Anatoxin-a Fails to Show Allelopathic Activity in the Presence of Cyanobacteria or Green Algae

50 Pages·2017·0.83 MB·English

Allegheny College
Allegheny College DSpace Repository http://dspace.allegheny.edu
Projects by Academic Year Academic Year 2016-2017

2017-04-27

Anatoxin-a Fails to Show Allelopathic Activity
in the Presence of Cyanobacteria or Green
Algae

Rzodkiewicz, Lacey

http://hdl.handle.net/10456/42770

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of the U.S. Code.
Anatoxin-a Fails to Show Allelopathic Activity in the
Presence of Cyanobacteria or Green Algae
By
Lacey D. Rzodkiewicz
April 3, 2017
Allegheny College
Department of Biology
Department of Modern Languages

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Table of Contents:
Acknowledgements………………………………………………………………………....4
Abstract…………………………………………………………………………………..…5
Introduction……………………………………………………………………………..…..5
Methods………………………………………………………………………………..…..11
Algal Cultures……………………………………………………………………..11
Estimation of growth……………………………………………………………...12
Statistical analysis………………………………………………………………...13
Results…………………………………………………………………………………….13
Cyanobacteria……………………………………………………………………..13
Green Algae…………………………………………………………....………….16
Discussion……………………………………………………………………….………...20
Conclusions…………………………………………………………………………….….24
References………………………………………………………………………………....25
Epilogue: Les Dangers économiques et de la santé posé par les algues en France……….28
Introduction………………………………………………………………………..28
La cause des fleurs d’eau………………………………………………………….29
Types des algues dangereuses……………………………………………………..32
Les algues vertes…………………………………………………...……...32
Les algues bleues…………………………………………………………..34
Solutions proposées………………………………………………………………..38
Limitation des phosphates des lessives……………………………………38
Limitation des engrais……………………………………………………..39
Une histoire du succès en Corse…………………………………………...41
Considérations économiques………………………………………………43
Conclusions………………………………………………………………………..45
Références bibliographiques………………………………………………………46
Tables and Figures:
Figure 1…………………………………………………………………………………..15
Figure 2…………………………………………………………………………………..16
Figure 3…………………………………………………………………………………..31
Figure 4…………………………………………………………………………………..39
Figure 5…………………………………………………………………………………..43
Table 1…………………………………………………………………………………...18
Table 2…………………………………………………………………………………...19
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Acknowledgements:
I would like to thank the Department of Biology at Allegheny College for allowing me to
conduct this research, particularly Dr. Milt Ostrofsky for his constant guidance throughout the
process, from the experimental design to final statistics. Additionally, I would like to thank the
Department of Modern Languages for their part in this joint venture between the two disciplines
for being understanding of the collaborative nature of my project, especially Professor Phillip
Wolfe, my advisor in French. I would also like to thank Melissa Mattwig, Melissa Rusczyk, and
Erika Levy for suggestions during experimental design and editing. Not to be forgotten, I would
like to recognize Shane Ostrom, Ross Carter, Evan Eshenaur, Leah Krainz, and, once again,
Melissa Mattwig for their assistance in data collection. Without all of these individuals, this
study would not have been possible.
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Abstract:
Harmful algal blooms (HABs) are often the result of cyanobacteria producing dangerous toxins
that contaminate the water. One such toxin, anatoxin-a, acts upon mammalian nicotinic
acetylcholine receptors, resulting in symptoms such as gastro-enteritis, seizures, or even death.
The evolutionary reason for anatoxin-a production may be 1) a byproduct of metabolism, 2) a
defense against herbivory, or 3) an allelopathic agent. No such metabolic pathways have been
found, and little evidence points towards a defense to herbivory beyond the mammalian effects.
With mammals not being the primary consumers, this leaves allelopathy as the most likely
option. This study investigated this third option through exposing cyanobacteria (Anabaena flos
aquae, Microcystis aeruginosa, and Oscillatoria agardhii) and green algae (Pediastrum duplex,
Scenedesmus quadricauda, and Staurastrum paradoxum) to varying concentrations of anatoxin-a
from low environmental levels to those denoted as too dangerous for human contact. Despite
anticipated enhancement of cyanobacterial growth and suppression of green algal growth, no
growth rates were found to have a significant enhancement or suppression by the toxin (p<0.05).
Results did not give clear support for the use of anatoxin-a as an allelopathic agent of
cyanobacteria.

Introduction:
Buildup of algae into harmful algal blooms (HABs) has been increasing in recent years.
This augmentation of HABs is linked to climate change and an increase in nutrient loading from
the surrounding environment. First identified for their danger in 1878 when farm animals began
to suffer after drinking from waters with an HAB, the hazards seem to have only increased in
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recent years (Francis, 1878). Now, water supplies are being shut down to prevent negative
effects on humans; for instance, the water supply in Toledo, OH was shut down for extended
periods of time in the years 2013, 2014, and 2015 to avoid drinking contaminated waters (“Algal
blooms lead to 'impaired' designation,” 2016). Specifically, cyanobacteria, more commonly
known as blue-green algae, are at the root of the danger posed by HABs. Multiple species of
cyanobacteria have been identified as possible producers of hepatotoxins, endotoxins, or
neurotoxins. The most heavily studied of these toxins has long been microcystin, a product of
Microcystis species (Sabart et al., 2015). However, new emphasis is being placed on the
neurotoxic substances, specifically one that is produced by multiple species of the genera
Anabaena and Oscillatoria and others including Aphanizomenon flos-aquae, Raphidiopsis
mediterranae, Planktothrix rubescens, and Phormidium favosum (Gugger et al., 2005).
These cyanobacteria are capable of producing anatoxin-a, a neurotoxin specific to blue-
green algae (Ettoumi et al., 2011). The list of species that are able to produce this toxin was once
thought to be limited to a few planktonic genera, but benthic algal species have recently been
indicated as producers as well (Gugger et al., 2005). With many possible sources that are each
adapted to different environments, anatoxin-a and its isomer have been reported in many
waterbodies in Europe, including rivers and lakes in France (Cadel-Six et al., 2007; Sabart et al.,
2015), reservoirs in Spain (Carrasco et al., 2007), and lakes in Italy (Messineo et al., 2009).
However, anatoxin-a is not limited to European waters or any specific hemisphere. An instance
of the toxin was reported in the United States in 2003 (Behm, 2003). Species that produce the
toxin have been isolated in New Zealand, as well as three countries in Asia and several more in
Africa (Gugger et al., 2005; Cadel-Six et al., 2007).
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A majority of these regions have reported some instance of mammalian or avian death
from the severe neurological effects of the toxin. The effects of anatoxin-a 1-(9-
azabicyclo[4.2.1]non-4-en-5-yl)ethanone) are often compared to the effects to nicotine.
Anatoxin-a has the ability to act on several binding sites in a similar manner to nicotine,
including the a4ß2 site that activates the release of dopamine; however, perhaps more
importantly, it acts on the same ion channel receptors as nicotine that cause muscles to contract
as they would if acetylcholine had bound to the receptor (MacPhail & Jarema, 2005). The toxin
binds with greater affinity than acetylcholine, allowing anatoxin-a to bind in lower
concentrations and outcompete acetylcholine (Gugger et al., 2005). Normally, when activated,
the ion channel causes muscle contractions, and extra acetylcholine is broken down by
acetylcholinesterase. However, anatoxin-a is not able to be broken down by this enzyme; in fact,
its congener anatoxin-a (s) acts to repress actions of acetylcholinesterase. Thus, when anatoxin-a
binds to the channel, the ion channel opens and remains open, causing muscle spasms, seizures,
and, if at high enough concentrations, mortality (Macphail & Jarema, 2005).
Deaths of animals including cattle (Rantala-Ylinen et al., 2011) and dogs (Gugger et al.,
2005; Cadel-Six et al., 2007) from drinking or bathing in infected waters have been reported due
to these neurological effects. These deaths normally are within minutes to a few hours depending
on the size of the animal after the onset of clinical symptoms, which include coma, convulsions,
rigors, and limb-twitching (Cadel-Six et al., 2007). In multiple instances, deaths were confirmed
to have occurred due to the cyanotoxin and not herbicide or pesticide exposure that may produce
similar clinical symptoms (Gugger et al., 2005; Cadel-Six et al., 2007). Few case studies of
human death from cyanoblooms have been reported in addition to the animal deaths outlined
above. A recent case in the United States cited the possibility of anatoxin-a causing a seizure
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related death in a teen after recreating in water with high levels of the toxin; the toxin was found
in both blood and tissue samples of the deceased (Behm, 2003). Lethal dose for 50% of the
population has been calculated based on mouse studies to be 200 µg/kg; these studies have since
been extrapolated and hold true for the application to larger organisms (Gugger et al., 2005).
Danger still persists for those that are not directly exposed to lethal doses of the toxin. In
several case studies presenting test animals with nonlethal doses, motor activity is significantly
impaired or diminished completely; this indicates a danger for animals drinking or bathing in
waters that are below the allowed or lethal toxin concentrations (Jarema et al., 2007). In the case
of human health, outbreaks of gastroenteritis have occurred following the contamination of
drinking water or infection originating from dialysis in medical procedures when anatoxin-a
levels fell below lethal doses (Messineo et al., 2009).
Of course, anatoxin-a may have devastating effects within its own ecosystem in addition
to the neurological effects it has upon mammals. The possibility also remains that
concentrations could travel up the trophic levels and act on levels beyond the primary consumers
of the cyanobacteria (Ettoumi et al., 2011). Reports have indicated the death of piscivorous fish
in French waters suffering from blooms producing anatoxin-a despite not ingesting the toxin
directly through algal consumption (Bélingard, 2016). Furthermore, the toxin may also have an
effect by preventing the development of juvenile organisms particularly sensitive to neurotoxins.
Anatoxins have been shown to cause the death of juvenile carp (Cyprinus carpio) within 29
hours of exposure to the toxin (Ettoumi et al., 2011).
Despite the concerning effects, the advantage of investing in anatoxin-a production is yet
unknown. It is unlikely the algae developed the toxin as a defense against mammals that would
not normally be in contact with phytoplankton or act as their primary consumers. In general,
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three possibilities are proposed: 1) the toxin is a byproduct of a metabolic function and an
unintentional secondary metabolite, 2) the toxin is a protection against herbivory, and 3) the
toxin is an allelopathic agent. No such metabolic pathways have been found (Burja et al., 2001).
While the toxin has been found to depress predatory rotifer populations, no other zooplankton
have demonstrated inhibition in their presence (Snell, 1980).
A growing body of literature indicates the third hypothesis, allelopathy, may be more
likely. Allelopathy can be defined as the ability of some plant species to communicate with one
another using chemical signalling. Signals may be sent to enhance the behavior and growth of
cohorts or repress the behavior or growth of competitors. In the case of phytopklankton, such
allelopathic signals from predecessors may be superior to other signals, including abiotic factors
like pH; success may require a balance of many factors that cannot all be detected, but an
allelopathic signal could indicate prior success and senescence of the species in the area and
hence the possibility of an ideal environment (Lewis, 1986). The hypothesis of allelopathy has
been studied in the past in relation to blue-green algal species. Keating (1977) investigated this
prospect through studying the successions of algal blooms in Linsley pond, a eutrophic lake; the
presence of some algal species now associated with anatoxin-a appeared to have an enhancing
effect on the succeeding populations and an inhibitory effect on predecessors. After bloom
senescence, the presence of an unknown toxin, likely anatoxin-a, increased. The next generation
of algae was composed primarily of genera that may produce anatoxin-a; similar cycles occurred
with increased presence of microcystin. Various studies have noted an increase in anatoxin-a
only when multiple species that are capable of producing the toxin are present, but not when a
single possible producer dominates the system (Cadel-Six et al., 2007; Carrasco et al., 2007;
Messineo et al., 2009; Sabart et al., 2015). The toxin appears to be found less frequently in