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.

Infection levels of Gnathostomatidae (Nematoda) larvae in commercial fishes in north-eastern Australian waters and related food safety concerns.

The majority of research on the safety of marine edible fish has primarily focused on anisakid nematodes, neglecting the potential risks posed by other parasites, including those belonging to the family Gnathostomatidae. In Australia, there have been few reported cases of human infections with gnathostomatid parasites since 2011. However, due to the absence of a standardized diagnostic test in the country, it is believed that the actual number of infections is higher than reported. This study aimed to assess the occurrence and prevalence of infectious gnathostomatid parasites in selected commercial fish species in Australia. A total of 1947 marine fish from northern Australia, representing 9 families, 16 genera, and 30 species, were examined for gnathostomatid nematode infections. Overall, 12.3 % of the fish were found to be infected with at least one gnathostomatid larva. Among the species examined, the yellow-dabbled flounder (Branchypleura novaezeelandiae) exhibited the highest prevalence (83.3 %; n = 6) and the largest number of gnathostomatid larvae. The identification of the gnathostomatid larvae was confirmed as belonging to the genus Echinocephalus based on both morphological characteristics and sequence data. No significant correlation was observed between the prevalence, mean abundance, and mean intensity of infection with the length or weight of the examined fish species. Notably, several of the infected fish species are considered popular choices in the Australian market. Hence, it is imperative to raise awareness among relevant food safety authorities regarding the occurrence of these parasites. The findings from this study should be taken into consideration for the revision of current seafood safety protocols in the country.

Occurrence and molecular identification of nematodes from blue mackerel Scomber australasicus Cuvier in Australian waters.

This study determines the occurrence and molecular identification of nematodes from blue mackerel Scomber australasicus Cuvier (Perciformes: Scombridae), an edible fish from Australian waters. A total of 150 fish were sourced from the New South Wales and Victorian coasts. Nematodes were initially classified morphologically as 10 morphotypes belonging to the families Anisakidae (Anisakis morphotypes I and II, Contracaecum morphotype II, Terranova morphotypes I and II), Raphidascarididae (Hysterothylacium morphotypes IV, VI, VIII, and XIV), and Capillariidae (Capillaria sp.), followed by molecular identification through sequencing of their internal transcribed spacer (ITS-1, 5.8S, ITS-2) region. Anisakis morphotype I was confirmed as A. pegreffii Campana-Rouget & Biocca, 1955 and A. berlandi Mattiucci, Cipriani, Webb, Paoletti, Marcer, Bellisario, Gibson & Nascetti, 2014. Anisakis morphotype II and Contracaecum morphotype II were confirmed as A. physeteris Baylis, 1923 and C. ogmorhini Johnston & Mawson, 1941, respectively. Terranova morphotypes I and II were identified as Pulchrascaris australis Shamsi, Barton & Zhu, 2020 and Euterranova pectinolabiata n. comb. (Shamsi, Barton & Zhu, 2019) Moravec & Justine, 2020, respectively. The specific identification of Hysterothylacium morphotypes IV, VI, and VIII was not possible as no comparable adult Hysterothylacium species sequences were available in GenBank, with the exception of morphotype XIV which was confirmed as H. persicum Shamsi, Ghadam, Suthar, Mousavi, Soltani & Mirzargar, 2016. Seven nematode morphotypes were identified for the first time in Australian blue mackerel. The outcomes of the study provide a basis for future research into the community structure, life cycles, and distribution of nematode species in Australian mackerel and to analyse and clarify their importance for public health.

Allopodocotyle palmi sp. nov. and Prosorhynchus maternus Bray & Justine, 2006 (Digenea: Opecoelidae & Bucephalidae) from the Orange-Spotted Grouper Epinephelus coioides (Hamilton, 1822) off Bali, Indonesia, Described Using Modern Techniques.

BACKGROUND: The most convincing species of Allopodocotyle Pritchard, 1966 (Digenea: Opecoelidae) are known overwhelmingly from groupers (Serranidae: Epinephelinae). Six species of Allopodocotyle have been reported, collectively, from species of Cromileptes Swainson, 1839, Epinephelus Bloch, 1793 and Plectropomus Oken, 1817. These are A. epinepheli (Yamaguti, 1942), A. heronensis Downie & Cribb, 2011, A. manteri (Saoud & Ramadan, 1984), A. mecopera (Manter, 1940), A. plectropomi (Manter, 1963) and A. serrani (Yamaguti, 1952). In addition, a not yet fully described and unnamed seventh species, morphologically and phylogenetically close to A. epinepheli, was isolated from the orange-spotted grouper Epinephelus coioides (Hamilton, 1822) off Bali, Indonesia in 2016. An eighth species, again from E. coioides off Bali is described herein. METHODS: Morphological and phylogenetic analyses justify the recognition of A. palmi sp. nov., which is also genetically different from the as yet unnamed congener from the same host and locality. For the first time, 3D confocal laser scanning microscopy was applied to study and distinguish Digenea taxonomically. We introduce the 'Palm pattern', a new simplified way to visualise morphometric differences of related digenean taxa. RESULTS: Allopodocotyle palmi sp. nov. is distinguished from its congeners that infect groupers by its elongate body with a size > 2.7 mm and diagonal testes. The ovary is located mainly, and the anterior testis completely, in the posterior half of the body; the uterine coils are in the fourth eighth of the body. The cirrus-sac is 0.75-1.4 (1.1) mm long, its posterior extremity is well separated from the anterior extent of the vitelline fields, just reaching the anterior border of uterine coils. In addition, Prosorhynchus maternus Bray & Justine, 2006 (Bucephalidae) was isolated from E. coioides, representing the first record in Indonesia and the third record for this fish species. CONCLUSION: The biodiversity research in Indonesia is enhanced with a new species description based on modern and newly applied techniques.

Echinocephalus caniculus n. sp. (Nematoda: Gnathostomatidae Railliet, 1895) from the lesser spotted dogfish Scyliorhinus canicula (L.) (Elasmobranchii: Scyliorhinidae Gill, 1862) off Tunisia, with a key to species of the genus Echinocephalus.

Echinocephalus caniculus n. sp. (Nematoda, Gnathostomatidae Railliet, 1895) was isolated from the spiral valve of the lesser spotted dogfish Scyliorhinus canicula (L.) from the waters off Kalaat El Andalous, North East Tunisia. This new species is mainly characterized by a cephalic bulb armed with 31-39 transverse rows of uncinated hooks, a comparatively long oesophagus, short spicules and the presence of a gubernaculum. The new species differs from its congeners by having four cervical sacs of almost equal length, a higher oesophagus/body length ratio, the arrangement of the caudal papillae, the absence of a medioventral preanal organ and numerous scattered `pores´ limited to the lateral side of the posterior part of the body. This is the first report of a member of the genus Echinocephalus Molin, 1858 from the Tunisian coast, and a new host and locality record for the Gnathostomatidae. A key to the species of Echinocephalus is provided.

Fish Parasite Community of Three Lakes with Different Trophic Status in Mecklenburg-Western Pomerania, Germany.

PURPOSE: The present study investigates the fish parasite fauna from Lake Tollense, Mecklenburg-Western Pomerania, Germany. METHODS: A total of 117 perch (Perca fluviatilis), bream (Abramis brama) and roach (Rutilus rutilus) were sampled for parasites during 2018 and 2019 from Lake Tollense and compared with earlier data from Lake Malchin and Lake Hohen Sprenz in 2011 and 2014, respectively. Parasites were identified based on morphological and molecular characters. RESULTS: A total of 32 parasite species were isolated from fishes of Lake Tollense, predominated by digeneans. Diplostomum baeri was found only in perch while D. spathacaeum was isolated from bream and roach. Parasite comparison of similar hosts between lakes revealed highly abundant Ichthyocotylurus spp. in perch and bream of Lake Malchin while Aspidogaster limacoides was most common in roach from Lake Tollense. Diversity indices of roach showed significant variation between localities. However, NMDS graph revealed separation of the three freshwater habitats based on the parasite fauna of perch, bream and roach. CONCLUSION: Based on the multivariate statistical analysis, the three natural inland water bodies could be distinguished based on the parasite communities of perch, bream and roach. The potential to utilise fish parasites as biological indicators in freshwater ecosystem is discussed.

Redescription of Aspidogaster limacoides Diesing, 1834 (Aspidogastrea: Aspidogastridae) from freshwater fishes of northern Germany.

Aspidogaster limacoides Diesing, 1834 (Aspidogastridae) is redescribed based on light and scanning electron microscopy of specimens from the stomach and intestine of Abramis brama, Rutilus rutilus and Scardinius erythrophthalmus (Actinopterygii: Cyprinidae). The fishes were sampled during 2018 and 2019 at Lake Tollense in Mecklenburg-Western Pomerania, Germany. The prevalence of A. limacoides was highest in R. rutilus (61.7%) followed by Scardinius erythrophthalmus (7.7%) and A. brama (2.9%), while it was absent in Perca fluviatilis from the same lake. The following structures of A. limacoides are described for the first time: a depression on the ventral side of the neck, variations in the number and the arrangement of alveoli, numerous pits scattered all over the body surface, the presence of a few papillae-like structures posterior lateral to the mouth, the number of marginal organs represented by openings of exocrine multicellular glands as shown in histology and the subterminal position of the excretory pore. These characters can be used to distinguish three species of Aspidogaster, namely, A. ijimai, A. conchicola and A. limacoides, suggesting that SEM is a useful and promising tool in differentiating Aspidogaster species. Comparison of molecular data of the ITS1-5.8S-ITS2 regions showed a 94% similarity to A. limacoides from the European part of Russia. Phylogenetic analysis showed that the present specimens clustered in the same clade with A. limacoides sensu stricto, forming a distinct group to the exclusion of congeners.

The occurrence and abundance of infective stages of zoonotic nematodes in selected edible fish sold in Australian fish markets.

Seafood is nutritious and a healthy source of proteins and its regular consumption is highly recommended by medical professionals and dieticians. Owing to this, the global consumption of seafood per capita has been significantly increasing since the 1960s. Consequently, seafood-borne pathogens, including parasites, have also become more widely known and recognised. In Australia, a vast island country, information about such parasites is extremely limited. This study aimed to determine the prevalence and abundance of zoonotic parasites, including anisakid nematodes, in selected Australian edible fish. Four species of fish, namely tiger flathead, Platycephalus richardsoni (n = 43), blue mackerel, Scomber australasicus (n = 117), snapper, Pagrus auratus (n = 11) and school whiting, Sillago flindersi (n = 90) were purchased from a fish market. Although a range of parasites was found, due to their significance for human health, the focus of this study was on nematodes whose infectious stage was found in these fish. The prevalence of nematodes in these fish species was 86.05%, 64.10%, 45.45% and 56.67%, respectively. Among the parasites found, Anisakis spp., Contracaecum spp. and Hysterothylacium spp. in tiger flathead, blue mackerel and school whiting, might be of zoonotic importance. Our findings suggest there is a need to revise current seafood safety protocols and develop educational campaigns for seafood industries stakeholders.

Occurrence of ascaridoid nematodes in selected edible fish from the Persian Gulf and description of Hysterothylacium larval type XV and Hysterothylacium persicum n. sp. (Nematoda: Raphidascarididae).

Despite several reports on the presence of the potentially zoonotic nematodes among edible fishes in the Persian Gulf, there is still no study on the specific identification of these parasites or their genetic characterisation. In the present study, a total of 600 fish belonging to five popular species of fish in the region, including Otolithes ruber, Psettodes erumei, Saurida tumbil, Scomberomorus commerson and Sphyraena jello were examined for infection with nematode parasites. Detailed microscopy of nematodes found in the present study followed by characterisation of the first and second internal transcribed spacers (ITS-1 and ITS-2, respectively) showed that they belong to five distinct taxa that could be potentially zoonotic. Anisakis type I was found in four species of fish, had identical ITS sequences as Anisakis typica previously reported in Australian waters and was different from those reported in the Nearctic. Hysterothylacium type VI in the present study was morphologically similar to those previously described from Australasian waters and ITS sequences were identical among Australian specimens and those found in the present study. Another Hysterothylacium larval type was also found in the present study which had identical ITS sequences and similar morphology to those previously reported and identified as H. amoyense in China Sea. Since no ITS sequence data from a well identified adult H. amoyense with an identifiable museum voucher number is yet available and due to some other issues discussed in the article we suggest assignment of this larval type from the China Sea and the Persian Gulf to H. amoyense is doubtful until future studies on a well identified male specimen of H. amoyense or other species reveals the specific identity of this larval type. We propose to refer to this larval type as Hysterothylacium larval type XV. In the present study we also describe a new species, Hysterothylacium persicum and discuss how to differentiate it from closely related species. We also found some adult females with distinct morphology and ITS sequence but due to lack of male specimens they have been referred as Hysterothylacium sp. in this paper. They had the same ITS sequence data as Hysterothylacium larval type VI. This study shows the presence of a relatively broad diversity of potentially zoonotic nematodes in edible fish of the Persian Gulf. Therefore educational campaigns for public and local health practitioners are suggested to protect consumers from becoming infected with these parasites.

A revised method of examining fish for infection with zoonotic nematode larvae.

The infection of fish with zoonotic nematodes, particularly anisakid nematodes is of great interest to many researchers who study food safety, human or animal health or who use them as biological tags for stock assessment studies. Accurate examination of fish for infection with anisakid larvae is crucial in making accurate estimates of their occurrence, abundance and prevalence in their fish hosts. Here we describe a new method of examining fish for infection with these parasites. In 2015, a total of 261 fish were purchased from a fish market in New South Wales, Australia. All fish were first examined by routine visual examination for infection with zoonotic nematode larvae and all data were recorded. Subsequently all internal organs were placed in a container and filled with water and incubated in the room temperature overnight. The prevalence, mean intensity and mean abundance of anisakids were significantly higher (p<0.05) when the revised method of examination, i.e., combining visual examination and overnight incubation in room temperature, was employed (63.98, 8.23 and 5.27, respectively) compared to routine visual examination with or without the aid of a microscope (8.81, 3.78 and 0.33, respectively). The proposed method is effective and has several advantages, such as: not using UV or HCl for fish examination, allowing the examination of a larger number of fish in shorter time; larval specimens collected being suitable for both morphological and DNA sequencing; and being simple and inexpensive. The disadvantages would be the odour of the specimens after overnight incubation as well as not being suitable for use with frozen fish. We suggest that results, conclusions or recommendations made in studies that claim no anisakid/ascaridoid larvae were found in a fish should be approached carefully if it is only based on visual examination of the fish.

Occurrence of Terranova larval types (Nematoda: Anisakidae) in Australian marine fish with comments on their specific identities.

Pseudoterranovosis is a well-known human disease caused by anisakid larvae belonging to the genus Pseudoterranova. Human infection occurs after consuming infected fish. Hence the presence of Pseudoterranova larvae in the flesh of the fish can cause serious losses and problems for the seafood, fishing and fisheries industries. The accurate identification of Pseudoterranova larvae in fish is important, but challenging because the larval stages of a number of different genera, including Pseudoterranova, Terranova and Pulchrascaris, look similar and cannot be differentiated from each other using morphological criteria, hence they are all referred to as Terranova larval type. Given that Terranova larval types in seafood are not necessarily Pseudoterranova and may not be dangerous, the aim of the present study was to investigate the occurrence of Terranova larval types in Australian marine fish and to determine their specific identity. A total of 137 fish belonging to 45 species were examined. Terranova larval types were found in 13 species, some of which were popular edible fish in Australia. The sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2 respectively) of the Terranova larvae in the present study showed a high degree of similarity suggesting that they all belong to the same species. Due to the lack of a comparable sequence data of a well identified adult in the GenBank database the specific identity of Terranova larval type in the present study remains unknown. The sequence of the ITS regions of the Terranova larval type in the present study and those of Pseudoterranova spp. available in GenBank are significantly different, suggesting that larvae found in the present study do not belong to the genus Pseudoterranova, which is zoonotic. This study does not rule out the presence of Pseudoterranova larvae in Australian fish as Pseudoterranova decipiens E has been reported in adult form from seals in Antarctica and it is known that they have seasonal presence in Australian southern coasts. The genetic distinction of Terranova larval type in the present study from Pseudoterranova spp. along with the presence of more species of elasmobranchs in Australian waters (definitive hosts of Terranova spp. and Pulchrascaris spp.) than seals (definitive hosts of Pseudoterranova spp.) suggest that Terranova larval type in the present study belong to either genus Terranova or Pulchrascaris, which are not known to cause disease in humans. The present study provides essential information that could be helpful to identify Australian Terranova larval types in future studies. Examination and characterisation of further specimens, especially adults of Terranova and Pulchrascaris, is necessary to fully elucidate the identity of these larvae.