Integrative phylogenetic, phylogeographic and morphological characterisation of the Unio crassus species complex reveals cryptic diversity with important conservation implications.

The global decline of freshwater mussels and their crucial ecological services highlight the need to understand their phylogeny, phylogeography and patterns of genetic diversity to guide conservation efforts. Such knowledge is urgently needed for Unio crassus, a highly imperilled species originally widespread throughout Europe and southwest Asia. Recent studies have resurrected several species from synonymy based on mitochondrial data, revealing U. crassus to be a complex of cryptic species. To address long-standing taxonomic uncertainties hindering effective conservation, we integrate morphometric, phylogenetic, and phylogeographic analyses to examine species diversity within the U. crassus complex across its entire range. Phylogenetic analyses were performed using cytochrome c oxidase subunit I (815 specimens from 182 populations) and, for selected specimens, whole mitogenome sequences and Anchored Hybrid Enrichment (AHE) data on âˆ¼ 600 nuclear loci. Mito-nuclear discordance was detected, consistent with mitochondrial DNA gene flow between some species during the Pliocene and Pleistocene. Fossil-calibrated phylogenies based on AHE data support a Mediterranean origin for the U. crassus complex in the Early Miocene. The results of our integrative approach support 12 species in the group: the previously recognised Unio bruguierianus, Unio carneus, Unio crassus, Unio damascensis, Unio ionicus, Unio sesirmensis, and Unio tumidiformis, and the reinstatement of five nominal taxa: Unio desectusstat. rev., Unio gontieriistat. rev., Unio mardinensisstat. rev., Unio nanusstat. rev., and Unio vicariusstat. rev. Morphometric analyses of shell contours reveal important morphospace overlaps among these species, highlighting cryptic, but geographically structured, diversity. The distribution, taxonomy, phylogeography, and conservation of each species are succinctly described.

Personality Influences Risk of Parasitism in Fish.

Influence of fish personality on infection rate is poorly studied. In the experiments on young-of-the-year Oncorhynchus mykiss and cercariae of the trematode Diplostomum pseudospathaceum, we tested the hypothesis that infection rate differs between more and less active ("bold" and "shy") fish. Will individual differences in infection persist upon re-infection? Fish serve as a second intermediate host for this trematode. Positive correlation was found between the results of consecutive infections. Accumulation of parasites with successive infections leads to an aggregated distribution of D. pseudospathaceum among the hosts, affecting individual fitness and polymorphism in fish populations. Persistent individual differences in parasite burden among fish and, as a result, vulnerability for predators confirms the role of parasites as an important factor of natural selection.

Freshwater mussels (Anodonta anatina) reduce transmission of a common fish trematode (eye fluke, Diplostomum pseudospathaceum).

Recent results suggest that bivalves can play an important role in restraining the spread of various aquatic infections. However, the ability of mussels to remove free-living stages of macroparasites and reduce their transmission is still understudied, especially for freshwater ecosystems. We investigated the influence of the common freshwater mussel (Anodonta anatina) on the transmission of a trematode (eye fluke, Diplostomum pseudospathaceum), which frequently infects fish in farms and natural habitats. In our experiments, mussels caused a significant decrease (P < 0·001) in the abundance of trematode free-living stages, from 6520 to 1770 cercariae L-1 on average (about 4-fold in 2 h). Individual clearance rates of mussels were 0·6‒3·7 L per hour (mean 1·9). These tests were followed by experimental infections of rainbow trout (Oncorhynchus mykiss) with different doses of D. pseudospathaceum cercariae in the presence or absence of mussels. Exposure of fish to cercariae in the presence of mussels significantly (P < 0·05) reduced the infection intensities in fish (by 30-40%) at all exposure doses. Our results indicate that freshwater bivalves can markedly reduce local cercariae densities and could be useful in mitigation of trematodoses harmful to fish farming.

Factors affecting between-lake variation in the occurrence of epidermal papillomatosis in roach, Rutilus rutilus (L.).

The theory of island biogeography predicts that the probability of a species occupying an island depends on a dynamic equilibrium between extinction and colonization. Epidermal papillomatosis is a disease manifesting as skin tumours on fish. We studied the factors affecting the occurrence of the disease in roach, Rutilus rutilus (L.), in 34 lakes. The results of discrimination analysis suggest that maximum depth, percentage of the drainage area of the lake covered by lakes in the vicinity and altitude best identified diseased lakes. Comparison of diseased and non-diseased lakes revealed that lake area could also be regarded as a variable contributing to the occurrence of the disease. The sampling date, proportion of males and mean length of fish did not discriminate between the lakes. The probability of the disease occurring was highest in large, deep, low-altitude lakes which had a high percentage of lakes in their vicinity. Thus, the results indicate that the colonization and extinction processes probably contribute to the occurrence of papillomatosis in roach, as predicted by the theory. Furthermore, the large natural variation in the occurrence of the disease could mask possible environmental effects and between-lake comparisons should be interpreted with caution when using the fish-papillomatosis system as an indicator of environmental stress.

The effects of treated effluents on the intensity of papillomatosis and HSP70 expression in roach.

Epidermal papillomatosis in fish has been proposed as an indicator of environmental stress but experimental evidence of connection between contaminants and papillomatosis in fish is scarce. We studied changes in the intensity of epidermal papillomatosis and the expression of heat shock protein 70 (HSP70) in roach, Rutilus rutilus, exposed to treated pulp mill and municipal effluents. In male roach, the increase in papillomatosis intensity was higher in fish exposed to 15% than in fish exposed to 1.5% concentration of municipal effluent. No differences were observed in papillomatosis development in females, or in HSP70 expression. In all the experiments conducted, the increasing effect of effluents seemed to be more pronounced in male fish suggesting that sex-related factors affected the intensity of papillomatosis after exposure to effluents. The present results indicate that environmentally relevant concentrations of municipal effluents may be contributing to the development of papillomatosis in fish.

Two Novel Microsporidia in Skeletal Muscle of Pike-Perch Sander lucioperca and Burbot Lota lota in Finland.

Two new species of Microsporidia were recognized in skeletal muscle of freshwater fishes from Finland. Myosporidium spraguei n. sp. from pike-perch Sander lucioperca occurred as mature spores within sporophorous vesicles (SPVs) within a xenoma. The ovoid spores were 3.8 µm long and 2.4 µm wide, based on transmission electron micrographs (TEM). The exospore and endospore were equally thick, the nucleus was monokaryotic and the polar filament was isofilar with 12 coils in a single rank, entirely adjacent to the prominent posterior vacuole. Small subunit (SSU) rDNA sequence confirmed the presence of M. spraguei n. sp. in burbot Lota lota . The second species, Microsporidium luciopercae n. sp., also from pike-perch, occurred within SPVs that occupied only a fraction of the volume of the otherwise intact myocyte; no xenoma was produced. Myocyte degeneration and necrosis occurred as mature spores dispersed into direct contact with the sarcoplasm. The ovoid spores were 4.6 µm long and 2.8 µm wide (based on TEM); they were monokaryotic and the polar filament was isofilar with 25 coils in a single rank in the posterior of the spore. The exospore was relatively thin with an irregular profile. Neither infection elicited an inflammatory response, although degenerate spores were observed within host cells, suggesting phagocytosis. Phylogenetic analysis of SSU sequences placed both organisms on distinct clades within the Marinosporidia.

Association between environmental stress and epidermal papillomatosis of roach Rutilus rutilus.

We studied the association between environmental stress and epidermal papillomatosis of roach Rutilus rutilus L. in Finnish waters using a 'matched pairs' design. Populations impacted by industrial and/or sewage effluents were compared to reference populations from pristine sites. We examined both the prevalence (proportion of diseased fish) and intensity (number of scales covered by tumors) of the disease. Results of Generalized Linear Mixed Models (GLMM) indicated that the risk of papillomatosis was 7.5 times higher in males than females, and increased 1.3 times for every 10 mm increment in fish length. We controlled for the possible effects of fish size, sex and temporal variation through sampling procedures and statistical analyses. Mean prevalence of epidermal papillomatosis was 16.6 and 5.8% in impact and reference populations, respectively (10 population pairs; nfish = 1714). Results of GLMM suggested that the risk of being diseased was 2.7 times higher in the impact than reference populations. Thus, the prevalence of epidermal papillomatosis in roach can be used as an indicator of environmental stress. Results of Linear Mixed Models indicated no difference in the intensity of the disease between impact and reference populations (5 population pairs; nfish = 73; mean+/-SE 10.7+/-1.8 and 11.7+/- 2.9 scales, respectively), although prevalence was higher in impact populations in those 5 population pairs. The possible relationship between environmental stress and intensity of epidermal papillomatosis in natural roach populations remains to be demonstrated.

Enzyme-assisted synthesis of a bivalent high-affinity dodecasaccharide inhibitor of mouse gamete adhesion. The length of the chains carrying distal alpha 1,3-bonded galactose residues is critical.

Proposing to study the molecular mechanisms of mouse gamete adhesion with the aid of high affinity adhesion inhibitors of saccharide nature, we report here the enzymatic synthesis of a bivalent oligosaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (4), consisting of two long arms that link together two distal alpha 1,3-galactose residues. Binding data reported elsewhere (E. Litscher et al., Biochemistry, 1995, 34, 4662-4669) show that 4 is a high affinity inhibitor of mouse gamete adhesion in vitro (IC50 = 9 microM), while a related octasaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc, consisting of two short arms is of very low inhibitory activity. The data highlight the importance of the two alpha-galactose residues of 4, and the length of the sugar chains joining them.

Immunocompetence, developmental stability and wingspot size in the damselfly Calopteryx splendens L.

Calopteryx splendens males exhibit a remarkable variation in wing pigmentation both within and between populations. In this study, we examined whether the wingspots of male C. splendens are related to male quality. We measured the nylon implant encapsulation rate for 85 males and found that males with larger wingspots had a faster encapsulation rate, indicating a better immunocompetence. We also found that the encapsulation rate was positively correlated with the density of haemocytes in the haemolymph. Another measurement of male quality, fluctuating asymmetry of wingspots, correlated negatively with the size of the wingspots. Males with asymmetrical wingspots also had lower encapsulation rates than more symmetrical males. Our results suggest that the size of wingspot is an indicator of male quality in C. splendens.

Influence of trematode parasitism on the growth of a bivalve host in the field.

Trematode-induced gigantism of molluscs, enhanced growth of trematode-parasitised individuals, has been observed in many laboratory studies. This study reports the effect of the sterilising trematode, Rhipidicotyle fennica, on the growth of the freshwater clam Anodonta piscinalis under field conditions. In addition to single infections (prevalence 44%), a few clams (3%) were infected with both R. fennica and Rhipidicotyle campanula. Parasite-induced gigantism was not found; parasites lowered host growth. The decreased in growth was correlated with the quantity of parasite material. Clams with double infections grew the least, although they did not differ significantly from hosts with a heavy single species infection. The growth of the experimental clams was lower than that of undisturbed control clams. Trematode-induced gigantism of molluscs in the field, in general, remains undemonstrated.

Quantity of sporocysts and seasonality of two Rhipidocotyle species (Digenea: Bucephalidae) in Anodonta piscinalis (Mollusca: Bivalvia).

The prevalence of Rhipidocotyle campanula in Lake Kuivasjärvi, northern Finland, was 5.7% (n = 1486) during 1989. The prevalences of R. campanula and R. fennica in Lake Saravesi, central Finland, during 1987 to 1989 were 1.0 and 33.2% (n = 1157), respectively. No clear seasonality in prevalence was found in either species, although miracidia of these digeneans are present only in late summer and autumn. The proportion of infected clams in which the gonad was full of sporocyst tubules was on average 90% for R. campanula and 30% for R. fennica. Clams with only few sporocyst tubules in their gonad were observed most frequently in spring. Old; empty sporocyst ribbons of R. fennica first appeared in the clams during August after the beginning of the cercarial emergence and disappeared slowly thereafter. However, in both species viable sporocyst ribbons with early developmental stages of cercariae were found throughout the year. The emergence of R. fennica cercariae began during July and continued throughout September in Lake Saravesi. The duration of cercariae emergence was found to be up to 72 days in the laboratory. R. campanula cerariae in the northern Lake Kuivasjärvi emerged from mid-June until mid-August.

Mass spectrometric and tandem mass spectrometric behavior of nitrocatechol glucuronides: a comparison of atmospheric pressure chemical ionization and electrospray ionization.

The mass spectrometric (MS) and tandem mass spectrometric (MS/MS) behavior of six nitrocatechol-type glucuronides using atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) was systematically studied, and the effect of operation parameters on the fragmentations are presented. The positive ion APCI- and ESI-MS spectra showed an intense protonated molecule and the respective negative ion spectra a deprotonated molecule with minimal fragmentation. The main fragment ions in the MS/MS spectra of the protonated and deprotonated molecules were [M + H - Glu]+ and [M - H - Glu]-, respectively, formed by the loss of the glucuronide moiety. The measured limits of detection indicated that ESI is a significantly more efficient ionization method than APCI in the negative and positive ion modes for the compounds studied. MS/MS was found to be less sensitive, but more reliable and simple than MS due to the absence of chemical noise.

Solid-phase bromination and Suzuki coupling of 2-carboxyindoles.

As part of an ongoing lead discovery project we have developed a convenient method for the modification and substitution of indole moieties at the 3-position. Selective bromination of three different 2-carboxyindoles was followed by Suzuki cross-coupling with aryl and heteroaryl boronic acids on a Merrifield resin solid-phase. After column chromatography, yields of the 3- substituted indoles ranged from 42-98%.

Effect of nitro groups and alkyl chain length on the negative ion tandem mass spectra of alkyl 3-hydroxy-5-(4'-nitrophenoxy) and alkyl 3-hydroxy-5-(2', 4'-dinitrophenoxy) benzoates

Tandem mass spectrometric behaviour was studied for a small combinatorial library of alkyl 3-hydroxy-5-(4'-nitrophenoxy) benzoates (A1-A5) and alkyl 3-hydroxy-5-(2', 4'-dinitrophenoxy) benzoates (B1-B5). The spectra were recorded by negative ion electrospray low-energy collision induced dissociation (CID) tandem mass spectrometry. The product ion spectra of [M - H](-) of the benzoates A1-A5 are similar, as are those of benzoates B1-B5. However, the spectra of the B series compounds differ significantly from those of the A series owing to the second electron-withdrawing nitro substituent in the B compounds. In addition, the length of the alkyl chain has an effect on the fragmentation. However, both series of compounds exhibit an abundant nitrophenoxy ion formed by the loss of 3-hydroxybenzoate. This is at m/z 138 in A1-A5 and at m/z 183 in B1-B5. A precursor ion scan of the nitrophenoxy ion provides a rapid method to identify the synthesised compounds in this type of combinatorial mixture. Copyright 1999 John Wiley & Sons, Ltd.

Production of human UDP-glucuronosyltransferases 1A6 and 1A9 using the Semliki Forest virus expression system.

Human UDP-glucuronosyltransferases (UGTs) 1A6 and 1A9 were expressed using Semliki Forest virus (SFV) vectors. Infection of chinese hamster lung fibroblast V79 cells with recombinant SFV-UGT viruses resulted in efficient protein expression as detected by metabolic labeling, Western blot analyses and immunofluorescence microscopy. The expression of UGT 1A6 and UGT1A9 in the SFV-infected cells was approximately two fold higher than in a stable V79 cell line. No UGT signal was detected in noninfected cells. In addition, SFV-UGT viruses also efficiently infected other mammalian cells, such as baby hamster kidney (BHK), chinese hamster ovary (CHO) and human lung (WI-26 VA4) cells leading to high production of recombinant enzyme. The measurement of enzyme activities and kinetic parameters using p-nitrophenol and nitrocatechol (entacapone) as substrates for UGT1A6 and UGT1A9, respectively, showed that the overall kinetic properties of the enzymes produced by the two systems were similar. We conclude that the SFV expression system represents an efficient, fast and versatile method for production of metabolic enzymes for in vitro assays.

Effect of starvation on parasite-induced mortality in a freshwater snail (Potamopyrgus antipodarum).

The level of host exploitation is expected, under theory, to be selected to maximise (subject to constraints) the lifetime reproductive success of the parasite. Here we studied the effect of two castrating trematode species on their intermediate snail host, Potamopyrgus antipodarum. One of the trematode species, Microphallus sp., encysts in the snail host and the encysted larvae "hatch" following ingestion of infected snails by birds. The other species, Notocotylus gippyensis, by contrast, releases swimming larvae; ingestion of the snail host is not required for, and does not aid, transmission to the final host. We isolated field-collected snails for 3 months in the laboratory, and followed the survival of infected and uninfected snails under two conditions: not fed and fed ad libitum. Mortality of the infected hosts was higher than mortality of the uninfected ones, but the response to starvation treatment was parasite species specific. N. gippyensis induced significantly higher mortality in starved snails than did Microphallus. Based on these results, we suggest that host exploitation by different species of trematodes may depend on the type of transmission. Encysting in the snail host may select for a reduced rate of host exploitation so as to increase the probability of transmission to the final host.

Neural network modeling for estimation of the aqueous solubility of structurally related drugs.

The ability of neural network models to predict aqueous solubility within series of structurally related drugs was evaluated. Three sets of compounds representing different drug classes (28 steroids, 31 barbituric acid derivatives, and 24 heterocyclic reverse transcriptase inhibitors) were studied. Topological descriptors (connectivity indices, kappa indices, and electrotopological state indices) were used to link the structures of compounds with their aqueous solubility. Separate models were built for each class of drugs using back-propagation neural networks with one hidden layer and five topological indices as input parameters. The effect of network size and training time on the prediction ability of the network was studied by the leave-one-out (LOO) procedure. In all three compound groups a neural network structure of 5-3-1 was optimal. To avoid chance effects, artificial neural network (ANN) ensembles (i.e.; averaging neural network predictions over several independent networks) were used. The cross-validated squared correlation coefficient (Q2) for 10 averaged predictions was 0.796 in the case of the steroid set. The corresponding standard error of prediction (SDEP) was 0.288 log units. For the barbiturates, Q2 and SDEP were 0.856 and 0.383, respectively, and for the RT inhibitors, these parameters were 0.721 and 0.401, respectively. The results indicate that neural networks can produce useful models of the aqueous solubility of a congeneric set of compounds, even with simple structural parameters.

Iron(III)-chelating properties of the novel catechol O-methyltransferase inhibitor entacapone in aqueous solution.

The iron(III) complex formation of entacapone, a novel catechol O-methyltransferase (COMT) inhibitor, has been studied at 25 degrees C in aqueous 0.1 mol/L NaCl solution by using the electromotive force titration method. Entacapone functions as a bidentate ligand chelating through the catecholate oxygen atoms and forms stable iron(III) complexes with the formation constant of a tris complex: log beta-613 ([FeL3(3-)][H]6+/[Fe3+][H2L]3) = -6.9 +/- 0.1. Distribution curves show that entacapone is highly effective for iron(III) in moderately dilute solution (10(-3) mol/L) whereas in very dilute solution (10(-6) mol/L) the iron hydroxo complexes together with FeL3(3-) dominate under physiological pH 7.4. Comparison of iron(III) species distribution in a competitive two-ligand entacapone-catechol system reveals that the complexation of entacepone is favored at high and low dilution.

Determination of a catechol-O-methyltransferase inhibitor, nitecapone, in human plasma and urine by liquid chromatography.

Methods based on reversed-phase liquid chromatography with amperometric detection have been developed for determination of nitecapone, 3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione, a COMT inhibitor, in human plasma and urine. Nitecapone was extracted with ethyl acetate-hexane mixtures from plasma after acidification with hydrochloric acid and from urine as the tetrabutylammonium ion-pair of its diphenylborate derivative. The recoveries of both methods exceeded 70% and the relative standard deviations for within-day precision were less than 4% and 8% at 50 ng ml-1 and at the quantitation limits, respectively. The methods are selective, sensitive and precise enough for determination of 4-5 ng ml-1 of nitecapone in plasma and urine and are thus suitable for the kind of pharmacokinetic studies exemplified in this paper.

Relationship between immobilised artificial membrane chromatographic retention and the brain penetration of structurally diverse drugs.

Retention factors were determined for a set of 26 drugs, for which brain/blood concentration data are available, using immobilised artificial membrane (IAM) chromatography. The compound set represented acidic, basic and neutral drugs from various structural classes. The relationship between IAM retention and lipophilicity (n-octanol-water partition coefficient Koct), molecular size and acid/base character of the drugs and the relationship between brain distribution and IAM retention and the other parameters were analysed. IAM retention was increased with increases in lipophilicity and solute size, and decreased by the ionisation of acidic groups. Ionisation of basic groups had no significant effect. A three-parameter regression model with log Koct, molecular weight and an indicator parameter for the presence of carboxyl group explained 93% of the variation in log kIAM. The concentration ratio between brain and blood (log BB) was only weakly correlated with the IAM chromatographic retention or n-octanol-water partitioning. Three-parameter models taking ionisation and size into account, in addition to either log Koct or log kIAM, explained about 85% of the variation of log BB in the test set. Although IAM chromatography offers no advantage in these models, it seems to provide a better model than n-octanol-water partitioning for the membrane distribution of ionised compounds.