Peptidylarginine Deiminase (PAD) and Post-Translational Protein Deimination—Novel Insights into Alveolata Metabolism, Epigenetic Regulation and Host–Pathogen Interactions

The alveolates (Superphylum Alveolata) comprise a group of primarily single-celled eukaryotes that have adopted extremely diverse modes of nutrition, such as predation, photoautotrophy and parasitism. The alveolates consists of several major phyla including the apicomplexans, a large group of unicellular, spore forming obligate intracellular parasites, and chromerids, which are believed to be the phototrophic ancestors of the parasitic apicomplexans. Molecular pathways involved in Alveolata host–pathogen interactions, epigenetic regulation and metabolism in parasite development remain to be fully understood. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family which causes post-translational protein deimination, affecting protein function through the conversion of arginine to citrulline in a wide range of target proteins, contributing to protein moonlighting in physiological and pathological processes. The identification of deiminated protein targets in alveolate parasites may therefore provide novel insight into pathogen survival and host-pathogen interactions. The current study assessed PAD homologues and deiminated protein profiles of two alveolate parasites, Piridium sociabile (Chromerida) and Merocystis kathae (Apicomplexa). Histological analysis verified strong cytoplasmic PAD expression in both Alveolates, detected deiminated proteins in nuclear and cytoplasmic compartments of the alveolate parasites and verified the presence of citrullinated histone H3 in Alveolata nucleus, indicating roles in epigenetic regulation. Histone H3 citrullination was also found significantly elevated in the host tissue, indicative of neutrophil extracellular trap formation, a host-defence mechanism against a range of pathogens, particularly those that are too large for phagocytosis. Proteomic analysis of deiminated proteins from both Alveolata identified GO and KEGG pathways strongly relating to metabolic and genetic regulation, with some species-specific differences between the apicomplexan and the chromerid. Our findings provide novel insights into roles for the conserved PAD/ADI enzyme family in the regulation of metabolic and epigenetic pathways in alveolate parasites, possibly also relating to their life cycle and host–pathogen interactions

Experimental challenges with Renibacterium salmoninarum in Arctic charr Salvelinus alpinus

Arctic charr Salvelinus alpinus L. is an important species in Icelandic aquaculture and the most common wild salmonid in Iceland. A study on the course of infection with the bacterium Renibacterium salmoninarum was conducted using 3 different challenge methods in brackish and fresh water. Bacterial isolation, ELISA and PCR tests were used for detection of the bacterium in multiple organ samples. In an experiment, run for 34 wk in brackish water, infection was established by intraperitoneal injection with 5 × 106 colony forming units (CFU) fish-1. There were external and internal symptoms of bacterial kidney disease (BKD) and mortalities between 6 and 13 wk after injection. A cohabitation trial was run simultaneously and infection was well established after 4 wk, as demonstrated by the detection methods applied. Symptoms of BKD were not seen and all but 1 cohabitant survived. In a separate experiment, infection was established by pumping a fixed amount of water from a tank with fingerlings infected by intraperitoneal injection into tanks with naïve fish, in fresh or brackish water, for 6 wk. Fish in the inflow tanks were reared for an additional 3 wk. There were neither macroscopic symptoms nor mortalities. ELISA and PCR tests showed that infection started to take hold after 3 wk. The challenge trials demonstrated that Arctic charr is susceptible to R. salmoninarum. Cohabitation and inflow of water from tanks with infected fish provide useful models for further studies on R. salmoninarum infection acquired in a natural way in Arctic charr.The authors are indebted to Islands-bleikja at Grindavik (Samherji hf., Akureyri, Iceland) for providing the fish used in the study, to the staff at Sudurnes Science and Learning Centre for attendance to the experimental fish and to Sigurour Snorrason and Asthildur Erlingsdottir for constructive criticism on the manuscript. This work received grants from the AVS R&D Fund of Ministry of Fisheries and Agriculture in Iceland no. R 10 0093-10 and R 13 074-13.Peer Reviewe

Harmless sea snail parasite causes mass mortalities in numerous commercial scallop populations in the northern hemisphere

Publisher's version (útgefin grein)Apicomplexans comprise a group of unicellular, often highly pathogenic, obligate parasites exploiting either one or two hosts to complete a full reproductive cycle. For decades, various scallop populations have suffered cyclical mass mortality events, several of which shown to be caused by apicomplexan infections. We report the first dual mollusc life cycle for an apicomplexan: a species highly pathogenic in various pectinid bivalve species, but apathogenic when infecting the common whelk as Merocystis kathae. The sympatric distribution of the common whelk and scallops in the North Atlantic makes transmission extremely effective, occurring via the gastrointestinal tract, by scavenging and predation in whelks and unselective filter feeding in scallops. Infective sporozoites from whelks utilize scallops´ haemocytes to reach muscular tissue, where asexual reproduction occurs. Phylogenetically, this apicomplexan is robustly placed within the Aggregatidae and its inclusion in analyses supports a common ancestry with other basal invertebrate apicomplexans. Scallops seem able to regulate lowlevel infections of M. kathae as they exist in normal populations while epizootics occur during high levels of exposure from locally infected whelks. A targeted removal of whelks from valuable scallop grounds would be advantageous to minimize the occurrence of M. kathae epizootics and prevent damaging economic losses.We acknowledge the staff at the Marine and Freshwater Research Institute in Iceland and Grant Campell at Scothatch scallop ranch, for sampling of research material.Peer Reviewe

Redescription and phylogenetic position of Myxobolus ‘eglefini’ and Myxobolus platessae n. comb. (Myxosporea), parasites in the cartilage of some North Atlantic marine fishes, with notes on the phylogeny and classification of the Platysporina

publishedVersio

Multiple Independent Origins of Apicomplexan-Like Parasites

The apicomplexans are a group of obligate animal pathogens that include Plasmodium (malaria), Toxoplasma (toxoplasmosis), and Cryptosporidium (cryptosporidiosis) [1]. They are an extremely diverse and specious group but are nevertheless united by a distinctive suite of cytoskeletal and secretory structures related to infection, called the apical complex, which is used to recognize and gain entry into animal host cells. The apicomplexans are also known to have evolved from free-living photosynthetic ancestors and retain a relict plastid (the apicoplast), which is non-photosynthetic but houses a number of other essential metabolic pathways [2]. Their closest relatives include a mix of both photosynthetic algae (chromerids) and non-photosynthetic microbial predators (colpodellids) [3]. Genomic analyses of these free-living relatives have revealed a great deal about how the alga-parasite transition may have taken place, as well as origins of parasitism more generally [4]. Here, we show that, despite the surprisingly complex origin of apicomplexans from algae, this transition actually occurred at least three times independently. Using single-cell genomics and transcriptomics from diverse uncultivated parasites, we find that two genera previously classified within the Apicomplexa, Piridium and Platyproteum, form separately branching lineages in phylogenomic analyses. Both retain cryptic plastids with genomic and metabolic features convergent with apicomplexans. These findings suggest a predilection in this lineage for both the convergent loss of photosynthesis and transition to parasitism, resulting in multiple lineages of superficially similar animal parasites

Outbreak of viral haemorrhagic septicaemia (VHS) in lumpfish (Cyclopterus lumpus) in Iceland caused by VHS virus genotype IV

Publisher's version (útgefin grein)A novel viral haemorrhagic septicaemia virus (VHSV) of genotype IV was isolated from wild lumpfish (Cyclopterus lumpus), brought to a land-based farm in Iceland, to serve as broodfish. Two groups of lumpfish juveniles, kept in tanks in the same facility, got infected. The virus isolated was identified as VHSV by ELISA and real-time RT-PCR. Phylogenetic analysis, based on the glycoprotein (G) gene sequences, may indicate a novel subgroup of VHSV genotype IV. In controlled laboratory exposure studies with this new isolate, there was 3% survival in the I.P. injection challenged group while there was 90% survival in the immersion group. VHSV was not re-isolated from fish challenged by immersion. In a cohabitation trial, lumpfish infected I.P. (shedders) were placed in tanks with naïve lumpfish as well as naïve Atlantic salmon (Salmo salar L.). 10% of the lumpfish shedders and 43%–50% of the cohabiting lumpfish survived after 4 weeks. 80%–92% of the Atlantic salmon survived, but no viral RNA was detected by real-time RT-PCR nor VHSV was isolated from Atlantic salmon. This is the first isolation of a notifiable virus in Iceland and the first report of VHSV of genotype IV in European waters.H2020 SFS-2014-2 ParaFishControl, Grant/ Award Number: 634429; European Union Reference Laboratory for Fish Diseases Grant Decision SI2.725290Peer Reviewe

Eðli og meinvirkni sníkjudýra af fylkingu Apicomplexa í tengslum við stórfelld afföll í stofnum hörpudisks (Bivalvia: Pectinidae) í Norður Atlantshafi

Apicomplexans comprise a group of unicellular, often highly pathogenic, obligate parasites infecting both vertebrates and invertebrates, exploiting either one (monoxenous) or two hosts (heteroxenous) to complete a full reproductive life cycle. Their pathogenicity varies considerably between species and/or their hosts but as most species are obligate, intracellular parasites they cause some level of pathology in all cases. In relation to abnormal mortality events experienced in scallop populations in the North Atlantic, four different species of scallops, i.e. Iceland scallop, queen scallop, king scallop and sea scallop, were examined for infectious agents, with the aim of shedding light on these events. Most emphasis was made on the Iceland scallop stock in Icelandic waters that unexpectedly collapsed during the 2000s. Its health status was annually monitored for 14 years. In addition to the scallops, whelks and other mollusc species were examined at later stages in the study, in context with presumable life cycle of a pathogen observed. Two different apicomplexan species, both of which appeared to be previously unknown, were identified: 1) Margolisiella islandica, a novel species infecting the heart auricles and highly prevalent in Iceland scallop, without causing any significant pathology, but absent in the three other scallop species. It is a monoxenous species, with all life stages present in a single host, i.e. the Iceland scallop. 2) Initially anonymous species, infecting muscular- and connective tissues, was found in all four scallop species examined. Except for scallops from the UK, infections were generally heavy, causing severe histopathological changes leading to significant reduction in the general condition of the scallops, especially adductor muscles, which become abnormally reduced and discoloured. Furthermore, it hampers normal gonad development, at least in the Iceland scallop. Molecular studies and in situ hybridization, revealed that it is conspecific with Merocystis kathae, an apicomplexan described from the common whelk, Buccinum undatum, more than 100 years ago. Consequently, its life cycle is heteroxenous, involving the common whelk as a definite host and scallops as intermediate hosts. This is the first dual mollusc life cycle described for an apicomplexan. M. kathae forms a fully resolved monophyletic clade with Aggregata spp. This aggregatid clade is associated with a sister clade containing Filipodium phascolosomae and Platyproteum vivax (Archigregarinorida (Squirmida)) from sipunculids. M. islandica and Pseudoklossia pectinis do not form part of this clade, but form a group Rhytidocystid species, infecting marine bivalves and polychaetes. This entire group forms a weakly supported clade of apicomplexans that are found in marine invertebrates. The results strongly suggest that M. kathae played a major role in the collapse of the Iceland scallop stock in Breidafjordur, Iceland. Furthermore, it caused abnormal condition of Faroese queen scallops and is a suspected aetiological agent behind mass mortality events of sea scallops in the eastern USA as well as a number of unresolved mass mortalities and abnormal condition experienced in various other populations of scallops. Scallops seem able to regulate low-level infections of M. kathae, as they exist in normal scallop populations. However, during prolonged or high levels of exposure from localised infected whelks, disease outbreaks occur. Hence, reasonable fisheries from both whelk and scallop stocks could lower infectious load and minimize the occurrence of M. kathae epidemics and prevent damaging economic losses.Fylking Apicomplexa, samanstendur af hópi sjúkdómsvaldandi, einfruma sníkjudýra sem sýkja bæði hryggdýr og hryggleysingja. Til þess að ljúka lífsferli sínum þarfnast þau ýmist eins hýsils (monoxenous) eða tveggja hýsla (heteroxenous). Meinvirkni þessa hóps sníkjudýra er mismikil, eftir tegundum og/eða hýslum þeirra. Þar sem þetta eru innanfrumusýklar, valda þeir þó einhverjum vefjaskemmdum í öllum tilfellum. Í tengslum við óeðlilega umfangsmikinn náttúrulegan dauða í ýmsum stofnum og tegundum hörpudiska í Norður-Atlantshafi, voru fjórar tegundir hörpudisks, frá Íslandi, Færeyjum, Bandaríkjunum og Bretlandi, rannsakaðar m.t.t. sjúkdóma, með það að markmiði að varpa ljósi á ástæður þessa mikla náttúrulega dauða. Megináherslan var lögð á stofn íslenska hörpudisksins í Breiðafirði, en algert hrun varð í stofninum á fyrstu árum þessarar aldar. Íslenski stofninn var vaktaður árlega í samfellt 14 ár. Til viðbótar hörpudiskum, voru beitukóngur, auk fleiri tegunda lindýra, rannsökuð á síðari stigum í tengslum við lífsferil sníkjudýrs sem greindist. Tvær tegundir af fylkingu Apicomplexa greindust í hörpuskeljunum, báðar að því er virtist áður óþekktar: 1) Tegund sem fékk nafnið Margolisiella islandica sem sýkir hjarta og fannst eingöngu í íslensku hörpuskelinni. Smittíðnin var há, en vefjaskemmdir samfara sýkingum voru óverulegar. 2) Tegund sem í fyrstu virtist áður óþekkt; sýkir vöðva og stoðvefi (connective tissues) og fannst í öllum fjórum hörpudiskstegundunum. Að frátöldum skeljum frá Bretlandseyjum, voru sýkingar almennt umfangsmiklar og ollu svæsnum vefjaskemmdum. Sýkingar hafa verulega neikvæð áhrif á almennt ástand skelja, einkum aðdráttarvöðva, sem verða óeðlilega rýrir auk þess að verða grá/brúnleitir og lausir í sér miðað við þétta, ljósa, heilbrigða vöðva. Að auki, valda sýkingar verulegum skemmdum á kynkirtlum sem leiðir til þess að þeir þroskast ekki með eðlilegum hætti. Frekari greiningar sýndu fram á að tegundin reyndist vera Merocystis kathae sem lýst var úr nýra beitukóngs fyrir meira en 100 árum en veldur sniglinum óverulegu heilsutjóni. M. kathae nýtir þ.a.l. tvo hýsla til að ljúka lífsferli sínum, hörpudisk og beitukóng. Þetta er fyrsti lífsferlill sem lýst er fyrir tegund af fylkingu Apicomplexa sem felur í sér tvær mismunandi tegundir lindýra.Þróunarfræðilega, myndar M. kathae einættaða (monophyletic) grein/hóp (clade) með Aggregata tegundum. Systur-grein/hópur M. kathae og Aggregata tegundanna samanstendur af Filipodium phascolosomae og Platyproteum vivax, tegundum af ættbálki Archigregarinorida sem sýkja liðorma, möttuldýr, akarnorma og sæbelgi. M. islandica og skyld tegund sem sýkir tegund hörpudisks, Pseudoklossia pectinis, tilheyra ekki þessari grein/hópi, en mynda sérstaka grein/hóp með Rhytidocystis tegundum, sem sýkja burstaorma og tveimur ónefndum tegundum úr sjávarsamlokum. Allar áðurnefndar greinar/hópar mynda svo saman grein/hóp tegunda sem sýkja sjávarhryggleysingja. Niðurstöður þessara rannsókna benda eindregið til þess að M. kathae sé meginástæða hruns íslenska hörpudisksstofnsins og slæmu ástandi hörpudiskstegundarinnar við Færeyjar. Auk þessa, eru vísbendingar um að þetta sníkjudýr valdi, og hafi valdið, stórfelldum náttúrulegum dauða í skeljastofnum við austurströnd Bandaríkjanna og mögulega miklum og óútskýrðum afföllum í öðrum stofnum ýmissa tegunda hörpudisks í NorðurAtlantshafi í gegnum tíðina. Þar sem M. kathae sýkingar finnast í heilbrigðum hörpudisksstofnum, virðist sem skeljar nái að höndla vægar sýkingar. Hins vegar, þegar mikið og langvarandi smitmagn berst frá smituðum beitukóngum á búsvæðum hörpudisks, geta komið upp faraldrar. Þar af leiðandi, gætu hófsamar veiðar, á bæði hörpudiski og beitukóngi, minnkað smitpressu og dregið úr líkum á uppkomu sjúkdómsfaraldra og tilheyrandi fjárhagstjóni samfara þeim.Ministry of Agriculture and Fisheries in Iceland, University of Malaya, Ross University School of Veterinary Medicine, St. Kitts West Indies, SMAST at the University of Massachusett

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

Abstract 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