Trematode Parasites of Otago Harbour (New Zealand) Soft-Sediment Intertidal Ecosystems: Life Cycles, Ecological Roles and DNA Barcodes

LeungNew Zealand et al.—Trematodes Journal of Marine of Otago and Harbour Freshwater Research, 2009, Vol. 43: 857–865 857 0028–8330/09/4304–0857 © The Royal Society of New Zealand 2009

Trematode parasites of Otago Harbour (New Zealand) soft-sediment intertidal ecosystems: life cycles, ecological roles and DNA barcodes

Tommy L.F. Leung1 INTRODUCTION 1 Kirsten M. Donald Parasites, though often unnoticed by most researchers, Devon B. Keeney2 are an integral part of intertidal ecosystems (Sousa Anson V. Koehler1 1991; Mouritsen & Poulin 2002). Trematodes 1 (Digenea), in particular, are common parasites of Robert C. Peoples most animal taxa occurring in intertidal communities Robert Poulin1 (Sousa 1991). Typically, intertidal trematodes use 1Department of Zoology molluscs (sometimes bivalves, but most often snails) University of Otago as first intermediate hosts (see Fig. 1) (Kearn 1998). P.O. Box 56 Within the molluscan host, trematodes replace Dunedin, New Zealand host tissue such as gonads and eventually occupy email: [email protected] a significant portion of the volume within the 2Department of Biological Sciences shell; the outcome of infection for the mollusc is Le Moyne College almost invariably castration (Mouritsen & Poulin Syracuse, NY, United States 2002). After developing as sporocysts or rediae and multiplying asexually within the mollusc host, trematodes leave the mollusc as free-swimming and Abstract Parasites, in particular trematodes short-lived cercariae that seek a second intermediate (Platyhelminthes: Digenea), play major roles in host, in which they encyst as metacercariae (Kearn the population dynamics and community structure 1998; Poulin 2006). Depending on the trematode of invertebrates on soft-sediment mudflats. Here, species, a range of animals can serve as second we provide a list of the 20 trematode species intermediate hosts, including crustaceans, molluscs, currently known to infect molluscs, crustaceans polychaetes, and fish (Kearn 1998). Predation on and polychaetes from Otago Harbour (New Zealand) infected second intermediate hosts by definitive soft-sediment intertidal areas, as well as information hosts (birds or fish, depending on the trematode on their transmission modes, life cycles, and known species) completes the life cycle (Kearn 1998). Adult ecological impacts. Several of the host-parasite worms in the definitive hosts then release their eggs, species combinations recorded here are reported for usually in host faeces. Depending on the species, the first time. We also provide DNA barcodes, based eggs are either ingested by suitable molluscs, or they on sequences of the cytochrome oxidase subunit 1 hatch into free-swimming miracidia that seek and (CO1) gene, for 19 of the 20 trematode species, to penetrate the mollusc first intermediate host to start facilitate future identification of these parasites in the cycle anew (Fig. 1). Because trematodes occur marine ecological studies. at relatively high abundance in intertidal zones, and because of their well-documented impacts on the growth, survival, reproduction and behaviour of Keywords crustaceans; molluscs; parasitism; their hosts (e.g., Curtis 1987; Huxham et al. 1993; cytochrome oxidase subunit 1 gene; intertidal Lafferty 1993; Probst & Kube 1999; Ferreira et al. mudflats 2005; Thieltges 2006), they can be important drivers of host population dynamics, community structure, and foodweb stability (Sousa 1991; Mouritsen & Poulin 2002; Thompson et al. 2005; Lafferty et al. 2006; Wood et al. 2007). M09011; Online publication date 15 July 2009 an important hurdle for most marine ecologists Received 23 February 2009; accepted 15 April 2009 encountering trematodes is the lack of basic 858 New Zealand Journal of Marine and Freshwater Research, 2009, Vol. 43

future studies. DNA barcoding provides a simple and reliable method of distinguishing between species (see Valentini et al. 2009).

METHODS Trematode samples have been collected from invertebrate hosts on soft-sediment shores within Otago Harbour (45°50′S, 170°40′E) from 2004 to 2008. These samples were obtained as part of studies on parasites of benthic invertebrates (see Donald et al. 2004; Fredensborg et al. 2005; Mouritsen & Poulin 2005b; Martotelli et al. 2008; Leung et Fig. 1 Typical three-host life cycle of a trematode. The different trematode life stages are indicated in italics, and al. 2009). Trematodes were identified to family or hosts are shown as boxes. The likely taxonomic identity genus level based on morphology (Schell 1970). of each host is indicated below each box. When possible, DNA sequences were obtained for each species from both the asexual parthenital stages (sporocysts or rediae) in the molluscan host, and the biological information about these parasites, and metacercarial stages. However, for some species, the lack of rigorous methods of identification only the parthenitae or metacercariae were available, not requiring taxonomic expertise. The latter whereas for one species, no specimens were problem is exacerbated as taxonomic descriptions available for DNA sampling. Individual parthenitae are often restricted to adult stages, whereas the or metacercariae were isolated from host tissue and most commonly found larval stages of trematodes transferred into a Petri dish containing 0.22 µm- (sporocysts or rediae, cercariae, and metacercariae) filtered sea water. They were then transferred into have generally not been described. The lack of another Petri dish also containing filtered sea water, information is a problem in parts of the world, such to rinse off residual host material. The parasites were as New Zealand, without a long tradition in marine then placed individually into a 1.5 ml Eppendorf tube parasitology. Although records of adult trematodes for DNA extraction. DNA was extracted in 500 µl and other parasites in New Zealand bird and fish of 5% chelex containing 0.1 mg/ml proteinase K, hosts have been compiled (Hewitt & Hine 1972; incubated at 60°C for 4 h and boiled at 100°C for Weekes 1982; McKenna 1998; Hine et al. 2000), 8 min. there is no similar synopsis of the occurrence of the The cytochrome oxidase subunit 1 gene, or CO1 same parasite species in their larval stages within gene, of each individual was amplified using the invertebrates. JB3 (5’-TTTTTTGGGCATCCTGAGGTTTAT- Over the past several years, numerous studies 3’) of Bowles et al. (1993) as the forward primer. have documented the presence of trematode parasites The reverse primer used was trem.cox1.rrnl in New Zealand soft-sediment intertidal ecosystems, (5’AATCATGATGCAAAAGGTA-3’) of Králová- especially in the Otago region, South Island (e.g., Hromadová et al. (2001). Martorelli et al. 2004, 2006; Leung et al. 2009). Most all PCRs (polymerase chain reactions) were of these species were unknown previously, and are run in 30 µl reaction mixtures. The optimal cycling likely widespread throughout New Zealand. These parameters included an initial denaturation step of studies have also addressed the ecological impact 95°C (2 min), followed by 40 cycles of 95°C (30 s), of these trematodes on natural communities (e.g., 48°C (40 s) and 72°C (1 min), followed by a final Mouritsen & Poulin 2005a,b; Thompson et al. 2005). extension phase at 72°C (10 min). PCR products Our first goal here was to synthesise both published were cut out of 1% agarose gels, gel-extracted and unpublished information, to provide a summary and�� purified using Purelink™ G �el�� Ex��traction�� kits of the known biology and life cycles of trematode (Invitrogen, United States), sequenced using the species from Otago Harbour, and of their ecological BigDye Terminator Cycle Sequencing Kit (Applied importance. Our second objective was to provide Biosystems, United States), and resolved with an DNA barcodes for all species identified to date, to ABI PRISM 3730 Genetic Analyser (Applied facilitate the precise identification of these species in Biosystems). Leung et al.—Trematodes of Otago Harbour 859

PCR of the CO1 gene yielded products with these heavily-parasitised cockles on the sediment 607–917 base pairs of readable sequence. Alignment surface are then subjected to increased predation by of sequences for comparison between specimens was oystercatchers (Thomas & Poulin 1998; Mouritsen performed using the MEGA v.3.1 genetic analysis 2002). Although impairment of cockle burrowing programme (Kumar et al. 2004); however, sequences apparently benefits the parasites by increasing their from the same species showed no within-species transmission success, surface-stranded cockles are variation. The sequences were deposited at GenBank also more likely than buried cockles to have their (see Table 1 for accession numbers). foot cropped by fish (Mouritsen & Poulin 2003). The metacercariae lodged in the tip of the foot are thus often ingested by fish, which are dead-end hosts for RESULTS the parasites. The adaptiveness for these parasites of inducing cockles to lie on the sediment surface is A total of 20 trematode species were identified from therefore unclear (Tompkins et al. 2004). Other second soft-sediment intertidal areas of Otago Harbour intermediate hosts that play roles in the life cycles of (Table 1). We obtained sequences for 19 of the 20 these echinostome trematodes include wedge shells, trematode species. No DNA could be obtained from Macomona liliana, and polychaetes (see Table 1). the remaining trematode species. Metacercariae of two species are found in the foot of M. liliana (Leung et al. 2009), whereas metacercariae of Acanthoparyphium sp. B have been found in the DISCUSSION mouthparts of a nereid polychaete (R. Peoples unpubl. data). The second intermediate host(s) of the other Several of the host-parasite species combinations Curtuteria sp. and Acanthoparyphium sp. C and D recorded are reported here for the first time. are unknown, though their taxonomic affinity suggests that they probably also use A. stutchburyi and M. Echinostomatidae and Gymnophallidae liliana as the second intermediate host. The echinostome trematode Curtuteria australis echinostome species found in cockles have has been described by Allison (1979), whereas the broader effects on the intertidal community. By sympatric echinostome Acanthoparyphium sp. has causing a part of the cockle population to lie on the recently been described (Martorelli et al. 2006). sediment surface, they create new microhabitats Molecular evidence has revealed that both C. and facilitate the coexistence of epibiotic organisms australis and Acanthoparyphium sp. are cryptic living attached to cockle shells (Thomas et al. 1998). species complexes, with Curtuteria consisting of Also, the presence of many cockles on the sediment two species and Acanthoparyphium consisting of surface resulting from echinostome infection alters four (Leung et al. 2009). seabed hydrodynamics and sedimentation patterns as first intermediate host, Curtuteria spp. (Mouritsen & Poulin 2005b), leading to greater use the whelk Cominella glandiformis whereas physical heterogeneity in the mudflat habitat. The Acanthoparyphium spp. use the mudsnail Zeacumantus outcome is that the zonation patterns of some subcarinatus (Allison 1979; Martorelli et al. 2006). key non-host invertebrates is indirectly altered by The cercariae of all species leave their respective parasitism (Mouritsen & Poulin 2005a). In addition, snail hosts to penetrate their second intermediate host. more benthic species (i.e., polychaetes) choose to The cercariae of C. australis and Acanthoparyphium settle in patches of mudflats with high densities sp.A have been found to infect cockles, Austrovenus of surface cockles, and thus the density, biomass stutchburyi, by entering through the inhalant and diversity of the entire benthic community are siphon, and encyst as metacercariae in the cockle’s also increased (Mouritsen & Poulin 2005b, 2006). foot (Allison 1979; Martorelli et al. 2006). From Overall, these parasites act as ecosystem engineers this point on, these species are almost equivalent (Thomas et al. 1998), modifying by their action from an ecological perspective (Babirat et al. 2004) the physical properties of the intertidal habitat with until they reach their oystercatcher (Haematopus community-wide repercussions. spp.) definitive hosts. Cockles can accumulate large another trematode, Gymnophallus sp., uses numbers of metacercariae, up to several hundred per cockles (and to a lesser extent M. liliana) as second cockle in some localities (Thomas & Poulin 1998). intermediate hosts (Leung & Poulin 2007). Its Heavily-parasitised cockles eventually lose their metacercariae do not form cysts, but lie freely on the ability to crawl or burrow into the sediment, and outer mantle epithelium of cockles. In some localities, 860 New Zealand Journal of Marine and Freshwater Research, 2009, Vol. 43

FJ765496–FJ765500 FJ765494–FJ765495 FJ765472–FJ765476 FJ765485–FJ765488 FJ765501–FJ765503 Not available FJ765510 FJ765509 FJ765489 FJ765490–FJ765493 FJ765511 Accession no. FJ765477–FJ765484 FJ765504–FJ765508 FJ765453–FJ765455 FJ765456 FJ765457–FJ765459 FJ765460–FJ765462 FJ765463–FJ765464 FJ765465–FJ765466 FJ765467–FJ765471 spp. spp. spp. spp. spp. spp. spp. Haematopus Haematopus Haematopus Haematopus Haematopus Haematopus Haematopus spp., maybe other birds spp. Larus Larus fish fish oystercatchers, fish unknown piscivorous birds shore birds oystercatchers, gulls, piscivorous birds or marine mammals shore birds unknown oystercatchers, oystercatchers, oystercatchers, Definitive host gulls, shore birds fish oystercatchers, oystercatchers, ,

;

; M. liliana M. liliana

, , Hemigrapsus ; , ; Halicarcinus whitei Halicarcinus

(?) ; wedge shell, (?) ; wedge shell, H. crenulatus , ungulata

Lysiosquilla spinosa Lysiosquilla Austrohelice crassa Austrohelice , Macomona liliana Paracalliope novizealandiae H. sexdentatus, , Paridotea Austrovenus stutchburyi Austrovenus A. stutchburyi A. stutchburyi A. stutchburyi A. stutchburyi A. stutchburyi Macrophthalmus hirtipes Macrophthalmus Mac. hirtipes unknown crustacean (?) cockle, unknown crustacean (?) Ha. varius H. sexdentatus Cyclograpsus lavauxi unknown crustacean (?) unknown cockle, fish unknown crustacean Nereid polychaetes cockle, cockle, amphipods, crabs, surfaces of mollusc shells or crustacean fish bivalves or fish unknown Second intermediate host wedge shell, cockle, mantis shrimps, crabs, Capitellid polychaetes cockle, crenulatus carapaces isopods,

aethiops )

Amphibola crenata G enBank accession numbers for species-specific DNA barcodes, of Otago Harbour (New Zealand) intertidal Melagraphia ( Zeacumantus subcarinatus Z. subcarinatus Z. subcarinatus Z. subcarinatus Z. subcarinatus Z. subcarinatus Z. subcarinatus Z. subcarinatus Z. subcarinatus Diloma subrostrata D. A. stutchburyi Cominella glandiformis Co. glandiformis Co. glandiformis Co. glandiformis Co. glandiformis mudsnail, mudsnail, mudsnail, mudsnail, mudsnail, whelk, whelk, mudsnail, cockle, mudsnail, mudsnail, mudsnail, First intermediate host unknown bivalve topshell, topshell, whelk, unknown snail whelk, pulmonate snail, whelk,

Philophthalmidae E chinostomatidae E chinostomatidae M icrophallidae undetermined Renicolidae Heterophyidae Family Opecoelidae Opecoelidae Opecoelidae Opecoelidae Strigeidae undetermined

a sp. a sp.B e chinostomatidae sp.C e chinostomatidae sp.D e chinostomatidae e chinostomatidae

sp. sp. sp. g ymnophallidae Intermediate and definitive hosts, and sp. m icrophallidae

sp. A

sp. 1 Table Trematode Curtuteria australis Curtuteria Acanthoparyphium Acanthoparyphium Acanthoparyphium Acanthoparyphium Gymnophallus novae- Maritrema zealandensis Microphallus Philophthalmus Galactosomum Renicola opecoelid sp. A * opecoelid sp.C* opecoelid sp.D opecoelid sp. E strigeid sp. microphallid sp. pectinata Cercaria unidentified m icrophallidae * Opecoelid sp.B of Donald et al. (2007) does not occur in Otago Harbour. trematodes. Leung et al.—Trematodes of Otago Harbour 861 all cockles are parasitised by Gymnophallus sp., addition, either because of local adaptation or other some containing over 100 metacercariae; in other selective process, there are life history differences localities 2–5 km distant, the parasite appears absent. between snail populations linked with trematode Individual cockles that contain large numbers of parasitism: juvenile snails grow at higher rates and echinostome metacercariae in their foot also harbour mature at smaller sizes in populations exposed to numerous Gymnophallus metacercariae (Poulin et high trematode prevalence than in localities where al. 2000; Leung & Poulin 2007). The exact causes trematode infections are rare (Fredensborg & Poulin of this pattern are unclear, since the relationship is 2006). independent of the size or location of cockles on The impact of Ma. novaezealandensis is not the mudflat (Leung & Poulin 2007); therefore, it is limited to the first intermediate host, but also xe tends not merely owing to the passive accumulation of to its second intermediate hosts. In the laboratory, all types of metacercariae over time. No harmful infection by Ma. novaezealandensis cercariae effect of Gymnophallus infection on cockle growth, causes changes in the swimming behaviour of the reproduction, or survival has been documented to amphipod Paracalliope novizealandiae (Leung & date. Furthermore, this trematode’s life cycle has Poulin 2006). More importantly, infection by Ma. not yet been resolved. Like other members of the novaezealandensis leads to increased mortality of family Gymnophallidae, its first intermediate host is amphipods, especially when several cercariae infect almost certainly a bivalve, with the wedge shell M. the host more or less simultaneously (Fredensborg et liliana being the most likely since there are no other al. 2004b). Molecular evidence from field-infected abundant bivalve species in Otago Harbour mudflats; amphipods confirms that in nature, infection can oystercatchers and other shore birds probably serve occur in “waves” of cercariae from the same snail as definitive hosts. penetrating the same amphipod in synchrony (Keeney et al. 2007b). This trematode may thus be an Microphallidae important factor in amphipod population dynamics. The microphallid trematode Maritrema novaezea The finding that it is a generalist capable of infecting landensis is one of the most common parasites of a wide array of crustacean second intermediate hosts the mudsnail Z. subcarinatus. Its life cycle, with full (see Table 1) also suggests that this trematode is an description of all developmental stages, has been important component of the Otago Harbour foodweb elucidated by Martorelli et al. (2004). After leaving (Thompson et al. 2005). their snail host, cercariae of this parasite species One other common trematode species from the penetrate and encyst within small crustaceans, family Microphallidae occurs on Otago Harbour including amphipods, isopods and crabs (see Table mudflats. This trematode,Microphallus sp., has only 1), to await ingestion by a suitable definitive host. To recently been described (Martorelli et al. 2008). It also date, the red-billed gull Larus novaehollandiae has uses the mudsnail Z. subcarinatus as first intermediate been confirmed as a definitive host (Fredensborg et host, but it is relatively rare, not occurring in more al. 2004a), but it is likely that other shore birds also than 1–2% of snails in local populations (T.L.F. host this trematode. Leung unpubl. data). Its metacercariae are markedly in some localities within Otago Harbour, such larger than those of Ma. novaezealandensis, and as Lower Portobello Bay (mid-harbour), up to they have been found in the body cavity of several two-thirds of snails have been found to be infected crab species including Macrophthalmus hirtipes by Ma. novaezealandensis (Fredensborg et al. and Hemigrapsus crenulatus (see Table 1); its 2005). The same individual snail can therefore be definitive hosts are most probably shore birds. This infected multiple times, resulting in many snails parasite had earlier been misidentified as belonging simultaneously harbouring multiple clones of Ma. to the genus Levinseniella (Poulin et al. 2003), but novaezealandensis (Keeney et al. 2007a). The DNA sequences from metacercariae in crabs match high prevalence of this parasite, combined with those from Microphallus sp. in mudsnails. The that of all other castrating trematodes infecting Z. metacercariae, inside the body cavity of crabs, act subcarinatus (Table 1), has serious implications synergistically with other parasites to induce altered for snail populations. The density and biomass of serotonin levels in crab brains, and thus alterations in Z. subcarinatus populations covary negatively with crab behaviour (Poulin et al. 2003). These changes trematode prevalence: in localities where prevalence induced in crab hosts may serve to enhance the is high, the snails occur at low density and low overall parasites’ transmission success, by increasing the biomass, and vice versa (Fredensborg et al. 2005). In susceptibility of crabs to bird predation. 862 New Zealand Journal of Marine and Freshwater Research, 2009, Vol. 43

Other trematodes the life cycle of the Otago renicolid follows this Second in prevalence only to Ma. novaezealandensis, pattern. the other trematode species commonly found Three species of the family Opecoelidae, in the mudsnail Z. subcarinatus within Otago genetically distinct though not distinguishable from Harbour is Philophthalmus sp. First reported by each other based on morphology, use topshells of the Howell (1965) from the Wellington (North Island) genus Diloma as first intermediate hosts (Donald coast, it has recently been described in detail by et al. 2004, 2007), with two of these species being Martorelli et al. (2008), who suggest that, based found in the topshells of Otago Harbour. First on its morphology, it may be P. burrili, a species reported and described by Clark (1958) in Diloma known from Australia (Howell & Bearup 1967). (Melagraphia) aethiops, molecular data now reveal Whereas Ma. novaezealandensis is the most that one of the distinct species, opecoelid sp.A, prevalent trematode in small-sized Z. subcarinatus, infects only D. subrostrata, whereas the second, it is gradually replaced by Philophthalmus sp. in opecoelid sp.C, is also found in other topshell species, larger snails, possibly because Philophthalmus sp. is mainly D. aethiops, but has not been found in D. the dominant antagonist whenever the two parasites subrostrata (Donald et al. 2004, 2007). Like most share the same host (Keeney et al. 2008). In addition other gastropods infected by trematodes, topshells to castrating their snail host, Philophthalmus sp. and hosting larval stages of these opecoelid trematodes Ma. novaezealandensis also appear to change the are castrated. In addition, their mobility and dispersal snail’s growth patterns: snails hosting both parasite on the mudflat are reduced compared with uninfected species typically have a much wider shell base than conspecifics (Miller & Poulin 2001). The cercariae uninfected snails or snails infected by only one of released by infected topshells must encyst in second the two species (Hay et al. 2005). Double infection intermediate hosts, probably crustaceans, to await by both parasites in the same snail results in a greater predation by fish definitive hosts; however, the diameter of shell whorls grown post-infection. identity of these additional host species is yet to be Cercariae of Philophthalmus sp. produced in the determined. snail leave the host and quickly encyst on any hard metacercariae of a species of opecoelid (species substrate, such as the external surfaces of molluscs E in Table 1) have also been found encysted in the or crustaceans (or the bottom of a Petri dish in the body cavity of capitellid polychaetes, however these laboratory) (Martorelli et al. 2008). There, they await were found to be genetically distinct from the species eventual ingestion by gulls or other shore birds, found in the topshells (R. Peoples unpubl. data); which serve as definitive hosts. the identity of their first intermediate host is thus The final two trematode species recorded from unknown, as is the remainder of their life cycle. the mudsnail Z. subcarinatus in Otago Harbour A further five species of trematodes are known from both occur at relatively low prevalences (less than Otago Harbour mudflats. Three of these species use 5% of snails in all localities sampled; T.L.F. Leung the mud whelk Co. glandiformis as first intermediate unpubl. data). The first,Galactosomum sp., produces host. The first species is an undescribed member of large cercariae, visible to the naked eye and strongly the family Strigeidae occurring at low prevalence in photophilic (Martorelli et al. 2008). Although the Co. glandiformis. Presumably, it uses fish as second life cycle of this particular species is unknown, in intermediate hosts and fish-eating birds as definitive other members of the genus Galactosomum, fish hosts, like other members of its family. In addition, serve as second intermediate hosts; fish actively an undescribed species of opecoelid (species D in feed on swimming cercariae, and the latter encyst as Table 1) and an undescribed species of microphallid metacercariae within fish tissues (Kearn 1998). The are also found in Co. glandiformis. The remaining cycle is completed when an infected fish is consumed two trematode species currently known from Otago by either a fish-eating bird like a heron or shag, or, Harbour include an unidentified species that uses in some species, by a marine mammal, such as a cockles, A. stutchburyi, as first intermediate host. seal. The other trematode in the mudsnail, Renicola Originally described and named Cercaria pectinata sp., produces cercariae equipped with a penetration by Chilton (1905), its taxonomic affiliations or life stylet (Martorelli et al. 2008). In other species of the cycle are unknown. It has been reported from Otago genus Renicola, cercariae penetrate either a fish or a Harbour since its original description (e.g., Poulin et bivalve such as an oyster, where they encyst to await al. 2000), but always at very low (<1%) prevalence. ingestion by their avian definitive host, normally a The final species, also undescribed and of unknown shore bird such as a gull; we, therefore, assume that taxonomic position, occurs at low prevalence in the Leung et al.—Trematodes of Otago Harbour 863 pulmonate snail Amphibola crenata. It was reported Curtis LA 1987. Vertical distribution of an estuarine snail by W. Farnie almost a century ago (unpublished altered by a parasite. Science 235: 1509–1511. presentation, 1917 Meeting of the Otago Institute), Donald KM, Kennedy M, Poulin R, Spencer HG 2004. but nothing has been documented about its ecology Host specificity and molecular phylogeny of larval or transmission cycle. Digenea isolated from New Zealand and Australian topshells (Gastropoda: Trochidae). International Journal for Parasitology 34: 557–568. CONCLUSIONS Donald KM, Sijnja A, Spencer HG 2007. Species assignation amongst morphologically cryptic The current list of 20 trematode species presented here larval Digenea isolated from New Zealand is unlikely a complete inventory of the species that topshells. Parasitology Research 101: 433–441. occur on the mudflats of Otago Harbour. In addition, Ferreira SM, Jensen KT, Martins P, Sousa SF, Marques the same host organisms are likely to host slightly JC, Pardal MA 2005. Impact of microphallid different trematode faunas in other areas. 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