Ontogeny of the Digestive and Feeding Systems in the Anemonefish Amphiprion Melanopus

Ontogenetic growth and development in the anemonefish Amphiprion melanopus (Pomacentridae) is very rapid when compared to other tropical and temperate fish species. A. melanopus hatched with a highly differentiated digestive tract and the ability to capture and ingest prey items. Their alimentary tract changes rapidly throughout the larval period. Concurrent with yolksac absorption at three days after hatching was the development of the stomach followed by calcification of the jaw structures. This period of acute structural change may be a critical period in their development. Metamorphosis coincided with settlement at 8 days after hatching and was marked by calcification of fins and acquirement of adult coloration. The rapid development found in this species may be a specialisation to enhance the return of larvae to a patchy and highly specific settlement habitat.     

Lysine 77 is a Key Residue in Aggregation of Equinatoxin II, a Pore-forming Toxin from Sea Anemone Actinia equina

Among eighteen point mutants of equinatoxin II produced in E. coli, containing a single cystein substitution at variable position, EqtIIK77C was chosen for its peculiar properties. It was almost 100 times less hemolytic than the wild-type, but its hemolytic activity could be restored by chemical modification of the thiol group, provided that a positive charge was reintroduced. This indicates that a positive charge at this position is necessary for toxin activity. The mutant formed larger pores as compared to the wild type, but displayed the same cation selectivity. The pores reverted to normal size upon reintroduction of the positive charge. The conformation of EqtIIK77C and its binding to lipid membranes, either vesicles or red blood cells, was almost normal. However the kinetics of calcein release from lipid vesicles was substantially slower than that of the wild-type. Taken together with the different size of the pore formed, this is an indication that mutation of Lys77 → Cys influences the normal development of the aggregate which is required for assembling the functional pore. Received: 18 May 1999/Revised: 18 September 1999     

Searching for a photocycle of the cryptochrome photoreceptors

The initial photochemistry of plant cryptochromes has been extensively investigated in recent years. It is hypothesized that cryptochrome photoexcitation involves a Trp-triad-dependent photoreduction. According to this hypothesis, cryptochromes in the resting state contain oxidized FAD; light triggers a sequential electron transfer from three tryptophan residues to reduce FAD to a neutral semiquinone (FADH*); FADH* is the presumed signaling state and it is re-oxidized to complete the photocycle. However, this photoreduction hypothesis is currently under debate. An alternative model argues that the initial photochemistry of cryptochromes involves a photolyase-like cyclic electron shuttle without a bona fide redox reaction mediated by the Trp-triad residues, leading to conformational changes, signal propagation, and physiological responses.     

Contributions to a molecular phylogeny and systematics of Anemone and related genera (Ranunculaceae-Anemoninae)

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Contributions to a molecular phylogeny and systematics of Anemone and related genera (Ranunculaceae-Anemoninae)

Plastid atpB/rbcL intergenic spacer sequences were obtained from 21 selected taxa and one hybrid of Anemoninae (Anemone, Pulsatilla, Hepatica) and compared with Ficaria (Ranunculinae) as an outgroup. From the resulting matrix (1226 bp) a single most parsimonious tree was obtained (Fig. 1 ). The branching of this tree is confirmed by many informative indels and appears largely congruent with past plastid restriction analyses. Several new taxa are added. The monophyly of the Anemoninae and their early split into two major clades is supported: clade I with the chromosome base number x = 8, clade Ⅱ with the reduced x = 7. Clade Ⅰ is made up of the basal Pulsatilla and the Rivularis + Vitifolia groups. The Multifida group links to the crown groups Coronaria, Blanda and Nemorosa. Clade Ⅱ consists of the basal Dichotoma group, followed by Hepatica, and finally by the N. Hemisphere Narcissiflora and the S. Hemisphere Antucensis groups as sisters. The problems of the Anemoninae ancestry, phylogenetic differentiation, and recent attempts for systematic classification are critically discussed. In view of the still incomplete sampling of DNA data, aconservative and informal approach to classification problems is recommended.     

Harnessing Evolution to Elucidate the Consequences of Symbiosis

Many organisms harbor microbial associates that have profound impacts on host traits. The phenotypic effect of symbionts on their hosts may include changes in development, reproduction, longevity, and defense against natural enemies. Determining the consequences of associating with a microbial symbiont requires experimental comparison of hosts with and without symbionts. Then, determining the mechanism by which symbionts alter these phenotypes can involve genomic, genetic, and evolutionary approaches; however, many host-associated symbionts are not amenable to genetic approaches that require cultivation of the microbe outside the host. In the current issue of PLOS Biology, Chrostek and Teixeira highlight an elegant approach to studying functional mechanisms of symbiont-conferred traits. They used directed experimental evolution to select for strains of Wolbachia wMelPop (a bacterial symbiont of fruit flies) that differed in copy number of a region of the genome suspected to underlie virulence. Copy number evolved rapidly when under selection, and wMelPop strains with more copies of the region shortened the lives of their Drosophila hosts more than symbionts with fewer copies. Interestingly, the wMelPop strains with more copies also increase host resistance to viruses compared to symbionts with fewer copies. Their study highlights the power of exploiting alternative approaches when elucidating the functional impacts of symbiotic associations.Symbioses, long-term and physically close interactions between two or more species, are central to the ecology and evolution of many organisms. Though “Symbiosis” is more often used to define interactions that are presumed to be mutually beneficial to a host and its microbial partner, a broader definition including both parasitic and mutualistic interactions recognizes that the fitness effects of many symbioses are complex and often context dependent. Whether an association is beneficial can depend on ecological conditions, and mutation and other evolutionary processes can result in symbiont strains that differ in terms of costs and benefits to hosts (Fig. 1).Open in a separate windowFig 1The symbiosis spectrum.The costs and benefits of symbiosis for hosts are not bimodal but span a continuum. The benefit to cost ratio is mediated both by environmental conditions and by the strain of symbiont. For example, the bacteria Hamiltonella defensa increases aphid resistance to parasitoid wasps. When Hamiltonella loses an associated bacteriophage, protection is lost. Also, in aphids, Buchnera aphidicola is a bacterial symbiont that provisions its hosts with critical nutritional resources. However, alterations of the heat shock promoter in Buchnera lessen the fitness benefit of symbiosis for the hosts under elevated temperatures. Amplification of a region of the Wolbachia genome known as Octomom causes the bacteria to shorten the lifespan of its Drosophila fly hosts.Elucidating the effects of host-associated microbes includes, when possible, experiments designed to assay host phenotypes when they do and do not have a particular symbiont of interest (Fig. 2). In systems in which hosts acquire symbionts from the environment, hosts can be reared in sterile conditions to prevent acquisition [1]. If symbionts are passed internally from mother to offspring, antibiotic treatments can sometimes be utilized to obtain lineages of hosts without symbionts [2]. The impacts of symbiont presence on survival, development, reproduction, and defense can be quantified, with the caveat that these impacts may be quite different under alternative environmental conditions. While such experiments are sometimes more tractable in systems with simple microbial consortia, the same experimental processes can be utilized in systems with more complex microbial communities [3,4].Open in a separate windowFig 2Approaches to functionally characterize symbiont effects.The first step in functionally characterizing the phenotypic impacts of a symbiont on its host is to measure phenotypes of hosts with and without symbionts. Any effects need to be considered in the light of how they are modified by environmental conditions. Understanding the mechanisms underlying symbiont alteration of host phenotype can involve, and often combines, genomic, genetic, and evolutionary approaches. Solid arrows indicate the path leading to results highlighted in Chrostek and Teixeira’s investigation of Wolbachia virulence in this issue of PLoS Biology.Once a fitness effect of symbiosis is ascertained, determining the mechanistic basis of this effect can be challenging. A genomics approach sometimes provides informative insight into microbial function. Sequencing of many insect-associated symbionts, for example, has confirmed the presence of genes necessary for amino acid and vitamin synthesis [5–8]. These genomic revelations, in some cases, can be linked to phenotypic effects of symbiosis for the hosts. For example, aphids reared in the absence of their obligate symbiotic bacteria, Buchnera aphidicola, can survive when provisioned with supplemental amino acids but cannot survive without supplementation, suggesting that Buchnera’s provisioning of amino acids is critical for host survival [9,10]. The Buchnera genome contains many of the genes necessary for amino acid synthesis [5].Linking genotype to phenotype, however, can be complicated. Experiments are necessary to functionally test the insights garnered from genome sequencing. For example, just because a symbiont has genes necessary for synthesis of a particular nutrient does not mean that the nutrient is being provisioned to its host. Furthermore, in many systems we do not know what genetic mechanisms are most likely to influence a symbiont-conferred phenotype. For example, if hosts associated with a given microbe have lower fitness than those without the microbe, what mechanism mediates this phenotype? Is it producing a toxin? Is it using too many host resources? In these cases, a single genome provides even less insight.Comparative genomics can be another approach. This requires collection of hosts with alternative symbiont strains and then testing these strains in a common host background to demonstrate that they have different phenotypic effects. Symbiont genomes can then be sequenced and compared to identify differences. This approach was utilized to compare genomes of strains of the aphid bacterial symbiont Regiella insecticola that confer different levels of resistance to parasitoid wasps [11]; the protective and nonprotective Regiella genome differed in many respects. Comparing the genomes of Wolbachia strains with differential impacts on fly host fitness [12,13] revealed fewer differences, though none involved a gene with a function known to impact host fitness. Comparative genomics rarely uncovers a holy grail as the genomes of symbiont strains with alternative phenotypic effects rarely differ at a single locus of known function.Another approach, which is at the heart of studies of microbial pathogens, is to use genetic tools to manipulate symbionts at candidate loci (or randomly through mutagenesis) and compare the phenotypic effects of genetically-manipulated and unmanipulated symbionts. Indeed, this approach has provided insights into genes underlying traits of both pathogenic [14] and beneficial [15,16] microbes. There is one challenge. Many host-associated symbionts are not cultivable outside of their hosts, which precludes utilization of most traditional genetic techniques used to modify microbial genomes.An alternative approach to studying symbiont function leverages evolution. Occasionally, lineages that once conferred some phenotypic effect, when tested later, no longer do. If symbiont samples were saved along the way, researchers can then determine what in the genome changed. For example, pea aphids (Acyrthosiphon pisum) harboring the bacteria Hamiltonella defensa are more resistant to parasitoid wasps than those without the bacteria [17,18]. Toxin-encoding genes identified in the genome of a Hamiltonella-associated bacteriophage were hypothesized to be central to this defense [18,19]. However, confirmation of the bacteriophage’s role required comparing the insects’ resistance to wasps when they harbored the same Hamiltonella with and without the phage. No Hamiltonella isolates were found in nature without the phage, but bottleneck passaging of the insects and symbionts generation after generation in the laboratory led to the loss of phage in multiple host lineages. Experimental assays confirmed that in the absence of phage, there was no protection [20]. Similarly, laboratory passaging of aphids and symbionts serendipitously led to spread of a mutation in the genome of Buchnera aphidicola, the primary, amino acid-synthesizing symbiont of pea aphids. The mutation, a single nucleotide deletion in the promoter for ibpA, a gene encoding for a heat-shock protein, lowers aphid fitness under elevated temperature conditions [21]. The mutation is found at low levels in natural aphid populations, suggesting that laboratory conditions facilitate maintenance of the genotype.In the above cases, evolution was a fortunate coincidence. In this issue of PLoS Biology, Chrostek and Teixeira (2014) illustrate another alternative, directed experimental evolution. Previous work demonstrated that a strain of the symbiotic bacterium Wolbachia, wMelPop, is virulent to its Drosophila melanogaster hosts, considerably shortening lifespan while overproliferating inside the flies [22]. To investigate the mechanism of virulence, researchers compared the genomic content of an avirulent Wolbachia strain to that of the virulent wMelPop [12,13]. These comparisons revealed that the wMelPop genome contains a region with eight genes that is amplified multiple times; in avirulent strains there is only a single copy. This eight gene region was nicknamed “Octomom.” To functionally test whether Octomom mediates Wolbachia virulence, over successive generations, Chrostek and Teixeira selected for females with either high or low Octomom copy numbers to start the next generations. They found that copy number could evolve rapidly and was correlated with virulence. Flies harboring wMelPop with more copies of Octomom had shorter lifespans. This cost was reversed in the presence of natural enemies; flies harboring wMelPop with more copies of Octomom had higher resistance to viral pathogens. Thus, selection provided a functional link between genotype and phenotype in a symbiont recalcitrant to traditional microbial genetics approaches.In many respects, this is similar to the research on aphids and their symbionts, where protective phenotypes were lost through passaging of aphids and symbionts generation after generation, as part of standard laboratory maintenance. Chrostek and Teixeira simply used the tools of experimental evolution to select for altered symbionts in a controlled fashion. Comparison of the studies also highlights two potential approaches—select for a phenotype and determine the genotypic change, or select for a genotype of interest and determine the phenotypic effect.Why do we need to know the genetic mechanisms underlying symbiont-conferred traits? In terms of evolutionary dynamics, the maintenance of a symbiont’s effect in a population is predicated on the likelihood of it being maintained in the presence of mutation, drift, and selection. Symbiosis research often considers how ecological conditions influence symbiont-conferred traits but less often considers the instability of those influences due to evolutionary change. From the perspective of applied applications to human concerns, symbiont alteration of insect phenotypes are potential mechanisms to reduce vectoring of human and agricultural pathogens, either through directly reducing insect fitness or reducing the capacity of vectors to serve as pathogen reservoirs [23–28]. Short term field trials, for example, have demonstrated spread and persistence of Wolbachia in mosquito populations [29,30]. Because Wolbachia reduce persistence of viruses, including human pathogens, in insects [26,31–33], this is a promising pesticide-free and drug-free control strategy for insect-vectored diseases. Can we assume that Wolbachia and other symbionts will always confer the same phenotypes to their hosts? If the conferred phenotype is based on a region of the genome where mutation is likely (e.g., the homopolymeric track within the heat shock promoter of aphid Buchnera, the Octomom region in Drosophila wMelPop), then we have clear reason to suspect that the genotypic and phenotypic makeup of the symbiont population could change over time. We need to investigate how populations of bacterial symbionts evolve in host populations under natural ecological conditions, carefully screening for both changes in phenotype and changes in genotype over the course of such experimental observations. We then need to incorporate evolutionary changes when modeling symbiont maintenance and when considering the use of symbionts in applied applications.     

Response to underwater laser pointer in the Orange-finned Anemonefish Amphiprion chrysopterus and three-spot damselfish Dascyllus trimaculatus

Response of orange-finned anemonefish Amphiprion chrysopterus and three-spot damselfish Dascyllus trimaculatus to red laser-pointer light was studied in Mo'orea, French Polynesia. Four magnificent anemones Heteractis magnifica and their resident fish were observed for typical behaviours (biting, chasing, hiding, posing, lunging and retreating) with and without exposure to laser-pointer light. Lunging behaviour increased significantly for both fish species upon exposure to laser-pointer light; none of the other behaviours changed significantly. We advance the hypothesis that orange-finned anemonefish and three-spot damselfish interpret laser pointer stimulation as a territorial threat.     

Sensitivity of the woodland herb Anemone hepatica to changing environmental conditions

Abstract. The decline of deciduous woodland populations of Anemone hepatica L. in southern Sweden is documented and possible causes are discussed. The study was based on (1) re‐investigations of 6.25 km² grid‐squares first studied in 1938–1970, (2) distribution of A. hepatica in woodland sites with well‐known soil chemical properties and (3) a detailed study over 12 consecutive years into the relationships between biological characteristics of the species (number of individuals, vegetative development, flowering frequency) and environmental variables (temperature, precipitation), soil chemistry and time. There was a close relationship between soil acidity (pH, solubility of Al³⁺) and both distribution and biological characteristics. The biological variables declined significantly over time but were not related to climatic variability. Increasing soil acidity and Al³⁺ solubility are concluded to be the main factors responsible for the decline of A. hepatica in S. Swedish deciduous woodlands.     

Barriers to Industrial Symbiosis: Insights from the Use of a Maturity Grid

The concept of industrial symbiosis (IS) over the last 20 years has become a well‐recognized approach for environmental improvements at the regional level. Many technical solutions for waste and by‐product material, water, and energy reuse between neighboring industries (so‐called synergies) have been discovered and applied in the IS examples from all over the world. However, the potential for uptake of new synergies in the regions is often limited by a range of nontechnical barriers. These barriers include environmental regulation, lack of cooperation and trust between industries in the area, economic barriers, and lack of information sharing. Although several approaches to help identify and overcome some of the nontechnical barriers were examined, no methodology was found that systematically assessed and tracked the barriers to guide the progress of IS development. This article presents a new tool—IS maturity grid—to tackle this issue in the regional IS studies. The tool helps monitor and assess the level of regional industrial collaboration and also indicates a potential path for further improvements and development in an industrial region, depending on where that region currently lies in the grid. The application of the developed tool to the Gladstone industrial region of Queensland, Australia, is presented in the article. It showed that Gladstone is at the third (active) stage of five stages of maturity, with cooperation and trust among industries the strongest characteristic and information barriers the characteristic for greatest improvement.     

Territorial Behavior and Ecology of the Anemonefish Amphiprion melanopus on Guam1)

Field observations and experiments on the Indo-Pacific anemonefish Amphiprion melanopus at Guam show that it colonizes the aggregating sea anemone Physobrachia douglasi extensively and nearly exclusively. There are an average of four fish per colony. The total standard length of anemonefish at each aggregation is highly correlated with the total area covered by resident anemones, suggesting both a carrying capacity and some form of social control of growth of anemonefish²). Inter-colony migration of juveniles, as well as larval recruitment, may contribute to maintenance of the optimum colony size. Adults do not migrate. Both juveniles and adults defend territory. Juvenile territories are mutually exclusive areas within the confines of the anemone aggregation, while adult territories are considerably larger than the area covered by the anemone aggregation. Though territories of mated ♀♀ and ♂♂ overlap completely, female emphasis is peripheral relative to the ♂. Adult conspecific intruders are attacked more heavily and at greater distances than are juveniles. Intraspecific territoriality in juveniles probably reflects the limited availability of critical habitat. In adults it may function to protect the pair bond. Interspecific aggression is less intense and appears to protect both the spawn and host anemones from various predators.     

Industrial Symbiosis Dynamics and the Problem of Equivalence: Proposal for a Comparative Framework

Industrial symbiosis (IS), one of the founding notions within the field of industrial ecology, has diffused throughout significant parts of the world as a practice that can reduce the ecological impact of the industrial processes of groups of firms. In this article, we propose a fresh look at this research topic, building on the considerable advances that have been made in the last 15 years in understanding how IS comes about. We propose a conceptual and theoretical framework for taking on the challenge of comparative analysis at a global level. This requires developing an approach to address a solution to the problem of equivalence: the difficulty of comparing instances of IS across different institutional contexts. The proposed framework emphasizes IS as a process and attempts to address the obstacles to comparative study by (1) identifying terminology to examine IS variants, (2) providing a typology of IS dynamics, and (3) formulating key research questions to illuminate a way forward. In developing our argument, we build on the collective experiences of collaborative research efforts in North America, Europe, and Asia as evidenced in recent overviews of the literature.     

Symbiosis and the origin of life

The paper uses chemical kinetic arguments and illustrations by computer modelling to discuss the origin and evolution of life. Complex self-reproducing chemical systems cannot arise spontaneously, whereas simple auto-catalytic systems can, especially in an irradiated aqueous medium. Self-reproducing chemical particles of any complexity, in an appropriate environment, have a self-regulating property which permits long-term survival. However, loss of materials from the environment can lead to continuing decay which is circumvented by physical union between different kinds of self-reproducing particles. The increasing complexity produced by such unions (symbioses) is irreversible so that the chemical system evolves. It is suggested that evolution by successive symbioses brough about the change from simple, spontaneously arising, auto-catalytic particles to complex prokaryotic cells.     

Symbiosis and the evolution of prokaryotes

It is postulated, with support from kinetic modelling, that a succession of symbioses was the major process of evolution during the early stages of life. The process became less effective with the passage of time, while evolution by the natural selection of variants became more effective. The postulate may contribute usefully to discussions on the evolution of biochemical complexity and the structure of cells.     

Symbiosis and the Regulation of Communities

Ecologists have long been interested in factors that controlthe structure of communities and the relative importance oftop-down effects of predators versus bottom-up effects of resources.There is a growing body of evidence that microbial symbiosesare important determinants of plant community structure andindirectly affect herbivore and predator trophic levels. Studieswith mycorrhizal fungi, N-fixing bacteria and endophytes ofgrasses have demonstrated that they can affect competition,coexistence, soil nutrient dynamics and plant-herbivore interactions.Long-term field experiments with one grass/endophyte interactionsuggest that grassland community structure is determined bythe fungus. While total plant productivity of experimental plotswas similar, the composition of the vegetation was altered byendophyte symbiosis. The host grass tall fescue (Festuca arundinacea)dominated plots when infected while other grasses greatly increasedin uninfected plots. Indirect evidence suggests that changesin prairie vole (Microtus ochrogaster) grazing patterns andreproductive physiology may be in part responsible for vegetationalchanges. These results provide evidence that, in addition tobottom-up and top-down forces, microbial symbionts of plantsare important determinants of community structure.