Marianne K. Litvaitis

Phylogeny of Gastrotricha: a morphology-based framework of gastrotrich relationships

Currently, the phylum Gastrotricha is divided into the orders Macrodasyida and Chaetonotida, with the structure of the myoepithelial pharynx being an important distinguishing feature. Macrodasyida currently has six recognized families, and Chaetonotida comprises seven families. However, within-group relationships are poorly understood. To arrive at a better understanding of gastrotrich systematics and phylogeny, we performed the first cladistic analysis of nearly all known gastrotrich genera using 71 morphological characters. Results suggest that the Gastrotricha is a monophyletic group (supported by 82% of bootstrap replications) with its most primitive taxa distributed among the families Dactylopodolidae and Neodasyidae. Monophyly of Macrodasyida and Chaetonotida was supported by 90% and 52% bootstrap replications, respectively. Within the Macrodasyida, the families Dactylopodolidae, Turbanellidae, Macrodasyidae, and Thaumastodermatidae all formed monophyletic clades. The families Planodasyidae and Lepidodasyidae were paraphyletic. Among the Chaetonotida, the marine family Xenotrichulidae was monophyletic, supported by 51% of bootstrap replications. A second clade containing all freshwater families was supported by 62% bootstrap values. However, Chaetonotidae were paraphyletic. Using this analysis as a framework, we now can explore possible patterns of evolution within it, and arrive at a consensus of the gastrotrich ground pattern. Moreover, in future molecular studies of metazoan phylogeny, we will be able to select gastrotrich species that are more appropriate representatives of the phylum.

A molecular test of cyanobacterial phylogeny: inferences from constraint analyses

Parsimony and neighbor-joining analyses of 16S rDNA nucleotide sequences of 68 species and strains of cyanobacteria and prochlorophytes supported a monophyletic Nostocales, a monophyletic Stigonematales, three independent lineages of prochlorophytes within the cyanobacteria, and a paraphyletic Chroococcales (p<0.0001) and Oscillatoriales (p = 0.0147). Within the Oscillatoriales, the genus Oscillatoria formed an unnatural taxon (p<0.0001) and needs major revisions. Using constraint analysis, the genus Microcystis was found to be monophyletic and the paraphyletic positions of Microcystis elabens and M. holsatica are probably due to long-branch attraction. Further, a separation of Chroococcales based on varying levels of polyunsaturated fatty acids is more consistent with nucleotide-based phylogenies than with existing morphological groupings. It is proposed that Chroococcales be redefined to exclude the genus Microcystis, and that a new order be erected for Microcystis. Finally, it is more parsimonious to assume a common cyanobacterial/prochlorophyte ancestor, than to evoke de novo synthesis of chlb in each prochlorophyte lineage plus in the lineage leading to green chloroplasts. This common ancestor is proposed to have contained both chlorophyll a and b plus phycobilins. Subsequent multiple losses of chlb in cyanobacteria and the loss of chla and phycobilins in prochlorophytes, led to the currently observed pigment distribution. It is therefore, recommended that Prochlorales be reclassified as cyanobacteria.

Mass Culture and Characterization of Tumor Cells From a Naturally Occurring Invertebrate Cancer Model: Applications for Human and Animal Disease and Environmental Health

On the northeastern coast of the United States and Canada, Mya arenaria, the soft shell clam, develops a diffuse, hemopoetic tumor (a fatal leukemia-like cancer) resulting from inactivation of p53-like family member proteins.These malignant cells provide a model for an unrelated set of human cancer cells that are also characterized by mortalin-based cytoplasmic sequestration of wild-type p53 tumor suppressor protein (mortalin is the mitochondrial Hsp70 protein). Here we describe methods for mass culture and long-term storage of tumor cells from this cancer. These are the first successful efforts at maintaining malignant cells from any marine invertebrate in vitro. Following passage (subculture), these cultures undergo transition from primary cultures to non-immortalized cell lines that continue to proliferate and do not re-differentiate the normal hemocyte phenotype. We also characterize normal clam hemocytes and the pathology of cancerous clam hemocytes in vitro and in vivo using light and electron microscopy, cyto- and immunocytochemistry, molecular biology, and a phagocytosis assay. Our protocols provide biomedical and environmental researchers with ready access to this naturally occurring cancer model. We discuss the clam cancer model regarding (a) human health and disease; (b) animal health, disease, and aquaculture; (c) environmental health monitoring; and (d) future research directions.

A Muscular Double Helix in Gastrotricha

Abstract Studies on the muscular system of gastrotrichs revealed a new muscle arrangement. Helicoidal muscles, in right- and left-hand spirals, were found in all 9 species of Gastrotricha examined. The macrodasyidans Acanthodasys aculeatus , Tetranchyroderma papii and Thaumastoderma heideri had helicoidal muscles along the entire digestive tract (pharynx and intestine), whereas Macrodasys caudatus and Turbanella cornuta displayed helicoidal muscles mainly along the pharynx. Representatives of the order Chaetonotida, Chaetonotus aculifer , Draculiciteria tessalata, Lepidodermella squamata , and Xenotrichula intermedia , all had helicoidal muscles on the intestine, while pharyngeal helicoidal muscles were not observed in C. aculifer or L. squamata . Helicoidal muscles are the thinnest-diameter muscles in the gastrotrich body and generally surround splanchnic circular muscles and longitudinal bands. The proposed functions of helicoidal muscles include antagonization of radial dilations of the digestive tract during feeding, pressurization of the intestine and closure of the pharyngeal-intestinal valve in chaetonotidans, and prevention of intestinal kinking during changes in body shape. The presence of this unique muscle orientation may be a synapomorphy for Gastrotricha.

The muscular system of Dactylopodola baltica and other macrodasyidan gastrotrichs in a functional and phylogenetic perspective

The gastrotrich muscular system is characterized by band‐like muscles arranged in orientations that reflect both function and phylogeny. To better understand the evolution of the Dactylopodolidae, a putative primitive lineage and potential sister group to other extant macrodasyidans, we have used a fluorescent phalloidin stain to visualize muscle patterns in the marine gastrotrich Dactylopodola baltica and eight other species of Macrodasyida from four families. The musculature of D. baltica is arranged as a series of circular, helicoidal and longitudinal bands around the digestive tract. Circular muscles and longitudinal muscles were found in splanchnic and somatic positions. Helicoidal muscles, in 50–60° angles with respect to the longitudinal body axis, surrounded circular and longitudinal splanchnic muscles in a spiralling orientation. The largest longitudinal muscles were the ventrolateral bands composed of numerous cross‐striated myocytes arranged in parallel arrays. The overall arrangement of the muscular system of D. baltica showed several similarities to other macrodasyidan gastrotrichs, including the presence and location of circular, helicoidal and longitudinal muscles, their orientation with respect to the longitudinal body axis and their points of insertion. Unique to D. baltica is the anterior and posterior arrangement of the ventrolateral muscles and the orientation of muscle branches that supply the ventral and dorsal aspects of the pharynx. Muscle data from observations of D. baltica and eight additional species were coded as phylogenetic characters, mapped onto a cladogram and compared to an existing phylogeny of the order. The direction of evolutionary change in specific muscle groups was inferred, as was the ground pattern of muscles for the Macrodasyida.

Hexamethyldisilazane for Scanning Electron Microscopy of Gastrotricha

We evaluated treatment with hexamethyldisilazane (HMDS) as an alternative to critical-point drying (CPD) for preparing microscopic Gastrotricha for scanning electron microscopy (SEM). We prepared large marine (2 mm) and small freshwater (100 μm) gastrotrichs using HMDS as the primary dehydration solvent and compared the results to earlier investigations using CPD. The results of HMDS dehydration are similar to or better than CPD for resolution of two important taxonomic features: cuticular ornamentation and patterns of ciliation. The body wall of both sculpted (Lepidodermeila) and smooth (Dolichodasys) gastrotrichs retained excellent morphology as did the delicate sensory and locomotory cilia. The only unfavorable result of HMDS dehydration was an occasional coagulation of gold residue when the solvent had not fully evaporated before sputter-coating. We consider HMDS an effective alternative for preparing of gastrotrichs for SEM because it saves time and expense compared to CPD.

The musculature of Draculiciteria tessalata (Chaetonotida, Paucitubulatina) : implications for the evolution of dorsoventral muscles in Gastrotricha

The muscular system of the marine interstitial gastrotrich Draculiciteria tessalata (Chaetonotida, Paucitubulatina) was analyzed with fluorescent phalloidin. Muscles in circular, longitudinal, helicoidal and dorsoventral orientations were found. Circular muscles were present as discreet rings on the pharynx only. Five pairs of longitudinal muscles were found in dorsal, lateral and ventral positions. One of the two pairs of lateral muscles is newly described for the species. Helicoidal muscles, external to the circular muscles and some longitudinal bands, spiraled around the pharynx and anterior portion of the intestine. Two pairs of segmentally-arranged dorsoventral muscles were also present. Lateral dorsoventral muscles extended from the base of the pharynx to the anterior part of the caudal furca. Medial dorsoventral muscles extended from the pharyngeal-intestinal junction into each ramus of the caudal furca. A hypothesis on the evolution of dorsoventral muscles in D. tessalata is proposed which includes a splitting of circular muscles into separate somatic and splanchnic components with a further displacement of both muscle sets into a dorsoventral orientation.

A molecular framework for the phylogeny of the Pseudocerotidae (Platyhelminthes, Polycladida)

Systematic relationships within the cotylean family Pseudocerotidae were examined using nucleotide sequences of the D3 expansion segment of the 28S rDNA gene. A previously suggested separation of Pseudoceros and Pseudobiceros based on the number of male reproductive systems was confirmed. Regardless of the algorithm employed, Pseudoceros always formed a monophyletic clade. Pseudobiceros appeared to be paraphyletic; however, a constrained maximum parsimony tree was not significantly longer (2 steps, α = 0.05). Additionally, the genera Maiazoon, Phrikoceros and Tytthosoceros were validated as taxonomic entities, and their relationships to other genera within the family were determined. Molecular data also supported species separations based on colour patterns. An intraspecific genetic distance of 1.14% was found for Pseudoceros bifurcus, whereas the intrageneric distance was 3.58%. Genetic distances among genera varied, with the closest distance being 2.048% between Pseudobiceros and Maiazoon, and the largest distance (8.345%) between Pseudoceros and Tytthosoceros.

Functional morphology of muscles in Tetranchyroderma papii (Gastrotricha)

Abstract Movement in gastrotrichs is powered by an interaction of ventral cilia and muscles. In interstitial gastrotrichs, movement among sand grains often requires the additional use of adhesive tubules that allow for behaviors such as escape responses and changes in body position. In this study, we describe orientations and possible mechanical actions of muscles during locomotion in the gastrotrich, Tetranchyroderma papii (Macrodasyida). Fluorescently labeled phalloidin was used to stain F-actin of muscles and visualize muscle patterns. Muscles are arranged in circular, longitudinal, and helicoidal orientations. Circular muscles were in the form of discreet rings around the pharynx and intestine, and contribute to the structure of the oral hood. Longitudinal muscles are largely concentrated on the ventral and ventrolateral sides of the body, where they aid in body flexion, including directional changes during ciliary swimming, body torsion, and escape responses. Helicoidal muscles, present as myocytes in left- and right-hand orientations, lie external of the circular bands and some of the longitudinal bands, and are hypothesized to counteract dilations of the pharynx and intestine during feeding. Extraordinary muscle orientations with undetermined functions include a pair of crossover muscles and a single semicircular muscle band at the caudal end.

Bobcats (Lynx rufus) as a Model Organism to Investigate the Effects of Roads on Wide-Ranging Carnivores

We are using bobcats (Lynx rufus) as a model organism to examine how roads affect the abundance, distribution, and genetic structure of a wide-ranging carnivore. First, we compared the distribution of bobcat-vehicle collisions to road density and then estimated collision probabilities for specific landscapes using a moving window with road-specific traffic volume. Next, we obtained incidental observations of bobcats from the public, camera-trap detections, and locations of bobcats equipped with GPS collars to examine habitat selection. These data were used to generate a cost-surface map to investigate potential barrier effects of roads. Finally, we have begun an examination of genetic structure of bobcat populations in relation to major road networks. Distribution of vehicle-killed bobcats was correlated with road density, especially state and interstate highways. Collision models suggested that some regions may function as demographic sinks. Simulated movements in the context of the cost-surface map indicated that some major roads may be barriers. These patterns were supported by the genetic structure of bobcats. The sharpest divisions among genetically distinct demes occurred along natural barriers (mountains and large lakes) and in road-dense regions. In conclusion, our study has demonstrated the utility of using bobcats as a model organism to understand the variety of threats that roads pose to a wide-ranging species. Bobcats may also be useful as one of a group of focal species while developing approaches to maintain existing connectivity or mitigate the negative effects of roads.