In Vivo Characterization of Stonefish Venom Effects in Rats

1. IN VIVO CHARACTERIZATION OF THE BIOCHEMICAL AND HISTOLOGICAL CHANGES INDUCED BY THE STONEFISH (SYNANCEIA VERRUCOSA) VENOM IN RATS Ahmad Khalil*¹, Mohammad Wahsha², Khalid Abu Khadra¹, Maroof A Khalaf², Tariq Al-Najjar² ¹ Department of Biological Sciences, Yarmouk University, Irbid, JORDAN. ² Marine Science Station, University of Jordan, Aqaba Branch, JORDAN. * Correspondence: kahmad76@yahoo.com

2. Fish Venoms in the Literature A search in the UniProtKB databank reveals a large number of entries for scorpion, spider and snake protein toxins, while data on marine and aquatic animals — particularly fish — remain rather scarce.

3. History and Epidemiology • Fish venom research is underrepresented in the literature, and toxicity of most fish venoms is still unknown. A phylogenetic analysis suggests that up to 1,200 fishes including stingrays, scorpionfish, zebrafish, stonefish, and some species of shark, catfish, and blenny are venomous. The majority of venomous fish are sedentary, slow moving, and live in shallow, protected waters. Fish venoms are classified as defensive, and are thought to be used much less frequently than those employed by other venomous animals for predatory purposes. 

4. Venoms Animals Research • Given that fish venoms remain considerably poorly studied when compared to terrestrial venoms, • Due to the difficulty in obtaining, extracting, and storingtheir venom.  • Exploring fish venoms full potential opens a wide possibilities for the development of new drugs.

5. Venoms Animals Research • The Reef stonefish (Synanceia verrucosa) - Is the world’s most venomous fish ever encountered by man. - Has caused occasional human fatalities. • Because their venom is of medically- importantnature, • It has been studied in terms of its • pharmacology, • epidemiology, and • clinical aspects of envenomation

6. Stonefish is an unattractive fish with an irregular ‘skin’ • Usually brown or grey and may have patches of yellow, or orangey-red in color. You are Looking at the World's Most Venomous Fish ↘ ↘ • The fish grows to 35 cm in length, although 50 cm 'monsters' have been reported. • Females are larger, longer and broader than males. • Its small eyes are well-elevated on its head, facing upwards, separated by a bony ridge.

7. Distribution • Synanceia are primarily marine, mainly on or near coral reefs or in intertidal areas. • However, some species are known to live in rivers. ① ⑤ ③ ③ • Map of Indo-Pacific locations: (1) Red Sea(JORDAN), (2) Indian Ocean (KENYA), (3) Indian Ocean (SRI LANKA), (4) JAVA SEA (INDONESIA), (5) Western Pacific (CHINA) and (6) Australia ② ④ ⑥

8. The stonefish is a master of camouflage Eye Spines • Stonefish • usually live on coral bottoms, often under rocks or ledges, • are able to bury in sand using their large pectoral fins. • Unlike most other venomous creatures that have bright warning coloration, • a stonefish looks like a part of a coral or an ordinary sand-covered rock, camouflaged with its surroundings. • Only the dorsal portion of the head remains exposed. Mouth

9. Description • Unlike most fishes, stonefish • does not have scales on its body. • has 13sharp and stiff stout dorsal fin spines which can inject an extremely poisonous venom. • A pair of venom glands are found associated to each spine, one on either side of individual spine • They are merely just a defensive mechanism for the stonefish, against its possible predators such as sharks, rays and sea snakes. Venom Duct Venom Gland Integumentary Sheath

10. Dangers of the stonefish • Many people suffer theagony of a stonefish sting every year. • Stonefish can be a potential hazard in the natural environment, and • the venom is life-threatening to humansspecifically to those who are careless or unconscious • envenomation imposes significant socioeconomic costs, as the victims may require days to weeks to recover from their injuries

11. Precautions • The venom is delivered when the spine pierces the tissue of the victim, and the venom enters the wound. • When taking intertidal walks, one should be in covered shoes. • Walk slowly and watch his/her step • Avoid murky waters and try to drag feet instead of stepping down if cannot see where to go. • Avoid wading in bare feet,particularly at night, should be avoided. • Pull back immediately if s/he feels a prick through shoe and do not exert full body weight down.

12. Danger to Humans • Stonefish • are not aggressive, but if threatened they will erect their dorsal spines within 10 sec. • The amount of venom is proportional to the amount of pressure applied to it. • a perceived threat, usually stepping on the animals or harassing them.

13. Envenomation in natural environment • Shoes, diving booties, gloves, and wetsuits provide some protection, but they are easily penetrated by the spines. • Comprehensive data on fish envenomation frequencies is unavailable. • Case studies make it clear that it is not an uncommon problem, accidental exposure to waders, divers, fishermen, or visitors of intertidal areas and recreational beaches, as well as in the home setting (e.g., handling by unwary marine aquarists).

14. Number of Incidents • In 1965 there were 81 cases of envenomations reported from PulauBukom Hospital, Singapore just within 4 years. • 25 cases of the use of anti-venom for stonefish reported to Commonwealth Serum Laboratories for a one-year period between 1989 and 1990, with most from Queensland. • 8 cases were reported from the SingaporeGeneral Hospitalbetween 2001 to 2003. • 14 calls to the QueenslandPoisons Information Systemin 2008 regardingstonefish poison.

15. Injuries are a Seasonal Problem • Cases increase in the summer monthsamong unwary walkers. • 72% of patients or cases are visitors rather than locals at holidays, reflecting the ignoranceof the presence of such toxic fish and lack of awarenessmeasures.

17. Clinical Presentation • redness and swelling, bruising, and puncture in sting site. • Immediate severe pain at the sting site. • This pain may spread to involve the entire limb and regional lymph nodes, peaking at around 60-90 minutes and lasting up to 12 hours if untreated. • Mild subsequent pain may persist for days to weeks. • Non specific features of envenoming include nausea, muscle weakness, vomiting and dizziness • Cardiovascular signs such as hypotension, bradycardia, collapse, pulmonary edema and cyanosis are rarely reported. • A remnant of the spine(s) may be left in the wound

18. Clinical Manifestations • Classic envenomation reveals one or more puncture wounds, each discolored by a surrounding ring of bluish cyanotic tissue. • It also causes vascular leakage resulting in gross edema. • Effects of the venom include shock, convulsions and paralysis. • The body of the affected person bends backwards like a bow with the pain. • Sometimes it's so frightful his eyes literally fall out of their sockets.

19. Mode of Action • Stonefish venom • is a mix of enzymes and non-enzymatic proteins. • is unstable and can be denatured by heat. • mechanism of action is not completely understood.  • stimulates the release a neurotransmitter, acetylcholine, from the neuromuscular junction, similar to the mechanism of action of the excitatory neurotoxin from black widow spider venom. • However, there are other mechanisms of action as well, which include cytotoxic, myotoxic and other effects.

20. First aid for stonefish sting Hot Water Immersion Technique • Based on the proposed heat-labile characteristics of these proteins; • Heat treatment is widely recommended as effective initial treatment to inactivate the venom that might otherwise cause a severe systemic reaction. • to relieve the pain, the affected limb should be immersed in water no warmer than 114ºF or 45ºC. • A local anesthetic can reduce the pain he pain.

21. Fish venom research • Little research has focused on the pharmacological or compositional nature of fish venoms. • the challenges involved in extracting, isolating and storing the venommakes their study and exploration a task that only the most tenacious researchers can perform • A better knowledge is neede through further investigations that explore their toxicity, histological and biochemical effects. • This would have an impact in developing new envenomation treatments and examiningvenom components' potential as novel drugs.

22. Goals of the Present Research • The present research was planned since Aqaba is a tourist destination from all overall world. • The endeavors are that investigating the toxicity of stone fish venom living along the Jordanian coast of the Gulf of Aqaba is beneficial and critical to plan new strategies in order to reduce its effects. • Furthermore, understanding how this venom affects the body tissues will help the medical services to apply suitable treatment and develop national anti-venoms.

23. Sites of Stonefish Collection * • Map of the Red Sea and the Gulf of Aqaba showing location of samples collection. • 5 stonefish S. verrucosa specimens • collected by SCUBA diving

24. Stonefish Catch • Stonefish stings can occur on the beach, not just in the water, with It is less common for the fish to sting when it is picked up. • Fish samples were kept alive in oxygenated,temperature- and salinity- controlled seawater at the aquaculture unit in Marine Science Station.

25. “Milking” of the Fish (Venom Extraction) • Applying pressure to the glands, fluid is observed to pass from the glands to the tip of the spine through the ducts. • Inserting a syringe directly into the venom gland to remove venom.

26. Rat Treatment • The fresh venom extract was homogenized in phosphate-buffered saline. • The supernatant was centrifuged for 15 min at 6000 X gto remove the insoluble materials. • Venom concentration were expressed as μg venom protein. • 6 week-old male Sprague Dawley rats (average body weight 150-200g) were used.

27. Acute Toxicity • Crude venom solutioninjected intramuscularly (i. m) with 0.5 ml of at concentration of 10, 20, 30, 40 ,50 and 60 µg/kg (10 animals, each). • A control group was maintained in each case by injecting an equal volume of PBS. • Animals were observed for 24 h and symptoms of toxicity. • Treated rats experienced: 1) loss of energetic activity 2) respiratory distress 3) convulsions and spastic paralysis 4) deathin the late stage.

28. Preliminary toxicity testing typically uses LD50 value 100 50 Response (% Death) 0.0 60.0 40.0 0.0 20.0 Venom Concentration (μg/kg b.w.) Graph showing the 24 hi. m. LD50 value of Synanceia verrucosa venom in rats • was 38.0 μg/kg body weight

29. Changes in the blood serum biochemical markers after asingle i . m. injection of male rats (N=10) with stonefish venom atLD50 dose (38 μg/kg). Data are presented as means.T: Hours post treatment. †: Statistically significant atP ≤ 0.05.ANOVA test followed by Duncan’s Multiple Range Test.

30. Histopathology: Liver CV • Control rat revealing normal architecture of the liver with hepatocytes radiating from the central vein (CV) and sinusoidal spaces, SP. • (B) 12 h venom-treated rat showing parenchymal cells hypertrophy (thick arrow), dilatation of sinusoidal space (thin arrows) with moderate inflammatory cell infiltration. • 24 h venom-treated section showing lipid accumulation (short arrows) and extensive inflammatory cell infiltration (arrowhead). (H and E stain at 400X).

31. Histopathology: kidneys • Control rat showing the glomerulus (thick arrow) with different types of tubules around it. • 12 h venom-treated rat showing interstitial hemorrhage (thin arrow). • 24 h venom-treated rat showing an increase in the widening of the urinary space (arrow head) and the lumen of the tubules. The interstitial hemorrhage is still present. (H and E staining at 400X).

32. Histopathology: Heart • control rat showing normal muscular structure. • 12 h venom-treated rats with myofibrillar degeneration. • (C) 24 h venom-treated rats showing myofibrillar degeneration, blood clot and vacoulation. Green arrow: myofibrillar degeneration; red arrow: pycnotic nuclei; white arrow blood clot; yellow arrow: vacoulation. (H and E staining at 400X).

33. Histopathology: Lungs • control presenting pulmonary parenchyma with normal features; thin alveolar walls (thin black arrow), variable-sized and well-organized alveolar spaces with no infiltrating inflammatory cells. (*): Bronchus; thick black arrow: pulmonary artery. • Section 12 h following venom administration showing thickened interalveolar septa (Δ) and dense inflammatory leukocyte infiltrate. • 24 h venom-treated rat showing thickened alveolar walls (blue arrow), decreased number of alveoli and inflammatory exudates (double-headed arrow); a: alveolus; b: alveolar duct. (H and E stain at 200X).

34. Histopathology: Brain • (a) control rat showing normal architecture. • (b) venom-treated rats with • congested blood sinusoid B. pycnotic nuclei C. vaculation D. necrosis a

35. Conclusions/Lessons learned in serum : Marker Indication • creatinekinase and damage to heart muscles lactate dehydrogenase • creatinine impairment of renal function • alanine transaminase decreased activity and weakness • High-sensitivity inflammatory response. C-reactive protein

36. Conclusions/Lessons learned • Tissue damage caused leakage of marker enzymes into circulation and disturbed activity of these enzymes probably resulted in the observed stress. • The results may account for some of the clinical manifestations observed in victims of stonefish envenomation. • The presented data provide further in vivo evidence of the stonefish toxic effects that may threaten human life and call for the need for special measures to be considered.

37. Future Perspectives • Future studies aimed at investigating changes in the hematological parameters (blood cell count, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, and platelet count), blood sugar, urea, triglycerides, cholesterol, and total protein in rat are being followed up in our laboratories. • It is hoped that these findings will help the health care industry to develop an indigenous medicine that can be used as remedy for analgesic and neurological disorders.

38. This work has been published

39. QUESTIONS?? Thank You Stonefish Flesh Dish Resembles that of a crab Professor Ahmad Khalil 00 962 799 923 290 kahmad76@yahoo.com