1. Spine replacement in the round stingray, Urolophus halleri, as a consequence of a traumatic injury to the spine Author : Petra Johansson Mentor: Dr. Lowe Affiliation: CSULB

2. Outline Introduction Introduction Materials and methods Materials and methods Expected results Expected results Conclusion Conclusion Future experiments Future experiments

3. Introduction Urolophus halleri Urolophus halleri

4. Introduction Geographic range Geographic range Habitat Habitat –Sandy or muddy bays –Off shore to 21m depth

5. Introduction Venomous caudal spine Venomous caudal spine –Inner core of vasodentine –Enamel-like outer layer –Retroserrate teeth –Longitudinal ventral ridge –Longitudinal lateral grooves –venom secreting cells –Integumentary sheet –venom secreting cells

6. Introduction Why a venomous caudal spine? Why a venomous caudal spine? Self defense Self defense –Predators –Humans Human injuries? Human injuries? 474 injuries during a 7 month period (Russel 1953) 80-120 injuries between Jun-Aug in Seal Beach

7. Introduction Spine replacement Spine replacement –Multiple spines –Spines replaced seasonally In July, secondary spines appear posterior and ventral to primary spines In July, secondary spines appear posterior and ventral to primary spines In November, old primary spines exfoliate In November, old primary spines exfoliate –No studies have been performed that show spine replacements occurring as a result of damage to or removal of the spine

8. Introduction What will I do? What will I do? –Determine if spine replacement occurs as a result of a traumatic injury to the spine –Determine the location of the spine regeneration area Hypothesis Hypothesis –Stingrays with pulled spines will replace their spines immediately –Spine protects against predators and insures survival –Pulling of spine affects growth area –Stingrays with clipped spines will not replace their spines immediately –Spine still functional and venomous –Clipping does not affect growth area –Spinegrowth same in all treatment groups

9. Materials and methods Animal collection/sample size Animal collection/sample size Beach seining Beach seining 15 adult males 15 adult males

10. Materials and methods Tank care Tank care –Three static seawater tanks –sub-gravel filter –airlifts –air-stones –Water quality Animal care Animal care –Feeding –Trace element blocks and vitamins –Parasite control One hour dips in 4 mL of 37% formalin in 15 L seawater One hour dips in 4 mL of 37% formalin in 15 L seawater Praziquantel drug tank treatment (2 ppm for a 24 hour period) Praziquantel drug tank treatment (2 ppm for a 24 hour period) http://anicca.net/parafish/parasite_ima ges/parasite.php?img_id=142

11. Materials and methods Treatments and general procedures Treatments and general procedures –All stingrays fin-clipped –Three treatments Control Control Clipped spines Clipped spines Pulled spines Pulled spines –Stingrays anesthetized (125 mg MS-222/1L seawater) –Measurements taken from May to November 2002 Presence/absence of primordial spine buds monitored biweekly Presence/absence of primordial spine buds monitored biweekly Secondary spine lengths measured monthly Secondary spine lengths measured monthly

12. Materials and methods Histology Histology –To determine spine regeneration area –Samples collected from 12 rays over a period of 2 months Postfixation Dehydration Attachment to slides Embedment Stained TEM Tissue Fixation

13. Materials and methods Tissue fixation Tissue fixation Fixed for 3 days in 1.5 % glutaraldehyde, 1.5 % paraformaldehyde, and 0.1 M cacodylate buffer (pH 7.4) containing 1 drop of 0.5 % CaCl2 and 0.1 M EDTA Fixed for 3 days in 1.5 % glutaraldehyde, 1.5 % paraformaldehyde, and 0.1 M cacodylate buffer (pH 7.4) containing 1 drop of 0.5 % CaCl2 and 0.1 M EDTA Postfixation Postfixation 2 hours in 1 % osmiumtetraoxide, 0.1 M cacodylate buffer (pH 7.4), 5 % sucrose, and 1 drop of 0.5 % CaCl2. 2 hours in 1 % osmiumtetraoxide, 0.1 M cacodylate buffer (pH 7.4), 5 % sucrose, and 1 drop of 0.5 % CaCl2. Rinses after each fixation Rinses after each fixation 48 hours at 4 degrees centigrade in 0.1 M cacodylate buffer and 10 % sucrose. 48 hours at 4 degrees centigrade in 0.1 M cacodylate buffer and 10 % sucrose.

14. Materials and methods Dehydration Dehydration Graded series of ethanol rinses Graded series of ethanol rinses –30 %, 50 %, 70 %, 95 %, and four changes of 100 %. Embedment Embedment Spurrs resin Spurrs resin Slide attachment and staining Slide attachment and staining Heat attachment Heat attachment Stained with toluidine blue Stained with toluidine blue TEM TEM

15. Materials and methods Statistical analyses Statistical analyses –Spine bud initiation 3 logistic regression analyses 3 logistic regression analyses –To determine if there is a significant change in probability of spine initiation over time 3 modified t-tests 3 modified t-tests –To determine if onset of spine initiation between the three treatments are significantly different from each other –Secondary spine growth Data logit-transformed Data logit-transformed ANCOVA ANCOVA –To determine if there is a significant displacement of secondary spine growth over time between the three treatments –To determine if there is a significant difference in growth rates between the three treatments

16. Expected results Spine bud data Spine bud data

17. Expected results Spine bud statistical data Spine bud statistical data –Logistic regression –T-test

18. Expected results Histology Histology –Mitotic activity greater in spine- regeneration area than in nearby tissues –Spine bud regeneration begins posterior and ventral to the primary spine –Primordial spine bud similar in structure to elasmobranch scale primordium

19. Expected results Secondary spine growth data Secondary spine growth data

20. Expected results Statistical results of secondary spine growth data: ANCOVA Statistical results of secondary spine growth data: ANCOVA p = proportion of maximum spine length

21. Conclusion Traumatic loss of the primary caudal spine alters the normal spine replacement cycle Traumatic loss of the primary caudal spine alters the normal spine replacement cycle –Supported from an evolutionary point of view –Teleost scales regenerate fast after injury. Secondary spine growths same in all three treatment groups Secondary spine growths same in all three treatment groups –Physiological mechanism to compensate for faster growth

22. Future experiments Spine removals Spine removals Year round Year round On both male and female Urolophus halleri On both male and female Urolophus halleri On both young and adult U. halleri On both young and adult U. halleri Presence of a predator? Presence of a predator? Seasonality effects Seasonality effects Light intensity Light intensity Temperature Temperature Spine grafts Spine grafts To determine if cell growth is due to the cells themselves To determine if cell growth is due to the cells themselves

23. Acknowledgements Dr. Lowe Dr. Lowe Dr. Archie (for invaluable help with statistical analysis) Dr. Archie (for invaluable help with statistical analysis) Dr. Mason Dr. Mason Howard Huges Medical Institute Howard Huges Medical Institute