Project Description

Project description

Dinoflagellates and diatoms such as Alexandrium spp., Dinophysis spp. and Pseudonitzschia spp are widely distributed bloom-forming marine micro-algae. These organisms can produce potent toxins which cause different shellfish poisoning syndromes. Human exposure to these toxins is potentially deadly and occurs via the consumption of toxin-exposed filter-feeding shellfish. Understanding the factors which contribute to the formation of algal blooms and toxin production and identification of the genetic and environmental key drivers is of environmental, economic, and clinical importance. For example the dinoflagellate Alexandrium spp. contains a number of toxic as well as non-toxic species. The genetic and environmental factors inducing toxin production are not well understood. Due to the unusual complexity and size of dinoflagellate genomes, there are limited genome sequencing projects for these organisms, and transcriptome sequencing has become the major focus in this field. 

However, a serious limitation is our incomplete understanding of genetic and metabolic network regulation in harmful algae species. Dinoflagellates have a well-developed metabolism to survive even under extreme stress conditions in which gene silencing mechanisms have been reported to be involved in modulating metabolic responses to environmental stress. In particular, microRNAs (miRNAs), a class of endogenous non‐coding RNAs, play an essential role in post‐transcriptional gene regulation in plants, animals and dinoflagellates. These are key factors in stress response molecular pathways triggering cellular responses and metabolic adjustments to stress conditions that affect toxin production. The project proposes to identify and characterize marine algal blooms for example Alexandrium spp. RNAs and proteins and metabolite‐mediated responses to different stress conditions on the toxin production procedures. Both toxin-producing and non-toxin-producing species will be compared. The data will be analyzed to detect the major cascades triggered when the organism faces toxin-inducing environmental conditions.  

The project will involve the information transfer from these procedures for the design and production of a prototype diagnostic kit for the detection of the toxin genes specific to the species. Molecular probes are being applied to distinguish species-specific RNA and DNA sequences for the rapid identification of toxins produced by organisms.  The direction of this new technology is to develop rapid reliable screening methods for phycotoxins and the causative organisms to protect public health, aquaculture, and natural resources. An innovative sample preparation for toxin detection will be designed so that the device can be used in field. To date a novel method that can be applied on-site and by non-educated farm owners is needed to accurately detect the harmful algae and toxin as an early warning monitoring device. Such a kit must be financially affordable so that it can be used by farm owners. 

This can be undertaken using a two-fold approach:  Developing a sample preparation method away from the device but where no laboratory equipment is required and secondly the utilization of specific reagents particular for metabolite or nucleic acid amplification that can be built into a cartridge to provide a significant level of detection for both species and toxin analysis.  The system will be evaluated for culture analysis using cultures and real samples. 

Additional information

In this study, a systems strategy integrating transcriptomic analysis and liquid chromatography/mass spectrometry-based metabolomics will be applied. Three to four major environmental stressors inducing toxin production in harmful algae species for example Alexandrium spp will be selected based on the literature review. The organism will be in exposure to these parameters separately to trigger the molecular and biochemical cascades resulting in toxin production.  

Due to the unusual complexity and large size of microalgal genomes, there are only a small number of Genome sequencing projects for these organisms, and Transcriptome sequencing has become the major focus in this field. Transcriptome data of several microalgal species including Alexandrium spp. is available in Genbank and can be used as a reference dataset for further gene assembly and gene expression studies of this organism. 

The availability of this bioinformatic source will represent a new instrument for researchers for the implementation of marine biotechnological resources, techniques, and products. 

Based on existing Transcriptome and Genome data in Genbank, with the integration of new transcript and metabolite data, this project aims to identify transcripts, metabolites, and inducers involved in the induction of processes associated with PSP toxin production. Illumina and 454 Sequencing of toxic and non-toxic Alexandrium spp. strains under various environmental conditions can be employed for both de novo transcriptome assembly as well as gene expression analysis / identification of fold changes of known and novel genes relevant to environmental stress responses and toxin production. 

Metabolomics results can be integrated with high throughput gene expression and targeted PCR Gene level results. The combination of metabolite and gene responses as well as identification of epi-genomic regions upstream and downstream of identified genes of interest may help elucidate specific drivers of toxin production in these organisms and identification of corresponding genes and metabolites. Both toxic and non-toxic algal species will be studied under various environmental conditions and the data will then be compared using bioinformatic technologies. 

Some candidate genes for saxitoxin production have been introduced and used for the detection of toxic species of this organism. Another parallel aim of the project is to design a portable early warning monitoring device that shellfish farmers can use on-site at farms or on boats for the detection of this toxic gene’s expression. The project will involve the technology transfer of these procedures for the detection of the toxin gene expression specific to the species onto production of a diagnostic kit which help to revitalize the sample preparation methodology for metabolite, proteins, or nucleic acid detection so that the device can be used in the field. The technology platform for the project is based on protein, metabolite, or transcript detection using RAPID tests available for the detection of subtle toxic molecules. Such rapid tests are used for the detection of covid-19 trace molecules based on antibody interaction or nucleic acid amplification on site. Also, rapid field test kits have been developed to screen for paralytic shellfish poisoning toxins. Yet, not all of those are applicable in the field.  

After the detection of the toxin genes for the species responsible for toxin production, top chains with maximum foldchanges will be distinguished. One approach to implementing an immunoassay is lateral flow immuno-chromatography (LFI). In such an assay, all the components are incorporated into a test strip, so that it is only necessary to add a sample extract to initiate the sequence of reactions. As a result, LFI tests require no special expertise or laboratory equipment in their use. Consequently, this technology has found many applications, such as in-home pregnancy test kits and COVID diagnosis. LFDs for shellfish toxins have been developed but none in combination with the harmful organism recognition producing the toxin. In this proposal a prototype will be designed for the simultaneous detection of toxin genes from a species, marker proteins and toxin. 

To develop a rapid test kit for detecting toxins an adequate supply of purified toxins is essential, especially one involving antibodies where toxin-protein conjugates are required for both immunizations and to form capture lines on the test strips. Furthermore, the production of LFI test strips with reliable and uniform responses requires specialized equipment such as Scienion S3 arrayer to design such a systems biology multiplex device. Test and component parameters will be adjusted to give an optimal response for each assay and then combined on one device. Complete test strips will be mounted in plastic cassettes and be able to be read using handheld readers. The detection power will be compared to other kits previously produced for toxin testing only. 

The methods that will be used are thus, PCR- Mass spectrometry – HPLC – Scienion bioprinting, bioinformatic sources (GenBank, Galaxy, etc.) to analyze the data, which are all in the form of large matrix data, software designed that mostly operate in the MATLAB and R environments. Chemometrics acts as a complementary method in data analysis and interpretation if needed. 

Five separate chapters are thus defined for each year of studies: 

Ch1: Literature Review on Omics approaches for harmful algal blooms and toxin production 

Ch2: Screening for environmental toxin stimulators, selection of top stimulators, and characterization of biosynthesis pathways (genomics/transcriptomics/proteomics/metabolomics) including protective functions against environmental stress. 

Ch3: Comparison of the datasets produced for different selected environmental stressors, toxic and non-toxic species, and characterization of major toxin coding genes,  

Ch4: Design of systems biology prototype multiplex biochip and evaluation with real samples  

Ch5: Conclusions and future research  

Essential & desirable candidate skills

Essential: Applicants should have a primary degree (1st or 2.1) in an appropriate discipline (e.g., Genetics, Molecular biology, Biochemistry, Environmental Science). Some experience of PCR – bioinformatics – metabolomics – Transcriptomics – 

Desirable: M.Sc. in an appropriate discipline e.g., Molecular biology, Biochemistry, Environmental Science, Biotechnology. Some practical experience in bioanalytical analysis.  

Supervisors

Katrina Campbell

Primary Supervisor:

Profile: Katrina Campbell
Email: katrina.campbell@qub.ac.uk
Institution: Queen's University, Belfast
Department/School: School of Biological Sciences

Lenka Mbadugha

Secondary Supervisor:

Profile: Lenka Mbadugha
Email: lenka.mbadugha@abdn.ac.uk
Institution: University of Aberdeen
Department/School: School of Biological Sciences

References

Ho KC, Lee  TCH, Kwok OT and Lee FWF. (2012). Phylogenetic analysis on a strain of Alexandrium tamarense collected from Antarctic Ocean. Harmful Algae, 15: 100-108.

Cusick K and Sayler G S. (2013). An overview on the marine neurotoxin, saxitoxin: Genetics, molecular targets, methods of detection and ecological functions. Marine drugs, 11(4): 991-1018.

McLean TI. (2013). “Eco-omics”: A Review of the Application of Genomics, Transcriptomics, and Proteomics for the Study of the Ecology of Harmful Algae. Microbial ecology, 65: 901-915.

Research Methods

The methods that will be used are thus, PCR- Mass spectrometry – HPLC – Scienion bioprinting, bioinformatic sources (GenBank, Galaxy, etc.) to analyze the data, which are all in the form of large matrix data, software designed that mostly operate in the MATLAB and R environments. Chemometrics acts as a complementary method in data analysis and interpretation if needed. 

Impact

One of the main outcomes of this work will be a significant step toward a better comprehension of the general architecture of the miRNAome and genetic network of Alexandrium spp., which are still largely unexplored in the case of toxin production. The sequencing and bioinformatic approaches developed during this project will shed light on the new characteristic of specific marine dinoflagellates giving new ecological and biotechnological information for this important ecological issue. This thesis is expected to combine marine biology, molecular biology, biochemistry, biotechnology, bioanalytical chemistry and bioinformatics. This interdisciplinary approach will also provide new insights into the biotechnological exploitation of marine genetic resources for the production of value-added secondary metabolites and to reduce the harmful outcomes of algal toxins for industry and human health. 

In addition, the successful design and prototype development of the rapid detection kit, can help its wide application in shellfish, crab, oyster, sea urchins, and other filter-feeding animals to prevent damage caused by algal toxins. 

For the correct detection of the genes responsible for toxin production which may be sensitive to specific environmental stressors, diagnostic kits can be made to identify the type of environmental stressor as a marker and the relative production of a specific toxin can be predicted depending on the type of environmental stressor and harmful algae species.

Proposed Timetable

  • Literature review (months 1 – 3) 
  • Screen toxic and non-toxic harmful algae strains eg Alexandrium Tamarense exposing them to altered environmental conditions such as N, P and Salinity and environmental pollutants for growth and toxin production and analyse them using genomics/transcriptomics and metabolomics analysis (Month 4-12).  
  • Sequence for example A. tamarense using RNAseq and using de novo assembly and mapping techniques assemble, annotate and compare the transcriptome of the screened strains to establish phenotypic specific gene expression and single nucleotide polymorphisms (Month 4-12).  
  • Correlate the metabolomics results with the gene, transcript expression and SNPs. Identify if any of the metabolites, genes or transcripts expressed are associated with specific environmental triggers mentioned previously (Month 13-18) 
  • Primer design for amplification and sequencing of key genes that may be identified though out the process (Months 19-21).  
  • Identification of key environmental chemicals/contaminants / pathogen / toxin to design prototype model system biology multiplex chip (Months 19-21) 
  • Evaluation of different biosensors for suitability in multiplex detection and suitability to couple to smartphone apps  (months 21 – 24) 
  • Design of rapid sample preparation / concentration methods for key contaminants (months 25 – 27) 
  • Evaluate model biological system lab on a chip biosensor against collated data from cultures and water samples (months 28 – 30) 
  • Completion of PhD thesis, dissemination of results at conferences and manuscripts for publication (months 3 -36).  

QUADRAT Themes

  • environmental-management

Partners

A partnership within the Diagnostics industry is currently under discussion. An update will be provided in due course.

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