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Molecular evidence for Acanthocephala as a subtaxon of Rotifera.

Rotifers are free-living animals usually smaller than 1 mm that possess a characteristic wheel organ. Acanthocephalans (thorny-headed worms) are larger endoparasitic animals that use vertebrates and arthropods to complete their life cycle. The taxa Acanthocephala and Rotifera are considered separate phyla, often within the taxon Aschelminthes. We have reexamined the relationship between Rotifera and Acanthocephala using 18S rRNA gene sequences. Our results conclusively show that Acanthocephala is the sister group of the rotifer class Bdelloidea. Rotifera was nonmonophyletic in all molecular analyses, which supports the hypothesis that the Acanthocephala represent a taxon within the phylum Rotifera and not a separate phylum. These results agree with a previous cladistic study of morphological characters.

Acanthocephala

Histochemical investigations of Rotifera Bdelloidea. I. Localization of cholinesterase activity.

Cholinesterase activity has been investigated in Rotifera Bdelloidea (Philodina roseola, Philodina tubercolata, Rotaria rotatoria and other unidentified species) by histochemical methods and in vivo observations. Parallel histological studies have been carried out. The enzyme specificity was tested by employing different substrates and inhibitors. The effects in vivo of tubocurarin, bungarotoxin and acetylcholine were also observed. Acetylcholinesterase activity is localized in the nervous and muscular tissues, in sensory organs and in all the ciliated cells. Secretory cells (subcerebral, salivary and pedal glands) and gonad cells (nuclei of the syncytial vitellarium and follicular layer, oocytes and eggs) show both acetyl- and butyrylcholinesterase activities. The effects in vivo of cholinesterase inhibitors, as well as those of tubocurarin, bungarotoxin and acetylcholine, are consistent with the histochemical results, indicating a cholinergic system of transmission and acetylcholinesterase, as well as butyrylcholinesterase, activity.

Acetylcholine

Rapid toxicity assessment using an in vivo enzyme test for Brachionus plicatilis (Rotifera).

A 1-hr in vivo enzyme inhibition assay based on esterase activity has good potential for marine toxicity assessment. A test was developed for the rotifer Brachionus plicatilis based on the nonfluorescent substrate fluorescein diacetate (FDA), which is metabolized by esterases to a fluorescent product. Enzyme inhibition, as determined by reduced fluorescence, can be scored visually or quantified using a fluorometer. Quantification of fluorescence permits the calculation of NOEC, LOEC, chronic value, and IC20. The 1-hr esterase inhibition test has sensitivity comparable to that of 24-hr rotifer acute tests for several compounds. The toxicity of six compounds was examined using the quantified assay. The resulting IC20s were within a factor of 3 of the 24-hour LC50s. IC20 values ranged from 0.017 mg/l for tributyltin to 3.1 mg/l for zinc, with an average coefficient of variation of 17.8%. Electrophoretic analysis of rotifer homogenates suggested that a single C esterase (acetylesterase) was responsible for FDA metabolism in B. plicatilis. Several other aquatic species are capable of metabolizing FDA, including Brachionus calyciflorus, Mysidopsis bahia, Menidia beryllina, Pimephales promelas, Ceriodaphnia dubia, Daphnia pulex, Artemia salina, and Ophryotrocha sp. The esterase inhibition test is an attractive tool for assessing aquatic toxicity because of its speed, simplicity, sensitivity, and applicability to a broad range of aquatic species.

Animals

Isolation and purification of glutathione S-transferases from Brachionus plicatilis and B. calyciflorus (Rotifera).

1. The enzyme glutathione S-transferase (GST), a critical element in xenobiotic metabolism, was isolated from the marine rotifer Brachionus plicatilis and its freshwater congener B. calyciflorus. 2. In B. plicatilis, GST comprised 4.2% of cytosolic protein and was present as three separate isozymes with mol. wts 30,000, 31,400 and 33,700. Specific activity of crude homogenates was 56 nmol min-1 mg-1 protein, while that of affinity chromatography purified GST was 1850. 3. In B. calyciflorus, GST was present as two isozymes with mol. wts of 26,300 and 28,500, representing 1.0% of cytosolic protein. Crude GST specific activity was 1750 nmol min-1 mg-1 protein and purified was 72,400. 4. Rotifer GSTs are unusual because they are monomers whereas all other animals thus far investigated posses dimeric GSTs.

Animals

Glycosidases in Brachionus plicatilis (Rotifera).

1. Tests for glycosidases were performed in homogenates of Brachionus plicatilis. 2. Hydrolytic activity was detected with the following substrates: (a) with synthetic substrates (NP = 4-nitrophenyl): NP-alpha- and NP-beta-D-glucopyranoside, NP-alpha- and NP-beta-D-galactopyranoside, NP-N-acetyl-beta-D-glucosaminide, NP-N-acetyl-beta-D-galactosaminide, NP-alpha- and NP-beta-D-mannopyranoside and NP-alpha-L-fucopyranoside; (b) with disaccharides: sucrose, maltose, trehalose, isomaltose, cellobiose, gentiobiose and lactose; (c) with polysaccharides: laminarine, carboxymethyl-cellulose, avicel, Micrococcus luteus (for lysozyme) and 4-nitrophenyl-alpha-D-maltoheptaoside (for amylase). 3. The pH dependence of the glycosidase activities was determined. 4. The distribution of enzyme activities within fractions from the homogenate was studied in order to localize them within the cell. 5. Proteins from Brachionus homogenate were separated by SDS-gel electrophoresis and the positions of the following glycosidase activities were detected by assays performed on the gels (estimated molecular weights in parentheses): alpha-glucosidase (250,000); beta-glucosidase (200,000); beta-galactosidase (70,000); N-acetyl-beta-glucosaminidase (60,000).

Animals

Rotifer neuropharmacology--I. Cholinergic drug effects on oviposition of Philodina acuticornis (Rotifera, Aschelminthes).

The presence of acetylcholine in the culture medium was found to result in egg retention in the bdelloid rotifer Philodina acuticornis in a dose-dependent fashion, with no other discernible physiological effect. When six to eight eggs accumulate, the animals distend, burst and die. No other cholinergic agonist has been shown to produce a similar effect. The antagonistic effect of six anticholinergic drugs and five acetylcholinesterase-inhibitor insecticides was investigated on egg retention. All compounds were found to inhibit this phenomenon to varying degrees, but on the basis of their EC50 neuromuscular blockers appear to be most active in inhibiting egg retention. We suggest, therefore, that egg retention is caused by a spasm or increased tone of the cloacal sphincter rather than a paralysis of muscles needed in the peristaltic expulsion of the egg. Five acetylcholine-esterase inhibitory insecticides also inhibit egg retention.

Acetylcholine

Rotifer neuropharmacology--II. Synergistic effect of acetylcholine on local anesthetic activity in Brachionus calyciflorus (Rotifera, Aschelminthes).

A number of compounds showing general anesthetic action in the rotifer Brachionus calyciflorus were investigated in the presence of acetylcholine. Non-ionizing anesthetics, including tricaine, showed no interaction with acetylcholine. However, highly ionized compounds like the local anesthetics procaine and lidocaine, the muscarinic blocker and local anesthetic atropine, and the beta-adrenergic blocker propranolol showed a synergistic effect with acetylcholine. ACh increased the general anesthetic effect of these compounds in a statistically highly significant dose-dependent fashion. To account for the mechanism of this unusual and novel effect it is proposed that these compounds interact with the anesthetic binding site of the rotifer cholinoceptor ionophore in the open state. It is also proposed that non-ionizing compounds have a general membrane effect only. In addition to anesthesia, atropine and propranolol cause foot paralysis in B. calyciflorus. This other novel effect is also enhanced by acetylcholine as well as decamethonium, a neuromuscular blocker.

Acetylcholine

Gnathostomulida--an enigmatic metazoan phylum from both morphological and molecular perspectives.

On the basis of few and contentious morphological characters Gnathostomulids have been thought to be the sister-group of either the Platyhelminthes or the Syndermata (Rotifera + Acanthocephala). We provide a full 18S rDNA sequence for a species of Gnathostomula and attempt to resolve its position among the Metazoa, on the basis of molecular evidence. Sixty sequences, representing 30 nominal phyla and including new entoproct and gastrotrich sequences, were used to reconstruct phylogenies using maximum-parsimony, neighbor-joining, and minimum evolution models. We were unable to support either of the morphological hypotheses outright and, moreover, our data supported more strongly a third possible relationship with the gnathostomulids as a member of the Nematoda + Chaetognatha clade. Superficially, as active benthic, vermiform creatures with sclerotized cuticular jaws, they fit a predicted ancestral form of the Nematoda + Chaetognatha clade and, as such, would arguably be members of the Ecdysozoa. The molecular data at least call for a reevaluation of the morphological data and a denser sampling of the lesser phyla. Data from morphology and molecules act synergistically in estimating phylogeny; morphology alone provided limited phylogenetic signal and alternative phylogenetic hypotheses, whereas the molecular solution suggested an alternative topology which, when interpreted in the light of comparative anatomy, may suggest previously unconsidered possibilities.

Acanthocephala

Action of malathion plus lindane pesticide on crustacean populations.

The effects of malathion + lindane biocide on Moina and Acanthocyclops populations were tested under experimental conditions. The LD50,24 for Moina is 0.00034 ml.liter-1. Percentages of Acanthocyclops surviving different biocide doses within the range 0.0002-0.0006 ml.liter were not significantly different from each other. The biocide dose used by the farmers in rice fields in the Ebro River Delta (0.00057 ml.liter-1) against crustaceans is high enough to control cladoceran and copepod populations. Here it is suggested that a lower dose would also be successful. Recovery of the zooplankton populations in rice fields after biocide use is initially accomplished by Rotifera spp. and then by crustaceans. This takes place mainly through the irrigation channel.

Analysis of Variance

18S rRNA data indicate that Aschelminthes are polyphyletic in origin and consist of at least three distinct clades.

The Aschelminthes is a collection of at least eight animal phyla, historically grouped together because the absence of a true body cavity was perceived as a pseudocoelom. Analyses of 18S rRNA sequences from six Aschelminth phyla (including four previously unpublished sequences) support polyphyly for the Aschelminthes. At least three distinct groups of Aschelminthes were detected: the Priapulida among the protostomes, the Rotifera-Acanthocephala as a sister group to the protostomes, and the Nematoda as a basal group to the triploblastic Eumetazoa.

Acanthocephala

Studies on the biodegradation of nonionic surfactants applied in the polyester fiber industry. II. Effect of activated sludge load on microorganisms.

The effect of selected nonionic surfactants Cirrasol FP, Cirrasol SF 200 and Cirrasol TCS on activated sludge organisms purifying wastes from the polyestre fibre industry was studied. The toxic effect of the mentioned surfactants towards bacteria, protozoa, rotifera and nematoda occurred at loads exceeding 0.32 mg surfactant/mg d.w./day and in periods of large fluctuations of the employed loads. Morphological changes of activated sludge flocs are also observed in similar conditions.

Adaptation, Physiological

Light and electron microscopic studies on the excretory system of Macrobiotus richtersi Murray, 1911 (Eutardigrada).

The excretory system of Macrobiotus richtersi consists of one dorsal and two lateral components and shows a high degree of structural complexity. In each of these a tricellular external lobe and a column can be distinguished, the two parts being connected distally. The surface of the lobe cells is increased by deep basal infoldings and fingerlike processes which form a labyrinth next to the basal lamina. Their cytoplasm contains numerous mitochondria, a well developed rough endoplasmic reticulum, dictyosomes, and granules in amounts depending on the physiological state of the animal. Excretory crystals occur in caveolae located in the lobe: between the fingershaped processes of the cell and in the space enclosed by the basal lamina on one side and the column on the other. The column faces an extracellular channel meandering along its whole length which is surrounded on the outside by a basal lamina. Morphologically the column is similar to the protonephridial channel of Rotifera. At the ultrastructural level, the cytoplasm of the column shows numerous mitochondria, rough endoplasmic reticulum, lysosomes, and a well developed Golgi apparatus. The lumen of the channel is coated by glycocalyx. At the base of the column several small cells form the proximal part of a duct that communicates with the gut. The morphology and ultrastructure of the excretory system of M. richtersi have been compared with similar a system in Isohypsibius megalonyx (Greven, 1979), and on these grounds a proposal is put forward to call the excretory organs of Tardigrada "nephridia" instead of "Malpighian tubules" .

Animals