Introduction
Morphological characterization of life cycle stages with the establishment of the identity of the intermediate hosts provides an important complement to parasite taxonomy (Blasco-Costa & Poulin, Reference Blasco-Costa and Poulin2017). This is an integrative approach in taxonomy (Dayrat, Reference Dayrat2005). In order to understand the ecology and evolution of any parasite, the full characterization of its developing stages is essential. Although the biodiversity of parasites is being discovered at a high rate, very little efforts are being taken by researchers to elucidate their life cycles. We have elucidated the life cycle of Pleurogenoindes wayanadensis in nature and established the same through experimental infection studies in the laboratory.
The genus Pleurogenoides of the family Pleurogenidae Looss, 1899 was established by Travassos (Reference Travassos1921) to accommodate those species of the genus Pleurogenes Looss, 1896, and considered Pleurogenoides tener as its type species. On the basis of the length of intestinal caeca and position of genital pore, Mehra & Negi (Reference Mehra and Negi1928) divided the genus Pleurogenoides into two sub-genera, P. (Pleurogenes) and P. (Telogonella) and, later, Srivastava (Reference Srivastava1934) dropped the genus Pleurogenoides and transferred its species to Pleurogenes. Macy (Reference Macy1936) retained the genus Pleurogenoides and the retention was accepted by Kaw (Reference Kaw1943) and Mukherjee & Ghosh (Reference Mukherjee and Ghosh1970). Of the 32 species of Pleurogenoides recorded worldwide from amphibians, 16 species were from India. Singh (Reference Singh1977) recorded Pleurogenoides gastroporus from Hoplobatrachus tigerinus in Kerala. Recently, three new species of Pleurogenoides – Pleurogenoides cyanophlycti, Pleurogenoides euphlycti and Pleurogenoides wayanadensis – were described by Shinad & Prasadan (Reference Shinad and Prasadan2017, Reference Shinad and Prasadan2018a) from Euphlyctis cyanophlyctis (Schneider, 1799) and H. tigerinus (Daudin, 1803) of the Wayanad region of the Western Ghats, Kerala, India. Shinad & Prasadan (Reference Shinad and Prasadan2018b, Reference Shinad and Prasadan2019) recorded the prevalence, intensity of infection and the mean abundance of P. wayanadensis from E. cyanophlyctis and H. tigerinus, respectively.
The life cycles of six species of Pleurogenoides have been studied and established in the world (Mathias, Reference Mathias1924; Neuhaus, Reference Neuhaus1940; Buttner, Reference Buttner1951; Okabe & Shibue, Reference Okabe and Shibue1951; Shibue, Reference Shibue1953; Macy, Reference Macy1964; Madhavi et al., Reference Madhavi, Dhanumkumari and Ratnakumari1987; Janardanan & Prasadan, Reference Janardanan and Prasadan1991; Brinesh & Janardanan, Reference Brinesh and Janardanan2014). Reports are available on metacercariae from crabs, imago and naiads of both dragonflies and damselflies and larvae of other insects (Dissanaike & Fernando, Reference Dissanaike and Fernando1960; Muraleedharan & Pande, Reference Muraleedharan and Pande1967; Krasnolobova, Reference Krasnolobova1970; Prakash & Pande, Reference Prakash and Pande1970; Vojtkova, Reference Vojtkova1970; Janardanan et al., Reference Janardanan, Ramanandan and Usha1987; Dhanumkumari, Reference Dhanumkumari2000, Brinesh & Janardanan, Reference Brinesh and Janardanan2014; Bolek et al., Reference Bolek, Detwiler, Stigge, Toledo and Fried2019).
The objectives of the present study included: (1) the morphological characterization of the developing stages of the parasite and to trace the intermediate hosts so as to elucidate the complete life cycle in nature; and (2) the establishment of the life cycle in the laboratory through experimental infection studies. The metacercariae exhibited progenetic development – the sexual maturation in an organism still in a morphologically juvenile stage (Gould, Reference Gould1977). The present paper describes the morphology of the developing stages of P. wayanadensis obtained from natural infection from the definitive and intermediate hosts and the life cycle stages from egg to egg-producing adult elucidated and experimentally established in the laboratory.
Materials and methods
Study area
The study was carried out and the specimens were collected from different paddy fields and freshwater bodies of the Thirunelly and Payode villages of the Western Ghats, Wayanad region, Kerala. The Western Ghats is considered one of the ‘hottest hotspots of biodiversity due to its very rich biodiversity and high endemism’ UNESCO (2012).
Collection and examination of frogs, dragonfly naiads and freshwater snails
The common Indian skittering frogs E. cyanophlyctis (Schneider, 1799) were collected from the paddy fields and the nearby streams of Thirunelly and Payode villages of the Western Ghats, Wayanad region, from October and November 2019, and were subsequently brought to the laboratory and examined for digenetic trematodes. After narcotization with chloroform, the frogs were dissected and the intestines were transferred to physiological saline for the detection of P. wayanadensis under a stereo zoom microscope (binocular LABOMED Luxeo 4Z, LABO AMERICA INC). The bithynid snail Bithynia (Digoniostoma) pulchella (Benson, 1836) was collected from the same habitat during the same period. Live snails were transferred to the laboratory and maintained in groups of 10–20 in glass beakers containing tap water. Snails that shed cercariae were isolated and kept individually in separate beakers. Water in the beakers was checked regularly for cercariae. The collected dragonfly naiads of the family Libellulidae were brought alive to the laboratory, maintained in clean aquariums/glass containers and occasionally fed with small aquatic insects. Naiads were sacrificed and carefully observed under the stereo zoom microscope for metacercariae. All the three host specimens were deposited in the Parasite Host Collections, Department of Zoology, Kannur University.
Studies on developing stages of the trematode
The recovered P. wayanadensis (metacercariae from the abdomen and thorax of naiads and adults from the duodenum of frogs) were transferred to 0.75% saline. The metacercariae were excysted by rupturing the cyst wall with a fine needle. Isolated P. wayanadensis were examined under the Nikon phase contrast research microscope (ECLIPSE Ni-U, NIKON, JAPAN) without supravital staining or after staining with neutral red. Permanent whole mounts were prepared after fixing them in 5% formalin under slight cover glass pressure and staining with acetocarmine, following the procedure outlined by Cantwell (Reference Cantwell and Clark1981). Eggs recovered from the adult P. wayanadensis were isolated and the miracidia were released from these eggs with a pair of fine-pointed needles. Both these eggs and miracidia were studied with vital staining under a Nikon phase contrast research microscope. A few infected snails were later crushed and examined for intra-molluscan stages. Sporocysts recovered from the hepatopancreas of infected snails were studied after supravital staining with neutral red. Cercariae emerged from the infected snails were studied live with or without vital staining using the Nikon phase contrast research microscope. Genital primordia were observed by using lacto-acetic carmine stain.
Measurements, sketches and photographs
A Nikon Y-TV55 camera, NIKON, JAPAN attached to a Nikon ECLIPSE Ni-U, NIKON, JAPAN phase contrast research microscope was used to take photographs. The trematodes were measured using the Nikon NIS-Elements, NIKON, JAPAN imaging software. Measurements (in μm) were taken on heat-killed specimens. All measurements are in micrometers (μm), as range followed by mean in parentheses. Descriptions are based on the measurements of a minimum of ten specimens. Illustrations were made using the Nikon Y-IDT drawing tube attached to the Nikon ECLIPSE Ni-U microscope, and the details were added free-hand from observations made on live specimens.
Experimental infections/lifecycle study
Eggs recovered from the adult P. wayanadensis from E. cyanophlyctis were isolated. For the release of miracidia, slight pressure was applied on the cover slip and/or teased with a pair of fine, sharp needles. Viable eggs and miracidia were fed to the helminth/infection-free B. (D.) pulchella (five in number) maintained in the laboratory. The snails were examined periodically to observe the development of sporocysts and cercariae. Fed snails were crushed within 5–9 days of post-feeding; their hepatopancreas were smeared on slides and observed for sporocysts. Helminth-free odonate naiads were used for experimental infection studies. Fifteen dragonfly naiads were individually exposed to fresh cercariae emerged from snails and observed periodically (3–5 days after post infection) to determine the course of metacercarial development. Ten helminth-free frogs were then force-fed with 33-day-old metacercariae. The specimens were then dissected and observed at three days of periodic intervals (three days post infection) for developing flukes. Observations were made on fresh specimens.
Infection-free specimens
The naiads and frogs used for the experimental infection studies were collected from infection-free areas (specific areas in the neighbouring district from where the collected specimens did not show P. wayanadensis infection in the regular parasitic survey for the past two years). Such frog specimens were maintained in the laboratory and their faecal material was carefully analysed for any eggs of P. wayanadensis continuously for a period of three months. Five laboratory-reared frogs of the same species were used to confirm the results obtained in the experimental infection studies. The frogs that were free from P. wayanadensis infection were used for the study. The specimens were maintained separately in clean aquariums/glass containers in the laboratory. Laboratory-born B. (D.) pulchella were used as infection-free specimens for the experimental infection studies.
Observations
Egg
Eggs (fig.1a) ovoid (27.0 × 13.0), light yellow, operculate, numerous in numbers, contained fully developed miracidia. Eggs were directly collected from the adult. Attempts made to release miracidia were successful.
Fig. 1. Egg (a), miracidium (b), sporocyst (c1, c2), cercaria (d1, d2), stylet (d3) and 21-day-old excysted metacercaria (e1, e2) of P. wayanadensis.
Miracidium
Miracidia (fig. 1b) are small, oval, with a pointed anterior portion. The body surface is covered with backwardly directed, moderately long cilia that reaches from the anterior portion to about one third of the body length. Penetration glands elliptical, uninucleated and placed one on either side of the median apical gland. Six moderate, uninucleated germinal cells occur in the posterior half of body. Miracidia measured 24.8 × 11.1.
Sporocyst
First intermediate host. Bithynia (Digoniostoma) pulchella.
Site of infection. Hepatopancreas.
Accession number. Z-S/DP-42.
Body of sporocysts (fig. 1c1 and c2) white, ovoid, containing two to three germ balls; measured 357.5 × 116.8. After eight to nine days of exposure to snails, miracidia developed into sporocysts. Sporocysts were recovered from the hepatopancreas of the five B. (D.) pulchella examined.
Cercaria
Natural infection with cercariae was observed in 22 of 348 B. (D.) pulchella (6.3%). Other species of snails – 246 Indoplanorbis exustus and 186 Lymnaea luteola – collected from the same geographic locations were refractory to infection with this cercaria. Under experimental conditions, cercariae emerged within 11–13 days exposure of eggs and/or miracidia to the snail. All the exposed snails were found infected. Cercariae were positively phototactic. They emerged throughout the daytime and the change of water acted as a cue for their emergence.
Description
Virgulate xiphidiocercariae (fig. 1d1 and d2); spinose, oval, body measured 941.9 × 453.5. Ventrally attached aspinose tail has non-uniform plications. Tail 976.8 in length and 139.5 in width. Oral sucker large, sub-terminal, 232.6 × 220.9. Ventral sucker small, round, post-equatorial and 69.8 × 69.8 in diameter. Sharply pointed stylet (fig. 1d3) inserted into the dorsal wall of the oral sucker. Stylet, with thickening at the shoulders, measured 147.0 × 30.0. Large, bi-lobed, inverted-comma-shaped virgula organ (290.7 × 93.0) occupied major portion of oral sucker.
Mouth sub-terminal. Pre-pharynx short followed by muscular pharynx, 58.1 × 93.0. Four pairs of penetration glands located antero-lateral to ventral sucker, their ducts open at the base of stylet. Large, thin-walled, V-shaped excretory bladder at the posterior end measured 279.0 × 290.7.
Metacercaria
Second intermediate host. Dragonfly naiads (Orthetrum sp.) and damselfly naiads (Ischnura sp. and Copera sp.).
Site of infection. Thorax and abdomen (Orthetrum sp.) and eyes (Ischnura sp. and Copera sp.).
Accession number. Z-N/O-12.
In natural infections, metacercariae were found in the eyes of the damselfly naiads of the Ischnura sp. and Copera sp. collected from the paddy fields from where the infected B. (D.) pulchella were collected. In natural conditions, the prevalence of infection was 56.2% (nine out of 16 were infected) in Ischnura sp. and 100% (three specimens collected were infected) in Copera sp. The metacercariae were also found in the thorax of the dragonfly naiads Orthetrum sp. collected from the same paddy field. In natural conditions, the prevalence of infection was 33.3% (four out of 12 were infected) in Orthetrum sp. Experimental infection has been successfully established in 15 naiads of Orthetrum sp. exposed to cercariae. The encysted metacercariae developed in the thorax and abdomen. Nine cysts were obtained from a single naiad exposed to cercariae. On the 14th day post infection, encysted metacercariae were obtained from the thorax.
On the 21st day post infection, encysted metacercariae were recovered from the thoracic region of dragonfly naiads. Cysts round; developing cysts wall bi-layered and thin. Excysted metacercariae measured 1023.5 × 499.3. Tegument thin and spinose. Backwardly directed spines visible more clearly on live specimens. Stylet could still be seen in the oral sucker. The cephalic glands with ducts as a bunch were found on either sides of oral sucker. Oral sucker sub-terminal measured 139.1 × 163.0 and ventral sucker 110.5 × 114.7. Mouth sub-terminal followed by developing pharynx (32.6 × 38.5), oesophagus (86.3 × 11.6) and intestinal caeca (355.5 × 51.0). Testes primordia visible as two round bodies just below the ventral sucker; right primordial testis measured 117.8 × 123.5 and left 136.5 × 125.2 in size. Ovarian primordium (74.2 × 63.1 in size) located in between the right intestinal caecum and ventral sucker. A developing cirrus pouch visible, measured 375.2 × 94.2. The V-shaped excretory vesicle filled with excretory concretions present at the posterior part of body (fig. 1e1 and e2).
On the 24th day post infection, excysted metacercaria measured 889.5 × 440.3. The stylet disappeared, cephalic glands present. Cuticular spines more visible, prominent in fore body. Oral sucker 143.0 × 152.0 and ventral sucker 105.4 × 106.3 in size. Pharynx measured 39.7 × 70.7 and oesophagus 42.3 × 29.5 in size. Intestinal caeca extend laterally a short distance anterior to developing testes (325.4 × 58.2). Size of the genital primordium reduced. Right testis measured109.6 × 94.8, left testis 107.6 × 103.6 and ovary 58.9 × 36.4. Cirrus sac 261.3 × 66.1 in size (fig. 2a1 and a2).
Fig. 2. Twenty-four-day-old excysted metacercaria (a1, a2), 27-day-old excysted metacercaria (b1, b2), 30-day-old excysted metacercaria (c1, c2) and 33-day-old encysted progenetic metacercaria (d1, d2) of P. wayanadensis.
On the 27th day post infection, bi-layered cyst walls fully developed; outer layer transparent and thick; the inner layer thick and opaque. The excysted metacercaria measured 851.3 × 507.3. Oral sucker 139.7 × 156.9 and ventral sucker 115.3 × 121.6 in size. Pharynx globular, muscular, 39.4 × 62.6 and oesophagus 42.8 × 26.5 in size. Intestinal caeca measured 343.6 × 56.2. Right testis 101.7 × 99.5 and left testis 116.7 × 118.5 and ovary measured 56.3 × 52.6. Vasa efferentia, vas deferens, seminal vesicle and oviduct discernible. Uterine coils empty. Cirrus sac 326.6 × 68.7 in size (fig. 2b1 and b2).
On the 30th day post infection, metacercaria measured 934.6 × 469.5. The cyst wall well developed. Cuticular spines more visible; prominent in fore body. Oral sucker 156.5 × 147.8 and ventral sucker 121.7 × 126.1. Pharynx 39.1 × 52.2. Oesophagus 21.7 × 8.7 followed by intestinal caeca 313.0 × 47.8. Right testis measured 126.1 × 121.7, left testis 134.8 × 130.4 and ovary 52.2 × 47.8. Cirrus sac 326.0 × 65.2 in size (fig. 2c1 and c2).
On the 33rd day post infection, progenetic metacercaria recovered from the abdomen of the libellulid naiad. Cyst measured 1052.0 × 936.8 and metacercaria measured 1031.0 × 810.5. Oral sucker 157.9 × 168.4 and ventral sucker 136.8 × 157.9 in size. Pharynx 42.1 × 63.2 and intestinal caeca measured 463.1 × 73.7. Reproductive structures well developed. Right testis 168.4 × 152.6, left testis 178.9 × 176.8 and ovary measured 94.7 × 94.7. Cirrus sac 384.2 × 93.7 in size. Eggs present in initial coils of uterus, lying posterior to ovary (fig. 2d1 and d2).
Adult
Definitive host. Euphlyctis cyanophlyctis.
Site of infection. Duodenum.
Accession number. Z-F/E-21.
In natural infection, adults of P. wayanadensis were found in the duodenum of the common water skittering frog, E. cyanophlyctis. The prevalence of natural infection was 11.1% (two out of 18 were infected) in E. cyanophlyctis. Experimental infection has been successfully established in the frogs (ten in number) exposed to encysted metacercariae. The adult trematodes were developed in the duodenum of all exposed frogs.
On the third day post infection, immature adults were obtained from the duodenum of the frog. Body measured 999.8 × 647.7. Cuticular spines more prominent. Oral sucker 191.3 × 202.3 and ventral sucker 126.1 × 139.1. Pharynx 65.2 × 99.9 and oesophagus 56.5 × 17.4. Intestinal caeca measured 526.0 × 108.7. Right testis 121.7 × 121.7, left testis 136.9 × 147.8 and ovary measured 82.6 × 84.8. Cirrus sac 465.1 × 99.9 in size. Eggs measured 30.4 × 10.7 (fig. 3a1 and a2).
Fig. 3. Three-day-old adult (a1, a2) and 17-day-old adult (b1, b2) of P. wayanadensis.
On the 17th day post infection, mature adults of P. wayanadensis were recovered (fig. 3b1 and b2). Duodenum of E. cyanophlyctis, force-fed with 33-day-old metacercariae contained mature P. wayanadensis. The prepatent period was 14–19 days. Seventeen-day-old flukes measured 1071.4 × 571.4. Mature flukes were identical to those recovered from field-collected E. cyanophlyctis.
Discussion
Experimental infection studies can provide crucial information when certain life cycle stages cannot be found in nature and different life cycle stages suspected of belonging to the same species are found in the same habitat (Blasco-Costa & Poulin, Reference Blasco-Costa and Poulin2017). These studies can provide more proof, along with morphological matching of life cycle stages recovered from the naturally infected hosts. Thus, the experimental infection study is an important tool for life cycle resolution. Information on the life cycle studies of trematode parasites is scanty. This paper describes the life cycle of P. wayanadensis (fig. 4) from egg to egg-producing adult as elucidated during the course of the present study. Pleurogenoides wayanadensis has a three-host life cycle. The adult is found in the duodenum of E. cyanophlyctis. Eggs from the adult trematode released miracidia, which entered the snail B. (D.) pulchella, developed into sporocysts and then into cercariae. Sporocysts were found in the hepatopancreas of the snail. The emerged cercariae were then encysted to form metacercariae in the eyes, thorax and abdomen of the naiads. When the definitive host, frog, was fed on the encysted metacercariae, the metacercariae developed into adult parasites. The life cycle is completed in about 58–65 days: the molluscan phase in 11–13 days, 33 days in the naiad host, where the metacercariae become infective, and a 14–19-days prepatent period in the definitive host (fig. 5).
Fig. 4. Life cycle of P. wayanadensis. (A) egg; (B) miracidia; (C) snail host; (D) sporocyst; (E) cercaria; (F) naiad host; (G) 14-day-old encysted metacercaria; (H) 21-day-old excysted metacercaria; (I) 24-day-old excysted metacercaria; (J) 27-day-old excysted metacercaria; (K) 30-day-old excysted metacercaria; (L) 33-day-old encysted progenetic metacercaria; (M) frog host; (N) three-day-old adult; (O) 17-day-old adult.
Fig. 5. Milestones in the life cycle of P. wayanadensis.
The life cycle of P. wayanadensis is similar to that of Pleurogenoides medians (Mathias, Reference Mathias1924; Neuhaus, Reference Neuhaus1940; Buttner, Reference Buttner1951), Pleurogenoides japonicus (Okabe & Shibue, Reference Okabe and Shibue1951; Shibue, Reference Shibue1953), P. tener (Macy, Reference Macy1964), Pleurogenoides orientalis (Madhavi et al., Reference Madhavi, Dhanumkumari and Ratnakumari1987), Pleurogenoides ovatus (Janardanan & Prasadan, Reference Janardanan and Prasadan1991) and Pleurogenoides malampuzhensis (Brinesh & Janardanan, Reference Brinesh and Janardanan2014); all the seven species have three host life cycles, and differ only with regard to the morphology/morphometry of larvae (table 1) and the invertebrate hosts (table 2).
Table 1. Measurements of cercaria, metacercaria and adult of P. wayanadensis.
Table 2. Summary of hosts in the life cycle of the seven species of Pleurogenoides.
The virgulate xiphidiocercaria of P. wayanadensis is comparable with the cercariae of P. medians (Mathias, Reference Mathias1924; Neuhaus, Reference Neuhaus1940; Buttner, Reference Buttner1951), P. tener (Macy, Reference Macy1964), P. orientalis (Madhavi et al., Reference Madhavi, Dhanumkumari and Ratnakumari1987), P. japonicus (Okabe & Shibue, Reference Okabe and Shibue1951; Shibue, Reference Shibue1953), P. ovatus (Janardanan & Prasadan, Reference Janardanan and Prasadan1991) and P. malampuzhensis (Brinesh & Janardanan, Reference Brinesh and Janardanan2014). The present cercaria is distinguishable from other cercariae by the shape and structure of stylet, virgula organ and excretory bladder, position and size of ventral sucker, number of penetration glands and morphometry. The cercariae of P. wayanadensis were positively phototactic. They emerge throughout the day and the change of water acts as a cue for their emergence.
Habitat of the bithynid snail B. (D.) pulchella varies from permanent or temporary freshwater bodies, marshes, swamps and shallow ponds. They are mostly found in mud, on rocks or attached to grass or other aquatic vegetation. For the present study, the snails were collected from the paddy fields with loose soil and muddy substratum. The dragonfly naiads and the frogs were also seen on the same habitat where the snails existed. The cercaria larvae can easily get access to the dragonfly/damselfly naiads, the second intermediate host. To complete the life cycle successfully, nearly all digenetic trematodes choose a confined habitat/ecological niche where all the hosts are available.
The metacercaria of P. wayanadensis exhibited progenetic development. Progenesis is the phenomenon of sexual maturation in an organism still in a morphologically juvenile stage (Gould, Reference Gould1977). Among parasites, precocious egg production has been known since the report by Von Siebold (Reference Von Siebold1835). This phenomenon was reported in trematodes for the first time by Dollfus (Reference Dollfus1924) with his observations on egg production by the metacercariae of P. medians (Pleurogenidae). Facultative truncation of life cycle is only shown by some individuals within a species. Pleurogenoides wayanadensis exhibits facultative progenesis at the metacercarial stage with immature eggs in their initial coils of uterus. Normal and progenetic metacercariae were observed together in the thorax and abdomen of the dragonfly naiads. Progenesis has survival value and is adaptive (Janardanan & Prasadan, Reference Janardanan and Prasadan1991). Facultative progenesis is an option for metacercariae in second intermediate hosts, and it will be selected in any unstable or temporary habitat in which intermediate hosts become stranded in marginal pools where no definitive hosts are available for the completion of the life cycle of those trematodes (Poulin & Cribb, Reference Poulin and Cribb2002). Metacercariae of P. medians, P. japonicus, Pleurogenoides sitapurii and P. ovatus are the other species under the genus Pleurogenoides that exhibit progenesis and undergo considerable growth and development inside the second intermediate hosts. The progenetic development in the metacercariae of P. medians encysted in the dragonfly naiads was reported by Buttner (Reference Buttner1951). The metacercaria of P. japonicus was reported from shrimps (Okabe & Shibue, Reference Okabe and Shibue1951) and that of P. sitapurii in the crab, Paratelphusa ceylonensis (Dissanaike & Fernando, Reference Dissanaike and Fernando1960). Metacercaria of P. ovatus were recovered from the connective tissues, hepatopancreas and musculature of the freshwater crab, Paratelphusa hydrodromous (Janardanan & Prasadan, Reference Janardanan and Prasadan1991).
The relict hypothesis (Stunkard, Reference Stunkard1959; Riggs & Ulmer, Reference Riggs and Ulmer1983) explains progenesis of the metacercariae as a reminiscence of an ancestral two-host cycle, whereas, according to the novelty hypothesis, progenetic species we observe today are secondarily derived from an ancestral three-host life cycle.
In the earlier studies, phylogenetic reconstructions revealed that trematodes have a primitive cycle involving a mollusc as the first host and a predatory vertebrate as the definitive host. According to these studies, trematodes, after a very long time, had adjusted their developmental schedule so as to incorporate a trophic link, as a second intermediate host, to increase the chances of transmission toward the vertebrate host (Rohde, Reference Rohde1994; Ewald, Reference Ewald1995; Cribb et al., Reference Cribb, Bray, Olson and Littlewood2003).
Investigations following analysis of character convergence strongly support the idea that the two-host life cycles are derived from more ancient three-host life cycles (Carney & Brooks, Reference Carney and Brooks1991; Smythe & Font, Reference Smythe and Font2001). Progenesis must, therefore, be considered as a novelty in the evolution of trematode life cycles. It can be interpreted that a simpler life cycle is easier to complete and the phenomenon of progenesis is to reduce the transmission events. Earlier studies (Buttner, Reference Buttner1951; Grabda-Kazubska, Reference Grabda-Kazubska1976; Font, Reference Font1980; Poulin & Cribb, Reference Poulin and Cribb2002) revealed that a large number of metacercariae perish with their intermediate hosts in normal three-host life cycles. Therefore, this may be a strategy where all metacercariae become potential breeders if they adopt progenetic development.
In the present study, the metacercariae were found in the eyes of the field-collected damselfly naiads Ischnura sp. and Copera sp., and in the thorax of the dragonfly naiads Orthetrum sp. In the experimental studies, dragonfly naiads of Orthetrum sp. were infected with the metacercariae of P. wayanadensis. The P. medians (Mathias, Reference Mathias1924; Neuhaus, Reference Neuhaus1940; Buttner, Reference Buttner1951), P. tener (Macy, Reference Macy1964), P. orientalis (Madhavi et al., Reference Madhavi, Dhanumkumari and Ratnakumari1987) and P. malampuzhensis (Brinesh & Janardanan, Reference Brinesh and Janardanan2014) are the other species under the genus Pleurogenoides that used odonate naiads as second intermediate hosts. The present metacercaria is distinct from the metacercariae of other Pleurogenoides species in its morphological features and dimensions.
Here, the eggs produced by the adult P. wayanadensis get deposited in the faecal matter of the definitive host frog, E. cyanophlyctis, and released miracidia larvae in the surrounding water. The miracidia enters the snail host, B. (D.) pulchella, where it develops into sporocyst and cercaria. The cercaria larva becomes metacercaria in the damselfly naiads of the Ischnura sp. and Copera sp. and dragonfly naiads of the Orthetrum sp. that exist in the same ecological niche. Through trophic-level transfer, the metacercaria reaches the definitive host frog when the latter feeds on the infected damselfly and dragonfly naiad, develops into an adult parasite and completes the life cycle.
In this study, natural infection of cercariae was found only in B. (D.) pulchella, and all the other snail species of the area were negative to this cercarial infection. The metacercariae were found infected in both the dragonfly and damselfly naiads. Adult flukes were found in the duodenum of the frog species E. cyanophlyctis and H. tigerinus, and all other frog species of the area were negative to this infection.
Acknowledgements
The authors are grateful to the Kerala State Council for Science, Technology and Environment (KSCSTE), Government of Kerala, for providing financial assistance (Major Research Project, SRS/220/2015/KSCSTE-completed) to carry out this study. The author C.S. is grateful to the Department of Science and Technology (DST) INSPIRE program for providing the Inspire Fellowship. The permission accorded by the Department of Forest and Wildlife, Government of Kerala, order numbers WL10-63909/2016 and KFDHQ-6782/2019-CWW/WL10 for collecting frogs and naiads, respectively, from the Wayanad forest region is also gratefully acknowledged. The authors are indebted to Prof K.P. Janardanan (retired), renowned parasitologist, for critically reviewing the manuscript.
Financial support
This study was partially supported (major research project, number SRS/220/2015/KSCSTE) by the Kerala State Council for Science, Technology and Environment (KSCSTE), Government of Kerala (the project was completed in 2019).
Conflicts of interest
None.
Ethical standards
All applicable international, national and/or institutional guidelines for the care and use of animals were followed. All procedures performed in the study involving animals were in accordance with the ethical standards of the institution or practice at which the study was conducted.
Author contributions
P.K.P. designed the experiments and guided the study. His research scholars, K.S. (working on the trematode parasites of frogs), C.S. (conducting research on the metacercaria larvae of the Western Ghats) and K.A. (conducting research on the cercarial fauna of Wayanad Region of the Western Ghats) performed the experiments and studied the trematodes in detail. All authors contributed equally in the manuscript writing and analysis of the data.
