Reproductive pattern and some biological features of anadromous fish tenualosa ilisha (family: clupeidae) from Pakistan.

Indian Journal of Geo-Marine Sciences Vol. 40(5), October 2011, pp. 687-696 Reproductive pattern and some biological features of anadromous fish tenualosa ilisha (family: clupeidae) from Pakistan. Sher Khan Panhwar, Ghazala Siddiqui * & Zarrien Ayub, Centre of Excellence in Marine Biology, University of Karachi, Karachi-75270, Pakistan [E-mail: ghazala_siddiqui@hotmail.com] Received 7 April 2010; revised 18 August 2010 This study is the first to describe the reproductive pattern of anadroumous fish Tenualosa ilisha based on the histological examination of male and female gonads. The overall sex-ratio was close to 1:1 ratio. The five stages of oogenesis, nearly ripe, fully developed, running ripe, partially spent and spent and four distinct phases of ovarian atresis alpha, beta, gamma and delta were observed. Five stages of spermatogenesis developing, nearly ripe, ripe, partially spent and spent were identified. The male and female specimens in ripe and partially spent stages were considered to be in spawning condition. The spawning males were encountered during the period from May to October with the highest in July, August and October. While spawning females were also observed during the period from May to October, with almost all females being in spawning condition. The length-weight relationship in females of T. ilisha was nearly isometric and in males was allometric. The relationship between fecundity and other variables i.e. total length, body weight, girth and ovary weight in this species was significant. The age estimated on the basis of scales radii and length of the scales showed that the age classes comprised of 1+ to 3+ years. The mean scale length in different length groups was 7.5 to 11.5 mm. Introduction The shads are most common group of clupeid fishes, mainly found in coastal and estuarine waters. Some of them are migratory and they ascend upstream for spawning while others complete their life cycle within the coastal waters or estuaries. The anadromous fish T. ilisha, (Hilsa shad/indian shad) is found in Pakistan, and is locally known as Palla. This is a traditional fish of Sindh and there is a famous saying about this fish that whosoever, eats Palla never leaves Sindh. This species migrates upstream into the Indus River from the sea for spawning purpose. Very few individuals that refrain from migration, spend their life in fresh water and mature and spawn there. 1 Hilsa shad has been reported from Arabian Gulf, Bangladesh, Burma, China, India, Iran, and South Vietnam. General information on the biology of Hilsa shad has been well documented. 2-24 In Pakistan the studies were carried out to understand the migration pattern, problems related to fishery and on the fluvial migration after the construction of fish passes and fish ladders at Ghulam Muhammad Barrage on River Indus 25-27. Later on the spawning pattern and fecundity of this species was also studied. 28,29 A comprehensive review on the biology and fishery of this species has been written by Jafery. 30 The work on the reproductive biology based on macroscopic examination of gonads has been published, the age and growth of this fish from Indus River has also been described 31 and the land locked population of this species from Kenjhar Lake Thatta has also been studied 30 32. The present study was aimed to described the gametogenic pattern of males and females T. ilisha through the histological examination of gonads and also to assess the other biological features such as length weight relationship and fecundity which provide better understanding of reproductive development in this fish. Materials and Methods In all, 135 specimens of T. ilisha were obtained at monthly intervals from the fish market Thatta, Sindh. The specimens were available at the fish market from March 2004 to October 2004 this is the season when this fish migrates upstream into the Indus River and caught by local fishermen. Standard morphological measurements, total length (TL), fork length (FL), standard length (SL) and) body girth (BG) were recorded nearest to 0.1 mm of the all specimens. A gross examination of the gonads was used for initial sex assessment. The fishes were dissected and the gonads were removed carefully. Total weight and gonad weight were recorded to the nearest 0.01 gm with the help of an electronic balance. A gonadosometic index (GSI) = (gonad weight/somatic weight) 100) was calculated by using somatic weight (SW=TW-GW) for each sex (, SW=

688 INDIAN J. MAR. SCI., VOL. 40, NO. 5, OCTOBER 2011 somatic weight; TW= total weight; GW= gonad weight). The tissue samples were processed by using standard wax histology techniques. 34. For histological examination the central part of the gonads were taken and fixed in Davidson s fixative 35 for 48 hours. Following the standard method of dehydration they were embedded in paraffin and cut at 5 to 7µm on rotary microtome and stained with Delafield Haematoxylin and counter stained with Eosin. The length-weight relationship was computed by the general parabolic equation: W = al b where W represents weight, L length of the fish, a a constant and b an exponent. This equation logarithmically can be written as: Log W = log a + b log L. The age and growth was determined from body scales by the method described by Raj. 36 The age was determined from 17 specimens (102 scales) of T. ilisha measuring 110 to 250 mm intl. From each fish eight scales were cleaned and placed between two glass slides. Total scales length was measured nearest to 0.01mm with the help of a divider perpendicularly from its base to its posterior margin. The number of radii were counted on each scale under a binocular microscope and an average number of radii was estimated for each fish. The fecundity was determined by the method described by Islam and Talbot. 37 For this purpose ten (10) mature female specimens were selected. The whole ovary was removed from the fish and weighed Table 1 Monthly sex ratios and chi-square (x 2 ) distribution of T. ilisha. Month No. M F Proportion of males Apr 10 8 2 0.8 1.8* May 25 11 14 0.44 0.18* Jun 20 11 9 0.55 0.1 Jul 20 11 9 0.55 0.1 Aug 20 10 10 0.50 0* Sep 20 7 13 0.35 0.9* Oct 20 13 7 0.65 0.9* Total 135 71 64 3.84 0.2* Significant at 5% level (P=.05) Table 2 T. ilisha: Sex-ratios and chi-square (x 2 ) distribution in different size-classes Size-class (mm) No. Male Female Proportion of males x 2 200-250 54 26 28 0.481 0.074* 251-300 74 44 30 0.595 2.649* 301-350 7 1 6 0.143 3.571* *Significant at 5% level (P= >0.05) x 2 on an electronic analytical balance. Three sub samples of 0.1 gm each were taken from the middle region of the ovary and kept separately in 5% formalin for a period of one month to facilitate the separation of eggs. The jars were hand stirred occasionally to aid in separation of eggs. The eggs were carefully separated from the tissues and were counted in a counting tray under the binocular microscope. The total number of eggs in fish were calculated from the average count of these sub samples and were multiplied with the total weight of the ovary. Results Sex-ratio Sex-ratios were determined on the basis of histological examination of gonads. The over-all sexratio (x² = 0.36, P>0.05) was close to the expected 1:1 ratio. Monthly analysis of male to female ratio indicated that the males were slightly greater in numbers than the females throughout the sampling period (Table 1). Sex-ratio in all the size-classes was also significantly (P>0.05) close to expected 1:1 ratio (Table 2). Size-frequencies The length frequency histograms of T. ilisha are shown in Fig. 1. Specimens belonging to the size-class 200-250 mm were present in all the samples collected Fig. 1 Length frequency histograms based on the monthly samples of T. ilisha

PANHWAR et al: BIOLOGICAL FEATURES OF ANADROMOUS FISH 689 during the period from April to October. In October, no female was found in this size-class while males accounted for only 5% of the population. The individuals of 251-300 mm size were found throughout the study period. However, their highest abundance (90%) was observed in October followed by June (83.33%) and July (60%). This size-class was represented by both males and females in all the months. Specimens belonging to the size-class 301-350 mm occurred in June and from August to October. With the exception of October, this sizeclass was represented by females only (Fig. 1). Histological staging of female gonads In T. ilisha five stages of ovarian maturity, 1. nearly ripe, 2. fully-developed, 3. running ripe, 4. partially spent, and 5. spent were observed. Table 3. Further more four atretic stages, 1. alpha, 2. beta, 3. gamma 4. delta were also identified (Table 4, Fig. 3). Temporal variation in the gametogenic pattern in females In the present investigation females with immature stage were not encountered. Fully developed condition of the gonads appeared in May and lasted till September. The highest number (55.5%) of fishes, with fully-developed gonads were observed in July, while their lowest number (7.6%) was recorded in September. Ovaries with running ripe stage in females appeared in May and continued till October. Females with running ripe ovaries dominated the population in May (71.4%) June (77.7%), September (92.30) and October (71.4%). Partially spent condition in females was observed from May to August and then in October. Highest number (28.5%) of partially spent females was found in October. Females with gonads in spent stage appeared only in August constituting 10.0% of the total population (Fig 2 & 3). Histological staging of male gonads On the basis of histological examination, the male gonads of T. ilisha were divided into five stages, 1. developing, 2. nearly-ripe, 3. ripe, 4. partially spent, and 5. spent (Table 5; Fig. 4). Temporal variation in the gametogenic pattern in males Developing male stage appeared from April to June and their highest number (100%) was recorded in April. Male gonads with nearly-ripe stage were found in the population during May to August with peak (72.7%) in July and the lowest number (9.1%) was observed in June. Males with ripe gonads started to appear in May (9.1%) and remained in the population Table 3 Stages of gonadal maturity based on macroscopic and histological examination of ovary in T. ilisha. Modified from Lowerre- Barbieri et al. 37 Stages Macroscopic characteristics Histological criteria Nearly ripe Ovaries paired elongated, small and purple in colour Fully developed Ovaries median to large yellowish color. Running ripe Ovaries large to very large deep yellow colored, more or less cylindrical shaped and expended completely in the body cavity. Mostly partially yolked oocytes few primary growth oocytes and cortical alveoli present. Mostly advanced yolked oocytes with small oil vacuole in cytoplasm, rarely primary growth oocytes. Mostly advanced yolked oocytes with a large single oil vacuole and occasional postovulatory follicles present. Partially spent Ovaries large, light yellowish in colour Presence of germinal vesicle breaks down oocytes, rarely primary growth oocytes and occasionally postovulatory follicles. Spent Ovaries small and dark purple in color. Presence of primary growth oocytes, atretic oocytes and large amount of degenerated tissue (macrophage aggregates) and appearance of some postovulatory follicles. Table 4 Stages of atretic oocytes based on histological observations as described by Hunter and Macewics 38 S. No. Stages Description 1 Alpha Advanced oocytes undergoing atresia followed by less developed oocytes. The vitelline membrane of eggs and follicle wall also degenerate with the advancement of stage and the vacuoles starts appearing 2 Beta The atretic oocytes continue to reduce in size as they resorb. The vacuoles increase in size and combine to form single large vacuole within the oocyte. 3 Gamma The oocytes that were remained in the follicles further shrink in size and their outer layers folds inwardly. 4 Delta Most of the reabsorbing oocytes loose their cellular material and become yellow brown in color and the oocyte undergoing degeneration are reabsorbed.

690 INDIAN J. MAR. SCI., VOL. 40, NO. 5, OCTOBER 2011 till October. Their highest number (92.3%) was recorded in October. The partially spent stage in males was observed from May to June and in September and this stage observed to be highest in May constituting 45.4% of the total sample. Spent condition in males was found in May, July, September and October (Fig. 5). Subsequently the individuals of length group 301-340 mm (7-9 radii) and 341-380 mm (10-12 radii) were assigned to 2 + and 3 + age groups, respectively. Correlation between total length of the fish and scale length was found to be significant (r 2 = 0.798) Spawning season On the basis of histological examination of gonads the females with ovaries in fully-developed, running ripe and partially spent stages were considered as spawning females. Females appeared to spawn during the period from May to October, with almost all the females being in spawning condition (Fig 6). The GSI indicated the spawning in females which started in April and gradually increased till October. A peak (10.2) was observed in October, whereas, the lowest GSI (6.77) value was recorded in April (Fig. 6). Similarly the male gonads in ripe and partially spent stage were considered as spawning males. Such males remained in the population from May to October; however, their peak (100%) was recorded in August followed by October 92% and July 90%. The peak of GSI occurred in April (1.21) and its lowest (0.23) GSI value was found in June. Length-weight relationship Student s t-test was used to test possible significant difference for slope Zar. 40 The t-test revealed that the regression slopes were significantly different from the theoretical slope in males (r 2 =0.816, P<0.025) and non-significant in females (r 2 = 0.651, P>0.25) (Tables 6 & 7). Fecundity As shown in Table 8 the egg counts were made from 10 ripe females. The specimens belonging to the size-classes 200-250 mm, 251-300 mm and 301-350 mm, carried an average number of 113483, 382105 eggs and 572709 eggs, respectively. Highest number of 6,14,482 eggs, whereas, lowest number of 87,267 eggs were counted in August and May, respectively. Relationship between fecundity and other variables i.e. total length, body weight, girth and ovary weight was significant (r 2 > 0.800). Determination of age Age class determination was based on interpretation of radii on scales (Table 9). The age classes comprised of 1 + to 3 + years. The individuals having an average number of radii 4-6 or less than 300 mm in total length were assigned to age group 1 +. Fig. 2 Temporal variation in the gonadal development in females. NR-nearly ripe; FD-fully developed; RR-running ripe; Ps. Partially spent; S-spent.

PANHWAR et al: BIOLOGICAL FEATURES OF ANADROMOUS FISH 691 Fig. 3 Tenualosa ilisha: T.S of ovary showing stages of oogenesis A- nearly ripe stage (100X) B,C- fully developed showing alpha atresia (100x) D, F- running ripe showing beta atresia (D,F 100x; E-200x) G- partially spent showing gamma atresia (50x) H & I spent ovaries showing delta atresia (100x) Abbreviations: AO = atretic oocytes; GVBD = Germinal vesicles breakdown; GVM= Germinal vesicles migration; BB brown bodies; (macrophage aggregates); PG = Primary growth oocytes; POF = Postovulatory Follicles.

692 INDIAN J. MAR. SCI., VOL. 40, NO. 5, OCTOBER 2011 Table 5 Stages of gonadal maturity based on maroscopic and histological examination of testis in T. ilisha. Modified from Lowerre- Barbieri et al. 37 Stages Macroscopic characteristics Histological criteria Developing Testes paired and light pinkish in color Mostly spermatocystes and few spermatids Nearly ripe Testes large than the developing stage, light cream colour Mostly spermatids, few spermatocyles and poresence of few spermatozoa also Ripe Tested large more or less cylindrical, whitish colored, filled with a creamy colored fluid. Lumen densely packed with spermatocytes present Partially spent Testes small to large light purple in colour A large amount of connective tissues and few spermatozoa Spent Testes very small contracted and colorless Connective tissue abundant follicle contracted very few residual spermatozoa Discussion Over-all sex-ratio was slightly in the favour of males, being 1.1:1. Male to female ratio of T. ilisha in the Godavari River was reported to be 1:9, being highly in favour of males. 41 While from the Hoogly River and Chilka Lake the sex-ratio in this species was 1:1. 42 However, more males than females were found in the population from the Hoogly near Barrack Pore 43 Some of the other workers who found the sex-ratio to be 1:1 in T. ilisha are Pillay 6 and Shafi et al. 44 From Ganga and Jumuna rivers in India the sex-ratio in the population of T. ilisha was described to be 2:1 45,. In Pakistan 3:1 sex-ratio in favour of males was observed in the population of T. ilisha from Indus River near Kotri Barrage based on the macroscopic examination of gonads. 31 The females of T. ilisha appeared to spawn during the period from May to October, with almost 100% of females being in spawning condition. In males spawning was observed from May to October with peak (100%) recorded in August followed by October 92% and July 90%. Narejo 31 reported that the spawning season in T. ilsha is from July to August in Indus River. Their findings were based on the morphological examination of the gonads from the four months samples procured during June to September. Swarp 46 and Nair 47 have suggested two breeding seasons (one in winter and other in summer) in T. ilsha from Ganges river system, India. Similarly from Bangladesh waters two breeding seasons, one in winter and other in summer was recorded 10. According to the Bell 48 some marine fishes prior to spawning accumulate high concentration of oils mainly lipid deposits, primarily the triacylglycerols, which are subsequently mobilized to support gonad development and spawning migration. The presence of large numbers of oil vacuoles has been reported in herring by Hickling and Eutenberg 49, in speckled trout by Vladykov 50 and in Glossogobius giuris by Sreeramu and Rajalakshmi. 51 Fig. 4 Temporal variation in the gonadal development in males. NR-nearly ripe; FD-fully developed; R- ripe; PS. Partially spent; S-spent

PANHWAR et al: BIOLOGICAL FEATURES OF ANADROMOUS FISH 693 Fig. 5 Tenualosa ilisha: T.S of testis showing stages of spermatogenesis. A, B- nearly ripe (100x) C- ripe (100x) D- partially spent (100x) E- partially spent showing main sperm duct (50x). F- spent (100x). Abbreviations: MSD = Main sperm duct; ST = spermatids; SZ = spermatozoa; CT-connective tissue

694 INDIAN J. MAR. SCI., VOL. 40, NO. 5, OCTOBER 2011 Table 9 Relationship between total length (TL) and scale length and the number of radii on scales. Length groups (TL mm) Mean scale length (mm) Number of radii Age group assigned 261-300 7.5 4-6 1 + 300-340 9.5 7-9 2 + 341-380 11.5 10-12 3 + Fig. 6 Frequency polygon showing spawning season in males (M) and females (F). GSIF- gonadosomatic index females; GSIMgonadosomatic index males. Table 6 Regression coefficient for length weight relationship for T. ilisha, 95% C.I (confidence intervals) for a (intercept) and b (slope), r 2 values are given, N=number of specimens. Sex N Log a 95% C.I of Log a In the present study the examination of histological sections of T. ilisha gonad revealed that at the beginning of spawning season the oil vacuoles coalesce and eventually a large single oil vacuole is formed. The knowledge of ovarian atresia is useful for the timing of spawning. In the present study four stages of ovarian atretic process were observed which are similar to earlier developed classification system 49 based on the development of oil vacuoles. Our study is the first to describe the atretic stages in the anadroumus fish T. ilisha. Such process has been earlier reported in striped mullet, Mugil cephalus from South Carolina estuaries, USA 52. The fecundity in T. ilisha was 87,267 to 614,482 in the females ranging from 210 to 350 mm in total b 95% C.I of log b M 71-1.154-0.633 2.534 0.445 0.651 F 64-0.708-0.508 0.957 0.356 0.816 Table 7 Values for t-test for the slopes of the regression equation (data of the table 3) where fish length was an independent and weight a dependant variable. Sex Slope Standard error Theoretical of slope slope t-test r 2 Probability M 2.534 0.223 3.00-2.089 <0.025 F 2.957 0.178 3.00-0.241 >0.25 Table 8 Logarithmic relationship between fecundity/total length (TL), body weight (BW), Girth (G) anf ovary weight (OW) in T. ilisha Equation Constant Log a Slope b r2 F=a+b TL -0.900 4.346 0.835 F=a+b BW 1.884 1.359 0.839 F=a+b G 1.985 3.683 0.822 F=a+b OW 3.674 1.182 0.945 length. Narejo et al. 31 had reported the fecundity ranged from 64,608 to 1,153,383 in females of T. ilisha which was found in a size range from 201-408 mm in total length. The specimens of T. ilisha examined during the present study were estimated to belong to age groups, 1 +, 2 + and 3 + when their total length were between 261-300, 301-340 and 341-380 mm, respectively. Jafri et al., 33 had considered the specimens of T. ilisha of size range 151-200 mm in TL as belonging to the age group 0 +. Narejo et al. 32 while studying the age groups in this species had reported that fishes of less than 250 mm belongs to age group 0 + and the fishes of size-groups 251-300, 301-350, 351-400, 401-450 and 451-500 mm belonged to the age groups 1 +, 2 +, 3 +, 4 + and 5 +, respectively. In the present study the length-weight relationship in females of T. ilisha was nearly isometric, whereas, in males it was allometric. Jaferi 33 observed b value close to 3.00 in males and females of T. ilisha while studying the length-weight relationship. In specimens for fishes studied from Bangladesh waters, the regression co-efficient of regression ranged between 2.6-2.8, for length weight in T. ilisha. 9,10 However, in population of T. ilisha from Ashar Canal, Iraq 53 studied for length/weight relationship the regression coefficient was 3.16 in both the sexes for the length-weight relationship. In population of T. ilisha from Persian Gulf the regression co-efficient was 2.98 and 3.16 for males and females respectively. 12 The difference in the length-weight relationship in the individuals of same species have been reported to occur due to geographical and temporal variation. 54,55 Recently, Hussain et al 56 have found the regression coefficient to range from 2.2 to 3.5 in 41 fish species studied from Korangi Phitti Creek area, Pakistan. References 1 Jonsson, B & Jonsson N, Partial migration: Niche shift versus sexual maturation in fishes: Reviews in Fish Biology & Fisheries, 3: 1993, 348-365. 2 Hora, S L & Nair, K K, Further observation on the bionomics and fishery of the Indian shad, Hilsa ilisha in Bengal waters. Rec. Ind. Mus. 42: 1940, 35 50.

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