Across the tree of life, DNA damage response (DDR) proteins play a pivotal, yet dichotomous role in organismal development and evolution. Here, we present a comprehensive analysis of 432 DDR proteins encoded by 68 genomes, including that of Nucleospora cyclopteri, an intranuclear microsporidia sequenced in this study. We compared the DDR proteins encoded by these genomes to those of humans to uncover the DNA repair-ome across phylogenetically distant eukaryotes. We also performed further analyses to understand if organismal complexity and lifestyle play a role in the evolution of DDR protein length and conserved domain architecture. We observed that the genomes of extreme parasites such as Paramicrocytos, Giardia, Spironucleus, and certain microsporidian lineages encode the smallest eukaryotic repertoire of DDR proteins and that pathways involved in modulation of nucleotide pools and nucleotide excision repair are the most preserved DDR pathways in the eukaryotic genomes analysed here. We found that DDR and DNA repair proteins are consistently longer than housekeeping and metabolic proteins. This is likely due to the higher number of physical protein-protein interactions which DDR proteins are involved. We find that although DNA repair proteins are generally longer than housekeeping proteins, their functional domains occupy a relatively smaller footprint. Notably, this pattern holds true across diverse organisms and shows no dependence on either lifestyle or mitochondrial status. Finally, we observed that unicellular organisms harbour proteins that are tenfold longer than their human homologues, with the extra amino acids forming interdomain regions with a clearly novel albeit undetermined function.

The Apicomplexa are obligate unicellular parasites found in terrestrial and aquatic environments. Nematopsis are found infecting marine invertebrates in a life cycle involving mollusks and crustaceans. In the present study, mantle and muscle tissues from nerite gastropods were microscopically examined for Nematopsis using wet mounts and histology. Oocysts contained one single sporozoite surrounded by an ellipsoidal wall and were surrounded by a parasitophorous vacuole within a host phagocyte. Length and width of fresh oocysts were measured and compared between host species. DNA was extracted from infected tissue, and regions of the rRNA gene were amplified using novel primers. Nematopsis-infected tissues from scallops from Argentina and Scotland were used as controls. The sizes of oocysts observed in nerite hosts from Saint Kitts were not significantly different. DNA sequences of Nematopsis isolated from nerite hosts in this study were identical and phylogenetically related to sequences obtained from scallops. Bayesian and maximum likelihood phylogenetic analyses robustly place the Nematopsis DNA sequences from this study with Nematopsis from scallops in Florida and members of the family Porosporidae. We conclude that marine Nematopsis will group within this clade or within the Porosporidae. We have provided specific oligonucleotide primers to assist with the molecular study of the Porosporidae.

Five adult Greater Caribbean manatees Trichechus manatus manatus were found stranded on various coasts of Puerto Rico; 2 stranded alive and 3 stranded dead. Clinical signs observed in live-stranded manatees included emaciation, weakness, bradypnea, arrhythmia, and nasal mucus discharge. Postmortem examinations revealed serosanguinous, mucohemorrhagic, or suppurative exudate in bronchi associated with luminal adult Pulmonicola cochleotrema (range: 18-182 trematodes), accompanied by pulmonary abscesses in 2 cases. Histologically, we observed eosinophilic bronchopneumonia of varying severity (n = 4) and chronic erosive to eosinophilic tracheobronchitis (n = 4) with squamous metaplasia (n = 3) and intralesional trematodes and eggs. The trematode identity was confirmed and compared through molecular analysis for the amplified 18S rDNA fragment. Comorbidities included enteric chiorchosis (n = 5), gastric heterocheilosis (n = 4), malnutrition (n = 4), trauma related to watercraft collision (n = 3), systemic toxoplasmosis (n = 1), acute bacterial peritonitis (n = 1), and interstitial nephritis (n = 1), suggesting that immunosuppression was a predisposing factor for lower respiratory tract pulmonicolosis. Based on lesion severity, clinical signs, and the presence and absence of other findings to explain death, this condition was considered the primary cause of death in 1 manatee, a contributory cause of death in 3 manatees, and an incidental finding in 1 individual. These clinicopathological descriptions will facilitate the diagnosis and clinical management of pulmonicolosis in T. manatus, a species endangered with extinction.

A 1-year-old mixed breed dog initially presented with marked ascites due to a low-protein transudate resulting from portal hypertension. Laboratory evaluation revealed non-regenerative anemia, lymphopenia, thrombocytopenia, evidence of hepatic insufficiency [hypoalbuminemia, decreased urea, increased post-prandial bile acids, prolonged activated partial thromboplastin time (aPTT)] and Ehrlichia canis infection. Approximately a week later, the dog was declining and was euthanized. On autopsy, multifocal hepatic granulomas and acquired portosystemic shunts (APSS) were seen. Imprint cytology revealed fungal hyphae and pyogranulomatous inflammation in the liver and brain. Disseminated Cladophialophora bantiana phaeohyphomycosis was diagnosed by histologic examination, culture and PCR. Immunosuppression due to ehrlichiosis is suspected to have predisposed this animal to fungal infection. To the authors’ knowledge, this is the first report of C. bantiana in the West Indies.

Members of the genus Scaphanocephalus mature in accipitrids, particularly osprey, Pandion haliaetus , with metacercaria causing Black Spot Syndrome in reef fishes. In most of the world, only the type species, Scaphanocephalus expansus (Creplin, 1842) has been reported. Recent molecular studies in the Western Atlantic, Mediterranean and Persian Gulf reveal multiple species of Scaphanocephalus , but have relied on 28S rDNA, mainly from metacercariae, which limits both morphological identification and resolution of closely related species. Here we combine nuclear rDNA with mitochondrial sequences from adult worms collected in osprey across North America and the Caribbean to describe species and elucidate life cycles in Scaphanocephalus . A new species described herein can be distinguished from S. expansus based on overall body shape and size. Phylogenetic analysis of the whole mitochondrial genome of Scaphanocephalus indicates a close relationship with Cryptocotyle . We conclude that at least 3 species of Scaphanocephalus are present in the Americas and 2 others are in the Old World. Specimens in the Americas have similar or identical 28S to those in the Mediterranean and Persian Gulf, but amphi-Atlantic species are unlikely in light of divergence in cytochrome c oxidase I and the lack of amphi-Atlantic avian and fish hosts. Our results provide insight into the geographic distribution and taxonomy of a little-studied trematode recently linked to an emerging pathology in ecologically important reef fishes.

S tony coral tissue loss disease (SCTLD) is currently ram- paging through reef-building corals across the Caribbean region, with colony mortality rates as high as 99%, affecting nearly 30 different coral species.Scientists have yet to pinpoint the exact etiology, but multiple hypotheses have been proposed, including abiotic stressors, viruses, bacteria, or a combination of these.On the reefs surrounding the island of St. Kitts, home to Ross University School of Veterinary Medicine (RUSVM), the disease was first confirmed in January 2020.Following a government permit, researchers at the university's Research Center for Conservation Medicine and Ecosystem Health (CoMEH) started multiple longitudinal studies that contribute to surveillance and disease investigation.These include a combined holistic diagnostic approach including in-field monitoring and sampling, histopathology, and microbiome sequencing.Clear and consistent differences between healthy and diseased coral microbiomes were identified, regardless of loca-

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.

West Indian manatees ( Trichechus manatus ) harbor a variety of endoparasites, including the nasal trematode Pulmonicola cochleotrema , which infects the respiratory tract, especially the nasal passages. Previous studies have described and identified this digenean using morphological data only. This study presents the first molecular identification of P. cochleotrema in West Indian manatees from Puerto Rico and Florida. Samples of the trematode were collected from seven manatees found stranded dead at both locations. The small subunit ribosomal DNA (18S rDNA) was amplified from each sample using universal primers for different regions of the gene, resulting in a consensus sequence of 1871 base pairs. The phylogenetic reconstruction was carried out using DNA sequences of other species of digenean parasites from other hosts, including a trematode of the same taxonomic family from another sirenian species. Specimens collected from both locations show the same molecular identity using SSU rDNA sequence data. The identity of P. cochleotrema was confirmed using the Basic Local Alignment Search Tool from the National Center for Biotechnology Information database, yielding a high similarity of 98.8 % with Opisthotrema dujonis and 98.2 % with Lankatrema mannarense located in the same clade in our analysis. The latter two digeneans belong to the Opisthotrematidae as does P. cochleotrema and previous studies reported them infecting the Eustachian tubes, esophagus, and digestive tract in dugongs ( Dugong dugon ). These findings evidence that the nasal trematode of manatees in Florida and Puerto Rico and the dugong, all inhabiting and feeding in marine environments, will have a marine mollusk as an intermediate host, probably a gastropod. The question remains, which species of nasal trematode are harbored by lotic-dwelling manatees in other parts of their distribution like South America.

Tetracapsuloides bryosalmonae is a myxozoan parasite and the causative agent of proliferative kidney disease (PKD), a serious, temperature-dependent and emerging disease affecting salmonid fish. It was first identified in Iceland in 2008, from Arctic charr inhabiting a shallow lowland lake. The aim of this study was to investigate the distribution and prevalence of macroscopic and subclinical T. bryosalmonae infections in Icelandic salmonids and compare different time periods, in context with depths, volumes, altitudes and areas of the lakes and fish age. Arctic charr (Salvelinus alpinus) and brown trout (Salmo trutta) from 34 lakes, sampled between 1994-1998 and 2009-2017, were examined for macroscopic signs of PKD (n = 2,151) and the presence of T. bryosalmonae infections (n = 1,424). In the earlier period, 43% of lakes (10/23) harboured T. bryosalmonae -infected fish. The mean prevalence in those lakes was 62.1%, being most common in shallow lowland lakes whilst deeper lakes at high altitudes were all free from infection. Only a single fish from one lake showed macroscopic signs of PKD, a shallow lowland lake in southwestern Iceland. In the latter period, T. bryosalmonae was found in 16/18 lakes studied (89%), with a mean prevalence of 78-79% (excluding T.b. free lakes), being most common in the smaller, shallower lakes at lower alttudes. Macroscopic signs of PKD were observed in 11 of 18 of the lakes studied (61%) with prevalences up to 67%, most common in younger fish inhabiting small shallow lowland lakes. The results indicate that the distribution of T. bryosalmonae and the presence of PKD in Iceland have increased over the last few decades. The disease was almost non-existent in the 1990s but has become very common during the last decade or two. With further water temperature increases, as predicted by climate models, PKD is likely to increasingly affect wild salmonid populations in Iceland.

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.

Many white, elliptical to spherical cyst-like bodies (0.43-1.57 mm in diameter) were found in the liver of ayu Plecoglossus altivelis in Shimane Prefecture, Japan. Each cyst contained one or two (rarely three) nematodes, 1.11-1.20 mm in body length. In parasitological surveys of ayu conducted from 2008 to 2013, infection was confirmed in ayu from three out of six river systems in the prefecture, with prevalence and intensity of infection ranging widely from 0 to 100% and from 1 to 837 cysts/ fish, respectively. We believe that infection occurred in rivers, inferred from the fact that uninfected artificially produced ayu became infected after release to rivers. Morphologically, the nematodes were identified as third stage larvae. They stayed motionless inside the spherical bodies up to 24 h after capture of the host but started to move actively when exposed to physiological saline, where they survived for up to 81 h at room temperature of 22-25 degrees C, <12 h at 37 degrees C or <1 min at 80 degrees C. Molecular phylogenetic analysis using 18S rDNA revealed that the nematode was robustly placed in the monophyletic Cucullanidae Glade but could be identified as either Cucullanus or Dichelyne. The lifecycle remains unknown.

Weathervane scallop, Patinopecten caurinus, the largest scallop species in the world, is distributed from northern California, U.S.A., to the Bering Sea, and is only commercially harvested in Alaska. The fishery is considered well managed by the State of Alaska (U.S.A) Department of Fish and Game (ADF&G) and federal government, with many precautionary measures in place to avoid overharvest. There have been episodic declines in some management areas due to unknown causes. Fishermen also encounter scallops with abnormal adductor muscles, a condition colloquially termed "weak meat", characterized by the retention of muscle when shucked, an obvious darkened discoloration, and/or an abnormal texture making the product unacceptable for marketing. A similar syndrome in Atlantic sea scallops, Placopecten magellanicus, described as "gray meat", occurs in the eastern U.S. and Canada, and proposed causes include senescence, loss of bioenergetics due to chronic infestations, or a synergism of these factors. Recently a severe apicomplexan infection was found to cause a gray meat condition in Iceland scallops, Chlamys islandica, and the collapse of that stock. This parasite was subsequently detected in Atlantic sea scallops with the gray meat condition off the U.S. East Coast. Studies that followed identified the parasite as Merocystis kathae, previously described from the common whelk, Buccinum undatum, more than 100 years ago. In 2015 Bering Sea fishermen reported weak meat in their catch, so samples were submitted to ADF&G for diagnosis. Adductor muscles from all affected scallops had many large foci of an apicomplexan associated with necrosis, fibrosis, and muscular atrophy. Given the reduced quality, marketability, and possibly fitness of affected scallops, we performed a survey to estimate prevalence, intensity, and geographic distribution of this apicomplexan in Alaskan weathervane scallops. We sampled 180 scallops, from individual beds within each of the three major geographically broad scallop areas in Alaska. Overall prevalence was about 82%, ranging from 69 to 100% by district. Overall mean infection intensity, based on the number of parasite foci/section, was about 9 (range of 5-29, by location), with scallops from the Bering Sea and Southwest Kodiak being most severely infected. Molecular analyses confirmed that the Alaskan parasite is M. kathae, i.e., the same apicomplexan that caused the collapse of Icelandic scallops and a suspected cause for gray meat and mass mortality of Atlantic sea scallops in northeast North America.

In this study, we performed comprehensive pathology examinations on 83 Tripneustes ventricosus from 11 locations on St. Kitts to build baseline data necessary for disease diagnosis in this species. Gross abnormalities were observed in 23/83 (28%) urchins and included spine loss, visceral hyperpigmentation, test discoloration, and test ulceration. Ciliates were the only protists identified in this study via examination of tissue wet mounts and histology, documented in 50/83 (60%) urchins. Microscopic observations associated with visibly abnormal status included muscle necrosis, test and appendage inflammation, appendage (tube feet, spines, and pedicellariae) degeneration, severe coelomocytosis, and generalized hypermelanosis. Enterocyte intranuclear inclusion bodies, microbial aggregates, nerve pigmentation, enteric pigmentation, integument-associated crustaceans, and encysted metazoan parasites were of uncertain pathological significance. The etiology for any lesion was not microscopically apparent, contrasting literature implicating common marine bacteria in urchin diseases. This study highlights the importance of histopathology in urchin disease investigations and facilitates the recognition of disease in T. ventricosus.

Proliferative kidney disease (PKD) is a widespread temperature-dependent disease in salmonids caused by the myxozoan parasite, Tetracapsuloides bryosalmonae (Canning, Curry, Feist, Longshaw et Okamura, 1999) (Tb).Tb has a two-host life cycle, involving fish as an intermediate host and freshwater bryozoans as the definitive host.Although salmonids are acknowledged as hosts for the parasite, it is less clear which fish species are active hosts in the life cycle of Tb.Differences in infection dynamics have been observed between some fish species, which are thought to be related to the existence of two main Tb-strains, the American and European.Iceland, having three species of indigenous salmonids and positioned geographically between Europe and North America, is an ideal location to study the natural development of Tb in wild fish.The main aim of this study was to determine the genetic origin of Tb in Iceland and confirm whether mature spores are produced in Icelandic salmonids.In this study, Icelandic salmonids were infected with the European Tb-strain.In situ hybridisation revealed that intraluminal sporogonic stages, including mature spores, were commonly observed in all three salmonid species.The presence of intraluminal stages has previously been confirmed in brown trout Salmo trutta Linnaeus and Atlantic salmon S. salar Linnaeus in Europe, but they have only been observed in Arctic charr Salvelinus alpinus (Linnaeus) in North America, infected by the local strain.This is, therefore, the first time that sporogonic stages have been observed in Arctic charr in Europe, where fish are infected with the European Tb-strain.Our data strongly suggest that all the three salmonid species inhabiting Icelandic waters serve as active hosts in the life cycle of Tb.However, for full confirmation, transmission trials are needed.

The Caribbean spiny lobster Panulirus argus, a long-lived decapod crustacean, is fished throughout its geographical range and generates close to 1 billion US dollars annually. Fisheries across this range are fully- or over-exploited, spurring management strategies such as closed seasons and minimum size limitations. The objective of this study was to determine the minimum legal size and appropriate closed season for the lobster population of Saint Kitts and Nevis by assessing and comparing the functional, physiological and morphological determinants of sexual maturity. Such a comparison has never been attempted for this species. Females did not reach functional maturity until 87.8 mm carapace length (CL) based on the presence of a spermatophore and eggs, but reached morphological maturity based on the presence of ovigerous setae at 72.5 mm CL. Physiological maturity was intermediate at 82.8 mm CL. Determinants of functional maturity overestimate the size at sexual maturity, while the presence of ovigerous setae underestimates it. The gonadosomatic index, a reliable method of assessing physiological maturity in female P. argus, can be used to monitor changes in reproductive activity in both males and females. Gross assessment of the ovary may be used to determine size at physiological maturity, and functional maturity can be used to calculate peak periods of reproduction. The peak physiological and functional maturity for P. argus occurs from March to May. As a result, we propose a closed season from March to June and suggest assessment of reproductive dynamics in order to determine minimum legal size for harvesting P. argus.

Gastrointestinal myxosporean parasites from the genus Enteromyxum are known to cause severe disease, resulting in high mortalities in numerous species of cultured marine fishes globally. Originally described as Myxidium spp., they were transferred to a new genus, Enteromyxum, to emphasize their novel characteristics. Their retention in the family Myxidiidae at the time was warranted, but more comprehensive phylogenetic analyses have since demonstrated the need for a new family for these parasites. We discovered a novel Enteromyxum in wild fish from Malaysia and herein describe the fourth species in the genus and erect a new family, the Enteromyxidae n. fam., to accommodate them. Enteromyxum caesio n. sp. is described infecting the tissues of the stomach in the redbelly yellowtail fusilier, Caesio cuning, from Malaysia. The new species is distinct from all others in the genus, as the myxospores although morphologically similar, are significantly smaller in size. Furthermore, small subunit ribosomal DNA sequence data reveal that E. caesio is <84% similar to others in the genus, but collectively they form a robust and discrete clade, the Enteromyxidae n. fam., which is placed as a sister taxon to other histozoic marine myxosporeans. In addition, we describe, using transmission electron microscopy, the epicellular stages of Enteromyxum fugu and show a scanning electron micrograph of a mature myxospore of E. caesio detailing the otherwise indistinct sutural line, features of the polar capsules and spore valve ridges. The Enteromyxidae n. fam. is a commercially important group of parasites infecting the gastrointestinal tract of marine fishes and the histozoic species can cause the disease enteromyxosis in intensive finfish aquaculture facilities. Epicellular and sloughed histozoic stages are responsible for fish-to-fish transmission in net pen aquaculture systems but actinospores from an annelid host are thought to be necessary for transmission to fish in the wild.

The Caribbean spiny lobster, Panulirus argus (Latreille, 1804) is a highly commercial species and comprises the largest spiny lobster fishery in the world. Although populations have declined throughout its range, there is little known regarding its diseases and pathogens. The objectives of this study were to provide illustrated and standardized methods for postmortem examination, and to describe baseline gross and microscopic pathology for P. argus. From July 2017-March 2019, a postmortem examination including comprehensive histological assessment was performed on 313 fishery-caught lobsters. Epibionts and lesions observed include branchial cirriped infestation (69%), branchial encysted nemertean worm larvae (23%), tail fan necrosis (11%), skeletal muscle necrosis (7%), antennal gland calculi (6%), branchial infarction (2%), and microsporidiosis (0.6%). This report confirms the rare prevalence of microsporidiosis in P. argus and describes nemertean worm larvae in the gill. This study also reports a condition resembling excretory calcinosis in spiny lobster. The methods and data produced by this study facilitate disease diagnosis and sustainable stock management of P. argus.

The parasitic copepod, Sphyrion lumpi, causes a significant host response in the beaked redfish, Sebastes mentella. The response is characterised by granulomatous tissue surrounding the attachment site, with infiltration of leucocytes and degeneration of myofibrils. A whole SSU rDNA locus of S. lumpi was successfully sequenced, expectedly inferring its phylogenetic position within the fish parasitic Copepoda Glade.

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.

The data that support the findings of this study are available from the corresponding author upon reasonable request.