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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