Comparing and Informing Morphological Species Identifications and Boundaries in Arthropod Gut-dwelling Protists Using Molecular Phylogenetic Analyses

Trichomycetes are fungal and protistan symbionts of arthropods and have been found in marine, freshwater, and terrestrial habitats on every continent except Antarctica. Minimally, trichomycetes associate commensally with their immature aquatic hosts (including black flies, mayflies, stoneflies, isopods, and others) attaching to the chitinous lining of the mid or hindgut. Classified with trichomycetes are Paramoebidium, a group of protists which associate in an obligate manner with their hosts. To date, 16 species of Paramoebidium have been identified by morphological approaches. These descriptions have included characterization and discrimination of species based on thallus (body) length and width, differences in the holdfast (structure used to attach to the gut of the host), and the appearance of the asexual, motile amoebae they release. Several challenges arise from this method. Namely, Paramoebidium species can be unremarkable in appearance. Immature Paramoebidium can have large variation, and finding a specimen in the process of releasing amoebae is very difficult as live organisms must be dissected directly from host tissue. We have been using molecular and phylogenetic approaches (sequencing rDNA genes) to assess morpho-species boundaries and their potential to differentiate and infer evolutionary relationships among Paramoebidium species. To date, efforts to compare recent molecular findings to morphological data have not been done. We plan to perform a morphological comparison of specimens used in the molecular data to highlight the pros and cons of the current classification system for Paramoebidium

Assessing the Potential Effects of Fungicides on Nontarget Gut Fungi (Trichomycetes) and Their Associated Larval Black Fly Hosts

Fungicides are moderately hydrophobic and have been detected in water and sediment, particularly in agricultural watersheds, but typically are not included in routine water quality monitoring efforts. This is despite their widespread use and frequent application to combat fungal pathogens. Although the efficacy of these compounds on fungal pathogens is well documented, little is known about their effects on nontarget fungi. This pilot study, a field survey in southwestern Idaho from April to December 2010 on four streams with varying pesticide inputs (two agricultural and two reference sites), was conducted to assess nontarget impact of fungicides on gut fungi, or trichomycetes. Tissues of larval black flies (Diptera: Simuliidae), hosts of gut fungi, were analyzed for pesticide accumulation. Fungicides were detected in hosts from streams within agricultural watersheds but were not detected in hosts from reference streams. Gut fungi from agricultural sites exhibited decreased percent infestation, density and sporulation within the gut, and black fly tissues had elevated pesticide concentrations. Differences observed between the sites demonstrate a potential effect on this symbiotic system. Future research is needed to parse out the details of the complex biotic and abiotic relationships; however, these preliminary results indicate that impacts to nontarget organisms could have far-reaching consequences within aquatic ecosystems

A Zebrafish Model of Roberts Syndrome Reveals That Esco2 Depletion Interferes with Development by Disrupting the Cell Cycle

The human developmental diseases Cornelia de Lange Syndrome (CdLS) and Roberts Syndrome (RBS) are both caused by mutations in proteins responsible for sister chromatid cohesion. Cohesion is mediated by a multi-subunit complex called cohesin, which is loaded onto chromosomes by NIPBL. Once on chromosomes, cohesin binding is stabilized in S phase upon acetylation by ESCO2. CdLS is caused by heterozygous mutations in NIPBL or cohesin subunits SMC1A and SMC3, and RBS is caused by homozygous mutations in ESCO2. The genetic cause of both CdLS and RBS reside within the chromosome cohesion apparatus, and therefore they are collectively known as “cohesinopathies”. However, the two syndromes have distinct phenotypes, with differences not explained by their shared ontology. In this study, we have used the zebrafish model to distinguish between developmental pathways downstream of cohesin itself, or its acetylase ESCO2. Esco2 depleted zebrafish embryos exhibit features that resemble RBS, including mitotic defects, craniofacial abnormalities and limb truncations. A microarray analysis of Esco2-depleted embryos revealed that different subsets of genes are regulated downstream of Esco2 when compared with cohesin subunit Rad21. Genes downstream of Rad21 showed significant enrichment for transcriptional regulators, while Esco2-regulated genes were more likely to be involved the cell cycle or apoptosis. RNA in situ hybridization showed that runx1, which is spatiotemporally regulated by cohesin, is expressed normally in Esco2-depleted embryos. Furthermore, myca, which is downregulated in rad21 mutants, is upregulated in Esco2-depleted embryos. High levels of cell death contributed to the morphology of Esco2-depleted embryos without affecting specific developmental pathways. We propose that cell proliferation defects and apoptosis could be the primary cause of the features of RBS. Our results show that mutations in different elements of the cohesion apparatus have distinct developmental outcomes, and provide insight into why CdLS and RBS are distinct diseases

Mapping Millipedes and Their Microscopic Milieu Elevates Excitement for Eccrinales Endobiont Extremes by Tracking Trichomycete Traits Across Scattered States

Trichomycetes is an ecological group of microorganisms, with both fungi and protist members, associated with the guts of arthropods. It was a former class of Fungi, including four orders, with one, the Eccrinales, now recognized as a part of a smaller clade called Mesomycetozoea. Upon dissecting digestive tracts of infested hosts under a stereomicroscope, “eccrinids” appear as unbranched, hair-like thalli attached to the gut lining by a cement-excreting holdfast. Methodologically, gut linings and any attached thalli are wet mounted on microscope slides, imaged live and/or stained and preserved as vouchers. Qualitative and quantitative aspects (measurements of informative characters such as thalli, holdfasts, and spores) are the backbone of traditional morphotaxonomy and species identification. Ecologically and in terms of life history, asexual spores, the sporangiospores, are released and either germinate in the same gut or are excreted to the external environment. There they remain until ingested by the same or another individual, with varied infestation rates depending on the population and niche. With a tendency for some degree of host specificity, one common host group of “eccrinids” is Diplopoda, or millipedes. Candidate hosts were collected from sites across the continental USA as part of a preliminary survey, presented herein

Local Efforts to Expand the World’s Largest Trichomycete Collection

Trichomycetes (gut fungi) are obligate symbionts of various arthropods and have been found in marine, freshwater, and terrestrial habitats on every continent except Antarctica. Gut fungi associate commensally with their immature aquatic hosts (including black flies, mayflies, stoneflies, isopods, and others) attaching to the chitinous lining of the mid- or hindgut. Gut fungi are routinely isolated from the host’s digestive tract into axenic cultures, which provide a rich resource of genetic material for phylogenic analysis. Given the obligate endosymbiotic nature of the fungi, unique challenges are encountered upon culturing, thus only 30% of all trichomycetes have been successfully cultured. Our laboratory houses the world’s largest culture collection of trichomycetes. We have been endeavoring to expand the collection by formulating suitable media in which field collected trichomycete specimens can grow. This requires attention to variables such as competing bacterial growth, pH changes required for sporulation, agar density and proper nutrient balance within the media. The continued development and enhancement of culture media is intended to increase the number of isolates that previously were considered unculturable and offer more robust samples of genetic material for future analyses

New species and emendations of Orphella: taxonomic and phylogenetic reassessment of the genus to establish the Orphellales, for stonefly gut fungi with a twist

We consolidate and present data for the sexual stages of five North American species of Orphella, fungal members of trichomycetes previously classified within Harpellales. Three species emendations accommodate the newly recognized characters, including not only the coiled zygospores and accompanying cells but also other morphological traits not provided in the original descriptions for O. avalonensis, O. haysii, and O. hiemalis. We describe three new species, Orphella cataloochensis from both the Smoky Mountains in USA and two provinces in Canada as well as O. pseudoavalonensis and O. pseudohiemalis, both from the Cascade Range, in Oregon, USA. Key morphological features for all known species are summarized and reviewed, with illustrations of some of the North American taxa to update and supplement the literature. The entire suite of morphological characters is discussed, with emphasis on species relationships and hypotheses on possible vicariant origins. We also present a molecular phylogeny based on nuc rDNA 18S and 28S, which supports Orphella as a lineage distinct from Harpellales, and we establish a new order, Orphellales, for it. With the combination of sexual features, now known for 12 of the 14 species of Orphella, and new molecular data, the group is now better characterized, facilitating and hopefully also promoting future studies toward a better understanding of their relationships, origins, and evolutionary history as stonefly gut-dwelling fungi.Fil: White, Merlin M.. Boise State University; Estados UnidosFil: Guàrdia Valle, Laia. Universitat Autònoma de Barcelona; EspañaFil: Lichtwardt, Robert W.. Kansas State University; Estados UnidosFil: Siri, Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Limnología "Dr. Raúl A. Ringuelet". Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología; ArgentinaFil: Strongman, Douglas. Saint Mary's University; CanadáFil: William, Robert T.. Saint Mary's University; CanadáFil: Gause, W. Justin. Boise State University; Estados UnidosFil: Tretter, Eric D.. Boise State University; Estados Unido

Resolving Relationships at the Animal-Fungal Divergence: A Molecular Phylogenetic Study of the Protist Trichomycetes (Ichthyosporea, Eccrinida)

Trichomycetes is a group of microorganisms that was considered a class of fungi comprising four orders of commensal, gut-dwelling endosymbionts obligately associated with arthropods. Since molecular phylogenies revealed two of those orders (Amoebidiales and Eccrinales = “protist trichos”) to be closely related to members of the protist class Ichthyosporea (=Mesomycetozoea), trichomycetes have been considered an ecological association of both early-diverging fungi and protists. Understanding of the taxonomy, evolution, and diversity of the protist trichos is lacking largely due to the difficulties inherent in species collection that have contributed to undersampling and understudy. The most recent classification divides the protist trichos between two families, Amoebidiidae and Eccrinidae (suborder Trichomycina, order Eccrinida). However, there is no comprehensive molecular phylogeny available for this group and major questions about the systematics of protist trichos remain unanswered. Therefore, we generated 18S and 28S rDNA sequences for 106 protist tricho samples and combined them with publicly available Eccrinida sequences for phylogenetic analyses. We also sequenced a conserved protein-coding gene (heat-shock 70 protein) to obtain a multigene data set. We conducted ancestral state reconstruction (ASR) and Bayesian tip-association significance test (BaTS) analyses by mapping six morphological and ecological characters onto the resulting phylogenetic trees. Our results demonstrate: 1) several ecological and morphological character states (habitat, host type, host stage at time of infestation, location within host, spore production, and growth form) are significantly correlated with the phylogeny, and 2) two additional protist tricho families should be incorporated into the taxonomy to reflect phylogenetic relationships. Our data suggest that an integrated strategy that combines morphological, ecological, and molecular characters is needed to further resolve and clarify the systematics of the Eccrinida

Association of cohesin and Nipped-B with transcriptionally active regions of the Drosophila melanogaster genome

The cohesin complex is a chromosomal component required for sister chromatid cohesion that is conserved from yeast to man. The similarly conserved Nipped-B protein is needed for cohesin to bind to chromosomes. In higher organisms, Nipped-B and cohesin regulate gene expression and development by unknown mechanisms. Using chromatin immunoprecipitation, we find that Nipped-B and cohesin bind to the same sites throughout the entire non-repetitive Drosophila genome. They preferentially bind transcribed regions and overlap with RNA polymerase II. This contrasts sharply with yeast, where cohesin only binds between genes. Differences in cohesin and Nipped-B binding between Drosophila cell lines often correlate with differences in gene expression. For example, cohesin and Nipped-B bind the Abd-B homeobox gene in cells in which it is transcribed, but not in cells in which it is silenced. They bind to the Abd-B transcription unit and downstream regulatory region, and thus could regulate both transcriptional elongation and activation. We posit that transcription facilitates cohesin binding, perhaps by unfolding chromatin, and that Nipped-B then regulates gene expression by controlling cohesin dynamics. These mechanisms are likely involved in the etiology of Cornelia de Lange syndrome, in which mutation of one copy of the NIPBL gene encoding the human Nipped-B ortholog causes diverse structural and mental birth defects