Morphological and cytochemical characteristics of Varanus niloticus (Squamata, Varanidae) blood cells

Varanus niloticus is a lizard residing within the Varanidae family. To date no studies detailing its blood morphology and characteristics have been conducted. This study used histologically stained blood and bone marrow samples to visualize the cells and their characteristics. The erythrocytes were nucleated, these nuclei were located in the middle of the elliptical cells. Hemoglobin filled the erythrocyte cytoplasm. Eosinophils were large cells with lobed nuclei and spherical acidophilic granules. Large granulocytes called heterophils were present and characterized by their fusiform/pleomorphic cytoplasmic granules. Small spherical granulocytes, known as basophils, presented with round, deeply stained metachromatic granules that gave the cytoplasm a dusty or cobblestoned appearance which was able to cover the nucleus, which in turn had an unusual shape. Thrombocytes ranged in shape from ellipsoidal to fusiform. They featured an elliptical, centrally located nucleus and a pale cytoplasm, with small vacuoles, and fine acidophilic granulation. The smallest variety of non‐granular leukocytes was the lymphocytes. Their cytoplasm was sparse, finely granular, light blue, had tiny cytoplasmic projections, featuring a high nucleus: cytoplasm ratio. Larger and smaller sized populations of lymphocytes were distinguished, with the larger cells similar in size to azurophils. In general, the pleomorphic monocytes were the biggest mononuclear leucocytes, displaying cytoplasmic projections. Their nuclei were ovoid, kidney‐ or bean‐shaped, with vacuolated and granular cytoplasms. Round cells were common among the monocytic azurophils, and they had a granular cytoplasm, and their nuclei were typically eccentric. The present research identifies the cell types and morphologies within the Varanus niloticus.


| INTRODUCTION
There are currently over 9000 species of reptiles, which are coldblooded vertebrates (Pérez-García, 2022). The River Nile and Sub-Saharan Africa are home to Varanus niloticus (Bayless, 2002). It is a member of the Varanidae family and is the longest lizard in Africa.
Varanus niloticus is also commonly referred to as the Nile monitor and the African savanna monitor. As a result of the pet trade invasion, Varanus niloticus has spread to many other regions, including Catalonia, Spain and the United States, and has since established itself as a native species, (Soler & Martinez Silvestre, 2013;Wood et al., 2016).
There are known species differences in the hematological characteristics between mammals, birds, and reptiles. Similar to avian blood, which contains erythrocytes, thrombocytes, and leucocytes, reptilian blood also contains these cell types (Fontes Pinto et al., 2018). Reptilian blood also contains granulocytes such as lymphocytes and monocytes, and granulocytes including heterophils, eosinophils, and basophils (Arizza et al., 2014;Stacy et al., 2011). Blood cells have been used to help differentiate between different reptilian species and understand unique features affecting the physiology of each species.
For example, the mangrove-dwelling monitor (Varanus indicus) and the savannah monitor (Varanus exanthematicus), have both had their blood cell counts and sizes compared to their body sizes. The metabolic activities of blood cells have helped explain the variations between their size and number (Frydlova et al., 2013). Studying reptilian blood from a phylogenetic perspective also advances our understanding of the evolutionary development of both non-avian reptiles and the reptile clade as a whole.
The current study uses a variety of stains, including hematoxylin and eosin, Periodic Acid Schiff, toluidine blue, methylene blue, and Safranin O, to examine the morphology and cytochemistry of Varanus niloticus blood cells from blood smears and bone marrow.

| Ethics and sample collection
The conduct of this study and its methodologies were overseen by Institutional Aquatic Animal Care and Use Committee IAACUC of Kafrelsheikh University, Approval number: IAACUC-KSU-2022-27.
Six male Nile monitors were kept in a controlled environment for two days in the animal facilities within the Faculty of Veterinary Medicine's Histology Department laboratory, South Valley University's (SVU), Egypt. Euthanasia was conducted on the third day using ethically approved techniques by a trained veterinary professional using intraperitoneal administration of sodium pentobarbital 60-120 mg/kg (Shaker & Ibrahium, 2021). All of the animals were deemed healthy, based on visual health assessments. To confirm maturity, snout-tovent lengths <38 cm, and weights of 10 ± 2 kg were confirmed (Moustafa et al., 2013). Blood was taken from three Nile monitors, and femur bone marrow samples were taken from the other three for histological processing and embedding in paraffin.

| Blood sample collection
Alcohol was used to disinfect the collection site, and a syringe used to draw 2 mm of blood from the ventral coccygeal vein in accordance with the previously described protocol (Salakij et al., 2014). For further processing and staining, the collected blood was immediately transferred into anticoagulant coated tubes (potassium ethylenediamine tetra acetic acid) to enable morphological analysis (Moustafa et al., 2013).

| Cytomorphological analysis
For cytomorphological studies, blood smears were prepared immediately on grease-free slides, fixed using methanol and left to dry. Each slide was then stained with either hematoxylin and eosin (H&E), Periodic Acid Schiff (PAS), Safranin O, toluidine blue or methylene blue.
The smears were dehydrated through ascending grades of alcohol 70% 80%, 90%, and 100%, 3 min per concentrations. The protocols for stain were previously described (Suvarna et al., 2013). Each stained blood smear was examined using an oil-immersion lens under a Leitz Dialux 20 microscope (Germany), and pictures were taken with a Canon digital camera (Canon PowerShot A95, Japan).

| Bone marrow sample collection, staining and visualization
Femurs were removed and cut longitudinally, and Worbel-Moustafa fixative was applied for 24 h (Abd-Elhafeez et al., 2017a, 2017b. Postfixation, decalcification was undertaken in 10% EDTA (in 0.1 M Tris/HCl buffer, pH 7.4) for 30 days, with the EDTA changed every 7 days until the bone became soft. The femurs were then washed four times in 0.1 M sodium phosphate buffer (pH 7.2) for 15 min each. All of the components of each buffer were as described previously (Abd-Elhafeez et al., 2021;Suvarna et al., 2013).
The dehydrated fixed and decalcified specimens were cleared in methyl benzoate, embedded in paraplast paraffin, and dehydrated in an ascending series of ethanol alcohol (Sigma Aldrich), as described previously (Abd-Elhafeez et al., 2021). Hematoxylin and eosin and Safranin O stains were used of stain paraffin sections of each femur using techniques previously described (Suvarna et al., 2013).

| Morphometric evaluation and measurements
Evaluations were undertaken on 50 erythrocytes and 30 of each of the white blood cells from across the samples using a light microscope with an oil immersion lens (Leitz Dialux 20 microscope, Germany).
Image J (http://fiji.sc/Fiji) was used to measure the morphological parameters including nucleus and cell diameters, in addition to cell cross sectional areas. The outcomes were collated into Excel (Microsoft, USA) and presented as mean ± standard deviation (SD).
White blood cell and nuclei sizes were statistically compared using one-way Analysis of variance with post hoc testing.

| RESULTS
Qualitative and quantitative investigations were undertaken on the blood samples and femur bone marrow. The morphological measurements of the erythrocytes and leucocytes are shown in Tables 1 and 2, respectively.
The largest cells were the heterophils at 19.8 ± 1.9 μm, the eosinophils, azurophils, monocytes, basophils, and lymphocytes decreased in size through to the smallest cells which were the thrombocytes at 9.1 ± 1.3 μm. In terms of cell diameter, the basophils, eosinophils, heterophils, azurophils, lymphocytes, monocytes and thrombocytes were all significantly different in size to each other (p values ranged from 0.01 to 0.0001; Table 2).

T A B L E 1 Morphometric measurements of erythrocytes
Note: Presented largest through to smallest cell size. N = 30 cells and their nuclei measured per cell type. All measurements were expressed as mean ± SD μm. Numbers on brackets indicate minimum-maximum range.

| Morphological description of cell types
Erythrocytes: The Varanus niloticus had nucleated erythrocytes, with the nucleus located in the middle of the elliptical shaped erythrocytes.
Hemoglobin filled the homogeneous cytoplasm of erythrocytes ( Figure 1a). Erythrocyte cell and nuclear widths, lengths, crosssectional areas and nucleus: cell size ratio is presented in Table 1.
Heterophils: Fusiform/pleomorphic cytoplasmic granules were used to identify the large granulocytes known as heterophils (Figure 1c,d).
Thrombocytes: The majority of the thrombocytes were elliptical in shape, though some were occasionally circular. The thrombocytes also had small vacuoles and finite acidophilic granulation within their cytoplasms. The thrombocytes ranged in shape from ellipsoidal through to fusiform. They contained an elliptical, central nucleus with Basophils: Small spherical granulocytes, the basophils, had round, intensely stained metachromatic granules that were able to cover the nucleus and gave the cytoplasm a dusty or cobblestone appearance.
The basophils had irregular shaped nuclei. Toluidine blue: The heterophil cytoplasm with its granules was barely stained following exposure to toluidine blue, whereas the F I G U R E 1 Morphological features of granulocytic white blood cells using H&E. (a, b) Varanus niloticus erythrocytes (arrows) were nucleated, these were located within the center of the elliptically shaped cells Erythrocytes had a homogeneous cytoplasm that was filled with hemoglobin. Eosinophils (e) were large cells with spherical acidophilic granules and a lobed nucleus. Small lymphocytes were present (ly). (c, d) Heterophils (h) were large granulocytes that were identified by their fusiform/pleomorphic cytoplasmic granules. Monocytes (m) were large pleomorphic cells and had indented bean-shaped nuclei and a vacuolated cytoplasm. (e, f) Thrombocytes (t) were ellipsoidal to fusiform in shape, with a pale cytoplasm and an elliptical, centrally located nucleus

| Bone marrow characteristics
The hematopoietic tissue within the femur was investigated regarding their morphological characteristics. Hematopoietic tissues were located within the marrow cavity of each femur, which was surrounded by bone spicules (Figure 10a). In the hematopoietic tis-  (Emmel, 1924;Mueller et al., 2008;Wingstrand, 1956).
Reptile erythrocytes resemble bird erythrocytes in both appearance and function, but they are larger in reptiles (Claver & Quaglia, 2009). The erythrocytes of the Nile monitor were flattened with an elliptical shape, this observation concurred with studies carried out on other reptilian species (Parida et al., 2014;Salakij et al., 2014;Stacy et al., 2011). The nuclei with the erythrocytes were positioned in the middle of the cell and contained hemoglobin. It was previously reported that erythrocyte nuclei in reptiles appeared to be rounded with irregular margins (Claver & Quaglia, 2009), this was also observed in V. niloticus in the present study. The relatively large size of the V. niloticus erythrocyte in this study (at 18 μm long) was also consistent with the larger size seen in ectotherms (reptiles, fish, amphibians) compared to endotherms (bird, mammals;Hawkey et al., 1991). This is also thought to be due to lower oxygen demands and mass-specific resting metabolic rates seen in ectoderms. The erythrocytes of reptiles also contain hemoglobin tetramers, like those of other vertebrates, which aid in the exchange of oxygen and carbon dioxide in tissue and organs. In fact, the lower metabolic rates of reptiles has been highlighted as a possible reason for erythrocyte longevity, in excess of 600 days (Altland & Brace, 1962). This compares to birds such as the duck, pigeon, and chicken with their high metabolism with erythrocytes living just 35-45 days on average (Rodnan et al., 1957). Meanwhile mammalian red blood cells have an average longevity of 22 days in mice, 100 days in people, 120 days in dogs (Rodnan et al., 1957).
White blood cells from reptiles have been classified based on their shapes rather than their functions (Claver & Quaglia, 2009).
Eosinophils, heterophils, and basophils made up the Nile monitor's granulocytic leucocytes, while lymphocytes and monocytes made up the agranulocytes. In several species of reptiles and birds, the heterophils replaced the neutrophils, as previously reported (Blofield et al., 1992;Sacchi et al., 2020;Stacy et al., 2011).
In terms of blood morphometrics, several researchers have noted that reptiles are a heterogeneous group because they show significant variations between orders and even within the same family members  , 2015;Sykes & Klaphake, 2008). The Nile monitor is well adapted to both an aquatic and terrestrial lifestyle and is typically found near water streams of the Nile and its tributaries. A more terrestrial-adapted species is the desert monitor (V. griseus). Our findings were in line with earlier studies that claimed that the aquatic vertebrates have larger erythrocytes than terrestrial counterparts (Aldrich et al., 2006;Arıkan, 2014;Fleischle et al., 2019).
The Nile monitor (V. niloticus) has blood that is primarily made up of erythrocytes, leucocytes, thrombocytes, and liquid plasma, similar to the blood compositions observed in other reptiles. Despite the fact that mammalian plasma is typically colorless, reptiles have yellowishgreen plasma, which is likely due to higher concentrations of riboflavin and carotenoids (Dessauer, 1970). Non-mammalian thrombocytes function in a manner similar to platelets in mammalian vertebrates, thrombocytes therefore play a hemostatic role (Martin & Wagner, 2019;Stacy & Harr, 2021). In the bird thrombocytes are also round to oval, nucleated, with a pale cytoplasm, and smaller than erythrocytes (Campbell & Joshua Dein, 1984).
The present research showed that the Nile monitor (V. niloticus) also produced thrombocytes that were small, ellipsoidal to fusiform in shape, with a pale cytoplasm, containing an elliptical nucleus in the middle. Additionally, the cytoplasm contains small vacuoles and finite acidophilic granulations. In terms of function, the thrombocytes of lower vertebrates were analogous to the platelet of mammalian species (Levin, 2019;Peng et al., 2018). Nucleated thrombocytes were recorded previously in many reptilian species (Alleman et al., 1992;Arıkan & Çicek, 2014;Giori et al., 2020;Louei Monfared, 2014;Metin et al., 2006). Only mammals have exhibited enucleated platelets while reptiles and birds have nucleated thrombocytes, which are arguably functionally less efficient than mammalian platelets (Martin & Wagner, 2019;Schmaier et al., 2011). The platelets of mammals are relatively short-lived as the life spans range between 10 days in humans and 5 days in mice (Johnson et al., 2018;Lebois & Josefsson, 2016). Taking in our consideration the mean erythrocyte lifespan in reptiles ranges between 600 to 800 days while in humans it is only 120 days, thrombocytes have a longer lifespan of thrombocytes in reptiles (Louis et al., 2020;Olsson et al., 2020). It is assumed that the slow metabolic rate of reptiles is closely related to the exceedingly slow turnover of erythrocytes comparing to other vertebrates (Khalaf et al., 2020;Stacy et al., 2011). It is worth mentioning that the thrombocytes in fish, amphibians, reptiles, and birds have a membrane-bound canalicular system which plays an essential role in their function (Chamut & Osvaldo, 2018;Levin, 2019;Selvadurai & Hamilton, 2018). This system is also found in mammalian platelets and has been linked to their origin in the megakaryocyte cytoplasm (Levin, 2019;Martin & Wagner, 2019;Stalker et al., 2012). Our morphometric analysis in the Nile monitor (V. niloticus) indicated that the smallest cells were the thrombocytes (9.07 ± 1.33 μm) which helps facilitate its role in maintaining blood hemostasis, blood clotting formation and the initiation of wound healing (Martin & Wagner, 2019;Peng et al., 2018). Additionally, these cells may exhibit some endocytic and phagocytic abilities (Silva et al., 2005).
Primarily due to a lack of functional knowledge, reptilian leucocytes are categorized according to appearance rather than function, as such their nomenclatures are tentative (de Carvalho et al., 2017;Stacy et al., 2011). The present research showed that heterophils appeared to be the second-most prevalent cell type in the Nile monitor after erythrocytes. Their defining features were that they were relatively large cells with a transparent cytoplasm. Reptilian structural and cytochemical studies revealed that heterophils function in a similar way to mammalian neutrophils, phagocytizing foreign particles and bacterial invaders (Arıkan, 2014;Fingerhut et al., 2020;Zimmerman et al., 2010). It was also interesting to note that heterophils were not found in any of the five species of Turkish lacertid lizards (Arıkan et al., 2009). The eosinophils in the present study were relatively large cells with lobed nuclei and spherical acidophilic granules. Even though the primary role of eosinophils in reptiles remains unclear, recent research has shown that helminthes and protozoan parasitic infections cause an abnormal increases in eosinophil numbers (Bessa et al., 2020;Mendoza-Roldan et al., 2020). The basophils in the Nile monitor were small, spherical granulocytes with small, round, metachromatic granules that were deeply stained. Some species of reptiles, like the green turtle, loggerhead turtles, bobtail lizards, and other European lizards, only occasionally have these cells (Casal & Or os, 2007;Moller et al., 2016;Sacchi et al., 2011). The primary role of basophils is long-term protection against chronic illness and the inflammatory response it triggers (Hawkey et al., 1989). Additionally, they may aid in the recovery and elimination of hemoparasites such as trypanosomes and haemogregarines from the reptilian body (Strik et al., 2007).
The Nile monitor's (V. niloticus) lymphocytes resembled those of other vertebrates previously studied. The smallest variety of the nongranular leukocytes, they had a large nucleus to cytoplasm ratio, following staining the cytoplasm was a light blue color. Lymphocytes are involved in immune responses and hematopoietic growth factor production (Arıkan, 2014). The present investigations showed that the largest mononuclear leucocyte with cytoplasmic projections was the monocyte. Their nuclei were kidney-or bean-shaped or ovoid in shape. These cells have previously, in reptiles, exhibited phagocytic traits and become active under conditions of persistent inflammation (Stacy et al., 2011). Azurophils, a different kind of granulocyte only found in reptiles, were also discovered in V. niloticus in the present investigation. These cells have also been observed in a variety of reptilian species, including lizards, snakes, crocodiles, and occasionally turtles (García-De la Peña et al., 2020). They are similar to monocytes in many ways, but evidence from several studies has shown they exhibit stronger phagocytotic affinities toward bacterial particles (de Carvalho et al., 2017).
In conclusion, the Nile Monitor's (V. niloticus) blood cells have morphological and cytochemical characteristics that are similar to those of other reptilian species. There are, however, some speciesspecific variations, which likely account for variations in their environmental circumstances. These findings may help in develop appropriate conservation and identification strategies for protecting endangered species and informing evolutionary biology, as well as providing clinically relevant research to track pathological conditions of this lizard.