Bioluminescence and Photoreception in Unicellular Organisms: Light-Signalling in a Bio-Communication Perspective.

Bioluminescence, the emission of light catalysed by luciferases, has evolved in many taxa from bacteria to vertebrates and is predominant in the marine environment. It is now well established that in animals possessing a nervous system capable of integrating light stimuli, bioluminescence triggers various behavioural responses and plays a role in intra- or interspecific visual communication. The function of light emission in unicellular organisms is less clear and it is currently thought that it has evolved in an ecological framework, to be perceived by visual animals. For example, while it is thought that bioluminescence allows bacteria to be ingested by zooplankton or fish, providing them with favourable conditions for growth and dispersal, the luminous flashes emitted by dinoflagellates may have evolved as an anti-predation system against copepods. In this short review, we re-examine this paradigm in light of recent findings in microorganism photoreception, signal integration and complex behaviours. Numerous studies show that on the one hand, bacteria and protists, whether autotrophs or heterotrophs, possess a variety of photoreceptors capable of perceiving and integrating light stimuli of different wavelengths. Single-cell light-perception produces responses ranging from phototaxis to more complex behaviours. On the other hand, there is growing evidence that unicellular prokaryotes and eukaryotes can perform complex tasks ranging from habituation and decision-making to associative learning, despite lacking a nervous system. Here, we focus our analysis on two taxa, bacteria and dinoflagellates, whose bioluminescence is well studied. We propose the hypothesis that similar to visual animals, the interplay between light-emission and reception could play multiple roles in intra- and interspecific communication and participate in complex behaviour in the unicellular world.

The evolution of convex trade-offs enables the transition towards multicellularity.

The evolutionary transition towards multicellular life often involves growth in groups of undifferentiated cells followed by differentiation into soma and germ-like cells. Theory predicts that germ soma differentiation is facilitated by a convex trade-off between survival and reproduction. However, this has never been tested and these transitions remain poorly understood at the ecological and genetic level. Here, we study the evolution of cell groups in ten isogenic lines of the unicellular green algae Chlamydomonas reinhardtii with prolonged exposure to a rotifer predator. We confirm that growth in cell groups is heritable and characterized by a convex trade-off curve between reproduction and survival. Identical mutations evolve in all cell group isolates; these are linked to survival and reducing associated cell costs. Overall, we show that just 500 generations of predator selection were sufficient to lead to a convex trade-off and incorporate evolved changes into the prey genome.

Molecular and biochemical basis for the loss of bioluminescence in the dinoflagellate Noctiluca scintillans along the west coast of the USA.

The globally distributed heterotrophic dinoflagellate Noctiluca scintillans (Macartney) Kofoid & Swezy is well known for its dense blooms and prominent displays of bioluminescence. Intriguingly, along the west coast of the USA its blooms are not bioluminescent. We investigated the basis for the regional loss of bioluminescence using molecular, cellular and biochemical analyses of isolates from different geographic regions. Prominent differences of the non-bioluminescent strains were: (1) the fused luciferase and luciferin binding protein gene (lcf/lbp) was present but its transcripts were undetectable; (2) lcf/lbp contained multiple potentially deleterious mutations; (3) the substrate luciferin was absent, based on the lack of luciferin blue autofluorescence and the absence of luciferin derived metabolites; (4) although the cells possessed scintillons, the vesicles that contain the luminescent chemistry, electron microscopy revealed additional scintillon-like vesicles with an atypical internal structure; (5) cells isolated from the California coast were 43% smaller in size than bioluminescent cells from the Gulf of Mexico. Phylogenetic analyses based on the large subunit of rDNA did not show divergence of the non-bioluminescent population in relation to other bioluminescent N. scintillans from the Pacific Ocean and Arabian Sea. Our study demonstrates that gene silencing and the lack of the luciferin substrate have resulted in the loss of a significant dinoflagellate functional trait over large spatial scales in the ocean. As the bioluminescence system of dinoflagellates is well characterized, non-bioluminescent N. scintillans is an ideal model to explore the evolutionary and ecological mechanisms that lead to intraspecific functional divergence in natural dinoflagellate populations.

Simple and rapid sample preparation system for the molecular detection of antibiotic resistant pathogens in human urine.

Antibiotic resistance in urinary tract infections (UTIs) can cause significant complications without quick detection and appropriate treatment. We describe a new approach to capture, concentrate and prepare amplification-ready DNA from antibiotic resistant bacteria in human urine samples. Klebsiella pneumoniae NCTC13443 (bla CTX-M-15 positive) spiked into filtered human urine was used as a model system. Bacteria were captured using anion exchange diaethylaminoethyl (DEAE) magnetic microparticles and concentrated 200-fold within ~3.5 min using a custom, valve-less microfluidic chip. Eight samples were processed in parallel, and DNA was released using heat lysis from an integrated resistive heater. The crude cell lysate was used for real time Recombinase Polymerase Amplification (RPA) of the bla CTX-M-15 gene. The end to end processing time was approximately 15 min with a limit of detection of 1000 bacteria in 1 mL urine.

Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform.

The widespread dissemination of CTX-M extended spectrum ß-lactamases among Escherichia coli bacteria, both in nosocomial and community environments, is a challenge for diagnostic bacteriology laboratories. We describe a rapid and sensitive detection system for analysis of DNA containing the blaCTX-M-15 gene using isothermal DNA amplification by recombinase polymerase amplification (RPA) on a digital microfluidic platform; active matrix electrowetting-on-dielectric (AM-EWOD). The devices have 16,800 electrodes that can be independently controlled to perform multiple and simultaneous droplet operations. The device includes an in-built impedance sensor for real time droplet position and size detection, an on-chip thermistor for temperature sensing and an integrated heater for regulating the droplet temperature. Automatic dispensing of droplets (45 nL) from reservoir electrodes is demonstrated with a coefficient of variation (CV) in volume of approximately 2%. The RPA reaction is monitored in real-time using exonuclease fluorescent probes. Continuous mixing of droplets during DNA amplification significantly improves target DNA detection by at least 100 times compared to a benchtop assay, enabling the detection of target DNA over four-order-of-magnitude with a limit of detection of a single copy within ~15 minutes.

Molecular detection of bioluminescent dinoflagellates in surface waters of the Patagonian shelf during early austral 2008.

We investigated the distribution of bioluminescent dinoflagellates in the Patagonian Shelf region using "universal" PCR primers for the dinoflagellate luciferase gene. Luciferase gene sequences and single cell PCR tests, in conjunction with taxonomic identification by microscopy, allowed us to identify and quantify bioluminescent dinoflagellates. We compared these data to coincidental discrete optical measurements of stimulable bioluminescence intensity. Molecular detection of the luciferase gene showed that bioluminescent dinoflagellates were widespread across the majority of the Patagonian Shelf region. Their presence was comparatively underestimated by optical bioluminescence measurements, whose magnitude was affected by interspecific differences in bioluminescence intensity and by the presence of other bioluminescent organisms. Molecular and microscopy data showed that the complex hydrography of the area played an important role in determining the distribution and composition of dinoflagellate populations. Dinoflagellates were absent south of the Falkland Islands where the cold, nutrient-rich, and well-mixed waters of the Falklands Current favoured diatoms instead. Diverse populations of dinoflagellates were present in the warmer, more stratified waters of the Patagonian Shelf and Falklands Current as it warmed northwards. Here, the dinoflagellate population composition could be related to distinct water masses. Our results provide new insight into the prevalence of bioluminescent dinoflagellates in Patagonian Shelf waters and demonstrate that a molecular approach to the detection of bioluminescent dinoflagellates in natural waters is a promising tool for ecological studies of these organisms.

Diversity of the luciferin binding protein gene in bioluminescent dinoflagellates--insights from a new gene in Noctiluca scintillans and sequences from gonyaulacoid genera.

Dinoflagellate bioluminescence systems operate with or without a luciferin binding protein, representing two distinct modes of light production. However, the distribution, diversity, and evolution of the luciferin binding protein gene within bioluminescent dinoflagellates are not well known. We used PCR to detect and partially sequence this gene from the heterotrophic dinoflagellate Noctiluca scintillans and a group of ecologically important gonyaulacoid species. We report an additional luciferin binding protein gene in N. scintillans which is not attached to luciferase, further to its typical combined bioluminescence gene. This supports the hypothesis that a profound re-organization of the bioluminescence system has taken place in this organism. We also show that the luciferin binding protein gene is present in the genera Ceratocorys, Gonyaulax, and Protoceratium, and is prevalent in bioluminescent species of Alexandrium. Therefore, this gene is an integral component of the standard molecular bioluminescence machinery in dinoflagellates. Nucleotide sequences showed high within-strain variation among gene copies, revealing a highly diverse gene family comprising multiple gene types in some organisms. Phylogenetic analyses showed that, in some species, the evolution of the luciferin binding protein gene was different from the organism's general phylogenies, highlighting the complex evolutionary history of dinoflagellate bioluminescence systems.

Understanding Bioluminescence in Dinoflagellates-How Far Have We Come?

Some dinoflagellates possess the remarkable genetic, biochemical, and cellular machinery to produce bioluminescence. Bioluminescent species appear to be ubiquitous in surface waters globally and include numerous cosmopolitan and harmful taxa. Nevertheless, bioluminescence remains an enigmatic topic in biology, particularly with regard to the organisms' lifestyle. In this paper, we review the literature on the cellular mechanisms, molecular evolution, diversity, and ecology of bioluminescence in dinoflagellates, highlighting significant discoveries of the last quarter of a century. We identify significant gaps in our knowledge and conflicting information and propose some important research questions that need to be addressed to advance this research field.

DISTRIBUTION AND GENETIC DIVERSITY OF THE LUCIFERASE GENE WITHIN MARINE DINOFLAGELLATES(1).

Dinoflagellates are the most abundant protists that produce bioluminescence. Currently, there is an incomplete knowledge of the identity of bioluminescent species arising from inter- and intraspecific variability in bioluminescence properties. In this study, PCR primers were designed to amplify the dinoflagellate luciferase gene (lcf) from genetically distant bioluminescent species. One of the primer pairs was "universal," whereas others amplified longer gene sequences from subsets of taxa. The primers were used to study the distribution of lcf and assess bioluminescence potential in dinoflagellate strains representing a wide variety of taxa as well as multiple strains of selected species. Strains of normally bioluminescent species always contained lcf even when they were found not to produce light, thus demonstrating the utility of this methodology as a powerful tool for identifying bioluminescent species. Bioluminescence and lcf were confined to the Gonyaulacales, Noctilucales, and Peridiniales. Considerable variation was observed among genera, or even species within some genera, that contained this gene. Partial sequences of lcf were obtained for the genera Ceratocorys, Ceratium, Fragilidium, and Protoperidinium as well as from previously untested species or gene regions of Alexandrium and Gonyaulax. The sequences revealed high variation among gene copies that obscured the boundaries between species or even genera, some of which could be explained by the presence of two genetic variants within the same species of Alexandrium. Highly divergent sequences within Alexandrium and Ceratium show a more diverse composition of lcf than previously known.