Complex Vertebrate Research Articles

An evolutionarily conserved AnkyrinG-dependent motif clusters axonal K2P K+ channels.

The evolution of ion channel clustering at nodes of Ranvier enabled the development of complex vertebrate nervous systems. At mammalian nodes, the K+ leak channels TRAAK and TREK-1 underlie membrane repolarization. Despite the molecular similarities between nodes and the axon initial segment (AIS), TRAAK and TREK-1 are reportedly node-specific, suggesting a unique clustering mechanism. However, we show that TRAAK and TREK-1 are enriched at both nodes and AIS through a common mechanism. We identified a motif near the C-terminus of TRAAK that is necessary and sufficient for its clustering. The motif first evolved among cartilaginous fish. Using AnkyrinG (AnkG) conditional knockout mice, CRISPR/Cas9-mediated disruption of AnkG, co-immunoprecipitation, and surface recruitment assays, we show that TRAAK forms a complex with AnkG and that AnkG is necessary for TRAAK's AIS and nodal clustering. In contrast, TREK-1's clustering requires TRAAK. Our results expand the repertoire of AIS and nodal ion channel clustering mechanisms and emphasize AnkG's central role in assembling excitable domains.

Ultrastructural analysis and 3D reconstruction of the frontal sensory-glandular complex and its neural projections in the platyhelminth Macrostomum lignano.

The marine microturbellarian Macrostomum lignano (Platyhelminthes, Rhabditophora) is an emerging laboratory model used by a growing community of researchers because it is easy to cultivate, has a fully sequenced genome, and offers multiple molecular tools for its study. M. lignano has a compartmentalized brain that receives sensory information from receptors integrated in the epidermis. Receptors of the head, as well as accompanying glands and specialized epidermal cells, form a compound sensory structure called the frontal glandular complex. In this study, we used semi-serial transmission electron microscopy (TEM) to document the types, ultrastructure, and three-dimensional architecture of the cells of the frontal glandular complex. We distinguish a ventral compartment formed by clusters of type 1 (multiciliated) sensory receptors from a central domain where type 2 (collar) sensory receptors predominate. Six different types of glands (rhammite glands, mucoid glands, glands with aster-like and perimaculate granula, vacuolated glands, and buckle glands) are closely associated with type 1 sensory receptors. Endings of a seventh type of gland (rhabdite gland) define a dorsal domain of the frontal glandular complex. A pair of ciliary photoreceptors is closely associated with the base of the frontal glandular complex. Bundles of dendrites, connecting the receptor endings with their cell bodies which are located in the brain, form the (frontal) peripheral nerves. Nerve fibers show a varicose structure, with thick segments alternating with thin segments, and are devoid of a glial layer. This distinguishes platyhelminths from larger and/or more complex invertebrates whose nerves are embedded in prominent glial sheaths.

A One Health Epidemiological Approach for Understanding Ticks and Tick-Borne Pathogens.

Background: The control and prevention of ticks and tick borne diseases (TBDs) is often difficult, since it is necessary to disrupt a complex transmission cycle, involving ticks and vertebrate hosts, which interact in a changing environment, driven by constant environmental and ecological changes. Our view is that factors driving the spread of R. microplus are complex and intrinsically interconnected, something that has often been ignored in control strategies. Results: The aim of this review is to analyze the importance of the epidemiological surveillance of ticks and tick-borne diseases (TTBDs) for Public Health, with the One Health approach; emphasizing the knowledge, importance, and distribution of TTBDs. Conclusions: The key points for surveillance, and raising the scope and limitations of surveillance programs, to delay the emergence of acaricide resistance, to reduce toxic residues in food for human consumption and to protect animal, human, and environmental health, from a One Health perspective will require calling producers, veterinarians, academics, pharmaceutical industry, and decision makers to join efforts in order to mitigate the effects of ticks and TBDs affecting the cattle industry in Mexico.

Cephalopod behaviour

Underlying all animal behaviors, from the simplest reflexive reactions to the more complex cognitive reasoning and social interaction, are nervous systems uniquely adapted to bodies, environments, and challenges of different animal species. Coleoid cephalopods - octopuses, squid, and cuttlefish - are widely recognized as the most behaviorally complex invertebrates and provide exciting opportunities for studying the neural control of behaviour. These unusual molluscs evolved over 400 million years ago from slow-moving armored forms to active predators of coastal and open ocean ecosystems. In this primer we will discuss how, during cephalopod evolution, the relatively simple ganglion-based molluscan nervous system has been extensively transformed to control the complex bodies and process extensive visual, tactile, and chemical sensory inputs, and summarize some recent findings about their fascinating behaviors.

Biological Magnification of Microplastics: A Look at the Induced Reproductive Toxicity from Simple Invertebrates to Complex Vertebrates

The issue of microplastic (MP) pollution is one of the most pressing environmental problems faced today and for the future. Plastics are ubiquitous due to their exponential use and mismanagement, resulting in the accumulation of fragments across the world. Hence, the problem of MP pollution is aggravated when these plastic items disintegrate into smaller particles due to different physical, chemical, and environmental factors. The consumption of these MP pollutants by wildlife is a worldwide concern and a potentially crucial risk for all ecosystems. Consequently, MPs have caused a wide variety of problems for both public health and wildlife concerning vital life processes—specifically reproduction, which is critical to species’ survival in an ecosystem. Despite MPs’ detrimental effects on wildlife reproduction, it remains unclear how MPs can affect the hypothalamic–pituitary–gonadal (HPG) axis. This review highlights the significant reproductive toxicity of MPs in wildlife, with potentially devastating consequences for human health. The findings emphasize the urgency of developing effective solutions for mitigating the adverse effects of MP pollution on the reproductive systems of wildlife and preserving the integrity of aquatic and terrestrial habitats.

Rapid and precise genome engineering in a naturally short-lived vertebrate.

The African turquoise killifish is a powerful vertebrate system to study complex phenotypes at scale, including aging and age-related disease. Here, we develop a rapid and precise CRISPR/Cas9-mediated knock-in approach in the killifish. We show its efficient application to precisely insert fluorescent reporters of different sizes at various genomic loci in order to drive cell-type- and tissue-specific expression. This knock-in method should allow the establishment of humanized disease models and the development of cell-type-specific molecular probes for studying complex vertebrate biology.

Evolution of the glomerulus in a marine environment and its implications for renal function in terrestrial vertebrates

Nearly a century ago, Homer Smith proposed that the glomerulus evolved to meet the challenge of excretion of water in freshwater vertebrates. This hypothesis has been repeatedly restated in the nephrology and renal physiology literature, even though we now know that vertebrates evolved and diversified in marine (saltwater) environments. A more likely explanation is that the vertebrate glomerulus evolved from the meta-nephridium of marine invertebrates, with the driving force for ultrafiltration being facilitated by the apposition of the filtration barrier to the vasculature (in vertebrates) rather than the coelom (in invertebrates) and the development of a true heart and the more complex vertebrate vascular system. In turn, glomerular filtration aided individual regulation of divalent ions like magnesium, calcium, and sulfate compatible with the function of cardiac and skeletal muscle required for mobile predators. The metabolic cost, imposed by reabsorption of the small amounts of sodium required to drive secretion of these over-abundant divalent ions, was small. This innovation, developed in a salt-water environment, provided a preadaptation for life in freshwater, in which the glomerulus was co-opted to facilitate water excretion, albeit with the additional metabolic demand imposed by the need to reabsorb the majority of filtered sodium. The evolution of the glomerulus in saltwater also provided preadaptation for terrestrial life, where the imperative is conservation of both water and electrolytes. The historical contingencies of this scenario may explain why the mammalian kidney is so metabolically inefficient, with ∼80% of oxygen consumption being used to drive reabsorption of filtered sodium.

A common vesicle proteome drives fungal biofilm development

Extracellular vesicles mediate community interactions among cells ranging from unicellular microbes to complex vertebrates. Extracellular vesicles of the fungal pathogen Candida albicans are vital for biofilm communities to produce matrix, which confers environmental protection and modulates community dispersion. Infections are increasingly due to diverse Candida species, such as the emerging pathogen Candida auris, as well as mixed Candida communities. Here, we define the composition and function of biofilm-associated vesicles among five species across the Candida genus. We find similarities in vesicle size and release over the biofilm lifespan. Whereas overall cargo proteomes differ dramatically among species, a group of 36 common proteins is enriched for orthologs of C. albicans biofilm mediators. To understand the function of this set of proteins, we asked whether mutants in select components were important for key biofilm processes, including drug tolerance and dispersion. We found that the majority of these cargo components impact one or both biofilm processes across all five species. Exogenous delivery of wild-type vesicle cargo returned mutant phenotypes toward wild type. To assess the impact of vesicle cargo on interspecies interactions, we performed cross-species vesicle addition and observed functional complementation for both biofilm phenotypes. We explored the biologic relevance of this cross-species biofilm interaction in mixed species and mutant studies examining the drug-resistance phenotype. We found a majority of biofilm interactions among species restored the community's wild-type behavior. Our studies indicate that vesicles influence the development of protective monomicrobial and mixed microbial biofilm communities.

Permian hypercarnivore suggests dental complexity among early amniotes

The oldest known complex terrestrial vertebrate community included hypercarnivorous varanopids, a successful clade of amniotes with wide geographic and temporal distributions. Little is known about their dentition and feeding behaviour, but with the unprecedented number of specimens of the varanopid Mesenosaurus from cave deposits in Oklahoma, we show that it exhibited serrations on the tooth crowns, and exceptionally rapid rates of development and reduced longevity relative to other terrestrial amniotes. In contrast, the coeval large apex predator Dimetrodon greatly increased dental longevity by increasing thickness and massiveness, whereas herbivores greatly reduced tooth replacement rates and increased dental longevity. Insectivores and omnivores represented the primitive condition and maintained modest replacement rates and longevity. The varied patterns of dental development among these early terrestrial amniotes reveal a hidden aspect of dental complexity in the emerging diverse amniote community, very soon after their initial appearance in the fossil record.

ACTH-Modulated Membrane Guanylate Cyclase Signaling System: Origin and Creation.

The membrane guanylate cyclase (MGC) cellular signaling pathway consists of seven signaling pathways and is critical for the survival of prokaryotes eukaryotes, and highly complex vertebrate organisms. A sequel to the author's earlier comprehensive reviews, covering the field of MGC from its origin to its establishment to the year 2014, this article exclusively deals with the history of its development from the year 1963 to 1987. It narrates the efforts involved in building on small projects, brick by brick, and its emergence from the chasm of disbelief, through steady, continuous work. To make the presentation simple and chronologically continuous, the subject matters of the earlier reviews and publication of these authors have been freely borrowed with appropriate citations.

The Middle Triassic vertebrate deposits of Kupferzell (Germany): Palaeoenvironmental evolution of complex ecosystems

In 1977, within 3 months of excavation, a 500 m spanning road-cut near Kupferzell (southern Germany) produced a total of ~30,000 vertebrate remains from the Middle Triassic Lower Keuper. The bulk of the material stems from two temnospondyl amphibians, Gerrothorax pulcherrimus (~70%) and Mastodonsaurus giganteus (~30%), with the pseudosuchian archosaur Batrachotomus kupferzellensis ranging first among the other remains. Analyses of data collected during excavation, supplemented by new fieldwork, provide rich information on the sedimentary setting as well as the development of the fauna and their ecosystems. The sequence consists of: basal coaly mudstones (K1), massive siltstones (K2), green siliciclastic mudstones (K3), yellow to pale brown marlstones (K4), and yellow massive dolostones (K5). The deposits comprise a succession of similar water bodies that were emplaced on a lacustrine to floodplain setting in which carbonate muds dominated. Two main lake systems, with intermittent/periodical marine influence, as well as relatively stark periods of drought, harboured complex vertebrate ecosystems. These were dominated by a relatively high diversity of fishes and temnospondyl amphibians with a lesser, but notable, presence of archosaurs, forming the top predators of the trophic web. The sequence records alternating periods of flooding and desiccation, shaping a relatively complex environmental setting that was likely prone to the presence of life, eventually becoming an exceptional fossil lagerstätte. Kupferzell, together with nearby contemporaneous localities, represent relatively diverse and complex ecosystems (including several top predators) that allow understanding the evolution and palaeoecology of Middle Triassic vertebrate communities, including the groups that diversified during the Mesozoic era.

Functions of the FGF signalling pathway in cephalochordates provide insight into the evolution of the prechordal plate.

ABSTRACTThe fibroblast growth factor (FGF) signalling pathway plays various roles during vertebrate embryogenesis, from mesoderm formation to brain patterning. This diversity of functions relies on the fact that vertebrates possess the largest FGF gene complement among metazoans. In the cephalochordate amphioxus, which belongs to the chordate clade together with vertebrates and tunicates, we have previously shown that the main role of FGF during early development is the control of rostral somite formation. Inhibition of this signalling pathway induces the loss of these structures, resulting in an embryo without anterior segmented mesoderm, as in the vertebrate head. Here, by combining several approaches, we show that the anterior presumptive paraxial mesoderm cells acquire an anterior axial fate when FGF signal is inhibited and that they are later incorporated in the anterior notochord. Our analysis of notochord formation in wild type and in embryos in which FGF signalling is inhibited also reveals that amphioxus anterior notochord presents transient prechordal plate features. Altogether, our results give insight into how changes in FGF functions during chordate evolution might have participated to the emergence of the complex vertebrate head.

New Findings on LMO7 Transcripts, Proteins and Regulatory Regions in Human and Vertebrate Model Organisms and the Intracellular Distribution in Skeletal Muscle Cells.

LMO7 is a multifunctional PDZ–LIM protein that can interact with different molecular partners and is found in several intracellular locations. The aim of this work was to shed light on LMO7 evolution, alternative transcripts, protein structure and gene regulation through multiple in silico analyses. We also explored the intracellular distribution of the LMO7 protein in chicken and zebrafish embryonic skeletal muscle cells by means of confocal fluorescence microscopy. Our results revealed a single LMO7 gene in mammals, sauropsids, Xenopus and in the holostean fish spotted gar while two lmo7 genes (lmo7a and lmo7b) were identified in teleost fishes. In addition, several different transcripts were predicted for LMO7 in human and in major vertebrate model organisms (mouse, chicken, Xenopus and zebrafish). Bioinformatics tools revealed several structural features of the LMO7 protein including intrinsically disordered regions. We found the LMO7 protein in multiple intracellular compartments in chicken and zebrafish skeletal muscle cells, such as membrane adhesion sites and the perinuclear region. Curiously, the LMO7 protein was detected within the nuclei of muscle cells in chicken but not in zebrafish. Our data showed that a conserved regulatory element may be related to muscle-specific LMO7 expression. Our findings uncover new and important information about LMO7 and open new challenges to understanding how the diverse regulation, structure and distribution of this protein are integrated into highly complex vertebrate cellular milieux, such as skeletal muscle cells.

Giant hornet (Vespa soror) attacks trigger frenetic antipredator signalling in honeybee (Apis cerana) colonies.

Asian honeybees use an impressive array of strategies to protect nests from hornet attacks, although little is understood about how antipredator signals coordinate defences. We compared vibroacoustic signalling and defensive responses of Apis cerana colonies that were attacked by either the group-hunting giant hornet Vespa soror or the smaller, solitary-hunting hornet Vespa velutina. Apis cerana colonies produced hisses, brief stop signals and longer pipes under hornet-free conditions. However, hornet-attack stimuli—and V. soror workers in particular—triggered dramatic increases in signalling rates within colonies. Soundscapes were cacophonous when V. soror predators were directly outside of nests, in part because of frenetic production of antipredator pipes, a previously undescribed signal. Antipredator pipes share acoustic traits with alarm shrieks, fear screams and panic calls of primates, birds and meerkats. Workers making antipredator pipes exposed their Nasonov gland, suggesting the potential for multimodal alarm signalling that warns nestmates about the presence of dangerous hornets and assembles workers for defence. Concurrent observations of nest entrances showed an increase in worker activities that support effective defences against giant hornets. Apis cerana workers flexibly employ a diverse alarm repertoire in response to attack attributes, mirroring features of sophisticated alarm calling in socially complex vertebrates.

A likely association of damselflies with the habitat heterogeneity provided by the freshwater swamp lily, Ottelia ovalifolia, in Eyre Peninsula granite rock-holes, with a review of potential threats to this ephemeral habitat

ABSTRACT The granite rock-holes (sometimes called gnammas) across northern Eyre Peninsula (EP), South Australia, are a unique but poorly studied ephemeral freshwater habitat containing a complex invertebrate community. Macroinvertebrate predator occurrence is often sporadic, both spatially and temporally. We aimed to determine if environmental conditions might predict predator occurrence in EP rock-holes. A total of 14 rock-holes were sampled across five granite outcrops along the Eyre Highway. Extensive dip-net sampling was undertaken and nymphs of three damselfly species were recorded from the rock-holes: Austrolestes annulosus (Lestidae), Ischnura aurora, and Xanthagrion erythroneurum (both Coenagrionidae), all in a single rock-hole at Pildappa Rock. This sole rock-hole contained a prominent floating-leaved, rooted aquatic macrophyte: the swamp lily, Ottelia ovalifolia (Hydrocharitaceae), which forms a complex, three-dimensional vegetative structure. Damselflies were hypothesised to be associated with the presence of O. ovalifolia, possibly as a result of the plant providing both suitable oviposition sites for the adults and habitat for nymphs throughout the water column, opportunities not afforded by the aquatic vegetation present in shallow rock-holes. Our findings contribute to the limited ecological information regarding EP rock-holes. We also briefly review potential threats to these ecosystems, an understanding of which will be critical to their management and conservation.

Behavioral and neurophysiological evidence suggests affective pain experience in octopus

SummaryPain is a negative affective state arising from tissue damage or inflammation. Because pain is aversive and its relief is innately rewarding, animals may learn to avoid a context in which pain is experienced and prefer one where pain relief occurs. It is generally accepted that vertebrate animals experience pain; however, there is currently inconclusive evidence that the affective component of pain occurs in any invertebrate. Here, we show that octopuses, the most neurologically complex invertebrates, exhibit cognitive and spontaneous behaviors indicative of affective pain experience. In conditioned place preference assays, octopuses avoided contexts in which pain was experienced, preferred a location in which they experienced relief from pain, and showed no conditioned preference in the absence of pain. Injection site grooming occurred in all animals receiving acetic acid injections, but this was abolished by local anesthesia. Thus, octopuses are likely to experience the affective component of pain.

The struggle to equilibrate outer and inner milieus: Renal evolution revisited

The journey of life, from primordial protoplasm to a complex vertebrate form, is a tale of survival against incessant alterations in climate, surface topography, food chain, and chemistry of the external environment. Kidneys present with an ensemble embodiment of the adaptations devised by diverse life-forms to cope with such challenges and maintain a chemical equilibrium of water and solutes, both in and outside the body. This minireview revisits renal evolution utilizing the classic: From Fish to Philosopher; the story of our internal environment, by Prof. Homer W. Smith (1895–1962) as a template. Prof. Smith’s views exemplified the invention of glomeruli, or its abolishment, as a mechanism to filter water. Moreover, with the need to preserve water, as in reptiles, the loop of Henle was introduced to concentrate urine. When compared to smaller mammals, the larger ones, albeit having loops of Henle of similar lengths, demonstrated a distinct packing of the nephrons in kidneys. Moreover, the renal portal system degenerated in mammals, while still present in other vertebrates. This account will present with a critique of the current concepts of renal evolution while examining how various other factors, including the ones that we know more about now, such as genetic factors, synchronize to achieve renal development. Finally, it will try to assess the validity of ideas laid by Prof. Smith with the knowledge that we possess now, and understand the complex architecture that evolution has imprinted on the kidneys during its struggle to survive over epochs.

Metabolic physiology of the freshwater planaria Girardia dorotocephela and Schmidtea mediterranea: reproductive mode, specific dynamic action, and temperature.

Planarians are widely used animal models for studies in regeneration, developmental biology, neurobiology, and behavior. However, surprisingly little is known about other aspects of their basic biology, even though such information might help validate these flatworms as a general animal model. We hypothesized that planaria, although dependent on simple diffusion of O2 across the integument for O2 uptake, would nonetheless show changes in oxygen consumption (V̇o2) associated with reproductive mode (sexual or asexual), feeding (specific dynamic action; SDA), temperature (Q10 values), and photoperiod typical of those responses of more complex invertebrates. In the current experiments, routine V̇o2 was measured over the range of 13-28°C in Schmidtea mediterranea and Girardia dorotocephala. At the long-term maintenance temperature of 18°C, routine V̇o2 was ~13 µL O2·g-1·h-1 in the two asexual strains, but approximately twice as high (27 µL O2·g-1·h-1) in the sexual strain of S. mediterranea, suggesting a metabolic cost for sexual reproduction. Metabolic temperature sensitivity, measured by Q10, was about one to three for all three groups. All three groups showed a large (~2- to 3-fold) increase in V̇o2 within a day following feeding, suggesting a large SDA effect. Starvation, causing "degrowth" in some planaria, resulted in a loss of one-third of body mass in sexual S. mediterranea but no body mass loss in either asexual strains. Collectively, these data indicate that, while being a relatively simple flatworm with no dedicated respiratory or circulatory system, their metabolic physiological responses are quite similar to those shown by more complex invertebrates and vertebrates, contributing to their validation as an animal model.

Centromere-specific antibody-mediated karyotyping of Okinawan Oikopleura dioicasuggests the presence of three chromosomes.

Oikopleura dioica is a ubiquitous marine tunicate of biological interest due to features that include dioecious reproduction, short life cycle, and vertebrate-like dorsal notochord while possessing a relatively compact genome. The use of tunicates as model organisms, particularly with these characteristics, offers the advantage of facilitating studies in evolutionary development and furthering understanding of enduring attributes found in the more complex vertebrates. At present, we are undertaking an initiative to sequence the genomes of Oikopleura individuals in populations found among the seas surrounding the Ryukyu Islands in southern Japan. To facilitate and validate genome assemblies, karyotyping was employed to count individual animals' chromosomes in situ using centromere-specific antibodies directed against H3S28P, a prophase-metaphase cell cycle-specific marker of histone H3. New imaging data of embryos and oocytes stained with two different antibodies were obtained; interpretation of these data lead us to conclude that the Okinawan Oikopleura dioica has three pairs of chromosomes, akin to previous results from genomic assemblies in Atlantic populations. The imaging data have been deposited to the open-access EBI BioImage Archive for reuse while additionally providing representative images of two commercially available anti-H3S28P antibodies' staining properties for use in epifluorescent and confocal based fluorescent microscopy.

Cataloguing the dead: breathing new life into pseudokinase research.

Pseudoenzymes are present within many, but not all, known enzyme families and lack one or more conserved canonical amino acids that help define their catalytically active counterparts. Recent findings in the pseudokinase field confirm that evolutionary repurposing of the structurally defined bilobal protein kinase fold permits distinct biological functions to emerge, many of which rely on conformational switching, as opposed to canonical catalysis. In this analysis, we evaluate progress in evaluating several members of the ‘dark’ pseudokinome that are pertinent to help drive this expanding field. Initially, we discuss how adaptions in erythropoietin‐producing hepatocellular carcinoma (Eph) receptor tyrosine kinase domains resulted in two vertebrate pseudokinases, EphA10 and EphB6, in which co‐evolving sequences generate new motifs that are likely to be important for both nucleotide binding and catalysis‐independent signalling. Secondly, we discuss how conformationally flexible Tribbles pseudokinases, which have radiated in the complex vertebrates, control fundamental aspects of cell signalling that may be targetable with covalent small molecules. Finally, we show how species‐level adaptions in the duplicated canonical kinase protein serine kinase histone (PSKH)1 sequence have led to the appearance of the pseudokinase PSKH2, whose physiological role remains mysterious. In conclusion, we show how the patterns we discover are selectively conserved within specific pseudokinases, and that when they are modelled alongside closely related canonical kinases, many are found to be located in functionally important regions of the conserved kinase fold. Interrogation of these patterns will be useful for future evaluation of these, and other, members of the unstudied human kinome.