Reproductive cycle of the endemic and threatened fish Puntius shalynius ( Cypriniformes : Cyprinidae ) in Meghalaya , India

Puntius shalynius is a highly endemic freshwater minor carp that is economically important and is threatened because of its ornamental value. The present investigation evaluated this barb gonadal maturation, based on morphology and spawning of this species in the Umiam river, Meghalaya, India. The population of this indigenous fish has declined due to its fragmented distribution and exploitation as an ornamental fish. The reproductive cycle of P. shalynius was studied for the first time. A total of 609 fish samples were randomly collected from the river for a period of two years during January 2010 and December 2011. Five maturity phases (rest, primary growth, secondary growth, ripe and spent) were observed on the basis of ovarian and testicular macroscopic evaluation throughout the annual cycle. Peak spawning activity was observed in the month of June/ July and it coincided with the start of the monsoon season. The study showed that the fish spawns once in a year with single spawning peak and that the species is a low fecund fish. It is important to conserve this species for its unique ecological value and urgent management policies should promote its sustainable utilization. Rev. Biol. Trop. 65 (1): 255-265. Epub 2017 March 01.

The shalyni barb, Puntius shalynius (Yazdani & Talukdar, 1975), is an important hill stream, endemic and threatened ornamental fish of Meghalaya, India.This species has a very restricted geographical distribution in India (Manorama & Ramanujam, 2014) and inhabits high altitude water bodies above 1 000 msl.It is an omnivorous fish, with 5.60 cm of average length (Manorama & Ramanujam, 2011).The phenotypic appearance of P. shalynius is characterized by having short, compressed, fairly deep body and dorsal profile a little more convex than the ventral profile.In some males, minute white tubercles are present on the head.Barbels are absent and scales are fairly big, hexagonal with anterior margins distinctly wavy.Two spots on either sides of the tail in both sexes, the anterior one (situated nearly opposite the end of the anal) more distinct than the posterior (situated near the base of the caudal).There is a blue horizontal line which runs along the middle of body and presence of minute black spots on sides of head (Jayaram, 1999).It has been in the category of "vulnerable" according to IUCN (2015), and has considered it in threatened status.
The vulnerable aspects of life histories of threatened freshwater fishes are very important to be identified in order to add to their conservation measures (Angermeier, 1995).Furthermore, the knowledge of their reproductive biology is a critical component for sustainable management and policy definition; a complete understanding of the gonad development status of fish species is considered to be an important step for resource managers and fish culturists (Kohinoor, Islam, Mia, Rahman, & Hussain, 2003).An effective management strategy recognizes that species not only depend on the existence of a suitable habitat, but also on its availability in the right time and place (Naiman & Latterell, 2005), more especially for endemic species.
In view of its ecological and economic importance, the aim of the present study was to analyze the gonadal morphological development on P. shalynius, and define the successive maturation phase so that a clear understanding on its reproductive cycle can be gained including GSI, fecundity and ova-diameter.The data obtained can be utilized for future aquaculture planning and formulating conservation measures of this potential species which is regarded to be threatened, and whose natural habitats are being damaged and the population is rapidly declining due to anthropogenic activities in Meghalaya, India.

MATERIALS AND METHODS
Sample collection: P. shalynius were monthly collected with the help of local fishers, during daytime from the Umiam River, North-East India, between January 2010 to December 2011.The study area is situated at an altitude of 1 020 msl and falls at an intersection of 25 o 40' N -91 o 54'20" E. Collection of live fishes started in January 18, 2010 and continued to collect every month.Fish were collected using cage net and other locally made traps like landing net.The samples were transported alive to the Fish Laboratory at the Department of Zoology, North Eastern Hill University, Shillong, and kept in the aquarium until fish were analyzed for the experiment.
Macroscopic study: Total length (TL) of each individual was measured to the nearest 0.1 cm and the total body weight (BW) was recorded to the nearest 0.1 g before dissecting each fish.The gonads were carefully removed and all surrounding tissues removed for further investigation.The gonad weight (GW) of each specimen was also weighed (accuracy of 0.01 g), while the sex was determined by naked eye examination of the gonads.Fish length was measured with the help of a Vernier caliper (Mitutoyo Series 560) and weight was recorded using an Electronic Balance (A&D EJ 200 Digital Scale).
Sex ratio: A total of 609 individuals were examined for sex ratio determination.The ovaries and testes of each fish were examined by naked eye as the gonads can be easily distinguished, belonging to a range of different developmental phases proposed by Grier, Uribe and Patino (2009).Deviation from the expected 1:1 sex ratio was analyzed using Chi-square test (χ 2 ) (Corder & Foreman, 2009).For the entire statistical procedures, the level of significance was maintained at 0.05.
Fecundity: Fecundity was estimated from gravid females collected from the Umiam River throughout the study period, and was determined using the gravimetric method (Babiker & Ibrahim, 1979).The ovary from the ripe females was removed from the fish and weighed.Two sub-samples of each, right and left ovary lobes, were kept in Gilson's fluid and shaken periodically to loosen the oocytes.Those that remained attached were teased off from the tissues during counting.Since dependence on GSI alone is not enough to determine gonadal maturation, oocyte diameters were also measured.Oocytes were counted and measured under a binocular microscope (Olympus CX31) using a calibrated phase micrometer.The absolute fecundity (AF) was calculated as suggested by Grimes and Huntsmen (1980).It was obtained by using the following formula: where, N 1 and N 2 = number of eggs for each subsample, W = total weight of ovary and w 1 and w 2 = weight of the ovary for each sub-sample.
Additionally to the absolute fecundity, relative fecundity (RF) has also been calculated following Hardisty (1964): where, AF = Absolute fecundity, BW = Total body weight.
The regressions between fecundity and total length and body weight were calculated according to Bagenal (1978).

Meteriological information (rainfall and temperature):
The data of annual rainfall and temperature of the study area and period was measured and provided by the Agricultural Engineering Division (ICAR, Umiam), Meghalaya.These were used to be compared with the GSI values, and to observe the influence of rainfall in fish spawning behavior.

RESULTS
Monthly variation in sex ratio showed that out of 609 fish analyzed, 283 (46.5 %) were males and 326 (53.5 %) were females (Table 1).The sex ratio in the different months observed was slightly different from the expected ratio of 1:1 (male: female).The overall sex ratio was 1: 1.16.Monthly fluctuation of sex ratio ranged from 1:0.75 (September) to 1: 2.17 (July), and was found to be statistically significant (P ≤ 0.05) and skewed in favor of females.
The following gonadal maturation phase were established for males and females based on macroscopic study: I = Rest, II = Primary growth, III = Secondary growth, IV = Ripe, V = Spent (Table 2).
According to the monthly percentage composition of ovarian maturity phase (Fig. 1), females at phase I was observed in November and December, when the temperature decreased.The females at phase II were found in January and February as soon as the temperature starts rising.During March to May, as the temperature and rainfall increased, the gonad developed to the most advanced phase III.Phase IV specimens were recorded in June and July, that showed the peak of temperature and rainfall.The last phase V began to appear in the month of August onwards till October declaring completion of mature gonadal development, with the decline in temperature and rainfall.The testicular development of P. shalynius was similar to that of the ovary during the study period, having synchronicity.From December to February, most of the males were at phase I, followed by more advanced  2. In females, there was an overall increase in mean GSI from 4.71 ± 0.39 in February to a maximum value of 12.03 ± 0.47 in June, representing the peak of the spawning season.Subsequently, there was a decrease from August onwards with mean GSI value remained 6.38 ± 1.19.Mean GSI for males gradually increased from 0.99 ± 0.07 in January to 5.92 ± 1.54 in April and then declined to 1.65 ± 0.16 in May, and more dramatically to 0.87 ± 0.12 in October.The peak spawning season was observed once a year in June for females, and April for males.In the present study, males spawned earlier than females but breeding season extends until October for both sexes.It is worth mentioning that in many fishes, the peak reproductive activity did not coincide in males and females.The high value of GSI in females and males coincided with the high rainfall and temperature seasons (Fig. 2).Gravid females were found in June and declined at the end of July.The sample ranged from 4.00 to 6.60 cm in total length and 0.65 to 3.97 g in body weight.The gonad weight of the specimens varied from 0.047 to 0.639 g.The absolute and relative fecundity ranged from 47 to 1 240 eggs, and 42 to 633 eggs per individual, respectively.The relationship between fecundity (F) and total length (TL) and total body weight (BW) may be described by the following equations: F = 3.54 x 10 -1 TL 4.06 (r = 0.71) F = 2.09 x BW 1.31 (r = 0.72) The ova diameter progressively increased from 0.37 mm (March) to 0.90 mm (June) along with progression of maturity phase.Primary growth and mature ova were found in the ovaries during March -May (0.37-0.68 mm) respectively.Ripe ova were found during June (0.90mm) and July (0.74 mm) with the peak season in June (Fig. 3).The frequency of ripe ova fall down from August onwards till October and these were completely absent for four months during November to February when the fish is in resting phase, and indicated that the spawning season was over.Thereafter, their frequency began to rise from March.No eggs were observed in the months of December and January.
In January, rainfall used to be less (1.6 mm) with gradual increase in May (429.9 mm) and the maximum found in June (880.6mm) then it decreases in July (338.6 mm) till it drops in December (12.3 mm).Temperature was observed low in December with 12 o C and high during July/August with 24 o C (Fig. 2).

DISCUSSION
The present study is the first with detailed information provided on the gonadal maturity in P. shalynius collected from its natural population, and described the annual maturation phase in both sexes.Reproductive cycle of a species provides information for its utilization in breeding programme of an endemic and threatened fish, especially P. shalynius.The results obtained from this study revealed that the species spawns once in a year from March to June in females, and March-April for males, and is a low fecund wild barb.
Sex ratio in different months showed some variations between males and females in the population.According to Nikolsky (1963) and Alp, Kara and Buyukcapar (2003), sex ratio   of the fish may deviate from the normal 1:1 due to a number of factors.One possible reason for this skewed could be the differential habitat occupation of both sexes.Such differential habitat occupancy by sexes has been earlier observed in tropical fish (Lewis & Fontoura 2005;Macuiane, Kaunda, Jamu, & Kanyerere, 2009).Skewed ratios may also occur as a result of the differences in instantaneous natural mortality between sexes (Vincentini & Araujo, 2003).Cek, Bromage, Randal and Rana (2001)  dukai (Joshi & Joshi, 1989) and Labeo rohita (Alam & Pathak, 2010).Gonad development and reproductive strategy have been described in many teleost fish species in an effort to understand the time course and reproductive cycle of the fish.In this investigation, five different maturity phases (I-V) of gonads have been described based on the macroscopic study.It is important to know the different phase of maturation to understand the time course and reproductive cycle of the fish.Similar phase of maturity have also been reported in Gobiodes rubicundus (Kader, Bhuiyan, & Manzur-I-Khuda, 1988), Mystus montanus (Arockiaraj, Haniffa, Seetharaman, & Singh, 2004) and Astyanax fasciatus (Carvalho, de Paschoalini, Santos, Rizzo, & Bazzoli, 2009).
Gonadosomatic index can be used to determine spawning season because GSI increases with the maturation of fish, being maximum during the period of the peak of maturity and declining abruptly thereafter, when the fish becomes spent (Stoumboudi, Villwock, Sela, & Abraham, 1993).According to the present study, a GSI value of 12.03 was considered a reliable indicator of reproductive maturity for females.A high value of the GSI in June and its sharp decrease in the month of August showed that the fish have spawned during June/ July which is consistent with the result of that maturity stages.It can be concluded that the fish spawns once in a year with a single spawning peak.Mannan, Maridass and Thangarani (2010) observed that P. filamentosus spawns once as the maximum GSI value was found only in April.
Absolute fecundity of P. shalynius is extremely low when compared to other cyprinids such as Rasbora daniconius (Nagendran, Shakuntala, Natarajan, & Vasan, 1981), whose fecundity ranged from 580 to 11 040 eggs.In Barilius vagra, fecundity was observed with 800 to 3 400 eggs (Gaur & Pathani, 1996).Bhuiyan, Islam and Zaman (2006) obtained absolute fecundity of 2 254 to 6 964 eggs in P. gonionotus.Solomon et al. (2011) recorded absolute fecundity in P. denisonii from 376 to 1 098 eggs, while relative fecundity was between 36 to 94 eggs similar with the present findings.The present observation on P. shalynius fecundity revealed that it is a low fecund fish when compared to other barbs.It is possible to suggest that the lower number of eggs can be correlated with short development time and low mortality rate of fingerlings, which means a higher survival rate of the fish.Thus, less number of eggs does not prove to be a disadvantage in reproductive potential.
The correlation coefficient (r) was found to be quite high (0.711 and 0.716) indicating a high degree of positive correlation between fecundity and total length, and fecundity and body weight.Similar findings were reported for P. ticto by Hossain, Rahman and Abdallah (2012) with the correlation coefficient value of 0.484 for fecundity and total length, whereas 0.63 for fecundity and body weight.Fecundity is directly proportional to size and body weight (Lehman, 1953).According to Jonsson and Jonsson (1999), fecundity increases with body size because the amount of energy available for egg production and the body cavity accommodating the eggs increase with fish size.
The ova diameter variation is one of the important evidences used in the determination of fish reproductive strategy (Tomasini, Collart, & Quignard, 1996).The progressive change observed in the intra-ovarian diameter for a period not less than a year, can give an idea of the spawning periodicity of fish (Biswas, 1993).All the ova were found to be spherical and uniform in diameter indicating that the eggs were shed in a single batch.The ova diameter progressively increased from 0.375 mm (March) to 0.745 mm (July) along with progression of maturity phase.Ripe ova found in the month of July (0.745 mm) indicated that the fish spawn once in a year.In August, the mature eggs started to decline signaling the end of breeding season.There was no ova formation during November to February when the fish was in immature phase.Sundarabarathy, Edirisinghe and Dematawewa (2004) reported the ova diameter to be 0.628 to 0.681 mm in P. titteya.Abedi, Shiva, Mohammadi and Malekpour (2011) described that the ova diameter of Garra rufa ranged from 0.03 to 1.98 mm, with a mean of 0.67 mm.Saha (2011) observed that the ova diameter increased from 0.111 to 0.370 mm along with the progression of maturity phase in Salmophasia phulo.
It is important to understand the breeding pattern during the spawning season, in order to plan proper conservation strategies and to avoid fishing during the breeding season; this may consequently promote an increase in natural stocks.This specific characteristic is best done by monitoring the GSI and by observing the macroscopic features of gonad development.The gonads development both in females and males of P. shalynius was represented by five phases: (i) rest, (ii) primary growth, (iii) secondary growth, (iv) ripe and (v) spent.These phases were similar to the ones reported for P. sarana (Chakraborty, Mirza, Miah, Habib, & Chakraborty, 2007).
The reproductive activity for both sexes of P. shalynius was influenced by environmental factors such as rainfall and water temperature for high value of GSI concurred with high rainfall and temperature.Again, rainfall acts as a cue for P. shalynius to breed, so that offspring are produced at times of better growth and survival.In freshwater teleosts, the high rainfall and temperature have been associated with spawning activity (Parkinson, Philippart, & Baras, 1999).Many possible factors have been suggested by earlier researchers as influencing the spawning of freshwater fishes; some of these factors such as temperature, water flow, water level and precipitation, can serve as triggers for tropical fish reproduction (Wootton, 1995).In carps, it has been established that intensive flooding either caused by the rainfall or artificial means, capable of inundating shallow areas, are essential to induce spawning (Hora, 1945).Exogenous factors sometimes influence endogenous reproductive processes.
Studies on GSI and ova diameter suggests that the peak breeding season of P. shalynius is in June/July extending until October for both sexes with single spawning and the species is a low fecund fish.There is a need to develop successful management strategies so that fish stocks can be maintained for future generations.Fishes that do not meet adequate size limits are required to be released.To protect small fish of some species, minimum size limits can be set to allow juvenile fish to survive long enough to reproduce.Another measure is to initiate closed season for several threatened species for which harvest is prohibited, so as to protect fish during vulnerable time in its development or life history.The inference drawn from this study can be used as baseline information for planning effective conservation measures of this endangered and endemic species of North-East India.

ACKNOWLEDGMENTS
Authors are thankful to North Eastern Hill University, Shillong for financial support under UPE (University with Potential for Excellence) scheme of UGC to Life Sciences, Council of Scientific and Industrial Research (CSIR), Ministry of Environment and Forest (MoEF) and Head of Department of Zoology for giving permission to carry out the experiment and also for providing all the research facilities.Four anonymous referees and the administrative editor are thanked for helpful comments and constructive suggestions.

Fig. 1 .
Fig. 1.Monthly changes in the frequency of occurrence of various maturation phases in Puntius shalynius of the Umiam river, Meghalaya, India.

Fig. 2 .
Fig. 2. Annual changes in temperature and rainfall of Barapani, Ribhoi District, India (upper panel), and monthly variation of gonadosomatic index (GSI; lower panel) in male and female of Puntius shalynius of the Umiam river, Meghalaya, India.Each point symbol on the line indicates mean ± SEM of GSI.

TABLE 1
Monthly variation in sex ratio and chi-square test of Puntius shalynius in the Umiam river, Meghalaya, India M= Male, F= Female; * Significant at P ≤ 0.05.phaseII in March and April.Testes at phase III appeared in May and from June onwards, they reached phase IV till early August.Testes were observed at phase V by the end of August and thereafter.In both cases, the peak spawning activity was principally observed in June and July.Monthly variations in the mean GSI of males and females P. shalynius are presented in figure

TABLE 2
Macroscopic descriptions of gonadal maturation phase of Puntius shalynius in the Umiam river, Meghalaya, India