Pathological, morphological, and molecular characterization of the infection by Brachycladium goliath in humpback whale Megaptera novaeangliae.

The humpback whale (Megaptera novaeangliae) is a cosmopolitan migratory, seasonal mysticete that frequents the Brazilian coast. Strands of specimens may occur during the migratory stay in the country. In 2021 and 2022, three live humpback whales stranded on the coast of Rio Grande do Sul and Santa Catarina states in southern Brazil. After euthanasia, specimens were necropsied, and organs were thoroughly examined for lesions. Grossly, in all three cases, the liver exhibited multifocal, irregular, firm, white areas on the hepatic capsule, which extended into the parenchyma. On the cut surface, the livers were yellow to pale brown with orangish to greenish areas, the bile ducts were prominent, thickened, and severely dilated, and leaf-shaped flukes were found inside of them. Additionally, one case showed moderate atrophy of the right hepatic lobe. The histological findings included dilation of bile ducts, hyperplasia of the bile duct epithelium, marked inflammatory infiltration of lymphocytes, plasma cells, and eosinophils, and portal fibrosis. The parasite Brachycladium goliath was both morphologically and molecularly identified based on diagnostic key for trematodes and the original description of the species, and the amplification and sequencing of the ITS-2 region, respectively. Even though hepatic injury was not the primary cause of stranding, it may have contributed to the debilitation of the whales. To the authors' knowledge, this is the first study that reports M. novaeangliae as a definitive host of B. goliath and that describes the lesions caused by the parasite in cetaceans.

Prominent hepatic ductular reaction induced by Oschmarinella macrorchis in a Hubbs' beaked whale Mesoplodon carlhubbsi, with biological notes.

Beaked whales are among the least known group of cetaceans, and information regarding their pathology and parasitology is especially scarce. We describe a case of significant parasitism by a trematode found in the liver of an adult male Hubbs' beaked whale Mesoplodon carlhubbsi that stranded in Hokkaido, Japan. Post-mortem examinations revealed a localised area of discolouration restricted to the hilar region of the left hepatic lobe, where spindle-shaped trematodes occupied the dilated and hypertrophic bile ducts. Histologically, the intrahepatic bile ducts were characterised by adenomatous hyperplasia with goblet cell metaplasia of the biliary epithelium. Findings in the adjacent hepatic parenchyma included pseudocarcinomatous ductular reactions obliterating hepatocytes, a histomorphology not previously reported in marine mammals. Morphological identification of the trematode corresponded to Oschmarinella macrorchis, which has only been reported once in a Stejneger's beaked whale, M. stejnegeri. PCR amplification and sequencing analyses of the parasite's mtDNA ND3, 18S and 28S rRNA regions generated novel gene sequences. Environmental contaminant levels were measured to explore its potential relationship with the parasitism but there was no conclusive association. A high level of polychlorinated biphenyl (30000 ng g-1 lipid weight) was detected in the blubber of this individual, when compared to those of 3 other male Hubbs' beaked whales stranded in Japan. Stomach contents were also analysed, indicating the presence of various squid species and unidentified fish. Our results contribute to the knowledge of a little-known beaked whale and provide evidence for the first time of the pathobiological response caused by O. macrorchis.

A new Synthesium species (Digenea: Brachycladiidae) from the bottlenose dolphin Tursiops truncatus (Cetacea: Delphinidae) in Southwestern Atlantic waters.

A new species of Synthesium from the bottlenose dolphin Tursiops truncatus in South Brazilian waters is described. Morphological and molecular identification was performed, and phylogenetic analyses were carried out using the ribosomal small subunit and internal transcribed spacer 1 and the mitochondrial NDH dehydrogenase subunit 3 and cytochrome c oxidase subunit 1 genes. The main characteristics of the new species are the subterminal round-shaped oral sucker, the anterior distribution of vitellaria reaching the level of the ovary and the oval-shaped testes. The results obtained with the molecular markers supported the inclusion of the specimens into the genus Synthesium. The nucleotide divergence detected for the mitochondrial genes among the new species and others of the same genus supported the erection of a new species. This is the ninth species assigned to the genus and the third Synthesium species recorded in the South Atlantic Ocean.

Molecular investigation of endoparasites of marine mammals (Cetacea: Mysticeti, Odontoceti) in the Western Mediterranean.

Introduction: Whales, dolphins, and porpoises are susceptible to infections by protozoan and metazoan parasites. Methods: In this study, tissue samples, as well as flatworms and roundworms, were collected from a common bottlenose dolphin (Tursiops truncatus), three short-beaked common dolphins (Delphinus delphis), two striped dolphins (Stenella coeruleoalba), a harbor porpoise (Phocoena phocoena), a long-finned pilot whale (Globicephala melas), and a fin whale (Balaenoptera physalus). These samples were molecularly analyzed. Results: In one D. delphis, Toxoplasma gondii was detected in multiple organs, including the cerebellum. The cysts of the tapeworms Clistobothrium delphini and Clistobothrium grimaldii were identified in G. melas. Flukes collected from D. delphis belong to Brachycladium atlanticum, while those removed from S. coeruleoalba probably represent a new species. Four species of lungworms were also identified: Halocercus delphini in S. coeruleoalba, Halocercus sp. in T. truncatus, Stenurus globicephalae in G. melas, and a potentially new Pharurus sp. in P. phocoena. Conclusion: These findings show, to the best of our knowledge, for the first time, the presence of T. gondii DNA in D. delphis. The cerebellum of the animal was Toxoplasma-infected, which might be relevant to inadvertent stranding. In this study, new genetic markers were sequenced for several helminth parasites of marine mammals, possibly including undescribed species.

A first record of digenean parasites of the dwarf sperm whale Kogia sima with morphological and molecular information.

Two species of digenean trematodes of the family Brachycladiidae were obtained from two male dwarf sperm whales Kogia sima that stranded along the island of Kyushu, southern Japan in 2017. From the liver of the first animal, a single, large gravid specimen of a digenean species was collected. The morphological features were consistent with those of the genus Brachycladium. The worm had a large body and was characterized by anterior caeca without lateral diverticula, the shape of testes, ovary, and eggs. Molecular analyses using gene sequences of the 28S rRNA and the mitochondrial NADH dehydrogenase subunit 3 also supported the inclusion of this specimen into the genus Brachycladium. The identity of this worm is undetermined due to the lack of information on the genus and is reported as Brachycladium sp. From the cranial sinuses of the second animal, 33 specimens of digeneans were collected that were morphologically identified as Nasitrema gondo. This report documents a new host record for N. gondo, and the sequence information is provided for this digenean for the first time. This is the second record of digenean parasites for the family Kogiidae, and the first record with morphological and molecular information. The possibility of digenean infection in the liver and cranial sinus should be kept in mind during the necropsy of stranded kogiids.

First elucidation of the life cycle in the family Brachycladiidae (Digenea), parasites of marine mammals.

Digeneans of the family Brachycladiidae are cosmopolitan parasites restricted to marine mammals. Their life cycles are unknown. Phylogenetically, Brachycladiidae are closely related to Acanthocolpidae, parasites of marine teleost fishes. Acanthocolpida typically possess three-host life cycles with gastropods of the superfamily Buccinoidea acting as the first intermediate hosts for most species, and either fishes or bivalves acting as the second intermediate hosts. A few species previously identified as Neophasis differ from other Acanthocolpidae in having naticid gastropods as first intermediate hosts, and both fishes and bivalves as second ones. We assumed that this may indicate an incorrect life cycle description and revised previous data on rediae and cercariae of Neophasis spp. from Cryptonatica affinis (Naticidae) and metacercariae from cardiid bivalves at the White Sea using molecular and morphological approaches. Sequence comparison showed that rediae and cercariae from C. affinis resembling some representatives of the genus Neophasis and metacercariae from bivalves resembling Neophasis oculata belong to the brachycladiid species Orthosplanchnus arcticus. Thus, the life cycle of O. arcticus proceeds as follows: seals serve as the definitive host, C. affinis as the first intermediate host and cardiid bivalves as the second. We found one more type of redia and cercaria in C. affinis which, by molecular evidence, also belongs to Brachycladiidae and is closely related to O. arcticus. Here we refer to them as Brachycladiidae gen. sp. 1 WS. We suggest that Brachycladiidae gen. sp. 1 WS may belong to either Orthosplanchnus or Odhneriella, with beluga whales possibly being the definitive host. Morphological features of O. arcticus and Brachycladiidae gen. sp. 1 WS cercariae are summarised and matched with published data on putatively brachycladiid cercariae. We compare and discuss the diversity of life cycle patterns among Brachycladiidae and Acanthocolpidae, and show that they differ not only in the type of definitive host, but also in both intermediate hosts.

Revision of the taxonomic status of Synthesium elongatum (Ozaki, 1935) (Brachycladiidae), an intestinal digenean of narrow-ridged finless porpoise (Neophocaena asiaeorientalis).

Synthesium elongatum (Brachycladiidae) is an intestinal digenean described from the finless porpoise (Neophocaena asiaeorientalis) in Japan. Few records of this species exist and there is a remarkable morphological similarity between S. elongatum and S. tursionis, such that a synonymy between the species has been suggested previously. However, no morphological and/or molecular analysis has been carried out to clarify the taxonomic status of S. elongatum. In this study, we collected specimens of Synthesium sp. from N. asiaeorientalis in western Japan. The specimens possess lobed testes within the third quarter of the body, a round ovary, and vitellaria extending to level of uterine field, which are diagnostic characters for both S. elongatum and S. tursionis. They were morphologically identified to S. elongatum or S. tursionis due to the fact that the available morphometric data for both species overlap remarkably. A molecular analysis of the mitochondrial ND3 gene showed that the pairwise nucleotide distances between these specimens and S. tursionis were small, and phylogenetic analysis showed that these specimens and S. tursionis are in the same clade. Therefore, it was indicated that S. elongatum and S. tursionis are the same species and, consequently, S. elongatum is a synonym of S. tursionis.

Phenotypic plasticity of six unusual cercariae in nassariid gastropods and their relationships to the Acanthocolpidae and Brachycladiidae (Digenea).

Species-level relationships of a complex of six putative acanthocolpid cercariae with ventral keels from nassariid gastropods from Capricornia, Queensland (Cercaria capricornia I-VI) were explored using mitochondrial and nuclear DNA sequence data. Analysis of the large subunit of nuclear ribosomal DNA (rDNA), the internal transcribed spacer 2 rDNA and subunit 3 of the nicotinamide-adenine-dinucleotide dehydrogenase mitochondrial gene indicates that the six distinct morphotypes previously identified relate to only three discrete genotypes. Consequently, the six cercarial morphotypes that were inferred to represent six species and distinguished previously based on morphology and behaviour, are considered here to be comprised of three species (i.e. Cercaria capricornia I and II are considered the same species, C. capricornia types III to V are considered to belong to a second species and C. capricornia VI is considered to comprise the third species in this complex). This recognition of reduced diversity following molecular analysis runs contrary to the more frequently reported uncovering of cryptic diversity, especially when larval trematodes are examined. Phylogenetic analysis of these morphologically unusual cercariae supports their inclusion in the Acanthocolpidae. Bayesian inference analysis identifies three distinct clades: (a) Stephanostomum+Monostephanostomum; (b) the Brachycladiidae and Pleorchis; and (c) Tormopsolus and the present cercariae. Genus-level identification of these cercariae remains uncertain given the current lack of available comparative genetic data on nucleic acid databases.

Evolutionary relationships between digeneans of the family Brachycladiidae Odhner, 1905 and their marine mammal hosts: A cophylogenetic study.

Cophylogenetic studies examine the congruence between host and parasite phylogenies. There are few studies that quantify the relative contribution of coevolutionary events, i.e. duplication, loss, failure-to-diverge, host-switching and spreading in trophically-transmitted parasites at the marine realm. We addressed this issue in the Brachycladiidae, a cosmopolitan digenean family specific to marine mammals. We used, for the first time, distance-based and event-based methods to explicitly test the coevolutionary events that have shaped the current brachycladiid-marine mammal associations. Parasite phylogeny was constructed using mtDNA ND3 sequences of nine brachycladiid species, and host phylogeny using cytochrome b sequences of 104 mammalian species. A total of 50 host-parasite links were identified. Distance-based methods supported the hypothesis of a global non-random association of host and parasite phylogenies. Significant individual links (i.e., 24 out of 50) were those related to Campula oblonga, Nasitrema delphini, N. globicephalae and Brachycladium atlanticum and their associated taxa from the Delphinoidea. Regarding event-based methods, we explored 54 schemes using different combinations of costs for each potential coevolutionary event. Three coevolutionary scenarios were identified across all schemes and in all cases the number of loss events (87-156) was the most numerous, followed by failure-to-diverge (40), duplication (3-6), host-switching (0-3) and cospeciation (0-2). We developed a framework to interpret the evolution of this host-parasite system and confirmed that failure-to-diverge and colonization with or without subsequent diversification could have been decisive in the establishment of the associations between brachycladiids and marine mammals.