Chapter 4 OBSERVATIONS

Transcription

1 Chapter 4 OBSERVATIONS

2 T present study on electron microscopy and life cycle study of some cestode fish parasites in Kashmir Valley involves two distinct aspects and accordingly the observations are presented separately under two main headings. Morphology and Life Cycle M orpholo gy In order to understand the life cycle in right perspective, it was felt necessary to have a detailed morphological observations o f the parasites, both light microscopic and electron microscopic. During the present endeavour two cestode parasites, viz., Bothriocephalus and Adenoscolex were recorded from the fishes; brief account of these is presented in the following pages. 4.1.A. Bo^riocephalus acheiiognathi Order Pseudophyllidea Cams, 1863 Order diagnosis - Eucestoda; Scolex with two, dorsal and ventral grooves (bothria) or lobes (bothridia); neck conspicuous or not. Strobila with well marked external segmentation, often weak or lacking; proglottides anapolytic. commonly acraspedote and linear, each containing usually one set o f reproductive organs, sometimes two sets. Genital apertures surficial in some families, marginal or submarginaj in others. Testes follicular, numerous; eggs commonly but not invariably operculated, liberating coracidium. Procercoid larval stage in crustanceans, plerocercoid larval stage in fishes. Adults mainly parasitic in fishes. 56

3 Bothriocephalidae Blanchard, 1849 Family diagnosis - Pseudophyllidea; small to large forms. Scolex elongated, more or less rectangular, sometimes spherical, club- or heart-shaped, usually with apical disc which may bear marginal hooks occasionally. Bolhria longitudinally elongated. Neck lacking. Stiobilia with distinct segmentation, of)en with secondary segmentation. Proglottids craspedote, each with more or less distinct median furrow and indistinct marginal groove. Testes medullary, in two lateral fields, continuous from proglottis to proglottis and sometimes across median line. Cirrus pouch round, in median field. Ovary bilobed or not, in ventral median medulla. Seminal receptacle present or absent. Vitellaria usually cortical. Uterine pore midventral opposite and anterior to cirrovaginal pore; eggs operculate, not embryonated when laid. Longitudinal excretory stems medullary, in testicular fields or just lateral to them. Bothriocephalus Rudolphi, 1808 Generic diagnosis: Bothriocephalidae; Scolex elongated, sometimes spherical or enlarged posteriorly, with apicaj disc, the bothrial edges o f which are indented. Marginal surface of scolex convex or concave, often longitudinally grooved. Bothria longitudinally elongated, o f varying length and depth. Neck lacking. Segmentation complete, often v^nth secondary segmentation. Proglottides craspedote, anapolytic; anterior ones bell or funnel shaped, posterior ones rectangular. Longitudinal excretory stems 2 or 3 on each side in medulla, l estes in lateral medulla. Ovary compact, transversally elongated, bilobed or not. Seminal receptacle absent. Vitellaria entirely cortical. Hggs thin shelled, operculated, not embryonated when laid. 57

4 Bolhriocephalus acheilognathi'(amaguti, 1934 (Figs. 1-12; Phgs.18-20; Pmg. 1-12; 21-28) Pseudophyllidea Carus, 1863 Bothriocephalidae Blanchard Bothriocephaius (Rudolphi, 1808) Luhe, 1899 B. acheilognathi Yamaguti, 1934 Species diagnosis Bolhriocephalus acheilognathi, a pathogenic inteslinaj tapeworm, was obtained in the intestine o f fishes, mostly in the pyloric region. The classification o f the bothriocephajid worms is based primarily on the shape o f the scolex (Mashego, 1982). According to Pool (1984) identification o f adujts o f B. acheilognathi should be based on the heart shaped scolex and prominent square apical disc. Identification o f tapeworms in the current study was. therefore based on these characteristics o f the particular species. Cestodes found in the present study were compared to sketches provided by various authorities as well as the diagnosis of B. acheilognathi by Yeh (1955) as cited in Papema (1996). Following characteristics were found with the help of light microscopy: Worms variable in size and number of segments. Eggs operculate and premature when laid. Scolex club or heail-shaped. laterally fiat, with apical disc, lateral grooves (Bothria). Mature segments broader than long. Gravid segments longer than broad. Neck lacking. 58

5 Flg.1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Figs. 1>5 Eggs of B. acheilognathi at different developmental stages Fig. 6 Coracidium of B. acheilognathi

6 Fig. 7 Procercoid of S. acheifognathi 8.9 Immature segments of B..ch e lla g n a lh l Figs,

7 Fig.12 G ravid se g m e n ts of B. a c h e ilo g n a lh i

8 P h g. 2 0 P h g s H e a v y In fe c tio n o f B. a c h e ilo g n a th i in a c a rp, s h o w in g th e p a ra s ite s th rou gh the transparent blocked intestine

9 Pm g 1. Scole* of 8. och e ilogn ath i Pmg. 2. Scole* w ith apical disc P m g. 3. E a rly Im m a tu re seg m ents P m g. 4. Im m a tu re segm ents Pr g, 5. Late Im m a tu re se g m e n ts P m g. S.P o ste rio r reg io n o f im m a tu re segm ents

10 Pmg. 7. Early m ature segments Pmg. 8. Early m ature segmenu P m g. 9. U t 0 M a tu re se g m e n ts segm ents T I A «Pmg. 11. Gravid segments Pmg. 12. Fully gravid segments Pm gs Different m aturation stages of 8. a c h e ilo g n a th i

11 P m g. 18 P m g. 24 Pm g. 25 Pm gs Different stages o f egg d e velo pm ent of B. a c h e ilo g n a th i P m gs. 26 and 27 - C o ra cid iu m of 8. a c he ilo g n a th i Pmg Procercoid of B. a c he ilo g n a th i

12 Pmg. 13. Scolex showing bothria Pmg. 14. Body of Adult B. a c he ilo g n o th i iiiaamiiatmaa Pmg. 15. Scolex of B. a c h e ilo g n o th i Pmg. 16. Bothrium enlarged P m g. 17. B o th riu m sh o w in g m icro triches and tu m u li PrTJgs S c a n n in g E le c tro n M ic ro s c o p v o f B. a c h e ilo g n o th i

13 ObserDations The ovoid eggs (m easuring 0.058x0.062m m ) possessed a distinct operculum at the narrow er end. The SEM study o f coracidium showed the presence o f num erous cilia. It is the presence o f cilia which m akes the coracidium motile. The procercoids w ere elongate w ith the anterior part alm ost triangular. The procercoids possessed a large, w ell developed cercom er, containing rudimentary embryonic hooks at the posterior extremity. Scanning electron m icroscopy o f scolex revealed elongated, deep and pear<shaped bothria. A prom inent bilobed apical disc w as present. Tlie tegument o f scolex possessed num erous m icrotriches. The m icrotriches were more condensed w ithin the bothria. Tlie m icrotriches o f scolex were m orphologically dififerent from the m icrotriches o f the strobila. Presence o f tum uli was also observed by SEM study. The tum uli w ere m ore condensed on scolex and became less abundant posterioriy along the strobila. 4.I.B. Adenoscolex Order Caryophyllidea: Small forms possessing only one set o f reproductive organs. H oldfast undifterentialed. or w ith grooves with som etim es sufficiently broad to sim ulate bothria. G enital aperture and uterine aperture opening on the ventral surface o f the body. U terus and vagina com m only discharging into a com m on utero-vaginal canal. Y olk glands cortical or m edullary, or partly cortical and partly medullary, according to family. 60

14 Caryophyllaeidae Leuckait, 1878 Family diagnosis: Small forms with the holdfast end varying in shape. Genital apertures on the last fourth o f the ventral surface. Utero-vaginal auium present but w ithout a sphincter muscle. Longitudinal parenchymal muscles in two layers. Yolk glands medullary. Adenoscolex oreini Fotedar, 1958 Generic diagnosis: with a broadened, curled or folded holdfast end, without pseudobothrial depressions. Cirrus pouch opening into a shallow, noneversible atrium. Uterine coils never anterior to the cirrus pouch. No external seminal vesicle. Adenoscolex oreini Fotedar, 1958 ( P m g l;f ig s ) Caryophyllaeidae Leuckart, 1878 Capingentinae Hunter A denoscolex Fotedar, 1958 A. oreini Fotedar, 1958 Description This tapeworm was observed to inhabit the latter 1/3" part o f the intestine. Body elongated, dorsovenu^ly flattened with crenated margin posteriorly. Fully mature w orm s in posterior part o f the body w ere broader and thicker. Scolex smooth, not clearly marked o ff from remaining part o f the body, slightly wider than body width, showing variation in shape in differenl forms. Anterior border smooth and somewhat truncated, without any external frills, wrinkles, grooves or 61

15 bothria; gland cells developed extensively in scolex region, extended posteriorly in well developed colum ns for m ore than three quarters o f anterior body length. A nterior end mm wide. N eck short, not clearly demarcated, slightly narrower, followed by cylindrical portion o f body. A single set o f genitellia, restricted to posterior seventh o f body except for testes and vitelline follicles, which occupy a great proportion o f whole anterior body. M ale and female genital openings separate. Testes rounded or broadly oval, scattered medially throughout most o f the body, bounded by vitelline follicles, extending from a short distance posterior to base o f neck region up to anterior region o f cirrus sac. Vas deferens loosely convoluted tube, median, anterior to cinis sac. Single ovary, posterior, medullary, having the outline o f inverted A. lower horns bend inwards. O varian isl32 hmus more or less at middle. Vitellaria numerous, cortical as w ell as medullary, posterioriy forming post ovarian vitelline follicles. U terus well developed, com pactly coiled never extend beyond cirrus sac. W ell developed shell gland behind isthmus. Gland cells surrounding uterine coil, few in pre-isthmus region. Eggs oval or boat shaped and operculated. W ith the help o f electron microscopy, tw o types o f microtriches were observed. O ne was the cone-like m icrotrix o f the attachm ent type that prevailed on the scolex. The second type was the filamentous m icrotriches localised in the middle and posterior part o f the body. The second type was more densely arranged than the preceding type. 62

16 Figs Anterior, middle and posterior regions of an immature Adenosco lc.

17 Fig.16 A nterior region of aduit A denoscolex

18 ,1 7 posterior region of adult/>deno.co/«fig

19 Pm g. 29. Scolex of A d e n o s c o le x Pm g.30. Scolex of A d e n o s c o le x Pmg 31 M iddle portion of 4</p/>osco/e)r Pmg. 32 Iggi imide AdenoscoleM P m g. 33. P o s te rio r p o rtio n o f A d e n o s c o le x P m g A d u l t A d e n o s c o le x

20 y j. /r- P m g. 3 7 P m g. 39 P m g. 40 P m g s E g g s o f A d e n o s c o le x ; C o r a c i d i u m o f A d e n o s c o le x

21 Pm g. 34. SEM of adult Adenoscolex Pm g. 36. S E M o f A d u lt Adenoscolex show in g posterior region P m g s S c a n n in g E le c tro n M ic ro s c o p y of Adenoscolex

22 Obserpations Table 2. Table showing comparative characteristics o f Adenoscalex as present study Characteristics Fayaz, 1993 Present observations Body length 49.5 mm 51mm Maximum widdi 1.64 mm 1.58 mm Scolex mm mm Neck 1.0-I.lm m mm Testes x mm x mm Ovarian isthmus x mm x Wings of Ovary x mm x mm Eggs x mm * mm Host Schizothorax niger & S. esocinus Schizothorax spp. Locality Anchar Lake and Manasbal Lake Dal Lake and River Jhelum Life Cycle Study Life cycle study in the present investigation v^as carried out in natural a well as under experimental conditions. Accordingly this portion o f observations is presented under two separate headings of natural and experimental conditions. 4.II.A. Study under Natural Conditions To understand the life cycle in natural conditions, infection dynamics of B acheilognathi and Adenoscalex were observed in both the intermediate hosts as well as in the final fish host. 4.IIA1. Infection dynamics The infection dynamics has been divided into four main headings: 4. IU.1.3. Infection dynamics of B. ac/ie//ogna(/i/in different fish species 4.11.A.1.b A.1.C. Seasonal dynamics of S. ac/iez/ognatfi/in different fish species Infection dynamics of 8. acftei/ognaf/ii in the copepod intermediate host 4.11.A.1.d. Seasonal abundance of Adenoscalex in Schizothorax and Cypnnus 63

23 ObsetvaNons 4,11 A1.a. Infecbon dynamics of 6. acheilognathi in different fish species The prevalence, mean intensity and abundance of B. acheilogmlhi were examined in different species o f Schizothorai and Cypr'mus. A well marked variation was observed among the different species of both the types of fishes. The observations are presented below: 4.II.A.1.a.fl). Infection dynamics in Schizolhorax species O f the 353 Schizolhorax spp. sampled during the present smdy, 45 fishes were found to be infected with B. acheilogmlhi. These fishes harbored 772 worais of B. acheilognalhi. The results are presented in Table 3. Table 3. Percentage incidence, mean intensity and abundance of B. acheilognathi in Schizolhorax species Fish host Number examined Number infected Number of parasites Percentage incidence (%) Mean intensity Abundanc 6 Schaotew esocmus S. cutvifrons S. ttiger Total Fig. 18. Graph depicting the prevalence o f B acheilognalhi in four Schizolhorax spccies

24 Prevalence: (Fig. 18). The prevalence o f B. acheilognalhi in Schizalhorax was 12.74%. The highest and the lowest prevalence among the Schizalhorax was observed in Schizothorax esocinus (25%) and S. cunifrons (5.6%). Statistical analysis indicates that there is a significant difference (P-value=0.004) in the prevalence of B. acheilognalhi among the different species of Schizolhorax. Fig. 19. Graph depicting the Mean intensity of B. acheiloj;nalhi in four Schizolhorax species Mean inlensily: The lowest and highest values for mean intensity of B. acheilognalhi were observed to be opposite to the values as observed for the prevalence. The maximum mean intensity was observed in S. curvifrons (53.8) and the lowest in S esocinus (10.73); (Fig. 19). Statistical analysis indicates that there is a significant difference (P-value=0.005) in the mean intensity of S acheilognalhi among different species of Schizolhorax.

25 Ohseroations Fig. 20. Graph depicting the abundance of B. acheilognathi in four Schizothorax species Abundance: (Fig. 20). The abundance o f B. acheilognathi in Schizothorax species was found to be The maximum abundance (2.26) was observed in S. esosinus and lowest abundance (1.42) was observed in S. niger. The difference in the abundance of B. acheilognathi was statistically significant (P-value=0.000). 4.II.A.1.a.(ii). Infection dynamics In the Cyprinus species O f the 319 Cyprinus species sampled during the present study. 42 fish specimens were observed to be infected with B. acheilognathi (13.16%). 759 worms of B. acheilognathi were harbored by these fishes ( Table 4). Ta b le 4. Perceotage iocidedce, m eao intensity and abundance of B. acheuognaihi in Fish host Number examined Number infected Number of parasites Percentage incidence(%) Mean intensity Abundan ce C.c.communis C.c.specvlaris n Total J 7 66

26 Fig. 21. Graph depicting the prevalence o f B. acheilognathi in the two species o f Cyprinus Prevalence: The Cyprinus carpio specularis showed comparatively higher (13.77%) prevalence than Cyprinus carpio communis (12.66%). Statistical analysis indicated that the difference in prevalence between these two species was insignificant (Fig. 21). Fig. 22. Graph depicting the mean intensity of B. acheitonnalhi in the two species of Cyprinus Mean inlensily: (Fig. 22). The mean intensity was also higher in Cyprinus carpio specularis than Cyprinus carpio communis. The difference in the mean intensity of the B. acheilognathi among these two species is statistically insignificant (! = ).

27 S < 100 j 50 J 0 ONumbefexarr Abundance C.carpio cummunis Ccarpio specularts Fish species Fig. 23. Graph depicting the abundance of B. acheilognafhi in the two species o f Cyprinus Abundance: (Fig. 23). The abundance was also higher in Cyprinus carpio speculahs than Cyprinus carpio communis. The difference was statistically significant (P-value= 0.01). 4.II.A.1.a.(iii). Comparison of infection dynamics between two fish types In general the prevaiencc, mean intensity, and abundance of B acheilognalhi in 672 fish specimens collected during the present study were 12.94%, and 2.27 respectively. The Cyprinus species showed more infection (in terms o f prevalence, mean intensity and abundance) than Schizolhorax species. Statistically the difference in the infection status among the two types o f fishes was not significant (Table 5; Fig. 24, 25 and 26). 68

28 Table 5. Percentage incidedce, mean intensity and abundance o f B. acheuognathi in Schizothorax and Cyprinus carpio Fish host Number examined NumlMr infected Number of parasites Percentage incidence Mean intensity Abundance Schizolhorax spp C. carpio spp, Total ^ Fig. 24. Graph depkaing the peroenlage incidenoe of B acheihgnaihi in Schizolhorax spp. and Cypime a rjx i g ONumbef examined 200 * Schizothorax spp C cafpio spp Fi«h species Fig. 25. Graph depicting the mean intensity of B ac-heihnnaihi in Schizolhorax spp. and ( yprimis c a r p i o

29 Fig. 26. Graph depicting the abundance of B. acheilogm thi in Schizothorax spp. and Cyprinus carpio 4.lt.A.1.b. Seasonal dynamics of B. acheifognstfii It was necessary to study the seasonal dynamics o f adult cestode in natural conditions in order to understand its life cycle. Seasonal occurrence was observed in Schizothorax as well as in Cyprinus carpio. 4.II.A.1.b.(i) Seasonal dynamics of a acheilognathi in Schizothorax The present investigation reveals a definite seasonal dynamics o f B. acheilognathi in both types o f fishes, Schizothorax (Table 6). T able 6. Percentage incidence, mean intensity and abundance of Season Number examined Spring Number infected P-value Incidence {%) Number of parasites MeanlSD Abunda nee ± Summef ± Autumn ± Winter ± T o ta l , IS ±

30 Prevalence: (Fig. 27). In Schizothorax, the highest (23.07%) and lowest (6.66%) prevalence was observed in the autumn and winter seasons. Statistical analysis indicates that the variation in the prevalence among different seasons was significant (0.02). F igure 27. Graph depicting the incidence o f B. acheilognaihi in Schizothorax in four seasons Mean intensity: The highest (24.5) and the lowest (6.4) mean intensity was observed in autumn and winter seasons respectively (Fig. 28). The variation in the mean intensity among the different seasons in Schizothorax was observed to be statistically significant (P-value= 0.005). Fig. 28. Graph depicting the mean intensity of B. acheilognaihi in Schizothorax in four seasons

31 Abundance: (Fig. 29). Abundance also followed the same trend, being highest (5.64) and lowest (0.42) in autumn and winter seasons respectively. The difference in the abundance in different seasons was statistically significant (Pvaiue= 0.000) I 200 I ^ onumbef examined abundance Spring Summer Autumn winter Total Fig, 29. Graph depicting the abundance of B. acheilo^nathi in Schizolhorax in different seasons 4.II.A.1.b.(ii) Seasonal dynamics of S. acheilognathi in Cyprinus carpio Table 7. P ercentage incidence, mean intensity and abundance of B. acheilognathi in Cyprinus in four seasons Season Number examined Number infected P-value Incidence (%) Number of parasites Meao±SD Abundan ce Spring ± Summer ± Autumn ± Winter ± Total I8.07± Prevalence: In Cyprinus the highest (20.20%) and lowest (7.46%) prevalence of Bolhriocephalus infection was observed in autumn and winter respectively. The difference in the prevalence o f Bolhriocephalus infection among the different seasons was not statistically significant (P-Value=O.115) (Fig. 30).

32 ObserDations f T - T H b - f H Spring Summer Autumn winter Total Season I Number examined Numbef infeaed Fig. 30. Graph depicting the percentage incidence o f B. acheilognathi in Cyprinus carpio in four seasons Mean intensity: Mean intensity o f Bothrioceph alus infection in Cyprinus was observed to be highest (21.9) in summer and lowest (7.2) in winter season. Statistically the seasonal variation in the mean intensity was not significant (Fig. 31). Fig. 31. Graph depicting the mean intensity of B acheihgnalhi in Cyprinus carpio in four seasons Abundance: Abundance followed the same Uend as the prevalence i.e. highest (4.15) in autumn and lowest (0.53) in winter season. The seasonal variation in the abundance was observed to be significant statistically (P= 0.000) (Fig. 32). 73

33 = 150 < Spring Summer Autumn winter Total Season QNumber examined abundance 4.IIA1.b.(iii). Spring season Fig. 32. Graph depicting the abundance o f B. acheilognalhi in Cyprinus carpio in four seasons Comparison of seasonal infection dynamics in Sch/zotfiorax and Cyprinus In the spring season, out o f 175 fish specimens o f both S c h iz o th o ra x and Cyprinus were collected, 17 fishes were found to be infected with B. acheilognalhi, harboring 252 worms with a prevalence o f 9.71%, mean intensity of 14.8 and abundance of 1.44 (Table 8). Table 8. Percentage incidence, mean intensify and abundance of B. acheuognashi in spring season Fish type Number examined S ctiizo^om Number infected p. vaiue incidence Number Mear\iSD Abundance (%) (parasites) 10.75% ± Cypmu& % ± species Total % 252 I4.8±

34 ObserDations Prevalence: (Fig. 33). In the spring season, the prevalence o f B. acheilognathi was observed to be higher in Schizothorax (10.75%) than Cyprinus (8.53%). Statistical analysis indicates that the difference was not significant between the prevalence o f these two fish types. 33. G t ^ dqiictii^ die comporison of prevalence of R acheihfftmk in Offxraff o»pw and& to^onax in sprii^ season Mean intensity: Mean intensity o f this tapeworm was also found to be higher in Schizothorax (15.9) than Cyprinus (13.3). Statistically the difference was not significant (Fig. 34). 2- I 500 J ; Schizothorax spp C carpiospp Fish species ONomber exami'^eo Mean ifitensin Fig. 34. Graph depicting the comparison of mean intensity o f /i acheilagnalhi in Cyprinus and Schizolhorax in sprinj; season

35 Abundance: (Fig. 35). Higher value of abundance o f B. acheilognalhi in spring season was observed in Schizothorax (1.70) than Cyprinus (1.13). The difference in the abundance among the two fish types was statistically significant (0.04) I 150 ^ ^ 0 J I C.cafpio cummunis Ccarpio speculans QNumbef examined Abundance Fig. 35i Graph dspklmg the comparison of abundance of B. acheilognalhi in CJprinic and &teorfbraar in spring season Sum m er season In the summer season, o f the 167 fishes examined. 19 fishes were found to be infected with 286 worms of B. acheilognalhi-, thus showing a prevalence of 11.37%, mean intensity o f and abundance o f 1.17 (Table 9). Table 9. Percentage incidence, mean intensity and abundance of B. acheuognalhi in sum m er season fish type S chizothorax spccies Number examined Number infected P value Incidence (%) Number parasites MeanlSD Abu ndari ce ± C yp rin u s I.9 ± specics 0 T o ta l I5.0 5 ±

36 Prevalence: In the summer season, Cyprinus (13.69%) showed higher level of Bothriocephalus infection than the Schhothorax (9.57%). The difference was observed to be statistically insignificant (P= 0.45) (Fig. 36). Fig. 36. G i^depictii^ the comparison of pievalwce o f advilo^^iaihi'mcyphnuisn^ 5yiE «/»rar in summer season Mean intensity: Mean intensity o f this womi was also observed to be higher in Cyprinus (21.9) than Schizolhorax (7.4) in the summer season. Statistically the difference in the mean intensity between these two fishes was insignificant (Fig. 37) ] SchizoUiorax spp C carpio spp Fish species ONumber examined Mean intensity Fig. 37. Graph depiding the compmson of mean irtensity of R advili>i^tihi in and.sctert/m n: in summer season

37 Abundance: The abundance w as also higher in Cyprinus (3.0) than Schizothorca (0.17). The difference in abundance was observed to be statistically significant (0.000) in summer season (Fig. 38). Autum n season Fig. 38. Graph depicting the ccmparison of abundance of R adieilo^iathi in QpimeandSchizolhonicmsjrnniaseaBC^ In the autum n season, 190 fishes were examined in which 41 were found to be infected with 925 worms o f B. a c h e ilo g n a lh i. Thus showing a prevalence of %, mean intensity o f and abundance o f 4.86 (Table 10). T able 10. P ercentage iocidence, m ean intensity and abundance of B. acheuognathi in autum n season Fish type Number examined Number infected P value Incidence (%) Number of parasites Mean±SD Abund ance Scftizo/horax species C ypnm s species ± ± T o t a l ±

38 Prevalence: In the autumn season, Schnolhorax showed higher (23.07%) prevalence o f Bothriocephalus infection than Cyprinus (20.20%) (Fig.39). Statistically the difference was insignificant. Fig. 39. Gra ]h depicting the comparison of pp^v^ilence of R and in autumn season in Mean imensiry. Mean intensity also showed higher values in Schizoihorax (24.5) as compared to Cyprinus (20.5) in the autumn season (Fig. 40). The difference was statistically insignificant (0.58). Fi& 40. Gn^ih depicting the comparison of mean inlaisity of fi aim ignclh in ( are!.siacwarirar in autumn aascn

39 ObserDations Abundance: The abundance in Schizothorax (5.64) was observed to be higher as compared to Cyprinus (4.15) (Fig. 41). Statistical analysis indicates that the difference between abundance of these fishes was significant (0.05) I 0 Schizothorax Cyprinus species Total $peaes ONumber examined Atxindance Fish speciss Fig. 41.Graph depicting the comparison o f abundance o f B. acheilognalhi In Cyprinus and Schizolhorax in autumn season W in ter season In the w inter season 142 fishes were collected and a tk r examination. 10 fishes were found to be infected with 68 tapeworms o f Bothriocephalus acheilognalhi; thus showing a prevalence o f 7.04%, mean intensity o f 6.8 and abundance o f 0.47 (Table 11). T able I I. Percentage incidence, m ean intensity and abundance of B. acheilognathi in w inter season Fish type Number examined Number infected P value Incidence (%l Number (parasites) MeantSO Abund ance Schizothorax spp ± Cyprinus ±I 0.53 spp. T otal ±

40 Prevalence: In w inter season, Cyprinus (7.46%) showed higher levels of prevalence o f B. acheihgnathi as compared to Schizolhorax (6.66%). The difference in the prevalence among these fishes was not significant (Fig. 42). 42. depictir^ the comparison of inddenoe of II CKheihgnathmCyprinusapdScHzoOionix'mvnit^seasm Mean intensity: Mean intensity was higher in Cyprinus (7.2) than Schizothorax (6.4) in this season. Statistically the difference was not significant between the mean intensity o f these fishes (Fig. 43). F1& 43. Graph dqiictingth: comparison ofmean irtenatyoffl acheilii^mhi'mcypnnic^ ait?*:) and iscfeji/wnar in wirter season 81

41 Abundance: Abundance o f Bothriocephalus infection was also higher in Cyprinus (7.2) than Schizotkorax (6.4) (Fig. 44). The difference was not statistically significant (0.435) Number examined Abundance ScMzothorax spp Cypnnus spp Fish species Fig, 44. Graph depicting the comparison o f abundance o f B. acheilognalhi in Cyprinus and Schizothorax in winter season Thus, from the above observations, it was concluded that highest percentage incidence in lx)th types o f fishes was seen in autumn. It was 23.07% in Schizothorax species and 20.20% in Cyprinus carpio. Both types o f fishes showed least infection during winter season. 4.IU.1.b.(w ). Seasonal variation of different maturation stages of B. acheilognathi in tfte Schizothorax and Cyprinus Based on the stage o f maturation, B. acheilognathi obtained during the present investigation were divided into three groups, immature, mature and gravid worms (Table 12 and 13). In spring season, out o f 159 w o m s obtained from Schizothorax. 117 (73.58% ) were immature w oims, 33 worms (20.75%) were mature and only (5.6%) were gravid worms. In Cyprinus spp., out o f 93 worms obtamcd, 72

42 (77.4%) were immature, 17 (18.27%) were mature and only 4 (4.3%) worms were observed to be gravid. In summer season among 67 worms were obtained from Schizothorax, 53 (79.10%) were immature, 11 (16.41%) were mature and only 3 (4.47%) w om s were gravid. 219 worms were obtained ftom the Cyprinus spp. Among these 219 worms, 183 (83.56%) were inunature, 27 (12.32%) were mature and 9 (4.10) worms were gravid. In autumn season, out o f 514 worms obtained from Schizothorax, 23 (4.47%) were immature, 73 (14.20%) were mature and 418 (81.32%) were gravid worms. From Cyprinus spp. 411 worms were obtained during autumn season. Out o f these 411 worms, 46 (11.92%) were immature, 49 (11.92%) were mature and 316 (76.88%) were gravid worms. In winter season 32 worms were obtained from the Schizothorax, among these worms 11 (34.37%) were immature, 6 (18.75%) were mature and 15 (46.87%) were gravid worms. From Cyprinm spp. 36 worms were obtained in this season. Among these 15 (41.6%) were immature, 2 (5.5%) were mature and 19 (52.77%) were gravid worms. The percentage incidence o f different maturation stages is presented in the Tables 12 and 13 below: Table 12. Seasonal variatiod o f difterent maturation stages o f B. acheuognathi io the Schizothorax Seasons N um b e r o f parasites Number ImmaCure worms N um ber of mature worms N um ber of gravid worms Spring / 73.58% 33/20.75% 9/5.66% Summer 67 53/79.10% 11/16.41% 3/4.47% Autumn /4.47% 73/14.20% 418/81.32% Winter 32 11/34.37% 6/18.75% 15/46.87%

43 O bservations T able 13. S e c o n a l v aria tio n o f diffe re n t m a tu ratio n stages o f B. acheiiognathi in the Cyprinus carpio Spring Summer Autumn Winter N u m b e r o f parasites N u m b e r Im m a tu re w orm s 72/17.4% 183/83.56% 46/11.19% 15/41.6 N u m b e r of mature worms 17/18.27% 27/12.32% 49/1 1.92% 2/5.5% N u m b e r of gravid worm s 4/4.3% 9/4.10% 316/ /52.77% Thus at the end o f spring season and during the sum m er season most wom is obtained w ere young specim ens. During autum n season most o f the worms obtained w ere fully developed specim ens with gravid segments. In winter very low incidence o f infection w as observed. This trend was shown by both fish types, i.e., Schizothorax and Cyprinus carpio A l e. Infection dynamics of B. acheiiognathi in the copepod intemiediate host copepods {Cyclops) w ere collected from Septem ber 2005 to August Out o f these 69 copepods were found to be infected with 140 procercoids of B. acheiiognathi; thus showing a prevalence o f 3.46%, mean intensity o f 2.02 and abundance o f The results are presented in the Table 14. T able 14. Infection o f Cyclops spp. w ith procercoids o f B. acheiiognathi (u n d e r n a tu ra l h ab itats) Month Nurntor Cyctop* Cyclop* WttJl Infection Number procercoid* ktchlenc* (%) Mean AtMindan ce Mean Water temperature C September October November December January Febniary March April May J June July August y. - - J Total

44 In the months o f December to March, no infection was detected. The infection (in terms o f prevalence, mean intensity and abundance) o f Bolkriocephalus in Cyclops showed an increase with the uicrease in temperature, teaching a peak in July (4.9%, 2.84, and 0.14 respectively) when the temperature was 29 C (28-30 C) (Figs. 45,46 and 47). Fig. 45. Graph depicting the monthly percentage Incidence of B. acheilognalhi in the copepod intermediate host Fig. 46. Graph depicting the monlhly mean imeraty of B the oopepod intemiediale host in 85

45 Months N u m b e r Cyclops N um ber procercoids Fig, 47. Grai* dqdicting the mwithly abundance of B. acheilogmthi in copepod intennediate host Table 15. Seasooai variation o f B. acheuognathi infection in Cyclops spp. (udder natural habitats) Season Number of Cyclops Cyclops whh infection Number of procercoids Percentage incidence Mean intensity Abundan ce Spring Summer Autumn Winter N u m b e r of Cyclops C y c lo p s with infection Rg. 48l Graph depicting the aasonal variation in percentage incidence of II acheilognalhi in the copepod intennediate host

46 Fig. 49. Graph depicting the seasonal variation in mean intensity o f 5. acheilognathi in the copepod intermediate host N u m b e r of C yclops N u m b e r of p rocercoids 50. Graph depicdng the seasonaj vbiiaion in abundance of fl in the copepod irtomedialc hoa From the above observations it is concluded that B acheilo^naihi in natural conditions takes about one year to complete its life cycle. According to the present observations, recruitment o f fish hosts takes place from lale spnni; lo midsummer, when the intermediate hosts showed higher levels ol Bolhriocephalus infection. After infecting the final host, they develop and gro«in summer and autumn and most o f the worms obtained in autumn were tully matim: with gravid segments. Intemiediatc host started showing infection from

47 Obseroations the April i.e., during spring and the infection showed increase, reaching a peak in mid-summer. The present observations suggest that B. acheilognalhi overwinter in the egg stage (Figs. 48,4 9 and 50). Spring/midsunimcr Peak of Copepods infection " Copepods infected Summcr/autumn Peak of adult infection in fish Winter Eggs liberated Life cycle of B. acheuognathi under natural conditions 4. II. A.1.d. Seasonal abundance of Adenoscolex in Schizothorax and Cyprinus carpio Infection dynamics o f Adenoscolex was observed in diltercnt seasons in both Schizolhorax as well as in Cyprinus carpio. Cyprinus carpio did not show any infection o f Adenoscolex. In Schizolhorax spp., out o f 321 Schizolhorax spp., only 9 fishes showed infection o f this parasite, containing 74 tapeworms, fhus showmg prevalence o f 2.80%, mean intensity of 8.2 and abundance of 0.23.

48 Highest prevalence o f infection o f Adenoscolex was observed in spring season (6.9%). Mean intensity and abundance were also higher in the spring season (Mean intensity=8.6; abundance=0.52). In summer season the prevalence was observed to be 5.4%, mean intensity o f 7.75 and abundance o f In autumn and winter no infection o i Adenoscolex was detected (Table 16). T able 16. Percentage incidedce, mean intensity and abundance of Adenoscolex in Schizothorax in different seasons. Season Number examined Number infected Incidence (%) Number parashes Mean intensity Abundan ce Spring Summer Autumn Winter Total % II. B. Study under Experimental Conditions This part of study was important because o f experimental conditions the ettcct of various factors like temperature, density, etc. on the life cycle of fish cestodcs was observed. The development o f egg. larva and adult were studied under experimental conditions. The possible intermediate hosts were given int cciion experimentally. All hosts which were given infection, did not show infection but only the susceptible hosts, which accordingly are described as the intermediate hosts, got infection. These were then maintained under experimental conditions and were given infection at different time intervals. The observations obtamed are presented below:

49 4. II. B. 1. Bothriocephalus acheilognathi Observarions o f the life cycle stages of B. acheilognathi viz., egg, coracidium, proceicoid and adult, made during the present endeavor under experimental conditions, are presented below: 4.II.B.1.(a) Development of eggs: (Table 17). The development of eggs at different temperatures was observed. The development and time of hatching of coracidium was seen to be dependent on the water temperature. The development of the eggs was obsei^(ed to be effected by the season also as the eggs obtained in different seasons showed difference in development duration at the same temperature. The least time duration for the development of eggs was 3 days at 23-25'C. Table 17. Effect of tem perature on the coracidium development of B. acheilognathi (under experimental conditions) T em perature *C Experim ent First coracidium(days post infection) 2-5 r No development 2-7 II No development 7 III No development 9-13 IV 15 days 9-13 V 9 days VI 4 days VII 3 days 30 v n i No development IX 6 days 35 X No development While working out the effect of lemperature, il was seen that then: was no development at the temperature of 2-7'C. At 9-13 C, eggs obtained in Ihc month o f September, developed in 15 days. At the same lemperature (9-13 C). the development took place only in 9 days but the eggs were taken from diftercni month (July). At C, coracidium formation look placc in 4 days, when ihc 90

50 eggs were taken in the month May. At C, it took 3 days. At 20-23'C. development took 6 days but the eggs were taken from different month (October). No development was seen at the temperatures 30'C and 35'C. The eggs kept at the temperatures 2-7'C, were seen to remain viable as they developed nomially when placed at temperatures above 7'C. But the eggs kept at the temperatures above 30*C did not develop and died. 4.II.B.1.(b) Procercoid formation: Fully developed procercoids which were recovered from the haeraocoel, were formed after days post infection at C in all copepods. R e la tio iu h ip I>etween egg densities a n d m ean o n m b e r o f pro cerco id s p e r h o s t Relationship between egg densities and mean number of procercoids per host was observed. The mean number o f procercoids per copepod increased with the increase in the egg density, but after reaching a particular number (approximately 13 procercoids per copepod), there was no increase in the number o f procercoids, even when the egg density was increased (Table 18; Fig. 51) Table 18. Relation between egg densities o f B. acheuognathi and the mean number of procercoids per host Egg density (10*^ Mean ±SD ± ± ±.5 6 6±1 7 7± ±2.5 to I2±l 15 13±l.5 20 I3±l The correlation between the egg density o f ft acheilunnaihi and the mean number o f procercoids per copepod was statistically significant (r = < I ).

51 Egg density (10-3 ) 51. Graph depkling the oonelation between egg densities of fl acheilognaihi and the mean numbct of prococoids per host X-ads= eggdensity. Y<cds=memmm}Kr(rfprocercokkperhc)a Relationship behveeo the egg density and the host mortality The relationship between the egg density and the host mortality was analyzed. This relationship revealed that with the increase in egg densities, there was also an increase in the host mortality. The observations are statistically significant (r = , P = 0.00) (Table 19; Fig. 52). Table 19. Rdation between the densities of eggs and Ibc host morlalit> E g f i d e n s ity (1 0 " ^ Host m orta lltv (% )

52 -E g g density (10-3 ) Host mortajity {%) Fig. 52. G i ^ depicting the idalkkibetvs«3nlhe egg d e n ^ and ttie host mortality. A'-aay= H?a^Bsf^'. V-iOK^hcisirnricii^fii) Relationship between the egg density and the size of procercoids. The relationship between the egg density and the size o f procercoids showed that with the increase o f egg density, the size o f procercoids first showed an increase up to 1 then it showed a decrease. The results are significant statistically (r = , P= 0.008) (Table 20; Fig. 53). Table 20. Relation between the egg density and the size of Egg density (10-^ Size of procercoids (lo Vm ) '

53 -E g g density (1 0-3 ) -S iz e o f procercoids (1 0 3 u m } Rg. S3. Graph depicting the correlation between egg density and the size of procefooids in oopepods. X-ads= egg density. Y-ads= q/"prtxxrvoids in copepcxh R elationship between th e size o f procerciods and the mean num ber of procercoids. The relationship between the size o f proccrcoids and the mean number o f procercoids was analyzed and demonstrated that with the decrease in the size o f procercoids, the mean number o f procercoids per host showed an increase. The observations are statistically significant (P = 0.001) and negatively correlated (r = -0.85) (Table 21; Fig. 54). T able 21. Relation behveen the size of procercoids and (he mean num ber of procercoids per host Size of procercoids (lo'^^m) M eanisd ± ± ±.5 5 6±1 ] 3 7± ± ±1 2.5 I3±l-5,.i 2 13±1

54 ObserDations -S iz e o f procerco ids (103iim) -M e a n n o o f pro cerco id s Fig, 5 4 Graph depicting the relation bet\seen the size of procersoids and ihe mean mai^xyofprxxn»idsperhosla<im=5^(y>too TtT;jcA, >- aas=meajm*t^cfprocercokkperhusl Survival o f copepods after exposing to different egg densities. The survival o f copepods exposed to different egg densities was examined. In the present study three experiments were performed in which copepods w ere exposed to different egg densities o f and in the third experiment copepods were not exposed to eggs. The observations obtained in these experim ents showed that the copepods exposed to higher egg densilit-s survived less well than those exposed to lower egg densities and than those o f the controls (Table 22; Fig. 55).

55 T able 22. Dayipost InfecHon (DPO N um ber o f copepods survm ng after exposing to ees density of 6700,2900 and 0 Number of SuiYhtlrg copepods (after ezpomdtoegg demttyof6700) Humber of «urvtvlng copepodt (after exposed to egg density of 2900) Number of surviving copepods (not exposed to eggs) S S

56 -D ays post infection (DPI) -N u m b e r of Surviving copepods (after exposed to egg density of 6700) - Num ber of surviving copepods -N u m b e r of surviving Surviving copepods (not exposed to eggs) Fig. 55. G raph depicting the num ber o f copepods surviving after exposing to egg density o f 6700, 2900 and 0. X-axis= days post in/eciion, Y-axisnumber o f stjrviving copepods 4.II.B.1.(c) Experimental infection of B. acheilognathito tlieir final hosts In the latx>ratory five aquaria were maintained, each containing 10 fish. All the fishes w ere intubated w ith the infected copepods. The prevalence of infection w as observed to be 84%. A fter 15 days post infecuon (DPI), fifteen fishes (three firom each aquarium ) w ere exam ined and 34, small unsegmented w orm s w ith a w ell developed scolex containing two bothria were observed in 12 fishes. Three fishes were uninfected. A fter 35 DPI, 10 fishes (2 from each aquarium ) were examined and 8 fishes w ere found to be infected containing 40 worms. Among these fishes, 7 fishes contained im m ature, segm ented w onns; 2 fishes were uninfec.ed and one fish harbored unsegmented tapeworm only. 97

57 After 110 DPI 15 fishes were examined. 13 fishes were infected, containing 122 wonns. The cestodes with gravid segments were obtained in 9 of these fishes and in four fishes only immature and mature cestodes were found. The gravid proglottids of these cestodes were wider than long to almost rectangular. Two fishes were uninfected. After 120 DPI 10 fishes were examined and 9 fishes were infected with 86 worms. The fiilly developed cestodes with gravid segments were found. These cestodes released eggs spontaneously into the water and were much wider than long. One o f the fishes was uninfected. (Table 23; Fig. 56) Table 23. Prevalence, mean intensity and abundance of B. acheilognathi in eiperimentauy infected Tishes Days post infection (DPI) Number of fishes Infected fishes Number of parasites Prevalence of infection {%) Mean intensity IB Total Num ber of fishes Infected fishes Abundance Fig. 56. G raph depicting the prcvaicncc li Mhdlogmilhi m experimentally infected fishes

58 Thus from the present experimental study, it is evident that B. acheilognathi needs less than five months to complete life cycle at a temperature range o f C. Hatching o f coracidium was completed in 3 days at this temperature. After entering into the eopepods, procercoid formauon occurred in days. The copepods infected with procercoids were taken by fish hosts where fiilly developed worms with gravid segments developed after 120 days. Fully developed cestodes in the fish Procercoid in the copepods Eggs Life cycle of R acheilognathi under eiperim cntal conditions

59 4. II. B.2. Adanoscolex infection in the intermediate host The larvae o f the Adenoscoiex were observed in the body cavity of the Tubifex (Annelida). No free swimming larval stage was observed. Eggs hatched in the oligochaete s gut, where a non ciliated hexacanth was released. The hexacanth penetrated the intestinal wall and in the body cavity developed into a procercoid stage (Table 24). Table 24. Prevalence^ mean intensity and abundance of procercoids of Adenoscoiex in the Tubifex Tubifex obsenred Number Infected Incidencc (%) Numb«r of procercoids Mean intensity Abundance Out o f 356 Tubifex observed in the present study, 3 1 were found to be infected with 73 procercoids of Adenoscoiex. Thus showing a prevalence of 8.49%, mean intensity=2.35, and abundance =0.2. From the above results on the Adenoscoiex. it is observed that this para.site occurs during one part of the year only. Recruitment might be occuiring in autamn and winter. The parasite develops in the fish and in spring season Ihe fishes showed high percentage of this infection. The infection showed a slight decrease in summer and was completely absent in the autumn and winter seasons.