the mid-sagittal plane between both ceratohyals, no remarkable differences are observed compared with the previous stage related to the elements of the hyoid arch (Fig. 4c). In the branchial and pectoral region (Fig. 4b,d), no additional cartilaginous elements have developed compared with the previous stage. Gills are still absent (Fig. 4). Osteocranium The ossa maxillaria and the ossa dentalia are still the only bones present at this stage (Fig. 4c). The former are slender round bones oriented towards the mandibular joints. The latter are flat broader bones lying lateral against the Meckel’s cartilages. Each bone bears three long, needle-like teeth, which now have erupted from their surrounding epithelial tissue. Still, no bone is present underneath the most anterior pair of teeth in both upper and lower jaw. Myology No additional muscles have developed (Fig. 4b–d; Table 3). Small increases in muscle fiber length and/or volume have occurred in the precursor of the musculus mandibulae complex, the levator arcus palatini and the adductor arcus palatini. Different from the 12 dpf stage, the posterior fibers of the precursor of the musculus mandibulae complex now overlay the ventrolateral side of the musculus levator arcus palatini. The musculus protractor hyoidei doubled in volume but did not change much in muscle fiber length, while the © 2015 Anatomical Society

sternohyoideus did not change in volume but slightly decreased in mean fiber length. The anterior ends of the musculi sternohyoidei are connecting medially at the level of the first branchial arch. A similar connection is observed for the anterior ends of the musculi protractor hyoidei at the level of the intermandibularis. The intermandibularis doubled in volume but did not change in fiber length.

Bite model The results of the bite model for every age are represented graphically in Fig. 5d–i, showing total food (FRFs) and JRFs. In the two oldest larval stages (12 dpf and 15 dpf) for which different levels of hyosymplectic adduction was modeled, adducting the hyosymplectica decreased the FRFs approximately 5%, while the JRFs increase with 4% (Fig. 5e,f,h,i). Abducting the hyosymplectica, on the other hand, causes the FRFs to increase with about 1%, while the associated JRFs decrease with 2% (Fig. 5e,f,h,i). For the 9 dpf, 12 dpf and 15 dpf larvae, the maximum jaw depression angles are 15 °, 30 ° and 40 °, respectively. For each of these larvae, only the forces associated with biting at the level of the first (circle) and third (diamond) dentary tooth are shown in the graphs (Fig. 5). The results of the second tooth are not included, as these values were all intermediate to the results obtained at the first and third tooth. All forces are calculated over a range of lower jaw depression angles varying between 0 ° (closed mouth) and maximum depression angles determined for each of the larvae (open mouth; Fig. 5d–i). Over the entire range of depression angles, a gradual decrease in FRFs is observed with an increasing depression angle for every investigated stage. Looking at the magnitude of the maximum bite force at the level of the first and third dentary tooth (dent1/ dent3) for the 9 dpf (16 lN/23 lN), 12 dpf (29 lN/48 lN) and 15 dpf (44 lN/63 lN; Fig. 5d–f), an increase in total FRF of 175% is observed when going from endogenous to exogenous feeding. As a result, as larvae grow, a gradual increase in FRFs can be seen, with highest forces closer to the jaw joint and at smaller gape angles. Looking at the corresponding JRFs, increasing the lower jaw depression angle also increases the magnitude of the JRFs with an average of about 5% in all larvae (Fig. 5g–i). The magnitudes of the JRFs are higher at the level of the first dentary tooth compared with the magnitudes obtained at the third dentary tooth. With a depletion of yolk, the maximum JRF at 9 dpf (270 lN) decreases at 12 dpf (224 lN) only to increase again at 15 dpf (260 lN; Fig. 5g–i).

Kinematic analysis Based on six recordings of pre-feeding engyodontic leptocephali of the European eel, the following trends with respect to the movability of the feeding apparatus are observed (Fig 6a,b).

334 Bite performance of pre-feeding leptocephali, M. Bouilliart et al.

a

b

c

d

e

f

g

h

i

Fig. 5 Left lateral view on the original 3D reconstructions of (a) a 9 dpf, (b) a 12 dpf and (c) a 15 dpf larva of the European eel (Anguilla anguilla). Cartilage is represented in blue, bone in yellow and muscles in red/pink. Graphical representation of (d–f) food reaction forces (FRFs) and (g–i) joint reaction forces (JRFs) for (d, g) the 9 dpf, (e, h) the 12 dpf and (f, i) the 15 dpf larva. Circles and diamonds represent the force estimations at the level of the first and third dentary teeth, respectively. All forces are calculated over a range of depression angles between a completely opened and closed mouth of the larvae. This range of depression angles varies between 15 °, 30 ° and 40 °, respectively, for the three investigated larval stages (9 dpf, 12 dpf and 15 dpf). Calculations based on the first bite model (reconstruction) are depicted by symbols. A dotted and dashed line represents calculations of the second and third model, respectively.

During mouth opening, the lower jaw rotates ventrally over an average angle of approximately 16 ° (Fig. 6a). Although most of these observed rotation angles range between 13 ° and 18 °, a single depression angle of up to 27 ° is registered within a 12 dpf larva (Fig. 6a). Because the observed angles do not exceed the theoretical maximum depression angle of 40 °, all observed depression angles fall within the range of angles defined based on the 3D models. Quantitatively, angles between 13 ° and 18 ° correspond to: (i) an absolute distance of 329.37  44.42 lm (mean  SD) between the anterior tip of the neurocranium and the lower jaw; and (ii) an absolute distance of 101.67  59.86 lm (mean  SD) between the most anterior teeth in upper and lower jaw (Fig. 6b). Both Mec-

kel’s cartilages and the elements of the hyoid arch get depressed simultaneously. While the ceratohyals are depressed over an average angle of approximately 20 ° around the ventral ends of the interhyals, the latter rotate dorsocaudally around the processus on the hyosymplectica over an angle varying between 9 ° and 14 ° (Fig. 6a,c,d). During the entire mouth opening process, the mandibular joint and the ossa maxillaria remain immobile, as no change in the relative position of both compared with the neurocranium is observed (Fig. 6c,d). During mouth closing, all displaced musculoskeletal elements simultaneously return towards their initial positions (Fig. 6c,d). Because individual muscle contractions could not be marked for visualization in the obtained video-recordings, © 2015 Anatomical Society

Bite performance of pre-feeding leptocephali, M. Bouilliart et al. 335

a

c

b

d

Fig. 6 Graphical representation of the rotation angles of both the lower jaw and the hyoid arch during depression of (a) the jaw and (b) the corresponding induced gape distances between the upper and lower jaw and both pairs of anterior teeth of recorded in vivo larvae of the European eel (n = 6). Graphical illustration of the relative displacement of six digitized points of the feeding apparatus (Fig. 1b; Table 2) along (c) the x-axis and (d) the y-axis during the recorded mouth opening (frames 2–6) and closing (frames 6–9) activity of a single leptocephalus larva (ID A_anguilla_Lepto12). Error-bars indicate personal digitization errors.

no individual contribution to the mouth opening or closing movement can be credited to the muscles present.

Discussion Combining 3D reconstructions (Figs 2–4) and in toto stained specimens (bone and cartilage – data not shown) © 2015 Anatomical Society

with sensitivity analyses (Fig. 5) and musculoskeletal knowledge from the literature (Norman, 1926; Hulet, 1978), an accurate understanding of the ontogenetic changes in pre-feeding leptocephalus larvae of the European eel was possible. This series allowed us to investigate the influence of ontogenetic changes in the morphology of the larval feeding apparatus on its

336 Bite performance of pre-feeding leptocephali, M. Bouilliart et al.

performance related to prey capturing (specifically bite performance).

Ontogenetic changes chondrocranium

in

the

engyodontic

At present, Norman (1926) is the only study providing detailed morphological descriptions of the ontogenetic changes in the chondrocranium of leptocephalus larvae of the European eel. The three stages of engyodontic larvae (ranging in size between 6.6 mm and 7.1 mm) used in the current study correspond to Norman’s Stage I (5.0–9.0 mm). However, despite their similarity in size, the Stage I specimens described by Norman show a further state of development of the chondrocranium compared with the endogenous and first-feeding larvae of the current study. This developmental delay is primarily seen in the endogenously feeding larva (6.6 mm TL; Fig. 2), whose ventrally directed head does not correspond to the horizontal head orientation described by Norman. Nonetheless, precursors of all elements observed by Norman of the mandibular and hyoid arch (apart from the basihyal), as well as parts of the neurocranium and the first couple of branchial arches have already developed within this 9-dpf-old larva. With the depletion of yolk, the heads of the larvae in the current study were transformed and match the orientation described for the stage I larvae (Figs 3 and 4). During this transformation, the rostrum of the neurocranium was repositioned dorsally relative to the chorda dorsalis, while the elements of the mandibular and hyoid arches were shifted from a vertical to a more horizontal orientation. Moreover, the elements of both arches changed not only in orientation but also in shape. As a result of these shape changes, an articulating lower jaw was formed due to the formation and repositioning of the convex ventral parts of the quadrata and the concave caudal ends of the Meckel’s cartilages. At the time of a complete yolk depletion in the 15 dpf larva (7.1 mm; Fig. 4), the topography of the chondrocranium closely resembled Norman’s Stage I larva. Still, some differences were observed in the neurocranium, including the absence of a connection between the taenia marginales and the anterior part of the cartilagines oticales and the absence of the processus occipitales in the 15 dpf larva (Fig. 4). The biggest difference, however, was observed in the branchial region (Figs 3d and 4d), where the presence of four pairs of ceratobranchials, two pairs of hypobranchials and one basibranchial resemble more the configuration in Norman’s Stage II (11 mm) than in the Stage I larva. Compared with total body length, the branchial arches therefore developed at a smaller size, while all other chondrocranial elements developed at larger size in these artificially bred, engyodontic leptocephali compared with the ones Norman described. In addition to the chondrocranium, two pairs of dermal bones were observed (Fig. 4b). Being round and slender,

the ossa maxillaria were different from the ones described by Norman, although it did resemble maxillaries of other larval anguilliformes (e.g. 10 mm TL leptocephalus larva of the family Ophichthidae; Leiby, 1979, 1981). The ossa dentaria, on the other hand, corresponded morphologically to the ones described for both anguillid and ophichthid engyodontic leptocephali (Norman, 1926; Leiby, 1979, 1981). However, in contrast to these descriptions, these bones did not support the first pair of curved dentary teeth in the current larvae (Fig. 4b). In the upper jaw, a similar situation was observed at the level of the first pair of (praemaxillary) teeth (Fig. 4b). This observation is in line with the reporting of the absence of ossa praemaxillaria in other anguillid and ophichthid engyodontic leptocephali (Norman, 1926; Leiby, 1979, 1981).

Ontogenetic changes in the larval musculature No significant movement of the lower jaw was observed in European eel larvae younger than 11 dpf (Table 1). Comparing the muscular configuration of the 12 dpf larva with the 9 dpf ones, differences in musculature could explain the absence of these lower jaw movements in the younger stages. First, a change in attachment sites of the levator and adductor arcus palatini and the adductor mandibulae was observed in the 12 dpf stage (Figs 2b,c and 3b,c). Both arcus palatini muscles now attach to the neurocranium, thus allowing the indirect mediolateral movement of the jaw by the controlled abduction and adduction of the hyosymplectica. Additionally, combining the increase in attachment area of the adductor mandibulae with the appearance of an articulating joint (due to the reorientation of Meckel’s cartilage relative to the quadratum), a more controlled elevation of the lower jaw could be hypothesized within the older larval stage. Looking at the depression of the jaw, the appearance of a pair of mandibulo-hyoid ligaments in the 12 dpf stage enables the so-called hyoid four-bar system typically observed in early fish larvae (Surlemont et al. 1989; Hunt von Herbing et al. 1996a,b; Figs 3b and 7a). These ligaments now couple the depression of the Meckel’s cartilages to the depression of the ceratohyals. The video-recordings of the larvae supported this hypothesis, as a preliminary kinematic analysis showed a simultaneous depression of the mandibula and the hyoid arch, which characterizes this hyoid coupling mechanism (Fig. 6d; Hunt von Herbing et al. 1996b). To assess the efficiency of this single linkage system, its kinematic efficiency was calculated following Aerts & Verraes (1984) (Fig. 7b). According to this calculation, the recorded hyoid caudal retraction angles varying between 9 ° and 14 ° (Fig. 6a) would allow the lower jaw to be depressed over an angle between 9 ° and 15 °, respectively (Fig. 7b). However, measurements on the in vivo recordings proved that mouth floor depression angles mostly exceeded a value of 15 ° (Fig. 6a,d), suggesting the involvement of © 2015 Anatomical Society

Bite performance of pre-feeding leptocephali, M. Bouilliart et al. 337

additional (muscular) systems for extended mouth opening. Based on the available information, this could be generated by the protractor hyoidei. Interesting, this muscle’s line of action was located below the jaw joint in the 9 dpf stage (Fig. 2b), while the reorientation of the head at 12 dpf shifted the muscle’s line of action to above the joint (Fig. 8a), suggesting elevation of the jaw during contraction of the protractor at 12 dpf. However, analysis of the videorecordings revealed that the ventral rotation of the ceratohyals during mouth opening in the 12 dpf stage again shifted the protractor’s line of action to below the jaw joint, allowing the muscle to contribute to the jaw-opening mechanism (Figs 6c,d and 8b; Adriaens et al. 2001). Observing the morphometric data across the transition from endogenous (9 dpf) to exogenous (15 dpf) feeding (Table 3), the following changes were noted. Between 9 dpf and 12 dpf, most head muscles nearly doubled in volume and/or in average fiber length. From 12 dpf to 15 dpf, no significant changes were observed in the average a

b

Fig. 7 Illustration of the 15 dpf reconstructed leptocephalus larva of the European eel with indication of (a) the present hyoid four-bar system and (b) graphical representation of the coupled depression of the lower jaw (M) by the hyoid arch (H) and the kinematic efficiency of this hyoid coupling. The gray area highlights the in vivo hyoid retraction angles. © 2015 Anatomical Society

fiber lengths of the muscles present, while increases in volume were seen within all muscles. More relevant for the estimation of the larval bite performances is the relation between the increase in volume and fiber length of a muscle. When the PCSA of the muscle increases between developmental stages, an increase in contractile force of the muscle is obtained. As a result, the most dominant increase in contractile force was initially (9–12 dpf) observed in the adductor arcus palatini, while later on (12–15 dpf) increase was predominantly seen in the levator arcus palatini and the protractor hyoidei. It therefore appeared that within these larvae the performance in generating force was first improved for the muscles involved in reducing the buccal cavity, before increasing that of those responsible for expanding the buccal cavity.

Ontogenetic changes in feeding performance Looking at the only muscle responsible for closing the larva’s mouth, the contractile force of the adductor mandibulae increased between consecutive larval stages (9 dpf, 12 dpf and 15 dpf). Solely based on muscle force, an increase in bite force was expected during the transition from endoto exogenous feeding. Calculated bite forces did increase from 23 to 63 lN during the transition from an endogenously (9 dpf) to an exogenously (15 dpf) feeding engyodontic leptocephalus, while associated JRFs decreased from 270 to 260 lN, respectively (Fig. 5d–i). Abducting the hyosymplectica increased the output force with about 1%, while their adduction resulted in a 5% decrease in bite force (Fig. 5d–f). Because changes of < 5% were observed in the JRFs, abducting or adducting the jaws had little impact on the forces estimated in these engyodontic larvae (Fig. 5g–i). As a result, the musculoskeletal topography of the exogenously feeding larvae allowed them to exercise a larger output force, while experiencing a slightly lower reaction force onto their jaw joint compared with the endogenously feeding larvae. Nonetheless, for every Newton produced by the larval feeding apparatus, 1/5 of the force was converted into bite force at the level of the teeth, while 4/5 was exerted at the level of the jaw joints. This seemingly disproportion between actual biting forces and JRFs questions whether forceful biting would at all be possible in the first-feeding engyodontic leptocephalus larva. Moreover, taking the morphology and orientation of the prognathous needle-like teeth into account (Fig. 4a–c), large bending rotation forces will be induced at the base of the tooth when a force is applied at its tip. Therefore, such a tooth configuration suggests an inefficient system for transferring forces between the tip and the base of the tooth. The teeth not being oriented perpendicular to the jaw also indicates a rather non-functional system for efficiently penetrating food particles. Additionally, these teeth also limit the maximum gape angle. While an average gape of approximately 330 lm could be measured between

338 Bite performance of pre-feeding leptocephali, M. Bouilliart et al.

a

b

Fig. 8 Lateral view of a 12 dpf leptocephalus larva of the European eel (ID A_anguilla_Lepto12) (a) before and (b) during mouth opening. The jaw joint is indicated by a cross, the line-of-action of the musculus protractor hyoidei is visualized by a dashed line. Scale bar: 100 lm.

the anterior tip of the upper and lower jaw, the prognathous teeth decreased the functional gape distance to about 100 lm (Fig. 6b). This indicates that only food particles smaller than 100 lm could be taken up, while bigger food items would have to be chewed upon in some way. Although no dietary information is available for engyodontic exogenously feeding leptocephali, some research has been conducted on identifying prey items in the guts of euryodontic larvae. As a result, a selection of gelatinous zooplankton, Polycystinea and small Crustacea (Riemann et al. 2010), as well as larvacean houses and fecal pellets (Mochioka & Iwamizu, 1996), are assumed to be predated on by leptocephalus larvae. Forces between 100 and 750 lN have been suggested for crushing diatoms (Hamm et al. 2003), whereas forces bigger than 5000 lN would be needed for breaking the shells of 1-week-old mussel larvae (Gaylord et al. 2011). Although these organisms have not been proven to be a part of the leptocephalus larval diet, the obtained measurements provide a framework to evaluate the range of possibilities to crush the natural food items. Because the calculated maximum output force does not exceed the minimum force required for crushing animals surrounded by silica or calcified shell, engyodontic leptocephali are most probably only capable of biting soft unshelled organisms. Although this statement is in line with the retrieval of gelatinous zooplankton, larvacean houses and fecal pellets in the leptocephali guts, it does not account for the presence of remnants of hard prey (protected by a carapace or opaline silica shell; Riemann et al. 2010). One explanation for the presence of these hard organisms is that larvae from this study are much smaller than those investigated by Riemann et al. (2010). Euryodontic larvae are older than engyodontic larvae and have developed not only a different set of short, broad-based larval teeth but also a different musculoskeletal topography (Leiby, 1979). As all research on potential prey items only focuses on the euryodontic larval stage, it can be hypothe-

sized that engyodontic larvae do not feed on hard organisms yet, while developmental changes will probably allow the euryodontic leptocephali to successfully deal with hard-shelled prey organisms later in life. Alternatively, the presence of shelled organisms in the larval guts could be possible if food items are being grasped and swallowed as a whole, for which suction pressure would be required. However, to feed successfully through suction feeding, the buccal cavity has to expand by abducting the suspensoria and depressing the mouth floor, while effectively sealing off the opercular slits and preventing leak flow at the level of the mouth opening (Drost & Van Den Boogaart, 1986; Hunt von Herbing et al. 1996b). In a fish’s head, efficient expansion of the buccal cavity is usually achieved by the synchronous action of three musculoskeletal linkage systems (Wittenrich et al. 2009). Because no opercular bones, opercular muscles or opercular ligaments have already developed in the investigated leptocephali of the European eel, only the first (hyoid four-bar) system will act as a mouth opener together with the musculus protractor hyoidei (Figs 4 and 7a). However, missing two out of the three linkage systems, it can be hypothesized that suction feeding will be inefficient in the engyodontic firstfeeding leptocephali. A successful suction feeding action is also questioned by the analysis of video-recordings showing no movement of the maxillary bones during mouth opening in these larvae (Fig. 6c,d). Because leak flow during suction feeding in larval fishes is usually prevented by framing the mouth opening by rotating the maxillary bones (Hunt von Herbing et al. 1996b), the absence of significant maxillary displacements prevents water (and prey items) to be contained inside the buccal cavity. This apparent inability of leptocephali to prevent leak flow, combined with a rather inefficient system to create suction pressure, makes the idea of suction feeding in these engyodontic larvae highly unlikely. Nonetheless, hard-shelled prey organisms may still end up in the guts of these larvae even in the absence of suction. The prognathous, needle-like teeth of the larvae namely

© 2015 Anatomical Society

Bite performance of pre-feeding leptocephali, M. Bouilliart et al. 339

create a sieve-like construction, allowing particles to be sieved out of the water column and into the buccal cavity during larval swimming. The advantage of this feeding strategy is that no bite forces are required as long as prey items are small enough to fit between the gaps of the teeth.

Conclusion Using 3D reconstructions, musculoskeletal changes to the feeding apparatus of engyodontic leptocephali of the European eel are visualized and described during the transition from endogenous to exogenous feeding. Focusing on feeding performance, the observed changes in the musculoskeletal topography throughout the transition cause an increase in the larva’s theoretically estimated bite force (from approximately 25 lN to about 65 lN), but cannot prevent that most of the generated force is concentrated in the mandibular jaw joints. Combining this obtained bite force and this distribution of forces with an average gape angle of the first-feeding leptocephalus larva of about 100 lm, it can be hypothesized that these engyodontic leptocephali are anatomically only capable of feeding on rather small and/or soft food particles.

Acknowledgements This study contributes to the project ‘Reproduction of European Eel: Toward a Self-sustained Aquaculture’ (PRO-EEL) supported financially by the European Commission’s 7th Framework Programme, Grant Agreement no. 245257. This research and M. Bouilliart were also funded by the Special Research Fund (BOF) of Ghent University (Grant no. 01D23012). The authors thank € ger-Johnsen and Christian Graver for assistance. All fish Maria Kru were handled in accordance with the European Union regulations concerning the protection of experimental animals (Dir 86/609/EEC).

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