hErPETOZOa 27 (3/4): 123 - 135 123 Wien, 30. Jänner 2015

Blood cell profile of the Indian Tree maculatus (G ray , 1830), during larval development until metamorphosis (anura: )

Zum Blutbild von (G ray , 1830 ) während der Larvalentwicklung bis zur Metamorphose (anura: rhacophoridae)

MadhusMITa das & P ravaTI KuMarI MahaPaTra

KurZFassuNG die vorliegende arbeit beschreibt veränderungen in der Erythrozytenmorphologie und in der häufigkeit verschiedener Leukozytentypen bei dem rhacophoriden Polypedates maculatus (G ray , 1830 ) im verlauf der Lar- valentwicklung vom stadium der hinterbeinknospe bis zum abschluß der Metamorphose. die Erythrozyten- formen umfaßten sowohl normalgestaltig ovale/elliptische und runde aber auch unregelmäßige Zellen in der Form von Tränentropfen, Kommas oder mit welligem bis gezacktem rand. Erythrozytenaggregate waren in allen unter - suchten Larvalstadien zu beobachten. von den fünf klar unterscheidbaren Leukozytentypen waren die neutrophilen Granulozyten und Lymphozyten während der frühen Larvenstadien relativ zahlreich, während das für Monozyten und eosinophile Granulozyten in den fortgeschrittenen Entwicklungsstadien zutraf. der anteil basophiler Granulo- zyten nahm während der Larvalentwicklung stetig zu. Blutplättchen traten in Klumpen auf, was die autoren eben - sowenig als artefakt ansehen wie unregelmäßig geformte Erythrozyten und Erythrozytenaggregate. die untersuchung beschreibt erstmals das Blutbild der Larven von P. maculatus . aBsTracT changes in erythrocyte morphology and leukocyte profile were studied in the Indian Tree Frog Polypedates maculatus (G ray , 1830), during larval development, from hind limb bud appearance to completion of metamor - phosis. The shape of erythrocytes varied from normal oval/elliptical and round to irregular forms such as teardrop- shaped, comma-shaped, and crenulated cells which we do not think are artifacts. aggregation of erythrocytes was observed throughout the developmental period analyzed. Out of five types of leukocytes that were clearly identi - fied, and lymphocytes were comparatively numerous during the early larval stages, where - as and granulocytes were more numerous in advanced stages of larval development. Percentage of granulocytes increased gradually during the larval development. Blood were found in clusters which is not classified as artifact, just as erythrocyte aggregation and irregular forms. The pres - ent study is the first to provide information on the blood cell profile of tadpoles of P. maculatus . KEy WOrds amphibia: anura: rhacophoridae; Polypedates maculatus , leukocytes, erythrocytes, hematology, blood cell profile,

INTrOducTION

Metamorphosis involves structural (rOsENKILdE et al. 1994) and the numbers reorganization and major physiological of leukocytes change ( davIs 2009). The changes under the control of endo- and leukocyte components of the exogenous factors and often results in immune system were utilized in assessing changes in habitat use ( saNTELIcs & a L - the general health of individuals in stressful varadO 2006). In the case of anurans, the or polluted environment ( caBaGNa et al. aquatic tadpoles metamorphose to terrestri - 2005; r uThErFOrd et al. 2005) and erythro - al froglets. as a part of the metamorphic cyte morphology was observed to be affect - changes, blood cell populations are renewed ed by polluted environment ( BarNI et al. 124 M. d as & P. K. M ahaPaTra

2007). as anurans are considered to be & a LsPauGh 1964). variation in erythro - environmentally sensitive , changes cyte shape and size was studied in response in blood cell profile could indicate the avail - to the chemical stressors in the aquatic envi - ability of contaminants in the environment ronment in P. snk. esculentus (BarNI et al. (caBaGNa et al. 2005; raFFEL et al. 2005). 2007). Information on the hematology of Even though the anuran fauna in India anuran larvae is largely restricted to temper - is particularly diverse in that it includes ate zone species. development and ultra - about 300 species ( aNIL et al. 2011; BIJu et structure of eosinophil, basophil and het - al. 2011), the blood cell profile of tadpoles erophil (neutrophil) granulocytes in tad - has been reported only for dubois’s Tree poles of Pelophylax snk. esculentus (L IN- Frog, (d uBOIs , 1987) NaEus , 1758) were reported (F raNK 1989a, by das & M ahaPaTra (2012). Besides, 1989b). relative frequency of different hematological information on few adult types of leukocytes in wild-caught tadpoles Indian anuran species is available, namely of Lithobates catesbeianus (s haW , 1802) Duttaphrynus melanostictus (s chNEIdEr , were described by davIs (2009). during 1799) (BaNErJEE et al. 1980), Hoplobatra - thyroid-induced metamorphosis of L. cates - chus tigerinus (d audIN , 1802) (MIshra & beianus , changes in leukocytes were ob- BaNErJEE 1983) and Polypedates maculatus served in the tadpoles ( JOrdaN & s PEIdEL (G ray , 1830) (MahaPaTra et al. 2012). To 1923, 1924). Moreover, stress related add to the knowledge of anuran larval blood, changes in the number of leukocytes were the present study characterizes ontogenetic found in the tadpoles of P. snk. esculentus changes in the leukocyte profile and erythro - (BarNI et al. 2007), L. catesbeianus cyte morphology of laboratory-reared tad - (BENNETT & h arBOTTLE 1968) and Litho - poles of the rhacophorid Indian Tree Frog bates pipiens (s chrEBEr , 1782) (BENNETT Polypedates maculatus (Gray , 1934).

MaTErIaLs aNd METhOds

Fresh foam nests containing eggs of aLTIG 1999). Ten tadpoles each per devel - Polypedates maculatus were collected from opmental stage were selected for investiga - a natural pond inside utkal university cam - tion. The procedure followed was described pus in august 2012 at Bhubaneswar (28° in detail by das & M ahaPaTra (2012) and 18’N, 85°50’E), Odisha, India. The hatch - included anesthesia (Ms-222, Tricaine lings were reared in the laboratory follow - Methanesulfonate, 0.0003 g/L, see Maha- ing the procedure as described by MOhaNTy - PaTra & M OhaNTy -h EJMadI 1994); blood hEJMadI (1977). as the holding water was collection from mid-tail amputation (stages conditioned tap water, its quality was not 26 to 44) and heart puncture (stages 45 and analyzed for potential contamination. The 46); blood smears (push slide technique); air characteristics of the rearing conditions drying; staining (Giemsa); examination were as follows: mean temperature: 28.4 ºc, under light microscope (hund h500 Wetz- mean humidity: 89.2 %, stocking density: lar, Germany); photodocumentation (canon one tadpole reared in 100 ml of treated sup - EOs 450, attached to the micro scope by EF- plied tap water; water renewal rate: at 24 s 18-55 1s mounting kit); microscopic hours interval; lighting regime: 12 hours blood cell differentiation ( hadJI -a ZIMI et al. day and 12 hours night. The tadpoles were 1987; TurNEr 1988; hEaTLEy & J OhNsON fed with boiled Amaranthus leaves ad libi - 2009) in smear regions where cells were tum . Tadpoles from Gosner stages 26 to 46 closely associated but did not overlap (GOsNEr 1960) were selected for the present (dacIE & L EWIs 1984); leukocytes counts investigation. These stages are comparable (in each smear the leukocytes of 100 grid to the Taylor and Kollros stages I to XXv cells were counted); erythrocytes measure - (TayLOr & K OLLrOs 1946; McdIarMId & ments (length, width, aspect ratio and area Blood cells profile during amphibian larval development 125 of cells and nuclei; ocular micro meter stan - cytes and monocytes] examined separately. dardized against stage micrometer, 0.01 The relationship between developmental mm, Erma, Japan; formulae for size calcula - stages and blood cell profile shown in scat - tions following GrENaT et al. 2009). ter plots was subject to regression analysis. statistical analysis.– Twenty- Pearson’s coefficient of correlation was one developmental stages (Gosner 26-46) used to characterize the relationships with ten specimens per stage entered the between tadpole developmental stages and analysis. One-way analysis of variance erythrocyte parameters. Polynomial regres - was used to compare parameters across sion of the second order was used to analyze stages, with each parameter [ length, width, the relationship between tadpole develop - aspect ratio and area of erythrocyte cells and mental stage and leukocyte profile. nuclei; frequency (%) of lymphocytes, neu - analyses were performed using sPss v. 11 trophil, basophil and eosinophil granulo - software.

rEsuLTs

Blood cell morphology periphery (Figs. 2a, 2b). Mono cytes were round with their nuclei either centrally The tadpoles of Polypedates macula - placed and indented (Fig. 2c) or eccentrical - tus showed some variety in erythrocyte ly placed and round (Fig. 2d). some mono - (, rBc) morphology. cytes showed eccentrically placed indented round rBcs with centrally placed nuclei nuclei (Fig. 2e) as well. Few monocytes (Fig. 1a), oval rBcs with centrally and with irregular edges were observed (Fig. 2f). eccentrically placed nuclei (Fig. 1b) and In neutrophil granulocytes, the nuclei were elliptical rBcs with centrally placed nuclei either trilobate (Fig. 2g) or tetralobate (Fig. (Fig. 1c) were observed. Erythrocytes with - 2h). Eosinophil granulocytes had bilobate out nuclei (senile erythrocytes) (Fig.1d) nuclei, located at one end of the cells (Fig. were observed in tadpoles of stages 37 to 2i). Basophil granulocytes had large dark 41. Irregular forms such as teardrop (Fig. violet-stained granules scattered over the 1e) and comma-shaped cells (Fig. 1f) were entire cell, including the irregular nuclei observed in tadpoles of stages 36 to 40. (Fig. 2j). Blood platelets were found in clus - From stages 26 to 33, erythrocytes exhibit - ters (Fig. 2k). ed poikilocytosis, i.e., abnormally shaped Length, width and areas occupied of rBcs (Fig. 1g). dividing rBcs (Fig. 1h) the rBcs and their nuclei are shown in were observed in tadpoles of stages 37 to Table 1 for larval developmental stages 26 44. In climax metamorphic stages (stage 41 through 46. Frequency of the of leukocytes to 45) crenulated erythrocytes (Figs. 1i and types at larval developmental stages 26 1j) occurred. Larger erythrocytes (Fig. 1k) through 46 are shown in Table 2. were found in tadpoles of stages 36 to 42. cells undergoing division were seen in statistical analysis tad poles of stages 39 to 44 (Fig. 1l). aggregation of erythrocytes leading to oval statistical comparisons of the various (Fig. 1m) or elongate structures (Fig. 1n) study parameters across 21 developmental was observed in almost all developmental stages showed significant variation in all stages investigated. parameters (Table 3, Figs. 3 and 4). Length, The leukocytes identified included width and area of the erythrocytes were neg - lymphocytes (large and small), monocytes, atively correlated with developmental stage , and . The (Figs. 3a, 3b, 3d). aspect ratio, i.e., lymphocytes were round with the nuclei length/width ratio, of the cell was positively occupying the entire cell leaving a narrow correlated with developmental stage (Fig. rim of light violet cytoplasm towards the 3c). In case of the nucleus, all the parame - 126 M. d as & P. K. M ahaPaTra

Fig. 1: Erythrocytes of the tadpoles of Polypedates maculatus (G ray , 1830). a - round cell with centrally placed nucleus, b - oval cells with centrally and eccentrically placed nuclei, c - elliptical cell with centrally placed nucleus, d - senile erythrocytes (without nucleus), e - teardrop-shaped cell, f - comma-shaped cell, g - poikilocytosis, h - dividing erythrocyte, i and j - crenulated erythrocytes, k - large erythrocytes, l - erythrocyte undergoing cell division, m and n - aggregation of erythrocytes. Length of scale bar represents 10 µm. abb. 1: Erythrozyten der Larven von Polypedates maculatus (G ray , 1830). a - runde Zelle mit zentralem Kern, b - ovale Zellen mit zentral und exzentrisch gelegenen Kernen, c - elliptische Zelle mit zentralem Kern, d - senile (kernlose) Erythrozyten, e - tränenförmige Zelle, f - kommaförmige Zelle, g - Poikilozytose, h - Erythrozyt in Teilung, i und j -Erythrozyten mit gezackten oder welligen Zellgrenzen, k - große Erythrozyten, l - Erythrozyt in Zellteilung, m und n - Erythrozytenaggregate. die Balkenlänge entspricht 10 µm. Blood cells profile during amphibian larval development 127

Fig. 2: Leucocytes and platelets of the tadpoles of Polypedates maculatus (G ray , 1830). a - large lymphocyte, b - small lymphocyte, c - with centrally placed indented nucleus, d - monocytes with eccentrically placed round nuclei, e - monocyte with eccentrically placed indented nucleus, f - monocytes with irregular cell edges, g - neutrophil with trilobate nucleus, h - neutrophil granulocyte with tetra lobate nucleus, i - eosinophil granulocyte, j - basophil granulocyte, k - platelets in cluster. Length of scale bar represents 10 µm. abb. 2: Leukozyten und Blutplättchen der Larven von Polypedates maculatus (G ray , 1830). a - großer Lymphozyt, b - kleiner Lymphozyt, c - Monozyt mit zentral gelegenem, gekerbten Kern, d - Monozyten mit exzentrischen, runden Kernen, e - Monozyt mit exzentrischem, gekerbten Kern, f - Monozyten mit unregelmäßigen Zellgrenzen, g - Neutrophiler Granulozyt mit dreilappigem Kern, h - neutrophiler Granulozyt mit vierlappigem Kern, i - eosinophiler Granulozyt, j - basophiler Granulozyt, k - verklumpte Blutplättchen. die Balkenlänge entspricht 10 µm. 128 M. d as & P. K. M ahaPaTra . c i n t e e i d

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Table 2: Percentage of leukocytes in the blood of Polypedates maculatus (Gray , 1934), at 21 defined (GOsNEr 1960) larval developmental stages. arithmetic mean values ± one standard deviation (sd) are given. The number of leukocytes measured was 1000 cells per developmental stage. Tab. 2: Prozentsatz der Leukozyten im Blut von Polypedates maculatus (Gray , 1934) bei 21 definierten (GOsNEr 1960) larvalen Entwicklungsstadien. angegeben sind der arithmetische Mittelwert ± eine standard- abweichung (sd). die anzahl vermessener Leukozyten betrug 1000 je Entwicklungsstadium.

developmental stage Lymphocytes Neutrophil Monocytes Eosinophil Basophil GOsNEr TayLOr & granulocytes granulocytes granulocytes (1960) KOLLrOs (1946) Mean ± sd Mean ± sd Mean ± sd Mean ± sd Mean ± sd 26 I 69.9 ±0.96 22.1±0.68 1±0 5.8±0.01 1.2±0.10 27 II 65.3±0.89 22.6±0.68 2.1±0.12 6.4±0.11 3.6±0.10 28 III 63.2±0.99 23.9±0.85 2.1±0.11 7.3±0.12 3.5±0.12 29 Iv 62.9±0.56 24.2±0.67 2.1±0.13 8±0.25 2.8±0.01 30 v 63.5±1.2 21±0.56 2.7±0.11 7.6±0.55 5.2±0.15 31 vI 61.3±1.56 19.2±1.23 4.2±0.23 9.2±0.55 6.1±0.25 32 vII 62.1±0.97 18.8±0.59 4.1±0.23 8.1±0.75 6.9±0.25 33 vIII 63.1±1.3 18±0.55 4.8±0.25 8±1.2 6.1±0.35 34 IX 63.9±1.25 15.2±0.68 7.5±0.31 6.1±1.1 7.3±0.55 35 X 66.9±1.56 15.9±0.96 6.8±0.35 5.2±0.25 5.2±0.55 36 XI 65.1±0.59 16.9±1.23 6.1±0.35 5.1±0.11 6.8±0.85 37 XII 62.3±1.54 15±0.85 6.8±0.36 8.6±0.56 7.3±0.65 38 XIII 58.3±2.3 15.9±0.55 8.9±0.45 9.9±0.85 7±0.75 39 XIv 58.8±2.1 15.9±0.85 9.1±0.11 10.6±1.2 5.6±0.11 40 Xv-XvII 56.9±1.53 17.1±0.74 9±1.2 8.9±1.1 8.1±0.25 41 XvIII-XIX 57.6±1.25 18.3±0.75 6±0.36 10±1.2 8.1±0.55 42 XX 56.9±1.69 19.2±0.29 5.5±0.11 10.3±1.11 8.1±0.86 43 XXI 54.1±1.65 16.1±0.12 6.8±0.12 13.9±0.13 9.1±0.12 44 XXII 56.6±1.23 19.2±0.11 5.8±0.11 11.2±0.25 7.2±0.12 45 XXIII-XXIv 54.2±1.55 18.5±0.55 8.1±0.35 11.1±0.55 8.1±0.15 46 XXv 53.2±1.23 19.6±0.55 8.3±0.55 10.8±0.75 8.1±0.25

Table 3: statistics characterizing the numerical relationship between erythrocyte morphological parameters as well as leukocyte type frequencies versus developmental stage in tadpoles of Polypedates maculatus (G ray , 1830) . The data of 210 tadpoles of 21 developmental stages ( GOsNEr 1960) was analyzed. F(20,189) - F-test value; (p) - probability value of the F-test; ( r) - Pearson’s coefficient of correlation, p-value - significance of the proba - bility value of Pearson’s correlation coefficient, aNOva ( R2) - coefficient of determination, aNOva (F) - F-test. Tab. 3: statistiken zur Kennzeichnung der numerischen Beziehung zwischen morphologischen Erythro- zytenmerkmalen sowie der häufigkeit bestimmter Leukozytentypen einerseits und dem Entwicklungsstadium andererseits bei den Larven von Polypedates maculatus (G ray , 1830) . die daten von 210 Kaulquappen aus 21 Entwicklungsstadien ( GOsNEr 1960) wurden untersucht. F(20,189) - Wert des F-Tests; ( p) - Wahrscheinlichkeits- niveau für den Wert des F-Tests; ( r) - Pearson’s Korrelationskoeffizient, p-value - signifikanz des Wahrscheinlichkeitswerts für den Wert von Pearson’s Korrelationskoeffizienten, aNOva ( R2) - Bestimmtheitsmaß, aNOva (F) - F-Test.

Blood cell parameter measured F (20, 189) (p)(r) p-value aNOva aNOva (R2) (F) Erythrocyte cell length [µm] 20.99 ≤ 0.0001 -0.101 0.663 Erythrocyte cell width [µm] 50.67 ≤ 0.0001 -0.533 0.012 Erythrocyte cell area [µm²] 47.98 ≤ 0.0001 -0.409 0.065 Erythrocyte cell aspect ratio (length/width) 50.67 ≤ 0.0001 0.652 0.001 Erythrocyte nucleus length [µm] 10.20 ≤ 0.0001 -0.686 0.0005 Erythrocyte nucleus width [µm] 9.41 ≤ 0.0001 -0.680 0.0006 Erythrocyte nucleus area [µm²] 5.957 ≤ 0.0001 -0.042 0.856 Erythrocyte nucleus aspect ratio (length/width) 10.53 ≤ 0.0001 -0.718 0.0002 Lymphocytes frequency (%) 12.93 ≤ 0.0001 0.7815 67.95 Neutrophil granulocytes frequency (%) 5.17 ≤ 0.0001 0.7452 55.55 Basophil granulocytes frequency (%) 8.70 ≤ 0.0001 0.8097 80.84 Eosinophil granulocytes frequency (%) 18.60 ≤ 0.0001 0.5778 26.00 Monocytes frequency (%) 22.12 ≤ 0.0001 0.7881 70.66 130 M. d as & P. K. M ahaPaTra

Fig. 3: correlation between different morphometric values of erythrocytes and numeric developmental stage of tadpoles of Polypedates maculatus (G ray , 1830). abb. 3: Korrelation zwischen den untersuchten morphometrischen Erythrozytenmerkmalen und dem numerischen Entwicklungsstadium bei Larven von Polypedates maculatus (G ray , 1830). Blood cells profile during amphibian larval development 131

Fig. 4: Polynomial regression of the second order describing the relationship between percentage of different types of leucocytes and numerical developmental stage of tadpo les of Polypedates maculatus (G ray , 1830) . abb. 4: Polynomiale regression zweiter Ordnung zur Beschreibung der Beziehung zwischen dem Prozentsatz verschiedener Leukozytentypen und dem numerischen Entwicklungsstadium bei Larven von Polypedates maculatus (G ray , 1830) .

ters showed a negative correlation that granulocytes in creased throughout devel - increased with the tadpole development opmental period (Fig. 4c). The number of (Figs. 3d, 3e, 3f, 3g). eosinophil granulocytes increased gradual - The percentage of lymphocytes var - ly, attaining maximum values at climax ied throughout the developmental period stages of metamorphosis (Fig. 4d). Mono - (Fig. 4a). Neutrophil granulocytes per - cyte frequency increased gradually, until it centage varied throughout the develop - settled at a high level in tadpoles of stages mental period and was highest during early 34 to 46. stages (Fig. 4b). Frequency of basophil 132 M. d as & P. K. M ahaPaTra

dIscussION

Blood cell morphology Thus, the present findings suggest the stud - ied tadpoles to have passed through critical The erythrocytes were morphological - conditions of metabolic stress where ery - ly heterogeneous as they appeared in vari - throcytes underwent variation in shape. ous shapes such as round, oval or elliptical. another feature of the blood smears was the similarly, the position of the nuclei within aggregation of the erythrocytes (Figs. 1m, these cells also varied. The nuclei were 1n) that occurred throughout the larval peri - either placed eccentrically (Fig. 1b) or cen - od. trally (Figs. 1a, 1c) within the cells. about senile erythrocytes (without nuclei) 20 % of the erythrocytes showed deviations (Fig. 1d) were observed in the tadpoles of from the normal shape. such increased stages 37 to 41. Presence of senile erythro - variation in shape of the erythrocytes (poik - cytes during metamorphosis was reported in ilocytosis) was evident at the early larval tadpoles of Pseudacris crucifer (W IEd - developmental stages 26 to 33 (Fig. 1g) but NEuWIEd , 1838), Hyla versicolor LEcONTE , was no longer observed at later stages, sug - 1825 , Lithobates clamitans (L aTrEILLE , gesting poikilocytosis to be a normal phe - 1801), Anaxyrus americanus (h OLBrOOK , nomenon during the early larval period. 1836) (sPEIdEL 1926) and Polypedates some specified forms of erythrocytes such teraiensis (das & M ahaPaTra 2012). Ery- as teardrop-shaped cells (Fig. 1e) and throcytes undergoing division observed in comma-shaped cells (Fig. 1f) were observed the tadpoles from stages 39 to 44 were cor - at stages 36 to 40. crenulated cells (Figs. related with increased erythropoietic activi - 1i, 1j) characterized by protrusion of spiny ty during metamorphosis as reported earlier processes from the external surface were by MaNIaTIs & I NGraM (1971) in the tad - seen in the tadpoles of stages 41 to 45. poles of L. catesbeianus . The comparative - These cells resembled the echinocytes and ly large erythrocytes in stages 36 to 42 tad - acanthocytes seen in mammals ( FOGLIa poles of the present study resembled imma - 2010). similar crenulated cells were report - ture erythrocytes described from tadpoles of ed in Lithobates pipiens (hOLLyFIELd 1966) L. catesbeianus (MaNIaTIs & I NGraM and Polypedates teraiensis (das & 1971). similar large erythrocytes were also MahaPaTra 2012). In red blood cells, observed in tadpoles of stages 42 to 45 of P. metabolic stress produces crenulated cells; teraiensis (das & M ahaPaTra 2012). this type of transformation of shape is size (length and width) and area of reversible. small crenulated erythrocytes erythrocytes and their nuclei were negative - appear during metamorphosis, increase in ly correlated with developmental stage, i.e., number as the larval development proceeds decreased with the progress in larval devel - and gradually lose their wrinkled appear - opment (Figs. 3a, 3b, 3e, 3f) whereas the ance ( dEGruchy 2008). In thyroxin-treat - aspect ratio i.e., L/W increased. several ed tadpoles of Lithobates catesbeianus , works reported that during anuran metamor - vaNKIN et al. (1970) observed the rBc out - phosis larger larval cells were replaced by lines to be more irregular and crenulated smaller adult erythrocytes ( BrOyLEs 1981; with many cytoplasmic projections and cor - duELLMaN & T ruEB 1986). In the present related this phenomenon with anemic condi - study, the area of the erythrocytes decreased tions and death of the tadpoles during meta - along with the development of the tadpoles morphosis. In vertebrates, abnormally (Figs. 3d, h). a similar decrease in the area shaped red blood cells (echinocytes, acan - of the erythrocyte was reported in tadpoles thocytes, schistocytes, teardrop cells, and of L. catesbeianus (hOLLyFIELd 1966; comma-shaped cells) were reported to be BENBassaT 1970; h asEBE et al. 1999) and P. present during anemic conditions ( cOLLEGE teraiensis (das & M ahaPaTra 2012). OF aMErIcaN PaThOLOGIsTs 2012) that decrease in area of erythrocytes during ectothermic animals are capable to with - metamorphosis seems to be associated with stand for a long period without increased the transition from an aquatic to a terrestrial mortality ( FEdEr & B urGGrEN 1992). mode of life. Blood cells profile during amphibian larval development 133

Leukocyte profile the increase of cellular debris ( davIs 2009). Increase in the number of monocytes during The nonspecific immune system of an later stages of metamorphosis can be corre - organism is represented by the leukocyte lated with increase in cellular debris and population in which each cell type performs need of activity. a distinct function in the immune process The percentage of eosinophil granulo - (JaIN 1986, 1993). Out of five types of leuko- cytes gradually increased with the develop - cytes observed in the present study, lympho - ment of tadpoles advancing (Fig. 4d). cytes were the most abundant cells. With the Eosinophils are important in the immune onset of metamorphosis, the percentage of system because of their antiallergic role, lymphocytes decreased (Fig. 4a), which sug - phagocytic function and antiparasitic gests their particular role in the early devel - actions ( dEGruchy 2008). also they are opmental stages. In tadpoles of L. cates - involved in the production of a number of beianus and P. teraiensis , the number of chemical substances that initiate and modu - lymphocytes also declined with the onset of late the immune and inflammatory response metamorphosis ( davIs 2009; d as & (adaMKO et al. 2005; rOThENBErG & MahaPaTra 2012). Neutrophil granulocytes hOGaN 2006; davIs 2009). Thus, eosino - were the second-most abundant type of cells phil granulocytes are suggested to modulate after lymphocytes in the present study (Table the process of tissue lysis during metamor - 1). The percentage of neutrophils was high - phosis. similarly, the proportion of baso - est in the early larval period (stages 26 to 34) phil granulocytes increased in parallel with and gradually decreased towards the climax the development of the tadpoles (Fig. 4c). metamorphic stages. Neutrophil granulo - such an increase was also reported in tad - cytes provide the “first line of defense” poles of P. teraiensis (das & M ahaPaTra against invading pathogens, or any inciting 2012) and L. catesbeianus (davIs 2009), in inflammatory signals ( aP PELBErG 2006). which latter case the author suggested the The highest percentage of neutrophils was trend to be related to these cells’ formation observed in the early developmental stages, and their entrance into the circulatory sys - suggesting increased requirement of non- tem rather than a direct association with specific immunity at the initial developmen - metamorphosis. tal phase, similarly seen in tadpoles of L. The present investigation provides catesbeianus (davIs 2009) and P. teraiensis broad information on the blood cell profile (das & M ahaPaTra 2012). The percentage of a continuous series of tadpole develop - of monocytes gradually increased during mental stages of Polypedates maculatus development (Fig. 4e). at later develop - which can be referred to as a reference in mental stages, there occurs more and more future investigations of blood morphology larval structure remodeling which results in in anuran larval developmental stages.

acKNOWLEdGMENTs

Md would like to thank the university Grant Graduate department of Zoology, utkal university commissions for a research Fellowship (rFsMs). under PursE grant of the year 2012. PKM thanks department of science and Technology, all procedures were approved by the Government of India for financial support to the Post care review committee at utkal university.

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daTE OF suBMIssION: July 3, 2013 corresponding editor: heinz Grillitsch

auThOrs: Madhusmita das – cell and developmental Biology Laboratory, P. G. department of Zoology, utkal university, Bhubaneswar, Odisha-751 004, India; Pravati Kumari MahaPaTra (corresponding author < [email protected] >) – cell and developmental Biology Laboratory, P. G. department of Zoology, utkal university, Bhubaneswar, Odisha-751 004, India.