By: Amy Christine

1. Altered Histology of the Thymus and Spleen in Contaminant-Exposed Juvenile American Alligators (Alligator mississippiensis) By: Amy Christine

2. Background • Alligators in Florida have been extensively investigated for endocrine parameters. • Initially driven by a population crash, dramatically low hatching rates, and an unexplained increase in the mortality of juvenile and adult alligators in Lake Apopka.

3. Background Cont. • Lake Apopka is the 4th largest lake in Florida and historically had a large population of alligators • Also history of extensive, well documented contamination (linked to spillage of pesticides: DDT, DDD, DDE)

4. Background Cont. • Endocrine and reproductive abnormalities have been detected in neonatal and juvenile alligators from Lake Apopka • Data suggests that the endocrine system of Lake Apopka can be organizationally modified during development by exposure to EDCs.

5. Hypothesis • EDC-related abnormalities in Lake Apopka alligators suggest a hypothesis that other systems influenced by sex steroids could also be affected. • Strong endocrine-immune connections suggest that morphology of immune tissue and function would be compromised when the endocrine system is altered.

6. Histological examination of the spleen and thymus was undertaken to evaluate the immune system of alligators in relatively pristine reference lakes (Woodruff and Orange) and a highly contaminated lake (Apopka). The experiment was broken up into two studies. Methods

7. Study 1: wild alligators • Juvenile alligators were collected from the three lakes by hand or noose from May 5 through July 1, 1997. • Animals were secured, uniquely tagged, and within 2 h bled from the postcranial sinus. • Physical measurements consisted of total and snout-vent length. Blood samples were stored on ice for 8-12h and then centrifuged, the resulting plasma was then stored at –80*C for radio immunoassays.

8. Study 1: wild alligators • 15-18h after capture, animals were brought back to laboratory and killed by lethal injection. • Relevant portions of the thymus and spleen were removed and placed in Bouin’s fixative.

9. Study 2: Captive-reared juvenile female alligators • Eggs were laid on either Lake Woodruff or Lake Apopka and incubated in laboratory. • After hatching, females were housed in a 20x40 foot enclosure with hiding boxes and swimming area. Enclosure was cleaned daily and animals were checked 2x per week for health. • Animals were killed and tissues taken in October, 1997.

10. Histology • Caudal end of spleen and anterior end of the thymus were removed and fixed for light microscopy.

11. Analysis • All histological measurements were taken by a single researcher using single blind procedures to prevent researcher bias.

12. Thymus • Medullary and cortical boundaries and areas change with immunological ability as well as season in reptiles. • Measurements of the cortical and medullary areas were obtained to assess the immunological status of the thymus.

13. Spleen • T-cell associated lymphocyte sheaths: PALS and PELS were measured in thickness or width rather than the area. • Ten lymphocyte sheaths were measured per spleen. • Within each section, lymphocyte sheaths were chosen at random.

14. Radioimmunoassays • Plasma was assayed for testosterone (T). • The data are used here for analysis of possible relationships between plasma T and immunological data.

15. Results: Study 1 • Regression analyses indicated very weak and nonsignificant relationships between snout-vent length and spleen or thymus parameters; therefore, body size was excluded in further analysis. • Plasma T concentration did not correlate with any immune parameter in wild caught alligators.

16. Results: Study 1 Cont. • Gender didn’t influence medulla/cortex ratios of the thymus from a given lake. • Differences in thymic ratios were found among lakes:1.) Animals from Lake Apopka had smaller thymic ratios than animals from either of the reference lakes. 2.)It was found that the lake of origin influenced thymic cortical areas, and no difference existed in the medullary areas. Animals from Lake Apopka had larger cortical areas than animals from either reference lake.

17. Results: Study 1 Cont. • Lymphocyte sheaths in the spleen did not differ in width when males were compared to females from lakes Orange and Apopka. • Males from Lake Woodruff had smaller sheaths than the females. • Sheath width did differ in females from the three lakes, Lake Apopka having the smallest and Lake Orange having the largest. • Males from Lake Orange had larger sheaths compared to males from the other two lakes.

18. Results: Study 2 • Captive-reared alligators also had no relationship between snout-vent length and immune parameters. • In contrast to the wild, no difference was found in thymic medulla/cortex ratio of captive alligators. • However, captive alligators had larger thymic ratios than observed in wild animals, although direct statistical comparison cannot be made and stress of captivity may have influenced these values.

19. Results: Study 2 Cont. • Captive raised alligators from Lake Apopka and Woodruff displayed lake-associated differences in lymphocyte sheath width as observed in wild animals • Lake Apopka alligators had smaller lymphocyte sheath width than Woodruff alligators.

20. Discussion • Alligators from Lake Apopka had a smaller medulla to cortex thymic ratio • Difference in thymic ratios was a result of alterations in the cortical area of the thymus • Increased cortical area in the Apopka alligators is indicative of a shift in the number of developing T-lymphocytes in the Apopka alligators relative to animals from the other lakes.

21. Discussion Cont. • Inc. in cortical area could be from a more rapidly dividing T-lymphocyte population, or through longer retention times within the cortex. • Data from other studies suggesting reduced T-lymphocytes responses would support the hypothesis that maturation is delayed, thus giving animals from Apopka elevated cortical areas.

22. Discussion Cont. • Data also demonstrated a difference in the cortical region of the thymus that is assoc. more with living in a specific lake than having that lake as its origin. • Captive-reared alligators showed no differences in thymic ratios compared to each other raised under identical conditions. • Therefore, differences observed do not appear to be of genetic or development origin, but due to environmental factors prior to capture.

23. Discussion Cont. • Observations of the spleen morphology could be related to the observed thymic differences. • Alligators from Lake Apopka have the smallest lymphocytes sheaths, suggesting that they have a highly active thymus to counter a reduced T-lymphocyte presence in the spleen. • Captive-raised alligators only differed in sheath width, the lack of a lake difference in thymic ratios in the captive alligators suggests that the environment experienced by the wild alligators creates lake differences in these histological features.

24. How did histological samples/ techniques improve this article? • This experiment was solely based on analyzing histological samples taken from the alligators and the results heavily relied on the comparative aspect of these samples. • The actual histological samples are evidence of what contaminants can do to a population.

25. Why is this article important? What would you do different? • This article is important because it shows how the effects of contamination on an environment can alter animals health and development. • One thing I would do differently would be to sample both groups (wild and captive) during the same season.

26. References • Rooney, Andrew A., Dieldrich S, Bermudez, and Louis J. Guillette Jr. “Altered Histology of the Thymes and Spleen in Contaminant-Exposed Juvenile American Alligators.” Journal of Morphology. Vol. 256: 349-359. June 2003.