Morphological, Genetic, and Physiological Effects of Nutrient Manipulation on a Colonial Marine Hydroid.

AbstractThe availability of environmental nutrients is an existential constraint for heterotrophic organisms and is thus expected to impact numerous biochemical and physiological features. The continuously proliferative polyp stage of colonial hydroids provides a useful model to study these features, allowing genetically identical replicates to be compared. Two groups of colonies of Eirene sp., defined by different feeding treatments, were grown by explanting the same founder colony onto cover glass. Colonies of both treatments were allowed to grow continuously by explanting them onto new cover glass as they reached the edge of the existing surface. The nutrient-abundant polyps grew faster and produced more clumped or "sheet-like" colonies. Compared to the founder colony, the nutrient-abundant colonies exhibited more mutations (i.e., single-nucleotide polymorphisms) than the nutrient-scarce colonies. Nevertheless, these differences were not commensurate with the differences in growth. Using a polarographic electrode, we found that the nutrient-abundant colonies exhibited lower rates of oxygen uptake relative to total protein. The probe 2',7'-dichlorodihydrofluorescein diacetate and fluorescent microscopy allowed visualization of the mitochondrion-rich cells at the base of the polyps and showed that the nutrient-abundant colonies exhibited greater amounts of reactive oxygen species than the nutrient-scarce colonies. Parallels to the Warburg effect-aerobic glycolysis, diminished oxygen uptake, and lactate secretion-found in human cancers and other proliferative cells may be suggested. However, little is known about anaerobic metabolism in cnidarians. Examination of oxygen uptake suggests an anaerobic threshold at a roughly 1-mg/L oxygen concentration. Nutrient-abundant colonies may respond more dramatically to this threshold than nutrient-scarce colonies.

An Organismal Perspective on the Warburg Effect and Models for Proliferation Studies.

Interest in the physiology of proliferation has been generated by human proliferative diseases, i.e., cancers. A vast literature exists on the Warburg effect, which is characterized by aerobic glycolysis, diminished oxygen uptake, and lactate secretion. While these features could be rationalized via the production of biosynthetic precursors, lactate secretion does not fit this paradigm, as it wastes precursors. Forming lactate from pyruvate allows for reoxidizing cytosolic NADH, which is crucial for continued glycolysis and may allow for maintaining large pools of metabolic intermediates. Alternatively, lactate production may not be adaptive, but rather reflect metabolic constraints. A broader sampling of the physiology of proliferation, particularly in organisms that could reoxidize NADH using other pathways, may be necessary to understand the Warburg effect. The best-studied metazoans (e.g., worms, flies, and mice) may not be suitable, as they undergo limited proliferation before initiating meiosis. In contrast, some metazoans (e.g., colonial marine hydrozoans) exhibit a stage in the life cycle (the polyp stage) that only undergoes mitotic proliferation and never carries out meiosis (the medusa stage performs this). Such organisms are prime candidates for general studies of proliferation in multicellular organisms and could at least complement the short-generation models of modern biology.

The enigmatic Placozoa part 2: Exploring evolutionary controversies and promising questions on earth and in space.

The placozoan Trichoplax adhaerens has been bridging gaps between research disciplines like no other animal. As outlined in part 1, placozoans have been subject of hot evolutionary debates and placozoans have challenged some fundamental evolutionary concepts. Here in part 2 we discuss the exceptional genetics of the phylum Placozoa and point out some challenging model system applications for the best known species, Trichoplax adhaerens.

Reactive Oxygen Species and the Stress Response in Octocorals.

AbstractReactive oxygen species (ROS) may damage cellular components but may also contribute to signaling that mitigates damage. In this context, the role of ROS in the stress response that leads to coral bleaching was investigated in three series of experiments with octocorals Sarcothelia sp. and Sympodium sp. Using video and fluorescent microscopy, the first experiments examined ROS and symbiont migration. Colonies mildly stressed with increased temperature and light showed increases in both ROS and numbers of migrating symbionts compared with stress-free controls. Symbionts migrating in the gastrovascular lumen may escape programmed cell death and provide a reservoir of healthy symbionts once conditions return to normal. In the second series of experiments, colonies were mildly stressed with elevated temperature and light. During stress, treated colonies were incubated in seawater enriched with two concentrations of bicarbonate (1 and 3 mmol/L), while controls were incubated in normal seawater. Bicarbonate enrichment provides additional carbon for photosynthesis and at some concentrations diminished the ROS emissions of stressed colonies of Sympodium sp. and Sarcothelia sp. In all experiments, the latter species tended to exhibit more ROS. Sympodium sp. contains Cladocopium sp. symbionts, which are less tolerant of stress, while Sarcothelia sp. contains the more resistant Durusdinium sp. Indeed, in direct comparisons, Sarcothelia sp. experienced higher levels of ROS under stress-free conditions and thus is conditioned to endure the stress associated with bleaching. Generally, ROS levels provide important insight into the cnidarian stress response and should be measured more often in studies of this response.

The enigmatic Placozoa part 1: Exploring evolutionary controversies and poor ecological knowledge.

The placozoan Trichoplax adhaerens is a tiny hairy plate and more simply organized than any other living metazoan. After its original description by F.E. Schulze in 1883, it attracted attention as a potential model for the ancestral state of metazoan organization, the "Urmetazoon". Trichoplax lacks any kind of symmetry, organs, nerve cells, muscle cells, basal lamina, and extracellular matrix. Furthermore, the placozoan genome is the smallest (not secondarily reduced) genome of all metazoan genomes. It harbors a remarkably rich diversity of genes and has been considered the best living surrogate for a metazoan ancestor genome. The phylum Placozoa presently harbors three formally described species, while several dozen "cryptic" species are yet awaiting their description. The phylogenetic position of placozoans has recently become a contested arena for modern phylogenetic analyses and view-driven claims. Trichoplax offers unique prospects for understanding the minimal requirements of metazoan animal organization and their corresponding malfunctions.

Evidence for a Syncytial Origin of Eukaryotes from Ancestral State Reconstruction.

Modern accounts of eukaryogenesis entail an endosymbiotic encounter between an archaeal host and a proteobacterial endosymbiont, with subsequent evolution giving rise to a unicell possessing a single nucleus and mitochondria. The mononucleate state of the last eukaryotic common ancestor (LECA) is seldom, if ever, questioned, even though cells harboring multiple (syncytia, coenocytes, and polykaryons) are surprisingly common across eukaryotic supergroups. Here, we present a survey of multinucleated forms. Ancestral character state reconstruction for representatives of 106 eukaryotic taxa using 16 different possible roots and supergroup sister relationships, indicate that LECA, in addition to being mitochondriate, sexual, and meiotic, was multinucleate. LECA exhibited closed mitosis, which is the rule for modern syncytial forms, shedding light on the mechanics of its chromosome segregation. A simple mathematical model shows that within LECA's multinucleate cytosol, relationships among mitochondria and nuclei were neither one-to-one, nor one-to-many, but many-to-many, placing mitonuclear interactions and cytonuclear compatibility at the evolutionary base of eukaryotic cell origin. Within a syncytium, individual nuclei and individual mitochondria function as the initial lower-level evolutionary units of selection, as opposed to individual cells, during eukaryogenesis. Nuclei within a syncytium rescue each other's lethal mutations, thereby postponing selection for viable nuclei and cytonuclear compatibility to the generation of spores, buffering transitional bottlenecks at eukaryogenesis. The prokaryote-to-eukaryote transition is traditionally thought to have left no intermediates, yet if eukaryogenesis proceeded via a syncytial common ancestor, intermediate forms have persisted to the present throughout the eukaryotic tree as syncytia but have so far gone unrecognized.

Evolutionary conflict and coloniality in animals.

Despite considerable interest in the effects of evolutionary conflict in colonies of social insects, relatively little attention has been paid to this issue in clonal animals with modular construction, such as colonial ascidians, bryozoans, and cnidarians. These colonial animals are structural individuals, subdivided into repeated morphological modules, which can individually acquire, process, and share resources. While size-related selection favors colony formation, evolutionary conflicts remain a potent obstacle to such cooperation. These conflicts can occur at several levels and must be mediated for cooperation to emerge. Module-level conflicts potentially result in coalitions of genetically similar modules failing to share resources or monopolizing reproduction. Mediation occurs by a number of mechanisms including: (a) a single-module bottleneck at the initiation of colony formation, (b) allorecognition that limits colony fusion to close kin, (c) development of new modules from connective tissue, (d) synchronization of module budding, (e) programmed module death, (f) terminal differentiation of reproductive modules, and (g) architectural constraints. Effective mediation of module-level conflicts, however, may in some cases contribute to cell-level conflicts. Animal colonies typically have multipotent stem cells, and genetically variant stem cells can potentially monopolize gamete formation. Limiting colony fusion to close kin may not eliminate such conflict. Finally, in at least some taxa an association between photosymbiosis and coloniality is found. Allocation of photosynthate can lead to host-symbiont conflicts that can be mediated by housing symbionts intracellularly and using chemiosmotic mechanisms to detect defectors. Colonial animals thus serve as a living laboratory of evolutionary conflict and its mediation.

Can natural selection and druggable targets synergize? Of nutrient scarcity, cancer, and the evolution of cooperation.

Since the dawn of molecular biology, cancer therapy has focused on druggable targets. Despite some remarkable successes, cell-level evolution remains a potent antagonist to this approach. We suggest that a deeper understanding of the breakdown of cooperation can synergize the evolutionary and druggable-targets approaches. Complexity requires cooperation, whether between cells of different species (symbiosis) or between cells of the same organism (multicellularity). Both forms of cooperation may be associated with nutrient scarcity, which in turn may be associated with a chemiosmotic metabolism. A variety of examples from modern organisms supports these generalities. Indeed, mammalian cancers-unicellular, glycolytic, and fast-replicating-parallel these examples. Nutrient scarcity, chemiosmosis, and associated signaling may favor cooperation, while under conditions of nutrient abundance a fermentative metabolism may signal the breakdown of cooperation. Manipulating this metabolic milieu may potentiate the effects of targeted therapeutics. Specific opportunities are discussed in this regard, including avicins, a novel plant product.

Chemiosmosis, Evolutionary Conflict, and Eukaryotic Symbiosis.

Mutualistic symbiosis, in which individuals of different species cooperate and both benefit, has long been an evolutionary puzzle. Why should individuals of two different species cooperate? In this case, as in all others, cooperation is not automatic, but rather requires the mediation of evolutionary conflicts. In chemiosmosis, redox reactions produce a trans-membrane "proton-motive force" that powers energy-requiring reactions in most organisms. Chemiosmosis may also have a role in conflict mediation. Chemiosmosis rapidly produces considerable amounts of products, increasing the risk of end-product inhibition and the formation of dangerous by-products, such as reactive oxygen species. While several mechanisms can modulate chemiosmosis, potential negative effects can also be ameliorated by simply dispersing excess product into the environment. This "free lunch you are forced to make" can attract individuals of other species leading to groups, in which other organisms share the products that are released into the environment by the chemiosmotic cell or organism. Since the time of Darwin, evolutionary biology has recognized that groups are the key to the evolution of cooperation. With many small groups, chance associations of cooperators can arise, even if cooperation is selected against at the individual level. Groups of cooperators can then outcompete groups of defectors, which do not cooperate. Indeed, numerous symbioses may have arisen in this way, perhaps most notably the symbioses of host cells and chemiosmotic bacteria that gave rise to the eukaryotic cell. Other examples in which one partner relies on chemiosmotic products supplied by the other include lichens, corals or other metazoans and dinoflagellates, sap-feeding insects, and plant-rhizobia and plant-mycorrhiza interactions. More problematic are cases of gut microbiomes-for instance, those of termites, ruminants, and even human beings. Under some but not all circumstances, chemiosmosis can be co-opted into punishing defectors and enforcing cooperation, thus leading to mutualistic symbioses.

Variation and multilevel selection of SARS-CoV-2.

The evolution of SARS-CoV-2 remains poorly understood. Theory predicts a group-structured population with selection acting principally at two levels: the pathogen individuals and the group of pathogens within a single host individual. Rapid replication of individual viruses is selected for, but if this replication debilitates the host before transmission occurs, the entire group of viruses in that host may perish. Thus, rapid transmission can favor more pathogenic strains, while slower transmission can favor less pathogenic strains. Available data suggest that SARS-CoV-2 may follow this pattern. Indeed, high population density and other circumstances that favor rapid transmission may also favor more deadly strains. Health care workers, exposed to pathogenic strains of hospitalized patients, may be at greater risk. The low case fatality rate on the Diamond Princess cruise ship may reflect the founder effect-an initial infection with a mild strain. A vaccine made with one strain may confer limited immunity to other strains. Variation among strains may lead to the rapid evolution of resistance to therapeutics. Finally, if less pathogenic strains are largely associated with mild disease, rather than treating all SARS-CoV-2 positive individuals equally, priority could be focused on testing and contact tracing the most seriously symptomatic patients.

Metabolic Activation and Scaling in Two Species of Colonial Cnidarians.

Metabolic activation can have a profound impact, for instance, by more than compensating for the lower resting metabolic rates of large organisms compared to smaller ones. In some animals, activity can easily be judged by the rate of muscle-driven movement. In sessile organisms, however, judging activity is less straightforward, although feeding often results in metabolic activation. Two colonial cnidarians were examined in this context, using entirely lab-grown material to remove any artifactual effects of experimental manipulations. Hydractinia symbiolongicarpus is a carnivorous hydroid that uses active muscular contractions to drive its gastrovascular fluid. Sympodium sp., on the other hand, is an octocoral that hosts photosynthetic Symbiodinium and uses cilia to propel its gastrovascular fluid. Measures of oxygen uptake indicated that feeding activated metabolism in H. symbiolongicarpus. While light treatment had no effect on subsequent dark metabolism in Sympodium sp., stress activated metabolism to an extent comparable to H. symbiolongicarpus. In both taxa, different individual size measures or synthetic size measures derived from principal component analysis produced different scaling relationships between metabolism and size. On balance, the data suggest that scaling was negatively allometric in Sympodium sp. and nearly isometric in H. symbiolongicarpus; yet metabolic activation was comparable in the two species. Regardless of the size measure used, active and resting colonies of H. symbiolongicarpus exhibited similar scaling relationships. Colonial animals may lack the large difference between resting and active metabolic rates found in highly active animals, and this may be related to how their metabolism scales with size.

Why Do Corals Bleach? Conflict and Conflict Mediation in a Host/Symbiont Community.

Coral bleaching has attracted considerable study, yet one central question remains unanswered: given that corals and their Symbiodinium symbionts have co-evolved for millions of years, why does this clearly maladaptive process occur? Bleaching may result from evolutionary conflict between the host corals and their symbionts. Selection at the level of the individual symbiont favors using the products of photosynthesis for selfish replication, while selection at the higher level favors using these products for growth of the entire host/symbiont community. To hold the selfish lower-level units in check, mechanisms of conflict mediation must evolve. Fundamental features of photosynthesis have been co-opted into conflict mediation so that symbionts that fail to export these products produce high levels of reactive oxygen species and undergo programmed cell death. These mechanisms function very well under most environmental conditions, but under conditions particularly detrimental to photosynthesis, it is these mechanisms of conflict mediation that trigger bleaching.

The evolution of individuality revisited.

Evolutionary theory is formulated in terms of individuals that carry heritable information and are subject to selective pressures. However, individuality itself is a trait that had to evolve - an individual is not an indivisible entity, but a result of evolutionary processes that necessarily begin at the lower level of hierarchical organisation. Traditional approaches to biological individuality focus on cooperation and relatedness within a group, division of labour, policing mechanisms and strong selection at the higher level. Nevertheless, despite considerable theoretical progress in these areas, a full dynamical first-principles account of how new types of individuals arise is missing. To the extent that individuality is an emergent trait, the problem can be approached by recognising the importance of individuating mechanisms that are present from the very beginning of the transition, when only lower-level selection is acting. Here we review some of the most influential theoretical work on the role of individuating mechanisms in these transitions, and demonstrate how a lower-level, bottom-up evolutionary framework can be used to understand biological complexity involved in the origin of cellular life, early eukaryotic evolution, sexual life cycles and multicellular development. Some of these mechanisms inevitably stem from environmental constraints, population structure and ancestral life cycles. Others are unique to specific transitions - features of the natural history and biochemistry that are co-opted into conflict mediation. Identifying mechanisms of individuation that provide a coarse-grained description of the system's evolutionary dynamics is an important step towards understanding how biological complexity and hierarchical organisation evolves. In this way, individuality can be reconceptualised as an approximate model that with varying degrees of precision applies to a wide range of biological systems.

An Evolutionary Framework for Understanding the Origin of Eukaryotes.

Two major obstacles hinder the application of evolutionary theory to the origin of eukaryotes. The first is more apparent than real-the endosymbiosis that led to the mitochondrion is often described as "non-Darwinian" because it deviates from the incremental evolution championed by the modern synthesis. Nevertheless, endosymbiosis can be accommodated by a multi-level generalization of evolutionary theory, which Darwin himself pioneered. The second obstacle is more serious-all of the major features of eukaryotes were likely present in the last eukaryotic common ancestor thus rendering comparative methods ineffective. In addition to a multi-level theory, the development of rigorous, sequence-based phylogenetic and comparative methods represents the greatest achievement of modern evolutionary theory. Nevertheless, the rapid evolution of major features in the eukaryotic stem group requires the consideration of an alternative framework. Such a framework, based on the contingent nature of these evolutionary events, is developed and illustrated with three examples: the putative intron proliferation leading to the nucleus and the cell cycle; conflict and cooperation in the origin of eukaryotic bioenergetics; and the inter-relationship between aerobic metabolism, sterol synthesis, membranes, and sex. The modern synthesis thus provides sufficient scope to develop an evolutionary framework to understand the origin of eukaryotes.

Conflict and cooperation in eukaryogenesis: implications for the timing of endosymbiosis and the evolution of sex.

Roughly 1.5-2.0 Gya, the eukaryotic cell evolved from an endosymbiosis of an archaeal host and proteobacterial symbionts. The timing of this endosymbiosis relative to the evolution of eukaryotic features remains subject to considerable debate, yet the evolutionary process itself constrains the timing of these events. Endosymbiosis entailed levels-of-selection conflicts, and mechanisms of conflict mediation had to evolve for eukaryogenesis to proceed. The initial mechanisms of conflict mediation (e.g. signalling with calcium and soluble adenylyl cyclase, substrate carriers, adenine nucleotide translocase, uncouplers) led to metabolic homeostasis in the eukaryotic cell. Later mechanisms (e.g. mitochondrial gene loss) contributed to the chimeric eukaryotic genome. These integral features of eukaryotes were derived because of, and therefore subsequent to, endosymbiosis. Perhaps the greatest opportunity for conflict arose with the emergence of eukaryotic sex, involving whole-cell fusion. A simple model demonstrates that competition on the lower level severely hinders the evolution of sex. Cytoplasmic mixing, however, is beneficial for non-cooperative endosymbionts, which could have used their aerobic metabolism to manipulate the life history of the host. While early evolution of sex may have facilitated symbiont acquisition, sex would have also destabilized the subsequent endosymbiosis. More plausibly, the evolution of sex and the true nucleus concluded the transition.

Structure and signaling at hydroid polyp-stolon junctions, revisited.

The gastrovascular system of colonial hydroids is central to homeostasis, yet its functional biology remains poorly understood. A probe (2',7'-dichlorodihydrofluorescein diacetate) for reactive oxygen species (ROS) identified fluorescent objects at polyp-stolon junctions that emit high levels of ROS. A nuclear probe (Hoechst 33342) does not co-localize with these objects, while a mitochondrial probe (rhodamine 123) does. We interpret these objects as mitochondrion-rich cells. Confocal microscopy showed that this fluorescence is situated in large columnar cells. Treatment with an uncoupler (2,4-dinitrophenol) diminished the ROS levels of these cells relative to background fluorescence, as did removing the stolons connecting to a polyp-stolon junction. These observations support the hypothesis that the ROS emanate from mitochondrion-rich cells, which function by pulling open a valve at the base of the polyp. The open valve allows gastrovascular fluid from the polyp to enter the stolons and vice versa. The uncoupler shifts the mitochondrial redox state in the direction of oxidation, lowering ROS levels. By removing the stolons, the valve is not pulled open, metabolic demand is lowered, and the mitochondrion-rich cells slowly regress. Transmission electron microscopy identified mitochondrion-rich cells adjacent to a thick layer of mesoglea at polyp-stolon junctions. The myonemes of these myoepithelial cells extend from the thickened mesoglea to the rigid perisarc on the outside of the colony. The perisarc thus anchors the myoepithelial cells and allows them to pull against the mesoglea and open the lumen of the polyp-stolon junction, while relaxation of these cells closes the lumen.

The impact of mitochondrial endosymbiosis on the evolution of calcium signaling.

At high concentrations, calcium has detrimental effects on biological systems. Life likely arose in a low calcium environment, and the first cells evolved mechanisms to maintain this environment internally. Bursts of calcium influx followed by efflux or sequestration thus developed in a functional context. For example, in proto-cells with exterior energy-converting membranes, such bursts could be used to depolarize the membrane. In this way, proto-cells could maintain maximal phosphorylation (metabolic state 3) and moderate levels of reactive oxygen species (ROS), while avoiding the resting state (metabolic state 4) and high levels of ROS. This trait is likely a shared primitive characteristic of prokaryotes. When eukaryotes evolved, the α-proteobacteria that gave rise to proto-mitochondria inhabited a novel environment, the interior of the proto-eukaryote that had a low calcium concentration. In this environment, metabolic homeostasis was difficult to maintain, and there were inherent risks from ROS, yet depolarizing the proto-mitochondrial membrane by calcium influx was challenging. To maintain metabolic state 3, proto-mitochondria were required to congregate near calcium influx points in the proto-eukaryotic membrane. This behavior, resulting in embryonic forms of calcium signaling, may have occurred immediately after the initiation of the endosymbiosis. Along with ROS, calcium may have served as one of the key forms of crosstalk among the community of prokaryotes that led to the eukaryotic cell.