Blood Cell Profile of the Developing Tadpoles and Adults of the Ornate Frog , Microhyla ornata ( Anura : Microhylidae )

Metamorphosis happens to be an important event in the lifetime of amphibians. Our study offers a record of blood cell profile of laboratory reared tadpoles during development and metamorphosis (Gosner stage 26 to 46) and adults of Microhyla ornata. The larval erythrocytes were observed to be circular, oval, and elliptical in shape. However, other variations were distinct during the prometamorphic and metamorphic stages. Crenulated erythrocytes showed a pattern of appearance, and the crenulations varied from minute serrations to highly spiked projections. Correlations between the morphometric values of erythrocytes during the larval development were also determined. The leukocyte profile of the tadpoles showed a high percentage of lymphocytes during larval development while the percentage of monocytes, eosinophils, and neutrophils remained high during metamorphosis and were positively correlated with the developing stages. Blood thrombocytes of the tadpoles were small and were found in clusters. Elliptical erythrocytes were the most common in the adult frogs. However, few erythrocytes were also circular in shape. In adults, the percentage of lymphocytes was found to be more in comparison with the other leucocytes, and neutrophils showed various polymorphic forms. Thrombocytes were nucleated and spindle shaped.


Introduction
Metamorphosis in amphibians is often a time of dramatic developmental change affecting nearly the entire organism and has been a subject of investigation in several directions [1,2].During this time, a large proportion of the animal's structure changes, the larva, and adults are unrecognizable as being the same individual.Interestingly, these two phases have opposing effects on tissues as in the first phase, there is growth and development of tissues and increase in body size, and in the second phase, massive tissue reconstruction and breakdown leading to a reduction in overall body size occurs [3].This brings about changes morphologically, physiologically and behaviorally to prepare for a new mode of existence.
Herpetologists are becoming increasingly aware of the importance of hematological parameters for evaluating the welfare of their study animals [4].Interpretation of the hematological parameters offers valuable pieces of information concerning the health status of the organism [4].The erythrocyte size has also been described to be used in ploidy determination [5].The effects of the phenomenon of tissue growth and lysis on the relative distribution of white blood cells in circulation have been considered as an important biological phenomenon since the 1920s [3], and a growing number of ecologists are turning to the enumeration of white blood cells from blood smears to assess stress in animals [4].Recent interest in counts of leucocytes in amphibians for environmental monitoring emphasizes the need to understand how white blood cells naturally vary throughout larval life [6][7][8][9].There also exists considerable variation in cell morphology in amphibians due to variation in metabolism [10][11][12].
Since normal hematology of many species is poorly understood and reference values are scarce [13], the present study aims to investigate the blood cell profile of the laboratory reared tadpoles and adult frogs of Microhyla ornata (Duméril and Bibron, 1841) inhabiting, Nandankanan, Bhubaneswar (20 ∘ 24  10  N, 85 ∘ 48  13  E), Odisha, India.Microhyla ornata is a microhylid frog belonging to class anura.A considerable work on M. ornata has been done with respect to life history [14], red blood cell sizes of adults [12], toxicities of some heavy metals [15], morphological, and acoustic comparisons between M. ornata, M. fissipes, and M. okinavensis [16], taxonomic relationships using mtDNA analysis [17], and changes in polyamine contents [18].However, hematological study of both tadpoles and adult frogs remains unexplored in this species in India.Study of hematological parameters during premetamorphic, prometamorphic, and metamorphic stages is crucial to gain proper understanding of changes occurring in the blood cells during metamorphosis; as change in mode of life brings about several new challenges.This paper provides the record of blood cell profile during development and metamorphosis in the tadpoles and also adult frogs of M. ornata.

Collection of Egg Nests and Adult
Frogs.Egg nests and adult frogs of Microhyla ornata were collected from their natural habitat.The egg nests were kept in plastic tubs containing conditioned 20 mm deep tap water (tap water stored and aerated for 72 hours).The tadpoles were reared following standardized procedure [19] and were fed with yolk of boiled egg of Gallus gallus domesticus (breed White Leghorn) ad libitum.The adult frogs (Figure 1(a)) after collection were maintained in the terrarium for acclimatization to laboratory conditions.All procedures were approved by the Animal Care Review Committee at Utkal University.

Tadpoles Investigated.
For the study, tadpoles from Gosner stages 26 to 46 [20] were considered which were comparable to the Taylor and Kollros [21] stages I to XXV.Ten numbers of tadpoles for each stage were selected for investigation.The tadpoles were divided into three subgroups based on developmental periods that are, premetamorphic (stages 25 to 35) (Figures 1(b) and 1(c)), prometamorphic (stages 36 to 41) (Figures 1(d), 1(e), and 1(f)), and metamorphic (stages 42 to 46) (Figures 1(g), 1(h), 1(i), and 1(j)) according to McDiarmid and Altig [22].Prior to drawing blood for peripheral smear, snout to tail tip length (STL) of tadpoles and snout to vent length of metamorphosed froglets were measured.

Preparation of Blood Smears. Procedures described by
Das and Mahapatra [23] were followed.Blood samples for adult frogs were successfully collected following standardized methods [24] without harming the animals in the morning hours.Tadpoles were anesthetized by exposing them to 0.3% MS-222 (Tricaine Methane Sulphonate) solution.The blood of tadpoles from stage 26 to 44 was obtained from tail amputation through the middle of the tail.For stages 45 and 46, blood was collected from the heart using a 26 gauge syringe needle.Blood smears were prepared using push slide technique.The dried blood smears were stained with Giemsa's stain or Leishman's stain and were observed under light microscope (Hund H500).

Identification and
Counting of Blood Cells.Erythrocytes and their variations were identified following Hadji-Azimi et al. [25], Sood [26], and Thrall [27].The leucocytes were identified as lymphocytes, monocytes, eosinophils, neutrophils, and basophils, following Hadji-Azimi et al. [25] and Thrall [27].Slides were viewed in zigzag pattern, covering all parts of the blood smear, and all leucocytes were counted in each field of view until 100 cells were counted.Per blood smear, 150 fields of views were randomly selected to assess erythrocytes.Only field of views with even distribution of erythrocytes was used.For the morphometric analysis of erythrocytes, fifty cells per blood smears were measured.The size of erythrocytes and their nuclei was measured by an ocular micrometer which was standardized against a stage micrometer (ERMA, Japan).For morphometric analysis, the formula of Arserim and Mermer [28] was followed.Photographs of the leucocytes and erythrocytes were taken with the help of a Canon EOS 450 12.2-megapixel camera (EF-S 18-55 1S Kit) connected to Hund 500 WETZLAR microscope.

Blood Cells Profile of the
Of all the variations in the shape of the erythrocytes, the most remarkable was the presence of several kinds of crenulated erythrocytes that had strong resemblance with echinocytes and acanthocytes as reported in mammalian peripheral smears.The crenulations showed a pattern of appearance as they were found in the peripheral smear of tadpoles between Gosner stages 37 and 45.Initially crenulations appeared as fine serrations in the erythrocytes of tadpoles of Gosner stages 37-40 resembling echinocytes (Figures 3(a) and 3(b)).Subsequently, highly crenulated forms resembling acanthocytes appeared on erythrocytes towards the climax stage (Gosner stages 41 to 45) (Figures 3(c), 3(d), and 3(e)).However, the maximum number of such elaborately spiked cells was seen in stage 44 tadpoles.These cells were absent in the froglets of stage 46.The crenulated cells had approximately 8-10 spiny projections distributed uniformly all over (Figures 3(c) and 3(d)) while few had developed 3-4 projections only at one side (Figure 3(e)).Moreover, several degenerating erythrocytes (Figure 3(f)) were recorded in the blood smears of the tadpoles of stages 42 to 44.In these stages, many erythrocytes were in the state of division (Figures 3(g), 3(h), 3(i), and 3(j)).Aggregation of erythrocytes was evident throughout developmental stages of the tadpoles (Figure 3(k)).

Morphology of Leucocytes.
Leucocytes were of five different types, that is, lymphocytes, monocytes, eosinophils, neutrophils, and basophils.The lymphocytes, both large and small (Figures 4(a) and 4(b), resp.), were round in shape.Their nuclei were also rounded and occupied almost the entire cell leaving a narrow rim of light violet cytoplasm towards the periphery.Some lymphocytes showed irregular membrane (Figure 4(c)).Monocytes had eccentrically placed indented nuclei (Figure 4(d)).Eosinophils showed bilobed nuclei where the connections between the lobes were distinct (Figure 4(e)).Neutrophils (Figure 4(f)) with trilobed and tetralobed nuclei were also observed.Neutrophils with trilobed nuclei were the most common in tadpoles.Basophils were identified by the presence of dark violet stained granules over the nuclei as well as entire cells (Figure 4(g)).

Blood Thrombocytes.
The blood thrombocytes were small in size and were found in clusters of 8 to 25 cells (Figure 4(h)).
The highest percentage of basophils was recorded during stage 46 tadpoles.Their overall percentage was low in the peripheral blood smears of the tadpoles as their percentage fluctuated between 0 and 6.05 ± 0.2.International Journal of Zoology their values in adult females were found to 12.42 ± 1.63 m and 7.72 ± 1.78 m, respectively.The ratio between the area of nuclei of the erythrocytes to the area of the erythrocytes (  /A) was found to be 0.26 ± 0.08 in male frogs while it was 0.31 ± 0.08 in the females.The length to breadth ratio of the erythrocytes (L/B) was found to be 1.73 ± 0.34 and 1.62 ± 0.32 in the male and female frogs, respectively.Similarly, the length to breadth ratio of nuclei of the erythrocytes (  /  ) in adult males and females was recorded to be 1.86 ± 0.32 and 1.66 ± 0.31, respectively.

Differential Leucocyte Count.
The mean lymphocytes and monocytes percentage in adult males were found to be 50.42± 5.2 and 6.24 ± 1.2, respectively.In adult females the percentage was found to be 51.28 ± 6.3 and 5.28 ± 1.8, respectively.In adult males, mean eosinophil percentage was 10.21 ± 1.9 while it was 9.32 ± 1.9 in adult females (Table 2).Mean neutrophil percentage in adult males was 30.41 ± 6.8 and in adult females it was found to be 31.21±10.2.The mean basophil percentage in adult males was 2.72 ± 1.0 while it was 2.91 ± 0.7 in adult females.In the adult frogs, percentage of monocytes and eosinophils was higher in males than females, but percentages of lymphocytes, basophils, and neutrophils were higher in females than males (Table 2).

Discussion
The erythrocytes of the tadpoles of M. ornata were observed to be round, oval, or elliptical in shape (Figures 2(a), 2(b), and 2(c)).This observation is similar to previous studies on larval amphibians which suggest presence of two general forms of erythrocytes, larval and adult forms [30][31][32].The larval form is large and elongated while the adult form is smaller and rounder.Nucleus, a characteristic feature of amphibian erythrocytes, was found to be placed at the center of the erythrocytes in almost all peripheral smears.However, few variations were also recorded where nuclei were either eccentrically positioned or pushed to the periphery (Figure 2(d)).In tadpoles of stages 37 to 42, several erythrocytes were noticed to be either unusually larger in size or smaller than the normal erythrocytes (Figures 2(e) and 2(f)).Few erythrocytes lacked a distinct membrane (Figure 2(g)) while several others had irregular appearance (Figures 2(h), 2(i), and 2(j)).Poikilocytosis was noticed in the peripheral blood smears of the tadpoles during the early stages in patches only.Abnormal shape of the erythrocytes included several tear drop forms (Figure 2(k)) and comma-shaped erythrocytes (Figure 2(l)).Some erythrocytes were found to lack nuclei (Figure 2(m)) while in others the nuclei were indistinct (Figure 2(n)).Similar large sized erythrocytes and erythrocytes lacking nucleus (senile erythrocytes) have been reported in tadpoles of Polypedates teraiensis [23].Such senile erythrocytes have also been reported during metamorphosis in other anurans [33].Several smaller dark bodies surrounding regular erythrocytes (Figure 2 these stages few erythrocytes appeared large in size having vacuolated structures that pushed the nuclei to the periphery making them look inconspicuous (Figure 2(p)).Moreover, dark patches were found inside some intact erythrocytes (Figure 2(q)).In other such cells, the membrane appeared to be disintegrated (Figure 2(r)).Interestingly, a remarkable degree of crenulated erythrocytes was also noticed (Figures 3(a), 3(b), 3(c), 3(d), and 3(e)) towards the late prometamorphic and metamorphic stages.Crenulations have been previously reported in red blood cells of Rana pipiens after forelimb emergence [30].These cells resembling echinocytes and acanthocytes of mammalian red blood cells have also been reported earlier in thyroid-treated tadpoles of Rana catesbeiana where cells were irregular with many cytoplasmic projections [34] and in tadpoles of P. teraiensis [23].Such cells have been described to be present during anemic conditions [34,35], and the ectothermic animals are capable to withstand anemic conditions for a long period without mortality [23,36].Erythrocytes which had undergone degeneration were also observed (Figure 3(f)).The size and shape of erythrocyte give an indication of the surface available for the exchange of gases in respiratory functions [37].Morphometric measurement of the erythrocytes (Table 1) confirmed a negative correlation existing between the developmental stages with respect to the size of the erythrocytes and their nuclei.However, their aspect ratio (length/breadth) was found to be positive.A gradual decline in the area occupied by the erythrocytes was observed in the tadpoles with progress in development as a negative correlation existed between the developmental stages with respect to the area of the erythrocytes and their nuclei.The smallest erythrocytes were present in the metamorphosed froglets.Broyles [38], Duellman, and Trueb [39] have described replacement of larger larval erythrocytes by smaller adult erythrocytes during metamorphosis in several anurans.Several cells were found to be in different stages of division as nuclear division was distinct (Figures 3(g), 3(h), 3(i), and 3(j)).An earlier report of increase in erythropoietic activity during metamorphosis has also been reported in R. catesbeiana [40].Another interesting observation was aggregation of several erythrocytes during different developmental stages (Figure 3(k)).
Several trends with respect to the percentage of leucocytes during different developmental stages appeared consistent with the earlier observations [3,6,7,23].Lymphocytes were found to be most abundant during the growth phase of larval development.Their percentage decreased towards end of metamorphosis (Table 2).Davis has reported 70% of lymphocytes in the tadpoles of stages 30 to 33 in R. catesbeiana which decreased with onset of metamorphosis.The trend in monocyte which is a phagocytic cell remained at par with the earlier observations of Davis [3].Their number increased significantly during the metamorphic stages.The higher number of monocytes has been correlated with the increased cellular debris left over from the tissue lysis during  remodeling of larval structures [3], and a positive correlation was observed with developmental stages.However, an unusual spike of neutrophils was observed during the metamorphic climax in the present study unlike the earlier reports on Rana catesbeiana [3] and P. teraiensis [23] where these cells were least abundant during the metamorphic period.Neutrophils remained significantly positive with respect to the developmental stages.The rise in the level of neutrophil in this species is suggested to be related to physiological condition.Eosinophils remained positively correlated with the developmental stages of the tadpoles.Since these cells are known to produce a variety of chemical substances [41] and respond to tissue injury [42], an elevation in the level of eosinophils in the present study is suggested to act to modulate the process of lysis of tissue during metamorphosis.The percentage of basophils fluctuated during development.However, a positive correlation was observed between percentage of basophils and developmental stages, but the correlation was not significant.Increase in the basophil level has been reported in tadpoles of R. catesbeiana   [3] and Polypedates teraiensis [23].Thrombocytes were found to be in clusters or stacked with each other during different developmental stages.The erythrocytes of adult Microhyla ornata were ellipsoidal in shape with centrally placed nuclei, and only a few rounded erythrocytes were observed.The characteristic shape of anuran erythrocyte has been reported to be ellipsoidal [43].Tok et al. [44] have reported morphologically similar erythrocytes among various species of anurans.Ellipsoidal erythrocytes have also been reported in the blood smear of Polypedates maculatus [23].Erythrocytes being the most important carrier of oxygen and carbon dioxide, it has been suggested that an elliptical body is more efficient than a spherical one of the same volume as far as greater rate of exchange is concerned [37].Only a few tear drop shaped cells and comma-shaped cells were found in the peripheral smear.However, other variations in shapes of erythrocytes were not recorded in the adults as found in their larval counterparts.In the present study the surface area covered by erythrocytes was found to be greater in males than in females (Table 1).Similar reports of higher surface area of erythrocytes in males (243.15 ± 7.841 m 2 ) in comparison with females (210.58 ± 38.279 m 2 ) have been reported in adult frogs of P. maculatus [23].In the present study, the mean breadth of the erythrocytes and their nuclei was found to be slightly higher in females than of males (Table 1).Arserim and Mermer [28] have reported larger erythrocyte length and breadth in cases of females (23.03 m and 14.59 m resp.)than males (22.32 m and 13.65 m, resp.) in Rana macrocnemis.The size of erythrocytes of adult M. ornata was found to be higher in comparison with the erythrocytes length (15.4 m ± 1.04) and breadth (11.47 m ± 0.82) of the balloon frog Glyphogloossus molossus belonging to family microhylidae [45].
Leucocyte differential in the adult frogs showed percentage of monocytes and eosinophils to be slightly higher in males than females, but percentages of lymphocytes, basophils, and neutrophils were higher in females.Davis and Durso [4] have reported lymphocytes and neutrophils to be the most commonly seen cell types within amphibians.They have also reported the average of the white blood cells for anurans as 52.6 for lymphocytes, 29.1 for neutrophils, 7.0 for eosinophils, 7.5 for basophils, and 4.0 for monocytes.Comparable percentages of lymphocytes and neutrophils were observed in the present study.But, the percentages of basophils remained low while the percentages of eosinophils and monocytes remained higher.Thrombocytes in the present study were nucleated and spindle shaped.Earlier reports suggest amphibian thrombocytes to be nucleated and having spindle appearance [13,28].
Thus, our study provides the baseline information on blood cell profile of tadpoles and adults of the ornate frog, Microhyla ornata.Moreover, it describes the changes that take place in shape, size and number of blood cells during metamorphosis necessary for the aquatic tadpoles to adapt to a terrestrial environment as a froglet.

Tadpoles of M. ornata 3 . 1 . 1 .
Morphology of Erythrocytes.The erythrocytes in all stages of the tadpoles were found to be circular (Figure2(a)), oval (Figure2(b)), and elliptical in shape (Figure2(c)).Nuclei of these three types of erythrocytes were round in shape and placed mostly in the center.However, few erythrocytes had eccentric nuclei pushed to the periphery (Figure2(d)).

Figure 6 :
Figure 6: Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of the ornate frog, Microhyla ornata.

)Figure 7 :
Figure 7: Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of the ornate frog, Microhyla ornata.

Figure 8 :
Figure 8: Correlation between the percentages of different types of leucocytes with respect to the developmental stages of the tadpoles of the ornate frog, Microhyla ornata.

Table 1 :
3.2.4.Morphometry of Erythrocytes.The surface area covered by erythrocytes in adult males was found to be 254.39 ± 40.82 m 2 while it was recorded to be 238.29 ± 29.75 m 2 in adult females (Table1).Therefore, the surface area occupied by erythrocytes was more in males than females. Te mean length and breadth of erythrocytes were found to be 22.11 ± Morphometry of erythrocytes during the larval development and in the adults of the ornate frog, Microhyla ornata.
2.16 m and 13.18 ± 2.20 m, in adult males.In adult females their values were found to be 21.96 ± 2.11 m and 13.93 ± 2.22 m, respectively.Similarly, the mean length and breadth of nuclei of erythrocytes in adult males were recorded to be 12.27 ± 1.16 m and 6.81 ± 1.62 m, respectively, while STL: snout to tail tip length; SVL: snout to vent length; SD: standard deviation; m: micrometer.

Table 2 :
Percentage of leucocytes during larval development and in the adults of the ornate frog, Microhyla ornata.