International Journal of Fisheries and Aquatic Studies 2019; 7(4): 334-338 E-ISSN: 2347-5129 P-ISSN: 2394-0506 (ICV-Poland) Impact Value: 5.62 (GIF) Impact Factor: 0.549 IJFAS 2019; 7(4): 334-338 © 2019 IJFAS www.fisheriesjournal.com Received: 11-05-2019 Accepted: 15-06-2019 PK Prasadan Ecological Parasitology and Tropical Biodiversity Laboratory, Department of Zoology, Kannur University, Mananthavady Campus, Wayanad, Kerala, India Sherin Chacko Ecological Parasitology and Tropical Biodiversity Laboratory, Department of Zoology, Kannur University, Mananthavady Campus, Wayanad, Kerala, India Clinostomum cupani n. sp. metacercaria infecting the freshwater fish, Pseudosphromenus cupanus from the Western Ghats, Wayanad region, India PK Prasadan and Sherin Chacko Abstract Clinostomum cupani n. sp. (Digenea, Clinostomidae) from the operculum and body cavity of the freshwater fish Pseudosphromenus cupanus collected from water bodies in the Wayanad region of the Western Ghats is described and illustrated. C. cupani n. sp. is new to the genus and is separated from its congeners on the basis of differences in morphology and morphometry. Detailed comparison of the present species with C. complanatum Rudolphi, 1814 and C. marginatum Rudolphi, 1819 ismade in this study. C. cupani n. sp. differs from all other species of Clinostomum Leidy, 1856 in the shape of its uterus and the presence of pigmented eyespots. It is named after the host P. cupanus. The prevalence of infection of C. cupani n. sp. is 50%, the intensity of infection is 12.33 and the mean abundance is 6.16. Keywords: Clinostomum cupani n. sp., digenea, metacercaria, second intermediate host, fish 1. Introduction The genus Clinostomum Leidy, 1856 (Digenea: Clinostomidae) has heteroxenous life cycle. Adults are found in the buccal cavity and esophagus of birds, reptiles and mammals [1]. The freshwater snails are their first intermediate hosts and freshwater fishes or amphibians as their second intermediate hosts [2, 3]. The metacercariae of Clinostomum are usually known as yellow grubs. This fish-borne zoonotic trematode causes acute pharyngitis or laryngitis in those who eat raw fish meat with metacercarial infection [4]. Human infections have been reported by many workers [4- 9]. Because of the high degree of morphological inconsistency within the same species, Clinostomum had been subjected to several taxonomic reviews [1, 2, 10-13], of which Ukoli [1] was one of the pioneer reviewers, who synonymized 20 Clinostomum species with C. complanatum and documented 13 valid species. Several species were then added to or detracted from the genus Clinostomum with the support of molecular and conventional taxonomic studies. Now this cosmopolitan genus encompasses 29 valid species [14]. The present paper describes the morphological features and taxonomic status of a new species of digenetic trematode metacercaria, C. cupani n. sp. infecting operculum and body cavity of freshwater fish P. cupanus from the Wayanad region of the Western Ghats. 2. Materials and Methods 2.1 Study area The study was carried out in the Wayanad region of the Western Ghats, one of the hottest hotspots of biodiversity in the world due to its incredible biological diversity and a high degree of endemism. The geographical location of Wayanad region is 11° 27' to 15° 58' north latitude and 75° 47' to 70° 27' east longitude. Correspondence PK Prasadan Ecological Parasitology and Tropical Biodiversity Laboratory, Department of Zoology, Kannur University, Mananthavady Campus, Wayanad, Kerala, India 2.2 Methods Host fish specimens collected from different water bodies using sweep net were kept alive, brought to the laboratory, maintained in clean glass jars or aquarium and occasionally fed with cooked rice or fish food. Fish were sacrificed and body surface, fins, abdominal cavity, gill filaments, eyes and muscles were carefully examined for the presence of metacercariae. Internal organs were dissected out, placed in separate Petri dishes containing ~ 334 ~ International Journal of Fisheries and Aquatic Studies 0.75% saline, macerated and observed under the binocular LABOMED Luxeo 4Z stereo zoom microscope. The live encysted meta cercariae were transferred to 0.75% saline, excysted by rupturing the cyst wall with a fine needle. Studies on meta cercariae were carried out on live and fixed specimens, without supravital staining or staining with neutral red or methylene blue, under Nikon ECLPSE Ni-U research microscope. Permanent whole mounts were prepared after fixing them in 5% formalin under slight cover glass pressure, and then staining with acetocarmine, following the procedure outlined by Cantwell [15]. Measurements (in µm) were taken with the support of Nikon NIS Elements Imaging software. Sketches were prepared with the Nikon Y- IDT drawing tube attached to the Nikon ECLPSE Ni-U research microscope and Photographs were taken with Nikon Y-TV55 camera attached to a Nikon ECLPSE Ni-U research microscope with the support of Nikon NIS Elements Imaging Software. Prevalence, intensity and mean abundance of infection were measured following Bush et al. [16]. 1979.74-2957.25 ×125.52-274.13 (2506 × 181.65), broad at the postacetabular region, lateral pouches are not prominent, extending up to the posterior end of the body. Testes deeply lobed; anterior testis irregular, close to the right margin of left caecum. 143.36-242.49 × 142.93-301.07 (181.70 × 215.44). Posterior testis slightly triangular, inter-caecal, 74.24-228.43 × 106.80-366.26 (151.34 × 232.02). Distance between testes 107.11-178.96 (147.23). Cirrus pouch large, slightly ovoid, 113.48-254.90 × 77.05-166.86 (177.88 × 107.75), in the intertesticular space close to the left margin of the right caecum. Ovary small, slightly oval, 57.81-100.16 × 55.19-91 (78.65 × 69.01). Ootype at the inter-testicular space between ovary and the left intestinal caecum. Uterine sac with lateral evaginations, 209.08- 606.43 × 106.60-324.69 (429.96 × 218.69) partially covers the intercaecal space in that region. Excretory bladder V-shaped. 3. Results 3.1 Clinostomum cupani n. sp. (Fig. 1) Body linguiform, 2188.00-3090.00 (2716.36) long and 648.96-951.24 (815.73) wide. Slightly ovoid anterior part bears a small sub-terminal oral sucker, 138.14-207.55 × 149.34-222.57 (171.33 × 195.87), surrounded by oral collar 235.08-319.95 × 372.94-562.15 (270.14 × 443.66). Cuticle aspinose. Ventral sucker large, well developed, round at the posterior region, 296.61-551.09 × 306.48-524.04 (397.91 × 384.13). Distance between suckers 299.19-398.97 (342.04). Short pre-pharynx, 52.88-84.12 × 11.76-26.77 (69.74 × 18.32). Pharynx rudimentary, 58.32-85.48 × 40.78-74.68 (69.63 × 59.26). Fig 2: Eyespots of Clinostomum cupani n. sp. metacercaria marked with a rectangular box. Fig 1: Clinostomum cupani n. sp. Metacercaria (Photograph and line drawing). Eyespots at the level of the anterior part of pharynx, anterior to caecal shoulders. Esophageal bulb absent. Intestinal caeca 3.2 Taxonomic summary Type specimen: Holotype (No. Z-TM 42) deposited in the helminth parasite collections, Ecological Parasitology and Tropical Biodiversity Laboratory, Department of Zoology, Kannur University, Mananthavady Campus, Wayanad670645, Kerala, India. Type host: P. cupanus ((No. Z-FTM 147) deposited in the Ichthyology collections, Department of Zoology, Kannur University, Mananthavady Campus, Wayanad-670645, Kerala, India. Type locality: Moolithod, Wayanad District, Kerala, India. Site of infection: Operculum and body cavity. Period of collection: June 2018 to January 2019 Prevalence: Three of six fishes (50%) screened were infected. Intensity: Thirty seven C. cupani n. sp. recovered from three infected fish (12.33). Mean abundance: Thirty seven C. cupani n. sp. recovered from a total of six fishexamined and, therefore, the mean abundance is 6.16. Of the six P. Cupanus examined, three were found infected with a total of 37 parasites. The prevalence of infection was 50%, while the intensity of infection stood at 12.33. 4. Discussion The present study showing a detailed morphological description of the metacercaria of C. cupani n. sp., ~ 335 ~ International Journal of Fisheries and Aquatic Studies emphasizes the presence of pigmented eyespots (Fig. 2) and large uterus with lateral pouches. According to Ukoli [1] the significant characters which are not likely to vary within the species and which are, therefore, most reliable for the purposes of classification of Clinostomum species are the presence of lateral evaginations in the uterine sac and the anterior extent of the vitellaria. The characters such as the position of gonads, genital pore & cirrus pouch and the shape of testes, which vary between meta cercariae and adults and all other characters that are likely to change due to external influences. Table 1: Comparison of morphometric characters (in µm) of metacercariae of the C. marginatum Rudolphi, 1819, C. complanatum Rudolphi, 1814 [11] and C. cupani n. sp. Clinostomum complanatum Rudolphi, Clinostomum cupani n. sp. (n = 7) min–max (mean) 1814 (n = 11) min–max (mean) Stout, oval, elongate, wider in gonadal Oval, elongate, wider in gonadal region Oval, elongate region Body L × B 4215.00–6346.00 × 979.00–1532.00 2188.00–3090.00 × 648.96–951.24 4495.00–7874.00 × 1635.00–2434.00 (5402.00 × 1329. 00) (2716.36 × 815.73) (5741.00 × 1934.00) Body L/B 3.05–4.40 (4.03) 2.20–4.37 (2.99) 3.10–3.52 (3.29) Oral collar B 502.00–874.00 (721.00) 686.00–1030.00 (820.00) 372.94–562.15 (443.66) Oral sucker L × 238.00–599.00 × 194.00–586.00 (312.00 259.00–337.00 × 284.00–507.00 (294.00 × 138.14–207.55 × 149.34–222.57 (171.33 B × 290.00) 401.00) × 195.87) Oral sucker 0.82–1.05 (0.92) 1.06–1.67 (1.36) 0.22–0.27 (0.23) B/body B Ventral sucker Larger than oral 510.00–757.00 × Larger than oral 637.00–910.00 × 766.00– Larger than oral 296.61–551.09 × L×B 571.00–804.00 (669.00 × 708.00) 952.00 (795.00 × 839.00) 306.48–524.04 (397.91 × 384.13) Ventral sucker 0.99–1.12 (1.06) 1.78–2.69 (2.14) 1.70–2.46 (1.95) B/oral sucker B Ventral sucker 0.47–0.62 (0.53) 0.39–0.49 (0.44) 0.42–0.55 (0.465) B/body B Distance between 959.00–1491.00 (1243.00) 860.00–1115.00 (1020.00) 299.19–398.97 (342.04) suckers Anterior testis 231.00–413.00 × 264.00–498.00 (307.00 316.00–957.00 × 273.00–559.00 (484.00 × 143.36–242.49 × 142.93–301.07 (181.70 L×B × 389.00) 412.00) × 215.44) Anterior testis 0.68–1.72 (1.29) 0.46–1.22 (0.90) 0.76–1.34 (1.18) B/L Posterior testis 233.00–457.00 × 295.00–492.00 (327.00 245.00–441.00 × 408.00–602.00 (328.00 × 74.24–228.43 × 106.80–366.26 (151.34 × L×B × 405.00) 493.00) 232.02) Posterior testis 0.89–1.52 (1.25) 1.09–1.88 (1.54) 1.19–2.00 (1.52) B/L Distance 196.00–393.00 (320.00) 214.00–527.00 (353.00) 107.11–178.96 (147.23) between testes 85.00–142.00 × 62.00–97.00 (115.00 × 135.00–164.00 × 97.00–178.00 (149.00 × 57.81–100.16 × 55.19–91.00 (78.65 × Ovary L × B 80.00) 129.00) 69.01) Ovary B/L 0.43–0.97 (0.71) 0.59–1.09 (0.87) 0.65–1.05 (0.88) Well developed, crescent shaped, Well developed, extending from inter Large and slightly ovoid, in the intercompressed against left anterior margin of testicular space to posterior right margin of testicular space close to the left margin of Cirrus sac L× B anterior testis 172.00–343.00 × 119.00– anterior testis 209.00–405.00 × 124.00– right caeca 113.48–254.90 × 77.05– 153.00 (256.00 × 137.00) 197.00 (296.00 × 157.00) 166.86 (177.88 × 107.75) Cirrus sac 0.03–0.06 (0.05) 0.03–0.07 (0.05) 0.05–0.08 (0.06) L/body L Located at the level of the anterior part of Eyespots Not observed Not observed pharynx, anterior tothe caecal shoulders Large and wider uterus. Uterine sac with Uterine sac L × Tubular proximal uterus. No lateral Tubular proximal uterus. No lateral lateral evaginations B digitations digitations 209.08–606.43 × 106.60–324.69 (429.96 × 218.70) Rudimentary Pharynx Not observed Not observed 58.32–85.48 × 40.78–74.68 (69.63 × 59.27) Slightly long 52.88–84.12 ×11.76–26.77 Pre-pharynx Not observed Not observed (69.74 × 18.32) Oesophagus Very short. oesophageal bulb present Oesophageal bulb well developed Oesophagus and oesophageal bulb absent Lateral to ventral sucker, characterized by Lateral pouches in intestinal caeca are not Lateral to ventral sucker and genital Intestinal caeca visible intestinal pouches especially in prominent Caeca more bulbous at complex, with prominent lateral pouches postacetabular part postacetabular region Characters Clinostomum marginatum Rudolphi, 1819 (n = 10) min–max (mean) The present species has some general morphological similarities with the C. marginatum (Table 1). But it exhibits some distinct morphological features such as the presence of pigmented eyespots, large sacculated uterus, pre-pharynx, rudimentary pharynx; absence of esophagus, positioning of the cirrus pouch and absence of cuticular spines. The described species also exhibits changes in morphometry such as distance between suckers, the ratio between oral sucker ~ 336 ~ International Journal of Fisheries and Aquatic Studies width and body width and distance between testes. The present species displays some similarity with C. complanatum (Table 1). Detailed study revealed that the species differs from C. complanatum [11] on many morphological features and morphometry like presence of pigmented eyespots, pre-pharynx, rudimentary pharynx, difference in the distance between suckers, ratio between oral sucker width and body width, size of anterior testis, distance between testes and shape & size of uterine sac. Neither C. complanatum nor C. marginatum has lateral digitation of the uterine sac [17] and presence of pigmented eyespots distinguishes the described species from these two species. The species described under the genus Clinostomum from India documented by Pandey and Agrawal [18] in their work ‘The metacercarial fauna of India’ included C. piscidium Southwell and Prashad, 1918, C. prashadi Bhalerao, 1942, C. gideoni Bhalerao, 1942, C. dasi Bhalerao, 1942, C. indicum Bhalerao, 1943, Clinostomum sp. Srivastava, 1950, C. schizothoraxi Kaw, 1950, C. microstomum Singh, 1955, C. giganticum Agarwal, 1955, C. macrosomum Jaiswal, 1957, C. mastacembeli Jaiswal, 1957, C. progonum Jaiswal, 1957, C. orientale Mukherjee, 1967, C. lucknowensis Pandey, 1968, C. trichogasteri Pandey, 1969 and Clinostomum sp. Rekharani and Madhavi, 1985. The presence of pigmented eyespots and lateral evaginations in the uterine sac are not reported in any of these species. Concerning the other species described, at the metacercarial stage, the morphological features observed in the present species differ consistently from C. cutaneum, which has a unique Y-shaped uterine sac [10], C. phalacrocoracis metacercaria that bears fan-shaped testes and straight uterus running from ventral sucker to anterior testis [19]. Although the presence of lateral digitations in the uterine sac is a common feature in C. detruncatum and the present species [17] the former lacks eyespots. The present species varies from C. tilapiae in which body surface is armed with numerous cuticular spines and straight uterus running from ventral sucker to anterior testis [20]. The species described is different from C. caffarae on the nature of testes, from C. arquus on the shape of testes & nature of uterine sac and from C. Cichlidorum on the absence of pharyngeal bulb [14]. 5. Acknowledgments Sherin Chacko is grateful to the Department of Science & Technology (DST) INSPIRE program for providing Inspire fellowship to carry out this study. 6. References 1. Ukoli FMA. On Clinostomum tilapiae n. sp., and C. phalacrocoracis Dubois, 1931 from Ghana, and a discussion of the systematics of the genus Clinostomum Leidy, 1856. Journal of Helminthology. 1966; 40:187214. 2. Matthews D, Cribb TH. Digenetic trematodes of the genus Clinostomum Leidy, 1856 (Digenea: Clinostomidae) from birds of Queensland, Australia, including C. wilsoni n. sp. from Egretta intermedia. Systematic Parasitology. 1998; 39:199-208. 3. Dias ML, Eiras JC, Machado MH. The life cycle of Clinostomum complanatum Rudolphi, 1814 (Digenea, Clinostomidae) on the floodplain of the high Parani river, Brazil. Parasitology Research. 2003; 89(6):506-508. 4. Lee GS, Park SW, Kim J, Seo KS, You KW, Chung JH, et al. A case of endoscopically treated laryngopharyngitis 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. ~ 337 ~ resulting from Clinostomum complanatum infection. Korean Journal of Gastroenterology. 2017; 69:177-180. Kitagawa N, Oda M, Totoki T, Washizaki S, Oda M, Kifune T. Lidocaine spray used to capture a live Clinostomum parasite causing human laryngitis. American Journal of Otolaryngology. 2003; 24:341-343. Sohn WM. Fish-borne zoonotic trematode metacercariae in the Republic of Korea. Korean Journal of Parasitology. 2009; 47:S103-S113. Hara H, Miyauchi Y, Tahara S, Yamashita H. Human laryngitis caused by Clinostomum complanatum. Nagoya Journal of Medical Science. 2014; 76:181-185. Jung SC, Oh HJ, Kim DM, Park JH. A case of pharyngitis caused by Clinostomum complanatum. Korean Journal of Otorhinolaryngology-Head and Neck Surgery. 2015; 58(1):61-3. Song HB, Choi MH, Chung EJ. Human Laryngeal Infection by Clinostomum complanatum. American Journal of Tropical Medicine and Hygiene. 2018; 98(1):7-8. Gustinelli A, Caffara M, Florio D, Otachi EO, Wathuta EM, Fiovaranti ML. First description of the adult stage of Clinostomum cutaneum Paperna, 1964 (Digenea: Clinostomidae) from grey herons Ardea cinerea L. and a redescription of the metacercaria from the Nile tilapia Oreochromis niloticus niloticus (L.) in Kenya. Systematic parasitology. 2010; 76:39-51. Caffara M, Locke SA, Gustinelli A, Marcogliese DJ, Fiovaranti ML. Morphological and molecular differentiation of Clinostomum complanatum and Clinostomum marginatum (Digenea: Clinostomidae) metacercariae and adults. Journal of parasitology. 2011; 97(5):884-891. Sereno-Uribe AL, Pinacho-Pinacho CD, García-Varela M, de León GPP. Using mitochondrial and ribosomal DNA sequences to test the taxonomic validity of Clinostomum complanatum Rudolphi, 1814 in fish-eating birds and freshwater fishes in Mexico, with the description of a new species. Parasitology research. 2013; 112:2855-2870. Rosser TG, Alberson NR, Woodyard ET, Cunningham FL, Pote LM, Griffin MJ. Clinostomum album n. sp. and Clinostomum marginatum (Rudolphi, 1819), parasites of the great egret Ardea alba L. from Mississippi, USA. Systematic Parasitology. 2017; 94:35-49. Sereno-Uribe AL, García-Varela M, Pinacho-Pinacho CD, de León GPP. Three new species of Clinostomum Leidy, 1856 (Trematoda) from Middle American fisheating birds. Parasitology Research. 2018, 1-15. Cantwell GE. Methods for invertebrates. In: (Eds. Clark G.) Staining procedures. Baltimore, Williams and Wilkins, 1981, 255-280. Bush AO, Lafferty KD, Lotz JM, Shostak AW. Parasitology meets ecology on its own terms: Margolis et al. Revisited. Journal of Parasitology. 1997; 83(4):575583. Acosta AA, Caffara M, Fioravanti ML, Utsunomia R, Zago AC, Franceschini L et al. Morphological and molecular characterization of Clinostomum detruncatum (Trematoda: Clinostomidae) metacercaria infecting Synbranchus marmoratus. Journal of Parasitology. 2016; 102:151-156. Pandey KC, Agrawal N. Metacercarial Fauna of India. Records of Zoological Survey of India, 2013, 117-135. International Journal of Fisheries and Aquatic Studies 19. Caffara M, Davidovich N, Falk R, Smirnov M, Ofek T, Cummings D et al. Redescription of Clinostomum phalacrocoracis metacercariae (Digenea: Clinostomidae) in cichlids from Lake Kinneret, Israel. Parasite. 2014; 21(32):1-6. 20. Caffara M, Locke SA, Echi PC, Halajian A, Benini D, Luus-Powell WJ et al. A morphological and molecular study of Clinostomid metacercariae from African fish with a redescription of Clinostomum tilapiae. Parasitology. 2017, 1-11. ~ 338 ~