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Blood Cell Profile of the Indian Tree Frog Polypedates Maculatus (G Ray , 1830), During Larval Development Until Metamorphosis (Anura: Rhacophoridae)

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Blood Cell Profile of the Indian Tree Frog Polypedates Maculatus (G Ray , 1830), During Larval Development Until Metamorphosis (Anura: Rhacophoridae) hErPETOZOa 27 (3/4): 123 - 135 123 Wien, 30. Jänner 2015 Blood cell profile of the Indian Tree Frog Polypedates maculatus (G ray , 1830), during larval development until metamorphosis (anura: rhacophoridae) Zum Blutbild von Polypedates maculatus (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, neutrophil granulocytes and lymphocytes were comparatively numerous during the early larval stages, where - as monocytes and eosinophil granulocytes were more numerous in advanced stages of larval development. Percentage of basophil granulocytes increased gradually during the larval development. Blood platelets 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, India 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 amphibian 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 animals, 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, Polypedates teraiensis (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 - (red Blood cell, 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.
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