Introduction
Podiceps gallardoi Rumboll, 1974 (Podicipedidae), commonly known as the hooded grebe, is a waterbird with a restricted distribution in southern South America. This grebe spends the breeding season (October–April) on lakes of the high basaltic plateaus of western Santa Cruz and winters at a few estuaries on the Atlantic coast of this province (Roesler et al., Reference Roesler, Imberti, Casañas and Volpe2012a). Its presence has also been confirmed in southern Chile, including a small population resident in summer in the Torres del Paine National Park and Puerto Natales, and individuals reported occasionally on both sides of the Magellan Strait (Roesler, Reference Roesler2015). The hooded grebe is critically endangered (BirdLife International, 2017). Several threats have been proposed to explain the decline of hooded grebe populations, including depredation of eggs and chicks by the kelp gull, Larus dominicanus Lichtenstein, and the American mink, Neovison vison (Schreber), habitat modification by local climate changes and introduced fishes, mainly rainbow trout, Oncorhynchus mykiss (Walbaum) (Roesler et al., Reference Roesler, Imberti, Casañas, Mahler and Reboreda2012b, Reference Roesler, Fasola, Casañas, Hernández, de Miguel, Giusti and Reboreda2016). Until now, the helminth fauna of hooded grebes has been unknown.
The aim of this work was to determine the diversity of helminths in hooded grebes from natural environments.
Materials and methods
During a population census, Roesler et al. (Reference Roesler, Imberti, Casañas, Mahler and Reboreda2012b) found 33 dead hooded grebes in El Cervecero Lake, Buenos Aires plateau, Santa Cruz Province, Argentina. The grebes were killed by a single American mink, although only one was eaten by this predator (Roesler et al., Reference Roesler, Imberti, Casañas and Volpe2012a). Ten of these dead birds were recovered in good condition and dissected in the field by Roesler; their viscera were preserved in 10% formalin and transported to the laboratory for helminthological examination. Digenean specimens were removed, stored in 70% ethanol, stained with a 1:6 dilution in 96% ethanol of hydrochloric carmine, dehydrated and mounted between two microscope cover glasses in Canada balsam. The measurements below are given in micrometres (μm), as the range followed by mean in parentheses. Drawings were made with the aid of a drawing tube. The following abbreviations are used in the tables: ASl, angle spine length; ATl, anterior testis length; ATw, anterior testis width; Bl, body length; Bw, body width; Cl, collar length; CSl, cirrus sac length; CSw, cirrus sac width; Cw, collar width; DSl, dorsal spines length; El, egg length; Ew, egg width; Fo, forebody length; LSl, lateral spine length; Mel, metraterm length; Mew, metraterm width; Oe, oesophagus length; OSl, oral sucker length; OSw, oral sucker width; Ovl, ovary length; Ovw ovary width; Phl, pharynx length; Phw, pharynx width; PTf, post-testicular field length; PTl, posterior testis length; PTw, posterior testis width; S, number of collar spines; VSl, ventral sucker length; VSw, ventral sucker width; Bl/El, body length/egg length; OS/Ph, oral sucker width/pharynx width; OS/VS, sucker width ratio; VS/OS, sucker width ratio. The following relative proportions were calculated after Kostadinova (Reference Kostadinova, Jones, Bray and Gibson2005): BW (%), maximum body width as a proportion of body length; FO (%), length of the forebody as a proportion of body length; U (%), distance between posterior margin of ventral sucker and anterior margin of ovary, as a proportion of body length (used as an approximation for the uterine field); T (%), length of the post-testicular field as a proportion of body length.
Results
The helminthological analysis revealed the presence of five helminth taxa: one cestode, one nematode and three digeneans. The cestodes were found in the intestine with a prevalence of 100% and mean intensity of 86.4 (range: 4–246). The tetramerid nematode was found in the stomach with a prevalence of 10% and mean intensity of 1. The three digenean species comprised a notocotylid found in intestinal caeca, with a prevalence of 20% and mean intensity of 11 (10–12), and plagiorchid and echinostomatid species in the intestine. The latter two are new to science and are described below. Species determination of the cestode, tetramerid and notocotylid was not possible owing to the poor condition of specimens.
Plagiorchiidae: Plagiorchis patagonensis n. sp.
Description
Based on three mature specimens. Body small, elongate, 977–1514 (1204) long by 251–339 (305) wide (fig. 1). BW = 21–35% (26%). Tegument covered with numerous small spines. Oral sucker subterminal, large, more or less rounded, 193–203 × 179–213 (196 × 192). Ventral sucker round, significantly smaller than oral sucker, pre-equatorial, in second quarter of body, 95–116 × 95–121 (105 × 104). Oral sucker width:ventral sucker width ratio, 1: 1.76–1.93 (1:1.86). Forebody 386–469 (420) long, 31–42% (36%) of total body length. Prepharynx not observed. Pharynx well developed, round, 76–97 × 74–106 (85 × 86). Oral sucker width:pharynx width ratio 1:2–2.3 (2.2). Oesophagus very short, 24 long. Intestinal caeca long, extending to near the end of the body. Testes 2, post-ovarian, spherical to oval, entire, oblique; left testis anterior to right testis; anterior testis 131–145 × 82–119 (140 × 95), posterior testis 117–169 × 92–124 (151 × 107). Cirrus sac dorsal and posterodorsal to ventral sucker, very elongate and strongly muscular, its distal end usually curved around right margin of ventral sucker, and with proximal part always between ovary and ventral sucker, 286–302 × 55–90 (293 × 73); contains seminal vesicle divided into oval proximal part, 34–48 × 30–47 (41 × 38), and rounded distal part, 41 in diameter. Pars prostatica short; prostatic cells numerous, occupying entire cirrus sac. Cirrus long and unarmed. Genital pore median, just anterior to ventral sucker. Body length:cirrus sac length ratio 1:3.2–5.2 (1:4.1). Ovary oval or round, entire, 67–107 × 50–64 (83 × 57), near right side of proximal end of cirrus sac and just posterior to ventral sucker. Vitellarium well developed, consisting of two fields of small vitelline follicles on either side of body, extending from the caecal bifurcation to posterior extremity; vitelline fields confluent between caecal bifurcation and ventral sucker. Vitelline reservoir not seen. Mehlis' gland immediately posterior to ovary. Seminal receptacle and Laurer's canal present. Uterine loops intercaecal, with descending and ascending loops between testes and post-testicular region. Metraterm slightly shorter than cirrus sac. Eggs numerous, small, operculate, 29–38 × 17–26 (33 × 22). Excretory vesicle Y-shaped, with long stem and relatively short branches.
Plagiorchis patagonensis n. sp.: (a) entire worm, ventral view (holotype); (b) enlarged view of terminal genitalia, ventral view (holotype). Scale bars: 100 μm.
Taxonomic summary
Type host. Podiceps gallardoi Rumboll, 1974 (Podicipediformes, Podicipedidae) (hooded grebe).
Site of infection. Intestine.
Type locality. El Cervecero Lake, Buenos Aires plateau, Santa Cruz Province, Argentina (47°09′20″S, 71°16′32″W).
Date of collection. May 2011.
Type material. Holotype MLP–He 7392; paratypes MLP–He 7393 (two specimens).
Prevalence. 30%.
Mean intensity. 1.3 (range 1–2).
Etymology. The specific name refers to the geographical region where the parasite was collected.
Remarks
The genus Plagiorchis Lühe, 1899 (Plagiorchiidae), currently includes over 100 nominal species parasitic in amphibians, reptiles, birds and mammals worldwide. Seven species are known in the Neotropical region: Plagiorchis freitasi Vicente, 1978 and Plagiorchis vicentei Oliveira Rodrigues, 1994 in lizards from Brazil (Vicente, Reference Vicente1978; Oliveira Rodrigues, Reference Oliveira Rodrigues1994); Plagiorchis luehei Travassos, 1927 in ophidians from Brazil and Argentina (Travassos, Reference Travassos1928; Lunaschi & Drago, Reference Lunaschi and Drago2010); Plagiorchis parumbursatus Freitas & Dobbin, 1961 in bats from Brazil (Freitas & Dobbin, Reference Freitas and Dobbin1961); Plagiorchis didelphidis (Parona, 1896) Stossich, 1904 in marsupials from Brazil, Peru and Paraguay (Travassos et al., Reference Travassos, Freitas and Kohn1969; Masi Pallares & Benitez Usher, Reference Masi Pallares and Benitez Usher1971; Tantalean et al., Reference Tantalean, Sarmiento and Huiza1992); Plagiorchis rangeli Artigas & Zerpa, 1961 in anurans from Brazil (Fernandes & Kohn, Reference Fernandes and Kohn2014) and Plagiorchis sp. in gulls from Peru (Jara et al., Reference Jara, Barrionuevo and Ruiz1987). All Neotropical species of Plagiorchis can be distinguished easily from the new species by the non-confluent distribution of vitelline glands in the forebody, unlike in P. patagonensis n. sp. In the Mexican Transition Zone (Morrone, Reference Morrone2005), where Neotropical and Nearctic biotas overlap, four species of Plagiorchis occur, each with a wide geographical distribution: Plagiorchis muris (Tanabe, 1922) (syn. P. (Multiglandularis) muris), parasitizing Natalus stramineus mexicanus Miller (as N. mexicanus) (Chiroptera) from Mexico (Caballero, Reference Caballero1943), also reported in mammals and birds naturally and/or experimental infections from Oriental and Nearctic regions (Dollfus, Reference Dollfus1925; McMullen, Reference McMullen1937); Plagiorchis noblei Park, 1936 (syn. P. gonzalchavezi Zerecero, 1949) in Tyrannus sp. (Passeriformes) from Mexico (Zerecero, Reference Zerecero1949), also reported in mammals and birds from the Nearctic region (Park, Reference Park1936; Blankespoor, Reference Blankespoor1974; MacKenzie & McKenzie, Reference MacKenzie and McKenzie1980); Plagiorchis maculosus (Rudolphi, 1802) in Turdus migratorius (Passeriformes) from Mexico (Pérez Ponce de León et al., Reference Pérez Ponce de León, Garcia-Prieto and Mendoza-Garfias2007), also reported in birds and mammals from Holarctic, Ethiopian, Oriental and Australian regions (Bock & Janssen, Reference Bock and Janssen1987); and Plagiorchis vespertilionis (Mueller, 1784) Braun, 1900, recorded in bats from Mexico (Caballero, Reference Caballero1940), also reported in bats from Holarctic, Ethiopian and Oriental regions, although Tkach et al. (Reference Tkach, Pawlowski and Sharpilo2000) considered only European forms to be true representatives of P. vespertilionis. Plagiorchis maculosus and P. vespertilionis can be differentiated easily from P. patagonensis n. sp. by having vitelline glands not confluent in the forebody, and the oral sucker smaller than or sub-equal to the ventral sucker. Plagiorchis noblei, P. muris and the new species have a similar distribution of vitelline glands in the forebody. Plagiorchis noblei is known from a wide spectrum of phylogenetically unrelated hosts (mammals and birds), and has enormous morphological and morphometric variations. Such plasticity may be explained, in part, by the lack of host specificity (Blankespoor, Reference Blankespoor1974; MacKenzie & McKenzie, Reference MacKenzie and McKenzie1980). Blankespoor (Reference Blankespoor1974) examined numerous adult specimens of P. noblei obtained experimentally in 51 vertebrate species exposed to metacercariae, and found wide plasticity in most characters (i.e. extent of vitellaria, position of suckers, size of gonads, size and position of cirrus sac, among others); the only stable characters were the size-ratio of suckers and egg size. According to descriptions given by Park (Reference Park1936), Zerecero (Reference Zerecero1949), Blankespoor (Reference Blankespoor1974) and MacKenzie & McKenzie (Reference MacKenzie and McKenzie1980), P. noblei differs from the new species mainly by the size-ratio of suckers (1:1–1.4 vs. 1:1.7–1.9) (table 1). Plagiorchis muris differs from P. patagonensis n. sp. mainly by having a larger body size (up to 2670 vs. up to 1514) and smaller oral:ventral sucker ratio (1:1.1–1.7 vs. 1.7–1.9) (table 2).
Comparative measurements of main morphological characters of P. patagonensis n. sp. and P. noblei.
*Calculated from original descriptions.
§Experimental.
Comparative measurements of P. patagonensis n. sp. and P. muris.
*Calculated from original descriptions.
**Calculated from original drawings.
§Experimental hosts.
In Podiceps spp., only two species of Plagiorchis have been reported: Plagiorchis laricola Skrjabin, 1924 in Podiceps grisegena (Boddaert) and Podiceps nigricollis Brehm from the Palaearctic region, and P. maculosus parasitizing Po. grisegena from the Holarctic region (Storer, Reference Storer2000), which was discussed above. According to Rees (Reference Rees1952), P. laricola is characterized by having a larger ovary than ventral sucker, vitelline glands confluent in the forebody and the cirrus sac not extending beyond the posterior margin of the ovary. Therefore, P. laricola can be differentiated from the new species found in Po. gallardoi by the large size of its ovary relative to the ventral sucker.
This is the first species of Plagiorchis to be described from Neotropical birds.
Echinostomatidae: Euparyphium tobianum n. sp.
Description
Based on five mature specimens. Small worms of elongate body (BW = 14–20%), 1600–2800 × 324–411 (2300 × 383), with maximum width at level of ventral sucker (fig. 2). Forebody ventrally concave, with tegument densely spiny, short to long 401–532 (468) in length (FO = 16–26%).
Euparyphium tobianum n. sp.: (a) entire worm, ventral view (holotype); (b) head collar with 37 collar spines (paratype); (c) enlarged view of terminal genitalia, dorsal view (holotype); (d) enlarged view of ovarian complex, dorsal view (holotype). Scale bars: 100 μm.
Head collar reniform, well developed, with ventral ridge, 152–304 × 193–343 (218 × 273). Collar spines 37–39; 4 angle spines on each ventral lappet (2 dorsal and 2 ventral) longer than marginal spines, 4 lateral spines in a single row on each side; 21–23 dorsal spines in double row, aboral spines slightly longer than oral. Angle spines 48–81 × 12–17 (64 × 14); lateral spines smaller than the angle spines, 50–71 × 10–12 (60 × 12); dorsal spines in aboral row, 43–64 × 10–14 (57 × 11), dorsal spines in oral row, 31–60 × 10–12 (45 × 11).
Oral sucker subterminal, 79–112 × 89–129 (97 × 103). Ventral sucker large, deep, in the first quarter of body, 290–406 × 222–275 (354 × 253). Ventral sucker width:oral sucker width ratio 1:2–2.7 (1:2.5). Prepharynx short. Pharynx, elongate, shorter than oral sucker, 64–95 × 33–43 (79 × 39). Oral sucker width:pharynx width ratio 1:2.4–3.2 (1:2.7). Oesophagus 203–309 (272) long. Intestinal bifurcation anterior and close to ventral sucker; intestinal caeca end blindly, close to posterior extremity.
Testes tandem, contiguous, oval, elongate, of similar size, and situated in the third quarter of the body. Anterior testis, 179–314 × 106–193 (239 × 144). Posterior testis, 202–386 × 111–193 (288 × 137). Post testicular field long, 396–803 (574) (T = 21–29%).
Cirrus sac dorsal to ventral sucker, 119–238 × 67–76 (171 × 71), containing an undivided and oval internal seminal vesicle, an elongate and convoluted pars prostatica, and tubular unspined cirrus. Genital pore median, just posterior to intestinal bifurcation.
Ovary entire, spherical, 71–130 × 63–116 (102 × 82). Laurer's canal present. Mehlis' gland well developed, median, situated between ovary and anterior testis. Uterine seminal receptacle conspicuous. Vitellarium follicular, follicles large, distributed in two lateral fields from midway between ventral sucker and ovary to almost the posterior extremity of the body. Uterine field short, with intercaecal loops (U = 5–12%). Metraterm muscular, similar in size to cirrus sac, 250–314 × 39–44 (277 × 41). Eggs not numerous, up to 5, 90–119 × 55–61 (102 × 57). Body length:egg length ratio 1:20–29.
Taxonomic summary
Type host. Podiceps gallardoi Rumboll, 1974 (Podicipediformes, Podicipedidae) (hooded grebe).
Type locality. El Cervecero Lake, Buenos Aires plateau, Santa Cruz Province, Argentina (47°09′20″S, 71°16′32″W).
Date of collection. May 2011.
Site of infection. Intestine.
Type material. Holotype MLP–He 7394; paratypes MLP–He 7395 (four specimens).
Prevalence. 90%.
Mean intensity. 150 (range 3–307).
Etymology. The new species is named after the common name of the host, macá tobiano.
Remarks
The morphological characters and most body proportions of the specimens described here are similar to those reported by Kostadinova (Reference Kostadinova, Jones, Bray and Gibson2005) for the genus Euparyphium Dietz, 1909, except for the number of collar spines. The new species described here has 37–39 collar spines, whereas 27, 45 or 55 spines are known in Euparyphium (according to Kostadinova, Reference Kostadinova, Jones, Bray and Gibson2005). Nevertheless, the latter genus diagnosis likely reflects the small number of species currently included in Euparyphium. In light of other morphological and morphometric similarities between our specimens and species of Euparyphium, we consider that the number of collar spines in the generic diagnosis must be amended.
Kostadinova & Gibson (Reference Kostadinova and Gibson2002) considered the genus Euparyphium to be constituted by four species. The type species, Euparyphium capitaneum Dietz, 1909 (syn. Euparyphium anhingae Premvati, 1968) described from birds from Neotropical and Nearctic regions, and Euparyphium guerreroi Tubangui, 1931, Euparyphium murinum Tubangui, 1931 and Euparyphium albuferensis Esteban, Toledo, Sánchez & Muñoz-Antolí, 1997, from mammalian hosts from Palaearctic and Oriental regions.
Euparyphium capitaneum, a parasite of Anhinga anhinga (L.) (Suliformes, Anhingidae), was described briefly by Dietz (Reference Dietz1910) in Brazil. It was described later in Cuba by Pérez Vigueras (Reference Pérez Vigueras1944) and the USA by Premvati (Reference Premvati1968) (as E. anhingae), and redescribed by Kudlai et al. (Reference Kudlai, Tkach, Pulis and Kostadinova2015). This species mainly differs from E. tobianum n. sp. by having fewer spines in the cephalic collar (27 vs. 37–39), very elongated and wavy testes, larger body size, smaller eggs, greater body length:egg length ratio, shorter metraterm and larger angle spines (table 3).
Comparative measurements of Euparyphium tobianum n. sp. and Euparyphium spp.
*Calculated from original descriptions.
**Calculated from original drawings.
The three remaining species of Euparyphium, described from rodents, mainly differ from the new species in the number collar spines, i.e. 55 in E. guerreroi, 45 in E. murinum and E. albuferensis vs. 37–39 in E. tobianum n. sp. The species from mammalian hosts also differ in most metrical characters and relative proportions (table 3) (Tubangui, Reference Tubangui1931a, Reference Tubanguib; Esteban et al., Reference Esteban, Toledo, Sánchez and Muñoz-Antolí1997).
Discussion
The present report of cestodes, tetramerids, notocotylids, P. patagonensis n. sp. and E. tobianum n. sp. constitutes the first record of helminths in wild populations of Po. gallardoi. Four other species of Podicipedidae inhabit Patagonia, Argentina: Podiceps major (Boddaert), Podiceps occipitalis Garnot, Rollandia rolland (Quoy & Gaimard) and Podilymbus podiceps (Linnaeus). The digenean fauna of grebes from Patagonia is poorly known. There are only the reports of Stephanoprora argentinensis Sutton, Lunaschi & Topa, 1982 parasitizing Po. major and R. rolland from Pellegrini Lake, Río Negro Province. A few other records of helminths from grebes exist in central Argentina: Petasiger argentinensis Lunaschi & Drago 2010 (Echinostomatidae) in Po. major and R. rolland; Tylodelphys adulta Lunaschi & Drago, 2004 (Diplostomidae) and Stephanoprora uruguayense Holcman-Spector & Olague, 1989 (Echinostomatidae) in Po. major; and Levinseniella cruzi Travassos, 1920 in R. rolland (Microphallidae) from Buenos Aires Province (Lunaschi et al., Reference Lunaschi, Cremonte and Drago2007; Drago & Lunaschi, Reference Drago and Lunaschi2015).
Most life cycles of Plagiorchis spp. involve lymnaeid snails, such as Lymnaea spp., Radix spp. and Stagnicola spp., as first intermediate host. The metacercariae can develop inside the sporocyst without emerging from the snail; in other snail species; in larval aquatic insects, mainly chironomids, ceratopogonids and culicids; or in crustaceans (Rees, Reference Rees1952; Yamaguti, Reference Yamaguti1975; Blankespoor, Reference Blankespoor1977; Bock & Janssen, Reference Bock and Janssen1987; Zikmundová et al. Reference Zikmundová, Georgieva, Faltýnková, Soldánová and Kostadinova2014). In the life cycles of Euparyphium spp., the cercariae develop in pulmonate gastropods (Planorbidae) and the metacercariae develop in the same planorbid, or in lymnaeid or physid species (Esteban et al., Reference Esteban, Toledo, Sánchez and Muñoz-Antolí1997; Kostadinova, Reference Kostadinova, Jones, Bray and Gibson2005).
The diet of the hooded grebe is scarcely known. During the breeding season on lakes of western Santa Cruz, these grebes feed mainly on snails (Lymnaea diaphana King) and other aquatic invertebrates, such as amphipods, copepods, cladocerans, larvae of chironomids and water beetles, and leeches (Fjeldså, Reference Fjeldså1986). In wintering grounds on the estuaries of the Atlantic coast of the province, they consume fish, crustaceans, hydrozoans, algae and chitons (Torres & Vargas, Reference Torres and Vargas2005). In light of what is known of the life cycles of members of Plagiorchis and Euparyphium, with snails as the main route of infection, hooded grebes probably acquire both digenean species during the breeding season on lakes of western Santa Cruz.
The pathogenicity of Plagiorchis spp. and Euparyphium spp. seems to be low, except in cases of massive infections. Plagiorchis laricola can cause necrotic enteritis, affecting the whole of the small intestine and duodenum, which may cause the death of the birds in cases of high infections (Foggie, Reference Foggie1937). The low intensity of infection of P. patagonensis n. sp. found in this study does not indicate that these parasites pose a threat to hooded grebe populations.
Acknowledgements
The authors express their gratitude to Ignacio Roesler and to the team of the Proyecto Macá Tobiano (Aves Argentinas and Ambiente Sur) for assistance with collection of the hosts, and to Sean Locke for his help with linguistic revision.
Financial support
This work was supported by CIC (Res. N° 048/16) and UNLP (11/N751).
Conflict of interest
None.
