Electrophysiological and
Phylogenetic studies on the
toxicity of gar oocyte extract
Gary LaFleur, Jr
Nicole Broussard
Chad Loupe
Allyse Ferrara
Nicholls State University
Dept of Biological Sciences
Supported by NIH COBRE grant; LSUHSC
Walter Ingliss Anderson
What makes one fish more
persistent than another?
Maisey, 1996
Among ancient fishes, gar have
maintained a high species count
body armor
lung
egg protection
AGWG
2008
“There were five persons suffering in all
the agony that pain could inflict.”
AGWG
2008
Garfish Roe paddies
were prepared,
frozen and fed to
crawfish
some ate
some got mad
AGWG
2008
Whole Organism Effect
We confirmed
previoius reports by
T Burns
BJ Rodrigue
Paula Patterson
Injection of Spotted Gar Roe causes
disorientation, rigid paralysis in 60 sec
AGWG
2008
Approach
•Develop bioassay to test toxicity
•Determine whether egg envelope is
required for toxicity
•Determine what life stages harbor toxicity
•Determine species affected by toxicity
•Determine the mechanism of toxicity
•Isolate distinct toxic agent
•Determine whether garfish synthesizes
the toxin
AGWG
2008
Photo by C Johanning
Spotted Gar were collected in the
Atchafalaya River Basin
AGWG
2008
Isolation of Ovarian Roe
Thanks to Olivia Smith for recent gar ovaries!
CoBRE
2007
Making the Ichthyootoxin
Extract
• 2g roe/ 20ml Locke’s
Saline
• Tissue Tearor
• Homogenize with tubes
on ice bath
• Centrifuged in Sorvall at
10,000 rpm for 10 min
• Supernatant taken
• Frozen in -80˚ C Freezer
AGWG
2008
Saline
Control
AGWG
2008
Percent Survival
120
100
80
60
40
20
0
-20
N=2
saline
N=6
N=6
N=6
N=6
N=6
2.0
1.0
0.2
0.02
0.002
Choupique
Gar roe (mg / g body wt)
Injected Substance
LD 50 approximately
0.6 mg roe / g body wt
AGWG
2008
Gar induced to
spawn by
Dr. Ferrara
using
Ovaprim
AGWG
2008
Photo by R Hotard
Photo by R Hotard
Extracts isolated from ovulated eggs, fertilized eggs, hatched
embryos all retained toxic activity
AGWG
2008
Percent Survival
120
100
80
60
Gar
roe
40
20
0
-20
N=2
N=6
2.0
Saline Choupique
Unf
egg
Fert
egg
5-day
larva
N=2
1.0 mg / g body wt
Injected Substance
All stages tested contained toxicity
Chorions not necessary for effect
AGWG
2008
Paralysis Results
Toxin Sensitive
Uca panacea
Procambarus clarkii
Toxin Insensitive
Poecillia latipinna
Uca longisignalis
Fundulus grandis
Allyse suggested:
“why not use a crustacean with a clear carapace?”
AGWG
2008
Assaying Effect on Neurogenic Heart
Nicole Broussard MS student
18 sec
AGWG
2008
Crawfish Preps for Electrophysiology
Thanks Hamilton Farris, LSUHSC
neuron, tail muscle, and heart muscle preps tested
AGWG
2008
Voltage (arbitrary)
heart response to toxin
Pre Toxin
10 s
30 s
6 minutes
0
0.5
1
1.5
2
Tim e (m s)
Extracellular recordings suggest a disruption of
glutamate signaling at neuromuscular junctionsAGWG
2008
Phylogenetic tests
VERTEBRATES: YES
EXCEPT FOR TELEOST
•Sensitive Groups:
•Vertebrates (except
teleost)
•CRUSTACEANS
•Tested the toxin sensitivity
of other invertebrates,
within and without Phylum
Arthropoda
•We expected that the toxin
would affect both
the mollusk and the insect
CRUSTACEANS: YES
INSECTS … ?
SNAILS … ?
•MODEL SPECIES:
•Littorina irrorata
•Marsh Periwinkle (Snail)
•Gryllus sigillatus
•Tropical house cricket
Gilbert 2000
•Average Snail
Survival
l
.01
mg
/m
g/m
l
n= 2
.1m
l
.00
1g
/m
.01
g/m
l
.1g
/m
l
Five snails/group
100%
80%
60%
40%
20%
0%
Sa
lin
e
•Hourly observations
noted at 4, 8, and 20
hours post-injection
•Error bars denote
standard deviation.
Percent Survival
•Data compiled from
two trials consisting of
30 animals each.
Chad Loupe
BS 2007
Toxin Concentration
Littorarina irrorata
Positive Control in Crawfish = 10 mg / g body wt
Five Crickets/Group
N=4
100
Percent Survival
•Average Cricket
Survival
•Compiled data from
four cricket trials
comprising of 30
animals each.
•Hourly observations
noted at 4, 8, and 20
hours post-injection
•Error bars denote
standard deviation.
80
60
40
20
l
g/
m
.0
1m
g/
ml
.1
m
l
.0
01
g/
m
l
.0
1g
/m
l
.1
g/
m
Sa
lin
e
0
Toxin Concentration
Gryllus sigillatus
Positive Control in Crawfish = 10 mg / g body wt
• After six trials and over
120 total observation hours,
the cricket and snail had no
notable response to the
toxin
• Toxin had a profound
effect on crustaceans.
• Immediate paralysis
and eventual death.
• .
• Although it is known that
glutamate is used as a
neurotransmitter in insects
and snails,
the toxin had no effect.
• Although it is known that
glutamate is used as a
neurotransmitter in insects,
the toxin had no effect on
crickets.
• crustaceans may be the
only invertebrates affected
VERTEBRATES: YES
EXCEPT FOR TELEOST
CRUSTACEANS: YES
INSECTS: NO
SNAILS?
Conclusions
•
•
•
•
•
•
Paralysis and Cardiotoxicity Assay developed
All crustaceans tested are sensitive
The toxin does not affect any teleosts tested
Egg Envelopes not necessary, cytoplasmic localization
Electrophysiology prep suggests neuromuscular junction
Neither Snails, nor Insects sensitive to toxin
Unanswered
Neurotoxic mechanism of action
Specific molecular entity
Biosynthetic origin of extract
AGWG
2008
Possible Research Applications
• Research: Applications similar to other
neurotoxins for experimental manipulation
• Crustacean avoidance: The species selectivity of
the garfish toxin may provide anti-fouling agents
against barnacles.
• Ecology: concentrating the toxin in eggs may
have contributed to the extraordinary persistence
of this ancient fish group.
AGWG
2008
Questions?
Thanks Allyse and Quenton for a great meeting!
Walter Ingliss Anderson
Future Experiments
EXPERIMENT 2: Isolating the Toxic Constituent of the Extract
Hypothesis: The neurotoxic entity contained within the garfish roe
homogenate is a single compound that can be isolated
Design: Homogenate will be fractionated and each fraction tested. The
toxic fraction will be further separated by SDS gel electrophoresis
isolating single protein bands for further voltage clamp testing.
Rationale: If the neurotoxin is indeed heat-labile as previously shown,
then we should be able to identify it using protein electrophoresis.
Other toxins such as tetrodotoxin and ciguateratoxin are NOT
proteins, and so there is a remote possibility that this compound
will be a molecule other than a protein, wherein isolations may
depend on GC, Mass Spec or other chromatography methods
Future Experiments
EXPERIMENT 3: Elucidating the site of Synthesis of the Neurotoxin
Hypothesis: We hypothesize that the garfish is synthesizing this
proteinaceous neurotoxin, and secreting into the blood, where it is
subsequently concentrated in the roe.
Design: PCR primers and antibodies will be designed against the Nterminal sequence, allowing for RT-PCR cloning of the complete
cDNA coding for the protein.
Rationale: The site of synthesis can be shown by documenting RNA
levels in specific tissues. Additionally immunolabeling
cytoplasmic vesicles containing the protein would suggest
synthesis though it may also suggest storage, as in the ovary.
If no tissues in the garfish are found to contain RNA coding for the
neurotoxin, a microbial origin of the neurotoxin will be implicated.
Future Experiments using electrophys
EXPERIMENT 1: Which ion channel is perturbed by roe homogenate?
Hypothesis: The paralysis caused by garfish roe injection in crawfish
is due to the specific blocking of an ion channel
Design: Using a crawfish whole cell prep that Dr. Farris' lab has
already had experience with, we will test the whole homogenate,
and then separate fractions of the garfish roe extract
Rationale: If paralysis is caused by blocking of an ion channel we will
be able to record this and identify the channel using voltage
clamp techniques.
If the crawfish prep is not convenient, other crustacea will suffice, as
well as cell preps from tetrapods or cultured cells
AGWG
2008
Our plan for the next several months includes:
• (1) Collaborate with Dr. Farris at LSUHSC to document
the activity on isolated crustacean and molluscan
neurons.
• (2) Test whether garfish toxin affects other invertebrates
including insects and molluscs.
• (3) Fractionate oocyte extract and test in paralytic,
cardiotoxic and neurophysiological bioassays.
• (4) Obtain N-terminal and internal sequence
• (5) Design degenerate primers to isolate the cDNA.
AGWG
2008
Future Experiments
EXPERIMENT 4: Obtaining the Primary Sequence of the Neurotoxin
Hypothesis: By obtaining the N-terminal sequence of the neurotoxin
we will have information needed to design primers for further
cloning and sequencing of the cDNA, as well as information that
may suggest whether the molecule originates from the garfish or a
symbiotic microbe.
Design: Proteins are routinely N-terminally sequenced, often receiving
up to 20 amino acids
If the N-terminus of the protein is blocked, proteolytic cleavage
products can be submitted as well for “internal sequences”
Future Work
• Fractionation followed by
bioassays to identify the active
molecule
• N-terminal sequencing to test
whether conserved with other
agent neurotoxins
• Determine if toxin originates from
garfish or symbiont
• Behavioral studies of crawfish to
test whether it deters predation
Walter Ingliss Anderson
Paralysis, and death also oocurs in
U. panacea, and U. longisignalis
WAS ’07
Ancient
Fishes
B. Background and Significance
Crawfish paralyzed by roe injection
1851 Report of human poisoning
•Research: Applications similar to Tetrodotoxin for channel manipulation
•Patents: The species selectivity of the garfish toxin may provide antifouling agents against barnacles.
•Evolution: Garfish currently being considered for protection
•intriguing reproductive strategy may deter predation.
Real time visualization of
extract Injection into shrimp cephalothorax
50
45
seconds til arrest
40
35
30
25
20
15
10
5
0
n = 4 shrimp
Injection, tachycardia, arrest in 10 sec
AGWG
2008
Current Research Plan
1. How does roe cause paralysis?
N
3. Is toxin synthesized by garfish?
Symbiotic
microbe?
C
2. What is 1o structure of toxin?
AGWG
2008