Buoyancy control and air breathing in royal knifefish (Chitala blanci) and a new hypothesis for the early evolution of vertebrate air-breathing behaviors.

We present the first description of inspiration-first air breaths in royal knifefish, Chitala blanci, a ray-finned fish known to use four-stroke air breaths. Four-stroke breaths are used by nearly all ray-finned fish species that use their gas bladder to breathe air and are the ancestral breath type of ray-finned fishes. Interestingly, one such species, Amia calva, is known to perform two distinct breath types. Amia use four-stroke breaths when they need more oxygen and performs inspiration-first breaths to restore buoyancy. We observed that C. blanci also performs inspiration-first breaths and tested whether the two breath types are performed for the same functions in C. blanci as they are in Amia. We recorded the frequency of each breath type when exposed to aquatic hypoxia and two conditions of oxygen availability. We found that C. blanci performed more four-stroke breaths (81% ± 15% of total breaths) than inspiration-first breaths when exposed to aerial normoxia but performed more inspiration-first breaths (72% ± 40%) than four-stroke breaths when exposed to aerial hyperoxia. These patterns match those described for Amia and indicate that C. blanci performs four-stroke breaths in response to oxygen depletion and performs inspiration-first breaths to maintain buoyancy. Few studies have examined the role of air-breathing in buoyancy regulation. Decreasing buoyancy, rather than oxygen availability, to stimulate air breaths may reveal that inspiration-first breaths are more common among fishes than we are aware. We consider this possibility and present a new hypothesis for the origin and early evolution of air breathing in vertebrates.

Epaxial and hypaxial co-contraction: a mechanism for modulating strike pressure and accuracy during suction feeding in channel catfish.

Most fish species use concentric epaxial and hypaxial contractions to suction feed, whereby both muscle groups produce cranial expansion and negative intraoral pressures. In contrast, channel catfish (Ictalurus punctatus) suction feed with little to no cranial elevation and epaxial shortening, generating suction power primarily with hypaxial shortening and pectoral girdle retraction. We hypothesized that channel catfish (1) actively anchor the head via isometric contraction of the epaxials and (2) vary feeding performance by modulating the absolute and relative outputs of the co-contracting muscles. We used a combination of electromyography, intraoral pressure recordings and specimen manipulation, and developed a new dual-lever model to explore this idea. We detected epaxial and hypaxial co-contraction prior to suction force development in all strikes. Our model revealed that the differential between the co-contracting muscles may be used to modulate suction pressure and strike accuracy.

Long-Axis Rotation of Jaws of Bamboo Sharks (Chiloscyllium plagiosum) During Suction Feeding.

Long-axis rotation (LAR) of the jaws may be an important component of vertebrate feeding mechanisms, as it has been hypothesized to occur during prey capture or food processing across diverse vertebrate groups including mammals, ray-finned fishes, and sharks and rays. LAR can affect tooth orientation as well as muscle fiber direction and therefore muscle power during feeding. However, to date only a handful of studies have demonstrated this LAR in vivo. Here, we use XROMM to document LAR of the upper and lower jaws in white-spotted bamboo sharks, Chiloscyllium plagiosum, during suction feeding. As the lower jaw begins to depress for suction expansion, both the upper jaw (palatoquadrate) and lower jaw (Meckel's cartilage) evert, such that their toothed surfaces move laterally, and then they invert with jaw closing. Eversion has been shown to tense the dental ligament and erect the teeth in some sharks, but it is not clear how this tooth erection would contribute to suction feeding in bamboo sharks. Two recent XROMM studies have shown LAR of the lower jaws during mastication in mammals and stingrays and our study extends LAR to suction feeding and confirms its presence in shark species. Examples of LAR of the jaws are becoming increasingly widespread across vertebrates with unfused mandibular symphyses. Unfused lower jaws are the plesiomorphic condition for most vertebrate lineages and therefore LAR may be a common component of jaw mechanics unless the mandibular symphysis is fused.

Air Breathing and Suction Feeding Kinematics in the West African Lungfish, Protopterus annectens.

Research on the water-to-land transition tends to focus on the locomotor changes necessary for terrestriality. However, the evolution from water breathing to air breathing was also a necessary precursor to the invasion of land. Air is approximately 1000 times less dense and 50 times less viscous, and contains hundreds of times more oxygen than water. However, unlike the transition to terrestrial locomotion, breathing air does not require body weight support, so the evolution of air breathing may have necessitated smaller changes to morphology and function. We used X-ray reconstruction of moving morphology to compare the cranial kinematics of aquatic buccal pumping, such as that seen in suction feeding, with the aerial buccal pumping required for lung ventilation in the West African lungfish (Protopterus annectens). During buccal pumping behaviors, the cranial bones and associated soft tissues act as valves and pumps, and the sequence of their motions controls the pattern of fluid flow. Both behaviors are characterized by an anterior-to-posterior wave of expansion and an anterior-to-posterior wave of compression. We found that the pectoral girdle and cranial rib rotate consistently during air breathing and suction feeding, and that the muscle between them shortens during buccal expansion. Overall, we conclude that the major cranial bones maintain the same basic functions (i.e., acting as valves or pumps, or transmitting power) across aquatic and aerial buccal pumping. The cranial morphology that enables aquatic buccal pumping is well suited to perform air breathing and accommodates the physical differences between air and water.

Relaxed Feeding Constraints Facilitate the Evolution of Mouthbrooding in Neotropical Cichlids.

AbstractMultifunctionality is often framed as a core constraint of evolution, yet many evolutionary transitions involve traits taking on additional functions. Mouthbrooding, a form of parental care where offspring develop inside a parent's mouth, increases multifunctionality by adding a major function (reproduction) to a structure already serving other vital functions (feeding and respiration). Despite increasing multifunctionality, mouthbrooding has evolved repeatedly from other forms of parental care in at least seven fish families. We hypothesized that mouthbrooding is more likely to evolve in lineages with feeding adaptations that are already advantageous for mouthbrooding. We tested this hypothesis in Neotropical cichlids, where mouthbrooding has evolved four or five times, largely within winnowing clades, providing several pairwise comparisons between substrate-brooding and mouthbrooding sister taxa. We found that the mouthbrooding transition rate was 15 times higher in winnowing than in nonwinnowing clades and that mouthbrooders and winnowers overlapped substantially in their buccal cavity morphologies, which is where offspring are incubated. Species that exhibit one or both of these behaviors had larger, more curved buccal cavities, while species that exhibit neither behavior had narrow, cylindrical buccal cavities. Given the results we present here, we propose a new conceptual model for the evolution of mouthbrooding, integrating the roles of multifunctional morphology and the environment. We suggest that functional transitions like mouthbrooding offer a different perspective on multifunctionality: increasing constraints in one trait may release them for another, generating new evolutionary opportunities.

Royal knifefish generate powerful suction feeding through large neurocranial elevation and high epaxial muscle power.

Suction feeding in ray-finned fishes involves powerful buccal cavity expansion to accelerate water and food into the mouth. Previous XROMM studies in largemouth bass (Micropterus salmoides), bluegill sunfish (Lepomis macrochirus) and channel catfish (Ictalurus punctatus) have shown that more than 90% of suction power in high performance strikes comes from the axial musculature. Thus, the shape of the axial muscles and skeleton may affect suction feeding mechanics. Royal knifefish (Chitala blanci) have an unusual postcranial morphology, with a ventrally flexed vertebral column and relatively large mass of epaxial muscle. Based on their body shape, we hypothesized that royal knifefish would generate high power strikes by utilizing large neurocranial elevation, vertebral column extension and epaxial shortening. As predicted, C. blanci generated high suction expansion power compared with the other three species studied to date (up to 160 W), which was achieved by increasing both the rate of volume change and the intraoral subambient pressure. The large epaxial muscle (25% of body mass) shortened at high velocities to produce large neurocranial elevation and vertebral extension (up to 41 deg, combined), as well as high muscle mass-specific power (up to 800 W kg-1). For the highest power strikes, axial muscles generated 95% of the power, and 64% of the axial muscle mass consisted of the epaxial muscles. The epaxial-dominated suction expansion of royal knifefish supports our hypothesis that postcranial morphology may be a strong predictor of suction feeding biomechanics.

Modular lung ventilation in Boa constrictor.

The evolution of constriction and of large prey ingestion within snakes are key innovations that may explain the remarkable diversity, distribution and ecological scope of this clade, relative to other elongate vertebrates. However, these behaviors may have simultaneously hindered lung ventilation such that early snakes may have had to circumvent these mechanical constraints before those behaviors could evolve. Here, we demonstrate that Boa constrictor can modulate which specific segments of ribs are used to ventilate the lung in response to physically hindered body wall motions. We show that the modular actuation of specific segments of ribs likely results from active recruitment or quiescence of derived accessory musculature. We hypothesize that constriction and large prey ingestion were unlikely to have evolved without modular lung ventilation because of their interference with lung ventilation, high metabolic demands and reliance on sustained lung convection. This study provides a new perspective on snake evolution and suggests that modular lung ventilation evolved during or prior to constriction and large prey ingestion, facilitating snakes' remarkable radiation relative to other elongate vertebrates.

A new conceptual framework for the musculoskeletal biomechanics and physiology of ray-finned fishes.

Suction feeding in ray-finned fishes requires substantial muscle power for fast and forceful prey capture. The axial musculature located immediately behind the head has been long known to contribute some power for suction feeding, but recent XROMM and fluoromicrometry studies found nearly all the axial musculature (over 80%) provides effectively all (90-99%) of the power for high-performance suction feeding. The dominance of axial power suggests a new framework for studying the musculoskeletal biomechanics of fishes: the form and function of axial muscles and bones should be analysed for power production in feeding (or at least as a compromise between swimming and feeding), and cranial muscles and bones should be analysed for their role in transmitting axial power and coordinating buccal expansion. This new framework is already yielding novel insights, as demonstrated in four species for which suction power has now been measured. Interspecific comparisons suggest high suction power can be achieved in different ways: increasing the magnitude of suction pressure or the rate of buccal volume change, or both (as observed in the most powerful of these species). Our framework suggests that mechanical and evolutionary interactions between the head and the body, and between the swimming and feeding roles of axial structures, may be fruitful areas for continued study.

Motor control in the epaxial musculature of bluegill sunfish in feeding and locomotion.

Fishes possess an impressive repertoire of feeding and locomotor behaviors that in many cases rely on the same power source: the axial musculature. As both functions employ different skeletal systems, head versus body, integrating these functions would likely require modular motor control. Although there have been many studies of motor control in feeding or locomotion in fishes, only one study to date has examined both functions in the same individuals. To characterize bilateral motor control of the epaxial musculature in feeding and locomotion, we measured muscle activity and shortening in bluegill sunfish (Lepomis macrochirus) using electromyography and sonomicrometry. We found that sunfish recruit epaxial regions in a dorsal-to-ventral manner to increase feeding performance, such that high-performance feeding activates all the epaxial musculature. In comparison, sunfish seemed to activate all three epaxial regions irrespective of locomotor performance. Muscle activity was present on both sides of the body in nearly all feeding and locomotor behaviors. Feeding behaviors used similar activation intensities on the two sides of the body, whereas locomotor behaviors consistently used higher intensities on the side undergoing muscle shortening. In all epaxial regions, fast-starts used the highest activation intensities, although high-performance suction feeding occasionally showed near-maximal intensity. Finally, active muscle volume was positively correlated with the peak rate of body flexion in feeding and locomotion, indicating a continuous relationship between recruitment and performance. A comparison of these results with recent work on largemouth bass (Micropterus salmoides) suggests that centrarchid fishes use similar motor control strategies for feeding, but interspecific differences in peak suction-feeding performance are determined by active muscle volume.

Gastrocnemius Muscle Structural and Functional Changes Associated with Domestication in the Turkey.

Selection for increased muscle mass in domestic turkeys has resulted in muscles twice the size of those found in wild turkeys. This study characterizes muscle structural changes as well as functional differences in muscle performance associated with selection for increased muscle mass. We compared peak isometric force production, whole muscle and individual fiber cross-sectional area (CSA), connective tissue collagen concentration and structure of the lateral gastrocnemius (LG) muscle in wild and adult domestic turkeys. We also explored changes with age between juvenile and adult domestic turkeys. We found that the domestic turkey's LG muscle can produce the same force per cross-sectional area as a wild turkey; however, due to scaling, domestic adults produce less force per unit body mass. Domestic turkey muscle fibers were slightly smaller in CSA (3802 ± 2223 µm2) than those of the wild turkey (4014 ± 1831 µm2, p = 0.013), indicating that the absolutely larger domestic turkey muscles are a result of an increased number of smaller fibers. Collagen concentration in domestic turkey muscle (4.19 ± 1.58 µg hydroxyproline/mg muscle) was significantly lower than in the wild turkeys (6.23 ± 0.63 µg/mg, p = 0.0275), with visible differences in endomysium texture, observed via scanning electron microscopy. Selection for increased muscle mass has altered the structure of the LG muscle; however, scaling likely contributes more to hind limb functional differences observed in the domestic turkey.

A biomechanical paradox in fish: swimming and suction feeding produce orthogonal strain gradients in the axial musculature.

The axial musculature of fishes has historically been characterized as the powerhouse for explosive swimming behaviors. However, recent studies show that some fish also use their 'swimming' muscles to generate over 90% of the power for suction feeding. Can the axial musculature achieve high power output for these two mechanically distinct behaviors? Muscle power output is enhanced when all of the fibers within a muscle shorten at optimal velocity. Yet, axial locomotion produces a mediolateral gradient of muscle strain that should force some fibers to shorten too slowly and others too fast. This mechanical problem prompted research into the gearing of fish axial muscle and led to the discovery of helical fiber orientations that homogenize fiber velocities during swimming, but does such a strain gradient also exist and pose a problem for suction feeding? We measured muscle strain in bluegill sunfish, Lepomis macrochirus, and found that suction feeding produces a gradient of longitudinal strain that, unlike the mediolateral gradient for locomotion, occurs along the dorsoventral axis. A dorsoventral strain gradient within a muscle with fiber architecture shown to counteract a mediolateral gradient suggests that bluegill sunfish should not be able to generate high power outputs from the axial muscle during suction feeding-yet prior work shows that they do, up to 438 W kg-1. Solving this biomechanical paradox may be critical to understanding how many fishes have co-opted 'swimming' muscles into a suction feeding powerhouse.

Fishes can use axial muscles as anchors or motors for powerful suction feeding.

Some fishes rely on large regions of the dorsal (epaxial) and ventral (hypaxial) body muscles to power suction feeding. Epaxial and hypaxial muscles are known to act as motors, powering rapid mouth expansion by shortening to elevate the neurocranium and retract the pectoral girdle, respectively. However, some species, like catfishes, use little cranial elevation. Are these fishes instead using the epaxial muscles to forcefully anchor the head, and if so, are they limited to lower-power strikes? We used X-ray imaging to measure epaxial and hypaxial length dynamics (fluoromicrometry) and associated skeletal motions (XROMM) during 24 suction feeding strikes from three channel catfish (Ictalurus punctatus). We also estimated the power required for suction feeding from oral pressure and dynamic endocast volume measurements. Cranial elevation relative to the body was small (<5 deg) and the epaxial muscles did not shorten during peak expansion power. In contrast, the hypaxial muscles consistently shortened by 4-8% to rotate the pectoral girdle 6-11 deg relative to the body. Despite only the hypaxial muscles generating power, catfish strikes were similar in power to those of other species, such as largemouth bass (Micropterus salmoides), that use epaxial and hypaxial muscles to power mouth expansion. These results show that the epaxial muscles are not used as motors in catfish, but suggest they position and stabilize the cranium while the hypaxial muscles power mouth expansion ventrally. Thus, axial muscles can serve fundamentally different mechanical roles in generating and controlling cranial motion during suction feeding in fishes.

Locomotor rib kinematics in two species of lizards and a new hypothesis for the evolution of aspiration breathing in amniotes.

Most lizards walk and run with a sprawling gait in which the limbs are partly advanced by lateral undulation of the axial skeleton. Ribs and vertebrae are integral to this locomotor mode, but 3D motion of the axial skeleton has not been reported for lizard locomotion. Here, we use XROMM to quantify the relative motions of the vertebrae and ribs during slow treadmill locomotion in three savannah monitor lizards (Varanus exanthematicus) and three Argentine black and white tegus (Salvator merianae). To isolate locomotion, we selected strides with no concurrent lung ventilation. Rib rotations can be decomposed into bucket-handle rotation around a dorsoventral axis, pump-handle rotation around a mediolateral axis, and caliper rotations around a craniocaudal axis. During locomotion, every rib measured in both species rotated substantially around its costovertebral joint (8-17 degrees, summed across bucket, pump and caliper rotations). In all individuals from both species, the middle ribs rotated cranially through bucket and pump-handle motion during the propulsive phase of the ipsilateral forelimb. Axial kinematics during swing phase of the ipsilateral forelimb were mirror images of the propulsive phase. Although further work is needed to establish what causes these rib motions, active contraction of the hypaxial musculature may be at least partly responsible. Unilateral locomotor rib movements are remarkably similar to the bilateral pattern used for lung ventilation, suggesting a new hypothesis that rib motion during locomotion may have been an exaptation for the evolution of costal aspiration breathing in stem amniotes.

Dual function of epaxial musculature for swimming and suction feeding in largemouth bass.

The axial musculature of many fishes generates the power for both swimming and suction feeding. In the case of the epaxial musculature, unilateral activation bends the body laterally for swimming, and bilateral activation bends the body dorsally to elevate the neurocranium for suction feeding. But how does a single muscle group effectively power these two distinct behaviours? Prior electromyographic (EMG) studies have identified fishes' ability to activate dorsal and ventral epaxial regions independently, but no studies have directly compared the intensity and spatial activation patterns between swimming and feeding. We measured EMG activity throughout the epaxial musculature during swimming (turning, sprinting, and fast-starts) and suction feeding (goldfish and pellet strikes) in largemouth bass (Micropterus salmoides). We found that swimming involved obligate activation of ventral epaxial regions whereas suction feeding involved obligate activation of dorsal epaxial regions, suggesting regional specialization of the epaxial musculature. However, during fast-starts and suction feeding on live prey, bass routinely activated the whole epaxial musculature, demonstrating the dual function of this musculature in the highest performance behaviours. Activation intensities in suction feeding were substantially lower than fast-starts which, in conjunction with suboptimal shortening velocities, suggests that bass maximize axial muscle performance during locomotion and underuse it for suction feeding.

Intra-oropharyngeal food transport and swallowing in white-spotted bamboo sharks.

Despite the importance of intraoral food transport and swallowing, relatively few studies have examined the biomechanics of these behaviors in non-tetrapods, which lack a muscular tongue. Studies show that elasmobranch and teleost fishes generate water currents as a 'hydrodynamic tongue' that presumably transports food towards and into the esophagus. However, it remains largely unknown how specific musculoskeletal motions during transport correspond to food motion. Previous studies of white-spotted bamboo sharks (Chiloscyllium plagiosum) hypothesized that motions of the hyoid, branchial arches and pectoral girdle, generate caudal motion of the food through the long oropharynx of modern sharks. To test these hypotheses, we measured food and cartilage motion with XROMM during intra-oropharyngeal transport and swallowing (N=3 individuals, 2-3 trials per individual). After entering the mouth, food does not move smoothly toward the esophagus, but rather moves in distinct steps with relatively little retrograde motion. Caudal food motion coincides with hyoid elevation and a closed mouth, supporting earlier studies showing that hyoid motion contributes to intra-oropharyngeal food transport by creating caudally directed water currents. Little correspondence between pectoral girdle and food motion was found, indicating minimal contribution of pectoral girdle motion. Transport speed was fast as food entered the mouth, slower and step-wise through the pharyngeal region and then fast again as it entered the esophagus. The food's static periods in the step-wise motion and its high velocity during swallowing could not be explained by hyoid or girdle motion, suggesting these sharks may also use the branchial arches for intra-oropharyngeal transport and swallowing.

Pectoral and pelvic girdle rotations during walking and swimming in a semi-aquatic turtle: testing functional role and constraint.

Pectoral and pelvic girdle rotations play a substantial role in enhancing stride length across diverse tetrapod lineages. However, the pectoral and pelvic girdle attach the limbs to the body in different ways and may exhibit dissimilar functions, especially during locomotion in disparate environments. Here, we tested for functional differences between the forelimb and hindlimb of the freshwater turtle Pseudemys concinna during walking and swimming using X-ray reconstruction of moving morphology (XROMM). In doing so, we also tested the commonly held notion that the shell constrains girdle motion in turtles. We found that the pectoral girdle exhibited greater rotations than the pelvic girdle on land and in water. Additionally, pelvic girdle rotations were greater on land than in water, whereas pectoral girdle rotations were similar in the two environments. These results indicate that although the magnitude of pelvic girdle rotations depends primarily on whether the weight of the body must be supported against gravity, the magnitude of pectoral girdle rotations likely depends primarily on muscular activity associated with locomotion. Furthermore, the pectoral girdle of turtles rotated more than has been observed in other taxa with sprawling postures, showing an excursion similar to that of mammals (∼38 deg). These results suggest that a rigid axial skeleton and internally positioned pectoral girdle have not constrained turtle girdle function, but rather the lack of lateral undulations in turtles and mammals may contribute to a functional convergence whereby the girdle acts as an additional limb segment to increase stride length.

XROMM kinematics of ventilation in wild turkeys (Meleagris gallopavo).

The avian ribcage is derived relative to other amniotes, and is hypothesised to be constrained in its movements during ventilation. The double-headed ribs form two articulations with the vertebrae, and are thought to rotate about a strict anatomical axis. However, this costovertebral joint constraint has not been demonstrated empirically and was not found in other taxa with double-headed ribs (i.e. crocodilians). Here, we used X-ray reconstruction of moving morphology (XROMM) to quantify rib rotation in wild turkeys (Meleagris gallopavo) during breathing. We demonstrate that, as predicted from anatomy, the ribs do rotate in a hinge-like manner about a single axis. There is also evidence for elliptical motion of the sternum, as has been reported in other taxa. The evolution of the avian ribcage is closely related to the co-evolution of ventilation and flight, and these results are important for how we model ventilation mechanics in living and fossil birds.

The Multi-Scale, Three-Dimensional Nature of Skeletal Muscle Contraction.

Muscle contraction is a three-dimensional process, as anyone who has observed a bulging muscle knows. Recent studies suggest that the three-dimensional nature of muscle contraction influences its mechanical output. Shape changes and radial forces appear to be important across scales of organization. Muscle architectural gearing is an emerging example of this process.

Shearing overbite and asymmetrical jaw motions facilitate food breakdown in a freshwater stingray, Potamotrygon motoro.

Many species of fish process their prey with cyclic jaw motions that grossly resemble those seen in mammalian mastication, despite starkly different tooth and jaw morphologies. The degree of similarity between the processing behaviors of these disparate taxa has implications for our understanding of convergence in vertebrate feeding systems. Here, we used XROMM (X-ray reconstruction of moving morphology) to investigate prey processing behavior of Potamotrygon motoro, the ocellate river stingray, which has recently been found to employ asymmetrical, shearing jaw motions to break down its prey. We found that P. motoro modulates its feeding kinematics to produce two distinct types of chew cycles: compressive cycles and overbite cycles. The latter are characterized by over-rotation of the upper jaw relative to the lower jaw, past the expected occlusal limit, and higher levels of bilateral asymmetry as compared with compressive chews. We did not find evidence of the mediolateral shearing motions typical of mammalian mastication, but overbite cycles appear to shear the prey item between the upper and lower toothplates in a propalinal fashion. Additionally, comparison of hyomandibular and jaw motions demonstrates that the angular cartilages decouple jaw displacement from hyomandibular displacement in rostrocaudal and mediolateral directions. The multiple similarities between mammalian mastication and the dynamic processing behavior of P. motoro support the use of sub-family Potamotrygoninae as a model for studying evolutionary convergence of mastication-like processing.