Assessing Ecotoxicity in Marine Environment Using Luminescent Microalgae: Where Are We At?

Nowadays, microalgae are particularly used to assess the environmental impact of contaminants in aquatic systems. Naturally present in some algal species, bioluminescence is highly used in application fields related to environmental monitoring. Bioluminescent dinoflagellates have played a pivotal role in this domain. When exposed to heavy metals or toxic organic compounds, biolu-minescent dinoflagellates have the capacity to decrease light emission. In addition, new molecular tools allow the possibility to produce genetically modified microorganisms which are able to perform luminescence. Combined with the luciferase reporter gene, two main genetic constructions can be employed. Activation of a specific inducible promoter induces the luminescence gene transcription and this signal increases over time. Constitutive promoters result in a high basal expression level of the reporter gene. During exposure to a potential toxic pollutant, the basal expression level will decrease due to the toxic effect. Toxicity bioassays based on engineered luminescent Chlorophyta microalgae are among the most sensitive tests and are an invaluable complement to classical toxicity assays.


Introduction
Persistent organic and inorganic environmental pollutants can be toxic and accumulate in numbers of aquatic species.To predict the effect of environmental stressors on populations, communities and ecosystems, ecotoxicological tests using microorganisms are particularly efficient.Marine microalgae are a major constituent of the aquatic food chain and play an important role in coastal ecosystems [1].These unicellular cells tend to be more sensitive than animal species to a wide variety of both organic and inorganic pollutants [2].They are particularly used for the development of new toxicity bioassays and to assess the environmental impact of contaminants in aquatic systems [3].The use of flow cytometry is an important step in the development of growth inhibition tests.It offers the possibility to perform multiparameter analysis on a wide range of cell properties (algal cell densities, cell size and shape or granularity) [4].In addition, compared to traditional tests, a high growth rate and good level of biomass are reached in a short time.Microalgae are widely used in ecotoxicological tests to evaluate the toxicity effects of diverse pollutants: dichloromethane and dichloroethane on Chlorella vulgaris [5], nanoparticles on freshwater (Chlorococcum sp. and Scenedesmus rubescens) and marine species (Dunaliella tertiolecta and Tetraselmis suesica) [6]; pentachlorophenol on co-cultures of freshwater phytoplankton species (the cyanobacterium Microcystis aeruginosa and the microalgae Chlorella vulgaris) [7].Nowadays, new microalgae models continue to emerge like Phaeocystis antarctica, a species isolated from Antarctic coastal marine environments, which will be suitable to bring information on the sensitivity of Antarctic microalgae to metal contamination [8].
Bioluminescent microalgae are often responsible for large-scale phenomena known as the "shining seas" in marine waters.Basically, the reaction of bioluminescence requires an enzyme, the luciferase, to catalyze an oxidation reaction.The natural capacity to produce bioluminescence by different species of microalgae appears as an interesting asset.Dinoflagellate and bacteria are the main bioluminescent microorganisms in marine environment (for a review see [9]) and ecological aspects and molecular mechanisms that regulate bioluminescence phenomenon are well known in these microorganisms.Nowadays, naturally produced luminescence can be used to monitor environmental quality: some bioluminescent dinoflagellates have a unique capacity of decreasing light emission when are exposed to heavy metals or toxic organic compounds.In addition, new molecular tools allow the possibility to produce genetically modified microorganisms which are able to perform luminescence.Although an exhaustive description of all applications of bioluminescence is outside the scope of this review, we will present here how the bioluminescence has inspired scientists in the field of ecotoxicological tests.

Bioluminescent Genes Used as a Detection Tool
Detection of harmful and innocuous bioluminescent microorganisms in environmental sea water can be done using various molecular techniques.Direct search in global environmental databases is sometimes difficult because the majority of libraries are collections of 18S rDNA sequences.To overcome this problem, the development of species-specific probes (targeting the LuxA gene of the lux operon in bacteria) has helped to identify luminescent bacteria in both laboratory cultures [10] and seawater samples [11].In the same way, molecular methods helped to characterize bioluminescent microalgae species [12].A particular attention was given to the dinoflagellates luciferase gene."Universal" primers were designed for the development of PCR-oriented approaches to specifically detect bioluminescent dinoflagellates in environmental waters samples [13]- [15].The development of PCR primers targeting a longer gene sequence helped to assess bioluminescent potential in multiple strains of one selected species [15].The authors found that out of 34 tested dinoflagellate strains, 23 possessed the luciferase gene while only 18 were able to produce light in laboratory culture conditions [15].Several transcriptomics' studies also suggested other sequences that could serve as indicators of bioluminescent species possessing luciferin binding protein as this protein is highly expressed in Lingulodinium polyedrum [16] [17] and in Alexandrium tamarense [18], and is the most highly expressed protein in Alexandrium catenella [19] [20].Highly localized bioluminescence in marine environmement is sometimes considered as an early indicator of algal bloom development [21].A large number of harmful dinoflagellate taxa luminesce [22] and in the future, bioluminescence detection will be probably a useful tool to localize them and prevent vulnerability of human populations.

Bioluminescent Dinoflagellates in Ecotoxicological Tests
Bioluminescent microorganisms can be used as natural whole-cell biosensors to sense environmental signals because of their natural capacity of decreasing light emission when exposed to toxic conditions ("natural luminescence", Figure 1).Historically, biossays using bioluminescent bacteria were firstly developed.The Microtox bioassay [23] is available since 1990.It proposes the detection of over 2000 toxic chemicals thanks to the reduction of light emission from the bioluminescent marine bacteria Vibrio fischeri as a means for measuring toxicity.For this purpose, the toxicity of different chemicals is compared by classifying them according to the half maximal effective concentration (EC 50 ) parameter.This EC 50 parameter represents the concentration of a compound which causes 50% of its maximal effect (for example, a 50% decrease of luminescence).In other words, for a given compound, the lowest is the concentration, the most toxic is the compound.The Microtox bioassay has become the commonly accepted method in ecotoxicity assessments [24] and is currently used in a wide range of applications.This 15-min test allows the detection of products as diverse as cadmium chloride (CdCI 2 ) (EC 50 = 56.8mg•L −1 ± 8.46 mg•L −1 ) or mercury chloride (HgCl 2 ) (EC 50 = 0.060 or 0.093 mg•L −1 depending on studies) [25] [26].Another bioluminescent bacteria, Photobacterium phosphoreum, was proposed for detecting and signalling the presence of toxicants in water systems [27].Microalgae are also attractive systems for the development of new toxicity bioassays.Some bioluminescent dinoflagellates (Lingulodinium polyedrum, Pyrocystis lunula...) show reduced light emission when exposed to heavy metals or toxic organic compounds ("natural bioluminescence", Figure 1).When exposed to lead (EC 50 = 321 ppb) and copper (EC 50 = 23 ppb), Lingulodinium polyedrum bioluminescence was affected by the metals in a dose-dependent manner [28] and yielded accurate and more sensitive results when compared to traditional test like the crustacean Mysidopsis survival test (LC 50 = 3130 ppb and LC 50 = 120 -140 ppb respectively) [28] [29].In the Lumitox bioassay that uses a Pyrocystis lunula monospecific culture, recovery of luminescence is sometimes achieved by decreasing pH depending on the toxicants used [30].The ASTM-approved QwikLite bioassay, developed with Pyrocystis lunula and Lingulodinium polyedrum, utilizes only 300 cells per experimental condition.Four other microalgae species (Ceratocorys horrida, Pyrocystis noctiluca, P. fusiformis and Pyrophacus steinii) can be used in these bioassays.Cells are separately maintained in a Laboratory Plankton Test Chamber (LPTC) [29] and evaluated following a 24 h exposure period.Whatever the test used, assays generally result in similar toxicity rankings for the metal tested.However, comparison between QwikLite and Microtox tests revealed that QwikLite assay was generally one to two orders of magnitude more sensitive than the Microtox test.For example, after exposure to copper, L. polydreum EC 50 was only 0.090 ± 0.012 mg•L −1 while V. fischeri EC 50 was 0.397 ± 0.030 mg•L −1 and after exposure to lead, L. polydreum EC 50 was only 0.747 ± 0.088 mg•L −1 while V. fischeri EC 50 reached 34.6 ± 11.7 mg•L −1  Absence of toxic compound [26].In the same way, QwikLite is much more sensitive to total ammonia (10 ± 1.1 mg•L −1 ) than Microtox, in which the 15 min EC 50 for inhibition of bioluminescence is reported to be higher than 900 mg•L −1 [31] [32].In contrast, although QwikLite generally shows better precision than classical algal growth rate bioassays, it can be insensitive to other compounds like diuron [32].

Genetic Engineering with Non-Naturally Luminescent Organisms
Genes responsible for bioluminescence can be transferred and expressed in several microorganisms.Luminescent reporter genes are popular tools for the real-time monitoring of gene expression in living cells.Utilization of luminescence has become an important component of biomedical research [33] [34].Biological processes can be monitored in vivo.Pathogens can be tracked over time.Our understanding of the effect of infections and host immune responses was substantially improved thanks to the production of recombinant virus and bacteria [35]- [39].More recently, in food industry (for a review see [40]), luminescence tools permit to follow the survival of pathogenic microorganisms and the expression of bacterial toxins in foods.Interestingly, specific bacteriophages genetically transformed with the Lux genes allow the direct detection of food pathogens [41].Genetically modified whole-cell bioreporters using luminescence detection are practical tools for the detection and monitoring of contaminants in environmental context.In vitro toxicity assays using recombinant Escherichia coli (encoding a luciferase from Photorhabdus luminescens) was used as an alternative to the Vibrio fischeri Microtox test [42].

Genetic Transformation with a Luciferase Gene Reporter System
Toxicity bioassays sometimes require utilization of genetic engineering with non luminescent organisms [43].Different expression systems exist.Gene reporter systems using inducible or constitutive promoters are two ways currently used for the evaluation of sample toxicity.An inducible expression system is based on a specific inducible promoter.Inducible promoter activation depends on a substance of interest which induces promoter activity, expression of the reporter gene, and transcription of an adjacent resistance gene.Luciferase gene reporter gives the opportunity to follow the detection of the substance of interest by luminescent signal.This signal increases over time as a result of inducible promoter activity ("Induc.prom.", Figure 1).Using this approach, 20 genetically modified E. coli strains were designed and responded differently in the presence of diverse chemicals [44].In the same way, whole-cell biosensor assays for arsenic detection have been proposed.Various indicator tests were developed for the detection of this compound in the environment [45]- [47].On the opposite way, a constitutive expression system generally uses a gene promoter that is highly expressed under normal conditions.It results in a high basal expression level of the reporter gene (see the following section).During exposure to a pollutant, the basal expression level and the emission of luminescence decrease due to the toxic effect ("Const.prom.", Figure 1).This substrate-dependent reporter system sometimes requires luciferin addition when only luciferase is present in the genetic construction (Figure 1).The use of these technologies produces fast and economical high-throughput biosensor systems for detecting environmental pollution.

Towards New Models
As for bacteria, the integration of genetic material by injection or transformation can be performed in eukaryotic organisms.Recently, new genetically modified microorganisms have emerged.Discovered in a Mediterranean lagoon, the unicellular alga Ostreococcus tauri (Chlorophyta, Mamiellophyceae) is the smallest free-living eukaryotic cell known to date [48] [49] and its compact genome is entirely sequenced and published [50] [51].Although this photosynthetic green alga does not have the intrinsic ability to produce bioluminescence naturally, it can easily be cultivated and transformed in the laboratory thus allowing functional genomics approaches.In vitro molecular constructions using luciferase gene reporter system can be integrated in the genome of O. tauri, enabling this eukaryote to emit light ("Const.prom.", Figure 1).Expression of genes of interest can then be studied in vivo in the context of fundamental research.Cell division mechanisms [52] and circadian clock pathways [53] were thereby analysed.Very recently, recombinant O. tauri cells expressing firefly luciferase were used to monitor the impact of herbicides on this marine alga and may thus be valuable biosensors for environmental monitoring [54].More precisely, a luminescence assay using CDKA-luc biosensor (integration in O. tauri genome of CDKA gene fused with luciferase coding sequence) gave reasonable range of EC 50 recorded for two biocides, diuron (EC 50 = 5.65 ± 0.44 µg•L −1 ) and Irgarol 1051 (EC 50 = 0.76 ± 0.10 µg•L −1 ) after 48 hours [54] compared to standard growth inhibition assays using other phytoplankton strains.This assay, using CDKA-luc lines, detected lesser diuron concentrations to those using a QwikLite test using Pyrocystis lunula (19 ± 13 mg•L −1 ) [32].These O. tauri CDKA-luc lines detected similar Irgarol 1051 concentrations compared to the growth inhibition assay using the marine chlorophyte Dunaliella tertiolecta [55] but was about ten-fold more sensitive than using growth inhibition with the freshwater species Raphidocelis subcapitata [56].Moreover, for each of the two biocides, lower EC 50 were obtained from the luminescent marker approach when compared to wild-type O. tauri growth experiments with the same 48 hours exposure time (five-fold lower EC 50 ) [54], underlining the improved sensitivity of this biosensor.To our knowledge, the O. tauri luminescence assay is the most sensitive test of all whole cell luminescence biosensors for detecting diuron and Irgarol 1051.It was particularly sensitive to both biocides compared to the frequently used Vibrio fischeri Microtox luminescent test, with a difference of three to four orders of magnitude (Microtox gave EC 50 = 58 and 51 mg•L −1 for diuron and Irgarol 1051 respectively) [54] [57] [58].

Conclusions and Future Perspectives
The use of microorganisms provides easiness of use, rapid response and cost effectiveness.They can be used in diverse applied fields notably in environmental monitoring.Knowledge acquired from natural bioluminescence allows the development of sophisticated molecular tools.Luminescent genetically modified microorganisms are easy to manipulate and allow rapid and reproducible experiments.They fulfil the major requirements of whole cell miniaturized reporters and constitute a new generation of biosensors.They are among the most sensitive tests and will probably be intensively used in emerging environmental issues.Recently, the transfer of metal oxide nanoparticles from microalgae (Cricosphaera elongata) to sea urchin larvae (Paracentrotus lividus) was shown [59].Investigating the potential toxicity of such particles on aquatic organisms and their entry into the food chain has become an important issue.Microalgae like Ostreococcus tauri are thus good candidates to help to answer to these questions.

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In vivo gene expression• Association with fluorescence (BRET…)