First Genome of Labyrinthula sp., an Opportunistic Seagrass Pathogen, Reveals Novel Insight into Marine Protist Phylogeny, Ecology and CAZyme Cell-Wall Degradation.

Labyrinthula spp. are saprobic, marine protists that also act as opportunistic pathogens and are the causative agents of seagrass wasting disease (SWD). Despite the threat of local- and large-scale SWD outbreaks, there are currently gaps in our understanding of the drivers of SWD, particularly surrounding Labyrinthula spp. virulence and ecology. Given these uncertainties, we investigated the Labyrinthula genus from a novel genomic perspective by presenting the first draft genome and predicted proteome of a pathogenic isolate Labyrinthula SR_Ha_C, generated from a hybrid assembly of Nanopore and Illumina sequences. Phylogenetic and cross-phyla comparisons revealed insights into the evolutionary history of Stramenopiles. Genome annotation showed evidence of glideosome-type machinery and an apicoplast protein typically found in protist pathogens and parasites. Proteins involved in Labyrinthula SR_Ha_C's actin-myosin mode of transport, as well as carbohydrate degradation were also prevalent. Further, CAZyme functional predictions revealed a repertoire of enzymes involved in breakdown of cell-wall and carbohydrate storage compounds common to seagrasses. The relatively low number of CAZymes annotated from the genome of Labyrinthula SR_Ha_C compared to other Labyrinthulea species may reflect the conservative annotation parameters, a specialized substrate affinity and the scarcity of characterized protist enzymes. Inherently, there is high probability for finding both unique and novel enzymes from Labyrinthula spp. This study provides resources for further exploration of Labyrinthula spp. ecology and evolution, and will hopefully be the catalyst for new hypothesis-driven SWD research revealing more details of molecular interactions between the Labyrinthula genus and its host substrate.

Cryopreservation methods are effective for long-term storage of Labyrinthula cultures.

Marine heterotrophic protists of the Labyrinthulomycota are of interest for their biotechnological (e.g. thraustochytrid production of lipids) and ecological (e.g. wasting disease and rapid blight by pathogens of the genus Labyrinthula) applications; culture-based laboratory studies are a central technique of this research. However, maintaining such microorganism cultures can be labour- and cost-intensive, with a high risk of culture contamination and die-off over time. Deep-freeze storage, or cryopreservation, can be used to maintain culture back-ups, as well as to preserve the genetic and phenotypic properties of the microorganisms; however, this method has not been tested for the ubiquitous marine protists Labyrinthula spp. In this study, we trialled 12 cryopreservation protocols on 3 Labyrinthula sp. isolates of varying colony morphological traits. After 6 mo at -80°C storage, the DMSO and glycerol protocols were the most effective cryoprotectants compared to methanol (up to 90% success vs. 50% success, respectively). The addition of 30% horse serum to the cryoprotectant solution increased Labyrinthula sp. growth success by 20-30%. We expect that these protocols will provide extra security for culture-based studies, as well as opportunities for long-term research on key Labyrinthula sp. isolates.

The First Isolation and Characterisation of the Protist Labyrinthula sp. in Southeastern Australia.

As a result of anthropogenic influences and global climate change, emerging infectious marine diseases are thought to be increasingly more common and more severe than in the past. The aim of our investigation was to confirm the presence of Labyrinthula, the aetiological agent of the seagrass wasting disease, in Southeastern Australia and provide the first isolation and characterisation of this protist, in Australia. Colonies and individual cells were positively identified as Labyrinthula using published descriptions, diagrams, and photographs. Their identity was then confirmed using DNA barcoding of a region of the 18S rRNA gene. Species level identification of isolates was not possible as the taxonomy of the Labyrinthula is still poorly resolved. Still, a diversity of Labyrinthula was isolated from small sections of the southeast coast of Australia. The isolates were grouped into three haplotypes that are biogeographically restricted. These haplotypes are closely related to previously identified saprotrophic clades. The study highlights the need for further investigation into the global distribution of Labyrinthula, including phylogenetic pathogenicity and analysis of host-parasite interactions in response to stressors. Given the results of our analyses, it is prudent to continue research into disease and epidemic agents to better prepare researchers for potential future outbreaks.

Microbial players involved in the decline of filamentous and colonial cyanobacterial blooms with a focus on fungal parasitism.

In the forthcoming decades, it is widely believed that the dominance of colonial and filamentous bloom-forming cyanobacteria (e.g. Microcystis, Planktothrix, Anabaena and Cylindrospermopsis) will increase in freshwater systems as a combined result of anthropogenic nutrient input into freshwater bodies and climate change. While the physicochemical parameters controlling bloom dynamics are well known, the role of biotic factors remains comparatively poorly studied. Morphology and toxicity often - but not always - limit the availability of cyanobacteria to filter feeding zooplankton (e.g. cladocerans). Filamentous and colonial cyanobacteria are widely regarded as trophic dead-ends mostly inedible for zooplankton, but substantial evidence shows that some grazers (e.g. copepods) can bypass this size constraint by breaking down filaments, making the bloom biomass available to other zooplankton species. A wide range of algicidal bacteria (mostly from the Alcaligenes, Flavobacterium/Cytophaga group and Pseudomonas) and viruses (Podoviridae, Siphoviridae and Myoviridae) may also contribute to bloom control, via their lytic activity underpinned by a diverse array of mechanisms. Fungal parasitism by the Chytridiomycota remains the least studied. While each of these biotic factors has traditionally been studied in isolation, emerging research consistently point to complex interwoven interactions between biotic and environmental factors.

Resource seeking strategies of zoosporic true fungi in heterogeneous soil habitats at the microscale level.

Zoosporic true fungi have frequently been identified in samples from soil and freshwater ecosystems using baiting and molecular techniques. In fact some species can be components of the dominant groups of microorganisms in particular soil habitats. Yet these microorganisms have not yet been directly observed growing in soil ecosystems. Significant physical characteristics and features of the three-dimensional structures of soils which impact microorganisms at the microscale level are discussed. A thorough knowledge of soil structures is important for studying the distribution of assemblages of these fungi and understanding their ecological roles along spatial and temporal gradients. A number of specific adaptations and resource seeking strategies possibly give these fungi advantages over other groups of microorganisms in soil ecosystems. These include chemotactic zoospores, mechanisms for adhesion to substrates, rhizoids which can penetrate substrates in small spaces, structures which are resistant to environmental extremes, rapid growth rates and simple nutritional requirements. These adaptations are discussed in the context of the characteristics of soils ecosystems. Recent advances in instrumentation have led to the development of new and more precise methods for studying microorganisms in three-dimensional space. New molecular techniques have made identification of microbes possible in environmental samples.

Ecological roles of the parasitic phytomyxids (plasmodiophorids) in marine ecosystems - a review.

Phytomyxea (plasmodiophorids) is an enigmatic group of obligate biotrophic parasites. Most of the known 41 species are associated with terrestrial and freshwater ecosystems. However, the potential of phytomyxean species to influence marine ecosystems either directly by causing diseases of their hosts or indirectly as vectors of viruses is enormous, although still unexplored. In all, 20% of the currently described phytomyxean species are parasites of some of the key primary producers in the ocean, such as seagrasses, brown algae and diatoms; however, information on their distribution, abundance and biodiversity is either incomplete or lacking. Phytomyxean species influence fitness by altering the metabolism and/or the reproductive success of their hosts. The resulting changes can (1) have an impact on the biodiversity within host populations, and (2) influence microbial food webs because of altered availability of nutrients (e.g. changed metabolic status of host, transfer of organic matter). Also, phytomyxean species may affect their host populations indirectly by transmitting viruses. The majority of the currently known single-stranded RNA marine viruses structurally resemble the viruses transmitted by phytomyxean species to crops in agricultural environments. Here, we explore possible ecological roles of these parasites in marine habitats; however, only the inclusion of Phytomyxea in marine biodiversity studies will allow estimation of the true impact of these species on global primary production in the oceans.

Can zoosporic true fungi grow or survive in extreme or stressful environments?

Zoosporic true fungi are thought to be ubiquitous in many ecosystems, especially in cool, moist soils and freshwater habitats which are rich in organic matter. However, some of the habitats where these fungi are found may periodically experience extreme conditions, such as soils in extremely dry, hot and cold climates, acidic and alkaline soils, polluted rivers, anaerobic soil and water, saline soil and water, periglacial soils, oligotrophic soils, tree canopies and hydrothermal vents. It is clear that many ecotypes of zoosporic true fungi have indeed adapted to extreme or stressful environmental conditions. This conclusion is supported by studies in both the field and in the laboratory. Therefore, in our opinion, at least some true zoosporic fungi can be considered to be extremophiles.

Newly emerging diseases of marine turtles, especially sea turtle egg fusariosis (SEFT), caused by species in the Fusarium solani complex (FSSC).

Sea turtles are presently considered severely endangered species that are historically threatened by many environmental factors. Recently, additional threats to sea turtles from two pathogenic species of fungi in the Fusarium solani species complex (F. falciforme and F. keratoplasticum) have been identified. These species infect marine turtle eggs, causing sea turtle egg fusariosis, and kill their embryos, with recent reports of hatch-failure in seven globally distributed species of endangered sea turtles (Caretta caretta, Chelonia mydas, Dermochelys coriaceae, Eretmochelys imbricata, Lepidochelys olivacea, Lepidochelys kempi and Natator depressus). Mycelia and spores of pathogenic species of Fusarium are produced in disturbed terrestrial soils and are transported to the ocean in coastal run off. We propose that these fungi grow on floating particles of plant tissues (leaves and wood), animal tissues, silt and plastics, which are carried by wind and currents and the turtles themselves to the beaches where the turtles lay their eggs.

Recovery of growth of Hyphochytrium catenoides after exposure to environmental stress.

The survival of an isolate of Hyphochytrium catenoides collected from soil in the Blue Mountains in eastern New South Wales, Australia, was tested under extreme conditions in the laboratory. This isolate recovered growth after being subjected to drying on filter paper, to heat while desiccated, to hypersalinity, to strict anaerobic conditions, to freezing temperatures, and to a short period in solutions at pH 2.8-11.2. The capacity to survive under these conditions in the laboratory suggests adaptation to fluctuating conditions in the soil. The partial DNA sequence of the 28S ribosomal RNA gene in the isolate from New South Wales was 98% similar to that in an isolate from Arizona with a similar morphology.

Pathogenic Labyrinthula associated with Australian seagrasses: Considerations for seagrass wasting disease in the southern hemisphere.

Marine disease ecology is a growing field of research, particularly for host organisms negatively impacted by a changing climate and anthropogenic activities. A decrease in health and increase in susceptibility to disease has been hypothesised as the mechanism behind wide-spread seagrass die-offs related to wasting disease in the past. However, seagrass wasting disease and the causative pathogen, Labyrinthula, have been vastly understudied in the southern hemisphere. Our aim was to build on the current knowledge of Australian Labyrinthula descriptions and phylogeny, while also providing a first look at wasting disease ecology in Australia. Five seagrass species along a 750km stretch of coastline in southeastern Australia were sampled. The resulting 38 Labyrinthula isolates represented a diversity of morphotypes and five haplotypes of varying phylogenetic clade positions and virulence. The haplotypes clustered with previously-described phylogenetic clades containing isolates from Asia, USA and Europe. Pathogenicity tests confirmed, for the first time, the presence of at least two pathogenic haplotypes in Australia. While historically there have been no reports of wasting disease-related seagrass habitat loss, the presence of pathogenic Labyrinthula highlights the need for disease monitoring and research to understand seagrass wasting disease ecology in Australia.

The roles of endolithic fungi in bioerosion and disease in marine ecosystems. II. Potential facultatively parasitic anamorphic ascomycetes can cause disease in corals and molluscs.

Anamorphic ascomycetes have been implicated as causative agents of diseases in tissues and skeletons of hard corals, in tissues of soft corals (sea fans) and in tissues and shells of molluscs. Opportunist marine fungal pathogens, such as Aspergillus sydowii, are important components of marine mycoplankton and are ubiquitous in the open oceans, intertidal zones and marine sediments. These fungi can cause infection in or at least can be associated with animals which live in these ecosystems. A. sydowii can produce toxins which inhibit photosynthesis in and the growth of coral zooxanthellae. The prevalence of many documented infections has increased in frequency and severity in recent decades with the changing impacts of physical and chemical factors, such as temperature, acidity and eutrophication. Changes in these factors are thought to cause significant loss of biodiversity in marine ecosystems on a global scale in general, and especially in coral reefs and shallow bays.

The roles of endolithic fungi in bioerosion and disease in marine ecosystems. I. General concepts.

Endolithic true fungi and fungus-like microorganisms penetrate calcareous substrates formed by living organisms, cause significant bioerosion and are involved in diseases of many host animals in marine ecosystems. A theoretical interactive model for the ecology of reef-building corals is proposed in this review. This model includes five principle partners that exist in a dynamic equilibrium: polyps of a colonial coelenterate, endosymbiotic zooxanthellae, endolithic algae (that penetrate coral skeletons), endolithic fungi (that attack the endolithic algae, the zooxanthellae and the polyps) and prokaryotic and eukaryotic microorganisms (which live in the coral mucus). Endolithic fungi and fungus-like boring microorganisms are important components of the marine calcium carbonate cycle because they actively contribute to the biodegradation of shells of animals composed of calcium carbonate and calcareous geological substrates.

Key Ecological Roles for Zoosporic True Fungi in Aquatic Habitats.

The diversity and abundance of zoosporic true fungi have been analyzed recently using fungal sequence libraries and advances in molecular methods, such as high-throughput sequencing. This review focuses on four evolutionary primitive true fungal phyla: the Aphelidea, Chytridiomycota, Neocallimastigomycota, and Rosellida (Cryptomycota), most species of which are not polycentric or mycelial (filamentous), rather they tend to be primarily monocentric (unicellular). Zoosporic fungi appear to be both abundant and diverse in many aquatic habitats around the world, with abundance often exceeding other fungal phyla in these habitats, and numerous novel genetic sequences identified. Zoosporic fungi are able to survive extreme conditions, such as high and extremely low pH; however, more work remains to be done. They appear to have important ecological roles as saprobes in decomposition of particulate organic substrates, pollen, plant litter, and dead animals; as parasites of zooplankton and algae; as parasites of vertebrate animals (such as frogs); and as symbionts in the digestive tracts of mammals. Some chytrids cause economically important diseases of plants and animals. They regulate sizes of phytoplankton populations. Further metagenomics surveys of aquatic ecosystems are expected to enlarge our knowledge of the diversity of true zoosporic fungi. Coupled with studies on their functional ecology, we are moving closer to unraveling the role of zoosporic fungi in carbon cycling and the impact of climate change on zoosporic fungal populations.

Zoosporic parasites infecting marine diatoms - A black box that needs to be opened.

Living organisms in aquatic ecosystems are almost constantly confronted by pathogens. Nevertheless, very little is known about diseases of marine diatoms, the main primary producers of the oceans. Only a few examples of marine diatoms infected by zoosporic parasites are published, yet these studies suggest that diseases may have significant impacts on the ecology of individual diatom hosts and the composition of communities at both the producer and consumer trophic levels of food webs. Here we summarize available ecological and morphological data on chytrids, aphelids, stramenopiles (including oomycetes, labyrinthuloids, and hyphochytrids), parasitic dinoflagellates, cercozoans and phytomyxids, all of which are known zoosporic parasites of marine diatoms. Difficulties in identification of host and pathogen species and possible effects of environmental parameters on the prevalence of zoosporic parasites are discussed. Based on published data, we conclude that zoosporic parasites are much more abundant in marine ecosystems than the available literature reports, and that, at present, both the diversity and the prevalence of such pathogens are underestimated.

Copper (II) lead (II), and zinc (II) reduce growth and zoospore release in four zoosporic true fungi from soils of NSW, Australia.

This study examined the responses of a group of four zoosporic true fungi isolated from soils in NSW Australia, to concentrations of toxic metals in the laboratory that may be found in polluted soils. All isolates showed greatest sensitivity to Cu and least sensitivity to Pb. All isolates showed significant reduction in growth at 60 ppm (0.94 mmol m(-3)) for Cu, while three declined significantly at 60 ppm (0.92 mmol m(-3)) Zn. The growth of two isolates declined significantly at 100 ppm (0.48 mmol m(-3)) Pb and one at 200 ppm (0.96 mmol m(-3)) Pb. The rate of production of zoospores for all isolates was reduced when sporangia were grown in solid PYG media with 60 ppm Cu. Three isolates significantly declined in production at 60 ppm Zn and three at 100 ppm Pb. All isolates recovered growth after incubation in solid media with 60 ppm Zn or 100 ppm Pb. Two isolates did not recover growth after incubation in 60 ppm Cu. If these metals cause similar effects in the field, Cu, Pb, and Zn contamination of NSW soils is likely to reduce biomass of zoosporic true fungi. Loss of the fungi may reduce the rate of mineralisation of soil organic matter.

Current ecological understanding of fungal-like pathogens of fish: what lies beneath?

Despite increasingly sophisticated microbiological techniques, and long after the first discovery of microbes, basic knowledge is still lacking to fully appreciate the ecological importance of microbial parasites in fish. This is likely due to the nature of their habitats as many species of fish suffer from living beneath turbid water away from easy recording. However, fishes represent key ecosystem services for millions of people around the world and the absence of a functional ecological understanding of viruses, prokaryotes, and small eukaryotes in the maintenance of fish populations and of their diversity represents an inherent barrier to aquatic conservation and food security. Among recent emerging infectious diseases responsible for severe population declines in plant and animal taxa, fungal and fungal-like microbes have emerged as significant contributors. Here, we review the current knowledge gaps of fungal and fungal-like parasites and pathogens in fish and put them into an ecological perspective with direct implications for the monitoring of fungal fish pathogens in the wild, their phylogeography as well as their associated ecological impact on fish populations. With increasing fish movement around the world for farming, releases into the wild for sport fishing and human-driven habitat changes, it is expected, along with improved environmental monitoring of fungal and fungal-like infections, that the full extent of the impact of these pathogens on wild fish populations will soon emerge as a major threat to freshwater biodiversity.

Morphology, phylogeny, and ecology of the aphelids (Aphelidea, Opisthokonta) and proposal for the new superphylum Opisthosporidia.

The aphelids are a small group of intracellular parasitoids of common species of eukaryotic phytoplankton with three known genera Aphelidium, Amoeboaphelidium, and Pseudaphelidium, and 10 valid species, which form along with related environmental sequences a very diversified group. The phyla Microsporidia and Cryptomycota, and the class Aphelidea have recently been considered to be a deep branch of the Holomycota lineage forming the so called the ARM-clade which is sister to the fungi. In this review we reorganize the taxonomy of ARM-clade, and establish a new superphylum the Opisthosporidia with three phyla: Aphelida phyl. nov., Cryptomycota and Microsporidia. We discuss here all aspects of aphelid investigations: history of our knowledge, life cycle peculiarities, the morphology (including the ultrastructure), molecular phylogeny, ecology, and provide a taxonomic revision of the phylum supplied with a list of species. We compare the aphelids with their nearest relatives, the species of Rozella, and improve the diagnosis of the phylum Cryptomycota.

Ecological functions of zoosporic hyperparasites.

Zoosporic parasites have received increased attention during the last years, but it is still largely unnoted that these parasites can themselves be infected by hyperparasites. Some members of the Chytridiomycota, Blastocladiomycota, Cryptomycota, Hyphochytriomycota, Labyrinthulomycota, Oomycota, and Phytomyxea are hyperparasites of zoosporic hosts. Because of sometimes complex tripartite interactions between hyperparasite, their parasite-host, and the primary host, hyperparasites can be difficult to detect and monitor. Some of these hyperparasites use similar mechanisms as their parasite-hosts to find and infect their target and to access food resources. The life cycle of zoosporic hyperparasites is usually shorter than the life cycle of their hosts, so hyperparasites may accelerate the turnaround times of nutrients within the ecosystem. Hyperparasites may increase the complexity of food webs and play significant roles in regulating population sizes and population dynamics of their hosts. We suggest that hyperparasites lengthen food chains but can also play a role in conducting or suppressing diseases of animals, plants, or algae. Hyperparasites can significantly impact ecosystems in various ways, therefore it is important to increase our understanding about these cryptic and diverse organisms.

Three dimensional quantification of biological samples using micro-computer aided tomography (microCT).

MicroCT is increasingly being used to observe soft animal and plant tissues. Conventional electron and light microscope staining protocols used to enhance the contrast of soft tissues have the potential to be adapted for use in microCT. This would increase the versatility of the microCT beyond improving qualitative observations to facilitating quantitative analysis of soft tissues. This paper describes the development of a culture system and staining protocol which has successfully been used to obtain three dimensional (3-D) quantitative data of filamentous and zoosporic soil fungi. The fungi were grown in an artificial matrix that was developed to simulate the particulate nature of soil. The combination of high contrast staining protocol and use of an X-ray translucent matrix allowed for 3-D qualitative and quantitative analysis of fungal growth. A salient point raised by this study is that the effectiveness of a protocol is reliant on the tissue or cell culture system which includes the composition of the sample, the sampling vessel, the depth of a sample and the combination of stains used. The potential use of this method extends to other fields where distribution and growth patterns in 3-D need to be quantified.