MOLECULAR DETECTION OF HABs SPECIES :

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1 MOLECULAR DETECTION OF HABs SPECIES : How it complement to HAB MONITORING PROGRAM Chui Pin LEAW PhD IOES, UM

2 Species recognition Taxonomy is a fundamental research that ones could not neglect in any discipline with respect to the organisms. In HAB studies, this is essentially important, as NOT all species are harmful. Teng et al. (2014) Harmful Algae

3 VARIATION?! Morphologic characteristics etc.

4 VARIATION?! biomolecules

5 Molecular Tools for species detection An Avenue in HABs Monitoring

6 Detection methods Molecular detection technologies included: Fluorescence in situ hybridization (WC-FISH, TSA- FISH) coupled with epi fluorescence microscope, flow cytometer, FlowCAM, or ESP. Real time quantitative PCR (qpcr), MIDTAL Insulated isothermal PCR (iipcr), Digital PCR (dpcr)

7 Fluorescence in situ Hybridization (FISH) How it works?

8 Molecular probes for species identification These probes are short oligonucleotides of bp length. In hybridization experiments, the probes will bind to target s sequence and later detect by a probe-label (fluorescent dyes, Digoxigenin); thus identify species of interest.

9 Species-specific rdna probe of Alexandrium minutum Yek et al (Proceedings of SCS)

10 Which gene to select? Probe were normally selected from genes with large dataset, e.g. LSU and ITS of ribosomal RNA genes. Genes that widely used in phylogenetic studies. Taxonomic level of specificity. Sufficient target site for probe binding.

11 In silico probe design Alexandrium minutum species- to strain-specific probes

12 Common features of molecular probes Sequence signatures serve as suitable target sites for nucleic acid probing. Probe sequence must be 100% complement to target sequence. Ideally, the probe of interest shows at least a single base mismatch to all non-target microorganisms.

13 Probe specificity and accessibility In silico

14 F I S H W O R K F L O W Algal cultures In silico Probe design Sequence signature Probe stability and stringency Whole-cell FISH FISH optimization

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16 Species-specific rrna probe of Pseudo-nitzschia pungens Teng et al. (in prep)

17 Species-specific rdna probe of Chattonella subsalsa Lau et al. (in prep)

18 Detection Device FISH coupled with epi fluorescence microscope, flow cytometer, FlowCAM, or ESP. FlowCAM with fluorescence trigger channels From MBARI

19 From Greenfield et al. Limnology and Oceanography: Methods (in review)

20 Real time quantitative PCR (qpcr) How it works? Detection and analysis its key feature is that the amplicon is detected as the reaction progress in real time.

21 Development of qpcr assay In silico primer/probes design: genus- or species-specific, toxic species-specific Optimization of gene amplification Selection of chemistries to detect amplicons. 5 nuclease assay (TaqMan Probes) SYBR Green I dye

22 q P C R W O R K F L O W qpcr setup Running qpcr Data analysis

23 Detection of A. tamiyavanichii cells along the coast of Malaysian Borneo

24 Insulated isothermal PCR (iipcr) How it works?

25 i i P C R W O R K F L O W

26 Advantages Portable Cheaper costs in term of running and instrument Shorter running time (1 hr)

27 Single cell PCR How it works? Conventional PCR/qPCR Sequence analysis Single-cell isolation

28 Karlodinium bloom in Johor Strait Lim et al. (in review) Harmful Algae

29 Molecular classifier in species recognition Secondary structure of ITS2 RNA transcript Compensatory base changes (CBCs) Two taxa with even one CBC occurred in the relatively conserved paired region of ITS2 transcript are sexually incompatible, thus are biologically distinct (Coleman 2009).

30 ITS2 RNA transcript of Coolia Leaw et al. (in prep)

31 Genotyping of HABs species in the environment Pseudo-nitzschia (Bacillariophyceae) community structure in the eastern South China Sea Teng et al. (in prep) Distribution pattern and species richness of Pseudo-nitzschia spp.

32 P. delicatissima ribotype 2 (282 bp) and P. caciantha ribotype 2 (345b/346a) were most abundant; with P. delicatissima the most widely distributed throughout the sampling locations. Most of the locations consisted more than one species indicating that Pseudo-nitzschia spp. were co-existed in the region. Teng et al. (in prep) Clustering analysis revealed a degree of community structuring in Pseudo-nitzschia. The result showed two linkages, one cluster with locations being dominated by P. caciantha; and the other with locations where it absent.

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34 Summary Detection and enumeration capabilities that are independent from microscopic techniques. Molecular tools provide an avenue and support for HAB monitoring: Providing sensitive and precise techniques to substitute labor-intensive cell counts Providing an alternative approach for detecting toxic/harmful algae Providing an early warning system for HAB

35 Acknowledgements Malaysian Government for grants support: MOSTI ScienceFund MOE FRGS UNESCO IOC-WESTPAC HAB, TMO Fellow Research Associates: PT Lim, G Usup, Y Fukuyo, A Kodama, M Iwataki, S Bates, DV Ha etc. Graduate students: TH TAN, ST TENG, KS HII, NF KON, LH YEK, R RAZALI Thank you

36 SPECIES RECOGNITION How Molecular Diagnostic could help?

37 Molecular classifier in species recognition Secondary structure of ITS2 RNA transcript Compensatory base changes (CBCs) Two taxa with even one CBC occurred in the relatively conserved paired region of ITS2 transcript are sexually incompatible, thus are biologically distinct (Coleman 2009).

38 ITS2 RNA transcript of Coolia Leaw et al. (in prep)

39 Cryptic diversity in Pseudo-nitzschia Teng et al. (2013) Botanica Marina Lim HC et al. (2012) J Phycol

40 A new species of Pseudo-nitzschia was discovered.

41 Lim HC et al. (2012) J Phycol Fig. 8 Molecular signatures of Pseudo-nitzschia species found in Malaysian Borneo. (A) P. brasilliana, (B) P. dolorosa, (C) P. micropora, (D) P. pungens, and (E) P. circumpora. Rectangle showing the CBC while arrow indicate the hemi-cbc.

42 More new species of Pseudo-nitzschia from the region

43 More new species of Pseudo-nitzschia P. batesiana P. lundholmiae P. fukuyoi Lim HC et al. (2013) J. Phycol.

44 Morphological plasticity Many species exhibit great morphological plasticity, and sometimes lead to misinterpretation of species identities. Molecular data offers an alternative for situations in which morphology is inconclusive.

45 Alexandrium ostenfeldii/ A. peruvianum complex A. peruvianum from Malaysia produce spirolides /saxitoxins Morphological features : size s.a. 1 6 These descriptive characters are somehow plastic. Lim et al. (2005) Harmful Algae

46 Conspecific? Alexandrium ostenfeldii/peruvianum complex Kremp et al. (2014) J. Phycol.

47 Alexandrium ostenfeldii and A. peruvianum are conspecific Kremp et al. (2014) J. Phycol.

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49 Molecular-assisted techniques Species recognition DNA barcoding Systematics molecular phylogenetic reconstruction Biodiversity and Biogeography: genetic variability estimation

50 Facilitate identification and recognition of species Assessing cryptic species Discovering potentially new species

51 A cosmopoliton dinoflagellate, Coolia monotis???

52 Thecal plate arrangement of Coolia species C. malayensis C. monotis C. tropicalis C. areolata C. canariensis APC 1 3 L/W of 7 < 2 L/W of 7 = 4 L/W of 7 2 Leaw et al. (2010) J. Phycol.

53 Lineage of Coolia malayensis C. monotis Nuclear encoded LSU rdna C. malayensis C. canariensis

54 Coolia malayensis Leaw, Lim et Usup

55 Species delimitation by GMYC model further supports the six lineages Species boundary

56 Alexandrium tamarense complex Placement of tropical A. tamarense into the A. affine clade Paraphyly of A. tamaranse? Leaw et al. (2005) Phycologia

57 MOLECULAR SYSTEMATICS: How it contributes to the beta-taxonomy?

58 Morphological traits used in Alexandrium taxonomy Thecal plates tabulation

59 Phylogenetic relevance? Character State Evolution

60 Character state evolution Leaw et al. (2005) Phycologia

61 Posterior sulcal plate (S.p.) synapormorphy synapormorphy synapormorphy Fukuyo (1985) ~ S.p. as the most distinctive character The character correlated well with the phylogenetic circumscription Reliable phylogenetic marker Transition point

62 Phylogenetic relationship of Pyrodinium and Alexandrium Gessnerium group P. bahamense consistently emerged as an ingroup in the genus Alexandrium The placement may not be readily accepted (from the taxonomist s point of view)

63 Morphology of Pyrodinium bahamense var. compressum

64 Character state evolution Leaw et al. (2005) Phycologia

65 Character state evolution Leaw et al. (2005) Phycologia

66 Pseudo-nitzschia phylogeny

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69 BIODIVERSITY AND BIOGEOGRAPHY How genetic variability contributes to it?

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71 Intraspecific phylogeographic pattern of Ostreopsis ovata BI MSN O. ovata O. lenticularis Leaw et al. (2001) Mar. Biotech. 3:

72 Phylogeographic break of O. ovata in the Sunda Shelf gene flow Malacca Straits group South China Sea group Leaw et al. (2001) Mar. Biotech.

73 Intraspecific genetic diversity in Coolia malayensis ITS2 transcript ITS region of rdna Leaw et al. (in prep)

74 Phylogeographic pattern of C. malayensis Leaw et al. (in prep)

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76 Global diversification of Pseudo-nitzschia pungens Lim HC et al. (2014) Harmful Algae

77 Global diversification of Pseudo-nitzschia pungens Lim HC et al. (2014) Harmful Algae