72 HERBERT C. DESSAUER Wilson, J. W. (1939). Some physiological properties of reptilian blood. J. cell, comp. Physiol. 13, 315-326. Wintrobe, M. M. (1933/1934). Variations in the size and hemoglobin content of erythro- cytes in the blood of various vertebrates. Folia haemat. Lpz. 51, 32-49. Wistrand, P. and Whitis, P. (1959). Distribution of carbonic anhydrase in alligator. Effect of acetazolamide on blood and aqueous humor CO . Proc. Soc. exp. Biol. Med. 101 2 CHAPTER 2 674-676. Wolfe, Μ. R. (1939). Standardization of the precipitin technique and its application to studies of relationships in mammals, birds, and reptiles. Biol. Bull. mar. biol. Lab. Morphology of the Circulating Woods Hole 76, 108-120. Wright, A. and Jones, I. C. (1957). The adrenal gland in lizards and snakes. J. Endocr. 15 Blood Cells 83-99. Zain, B. K. and Zain-ul-Abedin, M. (1967). Characterization of the abdominal fat pads of MARIE-CHARLOTTE SAINT GIRONS a lizard. Comp. Biochem. Physiol. 23, 173-177. Zain-ul-Abedin, M. and Qazi, M. H. (1965). Blood sugar levels of some reptiles found in Museum National d'Histoire Naturelle, Pakistan. Can. J. Biochem. Physiol. 43, 831-833. Brunoy, France Zarafonetis, C. J. D. and Kalas, J. P. (1960). Some hematologie and biochemical findings in Heloderma horridum, the Mexican bearded lizard. Copeia 240-241. Zweig, G. and Crenshaw, J. W. (1957). Differentiation of species by paper electrophoresis of serum proteins of Pseudemys turtles. Science, N.Y. 126, 1065-1066. I. Introduction The earliest works on the blood of reptiles described only the structure of its various elements, often comparing them with those of other vertebrates. Treatises on hematology generally contain sections on reptilian blood; unfortunately these are often based on the study of far too few, principally European species. A few recent monographs consider single species and include, besides descriptions of the different circulating blood cells, other observations on various problems - parasites of the blood, seasonal or sexual variation in the numbers of corpuscles, hematopoiesis, and the like. The earliest published works are those of Mandl (1839) and Gulliver (1840) concerning the erythrocytes of crocodilians and turtles. Important studies of comparative morphology of the blood include those by Gulliver (1842, 1875), Milne-Edwards (1856, 1857), Hayem (1879), Pappenheim (1909), Werzberg (1910), Schulz and Krüger (1925), Loewenthal (1928, 1930), Babudieri (1930), Jordan (1938), Ryerson (1949), Altman and Dittmer (1961), and Pienaar (1962); the last work contains an important biblio- graphy. See Efrati et al. (1970) for a study of hemopoiesis in a lizard. Many questions remain unanswered, and the difficulty in determining the different cellular lineages appears to be one of the principal obstacles to a comparative study of reptilian blood. Only mature cells are considered in this chapter although various stem cells are found in the circulating blood. Pienaar (1962) gives a synonymy of the nomenclature used by the major previous authors (pp. 29-33) and attempts to standardize the names given to the cells of the circulating blood. I will use a slightly simplified version of his system of nomenclature. 73 2. MORPHOLOGY OF THE CIRCULATING BLOOD CELLS 75 74 MARIE-CHARLOTTE SAINT GIRONS The different types of mature cells in the circulating blood of reptiles are : cytes are chromophilic. The masses of chromatin are more or less visible, 1. Erythrocytes (red blood cells) depending on the age of the cells. The most critical staining permits the demonstration of granules within the cytoplasm (Hirschler, 1928; Ryerson, 2. Granulocytes (granular myeloid leucocytes) 1949) and of the Golgi apparatus (Bhattacharya and Brambell, 1924). a. Eosinophils b. Basophils (mast cells) The circulating blood contains immature cells of the erythrocytic series c. Azurophils (basophilic and polychromatophilic normoblasts) characterized by a rounded d. Neutrophils form, blue cytoplasm, and a large nucleus which is less chromophilic than that of a mature erythrocyte. These cells are especially common in young or 3. Lymphocytes (large, medium, or small lymphocytes) moulting animals (Saint Girons, 1961) or ones heavily infected by hemo- 4. Monocytes (large mononuclears) parasites (Pienaar, 1962). Mitotic figures are also present. These two 5. Plasma cells phenomena reflect the process of erythropoiesis, generally in association with 6. Thrombocytes. thyroid activity. The senile forms of the red blood cells are larger than the All the cells are nucleated. The names of the different types of granulocytes normal erythrocytes. Their cytoplasm stains weakly, and their nuclei are do not correspond exactly with their chemical composition or with their pycnotic and often irregularly shaped. In the final stage, the cytoplasm dis- particular staining properties. appears, and only the nucleus is visible in a smear. These different stages The problems concerning the monophyletic or polyphyletic origin of the have been studied in detail in one turtle (Terrapene carolina, Jordan and different types of cells have not been completely resolved. According to the Flippen, 1913), one lizard (Cordylus vittifer, Pienaar, 1962), and two snakes great majority of workers, all the cells in the circulating blood derive from (Vipera russelii and Python regius, Slonimski, 1934). one multipotent type of hemoblast capable of differentiating in the bone marrow or spleen to produce erythrocytes, granulocytes (eosinophils, baso- The presence of intracorpuscular parasites may alter the shape and size phils, azurophils, and neutrophils), monocytes, and lymphocytes. The last are of erythrocytes considerably. With infection of the red blood cells of Taren- the scarcely modified hemocytoblasts or stem cells which retain their multiple tola mauritanica by the sporozoan Pyrhemocyton tarentole, the erythrocytes potentialities and can give rise to all the other types of corpuscles. Throm- and their nuclei become more circular (Wood, 1935). In lizards from Mexico bocytes are said to derive from small lymphocytes. Some workers postulate and Florida, the presence of Plasmodium spp. within the corpuscles affected different origins, beginning with the hemoblasts, for the leucocytic series on the dimensions of the cells, causing a more or less marked increase in their the one hand and for the erythrocytes on the other. They thus do not accept size (Thomson and Huff, 1944a and b; Bergman, 1957; Reichenbach- all the multiple potentialities of the lymphocytes. Other hematologists believe Klinke, 1963). that the cells of the circulating blood have three distinct origins : monocytes A recent study (Saint Girons and Saint Girons, 1969) of 76 species of rep- derive from a stem cell of the reticulo-endothelial system ; granulocytic cor- tiles belonging to 29 families allows precise statements to be made concerning puscles, lymphocytes, and thrombocytes arise from a second type of stem the morphology of the erythrocytes in members of the four orders of reptiles cell, the myeoblast; and finally the erythrocytes arise from the erythroblasts. (Table I and Figs 1-5). The histological techniques for the study of blood and of hematopoietic The cryptodiran turtles have rather large erythrocytes with regular, rounded organs have recently been considered by Gabe (1968). nuclei. Their nucleo-cytoplasmic ratios (see Table I) are slightly less than the average for reptiles. Turtles of the genus Pelomedusa (Pleurodira) have small, II. Erythrocytes almost rounded nuclei, and thus an especially low nucleo-cytoplasmic ratio (Gulliver, 1875; Taylor and Kaplan, 1961; Pienaar, 1962; Saint Girons and The erythrocytes or red blood corpuscles of the circulating blood are Duguy, 1963 ; Saint Girons and Saint Girons, 1969). nucleated, oval cells, rounded at their ends (Figs 6-13, see facing p. 82-3). Their nuclei are also oval, more or less regular, and centrally located; their The erythrocytes of Sphenodon punctatus differ from those of all other long axes lie parallel to those of the cells.* In blood smears stained by the reptiles by their great size which allows the recognition of this species, with- classic May-Grünwald-Giemsa technique, the yellowish cytoplasm most out any doubt, from a simple blood smear. Otherwise they have no striking often appears translucent and homogeneous. The nuclei of mature erythro- characteristics, although their regularly shaped nuclei are slightly more rounded than in most reptiles (Saint Girons and Saint Girons, 1969). *In the lizard Acanthodactylus erythrums the long axes of more than 50% of the nuclei deviate from those of the cells in the only specimen of this form that I have studied. The erythrocytes of lizards vary greatly in size depending on the family 76 MARIE-CHARLOTTE SAINT GIRONS 2. MORPHOLOGY OF THE CIRCULATING BLOOD CELLS 77 TABLE I TABLE 1—cont.. List of Species with Erythrocyte Dimensions. .Erythrocytes Nuclei Ratios (GD - Greatest diameter, LD - Least diameter, S - Surface GD LD GD/ .S .GD .LD GD/ S N/C N/C - Nuclear surface/cell surface) LD LD 2 22 Erythrocytes Nuclei Ratios mcm (μ) (μ )2 (μ) mcm (μ ) GD LD GD/ S .GD LD GD/ .S .N/C Teiidae LD LD Ameiva ameiva 13..8 7,6 1.82 82.3 5.8 2.7 2.15 12-3 0.149 mcm mcm mcm2 (μ) (Μ) mcm. Cnemidophorus tigris 15..8 8,3 1.90 102.8 5.9 2.4 2.46 11.1 0.108 Cordylidae TESTUDINES Cordylus cordylus 17..3 9,8 1.77 133.4 6.3 4.5 1.40 22.2 0.166 Testudinidae Cordylus vittifer 17..2 9.5 1.81 128.0 7.1 4.6 1.54 25.6 0.200 Testudo graeca 18.5 10.6 1.75 153. 8 6.1 4.3 1..42 20..6 0.134 Scincidae Emydidae Egernia sp. 16..3 10.4 1.57 129.9 6.1 4.2 1.45 20.2 0.155 Clemmys caspica leprosa 19.0 10.9 1.74 162 .5 6.5 4.8 1..35 24..5 0.151 Chalcides mionecton 14..4 8.1 1.77 91.6 5.7 3.8 1.50 17.2 0.177 Emys orbicularis 19.9 11.7 1.70 182.