Multidisciplinary approach to the diagnosis of Contracaecum magnipapillatum infections in Australian black noddies, Anous minutus (Charadriiformes: Laridae)

We provide the incidental necropsy findings associated with anisakid nematode infections of black noddy terns, Anous minutus Boie, 1844 (Charadriiformes: Laridae), from offshore islands in the southern Great Barrier Reef, Queensland, Australia. Specimens collected from the proventriculi were identified morphologically as Contracaecum magnipapillatum Chapin, 1925 (Rhabditida: Anisakidae), using light and scanning electron microscopy (SEM). The entire nuclear ribosomal DNA internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) was amplified by polymerase chain reaction (PCR) and sequenced to provide reference sequences for morphologically well-identified voucher specimens. Interestingly, after an alignment with closely related taxa using BLAST, sequences of the ITS1 and ITS2 were 100% identical to the sequences assigned to Contracaecum septentrionale Kreis, 1955, from a razorbill, Alca torda Linnaeus, 1758 (Charadriiformes: Alcidae), from Spain. These results either raise questions about the ITS as a genetic marker for some members of Contracaecum, or the identity of the specimens assigned to C. septentrionale, given that no supporting morphological data was associated with them. We highlight the need for a combined morphological and molecular approach to parasite diagnostics and the use of multiple genetic loci to resolve the molecular taxonomy of cryptic species. Morphological identifications should be taxonomically robust, transparent and precede the deposition of molecular barcodes in public repositories. The gross and histopathological findings of our investigation concur with previous reports of widespread Contracaecum infections in black noddies and support the contention that Contracaecum spp. are an unlikely primary cause of mortality.


Introduction
The black noddy, Anous minutus, is a common piscivorous seabird with a tropic and subtropic marine distribution.Vegetated islands off the coast of Queensland, Australia, support a large breeding population (Hill et al. 1997).A series of mass morbidity and mortality events of black noddies along the Queensland coast from mid to late 2021 presented an opportunity to examine the gastrointestinal nematode fauna associated with this ubiquitous seabird.Previous reports of the parasitic nematodes affiliated with this avian host are limited to a few early collections of adult Contracaecum magnipapillatum (Syn.C. magnicollare Johnson and Mawson, 1941), Anisakis sp.(Syn.Stomachus sp.) and an unidentified regurgitated Acuariidae larva from the Pacific Ocean (Chapin 1925;Johnston and Mawson 1941;1951;Mawson et al. 1986;Hugot et al. 1991;Fagerholm et al. 1996).The pathological findings 90 Page 2 of 9 associated with infection of black noddies by C. magnipapillatum on Heron Island were described by Fagerholm et al. (1996) and remain the only detailed account of parasitism by Contracaecum spp. in this host.
This report presents the necropsy findings for three birds infected with C. magnipapillatum and provides the first molecular data for the species.We discuss the challenges of resolving the molecular taxonomy of this nematode and its northern hemispheric congener Contracaecum septentrionale.We argue for a multidisciplinary approach to the identification of parasites using morphology in combination with molecular techniques.

Methods
In October 2021, necropsy examinations were performed on black noddies from three offshore islands in the Great Barrier Reef (Fig. 1) as part of a disease investigation into mass morbidity and mortality events at these locations.Birds 1-3 were collected either moribund or freshly dead during October from Masthead, Lady Musgrave and Heron Islands, respectively.Birds 1 and 2 were adult males and bird 3 was a non-breeding adult female.Birds were submitted to the Biosecurity Sciences Laboratory for necropsy examinations.Nematodes were collected from the gastrointestinal tracts of three birds and fixed in 70% ethanol.A subset of 12, 3 and 9 nematode specimens from birds 1-3, respectively, were forwarded to the Shamsi's Parasitology Laboratory at Charles Sturt University (CSU) for identification.

Histopathology
Fresh tissue from the proventriculi was dissected and fixed in 10% neutral buffered formalin overnight at room temperature for histological processing.Tissue sections were paraffin-embedded using standard techniques, sectioned at 3 µm and stained with haematoxylin and eosin (HE).

Morphological examination
Prior to morphological examination of the ethanol-preserved specimens, DNA extractions were carried out by removing a small tissue sample of mid-body from each nematode following the methods described in Shamsi et al. (2016).Specimens were subsequently slide-mounted in lactophenol to clear and examined morphologically using a compound microscope (Olympus CX23, Olympus Corporation, Japan).Nematodes were grouped according to the morphological traits defined in previous studies (Fagerholm et al. 1996;Shamsi et al. 2008;Shamsi et al. 2009a;b): labial structure and morphometry, excretory pore and nerve ring position, oesophageal ventriculus, ventricular appendix, intestinal caecum and the tail.Drawings were made to scale using a microscope drawing tube (BX43 Olympus Microscope, Olympus Corporation, Japan).Voucher specimens were deposited at the Queensland Museum, South Brisbane (reference numbers G240585 -G240586).

Molecular identification
DNA extractions were performed on subsamples of the mid-body using a DNeasy Blood and Tissue Kit (QIAGEN) according to the manufacturer's protocol and as modified by Shamsi et al. (2018).The entire ITS region of nuclear ribosomal DNA (ITS1-5.8S-ITS2)was amplified by PCR using primer sets SS1 and NC2 according to the protocols described previously (Shamsi et al. 2019a).PCR products of sufficient strength were sent to the Australian Genome Research Facility (AGRF) for Sanger sequencing.Sequence quality was checked using SeqMan v8.0 (DNASTAR).The consensus sequence was assembled from forward and reverse reads using the BioEdit software v7.2.5 (Hall 1999).

Gross and histological findings
Macroscopically, birds 1 and 2 were in poor body condition with no subcutaneous or coelomic fat reserves and marked atrophy of the pectoral muscles.Bird 3 was in moderate to good nutritional condition with obvious subcutaneous and some coelomic fat reserves.In all birds, gastrointestinal content appeared reduced.In birds 1 and 2, approximately 10-20 nematodes were detected in the oesophagus, proventriculus and gizzard; many were associated with focal ulcers in the proventricular mucosa.In bird 3, several nematodes were present in the distal oesophagus and proventriculus where they were associated with a focal nodule of darkened mucosa and brown-black strands of haemorrhage, respectively.
Histologically, the focus of ulceration in bird 1 extended from the mucosa well into the submucosal muscle (Fig. 2).Multiple partial longitudinal and cross sections of nematode parasites were present in the affected area, either free within the lumen or embedded in fibrin surrounded by degenerate heterophils in a granulomatous matrix.Nematodes were characterised by a thick cuticle, pseudocoelom, coelomyarian musculature, prominent lateral chords with associated eosinophilic gland cells and an intestine composed of uninucleate columnar cells with a prominent brush border (Fig. 2).Aggregates of bacteria surrounded by fibrin, embedded in eosinophilic cellular debris and degenerate and viable heterophils were present multifocally within the ulcerated area.In bird 3, focal ulceration of the proventriculus was associated with extensive bacterial colonisation, mucosal haemorrhage and chronic, transmural inflammation that included a dense lymphocytic infiltrate in the subserosa.Nematodes were present at the mucosal surface and beneath the koilin layer at the proventricular-gizzard junction.In bird 2, marked autolysis of the proventricular tissue precluded a histological description.

Morphological identification
Nematodes were morphologically identified as belonging to the genus Contracaecum based on apomorphic characters such as oppositely-directed caecae and anteriorly-located excretory pores.A subset of 5, 3 and 9 nematodes from birds 1 to 3, respectively, were in sufficient morphological condition for a species-level diagnosis using the characters outlined in Table 1.All specimens were identified as adult C. magnipapillatum on the basis of general morphometry, genitalia, the finger-like interlabia which lack a distal bifurcation and the labia that possess lateral articles forming inwardly bent hook-like extensions (Figs. 3, 4, and 5).

Molecular identification
ITS sequence data were obtained for 5 males and 1 female specimen (bird 1 = 2 specimens, bird 2 = 3 specimens, bird 3 = 1 specimen).All specimens had 100% identical sequences.A BLAST search of the entire ITS region (ITS1-5.8S-ITS2) in GenBank yielded no identical hits, and those with the highest similarity differed to our specimens by at least 53 nucleotides (6%).However, when the ITS1 and ITS2 regions were searched independently from the 5.8S component, they matched sequences assigned to C. septentrionale with 100% identity (accession numbers: AJ634784 and AJ634787, respectively).These sequences were obtained from specimens described as "morphologically corresponding to C. septentrionale", but supporting morphological descriptions were not provided.A BLAST search of the highly conserved 5.8S region against members of Contracaecum returned percent identities ranging between 97.18 and 98.59% with 100% query cover, but sequences from this locus were not available for C. magnipapillatum nor C. septentrionale.

Discussion
Our study is the first to provide both morphological and molecular data for the anisakid nematode species, Contracaecum magnipapillatum.Accurate identification of morphologically cryptic parasites, particularly in the context To date, C. magnipapillatum has been collected from a relatively broad host range almost exclusively in the Pacific (Anous spp.and unpublished museum records of piscivorous birds from Procellariidae, Stercorariidae and Sulidae) (Johnston and Mawson 1941;Mawson et al. 1986;Hugot et al. 1991;Fagerholm et al. 1996).Beyond the Pacific, the only published accounts of C. magnipapillatum are from a Canadian albatross, Diomedea sp.(Mawson 1956) and a great cormorant, Phalacrocorax carbo Linnaeus 1758 from Egypt (Al-Bassel 2006).There are several morphological differences that reliably separate C. septentrionale from C. magnipapillatum including the interlabia and the significantly smaller, non-overlapping body and spicule lengths of the latter (Table 1).Contracaecum septentrionale was originally described from the European shag Phalacrocorax aristotelis Linnaeus 1761 from Iceland based on male spicule length and morphological features of the anterior end of adults (Kreis 1955).During mass beaching events of the same host in Spain between 1996 and 1997, larvae and adults of C. septentrionale were morphologically identified using light microscopy and SEM (Abollo et al. 2001).With the exception where male spicule length was considered (D'Amelio et al. 1990), all other reports of C. septentrionale derive from molecular studies where supporting morphological data were not provided (Cianchi et al. 1992;Nadler et al. 2000;Mattiucci et al. 2008;Lin et al. 2013;Mohammad and Hbaiel 2019).Multilocus enzyme electrophoresis (MEE) studies were conducted on specimens assigned to C. septentrionale ex.Phalacrocorax aristotelis and P. carbo carbo from Norway, Iceland, southeastern Canada and the northeast Atlantic (D'Amelio et al. 1990;Cianchi et al. 1992;Mattiucci et al. 2008).A phylogenetic study of Contracaecum spp.by Nadler et al. (2000) incorporated a partial nuclear-encoded large-subunit ribosomal DNA sequence (accession no.AF226588) obtained from a single Icelandic specimen of C. septentrionale ex P. carbo previously identified by isoenzyme analysis from  2005) sequenced the first and second internal transcribed spacers (ITS1 and ITS2) of specimens morphologically corresponding to C. septentrionale collected from Alca torda Linnaeus 1758, Spain, and deposited sequences for this species in GenBank (accession numbers: AJ634784 and AJ634787, respectively).More recently, Mohammad and Hbaiel (2019) amplified the ITS1 region of fourth-stage larvae from a black-crowned night heron, Nycticorax nycticorax Linneaus 1758 from Iraq and assigned the specimens to C. septentrionale because they matched > 99% of the reference sequence for this species (accession no.MK424799) (Li et al. 2005).
The ITS1 and ITS2 of eukaryotes are highly variable nuclear loci widely used as molecular markers to resolve species and phylogenetic relationships among closely related taxa.These markers have been used to distinguish between species within Contracaecum and other members of the Anisakidae (e.g.Li et al. 2005;Shamsi et al. 2008;Shamsi et al. 2009b;a;Roca-Geronès et al. 2023) and also investigating the life cycle and transmission patterns of these parasites (e.g.Shamsi et al. 2011Shamsi et al. , 2017Shamsi et al. , 2019b)).A recent phylogenetic study of the ITS1 and ITS2 regions of Contracaecum incorporating sequences obtained from C. septentrionale (Li et al. 2005) demonstrated phylogenetically distinct clades among congeners (Roca-Geronès et al. 2023).Given that Table 1 Comparison of morphometric characters between specimens of C. magnipapillatum from the present study and Fagerholm et al. (1996) and the type specimens of C. septentrionale (Kreis 1955).
Measurements are presented as ranges in mm unless otherwise stated.Morphometric averages and the number of specimens examined are provided in parentheses *Not provided in the reference; calculated in the present study ?In the original publication, it was noted as 2.23-0.96

Reference
Present study Fagerholm et al. 1996Kreis 1955 Host

Anous minutus Anous minutus Phalacrocorax aristotelis
Locality Queensland, Australia (Fig. 1 the ITS1 and ITS2 regions of our specimens morphologically confirmed as C. magnipapillatum were 100% identical to those assigned to C. septentrionale (Li et al. 2005), we raise the following possibilities: (1): the ITS region is conserved among some members of the Contracaecum genus as previously demonstrated for members of Contracaecum osculatum sensu lato (Rudolphi, 1802) Baylis, 1920 (Zhu et al. 2000a(Zhu et al. , 2000b)); (2) specimens of C. magnipapillatum from the present study and those assigned to C. septentrionale from Li et al. (2005), and possibly other cited studies, are the same species (i.e. C. magnipapillatum).Avian host range also differs except for the single shared host, P. carbo, in the northern hemisphere.
Sequencing additional loci such as the four mitochondrial DNA regions previously shown to discriminate between species of Contracaecum (Mattiucci et al. 2008;Lin et al. 2013;Caffara et al. 2023) would provide further insight into the molecular taxonomy of C. magnipapillatum and C. septentrionale.Mitochondrial sequences assigned to C. septentrionale are available for comparative analysis in GenBank (Lin et al. 2013) and were probably obtained from the same cohort of specimens collected from Alca torda, Spain by Li et al. (2005).Constraints of the current project and our inability to source suitable additional reference material of both species prevented the pursual of further molecular work.This represents a caveat to this study but provides an Gross and histopathological findings in the proventriculi of infected birds were consistent with previous descriptions of Contracaecum spp.infections in seabirds (Fagerholm et al. 1996;Abollo et al. 2001).Contracaecum spp.nematodes were detected at necropsy in about one-third of the black noddies investigated during the mortality event, but the nematodes were only occasionally associated with grossly visible mucosal ulcers.There was no clear association between the presence of nematodes or ulcers and subjective body condition score, with the proventricular lesions seen in some birds considered incidental and not the cause of mortality.Findings in this study support the contention that Contracaecum spp.are an unlikely primary cause of mortality in many seabird hosts (Abollo et al. 2001;Fagerholm and Overstreet 2008;Ladds 2009).
In conclusion, our research presents both morphological and molecular information on the anisakid nematode species known as Contracaecum magnipapillatum.When it comes to accurately identifying morphologically similar parasites, especially within the context of an extensive disease investigation, it is crucial to adopt a multidisciplinary approach.Published taxonomic keys and original descriptions should be the primary point of reference for verification of specimens.The deposition of voucher specimens in publicly accessible collections should supplement published literature so that researchers can access material for review or further testing.CSU for the map, Selina Ossedryver from BSL for assisting with the necropsy examinations and Natalie Leo, Craig Smith, Jane Oakey, Louise Jackson and Christine McCarthy from BSL for their project insights.This study had partial financial support from CSU. Authors also acknowledge Queensland Parks and Wildlife Service, Northern Beaches Vet Hospital (Mackay), Australia Zoo Wildlife Hospital (Beerwah) for submitting birds for necropsy.

Fig. 1
Fig. 1 Localities of the black noddies examined in the present study.Inset shows location of islands of interest within the southern Great Barrier Reef

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Page 4 of 9 of an expanded disease investigation, should incorporate a multidisciplinary approach utilising traditional morphological techniques such as microscopy (light and SEM), in combination with molecular methods (de Leon and Nadler 2010; Caffara et al. 2023).

Fig. 2 A
Fig. 2 A Multiple Contracaecum larvae are evident within a deep ulcer of the proventriculus in bird 1.The inflammatory infiltrate (arrow) containing aggregates of bacteria extends to deep into the muscularis layer (M).Glandular mucosa (G), proventricular lumen

Fig. 3
Fig. 3 Line drawing of C. magnipapillatum from the present study.a Anterior end of the parasite.b-d Labial views

Fig. 5
Fig. 5 Images of C. magnipapillatum using light microscopy.a Subventral labium of male.b Lateral view of male pre-cloacal papillae (arrows) towards posterior region.c Ventriculus and ventricu-