) ) DEVELOPMENT OF HYLA ANNECTANSJERDON, 1870 FROMNAGALAND, INDIA 1 J. Meren Ao2 and SabitryBordoloi3 (Withfourplates Key words: Hyla annectans, ontogenic stages, normal development, Nagaland, laboratory condition, metamorphosis Hyla annectans breeds during May to July in temporary rain pools, terraced fields and other waterlogged areas (depth 5-7 cm). Normal development has been studied from the egg through metamorphosis for four breeding seasons (1996-2000) under laboratory conditions. Thetimerequired forcompletion ofalifecyclevariesunderdifferentweather conditions in thefield. In the laboratory (16 22°C)the frog completed its lifecycle in 64 days, 14 hours. Introduction middle ofApril and laststill July end. The frogs breed in water logged places like temporary Studiesonthesuccessiveontogenicstages, ponds, rainpools, puddles and terraced paddy to record the normal developmental table, are fields. Embryonicdevelopmentwasobservedfor important in understanding the ecology of a a period offour breeding seasons (1996-2000). speciesandforplanningconservation measures. Laboratory rearing was carried out in the Various authors (Dutta and Mohanty- DepartmentofZoology,KohimaScienceCollege, Hejmadi 1976, Mohanty-Hejmadi and Dutta Kohima, Nagaland. 1977, Agarwal and Niazi 1977, Roy and Khare 1978, Mohanty-Hejmadi et al. 1979, 1980, Methodology KiyasetuoandKhare 1986,Duttaetal. (1990-91 have contributed to the study of normal Amplexing pairs were collected from the developmental tables ofAnuran amphibians in field and transferred to aquaria or glass India. containers with water, allowing only halfofthe InNagaland (25° .15'-27°4’ N, 93° 20'-95° body to be submerged. Amplexing lasts for 3-5 15' E), Hyla annectans is distributed along the hours. Eggs are laid between 0100-0400 hrs in m Borail range at various altitudes 1,400-2,440 the aquaria as well as in the field. Development above msl. Average atmospheric temperature stages were fixed in 5% formaldehyde solution; during breeding season ranges from 16.5- measurements were taken from preserved 26.6 °C. Relative humidity ranges from 74.33- specimens.PhotographsforPlates 1-3 weretaken 81.63%. Precipitation ranges from 128.23- frompreservedspecimens, whileforPlate4 live 428.33 mm. Water temperature varies from individuals were used. 14-28.5 °C. Breeding activity starts from the Observations 'AcceptedDecember,2000 departmentofZoology,KohimaScienceCollege, Breeding activity starts from the middle Kohima797001,Nagaland,India ofApril(12.3-26.6°C)withthemales’ breeding •EcologyLaboratory,DepartmentofZoology, CottonCollege,Guwahati781 001 Assam,India call. Females appear only after one or two , JOURNAL, BOMBAYNATURAL HISTORYSOCIETY, 98(2), AUG.2001 169 DEVELOPMENTOFUYLA ANNECTANS showers. In early May, amplexing pairs were Stage 5: Eight cell stage (Age 2.45 hrs; collectedandkeptinglasscontainersoraquaria. length 1.52 mm). The third cleavage is Eggs were laid in batches in the field. In the latitudinal, slightly above the equator, which aquarium, due to the absence of vegetation or forms eight blastomeres. The four smaller other substratum, thejelly capsules ofthe eggs micromeres of the animal pole are pigmented adhered together to form a single mass. The darkbrown,whereasthefourbiggermacromeres number ofeggs laidby one female ranged from ofthe vegetalpole are unpigmented. 590-650. Culture was maintained in clean Stage 6: Sixteen cellstage (Age 3.20 hrs; enamelled trays and 100 fertilized eggs were length 1.52 mm) (Plate 1, Fig. 4). The cleavage stockedineachtraytoavoidover-crowding.They furrow is vertical. First, the pigmented wererearedinthelaboratoryat 16-22°C. Larvae micromeresaredividedintoeightcells,resulting were fed with Spirogyra, which is common in in twelve cells (i.e. 8 micromeres and 4 the breeding habitats. Tadpoles were staged macromeres).Thisisfollowedbythedivisionof according to Gosner (1960). the four unpigmented macromeres as the The no. ofindividuals per tray (Stocking cleavage furrow reaches the vegetal pole, number = 100) was reduced with progressive resulting in 16 cells altogether. developmental stages. Stage7: Thirty-twocellstage(Age3.52hrs; length 1.56mm) (Plate 1,Fig. 5).Thelatitudinal Embryonic stage cleavage furrows of the micromeres and Fortysamplesweremeasuredtorecordthe macromeres bring about the formation of 16 mean size ofeach stage (1-46). micromeres and 16 macromeres. Stage8:Midbiastula(Age7-54hrs; length Fertilization stages 1.56 mm) (Plate 1, Fig. 6). The number ofcells Stage 1: Fertilizedegg (Age 0 hrs; length increasedtomorethan64cells. Theblastomeres 1.52 mm) (Plate 1, Fig. 1). The egg is spherical. aremorenumerousandsmallerthanbefore. The Theanimalpoleispigmenteddarkbrown,paling surface ofthe animalpoleresembles a clusterof to white at the vegetal pole. beads. Stage 2: One cell stage (Age 0.55 hrs; Stage 9: Late blastula (Age 12.10 hrs; length 1.52 mm). A lightly pigmented area, the length 1.56mm). The surface ofthe animalpole grey crescent appears between the animal and has a granular appearance, which gradually vegetalpoletowardsthepigmentedhemisphere. becomessmooth. Thepigmentedregionextends overthevegetalpole,whichmarksthebeginning Cleavage stages oftheepibolicmovementofthemicromeresonto Stage 3: Two cell stage (Age 1.50 hrs; the macromeres. length 1.52mm)(Plate 1,Fig.2).Themeridional cleavage furrow originating at the animal pole Gastrulation stages proceeds to the vegetalpole and divides the egg Stage 10: Crescent dorsal lip (Age 15.05 completely into two equalblastomeres. hrs; length 1.56mm)(Plate 1,Fig. 7).Thedorsal Stage 4: Four cell stage (Age 2.20 hrs; lip of blastopore has formed and is length 1.52 mm) (Plate 1, Fig. 3). The second crescent-shaped. The unpigmented zone of the meridional furrow, which starts at the animal vegetal hemisphere is reduced due to continued pole, extends to the vegetal pole at a right angle migrationofthepigmentedmicromerestowards to the first. the vegetal pole. 170 JOURNAL, BOMBAYNATURAL HISTORYSOCIETY, 98(2), AUG. 2001 DEVELOPMENTOFHYLA ANNECTANS Ao, J. Meren et al.:Hyla annectans Jerdon Plate 1 Fig.1 Fertilizedegg, Fig. 2Twocell stage, Fig. 3 Fourcell stage, Fig. 4 Sixteencell stage, Fig. 5 Thirty-twocell stage, Fig. 6 Midblastula. Fig. 7 Crescentdorsal lip, Fig. 8 Horse-shoe shapeddorsal lip, Fig 9. Neuralplate. JOURNAL, BOMBAY NATURAL HISTORY SOCIETY, 98(2), AUG. 2001 171 DEVELOPMENTOFHYLA ANNECTANS Fig. 10Closure ofneural fold, Fig. 11 Neuraltube. Fig. 12, 13 Tailbud stage, Fig. 14 Muscularresponse stage, Fig. 15 Opercularstage, Fig. 16 Opercularfoldofembryo closed onright side, Fig. 17 Operculum ofembryo closed on leftside 172 JOURNAL, BOMBAY NATURAL HISTORY SOCIETY, 98(2), AUG. 2001 DEVELOPMENTOFHYLA ANNECTANS Stage 11: Horse-shoe shaped dorsal lip ruptures at the anterior end. (Age 16.50 hrs; length 1.56 mm) (Plate 1, Fig. Stage 17: Tail bud stage (Age 42.50 hrs; 8). The epibolic migration ofmicromeres over Trunk 2.44 mm; Tail 0.47 mm) (Plate 2, Figs. the vegetal pole reduces the exposed area of 12, 13). Tailbud appears at the posterior end of unpigmented macromeres, which is surrounded theembryo. Itiswiderthanlong, directeddorso- by the lateral lips ofthe semicircular or horse- posteriorly and marked offfrom the body by a shoe shaped blastopore. ventral notch. Stomodeal groove depression is Stage 12: Smallyolkplug{Age 26.10 hrs; slightly marked by a darker area. Bulges ofthe length 1.56 mm). The ventral lip ofblastopore gill plates are distinct. shifts to the posterior end. The uninvaginated Stage 18: Muscular response stage (Age macromeres, surroundedbytheblastoporal lips, 60.00 hrs; Trunk 2.71 mm; Tail 0.75mm) (Plate protrude a little and constitute the yolk plug. 2, Fig. 14). Head region is well defined, with optic bulges and bulges of the gill plates. Oral Neurulation stages suckers are indicatedby two heavily pigmented Stage 13: Neural plate (Age 34.30 hrs; elongated areas joined medially by a narrow length 1.6 mm) (Plate 1, Fig. 9). The embryo is lightly pigmented band below the stomodeum. slightlyelongated.Thedorsalsurfaceisflattened Stomodeal depression is seen between the oral to form the neural plate, which is differentiated suckers.Duetogradualelongationoftheembryo, with the concentration of pigments along its the tail starts curving laterally to right or left, borders. within the contour ofthe vitelline membrane. Stage 14: Neuralfold (Age 36.20 hrs; Stage 19: Heartbeatstage(Age70.07hrs; length 1.65 mm). The neural fold becomes Trunk3.10mm;Tail 1.4mm). Pulsationofheart distinct, with broad cerebral and nairow spinal is seen below and behind gill bud. Small cordregionsoftheneuralplate. Theneuralfolds pigmented depression at anterior end marks gradually approach each other from blastopore olfactory pit. Stomodeal depression becomes to anterior region. somewhat triangular. External gill buds Stage 15: Closure ofneuralfold (Age prominent. Embryo coils to mechanical stimuli. 38.20 hrs; length 1.72 mm) (Plate 2, Fig. 10). Stage20: Gillcirculationstage(Age72.29 Theposteriorendoftheembryobecomesbroader. hrs;Trunk3.28mm;Tail 1.5mm). Gillsdistinct, The neural folds come closer and touch each rudimentarybranchingatdistalend. Oralsuckers other, both in the cerebral and spinal cord nipple-shaped. Stomodeal pit still a shallow regions, formingashallowneuralgroove,which triangular depression. Vitelline membrane is broader in the cerebral region. becomesthinandweak. Anteriorendofthehead Stage 16: Neural tube (Age 40.50 hrs; pressed against the vitelline membrane. mm length2.0 (Plate2,Fig. 11)Theneuralfolds have fused completely to form the neural tube, Post-embryonic development which is raised at the mid-dorsal ridge and Stage 21: Lama hatched(Age 102.29 hrs; demarcated by a darkly pigmented strand. The HeadandTrunk3.2mm;Tail3.2mm).Thehead head and trunk are well marked. Gill plates causes a bulge in the vitelline membrane at the appear as faint bulges. The increase in size of anteriorend. At this point the membrane breaks the embryo along with the associated vitelline and the larva emerges from the mass ofjelly. capsule, isnotaccompaniedbyasimilarincrease Tailstraight,tailfindusky.Olfactorypitdeepens, in the length of the first envelope, and thus it corneabeginstobetransparent. Stomodeumnow JOURNAL, BOMBAYNATURAL HISTORYSOCIETY, 98(2), AUG.2001 173 1 DEVELOPMENTOFHYLA ANNECTANS adeeptriangularpit, whose opening isthe larval Tail 16.0 mm) (Plate 3, Fig. 19). Distal end of mouth. limb bud slightly conical. Stage 22: Tailfin circulation stage (Age Stage 29: Length oflimb bud is equal to 1 17.29 hrs; Head and Trunk 3.4 mm; Tail 3.6 one and halftimes its diameter (Age 600 hrs; mm). Tailfincirculationstartsatbaseofanterior Head and Trunk 9.0 mm, Tail 18.0 mm). Distal part of dorsal fin, just above the trunk. Mouth end conical. slightly wider. Upper and lower labial fringes Stage 30: Length oflimb bud is equal to develop, but without papillation. twiceitsdiameter(Age684hrs;HeadandTrunk Stage 23: Opercular fold stage (Age 11.0 mm, Tail 19.1 mm). Distal halfofconical mm 143.59 hrs; Head and Trunk 3.6 Tail 4.5 limbbudslightlybentventrally.Nopigmentation mm) (Plate 2, Fig. 15). Operculumcoversbases on limb bud. ofexternalgills.Jawsnotkeratinised.Upperand Stage 31: Footpaddlestage(Age 748 hrs; lower labial fringes develop papillae and faint Head andTrunk 11.5 mm; Tail 20.1 mm) (Plate labialridges. Pigmentationontailbegins, cloaca 3,Fig. 20).Thedistalendoflimbbudisflattened not opened. mediolaterallyto forma footpaddle. Kneebend State 24: Opercularfoldofembryo closed is prominent. Light pigmentation starts at outer on rightside (Age 172.29 hrs; Head and Trunk base oflimb bud. 3.7 mm; Tail 5.0 mm) (Plate 2, Fig. 16). Stage 32: First indentation (Age 813 hrs; Operculumclosed on the right side. Labialjaws Head and Trunk 12.0 mm; Tail 23.0 mm). The formsupra- and infra-rostrodont. A faintrow of margin of the foot paddle becomes slightly upper supra-angularkeratodont develops. Other indented on the dorsal side, which marks the characters as in Stage 23. prominences ofthe future 4th and 5th toes. Stage 25: Operculum ofembryo closedon Stage 33: Second indentation (Age 853 left side (Age 185.29 hrs; Head and Trunk 4.2 hrs; Head and Trunk 12.8 mm; Tail 24.5 mm) mm; Tail 6.0 mm) (Plate 2, Fig. 17). Operculum (Plate 3, Fig. 21). The margin ofthe footpaddle closed and gills disappear; oral suckers becomes indentedontheventralside,behindthe diminishing. Spiracle formed. Tail lightly prominence of4thtoe, whichmarksthe 3rd, 4th pigmented.Analtubeopens,tadpolestartsfeeding. and 5th toes. Stage 26: Hind limb bud stage (Age 356 Stage34: Thirdindentation(Age890hrs.; hrs; Head and Trunk 7.0 mm; Tail 10.0 mm) Head andTrunk 13.2 mm; Tail 26.0mm) (Plate (Plate 3, Fig. 18). Appearance ofhind limb bud 3, Fig. 22). The margin offoot paddle becomes at a groove between the belly wall and base of indented, on the ventral side, behind the tail oneitherside ofcloacaltailpiece. Lengthof prominence of 3rd toe, which marks the limb bud less than half its diameter. Dental prominence of2nd, 3rd, 4th and 5th toes. formulabecomes 1:1 + 1/3.Pigmentationspreads Stage35:Fourth indentation(Age936hrs; to dorsal and anal fins. HeadandTrunk 13.4mm; Tail 26.4mm.)(Plate Stage 27: Length oflimb budequalto half 3, Fig. 23). The margin of the foot paddle is its diameter (Age 420 hrs; Head and Trunk 7.8 indented behind the 2nd toe demarcating the mm; Tail length 13.0 mm). Length oflimb bud prominence ofthe 1st toe. All the five toes are equal to half its diameter. The patches of separated from each other. pigmentation in the tail fin spread considerably. Stage 36: Margin of5th toe web directed Stage 28: Length oflimb bud equal to its towards the tip of2nd toe (Age 1019 hrs; Head diameter(Age514hrs;HeadandTrunk8.4mm; and Trunk 13.6 mm; Tail 27.2 mm) (Plate 3, 174 JOURNAL BOMBAYNATURAL HISTORYSOCIETY, 98(2), AUG. 200 DEVELOPMENTOFHYLA ANNECTANS Ao, J. Meren et al.’.Hyla annectans Jerdon Plate 3 Fig. 18 Hindlimbbud stage, Fig. 19Lengthoflimbbudequal toits diameter, Fig. 20Footpaddle stage, Fig. 21 Secondindentation, Fig. 22 Thirdindentation, Fig. 23 Fourth indentation, Fig. 24Margin of5th toe webdirectedtowards thetipof2ndtoe, Fig. 25 Marginof5th toewebdirectedtowards prehallux. JOURNAL, BOMBAY NATURAL HISTORY SOCIETY, 98(2), AUG. 2001 175 DEVELOPMENTOFHYLA ANNECTANS Ao, J. Meren et al.:Hyla annectans Jerdon Plate 4 Fig. 26, 27 Cloacaltailpiecereduced, Fig. 28 Both forelimbs emerged, Fig. 29 Angleofmouthreachedthe middle ofeye, Fig. 30 Angle ofmouthreachedposteriormargin oftheeye, Fig. 31 Metamorphosedfroglet. 176 JOURNAL, BOMBAY NATURAL HISTORY SOCIETY, 98(2), AUG. 2001 DEVELOPMENTOFHYLA ANNECTANS Fig.24).Themarginofthe5thtoewebisdirected labial fringe begins, however angular papillae towards the tip ofthe 2nd toe. stillremainas a small tuftonbothcomers ofthe Stage 37: Margin of5th toe web directed mouth, which starts widening. The homy beak towards the tip of1st toe (Age 1084 hrs; Head is shed. Tail starts darkening. The angle ofthe andTrunk 13.7mm; Tail28.0mm). Themargin mouth is level with the nostril. The cloacal tail ofthe 5th toe web is directed towards the tip of piecedisappearsatthisstage, leavingthecloacal the 1st toe. Pigmentation appears in the 4th and aperturefreebelow. 5th toes along the foot. Toes are longer. A Stage 43: Angle ofmouth between the eye rudiment of prehallux is indicated by a light and nostril (Age 1434 hrs; Head and Trunk 14 protuberancebehind the 1sttoe. Ventral surface mm; Tail 16.0 mm). The widening angle of ofthe foot is closer to the ventral fin. mouth has reached a point midway between Stage 38: Margin of5th toe web directed nostril and the anterior margin ofeye. The tail towards prehallux (Age 1136 hrs; Head and becomes still darker, the dorsal and ventral fins Trunk 14.0 mm; Tail 29.00 mm). (Plate 3, Fig. shrink and the length ofthe tail is reduced, but 25). The margin ofthe 5th toe web is directed still longer than the extended hind limb. towards prehallux. Inner metatarsal turbercle Stage 44: Angle ofmouth reached the appear as a small outgrowth. Pigmentation middle ofeye Age 1462 hrs; Head and Trunk ( appears in 3rd, 4th and 5th toe along the foot. 14.00 mm; Tail 5.0 mm (Plate 4, Fig. 29). The Stage 39: Appearance ofsubarticular widening angle of the mouth has reached the tubercles in the toes (Age 1210 hrs; Head and levelofthemiddleoftheeye. Dorsalandventral Trunk 14.3 mm; Tail 29.2 mm). Subarticular finshavedisappeared.Tailresorbedconsiderably tubercles appear onthe inner surface ofthe toes and is as long as the femur. as light patches. The inner metatarsal tubercle Stage 45: Angle of the mouth reached becomes a small oval outgrowth. posteriormargin oftheeye(Age 1498 hrs; Head Stage 40: Toe pads complete (Age 1274 andTmnk 13.5 mm; Tail 1.0mm) (Plate4, Fig. hrs; Head and Trunk 14.4 mm; Tail 29.2 mm). 30). The widening angle of the mouth has Distal ends of the toes thickened. Subarticular reachedtheposteriormarginofthe eye. The tail tubercles are clearly elevated. The cloacal tail is resorbed to a small triangular stub. piece is notreduced. Stage 46: Metamorphosedfroglet (Age Stage 41: Cloacal tailpiece reduced (Age 1550 hrs; Head and Tmnk 13.5 mm; Tail 0.0 1392 hrs; Head and Trunk 14.8 mm; Tail 30.0 mm.) (Plate 4, Fig. 31). The triangular tail stub mm. (Plate4, Fig. 26, 27). The cloacal tail piece with dark tissue disappears completely. getsreducedandonlyanarrowstripremainsover Total time taken for completion of and in between bases of the thigh still attached metamorphosis was 64 days 14 hours. withventralfindistally.Theforelimbsarevisible throughtheskin. Greenpigmentationofthedorsal Discussion surface begins. Tail not dark. Keratodonts start shedding. Oral papillae remain intact. Hamburger (1947), Gosner (1960), Rugh Stage 42: Bothfore limbs emerge (Age (1962), Nieuwkoop and Faber (1967) were 1422 hrs; Head and Trunk 14.5 mm; Tail 25.1 referred for preparing the normal table. Gosner mm) (Plate4, Fig. 28). Bothfore limbs emerge, (1960) proposed 46 stages, with simplified usuallytherightforelimbemergesfirst,followed criteria for staging developmental events in after a few hours by the left. Resorbtion ofthe Pelobatids, Bufonids, Ranids and Hylids. In the JOURNAL, BOMBAYNATURAL HISTORYSOCIETY, 98(2), AUG.200! 177 DEVELOPMENTOFHYLA ANNECTANS presentstudy,thedevelopmentofHylaannectans embryos occur in stage 21, whenthe corneajust was divided into these 46 stages. Certain begins to be transparent. However, the cornea variations have been noted in the development becomesfullyclearonlytowardstheendofstage ofHyla annectans. 22 and beginning ofstage 23. At stage 22, the Developmental, stages have been divided circulation in the tail fin begins, but the tail fin intofifteenmajorsubheadings(1)Fertilizationtwo isnottransparentasinGosnerseriesandremains stages(2)Cleavagesevenstages(3)Gastrulathree dusky. Narrow cloacal tail piece persists, in stages (4) Neurula four stages (5) Tail bud one Gosner series - 41 which, however, disappears stage (6) Muscular response one stage (7) Heart only in stage 42 when both forelimbs have beat one stage (8) Gill circulation one stage emerged. Rostrodont and keratodonts are shed (9) Cornea becoming transparent one stage completely at stage 42. (10)Tailfincirculationonestage(11)Operculum formation three stages (12) Hind limb bud five Acknowledgement stages(13)Identationanddevelopmentoftoesten stages (14) Cloacal tail piece reduced one stage This paper is dedicated to the memory of (15) Metamorphosis climax five stages. Late Prof. MahendraKumarKhare, who initiated Characteristic features of development research in Developmental Biology in the North includethetailbud,initiallyindicatedbyastrong Eastern Hill University, Shillong, Meghalaya, upward arching ofthe dorsum. Hatching ofthe India. ReferENCES Agarwal, S.K. & I.A. Niazi (1977): Normal table of embryology.UniversityPress,Chicago. developmentalstagesoftheIndianBullfrogRana Mohanty-Hejmadi, P. & S.K. Dutta (1977): Breeding tigrina Daud. (Ranidae: Anura,Amphibia)Proc. habits and development ofRana cyanophlyctis Nat. Acad. Sci., India47(B) II: 79-92. (Schneider).J. Bombaynat. Hist. Soc. 76(2):291- Dutta,S.K.&P.Mohanty-Hejmadi(1976):Breedingand 296. lifehistoryofthe Indian Bull frog,Ranatigerina Mohanty-Hejmadi,P.,S.K.Dutta&S.C.—Maluck(1979): (Daudin). Prakruti-Utkal Univ. J. Sci. 13 (1&2): Life history of Indian Frogs II the marbled 51-59. balloon frog Uperodon systoma (Schneider). Dutta,S.K.,S.Jena&P. Mohanty-Hejmadi(1990-91): J. Zool. Soc. 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