Ribosomal RNA phylogeny of bodonid and diplonemid flagellates and the evolution of euglenozoa

Euglenozoa is a major phylum of excavate protozoa (comprising euglenoids, kinetoplastids, and diplonemids) with highly unusual nuclear, mitochondrial, and chloroplast genomes. To improve understanding of euglenozoan evolution, we sequenced nuclear small-subunit rRNA genes from 34 bodonids (Bodo, Neobodo, Parabodo, Dimastigella-like, Rhynchobodo, Rhynchomonas, and unidentified strains), nine diplonemids (Diplonema, Rhynchopus), and a euglenoid (Entosiphon). Phylogenetic analysis reveals that diplonemids and bodonids are more diverse than previously recognised, but does not clearly establish the branching order of kinetoplastids, euglenoids, and diplonemids. Rhynchopus is holophyletic; parasitic species arose from within free-living species. Kinetoplastea (bodonids and trypanosomatids) are robustly holophyletic and comprise a major clade including all trypanosomatids and most bodonids ('core bodonids') and a very divergent minor one including Ichthyobodo. The root of the major kinetoplastid clade is probably between trypanosomatids and core bodonids. Core bodonids have three distinct subclades. Clade 1 has two distinct Rhynchobodo-like lineages; a lineage comprising Dimastigella and Rhynchomonas; and another including Cruzella and Neobodo. Clade 2 comprises Cryptobia/ Trypanoplasma, Procryptobia, and Parabodo. Clade 3 is an extensive Bodo saltans species complex. Neobodo designis is a vast genetically divergent species complex with mutually exclusive marine and freshwater subclades. Our analysis supports three phagotrophic euglenoid orders: Petalomonadida (holophyletic), Ploeotiida (probably holophyletic), Peranemida (paraphyletic).     

The evolutionary history of kinetoplastids and their kinetoplasts

Despite extensive phylogenetic analysis of small subunit ribosomal RNA (SSUrRNA) genes, the deep-level relationships among kinetoplastids remain poorly understood, limiting our grasp of their evolutionary history, especially the origins of their bizarre mitochondrial genome organizations. In this study we examine the SSUrRNA data in the light of a new marker--cytoplasmic heat shock protein 90 (hsp90) sequences. Our phylogenetic analyses divide kinetoplastids into four main clades. Clades 1-3 include the various bodonid kinetoplastids. Trypanosomatids comprise the fourth clade. SSUrRNA analyses give vastly different and poorly supported positions for the root of the kinetoplastid tree, depending on the out-group and analysis method. This is probably due to the extraordinary length of the branch between kinetoplastids and any out-group. In contrast, almost all hsp90 analyses place the root between clade 1 (including Dimastigella, Rhynchomonas, several Bodo spp., and probably Rhynchobodo) and all other kinetoplastids. Maximum likelihood and maximum likelihood distance analyses of hsp90 protein and second codon-position nucleotides place trypanosomatids adjacent to Bodo saltans and Bodo cf. uncinatus (clade 3), as (weakly) do SSUrRNA analyses. Hsp90 first codon- plus second codon-position nucleotide analyses return a slightly different topology. We show that this may be an artifact caused, in part, by the different evolutionary behavior of first- and second-codon positions. This study provides the most robust evidence to date that trypanosomatids are descended from within bodonids and that B. saltans is a close relative of trypanosomatids. A total reevaluation of the high-level systematics within kinetoplastids is needed. We confirm that the interlocking network organization of kinetoplast DNA seen in trypanosomatids is a derived condition within kinetoplastids but suggest that open-conformation minicircles may have arisen early in kinetoplastid evolution. Further understanding of the evolution of kinetoplast structure and RNA editing is hampered by a paucity of data from basal (i.e., clade 1) bodonids.     

Mitochondrial minicircles in the free-living bodonid Bodo saltans contain two gRNA gene cassettes and are not found in large networks

In trypanosomatids, the majority of the guide (g) RNAs that provide the information for U-insertion/deletion RNA editing are encoded by minicircles that are catenated into large networks. In contrast, in the distantly related cryptobiid Trypanoplasma borreli, gRNA genes appear to reside in large 180-kb noncatenated DNA circles. To shed light on the evolutionary history and function of the minicircle network, we have analyzed minicircle organization in the free-living bodonid Bodo saltans, which is more closely related to trypanosomatids than T. borreli. We identified 1.4-kb circular DNAs as the B. saltans equivalent of minicircles via sequence analysis of 4 complete minicircles, 14 minicircle fragments, and 14 gRNAs. We show that each minicircle harbors two gRNA gene cassettes of opposite polarity residing in variable regions of about 200 nt in otherwise highly conserved molecules. In the conserved region, B. saltans minicircles contain a putative bent helix sequence and a degenerate dodecamer motif (CSB-3). Electron microscopy, sedimentation, and gel electrophoresis analyses showed no evidence for the existence of large minicircle networks in B. saltans, the large majority of the minicircles being present as circular and linear monomers (85-90%) with small amounts of catenated dimers and trimers. Our results provide the first example of a kinetoplastid species with noncatenated, gRNA gene-containing minicircles, which implies that the creation of minicircles and minicircle networks are separate evolutionary events.     

Phylogenomic analysis of kinetoplastids supports that trypanosomatids arose from within bodonids

Kinetoplastids are a large group of free-living and parasitic eukaryotic flagellates, including the medically important trypanosomatids (e.g., Trypanosoma and Leishmania) and the widespread free-living and parasitic bodonids. Small subunit rRNA- and conserved protein-based phylogenies support the division of kinetoplastids into five orders (Prokinetoplastida, Neobodonida, Parabodonida, Eubodonida, and Trypanosomatida), but they produce incongruent results regarding their relative branching order, in particular for the position of the Trypanosomatida. In general, small subunit rRNA tends to support their early emergence, whereas protein phylogenies most often support a more recent origin from within bodonids. In order to resolve this question through a phylogenomic approach, we carried out massive parallel sequencing of cDNA from representatives of three bodonid orders (Bodo saltans -Eubodonida-, Procryptobia sorokini -Parabodonida-, and Rhynchomonas nasuta -Neobodonida-). We identified 64 well-conserved proteins shared by these species, four trypanosomatids, and two closely related outgroup species (Euglena gracilis and Diplonema papillatum). Phylogenetic analysis of a concatenated data set yielded a strongly supported tree showing the late emergence of trypanosomatids as a sister group of the Eubodonida. In addition, we identified homologues of proteins involved in trypanosomatid mitochondrial mRNA editing in the three bodonid species, suggesting that editing may be widespread in kinetoplastids. Comparison of expressed sequences from mitochondrial genes showed variability at U positions, in agreement with the existence of editing activity in the three bodonid orders most closely related to trypanosomatids (Neobodonida, Parabodonida, and Eubodonida). Mitochondrial mRNA editing appears to be an ancient phenomenon in kinetoplastids.     

Strains of the heterotrophic flagellate Bodo designis from different environments vary considerably with respect to salinity preference and SSU rRNA gene composition

The morpho species Bodo designis is widespread and abundant globally in highly contrasting terrestrial and aquatic ecosystems. Whether the forms of Bodo designis from contrasting environments are conspecific, i.e. largely genetically identical, or whether they merely share the external morphology is presently not known. We examined the ability of different strains of Bodo designis isolated from different environments at different geographical sites to survive and grow at a salinity range of 0.5-45%. The Bodo designis strains from marine, freshwater, and terrestrial environments showed a different ability to cope with altered physiological conditions. Most of the tested strains were only able to tolerate a small salinity range, whereas others were able to withstand all tested salinity levels. We further examined the phylogenetic relationship between the different strains by sequencing the small subunit (SSU) rRNA gene. The resulting phylogenetic analyses suggest a huge genetic variation within Bodo designis, and also imply that Dimastigella and Rhyncomonas are developed inside Bodo designis. If the biological species concept is used, the genetic differences as well as the physiological barriers between the different strains of Bodo designis, would suggest that they should be assigned to different species.     

Mixotrophic trade‐off under warming and UVR in a marine and a freshwater alga

Mixotrophic protists combine phagotrophy and phototrophy within a single cell. Greater phagotrophic activity could reinforce the bypass of carbon (C) flux through the bacteria‐mixotroph link and thus lead to a more efficient transfer of C and other nutrients to the top of the trophic web. Determining how foreseeable changes in temperature and UVR affect mixotrophic trade‐offs in favor of one or the other nutritional strategy, along the mixotrophic gradient, is key to understanding the fate of carbon and mineral nutrients in the aquatic ecosystem. Our two main hypotheses were: (i) that increased warming and UVR will divert metabolism toward phagotrophy, and (ii) that the magnitude of this shift will vary according to the organism's position along the mixotrophic gradient. To test these hypotheses, we used two protists (Isochrysis galbana and Chromulina sp.) located in different positions on the mixotrophic gradient, subjecting them to the action of temperature and of UVR and their interaction. Our results showed that the joint action of these two factors increased the primary production:bacterivory ratio and stoichiometric values (N:P ratio) close to Redfield's ratio. Therefore, temperature and UVR shifted the metabolism of both organisms toward greater phototrophy regardless of the original position of the organism on the mixotrophic gradient. Weaker phagotrophic activity could cause a less efficient transfer of C to the top of trophic webs.     

Molecular taxonomy of the suborder Bodonina (Order Kinetoplastida), including the important fish parasite,Ichthyobodo necator

Ichthyobodo necator is an important fish ectoparasite with a broad host and ecological range. A novel method, involving the use of an anesthetic, allowed the collection of large numbers of parasites from the skin and gills of hybrid striped bass (Morone saxatilis male x M. chrysops female). Genomic DNA from these samples was used to amplify and clone the 18S rRNA gene. The 18S rRNA gene was similarly cloned from Bodo caudatus, Bodo edax, Bodo saltans, an unidentified Bodo species, and Dimastigella trypaniformis. The resulting sequences were aligned with other representative kinetoplastid species using pileup and similarities in secondary structure. Phylogenetic relationships within the suborder Bodonina and representatives of the suborder Trypanosomatina were determined using maximum-likelihood statistics. The phylogenetic analyses strongly supported the order Kinetoplastida as a monophyletic assemblage consisting of at least two major lineages. One lineage consisted exclusively of L. necator, indicating that it may represent a new suborder. The second lineage consisted of all other kinetoplastid species. This second lineage appeared to contain at least 8 bodonine sublineages, none of which correlated with currently recognized families. For three sublineages, there was a close correspondence between the 18S phylogeny and the classical taxonomy of Dimastigella, Rhynchobodo, and Rhynchomonas. In contrast, Bodo and Cryptobia were polyphyletic, containing species in two or more sublineages that may represent separate genera.     

Food selection by bacterivorous protists: insight from the analysis of the food vacuole content by means of fluorescence in situ hybridization

A modified fluorescence in situ hybridization (FISH) method was used to analyze bacterial prey composition in protistan food vacuoles in both laboratory and natural populations. Under laboratory conditions, we exposed two bacterial strains (affiliated with beta- and gamma-Proteobacteria -- Aeromonas hydrophila and Pseudomonas fluorescens, respectively) to grazing by three protists: the flagellates Bodo saltans and Goniomonas sp., and the ciliate Cyclidium glaucoma. Both flagellate species preferably ingested A. hydrophila over P. fluorescens, while C. glaucoma showed no clear preferences. Differences were found in the digestion of bacterial prey with B. saltans digesting significantly faster P. fluorescens compared to two other protists. The field study was conducted in a reservoir as part of a larger experiment. We monitored changes in the bacterial prey composition available compared to the bacteria ingested in flagellate food vacuoles. Bacteria detected by probe HGC69a (Actinobacteria) and R-BT065 were negatively selected by flagellates. Bacteria detected by probe CF319a were initially positively selected but along with a temporal shift in bacterial cell size, this trend changed to negative selection during the experiment. Overall, our analysis of protistan food vacuole content indicated marked effects of flagellate prey selectivity on bacterioplankton community composition.     

Phagotrophic protists are a key component of microbial communities processing leaf litter under contrasting oxic conditions

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Marine invasions by non-sea snakes, with thoughts on terrestrial-aquatic-marine transitions

Few species of snakes show extensive adaptations to aquatic environments and even fewer exploit the oceans. A survey of morphology, lifestyles, and habitats of 2552 alethenophidian snakes revealed 362 (14%) that use aquatic environments, are semi-aquatic, or aquatic; about 70 (2.7%) of these are sea snakes (Hydrophiinae and Laticaudinae). The ancient and aquatic family Acrochordidae contains three extant species, all of which have populations inhabiting brackish or marine environments, as well as freshwater. The Homalopsidae have the most ecologically diverse representatives in coastal habitats. Other families containing species exploiting saline waters with populations in freshwater environments include: the Dipsadidae of the western hemisphere, the cosmopolitan Natricidae, the African Grayinae, and probably a few Colubridae. Species with aquatic and semi-aquatic lifestyles are compared with more terrestrial (fossorial, cryptozoic, and arboreal) species for morphological traits and life histories that are convergent with those found in sea snakes; this may provide clues to the evolution of marine snakes and increase our understanding of snake diversity.     

Freshwater foraminiferans revealed by analysis of environmental DNA samples

Sediment-dwelling protists are among the most abundant meiobenthic organisms, ubiquitous in all types of aquatic ecosystems. Yet, because their isolation and identification are difficult, their diversity remains largely unknown. In the present work, we applied molecular methods to examine the diversity of freshwater Foraminifera, a group of granuloreticulosan protists largely neglected until now. By using specific PCR primers, we detected the presence of Foraminifera in all sediment samples examined. Phylogenetic analysis of amplified SSU rDNA sequences revealed two distinct groups of freshwater foraminiferans. All obtained sequences branched within monothalamous (single-chambered), marine Foraminifera, suggesting a repeated colonization of freshwater environments. The results of our study challenge the traditional view of Foraminifera as essentially marine organisms, and provide a conceptual framework for charting the molecular diversity of freshwater granuloreticulosan protists.     

Mitochondrial and nuclear localization of topoisomerase II in the flagellate Bodo saltans (Kinetoplastida), a species with non-catenated kinetoplast DNA

We have studied topoisomerase II (topo II) in the cells of Bodo saltans, a free-living bodonid (Kinetoplastida). Phylogenetic analysis based on the sequence of the entire topo II gene, which is a single-copy gene, confirmed that B. saltans is a predecessor of parasitic trypanosomatids. Antibodies generated against either an overexpressed unique C-terminal region of topo II or a synthetic oligopeptide derived from the same region did not cross-react with cell lysates of related trypanosomatids, while they recognized a single specific band in the B. saltans lysate. Immunolocalization experiments using both antibodies showed that topo II is evenly dispersed throughout the kinetoplast. This is in striking difference from the localization of topo II in other flagellates, where it occurs in two antipodal centers flanking the kinetoplast disk. Moreover, the same topo II has a distinct localization in multiple loci at the periphery of the nucleus of B. saltans. With a minicircle probe derived from the conserved region we have shown that all relaxed non-catenated minicircles are confined to the globular kinetoplast DNA bundle. Therefore, in the mitochondrion of this primitive eukaryote topo II does not catenate relaxed DNA circles into a network in vivo, while a decatenating activity is present in partially purified cell lysates.     

Biological weighting functions for DNA damage in sea urchin embryos exposed to ultraviolet radiation

Laboratory experiments examining the effects of ultraviolet radiation (UVR, 290-400 nm) on DNA damage were carried out using the embryos of three species of sea urchins from different habitats; Strongylocentrotus droebachiensis from the Gulf of Maine, Sterechinus neumayeri from the Antarctic, and Evechinus chloroticus from New Zealand. All three species exhibited significant amounts of accumulated DNA damage, measured as cyclobutane pyrimidine dimers (CPD) photoproducts, when exposed to UVR in the laboratory. Biological weighting functions (BWFs) revealed that S. neumayeri has significantly higher sensitivity to UVR-induced DNA damage across most of the UVR spectrum compared to the other two species, and all species were observed to have weightings in the ultraviolet-A (UVA, 320-400 nm) portion of the spectrum. The increased sensitivity to ultraviolet-B (290-320 nm) and UVA in S. neumayeri is correlated with the lowest concentration of UVR absorbing compounds observed in the embryos of the three species of urchin used in this study. Sea urchin embryos and larvae in the respective habitats of the species tested are known to occur within 5 m of the surface of the ocean where both UVB and UVA wavelengths occur. Solar irradiances of UVR at a depth of 5 m, weighted using the urchin DNA damage BWFs, show that E. chloroticus receives the greatest amount of biologically effective UVR despite having the lowest wavelength dependent weightings for DNA damage when compared to the other two species.     

Rapid enzymatic response to compensate UV radiation in copepods

Ultraviolet radiation (UVR) causes physical damage to DNA, carboxylation of proteins and peroxidation of lipids in copepod crustaceans, ubiquitous and abundant secondary producers in most aquatic ecosystems. Copepod adaptations for long duration exposures include changes in behaviour, changes in pigmentation and ultimately changes in morphology. Adaptations to short-term exposures are little studied. Here we show that short-duration exposure to UVR causes the freshwater calanoid copepod, Eudiaptomus gracilis, to rapidly activate production of enzymes that prevent widespread collateral peroxidation (glutathione S-transferase, GST), that regulate apoptosis cell death (Caspase-3, Casp-3), and that facilitate neurotransmissions (cholinesterase-ChE). None of these enzyme systems is alone sufficient, but they act in concert to reduce the stress level of the organism. The interplay among enzymatic responses provides useful information on how organisms respond to environmental stressors acting on short time scales.     

Molecular phylogenetics reveals a pattern of biome conservatism in New World anchovies (family Engraulidae)

Evolutionary transitions between marine and freshwater biomes are relatively rare events, yielding a widespread pattern of biome conservatism among aquatic organisms. We investigated biome transitions in anchovies (Engraulidae), a globally distributed clade of economically important fishes. Most anchovy species are near-shore marine fishes, but several exclusively freshwater species are known from tropical rivers of South America and were previously thought to be the product of six or more independent freshwater invasions. We generated a comprehensive molecular phylogeny for Engraulidae, including representatives from 15 of 17 currently recognized genera. Our data support previous hypotheses of higher-level relationships within Engraulidae, but show that most New World genera are not monophyletic and in need of revision. Ancestral character reconstruction reveals that New World freshwater anchovies are the product of a single marine to freshwater transition, supporting a pattern of biome conservatism. We argue that competition is the principal mechanism that regulates aquatic biome transitions on a continental scale.     

Environmental DNA (eDNA): Powerful technique for biodiversity conservation

Environmental DNA metabarcoding is a non-invasive method for discovering and identifying rare and endangered species in a variety of ecosystems, including aquatic environments, based on the retrieval of genetic traces emitted into the environment by animals. Environmental (e) DNA research has grown in popularity over the last decade as a result of a rise in the number of studies that employ DNA taken from the environment, particularly in freshwater and marine ecosystems. In terms of detecting diversity patterns, we may claim that DNA retrieved from the environment (eDNA) is altering the game. For resource management in fisheries, information on species composition and biomass/abundance of commercially and noncommercially harvested species is critical. The eDNA is a truly non-invasive method that inflicts no damage on the species or habitats under study even during sampling, the eDNA technique never harms any ecosystems or threatened species. This novel molecular method never affects any endangered species or ecosystem during sampling. Environmental DNA analysis has become more widely accepted and is used in the detection of the presence and absence of aquatic macrofauna, such as freshwater and marine fish. This review study may aid researchers in better understanding the current state of eDNA technology. Despite the fact that various scientists have used eDNA to investigate the worldwide biodiversity of aquatic environments, no one in India is focusing on this new technology. We conclude that the eDNA technique has the potential to become a next-generation tool for biodiversity research and aquatic ecosystem conservation.     

Alogomyces tanneri gen. et sp. nov., a chytrid in Lobulomycetales from horse manure

The order Lobulomycetales contains chytrids from soil, freshwater and marine habitats; environmental DNA sampling has indicated that representatives of this order might be found in deep ocean localities. We describe Alogomyces tanneri as the first lobulomycetalean chytrid isolated from horse manure; A. tanneri is also the first species in the order to possess a rumposome in its zoospore. This species widens the range of habitats, ultrastructural variation and thallus morphology for Lobulomycetales.     

Predation on prokaryotes in the water column and its ecological implications

The oxic realms of freshwater and marine environments are zones of high prokaryotic mortality. Lysis by viruses and predation by ciliated and flagellated protists result in the consumption of microbial biomass at approximately the same rate as it is produced. Protist predation can favour or suppress particular bacterial species, and the successful microbial groups in the water column are those that survive this selective grazing pressure. In turn, aquatic bacteria have developed various antipredator strategies that range from simply 'outrunning' protists to the production of highly effective cytotoxins. This ancient predator-prey system can be regarded as an evolutionary precursor of many other interactions between prokaryotic and eukaryotic organisms.