Haplosporidium raabei n. sp. (Haplosporidia): a parasite of zebra mussels, Dreissena polymorpha (Pallas, 1771).

Extensive connective tissue lysis is a common outcome of haplosporidian infection. Although such infections in marine invertebrates are well documented, they are relatively rarely observed in freshwater invertebrates. Herein, we report a field study using a comprehensive series of methodologies (histology, dissection, electron microscopy, gene sequence analysis, and molecular phylogenetics) to investigate the morphology, taxonomy, systematics, geographical distribution, pathogenicity, and seasonal and annual prevalence of a haplosporidian observed in zebra mussels, Dreissena polymorpha. Based on its genetic sequence, morphology, and host, we describe Haplosporidium raabei n. sp. from D. polymorpha - the first haplosporidian species from a freshwater bivalve. Haplosporidium raabei is rare as we observed it in histological sections in only 0·7% of the zebra mussels collected from 43 water bodies across 11 European countries and in none that were collected from 10 water bodies in the United States. In contrast to its low prevalences, disease intensities were quite high with 79·5% of infections advanced to sporogenesis.

Chromosome analysis of Phyllodistomum folium (Trematoda, Gorgoderidae) infecting three European populations of zebra mussels.

The mitotic chromosome sets of the larval stages of Phyllodistomum folium infecting three European populations of Dreissena polymorpha were studied using conventional Giemsa staining and karyometrical analysis. The karyotype, described herein for the first time, consisted of nine chromosome pairs (2 n=18). The chromosomes were comparatively small and measured from 1.19 micro m to 5.22 micro m. The first and second longest chromosome pairs were, respectively, approximately 20% and 18% of the mean total chromosome set length and were clearly differentiated from the other seven pairs, which gradually decreased in size. Most chromosomes were biarmed with medially or submedially located centromeres; only pairs 1 and 4 contained subterminally located centromeres. No significant differences in the relative length values of corresponding chromosomes were observed between the three populations studied. The main interpopulation differences were detected in the centromeric index values of some corresponding chromosome pairs. These data, when analysed in conjunction with those of other Gorgoderidae, indicate that the karyotype of P. folium and other species in this family do not show any clear affinities with "typical" plagiorchiate chromosome sets.

Characterization of intracytoplasmic prokaryote infections in Dreissena sp. (Bivalvia: Dreissenidae).

This study characterizes intracytoplasmic infections with prokaryote microorganisms in Dreissena sp. (near Dreissena polymorpha) from northeastern Greece and represents the first report of such infections in freshwater bivalves. Light microscope observations of stained tissues revealed basophilic, cytoplasmic inclusion bodies in 87.5% (28/32) of the mussels sectioned. Inclusions in epithelial cells and connective tissues were noted, respectively, in 34.4 and 71.9% of the sample, with 5 mussels (15.6%) having both tissue types infected. Epithelial cell infections were observed in histological sections only in digestive gland tubules and ducts; within tubules, inclusions were present more often in secretory than digestive cells. Connective tissue infections, however, were systemic; among the 32 mussels sectioned, inclusions were found in the gills (65.6%), foot (12.5%), mantle (9.4%), labial palps (6.3%), digestive gland (6.3%), stomach (6.3%), and gonads (3.1%). Cytoplasmic inclusions (maximum dimension, 138 microm) were prominent enough in the gills to be visible in 17.0% of the 247 mussels dissected. Ultrastructurally, prokaryote cells in gill connective tissues were clearly characteristic of Chlamydiales-like organisms, with each intracytoplasmic inclusion containing a loosely packed mixture of elementary, reticulate, intermediate bodies, and blebs. Prokaryote colonies in digestive gland epithelial cells exclusively contained 1 of 4 morphological cell types and were considered Rickettsiales-like. Hexagonal, virus-like particles were present in the cytoplasm of the largest of these Rickettsiales-like prokaryotes. Although host stress was evident from localized cell necrosis and dense hemocyte infiltration, overall infection was fairly benign, with no major, adverse impact on body condition evident among sectioned or dissected mussels. A possible negative effect was partial constriction of gill water tubes, but at the infection intensity observed (typical range 1 to 7 inclusion bodies per section), significant interference with respiration and other metabolic functions of the gills was highly unlikely.

Structural determinants outside the PXDLS sequence affect the interaction of adenovirus E1A, C-terminal interacting protein and Drosophila repressors with C-terminal binding protein.

C-Terminal binding protein (CtBP) interacts with a highly conserved amino acid motif (PXDLS) at the C terminus of adenovirus early region 1A (AdE1A) protein. This amino acid sequence has recently been demonstrated in the mammalian protein C-terminal interacting protein (CtIP) and a number of Drosophila repressors including Snail, Knirps and Hairy. In the study described here we have examined the structures of synthetic peptides identical to the CtBP binding sites on these proteins using NMR spectroscopy. It has been shown that peptides identical to the CtBP binding site in CtIP and at the N terminus of Snail form a series of beta-turns similar to those seen in AdE1A. The PXDLS motif towards the C terminus of Snail forms an alpha-helix. However, the motifs in Knirps and Hairy did not adopt well-defined structures in TFE/water mixtures as shown by the absence of medium range NOEs and a high proportion of signal overlap. The affinities of peptides for Drosophila and mammalian CtBP were compared using enzyme-linked immunosorbent assay. CtIP, Snail (N-terminal peptide) and Knirps peptides all bind to mammalian CtBP with high affinity (K(i) of 1.04, 1.34 and 0.52 microM, respectively). However, different effects were observed with dCtBP, most notably the affinity for the Snail (N-terminal peptide) and Knirps peptides were markedly reduced (K(i) of 332 and 56 microM, respectively) whilst the Hairy peptide bound much more strongly (K(i) for dCtBP of 6.22 compared to 133 microM for hCtBP). In addition we have shown that peptides containing identical PXDLS motifs but with different N and C terminal sequences have appreciably different affinities for mammalian CtBP and different structures in solution. We conclude that the factors governing the interactions of CtBPs with partner proteins are more complex than simple possession of the PXDLS motif. In particular the overall secondary structures and amino acid side chains in the binding sites of partner proteins are of importance as well as possible global structural effects in both members of the complex. These data are considered evidence for a multiplicity of CtBPs and partner proteins in the cell.

Structural determinants in adenovirus 12 E1A involved in the interaction with C-terminal binding protein 1.

The interaction between the C-terminal binding protein 1 (CtBP-1) and purified Ad12 E1A protein has been examined through the use of a combination of biophysical techniques. A fragment equivalent to the 77 C-terminal amino acids of Ad12 E1A (Ad12 77-a.a. E1A) was generated by limited proteolysis of Ad12 266-a.a. E1A at Phe(187) and/or Tyr(189) using chymotrypsin. The impact of deletion of the 189 N-terminal amino acids from E1A on the equilibrium dissociation constant K(d) for binding to CtBP was assessed using ELISA in vitro binding assays and intrinsic fluorescence spectroscopy. Values of K(d) of 4.0 and 38 nM were determined for full-length and truncated forms of E1A, respectively. Circular dichroism spectroscopic studies revealed that the conformation adopted by these polypeptides is dependent on the surrounding environment, which is predominately randomly folded when free in solution, but adopting a more ordered alpha-helical secondary structure in the presence of trifluoroethanol. Using nuclear magnetic resonance (NMR) spectroscopy to examine the interaction between Ad E1A and CtBP it was observed that the chemical shift positions of individual backbone amide nitrogen atoms were well resolved in (15)N-(1)H-HSQC NMR spectra performed on samples of isotopically (15)N-labeled Ad12 77-a.a. E1A. In the presence of CtBP, signals of backbone amide nitrogen atoms displayed increased linewidth consistent with an increase in molecular mass upon binding CtBP. In addition, some signals that have been attributed to Val(254/256) and Leu(259), and reside within the binding site for CtBP on E1A, are shifted in the (15)N- and/or (1)H-dimensions, defining specific contacts between E1A and CtBP. These data suggest that structural determinants in the C-terminal PXDLS binding motif in the rest of exon 2 and in exon 1 all contribute to optimizing the conformation of the binding site on Ad12 E1A for CtBP. However, no interaction was observed between CtBP and truncated Ad12 E1A, which no longer contained the C-terminal binding motif.

The structure of the site on adenovirus early region 1A responsible for binding to TATA-binding protein determined by NMR spectroscopy.

Previous detailed mutational analysis has shown that the binding site on adenovirus (Ad) early region 1A (E1A) for TATA-binding protein (TBP) is located toward the N terminus of conserved region 3 (CR3). Here we demonstrate that synthetic peptides of between 15 and 22 amino acids, identical to amino acid sequences of CR3 present in the larger Ad5 E1A (13 S product) and in both the Ad12 E1A (13 and 12 S products) proteins that lie N-terminal to the zinc finger motif, can disrupt binding of E1A to TBP. These findings suggest that the peptides are biologically active in terms of interacting with TBP and must therefore comprise some, if not all, of the TBP binding site on E1A. The interaction between Ad12 E1A and TBP was confirmed by direct co-precipitation experiments. In 1H NMR studies of CR3 peptides, regular patterns of NOEs were observed from which their conformational preferences in aqueous solution were determined. Both Ad5 and Ad12 peptides were shown to contain regions of helical backbone structure in 50% trifluoroethanol. In each case, the type and intensities of NOE cross-peaks observed correlated best to alpha-helical turns. These helices are more extensive in larger peptides and extend from Glu141 to Val147 and from Arg144 to Pro152 in the full-length Ad5 and Ad12 13S E1A proteins, respectively. The structure of a 19-residue Ad5 CR3 peptide carrying the V147L mutation in the full-length protein that abolishes TBP binding was examined. No significant differences between the substituted and wild type peptides were observed, suggesting that this substitution in the intact protein may cause disruption of global rather than local structures.

Structural determinants present in the C-terminal binding protein binding site of adenovirus early region 1A proteins.

The C-terminal binding protein (CtBP) has previously been shown to bind to a highly conserved six-amino acid motif very close to the C terminus of adenovirus early region 1A (Ad E1A) proteins. We have developed an enzyme-linked immunosorbent assay that has facilitated the screening of synthetic peptides identical or similar to the binding site on Ad E1A for their ability to bind CtBP and thus inhibit its interaction with Ad12 E1A. It has been shown that amino acids both C-terminal and N-terminal to the original proposed binding site contribute to the interaction of peptides with CtBP. Single amino acid substitutions across the binding site appreciably alter the Kd of the peptide for CtBP, indicative of a marked reduction in the affinity of the peptide for CtBP. The solution structures of synthetic peptides equivalent to the C termini of both Ad5 and Ad12 E1A and two substituted forms of these have been determined by proton NMR spectroscopy. Both the Ad12 and Ad5 peptides dissolved in trifluoroethanol/water mixtures were found to adopt regular secondary structural conformations seen as a series of beta-turns. An Ad12 peptide bearing a substitution that resulted in only very weak binding to CtBP (Ad12 L258G) was found to be random coil in solution. However, a second mutant (Ad12 V256K), which bound to CtBP rather more strongly (although not as well as the wild type), adopted a conformation similar to that of the wild type. We conclude that secondary structure (beta-turns) and an appropriate series of amino acid side chains are necessary for recognition by CtBP.

Regeneration of the binding properties of adenovirus 12 early region 1A proteins after preparation under denaturing conditions.

Adenovirus 12 early region 1A (Ad12 E1A) was expressed in Escherichia coli. Protein was purified in good yield in the presence of 8 M urea and then renatured by dialysis against dilute NH4HCO3 buffer. The affinity of this protein for pRb, C-terminal binding protein (CtBP), TATA binding protein (TBP), and SUG1 was similar to, or greater than, that of Ad12 E1A prepared by immunoaffinity chromatography under nondenaturing conditions. While the binding of the 266- and 235-amino-acid (aa) E1A components to TBP showed similar characteristics the larger E1A protein had a higher affinity for CtBP, pRb, and SUG1. Using nuclear magnetic resonance (NMR) spectroscopy it was shown that structural perturbations occurred in the 266-aa protein in the presence of Zn2+ consistent with binding--no such changes were seen for the 235-aa protein. Limited proteolysis of the 266- and 235-aa E1A proteins gave rise to comparable polypeptide products, suggesting overall similarities in structure. However, the different affinities of the 266- and 235-aa proteins for the partner proteins and the differences seen in the NMR spectra from the two proteins suggested structural differences.

Field and laboratory studies of zebra mussel (Dreissena polymorpha) infection by the ciliate Conchophthirus acuminatus in the Republic of Belarus.

This study quantifies the infection prevalence and intensity of the European, commensal, host-specific ciliate Conchophthirus acuminatus (Scuticociliatida: Conchophthiridae) in five zebra mussel populations within the Republic of Belarus. Laboratory and field experiments were also conducted to assess variables affecting infection. C. acuminatus was found in zebra mussels in all five waterbodies sampled: Naroch, Myastro, and Lukomskoe Lakes, Skema Stream, and in the Svisloch River. Prevalence was always 100%, with the exception of shallow areas (

Synthesis, physical and biological properties of lithocholyl-lysyl-fluorescein: a fluorescent monohydroxy bile salt analogue with cholestatic properties.

We have synthesised and characterised a fluorescent monohydroxy bile salt analogue, lithocholyl-lysyl-fluorescein and compared its physical and biological properties with those of lithocholate, glycolithocholate, sulpholithocholate, lithocholic acid glucuronide and taurocholate. The synthetic method used excess N-epsilon-CBZ-L-lysine methyl ester hydrochloride and lithocholic acid via N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinolone (EEDQ) to give lithocholyl-lysine. Lithocholyl-lysyl-fluorescein (LLF) was then prepared using equimolar amounts of lithocholyl-lysine and fluorescein isothiocyanate (FITC) in bicarbonate buffer. LLF retained an apple green fluorescence, similar to that of fluorescein. Unlike lithocholate, the critical micellar concentrations (CMCs) of LLF, glycolithocholate (GLC), lithocholic acid glucuronide (LG) and sulpholithocholic acid (SLC) were similar. HPLC retention times (tRs) of LLF and GLC were similar with a ratio of LLF/GLC of 1.05. In contrast, the tR of SLC (6.52 min) but not of LG (21.2 min) was more comparable to that of taurocholate (5.73 min). In rats under pentobarbital anaesthesia, the plasma half-life (t(1/2alpha)) (min) was 4.5 +/- 1.3 (n = 6) for LLF, 2.9 +/- 0.4 (n = 5) for [14C]sulpholithocholate (14C-SLC) and 4.3 +/- 0.3 (min) for [14C]lithocholic acid glucuronide (14C-LG). Plasma clearances of 14C-SLC, LLF and 14C-LG were 15.5 +/- 2.2 (n = 6), 18.1 +/- 4.2 (n = 6) and 17.8 +/- 0.5 ml/min/kg (n = 6) (P = 0.15), respectively. Biliary excretion in bile-fistula rats gave cumulative 20 min biliary output as a percentage of injected dose as follows: LLF, 71.6 +/- 0.8% (n = 10); 14C-SLC, 75.5 +/- 2.8% (n = 6) and 14C-LG, 61.7 +/- 0.5% (n = 6) (P = NS). Peak biliary excretion rates, given as % dose/2 min, were 10.2 +/- 0.3 for LLF, 13.5 +/- 0.6 for 14C-SLC and 12.8 +/- 0.4 for 14C-LG. In another group of bile-fistula rats, a 3.0 micromol/500 microl saline i.v. bolus of LLF caused a 15.4 +/- 1.9% decrease in bile flow and, similarly, sodium lithocholate in a solution of albumin caused a 17.9 +/- 1.8% (P = NS) diminution in bile flow. Despite the similar cholestatic properties of LLF and lithocholate, LLF was more soluble than lithocholate, with a relative retention time on HPLC similar to that of GLC. LLF is a divalent 'unipolar' anionic fluorescent monohydroxy bile salt analogue with physical, biological and cholestatic properties that are similar to those of lithocholate, glycolithocholate and their derivatives and thus offers a potentially useful probe for studying mechanisms of monohydroxy bile salt-induced cholestasis at the hepatocellular level.

Use of stream width for determining the dosage rates of Bacillus thuringiensis var. israelensis for larval black fly (Diptera: Simuliidae) control.

Data from several operational black fly abatement programs using Bacillus thuringiensis var. israelensis (B.t.i.) in New York State's Adirondack Mountains were analyzed to determine what the concentration of formulation in the stream might have been if application rates were determined by a stream's width rather than its discharge. There was a high correlation (magnitude of R = 0.87) between discharge and width measured at 315 treatment sites. Had the applications been based upon the stream width, 96% of the actual concentrations in the streams would have been within plus or minus a factor of 5 and none exceeding a factor of 10, a range in which B.t.i. remains environmentally safe and effective.

Low affinity interactions of GDPbetaS and ribose- or phosphoryl-substituted GTP analogues with the heterotrimeric G protein, transducin.

We have examined the effects of three commonly used classes of guanine nucleotide analogues on the retinal G protein, transducin (Gt), and found them to be quite different from those that might be expected from results with other GTP-binding proteins. The most surprising results were with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS); rather than inhibiting activation of Gt, GDPbetaS addition activated Gt as a result of a trace contaminant. Even when the contaminant levels were reduced 5-fold by chromatography, its effects dominated those of GDPbetaS, which binds Gt at least 1500-fold more weakly than guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The affinity of Gt for GDP was found to be at least 300-fold weaker than for GTPgammaS, while the affinities of GTP and GTPgammaS were similar. Ribose-modified GTP analogues, including 2'(3')-O-(N-methylanthraniloyl) GTP (mantGTP), 2'(3')-O-[(2-aminoethyl)carbamyl] GTP (edGTP), and adducts of fluorescein 5-isothiocyanate and rhodamine B-isothiocyanate with edGTP, interacted extremely weakly, if at all, with the GTP binding site of the alpha subunit of Gt. They were neither effective activators of Gt nor effective inhibitors of activation by GTP or GTPgammaS. A gamma-phosphoryl-modified analogue, an adduct of GTPgammaS and (5-(2(iodoacetyl)aminoethyl)amino)naphthalene-1-sulfonic acid (dnsGTP), also activated Gt weakly, if at all, and did not inhibit its activation. The exclusion of these analogues points to the highly restrictive and specific nature of the GTP binding site of Gt, in contrast to those of numerous other GTP-binding proteins which are potently activated or inhibited by these analogues.

Ras binding to a C-terminal region of GAP.

Using fluorescence spectroscopy we have identified a binding region for Ras on the GTPase activating protein (GAP) lying within residues 715-753. A synthetic peptide Y922, corresponding to residues 716-753 of GAP binds to wild type Ras showing 3.3-fold higher affinity for the GTP- over the GDP-bound forms of Ras. Binding is stabilised by Mg2+, although Y922 does not stimulate the GTPase activity of Ras. Peptide binding to the Y32A and Y40F Ras mutants showed equal affinity for both GDP- and GTP-bound forms, with binding to Y32A.GDP abolished in the absence of Mg2+. These results suggest that Y922 mimics the in vivo interactions shown by the intact p120GAP protein and provide the first direct demonstration of Ras interaction with GAP in the region 715-753.

Conformational differences between complexes of elongation factor Tu studied 19F-NMR spectroscopy.

An analogue of elongation factor Tu (EF-Tu) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorotyrosine. The 19F-NMR spectra of the binary complexes of this protein with GDP, GTP and elongation factor Ts (EF-Ts) and the ternary complexes EF-Tu.GDP.aurodox and EF-Tu.GDP.EF-Ts were measured. EF-Tu contains ten tyrosine residues and all of the complexes studied gave complex 19F spectra with overlapping resonances. EF-Tu.GDP gave a spectrum in which two signals were markedly different from those shown by the other complexes, the two resonances being shifted downfield by at least 3.4 ppm and 0.9 ppm relative to their shifts in the other complexes. Such large downfield shifts can be explained by second-order electric field shielding effects resulting from these two tyrosine residues being in a sterically constrained environment in EF-Tu.GDP and with the steric restraints being released in all of the other complexes. The X-ray diffraction structure of EF-Tu.GDP shows that Tyr87 in the N-terminal domain (domain I) and Tyr309 in the C-terminal domain (domain III) are both buried within the protein and are close to each other: these residues are in regions of EF-Tu previously implicated in the structural changes between EF-Tu.GDP and EF-Tu.GTP by other workers. If these tyrosine residues correspond to the two downfield resonances of the spectra of EF-Tu.GDP, the results from the 19F-NMR would be consistent with these earlier indications that domain I interacts closely with domain III in EF-Tu.GDP and that the amino acids between Gly83 and Gly100 are an important part of this interaction. For all the other complexes studied, these tyrosines are in a less sterically crowded environment consistent with a weaker interaction between the two domains. The 19F-NMR spectrum of the trypsin-cleaved product of EF-Tu.GDP, from which the X-ray diffraction structural data have been obtained, shows no significant differences from the native protein so that trypsin cleavage causes no large changes in the protein's structure.

Impact of the black fly (Diptera: Simuliidae) control agent Bacillus thuringiensis var. israelensis on chironomids (Diptera: Chironomidae) and other nontarget insects: results of ten field trials.

Except for moderate mortality among filter-feeding chironomids, Rheotanytarsus spp., the results of 10 field trials with Bacillus thuringiensis var. israelensis (B.t.i.) indicated a wide margin of safety to the chironomid community and other stream nontarget insects. Mayflies, caddisflies and 2 other types of chironomids, i.e., tube-dwelling (Chironominae) and surface-dwelling, caseless larvae (mix of Chironominae, Diamesinae, Orthocladiinae, Tanypodinae), did not appear to be affected. The susceptibility of filter-feeding chironomids was considerably less than black flies; for example, 4 months of data collected during an operational black fly control program indicated a mean (+/- 95% CI) mortality among Rheotanytarsus larvae of 23 (15-32)% vs. 98 (97-99)% among black flies. Although clearly demonstrating the potential of adverse impact on filter-feeding chironomids in operational black fly programs, these trials also confirmed the narrow impact of B.t.i. on the overall stream insect community.

Mermithid parasitism of black flies (Diptera: simuliidae).

Mermithid nematodes are common parasites of black flies and play a significant role in the natural regulation of these medically important insects. Infection levels tend to he moderate and perennial, with epizootics rare and highly localized. Mermithid parasitism almost invariably results in the death of the black fly, and thus considerable attention has focused on the potential of these nematodes as biocontrol agents. Early instar black fly larvae appear most susceptible to infection, and integumental penetration hy mermithid preparasites is the only known mode of entry. Postparasitic nematodes typically emerge before host pupation. However, carryover of parasitism into adult simuliids is an important mechanism for local dispersal and recolonization of upstream areas. Following emergence, the mermithids molt to the adult stage. Copulation ensues, the females then laying eggs which eventually give rise to the next generation of infective preparasites. The number of described species is conservatively estimated at 35-40, with most species within the genera Mesomermis, Gastromermis, and Isomermis. The taxonomy of this group of mermithids is a challenging and little explored area. Host-specificity statements, therefore, must be made cautiously because of these systematic problems and others within the Simuliidae. In most instances, temporal and spatial factors limit the host range of these mermithids among simuliid species. Differential susceptibilities anmng larvae concurrently present within the same microhabitat probably reflect varying degrees of host attractiveness and behavioral-physiological resistance. Effects of parasitism on the host may include prevention of metamorphosis, sterility, intersexual development, and behavior modification. Evaluation of the technical feasibility of mermithid control of black flies has been stymied by the limitations of current inoculum-production technology. Continued advances in in vivo and in vitro culture methods are required to accelerate the research process.