MATERIALS AND METHODS

Nallah Sukhnag is one of the important tributary of River Jhelum and is among the five major inflows of the Wular Lake (Ramsar site). This Nallah runs through Budgam district and originates both from spring ^‘Sukhnag spring’ (Sokha Nag, the spring of solace) and high altitude glacier called Damdam in Damsar near Tossa Maidan. Nallah Sukhnag finally merges with the outlet of Hokersar wetland (Ramsar site) at Sozeith Narbal. This Nallah not only is a source of water for domestic and agricultural purposes, but also provides natural environment to aquatic biota particularly fish. The fish hosts were collected at 4 sites from Nallah Sukhnag from Feb, 2017-Jan, 2019 and brought alive to the Parasitological Research Laboratory by using small containers containing water. The sites were selected on the basis of flow of water, altitudes and the level of pollution. Sites selected were as:

The fish hosts were identified as Schizothorax plagiostomus Heckel, 1838 and Cyprinus carpio communis, Linnaeus, 1758. The sample size was calculated by using the formula given by Thrustfield¹²:

Hence, d = 0.05 and p = 0.5 (50%)

As no work was done on the selected study area, so the expected prevalence in the study area was taken as 50%¹³. Thus, the minimum desired sample size was calculated to 768. The fishes were then killed by a blow on head. Immediately after killing, fishes were examined externally and then thorough examination of the whole body surfaces was done with the help of stereomicroscope. For internal examination, fishes were dissected mid ventrally and whole body cavity was scanned for helminth parasites. Then visceral organs like alimentary canal, liver kidney and gall bladder were removed and scanned in separate petridishes containing normal saline for monogenetic and encysted digenetic trematodes.

Fig. 1:	Cestodes in Schizothorax intestine

Fig. 2:	Acanthocephalan in Schizothorax intestine

Few menthol crystals were added to the normal saline containing the parasite adhered to the intestinal wall which helped in their easy detachment from the intestinal wall.

In order to identify the recovered parasites (Fig. 1-3) permanent slide preparation of trematodes, cestodes and acanthocephalan worms was carried out.

Fig. 3:	Cestodes in Cyprinus

For this purpose worms were fixed in carnoy’s fixative, stained with acetoalum carmine, dehydrated in ascending grades of ethanol, cleared in Xylene and mounted in DPX. Photomicrography was taken with DP-12 Digital Camera attached to Olympus Research Microscope in the Department of Zoology, University of Kashmir, Srinagar. Identification of helminth parasites was then carried out by the keys given by Yamaguti¹⁴. Prevalence and mean intensity of helminth infection was calculated using formulas given by Margolis et al.¹⁵ and Gudivada et al.¹⁶ :

Data analysis: Data was fed into Microsoft excel and then analyzed by using Minitab Version 13. The effect of size, gender on the level of parasitism in fish hosts was analyzed by employing Chi-square test. The p-value is <0.05 (at 5% level of significance) was considered as significant.

RESULTS

Helminth parasites recovered during the current study were Diplozoon kashmirensis, Adenoscolex oreini and Pomphorhynchus kashmirensis from S. plagiostomus, Bothriocephalus acheilognathi and Pomphorhynchus kashmirensis from Cyprinus carpio communis (Fig. 4).

Fig. 4(a-g):

Posterior part of Diplozoon kashmirensis (Opisthaptor), (b) Anterior region of Adenoscolex oreini with smooth scolex, (c) Posterior part of Adenoscolex oreini showing inverted A shaped ovary (d) Scolex of Bothriocephalus acheilognathi heart shaped, (e) Gravid proglottid of Bothriocephalus acheilognathi, (f) Scolex of Pomphorhynchus kashmirensis bearing hooks and (g) Posterior end of Pomphorhynchus kashmirensis showing testes of male and cement glands

Table 1:	Gender wise prevalence of helminth parasites in S. plagiostomus and C. carpio communis

Table 2:	Size wise prevalence of helminth parasites in S. plagiostomus and C. carpio communis

Gender wise prevalence of helminth parasites in S. plagiostomus and in C. carpio communis: From February, 2017-January, 2019, out of 574 fish specimens of S. plagiostomus, 273 were males and 301 were females. Out of 273 males, 86 were infected with helminth parasites showing the prevalence of 31.5%. Out of 301 females, 59 were infected showing prevalence percentage of 19.6%. Prevalence of helminth parasites was higher in males than females. In C. carpio communis during the two years, out of 194 specimens, 100 were males out of which 16 were infected having prevalence of 16% and out of 94 females, 5 were infected giving the percentage prevalence of 5.31%. Significant differences were observed in both fish species in gender wise prevalence as p 0.05 (Table 1).

Size wise prevalence of helminth parasites in S. plagiostomus and C. carpio communis: During current study, out of 574 specimens of S. plagiostomus, 245 were in larger size and 329 were smaller. Out of 245 fishes of S. plagiostomus, 91 were infected giving the prevalence of 37.14% and out of 329, 54 were infected showing the prevalence percentage of 16.41%. Out of 194 specimens of C. carpio communis, 82 were larger in size and 112 were smaller. Out of 82, 15 were infected giving the prevalence of 18.29% and out of 112, 6 were infected giving the prevalence of 5.35%. Data is statistically significant at p<0.05 (Table 2).

Site wise prevalence of helminth infection: Out of 183 fishes belonging to S. plagiostomus collected from Site I (Beerwah), 34 were infected showing the prevalence percentage of 18.57%.

Table 3:	Site wise prevalence of helminth infection

Table 4:	Overall parasite wise prevalence and mean intensity of helminth infection

Table 5:	Parasite wise prevalence in S. plagiostomus and C. carpio communis

Out of 197 fishes from Site II (Makhama), 53 were infected showing the prevalence percentage of 26.9%. From Site III (Kanihama), 194 specimens of S. plagiostomus were collected, out of which 58 were infected showing percentage prevalence of 29.89%. From Site IV (Narbal), 194 fishes belonging to C. carpio communis were collected out of which 21 were infected with prevalence of 10.82%. Statistically significant differences were observed in site wise prevalence at p<0.05 (Table 3).

Overall parasite wise prevalence of helminth infection: During the current study, out of 768 fish specimens, 59 were infected with trematodes belonging to genus Diplozoon with a prevalence of 7.68% and mean intensity of 1.13, 68 infected with Adenoscolex oreini (cestode) with prevalence of 8.54% and mean intensity of 17.64, 9 were infected with Bothriocephalus acheilognathi (cestode) showing prevalence of 1.17% and mean intensity of 8 and 84 were infected with Pomphorhynchus kashmirensis showing prevalence of 10.93% and mean intensity of 9.98. The highest prevalence was of Pomphorhynchus kashmirensis and least was that of Bothriocephalus acheilognathi. Highest mean intensity was of Adenoscolex oreini followed by Pomphorhynchus kashmirensis then Bothriocephalus acheilognathi and lowest that of Diplozoon kashmirensis. Statistically significant differences were observed in parasite wise prevalence as well as mean intensity at p<0.05 (Table 4).

Parasite wise prevalence and mean intensity in S. plagiostomus and C. carpio communis: Table 5 showed the significant differences in parasite wise prevalence and mean intensity in S. plagiostomus and C. carpio communis at p<0.05. Out of 574 specimens of S. plagiostomus, 59 were infected with Diplozoon kashmirensis, 68 with Adenoscolex oreini and 72 with Pomphorhynchus kashmirensis showing prevalence percentage of 10.27, 11.84 and 12.54%, respectively. Mean intensities of Diplozoon kashmirensis was 1.13, Adenoscolex oreini 17.64 and Pomphorhynchus kashmirensis 10.18. Out of 194 fishes of C. carpio communis, 9 were infected with Bothriocephalus acheilognathi and 12 with Pomphorhynchus kashmirensis showed prevalence percentage of 4.63 and 6.18%, respectively. Mean intensities of Bothriocephalus acheilognathi and Pomphorhynchus kashmirensis in C. carpio communis was 8 and 8.33, respectively.

DISCUSSION

In current study significant variations were observed in helminth infection between males and females. Maximum helminth infection was reported in males in comparison to females that is in conformity with the study performed by Goselle et al.¹⁷, who reported maximum helminth infection in males of Clarias gariepinus and Tilapia zilli at Lamingo Dam, JOS, Nigeria. Mgbemena¹⁸ found a higher infection in male of C. lazera Emere¹⁹ reported differences in parasitic load between male and female fish and attributed it to differences in feeding habit of fishes as well as different degrees of resistance shown to infection. Bekele and Hussien²⁰ observed the highest infection rate of parasites in males of Oreochromis niloticus and Clarias gariepinus in Lake Ziway, Ethiopia. Our findings are in conformity with Gautam et al.⁴, who also observed highest helminth infection in males of Channa punctatus and Channa striatus. Khan et al.²¹ observed the seasonal variation of parasitic infections in fish Johniuss dussumieri where more males were infected as compared to female fishes that supports our results. However Adegbehingbe and Umezurike²² noticed higher infection of helminth parasites in females of Parachanna Obscura than males that is in contrast to our study. Adikwu and Ibrahim²³ reported high infection in females as compared to male in Clarias garienpinus, Nigeria. According to Emere and Egbe²⁴, the highest infection in female fishes is attributed to physiological state of most gravid females that could have reduced resistance to infection by parasites.

According to our study maximum helminth infection was observed in large sized fishes of both S. plagiostomus and C. carpio communis in both years that is supported by Dar et al.²⁵ also reported that increase in prevalence in Cyprinus and Schizothorx species with larger size. Ibraq and Fayaz²⁶ observed the maximum infection in large size fishes of both S. plagiostomus and C. carpio communis. As the fish grows older, there is greater possibility of parasitic larvae to get accumulated from year to year and may lead to increased parasitism in larger fishes²⁷. Maximum prevalence of parasites in larger fishes could be also attributed to the longer time of exposure to the surrounding environment by large body size²⁸.

Our findings clearly demonstrate that there exists a significant difference in the prevalence of infection in different study sites on the same water body (Table 3). The results of current study revealed the maximum prevalence of helminths in fishes at Site III (Kanihama) that may be due to pollution of water that have increased the development of intermediate hosts at that region and ultimately have increased the parasitic load in fishes²⁹. This rise in prevalence infection at Site III is in agreement with Khurshid and Ahmad³⁰, who reported the higher parasitic prevalence at highly polluted site in comparison to other sites in Shallabugh wetland. Our findings are also in agreement with Qayoom et al.³¹, who observed the maximum prevalence of helminth infection in cold water fishes of River Jhelum at polluted site.

During this study, significant differences were observed in overall parasite wise prevalence and mean intensities. Among acanthocephalans, only Pomphorhynchus kashmirensis was reported that showed highest prevalence which is inconsonance with the earlier findings^32,33. Ecological factors including feeding behavior, diet of the host, water temperature³⁴ and the wide host range³⁵ are the main factors for influencing the parasitic prevalence. Significant differences were also reported in parasite wise prevalence and mean intensities in S. plagiostomus and C. carpio communis in both years as p-value is <0.05. In current investigation, S. plagiostomus was abundantly infested with acanthocephalans followed by cestodes and then trematodes that is in accordance to Yousuf et al.⁷. This differences in parasitism in above fish species is attributed to the different preferences of fishes for food at different phases of life³⁶ and the parasitic resistance of the exotic host i.e., Cyprinus species.

CONCLUSION

This study concluded that prevalence of helminths was maximum in large sized and male fishes in comparison to smaller and females. In S. plagiostomus and C. carpio communis maximum prevalence was shown by P. kashmirensis. This study showed that S. plagiostomus were abundantly infected with helminths than C. carpio communis and prevalence of helminths mainly depends on fish feeding habits, pollution of water, sex and size of fish.

SIGNIFICANCE STATEMENT

To enhance the fish production, it is important to study their parasitic infestation as the pathogenicity of helminths is responsible for causing extensive damage to fish health. In order to prevent and eliminate such infections from the fish, it is extremely important to achieve early and correct diagnosis of the larval stages of the parasites. It is imperative to evaluate the parasitic load in fish as it serves as an essential tool that not only provides information concerning health status of fish, but also provides information on their own changes in diversity, prevalence and abundance related to different environmental stressors. The data of this study will give a baseline data for other studies.

ACKNOWLEDGMENT

Authors are highly thankful to Department of Zoology, University of Kashmir, for providing the equipments and other facilities for successfully carrying out this work.