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Evidence for the transfer of polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans from soil into biota.

The concentrations of six indicator and co-planar polychlorinated biphenyls (PCBs), 17 polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans (PCDD/Fs) were determined in soil and associated biota samples collected from a polluted and wasted farmland in southern China. The sum of six indicator PCBs (Nos 28, 52, 101, 138, 153 and 180) concentrations in biota samples were proportional to the total amount of PCBs that transferred from soil to the corresponding biota samples, and the sum six indicator PCB congeners in samples were about 20% of the total PCBs, respectively. The ratios of the sum six indicator PCBs and the total PCBs in samples collected in the same area were approximately equal. The sums of six indicator PCBs were proportional to the total PCBs that transferred from soil to plant, while the co-planar PCBs were not. Analysis of individual PCB and PCDD/F congener signatures in soil revealed the original pollutant source after transportation and biodegradation for 14 years using principal component analysis (PCA). The pattern of PCBs in soils and plants could reflect the original pollution source after transportation and biodegradation for 14 years, while the pattern of PCBs in the terrestrial animals could not reflect the primary source. The consumption foods in this region such as foraging chicken eggs, foraging duck eggs, and loach (Misgurnus anguillicaudatus) were highly contaminated, the total toxicity equivalent (TEQ) was up to 784 pg WHO-TEQ/g on basis lipid in foraging chicken eggs.

Animals↗

Do sewage treatment plant discharges substantially impair fish reproduction in polluted rivers?

Sewage treatment plants are frequently associated with the release of xenobiotics and, consequently, with alterations of the reproductive function induced by many of these substances in aquatic organisms. In order to assess the impacts of sewage treatment plant (STP) discharges in polluted rivers, two sentinel species (gudgeon Gobio gobio and stoneloach Barbatula barbatula) were caught during their reproductive cycle upstream and downstream two STPs (STP1--Goffontaine, STP2--Wegnez). Gonadosomatic index, histological (testicular and ovarian stages, atretic follicles, intersexuality) and endocrine (sex steroids, aromatase activity, alkali-labile phosphorus) parameters were assayed. In brief, the results revealed no systematic significant differences (p<0.05) between upstream and downstream sites, whatever the STP, species or sampling period. However, stoneloach females displayed some signs of reproductive impairment and endocrine disruption downstream STP1 (reduced GSI, oocyte diameter and ALP concentrations, increased proportion of atretic follicles) and STP2 (changes in gonadal aromatase activity and plasma levels of 11-KT and T). Few significant changes were observed for gudgeon males and females while there were no significant differences between upstream and downstream sites for stoneloach males. Moreover, plasma E(2) concentrations recorded in gudgeon males sampled in all sites were as high as in females and this was confirmed by high ALP levels. Besides, spermatogenesis of gudgeon males was delayed in STP1 upstream and downstream sites compared to the corresponding sites in STP2. These observations for gudgeon males do not seem related to STP discharge but to a probable estrogenicity of the river. Therefore, as shown by the results, stoneloach seemed more sensitive than gudgeon to STP discharges. In the present study, sewage treatment plant discharges do not substantially impair fish reproduction. In this respect, caution is required when generalising negative impacts of STP discharges.

Animals↗

Mechanism of apoptosis induced by a polysaccharide, from the loach Misgurnus anguillicaudatus (MAP) in human hepatocellular carcinoma cells.

The biological activities of the polysaccharide have attracted more and more attention in the biochemical and medical areas due to their anti-cancer effects. To estimate the anti-tumor mechanism of MAP, a novel polysaccharide from the loach, Misgurnus anguillicaudatus, the apoptosis effects of the polysaccharide on the human hepatocellular carcinoma cells (SMMC-7721 cells) were studied. The present studies showed that MAP could induce cell apoptosis which was closely accompanied with an increase of intracellular-free calcium concentration ([Ca2+]i), the enhancement of reactive oxygen species (ROS) level, dissipation of mitochondria membrane potential (MMP), up-regulation of p53 mRNA, increase expression of Bax mRNA, and decrease expression of Bcl-2 mRNA. These results suggested that cell apoptosis induced by MAP mainly was mediated by mitochondrial pathways, not involved death receptors (DRs) pathways. The mechanism possibly is that MAP acts on mitochondria and boosts ROS, ROS mediates a release of Ca2+ from the intracellular Ca2+ pool, increasing [Ca2+]i targets the cells a start-up of the apoptosis program. However, further research on the molecular mechanisms of MAP effecting on the cells' mitochondria is necessary.

Animals↗

Effect of DNA repair inhibitor (3-aminobenzamide) on genetic stability of loach (Misgurnus fossilis) embryos derived from cryopreserved sperm.

Semen cryopreservation is widely used in clinical medicine, agriculture, aquaculture and biomedical research, but it is an inefficient technique that induces extensive cytoplasmic damage and loss of fertilising ability. Whether any genetic damage (i.e. DNA strand breakage or mutation) is also induced is still unclear. However, previous data has indicated that this is likely. The present study was designed to explore this possibility further by using inhibitors of the DNA repair system to block DNA repair in embryos derived from cryopreserved spermatozoa. If cryopreservation causes strand breaks in sperm DNA it might be expected that inhibition of a repair enzyme such as poly(ADP-ribose) polymerase (PARP) would enhance any such negative effect of cryopreservation. To check this hypothesis 3-aminobenzamide (3-AB) was used as an inhibitor of PARP. Weather loach (Misgurnus fossilis) eggs were fertilised using cryopreserved as well as fresh spermatozoa. Embryos derived from cryopreserved spermatozoa were exposed to 10 mM 3-AB for 2 h after fertilisation. The experiments were carried out using 43,544 embryos from 5 females and 10 males. Embryo survival was evaluated at different stages until the hatching stage. Sperm cryopreservation significantly decreased embryo survival (53.6+/-2.79% compared to 76.97+/-2.79% of control; P<0.01). The addition of 3-AB to the medium with embryos derived from cryopreserved sperm further decreased embryo survival from 53.6+/-2.79% to 46.1+/-2.79% (P<0.01) whereas there was no adverse effect of 3-AB exposed embryos derived from fresh sperm (76.97+/-2.79% of control compared to 74.8+/-2.79% of control+3-AB). The effect of 3-AB provides indirect evidence that cryopreservation might induce instability in sperm DNA, and that such damage can be repaired by the oocyte repair system after fertilisation.

Animals↗

Phylogenetic relationships and biogeography of the genus Chondrostoma inferred from mitochondrial DNA sequences.

A phylogeny of the species of the nase genus Chondrostoma was constructed from a complete mitochondrial cytochrome b gene (1140 bp). Molecular phylogeny was used to revise the current systematics of this group, and to infer a biogeographical model of the Mediterranean area during the Cenozoic period. We confirmed the monophyly of the genus Chondrostoma, and defined seven different lineages within it: Polylepis, Arcasii, Lemmingii, Toxostoma, Nasus, C. genei, and C. soetta. The separation of main lineages within Chondrostoma occurred in the Middle-Upper Miocene, approximately 11 million years ago, while the greatest species radiation took place in the Pliocene close to the time the current drainages system were created. It is unlikely that this genus experienced an extensive dispersal during the Messinian, in the Lago-Mare Phase. Given the level of current knowledge, a biogeographical model constructed on the basis of vicariant events seems more realistic than does a dispersalist model.

Animals↗

Phylogenetic relationships among the Ibero-African cobitids (Cobitis, cobitidae) based on cytochrome b sequence data.

We estimated the phylogenetic relationships of all Ibero-African spined loaches of the genus Cobitis using the complete mitochondrial cytochrome b gene (1140bp). We analysed 93 individuals of seven cobitid species found in all the principal drainages of the Iberian Peninsula and North Africa. A molecular phylogeny was used to revise current systematics of the Ibero-African Cobitis species and to infer a biogeographical model for Cobitis in the Western Mediterranean area during the Cenozoic period. Phylogenetic analysis provided support for the monophyly of two mtDNA clades: Clade A or Italian Clade with the Italian species (C. bilineata, C. zanandreai), and Clade B or the Ibero-African Clade. The Ibero-African Clade included all species endemic for the Iberian Peninsula (C. vettonica, C. calderoni, and C. paludica) and North Africa (C. maroccana). The species C. paludica does not constitute a natural group, and could be divided into at least four monophyletic mtDNA lineages with moderate to high bootstrap values and posterior probability support. Phylogenetic relationships of the Ibero-African species were not resolved satisfactorily, but in all analyses C. calderoni from Northern Iberian Peninsula was basal. We have re-calibrated a molecular clock for the genus Cobitis (0.68% per million year by pairwise) using populations inhabiting both sides of the Gibraltar Strait. Application of this Cobitis mtDNA clock provides evidence that the Messinian salinity crisis played a primary role in the diversification of some Ibero-African cobitid species. The basal polytomies of the Ibero-African Clade might suggest that all mtDNA lineages diversified rapidly.

Animals↗

Molecular phylogeny of the Southeast Asian freshwater fish family Botiidae (Teleostei: Cobitoidea) and the origin of polyploidy in their evolution.

The freshwater fish family Botiidae is represented by seven genera on the Indian subcontinent and in East and Southeast Asia and includes diploid as well as evolutionary tetraploid species. We present a phylogeny of Botiidae including 33 species representing all described genera using the mitochondrial cytochrome b and 12s rRNA genes to reconstruct the phylogenetic relationships among the genera and to estimate the number of polyploidisation events during their evolution. Our results show two major lineages, the subfamilies Leptobotiinae with the genera Leptobotia and Parabotia and Botiinae with the genera Botia, Chromobotia, Sinibotia, Syncrossus, and Yasuhikotakia. Our results suggest that two species that were traditionally placed into the genus Yasuhikotakia form a monophyletic lineage with the species of Sinibotia. A review of the data on the ploidy level of the included species shows all diploid species to belong to Leptobotiinae and all tetraploid species to Botiinae. A single polyploidisation event can therefore be hypothesised to have occurred in the ancestral lineage leading to the Botiinae.

Animals↗

Vicariance, colonisation, and fast local speciation in Asia Minor and the Balkans as revealed from the phylogeny of spined loaches (Osteichthyes; Cobitidae).

We reconstruct the phylogeny of the morphologically diagnosable subgenera Bicanestrinia, Beysehiria, and Cobitis sensu stricto of the genus Cobitis from Asia Minor and the Balkans. We used the complete cytochrome b gene of 65 specimens in order to infer their evolutionary history in this zoogeographically interesting area. Our phylogeographic analysis did not evidence the previously suggested monophyly of the Bicanestrinia subgenus but revealed five monophyletic lineages in the area: the lineages Bicanestrinia I-IV including all species of Bicanestrinia plus the lineage Cobitis s. str. The monotypic subgenus Beysehiria from Lake Beysehir in Anatolia was closely related to the syntopic population of C. turcica and nested inside the lineage Bicanestrinia III. The strictly allopatric distribution of the four lineages of Bicanestrinia suggests that vicariance has played a major role in the diversification of Bicanestrinia. All analysed species of Cobitis s. str. from Asia Minor and Balkans were closely related to Cobitis s. str. from Central Europe, the Danube basin and the Caucasus, indicating at least two colonisation events into Asia Minor and the Balkans. A third, recent colonisation event led to the presence of C. strumicae, generally restricted to the Aegean Sea drainage, in the Danube basin. Besides the evidences of vicariance and colonisation events in the phylogenetic history of the genus Cobitis in Asia Minor and the Balkans, our analysis suggested also a rapid morphological evolution of C. bilseli in a lacustrine environment. Application of Cobitis mitochondrial cytochrome b clocks of 0.68% sequence divergence per million years (MY) suggest that the split between the five major lineages happened approximately 12.4-17.6 MYA, and according to the lack of basal resolution of this monophyletic group probably the split of all lineages happened within a narrow time window.

Animals↗

Detrimental effects of cryopreservation of loach (Misgurnus fossilis) sperm on subsequent embryo development are reversed by incubating fertilised eggs in caffeine.

Cryopreservation can cause changes to the genetic material of cells, but the mechanism and significance of these changes are still unknown. It has been suggested that some damage to the sperm genome could be repaired by the DNA repair system of the oocyte after fertilisation. Caffeine has been reported to be an inhibitor of such repair processes. In this study the effect of caffeine on the repair system of Loach (Misgurnus fossilis) oocytes was investigated. Loach eggs were fertilised using cryopreserved sperm. Embryos derived from cryopreserved sperm were exposed to 2.6mM caffeine for 1h after fertilisation. The experiments were carried out using 32313 embryos from four females and eight males. Embryo survival was evaluated for 46 h until the hatching stage. Reduction in embryo survival after 20th stage is generally believed to result from the failure in the genome function of embryos. Cryopreservation of sperm significantly decreased embryo survival (53.4+/-2.8% compared to 68.4+/-2.8% of control) after the 20th stage. However, the addition of caffeine to the embryos derived from cryopreserved sperm, in contrast to our expectation, significantly increased survival of loach embryos (70.9+/-2.8% compared to 53.4+/-2.8% of embryos derived from cryopreserved sperm in the absence of caffeine). The effect of individual donors of sperm and eggs on overall embryo survival was also studied. Whilst no significant differences were observed between males, the effect of individual females on embryo survival was significant. The analysis of embryo survival at different developmental stages showed that embryo survival both before and after 20th stage decreased with embryo development. When fresh sperm were used the decline of embryo survival with development was more pronounced compared with those embryos derived from cryopreserved sperm. Possible explanations of these effects are presented.

Animals↗

Preferential interaction of loach DNA polymerase delta with DNA duplexes containing single-stranded gaps.

We studied the interaction of DNA polymerase delta (pol delta) purified from the eggs of the teleost fish Misgurnus fossilis (loach) with DNA duplexes containing single-stranded gaps of 1-13 nucleotides (nt). In the absence of processivity factors (PCNA, RF-C, and ATP), pol delta elongated primers on single-stranded DNA templates in a distributive manner. However, the enzyme was capable of processive synthesis by filling gaps of 5-9 nt in DNA duplexes. These data suggest that, upon filling a small gap, pol delta interacts with the 5'-terminus downstream of the gap as well as with the 3'-terminus of the primer. Interaction of pol delta with the proximal 5'-terminus restricting the gap was confirmed by electrophoretic mobility shift assay. Analysis of the enzyme binding to DNA duplexes containing gaps of various sizes showed a much higher affinity of pol delta for duplexes with gaps of about 10 nt than for DNA substrates with primers annealed to single-stranded templates. The most efficient pol delta binding was observed in experiments with DNA substrates containing unpaired 3'-tails in primers. The data obtained suggest that DNA molecules with small gaps and single-stranded tails may serve as substrates for direct action of pol delta in the course of DNA repair.

Adenosine Triphosphate↗

A novel antimicrobial peptide from the loach, Misgurnus anguillicaudatus.

A novel antimicrobial peptide, named misgurin, was isolated and characterized from the loach (mudfish), Misgurnus anguillicaudatus. The 21-amino-acid peptide with a molecular mass of 2502 Da was purified to homogeneity using a heparin-affinity column and C18 reverse-phase and gel-permeation high-performance liquid chromatography. The complete amino acid sequence of misgurin, which was determined by an automated amino acid sequencer, was Arg-Gln-Arg-Val-Glu-Glu-Leu-Ser-Lys-Phe-Ser-Lys-Lys-Gly-Ala-Ala-Ala-Arg- Arg-Arg-Lys. Misgurin is a strongly basic peptide which has 5 arginine and 4 lysine residues. Comparison of the amino acid sequence with those of other known antimicrobial peptides revealed that misgurin was a novel antimicrobial peptide. Misgurin showed a strong antimicrobial activity in vitro against a broad spectrum of microorganisms without significant hemolytic activity and was about 6 times more potent than magainin 2. Scanning electron microscopy confirmed that the peptide caused damage to the cell membrane by a pore-forming mechanism similar to that of magainin 2. This damage occurred at the minimal inhibition concentration (MIC), but at higher concentration than MIC it lysed the cell.

Amino Acid Sequence↗

Ultrastructural changes in fish gills as biomarker to assess small stream pollution.

In order to verify the principal suitability of gill ultrastructure as a biomarker, semi-field studies with two endigoneous fish, trout (Salmo trutta f. fario) and loach (Barbatula barbatula), were performed. The fish were exposed in flow-through systems to one heavily polluted (Körsch) and one lightly polluted small stream (Krähenbach) in South-West Germany. Ultrastructural responses in gills were correlated with limnological and chemical data recorded over a 2 year period in each stream. After 8 weeks of exposure to the heavily polluted stream, fish showed ultrastructural changes in the gills, such as cell proliferation, dilation of the endoplasmic reticulum, hyperplasia, hypersecretion, and epithelial lifting in chloride, epithelial, and mucus cells. The results of the study demonstrate that ultrastructural reactions in the gills of fish kept under semi-field conditions are potentially useful biomarkers indicating small stream pollution.

Animals↗

The toxic effects of microcystin-LR on embryo-larval and juvenile development of loach, Misguruns mizolepis Gunthe.

Microcystin-LR, a specific and potent hepatotoxin, was tested for its effects on loach embryo-larval and juvenile development. The results of this study showed that loach embryos were more sensitive when exposed to microcystin-LR at a later than at an earlier stage of development. Juveniles were far less sensitive to MC-LR than were embryos and larvae. Mortality and developmental abnormality were proven to be dose-dependent and to be stage-specific sensitive. Among the abnormal changes noted were: pericardial edema and tubular heart, bradycardia, homeostasis, poor yolk resumption, small head, curved body and tail, and abnormal hatching. Liver and heart were the main targets of microcystin-LR toxicity. Ultrastructural analysis documented a complex set of sublethal effects of microcystin-LR on loach hepatocytes, chiefly including morphological alteration in nuclear and RER of loach liver cells. In addition, microcystin-LR was lethal to loach juvenile in the subacute (7 days) exposure (LC(50)=593.3 microg/l).

Animals↗

Hazard assessment of selenium to endangered razorback suckers (Xyrauchen texanus).

A hazard assessment was conducted based on information derived from two reproduction studies conducted with endangered razorback suckers (Xyrauchen texanus) at three sites near Grand Junction, CO, USA. Selenium contamination of the upper and lower Colorado River basin has been documented in water, sediment, and biota in studies by US Department of the Interior agencies and academia. Concern has been raised that this selenium contamination may be adversely affecting endangered fish in the upper Colorado River basin. The reproduction studies with razorback suckers revealed that adults readily accumulated selenium in various tissues including eggs, and that 4.6 microg/g of selenium in food organisms caused increased mortality of larvae. The selenium hazard assessment protocol resulted in a moderate hazard at the Horsethief site and high hazards at the Adobe Creek and North Pond sites. The selenium hazard assessment was considered conservative because an on-site toxicity test with razorback sucker larvae using 4.6 microg/g selenium in zooplankton caused nearly complete mortality, in spite of the moderate hazard at Horsethief. Using the margin of uncertainty ratio also suggested a high hazard for effects on razorback suckers from selenium exposure. Both assessment approaches suggested that selenium in the upper Colorado River basin adversely affects the reproductive success of razorback suckers.

Animals↗

Is chemical contamination responsible for the decline of the copper redhorse (Moxostoma hubbsi), an endangered fish species, in Canada?

The copper redhorse (Catostomidae: Moxostoma hubbsi) is an endangered fish species whose worldwide distribution is limited to the St. Lawrence River and three of its tributaries, in Canada. Severe reproductive impairment and lack of successful recruitment reported in this species have been hypothetically associated with water pollution. In order to obtain an initial description of contamination levels in copper redhorse, seven accidentally-killed specimens from the Richelieu River were analyzed for trace metals, organochlorine pesticides, chlorobenzenes, PAHs, PCBs, dioxins and furans. Fish varied between 9 and 33 years of age, which corresponds to mature individuals. The levels of contaminants analyzed in different body tissues were close to and often lower than levels reported in other catostomid fish species from nearby locations within the St. Lawrence River basin. Concentrations of total mercury, cadmium and co-planar PCBs increased with fish age. The types and concentrations of contaminants found suggested that the Richelieu River spawning population of copper redhorse would migrate and spend time in the St. Lawrence River. Concentrations of many contaminants were often highest in gonadal tissues, but levels were much lower than reported in the literature as causing reproductive impairment or egg and fry mortality in fish. Further research is needed to assess the potential link between contaminants and reproductive failure in this endangered fish species.

Age Factors↗

An efficient method for blocking the 1st mitotic cleavage of fish zygote using combined thermal treatment, exemplified by mud loach (Misgurnus mizolepis).

Blocking the first mitotic cleavage of the zygote is a key tool for chromosome-set manipulations in fish. We developed an improved method for inducing tetraploidy by blocking the mitosis with a combination of heat shock at 40.5 degrees C for 1, 2, or 3 min followed by cold shock at 1.5 degrees C for 30, 45, or 60 min. When applied during the first cleavage metaphase of mud loach (Misgurnus mizolepis) zygotes, the optimal combination was heat for 2 min followed by cold for 45 min. At 1 month, the frequency of 4N survivors and the yield from total eggs fertilized was 55.7 and 14.4%, respectively, compared to heat shock alone with 20.0% efficiency and 3.6% yield. The effectiveness of the procedure was confirmed by diploid mitotic gynogenesis using transgenic markers. The overall yield of homozygous diploids, 34.0%, was better than that for single heat shock, 17.3%. The tetraploids and homozygous diploids had higher early mortality than normal diploid controls. However, at 1 month, the viability of the tetraploids was the same as normal diploids. For gynogenetic diploids, the survival was similar to normal diploids after 3 months. The high efficiency of this new protocol extends the opportunity to study polyploidy in basic and applied research.

Animals↗

Common-source outbreak of acute infection due to the North American liver fluke Metorchis conjunctus.

BACKGROUND: We investigated an outbreak of acute clinical illness among 19 people who ate raw fish (sashimi) prepared from the white sucker, Catostomus commersoni, caught in a river north of Montreal, Canada. METHODS: We collected epidemiological, clinical, laboratory, and serological data on 19 individuals who ate the sashimi and six who did not. Because of the suggestive clinical picture, we set out to recover helminth parasites from uneaten fish. FINDINGS: The illness consisted of persistent upper abdominal pain, low grade fever, high blood eosinophil concentrations, and raised liver enzymes. After 10 days, opisthorchild-like eggs were found in stools. Symptoms persisted for 3 days to 4 weeks without treatment, but responded rapidly to praziquantel therapy. Necropsy of golden hamsters infected with metacercariae from uneaten fish revealed adult flukes identified as Metorchis conjunctus. INTERPRETATION: We describe an acute illness caused by the North American liver fluke M conjunctus. This is a new human disease and is the first report of a common-source outbreak of an acute illness caused by liver flukes of the family Opisthorchiidae.

Adult↗

Effects of food ration on survival and sublethal responses of lake chubsuckers (Erimyzon sucetta) exposed to coal combustion wastes.

Study organisms in chronic toxicological bioassays are often provided with excessive resources to remove food limitations as a confounding experimental variable. Under more ecologically realistic situations, resources are often less abundant and such restrictions may alter the responses of organisms to environmental contaminants. Here, we investigated the interaction between resource level and sediment toxicity in the lake chubsucker, Erimyzon sucetta. For 78 days we fed fish one of three ration levels (1X, 2X, 4X; uncontaminated food) that was grazed directly from either clean sand or coal ash-contaminated sediments. Despite provision of uncontaminated food, fish exposed to the contaminated sediments accumulated significant whole body concentrations of As, Se, Sr, and V. Food ration affected the pattern of Se accumulation, with lowest concentrations accumulated by fish supplied with the lowest rations (1X). Paradoxically, fish in the 1X-ash treatment were most adversely effected by ash-exposure, despite having Se burdens much lower than fish in the 2X- and 4X-ash treatments. Fish in the 1X-ash treatment exhibited higher mortality, lower proportional growth, and increased incidence of fin erosion compared to fish provided with higher rations. Such results may, in part, be explained by the apparent inability of fish with reduced rations to maintain positive energy balance, as evidenced by their higher standard metabolic rates compared to control fish fed similar rations. Our results underscore the importance of considering resource quantity and nutritional factors in chronic bioassays in order to draw more ecologically realistic conclusions about contaminant effects.

Animal Feed↗