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The Journal of Experimental Biology 211, 3826-3835 Published by The Company of Biologists 2008 doi:10.1242/jeb.014464

Feeding biomechanics of juvenile red snapper (Lutjanus campechanus) from the northwestern Gulf of Mexico

Janelle E. Case1, Mark W. Westneat2 and Christopher D. Marshall1,* 1Texas A&M University at Galveston, Departments of Wildlife and Fisheries Science and Marine Biology, 5007 Avenue U, Galveston, TX 77551, USA and 2Field Museum of Natural History 1400, S Lakeshore Dr., Chicago, IL 60605, USA *Author for correspondence (e-mail: [email protected])

Accepted 6 September 2008

SUMMARY Juvenile red snapper settle across several complex habitats, which function as nurseries for young fish. Little is known about their life history or feeding biomechanics during this time. However, recent studies have shown higher growth rates for juveniles located on mud habitats adjacent to low profile reefs, perhaps because of varied prey availability and abundance. To further investigate the habitat needs of juvenile red snapper and test hypotheses of feeding development, individuals were collected from a low profile shell ridge and adjacent mud areas on Freeport Rocks, TX, USA, and divided into three size classes (≤3.9, 4.0–5.9, ≥6.0 cm SL). Output from a dynamic lever model suggested an ontogenetic shift in feeding morphology. Biomechanical modeling also predicted that off-ridge juveniles would have slower, stronger jaws compared with on-ridge juveniles. Kinematic profiles obtained from actual feeding events validated the modelsʼ predictive ability. Analysis of prey capture events demonstrated that on-ridge juveniles exhibited larger jaw displacements than off-ridge juveniles. Shape analysis was used to further investigate habitat effects on morphology. Off-ridge juveniles differed from on-ridge juveniles in possessing a deeper head and body. Results from model simulations, kinematic profiles, behavioral observations and shape analysis all compliment the conclusion that on- ridge juveniles exhibited more suction feeding behavior, whereas off-ridge juveniles used more biting behavior. Habitat disparity and possibly available prey composition generated variations in juvenile feeding biomechanics and behavior that may affect recruitment. Key words: biomechanics, kinematics, feeding, Lutjanus campechanus, red snapper.

INTRODUCTION (Lutjanus campechanus Poey 1860), to investigate changes in skull Ecomorphological studies are predicated on identifying patterns development and feeding biomechanics to provide an among morphology, behavioral performance and ecology ecomorphological explanation of divergent early life history patterns. (Wainwright, 1994), and have been utilized to test a variety of Larval red snapper settle out of the water column at approximately hypotheses concerning the relationships between feeding 16mm (Rooker et al., 2004) and are attracted to complex habitats, performance and foraging ecology among teleosts (e.g. Clifton and which serve as essential nursery grounds for juveniles (Szedlmayer Motta, 1988; Wainwright, 1996). Ecomorphological studies are also and Howe, 1997). The settlement patterns within these habitats useful in examining the functional consequences of ontogenetic remain unclear. Significantly higher recruitment occurs on shell changes on morphology and diet shifts in teleosts (e.g. Osenberg et ridges (on-ridge) in the northeastern Gulf of Mexico (Szedlmayer al., 1988; Hyndes et al., 1997; Hunt von Herbing, 2001; Graeb et and Conti, 1999) and on adjacent mud habitats (off-ridge) in the al., 2005; Monteiro et al., 2005). Ontogenetic shifts reduce northwestern Gulf of Mexico (Rooker et al., 2004). Juvenile growth competition through intraspecific (Hernandez and Motta, 1997; rates are significantly higher in off-ridge areas around Freeport Hyndes et al., 1997; Soto et al., 1998) or interspecific (Mittelbach Rocks (Rooker et al., 2004; Geary et al., 2007), suggesting that et al., 1992; Huskey and Turingan, 2001) resource partitioning. available prey resources may differ between off-ridge mud bottoms Furthermore, such shifts can reduce predation (Werner and Gilliam, and on-ridge shell ridges. Therefore we asked, ‘Do juveniles 1984) and maximize growth rates (Olson, 1996; Post, 2003) of respond to prey availability by altering feeding morphology or teleosts during early life development. Fast growth is most modulating feeding behavior?’ advantageous during larval and juvenile stages when individuals Although red snapper larval development (Collins et al., 1980; are most vulnerable to predation (Werner and Gilliam, 1984; Post, Pothoff et al., 1988; Drass et al., 2000) and diet of both adults and 2003). Therefore, improving a juvenile’s ability to take advantage juveniles have been examined (Bradley and Bryan, 1976; Moran, of the most abundant or high energy food source over ontogeny 1988; Ouzts and Szedlmayer, 2003; Szedlmayer and Lee, 2004), may increase individual fitness and enhance recruitment potential their feeding mechanics and behavior have not been investigated. (Olson, 1996; Persson and Brönmark, 2002; Post, 2003). Therefore, this research explored the relationships between Early life history studies that focus on the interaction between morphology and feeding kinematics within the context of trophic skull development, feeding mechanics, and their ecological ecology of juvenile red snapper using biomechanical modeling, consequences, are an important way to address critical questions in kinematic behavioral performance tests and shape analysis. We the ecomorphology of fishes. This study used juvenile red snapper hypothesized that juvenile red snapper settling onto different habitats

THE JOURNAL OF EXPERIMENTAL BIOLOGY Juvenile red snapper feeding biomechanics 3827 would exhibit a divergence in skull morphology and/or feeding biomechanics that may be correlated to reported juvenile diet patterns (Szedlmayer and Lee, 2004). To test these hypotheses, we performed experiments involving juvenile red snapper collected from different habitats and carried out biomechanical modeling of their jaws. This allowed us to determine how interactions between feeding ecology and functional morphology may influence growth and settlement patterns by testing for significant differences in jaw morphology, lever mechanics, kinematics and phenotypic plasticity among juvenile red snapper across three size classes (≤3.9, 4.0–5.9, ≥6.0cmSL) and between two nursery habitats (on-ridge and off- ridge).

MATERIALS AND METHODS collection and analyses Juvenile red snapper were collected between June and September 2004, and in August 2005, on and off the Freeport rocks shell ridge (Freeport, TX, USA). On-ridge areas were characterized by abundant relic oyster shell; off-ridge sites were characterized by silt and mud. Juvenile red snapper (N=530) were collected using a 6-m otter trawl with 2cm mesh, 1.25cm inner mesh, 0.6cm link tickler chain, and 0.457ϫ0.914m doors. Trawls were made in 5-min increments at Fig. 1. Morphometric measurements used as inputs in the jaw lever model, 2.5 knots. Juveniles for kinematics studies (N=17) were sorted by MandibLever 3.2. habitat (on-ridge N=8 and off-ridge N=9) and kept in separate ‘live’ wells onboard the research vessel. Additional subjects were anesthetized then frozen and kept for jaw lever analyses (N=230), parameters can be used to make predictions regarding fish feeding and shape analyses (N=111). Mass (g) and standard length (SL; cm) kinematics. were recorded for all juveniles and assigned to the following size Morphometric measurements of the lower jaw and associated jaw classes, small (1.8–3.9cmSL), medium (4.0–5.9cmSL) or large closing muscles (the A2 and A3 subdivisions of the adductor (6.0–10.88cmSL). Collections were made under TAMU IACUC mandibulae; Fig.1, Table1) were taken to the nearest 0.01cm using Animal Use Protocol no. 2003-84 and Texas Park and Wildlife either a calibrated eye reticule on a Nikon SMZ1500 stereoscope, Permit no. SPR 0902-243. or with digital vernier calipers. The following 12 measurements were Prior to conducting parametric statistical tests, normality of all collected: (1) in-lever A2, from quadrate-articular joint to A2 data was tested using a Kolmogorov–Smirnov test. If normality was insertion point on ascending process of articular; (2) in-lever A3, not met, data were transformed. Levene’s test was used to test the from quadrate-articular joint to A3 insertion point on medial face assumption of homogeneity of variances. Bonferroni post-hoc tests of lower jaw; (3) in-lever Open, from quadrate-articular joint to were used when the assumption of equal variance was met; insertion of interoperculomandibular ligament on posteroventral Dunnett’s t3 post-hoc tests were used in cases where variances were margin of articular; (4) out-lever, from quadrate-articular joint to heteroscedastic. All statistical tests were conducted using SPSS 11 anterior most tip of dentary; (5) A2 length, from origin on ventral (SPSS, Chicago, IL, USA) for a Mac and JMP 6 (SAS, Cary, NC, margin on preopercle to insertion on ascending process of articular; USA). More specific statistical analyses are listed under each (6) A3 total length, from origin on preopercle and hyomandibula methodological subheading (model of lower jaw lever mechanics, to insertion on medial face of lower jaw; (7) A3 tendon length, from feeding kinematics, and phenotypic variation). origin on tapering end of A3 muscle to insertion on the medial face of the lower jaw; (8) A2–joint distance, distance from A2 origin to Model of lower jaw lever mechanics quadrate-articular joint; (9) A3–joint distance, distance from A3 Lever mechanics were used to calculate the trade off between origin to quadrate-articular joint; (10) A2–A3 ins, distance from A2 velocity and force (Wainwright and Richard, 1995; Westneat, 1994; insertion to A3 insertion; (11) LJtop length, from the tip of the Westneat, 2003), and make predictions about the feeding mode of coronoid process to the anterior jaw tip; and (12) LJBot length, from juvenile red snapper. The biomechanics of juvenile red snapper the posteroventral margin of the articular to the anterior jaw tip. feeding were modeled by investigating the anatomical arrangement Mass of the A2 and A3 muscles were recorded to the nearest 0.01g. of the lower jaw as a third order lever using the program Assumptions regarding jaw muscle contractile physiology were MandibLever 3.2 (Westneat, 2003). This model incorporates the made following Westneat (Westneat, 2003): maximum shortening –1 influence of closing muscles on lever ratio calculations and creates velocity, or Vmax (10Ls ), maximum isometric stress of muscle a set of dynamic output variables over the entire jaw closing. The contraction, or Pmax (200kPa), dynamic contraction velocity of use of a dynamic model is advantageous since static measurements muscle (0.05–0.8 of Vmax), isometric force per unit area of muscle usually overestimate mechanical advantage because the influence (0.05–0.79 of Pmax); and a peak jaw opening rotation value based of changing muscle insertion angles is not accounted for (Westneat, on juvenile red snapper kinematic data (57°). Jaw muscle contraction 2003). The model, therefore, calculates an effective mechanical percentage was calculated as the percent change in length from advantage (EMA), which is a more accurate measurement of force the open (stretched) position. Morphometric measurements and transmission from muscle to the lower jaw. The model also muscular assumptions were used as inputs in the biomechanical lever calculates a variety of other dynamic variables, such as bite force, model, available free on the web from the second author. A total angular velocity and percent muscle contraction, and these of 230 simulations of lower jaw closing were run to predict feeding

THE JOURNAL OF EXPERIMENTAL BIOLOGY 3828 J. E. Case, M. W. Westneat and C. D. Marshall

Table 1. Morphometric data used as input into the lever model, MandibLever 3.2, for juvenile red snapper from three size classes and two habitats Small (N=74) Medium (N=82) Large (N=74) On-ridge (N=113) Off-ridge (N=117) 1. In-lever A2 (cm) 0.13±0.01 0.21±0.01 0.29±0.01 0.20±0.01 0.22±0.01 2. In-lever A3 (cm) 0.19±0.01 0.31±0.01 0.42±0.01 0.32±0.01 0.29±0.01 3. In-lever open (cm) 0.08±0.003 0.12±0.003 0.18±0.004 0.13±0.005 0.12±0.004 4. Out-lever (cm) 0.59±0.01 0.87±0.01 1.21±0.01 0.95±0.02 0.83±0.03 5. A2 length (cm) 0.34±0.01 0.52±0.01 0.75±0.01 0.58±0.02 0.49±0.02 6. A3 total length (cm) 0.57±0.01 0.94±0.02 1.38±0.02 1.03±0.03 0.90±0.03 7. A3 tendon length (cm) 0.12±0.004 0.69±0.33 0.35±0.01 0.58±0.24 0.22±0.01 8. A2-joint distance (cm) 0.32±0.01 0.49±0.01 0.70±0.01 0.54±0.02 0.46±0.02 9. A3-joint distance (cm) 0.55±0.01 0.85±0.01 1.24±0.02 0.93±0.03 0.83±0.03 10. A2-A3Ins (cm) 0.12±0.004 0.21±0.004 0.29±0.01 0.21±0.01 0.20±0.01 11. LJtop length (cm) 0.51±0.01 0.73±0.01 1.02±0.01 0.82±0.02 0.69±0.02 12. LJBot length (cm) 0.65±0.01 0.96±0.01 1.33±0.02 1.04±0.03 0.92±0.03 13. A2 mass (g) 0.001±0.0001 0.003±0.0002 0.01±0.001 0.005±0.0004 0.003±0.0004 14. A3 mass (g) 0.001±0.0001 0.004±0.0002 0.02±0.003 0.01±0.002 0.01±0.001 Values are means ± s.e.m.

behavior of juvenile red snapper from three size classes and two habitats. To test the hypothesis that there was no significant difference in the morphology of the feeding apparatus of juvenile red snapper across ontogeny, output parameters were analyzed using multivariate analyses of variance (MANOVA) with size class as a fixed factor, and model output parameters as dependent variables. Significant differences among size classes were determined by post-hoc tests. To test for significant differences between habitats, output parameters were analyzed using multivariate analyses of covariance (MANCOVA) with habitat as a fixed factor, model output parameters as dependent variables and standard length as a covariate.

Feeding kinematics Feeding kinematic trials were used to validate the predictive biomechanical model output, and compare the feeding biomechanics of juvenile red snapper across size classes and between habitats. Juvenile red snapper were transported to the laboratory and housed in habitat-specific 38–189l saltwater tanks and maintained at 26°C, 32p.p.t. salinity, and pH8.2. Fish were allowed to acclimatize, and Fig. 2. (A) Points used for digitizing juvenile red snapper cranial kinematics then trained to feed from a stationary tube under 500W of light. from feeding trials. (B) Landmark configuration on juvenile red snapper During the first collection season, a mass mortality event occurred used in geometric morphometric analyses. because of an Amyloodinium ocellateum outbreak during a hurricane evacuation. Not enough individuals were available to investigate ontogenetic changes; therefore, only a habitat treatment was included the posterior-most point of the orbit of the eye, (H) the first dorsal in the kinematic analysis. Juveniles used in feeding kinematic trials spine origin, (I) the anterodorsal tip of the opercle at the junction (on-ridge N=8, off-ridge N=9) all fell within the medium size class with the preopercle and the hyomandibula, (J) the posterodorsal tip (4.0–5.9cmSL). of the opercle, (K) the origin of the first pectoral fin ray. These 11 Juveniles were positioned laterally in front of the camera using anatomical landmarks were used to calculate the following 14 a piece of Plexiglas with a 1cm2 grid as a reference and fed pieces kinematic variables: (1) maximum gape (cm), (2) time to maximum of squid, sized to 50% of the individual’s oral diameter, until satiated. gape (ms), (3) maximum gape angle (degrees), (4) time to maximum Feeding events were recorded using a Redlake PCI Motion Scope gape angle (ms), (5) maximum lower jaw rotation (degrees), (6) high-speed camera at 250framess–1. Three representative feeding time to maximum lower jaw rotation (ms), (7) maximum upper jaw events for each juvenile were selected for analysis. Juveniles were protrusion (cm), (8) time to maximum upper jaw protrusion (ms), then sacrificed with an overdose of methane tricaine sulphonate (MS- (9) maximum cranial rotation (degrees), (10) time to maximum 222). Feeding events were digitized frame by frame, starting with cranial rotation (ms), (11) maximum depression of the hyoid (cm), the onset of strike until mouthparts returned to their starting (12) time to maximum hyoid depression (ms), (13) maximum position, using Motus 8.2 (Vicon, Denver, CO, USA). Digitized maxillary rotation (degrees), and (14) time to maximum maxillary points (Fig.2A) included: (A) the anterior tip of the premaxilla, (B) rotation (ms). Angular velocities and phase timings were also the anterior tip of the dentary, (C) the dorsal most visible point of calculated. the maxilla, (D) the maxilla–premaxilla articulation, (E) the Scatter plots of gape distance and gape angle versus closing mandible–quadrate articulation, (F) the ventral floor of mouth, (G) duration were used to determine the predictive ability of the lever

THE JOURNAL OF EXPERIMENTAL BIOLOGY Juvenile red snapper feeding biomechanics 3829 model by comparing data from the lever model and live kinematics. A3 muscle force contribution to total bite force was two times greater To statistically test the model as an accurate predictor of feeding than the A2 muscle over ontogeny, with a substantial increase over behavior, we log-transformed the gape, gape angle, and time axes the A2 muscle in large juveniles, suggesting a possible mechanism to linearize the curvilinear relationships between time and for shifts in juvenile feeding mode (Fig.3C,D). As gape increased kinematics, and performed analysis of covariance (ANCOVA) to with size, A3 muscle jaw closing duration significantly decreased test whether slopes and/or y-intercepts of the model and video data 8.4% (A2 P=0.56, A3 P<0.001, MANOVA) resulting in an expected were significantly different. significant 16.2% increase in angular velocity of the A3 muscle (A2 Kinematic variables were also used to characterize and quantify P=0.34, A3 P=0.002, MANOVA; Fig.3E,F, Table2). The A2 the feeding behavior of juvenile red snapper between habitats. To muscle angular velocity was faster than the A3 muscle in small test the hypothesis that there was no significant difference in feeding juveniles but slower than the A3 muscle in medium and large behavior of juvenile red snapper between habitats (P≤0.05), juveniles (Fig.3F). Percent muscle contraction required to close the kinematic variables were analyzed using multivariate analysis of lower jaw generally decreased 2% over ontogeny for the A2 muscle variance (MANOVA) with habitat as a fixed factor and kinematic and significantly by 8.5% for the A3 muscle (A2 P=0.56, A3 variables as dependent variables. Kinematic profiles were generated P<0.001, MANOVA; Table2). The A3 muscle increased in size at for each variable to examine their relationship to one another and a faster rate than the A2 muscle, causing the A3 muscle morphology identify different phases over a complete feeding event. and function to change to a greater extent over ontogeny than the A2 muscle. As the A3 in-lever became longer, A3 force contribution Phenotypic variation increased as a result of an increase in mechanical advantage. As the Shape variables were collected to investigate differences in body A3 muscle became longer the angular velocity increased, resulting shape of juvenile red snapper from the on-ridge and off-ridge in shorter closing durations and less percent muscle contribution to habitats. Lateral images of juvenile red snapper (on-ridge N=56, close the lower jaw, resulting in the A3 muscle assuming the off-ridge N=55; small N=50, medium N=33, large N=28) were dominant role in lower jaw closing. captured using a digital camera. Two-dimensional coordinates were Trends in changes in muscle function were observed over recorded from the following 19 landmarks (Fig.2B) digitized ontogeny. To examine if these trends indeed led to an adult feeding around the juvenile body perimeter using the program tpsDig (v.2) mode, adult red snapper (N=3) were also modeled. These results (Rohlf, 2005a): (1) anterior tip of the dentary, (2) anterior tip of the from adults were only used to make general qualitative comparisons premaxilla, (3) anterior-most point of the eye orbit, (4) center of of the potential dynamic actions of the A2 and A3 muscles during the eye, (5) posterior-most point of the eye orbit, (6) anterior-most lower jaw closing. They were not used for any statistical point of the frontal bone, (7, 8) anterior and posterior insertions of comparisons. Overall, the A3 muscle was larger than the A2 in the dorsal fin, respectively, (9) dorsal origin of the caudal fin, (10) adults, both in length and cross-sectional area (Table3) according middle of caudal fin insertion where the lateral line terminates, (11) to the model. The A3 muscle contributed more force to overall bite ventral origin of the caudal fin, (12, 13) posterior and anterior force, had higher effective mechanical advantage, and thus a lower insertions of the anal fin, respectively, (14) anterior-most insertion velocity ratio than the A2 muscle (Table3). Total duration of the of the pelvic fin, (15) first branchiostegal ray at the body outline, A3 muscle in lower jaw closing was shorter than the A2 muscle (16) quadrate-articular joint, (17) origin of the first pectoral fin ray, (Table3). Since both muscles rotated through the same gape, the (18) posterodorsal tip of the opercle, (19) anterior-most point of the angular velocity of the A3 muscle was higher and the percent muscle lateral line. TpsRelw software (v.1.42) (Rohlf, 2005b) was used to contraction required to close the lower jaw was smaller than the A2 align shape data by rotating, translating and scaling the landmark muscle (Table3). The trends observed in large juvenile A2 and A3 coordinates, using least squares superimposition. Aligned data were muscle function are consistent with data from adult model used to calculate shape variables. Significant variations in shape simulations, suggesting that when juveniles reach approximately were tested using MANOVA with shape variables as dependent 6cm in length they switch to their adult feeding mechanism. variables, and habitat and size as fixed variables. An eigendecomposition of the effect sum of squares and cross-products Habitat (SSCP) matrix was performed and used to calculate the shape Model simulations were carried out in which a habitat effect was variance explained by habitat and allometry. In addition, associated tested and size classes were pooled to determine if A2 and A3 muscle eigenvectors were multiplied by shape variables to yield linear axis function differed between habitats. The cross-sectional areas of the scores. TpsRegr software (v.1.31) (Rohlf, 2003) produced thin-plate A2 and A3 muscles in juveniles did not differ significantly between spline transformation grids (Fig.6), which provided a visualization habitats (A2 P=0.32, A3 P=0.36, MANCOVA; Table2). Muscle of shape variation. length differed between habitats: the A2 muscle was 31.1% longer in off-ridge juveniles (1.03±0.06cm) and the A3 muscle was 10.7% RESULTS longer in on-ridge juveniles (1.21±0.04cm; Table2). Effective Lower jaw lever model mechanical advantage (EMA) was significantly less (6.3–20.8%; Ontogeny A2 P<0.001, A3 P=0.002, MANCOVA), and the velocity ratio was Model simulations demonstrated that for the A3 muscle effective significantly greater (3.3–20%; A2 P<0.001, A3 P=0.02, mechanical advantage increased 3% (A2 P=0.28, A3 P=0.02, MANCOVA) in on-ridge juveniles compared with off-ridge MANOVA) and velocity ratio decreased 6.7% (A2 P=0.22, A3 juveniles. The A2 muscle exhibited lower EMA and greater velocity P<0.001, MANOVA) as body size increased (Fig.3A,B, Table2). than the A3 muscle for both habitats (Fig.3G,H). The A3 muscle Muscle force contribution (A2 and A3 P<0.001, MANOVA) and force contribution to bite force (P=0.86, MANCOVA) and the total total bite force (P<0.001, MANOVA) significantly increased four bite force (P=0.59, MANCOVA) was not significant between to eight times with body size (Fig.3C,D, Table2), as expected with habitats. The A2 muscle force contribution to bite force was an associated increase in muscle cross-sectional area. The potential significantly greater (1.1ϫ) on-ridge (P=0.01, MANCOVA). Off- functional roles of the A2 and A3 muscles changed over ontogeny. ridge juveniles exhibited 12.2% smaller gapes (A2 P=0.17, A3

THE JOURNAL OF EXPERIMENTAL BIOLOGY 3830 J. E. Case, M. W. Westneat and C. D. Marshall

Ontogeny Habitat A2 A3 6 0.4 6 a a,b b A A B A G H 0.3 A A a b 5 0.3 B A A A 4 B 4 a 0.2 b b a b 0.2 A 3 EMA EMA 2 2 Velocity ratio Velocity 0.1 ratio Velocity 0.1 1 0 0 0 0 0.10 0.30 1.0 B b C c D C I A a 100 J a 0.08 0.25 0.8 A a A 80 0.20 0.06 0.6 60 C 0.15 0.04 b B 0.4 40

0.10 Gape (cm) Bite force (N) Bite force 0.02 a B A 0.2 (ms) Duration Total bite force (N) bite force Total 20 A 0.05 0 0 0 0

) 5 ) 6 25 a –1 –1 100 b b a B b E A A A b F A b K L 4 A 5 A 20 A 80 A a a 4 b 3 15 60 B 3 2 10 40 2 Duration (ms) Duration 20 1 1 5 Percent contraction Percent

0 ms (deg. Angular velocity 0 ms (deg. Angular velocity 0 0 Small Medium Large Small Medium Large On-ridge Off-ridge On-ridge Off-ridge

Fig. 3. Simulation results (mean ± s.e.m.) from the lever model, MandibLever 3.2, for the A2 and A3 muscles of juvenile red snapper from three size classes and two different habitats. Capital letters represent significant differences of A2 muscle output parameters and lower case letters represent significant differences of A3 muscle output parameters (P<0.05).

Table 2. MANOVA, MANCOVA results of A2 and A3 muscle parameters from MandibLever model simulations of lower jaw closing for juvenile red snapper over three size classes and two habitats Length (cm) CSA (cm2) EMA VR BiteF (N) Dur (ms) Gape (cm) AngVel (deg. ms–1) % Cont Ontogeny A2 Small 0.56±0.05 0.002±0.00 0.21±0.01 4.83±0.04 0.01±0.004 89.8±0.12 0.52±0.01 3.41±0.01 21.2±0.06 Medium 0.91±0.05 0.01±0.00 0.22±0.01 4.5±0.03 0.02±0.004 91.1±0.12 0.76±0.01 3.55±0.01 21.6±0.06 Large 1.14±0.04 0.01±0.00 0.22±0.01 4.56±0.04 0.04±0.004 87.7±0.12 1.06±0.01 3.82±0.01 20.8±0.06 F – – 1.29 1.52 169.7 0.58 572.5 1.08 0.58 P value – – 0.28 0.22 0.00* 0.56 0.00* 0.34 0.56

A3 Small 0.71±0.02 0.003±0.00 0.31±0.02 3.13±0.05 0.01±0.01 98.4±0.27 0.52±0.01 3.31±0.01 23.3±0.13 Medium 1.12±0.02 0.01±0.002 0.32±0.02 2.89±0.04 0.03±0.01 94.6±0.26 0.75±0.01 3.99±0.01 22.4±0.13 Large 1.62±0.02 0.02±0.002 0.32±0.02 2.92±0.05 0.08±0.01 90.1±0.27 1.05±0.01 3.95±0.01 21.3±0.13 F – – 3.8 8.4 151.4 8.4 517.4 6.4 8.4 P value – – 0.02* 0.00* 0.00* 0.00* 0.00* 0.002* 0.00* Habitat A2 On-ridge 0.71±0.02 0.01±0.00 0.19±0.01 5.15±0.12 0.02±0.001 79.4±1.67 0.83±0.004 4.25±0.01 18.8±0.39 Off-ridge 1.03±0.06 0.01±0.00 0.24±0.004 4.12±0.11 0.02±0.001 99.4±1.57 0.73±0.004 2.96±0.01 23.5±0.37 F – – 64.5 48.9 6.27 76.8 1.92 44.3 76.8 P value – – 0.00* 0.00* 0.013* 0.00* 0.17 0.00* 0.00*

A3 On-ridge 1.21±0.04 0.01±0.001 0.3±0.004 3.03±0.04 0.05±0.003 91.6±1.72 0.82±0.004 4.07±0.01 21.7±0.41 Off-ridge 1.08±0.04 0.01±0.001 0.32±0.004 2.93±0.04 0.03±0.003 97.2±1.62 0.72±0.004 3.46±0.01 23±0.38 F – – 9.61 5.35 0.03 4.09 2.86 4.83 4.09 P value – – 0.002* 0.022* 0.86 0.04* 0.092 0.03* 0.04* Length, muscle length; CSA, cross-sectional area; EMA, effective mechanical advantage; VR, velocity ratio; BiteF, muscle bite force contribution; Dur, closing duration; Gape, closing gape distance; AngVel, angular velocity; % Cont, percent muscle contraction required to close the lower jaw. Values are means ± s.e.m. *P<0.05, d.f.=1, 215.

THE JOURNAL OF EXPERIMENTAL BIOLOGY Juvenile red snapper feeding biomechanics 3831

Table 3. Results of A2 and A3 muscle parameters from lever model respectively; Fig.3K). From these results, the A2 muscle appears simulations of lower jaw closing for adult red snapper to be the dominant muscle in on-ridge juveniles for fast closing, A2 A3 whereas the A3 muscle appears to be the dominant muscle for fast closing in off-ridge juveniles. Muscle length (cm) 4.19 7.60 Cross-sectional area (cm2) 0.34 0.44 Model validation Muscle force exerted (N) 5.42 6.96 Bite force (N) 1.54 2.37 A comparison of model output to kinematic results tested the Effective mechanical advantage (EMA) 0.29 0.34 predictive accuracy of the jaw lever model. The overall relationship Velocity ratio (VR) 3.02 2.70 between jaw closing duration and gape displacement (Fig.4A), as Duration (ms) 110.5 95.7 well as a plot of the average model with several representative Gape (cm) 4.60 4.56 –1 individuals (Fig.4B), show a close agreement between model and Angular velocity (deg. ms ) 2.71 3.44 live kinematic data. Model and video data were not significantly Percent contraction 26.2 22.6 different for slope (P=0.21, ANCOVA) or intercept (P=0.08, ANCOVA). Similar plots of jaw closing duration against gape angle (Fig.4C,D) show that the rate of angle change was greater in the P=0.09, MANCOVA), closing durations of 5.8–20.1%, which were model simulations than in living kinematics, although, similar jaw significantly longer (A2 P<0.001, A3 P=0.04, MANCOVA), closing curve shape and gape angle values were observed, 15–30.4% slower angular velocities (A2 P<0.001, A3 P=0.03, particularly when several representative kinematic plots were MANCOVA), and 5.7–20% greater percent muscle contractions (A2 compared to the model (Fig.4D). For gape angle, model and video P<0.001, A3 P=0.04, MANCOVA) required to close the lower jaw data were not significantly different for slope (P=0.81, ANCOVA) compared to on-ridge juveniles for both muscles (Fig.3I–L). The but had significantly different intercepts (P=0.02, ANCOVA). closing duration and percent muscle contraction of the A2 muscle Overall, the lever model was an accurate predictor of gape was shorter in on-ridge juveniles (79.49±1.67ms and 18.82±0.39ms, displacement and gape angle in juvenile red snapper, with expected respectively) and larger in off-ridge juveniles (99.4±1.57ms and higher variability in the living fish kinematics. 23.5±0.37 ms, respectively) compared with the A3 muscle (Fig.3J,L). The angular velocity of the A2 muscle was greater than Feeding kinematics the A3 muscle in on-ridge juveniles (4.25±0.01deg.ms–1 and On-ridge juveniles (92%) would approach and engulf prey items 4.07±0.01deg.ms–1, respectively) and smaller than the A3 muscle from a distance using a single explosive jaw movement. Jaw in off-ridge juveniles (2.96±0.01deg.ms–1 and 3.46±0.01deg.ms–1, protrusion and hyoid depression began after mouth opening and

0.8 60 A Kinematics C 0.7 Kinematics Model 50 Model 0.6 40 0.5 0.4 30 0.3 20 0.2 10 0.1 0 0 0.8 80 B Model D Gape (cm) 0.7 Model 70 Rep fish 1

Rep fish 1 Gape angle (deg.) 0.6 60 Rep fish 2 Rep fish 2 Rep fish 3 0.5 50 0.4 40 0.3 30 0.2 20 0.1 10 0 0 0 20406080100 0 20406080100 Time (ms)

Fig. 4. Comparison of model predictions and video data for jaw closing parameters of juvenile red snapper. (A) Time plot of mean gape distance change as the jaws close, starting at peak gape, illustrating the tight relationship between the average model predictions and the average video kinematics. (B) Mean gape change of the model illustrated with two representative red snapper individuals. (C) Time plot of mean gape angle as the jaws close, starting at peak gape, showing that the living fish kinematics were slower than simulated kinematics, based on a Vmax of 10 muscle lengths per second contraction speed. (D) Mean gape change of the model illustrated with three representative red snapper individuals. Error bars on all plots in A and C are standard deviations of the mean.

THE JOURNAL OF EXPERIMENTAL BIOLOGY 3832 J. E. Case, M. W. Westneat and C. D. Marshall

0.6 IIIII I III IV III IV 0.4

Gape (cm) 0.2

0 1.2 1.1 1.0 0.9 0.8 0.7 Hyoid depression (cm) Hyoid 1.2 1.1 1.0 0.9 0.8 04080120 Jaw protrusion (cm) Jaw Time (ms)

0.6 III III II III 0.4

Gape (cm) 0.2

0 1.2 1.1 1.0 0.9 0.8 0.7 Hyoid depression (cm) Hyoid 1.2 1.1 1.0 0.9 0.8 0 100 200 300 400 Jaw protrusion (cm) Jaw Time (ms)

Fig. 5. Selected high-speed video frames and kinematic profiles of displacement variables from a representative prey capture event for one (A) on-ridge and (B) off-ridge juvenile red snapper. In the profiles, phases are indicated across the top by horizontal bars and labeled: I, preparatory; II, expansive; III, compressive; and IV, recovery. Vertical lines represent time of prey capture. reached their maxima after maximum gape was achieved (Fig.5A). jaw protrusion decreased and then increased again during the prey Maxillary rotation and cranial rotation began to increase at the transport gape, reaching a second maximum along with maximum beginning of the feeding event and reached their maxima after hyoid depression (Fig.5B). After the prey transport was achieved, maximum gape was achieved (Table4). The hyoid and jaw tips the mouth closed and the hyoid, jaw tips, cranium and maxillary returned to their original positions after the mouth had closed returned to their starting positions simultaneously (Fig.5B). (Fig.5A). On-ridge juvenile red snapper expressed larger and faster jaw Off-ridge juveniles (37%) generally captured the prey item and movement compared with off-ridge juveniles. Maximum momentarily held it between the jaws, resulting in a prey transport displacement variables were significantly greater in on-ridge cycle to move the prey to the pharyngeal jaws. The hyoid depression juveniles (all P<0.001, MANOVA). Maximum angular variables began increasing at approximately the same time as the initial gape were greater in on-ridge juveniles, significantly for maximum cranial displacement and increased to its maximum, which occurred after rotation (P=0.02, MANOVA) and maximum maxillary rotation the prey transport gape maximum (Fig.5B). After the initial gape, (P=0.02, MANOVA). Time to maximum displacement and angular

THE JOURNAL OF EXPERIMENTAL BIOLOGY Juvenile red snapper feeding biomechanics 3833

Table 4. Maximum kinematic variables for juvenile red snapper from two different habitats Variable On-ridge (N=8) Off-ridge (N=9) FPvalue Maximum gape (cm) 0.57±0.03 0.43±0.02 17.3 0.00* Time to maximum gape (ms) 117.0±12.7 98.9±8.85 1.05 0.31 Maximum hyoid depression (cm) 1.18±0.02 1.01±0.03 25.0 0.00* Time to maximum hyoid depression (ms) 152.2±17.7 150.5±15.5 0.002 0.97 Maximum jaw protrusion (cm) 1.28±0.02 1.08±0.03 26.4 0.00* Time to maximum jaw protrusion (ms) 158.3±17.8 150.9±15.6 0.04 0.85 Maximum gape angle (deg.) 60.2±2.73 54.2±8.12 3.35 0.07 Time to maximum gape angle (ms) 116.5±12.8 100.6±11.0 0.39 0.54 Maximum lower jaw rotation (deg.) 173.0±1.95 170.3±1.27 1.34 0.25 Time to maximum lower jaw rotation (ms) 117.0±12.6 110.3±16.0 0.15 0.70 Maximum cranial rotation (deg.) 66.07±0.85 63.61±0.71 5.66 0.02* Time to maximum cranial rotation (ms) 163.7±12.6 145.3±11.7 0.12 0.73 Maximum maxillary rotation (deg.) 103.9±1.04 108.4±1.41 5.49 0.02* Time to maximum maxillary rotation (ms) 128.8±22.0 123.3±0.01 0.01 0.92 Maximum gape velocity (deg. ms–1) 3.10±0.47 2.11±0.39 2.88 0.1 Maximum lower jaw rotation velocity (deg. ms–1) 1.65±13.9 0.98±0.16 5.29 0.03* Maximum cranial rotation velocity (deg. ms–1) 0.41±0.08 0.20±0.03 2.83 0.1 Maximum maxillary rotation velocity (deg. ms–1) 0.70±0.15 0.58±0.12 0.01 0.93 Time to prey capture (ms) 117.0±11.8 124.0±12.6 0.03 0.86 Values are means ± s.e.m. *P<0.05, d.f.=1, 48. variables did not differ significantly between habitats (all P>0.05, became slower and more forceful as size increased. The lever ratio MANOVA). Maximum angular velocities were faster in on-ridge values of small and large juvenile red snapper are consistent with juveniles for all angles, significantly for maximum lower jaw rotation values for suction feeders and biters, respectively. Measurements velocity (P=0.03, MANOVA). Prey capture time was shorter in on- of lever ratios have successfully predicted a diet of small, soft prey ridge juveniles (P=0.86, MANOVA). items and a diet of larger, harder prey items, respectively, in other teleosts (Barel, 1983; Westneat, 1994; Westneat, 2004; Wainwright Phenotypic variation and Richard, 1995). Therefore, this study strongly suggests that an Shape analysis further supported morphological and behavioral ontogenetic shift in morphology occurred that enabled large red differences in juvenile red snapper throughout ontogeny and between snapper juveniles to exploit harder prey types. Adult red snapper habitats. Lateral body morphology of juvenile red snapper lever ratios demonstrated that the slow but forceful lower jaw significantly differed across size (P<0.001, MANOVA) and also movement is indeed the mature feeding mode, which correlates well differed between the two habitats (P=0.01, MANOVA). Habitat with hard prey items of the adult diet (i.e. crabs and mollusks) effect accounted for 1.6% of the total morphological variation and (Bradley and Bryan, 1976; Moran, 1988; Ouzts and Szedlmayer, size effect accounted for 9.1%. The effect of habitat was small in 2003). Dietary data from studies of juvenile red snapper (Bradley magnitude, but high in significance. Thin plate spline transformation and Bryan, 1976; Szedlmayer and Lee, 2004) and other lutjanids grids illustrate changes along the shape axis (Fig.6). The habitat (Rooker, 1995) suggest a shift towards the adult feeding mode that effect axis indicated that off-ridge juveniles had a deeper head and corresponds with the morphological and biomechanical switch body than on-ridge juveniles (Fig.6). The size effect axis indicated observed in this study in juveniles at 6cmSL; it is known that a that as juvenile body size increased the head and body became change in diet of lutjanids initiates movement to deeper water deeper. (Rooker, 1995; Cocheret de la Morinière et al., 2003; Szedlmayer and Lee, 2004). Over ontogeny juveniles developed the DISCUSSION morphological capability to consume a broader range of prey, which Juvenile red snapper feeding ontogeny would allow juveniles to begin to occupy a wider ecological niche This study provides data suggesting a possible mechanism for a (King, 1971; Liem, 1980; Luczkovich et al., 1995), become more transition from a juvenile to an adult feeding mode. Modeling data opportunistic feeders and therefore could effectively move into adult suggests that the potential function of the A2 and A3 adductor populations in deeper waters. muscles changed ontogenetically. In small juveniles, the A2 muscle dominated lower jaw movement, whereas the A3 dominated in large Habitat effects on juvenile red snapper feeding juveniles. The switch from A2 muscle dominance to A3 muscle Juvenile red snapper from different habitats (on-ridge vs off-ridge) dominance at 6cmSL in juvenile red snapper marks the transition exhibited differences in their feeding mechanics, feeding behavior, to an adult feeding mode. This ontogenetic change of the A2 and as well as their head and body morphology. The lower jaw lever A3 muscle function is supported by the fact that the A3 muscle in model of juvenile red snapper from the two habitats demonstrated adult red snapper is also the dominant lower jaw closing muscle. significant differences in their potential feeding capabilities. The The A2 and A3 muscle function in large red snapper juveniles was mechanical advantage of the lower jaw from on-ridge juveniles similar to data from a benthic foraging wrasse (Cheilinus trilobatus) was similar to values from suction feeders, whereas mechanical (Westneat, 2003). The jaw lever model data also suggests that advantage of the lower jaw from off-ridge juveniles was similar to ontogenetic changes in morphology had an impact on feeding values from biters (Wainwright and Richard, 1995; Westneat, kinematics of juvenile red snapper and possibly diet. Lever ratio 2004). Therefore, the lower jaw lever model prediction that off- data demonstrated that, over ontogeny, the lower jaw movement ridge juveniles have an increased capability to crush harder prey

THE JOURNAL OF EXPERIMENTAL BIOLOGY 3834 J. E. Case, M. W. Westneat and C. D. Marshall

Habitat axis scores Fig. 6. Morphological responses by juvenile red snapper 0.01 to habitat. Habitat axis scores plotted with transformation Off-ridge grids for illustration. Grids are magnified 10ϫ to enhance habitat effect. 0.005

0 On-ridge

–0.005

–0.01 types, or bite off pieces of larger prey, whereas on-ridge juveniles this point moves in the same direction in red drum (Scianenops have an increased suction capability to capture small, soft prey ocellatus) fed soft prey items (Ruehl and DeWitt, 2007). Therefore, appears to be supported. In addition, results from feeding kinematic differences in juvenile red snapper head and body shape further trials of juvenile red snapper also supported predictions of the jaw supports the hypothesis that on-ridge juveniles consume softer prey lever model. Actual feeding events of juvenile red snapper items, or smaller items that are ingested whole, most probably using demonstrated that on-ridge juveniles expressed kinematic profiles suction. Concomitantly, shape analysis of off-ridge juvenile snapper typical of suction feeders (e.g. Liem, 1980; Van Leeuwen and supports the hypothesis that deeper bodied off-ridge juveniles Muller, 1984; Svanbäck et al., 2002), whereas off-ridge juveniles consume harder prey types, or pieces of larger prey, using biting. exhibited a more manipulative, biting behavior. Furthermore, in These differences in body shape between habitats also suggest that captivity, off-ridge juveniles were observed actively biting the prey off-ridge juveniles have reached a more developed ontogenetic state given to them, as well as each other. Captive off-ridge juveniles faster, since they have taken the shape of larger juveniles. On-ridge would approach other juveniles and bite them to remove large pieces juveniles were slower to reach the near-adult ontogenetic stage; of flesh, or completely bite them in half. Anecdotally, we observed similar patterns have been observed in sharpsnout seabream that fish prey identified in off-ridge juvenile stomach contents were (Diplodus puntazzo) (Kouttouki et al., 2006). large pieces of fishes, not whole fishes. This is consistent with the By integrating morphological modeling, kinematic behavioral behavior observed in captivity. Captive on-ridge juveniles were performance testing and shape analysis, this study has provided new observed using suction to capture prey given, and were rarely seen insight into data on the biomechanical development of juvenile red biting each other. snapper during early life history stages, and its possible trophic Juvenile red snapper also exhibited phenotypic plasticity in consequences. It is probable that developmental modifications in response to differences between habitats. Shape analysis has been feeding ability of juvenile red snapper, primarily in the ontogenetic used previously to demonstrate the expression of phenotypic changes in A2 and A3 muscle function, resulted in size-related diet plasticity of an organism induced by varying environmental factors shifts, and the capability to consume harder prey types that are more (Robinson and Wilson, 1996; Robinson et al., 1996; Svanbäck and typical of adult diets. The pivotal size for juvenile red snapper Eklöv, 2002; Svanbäck and Eklöv, 2003; Doughty and Reznick, development appears to be at 6–7cm. At this size the hyoid and 2004; Parsons and Robinson, 2007). Habitat has been shown to mandibular arches have fully ossified (Potthoff et al., 1988) and generate resource polymorphism in fish. For example, where fish juveniles have attained the morphological capability to fill a wider of the same occupy different habitats some species developed ecological niche. This allows juveniles to successfully begin different body shapes and the ability to consume different prey types competing with larger juveniles and adults, and they can therefore (Lavin and McPhail, 1986; Ehlinger and Wilson, 1988; Malmquist, effectively move into the adult population. However, habitat may 1992; Robinson et al., 1996; Svanbäck and Eklöv, 2002). In this influence the transition between these ontogenetic stages. Off-ridge study, off-ridge juvenile red snapper had deeper heads, whereas on- juveniles in this study possessed the morphological capability to ridge juveniles had more streamlined heads. Studies of consume harder prey types, but more importantly fish, earlier than polymorphism suggest that streamlined bodies are associated with on-ridge juveniles at the same body size. By developing a stronger midwater feeders and are optimal for high velocity prey capture of bite, off-ridge juveniles may compensate for any gape limitations by elusive prey. By contrast, deeper bodies are associated with low biting pieces of prey larger than their mouth. A fish diet is high in velocity and high maneuverability, and this is optimal for benthic caloric value, so an earlier switch to piscivory promotes faster growth foragers that feed on hard prey (Ehlinger and Wilson, 1988; and survival (Persson and Brönmark, 2002; Post, 2003; Graeb et al., Malmquist, 1992; Motta et al., 1995; Robinson and Wilson, 1996; 2005), which may explain the higher growth rates of juvenile red Robinson et al., 1996; Walker, 1997; Hjelm et al., 2003; Svanbäck snapper reported in off-ridge areas (Rooker et al., 2004; Geary et al., and Eklöv, 2003). Controlled experiments in which prey items were 2007). In general, faster growing fish resulting from an early switch manipulated demonstrated a morphological difference in head to piscivory represent the population majority within the cohort, and shape, streamlined versus deep, when fish were fed small, soft prey therefore contribute more individuals to the adult population (Olson, versus harder prey types, respectively (Meyer, 1987; Wimberger, 1996; Ludsin and DeVries, 1997; Persson and Brönmark, 2002). 1991; Wimberger, 1992; Hegrenes, 2001; Parsons and Robinson, 2007). Among the selected landmarks from this study, the The authors gratefully thank Drs Thomas DeWitt and Jay Rooker for their contributions throughout the study. We thank Jay Rooker for assistance in branchiostegal ray point (15) moved anteriorly, producing a more collecting juvenile red snapper. We also thank Joe Mikulas and Ryan Schloesser streamlined head in on-ridge juveniles. Previous studies showed that for their help in the collection and maintenance of juvenile fish. This work was

THE JOURNAL OF EXPERIMENTAL BIOLOGY Juvenile red snapper feeding biomechanics 3835 supported by the Texas A&M at Galveston Department of Marine Biology and the Motta, P. J., Clifton, K. B., Hernandez, P. and Eggold, B. T. (1995). Luke and Erma Lee Mooney Student Travel Grant. The development of Ecomorphological correlates in ten species of subtropical seagrass fishes: diet and MandibLever software was supported by NSF grant 0235307. microhabitat utilization. Environ. Biol. Fish. 44, 37-60. Olson, M. H. (1996). Ontogenetic niche shifts in largemouth bass: variability and consequences for first-year growth. Ecology 77, 179-190. Osenberg, C. W., Werner, E. E., Mittelbach, G. G. and Hall, D. J. (1988). Growth REFERENCES patterns in bluegill (Lepomis macrochirus) and pumpkinseed (L. gibbosus) sunfish: Barel, C. D. N. (1983). Towards a constructional morphology of the cichlid fishes environmental variation and the importance of ontogenetic niche shifts. Can. J. Fish. (Teleostei, ). Neth. J. 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