A Study on the Toxin: Atracotoxin AKA (spider toxin) Australian Funnel Web Spider Presented by: Anu Bhalla, Patrick Manzanares, & Reshawn Jackson

Why Examine? The Australian Funnel-Web Spider releases the atracotoxin on non-dry bite victims (venom secreted). As spiders, they are among the third most venomous in the world. By studying them we are able to understand how to properly behave/react around them, and what to expect if bitten by them.

Species producing Atracotoxin Hadronyche Atrax Robustus

Hadronyche vs. Atrax Robustus Similarities Between Two Species –Body lengths ranging from 1 cm to 5 cm –Located in Australia: Eastern Seaboard –Both can be lethal to humans –Fatalities occur from 15 minutes- 6 days –Males have significantly shorter life spans then females –Male toxin is 5x stronger than female toxin –Toxins from both species are almost similar in structure and mechanisms. Differences Between Two Species –Hadronyche are slightly more lethal then Atrax Robustus –Atrax Robustus venom causes sustained hypotension, which is not seen in Hadronyche.

Toxin Characteristics % Dry Bites –10%-25% produce toxicity Lethal Dose in Observed Species: –Monkey: 0.2mg/kg –Mice: 1.5ug/kg

Human Fatalities (55-58) know deaths cases per year seeking treatment Lethal in man/primates/mice but not lethal in some other lab animals unless given extremely high doses (literature does not specify genus types, or threshold that high dosage must reach).

Toxin Characteristics Biochemistry & Mechanism –Active component: Robustoxin –Contains high levels of hyaluronidase (responsible for toxin spreading through tissues). Hyaluronidase lowers the viscosity of hyaluronic acid, thereby increasing tissue permeability, which allows toxin to spread into inner layer of stated tissue. Once inside, toxin has greater potential to cause damage.

Toxin Characteristics Biochemistry & Mechanism (Cont) Slows inactivation of voltage gated Na+ channels. Creates surplus of Na+ ions in cell Inhibition and spontaneous transmitter release Results in a continued firing of action potentials Such behavior causes the muscles or other tissue to become ‘over-excited.’

Biochemistry and Mechanisms (Cont) –As a result of excessive Na intake, both the Presynaptic neurotransmitter of autonomic and motor neurons are targeted for disruption. –Causes inhibition of the release of transmitters (Ach, noradrenalin) as well as increase in spontaneous transmitter release. Muscle Stiffness and/or epileptic seizures may result due to spontaneous release. Similar action on the neuromuscular junction has been postulated. Below Image: Voltage Sensitive Na Channel

Symptoms of Toxins Eyes, ears, nose, and throat Drooping eyelids Double vision Swallowing difficulty Tongue spasms Heart and blood Collapse High blood pressure Rapid heart rate Lungs Difficulty breathing Pulmonary Oedema Muscles and joints Joint pain Severe muscle spasms -- usually in the legs and belly area Nervous system Chills Coma Headache Numbness of mouth and lips Skin Redness around the site of bite Sweating -- excessive Piloerection Stomach and intestines Diarrhea Nausea Vomiting

Symptoms (cont) Cardiovascular- initial fall in blood pressure, then rise to hypertensive levels. Acid-base disturbances- acute metabolic acidosis and respiratory acidosis. Intracranial hypertension Temperature- hyperthermia

Factors Affecting Toxicity and Symptoms of Spider Bite Quantity of venom Number of times bitten Species of spider Sex- M/F Maturity- age of spider Attack position Bacterial flora on fangs, mouth parts, and adjacent structures. –Causing other infections Length of time for each bite –Allowing more/less concentration Season: –Venom concentration change with seasons. Age of victim –Younger/older victims are more susceptible Size of victim Location of bite on victim Pre existing health conditions Allergic rxns

Treatments Pressure immobilization bandage: –Slows venom movement & can inactivate venom Steroid and diuretic treatments: Get toxin out of system faster. Antivenom Treatment: –Antivenom is prepared by hyperimmunising rabbits with male A robustus venom and is therefore a rabbit immunoglobin. –Serum can then be injected into infected person. –The average quantity per ampule is 100mg of purified rabbit IgG, which is enough to neutralize in vitro four adult male spider bites.

Does the biochemistry of spiders toxin change if spider is attacking its prey vs. defending its life? Toxin biochemistry does vary from attack mode to defensive mode (Difference between exact peptide composition unknown). Reasons for the change in biochemistry Limits the cost of toxin produced. Energy can be spent on survival and reproduction Insect-selective neurotoxins (discussed further in slide 16) –Spiders seen to release neurotoxin on insect (prey). –Cause of secreting neurotoxin opposed to cytotoxin: Some insects have been shown to develop resistance to toxin (insects not specified). –Acts as possible reflection of co-evolution between predator (spider) and prey (insect).

Possible reasons for Divergence of Toxin Divergence: Notion that two toxins once had a common ancestor but separated at some point in the past. Literature shows some evidence that the toxin did evolve from a single species (the common ancestor) into 2 separate species. Toxins from both species are almost similar in structure and mechanisms. –Atrax Robustus venom, the difference being the sustained hypotension which is not seen in Hadronyche. Spider had to evolve prey’s resistance to toxin. Possible signs of “Red Queen Hypothesis.” Through differential survival and reproduction, prey become resistant to toxin. Similarly, through differential survival and reproduction, the properties of the predator’s toxin becomes resistant to prey’s immunity. Co-evolution of predator (spider) and prey (insect) may have resulted in a possible cytotoxin evolving to the observed neurotoxin.

Applications for Toxin Pesticides Specific for insects such as cockroaches, crickets, fruit-flies and the Helicoverpa armigera moth which destroys cotton crops. Targeting specific species prevents the accidental killing of other insects. This selectivity also means that the pesticide is harmless to other organisms so there would be no danger if it entered the food chain. The compounds in venom are environmentally friendly and the development of resistance to a spider venom pesticide would be slow. Traditional chemical pesticides do not tend to be species specific, are toxic to humans in large amounts and insects develop resistance towards them relatively fast so it is easy to see why pesticides based on spider venom are attractive.

Application for Toxin (cont) Prevention of Brain Damage Oxygen deprivation caused stroke or excessive smoke inhalation can result in nerve cell damage in the brain. Glutamate is a neurotransmitter in the human brain and large amounts of it are released by these damaged neurons causing the death of neighboring nerve cells. The funnel-web spider produces a venom containing the active ingredient HF-7 which blocks receptors on the nerve cell membranes and prevents glutamate production. A drug developed using this compound could therefore limit brain damage for stroke victims.

References Isbister, Geoffrey; Graudins, Andis; White, Julian; Warrell, David. Journal of Toxicology -- Clinical Toxicology, Apr2003, Vol. 41 Issue 3, p291, 10p; (AN ) Arachnid toxinology in Australia: From clinical toxicology to potential applications.Authors: Dec2006, Vol. 48 Issue 7, p , 27p Handbook of clinical toxicology of animal venoms and poisons, By Jürg Meier, Julian White Textbook of Biochemistry with Clinical Correlations, By Thomas Devlin, 3 rd Ed, p 329.