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Morphological and molecular studies of bivalves to assess the hidden diversity of protistan parasites: Investigations of cryptic diseases in shellfish
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Systematic Biology. (Burki lab)ORCID iD: 0000-0003-1733-4085
2024 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis investigates the host - parasitic relationships in mussel, started by investigating unexplained massmortalities in freshwater mussels, Margaritifera margaritifera. By compiling geodata with symptoms and histopathology (Paper 1). In Paper 2, I continued on these investigations, describing a parasitic gregarines in host. Using in situ hybridization (ISH) and ultrastructural analysis, we linked this gregarine to host detected lesions. To investigate fixed sampled, we used CARD-FISH demonstrating new patterns of parasitic infiltration of gonads. (Paper 3). In marine environments, protist parasites have been throughly documented, though several lesions remain unexplained. Using cell culture, we isolated Hexamita nelsoni linking this protist with blue mussels lesions in muscle & gonads, using CARD-FISH (Paper 4). The largest threat towards marine mussel aquaculture are Ascetosporean parasites, not culturable, requiring alternative methods for genomic studies. We used, density gradient filtration to concentrate Marteilia pararefringens and Bonamia ostreae from infected tissues, enabling these studies, Paper 5.

Abstract [sv]

Mitt doktorandsarbete undersöker relationen mellan musslan och dessa parasiter. Arbetet startade med studier av oförklarade massdödligheter bland sötvattens musslor, Margaritifera margaritifera, genom sammanställning av geologiska data och observerade sjukdomssymtom (Paper 1). In nästa studie (Paper 2), fortsätter vi detta genom att beskriva en ny gregarin (Apicomplexa) associerade med organförändringar. Med In situ hybridisering (ISH) och elektronmikroskopi, kunde vi koppla dessa till observerade vävnadsskador. Vidare påvisade vi olika arter av gregariner genom CARD-FISH metodik i histologiska prover och demonstrera därigenom nya mönster av parasitär infiltration (Paper 3). Mussel parasiter i marin miljö är  väldokumenterade, även om vissa organskador förblivit oförklarade. Genom cellkultur från blåmussla isolerades Hexamita nelsoni, och kopplades till observerade organskador, i muskel och gonadvävnad, med CARD-FISH (Paper 4). Det största hoten mot vattenbruk av Bivalver är Ascetosporea parasiter, som ej går at odla. För genomisk undersökning krävs andra alternativ. Med gradient filtrering  möjliggjordes koncentration av dessa celler och genomiska studier av Marteilia och Bonamia.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2024. , p. 69
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 2450
Keywords [en]
Margaritifera margaritifera, Freshwater Pearl Mussel, Mass mortality events, Gregarine, Pathology, CARD-FISH, ISH, Mytilus edulis, blåmussla, Hexamita nelsoni, phylogeny, histology
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-538623ISBN: 978-91-513-2233-9 (print)OAI: oai:DiVA.org:uu-538623DiVA, id: diva2:1898818
Public defence
2024-11-01, Lindahlsalen, 05.01058, Evolutionsbiologiskt centrum (EBC), Norbyvägen 14, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2024-10-11 Created: 2024-09-18 Last updated: 2024-10-31
List of papers
1. Mass Mortality Events in Freshwater Pearl Mussel (Margaritifera margaritifera) Populations in Sweden: An Overview and Indication of Possible Causes
Open this publication in new window or tab >>Mass Mortality Events in Freshwater Pearl Mussel (Margaritifera margaritifera) Populations in Sweden: An Overview and Indication of Possible Causes
2019 (English)In: Freshwater Mollusk Biology and Conservation, ISSN 2472-2944, Vol. 22, no 2, p. 61-69Article in journal (Refereed) Published
Abstract [en]

The freshwater pearl mussel Margaritifera margaritifera is an endangered species in Sweden with more than 600 known populations distributed in 16 out of 21 counties. Only approximately one-third of these populations are considered viable and healthy with signs of recent juvenile recruitment. From 2011 to 2017, research documented an increased mortality in this species, of up to 100% in some populations, but no etiological cause of these mortalities has been identified. With this paper, we provide current knowledge of locations where mass mortality of freshwater pearl mussel has been found in Sweden and discuss possible causes. Postmortem sampling and histopathological findings from two counties in 2016–17 detected lesions in digestive glands indicating a reduced capacity for nutrient uptake. Results from these macroscopic and microscopic investigations also indicate a reduction in, or a lack of, reproductive output compared with reference populations.

Place, publisher, year, edition, pages
Freshwater Mollusk Conservation Society, 2019
Keywords
Margaritifera margaritifera, die-offs, mortality, pathology, emaciated
National Category
Ecology
Identifiers
urn:nbn:se:uu:diva-538618 (URN)10.31931/fmbc.v22i2.2019.61-69 (DOI)
Note

Niklas Wengström and Anders Alfjorden contributed equally to this work

Available from: 2024-09-18 Created: 2024-09-18 Last updated: 2024-09-19Bibliographically approved
2. Identification of a new gregarine parasite associated with mass mortality events of freshwater pearl mussels (Margaritifera margaritifera) in Sweden
Open this publication in new window or tab >>Identification of a new gregarine parasite associated with mass mortality events of freshwater pearl mussels (Margaritifera margaritifera) in Sweden
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2024 (English)In: Journal of Eukaryotic Microbiology, ISSN 1066-5234, E-ISSN 1550-7408, Vol. 71, no 3, article id e13021Article in journal (Refereed) Published
Abstract [en]

Freshwater bivalves play key ecological roles in lakes and rivers, largely contributing to healthy ecosystems. The freshwater pearl mussel, Margaritifera margaritifera, is found in Europe and on the East coast of North America. Once common in oxygenated streams, M. margaritifera is rapidly declining and consequently assessed as a threatened species worldwide. Deterioration of water quality has been considered the main factor for the mass mortality events affecting this species. Yet, the role of parasitic infections has not been investigated. Here, we report the discovery of three novel protist lineages found in Swedish populations of M. margaritifera belonging to one of the terrestrial groups of gregarines (Eugregarinorida, Apicomplexa). These lineages are closely related–but clearly separated–from the tadpole parasite Nematopsis temporariae. In one lineage, which is specifically associated with mortality events of M. margaritifera, we found cysts containing single vermiform zoites in the gills and other organs of diseased individuals using microscopy and in situ hybridization. This represents the first report of a parasitic infection in M. margaritifera that may be linked to the decline of this mussel species. We propose a tentative life cycle with the distribution of different developmental stages and potential exit from the host into the environment.

Place, publisher, year, edition, pages
John Wiley & Sons, 2024
Keywords
freshwater perl mussel, gregarine, ISH, Margaritifera margaritifera, mass mortality, mortality, Nematopsis, pathology, phylogeny, TEM
National Category
Ecology
Identifiers
urn:nbn:se:uu:diva-446934 (URN)10.1111/jeu.13021 (DOI)001184534000001 ()
Funder
Science for Life Laboratory, SciLifeLabSwedish Research Council Formas, 2017-01197Swedish Research Council, 2018-0597Swedish Research Council, 2017-04563
Note

Authors and title in the list of papers of Mahwash Jamy's thesis: Alfjorden, A., Brännström, I. O., Wengström, N., Jamy, M., Kristmundson, A., Jansson, E., Burki, F. Identification of a new gregarine parasite [Apicomplexa, Alveolata] in mass mortality events of freshwater pearl mussels (Margaritifera margaritifera)

Available from: 2021-06-22 Created: 2021-06-22 Last updated: 2024-09-20Bibliographically approved
3. Morphological and pathological description of a new terrestrial eugregarine lineage infecting freshwater pearl mussels (Margaritifera margaritifera) in western Sweden
Open this publication in new window or tab >>Morphological and pathological description of a new terrestrial eugregarine lineage infecting freshwater pearl mussels (Margaritifera margaritifera) in western Sweden
(English)Manuscript (preprint) (Other academic)
Abstract [en]

 Here, we report on the investigation of newly discovered gregarine lineages found infecting western Swedish populations of the freshwater mussel, M. margaritifera. This protist lineage, belonging to one of the terrestrial groups of gregarines (Eugregarinorida, Apicomplexa) was previously detected in a population of freshwater mussels that had been selected as control group in a previous study of mussel mortality events (MME). The MME had decreased the population of freshwater pearl mussels (FPM) in its sister population, by more than 60% within the same water catchment area. Even though no mortalities were reported from this control population, the population were not showing signs of ongoing recruitment, even if a there was trout population that could support a successful recruitment and distribution of FPM larvae. Our investigation indicated pathologies within the gonads and that these changes were associated to at least one type of gregarine infection, previously reported as lineage C from this creek. We describe the parasitic gregarine cell morphologies within gonads, foot and digestive gland and used CARD-FISH to link Lineage C sequence to protist cells and lesion associated in the host. This provides a promising method to infer disease associated lesions with environmental sequences.

Keywords
Margaritifera margaritifera, Freshwater Pearl Mussel, Gregarine, reproductive disorder, vertical transmission, phylogeny, pathology, CARD-FISH
National Category
Natural Sciences
Research subject
Biology with specialization in Systematics
Identifiers
urn:nbn:se:uu:diva-538791 (URN)
Funder
Swedish Research Council Formas, 2017-01197
Available from: 2024-09-20 Created: 2024-09-20 Last updated: 2024-09-20
4. First report of Hexamita nelsoni in blue mussel (Mytilus edulis): morphology, phylogeny, and host-parasite interaction
Open this publication in new window or tab >>First report of Hexamita nelsoni in blue mussel (Mytilus edulis): morphology, phylogeny, and host-parasite interaction
(English)Manuscript (preprint) (Other academic)
Abstract [en]

 Bivalve diseases caused by protozoan parasitic infection are monitored by coordinated surveillance programs across European member countries. These routine monitorings of bivalve health, however, only survey a few relatively well known parasites, which may leave undetected a range of lesser known opportunistic protozoan agents. Under increased stress, for example due to shifting environmental conditions affecting water quality and nutrient access, these opportunistic parasites may develop pathogenicity impacting reproduction or growth and therefore decreasing the success and future development of aquaculture or wild population sustainability. During routine histopathological surveillance of blue mussels in Sweden, we noticed cryptic lesions in the adductor muscle in high prevalence over a period ranging from 2017 to 2022. These lesions suggested cell infiltration and degenerative changes in the muscle tissues. In this study, we report on the isolation, identification, and culture of the protistan cells likely responsible for at least some of these lesions. Using histology and cytology, molecular phylogeny, and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), we show that the cells correspond to the H. nelsoni sequence initially isolated from oysters. We also reveal the presence of H. nelsoni in the digestive glands and mantel, and that this parasite might be horizontally transmitted from adult to larvae via infections within gonads, ovaries. This study represents the first report of H. nelsoni in blue mussel.

Keywords
Mytilus edulis, parasite, protists, Diplomonada, Hexamita nelsoni, adductor muscle, ovarie, digestive gland, mantel, histology, CARD-FISH, phylogeny
National Category
Natural Sciences
Research subject
Biology with specialization in Systematics
Identifiers
urn:nbn:se:uu:diva-538803 (URN)
Funder
Swedish Research Council Formas, 2017-01197
Available from: 2024-09-20 Created: 2024-09-20 Last updated: 2024-09-20
5. Comparative genomics of Ascetosporea gives new insight into the evolutionary basis for animal parasitism in Rhizaria
Open this publication in new window or tab >>Comparative genomics of Ascetosporea gives new insight into the evolutionary basis for animal parasitism in Rhizaria
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2024 (English)In: BMC Biology, E-ISSN 1741-7007, Vol. 22, no 1, article id 103Article in journal (Refereed) Published
Abstract [en]

Background

Ascetosporea (Endomyxa, Rhizaria) is a group of unicellular parasites infecting aquatic invertebrates. They are increasingly being recognized as widespread and important in marine environments, causing large annual losses in invertebrate aquaculture. Despite their importance, little molecular data of Ascetosporea exist, with only two genome assemblies published to date. Accordingly, the evolutionary origin of these parasites is unclear, including their phylogenetic position and the genomic adaptations that accompanied the transition from a free-living lifestyle to parasitism. Here, we sequenced and assembled three new ascetosporean genomes, as well as the genome of a closely related amphizoic species, to investigate the phylogeny, origin, and genomic adaptations to parasitism in Ascetosporea.

Results

Using a phylogenomic approach, we confirm the monophyly of Ascetosporea and show that Paramyxida group with Mikrocytida, with Haplosporida being sister to both groups. We report that the genomes of these parasites are relatively small (12–36 Mb) and gene-sparse (~ 2300–5200 genes), while containing surprisingly high amounts of non-coding sequence (~ 70–90% of the genomes). Performing gene-tree aware ancestral reconstruction of gene families, we demonstrate extensive gene losses at the origin of parasitism in Ascetosporea, primarily of metabolic functions, and little gene gain except on terminal branches. Finally, we highlight some functional gene classes that have undergone expansions during evolution of the group.

Conclusions

We present important new genomic information from a lineage of enigmatic but important parasites of invertebrates and illuminate some of the genomic innovations accompanying the evolutionary transition to parasitism in this lineage. Our results and data provide a genetic basis for the development of control measures against these parasites.

Place, publisher, year, edition, pages
BioMed Central (BMC), 2024
Keywords
Genome reduction, Reductive evolution, Evolutionary transition, Phylogeny, Protozoa, Intracellular parasite, Bonamia, Marteilia, Paramarteilia, Mikrocytos, Paramikrocytos
National Category
Evolutionary Biology Genetics
Identifiers
urn:nbn:se:uu:diva-538616 (URN)10.1186/s12915-024-01898-x (DOI)001214191700002 ()38702750 (PubMedID)
Funder
Swedish Research Council, 2017-04563Science for Life Laboratory, SciLifeLabSwedish Research Council Formas, 2017-01197Stockholm University
Available from: 2024-09-18 Created: 2024-09-18 Last updated: 2024-09-18Bibliographically approved

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