A combined diagnostic approach for the investigation of lesions resembling aspergillosis in Caribbean sea fans (Gorgonia spp.).

Aspergillosis of gorgonian sea fans is a Caribbean-wide disease characterized by focal, annular purple pigmentation with central tissue loss. We applied a holistic diagnostic approach including histopathology and a combination of culture and direct molecular identification of fungi to evaluate these lesions with the goal of determining the diversity of associated micro-organisms and pathology. Biopsies were collected from 14 sea fans without gross lesions and 44 sea fans with lesions grossly consistent with aspergillosis in shallow fringing reefs of St. Kitts. Histologically, the tissue loss margin had exposure of the axis and amoebocyte encapsulation with abundant mixed micro-organisms. Polyp loss, gastrodermal necrosis, and coenenchymal amoebocytosis were at the lesion interface (purpled area transitioning to grossly normal tissue) with algae (n = 21), fungus-like hyphae (n = 20), ciliate protists (n = 16), cyanobacteria (n = 15), labyrinthulomycetes (n = 5), or no micro-organisms (n = 8). Slender, septate hyaline hyphae predominated over other morphological categories, but were confined to the axis with little host response other than periaxial melanization. Hyphae were absent in 6 lesioned sea fans and present in 5 control biopsies, questioning their pathogenicity and necessary role in lesion causation. From cultivation, different fungi were isolated and identified by sequencing of the nuclear ribosomal internal transcribed spacer region. In addition, 2 primer pairs were used in a nested format to increase the sensitivity for direct amplification and identification of fungi from lesions, thereby circumventing cultivation. Results suggest mixed and opportunistic infections in sea fans with these lesions, requiring longitudinal or experimental studies to better determine the pathogenesis.

A phylogenetically unresolved apicomplexan (APXSc) causing swirl lesions in the Tehuelche scallop, Aequipecten tehuelchus, from the Southwest Atlantic coast.

The study reports a previously unknown apicomplexan (APXSc) parasite infecting wild scallops Aequipecten tehuelchus (d'Orbigny, 1842) from two separate areas (La Tapera and Punta Conos) of the San José gulf, in Patagonia Argentina. Histology, transmission electron microscope, molecular analyses and in situ hybridization were performed to describe the morphology of APXSc, and confirm its phylogenetic status. The prevalence of APXSc infection was 24% and 72% in scallops from La Tapera and Punta Conos, respectively. Seasonal variation was observed for scallops from La Tapera, recording highest prevalence in summer. A positive relationship between the presence of the APXSc and the size of the scallops was observed. A SSU rDNA consensus sequence of 1758 base pairs was generated which has a 94.8% identity to sequences obtained from a pathogenic apicomplexan parasite infecting Ostrea chilensis in New Zealand, but not closely related to other apicomplexans. The asexual reproduction, i.e. merogony, occurs in the Tehuelche scallop whilst the gamogonic and sporogonic stages were absent, suggesting a yet unknown definitive host. Severe host inflammation response involving fibroblast-like hemocytes surrounding the APXSc in the form of granuloma-like "swirls" is characteristic for this apicomplexan infection. Further studies are needed to reveal the life cycle, and presumable pathogenicity of APXSc.

Pathology of multifocal purple spots, a nonspecific lesion morphology of Caribbean sea fans Gorgonia spp.

Disease is contributing to the decline of coral reefs globally, but the cause and pathogenesis of most coral diseases are poorly understood. Using Gorgonia ventalina and G. flabellum as a model for coral disease diagnosis, we histologically and microbiologically examined 45 biopsies of lesions resembling Gorgonia multifocal purple spots (MFPS) with the aim of forming a comprehensive case definition based on gross and microscopic morphologic descriptions and associated etiologies. Macroscopically, all lesions were small circular areas of purple pigmentation. Gross morphologies included pigmentation only (4/45, 9%), or pigmentation with branchlet expansion and fusion (19/45, 22%), sessile masses (17/45, 38%), or hard nodules (5/45, 9%). Histological morphologic diagnoses included amoebocyte encapsulation (9/45, 20%), coenenchymal amoebocytosis (6/45, 13%), melanin (17/45, 38%), and gorgonin deposition (13/45, 29%). Sixty-four percent of instances of fungi and 86% of labyrinthulomycetes were localized to grossly normal portions of the biopsy, whereas barnacles were only within lesions, and 87% of instances of algae and 82% of cyanobacteria were within lesioned area of the biopsy. Penicillium (n = 12) was the predominant genus of fungi isolated from biopsies. Barnacles were identified as Conopea sp. using molecular techniques. The pathology and etiology underlying MFPS lesions are diverse, consistent with a highly nonspecific lesion pattern rather than a specific disease. This study demonstrates the importance of microscopic examination of tissues for accurate classification of coral diseases and lesion patterns.

Scaphanocephalus-associated dermatitis as the basis for black spot disease in Acanthuridae of St. Kitts, West Indies.

Acanthurus spp. of St. Kitts and other Caribbean islands, including ocean surgeonfish A. bahianus, doctorfish A. chirurgus, and blue tang A. coeruleus, frequently show multifocal cutaneous pigmentation. Initial reports from the Leeward Antilles raised suspicion of a parasitic etiology. The aim of this study was to quantify the prevalence of the disease in St. Kitts' Acanthuridae and describe its pathology and etiology. Visual surveys demonstrated consistently high adjusted mean prevalence at 3 shallow reefs in St. Kitts in 2017 (38.9%, 95% CI: 33.8-43.9) and 2018 (51.5%; 95% CI: 46.2-56.9). There were no differences in prevalence across species or reefs, but juvenile fish were less commonly affected than adults. A total of 29 dermatopathy-affected acanthurids were sampled by spearfishing for comprehensive postmortem examination. Digenean metacercariae were dissected from <1 mm cysts within pigmented lesions. Using partial 28S rDNA sequence data they were classified as Family Heterophyidae, members of which are commonly implicated in black spot disease of other fishes. Morphological features of the parasite were most typical of Scaphanocephalus spp. (Creplin, 1842), and 2 genetic profiles were obtained suggesting more than 1 digenean species. Histologically, pigmented lesions had mild chronic perivascular dermatitis and increased melanophores and melanin density, often centered on encysted digenean metacercariae. In 1 affected A. chirurgus, similar metacercariae were histologically identified in skeletal and cardiac muscle. Further research is needed to clarify impact on host fitness, establish the number of heterophyid digenean species that cause black spots on Caribbean fishes and to determine the intermediate and definitive host species.

Extraintestinal Acanthocephalan Oncicola venezuelensis (Oligacanthorhynchidae) in Small Indian Mongooses (Herpestes auropunctatus) and African Green Monkeys (Chlorocebus aethiops sabaeus).

We identified multiple extraintestinal cystacanths during routine postmortem examination of 3 small Indian mongooses and 2 African green monkeys from the Caribbean island of St. Kitts. In mongooses, cystacanths were encysted or free in the subcutaneous tissue, skeletal muscle, or peritoneal or pericardial cavities, whereas in the monkeys, they were in the cavity and parietal layer of the, tunica vaginalis, skeletal muscle, and peritoneal cavity. Morphological, histological, and molecular characterization identified these cystacanths as Oncicola venezuelensis (Acanthocephala: Oligacanthorhynchidae). There was minimal to mild lymphoplasmacytic inflammation associated with the parasite in the mongooses and moderate inflammation, mineralization, hemorrhage, and fibrosis in the connective tissue between the testis and epididymis in 1 monkey. We identified a mature male O. venezuelensis attached in the aboral jejunum of a feral cat, confirming it as the definitive host. Termites serve as intermediate hosts and lizards as paratenic hosts. This report emphasizes the role of the small Indian mongoose and African green monkey as paratenic hosts for O. venezuelensis.

X-Cells Are Globally Distributed, Genetically Divergent Fish Parasites Related to Perkinsids and Dinoflagellates.

"X-cells" have long been associated with tumor-like formations (xenomas) in marine fish, including many of commercial interest. The name was first used to refer to the large polygonal cells that were found in epidermal xenomas from flatfish from the Pacific Northwest [1]. Similar looking cells from pseudobranchial xenomas had previously been reported from cod in the Atlantic [2] and Pacific Oceans [3]. X-cell pathologies have been reported from five teleost orders: Pleuronectiformes (flatfish), Perciformes (perch-like fish), Gadiformes (cods), Siluriformes (catfish), and Salmoniformes (salmonids). Various explanations have been elicited for their etiology, including being adenomas or adenocarcinomas [4, 5], virally transformed fish cells [6-8], or products of coastal pollution [9, 10]. It was hypothesized that X-cells were protozoan parasites [1, 11-13], and although recent molecular analyses have confirmed this, they have failed to place them in any phylum [14-18], demonstrating weak phylogenetic associations with the haplosporidians [16] or the alveolates [15]. Here, we sequenced rRNA genes from European and Japanese fish that are known to develop X-cell xenomas. We also generated a metagenomic sequence library from X-cell xenomas of blue whiting and Atlantic cod and assembled 63 X-cell protein-coding genes for a eukaryote-wide phylogenomic analysis. We show that X-cells group in two highly divergent clades, robustly sister to the bivalve parasite Perkinsus. We formally describe these as Gadixcellia and Xcellia and provide a phylogenetic context to catalyze future research. We also screened Atlantic cod populations for xenomas and residual pathologies and show that X-cell infections are more prevalent and widespread than previously known.

Morphological, ultrastructural, and molecular description of Unicapsula fatimae n. sp. (Myxosporea: Trilosporidae) of whitespotted rabbitfish (Siganus canaliculatus) in Omani waters.

Investigations regarding the parasite fauna of wild whitespotted rabbitfish (Siganus canaliculatus) Park, 1797 revealed white, spherical, loosely attached cysts measuring 896 (375-1406) µm in diameter in the inner endothelial wall of the esophagus and stomach. Mature spores inside these cysts corresponded to the original description of spores belonging to the genus Unicapsula Davis, 1924. Unicapsula fatimae n. sp. spores were 6.23 (5.60-6.60) µm in length and 6.80 (6.12-7.39) µm in width. The length of large polar capsule was 2.62 (2.18-2.97) µm and width was 2.65 (2.32-2.90) µm, and the extended large polar capsule filament length was 15.50 (11.71-19.99) µm. Transmission electron microscope images of the plasmodia revealed a complex cyst structure that was unique among other Unicapsula spp. Ultrastructural details of the host-parasite interface and developmental stages of a species from the Unicapsula genus are described for the first time. Histology of an infected esophagus revealed some abnormalities and changes in the host tissue around the infection site, including hypertrophy of host esophagus epithelial cells and hyperplasia of host glandular tubules. The parasite presented here has been added to the genus Unicapsula using comparative morphological analysis and ultrastructural investigations supported by 18S small subunit ribosomal DNA molecular analysis.

Histozoic myxosporeans infecting the stomach wall of elopiform fishes represent a novel lineage, the Gastromyxidae.

BACKGROUND: Traditional studies on myxosporeans have used myxospore morphology as the main criterion for identification and taxonomic classification, and it remains important as the fundamental diagnostic feature used to confirm myxosporean infections in fish and other vertebrate taxa. However, its use as the primary feature in systematics has led to numerous genera becoming polyphyletic in subsequent molecular phylogenetic analyses. It is now known that other features, such as the site and type of infection, can offer a higher degree of congruence with molecular data, albeit with its own inconsistencies, than basic myxospore morphology can reliably provide. METHODS: Histozoic gastrointestinal myxosporeans from two elopiform fish from Malaysia, the Pacific tarpon Megalops cyprinoides and the ten pounder Elops machnata were identified and described using morphological, histological and molecular methodologies. RESULTS: The myxospore morphology of both species corresponds to the generally accepted Myxidium morphotype, but both had a single nucleus in the sporoplasm and lacked valvular striations. In phylogenetic analyses they were robustly grouped in a discrete clade basal to myxosporeans, with similar shaped myxospores, described from gill monogeneans, which are located at the base of the multivalvulid clade. New genera Gastromyxum and Monomyxum are erected to accommodate these myxosporean taxa from fish and gill monogeneans respectively. Each are placed in a new family, the Gastromyxidae with Gastromyxum as the type genus and Monomyxidae with Monomyxum as the type genus. CONCLUSIONS: To improve modern systematics of the myxosporeans it is clear that a combination of biological, ecological, morphological and molecular data should be used in descriptive studies, and the naming and redistribution of taxa and genera is going to be necessary to achieve this. Here we demonstrate why some Myxidium-shaped myxospores should not be included in the family Myxidiidae, and create two new families to accommodate them based on their site of infection, host biology / ecology, DNA sequence data and morphological observations. Subsequent descriptive works need to follow a similar course if we are going to create a prevailing and workable systematic structure for the Myxosporea.

Zoothamnium duplicatum infestation of cultured horseshoe crabs (Limulus polyphemus).

An outbreak of the sessile peritrich Zoothamnium duplicatum in a pilot, commercial-scale Limulus polyphemus hatchery resulted in the loss of ∼96% (40,000) second/third instar larvae over a 61day period. peritrich growth was heavy, leading to mechanical obstruction of the gills and physical damage. The peritrichs were controlled without resultant loss of juvenile crabs by administering 10ppm chlorine in freshwater for 1h and the addition of aquarium grade sand; a medium into which the crabs could burrow and facilitate cleaning of the carapace. Peritrich identity was confirmed from a partial SSU rDNA contiguous sequence of 1343bp (99.7% similarity to Z. duplicatum).

Multiple gene analyses of caligid copepods indicate that the reduction of a thoracic appendage in Pseudocaligus represents convergent evolution.

BACKGROUND: The Caligidae is a family of parasitic copepods containing over 30 recognised genera. They are commercially important parasites as they cause disease in numerous finfish aquaculture facilities globally. Morphological features are used to distinguish between the genera and Pseudocaligus has traditionally been differentiated from Caligus solely by the presence of a much reduced form of the fourth thoracic leg. Currently there are numerous DNA sequences available for Caligus spp. but only the type species, Pseudocaligus brevipedis, has molecular data available, so systematic studies using molecular phylogenetic analyses have been limited. METHODS: Three gene regions, SSU rDNA, 16S and CO1, for Pseudocaligus fugu from puffer fish from Japan and Pseudocaligus uniartus from rabbit fish from Indonesia are sequenced and molecular phylogenetic analyses performed in order to infer phylogenetic relationships between Pseudocaligus and other caligid copepods. RESULTS: The analysis revealed that there was no discrete grouping of Pseudocaligus spp. and that they had a polyphyletic distribution within Caligus taxa. Pseudocaligus fugu grouped with Caligus elongatus and contained a unique synapomorphy in the SSU rDNA region only seen in members of that clade. Pseudocaligus uniartus formed a well-supported group, in the SSU rDNA analyses, with a Caligus sp. that also infects rabbit fish, but was unresolved in the other analyses. Pseudocaligus brevipedis consistently and robustly grouped with Caligus curtus and C. centrodonti in all analyses. The majority of Lepeophtheirus spp. form a monophyletic sister group to the Caligus clade; however, L. natalensis is unresolved in all analyses and does not form part of the main Lepeophtheirus clade. CONCLUSIONS: These findings do not support the morphological-based distinction between Pseudocaligus and Caligus, suggesting that the reduced fourth leg is a feature that has evolved on multiple occasions throughout caligid evolution. Congruent molecular phylogenetic data support groupings based on the presence of morphological features, such as lunules, geography and host fish type rather than appendage morphology. Therefore, we support the synonymy of Pseudocaligus with Caligus.

Phylogenetic position of Sphaerospora testicularis and Latyspora scomberomori n. gen. n. sp. (Myxozoa) within the marine urinary clade.

An amendment of the family Sinuolineidae (Myxosporea) is proposed in order to include a newly described genus Latyspora n. gen. The type species Latyspora scomberomori n. gen. n. sp. is a coelozoic parasite in the kidney tubules of Scomberomorus guttatus. In addition to the morphological and molecular characterization of L. scomberomori n. gen. n. sp., we also present novel SSU rDNA data on Sphaerospora testicularis, a serious parasite of Dicentrarchus labrax. Performed phylogenetic analyses revealed that both species cluster within the marine urinary clade encompassing the representatives with a shared insertion within their V4 SSU rRNA region and grouping according to the shape of their spores' sutural line and their similar tissue tropism in the host. Sphaerospora testicularis is the closest relative to Parvicapsula minibicornis within the Parvicapsula subclade and L. scomberomori n. gen. n. sp. is the basal species of the Zschokkella subclade. The phylogenetic position of S. testicularis, outwith the basal Sphaerospora sensu stricto clade, and its morphology suggest it being a non-typical Sphaerospora. The sequence data provided on S. testicularis can help in future revisions of the strongly polyphyletic genus Sphaerospora. We recommend re-sequencing of several sphaerosporids as an essential step before such taxonomic changes are accomplished.

Myxobolus albi n. sp. (Myxozoa) from the Gills of the Common Goby Pomatoschistus microps Krøyer (Teleostei: Gobiidae).

A recent investigation into the myxozoan fauna of common gobies, Pomatoschistus microps, from the Forth Estuary in Scotland, revealed numerous myxosporean cysts within the gill cartilage. They were composed of polysporous plasmodia containing myxobolid spores that were morphologically different from the other known species of Myxobolus and from the myxosporeans previously recorded from this host (i.e. the ceratomyxid Ellipsomyxa gobii, infecting the gall bladder, and the kudoid Kudoa camarguensis, infecting the muscle tissues). Spores were ovoid, 9.4 x 9.1 microm with a thickness of 6.6 microm, with two pyriform polar capsules, the polar filaments of which had four to five turns. Molecular analysis of the parasite's small subunit rDNA region, based upon a contiguous sequence of 1,558 base pairs, discriminated it from other myxosporean species that have been characterized so far. A comparison of the spore morphology and the molecular sequences determined for this new isolate with other myxozoans described to date, confirmed its identity as a previously unknown myxobolid supporting the proposal that this isolate be elevated to the species level as a new species within the genus Myxobolus. A phylogenetic analysis places this new myxobolid, Myxobolus albi n. sp., in a basal position of a clade containing the majority of Henneguya spp. sequenced to date and various Myxobolus spp.

Spinal curvature of cultured Japanese mackerel Scomber japonicus associated with a brain myxosporean, Myxobolus acanthogobii.

Skeletal deformities were found in the cultured Japanese mackerel Scomber japonicus. External and radiographical observations showed the deformed fish to exhibit a dorso-ventral spinal curvature (kyphosis) without fracture or dislocation of the vertebrae. Numerous myxosporean cysts, ca. 0.3 to 1.0 mm in diameter, formed in the 4th ventricle, the cavity of the optic tectum, the surface of the olfactory lobe and bulb, the optic lobe and the inferior lobe of the brain. Spore morphology and molecular analysis of the small subunit ribosomal RNA gene sequence identified the myxosporean parasite as Myxobolus acanthogobii, a parasite which also causes scoliosis in yellowtail Seriola quinqeradiata. Histopathological observation showed that the myxosporean cysts were encapsulated within the host's collagenous layer although some had disintegrated to disperse mature spores into the cranial cavity. Occasionally, lymphocytic infiltration and local granulomatous inflammation were found to be associated with spore dispersion.