Dentition patterns and molecular diversity of Mastophorus muris (Gmelin, 1790) (Nematoda: Spiruroidea) support a host-associated subdivision.

Mastophorus muris (Gmelin, 1790) is a globally distributed parasitic nematode of broad range mammals. The taxonomy within the genus Mastophorus and the cryptic diversity among the genus are controversial among taxonomists. This study provides a detailed morphological description of M. muris from Mus musculus combined with a molecular phylogenetic approach. Moreover, descriptions and molecular data of M. muris from non-Mus rodents and wildcats complement our findings and together provide new insights into their taxonomy. The analysis of M. muris was based on light microscopy and scanning electron microscopy. The morphological description focused on the dentition pattern of the two trilobed pseudolabia. Additionally, we described the position of the vulva, arrangement of caudal pairs of papillae, spicules and measured specimens from both sexes and the eggs. For the molecular phylogenetic approach, we amplified the small subunit ribosomal RNA gene and the internal transcribed spacer, and the cytochrome c oxidase subunit 1. Mastophorus morphotypes based on dentition patterns and phylogenetic clustering indicate a subdivision of the genus in agreement with their host. We recognize two groups without a change to formal taxonomy: One group including those specimens infecting Mus musculus, and the second group including organisms infecting non-Mus rodents. Our genetic and morphological data shed light into the cryptic diversity within the genus Mastopohorus. We identified two host-associated groups of M. muris. The described morphotypes and genotypes of M. muris allow a consistent distinction between host-associated parasites.

The oral sensory organs in Bathochordaeus stygius (Tunicata Appendicularia) are unique in structure and homologous to the coronal organ.

BACKGROUND: Appendicularia consists of approximately 70 purely marine species that belong to Tunicata the probable sister taxon to Craniota. Therefore, Appendicularia plays a pivotal role for our understanding of chordate evolution. In addition, appendicularians are an important part of the epipelagic marine plankton. Nevertheless, little is known about appendicularian species, especially from deeper water. RESULTS: Using µCT, scanning electron microscopy, and digital 3D-reconstruction techniques we describe three pairs of complex oral sensory organs in the mesopelagic appendicularian Bathochordaeus stygius. The oral sensory organs are situated at the anterior and lateral margin of the mouth and inside the mouth cavity. A single organ consists of 22-90 secondary receptor cells that project apical cilia through a narrow hole in the epidermis. The receptor cells are innervated by branches of the second brain nerve. CONCLUSIONS: Based on position, morphology, and innervation we suggest that the oral sensory organs are homologues of the coronal organs in other tunicates. We discuss the hypothesized homology of coronal organs and the lateral line system of primary aquatic vertebrates. The complex oral sensory organs of B. stygius are unique in tunicates and could be adaptations to the more muffled environment of the mesopelagic.

A lipocalin mediates unidirectional heme biomineralization in malaria parasites.

During blood-stage development, malaria parasites are challenged with the detoxification of enormous amounts of heme released during the proteolytic catabolism of erythrocytic hemoglobin. They tackle this problem by sequestering heme into bioinert crystals known as hemozoin. The mechanisms underlying this biomineralization process remain enigmatic. Here, we demonstrate that both rodent and human malaria parasite species secrete and internalize a lipocalin-like protein, PV5, to control heme crystallization. Transcriptional deregulation of PV5 in the rodent parasite Plasmodium berghei results in inordinate elongation of hemozoin crystals, while conditional PV5 inactivation in the human malaria agent Plasmodium falciparum causes excessive multidirectional crystal branching. Although hemoglobin processing remains unaffected, PV5-deficient parasites generate less hemozoin. Electron diffraction analysis indicates that despite the distinct changes in crystal morphology, neither the crystalline order nor unit cell of hemozoin are affected by impaired PV5 function. Deregulation of PV5 expression renders P. berghei hypersensitive to the antimalarial drugs artesunate, chloroquine, and atovaquone, resulting in accelerated parasite clearance following drug treatment in vivo. Together, our findings demonstrate the Plasmodium-tailored role of a lipocalin family member in hemozoin formation and underscore the heme biomineralization pathway as an attractive target for therapeutic exploitation.

An apicoplast-resident folate transporter is essential for sporogony of malaria parasites.

Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co-factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2-deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single-membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2-deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector.

Common plant flavonoids prevent the assembly of amyloid curli fibres and can interfere with bacterial biofilm formation.

Like all macroorganisms, plants have to control bacterial biofilm formation on their surfaces. On the other hand, biofilms are highly tolerant against antimicrobial agents and other stresses. Consequently, biofilms are also involved in human chronic infectious diseases, which generates a strong demand for anti-biofilm agents. Therefore, we systematically explored major plant flavonoids as putative anti-biofilm agents using different types of biofilms produced by Gram-negative and Gram-positive bacteria. In Escherichia coli macrocolony biofilms, the flavone luteolin and the flavonols myricetin, morin and quercetin were found to strongly reduce the extracellular matrix. These agents directly inhibit the assembly of amyloid curli fibres by driving CsgA subunits into an off-pathway leading to SDS-insoluble oligomers. In addition, they can interfere with cellulose production by still unknown mechanisms. Submerged biofilm formation, however, is hardly affected. Moreover, the same flavonoids tend to stimulate macrocolony and submerged biofilm formation by Pseudomonas aeruginosa. For Bacillus subtilis, the flavonone naringenin and the chalcone phloretin were found to inhibit growth. Thus, plant flavonoids are not general anti-biofilm compounds but show species-specific effects. However, based on their strong and direct anti-amyloidogenic activities, distinct plant flavonoids may provide an attractive strategy to specifically combat amyloid-based biofilms of some relevant pathogens.

Phylogenetic analysis of phenotypic characters of Tunicata supports basal Appendicularia and monophyletic Ascidiacea.

With approximately 3000 marine species, Tunicata represents the most disparate subtaxon of Chordata. Molecular phylogenetic studies support Tunicata as sister taxon to Craniota, rendering it pivotal to understanding craniate evolution. Although successively more molecular data have become available to resolve internal tunicate phylogenetic relationships, phenotypic data have not been utilized consistently. Herein these shortcomings are addressed by cladistically analyzing 117 phenotypic characters for 49 tunicate species comprising all higher tunicate taxa, and five craniate and cephalochordate outgroup species. In addition, a combined analysis of the phenotypic characters with 18S rDNA-sequence data is performed in 32 OTUs. The analysis of the combined data is congruent with published molecular analyses. Successively up-weighting phenotypic characters indicates that phenotypic data contribute disproportionally more to the resulting phylogenetic hypothesis. The strict consensus tree from the analysis of the phenotypic characters as well as the single most parsimonious tree found in the analysis of the combined dataset recover monophyletic Appendicularia as sister taxon to the remaining tunicate taxa. Thus, both datasets support the hypothesis that the last common ancestor of Tunicata was free-living and that ascidian sessility is a derived trait within Tunicata. "Thaliacea" is found to be paraphyletic with Pyrosomatida as sister taxon to monophyletic Ascidiacea and the relationship between Doliolida and Salpida is unresolved in the analysis of morphological characters; however, the analysis of the combined data reconstructs Thaliacea as monophyletic nested within paraphyletic "Ascidiacea". Therefore, both datasets differ in the interpretation of the evolution of the complex holoplanktonic life history of thaliacean taxa. According to the phenotypic data, this evolution occurred in the plankton, whereas from the combined dataset a secondary transition into the plankton from a sessile ascidian is inferred. Besides these major differences, both analyses are in accord on many phylogenetic groupings, although both phylogenetic reconstructions invoke a high degree of homoplasy. In conclusion, this study represents the first serious attempt to utilize the potential phylogenetic information present in phenotypic characters to elucidate the inter-relationships of this diverse marine taxon in a consistent cladistic framework.

High-precision morphology: bifocal 4D-microscopy enables the comparison of detailed cell lineages of two chordate species separated for more than 525 million years.

BACKGROUND: Understanding the evolution of divergent developmental trajectories requires detailed comparisons of embryologies at appropriate levels. Cell lineages, the accurate visualization of cleavage patterns, tissue fate restrictions, and morphogenetic movements that occur during the development of individual embryos are currently available for few disparate animal taxa, encumbering evolutionarily meaningful comparisons. Tunicates, considered to be close relatives of vertebrates, are marine invertebrates whose fossil record dates back to 525 million years ago. Life-history strategies across this subphylum are radically different, and include biphasic ascidians with free swimming larvae and a sessile adult stage, and the holoplanktonic larvaceans. Despite considerable progress, notably on the molecular level, the exact extent of evolutionary conservation and innovation during embryology remain obscure. RESULTS: Here, using the innovative technique of bifocal 4D-microscopy, we demonstrate exactly which characteristics in the cell lineages of the ascidian Phallusia mammillata and the larvacean Oikopleura dioica were conserved and which were altered during evolution. Our accurate cell lineage trees in combination with detailed three-dimensional representations clearly identify conserved correspondence in relative cell position, cell identity, and fate restriction in several lines from all prospective larval tissues. At the same time, we precisely pinpoint differences observable at all levels of development. These differences comprise fate restrictions, tissue types, complex morphogenetic movement patterns, numerous cases of heterochronous acceleration in the larvacean embryo, and differences in bilateral symmetry. CONCLUSIONS: Our results demonstrate in extraordinary detail the multitude of developmental levels amenable to evolutionary innovation, including subtle changes in the timing of fate restrictions as well as dramatic alterations in complex morphogenetic movements. We anticipate that the precise spatial and temporal cell lineage data will moreover serve as a high-precision guide to devise experimental investigations of other levels, such as molecular interactions between cells or changes in gene expression underlying the documented structural evolutionary changes. Finally, the quantitative amount of digital high-precision morphological data will enable and necessitate software-based similarity assessments as the basis of homology hypotheses.

Neurogenesis in directly and indirectly developing enteropneusts: of nets and cords.

Concerning the evolution of deuterostomes, enteropneusts (acorn worms) occupy a pivotal role as they share some characteristics with chordates (e.g., tunicates and vertebrates) but are also closely related to echinoderms (e.g., sea urchin). The nervous system in particular can be a highly informative organ system for evolutionary inferences, and advances in fluorescent microscopy have revealed overwhelming data sets on neurogenesis in various clades. However, immunocytochemical descriptions of neurogenesis of juvenile enteropneusts are particularly scarce, impeding the reconstruction of nervous system evolution in this group. We followed morphogenesis of the nervous system in two enteropneust species, one with direct (Saccoglossus kowalevskii) and the other with indirect development (Balanoglossus misakiensis), using an antibody against serotonin and electron microscopy. We found that all serotonin-like immunoreactive (LIR) neurons in both species are bipolar ciliary neurons that are intercalated between other epidermal cells. Unlike the tornaria larva of B. misakiensis, the embryonic nervous system of S. kowalevskii lacks serotonin-LIR neurons in the apical region as well as an opisthotroch neurite ring. Comparative analysis of both species shows that the projections of the serotonin-LIR somata initially form a basiepidermal plexus throughout the body that disappears within the trunk region soon after settlement before the concentrated dorsal and ventral neurite bundles emerge. Our data reveal a highly conserved mode of neurogenesis in enteropneusts that is independent of the developing mode and is inferred to be a common feature for Enteropneusta. Moreover, all detected serotonin-LIR neurons are presumably receptor cells, and the absence of serotonin-LIR interneurons from the enteropneust nervous system, which are otherwise common in various bilaterian central nervous systems, is interpreted as a loss that might have occurred already in the last common ancestor of Ambulacraria.

Cell lineages and fate maps in tunicates: conservation and modification.

Comparison of features of the cell lineages and fate maps of early embryos between related species is useful in inferring developmental mechanisms and amenable to evolutionary considerations. We present cleavage patterns, cell lineage trees, and fate maps of ascidian and appendicularian embryos side by side to facilitate comparison. This revealed a number of significant differences in cleavage patterns and cell lineage trees, whereas the fate maps were found to be conserved. This fate map similarity can be extended to vertebrates, thus representing the fate map characteristics of chordates. Cleavage patterns and cell lineages may have been modified during evolution without any drastic changes in fate maps. Selective pressures that constrain developmental mechanisms at early embryonic stages might not be so strong as long as embryos are still able to generate a chordate-type fate map. Aquatic chordates share similar fate maps and morphogenetic movements during gastrulation and neurulation, eventually developing into tadpole-shaped larvae. As swimming by tail beats, and not by cilia, is advantageous, selective pressure may maintain the basic elements of the tadpole shape. We also discuss the evolutionary origin of the vertebrate neural crest and the embryonic origin of the appendicularian heart to illustrate the usefulness of cell lineage data. From an evolutionary standpoint, cell lineages behave like other characteristics such as morphology or protein sequences. Both novel and primitive features are present in extant organisms, and it is of interest to identify the relative degree of evolutionary conservation as well as the level at which homology is inferred.

Evolutionary Insights from the Mitochondrial Genome of Oikopleura dioica: Sequencing Challenges, RNA Editing, Gene Transfers to the Nucleus, and tRNA Loss.

Sequencing the mitochondrial genome of the tunicate Oikopleura dioica is a challenging task due to the presence of long poly-A/T homopolymer stretches, which impair sequencing and assembly. Here, we report on the sequencing and annotation of the majority of the mitochondrial genome of O. dioica by means of combining several DNA and amplicon reads obtained by Illumina and MinIon Oxford Nanopore Technologies with public RNA sequences. We document extensive RNA editing, since all homopolymer stretches present in the mitochondrial DNA correspond to 6U-regions in the mitochondrial RNA. Out of the 13 canonical protein-coding genes, we were able to detect eight, plus an unassigned open reading frame that lacked sequence similarity to canonical mitochondrial protein-coding genes. We show that the nad3 gene has been transferred to the nucleus and acquired a mitochondria-targeting signal. In addition to two very short rRNAs, we could only identify a single tRNA (tRNA-Met), suggesting multiple losses of tRNA genes, supported by a corresponding loss of mitochondrial aminoacyl-tRNA synthetases in the nuclear genome. Based on the eight canonical protein-coding genes identified, we reconstructed maximum likelihood and Bayesian phylogenetic trees and inferred an extreme evolutionary rate of this mitochondrial genome. The phylogenetic position of appendicularians among tunicates, however, could not be accurately determined.