Expression of 20 SCPP genes during tooth and bone mineralization in Senegal bichir.

The African bichir (Polypterus senegalus) is a living representative of Polypteriformes. P. senegalus possesses teeth composed of dentin covered by an enameloid cap and a layer of collar enamel on the tooth shaft, as in lepisosteids. A thin layer of enamel matrix can also be found covering the cap enameloid after its maturation and during the collar enamel formation. Teleosts fish do not possess enamel; teeth are protected by cap and collar enameloid, and inversely in sarcopterygians, where teeth are only covered by enamel, with the exception of the cap enameloid in teeth of larval urodeles. The presence of enameloid and enamel in the teeth of the same organism is an opportunity to solve the evolutionary history of the presence of enamel/enameloid in basal actinopterygians. In silico analyses of the jaw transcriptome of a juvenile bichir provided twenty SCPP transcripts. They included enamel, dentin, and bone-specific SCPPs known in sarcopterygians and several actinopterygian-specific SCPPs. The expression of these 20 genes was investigated by in situ hybridizations on jaw sections during tooth and dentary bone formation. A spatiotemporal expression patterns were established and compared with previous studies of SCPP gene expression during enamel/enameloid and bone formation. Similarities and differences were highlighted, and several SCPP transcripts were found specifically expressed during tooth or bone formation suggesting either conserved or new functions of these SCPPs.

Ancient fish lineages illuminate toll-like receptor diversification in early vertebrate evolution.

Since its initial discovery over 50 years ago, understanding the evolution of the vertebrate RAG- mediated adaptive immune response has been a major area of research focus for comparative geneticists. However, how the evolutionary novelty of an adaptive immune response impacted the diversity of receptors associated with the innate immune response has received considerably less attention until recently. Here, we investigate the diversification of vertebrate toll-like receptors (TLRs), one of the most ancient and well conserved innate immune receptor families found across the Tree of Life, integrating genomic data that represent all major vertebrate lineages with new transcriptomic data from Polypteriformes, the earliest diverging ray-finned fish lineage. Our analyses reveal TLR sequences that reflect the 6 major TLR subfamilies, TLR1, TLR3, TLR4, TLR5, TLR7, and TLR11, and also currently unnamed, yet phylogenetically distinct TLR clades. We additionally recover evidence for a pulse of gene gain coincident with the rise of the RAG-mediated adaptive immune response in jawed vertebrates, followed by a period of rapid gene loss during the Cretaceous. These gene losses are primarily concentrated in marine teleost fish and synchronous with the mid Cretaceous anoxic event, a period of rapid extinction for marine species. Finally, we reveal a mismatch between phylogenetic placement and gene nomenclature for up to 50% of TLRs found in clades such as ray-finned fishes, cyclostomes, amphibians, and elasmobranchs. Collectively, these results provide an unparalleled perspective of TLR diversity and offer a ready framework for testing gene annotations in non-model species.

Sensorimotor control of swimming Polypterus senegalus is preserved during sensory deprivation conditions across altered environments.

Control of locomotion involves the interplay of sensory signals and motor commands. Sensory information is essential for adjusting locomotion in response to environmental changes. A previous study using mathematical modelling of lamprey swimming has shown that, in the absence of sensory feedback, increasing fluid viscosity constrains swimming kinematics, limiting tail amplitude and body wavelength, resulting in decreased swimming speed. In contrast, previous experiments with Polypterus senegalus reported increased magnitude swimming kinematics (increased body curvature, body wave speed and frequency, and pectoral fin frequency) in high viscosity water suggesting that sensory information is used to adjust swimming form. It is not known what sensory systems are providing the necessary information to respond to these environmental changes. We tested the hypothesis that lateral line and visual input are responsible for the sensory-driven increase in swimming kinematics in response to experimentally increased fluid viscosity. The kinematics of five P. senegalus were recorded in two different viscosities of water while removing lateral line and visual sensory feedback. Unlike the mathematical model devoid of sensory feedback, P. senegalus with lateral line and/or visual senses removed did not reduce the magnitude of swimming kinematic variables, suggesting that additional sensory feedback mechanisms are present in these fish to help overcome increased fluid viscosity. Increases in swimming speed when both lateral line and visual sensory feedback were removed suggest that lateral line and visual information may be used to regulate swimming speed in P. senegalus, possibly using an internal model of predictions to adjust swimming form.

Behaviour and muscle activity across the aquatic-terrestrial transition in Polypterus senegalus.

Amphibious fishes moving from water to land experience continuous changes in environmental forces. How these subtle changes impact behavioural transitions cannot be resolved by comparisons of aquatic and terrestrial locomotion. For example, aquatic and terrestrial locomotion appear distinct in the actinopterygian fish Polypterus senegalus; however, it is unclear how gradual water level changes influence the transition between these locomotor behaviours. We tested the hypothesis in P. senegalus that swimming and walking are part of an incremental continuum of behaviour and muscle activity across the environmental transition from water to land rather than two discrete behaviours, as proposed by previous literature. We exposed P. senegalus to discrete environments from fully aquatic to fully terrestrial while recording body and pectoral fin kinematics and muscle activity. Anterior axial red muscle effort increases as water depth decreases; however, a typical swimming-like anterior-to-posterior wave of axial red muscle activity is always present, even during terrestrial locomotion, indicating gradual motor control changes. Thus, walking appears to be based on swimming-like axial muscle activity whereas kinematic differences between swimming and walking appear to be due to mechanical constraints. A discrete change in left-right pectoral fin coordination from in-phase to out-of-phase at 0.7 body depths relies on adductor muscle activity with a similar duty factor and adductor muscle effort that increases gradually as water depth decreases. Thus, despite distinct changes in kinematic timing, neuromuscular patterning is similar across the water depth continuum. As the observed, gradual increases in axial muscle effort reflect muscle activity changes between aquatic and terrestrial environments observed in other elongate fishes, a modified, swimming-like axial muscle activity pattern for terrestrial locomotion may be common among elongate amphibious fishes.

von Willebrand factor D and EGF domains is an evolutionarily conserved and required feature of blastemas capable of multitissue appendage regeneration.

Regenerative ability varies tremendously across species. A common feature of regeneration of appendages such as limbs, fins, antlers, and tails is the formation of a blastema-a transient structure that houses a pool of progenitor cells that can regenerate the missing tissue. We have identified the expression of von Willebrand factor D and EGF domains (vwde) as a common feature of blastemas capable of regenerating limbs and fins in a variety of highly regenerative species, including axolotl (Ambystoma mexicanum), lungfish (Lepidosiren paradoxa), and Polpyterus (Polypterus senegalus). Further, vwde expression is tightly linked to the ability to regenerate appendages in Xenopus laevis. Functional experiments demonstrate a requirement for vwde in regeneration and indicate that Vwde is a potent growth factor in the blastema. These data identify a key role for vwde in regenerating blastemas and underscore the power of an evolutionarily informed approach for identifying conserved genetic components of regeneration.

Comparative iTRAQ proteomics revealed proteins associated with lobed fin regeneration in Bichirs.

BACKGROUND: Polypterus senegalus can fully regenerate its pectoral lobed fins, including a complex endoskeleton, with remarkable precision. However, despite the enormous potential of this species for use in medical research, its regeneration mechanisms remain largely unknown. METHODS: To identify the differentially expressed proteins (DEPs) during the early stages of lobed fin regeneration in P. senegalus, we performed a differential proteomic analysis using isobaric tag for relative and absolute quantitation (iTRAQ) approach based quantitative proteome from the pectoral lobed fins at 3 time points. Furthermore, we validated the changes in protein expression with multiple-reaction monitoring (MRM) analysis. RESULTS: The experiment yielded a total of 3177 proteins and 15,091 unique peptides including 1006 non-redundant (nr) DEPs. Of these, 592 were upregulated while 349 were downregulated after lobed fin amputation when compared to the original tissue. Bioinformatics analyses showed that the DEPs were mainly associated with Ribosome and RNA transport, metabolic, ECM-receptor interaction, Golgi and endoplasmic reticulum, DNA replication, and Regulation of actin cytoskeleton. CONCLUSIONS: To our knowledge, this is the first proteomic research to investigate alterations in protein levels and affected pathways in bichirs' lobe-fin/limb regeneration. In addition, our study demonstrated a highly dynamic regulation during lobed fin regeneration in P. senegalus. These results not only provide a comprehensive dataset on differentially expressed proteins during the early stages of lobe-fin/limb regeneration but also advance our understanding of the molecular mechanisms underlying lobe-fin/limb regeneration.

Polypterus and the evolution of fish pectoral musculature.

Polypterus, a member of the most primitive living group of ray-finned fishes, has demonstrated the ability to perform fin-assisted terrestrial locomotion, a behavior that indicates a complex pectoral musculoskeletal system. Review of the literature reveals that many aspects of the pectoral muscular anatomy of Polypterus are still unclear, with a number of conflicting descriptions. We provide a new interpretation of the pectoral musculature using soft tissue-enhanced microCT scanning and gross anatomical dissection. The results demonstrate a complex musculature, with six independent muscles crossing the glenoid-fin joint. Comparisons with other bony-fish (Osteichthyes), including both ray-finned (Actinopterygii) and lobed-fin (Sarcopterygii) fish, indicate the presence of novel muscles within Polypterus: coracometapterygialis I+II and the zonopropterygialis medialis. Examination of these muscular additions in the context of osteichthyan phylogeny indicates that this represents a previously unrecognized event in the evolution of pectoral musculature in Osteichthyes. Despite its phylogenetic position as a basal actinopterygian, the musculature of Polypterus has more similarities both anatomically and functionally with that of sarcopterygians. This anatomy, along with other features of Polypterus anatomy such as lobed fins, ventral paired lungs, and a large spiracle, may make it a good model for inferences of stem tetrapod locomotion.

Plastic loss of motile cilia in the gills of Polypterus in response to high CO2 or terrestrial environments.

The evolutionary transition of vertebrates from water to land during the Devonian period was accompanied by major changes in animal respiratory systems in terms of physiology and morphology. Indeed, the fossil record of the early tetrapods has revealed the existence of internal gills, which are vestigial fish-like traits used underwater. However, the fossil record provides only limited data on the process of the evolutionary transition of gills from fish to early tetrapods. This study investigated the gills of Polypterus senegalus, a basal ray-finned/amphibious fish which shows many ancestral features of stem Osteichthyes. Based on scanning electron microscopy observations and transcriptome analysis, the existence of motile cilia in the gills was revealed which may create a flow on the gill surface leading to efficient ventilation or remove particles from the surface. Interestingly, these cilia were observed to disappear after rearing in terrestrial or high CO2 environments, which mimics the environmental changes in the Devonian period. The cilia re-appeared after being returned to the original aquatic environment. The ability of plastic changes of gills in Polypterus revealed in this study may allow them to survive in fluctuating environments, such as shallow swamps. The ancestor of Osteichthyes is expected to have possessed such plasticity in the gills, which may be one of the driving forces behind the transition of vertebrates from water to land.

New Data on Nephron Microanatomy and Ultrastructure of Senegal Bichir (Polypterus senegalus).

This study presents new data on the microanatomy and ultrastructure of Polypterus senegalus nephrons. It was shown that the diameter and ultrastructure of renal corpuscles, a well-developed neck segment, and ultrastructure of two types of epithelial cells of the proximal tubule are ancestral signs of the modern population of P. senegalus associated with habitat conditions in the aquatic environment. The outer diameter of the tubules, the height of the epitheliocytes, the presence of two types of epithelial cells of the intermediate and distal tubules of the corresponding ultrastructure, and a large area of nephrogenic tissue are progressive features of the modern population of P. senegalus, associated with adaptation to air respiration and periodic terrestrial migrations, which were formed at the early stages of evolution of P. senegalus convergently with lungfish and amphibians.

Effect of cooking and smoking methods on proximate composition, lipid oxidation and mineral contents of Polypterus bichir bichir fish from far-north region of Cameroon.

The objective of this study was to evaluate the effect of different cooking and smoking treatments on proximate composition, lipid quality and mineral content of Polypterus bichir bichir, a fish consumed in Far-North Region of Cameroon. Results revealed that the proximate composition was significantly (P < 0.05) affected by treatments: except boiling, all the others treatment reduced significantly (P < 0.05) moisture content of fish while lipid and protein were significantly increased. After processing, the free fatty acids, peroxide and thiobarbituric acid reactive substances values (TBARS) were increased. Iodine value of all treated samples was significantly (P < 0.05) decreased. The combined treatments (frying + boiling and smoking + boiling) negatively affected the lipid quality of fish. Boiling caused significant losses in the mineral contents of fish while smoking treatment led to an important increase of its mineral contents. Steaming appeared to be the best processing method for cooking fish concerning the lipid stability.

Morphological analysis of the cerebellum and its efferent system in a basal actinopterygian fish, Polypterus senegalus.

Although all vertebrate cerebella contain granule cells, Purkinje cells, and efferent neurons, the cellular arrangement and neural circuitry are highly diverse. In amniotes, cerebellar efferent neurons form clusters, deep cerebellar nuclei, lie deep in the cerebellum, and receive synaptic inputs from Purkinje cells but not granule cells. However, in the cerebellum of teleosts, the efferent neurons, called eurydendroid cells, lie near the cell bodies of Purkinje cells and receive inputs both from axons of Purkinje cells and granule cell parallel fibers. It is largely unknown how the cerebellar structure evolved in ray-finned fish (actinopterygians). To address this issue, we analyzed the cerebellum of a bichir Polypterus senegalus, one of the most basal actinopterygians. We found that the cell bodies of Purkinje cells are not aligned in a layer; incoming climbing fibers terminate mainly on the basal portion of Purkinje cells, revealing that the Polypterus cerebellum has unique features among vertebrate cerebella. Retrograde labeling and marker analyses of the efferent neurons revealed that their cell bodies lie in restricted granular areas but not as deep cerebellar nuclei in the cerebellar white matter. The efferent neurons have long dendrites like eurydendroid cells, although they do not reach the molecular layer. Our findings suggest that the efferent system of the bichir cerebellum has intermediate features between teleosts and amniote vertebrates, and provides a model to understand the basis generating diversity in actinopterygian cerebella.

Analysis of pallial/cortical interneurons in key vertebrate models of Testudines, Anurans and Polypteriform fishes.

The organization of the pallial derivatives across vertebrates follows a comparable elementary arrangement, although not all of them possess a layered cortical structure as sophisticated as the cerebral cortex of mammals. However, its expansion along evolution has only been possible by the development and coevolution of the cellular networks formed by excitatory neurons and inhibitory interneurons. Thus, the comparative analysis of interneuron types in vertebrate models of key evolutionary significance will provide important information, due to the extraordinary anatomical sophistication of their interneuron systems with simpler behavioral implications. Particularly in mammals, the main consensus for classifying interneuron types is based on non-overlapping markers, which do not form a single population, but consist of several distinct classes of inhibitory cells showing co-expression of other markers. In our study, we analyzed immunohistochemically the expression of the main markers like somatostatin (SOM), parvalbumin (PV), calretinin (CR), calbindin (CB), neuropeptide Y (NPY) and/or nitric oxide synthase (NOS) at the pallial regions of three different models of Osteichthyes. First, we selected two tetrapods, one amniote from the genus Pseudemys belonging to the order Testudine, at the base of the amniote diversification and with a three-layered simple cortex, and the Anuran Xenopus laevis, an anamniote tetrapod with a non-layered evaginated pallium, and finally the order Polypteriform, a small fish group at the base of the actinopterygian diversification with an everted telencephalon. SOM was the most conserved interneuron type in terms of its distribution and co-expression with other markers such as CR, in contrast to PV, which showed a different pattern between the models analyzed. In addition, the SOM expression supports a homological relationship between the medial pallial derivatives in all the models. CR and CB expressions in the tetrapods were observed, particularly, CR expressing cells were detected in the medial and the dorsal pallial derivatives, in contrast to CB, which appeared only in discrete scattered populations. However, the pallium of Polypteriforms fishes was almost devoid of CR cells, in contrast to the important number of CB cells observed in all the pallial regions. The NPY immunoreactivity was detected in all the pallial domains of all the models, as well as cells coexpressing CR. Finally, the pallial nitrergic expression was also conserved, which allows to postulate the homological relationships between the ventropallial and the amygdaloid derivatives. In summary, even in basal pallial models the neurochemically characterized interneurons indicate that their first appearance took place before the common ancestor of amniotes. Thus, our results suggest a shared pattern of interneuron types in the pallium of all Osteichthyes.

Bichirs employ similar genetic pathways for limb regeneration as are used in lungfish and salamanders.

Bichirs are a sister group to sarcopterygian and tetrapods that can fully regenerate their endochondral-skeleton-fins. Histological and transcriptomic comparison approaches have been used to investigate the morphology and genetic basis of bichir lobe-fin regeneration, with strong down-regulation of muscle-related genes and up-regulation of ECM-related genes and developmental genes being observed. Bichir limb regeneration involves similar cellular processes to those employed by lungfish and salamander, with MARCKS-like protein (MLP) that is known to be a putative regeneration-initiating molecule in salamander, also up-regulated in the early stages of bichir lobe-fin regeneration. These gene expression results suggest that limb regeneration pathways in these amphibians have a common ancestral inheritance, consistent with evolution from endochondral-skeleton-fin structures to endochondral-skeleton-limb structures of vertebrates.

Lungs and gas bladders: Morphological insights.

This paper summarizes the main morphological tracts exhibited by lungs and gas bladders in fishes. The origin and organ location, the presence of a glottal region, the inner architecture, the characteristics of the exchange barrier and the presence of pulmonary arteries have been reviewed in the two types of air-breathing organs. With the exception of the dorsal (bladders) or ventral (lungs) origin from the posterior pharynx, none of the morphological traits analyzed can be considered specific for either lungs or gas bladders. This is exemplified by analysis of the morphology of the lung of the Dipnoii and Polypteriformes and of the bladder of the Lepisosteiformes. All of them are obligate air-breathers and show a lung-like (pulmonoid) air-breathing organ. However, while the lungfish lung and the bladder of the Lepisosteiformes occupy a dorsal position and are highly trabeculated, the polypterid lung occupies a ventral position and shows a smooth inner surface. Structural and ultrastructural differences are also highlighted. Noticeably, a large part of the inner surface area of the lung of the Australian lungfish is covered by a ciliated epithelium. A restricted respiratory surface area may help to explain the incapability of this species to aestivate. The respiratory bladder of basal teleosts displays a more complex morphology than that observed in more primitive species. The bladder of basal teleosts may appear divided into respiratory and non-respiratory portions, exhibit intricate shapes, invade adjacent structures and gain additional functions. The increase in morphological and functional complexity appears to prelude the loss of the respiratory functions.

Locomotion in elongate fishes: A contact sport.

Despite the physical differences between water and air, a number of fish lineages are known to make terrestrial excursions on land. Many of these fishes exhibit an elongate body plan. Elongation of the body can occur in several ways, the most common of which is increasing the number of vertebrae in one or both regions of the axial skeleton--precaudal and/or caudal. Elongate species are often found in three-dimensionally complex habitats. It has been hypothesized that elongate fishes use this structure to their locomotor advantage. In this study, we consider how elongation and differences in vertebral regionalization correspond with the use of wooden pegs, which are provided as analogs to vertically oriented substrate, structures that protrude above the ground. We compare aquatic and terrestrial locomotor behaviors of Polypterus senegalus, Erpetoichthys calabaricus, and Gymnallabes typus as they move through a peg array. When considering axial elongation we find that the highly elongate species, E. calabaricus and G. typus, contact more pegs but on average move slower in both environments than P. senegalus. When considering axial regionalization, we find that the precaudally elongate species, P. senegalus and E. calabaricus, differ in the patterns of peg contact between the two environments whereas the caudally elongate species, G. typus, exhibits similar peg contact between the two environments. Our study highlights the importance of incorporating body shape and vertebral regionalization to understand how elongate fishes move in water and on land.

Finite element modeling of multilayered structures of fish scales.

The interlinked fish scales of Atractosteus spatula (alligator gar) and Polypterus senegalus (gray and albino bichir) are effective multilayered armor systems for protecting fish from threats such as aggressive conspecific interactions or predation. Both types of fish scales have multi-layered structures with a harder and stiffer outer layer, and softer and more compliant inner layers. However, there are differences in relative layer thickness, property mismatch between layers, the property gradations and nanostructures in each layer. The fracture paths and patterns of both scales under microindentation loads were different. In this work, finite element models of fish scales of A. spatula and P. senegalus were built to investigate the mechanics of their multi-layered structures under penetration loads. The models simulate a rigid microindenter penetrating the fish scales quasi-statically to understand the observed experimental results. Study results indicate that the different fracture patterns and crack paths observed in the experiments were related to the different stress fields caused by the differences in layer thickness, and spatial distribution of the elastic and plastic properties in the layers, and the differences in interface properties. The parametric studies and experimental results suggest that smaller fish such as P. senegalus may have adopted a thinner outer layer for light-weighting and improved mobility, and meanwhile adopted higher strength and higher modulus at the outer layer, and stronger interface properties to prevent ring cracking and interface cracking, and larger fish such as A. spatula and Arapaima gigas have lower strength and lower modulus at the outer layers and weaker interface properties, but have adopted thicker outer layers to provide adequate protection against ring cracking and interface cracking, possibly because weight is less of a concern relative to the smaller fish such as P. senegalus.

The anatomical components of the cardiac outflow tract of the gray bichir, Polypterus senegalus: their evolutionary significance.

It has been reported that in chondrichthyans the cardiac outflow tract is composed of the myocardial conus arteriosus, while in most teleosteans it consists of the nonmyocardial bulbus arteriosus. Classical studies already indicated that a conus and a bulbus coexist in several ancient actinopterygian and teleost groups. Recent work has shown that a cardiac outflow tract consisting of a conus and a bulbus is common to both cartilaginous and bony fishes. Nonetheless and despite their position at the base of the actinopterygian phylogenetic lineage, the anatomical arrangement of the cardiac outflow tract of the Polypteriformes remained uncertain. The present study of hearts from gray bichirs was intended to fill this gap. The cardiac outflow tract of the bichir consists of two main components, namely a very long conus arteriosus, furnished with valves, and a short, intrapericardial, arterial-like bulbus arteriosus, which differs from the ventral aorta because it is covered by epicardium, shows a slightly different spatial arrangement of the histological elements and is crossed by coronary arteries. Histomorphologically, the outflow tract consists of three longitudinal regions, distal, middle and proximal, an arrangement which has been suggested to be common to all vertebrates. The distal region corresponds to the bulbus, while the conus comprises the middle and proximal regions. The present findings reinforce the notion that the bulbus arteriosus of fish has played an essential role in vertebrate heart evolution as it is the precursor of the intrapericardial trunks of the aorta and pulmonary artery of birds and mammals.

A study of the relict fish fauna of northern Chad, with the first records of a polypterid and a poeciliid in the Sahara desert.

Seventeen species and sub-species of fishes belonging to four families (Cyprinidae, Clariidae, Aplocheilidae, Cichlidae) were known to occur in perennial bodies of water in the Sahara desert. The study of fishes collected in Lake Boukou near Ounianga Serir (Borkou, northern Chad) shows, for the first time, the occurrence in the Sahara desert of relict populations of Polypterus senegalus (Polypteridae) and Poropanchax normani (Poeciliidae). The Cichlidae Tilapia zilli was also collected in this lake. With these new records, the relict fish fauna currently known in lakes and gueltas of the Borkou plateaus comprises six species. In the Ennedi Mountains, where the specific status of Barbus populations was unclear, B. macrops was collected in Bachikere guelta. The toad Amietophrynus regularis was collected in Ounianga Kebir.