Introduction
Linton (Reference Linton1910) established the genus Hapladena with H. varia infecting some acanthurid fishes in Florida as the type. Yamaguti (Reference Yamaguti1971) considered Deredena Linton, Reference Linton1910 and Hairana Nagaty, 1948 as junior synonyms of Hapladena and Overstreet & Curran (Reference Overstreet, Curran, Jones, Bray and Gibson2005) agreed with this arrangement. In addition to the type, Yamaguti (Reference Yamaguti1971) listed nine species, H. ovalis (Linton, Reference Linton1910) Manter, 1947; H. leptotelea Manter, 1947; H. sohali (Nagaty, 1948); H. magna (Nagaty, 1948); H. megatyphlon Perez Vigueraz, 1957; H. acanthuri Siddiqi & Cable, Reference Siddiqi and Cable1960; H. spinosa Manter & Pritchard, Reference Manter and Pritchard1961; H. tanyorchis Manter & Pritchard, Reference Manter and Pritchard1961; H. nasonis Yamaguti, 1970, under the genus. Since then, Machida & Uchida (Reference Machida and Uchida1990) synonymized H. nasonis with H. tanyorchis, and Nahhas & Carlson (Reference Nahhas and Carlson1994) treated H. megatyphlon as a synonym of H. leptotelea. Thus, at present there are eight valid species under the genus Hapladena. As far as is known, the life cycle of the genus Hapladena has not previously been established. The only available information is the description of a species of cercaria, Cercaria caribbea LII infecting the snail, Zebrina browniana in Curcao, The Netherlands Antilles, and its metacercaria which encysts in the open, by Cable (1962), who considered the cercaria and metacercaria as the probable life history stages of H. varia, a common parasite of surgeon fishes in Curcao.
While examining the bald glassy perchlet Ambassis gymnocephalus from Chaliyar and Kadalundi rivers in Kozhikode district, specimens of a species of Hapladena were found in the intestine. Further studies proved that it is a new species and is named Hapladena gymnocephali sp. nov. The gymnocephalous, bi-ocellate cercariae of H. gymnocephali were recovered from the snail, Gabbia travancorica, from Kadalundi and Eranjipalam in Kozhikode district during the months of September–December. Cercariae ingested by the host fish, developed into adult worms in the intestine, without undergoing a metacercarial stage of development.
The recovery of H. gymnocephali forms the first report of the genus in India; this is also the first report of the life cycle of the genus Hapladena.
Materials and methods
Ambassis gymnocephalus from the Chaliyar and Kadalundi rivers in Kozhikode district were examined for digenetic trematodes, which were studied using a phase-contrast microscope with or without vital staining. Those used for permanent preparations were fixed in 10% formalin under slight cover glass pressure and stained in alum carmine, following the procedure outlined by Cantwell (Reference Cantwell and Clark1981).
Specimens of the freshwater snail Gabbia travancorica were collected from Kadalundi and Eranjipalam during September–December, 2004. Snails were screened in the laboratory for cercariae, which on emergence were supra-vitally stained with neutral red and studied alive. Genital primordia were observed in lacto acetic carmine-stained cercariae. Measurements were made on heat-killed cercariae. A few infected snails were later crushed and examined for intra-molluscan stages of development.
In order to establish experimental infections in fish, fingerlings of A. gymnocephalus, collected from freshwater streams, where Hapladena infections do not occur, were kept in beakers containing cercariae and observed for their feeding activity. Fish, which were observed actively ingesting the cercariae, were maintained in aquarium tanks containing river water and sacrificed at intervals to study the course of development of the adult. Eggs released by naturally infected adults were maintained in filtered river water for examination of miracidial development. Newly emerged miracidia were studied under a phase-contrast microscope for structural details.
Descriptions of larvae and adult stages are based on measurements of a minimum of 10 specimens fixed in 10% formalin. Measurements are presented in micrometres; the range is followed by mean values in parentheses. Figures were drawn with the aid of a camera lucida, and details added freehand from observations made on live specimens.
Results
Egg
Eggs (fig. 1a) yellow, reniform, operculate; 52–56 × 36–39 (54 × 38).
Fig. 1 Life cycle stages of Hapladena gymnocephali sp. nov. (a) egg; (b) miracidium; (c) redia; (d) freshly emerged cercaria; (e) cercaria with tail expanded and elongated; (f) adult; (g) ootype complex.
Miracidium
Miracidia (fig. 1b) hatched after 5 days of incubation; ovoid, with single eye-spot and many germinal cells; 57–60 × 37–39 (58 × 38).
Redia
Rediae (fig. 1c) develop in the hepatopancreas of Gabbia travancorica. Body elongate, saccular, light-brown, filled with a few fully developed cercariae, numerous developing cercariae and germinal balls at different stages of development; 462–669 × 100–154 (524 × 129).
Cercaria
Natural infections were found in 20 of 1370 (1.4%) Gabbia travancorica collected from Kadalundi and Eranjipalam in Kozhikode district during a 4-month period, from September to December 2004. Cercariae (fig. 1d and e) emerged diurnally and exhibited positive phototaxis. Freshly emerged cercariae swim actively with lashing movements of their tails. After a few minutes of exposure to water, the tails of cercariae start to swell and elongate forming a prominent, white, opaque structure. At rest the cercaria remains suspended in the water column with the tail directed upward and bent at the base, directing the body also upward.
Description
Gymnocephalous, bi-ocellate, distome cercaria. Body spinose, pyriform, heavily pigmented, 204–290 (249) long, 102–138 (114) wide. Tail narrow, short, translucent in freshly emerged cercariae; 180–210 × 70–87 (194 × 76). Within a few minutes, the tail becomes elongated and expanded into a white, opaque, irregularly wrinkled structure, measuring 495–624 × 109–140 (579 × 124).
Oral sucker sub-terminal, round, 34–41 (39) in diameter. Ventral sucker round, equatorial, 33–41 (37). Prepharynx 9–29 (20) long; pharynx globular, 19–23 (20) in diameter. Oesophagus 71–95 (81) long, bifurcates postero-lateral to ventral sucker. Caeca 34–59 (49) long, extend to near posterior quarter of body. Eye-spots prominent, on either side of pharynx; 9–13 × 9-15 (10 × 11). Distance between eye-spots 23–34 (31). Testis rudimentary, transversely oval, composed of a single mass of cells; 18–33 × 29–42 (24 × 34). Ovary also rudimentary sub-spherical, pre-testicular, 12–14 × 19–27 (13 × 22). Rudimentary hermaphroditic sac ovoid, antero-lateral to ventral sucker, 31–46 × 12–15 (38 × 13).
Excretory bladder I-shaped, 31–34 (32) long; main collecting ducts ascend from antero-lateral walls of bladder, extend to the region of oral sucker, then run backwards to the level of the caecal bifurcation and bifurcate to form anterior and posterior collecting ducts. Excretory pore median at posterior end of body. Flame cells numerous, pattern not determined.
Adult
Development of flukes was observed in experimentally infected Ambassis gymnocephalus. Uninfected fish were exposed to cercariae that emerged from Gabbia travancorica. Cercariae, actively ingested by the fish, enter its stomach. Cercariae with fragmented tails were found in the stomach 1 h post-feeding. After 24 h, cercarial bodies, which increased slightly, were recovered from the intestine of the fish. These were structurally similar to the cercariae. On the second day, developing adults (fig. 1f and g) were 288–320 × 110–152 (301 × 131), the oral sucker was 39–49 (44), and the ventral sucker was 34–39 (36) in diameter. Eye-spots had started to disintegrate. On the fourth day post-feeding, immature adults had grown to 330–366 × 171–184 (348 × 178), the oral sucker was 42–64 (52) and the ventral sucker was 39–52 (47) in diameter. Eye-spots were lost, and the hermaphroditic sac had increased in size, 52–72 × 49–66 (60 × 57). On the sixth day, the hermaphroditic sac was 56–81 × 49–74 (76 × 64), testis 36–44 × 52–59 (39 × 55), ovary 16–19 × 20–24 (17 × 22), and the body did not show much increase in size. Eggs developed in 7-day-old adults, measuring 346–388 × 178–189 (363 × 182) with an oral sucker 49–62 (55), ventral sucker 49–59 (51), testis 42–52 × 63–69 (47 × 66), ovary 19–23 × 25–29 (20 × 26), and hermaphroditic sac 69–99 × 52–98 (84 × 89).
Natural infections with adult flukes were found in 171 of 249 (68.67%) Ambassis gymnocephalus collected from Chaliyar and Kadalundi rivers. The intensity of infection varied from 1 to 15. Naturally infected flukes were identical with those recovered from experimentally infected fish.
Description
Body spinose, pyriform, unpigmented, 361–547 × 178–303 (414 × 236). Body spines 3–8 (5) long. Eye-spot pigment scattered in the region postero-lateral to oral sucker. Oral sucker round, sub-terminal, 51–77 (65) in diameter. Ventral sucker round, muscular, 48–69 (55) in diameter. Sucker ratio 1: 0.77–0.90 (1: 0.85). Fore-body 102–214 (134) long, 31–39% (35%) of body length. Prepharynx 3–15 (8) long; pharynx sub-globular, 23–37 × 27–49 (34 × 38). Oral sucker/pharynx width ratio, 1: 0.5–0.6 (1: 0.57). Oesophagus 62–192 (126) long, and bifurcates postero-laterally to the ventral sucker. Caeca 69–184 (96) long, and terminate blindly 56–136 (85) from posterior end of body.
Testis single, reniform, 36–105 × 99–148 (69 × 112), 21–69 (42) posterior to ventral sucker. Post-testicular field 46–102 (75) long, 13–22% (17%) of body length. Seminal vesicle bipartite; distal portion enclosed within the hermaphroditic sac; internal seminal vesicle sub-globular, 29–74 × 26–62 (41 × 39) and external seminal vesicle 52–99 × 42–52 (72 × 48) in size.
Ovary ovoid, pre-testicular, 21–49 × 39–102 (36 × 71), 3–34 (21) posterior to ventral sucker. Seminal receptacle ovoid, sinistral to ovary, 27–33 × 36–39 (37). Laurer's canal well developed, opens near anterior margin of ovary. Ootype complex postero-lateral to ovary. Uterus extends between ovary and hermaphroditic sac, containing 1–6 eggs and numerous sperm. Hermaphroditic sac anterior to ventral sucker, enclosed metraterm, hermaphroditic duct and internal seminal vesicle 36–138 × 52–115 (65 × 77). Hermaphroditic duct tubular, muscular and eversible. Genital pore median, 12–79 (59) anterior to ventral sucker. Vitelline follicles large, extending from the middle level of the hermaphroditic sac to the post-testicular region, where they are not confluent. Vitelline ducts run transversely at the mid-testicular region to meet the ootype.
Excretory bladder I-shaped, 64–95 (77) long, extends to posterior end of testis, where it receives two collecting tubes. Two pairs of lymph vessels extend along the antero-lateral margins of the body.
Taxonomic summary
Genus: Hapladena Linton, Reference Linton1910.
Species: Hapladena gymnocephali sp. nov.
Type host: Ambassis gymnocephalus Lacepede, Ambassidae.
Site: Intestine.
Molluscan host: Gabbia travancorica (Benson); Bithyniidae.
Type locality: India, Kerala, Kozhikode district, Chaliyar and Kadalundi rivers.
Holotype: Deposited in the Department of Zoology, University of Calicut, Kerala, India. No. Z./Par./Dig.-2004-1a
Paratypes: Z./ Par./ Dig.-2004-1b-c.
Date of collection: 12 June 2004.
Etymology: Named after the species name of the type host.
Discussion
The genus Hapladena Linton, Reference Linton1910 belongs to the subfamily Megasoleninae Manter, 1935 of the family Haploporidae Nicoll, 1914. Of the eight valid species, the present form is comparable to H. acanthuri Siddiqi and Cable, Reference Siddiqi and Cable1960 and H. spinosa Manter and Pritchard, Reference Manter and Pritchard1961 in having sucker ratios and egg size which are similar, and in having a median genital pore. A comparison of characters of the three species, presented in table 1, shows that both H. acanthuri and H. spinosa are distinctly different from the present form in having an elongated body, longer than broad testis, a vitelline field which extends posterior to the ventral sucker and a longer excretory bladder which reaches up to the level of ovary. Hapladena acanthuri differs further in having a longer hermaphroditic sac which extends slightly posterior to the ventral sucker, and H. spinosa differs in the nature of Laurer's canal and internal seminal vesicle and in the presence of a sphincter around the genital pore. Based on the differences of the present fluke with its closely related forms, it would appear reasonable to treat the present form as a new species of Hapladena. It is designated H. gymnocephali sp. nov. after the specific name of the type host, Ambassis gymnocephalus.
Table 1 Comparison of characters of Hapladena gymnocephali sp. nov. with related species; measurements in micrometres.
The only available information on the life cycle of the genus Hapladena is the description of Cercaria caribbea LII, a large gymnocephalous, bi-ocellate cercaria belonging to the family Haploporidae, infecting the snail Zebrina browniana in Curacao, The Netherlands Antilles, and its metacercaria, which encysts in the open (Cable, 1962). Based on the morphology and ecology of the larval stages, Cable considered them to be the life history stages of Hapladena varia, a common parasite of surgeon fishes in that region. No experimental attempts were made to substantiate this contention. The cercaria of H. gymnocephali is different from other known gymnocephalous, bi-ocellate, distome cercariae in its dimensions, the development of the testis, ovary and other reproductive structures, and in the absence of a cyst-forming ability. However, it needs comparison with C. caribbea LII, which is supposed to be the larval stage of Hapladena varia. The cercaria of H. gymnocephali is distinctly different from C. caribbea LII in the nature of the tail and in having differentiated testis, ovary and other reproductive structures. Further, it never encysts in the open to form a metacercarial stage of development.
As far as is known, the life cycles of only ten haploporid species are known: Pseudohapladena pearsoni (Martin, Reference Martin1973) Overstreet & Curran, Reference Overstreet, Curran, Jones, Bray and Gibson2005 by Martin (Reference Martin1973); Saccocoelium obesum Looss, 1902, S. tensum Looss, 1902 and Haploporus benedeni (Stossich, 1887) Looss, 1902 by Fares & Maillard (Reference Fares and Maillard1974); Carassotrema koreanum Tang & Lin, Reference Tang and Lin1979 and C. wui Tang & Lin, Reference Tang and Lin1979 by Tang & Lin (Reference Tang and Lin1979); Saccocoelioides carolae Lunaschi, 1984 by Martorelli (Reference Martorelli1988); S. tarpazensis Diaz & Gonzalez, Reference Diaz and Gonzalez1990 by Diaz & Gonzalez (Reference Diaz and Gonzalez1990) and Pseudohapladena martini (Madhavi, 1979) Overstreet & Curran, Reference Overstreet, Curran, Jones, Bray and Gibson2005 and Carassotrema bengalense Rekharani & Madhavi, 1985 by Shameem & Madhavi (Reference Shameem and Madhavi1991). For all these species, the cercaria is a gymnocephalous, bi-ocellate and distome type, infecting snails belonging to the superfamily Rissooidea and encysting on any available substratum or surface film of water. Definitive hosts are fish and infection is through ingestion of metacercariae. Hapladena gymnocephali sp. nov. also possesses a two-host life cycle, but the actively swimming, and structurally different cercariae emerging from the snail, Gabbia travancorica, are ingested by Ambassis gymnocephalus and develop into adult flukes in its intestine. Cercariae are positively phototactic, and on exposure to water their tails swell and elongate to become prominent, white, opaque structures. This may be an adaptive behaviour shown by the cercaria to attract the definitive host, which is a surface-feeding fish. This is an example of a parasite drawing the attention of the definitive host toward its infective stage for successful transmission. The direct pathway exhibited by H. gymnocephali for completion of its life cycle has survival value and is adaptive, as the cercaria is the infective stage developing directly into the adult in the definitive host.
Acknowledgements
Our sincere thanks are due to Professor Robin M. Overstreet, for determining the taxonomic position of the genus Hapladena. We are also thankful to the Head of the Department of Zoology, University of Calicut, for facilities provided. One of us (P.S.) is grateful to the CSIR, New Delhi, for the financial support in the form of a Research Fellowship.
