DEVELOPMENTAL STUDY OF SCHIZOTHORAX PLAGIOSTOMUS FROM PRE TO POST HATCHING.

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Author: S.N.Bahuguna*, R.S.Negi, P.Bahuguna and M. K. upadhyay

INTRODUCTION

Garhwal Himalaya is very rich by means of Snowfed Rivers and tributaries. These rivers and tributaries have great diversity of the fish fauna. Schizothorax and Tor species are the two most important fish genera in respect to food and commercial production in Garhwal hillstreams. Geographically Schizothorax species inhibited in different rivers, lakes, tributaries throught Himalayan and sub Himalayan region extending to confines of China, eastern Afghanistan, Pakistan, eastern Turkistan, Nepal, Ladakh, Tibet, Bhutan, north-east India, including Kashmir, Himanchal Pradesh, Uttaranchal (Garhwal and Kumaun region) and Assam1. Snow trouts divided in two genera (i) Schizothorax (Heckel) and (ii) Schizothoracichtys. The term 'trout' refer to some member of family salmonaidae but differ from other salmonids2. The word trout is sometimes used as a synonym for brown trout (salmon trout fario, linnaeous) and rainbow trout (Salmo gairdneri, Richardson). In Garhwal trout occur in water among the snow trouts Schizothorax richardsonii (Gray) and Schizothorax plagiostomus are the important food fish of Garhwal Himalaya and these snow trout is highly economical indigenous food fish of Garhwal region3. Schizothorax is locally known as 'maseen' and having maximum recorded weight 2.3 kg and 50.00 cm in length, with 85% total catchments4. According to Raizada5 (1985) Schizothorax plagiostomus species gains weight upto 2.5 kg and 60.00 cm in length. The female fishes spawn natural as well as in artificial environment in two seasons viz. September-October and second March - April. Sexually matured S. plagiostomus (when they reach 18-24 cm length) spawn naturally in clear water on gravelly / stony ground or on fine pebbles at 10-30 cm depth. Water current of 2.8-4m/sec, pH 7.5, dissolved oxygen concentration of 10-15 mg/L and gravel size of 50-60 mm are the optimum conditions for spawning6. The present study has been under taken to know the embryonic development in pre to post flexion stages of the Schizothorax plagiostomus larvae. A few studies on pre to post flexion morphological development of marine fishes carried out by Yabe7 et al., (1966); Kaji8 et al.,(1999); etc. In recent years different aspect of this trout have been studied by researchers in India and abroad9, 10, 11, 12,13,14,15. Therefore, in the present study an attempt has been made to find out the morphological development from pre and post flexion stages in the larvae of Garhwal Himalayan snow trout Schizothorax plagiostomus (Heckel). This study would be more important because S. plagiostomus (Heckel) is an economically important and could be cultivable fish in hill area. Therefore the study is important especially to those who want to change this trout into cultivable fish.

MATERIAL AND METHODS

For for breeding experiment live Schizothorax plagiostomus (Heckel) were collected from the glacierfed river Alaknanda at and its springfed tributary, the khanda gad at near khanda Chatti by using "cast gill net". These stations were located between latitude 30010' to 30015' N and longitute 78045' to 78050'E. There elevation was 500 and 603 meters above mean sea level. Induce breeding experiments were carried out during October-November at the bank of Alaknanda River by stripping method. Mature male and female (Brooders) were stripped in the ratio of 2 male and 1 female. The eggs and milt were taken out by applying the slight pressure on the side of mature male and female (brooders) abdomen. The eggs and milt were mixed with the help of birds feathers in a cleaned enameled tray for five to ten minutes15. The excess of milt was washed off by adding and changing the water of enameled trays. Then a small quantity of water was added to the tray, to keep the eggs to get wet and hardened. After that fertilized eggs were brought to the laboratory in plastic bucket and kept in glass jar hatchery, aquarium, flour sieve on hatching tub, hatching trays and in enameled tray. Some of the fertilized eggs were kept in flour sieve and in hatching tray etc., the process of development were examined properly. After hatching to till post flexion stages at 4-8 hrs intervals and 5-10 larvae were fixed in different fixatives viz. 4% formalin, 70% alcohol, aqueous alcoholic bouins etc., for morpho-histological study. For the Morphological study Schizothorax plagiostomus (Heckel) were classified into three groups on the basis of their developmental stages viz. pre flexion, flexion and post flexion. In Pre flexion stage the larvae of S. plagiostomus has been put just after hatching to 3rd day. In Flexion stage most of the internal organs are developed and some of them become functionally active. The age of larvae is 10th days after hatching. The post flexion stage in Schizothorax plagiostomus begins after 12th days of hatching. For the morphological study of different developmental stages and technique were followed as given by Taylor16 (1967), Pearse17 (1975), Dingerkus and Uhler18 (1977) and Kaji, et.al.19 (1996) with some modifications were applied according to the local condition15. Fixation of the yolk sac larvae just started after 4 -8 hrs of the hatching in different solution i.e. 70% alcohol and 4% formalin for subsequent morphological evaluations till post flexion stages (upto 15 day after hatching). After completion of fixation whole larvae were washed 2-3 times in distilled water and alcohol, than larvae were preserved in 70% alcohol till further dehydration etc. After proper dehydration and slide preparation (W.M.) they were examined under the optic and research microscope than photomicrographs of some prepared (W.M.) slides were taken with the help of PM-6 and PM -10 Olympus- photomicroscopy systems.

OBSERVATION

Released eggs of S. plagiostomus are characterized as soft, pulpy and small. Outer most egg membrane is very thin and delicate and can easily be punctured by picking with forceps. Perivitelline space is a space between vitelline membrane and egg proper in the centre of egg occupies large space towards the upper side of egg body. The perivitelline space is filled with perivitelline fluid having yolk sac with germinal disc towards larger side. After the fertilization of snow trout eggs, they swell up considerably (measuring 1.44-1.8mm) within 10-20 min. Most probably this is due to the absorption of water by the eggs through their vitelline membrane. These eggs are highly adhesive in nature and due to osmotic pressure they attach to the substrate. Symmetrically the eggs are round in shape with glassy bead in appearance. Along with the development of embryo, constriction appears in the yolk; Yolk further gets elongated to form tube shape structure and ultimately acquires the shape of a posteriorly elongated yolk sac (Plate I).

EMBRYONIC DEVELOPMENT BEFORE HATCHING:

The fertilized egg of S. plagiostomus undergoes a series of developmental process. After fertilization the egg bears a small embryo, Firstly the germinal disc appears, at the animal pole and then many cells are formed resulting by usual division (Plate I b-d). After two hours of fertilization, first cleavage in the egg was seen, resulting in the division of egg leading to formation of two blastomeres of almost equal size (Plate I.e). Second cleavage right angle to the first was noticed another two hours i.e. the egg reached for four-celled stage (Plate I. f). As the four-celled stage is reached, the division becomes more rapid. Two parallel cleavages parallel to first cleavage appears after another 2 hrs and resulting in 8-celled stage (Plate I.g). After 9 hrs of fertilization, two more cleavage parallel to second cleavage appears leading to formation of 16 blastomeres i.e. 16-celled stage is attained. Subsequent cleavage resulted in the formation of 64-celled stage. Morula stage is attained after 10 to 12 hrs of fertilization. This morula stage was characterized by small sized blastomeres and spreading of marginal cells began to spread over the surface of yolk (Plate I h). After 3-4 hrs of morula stage a slight thickening in the form of germ ring appeared in the deeper layer at the periphery of blastoderm and becoming more clear subsequently, and the yolk, almost completely covered with blastoderm except for the 'yolk plug stage' in, which outline of the developing embryo was visible through egg membrane (Plate I i). As embryo becomes 56 hrs old, it began to attain shape and appeared as circular tubular folds in yolk. Cephalic end in the form of oval expansions started. By this stage embryo appears like "pea shape" and further pre cephalic movement by the embryo in the perivitelline fluid also noticed (Plate I j-k). By now embryo begins to elongate over the yolk, and formation of small head and tapering tail on the other side of the yolk was seen. Development of eye in the form of eyespot was visible and vertebral segment marked fin fold started appearing (Plate I .l). At 90 hrs old stage, the embryos clearly exhibited twisting movement such that the tail almost touched the head. Development of eyes advances leading to optic evaginations, which were evident laterally. The optic capsule was present behind the eye where different lobes of brain are visible. The posterior region is with tail and myotomes. Notochord formed, from optic lobs to the tail. Embryonic fin fold continues to increase in size (Plate I m). By this time embryo becomes active moving freely in the perivitelline space exhibiting twitching movement. The constriction of yolk sac pronounces, forming small portion towards the tail and larger towards the head end. Both cephalic and caudal portions were almost free from yolk mass. V-shaped myotomes ranging from 18-28 notochords extending up to the tail end. The optic cups having rudimentary lens where a black spots (eye) visible in this vesicles. Blood was clear but hemoglobin was almost ansen and Heart beats very rapid. Vigorous movement noticed by tail. Embryo started filling the perivitelline space (Plate I n). The embryo further elongates and gradually differentiated. Number of myotomes makes vigorous twitching movement and frequently changing position. Within next 4-8 hrs the embryo becomes completely differentiated and ready for hatching (Plate I o-p).

EMBRYONIC DEVELOPMENT AFTER HATCHING:

Hatching in the eggs takes place 110 to 115 hrs after fertilization in laboratory by the lashing movement of tail leading to rupture of egg membrane, and tail coming out first from the egg shell where fertilized eggs put in the laboratory. The body of newly hatched larvae was transparent lacking pigmentation. The club shaped yellowish colour yolk present. In this stage eyes and otocysts was clearly visible. The striopore (the future anus) was noticed in rudimentary form where the yolk sac ended. Newly hatched larvae were in active lying at the bottom of tray and moving vigorously with tactile stimulus. A continuously median fin fold extends along covering the entire trunk and tail region up to the demarcation of yolk sac. Eyes were unpigmented and auditory capsules were in the form of two black auditory vesicles and myotomes are visible (Plate II a & Plate III a-b). Hatchlings were thin and yellow coloured yolk sac bulbous in appearance. Oil globule absent on yolk sac or body region and notochord straight extending up to tail end. Two chambered heart with yellow colored blood which circulating in body is visible. Median transparent fin-fold has increased in width, anal pore become a distinct aperture neural material and eye lens still pigmented (Plate II a & Plate III c-d). The 2nd day larvae were more active, but resting most of time at the bottom showing occasional movements. Eyes start to develop with a pigmentation and yolk sac started to reduce in size. Rudimentary pectoral fins appeared and cephalic regions well marked. Pericardial cavity lying between yolk sac and head becomes well defined and conspicuous. In this stage well-developed pulsating heart with well defined auricle and ventricle was seen. Blood still yellow coloured, myotomes clearly visible. Three parts of brain were demarcated (Plate III e). 3rd day larvae were still inactive lying at the bottom. Yolk sac absorbed especially towards anterior end. Blood circulation was visible from cephalic region to tail. Blood still yellowish fin fold well demarcated especially on ventral side. Opercular area in head region bearing gill clefts becomes clearly demarcated. Pectoral fins started to develop in this stage (Plate II c). Myotomes increase in number and body movement of larva was also absorved ofter causing the distervance. Development of fin fold started dorsally, encircling the tail and ended at the demarcation of head and trunk regions ventrally. Opercular area was more defined. Pectoral fin still non-functional and faint fin-ray could be seen in transparent fin fold. Eyes become pigmented and rudimentary jaws appeared (Plate II d & Plate III e-g). Most of the yolk sac gets absorbed. Yolk sac lost its roundness and become long tubular form. Heart well differentiated with various vessels carrying blood throughout the body. Blood becomes slightly reddish and heart was blood red in colour. Heart when viewed laterally showed rudimentary gill arches. Both jaws formed, the lower one moved occasionally. Anal opening was distinct and development of alimentary canal commenced. On the 6th-8th day Yolk was reduced and mostly absorbed, jaws and operculum well developed, formation of alimentary canal was completed, anterior end of gut becomes slightly broad to form intestinal bulb, swim bladder rudimentary. On the 9th-11th day Caudal fin rays visible, ventral fin fold elongated upto trunk region, alimentary canal differentiated into intestinal bulb, intestine and rectum, intestinal bulb was clear and broad, Swim bladder was more defined and inflated. On 11th - 13th days larvae, Caudal fin was more develop and functional, fin rays clearly visible, Intestinal bulb having comparatively large and slightly more mucosal folds than comparison of other parts, posterior chamber elongated and cavity of anterior chamber increased in size in swim bladder. On the 13-15th days larvae, Jaws well developed and functional, having good movement, operculum well develop and functional, pectoral fin elongated and functional, dorsal fin well develop and dorsal fin fold start to reduce toward tail region, caudal fin round in appearance and functional well differentiated and functional and fin rays clearly visible, intestinal bulb well develop and comparatively larger.

DISCUSSION:

The eggs of Schizothorax plagiostomus are round, soft, transparent with yellowish ting yolk and adhesive in nature after spawning. Same observation was given by Raizaida5, (1985) in Schizothorax plagiostomus from Himachal Pradesh and by Joshi and Sunder11, (1995) from Kumaon lakes in Schizothorax richardsonii. The ova diameter of fertilized egg was measured in different snow trout fishes by Gupta and Subla20, (1985) 1.9-2.7 mm in Schizothorax curvifrons, Sunder21 (1984) in Schizothorax longipinnis, it was 2.1 mm, while in S. richardsonii from Kumaon lakes the ova diameter was recorded 2.3-3.0 mm11. In present study the ova diameter of S. plagiostomus was measured 1.44-1.6 mm, swells up to 1.8 mm after fertilization, which is very close to ova diameter 1.2-1.8mm in S. plagiostomus from river Alaknanda as reported by Agarwal22, (1996). The variation in ova diameter may be due to genetic variation or habitat effects in different snow trout fishes. However, after fertilization the cellular division inside the egg was similar as other snow trout fishes, yet the differences were observed in incubation period from the different habitat. In S. plagiostomus from the river Alaknanda around Srinagar Garhwal incubation period takes place at 110-115 hrs. after fertilization in laboratory condition when the water temperature ranged from 21-240C. Approximately similar incubation timings were reported in various Schizothorax spp. by the different authors. In S. plagiostomus from Kumaon lakes incubation period reported 196 hrs when the water temperature was ranged 14.5-190C11. In Kashmir fishes this hatching was ranged from 192-360 hrs in S. niger, S. micropogon, S. curvifrons, S. esocinus and S. richardsonii when water temperature ranged from 9-180C23,24, while Gupta and Subla20 (1985) reported 108 hrs of incubation at 15-200C in S. curvifrons. Therefore, not only water temperature was responsible to effect the incubation/ hatching period but along with this some other physico-chemical factors of the hatching ground was responsible for this biological phenomenon e.g bottom of the river, movement area of the fish before spawning etc. Before hatching considerable changes were take place inside the egg of S. plagiostomus, as after 2 hrs. of fertilization blastodisc was formed and than morula stage was developed after 10-12 hrs of fertilization. After 56-74 hrs of fertilization the embryo had begun to take shape and the beginning of the cephalic end was clear, as an oval expansion of embryo is more or less pea-shaped in this stage. At 82hrs embryo elongates over the yolk with small head with tapering tail. The further elongation and differentiation of embryo takes gradually at 106 hrs. after fertilization. Later on the number of myotomes was increased and the movement of embryo was quite vigorous and ready to hatch 110-115 hrs. in laboratory after fertilization. Almost the same observations were made by Gupta and Subla20 (1985) in S. curvifrons from the river Jhelum (Kashmir), Raizaida5 (1985) in S. plagiostomus from Himachal Pradesh and Joshi and sunder11 (1995) in S. richardsonii from Kumaon Lake. The difference in development or in incubation timing of various organs inside the eggs may be due to genetic and phyisco-chemical nature of environment of different Schizothorax spp. The further larval development in newly hatched larvae of S. plagiostomus was almost same as reported in other snow trout fishes by Gupta and Subla20 (1985); Raizada5 (1985); Joshi and Sunder11 (1995). All developing larvae were not hatched in same time may be due to eco-physiological condition of the environment, while the steps of metamorphic changes was the same in as in other species of snow trout as reported by other workers 20,5. Absorption of yolk sac was more rapid in larvae this may be due to the effect of water temperature and dissolved oxygen of the environment where the hatched larvae were growing. Rapid absorption of yolk was cause of the more mortality than the larvae where the absorption of yolk was slow. This may also effect the development of different organs viz. air bladder, liver kidney, brain, pancreas, gal bladder etc. On the fifth day after hatching the larvae of S. plagiostomus the yolk was absorbed. The same was also reported in same other snow trout fishes by Gupta and Subla20, (1985).

ACKNOWLEDGEMENT

The authors are highly thankful to ICAR, New Delhi, for funding assistant through project section No. 4(16) 2000/ASR-I/2003-2006.

REFERENCE

1. Day, F. (1958): The Fishes of India: Being a natural history of the fishes known to inhabit the seas and fresh waters of India, Burma and Ceylon. Vol. 1 (text). William Dawson and Sons Ltd., London. 564. 2. Jhingran, V.G. and Sehgal, K.L. (1978). Proc. Nat. Sem. Res. Dev. Env. Himalayas. DST, New Delhi, 239-247. 3. Bahuguna, S.N. (2002). Final Technical Report. Submitted to ICAR, Krishi Anusandhan Bhawan, New Delhi. 4. Singh, H.R., S.P. Badola and A.K. Dobriyal (1987): J. Bombay Nat. Hist. Soc. 84 (1): April. 5. Raizaida, S.B. (1985). J. Bombay Nat. Hist. Soc. 82(4): 130-137. 6. Shrestha, T.K. and S.S. Khanna (1979): Matsya. 5: 23-34. 7. Yabe, H., Ueyanagi, S. and Watanabe, H. (1966): T. maccoyii. Report of Nanakai Regional Fisheries Research Laboratory. 23: 95-129. 8. Kaji, T., Tananka, M., Oka, M., Ohsumi, S., Teruya, K. and Hirokawa, J. (1999): Fish Sci. 65: 700-707. 9. Khaitov, KH. Kh., Ibragimov, I.I. and Omorov, E.O.(1991): Gidrobiologich. Zhu. 27(4): 25-32. 10. Singh, N., Bahuguna, S.N., and Bhatt, K.C. (1993): Acta Icthologica 23 (1): 1-30. 11. Joshi, C.B. and sunder, S. (1995): Uttar Pradesh J. Zool. 15 (2): 136-140. 12. Fang, J. and Zhou, Yi. (1997: Acta. Hydrobiol. Sinica. 19(1): 70-74. 13. Maithani, S. (2000): D. Phil. Thesis awarded from H.N.B.Garhwal University, Srinagar (Garhwal). 14. Bahuguna, S.N. and Maithani, S.(2005). Proc. Nat. Seminar, Chandigarh. Feb. 16-18, 2005: 69-77. 15. Bahuguna, S.N.(2006). Final technical report submitted and adapted ICAR, Krishi Bhawan new Delhi. 16. Taylor, W.R. (1967): Proc. United State National Museum, 122: 1-17. 17. Pearse, A.E. (1975): Histochemistry, theoretical and applied. Vol. I, Churchill living Stone, Edinburgh. 18. Dingerkus, G. and Uhler, L.D. (1977): Enzyme clearing of alcian blue stained whole small vertebrates for demonstration of cartilage. Stain technology,52:229-232 19. Kaji, T., Tanaka, M., Takahashi, Y., Oka, M. and Ishibashi, N. (1996): Mar. Mar. Freshwater Res. 47: 261-269. 20. Gupta, S.S and Subla, B.A. (1985): Indian J. Fish., vol. 32(1): 101-111. 21. Sunder, H. (1984): J. Himalayan. Res. & Dev. 3:17-23. 22. Agarwal, N. K. (1996). Fish reproduction A.P.H. Publishing Corporation 5, Darya Ganj, Ansari Road. New Delhi-02. 23. Raina, H.S., Vass, K.K., Sunder, S., Bhagat, M.J., Bali, U. and Langer, R.K. (1985 a): Zool. Orientalis. 2 (1-2): 24-30. 24. Raina, H.S., Vass, K.K., Sunder, S., Moza, U. and Langer, R.K. (1985 b): Bull. Env. Sci. 2(1): 27 - 33. 25. Heckel, J.J. (1838): Fische aus Caschmir. Wien. pp 11-52.Pls I-IX. Abbreviations use in Figure H=head, T=Tail, Ys=Yolk sac, E=eye