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.

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.

Parasite-induced aggression and impaired contest ability in a fish host.

BACKGROUND: Success of trophically transmitted parasites depends to a great extent on their ability to manipulate their intermediate hosts in a way that makes them easier prey for target hosts. Parasite-induced behavioural changes are the most spectacular and diverse examples of manipulation. Most of the studies have been focused on individual behaviour of hosts including fish. We suggest that agonistic interactions and territoriality in fish hosts may affect their vulnerability to predators and thus the transmission efficiency of trophically transmitted parasites. The parasite Diplostomum spathaceum (Trematoda) and juvenile rainbow trout, Oncorhynchus mykiss, were used to study whether infection can alter aggression rates and territorial behaviour of intermediate fish hosts. RESULTS: The changes in behaviour of rainbow trout, Oncorhynchus mykiss, infected with an eye fluke Diplostomum spathaceum (Trematoda), was monitored over the course of an experimental infection for 1.5 months. At the beginning of their development, not yet infective D. spathaceum metacercariae decreased the aggressiveness of rainbow trout. By the time that metacercariae were fully infective to their definitive hosts, the aggressiveness increased and exceeded that of control fish. Despite the increased aggressiveness, the experimentally infected fish lost contests for a territory (dark parts of the bottom) against the control fish. CONCLUSIONS: The results obtained indicate that the parasitized fish pay the cost of aggressiveness without the benefit of acquiring a territory that would provide them with better protection against predators. This behaviour should increase transmission of the parasite as expected by the parasite manipulation hypothesis.

Penetration and migration success of Diplostomum spp. cercariae in Arctic charr.

We exposed 2 groups of young-of-the-year Arctic charr (Salvelinus alpinus) singly to 54 +/- 2 (mean +/- SE) Diplostomum spp. cercariae that had emerged from 4 randomly sampled snail hosts (Lymnaea stagnalis). The rearing tanks of the fish received Diplostomum spp. cercariae via the incoming water; therefore, 18 of the 36 fish had parasites in their eyes before the experimental exposure. Of the Diplostomum spp. cercariae presented to the fish, 19% penetrated and 46% of those that had penetrated the fish migrated successfully to the lens of the fish eye. The migration success of Diplostomum spp. from the site of penetration to the fish eye lens was lower in the previously parasitized (37.0 +/- 8.7% [mean +/- SE] adjusted with the number of penetrated cercariae) than in the unparasitized fish (55.3 +/- 8.8%) and differed between the individual snail host from which the cercariae had emerged. In addition, the migration success of Diplostomum spp. decreased with an increase in the number of the cercariae that penetrated the fish. At the individual snail host level, there seemed to be a trade-off between penetration and migration ability of the cercariae. The results indicate that success of Diplostomum spp. in penetration and especially in migration to the fish eye is affected by both the molluscan first intermediate host and the piscine second intermediate host.

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.

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.

Burrowing behaviour affects Paraergasilus rylovi abundance in Anodonta piscinalis.

Burrowing depth may affect predation rate, feeding ability and reproduction in bivalve clams. We studied the effect of burrowing depth on the abundance of the ergasilid Paraergasilus rylovi in the freshwater bivalve clam Anodonta piscinalis. We transplanted uninfected clams to a lake where they were allowed to choose their preferred burrowing depth, and were exposed naturally to copepodids of the parasite. There was a significant positive correlation between proportionate burrowing depth (PBD) and the abundance of P. rylovi at the end of the 17-day experiment, the deeper-burrowed clams harbouring more P. rylovi. Original PBD (0%, 50%, 100%) did not influence the final PBD or parasite abundance. Clam length affected PBD, smaller clams burrowing deeper, but it did not affect parasite abundance. Infected experimental clams and naturally-burrowed uninfected clams, both originating from the same lake, did not differ in their mean PBD. This indicated that burrowing of the experimental clams affected parasitism rather than the parasites altering burrowing of the clams. In line with the experimental result, we observed a significant positive correlation between PBD and the abundance of P. rylovi also among clams collected from 2 natural A. piscinalis populations.

Factors affecting the intensity of epidermal papillomatosis in populations of roach, Rutilus rutilus (L.), estimated as scale coverage.

Studies of epidermal papillomatosis in fish populations have only rarely focused on the intensity of the disease, i.e. the number and size of papillomas. Furthermore, the methods used to evaluate the intensity of papillomatosis have not been standardized. We tested the reliability of a method based on counting of scales covered by papilloma tumours in roach, Rutilus rutilus (L). In addition, we studied the frequency distributions of the number of scales covered by papillomas within populations, evaluated the correlation between the prevalence and mean intensity of the disease among populations and examined the intensity of papillomatosis in roach with respect to sex and size of fish. Reliability of the scale coverage method was high. Therefore, the method could offer an effective way to determine the intensity of papillomatosis in fish species with large scales. The frequency distribution of the scale coverage of papillomas was highly aggregated within all populations studied. The mean intensity of papillomatosis increased with the size of the fish and was higher in males than in females. However, there was no correlation between the mean intensity and prevalence of the disease among the 19 roach populations studied.

Long-lasting effect of stress on susceptibility of a freshwater clam to copepod parasitism.

The question whether a stress event can have a long-lasting effect on susceptibility to parasites was studied using a freshwater bivalve clam and its crustacean parasite as a model system. Anodonta piscinalis clams were collected from 2 populations during August-September 2002. Clams were transported to the laboratory and marked. The stressed clams were subjected to low oxygen for 25 days, while the unstressed control clams were caged in their lakes of origin for the same period. Then the clams were transported to a third lake where they were exposed to natural infections by the ergasilid copepod, Paraergasilus rylovi, 11 months after the stress event. The stressed clams were more intensively parasitized. They also showed lower growth, lower reproduction and lower survival than the unstressed control clams. The results indicate that susceptibility of A. piscinalis to P. rylovi infection may be condition dependent, and that stress may have a long-lasting, increasing effect on host susceptibility to parasitism in natural populations.

Aspects of the ecology and natural history of Paraergasilus rylovi (Copepoda, Ergasilidae) parasitic in unionids of Finland.

The distribution of Paraergasilus rylovi in 17 populations of unionids was investigated. In 1 unionid population, the parasite was studied regarding host age, size, sex, and the reproductive period (occurrence of egg sacs). Results from pooled material from the years 1987--1989 and 1996 (southern Finland, 11 populations) indicated that Anodonta piscinalis (n = 1,359) is the main host (total mean prevalence 71% and intensity +/-SE of infection 16.4+/-0.6). Pseudanodonta complanata (n = 106) was infected occasionally (3% and 1.3+/-0.3), whereas Unio pictorum (n = 108) and U. tumidus (n = 17) were not infected. Results from 17 A. piscinalis populations showed that P. rylovi occurs in southern Finland but not in northern Finland. In A. piscinalis, the mean intensity of infection was higher in lake populations than in river populations. Both host age and length had a negative relationship with the intensity of P. rylovi infection. Host sex did not affect the intensity of infection. Egg sacs of P. rylovi were found from June to August. There was a tendency for higher intensities of infection in autumn. Infection by the digenean Rhipidocotyle fennica had no effect on the intensity of P. rylovi infection.

Reduction in the level of infection of the bivalve Anodonta piscinalis by the copepod Paraergasilus rylovi using high temperature and low oxygen.

The aim of this study was to develop a method to kill or expel the gill-dwelling crustacean parasite Paraergasilus rylovi from a common freshwater clam, Anodonta piscinalis. Naturally infected clams were exposed to different water-quality treatments and monitoring in the laboratory. In a high-temperature treatment (26 C vs. control 18 C), the mean abundance of the parasite decreased to near zero in 7 days. Because only 2 clams of 72 died in this treatment during the 14-day experiment, the survival of the host was not seriously at risk at the high temperature. 'Low oxygen, no water change' (18 C) was the second most effective treatment, followed by a 'low-oxygen, water-flow' (18 C) treatment. At the end of the experiment, the mean parasite abundance was significantly lower in all the treatments than in the control clams (18 C). A few P. rylovi individuals abandoned the host at 26 C but died in a couple of days outside the host. However, the parasites lived on average (+/-SE) 12.7 +/- 0.9 days outside the clam, and were also shown to be capable of infecting another uninfected host individual, at 18 C. The results of the present study suggest that high temperature provides an effective, ecologically sustainable method to manipulate the intensity of P. rylovi infection.

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%.

Quantitation of entacapone glucuronide in rat plasma by on-line coupled restricted access media column and liquid chromatography-tandem mass spectrometry.

A column-switching liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS-MS) method was developed for the direct analysis of entacapone glucuronide in plasma. The plasma samples (5 microl) were injected onto a C18-alkyl-diol silica (ADS) column and the matrix compounds were washed to waste with a mixture of 20 mM ammonium acetate solution at pH 4.0-acetonitrile (97:3). The retained analyte fraction containing (E)- and (Z)-isomers of glucuronides of entacapone and tolcapone glucuronide (internal standard) was backflushed to the analytical C18 column, with a mixture of 20 mM ammonium acetate-acetonitrile (85:15) for the final separation at pH 7.0. The eluate was directed to the mass spectrometer after splitting (1:100). The mass spectrometer was operated in the negative ion mode and the deprotonated molecules [M-H]- were chosen as precursor ions for the analytes and internal standard. Collisionally induced dissociation of [M-H] in MS-MS resulted in loss of the neutral glucuronide moiety and in the appearance of intensive negatively charged aglycones [M-H-Glu]-, which were chosen as the product ions for single reaction monitoring. Quantitative studies showed a wide dynamic range (0.0025-100 microg/ml) with correlation coefficients better than 0.995. The method was repeatable within-day (relative standard deviation, RSD<7%) and between-day (RSD<14%) and the recovery (78-103%) was better than with the traditional, laborious pretreatment method. The use of tandem mass spectrometry permitted low limits of detection (1 ng/ml of entacapone glucuronide). The method was applied for the quantitation of (E)- and (Z)-isomers of entacapone glucuronide in plasma of rats used in absorption studies.

Glucuronidation of 1-hydroxypyrene by human liver microsomes and human UDP-glucuronosyltransferases UGT1A6, UGT1A7, and UGT1A9: development of a high-sensitivity glucuronidation assay for human tissue.

Human UDP-glucuronosyltransferases (UGT, EC 2.4.1.17) involved in the biotransformation of pyrene were investigated by a sensitive fluorometric high-performance liquid chromatography (HPLC)method developed for determining activities toward 1-hydroxypyrene. The endpoint metabolite of pyrene, 1-pyrenylglucuronide, is a well-known urinary biomarker for the assessment of human exposure to polycyclic aromatic hydrocarbons. 1-Pyrenylglucuronide was synthesized using rat liver microsomes as biocatalyst. The yield was satisfactory, 22%. 1-Pyrenylglucuronide, identified by (1)H NMR and by electrospray mass spectrometry, was used for method validation and calibration. The HPLC assay was very sensitive with a quantitation limit of 3 pg (8 fmol) for 1-pyrenylglucuronide. The assay was precise, showing a relative standard deviation of 5% or less at 0.1 to 300 microM 1-hydroxypyrene. Only 2 microg of microsomal protein was required for the assay in human liver. The glucuronidation of 1-hydroxypyrene was catalyzed at high rates in microsomes from pooled or three individual liver samples, showing comparable apparent K(m) values. The formation of 1-pyrenylglucuronide was catalyzed by recombinant human UGT1A6, UGT1A7, and UGT1A9, the K(m) values being 45, 12, and 1 microM, respectively. The apparent K(m) values in human liver microsomes, ranging from 6.9 to 8.6 microM, agreed well with these results. The method provides a sensitive tool for measuring extremely low UGT activities and a specific means for assessing interindividual differences in 1-hydroxypyrene-metabolizing UGT activities in human liver and other tissues.

High-performance liquid chromatographic method combining radiochemical and ultraviolet detection for determination of low activities of uridine 5'-diphosphate-glucuronosyltransferase.

A novel reversed-phase high-performance liquid chromatographic method was developed to measure UDP-glucuronosyltransferase (UGT) activity. Radiochemical and UV detection were combined in this UDP-[(14)C]glucuronic acid-utilizing method which was especially aimed at determination of low activities typical of N-glucuronidation of various amines and heterocycles. 4-Nitrophenol and levomedetomidine were used as substrates to validate this method, and applicability was tested with commonly used model substrates of N-glucuronidation, 4-aminobiphenyl and amitriptyline, and several 4-arylalkyl-1H-imidazole compounds. Detection limits were very low, 0.5-10 pmol, corresponding to UGT activities from 0.04 to 0.8 pmol/min/mg protein depending on UV absorbance of the glucuronide conjugate. The sensitivity was 10- to 100-fold compared with earlier HPLC assays using radiochemical detection. This method enabled quantitation without a reference glucuronide, and its high sensitivity allows for characterization of N-glucuronidation kinetics of various substrates. Using this method, human liver microsomal UGT activity was determined for a series of 4-arylalkyl-1H-imidazoles. Of these compounds, levomedetomidine was glucuronidated at the highest rate, 1.69 nmol/min/mg protein, using a 500 microM substrate concentration. In comparison, activities for the commonly used UGT substrates, 4-nitrophenol, 4-aminobiphenyl, and amitriptyline were 18.80, 3.23, and 0.23 nmol/min/mg protein, respectively.

Molecular mechanisms controlling the rate and specificity of catechol O-methylation by human soluble catechol O-methyltransferase.

Molecular mechanisms determining the turn-over rate and specificity of catechol O-methylation were studied by combining enzyme kinetic measurements, computational modeling of substrate properties and fitting ligands in a 3D model of the active site of the enzyme. Enzyme kinetic measurements were carried out for 46 compounds, including most clinically used catechol drugs, by using recombinant human soluble catechol O-methyltransferase (COMT). The most important mechanism decreasing the turnover rate and increasing affinity was the electron withdrawing effect of substituents. Several other mechanisms by which substituents affected reactivity and affinity were identified. Highest turnover rates were determined for unsubstituted catechol and pyrogallol. Pyrogallol derivatives generally seemed to be more specific substrates than catechols. Catecholestrogens were the most specific endogenous substrates, whereas catecholamines were rather poor substrates. Among the catechol drugs used in the L-DOPA treatment of Parkinson's disease, the COMT inhibitors entacapone and tolcapone were not methylated, whereas the DOPA decarboxylase inhibitor benserazide was 15 times more specific substrate than L-DOPA, the target of COMT inhibition. The structure-activity relationships found allow the prediction of reactivity, affinity, and specificity with useful accuracy for catechols with a wide range of structures and properties. The knowledge can be used in the evaluation of metabolic interactions of endogenous catechols, drugs and dietary catechols, and in the designing of drugs with the catechol pharmacophore.