Taxonomy and nomenclature of Oophila amblystomatis (Chlorophyceae, Chlamydomonadales).

The unicellular green alga Oophila amblystomatis was named by Lambert in 1905 based upon its association with egg masses of the spotted salamander Ambystoma maculatum. We collected algal cells from Lambert's original egg capsule preparations that were contributed to Phycotheca Boreali-Americana (PBA) in 1905 and subjected them to DNA extraction and PCR with O. amblystomatis-specific 18S rRNA gene primers. DNA amplified from these preparations was cloned and nine clones were sequenced. Along with representative sequences from the Oophila clade and Chlorophyceae, a phylogenetic tree was inferred. Seven sequences clustered within the Oophila clade and two clustered with Chlamydomonas moewusii, which is included in a sister clade to Oophila. By sequencing algal material from the egg capsules of representative type material we can unambiguously characterize O. amblystomatis and define a monophyletic clade centered on this type material. Accordingly, we reject a recent proposal that this species be transferred to Chlorococcum.

Bacterial Diversity in Egg Capsular Fluid of the Spotted Salamander Ambystoma maculatum Decreases with Embryonic Development.

Egg capsules within egg masses of the spotted salamander Ambystoma maculatum host a symbiosis with the unicellular green alga Oophila amblystomatis. However, this alga is not the only microbe to inhabit those capsules, and the significance of these additional taxa for the symbiosis is unknown. Spatial and temporal patterns of bacterial diversity in egg capsules of A. maculatum have recently begun to be characterized, but patterns of bacterial diversity as a function of embryonic development are unknown. We sampled fluid from individual capsules in egg masses over a large range of host embryonic development in 2019 and 2020. We used 16S rRNA gene amplicon sequencing to examine how diversity and relative abundance of bacteria changed with embryonic development. In general, bacterial diversity decreased as embryos developed; significant differences were observed (depending on the metric) by embryonic development, pond, and year, and there were interaction effects. The function of bacteria in what is thought of as a bipartite symbiosis calls for further research.

(Epi)genomic adaptation driven by fine geographical scale environmental heterogeneity after recent biological invasions.

Elucidating processes and mechanisms involved in rapid local adaptation to varied environments is a poorly understood but crucial component in management of invasive species. Recent studies have proposed that genetic and epigenetic variation could both contribute to ecological adaptation, yet it remains unclear on the interplay between these two components underpinning rapid adaptation in wild animal populations. To assess their respective contributions to local adaptation, we explored epigenomic and genomic responses to environmental heterogeneity in eight recently colonized ascidian (Ciona intestinalis) populations at a relatively fine geographical scale. Based on MethylRADseq data, we detected strong patterns of local environment-driven DNA methylation divergence among populations, significant epigenetic isolation by environment (IBE), and a large number of local environment-associated epigenetic loci. Meanwhile, multiple genetic analyses based on single nucleotide polymorphisms (SNPs) showed genomic footprints of divergent selection. In addition, for five genetically similar populations, we detected significant methylation divergence and local environment-driven methylation patterns, indicating the strong effects of local environments on epigenetic variation. From a functional perspective, a majority of functional genes, Gene Ontology (GO) terms, and biological pathways were largely specific to one of these two types of variation, suggesting partial independence between epigenetic and genetic adaptation. The methylation quantitative trait loci (mQTL) analysis showed that the genetic variation explained only 18.67% of methylation variation, further confirming the autonomous relationship between these two types of variation. Altogether, we highlight the complementary interplay of genetic and epigenetic variation involved in local adaptation, which may jointly promote populations' rapid adaptive capacity and successful invasions in different environments. The findings here provide valuable insights into interactions between invaders and local environments to allow invasive species to rapidly spread, thus contributing to better prediction of invasion success and development of management strategies.

Physiological benefits and latent effects of an algal-salamander symbiosis.

Embryos of the salamander Ambystoma maculatum (Shaw) and the uni-cellular green alga Oophila amblystomatis (Lambert ex Wille) have evolved a resource exchange mutualism. Whereas some of the benefits of the symbiosis to embryos are known, the physiological limitations of the relationship to embryos and carry over or latent effects on larvae are not. To determine the impact of the relationship across life history stages, we measured the growth, survival, and metabolic rate in response to hypoxia of salamander embryos reared under 0-h light (algae absent), 14-h light (control - algae present, fluctuating light conditions) and 24-h light (algae present, chronic light conditions) and the resulting larvae two-weeks post hatch. Embryos reared under 0-h light demonstrated decreased growth and survival compared to 14- and 24-h light, with no effect on metabolic rates or the response of metabolic rates to declining oxygen partial pressure (pO2). Conversely, larvae from embryos reared under 0-h light exhibited compensatory growth during the two-week larval rearing period, with body sizes matching those from the 14-h light treatment. Larvae from embryos reared under 24-h light had lower wet body mass and LT50 values upon starvation compared to those reared under 14-h light. Coupled with the lowest metabolic rates under normoxic pO2 levels, this indicates the presence of negative latent effects. We discuss the findings in relation to the effect of the symbiotic relationship on hypoxia tolerance and larval fitness with respect to the presence of compensatory growth and negative latent effects.

Differences in Larval Arm Movements Correlate with the Complexity of Musculature in Two Phylogenetically Distant Echinoids, Eucidaris tribuloides (Cidaroidea) and Lytechinus variegatus (Euechinoidea).

Within a common body plan, echinoid planktotrophic larvae are morphologically diverse, with variations in overall size, the length, and number of arms and the presence or absence of epidermal structures. In this report, we are interested in variation in larval arm-flexing behavior and correlated differences in larval musculature. Larvae of the cidaroid Eucidaris tribuloides exhibit conspicuous and regular arm-flexing behavior. In contrast, Lytechinus variegatus, a representative of the euechinoid clade, does not exhibit this behavior. We hypothesized that there were differences in musculature that correlated with this behavioral contrast and compared the development and structure of larval muscles between these species. We report substantial differences in some aspects of larval musculature. In addition to previously described oral musculature, both larvae possessed polygon-shaped musculature at the basal end of the larva. However, larval musculature in E. tribuloides was larger and contained additional muscles not observed in larvae of L. variegatus. Therefore, a conspicuous larval behavior consisting of repeated flexing of the postoral and posterodorsal larval arms was correlated with a larger, more complex musculature. This simple contrast indicates that larval musculature not associated with endoderm evolves in a manner that relates to differences in larval behavior and that additional comparisons are warranted.

Fouling-release and chemical activity effects of a siloxane-based material on tunicates.

The antifouling performance of a siloxane-based elastomeric impression material (EIM) was compared to that of two silicone fouling-release coatings, Intersleek 757 and RTV-11. In field immersion trials, the EIM caused the greatest reduction in fouling by the solitary tunicate Ciona intestinalis and caused the longest delay in the progression of fouling by two species of colonial tunicate. However, in pseudobarnacle adhesion tests, the EIM had higher attachment strengths. Further laboratory analyses showed that the EIM leached alkylphenol ethoxylates (APEs) that were toxic to C. intestinalis larvae. The EIM thus showed the longest duration of chemical activity measured to date for a siloxane-based coating (4 months), supporting investigations of fouling-release coatings that release targeted biocides. However, due to potential widespread effects of APEs, the current EIM formulation should not be considered as an environmentally-safe antifoulant. Thus, the data also emphasize consideration of both immediate and long-term effects of potentially toxic constituents released from fouling-release coatings.

In Situ Confocal Raman Microscopy of Hydrated Early Stages of Bacterial Biofilm Formation on Various Surfaces in a Flow Cell.

Bacterial biofilms are precursors to biofouling by other microorganisms. Understanding their initiation may allow us to design better ways to inhibit them, and thus to inhibit subsequent biofouling. In this study, the ability of confocal Raman microscopy to follow the initiation of biofouling by a marine bacterium, Pseudoalteromonas sp. NCIMB 2021 (NCIMB 2021), in a flow cell, using optical and confocal Raman microscopy, was investigated. The base of the flow cell comprised a cover glass. The cell was inoculated and the bacteria attached to, and grew on, the cover glass. Bright field images and Raman spectra were collected directly from the hydrated biofilms over several days. Although macroscopically the laser had no effect on the biofilm, within the first 24 h cells migrated away from the position of the laser beam. In the absence of flow, a buildup of extracellular substances occurred at the base of the biofilm. When different coatings were applied to cover glasses before they were assembled into the flow cells, the growth rate, structure, and composition of the resulting biofilm was affected. In particular, the ratio of Resonance Raman peaks from cytochrome c (CC) in the extracellular polymeric substances, to the Raman phenylalanine (Phe) peak from protein in the bacteria, depended on both the nature of the surface and the age of the biofilm. The ratios were highest for 24 h colonies on a hydrophobic surface. Absorption of a surfactant with an ethyleneoxy chain into the hydrophobic coating created a surface similar to that given with a simple PEG coating, where bacteria grew in colonies away from the surface rather than along the surface, and CC:Phe ratios were initially low but increased at least fivefold in the first 48 h.

One rhinophore probably provides sufficient sensory input for odour-based navigation by the nudibranch mollusc Tritonia diomedea.

Tritonia diomedea (synonymous with Tritonia tetraquetra) navigates in turbulent odour plumes, crawling upstream towards prey and downstream to avoid predators. This is probably accomplished by odour-gated rheotaxis, but other possibilities have not been excluded. Our goal was to test whether T. diomedea uses odour-gated rheotaxis and to simultaneously determine which of the cephalic sensory organs (rhinophores and oral veil) are required for navigation. In a first experiment, slugs showed no coherent responses to streams of odour directed at single rhinophores. In a second experiment, navigation in prey and predator odour plumes was compared between animals with unilateral rhinophore lesions, denervated oral veils, or combined unilateral rhinophore lesions and denervated oral veils. In all treatments, animals navigated in a similar manner to that of control and sham-operated animals, indicating that a single rhinophore provides sufficient sensory input for navigation (assuming that a distributed flow measurement system would also be affected by the denervations). Amongst various potential navigational strategies, only odour-gated positive rheotaxis can produce the navigation tracks we observed in prey plumes while receiving input from a single sensor. Thus, we provide strong evidence that T. diomedea uses odour-gated rheotaxis in attractive odour plumes, with odours and flow detected by the rhinophores. In predator plumes, slugs turned downstream to varying degrees rather than orienting directly downstream for crawling, resulting in greater dispersion for negative rheotaxis in aversive plumes. These conclusions are the first explicit confirmation of odour-gated rheotaxis as a navigational strategy in gastropods and are also a foundation for exploring the neural circuits that mediate odour-gated rheotaxis.

Neural development in Eucidaris tribuloides and the evolutionary history of the echinoid larval nervous system.

The structure and development of the larval nervous systems of all classes of echinoderms have been described and details of embryonic signaling mechanisms patterning neurogenesis have been revealed experimentally in sea urchins. Several features of neuroanatomy and neural development indicate that echinoids are the most derived group. Here we describe the development and organization of the nervous system of a cidaroid, Eucidaris tribuloides. The cidaroids are one of two major clades of echinoids, and are considered to have features of anatomy and development that represent the common ancestor to all echinoids. The embryos of E. tribuloides lack a thickened animal plate and serotonergic neurons arise laterally, associated with the ciliary band. Although lacking a discrete apical organ, plutei have serotonergic neurons associated with the pre-oral ciliary band joined by a few diffusely arranged connecting axons. Chordin and Hnf6, early markers for oral ectoderm and ciliary band, are expressed in similar patterns to euechinoids. However, an animal pole domain marker, Nk2.1, is expressed in a broader region of anterior ectoderm than in euechinoids. Six3, a proneural marker that is restricted to the animal plate of euechinoids, is expressed laterally in the preoral ciliary band at the same location as the serotonergic neurons. We conclude that the organization and development of the larval nervous system of E. tribuloides retains features shared with other echinoderm larvae, but not with euechinoids. These data support a model in which several distinctive features of euechinoid neural organization are derived, having arisen after the divergence of the two clades of echinoids about 265 million years ago. We hypothesize that differences in the developmental mechanisms that restrict neurogenesis to the animal pole forms the basis for the distinctive neuroanatomy of euechinoids.

Intracellular invasion of green algae in a salamander host.

The association between embryos of the spotted salamander (Ambystoma maculatum) and green algae ("Oophila amblystomatis" Lamber ex Printz) has been considered an ectosymbiotic mutualism. We show here, however, that this symbiosis is more intimate than previously reported. A combination of imaging and algal 18S rDNA amplification reveals algal invasion of embryonic salamander tissues and cells during development. Algal cells are detectable from embryonic and larval Stages 26-44 through chlorophyll autofluorescence and algal 18S rDNA amplification. Algal cell ultrastructure indicates both degradation and putative encystment during the process of tissue and cellular invasion. Fewer algal cells were detected in later-stage larvae through FISH, suggesting that the decline in autofluorescent cells is primarily due to algal cell death within the host. However, early embryonic egg capsules also contained encysted algal cells on the inner capsule wall, and algal 18S rDNA was amplified from adult reproductive tracts, consistent with oviductal transmission of algae from one salamander generation to the next. The invasion of algae into salamander host tissues and cells represents a unique association between a vertebrate and a eukaryotic alga, with implications for research into cell-cell recognition, possible exchange of metabolites or DNA, and potential congruence between host and symbiont population structures.

HSP90 expression in two migratory cell types during ascidian development: test cells deposit HSP90 on the larval tunic.

Heat shock protein 90 (HSP90) is a ubiquitously expressed molecular chaperone that controls the folding, assembly and activity of proteins, many of which are involved in signal transduction. Recent work has shown that HSP90 is present extracellularly, indicating a heretofore under-appreciated requirement for extracellular chaperoning, particularly among migratory cells. We applied immunological and surgical techniques to document the differential expression of HSP90 during ascidian development. Relative to other cell types during development, test cells and trunk lateral cells (TLCs), two migratory cell types in the ascidian Boltenia villosa, express elevated levels of HSP90. Late in embryogenesis, test cells deposit HSP90 onto the tunic, the second report of extracellular HSP90 during animal development. The pyurid ascidian Halocynthia igaboja and the styelids Cnemidocarpa finmarkiensis and Botrylloides violaceus all express HSP90 at elevated levels in larval mesenchyme, suggesting that this pattern of expression is widespread in the Ascidiaceae. We show that HSP90 expression in TLCs and test cells is coincident with the presence of HNK-1. Whereas in B. violaceous, cell populations expressing elevated levels of HSP90 are distinct from those expressing HNK-1, in B. villosa both these antigens are present in the TLCs. We evaluate existing hypotheses about test cell function and, in reference to the similarities between test cells and some of the descendants of TLCs, hypothesize that test cells are TLC descendants. Implications for the proposed evolutionary relationship between TLCs and neural crest are briefly discussed.

Sniffing out new data and hypotheses on the form, function, and evolution of the echinopluteus post-oral vibratile lobe.

The performance requirements of ciliary band feeding explain the convoluted forms of many marine invertebrate larvae. Convolutions increase surface area and therefore feeding rates per unit body volume. We review recent advances in morphology, neural development, and behavior at settlement of the echinoid Lytechinus pictus and provide new ultrastructural and expression data on larvae of its congener, L. variegatus. Larvae of the echinometrid Colobocentrotus atratus contain neurons identified by their expression of nitric oxide synthase (NOS), indicating that this character is not unique to Lytechinus. We hypothesize that in some echinoids the convoluted shape of the post-oral vibratile lobe (POVL) covaries with the distribution of identified sensory neurons to enable olfaction during settlement. An analysis of variation in structural elaboration of the post-oral transverse ciliary band (PTB) within Echinoida and in feeding larvae of other echinoderm classes indicates that only echinoids, but not all echinoids, possess this novel character; larvae that do are distributed heterogeneously within the class. In recognition of this specialized function for the POVL and surrounding ectoderm, and because it is lobate and grows toward the mouth, we propose naming this structure the adoral lobe.

Analysis of nitric oxide-cyclic guanosine monophosphate signaling during metamorphosis of the nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia).

The gas nitric oxide (NO), and in some cases its downstream second messenger, cyclic guanosine monophosphate (cGMP) function in different taxa to regulate the timing of life-history transitions. Increased taxonomic sampling is required to foster conclusions about the evolution and function of NO/cGMP signaling during life-history transitions. We report on the function and localization of NO and cGMP signaling during metamorphosis of the nudibranch Phestilla sibogae. Pharmacological manipulation of NO or cGMP production in larvae modulated responses to a natural settlement cue from the coral Porites compressa in a manner that suggest inhibitory function for NO/cGMP signaling. However, these treatments were not sufficient to induce metamorphosis in the absence of cue, a result unique to this animal. We show that induction of metamorphosis in response to the settlement cue is associated with a reduction in NO production. We documented the expression of putative NO synthase (NOS) and the production of cGMP during larval development and observed no larval cells in which NOS and cGMP were both detected. The production of cGMP in a bilaterally symmetrical group of cells fated to occupy the distal tip of rhinophores is correlated with competence to respond to the coral settlement cue. These results suggest that endogenous NO and cGMP are involved in modulating responses of P. sibogae to a natural settlement cue. We discuss these results with respect to habitat selection and larval ecology.

Ontogeny of the holothurian larval nervous system: evolution of larval forms.

Echinoderm larvae share numerous features of neuroanatomy. However, there are substantial differences in specific aspects of neural structure and ontogeny between the dipleurula-like larvae of asteroids and the pluteus larvae of echinoids. To help identify apomorphic features, we have examined the ontogeny of the dipleurula-like auricularia larva of the sea cucumber, Holothuria atra. Neural precursors arise in the apical ectoderm of gastrulae and appear to originate in bilateral clusters of cells. The cells differentiate without extensive migration, and they align with the developing ciliary bands and begin neurogenesis. Neurites project along the ciliary bands and do not appear to extend beneath either the oral or aboral epidermis. Apical serotonergic cells are associated with the preoral loops of the ciliary bands and do not form a substantial commissure. Paired, tripartite connectives form on either side of the larval mouth that connect the pre-oral, post-oral, and lateral ciliary bands. Holothurian larvae share with hemichordates and bipinnariae a similar organization of the apical organ, suggesting that the more highly structured apical organ of the pluteus is a derived feature. However, the auricularia larva shares with the pluteus larva of echinoids several features of neural ontogeny. Both have a bilateral origin of neural precursors in ectoderm adjacent to presumptive ciliary bands, and the presumptive neurons move only a few cell diameters before undergoing neurogenesis. The development of the holothurian nervous systems suggests that the extensive migration of neural precursors in asteroids is a derived feature.

Development of nitric oxide synthase-defined neurons in the sea urchin larval ciliary band and evidence for a chemosensory function during metamorphosis.

We previously reported that initiation of metamorphosis of larvae of Lytechinus pictus is negatively regulated by nitric oxide (NO) and cGMP. We have examined the expression of nitric oxide synthase (NOS) and cGMP in cells of the developing larva. A section of the post-oral ciliary band of feeding larvae includes neural cells defined by their expression of both NOS and the echinoderm neural-specific antibody 1E11. These neurons project processes to the pre-oral neuropile during larval development. Larvae regenerated this section of the ciliary band after its excision, complete with NOS-defined neurons that projected again to the pre-oral neuropile. Excision of ectoderm containing the post-oral ciliary band prevented a behavioral and morphogenetic response of competent larvae to biofilm, and delayed initiation of metamorphosis. Elevated cGMP levels were detected in several larval and juvenile cell types prior to metamorphosis. Treatment of larvae with ODQ, an inhibitor of soluble guanylate cyclase, decreased cGMP levels and induced metamorphosis while a generator of NO counteracted this effect, indicating inhibition of metamorphosis by NO operates via interaction with soluble guanylate cyclase. We discuss these observations, proposing that the NOS-defined neurons in the post-oral ciliary band have a chemosensory function during settlement and metamorphosis that involves morphologically specialized ectoderm and manipulation of fluid flow. We provide a tentative cellular model of how environmental signals may be transduced into a metamorphic response.

Interspecific variation in metamorphic competence in marine invertebrates: the significance for comparative investigations into the timing of metamorphosis.

Metamorphosis in marine invertebrate larvae is a dynamic, environmentally dependent process that integrates ontogeny with habitat selection. The capacity of many marine invertebrate larvae to survive and maintain metamorphic competence in the absence of environmental cues has been hypothesized to be an adaptive convergence (Hadfield and others 2001). A survey of the literature reveals that a single generalized hypothesis about metamorphic competence as an adaptive convergence is not sufficient to account for interspecific variation in this character. In an attempt to capture this variation, we discuss the "desperate larva hypothesis" and propose two additional hypotheses called the "variable retention hypothesis" and the "death before dishonor hypothesis." To validate these additional hypotheses we collected data on taxa from the published literature and performed a contingency analysis to detect correlations between spontaneous metamorphosis, habitat specificity and/or larval life-history mode, three characters relevant to environmentally induced settlement and metamorphosis. In order to account for phylogenetic bias in these correlations, we also constructed a phylogeny of these taxa and again performed a character-correlation analysis. Both these tests suggest that juvenile habitat specificity is correlated to the capacity of individuals to retain the competent larval state in the absence of substrate cues and therefore validate the existence of more than one hypothesis about metamorphic competence. We provide new data from the sea urchin Lytechinus pictus that suggest that nitric oxide (NO) and thyroxine hormone signaling interact to determine the probability of settlement in response to a settlement cue. Similarly, we provide evidence that thyroxine signaling in the sand dollar Dendraster excentricus increases spontaneous metamorphosis in the absence of cues from adult conspecifics in a manner that is independent of larval age.

On nitric oxide signaling, metamorphosis, and the evolution of biphasic life cycles.

Complex life cycles are ancient and widely distributed, particularly so in the marine environment. Generally, the marine biphasic life cycle consists of pre-reproductive stages that exist in the plankton for various periods of time before settling and transforming into a benthic reproductive stage. Pre-reproductive stages are frequently phenotypically distinct from the reproductive stage, and the life cycle transition (metamorphosis) linking the larval and juvenile stages varies in extent of change but is usually rapid. Selection of suitable adult sites apparently involves the capacity to retain the larval state after metamorphic competence is reached. Thus two perennial and related questions arise: How are environmentally dependent rapid transitions between two differentiated functional life history stages regulated (a physiological issue) and how does biphasy arise (a developmental issue)? Two species of solitary ascidian, a sea urchin and a gastropod, share a nitric oxide (NO)-dependent signaling pathway as a repressive regulator of metamorphosis. NO also regulates life history transitions among several simple eukaryotes. We review the unique properties of inhibitory NO signaling and propose that (a) NO is an ancient and widely used regulator of biphasic life histories, (b) the evolution of biphasy in the metazoa involved repression of juvenile development, (c) functional reasons why NO-based signaling is well suited as an inhibitory regulator of metamorphosis after competence is reached, and (d) signaling pathways that regulate metamorphosis of extant marine animals may have participated in the evolution of larvae.

HSP90 function is required for morphogenesis in ascidian and echinoid embryos.

Treatment of embryos of the ascidians Boltenia villosa and Cnemidocarpa finmarkiensis and the sea urchin Strongylocentrotus purpuratus with the anti-HSP90 drugs geldanamycin and radicicol caused morphogenetic arrest. All embryonic stages during which obvious morphogenesis was observed were sensitive to treatment, including formation of the sea urchin blastular epithelium. Arrested embryos were viable for many hours to days post-treatment, indicating a low general toxicity of these drugs. Morphogenetic movements including gastrulation and migration (but not ingression) of sea urchin primary and secondary mesenchyme cells were arrested 8-10 h after treatment began. Cell division and developmentally regulated expression of some genes continued after morphogenesis was arrested. Anti-HSP90 drugs cause selective inactivation or degradation of proteins with which the protein chaperone HSP90 interacts. Therefore, morphogenetic arrest subsequent to the disruption of HSP90 function may result from the reduction in concentration, or activity, of client proteins required for morphogenetic movements of cells. The use of these drugs may provide a means to identify novel activities or proteins involved in morphogenesis.