Three projects and three goals for the next 6 months

(1) Alexandrium project

Research question: What are environmental drivers for blooms of Alexandrium in Bellingham Bay? 

Goal 6 months:

Question: Can we use already designed probes to quantify Alexandrium?

Use a TaqMan probe designed for identification of Alexandrium at the genus level to quantify Alexandrium fundyense and Alexandrium tamarense from culture. Then, use that same probe to quantify Alexandrium in Bellingham Bay.

(2) Pseudo-nitzschia project

Research question: What are environmental drivers for blooms of Pseudo-nitzschia in Bellingham Bay? 

Goal 6 months: 

Question: Can we use already designed probes to identify and quantify Pseudo-nitzschia?

Use rRNA-targeted probes in whole cell hybridization to enumerate cells in Bellingham Bay.

And, use Scanning Electron Microscopy (SEM) to quantify the cells.

(3) Longfin smelt project

Goal 6 months:

Can we use TaqMan probes to quantify Longfin smelt in the Nooksack River?

Sigma-Aldrich schematic for real-time PCR

 

How TaqMan Works -- Ask TaqMan® Ep. 13 by Life Technologies

Webinar with Dr. Gregory Cajete

Today I attended a webinar with 1100 participants, including 137 different tribes. 

It is the 4th webinar in the series: "Indigenous Perspectives on Earth and Sky" 

"Native Astronomy through Native Eyes"  was presented by Dr. Gregory Cajete" 

Nancy Maryboy introduced the webinar series.

The webinar is part of a series that is planned by Indigenous Education Institute San Juan Island National Monuments, San Juan Island National Historical Park and BLM Collaborative Action and Dispute Resolution.

Description of webinar is here: Dr. Cajete is Professor Emeritus and former Director of the Native American Studies program at the University of New Mexico. He is a renowned author and artist from Santa Clara Pueblo, New Mexico. He has pioneered reconciling Indigenous perspectives in science with a western academic setting. His focus is on teaching culturally based science, with its emphasis on health and wellness. 

New day new Alexandrium

Currently, we do not have cultures of Alexandrium catenalla - therefore we will try to purchase A. fundyense. This means I need to find probes for A. fundyense. I learned this morning that A. fundyense and A. tamarense are closely related, while mating compatibilities even suggest them to be varieties of a single heterothallic species. 

Image of A. fundyense found at: https://microbewiki.kenyon.edu/index.php/Alexandrium_fundyense_NEU2011

 

Light microscope and Scanning Electron Microscopy images of Alexandrium species (Kim et al., 2017)

 

How will closely related species affect probe design? 

I read yesterday that a recent study couldn't make species-specific primers for the ITS1-5.8S-ITS2 rRNA gene region for Alexandrium catenalla. This was due to high nucleotide similarilty both with A. tamarense and A. fundyense (Galluzzi et al., 2010).

Well, now I need to know the difference between ITS1-5.8S-ITS2 rRNA gene and hypervariable D1-D2 domain. The diagram below helps me visualize where the regions are located. However, why use ITS rather than D1? Vandersea et al., 2017 reviewed two decades of investigations of Alexandrium species and found molecular studies designed species-specific assays for Alexandrium commonly targeted the hypervariable region in the LSU DI-D2 domain. 

Schematic representation of nuclear ribosomal DNA regions (Stockinger et al., 2010).

Vandersea et al., 2017 developed a species-specific PCR assay for A. fundyense Group  - not a TaqMan assay unfortunately - for which I am looking. 

Let me try explain using these figures:

TaqMan assay requires a forward and a reverse primer, as well as a reporter probe.

Shown here is the forward and reverse primers, as well as the TaqMan probe - with a "Q" quencher. The fluorescence occurs when the "R" is removed and leaves behind the "Q."

Hatfield et al., 2019 used a Taqman qPCR assay to target multiple species of Alexandrium. The assay targets a 125bp region of the 18S rDNA gene and was developed by de novo alignment of 25 Alexandrium species.They noted limitations to the assay - no resolution of taxonomy beyond Genus level. This is not useful for me - I want to identify species - I cannot use this assay. 

 

I will continue to look for primers and probes for A. fundyens. In the meantime, here are some figures I found today that show the interior of A. fundyense as well as the life cycle of A. fundyense.

 

Figure showing the interior of Alexandrium fundyense as it is infected by the parasite
Amoebophrya (Lu et al., 2016). This is a nice drawing of the interior of the Alexandrium
fundyense cells.

Diagram of Alexandrium life cycle found at: https://microbewiki.kenyon.edu/index.php/Alexandrium_fundyense_NEU2011

 

Galluzzi, L., Bertozzini, E., Penna, A. et al. Analysis of rRNA gene content in the Mediterranean dinoflagellate Alexandrium catenella and Alexandrium taylori: implications for the quantitative real-time PCR-based monitoring methods. J Appl Phycol 22, 1–9 (2010). https://doi.org/10.1007/s10811-009-9411-3

Lu, Yameng & Wohlrab, Sylke & Groth, Marco & Glöckner, Gernot & Guillou, Laure & John, Uwe. (2016). Transcriptomic profiling of Alexandrium fundyense during physical interaction with or exposure to chemical signals from the parasite Amoebophrya. Molecular ecology. 10.1111/mec.13566. 

Kim, Eun & LI, Zhun & Oh, Seok & Ho, Yoon & Shin, H H. (2017). Morphological identification of Alexandrium Species (Dinophyceae) from Jinhae-Masan Bay, Korea. Ocean Science Journal. 52. 1-11. 10.1007/s12601-017-0031-6. 

Stockinger, H., Krüger, M. and Schüßler, A. (2010), DNA barcoding of arbuscular mycorrhizal fungi. New Phytologist, 187: 461-474. doi:10.1111/j.1469-8137.2010.03262.x

Robert G. Hatfield, Timothy Bean, Andrew D. Turner, David N. Lees, James Lowther, Adam Lewis, Craig Baker-Austin, 2019, Development of a TaqMan qPCR assay for detection of Alexandrium spp and application to harmful algal bloom monitoring, Toxicon: X, vol. 2, https://doi.org/10.1016/j.toxcx.2019.100011.

Sequencing primers and conditions

I am on the lookout for properties of sequencing primers for the A. catenella hypervariable D1-D2 domain, and primers for the qPCR analysis targeting this domain. I am also looking for the qPCR cycling conditions.

Garneau et al., 2011 selected primers UScatF (5-AACAGACTTGATTTGCTTGG-3) and UScatR (5-CACAGGAGACTTATCATTCATG-3) and produced a predicted PCR amplicon length of 141 bp.

The final qPCRs were  carried out in 50-l volumes containing:

1.  10 ul of 1:100 crude lysate (5 to 10 ng of environmental DNA), 
2. a 300 nM concentration of each primer, 
3. and a 400 nM concentration of fluorogenic probe. 
4. 1ul PCR colorless GoTaq Flexi buffer, 
5. 7 mM Mg2,
6. 200 M dNTP mix, 
7. and 2.5 units of GoTaq DNA polymerase
   

All reagents, samples, and standards were prepared on ice prior to thermal cycling. PCRs and a blank control (no DNA added) were set up in triplicate in 96-well PCR plates sealed with flat strip caps (Bio-Rad; 2239441) and centrifuged briefly to remove bubbles. The qPCR thermal cycling conditions were as follows: 1 cycle of heating at 95°C for 3 min and then 40 cycles of 94°C for 15 s (denaturation), 53.2°C for 30 s (annealing), and 72°C for 30 s (extension). Thermal cycling and real-time data collection at the annealing step were performed using an iCycler iQ realtime PCR detection system (Bio-Rad Laboratories)  

Kamikawa et al., 2007 used primers specific to A. catenella: catF (50-CCTCAGTGAGATTGTAGTGC-30) and catR (GTGAAAGGTAATCAAATGTCC-30) and the
probewasTaqman cat (50-FAM-ATGGGTTTTGGCTGCAAGTGCA-
TAMRA-30).  

 PCR cycles were carried out using 10 ml volumes that consisted of:

1. 1 ml of temperate DNA, 
2. 0.3 mM of each primer pair, 
3. 0.2 mM of the probe, 
4. 1 LC FastStart DNA Master Hybridization Probes (containing PCR buffer, dNTP, MgCl2, and Taq polymerase) (Roche Diagnostics GmbH), 
5. Mg2+ to a final concentration of 3 mM(Roche Diagnostics GmbH), 
6. and PCR grade water to a final volume of 10 ml (Roche Diagnostics GmbH). 

The cycling conditions were as follows: one cycle at 95 8C for 1 min; 50 cycles at 94 8C for 15 s, 56 8C for 30 s, and 72 8C for 30 s.

First afternoon on the boat

 Today we took the 26’ aluminum chambered boat to Bellingham Bay to collect a Solid Phase Adsorption Toxin Testing (SPATT) sample (from the Se'lhaem buoy), YSI data (T, pH, Cond. dissolved oxygen, salinity data), and a tow (~5 m depth). 

Thayne with the Buoy in background.

A week-old SPATT sampler was attached to Se'lhaem .We cut it off and replaced it with a new SPATT sampler. The SPATT samplers are kept in the -80 freezer until LCMS-MS analysis.

Here is a link to video of Thayne setting up the YSI that collects environmental data. He is also collecting a VanDoren sample from 5 feet and 10 feet from the ocean surface.