(Very) long-term transport of Silurus glanis, Carcharhinus melanopterus, Scomber colias, Trachurus picturatus, Polyprion americanus, Rhinoptera marmoratus, Salmo salar, Scomber scombrus, Sardina pilchardus, and others, by land, water and air.

In this paper, we cover 4 years of live fish transports that ranged from 14 to 200 h (8 days), and bioloads from 3.8 to 76.9 kg/m3 . The key ingredients for success in all trips, where virtually no mortality occurred, was atributed to (1) pre-buffering the water with sodium bicarbonate and sodium carbonate at 50 g/m3 (each)-and/or ATM Alka-HaulTM at 25 g/m3 -and applying additional (partial or full) doses throughout each transport, whenever the tanks were accessible; (2) pre-quenching ammonia with ATM TriageTM at 32 g/m3 , and applying additional (partial or full) doses throughout each transport, whenever the tanks were accessible; (3) keeping the dissolved oxygen saturation rate above 100%, ideally above 150%; (4) Keeping temperature on the lower limit of each species' tolerance range; (5) Using foam fractionators to effectively eliminate organic matter from the water and (6) Using pure sine wave inverters, which allows for a steady supply of electrical current throughout the transport. The use of a 'preventive' versus 'corrective' pH buffering philosophy is also discussed.

GABAergic modulation of olfactomotor transmission in lampreys.

Odor-guided behaviors, including homing, predator avoidance, or food and mate searching, are ubiquitous in animals. It is only recently that the neural substrate underlying olfactomotor behaviors in vertebrates was uncovered in lampreys. It consists of a neural pathway extending from the medial part of the olfactory bulb (medOB) to locomotor control centers in the brainstem via a single relay in the caudal diencephalon. This hardwired olfactomotor pathway is present throughout life and may be responsible for the olfactory-induced motor behaviors seen at all life stages. We investigated modulatory mechanisms acting on this pathway by conducting anatomical (tract tracing and immunohistochemistry) and physiological (intracellular recordings and calcium imaging) experiments on lamprey brain preparations. We show that the GABAergic circuitry of the olfactory bulb (OB) acts as a gatekeeper of this hardwired sensorimotor pathway. We also demonstrate the presence of a novel olfactomotor pathway that originates in the non-medOB and consists of a projection to the lateral pallium (LPal) that, in turn, projects to the caudal diencephalon and to the mesencephalic locomotor region (MLR). Our results indicate that olfactory inputs can induce behavioral responses by activating brain locomotor centers via two distinct pathways that are strongly modulated by GABA in the OB. The existence of segregated olfactory subsystems in lampreys suggests that the organization of the olfactory system in functional clusters may be a common ancestral trait of vertebrates.

Comparative morphology of snake (Squamata) endocasts: evidence of phylogenetic and ecological signals.

Brain endocasts obtained from computed tomography (CT) are now widely used in the field of comparative neuroanatomy. They provide an overview of the morphology of the brain and associated tissues located in the cranial cavity. Through anatomical comparisons between species, insights on the senses, the behavior, and the lifestyle can be gained. Although there are many studies dealing with mammal and bird endocasts, those performed on the brain endocasts of squamates are comparatively rare, thus limiting our understanding of their morphological variability and interpretations. Here, we provide the first comparative study of snake brain endocasts in order to bring new information about the morphology of these structures. Additionally, we test if the snake brain endocast encompasses a phylogenetic and/or an ecological signal. For this purpose, the digital endocasts of 45 snake specimens, including a wide diversity in terms of phylogeny and ecology, were digitized using CT, and compared both qualitatively and quantitatively. Snake endocasts exhibit a great variability. The different methods performed from descriptive characters, linear measurements and the outline curves provided complementary information. All these methods have shown that the shape of the snake brain endocast contains, as in mammals and birds, a phylogenetic signal but also an ecological one. Although phylogenetically related taxa share several similarities between each other, the brain endocast morphology reflects some notable ecological trends: e.g. (i) fossorial species possess both reduced optic tectum and pituitary gland; (ii) both fossorial and marine species have cerebral hemispheres poorly developed laterally; (iii) cerebral hemispheres and optic tectum are more developed in arboreal and terrestrial species.

Odorant organization in the olfactory bulb of the sea lamprey.

Olfactory sensory neurons innervate the olfactory bulb, where responses to different odorants generate a chemotopic map of increased neural activity within different bulbar regions. In this study, insight into the basal pattern of neural organization of the vertebrate olfactory bulb was gained by investigating the lamprey. Retrograde labelling established that lateral and dorsal bulbar territories receive the axons of sensory neurons broadly distributed in the main olfactory epithelium and that the medial region receives sensory neuron input only from neurons projecting from the accessory olfactory organ. The response duration for local field potential recordings was similar in the lateral and dorsal regions, and both were longer than medial responses. All three regions responded to amino acid odorants. The dorsal and medial regions, but not the lateral region, responded to steroids. These findings show evidence for olfactory streams in the sea lamprey olfactory bulb: the lateral region responds to amino acids from sensory input in the main olfactory epithelium, the dorsal region responds to steroids (taurocholic acid and pheromones) and to amino acids from sensory input in the main olfactory epithelium, and the medial bulbar region responds to amino acids and steroids stimulating the accessory olfactory organ. These findings indicate that olfactory subsystems are present at the base of vertebrate evolution and that regionality in the lamprey olfactory bulb has some aspects previously seen in other vertebrate species.

Endocast, brain, and bones: Correspondences and spatial relationships in squamates.

Vertebrate endocasts are widely used in the fields of paleoneurology and comparative neuroanatomy. The validity of endocranial studies is dependent upon the extent to which an endocast reflects brain morphology. Due to the variable neuroanatomical resolution of vertebrate endocasts, direct information about the brain morphology can be sometimes difficult to assess and needs to be investigated across lineages. Here, we employ X-ray computed tomography (CT), including diffusible iodine-based contrast-enhanced CT, to qualitatively compare brains and endocasts in different species of squamates. The relative position of the squamate brain within the skull, as well as the variability that may exist in such spatial relationships, was examined to help clarify the neurological regions evidence on their endocasts. Our results indicate that squamate endocasts provide variable representation of the brain, depending on species and neuroanatomical regions. The olfactory bulbs and peduncles, cerebral hemispheres, as well as the medulla oblongata represent the most easily discernable brain regions from squamate endocasts. In contrast, the position of the optic lobes, the ventral diencephalon and the pituitary may be difficult to determine depending on species. Finally, squamate endocasts provide very limited or no information about the cerebellum. The spatial relationships revealed here between the brain and the surrounding bones may help to identify each of the endocranial region. However, as one-to-one correspondences between a bone and a specific region appear limited, the exact delimitation of these regions may remain challenging according to species. This study provides a basis for further examination and interpretation of squamate endocast disparity.

A landmarking protocol for geometric morphometric analysis of squamate endocasts.

Landmark-based geometric morphometrics is widely used to study the morphology of the endocast, or internal mold of the braincase, and the diversity associated with this structure across vertebrates. Landmarks, as the basic unit of such methods, are intended to be points of correspondence, selected depending on the question at hand, whose proper definition is essential to guarantee robustness and reproducibility of results. In this study, 20 landmarks are defined to provide a framework to analyze the morphological variability in squamate endocasts. Ten species representing a cross-section of the diversity of Squamata from both phylogenetic and ecological (i.e., habitat) perspectives were considered, to select landmarks replicable throughout the entire clade, regardless of the degree of neuroanatomical resolution of the endocast. To assess the precision, accuracy, and repeatability of these newly defined landmarks, both intraobserver and interobserver error were investigated. Estimates of measurement error show that most of the landmarks established here are highly replicable, and preliminary results suggest that they capture aspects of endocast shape related to both phylogenetic and ecologic signals. This study provides a basis for further examinations of squamate endocast disparity using landmark-based geometric morphometrics.

The function of oscillatory tongue-flicks in snakes: insights from kinematics of tongue-flicking in the banded water snake (Nerodia fasciata).

Tongue-flicking is an important sensory behavior unique to squamate reptiles in which chemical stimuli gathered by the tongue are delivered the vomeronasal organ situated in the roof of the mouth. Because tongue-flick numbers can easily be quantified, this behavior has been widely used as a measure of vomeronasal sampling in snakes using related variables such as tongue-flick rate or tongue-flick/attack score. Surprisingly, the behavior itself and especially the function of the oscillatory tongue-flicks remains poorly understood. To describe the overall kinematics of tongue-flicking in the colubrid snake Nerodia fasciata and to test predictions on the function of oscillatory tongue-flicks, we filmed the tongue-flicks of 8 adult Nerodia fasciata using 4 synchronized high-speed cameras. Three-dimensional kinematic and performance variables were extracted from the videos in order to quantify tongue movements. Based on the kinematic analysis, we demonstrate the existence of 2 functional and behavioral tongue-flick categories. Tongue-flicks with oscillations meet all the criteria for being adapted to the collection of odorants; simple downward extensions appear better suited for the rapid pick up of nonvolatile chemical stimuli from the substrate or a food item. External stimuli such as tactile and/or vomeronasal stimulation can induce a shift between these categories.

Assisted walking in Malagasy dwarf chamaeleons.

Chamaeleons are well known for their unique suite of morphological adaptations. Whereas most chamaeleons are arboreal and have long tails, which are used during arboreal acrobatic manoeuvres, Malagasy dwarf chamaeleons (Brookesia) are small terrestrial lizards with relatively short tails. Like other chamaeleons, Brookesia have grasping feet and use these to hold on to narrow substrates. However, in contrast to other chamaeleons, Brookesia place the tail on the substrate when walking on broad substrates, thus improving stability. Using three-dimensional synchrotron X-ray phase-contrast imaging, we demonstrate a set of unique specializations in the tail associated with the use of the tail during locomotion. Additionally, our imaging demonstrates specializations of the inner ear that may allow these animals to detect small accelerations typical of their slow, terrestrial mode of locomotion. These data suggest that the evolution of a terrestrial lifestyle in Brookesia has gone hand-in-hand with the evolution of a unique mode of locomotion and a suite of morphological adaptations allowing for stable locomotion on a wide array of substrates.

Sensory cutaneous papillae in the sea lamprey (Petromyzon marinus L.): II. Ontogeny and immunocytochemical characterization of solitary chemosensory cells.

Solitary chemosensory cells (SCCs) and their innervating fibers are located in the respiratory system of many vertebrates, including papillae on lamprey gill pores. In order to gain stronger insight for the role of these chemosensory cells, we examined immunocytochemical and innervation characteristics, as well as abundance at the different stages of the lamprey life cycle. The SCCs were distinguished from the surrounding epithelial cells by calretinin and phospholipase C140 immunoreactivity. Nerve fibers extended into the gill pore papillae, as far as the SCCs and serotonergic fibers extended from the underlying dermis into the papillar base. Gill pore papillae were absent and SCCs were sparse during the larval stage and in newly transformed lamprey. Few SCCs were located on small nub-like papillae during the parasitic juvenile stage, but SCCs were abundant on prominent papillae in migrating and in spawning adults. These findings show similarities between the SCCs in lampreys and other vertebrates and suggest that gill SCC function may be important during the feeding juvenile and the adult stages of the lamprey life cycle.

Dopaminergic modulation of olfactory-evoked motor output in sea lampreys (Petromyzon marinus L.).

Detection of chemical cues is important to guide locomotion in association with feeding and sexual behavior. Two neural pathways responsible for odor-evoked locomotion have been characterized in the sea lamprey (Petromyzon marinus L.), a basal vertebrate. There is a medial pathway originating in the medial olfactory bulb (OB) and a lateral pathway originating from the rest of the OB. These olfactomotor pathways are present throughout the life cycle of lampreys, but olfactory-driven behaviors differ according to the developmental stage. Among possible mechanisms, dopaminergic (DA) modulation in the OB might explain the behavioral changes. Here, we examined DA modulation of olfactory transmission in lampreys. Immunofluorescence against DA revealed immunoreactivity in the OB that was denser in the medial part (medOB), where processes were observed close to primary olfactory afferents and projection neurons. Dopaminergic neurons labeled by tracer injections in the medOB were located in the OB, the posterior tuberculum, and the dorsal hypothalamic nucleus, suggesting the presence of both intrinsic and extrinsic DA innervation. Electrical stimulation of the olfactory nerve in an in vitro whole-brain preparation elicited synaptic responses in reticulospinal cells that were modulated by DA. Local injection of DA agonists in the medOB decreased the reticulospinal cell responses whereas the D2 receptor antagonist raclopride increased the response amplitude. These observations suggest that DA in the medOB could modulate odor-evoked locomotion. Altogether, these results show the presence of a DA innervation within the medOB that may play a role in modulating olfactory inputs to the motor command system of lampreys.

Sensory cutaneous papillae in the sea lamprey (Petromyzon marinus L.): I. Neuroanatomy and physiology.

Molecules present in an animal's environment can indicate the presence of predators, food, or sexual partners and consequently, induce migratory, reproductive, foraging, or escape behaviors. Three sensory systems, the olfactory, gustatory, and solitary chemosensory cell (SCC) systems detect chemical stimuli in vertebrates. While a great deal of research has focused on the olfactory and gustatory system over the years, it is only recently that significant attention has been devoted to the SCC system. The SCCs are microvillous cells that were first discovered on the skin of fish, and later in amphibians, reptiles, and mammals. Lampreys also possess SCCs that are particularly numerous on cutaneous papillae. However, little is known regarding their precise distribution, innervation, and function. Here, we show that sea lampreys (Petromyzon marinus L.) have cutaneous papillae located around the oral disk, nostril, gill pores, and on the dorsal fins and that SCCs are particularly numerous on these papillae. Tract-tracing experiments demonstrated that the oral and nasal papillae are innervated by the trigeminal nerve, the gill pore papillae are innervated by branchial nerves, and the dorsal fin papillae are innervated by spinal nerves. We also characterized the response profile of gill pore papillae to some chemicals and showed that trout-derived chemicals, amino acids, and a bile acid produced potent responses. Together with a companion study (Suntres et al., Journal of Comparative Neurology, this issue), our results provide new insights on the function and evolution of the SCC system in vertebrates.

Sensory Activation of Command Cells for Locomotion and Modulatory Mechanisms: Lessons from Lampreys.

Sensorimotor transformation is one of the most fundamental and ubiquitous functions of the central nervous system (CNS). Although the general organization of the locomotor neural circuitry is relatively well understood, less is known about its activation by sensory inputs and its modulation. Utilizing the lamprey model, a detailed understanding of sensorimotor integration in vertebrates is emerging. In this article, we explore how the vertebrate CNS integrates sensory signals to generate motor behavior by examining the pathways and neural mechanisms involved in the transformation of cutaneous and olfactory inputs into motor output in the lamprey. We then review how 5-hydroxytryptamine (5-HT) acts on these systems by modulating both sensory inputs and motor output. A comprehensive review of this fundamental topic should provide a useful framework in the fields of motor control, sensorimotor integration and neuromodulation.

Chemosensory-induced motor behaviors in fish.

Chemical sensory signals play a crucial role in eliciting motor behaviors. We now review the different motor behaviors induced by chemosensory stimuli in fish as well as their neural substrate. A great deal of research has focused on migratory, reproductive, foraging, and escape behaviors but it is only recently that the molecules mediating these chemotactic responses have become well-characterized. Chemotactic responses are mediated by three sensory systems: olfactory, gustatory, and diffuse chemosensory. The olfactory sensory neuron responses to chemicals are now better understood. In addition, the olfactory projections to the central nervous system were recently shown to display an odotopic organization in the forebrain. Moreover, a specific downward projection underlying motor responses to olfactory inputs was recently described.

Chemical basis of prey recognition in thamnophiine snakes: the unexpected new roles of parvalbumins.

Detecting and locating prey are key to predatory success within trophic chains. Predators use various signals through specialized visual, olfactory, auditory or tactile sensory systems to pinpoint their prey. Snakes chemically sense their prey through a highly developed auxiliary olfactory sense organ, the vomeronasal organ (VNO). In natricine snakes that are able to feed on land and water, the VNO plays a critical role in predatory behavior by detecting cues, known as vomodors, which are produced by their potential prey. However, the chemical nature of these cues remains unclear. Recently, we demonstrated that specific proteins-parvalbumins-present in the cutaneous mucus of the common frog (Rana temporaria) may be natural chemoattractive proteins for these snakes. Here, we show that parvalbumins and parvalbumin-like proteins, which are mainly intracellular, are physiologically present in the epidermal mucous cells and mucus of several frog and fish genera from both fresh and salt water. These proteins are located in many tissues and function as Ca(2+) buffers. In addition, we clarified the intrinsic role of parvalbumins present in the cutaneous mucus of amphibians and fishes. We demonstrate that these Ca(2+)-binding proteins participate in innate bacterial defense mechanisms by means of calcium chelation. We show that these parvalbumins are chemoattractive for three different thamnophiine snakes, suggesting that these chemicals play a key role in their prey-recognition mechanism. Therefore, we suggest that recognition of parvalbumin-like proteins or other calcium-binding proteins by the VNO could be a generalized prey-recognition process in snakes. Detecting innate prey defense mechanism compounds may have driven the evolution of this predator-prey interaction.

Shake rattle and roll: the bony labyrinth and aerial descent in squamates.

Controlled aerial descent has evolved many times independently in vertebrates. Squamates (lizards and snakes) are unusual in that respect due to the large number of independent origins of the evolution of this behavior. Although some squamates such as flying geckos of the genus Ptychozoon and the flying dragons of the genus Draco show obvious adaptations including skin flaps or enlarged ribs allowing them to increase their surface area and slow down their descent, many others appear unspecialized. Yet, specializations can be expected at the level of the sensory and neural systems allowing animals to maintain stability during controlled aerial descent. The vestibular system is a likely candidate given that it is an acceleration detector and is well-suited to detect changes in pitch, roll and yaw. Here we use conventional and synchrotron µCT scans to quantify the morphology of the vestibular system in squamates able to perform controlled aerial descent compared to species characterized by a terrestrial or climbing life style. Our results show the presence of a strong phylogenetic signal in the data with the vestibular system in species from the same family being morphologically similar. However, both our shape analysis and an analysis of the dimensions of the vestibular system showed clear differences among animals with different life-styles. Species able to perform a controlled aerial descent differed in the position and shape of the inner ear, especially of the posterior ampulla. Given the limited stability of squamates against roll and the fact that the posterior ampulla is tuned to changes in roll this suggests an adaptive evolution of the vestibular system in squamates using controlled aerial descent. Future studies testing for similar differences in other groups of vertebrates known to use controlled aerial descent are needed to test the generality of this observation.

Effect of locomotor approach on feeding kinematics in the green anole (Anolis carolinensis).

Squamates are well-known models for studying to examine locomotor and feeding behaviors in tetrapods, but studies that integrate both behavioral activities remain scarce. Anolis lizards are a classical lineage to study the evolutionary relationships between locomotor behavior and complex structural features of the habitat. Here, we analyzed prey-capture behavior in one representative arboreal predator, Anolis carolinensis, to demonstrate the functional links between locomotor strategies and the kinematics of feeding. A. carolinensis uses two strategies to catch living insects on perches: Head-Up Capture and Jump Capture. In both cases, lizards use lingual prehension to capture the prey and the kinematic patterns of the trophic apparatus are not significantly influenced by the selected strategies. Therefore, to capture one prey type, movements of the trophic structures are highly fixed and A. carolinensis modulates the locomotor pattern to exploit the environment. Predation behavior in A. carolinensis integrates two different behavioral patterns: locomotor plasticity of prey-approach and biomechanical stereotypy of tongue prehension to successfully capture the prey.