Two new acoels (Acoelomorpha) of the genus Haplogonaria from the northwest Atlantic.

Two previously unknown species of Haplogonaria (Acoela), H. schillingi sp. nov. and H. baki sp. nov., are described from the coastline of Maine, USA. The two species are morphologically similar to each other but H. schillingi can be distinguished from H. baki by its red pigmentation, its possession of a large genital atrium that branches posteriorly to the seminal vesicle and anteriorly to the vagina, a seminal vesicle that is more ellipsoid-shaped than spherical, and a well-defined wall in the seminal bursa. We provide a description of the new species using live observation, light microscopy of serial sagittal sections, and confocal microscopy imaging of F-actin. We compare the morphology of the new species with other members of the genus and discuss the phylogenetic position of H. schillingi in light of conflicting morphological and molecular data.

The Acoela: on their kind and kinships, especially with nemertodermatids and xenoturbellids (Bilateria incertae sedis).

Acoels are among the simplest worms and therefore have often been pivotal in discussions of the origin of the Bilateria. Initially thought primitive because of their "planula-like" morphology, including their lumenless digestive system, they were subsequently dismissed by many morphologists as a specialized clade of the Platyhelminthes. However, since molecular phylogenies placed them outside the Platyhelminthes and outside all other phyla at the base of the Bilateria, they became the focus of renewed debate and research. We review what is currently known of acoels, including information regarding their morphology, development, systematics, and phylogenetic relationships, and put some of these topics in a historical perspective to show how the application of new methods contributed to the progress in understanding these animals. Taking all available data into consideration, clear-cut conclusions cannot be made; however, in our view it becomes successively clearer that acoelomorphs are a "basal" but "divergent" branch of the Bilateria.

Antimicrobial action of the American cranberry constituents; phenolics, anthocyanins, and organic acids, against Escherichia coli O157:H7.

We investigated the antimicrobial effect of constituents of the American cranberry (Vaccinium macrocarpon); sugar plus organic acids, phenolics, and anthocyanins, against Escherichia coli O157:H7. Each fractional component was assayed over a 24-h period with 5-log initial inocula to determine the minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and log CFU/ml reductions, at their native pH and neutral pH. Each fraction produced significant reductions (P<0.05) at the native pH: MICs for sugars plus organic, phenolics, and anthocyanins were 5.6/2.6 Brix/acid (citric acid equivalents) 2.70g/L (gallic acid equivalent), and 14.80mg/L (cyanidin-3-glucoside equivalent), respectively. Sugars plus organic acids at native pH (3) produced a reduction below detectable limits (<1 log CFU/ml) compared to the control at 24h for 11.3/5.2 and 5.6/2.6 Brix/acid. Phenolics at native pH (4) produced reductions below detectable limits compared to the control at 24h and initial inocula for treatments of 5.40 and 2.70g/L. Anthocyanins at native pH (2) produced reductions below detectable limits for treatments of 29.15 and 14.80mg/L cyanidin-3-glucoside equivalents. Neutralized phenolics and anthocyanins had the same MIC and MBC as those at their native pH. Neutralized sugars plus organic acids did not inhibit bacterial growth compared to the control. Neutralized phenolics reduced bacteria below detectable limits in treatments of 5.40g/L and 2.70g/L compared to the control. Neutralized anthocyanins reduced bacterial growth below detectable limits at the concentration of 29.15mg/L, but at 14.80mg/L there was no significant reduction. Stationary-phase cells of E. coli O157:H7 were treated with 5% of each fraction in 0.8% NaCl for 20min and viewed under transmission electron microscopy. All fractions caused significant damage compared the control. Sugars plus organic acids caused visible osmotic stress, while phenolics and anthocyanins caused disintegration of the outer membrane.

To be or not to be a flatworm: the acoel controversy.

Since first described, acoels were considered members of the flatworms (Platyhelminthes). However, no clear synapomorphies among the three large flatworm taxa -- the Catenulida, the Acoelomorpha and the Rhabditophora -- have been characterized to date. Molecular phylogenies, on the other hand, commonly positioned acoels separate from other flatworms. Accordingly, our own multi-locus phylogenetic analysis using 43 genes and 23 animal species places the acoel flatworm Isodiametra pulchra at the base of all Bilateria, distant from other flatworms. By contrast, novel data on the distribution and proliferation of stem cells and the specific mode of epidermal replacement constitute a strong synapomorphy for the Acoela plus the major group of flatworms, the Rhabditophora. The expression of a piwi-like gene not only in gonadal, but also in adult somatic stem cells is another unique feature among bilaterians. These two independent stem-cell-related characters put the Acoela into the Platyhelminthes-Lophotrochozoa clade and account for the most parsimonious evolutionary explanation of epidermal cell renewal in the Bilateria. Most available multigene analyses produce conflicting results regarding the position of the acoels in the tree of life. Given these phylogenomic conflicts and the contradiction of developmental and morphological data with phylogenomic results, the monophyly of the phylum Platyhelminthes and the position of the Acoela remain unresolved. By these data, both the inclusion of Acoela within Platyhelminthes, and their separation from flatworms as basal bilaterians are well-supported alternatives.

Morphology and ultrastructure of the pharynx in Solenofilomorphidae (Acoela).

The homology of pharynges within the mostly pharynx-less Acoela has been a matter of discussion for decades. Here, we analyze the pharynges of three members of the Solenofilomorphidae, Myopea sp. and two species of the genus Solenofilomorpha, by means of light and transmission electron microscopy. Special focus is placed on the ultrastructure of the pharyngeal musculature, epidermis surrounding the mouth, pharyngeal epithelium, and junction with the digestive parenchyma. The main goal of this study was to evaluate the usefulness of certain characters for broader comparisons within the Acoela. Among the three species, characters relating to position of the mouth, presence and elaboration of sphincter muscles, presence of pharyngeal glands, and ultrastructure of epitheliosomes proved to be variously species- and genus-specific. The arrangement of pharyngeal muscles and their connection with body wall musculature, ultrastructure of receptor cells, and morphology of a nonciliated glandular region in the posterior pharynx, in contrast, appear to be characteristic of the family Solenofilomorphidae and thus of predominant interest for comparisons with other acoel families.

Comparative morphology of the bursal nozzles in acoels (Acoela, Acoelomorpha).

Systematics of the Acoela is particularly difficult because of the paucity of readily discernible morphological features. In other soft-bodied worms, sclerotized structures, such as copulatory stylets, provide important characters that can be seen in whole mounts, but acoels generally lack such features. Among the few sclerotized structures in acoels are bursal nozzles-tubiform outlets on the seminal bursae that are believed to be conduits (spermatic ducts) through which allosperm are transported to the oocytes. Early classifications of the Acoela used features of the female reproductive system, including bursal nozzles, for distinguishing major groups, but the current system essentially ignores them as too plastic to provide higher-level distinctions. We used confocal and electron microscopy to further characterize bursal nozzles in five acoel species, and found all composed of actin-reinforced extensions of stacked, flat mesenchymal cells. In Notocelis gullmarensis, Aphanostoma bruscai, and Daku woorimensis, the nozzle is a stiffened region of the same cells forming the wall of the bursa. By contrast, in Wulguru cuspidata cells forming the nozzle are distinct from those of the bursa. The so-called bursal cap of A. bruscai and D. woorimensis has small sclerotized disjunct units within it, also composed of stacked, flat, actin-reinforced cells. The nozzle of W. cuspidata, prominent like that of other convolutid acoels, is relatively complex, its actin-reinforced cells sandwiched with secretory cells and its base bearing a "sorting apparatus" of egg-shaped cells that send narrow processes inside the spermatic duct. Cases of sperm inside the nozzle corroborate its assumed role in reproduction. Whereas most nozzles sit at the end of the bursa facing the ovary, in species of Pseudmecynostomum and purportedly in a few other acoels, they sit between the female pore and the bursa, constituting what we call a vaginal nozzle. All bursal nozzles of acoels show a common ground pattern indicating common ancestry, but certain features discerned through electron and confocal microscopy show promise of providing synapomorphies for grouping some species.

Concordance of molecular and morphological data: The example of the Acoela.

Morphological features of the Acoela appear to be quite plastic, including those of the copulatory organs, which provide the principle characteristics used for the systematics of this group. Consequently, classification schemes of the Acoela comprise numerous polyphyletic groupings. In this review, we detail recent revisions of acoel systematics using molecular sequence data and new and reevaluated morphological characteristics. Gene trees are discordant with traditional systematic schemes but strongly concordant with new morphological characteristics obtained through the use of transmission electron microscopy and confocal laser scanning microscopy, namely, characteristics of body-wall and copulatory organ musculature, sperm, sperm ducts, sagittocysts, and immunocytochemistry of the nervous system. This merger of molecular and morphological data has led to significant changes in acoel classification, including a major emendation of the largest family of the Acoela, the Convolutidae, whereby half of its members were transferred to a newly created family, the Isodiametridae.

Epithelium--the primary building block for metazoan complexity.

In simplest terms, the complexity of the metazoan body arises through various combinations of but two tissue types: epithelium and mesenchyme. Through mutual inductions and interactions, these tissues produce all of the organs of the body. Of the two, epithelium must be considered the default type in the Eumetazoa because it arises first in embryonic development and because mesenchyme arises from it by a switching off of the mechanisms that underly differentiation and maintenance of epithelial cells. In the few model metazoans whose epithelia have been studied by molecular techniques (largely Drosophila, Caenorhabditis, mouse), the molecular mechanisms underlying differentiation of epithelia show remarkable similarity. Extrapolating from these studies and from comparisons of the morphology of epithelia in lower metazoans, I propose how epithelia arose in the stem metazoan. Steps in epithelial differentiation include 1) establishment of cell polarity by molecular markers confined to either apical or basolateral domains in the plasma membrane; 2) aggregation of cells into sheets by localization of cell-adhesion molecules like cadherin to the lateral membrane; 3) formation of a zonula adherens junction from the cadherins by their localization to a discrete belt; 4) cell-to-cell linking of certain transmembrane proteins (primitively in the septate junction) to produce gates that physiologically isolate compartments delimited by the cells; and 5) synthesis of a basal lamina and adaptation of receptors (integrins) to its components. Despite morphological differences in the variety of cell junctions evident in various epithelia, the underlying molecular markers of these junctions are probably universally present in all eumetazoan epithelia.

Molecular systematics of the Acoela (Acoelomorpha, Platyhelminthes) and its concordance with morphology.

The phylogenetic relationships of the lower worm group Acoela were investigated using newly obtained nuclear 18S rDNA sequences from 16 acoels in combination with 16 acoel sequences available on GenBank from other laboratories. Parsimony and maximum likelihood analyses of the molecular data supported the concept that the Acoela is monophyletic; however, the gene tree produced by these analyses conflicts with the current taxonomic system for the Acoela in several family-level groupings. Most notable is the apparent polyphyly of the largest family of acoels, the Convolutidae. DNA analysis grouped together species of small-bodied convolutids in one clade, while large-bodied convolutids grouped in a separate clade with other large-bodied acoels. Despite such conflicts, the branching pattern in the gene tree is well supported by morphological characters of sperm and body-wall musculature.

Hopping locomotion in a nematode: Functional anatomy of the caudal gland apparatus of Theristus caudasaliens sp. n.

The interstitial nematode Theristus caudasaliens n. sp. normally locomotes by hopping on the left side of its tail tip, a mode of locomotion that is unique among nematodes. The animal uses its caudal glands and caudal musculature to perform the hops, attaching itself momentarily between hops with the glands and executing the hops by straightening and curling the posterior part of its body. The caudal gland apparatus can be seen by electron microscopy to consist of five gland cells of two different types. Three of these cells, termed viscid glands, are involved in adhesion of the animal to substrates and produce ovoid granules with a central dense band. The other two cells are characterized by smaller, lessdense granules and presumably function in releasing the animal from substrates. The ducts of both gland types extend to the tail tip where they terminate in a common crescent-shaped space. Their secretions are released to the outside through two pores on the left side of the tail tip. There is no spinneret valve in this nematode. The muscles of the tail and of the mid-body region are developed to the same extent. The caudal gland apparatus can be compared with the duo-gland adhesive organs of other interstitial animals, but its homology with either these organs or the caudal glands of other nematodes is uncertain. Theristus caudasaliens is described as a new species.