New gregarine species (Apicomplexa) from tunicates show an evolutionary history of host switching and suggest a problem with the systematics of Lankesteria and Lecudina.

Apicomplexa (sensu stricto) are a diverse group of obligate parasites to a variety of animal species. Gregarines have been the subject of particular interest due to their diversity, phylogenetically basal position, and more recently, their symbiotic relationships with their hosts. In the present study, four new species of marine eugregarines infecting ascidian hosts (Lankesteria kaiteriteriensis sp. nov., L. dolabra sp. nov., L. savignyii sp. nov., and L. pollywoga sp. nov.) were described using a combination of morphological and molecular data. Phylogenetic analysis using small subunit rDNA sequences suggested that gregarines that parasitize ascidians and polychaetes share a common origin as traditionally hypothesized by predecessors in the discipline. However, Lankesteria and Lecudina species did not form clades as expected, but were instead intermixed amongst each other and their respective type species in the phylogeny. These two major genera are therefore taxonomically problematic. We hypothesize that the continued addition of new species from polychaete and tunicate hosts as well as the construction of multigene phylogenies that include type-material will further dissolve the currently accepted distinction between Lankesteria and Lecudina. The species discovered and described in the current study add new phylogenetic and taxonomic data to the knowledge of marine gregarine parasitism in ascidian hosts.

Author Correction: Epigenetic patterns associated with an ascidian invasion: a comparison of closely related clades in their native and introduced ranges.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

No evidence for an association between Clock gene allelic variation and migration timing in a long-distance migratory shorebird (Limosa lapponica baueri).

The gene Clock is a key part of the Core Circadian Oscillator, and the length of the polyglutamine (poly-Q) repeat sequence in Clock (ClkpolyQcds) has been proposed to be associated with the timing of annual cycle events in birds. We tested whether variation in ClkpolyQcds corresponds to variation in migration timing in the bar-tailed godwit (Limosa lapponica baueri), a species in which individuals show strong annual consistency in their migration timing despite the New Zealand population migrating across a 5-week period. We describe allelic variation of the ClkpolyQcds in 135 godwits over-wintering in New Zealand (N.Z.) and investigate whether polymorphism in this region is associated with northward migration timing (chronophenotype) from N.Z. or (for 32 birds tracked by geolocator) after the primary stopover in Asia. Six Clock alleles were detected (Q7‒Q12) and there was substantial variation between individuals (heterozygosity of 0.79). There was no association between ClkpolyQcds polymorphism and migration timing from N.Z. The length of the shorter Clock allele was related to migration timing from Asia, though this relationship arose largely from just a few northern-breeding birds with longer alleles. Other studies show no consistent associations between ClkpolyQcds and migration timing in birds, although Clock may be associated with breeding latitude in some species (as an adaptation to photoperiodic regime). Apparent relationships with migration timing could reflect latitude-related variation in migration timing, rather than Clock directly affecting migration timing. On current evidence, ClkpolyQcds is not a strong candidate for driving migration timing in migratory birds generally.

Epigenetic patterns associated with an ascidian invasion: a comparison of closely related clades in their native and introduced ranges.

Environmentally induced epigenetic modifications have been proposed as one mechanism underlying rapid adaptive evolution of invasive species. Didemnum vexillum is an invasive colonial ascidian that has established in many coastal waters worldwide. Phylogenetic analyses have revealed that D. vexillum populations consist of two distinct clades; clade B appears to be restricted to the native range (Japan), whereas clade A is found in many regions throughout the world, including New Zealand. The spread of D. vexillum clade A suggests that it might be intrinsically more invasive than clade B, despite low levels of genetic diversity compared to populations from the native region. This study investigated whether D. vexillum clade A exhibits epigenetic signatures (specifically differences in DNA methylation) associated with invasiveness. Global DNA methylation patterns were significantly different between introduced clade A colonies, and both clades A and B in the native range. Introduced colonies also showed a significant reduction in DNA methylation levels, which could be a mechanism for increasing phenotypic plasticity. High levels of DNA methylation diversity were maintained in the introduced population, despite reduced levels of genetic diversity, which may allow invasive populations to respond quickly to changes in new environments. Epigenetic changes induced during the invasion process could provide a means for rapid adaptation despite low levels of genetic variation in introduced populations.

PCR and histology identify new bivalve hosts of Apicomplexan-X (APX), a common parasite of the New Zealand flat oyster Ostrea chilensis.

Apicomplexan-X (APX) is a significant pathogen of the flat oyster Ostrea chilensis in New Zealand. The life cycle and host range of this species are poorly known, with only the zoite stage identified. Here, we report the use of molecular approaches and histology to confirm the presence of APX in samples of green-lipped mussels Perna canaliculus, Mediterranean mussels Mytilus galloprovincialis and hairy mussels Modiolus areolatus collected from widely distributed locations in New Zealand. The prevalence of APX infection estimated by PCR was 22.2% (n = 99) and 50% (n = 30) in cultured green-lipped mussels from Nelson and Coromandel, respectively; 0.8% (n = 258), 3.3% (n = 150) and 35.3% (n = 17) in wild Mediterranean mussels from Nelson, Foveaux Strait and Golden Bay, respectively; and 46.7% (n = 30) in wild hairy mussels from Foveaux Strait. Histology detected all cases of PCR that were positive with APX and appeared to be more sensitive. The prevalence of APX estimated by histology in green-lipped mussels from Coromandel was 60% versus 50% by PCR, and 4.3%, 10.7% and 52.9% by histology versus 0.8%, 3.3% and 35.3% by PCR in wild Mediterranean mussels from Nelson, Foveaux Strait and Golden Bay, respectively. The specific identity of the parasite found in mussels was determined by sequencing PCR products for a portion (676 bp) of the 18S rRNA gene; the resulting sequences were 99-100% similar to APX found in flat oysters. Phylogenetic analyses also confirmed that all isolates from green-lipped, Mediterranean and hairy mussels grouped with APX isolates previously identified from flat oysters. This study indicates the wide geographical distribution of APX and highlights the potentially multi-host specific distribution of the parasite in commercially important bivalve shellfish.

PCR test to specifically detect the apicomplexan 'X' (APX) parasite found in flat oysters Ostrea chilensis in New Zealand.

Described here is a polymerase chain reaction (PCR) test to detect the apicomplexan-X (APX) parasite of a flat oyster species, Ostrea chilensis, endemic to New Zealand. The test primers target sequences in the in situ hybridisation probes identified to bind specifically to APX 18S rRNA and amplify a 723 bp DNA product. The test did not amplify 18S rRNA gene sequences of other apicomplexan species, including Toxoplasma gondii, Neospora caninum, Selenidium spp., Cephaloidophorida spp., Lecudina spp. and Thiriotia sp. Of 73 flat oysters identified by histology to be infected with APX at different severities, 69 (95%) tested PCR-positive. Failure to amplify an internal control indicated the presence of PCR inhibitors in the 4 PCR-negative samples. The high analytical sensitivity, specificity and speed of the PCR test should make it a useful tool for detecting APX.

Effects of temperature and salinity stress on DNA methylation in a highly invasive marine invertebrate, the colonial ascidian Didemnum vexillum.

Environmentally induced epigenetic changes may contribute to phenotypic plasticity, increase adaptive potential in changing environments, and play a key role in the establishment and spread of invasive species in new habitats. In this study, we used methylation-sensitive amplified polymorphism (MSAP) to assess environmentally induced DNA methylation changes in a globally invasive clonal ascidian, Didemnum vexillum. We tested the effect of increasing temperature (19, 25 and 27 °C) and decreasing salinity (34, 32, 30, 28 and 26 practical salinity units (PSU)) on global DNA methylation, growth and survival rates. Exposure to 27 °C resulted in significant changes in DNA methylation over time. Growth also decreased in colonies exposed to high temperatures, suggesting they were under thermal stress. In contrast, no differences in growth nor DNA methylation patterns were observed in colonies exposed to a decreasing salinity gradient, potentially due to prior adaptation. The results of this study show that environmental stress can induce significant global DNA methylation changes in an invasive marine invertebrate on very rapid timescales, and that this response varies depending on the type, magnitude, and duration of the stressor. Changes in genomic DNA methylation and the rate of growth may act to 'buy survival time' under stressful conditions, expanding the distribution limits of this globally invasive species.

Efficient dispersal and substrate acquisition traits in a marine invasive species via transient chimerism and colony mobility.

Over the past three decades the colonial ascidian Didemnum vexillum has been expanding its global range, significantly impacting marine habitats and aquaculture facilities. What biological features make D. vexillum so highly invasive? Here, we show that juxtaposed allogeneic D. vexillum colony fragments ('ramets') may, initially, form chimeric entities. Subsequently, zooids of the differing genotypes within such chimeras coordinately retreat away from fusion zones. A few days following such post-fusion retreat movements there is further ramet fission and the formation of zooid-depauperate tunic zones. Using polymorphic microsatellite loci to distinguish between genotypes, we found that they were sectorial at the fusion zones and the subsequent ramet movements resulted in further spatial separation of the paired-genotypes indicating that the fusion events observed did not lead to formation of long-term, stable chimeras. Thus, movements of D. vexillum colony ramets from initial fusion zones lead to progressive segregation of genotypes probably minimizing potential somatic/germ-cell competition/parasitism. We speculate that relatively fast (≤10 mm/day) movement of D. vexillum colonies on substrates along with frequent, and perhaps unrestrained, transient allogeneic fusions play significant roles in this species' striking invasiveness and capacity to colonize new substrates.

Partial 18S rRNA sequences of apicomplexan parasite 'X' (APX), associated with flat oysters Ostrea chilensis in New Zealand.

Apicomplexa is a large phylum of parasitic protists renowned for significant negative health impacts on humans and livestock worldwide. Despite the prevalence and negative impacts of apicomplexans across many animal groups, relatively little attention has been given to apicomplexan parasites of invertebrates, especially marine invertebrates. Previous work has reported an apicomplexan parasite 'X' (APX), a parasite that has been histologically and ultrastructurally identified from the commercially important flat oyster Ostrea chilensis in New Zealand. This apicomplexan may exacerbate host vulnerability to the infectious disease bonamiosis. In this study, we report 18S rRNA sequences amplified from APX-infected O. chilensis tissues. Phylogenetic analyses clearly established that the 18S sequences were of apicomplexan origin; however, their detailed relationship to known apicomplexan groups is less resolved. Two specific probes, designed from the putative APX 18S rRNA sequence, co-localised with APX cells in in situ hybridisations, further supporting our hypothesis that the 18S sequences were from APX. These sequences will facilitate the future development of inexpensive and sensitive molecular diagnostic tests for APX, thereby assisting research focussed on the biology and ecology of this organism and its role in morbidity and mortality of O. chilensis.

Thickness-Controlled Quasi-Two-Dimensional Colloidal PbSe Nanoplatelets.

We demonstrate controlled synthesis of discrete two-dimensional (2D) PbSe nanoplatelets (NPLs), with measurable photoluminescence, via oriented attachment directed by quantum dot (QD) surface chemistry. Halide passivation is critical to the growth of these (100) face-dominated NPLs, as corroborated by density functional theory studies. PbCl2 moieties attached to the (111) and (110) of small nanocrystals form interparticle bridges, aligning the QDs and leading to attachment. We find that a 2D bridging network is energetically favored over a 3D network, driving the formation of NPLs. Although PbI2 does not support bridging, its presence destabilizes the large (100) faces of NPLs, providing means for tuning NPL thickness. Spectroscopic analysis confirms the predicted role of thickness-dependent quantum confinement on the NPL band gap.

Spectroscopic and Device Aspects of Nanocrystal Quantum Dots.

The field of nanocrystal quantum dots (QDs) is already more than 30 years old, and yet continuing interest in these structures is driven by both the fascinating physics emerging from strong quantum confinement of electronic excitations, as well as a large number of prospective applications that could benefit from the tunable properties and amenability toward solution-based processing of these materials. The focus of this review is on recent advances in nanocrystal research related to applications of QD materials in lasing, light-emitting diodes (LEDs), and solar energy conversion. A specific underlying theme is innovative concepts for tuning the properties of QDs beyond what is possible via traditional size manipulation, particularly through heterostructuring. Examples of such advanced control of nanocrystal functionalities include the following: interface engineering for suppressing Auger recombination in the context of QD LEDs and lasers; Stokes-shift engineering for applications in large-area luminescent solar concentrators; and control of intraband relaxation for enhanced carrier multiplication in advanced QD photovoltaics. We examine the considerable recent progress on these multiple fronts of nanocrystal research, which has resulted in the first commercialized QD technologies. These successes explain the continuing appeal of this field to a broad community of scientists and engineers, which in turn ensures even more exciting results to come from future exploration of this fascinating class of materials.

Geographically conserved microbiomes of four temperate water tunicates.

Tunicates are useful models for exploring microbiomes because they have an innate immune system resembling that of chordates. Automated ribosomal RNA intergenic spacer analysis and High-Throughput Sequencing were used to compare the tunic microbiomes of Ciona robusta (formerly Ciona intestinalis type A), Ciona savignyi, Botrylloides leachi and Botryllus schlosseri sampled from three distinct locations with limited genetic connectivity. Bacterial phylotype profiles were conserved within each species, and there were no detectable differences between tunic and tunic + cuticle subsamples from an individual. Bacterial operational taxonomic unit (OTU) diversity was lowest for C. savignyi (320 ± 190 OTUs) and highest for B. schlosseri (1260 ± 190 OTUs). Each species had a distinct set of bacterial OTUs (pseudo-F = 3.0, p > 0.001), with the exception of B. leachi and B. schlosseri from one sampling location (t = 1.2, p = 0.09). Of note were OTUs assigned to Alphaproteobacteria from C. robusta plus Phyllobacteriaceae and Endozoicomonas from C. savignyi. These OTUs contributed 51, 22 and 10% of sequence reads, respectively, and are related to known bacterial symbionts. The within-species conservation of core OTUs across three distinct and co-occurring populations of tunicates provides compelling evidence that these tunicates foster defined microbiomes.

Shape-Controlled Narrow-Gap SnTe Nanostructures: From Nanocubes to Nanorods and Nanowires.

The rational design and synthesis of narrow-gap colloidal semiconductor nanocrystals (NCs) is an important step toward the next generation of solution-processable photovoltaics, photodetectors, and thermoelectric devices. SnTe NCs are particularly attractive as a Pb-free alternative to NCs of narrow-gap lead chalcogenides. Previous synthetic efforts on SnTe NCs have focused on spherical nanoparticles. Here we report new strategies for synthesis of SnTe NCs with shapes tunable from highly monodisperse nanocubes, to nanorods (NRs) with variable aspect ratios, and finally to long, straight nanowires (NWs). Reaction at high temperature quickly forms thermodynamically favored nanocubes, but low temperatures lead to elongated particles. Transmission electron microscopy studies of reaction products at various stages of the synthesis reveal that the growth and shape-focusing of monodisperse SnTe nanocubes likely involves interparticle ripening, while directional growth of NRs and NWs may be initiated by particle dimerization via oriented attachment.

Carrier multiplication detected through transient photocurrent in device-grade films of lead selenide quantum dots.

In carrier multiplication, the absorption of a single photon results in two or more electron-hole pairs. Quantum dots are promising materials for implementing carrier multiplication principles in real-life technologies. So far, however, most of research in this area has focused on optical studies of solution samples with yet to be proven relevance to practical devices. Here we report ultrafast electro-optical studies of device-grade films of electronically coupled quantum dots that allow us to observe multiplication directly in the photocurrent. Our studies help rationalize previous results from both optical spectroscopy and steady-state photocurrent measurements and also provide new insights into effects of electric field and ligand treatments on multiexciton yields. Importantly, we demonstrate that using appropriate chemical treatments of the films, extra charges produced by carrier multiplication can be extracted from the quantum dots before they are lost to Auger recombination and hence can contribute to photocurrent of practical devices.

Tunicate pregnane X receptor (PXR) orthologs: transcript characterization and natural variation.

Vertebrate pregnane X receptor (PXR, NR1I2), a ligand-activated nuclear receptor (NR), regulates expression of detoxification genes. Vertebrate PXR orthologs may adaptively evolve to bind deleterious/toxic xenobiotics typically encountered by organisms from their diet. Tunicates (phylum Chordata) are marine filter-feeders that form a sister clade to the Vertebrata. Genomes of two tunicate taxa, Ciona intestinalis and Botryllus schlosseri, encode at least two PXR orthologs (abbreviated VDR/PXRα and ß). Here we report characterization of the transcript structures and sequence variation of three tunicate PXR orthologs: C. intestinalis VDR/PXRα and ß, and B. schlosseri VDR/PXRα. The three predicted proteins consist of both DNA-binding (DBD) and ligand-binding (LBD) domains typical of NRs. The C. intestinalis VDR/PXRß LBD may be significantly larger than that of the VDR/PXRα orthologs. In both tunicate taxa, the mRNAs were characterized by high frequencies of single nucleotide polymorphisms (SNPs, ca. 3 SNPs/100 base pairs). The majority of SNPs were synonymous and standard tests (Tajima's D, dN/dS ratios) indicated strong purifying selection. However, one base pair frameshift allelic variants were found in the C. intestinalis VDR/PXRα and ß genes. The predicted proteins consisted of a DBD but lacked an LBD. The persistence of these variants may possibly reflect constitutive expression of detoxification genes as a selective advantage in the marine environment. These results provide a foundation for further investigations into the molecular evolution, population genetics and functioning of tunicate receptors involved in detection of marine bioactive compounds.

Detection of marine microalgal biotoxins using bioassays based on functional expression of tunicate xenobiotic receptors in yeast.

Marine microalgae can produce biotoxins that cause widespread poisoning in marine ecosystems and may also affect human health. While established microalgal biotoxins are detectable using chemical methods, a need remains for robust, inexpensive bioassays. Ligand-binding domains (LBDs) from a tunicate nuclear receptor, VDR/PXRα, which is orthologous to both the vertebrate pregnane X receptor (PXR) and the vitamin D receptor (VDR), can be activated by microalgal biotoxins when expressed in mammalian cell lines. Building on this observation, we developed a generic recombinant yeast bioassay platform that expresses chimeric proteins containing tunicate VDR/PXRα LBDs which mediate ligand-dependent transcription of a reporter gene (lacZ) encoding an easily assayed enzyme (ß-galactosidase). Recombinant yeast strains expressing VDR/PXRα LBDs from two tunicate species, Ciona intestinalis and Botryllus schlosseri, were exposed to both synthetic and natural toxins. Structurally simple synthetic chemicals (n-butyl-p-aminobenzoate, carbamazepine, p-aminobenzoic acid, and bisphenol-A) generated EC50 values in the µM range, while more structurally complex marine biotoxins (okadaic acid, pectenotoxin-11, and portimine) activated the assays in the nM range. Given the large number of tunicate species, we propose that tunicate VDR/PXR LBDs may be used as 'sensor elements' in similar yeast-based high-throughput bioassays for detection of established microalgal biotoxins and uncharacterised marine bioactive compounds.

Comparison of whole mitochondrial genome sequences from two clades of the invasive ascidian, Didemnum vexillum.

The mitochondria are the main source of cellular energy production and have an important role in development, fertility, and thermal limitations. Adaptive mitochondrial DNA mutations have the potential to be of great importance in determining aspects of the life history of an organism. Phylogenetic analyses of the globally invasive marine ascidian Didemnum vexillum using the mitochondrial cytochrome c oxidase 1 (COX1) coding region, revealed two distinct clades. Representatives of one clade (denoted by 'B') are geographically restricted to D. vexillum's native region (north-west Pacific Ocean, including Japan), whereas members of the other clade (denoted by 'A') have been introduced and become invasive in temperate coastal areas around the world. Persistence of clade B's restricted distribution may reflect it being inherently less invasive than clade A. To investigate this we sought to determine if the two clades differ significantly in other mitochondrial genes of functional significance, specifically, alterations in amino acids encoded in mitochondrial enzyme subunits. Differences in functional mitochondrial genes could indicate an increased ability for clade A colonies to tolerate a wider range of environmental temperature. Full mitochondrial genomic sequences from D. vexillum clades A and B were obtained and they predict significant sequence differences in genes encoding for enzymes involved in oxidative phosphorylation. Diversity levels were relatively high and showed divergence across almost all genes, with p-distance values between the two clades indicating recent divergence. Both clades showed an excess of rare variants, which is consistent with balancing selection or a recent population expansion. Results presented here will inform future research focusing on examining the functional properties of the corresponding mitochondrial respiration enzymes, of A and B clade enzymes. By comparing closely related taxa that have differing distributions it is possible to identify genes and phenotypes suited to particular environments. The examination of mitochondrial genotypes, and associated enzyme functioning, across populations may aid in our understanding of thermal tolerance and environmental adaptation.

Marine invertebrate xenobiotic-activated nuclear receptors: their application as sensor elements in high-throughput bioassays for marine bioactive compounds.

Developing high-throughput assays to screen marine extracts for bioactive compounds presents both conceptual and technical challenges. One major challenge is to develop assays that have well-grounded ecological and evolutionary rationales. In this review we propose that a specific group of ligand-activated transcription factors are particularly well-suited to act as sensors in such bioassays. More specifically, xenobiotic-activated nuclear receptors (XANRs) regulate transcription of genes involved in xenobiotic detoxification. XANR ligand-binding domains (LBDs) may adaptively evolve to bind those bioactive, and potentially toxic, compounds to which organisms are normally exposed to through their specific diets. A brief overview of the function and taxonomic distribution of both vertebrate and invertebrate XANRs is first provided. Proof-of-concept experiments are then described which confirm that a filter-feeding marine invertebrate XANR LBD is activated by marine bioactive compounds. We speculate that increasing access to marine invertebrate genome sequence data, in combination with the expression of functional recombinant marine invertebrate XANR LBDs, will facilitate the generation of high-throughput bioassays/biosensors of widely differing specificities, but all based on activation of XANR LBDs. Such assays may find application in screening marine extracts for bioactive compounds that could act as drug lead compounds.

Persistent Inter-Excitonic Quantum Coherence in CdSe Quantum Dots.

The creation and manipulation of quantum superpositions is a fundamental goal for the development of materials with novel optoelectronic properties. In this letter, we report persistent (~80 fs lifetime) quantum coherence between the 1S and 1P excitonic states in zinc-blende colloidal CdSe quantum dots at room temperature, measured using Two-Dimensional Electronic Spectroscopy. We demonstrate that this quantum coherence manifests as an intradot phenomenon, the frequency of which depends on the size of the dot excited within the ensemble of QDs. We model the lifetime of the coherence and demonstrate that correlated interexcitonic fluctuations preserve relative phase between excitonic states. These observations suggest an avenue for engineering long-lived interexcitonic quantum coherence in colloidal quantum dots.

Dynamic localization of electronic excitation in photosynthetic complexes revealed with chiral two-dimensional spectroscopy.

Time-resolved ultrafast optical probes of chiral dynamics provide a new window allowing us to explore how interactions with such structured environments drive electronic dynamics. Incorporating optical activity into time-resolved spectroscopies has proven challenging because of the small signal and large achiral background. Here we demonstrate that two-dimensional electronic spectroscopy can be adapted to detect chiral signals and that these signals reveal how excitations delocalize and contract following excitation. We dynamically probe the evolution of chiral electronic structure in the light-harvesting complex 2 of purple bacteria following photoexcitation by creating a chiral two-dimensional mapping. The dynamics of the chiral two-dimensional signal directly reports on changes in the degree of delocalization of the excitonic states following photoexcitation. The mechanism of energy transfer in this system may enhance transfer probability because of the coherent coupling among chromophores while suppressing fluorescence that arises from populating delocalized states. This generally applicable spectroscopy will provide an incisive tool to probe ultrafast transient molecular fluctuations that are obscured in non-chiral experiments.