INVESTIGATING THE EFFECTS OF TEMPERATURE ON THE GROWTH AND TOXIN PRODUCTION OF , ANATOXIN AND CYLINDROSPERMOPSIN- PRODUCING

Emily N. Beers

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

August 2020

Committee:

Timothy Davis, Advisor

George Bullerjahn

Robert McKay © 2020

Emily Beers

All Rights Reserved iii ABSTRACT

Timothy Davis, Advisor

Cyanobacterial harmful algal blooms (cHABs) are a growing global issue that influences

the health and socioeconomic status of those dependent on impacted water bodies. Variation in

water surface temperatures driven by changing global climate has allowed for increases in the

growth of toxigenic HAB-forming cyanobacteria. It has been documented that a range of some

genera have shifted with increasing temperature, causing some strains to be able to invade new

aquatic environments. While previous studies have investigated the effects of temperature on

microcystin-producing cyanobacteria, in the current study, we test the effects of temperature on individual cyanobacterial strains capable of producing either , cylindrospermopsins or anatoxin-a across a thermal gradient of 17 to 30 ºC under common light and replete nutrient conditions. The goal is to determine the strain specific optimal temperatures and limits for growth and toxin production in these HAB-forming cyanobacteria. Using this framework, we tested several different genera including, Aphanizomenon, Dolichospermum,

Cylindrospermopsis, and . Warmer temperatures favored cell growth while cooler

temperatures showed higher toxin production per cell. Overall, the most toxins produced by

samples at the end of the experiments were produced where there were the fastest growth rates. iv To my parents Jay and Alesa Beers, as well as, the rest of my friends and family thank you so

much for all of your support and love.

All of my lab mates and friends that I’ve made at BGSU, I never thought I’d find myself in NW

Ohio but I’m exceeding glad that I did. I will miss you all! v ACKNOWLEDGMENTS

I would like to thank: My advisor Dr. Timothy Davis and my committee members Dr.

George Bullerjahn and Dr. Michael Mckay for allowing me to work with you and further my education and love for science.

William Cody for helping me with cellular enumerations.

My undergraduate helpers Katherine Wolf, Dakota Jenkins and Alexis Heath for counting endless amounts of cultures for me.

My undergraduate professor Dr. Courtney Wigdahl-Perry for always having faith in me and suggesting I apply to BGSU to be in Dr. Davis’ lab.

ODHE and Ohio Sea Grant for their funding.

ANASS, Cawthron Institute and CSIRO for their cultures.

The entire BGSU lab Katelyn McKindles, Michelle Neudeck, Callie Nauman, Mathew

Kennedy, Laura Reitz, Dr. Paul Matson, Daniel Peck, Jonathan DeMarco, Seth Buchholz,

Christina Moore and Kari Lane Shupe. vi

TABLE OF CONTENTS

Page

CHAPTER 1: INTRODUCTION ...... 1

1.1: Hypothesis...... 5

CHAPTER 2: MATERIALS & METHODS ...... 7

2.1: Culturing...... 7

2.2: Temperature Gradient Chamber...... 8

2.3: Experimental Setup...... 10

2.4: Sampling...... 11

CHAPTER 3: RESULTS ...... 13

3.1: Dolichospermum circinale ...... 13

3.2: Aphanizomenon ...... 18

3.3: Cylindrospermopsis raciborskii...... 19

3.4: Growth Rates...... 21

CHAPTER 4: DISCUSSION ...... 22

4.1: Future Work ...... 24

LITERATURE CITED ...... 26 vii

LIST OF FIGURES

Figure Page

1 Interior of the temperature gradient chamber showing hydrated air bubbling system.

The temperature gradient runs from 17.1 to 30.1oC from left to right...... 9

2 Dolichospermum circinale strain CS-337/01 growth curves from 17.1 to 27.2oC..... 13

3 Dolichospermum circinale strain CS-337/01 saxitoxin quota from 17.1 to 27.2oC..... 14

4 Dolichospermum circinale strain CS-541/06 growth curves from 17.1 to 30.1oC...... 15

5 Dolichospermum circinale strain CS-541/06 saxitoxin quota from 17.1 to 30.1oC..... 15

6 Dolichospermum circinale strain CS-1031 growth curves from 17.1 to 27.2oC...... 16

7 Dolichospermum circinale strain CS-1031 saxitoxin quota from 17.1 to 27.2oC...... 17

8 Aphanizomenon strain CAWBG01growth curves from 18.6 to 28.9oC...... 18

9 Aphanizomenon strain CAWBG01saxitoxin quota from 18.6 to 28.9oC...... 18

10 Cylindrospermopsis raciborskii strain CS-506 growth curves from 21.7 to 30.1oC...... 19

11 Cylindrospermopsis raciborskii strain CS-506 cylindrospermopsin quota from 21.7 to

30.1oC...... 20 viii

LIST OF TABLES

Table Page

1 List of samples by strain, toxin produced and source of the strain ...... 8

2 Temperature (oC) of each sample within the temperature gradient...... 10

3 Growth rates for the 5 strains at each temperature they were run at...... 21

4 List of optimal temperatures for both growth and toxin quota of each strain...... 21 1

CHAPTER 1: INTRODUCTION

Harmful algal blooms can occur in both salt and freshwater, plaguing our lakes and oceans, although algae are natural micro-organisms their affects have been exacerbated through eutrophication and global climate change (Anderson, et al., 2002).As global climate change continues to cause perturbations to freshwater ecosystems from increased temperature, larger and more frequent cyanobacterial harmful algal blooms are probable (O’Neill et al., 2012; Savadova, et al., 2018). Changes in species abundance and distribution can be attributed to warming in freshwater systems as well as disruptions in diel migration of phyto- and zooplankton (Rosenzweig, et al., 2007). Lakes across the northern hemisphere, where climate change is predicted to a have higher impact (Stocker, et al., 2013), have shown an overall increase in surface temperate from 0.2 to 2oC across

North American and Europe since the mid 20th century (Rosenzweig, et al., 2007). Even

deep-water temperatures of some African rift lakes have shown increases in typically

homothermal regions of the water column (O’Reilly, et al., 2003).Warming temperatures

will also influence summer stratification; increasing the length of the time the lake

remains stratified, which could favor cyanobacterial growth and further extend the bloom

season (Stasio Jr., et al., 1996; O’Neil et al., 2012).

Warming in lake surface temperatures could change the habitat suitability for

temperate cyanobacteria allowing niches to open and subtropical, invasive cyanobacterial strains to gain a foothold (Sukenik, et al., 2012). Cyanotoxins produced by cyanobacteria harmful algal blooms (cHAB) are threat to the world’s freshwater systems and have various detrimental impacts on animals and humans exposed whether through ingestion or dermal contact (Loftin, et al., 2016). Environmental and health organizations such at 2 the United States EPA, Environment and Climate Change Canada and the World Health

Organization have set guidelines for exposure and drinking water limits to attempt to minimize the number of people affected by these harmful cyanotoxins. Global climate change and eutrophication coupled with rising populations has put additional stressors on our surface waters resulting in increased bloom activity and toxicity in the Americas,

Europe and Australia, that has fueled a rise in human health risk and animal death

(Falconer, et al., 2005). Increases in bloom activity can also negatively influence the economy through unsightly scums, sulfurous smells and fish kills that can lead to decreases in lakefront property value and beach closings.In 2014, the Toledo Water Crisis struck northwest Ohio leaving thousands without water and an estimated $65 million in damages and lost revenue (Bingham, et al. 2015).It was estimatedin 2015, Canada could lose up to $272 million CDN in recreation and tourism revenue in the following decade due to Lake Erie impairment alone (Smith, 2019).

There are over 40 known genera of cyanobacteria that are capable of producing toxins, including but not limited to cylindrospermopsin, anatoxin and saxitoxin

(Carmichael, 2012). Saxitoxins, a potent alkaloid neurotoxin, make up the more commonly known paralytic shellfish toxins (PSTs) that block sodium ion channels in the nervous system which can lead to paralysis, respiratory failure and in extreme cases, death (Wiese, et al., 2010). The Food and Drug Administration limits saxitoxin exposure to 80 μg/100g of saxitoxin. Cylindrospermopsin, a hepatotoxin, is thought to be a potential carcinogen due to its ability to cleave DNA through its reactive guanidine and sulfate groups (Shen, et al., 2002). The US Environmental Protection Agency limits cylindrospermopsin exposure to 3 ppb for adults in drinking water. Anatoxin, a 3 neurotoxin, was first isolated in the 1970’s from an Anabaena flos-aquae strain and was discovered to mimic acetylcholine while some other forms can act by inhibiting acetylcholinesterase causing depletion of energy stores, respiratory failure and death if consumed in high enough doses (Mejean, et al., 2014). Anatoxins posses a risk to human health through recreation, drinking water contamination and cyanobacterial-based dietary supplements, and stricter regulations for anatoxin detection are needed to reduce exposure.

In a study done on Microcystis aeruginosa, it was shown that lower temperatures yielded slower growth rates but had high toxin quotas per cell of microcystin (Peng, et al., 2018). Will other cyanotoxins follow a similar pattern and what could this mean for our freshwater systems with increasing stress brought about by global climate change?

Dolichospermum circinale, a filamentous cyanobacterium, is responsible for creating potent saxitoxin-producing blooms throughout Australia. Each strain isolated produced varying amounts of saxitoxins and some of its 57 analogues (Pereyra, et al., 2017). Other than Cylindrospermopsis and Dolichospermum, Aphanizomenon, Planktothrix, Lyngbya and Scytonema are also known saxitoxin-producing cyanobacteria (Wiese, et al., 2010).

Australia suffers from cylindrospermopsin-producing blooms of

Cylindrospermopsis/Raphidiopsis raciborskii that have caused many gastrointestinal, liver and kidney malfunction, the worst case being in 1979 with 138 illnesses reported

(Hawkins, et al., 1997; Buford et al., 2016). In 2006, cylindrospermopsin was detected in

France for the first time when 6 of the 11 tested lakes tested positive by LCMS-MS coincident with the presence of Aphanizomenon and Anabaena. Other genera known to 4 produce cylindrospermopsin include, Cylindrospermopsis, Raphidiopsis, Lyngbya, and

Umezakia (Brient, et al., 2008).

Cylindrospermopsis is known for its ability to invade freshwater systems due to its ability to withstand a wide range of light and temperatures, as well as being capable of fixing atmospheric nitrogen through the heterocyst cells (Antunes, et al., 2015). A subtropical C. raciborskii strain likely originating from South America has been found in the Laurentian Great Lakes with akinete formations allowing the strain to overwinter during cold temperatures (NOAA GLERL). In a study on C. raciborskii strain C10, growth was 3× greater at 25 oC than at 19oC although saxitoxin quota was not affected,

but this could change by species as well as strain. However, dcSTX was produced at 25

oC but not 19oC in culture during the experiment (Castro, et al., 2004). While C.

raciborskii is established but not currently dominating in the Great Lakes, temperature

shifts to warmer waters could favor the growth over current phytoplankton species.

Worrying trends have been discovered in the proliferation of C. raciborskii in correlation

to global climate change trends, showing increased bloom activity in temperate lakes

across the globe of C. raciborskii strains (Sinha, et al., 2012).Aphanizomenon has also

been documented to expand its habitat into temperate European lakes; both

Cylindrospermopsis and Aphanizomenon are known cyanotoxin producers of

cylindrospermopsins and saxitoxins (Sukenik, et al., 2012). For hepatotoxic

cyanobacteria, warmer temperatures may favor their growth and intracellular toxicity

through grazing and other phytoplankton competition (El-Shehawy, 2012).However,

increased temperature was shown to reduce the amount of toxin produced in

Aphanizomenon and Anabaena regardless of growth rates in culture (Rapala, et al., 1993). 5

In a study done on Aphanizomenon gracile, saxitoxin production and release was stable across temperatures ranging from 15 oC to 28oC, with slightly lower concentrations at

15oC, but it was shown that the growth rate was not directly correlated to toxin

production in that experiment (Casero, 2014). However, in a similar experiment using A. gracile, saxitoxin production was greatest at 30oC compared to the cooler temperatures of

12 oC and 23oC (Cires, 2017).

There are many occurrences across the United States where toxins other than

microcystins have been produced by cyanobacterial harmful algal blooms. For instance,

there are cases of cattle, waterfowl, wildlife and dog deaths that have been reported due

to anatoxin poisoning likely through surface water ingestion (Devlin, et al., 1976). In a

report on the use of cyanobacteria as a dietary supplement, 7.7% of samples tested

contained measurable levels of anatoxin up to 33 ug/g in Aphanizomenon and Spirulina-

containing powders (Rellan, et al., 2009). In Salem, Oregon a drinking water advisory was established for vulnerable populations in the summer of 2018 due to hepatotoxins being found in the water supply above EPA standards for the young, elderly and immuno-compromised (City of Salem, 2018). Harmful algal blooms by cyanobacteria

Aphanizomenon(Nobel 1997), Dolichospermum (Yema, 2016)and Cylindrospermopsis

(Bai, 2014) have the ability to persist in nutrient poor conditions due to their ability to fix

nitrogen gas.

1.1 Hypothesis

Will temperature have an effect on growth rates and cyanotoxin production quota

for strains producing saxitoxin-a, cylindrospermopsin and anatoxin? Based on previous

studies, I hypothesize that toxigenic cyanobacterial strains will favor warmer 6 temperatures for growth and toxin production but that the optimal temperature for toxin production will be lower than that of the optimal temperature for growth rate. In this research, I hope to discover the peak temperatures for both growth and intracellular toxin production for each strain. 7

CHAPTER 2: MATERIALS & METHODS

2.1 Culturing

After samples were received from other sources, each strain was grown in the 5 media that the lab used to culture each organism to determine which would be most suitable for that strain. Each strain also came with a guide that listed the medium, light and temperature levels in which they were previously grown. Once the media yielding the highest growth rate was identified, each strain was transferred to that medium, and incubated on a 12:12 hour light:dark schedule with a light intensity of 10 μmol m-2s-1 and temperature of 23oC. These conditions were ideal for all but 2 strains that grew best at a slightly lower light intensity of 5μmol m-2s-1. Strains that preferred 5 over 10 μmol m-2s-1

light intensity were covered with mesh to reduce the incident light. Experimental strains

were grown in Jaworski’s Medium (JM) (Culture College of Algae and Protozoa,

seehttps://www.ccap.ac.uk/pdfrecipes.htm). 8

Table 1: List of samples by strain, toxin produced and source of the strain.

CYN: cylindrospermopsin cyanotoxin, STX: saxitoxin cyanotoxin, ANA: anatoxin cyanotoxin

Genus Species Strain Obtained from Medium Toxin

Cylindrospermopsis raciborskii CS-506 ANASS JM CYN

Dolichospermum (Anabaena) circinale CS-337/01 ANASS JM STX

Dolichospermum (Anabaena) circinale CS-537/02 ANASS JM STX

Dolichospermum (Anabaena) circinale CS-541/06 ANASS JM STX

Dolichospermum (Anabaena) circinale CS-1031 ANASS JM STX

Aphanizomenon sp. CAWBG01 Cawthron, NZ JM STX

Dolichospermum (Anabaena) flos-aquae SAG 30.87 SAG JM ANA

2.2 Temperature Gradient Chamber

The temperature gradient chamber was modeled closely after a chamber built at

Woods Hole Oceanographic Institute that was designed to test the influence temperature and salinity had on the growth of different dinoflagellate species (Watras et al., 1982).

There are heating and cooling mechanisms on either end. The cooling system pumped cold water through tubes to the left end of the insulated bar using a temperature controlled water cooler. The heating element on the right side of the chamber was controlled electrically. Both sources contacted the aluminum bar to createa thermal gradient.

Each temperature gradient chamber held 2 insulated aluminum bars, accommodating 30 samples each. The temperature gradient ran from 17 to 30oC with a 9 total of 10 different temperatures with each temperature in triplicate. Each sample was housed in a 50 ml Falcon tube that rested in the aluminum bar with the bottom portion of the tube exposed to the light source below. The lights were kept on a 12:12 hour light:dark schedule to replicate the light regimes the incubator stocks. To induce mixing and remove stratification within the tube, hydrated air was used to lightly bubble the samples. The hydrated air system was composed of pumps that pushed air through flasks filled with deionized water. Each sample had its own pinch clamp to control the amount of airflow allowed into the tube and a needle valve on the end of each group of 6 samples to reduce the amount of backpressure on the air pumps.

Figure 1: Interior of the temperature gradient chamber showing the hydrated air bubbling system. The temperature gradient runs from 17.1 to 30.1oC from left to right. 10

Table 2: Temperature (°C) of each sample within the temperature gradient chamber

1 2 3 4 5 6 7 8 9 10

A 17.1 18.6 20.2 21.7 23 24.3 25.9 27.2 28.9 30.0

B 17.0 18.6 20.2 21.7 23.1 24.3 25.9 27.1 28.9 30.1

C 17.1 18.6 20.2 21.7 23.1 24.3 25.9 27.1 28.9 30.1

Both of the C.raciborskii strains CS-506 and T3 as well as the D.circinalis strain

131C were grown at a lower light level of 5 μmol m-2s-1whereas the other 6 strains were

grown at a light intensity of 10 μmol m-2s-1.

2.3 Experimental Setup

Preliminary growth curves were conducted on each strain to acclimate it to the bubbling and temperature regime over the course of the experiment. Each strain was diluted to a starting preliminary density of 250,000 cells/mL in each of the 30 Falcon tubes and left to grow and adapt for 4-6 weeks depending on growth rate. Strains were monitored weekly through the preliminary phase for bleaching and to ensure growth by

retrieving 1 mL from each temperature and checking cell counts. To begin the

experimental phase, samples were combined and diluted at each temperature to 250,000

cells/mL and sampled for cell enumeration and toxin analysis. Temperatures in which the

3 combined samples did not reach at least 500,000 cells/mL during the preliminary

growth rounds were not included in the experimental round due to their failure to

acclimate to that temperature. Bubbling rate was monitored daily while temperature was

checked every 5 days at each sample point. 11

2.4 Sampling

During the experimental phase, each strain was sampled at5 day intervals for total toxin, dissolved toxin and cell enumeration. One mL aliquots were removed from all 3 samples at every temperature using a micropipette. The experimental phases continued for 30 days for a total of 7 time points for each strain. However, C. raciborskii strain CS-

506 toxin samples were spun down into pellets so that the media and any suspended, extracellular toxin could be removed. Once the toxin samples were run using an ELISA kit, 1 mL of deionized water was added back in and mixed. Due to the positive buoyancy and large gas vesicles of other strains, they were sampled as followed. One mL samples for cell enumerations were preserved with 20 μL of Lugol’s iodine and whole toxin samples were stored in a -80oC Cryocube. For dissolved toxin, 1 mL was passed through

a 0.2 μm nylon syringe filter with a 3 mL syringe and stored in a -200C freezer. After

sampling, 3 mL of Jaworski’s Medium (JM) was added back into the cultures to account

for what was removed and to ensure that the culture remained nutrient replete.

Samples were sent out to Bill Cody(Aquatic Specialists, Malinta, OH)

to establish an average number of cells per filament/trichome. Enumerations were

conducted on an inverted microscope using a hemocytometer to determine the count of

filaments and thus the cell density (Munawar, 1976). Toxin analysis was conducted after

3 freeze-thaw cycles using Abraxis ELISA (Warminster, PA) kits for saxitoxin and

cylindrospermopsin analysis. For strain CS-506, the pelleted samples were resuspended in deionized water and analyzed by the ELISA cylindrospermopsin kit for intracellular toxin. The other 6 strains sampled by collecting for both total and dissolved toxins were

processed and dissolved values were subtracted from total toxin values to determine the 12 amount of intracellular toxin. The anatoxin producer A. flos-aquae will be sent to a third party for anatoxin measurements by LC MS/MS. The toxin quota was calculated using cell abundance data and intracellular toxin values to give values expressed in femtograms

(fg) per cell. 13

CHAPTER 3: RESULTS

Due to time constraints, lab closings and shipment issues because of COVID-19,

the results from strains Dolichospermum flos-aquae SAG 30.87 and Dolichospermum

circinale CS-537 will not be shown in this thesis.

3.1 Dolichospermum circinale

2.E+06 2.E+06 2.E+06 1.E+06 17.1 1.E+06 18.6 1.E+06 20.2 21.7 8.E+05 23.1 CS-337 (cells/mL) 6.E+05 24.3 4.E+05 25.9 2.E+05 27.2 0.E+00 0 5 10 15 20 25 30 Day

Figure 2: Dolichospermum circinale strain CS-337/01 growth curves from 17.1 to

27.2oC 14

45

40

35

30 17.1 18.6 25 20.2 20 21.7 23.1 15 24.3 10

CS-337 Saxitoxin Quota (fg/cell) Saxitoxin CS-337 25.9 5 27.2

0 0 5 10 15 20 25 30 Day

Figure 3: Dolichospermum circinale strain CS-337/01 saxitoxin quota from 17.1 to

27.2oC

D. circinale CS-337 had an optimal growth temperature of 20.2oC but started to

bleach after 25 days where 21.7oC ended up being the most dense at the end of the 30 day

experimental trial. At 17.1oC,the highest saxitoxin quota was detected consistently

throughout the duration of the trial, with an ending quota of over 40 fg/cell. However,

there was little to no growth by the end of the experiment at that temperature. Where

growth rate was the highest at 20.2oC, saxitoxin production is quite low throughout the

duration of the experiment, producing the second lowest amount at 5.16 fg/cell. 15

7.E+05

6.E+05 17.1 5.E+05 18.6 20.2 4.E+05 21.7 3.E+05 23.1 24.3

CS-541 cells/mL 2.E+05 25.9

1.E+05 27.2 28.9 0.E+00 30.1 0 5 10 15 20 25 30 Day

Figure 4: Dolichospermum circinale strain CS-541/06 growth curves from 17.1 to

30.1oC

90

80

70 17.1 18.6 60 20.2 50 21.7 40 23.1

30 24.3 25.9 20 27.2 CS-541 Saxitoxin Quota (fg/cell) Saxitoxin CS-541 10 28.9 0 30.1 0 5 10 15 20 25 30 Day

Figure 5: Dolichospermum circinale strain CS-541/06 saxitoxin quota from 17.1 to

30.1oC 16

D. circinale strain CS-541had almost no intracellular toxin production at the

beginning of the experimental trial at the warmest four temperatures from 25.9 to 30.1oC

even though the optimal growth temperature was 25.9oC. Toxin production varied

throughout the experiment but ultimately the lowest temperature of 17.1oC was favored,

where over 70 fg/cell of saxitoxin was produced by day 30. Again, the temperature where

peak growth occurred had some of the lowest saxitoxin production within the cells with

34.22 fg/cell of saxitoxin produced by the end of the experiment at 25.9oC.

1.E+06

9.E+05

8.E+05

7.E+05 17.1 6.E+05 18.6

5.E+05 20.2 21.7 4.E+05 23.1 CS-1031 (cells/mL) CS-1031 3.E+05 24.3 2.E+05 25.9 1.E+05 27.2

0.E+00 0 5 10 15 20 25 30 Day

Figure 6: Dolichospermum circinale strain CS-1031 growth curves from 17.1 to 27.2oC 17

100

90

80

70 17.1°C 60 18.6

50 20.2 21.7 40 23.1 30 24.3 20 25.9 CS-1031 Saxitoxin Quota (fg/cell) Saxitoxin CS-1031 10 27.2°C

0 0 5 10 15 20 25 30 Day

Figure 7: Dolichospermum circinale strain CS-1031 saxitoxin quota from 17.1 to 27.2oC

Optimal growth temperature varied throughout the experiment of D. circinale

strain CS-1031 from21.7 and 24.3oC; however, samples grown at 24.3oC ended up with

the most growth at the end of the experiment with over 900,000 cells/mL. The lowest two

temperatures of 17.1 and 18.6oC had the highest saxitoxin quotas with samples incubated

at 17.1oC producing up to 70 fg/cell of saxitoxin at the end of the trial. At the end of the

30 days, 24.3oC, had the lowest amount of intracellular saxitoxin produced at 24.03

fg/cell. 18

3.2 Aphanizomenon

2.E+06 2.E+06 2.E+06 2.E+06 18.6°C 1.E+06 20.2 1.E+06 21.7 1.E+06 23.1 24.3 8.E+05 25.9 CAWBG01 (cells/mL) 6.E+05 27.2 4.E+05 28.9°C 2.E+05 0.E+00 0 5 10 15 20 25 30 Day

Figure 8: Aphanizomenon strain CAWBG01growth curves from 18.6 to 28.9oC

10

9

8 18.6°C 20.2 7 21.7 6 23.1 5 24.3 25.9 4 27.2 3 28.9°C 2 CAWBG01 Saxitoxin Quota (fg/cell) Saxitoxin CAWBG01 1

0 0 5 10 15 20 25 30 Day

Figure 9: Aphanizomenon strain CAWBG01saxitoxin quota from 18.6 to 28.9oC 19

The CAWBG01 Aphanizomenon strain had an optimal growth temperature

consistently at 24.3oC, which was also the temperature that had the lowest saxitoxin

quota of 1.3 fg/cell at the end of the experiment. Initially, saxitoxin quotas were higher at

the start of the experiment in the cooler temperatures from 18.6 to 20.2oC but the highest

saxitoxin quota was at the warmest temperature of 28.9oC for the majority of the

experiment ending at 7.57 fg/cell. However, there was little to no growth in the samples

incubated at 28.9oC.

3.3 Cylindrospermopsis raciborskii

9.E+05

8.E+05

7.E+05

6.E+05 21.7 5.E+05 23.1 24.3 4.E+05 25.9 3.E+05 CS-506 (cells/mL) 27.2 2.E+05 28.9 1.E+05 30.1

0.E+00 0 5 10 15 20 25 30 Day

Figure 10: Cylindrospermopsis raciborskii strain CS-506 growth curves from 21.7 to

30.1oC 20

300

250

200 21.7°C 23.1 150 24.3 25.9 100 27.2 28.9 50

Cylindrospermopsin Quota (fg/cell) 30.1°C

0 0 5 10 15 20 25 30 Day

Figure 11: Cylindrospermopsis raciborskii strain CS-506 cylindrospermopsin quota from

21.7 to 30.1oC

Initially, in the C. raciborskii CS-506 strain, samples favored the warmer

temperatures for most rapid growth, but by day 15 of the experimental samples grown at

24.3oC grew more quickly than the others. Temperatures ranging from 24.3 to 25.9oC

held samples with the highest growth by the end of the trial, having over 800,000

cells/mL. At day 5, cylindrospermopsin quota seemed to peak at 270.09 fg/cell when

samples were incubated at 21.7oC but by the end of the experiment toxin quota was

greatest at temperature 23.1oC, producing 103.25 fg/cell. Some of the lowest

cylindrospermopsin quotas were in samples grown at the warmest temperature of 30.1oC. 21

3.4 Growth Rates

Table 3: Growth rates for the 5 strains at each temperature they were run at nd = no data

Temp CS-506 CAWBG01 CS-541 CS-1031 CS-337 17.1 nd nd 9.70e-3 4.50e-3 3.90e-3 18.6 nd 4.96e-2 1.16e-2 2.00e-2 1.25e-2 20.2 nd 6.93e-2 1.79e-2 3.01e-2 5.86e-2 21.7 8.12e-2 6.07e-2 1.75e-2 5.09e-2 6.96e-2 23.1 6.51e-2 6.24e-2 2.17e-2 4.42e-2 5.53e-2 24.3 9.10e-2 7.93e-2 2.71e-2 4.65e-2 4.56e-2 25.9 9.51e-2 5.78e-2 3.35e-2 3.81e-2 3.47e-2 27.2 8.74e-2 4.03e-2 2.49e-2 2.61e-2 1.13e-2 28.9 7.68e-2 1.74e-2 1.81e-2 nd nd 30.1 4.68e-2 nd 1.08e-2 nd nd

Table 4: List of optimal temperatures for both growth and toxin quota of each strain

Strain Growth °C Toxin Quota °C CS-337 21.7 17.1 CS-541 25.9 17.1 CS-1031 21.7 17.1 CAWBG01 24.3 28.9 CS-506 25.9 23.1

Even though each strain’s optimal temperature for growth and toxin production is

different, the most total toxin produced in the cultures is at the optimal growth

temperature. The smaller amounts of toxin produced per cell at optimal temperatures still

had the most overall toxin produced by the end of the experiment. Based of these

experiments, rising temperatures will not only favor more rapidly growing blooms but

higher amounts of toxin produced. 22

CHAPTER 4: DISCUSSION

While temperature and light levels were checked every 5 days when samples were taken during the experiment, there were instances when temperatures would need to be adjusted back to their original settings. Due to fluctuations in laboratory temperature and humidity, the interior of the temperature gradient chambers were sometime influenced which could skew sample temperature. There was also 2 different temperature gradient chambers used throughout the experiments of the 7 strains. While both chambers were built according to identical specifications (while also using the same materials) one was painted while the other was not, which influenced the humidity inside of the temperature gradient chamber. The painted chamber also had a viewing window, which allowed for more consistent bubbling to occur because the bottom of the sample tube could be viewed. Strains CAWBG01, CS-541 and CS-1031 were incubated in the painted box during their experiments.

Temperature affected both growth rate and toxin quota for each of the strains.

When cultures were grown at temperatures where rapid growth was initially observed, growth rates ultimately slowed or cultures died by the end of the experiment (see Fig.9, temperature 30.1oC for example). Comparatively, temperature promoting slower growth yielded steady increases in cell density throughout the duration of the experiment (see

Fig. 5, temperature 27.2oC). Each of the D. circinale strains could withstand the coldest

temperature of 17.1oC; however, Aphanizomenon CAWBG01and C. raciborskii CS-506

could not grow enough through preliminary phases for 17.1 oC to be included in the

experimental trials. While samples do seem to favor higher temperatures initially, each

optimal temperature of the 5 strains hovered around the middle of the gradient towards 23 the end of the experiment. This could pose a problem for short-term blooms in nature when temperatures spike and when nutrients/weather conditions are conducive for bloom formation, it could cause rapid growth followed by swift decay. Those types of scenarios create hypoxic conditions, resulting in fish kills and mass toxin release when the cells lyse.

Where growth was optimal, toxin production seemed to be lowest (see Fig. 7 and

8, temperature 24.3oC). It’s possible that energy is expended on intracellular toxin

production instead of on cell reproduction and overall bloom density when temperature is

not favorable for growth. Shown in Table 3, optimal temperatures for growth and toxin

quota are decoupled and with the exception of strain CAWBG01, cultures have lower

optimal toxin quota temperatures than growth temperatures. Toxin quota overall was

lowest in Aphanizomenon CAWBG01 but this strain grew to the highest density. The

highest overall toxin quota was in C. raciborskii CS-506. High toxin quotas, coupled with

lower bloom densities pose a threat to public health. Even with lower temperatures and

cell densities below ideal bloom densities, the water can still pose a risk and toxin levels

could be above EPA contact standards.

D. circinale strain CS-541 successfully grew at all 10 temperatures across the

gradient, but it was also the slowest grower and had the lowest cell density amassed after

the 30 day trial. However, CS-541 had a higher saxitoxin quota than 2 other saxitoxin-

producing strains despite its reduced growth throughout the experiment. C. raciborskii

CS-506 had to run through 2 preliminary trials after the first one bleached in under 2

weeks. The second trial was lengthy and lasted just over 7 weeks before it was ready to be diluted for the experimental trial and even then samples were diluted down to a 24 starting density 100,000 rather than 250,000 cells/mL. Due to C. raciborkii’s negative buoyancy it’s most likely that the CS-506 strain did not tolerate bubbling, which forces the cells to circulate around the tube and near the top. Sample B7 of strain CS-337 also bleached on November 24, 2019 at temperature 25.9oC so averages after that date for the

duration of the CS-337 trial were measured from 2 samples instead of the standard 3. The

CS-541 strain was also the first strain to be run where spinning down the samples did not

work using a centrifuge, which resulted in dissolved toxin samples being taken after the

completion of the experiment, with samples being grown in the incubator at the same

light and median temperature conditions.

4.1: Future Work

Final analysis of the Dolichospermum CS-537 and SAG 30.87 strains will be

conducted. In the future, benthic strains could be tested to see if they behave similarly as

pelagic strains, as well as testing cultures grown at different light intensities. Experiments

show that light levels can influence intracellular toxin accumulation of toxigenic

cyanobacteria (Sivonen, 1990). Running simultaneous experiments at the same

temperatures but different irradiances on the same strain could help better forecast bloom

and toxicity events due to global climate change. Higher temperatures coupled with

stronger light intensities could increase cyanobacterial harmful algae blooms on our

freshwater lakes. By taking experiments done on temperature, light intensity and nutrient

loading and combining it with weather data, predictive models came be made for bloom

intensity and toxin production. It would be most useful to be able to forecast strains for

each lake around the world based on their conditions. This would vastly improve public 25 health and water treatment for those who struggle with recurrent cyanobacterial harmful algae blooms.

Another experiment to that could be conducted would be to look at energy allotted to cellular division against toxin production. We saw during the experiment that even when growth rates were low, toxin production per cell could reach high levels.

However, was this due to the strain not being held in optimal conditions for growth or for another reason? If so, why expend energy on producing toxins when there is minimal growth, to possibly ward off grazing or filter feeding? Comparing growth rates of toxic and non-toxic strains at the same temperature would also be interesting to see if the energy used in producing cyanotoxins slows the growth of the strain and by how much. 26

LITERATURE CITED

Antunes, Jorge T., Pedro N. Leão, and Vítor M. Vasconcelos. 2015. "Cylindrospermopsis Raciborskii: Review of the Distribution, Phylogeography, and Ecophysiology of a Global Invasive Species." Frontiers in Microbiology 6: 473. doi:10.3389/fmicb.2015.00473. https://www.ncbi.nlm.nih.gov/pubmed/26042108.

Bai, Fang, Rui Liu, Yanjun Yang, Xiaofei Ran, Junqiong Shi, and Zhongxing Wu. 2014. "Dissolved Organic Phosphorus use by the Invasive Freshwater Diazotroph Cyanobacterium, Cylindrospermopsis Raciborskii." Harmful Algae 39: 112-120. doi:10.1016/j.hal.2014.06.015. https://search.datacite.org/works/10.1016/j.hal.2014. 06.015.

Brient, Luc, Marion Lengronne, Myriam Bormans, and Jutta Fastner. 2009. "First Occurrence of Cylindrospermopsin in Freshwater in France." Environmental Toxicology 24 (4) (Aug): 415-420. doi:10.1002/tox.20439. https://onlinelibrary.wiley.com/doi/abs/10.1002/tox.20439.

Carmichael, Wayne W. 2001. "Health Effects of Toxin-Producing Cyanobacteria: “The CyanoHABs”." Human and Ecological Risk Assessment: An International Journal 7 (5): 1393-1407. doi:10.1080/20018091095087. https://doi.org/10.1080/20018091095087.

Casero, María Cristina, Andreas Ballot, Ramsy Agha, Antonio Quesada, and Samuel Cirés. 2014. "Characterization of Saxitoxin Production and Release and Phylogeny of Sxt Genes in Paralytic Shellfish Poisoning Toxin-Producing Aphanizomenon Gracile." Harmful Algae 37 (Jul): 28-37. doi:10.1016/j.hal.2014.05.006. http://dx.doi.org/10.1016/j.hal.2014.05.006.

Castro, Daniela, Diana Vera, Néstor Lagos, Carlos Garcı́a, and Mónica Vásquez. 2004. "The Effect of Temperature on Growth and Production of Paralytic Shellfish Poisoning Toxins by the Cyanobacterium Cylindrospermopsis Raciborskii C10." Toxicon 44 (5): 483-489. doi:10.1016/j.toxicon.2004.06.005. https://www.sciencedirect.com/science/article/pii /S0041010104002594. 27

Cirés, Samuel, Adrián Delgado, Miguel González-Pleiter, and Antonio Quesada. 2017. "Temperature Influences the Production and Transport of Saxitoxin and the Expression of Sxt Genes in the Cyanobacterium Aphanizomenon Gracile." Toxins 9 (10) (Oct 13,): 322. doi:10.3390/toxins9100322. https://www.ncbi.nlm.nih.gov/pubmed/29027918.

De Stasio, Bart T., David K. Hill, Julie M. Kleinhans, Nathan P. Nibbelink, and John J. Magnuson. 1996. "Potential Effects of Global Climate Change on Small North temperate Lakes: Physics, Fish, and Plankton." Limnology and Oceanography 41 (5) (Jul): 1136-1149. doi:10.4319/lo.1996.41.5.1136. https://onlinelibrary.wiley.com/doi/abs/10.4319/lo.1 996.41.5.1136.

El-Shehawy, Rehab, Elena Gorokhova, Francisca Fernández-Piñas, and Francisca F. del Campo. 2012. "Global Warming and Hepatotoxin Production by Cyanobacteria: What can we Learn from Experiments?" Water Research 46 (5) (Apr 1,): 1420-1429. doi:10.1016/j.watres.2011.11.021. https://www.sciencedirect.com/science/article/pii/ S0043135411007056.

Falconer, Ian R. and Andrew R. Humpage. 2005. "Health Risk Assessment of Cyanobacterial (Blue-Green Algal) Toxins in Drinking Water." International Journal of Environmental Research and Public Health 2 (1) (Apr): 43-50. doi:10.3390/ijerph2005010043. https://www.ncbi.nlm.nih.gov/pubmed/16705800.

Hawkins, Peter R., Nimal R. Chandrasena, Gary J. Jones, Andrew R. Humpage, and Ian R. Falconer. 1997. "Isolation and Toxicity of Cylindrospermopsis Raciborskii from an Ornamental Lake." Toxicon 35 (3): 341-346. doi:10.1016/S0041-0101(96)00185- 7. https://www.sciencedirect.com/science/article/pii/S0041010196001857.

Loftin, Keith A., Jennifer L. Graham, Elizabeth D. Hilborn, Sarah C. Lehmann, Michael T. Meyer, Julie E. Dietze, and Christopher B. Griffith. 2016. "Cyanotoxins in Inland Lakes of the United States: Occurrence and Potential Recreational Health Risks in the EPA National Lakes Assessment 2007." Harmful Algae 56 (Jun): 77-90. 28

doi:10.1016/j.hal.2016.04.001. https://www.sciencedirect.com/science/article/pii/S1 568988315300883.

Méjean, Annick, Guillaume Paci, Valérie Gautier, and Olivier Ploux. 2014. "Biosynthesis of Anatoxin-a and Analogues (Anatoxins) in Cyanobacteria." Toxicon 91 (Dec 1,): 15-22. doi:10.1016/j.toxicon.2014.07.016. https://www.sciencedirect.com/science/article/pii /S0041010114002165.

Menzel, A., C. Rosenzweig, D. J. Karoly, P. Tryjanowski, C. Liu, G. Casassa, A. Imeson, T. L. Root, S. Rawlins, and B. Seguin. 2007. Assessment of Observed Changes and Responses in Natural and Managed Systems Cambridge University Press. doi:10.5167/UZH-33180. https://search.datacite.org/works/10.5167/UZH-33180.

Munawar, Mohiuddin and Iftekhar F. Munawar. 1976. "A Lakewide Study of Phytoplankton Biomass and its Species Composition in Lake Erie, April-December 1970." Journal of the Fisheries Board of Canada 33 (3) (Mar 1,): 581-600. doi:10.1139/f76-075. http://www.nrcresearchpress.com/doi/abs/10.1139/f76-075.

O'Reilly, Catherine M., Brent A. McKee, Pierre-Denis Plisnier, Andrew S. Cohen, and Simone R. Alin. 2003. "Climate Change Decreases Aquatic Ecosystem Productivity of Lake Tanganyika, Africa." Nature 424 (6950) (Aug 14,): 766-768. doi:10.1038/nature01833. http://dx.doi.org/10.1038/nature01833.

Pereyra, Joao P. A., Paul M. D'Agostino, Rabia Mazmouz, Jason N. Woodhouse, Russell Pickford, Ian Jameson, and Brett A. Neilan. 2017. "Molecular and Morphological Survey of Saxitoxin-Producing Cyanobacterium Dolichospermum Circinale (Anabaena Circinalis) Isolated from Geographically Distinct Regions of Australia." Toxicon 138 (Nov): 68-77. doi:10.1016/j.toxicon.2017.08.006. https://www.sciencedirect.com/science/article/pii /S0041010117302428.

Rapala, Jarkko, Kaarina Sivonen, Raija Luukkainen, and Seppo I. Niemelä. 1993. "Anatoxin-a Concentration inAnabaena andAphanizomenon Under Different Environmental Conditions and Comparison of Growth by Toxic and Non- 29

toxicAnabaena-Strains — a Laboratory Study." Journal of Applied Phycology 5 (6): 581-591. doi:10.1007/BF02184637. https://doi.org/10.1007/BF02184637.

Rellán, Sandra, Joana Osswald, Martin Saker, Ana Gago-Martinez, and Vitor Vasconcelos. 2009. First Detection of Anatoxin-a in Human and Animal Dietary Supplements Containing Cyanobacteria. Vol. 47. doi:https://doi.org/10.1016/j.fct.2009.06.004. http://www.sciencedirect.com/science/ article/pii/S0278691509002762.

Savadova, Ksenija, Hanna Mazur-Marzec, Jūratė Karosienė, Jūratė Kasperovičienė, Irma Vitonytė, Anna Toruńska-Sitarz, and Judita Koreivienė. 2018. "Effect of Increased Temperature on Native and Alien Nuisance Cyanobacteria from Temperate Lakes: An Experimental Approach." Toxins 10 (11) (Oct 30,): 445. doi:10.3390/toxins10110445. https://www.ncbi.nlm.nih.gov/pubmed/30380769.

Shen, Xiaoyun, P. K. S. Lam, G. R. Shaw, and W. Wickramasinghe. 2002. "Genotoxicity Investigation of a Cyanobacterial Toxin, Cylindrospermopsin." Toxicon 40 (10): 1499-1501. doi:10.1016/S0041-0101(02)00151- 4. https://www.sciencedirect.com/science/article/pii/S0041010102001514.

Smith, Robert B., Brad Bass, David Sawyer, David Depew, and Susan B. Watson. 2019. "Estimating the Economic Costs of Algal Blooms in the Canadian Lake Erie Basin." Harmful Algae 87 (Jul): 101624. doi:10.1016/j.hal.2019.101624. http://dx.doi.org/10.1016/j.hal.2019.101624.

Stavric, B., N. R. Hunter, R. K. Pike, O. E. Edwards, J. P. Devlin, and P. R. Gorham. 1977. "Anatoxin-a, a Toxic Alkaloid from Anabaena Flos-Aquae NRC- 44h." Canadian Journal of Chemistry 55 (8) (Apr 15,): 1367-1371. doi:10.1139/v77- 189. http://www.nrcresearchpress.com/doi/abs/10.1139/v77-189.

Sukenik, Assaf, Ora Hadas, Aaron Kaplan, and Antonio Quesada. 2012. "Invasion of (Cyanobacteria) to Subtropical and Temperate Freshwater Lakes – Physiological, Regional, and Global Driving Forces." Frontiers in Microbiology 3: 86. doi:10.3389/fmicb.2012.00086. https://www.ncbi.nlm.nih.gov/pubmed/22408640 30

Watras, Carl J. & Anderson, Donald M. “Regulation of Growth in an Estuarine Clone of Gonyaulax tamarensis Lebour: Salinity-Dependent Temperature Responses. March 1982. vol. 62. pp. 25-37.

Wagner, Carola and Rita Adrian. 2009. "Cyanobacteria Dominance: Quantifying the Effects of Climate Change." Limnology and Oceanography 54 (6): 2460-2468. doi:10.4319/lo.2009.54.6_part_2.2460. https://doi.org/10.4319/lo.2009.54.6_part_2. 2460.

Wiese, Maria, Paul M. D'Agostino, Troco K. Mihali, Michelle C. Moffitt, and Brett A.

Neilan. 2010. "Neurotoxic Alkaloids: Saxitoxin and its Analogs." Marine Drugs 8

(7) (Jul 20,): 2185-2211. doi:10.3390/md8072185. https://www.ncbi.nlm.nih.gov/pubmed/20714432.