Parasitol Res DOI 10.1007/s00436-014-3809-9
ORIGINAL PAPER
Two new species of Maritrema Nicoll, 1907 (Digenea: Microphallidae) from New Zealand: morphological and molecular characterisation Bronwen Presswell & Isabel Blasco-Costa & Aneta Kostadinova
Received: 10 November 2013 / Accepted: 2 February 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Two new species of the microphallid genus Maritrema (Maritrema) Nicoll, 1907 are described from freshwaters in the South Island of New Zealand. Maritrema deblocki n. sp. occurs as an adult in the mallard Anas platyrhynchos (L.); Maritrema poulini n. sp. is found as sporocysts/cercariae in Potamopyrgus antipodarum (Gray) and as metacercariae in two species of amphipod and two species of isopod. We use morphological and molecular characterisation to distinguish between the two species, and compare them to their four morphologically closest congeners. M. deblocki n. sp. and M. poulini n. sp. are distinguished from each other by the relative sucker size, the positions of the genital pore and ovary, the convergence of the vitelline ribbons, and overall size. With the aid of molecular data, we matched life cycle stages of M. poulini n. sp. and assessed its use of multiple second intermediate hosts. Phylogenetic analyses included sequences for the two new species and the available microphallid sequences for the large ribosomal subunit and the internal transcribed spacer 1 of the ribosomal RNA gene. Closer to each other than to any other species, the sister species together with Maritrema novaezealandense Martorelli, Fredensborg, Mouritsen & Poulin, 2004, Maritrema heardi (Kinsella & Deblock, 1994), Maritrema eroliae Yamaguti, 1939 and Maritrema oocysta (Lebour, 1907) formed a well-supported clade. In addition, we clarify Bronwen Presswell and Isabel Blasco-Costa contributed equally to this work. B. Presswell (*) : I. Blasco-Costa Department of Zoology, University of Otago, PO box 56, Dunedin, New Zealand e-mail:
[email protected] A. Kostadinova Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, Branišovská 31, 370 05 České Budějovice, Czech Republic
the taxonomic identity of several unidentified Microphallus spp. in the recent ecological literature from New Zealand and propose corrected spellings for a number of Maritrema species epithets. Keywords Maritrema . Microphallidae . taxonomy . phylogeny . New Zealand
Introduction The genus Maritrema Nicoll, 1907 (Digenea: Microphallidae) contains about 65 species, predominantly parasitic in birds of freshwater, brackish, and marine ecosystems. The life cycles of Maritrema spp. involve gastropods and crustaceans as first and second intermediate hosts, respectively. Subgenus Maritrema (Maritrema) Nicoll, 1907 was formed by default when Deblock (1973) erected the subgenus Maritrema (Atriospinosum) Deblock, 1973 to include the species with a spinous atrioacetabular plate adjacent to the genital pore in contrast to species of Maritrema (Maritrema) which are characterised by a simple, slightly differentiated, unarmed genital pore (Deblock 2008). Two species have previously been reported from New Zealand: Maritrema gratiosum Nicoll, 1907 in the eastern bar-tailed godwit Limosa lapponica baueri Nauman (see Allison 2001) and South Island pied oystercatcher Haematopus finschi Martens (as Haematopus ostralegus finschii, see Allison 2000) and Maritrema novaezealandense Martorelli, Fredensborg, Mouritsen & Poulin, 2004 in the redbilled gull Chroicocephalus scopulinus (Forster) (as Larus novaehollandiae scopulinus, see Martorelli et al. 2004). There are ten records of eight species from Australia: Maritrema brevisacciferum Shimazu & Pearson, 1991; Maritrema calvertense Smith, 1974; Maritrema eroliae Yamaguti, 1939; Maritrema oocysta (Lebour, 1907); Maritrema ornithorhynci Hickman, 1955; Maritrema rubeum Deblock & Canaris, 1996; Maritrema spinulosum, Deblock &
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Canaris, 1996; and Maritrema sp. (Hickman 1955; Smith 1974, 1983; Deblock & Pearson 1968; Shimazu and Pearson 1991; Deblock and Canaris 1996). Species delimitation has benefited enormously from the use of molecular data facilitating unambiguous diagnoses of problematic taxa and discovery of cryptic species. In the case of parasites with complex life cycles, such as digeneans, the application of molecular tools has also favoured studies aimed at matching life cycle stages and uncovering host–parasite associations (Pérez-Ponce de León and Nadler 2010). To date, few studies have used this approach for freshwater trematode species (e.g., Georgieva et al. 2012, 2013) and, to the best of our knowledge, only one for microphallids (Galaktionov et al. 2012). Despite the large diversity of the species of Maritrema and the many synonyms hampering reliable species discrimination based solely on morphological grounds, the application of molecular taxonomy has rarely been employed for Maritrema spp. To date, ribosomal sequences of the 28S and ITS rDNA regions are available for eight named species (Tkach et al. 2000, 2003; Al-Kandari et al. 2011; Pina et al. 2011; Galaktionov et al. 2012). Here, we describe morphologically, and characterise genetically, two new species of Maritrema from fresh and brackish waters of New Zealand, which lack a spinous atrio-acetabular plate near the genital pore and are placed in the subgenus Maritrema (Maritrema). An adult morphotype of Maritrema was recovered during a survey of adult mallard ducks (Anas platyrhynchos L.). In addition, sporocysts and cercariae were found in the mollusc Potamopyrgus antipodarum (Gray), and metacercariae were found in the isopod Austridotea annectens Nicholls and amphipods Paracalliope fluviatilis (Thomson) and Paracorophium excavatum (Thomson), all of which were identical genetically, but differed from the adults in the mallard. In order to describe the adult characters of the second species, we grew metacercariae in vitro. In addition, we clarify here the taxonomic identity of several unidentified Microphallus sp. in the recent ecological literature from New Zealand and propose corrected spellings for a number of Maritrema spp. epithets.
Materials and methods Specimens Adult mallard ducks were shot at Karitane Estuary, Otago, South Island, New Zealand in May 2011 during the official hunting season, under licence in accordance with the Fish & Game New Zealand regulations governing the region of Otago. The viscera were dissected and all intestinal worms preserved in 96 % ethanol for molecular analyses or 70 % for whole mounts. Isopods and amphipods were collected from macrophytes along the shoreline of Lake Waihola and
molluscs were sampled from Tomahawk lagoon using dip nets at various times of year between 2011 and 2013. Invertebrates were kept alive in aerated lake water until examined. Metacercariae, extracted from the body cavity of the crustaceans, were fixed in either 96 % ethanol for molecular analyses or 70 % for whole mounts. Live metacercariae, gathered from the body cavity of isopods, excysted naturally when placed in a culture medium of NCTC 109 (Sigma, Auckland, New Zealand) supplemented with 20 % chicken serum (deactivated at 56 °C for 30 min), and incubated at 40 °C (adapted from Lloyd and Poulin 2011). Incubation temperature was based upon the body temperature of the assumed bird definitive host. A mixture of penicillin (120 mg/ml), streptomycin (100 mg/ml), and fish fungicide (Prefuran, Argent Laboratories, New York, NY, USA; 0.2 mg/ml) was added to the medium to prevent bacterial and fungal contamination. Thirteen specimens were cultured for 48 h before being killed in hot water and fixed in 70 % ethanol. There was no evidence of ovigenesis in these specimens. A further 23 specimens were grown for 72 h at which time 50 % contained eggs. These did not fix successfully and were therefore only used for measuring egg size. These specimens were not included as type material. Infected P. antipodarum were identified by placing about 40 snails each in a number of plastic Petri dishes filled with lake water, incubating them at 25 °C under intense light, and screening for cercariae after 24 h. Snails from Petri dishes containing shedding snails were individually isolated into 12-well plates in order to identify infected snails. Cercariae were collected for live observation, digital microphotography, and fixed with 96 % ethanol for subsequent DNA extraction. Infected snails were later dissected to obtain sporocysts. Morphological data Adult specimens from ducks and metacercariae grown in vitro were stained using iron acetocarmine, dehydrated through a graded ethanol series, cleared in clove oil, and examined as permanent mounts in Canada balsam. Figures were made using a drawing tube mounted on an Olympus light compound microscope at ×1,000 magnification. Measurements of adults and in vitro grown metacercariae were taken from drawings at ×400 magnification. Live cercariae were stained with Neutral Red and examined as wet mounts under a light compound microscope at ×1,000 magnification. Visualising flame cells was facilitated with the addition of urea. Measurements were taken from digital photographs using ImageJ software (Wayne Rasband, NIH, USA). Metacercariae used for ‘adult’ measurements for Maritrema poulini included both newly excysted metacercariae and individuals that had been cultured in vitro for 48 h. T-tests performed on log-transformed metrical data for both newly excysted and in vitro-grown specimens revealed no significant differences;
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therefore, the metrical data from these two sets of specimens were combined. Drawing of the cercaria is a composite from a series of photographs. All measurements in the text are in micrometres unless otherwise stated and are given as the range followed by the mean±standard deviation and the number of measurements in parentheses. The type material is deposited in the British Museum (Natural History) Collection at the Natural History Museum, London (NHMUK) and in the Otago Museum, Dunedin, New Zealand. Comparative material examined comprised type specimens of M. calvertense ex Anas castanea (Eyton) from Tasmania from the collection of the Tasmanian Museum and Art Gallery; accession numbers K250 (holotype) and K249, K251, and K252 (paratypes). Molecular data generation Four adult isolates from A. platyrhynchos, eight isolates of five to ten pooled cercariae from individual molluscs, and 11 metacercarial isolates (three ex A. annectens, four ex P. fluviatilis, and three ex P. excavatum) were characterised molecularly (Table 1). Two isolates of M. novaezealandense (sporocysts) from Zeacumantus subcarinatus (Sowerby) were also used for comparative purposes. Genomic DNA was extracted from ethanol-fixed isolates in 200 μL of a 5 % suspension of Chelex® in deionised water and containing 0.1 mg/ml proteinase K followed by incubation at 56 °C for 5 h, boiling at 90 °C for 8 min, and centrifugation at 14,000 g for 10 min. Partial fragments of the ribosomal RNA gene were amplified: the large ribosomal subunit (28S) [1,800 bp; primers U178F: 5′-GCA CCC GCT GAA YTT AAG-3′ and L1642R: 5′-CCA GCG CCA TCC ATT TTC A-3′ (Lockyer et al. 2003)] and internal transcribed spacer 1 (ITS1) [900 bp; primers M1780F: 5′-ACA CCG CCC GTC GCT ACT A-3′ and M5·8R: 5′-GGC TGC GCT CTT CAT CGA CA-3′ used by Galaktionov et al. (2012)]. Polymerase chain reaction (PCR) amplifications were performed in 25 μL reactions containing 2.5 μL of extraction supernatant, 1× PCR buffer (16 mM (NH4)2SO4, 67 mM Tris–HCl at pH 8.8), 2 mM MgCl2, 200 μM of each dNTP, 0.5 mM each primer, and 0.7 units BIOTAQ™ DNA polymerase (Bioline Ltd.). Thermocycling conditions used for amplification of the rDNA regions follow Blasco-Costa et al. (2009) for the 28S fragment and Galaktionov et al. (2012) for ITS1. PCR amplicons were purified prior to sequencing using exonuclease I and shrimp alkaline phosphatase enzymes (Werle et al. 1994). Amplicons were cycle-sequenced from both strands using PCR primers and an internal primer for the 28S fragment (L1200R: 5′-GCA TAG TTC ACC ATC TTT CGG-3′ (Littlewood et al. 2000)); employing BigDye® Terminator v. 3.1 Ready Reaction Cycle Sequencing Kit, alcohol-precipitated and run on an ABI 3730XL Analyser (Applied Biosystems, Foster City, CA, USA). Contiguous sequences were assembled
and edited using Sequencher™ (GeneCodes Corp. v. 5) and submitted to GenBank (see accession numbers in Table 1). Molecular analyses Newly generated sequences for the 28S rDNA and the ITS1 fragments (23 and 25 sequences, respectively) were aligned in two independent datasets together with published sequences of microphallids from Genbank using MUSCLE implemented in MEGA v5 (Tamura et al. 2011). The extremes of the alignments were trimmed to match the shortest sequence prior to phylogenetic analyses. The 28S dataset (1,132 bp long) included eight representative sequences of Maritrema spp. and 12 of Microphallus spp. retrieved from GenBank (Table 1). Three sequences of species belonging to families sister to the Microphallidae, i.e., Pleurogenidae, Phaneropsolidae, and Prostogonimidae, were included as outgroups. The ITS1 dataset (422 bp long) included five representative sequences of Maritrema spp. and ten Microphallus spp. The phylogenetic analyses were run on the two datasets individually and combined under the maximum likelihood (ML) and Bayesian inference (BI) criteria, employing the nucleotide substitution model GTR+Γ. ML analyses were conducted using the program RAxML v. 7.3 (Stamatakis 2006; Stamatakis et al. 2008). All model parameters and bootstrap nodal support values (1,000 repetitions) were estimated using RAxML. BI trees were constructed using MrBayes v. 3.2 (Ronquist et al. 2012), running two independent MCMCMC runs of four chains for 107 generations and sampling tree topologies every 103 generation. Burn-in periods were set to 106 generations according to the standard deviation of split frequencies values (<0.01). A consensus topology and nodal support estimated as posterior probability values (Huelsenbeck et al. 2001) were calculated from the remaining trees. All MrBayes and RAxML computations were performed on the freely available computational resource University of Oslo Bioportal (http://www.bioportal.uio.no/). Note on orthography Diaz et al. (2012) pointed out that the name Maritrema is neuter and proposed corrected spellings for several species under article 34.2 of the International Code of Zoological Nomenclature. We add to their list the following species: Maritrema calvertensis Smith, 1974; Maritrema carpathica Matskasi, 1984; Maritrema erpobdellicola Timon-David, 1963; Maritrema inusitata Leonov & Tchimbaliouk, 1963; Maritrema jebuensis Chung, Lee, Sohn, Lee, Park, Oh, Chai & Seo, 2010; Maritrema laricola Ching, 1963; Maritrema linguilla Jägerskiöld, 1908; Maritrema majestova Ke 1976; Maritrema metrastephanus Ke, 1985; Maritrema micrograpsa Ke, 1985; Maritrema novaezealandensis Martorelli, Fredensborg, Mouritsen & Poulin, 2004; Maritrema pacifica Ching, 1974; Maritrema parainusitata
Metacercaria
Metacercaria
Sporocyst
Metacercaria
M. poulini n. sp.
M. poulini n. sp.
M. novaezealandense
M. arenariac
Clypeomorus bifasciatus Oryzomys palustris Neomys anomalus Hydrobia ulvae Carcinus maenas Oryzomys palustris Hydrobia ulvae
Semibalanus balanoides
Zeacumantus subcarinatus
Paracorophium excavatum
Paracalliope fluviatilis
Sporocyst Hydrobia ulvae Sporocyst containing metacercariae Onoba aculeusa, Littorina saxatilisb Sporocyst containing metacercariae Littorina saxatilis Adulta; Metacercariab Larus schistisagusa; Carcinus maenasb
M. primas M. pseudopygmaeus
M. pygmaeus M. similis
M. piriformes
Oryzomys palustris Littorina sitkana Hydrobia ulvae Somateria mollissima v. nigrum Sporocyst containing metacercariae Littorina saxatilis
M. basodactylophallus M. calidris M. fusiformis M. kurilensis
Adult Sporocyst containing metacercariae Sporocyst Adult
Locality
Grindavik, SW Iceland; Impoveem, Sea of Okhotsk, Russiaa; Belfast Lough, Northern Irelandb
Belfast Lough, Northen Ireland Kandalaksha Bay, White Sea, Russia
Grindavik, SW Iceland
Cedar Key, Florida, USA Sea of Okhotsk, Sakhalin, Russia Belfast Lough, Northen Ireland Yamskaya Bay, N Sea of Okhotsk, Russia
Belfast Lough, Northen Ireland
Shuwaikh Bay, Kuwait Florida, USA Rakhiv, Zakarpatskan Region, Ukraine Belfast Lough, Northen Ireland São Jacinto channel, Aveiro estuary, Portugal Cedar Key, Florida, USA Kandalaksha Bay, White Sea, Russia
Belfast Lough, Northen Ireland
Portobello Bay, Dunedin, New Zealand
Lake Waihola, Waihola, New Zealand
Lake Waihola, Waihola, New Zealand
Karitane Estuary, Otago, New Zealand Potamopyrgus antipodarum Tomahawk Lagoon, Dunedin, New Zealand Austridotea annectens Lake Waihola, Waihola, New Zealand
Anas platyrhynchos
Host
Sporocyst containing metacercariae Hydrobia ulvae
Sporocyst and metacercaria Adult Adult Sporocyst containing metacercariae Metacercaria Adult Sporocyst
Metacercaria
M. poulini n. sp.
M. cf. eroliae M. heardi M. neomi M. oocysta M. portucalense M. prosthometra M. subdolum Microphallus spp. M. abortivus
Cercaria
Adult
M. poulini n. sp.
Maritrema spp. M. deblocki n. sp.
Life cycle stage
HM584133/HM584153 HM584137a/HM584156a; HM584155b
AY220627/– HM584126a/HM584147b
HM584122/HM584154
AY220628/– HM584125/HM584151 AY220633/– HM584140/HM584169
AY220626/HM584159
JF826247/HQ650133 AY220632/– AF151927 AY220630/HM584143 –/HQ993044 AY220631/– HM584135/HM584145
KJ144173 (2 isolates)/ KJ144179 (5 isolates) KJ144174 (8 isolates)/ KJ144180 (7 isolates) KJ144175 (3 isolates)/ KJ144181 (3 isolates) KJ144176 (4 isolates)/ KJ144182 (4 isolates) KJ144177 (3 isolates)/ KJ144183 (3 isolates) KJ144178 (2 isolates)/ KJ144184 (4 isolates) AY220629/HM584144
Galaktionov et al. (2012) Galaktionov et al. (2012)
Tkach et al. (2003) Galaktionov et al. (2012)
Galaktionov et al. (2012)
Tkach et al. (2003); Galaktionov et al. (2012) Tkach et al. (2003) Galaktionov et al. (2012) Tkach et al. (2003) Galaktionov et al. (2012)
Tkach et al. (2003); Galaktionov et al. (2012) Al-Kandari et al. (2011) Tkach et al. (2003) Tkach et al. (2000) Galaktionov et al. (2012) Pina et al. (2011) Tkach et al. (2003) Galaktionov et al. (2012)
Present study
Present study
Present study
Present study
Present study
Present study
GenBank acc. nos. (28S/ITS1) Source
Table 1 List of the taxa included in the phylogenetic analyses with data on the life cycle stage, host, locality, and GenBank accession numbers for the sequences used
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Tkach et al. (2003) Tkach et al. (2003) Tkach et al. (2003)
Galaktionov et al. (2012) HM584139/HM584163
Kulkina & Beljakova, 1983; Maritrema portucalensis Pina, Russell-Pinto & Rodrigues, 2011; Maritrema pulcherrima Travassos, 1929; Maritrema pyrenaica Deblock & Combes, 1965; and Maritrema setoensis Bridgman, 1971 must be corrected to M. calvertense, M. carpathicum, M. erpobdellicolum, M. inusitatum, M. jebuense, M. laricolum, M. linguillum, M. majestovum, M. metrastephanum, M. micrograpsum, M. novaezealandense, M. pacificum, M. parainusitatum, M. portucalense, M. pulcherrimum, M. pyrenaicum, and M. setoense.
Results AY220622 AY220618 AY220634
Galaktionov et al. (2012) HM584142/HM584160
GenBank acc. nos. (28S/ITS1) Source
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Maritrema (Maritrema) deblocki n. sp.
Type host: Anas platyrhynchos L. (Aves: Anseriformes: Anatidae). Type locality: Karitane Estuary, Otago, New Zealand (45°37′ 28″S, 170°38′10″E, brackish, sea level). Site in host: Lower intestine and caeca. Type material: Holotype (NHMUK 2014.1.10.1); paratypes (NHMUK 2014.1.10.2-16; Otago Cat. No. IV43216). First and second intermediate hosts: unknown. Prevalence: 20 % (in two out of ten birds dissected). Intensity: 27 in one bird, hundreds in second bird. Etymology: This species is named for Professor Stéphane Deblock, who has contributed more than anyone to our knowledge of the Microphallidae.
b
a
Adult Adult Adult Outgroups Brandesia turgida Parabascus duboisi Prosthogonimus cuneatus
Data associated with given sequence Data associated with given sequence c Junior synonym of M. gratiosum (name herein given as per GenBank metadata)
Ukraine Ukraine Ukraine
Adult M. triangulatus
Rana lessonae Myotis daubentoni Sturnus vulgaris
Cape Taygonos, Sea of Okhotsk, Russia
Somateria mollissima v. nigrum Somateria mollissima v. nigrum Adult
Yamskaya Bay, Sea of Okhotsk, Russia
Locality
Microphallus sp.
Table 1 (continued)
Life cycle stage
Host
Taxonomic summary
Description of adult Based on whole mounts of 25 ovigerous adults from the intestines of two mallard ducks (Fig. 1; Table 2). Body minute, 196 −299 (239±29)×127−190 (156±15), dorso-ventrally flattened, pyriform, and widest at level of testes (Fig. 1a). Tegument covered entirely with minute spines. Oral sucker subterminal, 26−41 (33±4)×25−40 (32±3) (n=25). Ventral sucker at about midbody length, 25−39 (32± 4)× 23−45 (35±6) (n= 24). Suckers almost equal in size, sucker length ratio 1:0.79−1.27 (1:0.98±0.12), sucker width ratio 1:0.67−1.48 (1:0.98±0.18). Prepharynx short, 1−21 (10±5) (n=19). Pharynx muscular, round to elongate oval, 18−26 (22±2)×16−24 (20±2) (n=24). Oesophagus equal to or shorter than prepharynx, 3−16 (9±4) (n=19), with epithelial lining. Intestinal bifurcation in anterior body third. Caeca short, with epithelial lining, diverge at approximately 130°, reach to level of posterior margin of ventral sucker, but do not extend beyond ends of cirrus-sac; right and left branches of equal length, 55–102 (75±11) (n=36). Testes postovarian, lateral, symmetrical, round to transversely-elongate, smooth or slightly irregular in outline, often obscured by uterine loops filled with eggs; right testis 27–38
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(more posterior in holotype), transversely-elongate, triangular to subtriangular, smooth or with tri-lobed posterior margin in six specimens, separated from cirrus-sac. Seminal receptacle, oötype, Mehlis’ gland, and Laurer’s canal not observed, obscured by eggs. Uterus postcaecal, with extra-testicular coils. Metraterm thin-walled, often not visible. Eggs operculate, numerous, 15−21 (18±1)×7−11 (9±1) (n=57). Vitellarium in hindbody, comprised of numerous small follicles forming two symmetrical ribbons encircling uterine coils and testes; ribbons converge anteriorly to testes and interrupted posteriorly (horseshoe-shaped vitellarium sensu Deblock 2008). Excretory vesicle short, Y-shaped, arms reach to mid-level of testes, pore terminal. Maritrema (Maritrema) poulini n. sp. Syns Microphallus sp. of Coats et al. (2010), Hansen and Poulin (2005, 2006), Lagrue and Poulin (2008a), Lefebvre et al. (2005), Lefebvre and Poulin (2005), Luque et al. (2007), Rauque et al. (2011), Ruiz-Daniels et al. (2013); Microphallus sp. ‘poulini’ of Hechinger (2012). Taxonomic summary
Fig. 1 Maritrema deblocki n. sp. Holotype ex Anas platyrhynchos. a Ventral view, b Terminal genitalia, ventral view. Scale bars 50 μm
(32±8)×24–29 (26±3) (n=2); left testis 28–48 (37±7)×24– 40 (32±6) (n=17). Cirrus-sac (Fig. 1b) intercaecal, 97–143 (127±11) long, with maximum width at base 12–29 (25±4) [body length to cirrus-sac ratio 1:0.45−0.63 (1:0.53±0.05)], with shape of inverted “J”, located between intestinal caeca and ventral sucker, overlapping dorsally ventral sucker, with a very thick, muscular wall (6–7 thick), fibres in the form of a spiral around sac. Seminal vesicle elongate, occupies one third to half length of cirrus-sac. Pars prostatica not clearly delimited, prostatic cells small. Ejaculatory duct convoluted. Invaginated cirrus glabrous, poorly developed, evaginated cirrus not observed. Genital atrium small; genital pore simple, unarmed, adjacent and sinistral to ventral sucker in most specimens, but overlapped by ventral sucker in the holotype. Ovary dextral, 13−29 (19±4)×21−42 (28±5) (n=18), at level of posterior half of ventral sucker but not overlapped
Type host: Austridotea annectens Nicholls (Crustacea, Isopoda) (second intermediate host). Other second intermediate hosts: Austridotea lacustris (Thomson) (Crustacea: Isopoda); Paracalliope fluviatilis (Thomson) (Crustacea: Amphipoda); Paracorophium excavatum (Thomson) (Crustacea: Amphipoda). Type locality: Lake Waihola, Otago, New Zealand (46°01′12″ S, 170°05′42″E, brackish, sea level). First intermediate host: Potamopyrgus antipodarum (Gray) (Mollusca: Gastropoda). Definitive host: Unknown, most likely water birds of the family Anatidae. Other localities: Tomahawk Lagoon, Otago New Zealand (45°54′02″S, 170°32′40″E, freshwater/brackish, sea level); Creek off Taeri River, Otago, New Zealand (45°06′45″S, 170°15′18″E, freshwater; elevation, 425 m). Site in host: Metacercariae in isopod pleopods. Type material: Holotype (cultured, NHMUK 2014.1.10.17); paratypes (NHMUK 2014.1.10.18-33; Otago Cat. No. IV43217). Prevalence: In P. antipodarum (Tomahawk Lagoon): <0.1 %; prevalence in crustaceans from Lake Waihola as follows: A. annectens, 100 % (n=22); A. lacustris, one out of two specimens; P. fluviatilis, 42 % (n=52); P. excavatum, 23 % (n=13). Mean intensity: In A. annectens, 25.1; in A. lacustris, 5.0; in P. fluviatilis, 2.2; and in P. excavatum, 7.3. Etymology: This species is named for Professor Robert Poulin, ecological parasitologist mainly responsible for the
No data
No data
Freshwater/brackish
239 (196−299)
156 (127−190) 1:1.54 (1:1.30−1.76) 33 (26−41) 32 (25−40) 32 (25−39) 35 (23−45) 1: 0.98 (1:0.67−1.48) 10 (1−21) 22 (18−26) 20 (16−24) 9 (3−16) 75 (55−102) Reach to level of posterior margin of ventral sucker Round to elongate-oval 37 (28−48) 32 (24−40) 32 (27−38)
First intermediate host
Second intermediate host
Habitat
Body length (BL)
Body width Body width:length ratio Oral sucker length Oral sucker width Ventral sucker length Ventral sucker width Sucker width ratio Prepharynx length Pharynx length Pharynx width Oesophagus length Caeca length Caeca position
Testes shape Left testis length Left testis width Right testis length
New Zealand
Anas platyrhynchos
Definitive host
Present study
Source
Locality
M. deblocki n. sp.
Species
Travassos (1920); Deblock (1972)c Brazil
Smith (1974); present studya Tasmania
M. nicolli Travassos, 1920
M. calvertense Smith, 1974
122 (106−144) 1:1.53 (1:1.29−1.93) 25 (21−29) 26 (23−30) 28 (22−31) 27 (21−30) 1:0.94 (1:0.82−1.16) 4 (0−20) 17 (15−21) 14 (12−17) 9 (2−16) 55 (41−66) Reach to mid-level of ventral sucker Round to oval 29 (21−37) 27 (22−33) 29 (23−37)
187 (164−227) 144 (124−187) 1:1.4d 29 (23−34) − 29 (27−31) − 1:1.0d 0−short 20 (18−22) 20 (18−22) <10a 50−55a Reach to anterior margin of ventral sucker Elongate to oval − 40 (36−47) −
215 (191−268)
177−213 1:1.5d 28−32 − 28 − 1:0.8d 12−14 28 10 35 78 Not reaching to ventral sucker Round/subround − 30 −
156−241
Anas castanea (Eyton) (as Mareca), Anas bahamensis Thinornis cucullatus (Vieillot) (L.) (as Daphila)b (as Charadrius), Elseyornis melanops (Vieillot) (as Charadrius)b Potamopyrgus antipodarum Coxiella badgerensis No data (Gray) (Johnston) Austridotea annectens Austrochiltonia australis (Sayce) No data Nicholls, A. lacustris (Thomson), Paracalliope fluviatilis (Thomson), Paracorophium excavatum (Thomson) Freshwater/brackish Brackish Marine/brackish
No data
New Zealand
Present study
M. poulini n. sp.
Pusa caspica (Gm.) (as Phoca)b
Caspian Sea
Kurochkin (1962)
M. sobolevi Kurochkin, 1962
120–160 1:1.9d − 25–31 − 30–34 1:1.0d 19 19 10 40–50 80–100 Reach to anterior margin of ventral sucker Transversely-oval − − −
280–400
Marine
140−170 1:1.9d 25−40 32−41 30 50 1:0.75d 18−24 18−20 20−24 6−11 − Reach to anterior margin of ventral sucker Oval 28−44 − −
250−290
Marine
No data Heleobia stagnorum (Gm.) (as Paludestrina stagnalis (Baster))b Obesogammarus first intermediate host crassus (Sars) (as Pontogammarus)b
Tringa totanus (L.) (as Totanus calidris)b
Nicoll (1907) (as M. humile) Europe; Australia
M. oocysta (Lebour, 1907)
Table 2 Comparative morphometric data for Maritrema deblocki n. sp. and M. poulini n. sp. and morphologically similar species of Maritrema. Data from original descriptions except where noted. All measurements are in micrometres and given in the form: mean (range) or range only
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28 (21−42) Horseshoe-shaped with posteriorly directed opening 18 (15−21) 9 (7−11)
Ovary width Vitellarium
17 (15−21) 10 (8−11)
30 (26−36) Incomplete anteriorly and posteriorly
27 (25−30) Horseshoe-shaped with posteriorly directed opening 21 (18−23) 12 (10−15)
16a
b
− 28−31 Horseshoe-shaped with posteriorly directed opening 17−18 10−11
− − Complete
16−18 8−11
48 Complete
12; 15−16c 8
− 160–170 1:0.5e 30–33 Thick (9 μm) No
M. sobolevi Kurochkin, 1962
Round; dextral; level with posterior half of ventral sucker
− 93 1:0.33–0.5e 32 Thick No
M. oocysta (Lebour, 1907)
Tri-lobed; median/ dextral; almost below level of ventral sucker
− 115c 1:0.5e 28c Thick No (sensu Deblock 1971) Round/lobed; dextral; level with posterior half of ventral sucker 32
− 90−100a 1:0.5a 13−15a Thick Yes Ovoid/subtriangular; dextral; level with ventral sucker
M. nicolli Travassos, 1920
M. calvertense Smith, 1974
Data from re-examination of type-specimens Host names are currently valid names; generic names given in the source reference are given in parentheses c Data from the redescription of the type-material by Deblock (1972) d Ratios calculated from measurements of original illustrations e Data from Deblock (1971)
a
Egg-length Egg-width
Ovary length
Triangular to subtriangular; Triangular, pointing dextral, at level of downwards; dextral, posterior half of level with ventral ventral sucker or sucker more posterior 19 (13−29) 18 (14−25)
Ovary shape and position
24 (18–29) 98 (78−118) 1:0.54 (1:0.45−0.65) 18 (15−23) Thick (4−6 μm) No
26 (24−29) 127 (97−143) 1:0.53 (1:0.45−0.63) 25 (12−29) Thick (6−7 μm) No
Right testis width Cirrus-sac length (CSL) Ratio BL:CSL Cirrus-sac width at base Cirrus-sac wall Cirrus spined
M. poulini n. sp.
M. deblocki n. sp.
Species
Table 2 (continued)
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discovery of this species and enabler of this project. In the paper by Hechinger (2012) on the cercariae found in P. antipodarum, the author called the cercaria that we believe to represent this species “Microphallus sp. ‘poulini’”, and we herewith adopt his informal appellation as a species epithet, to indicate that they are the same species. Description of adult Based on whole-mounts of 14 freshly excysted metacercariae from Lake Waihola and nine specimens cultured for 48 h (Fig. 2b; Table 2). Body minute, 164–227 (187±18)×106– 144 (122±10), dorso-ventrally flattened, pyriform, widest at level of testes (Fig. 2b). Tegument covered entirely with minute spines. Oral sucker subterminal, 21−29 (25±2)×23−30 (26±2) (n=23). Ventral sucker at about mid-body length, 22−31 (28 ±2)×21−30 (27±2) (n=23). Oral sucker slightly smaller than ventral; sucker length ratio 1:0.72−1.03 (1:0.90 ± 0.08); sucker width ratio 1:0.82−1.16 (1:0.94 ± 0.09). Prepharynx short, 0−20 (4 ± 5) (n = 20) (0 in holotype). Pharynx muscular, round to elongate oval, 15−21 (17±2)× 12−17 (14±1) (n=22). Oesophagus equal to or longer than prepharynx, 2−16 (9 ± 4) (n = 18), with epithelial lining.
Fig. 2 Maritrema poulini n. sp. a Cercaria, ventral view; b holotype (excysted metacercaria ex Austridotea annectens), ventral view. Scale bar 50 μm
Intestinal bifurcation in anterior body third. Caeca short, diverge at approximately 130°, reach to mid-level of ventral sucker, but do not extend beyond ends of cirrus-sac; right and left branches of equal length, 41−66 (55±7) (n=37). Testes postovarian, lateral, symmetrical, round, or obliquelyoval; right testis 23−37 (29±4)×18−29 (24±3) (n=22), left testis 21−37 (29 ± 4) × 22−33 (27 ± 3) (n = 21). Cirrus-sac intercaecal, 78–118 (98±10) long, with maximum width at base 15−23 (18±2) [body length to cirrus-sac ratio 1:0.45−0.65 (1:0.54±0.05)], with shape of inverted “J”, located between intestinal caeca and ventral sucker, not overlapping it dorsally, with a thick, muscular wall (4−6 thick), fibres in the form of a spiral around sac. Seminal vesicle elongate, occupies about half length of cirrus-sac. Ejaculatory duct slightly convoluted. Invaginated cirrus glabrous, evaginated cirrus not observed. Genital atrium shallow; genital pore simple, unarmed, sinistral, at mid-level of but well separated from ventral sucker. Ovary dextral, 14−25 (18±3)×26−36 (30±2) (n=23), at level of posterior half of ventral sucker, slightly overlapping it dorsally, transversely-elongate, triangular to subtriangular; contiguous with or slightly overlapping cirrus-sac dorsally. Seminal receptacle (10 μm in diameter); oötype posterior to ovary; Mehlis’ gland and Laurer’s canal not observed. Uterus post-caecal. Metraterm thick-walled. Eggs operculate,
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numerous, 15−21 (17±1)×8−11 (10±1) (n=25). Vitellarium in hindbody, comprised of numerous large follicles forming two symmetrical ribbons, encircling uterus and testes; ribbons interrupted anteriorly and posteriorly. Excretory vesicle Y-shaped, arms reach to mid-level of testes; pore terminal.
mass of cells in posterior third of body, 17−32 (30±2) (n=9) in diameter. Excretory vesicle Y-shaped. Flame-cell formula 2[(2+2)+ (2+2)]=16. Cercarial movement is more or less continuous, by rapid beating of the tail and contractions of the body, making for a slow, meandering, or circular path through the water, usually close to, but not settling on the substrate.
Description of cercaria Description of metacercaria Based on 18 live specimens ex P. antipodarum from Tomahawk Lagoon. Small distome xiphidiocercariae developing in elongate sporocysts c.900 × 200, containing c.20 cercariae and c.25 germ balls. Body elongate-oval, 83−131 (108±11) long, with maximum width at midbody, 41−66 (57 ± 7) (Fig. 2a). Entire tegument covered with minute spines. Tail simple, shorter than body, 60−119 (83 ±15) long and 11−17 (14±2) (n=13) wide at base, with transverse tegumental striations. Oral sucker subterminal, with poorly defined musculature, 20−27 (22±2)×20−27 (22±2) (n=13). Stylet apical, symmetrical, straight, lanceolate, slightly wider at base, with lateral pointed thickenings at about mid-length, 13−16 (14±1) long and 2−3 wide at widest point (n=17). Ventral sucker primordial, indistinct, 14−22 (17±3) (n=13) in diameter. Digestive system is not developed. Penetration gland-cells four pairs, filled with granular content of different density, located in second third of body. First two pairs larger, with large nuclei; second pair much smaller with denser granular content staining more intensely; ducts open latero-dorsally to stylet. Genital primordium an oval
Fig. 3 Exopodal segment of the pleopod of Austridotea annectens showing small and medium sized (left and centre arrows) pre-encysted metacercariae of Maritrema poulini n. sp. in the peripheral sinus and encysted metacercaria in the central sinus (right arrow). Before encysting in the central sinus, the metacercariae move around the periphery growing larger until migrating to the centre where they encyst. Scale bar 200 μm
Based on three live encysted specimens from A. annectens. Metacercariae in small spherical, translucent cysts, 206−208 (207±5) in diameter. Cyst wall consisting of two hyaline layers, outer layer 7−8 (8) thick, inner layer 6−8 (7) thick. Metacercaria folded within cyst; excysted metacercaria lanceolate to pyriform, with tegument covered with numerous spines, distributed throughout the entire body surface and morphology closely resembling that of adults except for the lack of eggs. Body and organ sizes not significantly different between newly excysted metacercariae and specimens cultured for 48 h. Metacercariae encysted in body cavity of amphipods and the pleopodal chamber of isopods. In isopods, pre-encystment metacercariae were found embedded within the peripheral sinuses of the pleopod, and encysted metacercariae were found in the central sinuses (Fig. 3) (see also Benjamin and James 1987 who discovered this migration for M. linguillum).
Molecular results Comparative sequence analysis of the newly characterised isolates revealed three unique genotypes for each of the 28S and ITS1 rDNA regions. The isolates of adult Maritrema deblocki n. sp. from A. platyrhynchos comprised one unique genotype for each marker. Cercarial isolates from the mollusc P. antipodarum and metacercarial isolates from A. annectens, P. fluviatilis, and P. excavatum showed identical sequences for each of the genetic markers employed. This result allowed us to assign these larval stages to M. poulini n. sp. The third genotype corresponded to the cercarial isolates of M. novaezealandense from the mollusc Z. subcarinatus. Divergence within the genus Maritrema ranged from 1.0 to 9.3 % in the 28S and from 2.6 to 17.1 % in the ITS1. Among the species compared here, the lowest divergence was observed between M. deblocki and M. poulini (1.0 %) for the 28S fragment, and between M. eroliae and M. novaezealandense (2.6 %) for the ITS1. M. oocysta was somewhat divergent from both M. deblocki and M. poulini (28S, 3.2 and 3.5 %; ITS, 9.1 and 8.8 %, respectively). M. deblocki and M. poulini diverged by 3.9 % in the ITS1,
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Figure 4 shows the phylogenetic hypotheses depicted by BI and ML analyses of the 28S and ITS1 individual and combined datasets. The genera Maritrema and Microphallus were found to be reciprocally monophyletic (Fig. 4a). Because no outgroup was used in the analyses based on the ITS1 and the combined dataset, a mid-point rooting was applied to enforce the monophyly of Maritrema and Microphallus (as obtained in the analyses based on the 28S; Fig. 4a) in the illustrated consensus trees (Fig. 4b, c). Regardless of the variation in the species included in each analysed dataset, the relationships among the species of interest here were largely congruent. M. deblocki n. sp. and M. poulini n. sp. appeared as well-supported closely related sister species in all analyses (Fig 4a–c), clustering together with Maritrema neomi Tkach, 1998 when this species was included (Fig. 4a), whereas M. novaezealandense clustered together with Maritrema heardi (Kinsella & Deblock, 1994) and M. eroliae. The clade formed by the two newly described species clustered together with M. novaezealandense, M. heardi, and M. eroliae (Fig. 4a) or with M. oocysta, M. novaezealandense, and M. eroliae (Fig. 4b, c). M. oocysta showed a labile placement, either as early divergent to the clades containing the New Zealand species (Fig. 4a) or as sister to the clade formed by M. novaezealandense and M. eroliae, with low support (Fig. 4b, c). The earliest divergent species within the Maritrema clade was either M. subdolum Jägerskiöld, 1909 or M. gratiosum, depending on the dataset used.
Discussion Species distinction
Fig. 4 Bayesian inference phylograms derived from 28S (a), ITS1, (b) and 28S+ITS1 (c) rRNA gene sequences with posterior probability and bootstrap percentage values above and below the branches (posterior probabilities <0.90 and bootstrap values <60 not reported). The scale bars indicate the number of substitutions per site. Maritrema spp. clade surrounded with dashed line. Taxa in bold were newly sequenced in this study
which is considerably higher than the minimum genetic distance between species of Maritrema investigated here. Thus, the genetic differences observed for both 28S and ITS1 rDNA fragments support the distinct species status of M. deblocki and M. poulini.
Using the key of Deblock (1971), the two new species appear closest to his “three strongly related species”: Maritrema nicolli Travassos, 1920 described from Anas bahamensis (L.) in Brazil, M. oocysta described from Heleobia stagnorum (Gm.) in Britain, and Maritrema sobolevi Kurochkin, 1962 described from Pusa caspica (Gm.) and Obesogammarus crassus (Sars) in Russia (Lebour 1907; Travassos 1920; Kurochkin 1962). These species, which constitute the “oocysta” species complex of Deblock and Combes (1965) share with M. deblocki n. sp. and M. poulini n. sp.: small body size (<300 μm), suckers of equal size, thick-walled cirrus-sac, and glabrous cirrus. The only other species described since the work of Deblock (1971) that shares these features is M. calvertense described from A. castanea, Thinornis cucullatus (Vieillot), and Elseyornis melanops (Vieillot) in Tasmania (Smith 1974). The original description does not
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include detail on the nature of the cirrus, and subsequently it was assumed to be unarmed (Gracenea et al. 1993; Shimazu and Pearson 1991). However, re-examination of the type specimens of M. calvertense revealed that the cirrus does in fact possess spines. While this character serves to separate M. calvertense from the two new species herein, we have included it in our comparison below as it is similar in all other respects and previous literature has listed it as without cirrus spines. Table 2 contains the available comparative morphometric data for these species. In addition to the presence of a spined cirrus, M. calvertense differs from M. deblocki in having shorter caeca reaching to the level of the anterior margin of the ventral sucker, elongate-oval testes, smaller cirrus-sac, and overall larger eggs (mean, 21×12 vs. 18×9 μm). M. calvertense can be distinguished from M. poulini by its on average larger size of body (mean, 215× 144 vs. 187×122 μm), oral sucker (mean diameter, 29 vs. 25 μm), pharynx (mean, 20×20 vs. 17×14 μm), and eggs (21×12 vs. 17×10 μm); caeca reaching to the level of the anterior margin of the ventral sucker; elongate-oval testes that are also distinctly wider; narrower cirrus-sac; and spined cirrus (see Table 2). M. nicolli can be distinguished from M. deblocki and M. poulini by its more elongate pharynx (mean 35 vs. 22 and 17 μm, respectively), longer oesophagus (mean 35 vs. 9 and 9 μm, respectively), distinctly larger ovary (mean 32×48 vs. 19×28 and 18×30 μm, respectively), somewhat smaller eggs (12 μm long in the original description, vs. 18 and 17 μm, respectively), and the structure of the genital atrium (see Table 2). It is worth noting that Deblock (1972) suggested that the indistinct, but apparently voluminous atrial formation places M. nicolli in a different genus (although he did not specify a genus or name a new one). M. oocysta differs from both species in its more elongate body shape (body width to length ratio of 1:1.9), shorter cirrus-sac in relation to body length (body length to cirrussac length ratio 1:0.33–0.50 vs. 1:0.45−0.63 in M. deblocki and 1:0.45−0.65 in M. poulini), narrower pharynx (mean, 10 vs. 20 and 14 μm, respectively) and much longer oesophagus (mean, 40–50 vs. 9 and 9 μm, respectively; see Table 2). M. oocysta has an abbreviated two-host life cycle with metacercariae encysting in the sporocysts, and thus differs distinctly from M. poulini which, as shown above, utilises a three-host life cycle. Most importantly, the genetic divergence between M. deblocki and M. poulini and M. oocysta observed for both 28S and ITS1 is consistent with interspecific distinction; this is supported by the distinct positions of the three species in phylogenies (Fig. 4). M. sobolevi differs from both new species described above in its massive cirrus-sac (160−170×30−33 vs. 97−143×12 −29 μm in M. deblocki and 78−118×15−23 μm in M. poulini) which also has a thicker wall (9 vs. 6−7 and 4−6 μm, respectively), the presence of metraterm with a muscular sphincter,
and the round shape of the ovary (see Table 2). This species can be further distinguished from M. poulini in its distinctly greater size of the body and almost all organs and the typical horseshoe-shaped vitellarium, and from M. deblocki by the extent of the caeca (reaching to anterior vs. posterior margin of ventral sucker), the mammalian host and geographically distant type locality (M. sobolevi is known only from its original description; Kurochkin 1962). M. deblocki and M. poulini appear to represent an example of sibling species (i.e., morphologically similar and genetically closely related). They are distinguishable from each other by the relative body size (see Table 2). In M. deblocki, the suckers are of subequal size on average, whereas in M. poulini the oral sucker is slightly smaller than the ventral (mean sucker width ratio, 1:1.06 vs. 1:0.94). The difference in the thickness of the cirrus-sac wall is greater than would be expected simply from size difference (6−7 μm in M. deblocki vs. 4−6 μm in M. poulini). In M. deblocki, the genital pore is adjacent to the ventral sucker, whereas in M. poulini it is well separated from the latter. In M. deblocki, the ovary does not overlap dorsally the ventral sucker and is separated from the cirrus-sac, but in M. poulini the ovary is slightly overlapping the ventral sucker and is contiguous with or slightly overlapping dorsally the cirrus-sac. In M. deblocki, the vitellarium is in the form of two ribbons converging anteriorly (horseshoeshaped vitellarium) whereas in M. poulini they are interrupted both anteriorly and posteriorly; the size of the vitelline follicles in the latter species also appear larger. The size of the ovary in the two new species is the same, notwithstanding the differences in body size, so that the ovary of M. poulini is considerably larger related to body size. Finally, the genetic differences observed for both 28S and ITS1 rDNA also support the differentiation of the two species of Maritrema described here. All of the above comparisons support the distinct species status of M. deblocki n. sp. and M. poulini n. sp. The discovery of two morphologically similar, yet genetically distinct Maritrema species, in common and well-studied host species from southern South Island, New Zealand, suggests that the number of microphallid species reported may be greatly underestimated. To date, only a small fraction of species within the Microphallidae have been characterised molecularly as part of taxonomic or phylogenetic studies (Al-Kandari et al. 2011; Cribb et al. 2001; Galaktionov et al. 2012; Littlewood and Olson 2001; Pina et al. 2011; Tkach et al. 2000, 2003) and some unidentified isolates have been sequenced as part of a larger barcoding study (Leung et al. 2009). The application of a combined morphological and molecular approach to the taxonomic study of this group will likely unravel a larger species diversity in this already diverse and globally distributed family with 50 genera (Deblock 2008), as has been the case in other trematode families (e.g., BlascoCosta et al. 2010; Bott et al. 2013; Miller and Cribb 2008).
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Given the morphological similarity between otherwise genetically distinct species found in this study, it seems likely that additional examination of material from the type hosts and localities of those species historically synonymised with M. eroliae, i.e., M. echinocirrata Leonov, 1958 (see Deblock and Pearson 1968); Maritrema kitanense Shibue, 1953 (see Deblock 1975); Maritrema magnicirrus Belopolskaia, 1952 (see Deblock and Pearson 1968); Maritrema urayense sensu Ogata 1951 (see Deblock 1975), with the application of combined morphological and molecular methods may uncover closely related, but genetically distinct species. In addition, the materials described as M. eroliae by Bridgman et al. (1972), Deblock and Pearson (1968), Ogata (1951), and Smith (1971), but which were considered by Deblock and Canaris (1992) to differ from the nominal species, also deserve further examination. Another species that would benefit from such a combined approach is the type-species of Maritrema (Maritrema), M. gratiosum (syn. M. arenaria), which has been reported by several authors with a wide range of measurements, from at least 13 different hosts and from localities all over the northern hemisphere (Belopolskaia 1953; Ching 1978; Deblock and Rausch 1972; Hadley and Castle 1940; Ke 1976 inter alia). Bearing in mind the experience from our study, it would also be interesting to re-examine or collect new specimens of M. calvertense from different bird hosts, since Smith (1974) stated that worms from different hosts varied in size. However, the type specimens deposited by the author were all from the same host (A. castanea), so it was not possible to re-evaluate host-related differences in size. Synonymies for records from New Zealand For any parasite with a complex life cycle, it is not possible to ascertain based on morphology alone, that various life cycle stages found in different hosts are the same species. Now, our ability to use DNA sequence data to match cercariae with metacercariae and adults enables elucidation of life cycles more quickly and with confidence. Matching different life cycle stages of M. poulini was possible through the use of molecular markers as done for other taxa (Galaktionov et al. 2012; Georgieva et al. 2012, 2013) and in this case we were also able to identify three different second intermediate hosts with, perhaps, different susceptibilities. Maritrema (M.) poulini has been known for some time, but erroneously named “Microphallus sp.” in several ecological and host–parasite interaction studies (Coats et al. 2010; Hansen and Poulin 2005, 2006; Lagrue and Poulin 2008a; Lefebvre et al. 2005; Lefebvre and Poulin 2005; Luque et al. 2007; Rauque et al. 2011; Ruiz-Daniels et al. 2013). In the review by Hechinger (2012) on the trematodes found in P. antipodarum in New Zealand, this is the species named “Microphallus sp. ‘poulini’”. In the interests of clarity, we would add that in the literature there are two further unnamed
New Zealand microphallids that may be confused with the “Microphallus sp.”. mentioned above: Microphallus sp. of Lively and colleagues in numerous publications since the 1980s (also mentioned in Lagrue et al. 2007; Lagrue and Poulin 2008b), which is, indeed, a species of Microphallus and which has a two-host life cycle, being found as a metacercaria in P. antipodarum [Hechinger’s “Microphallus sp. ‘livelyi’” (see Hechinger 2012)]; and Microphallus sp. in Z. subcarinatus (see Dittmer et al. 2011; Fredensborg et al. 2006; Keeney et al. 2007; Koehler and Poulin 2010; Leung et al. 2009; Martorelli et al. 2008) which is a marine species found on the shores of Otago Peninsula in the amphipod Paracalliope novizealandiae (Dana) and crabs Austrohelice crassa (Dana), Cyclograpsus lavauxi (Milne-Edwards), Hemigrapsus crenulatus (Milne-Edwards), Hemigrapsus sexdentatus (Milne-Edwards), and Macrophthalmus hirtipes (Jacquinot). Ecological aspects Hechinger (2012) found a prevalence for Microphallus sp. ‘poulini’ of 2% in P. antipodarum “from all over New Zealand”. Our data for the prevalence of M. poulini in the first intermediate host in Tomahawk Lagoon are 20-fold lower. Other studies of “Microphallus sp.” in second intermediate hosts have recorded prevalence (and mean intensities) of up to 43% (1.5 individuals per host) in P. fluviatilis (Swin Burn Stream, South Island; Rauque et al. 2011), 65−80% (1−18 individuals per host) in P. excavatum (Lake Waihola; Luque et al. 2007), and 43−58% (up to 51 individuals per host) in A. annectens (Lake Waihola; Hansen and Poulin 2005, 2006). These values are close to the infection parameters for M. poulini reported here. We found far higher prevalence and intensity of this species in the isopod A. annectens, than in the amphipods, suggesting that transmission of the cercariae to the isopod is highly successful. This may indicate that A. annectens is a preferred second intermediate host for M. poulini, perhaps due to the larger body size, relatively slow movement, and adherence to the substrate of this host species. In addition to A. annectens, metacercariae of M. poulini were also found in the closely related, A. lacustris (Thomson). Metacercariae from this second isopod were not genetically identified, as specimen numbers were low, but they were similar to M. poulini in the morphology and size of the cysts and the location in the host body. Chadderton et al. (2003) noted that the two species of isopod, A. lacustris and A. annectens, although utilising the same habitats, were rarely found together; they also never found them co-occurring in Lake Waihola. During dip netting for this study, however, the two species were collected in the same sample, although the density of A. annectens was far greater than that of A. lacustris. Interestingly, specimens for this study were collected from the south margin of Lake Waihola, and collections for another
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study from the north margin have yielded a higher density of A. lacustris as opposed to A. annectens (C. Lagrue, personal communication). Information from this latter study should reveal the infection rate of A. lacustris in this part of the lake. In our sample, although the specimens of A. lacustris were large (up to 15-mm long) prevalence and intensity were low, suggesting that either these large specimens have developed an immunity to infection, or that A. lacustris is not an ideal host for M. poulini possibly because it has a more cryptic lifestyle than A. annectens, preferring the underside of rocks to open sand or mud substrate (C. Lagrue, personal communication). Studies of “Microphallus sp.” (i.e., M. poulini n. sp.) by Hansen and Poulin (2005, 2006) used as models the metacercariae in A. annectens and P. fluviatilis and the cercariae in P. antipodarum. They found that isopods infected with “Microphallus sp.” were more active swimmers and showed less evasive behaviour in the presence of predators than uninfected ones, thus increasing their chances of being eaten by a definitive predatory host. They also found that, at a local scale, infection levels of “Microphallus sp.” depend upon the densities of local isopod intermediate hosts. M. deblocki n. sp., and, almost certainly, M. poulini n. sp., use dabbling ducks and/or other bottom feeding water birds as their definitive hosts; therefore, infection of the definitive hosts is probably facilitated by the behavioural modification of their second intermediate hosts (Hansen and Poulin 2005). In conclusion, finding new species in well-studied systems is still surprising although far from rare. The application of molecular methods has corroborated morphological evidence to distinguish between the two sibling species of Maritrema in New Zealand and has allowed us to match two life cycle stages of M. poulini and to detect several second intermediate hosts of this species. Our work highlights how ecological studies would benefit from biodiversity and taxonomic studies by gaining a better background knowledge on the systems, eliminating unexplained variation and thus, strengthening their conclusions. Acknowledgements The authors would like to thank Martin Corbett who shot the ducks, Colin MacLeod for providing M. novaezealandense specimens, and Clément Lagrue for collecting at L. Waihola. Special thanks are due to Dr. Leslie Chisholm of the South Australia Museum for lending material from their collection. Furthermore, the authors would like to express gratitude to Thomas Köntges of the Classics Department, University of Otago, and to Dr. Patricia Jeskins, for assistance with Latin. This work has been supported indirectly by the Marsden Fund (Royal Society of New Zealand) and a Zoology Department PBRF Research Enhancement grant to Professor Robert Poulin; and a Marie Curie Outgoing International Fellowship within the 7th European Community Framework Programme (IB-C, Grant PIOF-GA-2009-252124).
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