Annotated checklist and genetic data for parasitic helminths infecting New Zealand marine invertebrates

Parasitic helminths with complex life cycles require multiple hosts in a particular order to complete their life cycles. Although almost all helminths infect invertebrates at some point in their life cycle, we know very little about which species of invertebrates harbor parasites compared with what is known for vertebrates. In New Zealand, <1% of marine invertebrates that may be expected to host parasites have records of parasite infections. This is a strong indication that our knowledge of invertebrate parasites within marine ecosystems is highly limited. Here, we provide the first comprehensive parasite – host checklist including data from the literature and newly discovered infections of parasitic helminths infecting marine invertebrates in New Zealand. Including both pre-existing and newly found data from our survey, we present data on 73 parasite taxa (five acanthocephalans, 13 cestodes, nine nematodes, and 46 trema-todes) infecting 62 marine invertebrate species in New Zealand. In addition, we compile existing and new genetic data for many of these parasites, as a useful tool for future studies of parasite biodiversity and phylogenetics.


| INTRODUCTION
Parasitic helminths exhibit diverse and fascinating life histories.Most have complex life cycles, which require that parasites pass through multiple host species in a particular order to mature, reproduce, and complete one generation.The larval stages of such helminths are found in one or more intermediate hosts (typically invertebrates), and the adult stage, in which the worm matures and sexually reproduces, infects a definitive host (typically a vertebrate).Despite the fact that helminths with complex life cycles almost invariably use invertebrates at some point, our knowledge regarding parasites in invertebrates is largely unknown compared with that of vertebrates (Leung et al., 2015).In New Zealand, a recent review of marine parasites revealed that <1% of all invertebrate species that may be expected to host parasites actually have records of infection in the literature (Bennett, Presswell, et al., 2021).This is a strong indication that our knowledge of invertebrate parasites in New Zealand's marine ecosystem is extremely limited.
Although clearly overlooked and underrepresented in past studies, parasites that infect invertebrates are important for several reasons.First, parasites of invertebrates can significantly impact commercial fisheries and aquaculture (Shinn et al., 2015), and damage may include host castration and lowered growth and survival rates (e.g., Bower et al., 1994).Second, the matching of the identity of a larval parasite in an invertebrate intermediate host with its adult counterpart from a definitive host provides evidence for predator-prey interactions between the two hosts (e.g., Bennett et al., 2019).The marine realm is a notoriously difficult environment in which to study predator-prey interactions between animals, and helminth life cycle data can thus help, indirectly, to fill in the gaps in our knowledge of marine food webs.Third, knowledge of larval helminths within intermediate hosts has huge value to taxonomy.A species is characterized by its whole life cycle and not just its adult form; without identifying each life stage within each host, our knowledge of parasite biodiversity is incomplete (Blasco-Costa & Poulin, 2017).Last, in some marine ecosystems, the total biomass of larval parasites is comparable with that of top predators (Kuris et al., 2008).Some helminths such as trematodes have a free-living stage after emergence from their invertebrate hosts and constitute an important food source for animals that are not suitable hosts (e.g., Thieltges et al., 2013).Thus, parasites account for a significant portion of the overall biomass in a way that can affect the structure and functioning of whole ecosystems.Without a basic knowledge of which parasites are present within an ecosystem, our understanding of how natural systems are structured and how they function is limited.
The way forward to fully document parasite biodiversity in invertebrate hosts is seemingly straightforward: recover and identify parasites that infect marine macroinvertebrates.The main limitation here is that most larval parasites cannot be reliably identified to species, genus, or sometimes even family level using morphological characteristics alone.This also means that they usually cannot be matched to parasite species already known from their adult forms.Genetic tools are increasingly overcoming this limitation, allowing parasitologists to match larval forms from intermediate hosts with adult forms from definitive hosts (e.g., Bennett & Presswell, 2019;Presswell & Bennett, 2020).This is a crucial next step in the discovery of invertebrate parasite biodiversity, which will allow resolution of complete life cycles, provide appropriate taxonomic identifications, and compare between closely related species, geographic areas, and other life stages within multiple hosts (Blasco-Costa & Poulin, 2017).This can only be achieved through large-scale biodiversity surveys and through the use of integrative taxonomic methods that combine genetic and morphological data to form an overall picture of which invertebrates within a given ecosystem host particular parasite species.
Another important step to enhance our understanding of the parasites of invertebrates is to compile data on parasite infections within a given ecosystem.Synthetic checklists are essential tools to fully harness knowledge of host-parasite interactions and answer broader questions about biodiversity and ecosystem functioning.In New Zealand, readily available comprehensive parasite checklists are available for all vertebrate groups including birds (McKenna, 2010(McKenna, , 2018)), mammals (Lehnert et al., 2019;McKenna, 2010McKenna, , 2018)), and fish (Hewitt & Hine, 1972;Hine et al., 2000).There are, however, no checklists for parasites of New Zealand invertebrates, despite there being more invertebrate species present in New Zealand's marine environment than all vertebrate species combined (Gordon, 2009;Gordon et al., 2010).
The objective of this study is to provide the first comprehensive checklist of parasitic helminths (acanthocephalans, cestodes, nematodes, and trematodes) currently known to infect New Zealand marine invertebrates.We focused on New Zealand marine invertebrates because the coast has been the focus of various parasitological surveys in the past two decades (e.g., Donald & Spencer, 2016;Koehler & Poulin, 2010;Leung, Donald, et al., 2009) but never have all published data been brought together in a succinct form.We also include new records of infections from a recent and extensive parasite biodiversity survey undertaken between June 2019 and August 2021 from the Otago Coast, South Island.In addition, we compile and present existing publicly available genetic data of these parasites and complement this by adding newly generated genetic data following our survey.Our study represents a one-stop repository of all available information on helminth parasites of New Zealand marine invertebrates.

| METHODS
For existing records of parasites infecting marine invertebrates in New Zealand, data were assembled from primary publications found on Google Scholar using combinations of relevant keywords, plus searches of the reference lists in those publications.In total, 138 relevant publications were found, each providing at least one record of a host-parasite association between a larval helminth and an invertebrate host.
With regard to new records, 6295 invertebrates from 87 invertebrate species were sampled from the Otago Harbour, South Island, New Zealand, between June 2019 and August 2021.Samples were opportunistically collected from a range of activities including hand sampling along the intertidal zone, plankton sampling on surface water, and box dredge on benthic substrates as part of a larger survey of parasites infecting marine animals in the Otago Harbour (unpubl.data).In addition, specimens of one cephalopod, Moroteuthopsis ingens, were provided from a recent trawl survey by the National Institute for Water and Atmospheric Research and collected from the Chatham Rise off the east coast of New Zealand in January 2020.
Host species were identified to the lowest taxonomic level possible using invertebrate keys and guides such as Spencer et al. (2016) and Carson and Morris (2017).Upon necropsy of host individuals, parasites were recovered and stored in 70% EtOH for genetic and morphological analysis.
Genomic DNA was extracted from parasites using DNeasy(R) Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.For nematodes, the partial 18S rRNA gene was amplified using primers Nem18SF and Nem18SR and conditions from Wood et al. (2013).For trematodes, cestodes, and acanthocephalans, a partial 28S rRNA gene was targeted using primers T16 and T30 (Harper & Saunders, 2001) under conditions from Bennett et al. (2019).Additionally, cox1 was targeted for some trematode and nematode taxa, using either primers JB3 (Bowles et al., 1993) andTrem. cox1.rrnl (Králová-Hromadová et al., 2008) or universal Folmer et al. (1994) primers LCO1490 and HCO2198, under PCR conditions described in Bennett and Presswell (2019).The resulting PCR product was cleaned using EXO-SAP™-Express PCR Product Cleanup Reagent (USB Corporation, Cleveland, OH, USA) following manufacturer's instructions.Sanger sequencing by capillary electrophoresis was performed by the Genetic Analysis Service, Department of Anatomy, University of Otago (Dunedin, New Zealand).Newly generated sequences were imported into Geneious Prime(R)v2021.1.1,trimmed using the trim function with default parameters, and manually edited for incorrect or ambiguous bases.The resulting sequences were submitted to GenBank under accession numbers ON661298-ON661331, ON661074-ON661076, and ON656399-ON656400.To achieve lowest taxonomic resolution possible, we used both BLASTn searches on GenBank to confirm species, identity if the sequences matched 100% with existing taxa, or to establish preliminary identification.Larval stages of some taxa (particularly trematodes) had morphological features that aided in identification, in which case extra specimens were stained or cleared for examination.Further taxonomic resolution was sought when number of individuals and preservation condition allowed, with the aid of morphological keys or original descriptions such as Schell (1970), Allison (1979), Brockerhoff andSmales (2002), Martorelli et al. (2004), Martorelli et al. (2006), and O 'Dwyer, Blasco-Costa, et al. (2014).We acknowledge the importance of morphological vouchers for validation of the sources of DNA sequence data and have submitted morphological vouchers where possible.Unfortunately, most of the parasites found in this study were too small to be divided for molecular and morphological reference and were therefore sacrificed for DNA sequencing.Extra specimens were reserved for future study when genetically comparable adults become available.
Within the parasite-host list, each record contains information about the taxonomic authority, host species, and their common name (labeled A, B, C, and so on for more than one host), locality (either harbor, coastal city, or region within New Zealand), site of infection within host, life stage of parasite within host, and all bibliographical references associated with the parasite and host pair.Hosts within each parasite species record may have a "Remarks" subheading, under which are listed synonyms or notes pertaining to the specific hostparasite pair.Additionally, each parasite species may have an "Other remarks" subheading, which pertains to relevant information of interest, including definitive host species if known and location of type material for taxonomically described species.For host taxonomic names, we referred to WoRMS Editorial Board (2021) or the most upto-date primary taxonomic literature (e.g., Caira & Jensen, 2017).The parasites are presented in alphabetical order by family within their relevant phylum, class, and order.Within families, species are listed in alphabetical order.Hosts of each parasite species are listed in alphabetical order by family and species.The host-parasite list is ordered alphabetically by host taxonomy.

| RESULTS
A majority of records of host-parasite associations in the literature come from the last 20 years of research in parasitology, although the first record was published in 1903 by Haswell (Figure 1B).In total, including both pre-existing records and those newly found in our survey, we present data on 73 parasite taxa (five acanthocephalans, 13 cestodes, nine nematodes, and 46 trematodes) infecting 62 invertebrate species (six polychaetes, five amphipods, 16 decapods, one chaetognath, one cnidarian, one euphausiid, three isopods, two stomatopods, one barnacle, six bivalves, four cephalopods, and 16 gastropods).Of the 73 parasite taxa, 20 obtained from our survey are reported for the first time in New Zealand marine invertebrates (one acanthocephalan, 10 cestodes, eight trematodes, and one nematode).
Additionally, for 13 invertebrate host species, this is the first time they are reported as hosts to parasitic helminths.Of the parasite species, 80% (58/73) have associated genetic data, and of those, 40% (23/58) are newly produced from this study (Table 1).Most parasites are only known from one host, and most hosts are only known to be infected with one parasite species (Figure 1A).The host with the highest number of parasite records is Zeacumantus subcarinatus with 11 helminth species.The trematode Maritrema novaezealandense infects the highest number of different host species (14).
We also provide video data on three parasite species recov- Below, we present the parasite-host checklist of helminth parasites recorded in invertebrate species in New Zealand, representing an up-to-date summary of the current knowledge.We also present a host-parasite list of helminth parasites reported in invertebrate species in New Zealand in Table 2.

Other remarks
The adult stage of this species has been described from the variable oystercatcher, Haematopus unicolor, and from the South Island pied oystercatcher, H. finschi (Smales, 2002).This host species was originally identified as Transorchestia chilensis (MILNE-EDWARD 1840) but has recently been identified from molecular data as T. serrulata
Remarks.This is the first record of a cystacanth of B. balaenae in New Zealand.4

Other remarks
The only previous record of B. balaenae from New Zealand is of a subadult in an accidental host, little penguin (Eudyptula novaehollandiae) from Otago, New Zealand (Bennett, McPherson, et al., 2021).
Infection site.Body cavity.
Remarks.All of the above authors used host name Macrophthalmus hirtipes.

Other remarks
In New Zealand, adults of P. antarcticus are found in bar-tailed godwit, Limosa lapponica, and in South Island pied oystercatcher, Haematopus finschi (Brockerhoff & Smales, 2002), as well as in kelp gull, Larus dominicanus (Latham & Poulin, 2002c; these authors used the vernacular name "southern black-backed gull").There is genetic sequence available for this species (from off the coast of Chile from Hemigrapsus
Remarks.All of the above authors used host name Macrophthalmus hirtipes.
Infection site.Body cavity.
References.This study, new record.Localities.Chatham Rise.
References.This study, new record.
Infection site.Body cavity.
References.This study, new record.
Infection site.Body cavity.
References.This study, new record.
Infection site.Body cavity.
References.This study, new record.
Infection site.Body cavity.
Infection site.Body cavity.
References.This study, new record.
Infection site.Body cavity.
References.This study, new record.Localities.Karamea Bight, Tasman Bay and Cape Egmont, Taranaki.
Infection site.Visceral cavity and muscle.

Other remarks
We have since recovered Nybelinia sp. from arrow squid and assume that our specimens and those of Smith et al. (1981) represent the same species.
References.This study, new record.
References.This study, new record.
References.This study, new record.Infection site.Gonad.
Other remarks Howell (1966Howell ( , 1967) ) and Jones (1975) refer to A. longicornutus as Bucephalus longicornutus.An adult was described and documented in New Zealand from a giant stargazer, Kathetostoma giganteum (Manter, 1954).The type specimen is held at the U.S. National Museum helminth collection under number 49116.Localities.Otago Harbour.

Other remarks
The cercarial stage was named Cercaria pectinata HUET, 1891 in the above references.This cercaria has been assigned to Bacciger bacciger (RUDOLPHI 1819) NICOLL 1914 by various sources (Bray & Gibson, 1980;Palombi, 1934) but not for New Zealand specimens.We have therefore taken a cautious approach in assigning the records from New Zealand to family level.Localities.Not specified.
Localities.Not specified.

Other remarks
Infection site.Embedded in wall of stomach.

Other remarks
The specimens of Overstreet and Hochberg (1975) are reported as immature adults, suggesting that the octopus is acting as paratenic host, with a probable fish as definitive host.
References.New record.
References.New record.

Other remarks
We provide a video file of metacercarial movement from this chiton host in Video S1.
Infection site.Foot.
Localities.Widespread, South Island and North Island.
Infection site.Gonad and digestive gland.
Infection site.Not specified.

References. Koppel et al. (2011).
Remarks.This host is likely to be a dead-end host (Koppel et al., 2011).

Other remarks
The name used here, Acanthoparyphium sp.A, follows Leung, Keeney, and Poulin (2009)) and is used mostly in the literature thereafter.Prior to that paper, Babirat et al. (2004) reported on a "23-spine echinostome"; Poulin and Mouritsen (2004)  Localities.Otago Harbour.
Infection site.Found in the mouth parts.
Remarks.The polychaete is assumed to be an accidental host of this Acanthoparyphium species (Peoples et al., 2012).
References.New record.

Other remarks
Based on DNA sequencing of a partial 28S sequence, this is not Acanthoparyphium sp.A previously known to infect N. scapha (Koppel et al., 2011).It is likely to be one of the above Acanthoparyphium spp.
B-E, but without further DNA analysis, we are unable to assign it to one of them, and have therefore left it undesignated.
Localities.North and South Islands.
Localities.Throughout South Island.
Other remarks Keeney et al. (2015) listed this in the subfamily Himasthlinae but this is not currently accepted according to WoRMS (marinespecies.org).

Stage. Foot.
References.This study, new record.
References.This study, new record.
References.This study, new record.
Infection site.Foot.
Infection site.Foot.
Infection site.Host tissue.
Infection site.Within host tissue and coelom.

Other remarks
Adults of C. australis are known from the South Island pied oystercatcher, Haematopus finschi (Allison, 1979).Type specimens are held at Te Papa Museum, New Zealand.
Infection site.Foot.

Other remarks
Leung, Keeney, and Poulin (2009) originally detected this species molecularly as a cryptic sister species to C. australis, which we name Curtuteria sp.A for future comparison.Localities.Otago Peninsula.
References.New record, this study.
Infection site.Gills, hepatopancreas, appendages, or free within the body cavity.
Infection site.Gills, hepatopancreas, appendages, or free within the body cavity.
Infection site.Gills, hepatopancreas, appendages, or free within the body cavity.
Infection site.Gills, hepatopancreas, appendages, or free within the body cavity.
Infection site.Gills, hepatopancreas, appendages, or free within the body cavity.
Infection site.Gills, hepatopancreas, appendages or free within the body cavity.
Remarks.All above authors used host name Macrophthalmus hirtipes.
Remarks.Above authors used host name Lysiosquilla spinosa.
Infection site.Body cavity.
References.New record.
Infection site.Body cavity and appendages.
Infection site.Body cavity.
References.New record.

Other remarks
This species and life cycle was described by Martorelli et al. (2004) from red-billed gulls in Otago.References prior to this paper used the name Maritrema sp.Originally described as M. novaezealandensis, the name was corrected to M. novaezealandense by Presswell et al. (2014).
Type specimens are held at Museo de La Plata, La Plata, Argentina, Helminthological Collection under numbers 5279 and 5280.
Remarks.Latham et al. (2003) state that the metacercariae are ingested by scavenging freshly dead crabs (H.hirtipes, H. crenulatus, and H. sexdentatus) infected with Maritrema sp. and that the whelk is a temporary paratenic host.

Other remarks
As this is not mentioned in the description of Maritrema novaezealandense from Martorelli et al. (2004), we are cautiously assuming that this is a different species until such time as its identity can be confirmed.Localities.Otago Peninsula; Otago Harbour; Marlborough, Auckland.

Other remarks
Although there is no genetic evidence, we assume that the references above refer to the same species of microphallid.We have named this sp. 1 for convenience.The GenBank accession number listed in Leung, Donald, et al. (2009) is incorrect; the number given, FJ765509, should read FJ765510.
Infection site.Digestive gland and gonad.

Other remarks
This is either Microphallus sp. or Megalophallus sp., according to Martorelli et al. (2008).It is possible that this species is the same as Microphallus sp. reported from Z. subcarinatus by other authors (see below).
We have named this sp. 2 for convenience.
Remarks.All above authors use host name Macrophthalmus hirtipes.
Stage.Sporocyst, cercaria.Remarks.It is possible that this Microphallus sp. is the same as the microphallid described by Martorelli et al. (2008) (see above).

Other remarks
This was listed as Microphallus sp. in the above references.We have given it the appellation "sp.1" to make it comparable in future studies.Localities.Otago Harbour.
Infection site.Not specified.
Infection site.Not specified.
Infection site.Not specified.

Other remarks
The redia and cercaria of this species were described fully by O 'Dwyer, Blasco-Costa, et al. (2014) Localities.Kaikoura.
Infection site.Not specified.
Stage.Not known.

Other remarks
This species was detected by genetic signal only.No morphological data are available (O 'Dwyer, Blasco-Costa, et al., 2014).
Infection site.Lobes of the kidney and in the kidney coelom. Stage.Adult.
Remarks.All above authors used host name Octopus maorum.
Infection site.Renal sacs and beneath nearby membranes of other viscera. Stage.Adult.
Infection site.Soft tissues.
Infection site.Host tissues.

Other remarks
Infection site.Soft tissues.
Infection site.Soft tissues.
Infection site.Soft tissues.
Infection site.Soft tissues.

Other remarks
Infection site.Not specified.
Infection site.Not specified.
Infection site.Not specified.
Other remarks Donald and Spencer (2016) isolated a further species from whelks, which they called Opecoelidae sp.D. Thieltges et al. (2009) did not name their opecoelid, but, based on host identity, we assume their specimens were also Opecoelidae sp.D.
Infection site.Muscular tissue and coelom, throughout host.
Infection site.Muscular tissue and coelom.
Infection site.Muscular tissue and coelom, anterior portion of host.
Infection site.Body cavity.
References.This study, new record.

Other remarks
This species, defined by genetic identity, has been named Opecoelidae sp.F for the sake of consistency with the foregoing published species.
Infection site.Body cavity.

Other remarks
Based on cox1 genetic data, this is not the same as Renicola sp.NZ of O 'Dwyer, Blasco-Costa, et al. (2014).Localities.Otago Harbour.
References.New record, this study.

Other remarks
We provide a video file of metacercaria movement from within a euphausiid host in Video S3 and an individual metacercaria ex situ in Video S4.We have deposited a hologenophore specimen to Te Papa museum under accession W.003620. Localities.Otago Harbour.
Infection site.Body cavity.
References.New record, this study.

Other remarks
Based on 28S genetic data, this schistosome is probably a sister species to that found by Brant et al. (2017)   Localities.Otago Harbour, Otago Peninsula.

Other remarks
We have named this species Strigeidae gen.sp. 1 for convenience.
This species is referred to as "strigid" throughout Thieltges et al.Localities.Otago Harbour.
Infection site.Host tissue and coelom.

Other remarks
We have named this species Strigeidae gen.sp. 2 for convenience.
Infection site.Not specified.
Stage.Larva, stage not specified.
Infection site.Not specified.
Stage.Larva, stage not specified.
Infection site.Not specified.
Stage.Larva, stage not specified.
Infection site.Not specified.
Stage.Larva, stage not specified.
Infection site.Body cavity.

Other remarks
The endemic mermithid T. zealandica was described by Poinar et al. (2002), and type material is accessioned at Museum of New Zealand Te Papa Tongarewa, Wellington, New Zealand: ZW1509-10.

| DISCUSSION
This study provides both the first comprehensive checklist of parasitic helminths infecting New Zealand's marine invertebrates and insights into their ecological interactions.We present data on 73 parasite taxa and their 62 invertebrate hosts.All taxa are recorded as larval stages, with the exception of Neolebouria maorum, a trematode that completes its life cycle within two octopus species, Macroctopus maorum and Octopus australis.This study documents 20 new parasite taxa infecting marine invertebrates and adds 13 invertebrates as hosts that previously had no records of parasitic helminths.We present only the second record of chitons as trematode hosts and the third record of limpets as hosts to schistosome parasites, anywhere in the world.Below, we give a brief history of invertebrate parasitology in New Zealand and highlight some of the unusual or interesting findings that have increased our knowledge of the New Zealand fauna.
Historically, the first mention of a helminth infection in a New Zealand marine invertebrate was by Haswell (1903) The mud snail Zeacumantus subcarinatus harbors the largest complement of helminth parasites and has been widely used as a model organism to test ecological and evolutionary hypotheses (e.g., hostparasite co-evolution, Keeney, King, et al., 2009;behavioral manipulation, Thomas & Poulin, 1998;host specificity, Keeney et al., 2015).
The large number of parasites known from Z. subcarinatus may simply be the product of the extensive sampling conducted on this snail.
Alternatively, the high parasite count may be indicative of the central role that this species plays in the mudflat ecosystem.Other host species are typically found to be infected with a single parasite species (Figure 1A).Further sampling of these hosts on a broader geographic scale will be necessary to determine whether a single parasite species per host is the norm.Indeed, parasite species richness per host generally correlates with sampling effort (Walther et al., 1995).
Similarly, the trematode Maritrema novaezealandense, with the broadest host spectrum, is another species that has been well studied.
Is it truly a generalist, or is its large number of recorded hosts a result of sheer study effort?This question could only be answered by exhaustive, ecosystem-wide surveys of all free-living taxa.
We present only the second documented record worldwide of chitons (Polyplacophora) hosting trematodes.The single previous record was by Prévot (1965), who reported finding Proctoeces macula- With 56 polyplacophoran species in New Zealand waters (Wassilieff, 2006), members of the class clearly require further investigation.
Parasites can provide insights into the evolutionary history and historical distribution of parasite taxa and, sometimes, their hosts.contention that members of this clade exhibit a Southern Hemisphere distribution, while showing that dispersal within the region is greater than expected (Brant et al., 2017).
New Zealand and the Southern Pacific Ocean are one of the main regions particularly lacking in cephalopod parasite research (Tedesco et al., 2020).It is therefore not surprising that only three of the 100 + cephalopod species in New Zealand waters (Gordon et al., 2010) currently have records of parasitic helminths.Next to nothing is known about which cephalopods are important intermediate hosts and trophic connectors for parasite life cycles.In this study, we recovered six new parasites infecting the arrow squid Nototodarus sloanii, all of which are larval cestodes never reported in this host species before.Surveys of other cephalopod species may yet yield equally diverse infections.Further studies on cephalopod parasite assemblages would shed greater light on parasite life cycles, especially because cephalopods hold varying positions within marine food webs as both predators and prey (Navarro et al., 2013).
The commercial fishing industry is worth over $4 billion to the New Zealand economy, over $1.2 billion of which comes from invertebrates (shellfish and arrow squid) (Williams et al., 2017).It is therefore essential to identify and monitor the parasites that can have an influence on fisheries.The green-lipped mussel Perna canaliculus, for example, is New Zealand's most valuable aquaculture species (Castinel et al., 2019).Green-lipped mussels are known to host two trematode cercariae, Tergestia agnostomi and Alcicornis longicornutus, with prevalence in New Zealand populations ranging 1%-16% in some areas (Linzey, 1971).Unfortunately, their impact on the production of the mussel populations remains unclear (Castinel et al., 2019), although trematodes at this stage of their life cycle often castrate their molluscan host and affect its growth.Similarly, arrow squid constitute 11% of the deep water fisheries catch (Williams et al., 2017) and have played a huge role in the fisheries industry since the 1970s.Early in the history of the squid fishery, Smith et al. (1981) was able to differentiate two populations into different species, in part, using the prevalence of infections of Anisakis sp. and Nybelinia sp.This led to the suggestion that fisheries set individual quotas for each species to prevent overfishing of one species and effectively manage resources.
There are over 80 adult cestode species known to infect marine animals in New Zealand (Bennett, Presswell, et al., 2021), most, if not all, of which must use invertebrates in at least one stage of their life cycles.Prior to this study, however, there were only two records of larval cestodes in marine invertebrates, and the first and second records did not appear until 1981 and 2016, respectively (Lagrue et al., 2016;Smith et al., 1981).This is not surprising, as data on cestode life cycles are generally lacking, primarily due to the fact that larval and adult stages cannot easily be matched using morphological characteristics alone.DNA sequencing, however, is improving this sit- Parasitic helminths comprise a number of phylogenetically distinct taxa, and inevitably, a variety of molecular markers have been employed in their study over the last 20 years, both in New Zealand and worldwide.For example, larval nematodes are more often characterized using 18S or cox1 sequence data, whereas acanthocephalans have been typically characterized with 28S sequences.In addition, molecular markers used can vary between studies depending on their goal.For example, opecoelid trematodes have been the subject of population level discrimination and therefore the highly variable 16S and ITS1 markers have been employed (Donald & Spencer, 2016).On the other hand, himasthlid trematodes are typically the focus of surveys aiming to place them into a higher phylogenetic context (e.g., Tkach et al., 2016).Additionally, choice of molecular markers is often a product of the authors' personal preference.Consequently, much of the genetic data available is not comparable, and taxa from different sources cannot easily be matched.If these data were comparable, future researchers could readily explore the genetic diversity of parasites that infect invertebrates and identify biogeographic, evolutionary, and ecological patterns.Notwithstanding pleas to the contrary (e.g., Zimmermann et al., 2014), consistency of molecular marker use remains a distant goal.
Parasites of marine invertebrates have long been overlooked though their ecological role in influencing host health, trophic interactions, and energy flow within natural systems is undeniable (Kuris et al., 2008;Mouritsen & Poulin, 2005).Here, in the first of its kind for any country, we have provided a checklist of all known records of helminth parasites in New Zealand marine invertebrates and highlighted a few instances in which knowledge of parasites impacts directly on our knowledge of invertebrate ecology, economics, and taxonomy.Many marine invertebrate host groups play key roles in ecological communities, with high socioeconomic value for humans (e.g., arrow squid and green-lipped mussel), so data about their parasites are essential.Our study, by collecting new data and organizing known parasite-host associations and their genetic data, establishes a baseline for future advances in this discipline.
ered in this survey as examples for nonparasitologists of what larval parasites within invertebrate hosts can look like.These include the trematode Acanthoparyphium sp.A from a chiton host (Supporting Information Video S1), cercariae of Schistosomatidae gen.sp.being shed from a limpet host (Video S2) and the trematode Copiatestes thyrsitae within a euphausiid host (Video S3) and ex situ (Video S4).
showed genetic distinction between Microphallus sp.NZ and Microphallus sp. 1 above.Voucher material is deposited at Institute of Parasitology, Academy of Sciences of the Czech Republic, under accession number HCIP D-702.
described this species as Plagioporus maorum and placed it in family Allocreadiidae.The type specimens are in the Canterbury Museum, and we have deposited paragenophore specimens to Te Papa museum under accession W.003619. 3.20.2| Opecoelidae gen.sp.A Host A Diloma subrostratum (GRAY 1835).Trochidae, mudflat top shell.
Donald et al. (2004) used molecular phylogenies to show that a single morphotype of opecoelid species found in New Zealand could be separated into three clades.Their "Clade 2" species, infecting only Diloma subrostratum in Otago and Southland, was subsequently called Opecoelidae sp.A in their paper of 2007.Koppel et al. (2011) subsequently found specimens in limpets that matched genetically with Opecoelidae sp. A. 3.20.3| Opecoelidae gen.sp.B Host A Diloma subrostratum.Trochidae, mudflat top shell.
used the incorrect host name D. subrostrata.Other remarks Donald et al. (2004) used molecular phylogenies to show that a single morphotype of opecoelid species found in New Zealand could be separated into three clades.Their "Clade 3a" species, infecting only Diloma subrostratum throughout New Zealand, was subsequently called Opecoelidae sp.B in their paper of 2007.3.20.4| Opecoelidae gen.sp.C Host A Diloma aethiops (GMELIN 1791).Trochidae, spotted top shell.
Donald et al. (2004) used the host name Diloma nigerrima.Donald et al. (2004) used molecular phylogenies to show that a single morphotype of opecoelid species found in New Zealand could be separated into three clades.Their "Clade 3b" species, infecting three Diloma species throughout New Zealand (but not Diloma subrostratum), was subsequently called Opecoelidae sp.C in their paper of 2007.Clark (1958) described the opecoelid found in Diloma aethiops as Cercaria melagraphia, and Donald et al. (2004) recognized the cercarial species as their species C.3.20.5 | Opecoelidae gen.sp.D Host A Cominella glandiformis, Cominellidae, mudflat whelk.
gave the name Opecoelidae sp.E to opecoelids collected from polychaetes by R. Peoples in order to keep consistency in the naming of these larval stages.Peoples and Poulin (2011) and Peoples et al. (2012) later wrote about them in two studies, retaining the name, Opecoelidae sp.E. 3.20.7 | Opecoelidae gen.sp.F Host A Trizocheles spinosus (HENDERSON 1888).Pylochelidae, hermit crab.
in the false limpet Siphonaria lessoni and in the gull Larus dominicanus in Argentina, as well as in the penguin Spheniscus demersus in South Africa.We provide a video file of schistosome cercaria shedding from the limpet host in Video S2 and have deposited ethanol-preserved cercarial paragenophore specimens to Te Papa museum under accession W.003622.
tus(LOOSS 1901) ODHNER 1911 (Fellodistomidae)  in Acanthochitona ("Acanthochites") discrepens in the Mediterranean.We collected trematode parasites belonging to Acanthoparyphium sp.A (Himasthlidae) from two species of chiton, Sypharochiton pelliserpentis and Chiton glaucus.These metacercariae were recovered from the mantle cavity and were molecularly identified as Acanthoparyphium sp.A (ofLeung,   Keeney, & Poulin, 2009) using a partial 28S gene sequence that matched 100% in a BLASTn search to Acanthoparyphium sp.A haplotype 24 (accession KJ956275).That the encysted metacercariae were clearly alive and well within the chitons was evident by their movement (See Video S1).Acanthoparyphium sp.A has been recovered from six invertebrate intermediate hosts indicating low host specificity at this life stage.The definitive hosts are thought to be oystercatchers, and according toMarchant and Higgins (1993), New Zealand oystercatchers often consume chitons.It is therefore likely that chitons contribute to the transmission of this parasite and completion of its life cycle.Chitons are common, distinctive, and cosmopolitan, so it is surprising that there are no more reports of their parasites in the literature.It is not clear whether the lack of reports is simply due to insufficient study or whether they are rarely infected.
Avian schistosomes (Trematoda) evolved from a basal clade of marine forms which branched into a large number of freshwater forms.Brant et al. (2017) discovered avian schistosomes comprising a distinct marine clade within the larger freshwater clade, suggesting a secondary return of the lineage to a marine environment.Their specimens were from the false limpet Siphonaria lessoni, the gull Larus dominicanus from Argentina, and the penguin Spheniscus demersus from South Africa.Here, we have recovered specimens from the limpet Patelloida corticata which match 98.9%-99.7%withBrant et al.'s (2017) sequences (28S rDNA) and which we consider to be conspecific.This is the first time a schistosome from this clade has been identified in New Zealand waters, supportingBrant et al.'s (2017) uation.For example,Bennett et al. (2019)  matched larval and adult forms of the cestode species Acanthobothrium sp. 1 infecting the red swimming crab Nectocarcinus antarcticus and the rough skate Zearaja nasuta using 28S sequence data.The application of molecular tools to resolve parasite life cycles can also greatly enhance food web reconstruction in marine environments(Blasco-Costa & Poulin, 2017).