Phylogeny of freshwater parasitic copepods in the Ergasilidae (Copepoda : Poecilostomatoida) based on 18S and 28S rDNA sequences

The phylogenetic relationships among the Ergasilidae genera are poorly understood. In this study, 14 species from four genera in the Ergasilidae including Sinergasilus, Ergasilus, Pseudergasilus, and Paraergasilus were collected in China, and their phylogenetic relationships were examined using neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian inference methods based on partial sequences of 18S and 28S ribosomal deoxyribonucleic acid, respectively. All the analyses suggest that the Sinergasilus and Paraergasilus are both monophyletic, but the Ergasilus is polyphyletic rather than monophyletic. Considering the relationships among the four genera, the phylogenetic analyses and subsequent hypothesis tests all suggest that Pseudergasilus clustered with some Ergasilus species may have a closer relationship with Sinergasilus rather than with Paraergasilus. It is proposed that the Sinergasilus and the Pseudergasilus species might have evolved from Ergasilus species.

Morphological description of two bucephalid trematodes collected from freshwater fishes in the Uji River, Kyoto, Japan

The morphology of two species of bucephalids (Bucephalidae; Digenea; Trematoda), which since 1999 has caused a fish disease at the Uji River, Kyoto Prefecture, Japan, is described. Parabucephalopsis parasiluri Wang, 1985 was first recorded in the Uji River in 2000, and Prosorhynchoides ozakii (Nagaty, 1937) in 2005. The definitive host of both species is the Lake Biwa catfish (Silurus biwaensis), and the second intermediate hosts include many fish species from several families. P. parasiluri is an introduced parasite that invaded with its first intermediate host, golden mussels (Limnoperna fortunei), from the Asian continent. P ozakii may also be an introduced species, although its first intermediate host has not been identified. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Icefish (Salangidae) as an indicator of anthropogenic pollution in freshwater systems using nitrogen isotope analysis

We investigated differences in delta N-15 of seston and icefishes from seven freshwater ecosystems with different trophic states in China. An increase of seston delta N-15 values was accompanied by an increase of total nitrogen and phosphorus concentrations. Significantly positive correlations were observed between delta N-15 of icefishes and delta N-15 of seston, total nitrogen and phosphorus concentrations. This study demonstrated that icefishes could be preferred indicators of anthropogenic contamination in test systems because they integrated waste inputs over long time periods and reflected the movement of waste through the pelagic food chain.

Morphological and genetic differences of Trypanosoma in some Chinese freshwater fishes: difficulties of species identification

Blood smears and purified trypanosome from freshwater fishes yellow catfish (Pseudobagras fulvidraco) and common carp (Cyprinus carpio) captured from Niushan Lake, Hubei Province were examined to determine whether all of their trypanosomes were Trypanosoma pseudobagri, a species of supposed host specificity and widespread existence across China. Trypanosomes occurred in 16/16 blood smears, and morphometric character analysis of trypanosomes from these smears showed that there were three morphospecies, Trypanosoma sp Carpio, T. sp Pseudobagri, and T. sp. 18S rDNA sequences of trypanosomes from 16 samples revealed three genetic groups among these fish trypanosomes. Group 1 was from C. carpio containing T. sp Carpio; groups 2 and 3 were from P. fulvidraco containing T. sp Pseudobagri and T. sp, respectively. The high similarity of morphometric characters and 18S rDNA sequences showed that T. sp Carpio and T. siniperca probably were the same species. T. sp Pseudobagri was the first occurrence in China. Sequence comparison showed that T. sp Pseudobagri sequence was most similar to that of clone Marv, whereas T. sp sequence differ from those of T. sp Carpio and T. sp Pseudobagri by 5.4 and 5.8%, respectively, and tentatively identified as T. pseudobagri. It was concluded that three species of trypanosomes, at least three genotypes occur in Niushan Lake fishes, and P. fulvidraco in this region appear to contain both types, although the identification of T. pseudobagri remains a problem.

Vertical mixing within the epilimnion modulates UVR-induced photoinhibition in tropical freshwater phytoplankton from southern China

1. The importance of vertical mixing in modulating the impact of UVR on phytoplankton photosynthesis was assessed in a tropical, shallow lake in southern China from late winter to mid-spring of 2005. 2. Daily cycles of fluorescence measurements (i.e. photosynthetic quantum yield, Y) were performed on both 'static' and in situ samples. Static samples were of surface water incubated at the surface of the lake under three radiation treatments - PAB (PAR + UVR, 280-700 nm), PA (PAR + UV-A, 320-700 nm) and P (PAR, 400-700 nm). In situ samples were collected every hour at three different depths - 0, 0.5 and 1 m. 3. The general daily pattern was of a significant decrease in Y from early morning towards noon, with partial recovery in the afternoon. Samples incubated under static conditions always had lower Y than those under in situ conditions at the same time of the day. 4. Under stratified conditions, no overall impact of UVR impact could be detected in situ when compared with the static samples. Further rapid vertical mixing not only counteracted the impact of UVR but also stimulated photosynthetic efficiency. 5. Based on these measurements of fluorescence, the mixing speed of cells moving within the epilimnion was estimated to range between 0.53 and 6.5 cm min(-1). 6. These data show that mixing is very important in modulating the photosynthetic response of phytoplankton exposed to natural radiation and, hence, strongly conditions the overall impact of UVR on aquatic ecosystems.

Fish ES cells and applications to biotechnology

ES cells provide a promising tool for the generation of transgenic animals with site-directed mutations. When ES cells colonize germ cells in chimeras, transgenic animals with modified phenotypes are generated and used either for functional genomics studies or for improving productivity in commercial settings. Althought the ES cell approach has been limited to, mice, there is strong interest for developing the technology in fish.. We describe the step-by-step procedure for developing ES cells in fish. Key aspects include avoiding cell differentiation, specific in vitro traits of pluripotency, and, most importantly, testing for production of chimeric animals as the main evidence of pluripotency. The entire process focuses on two model species, zebrafish and medaka, in which most work has been done. The achievements attained in these species, as well as their applicability to other commercial fish, are discussed. Because of the difficulties relating to germ line competence, mostly of long-term fish ES cells, alternative cell-based approaches such as primordial germ cells and nuclear transfer need to be considered. Although progress to date has been slow, there are promising achievements in homologous recombination and alternative avenues yet to be explored that can bring ES technology in fish to fruition.

Induction of oxidative stress and apoptosis by PFOS and PFOA in primary cultured hepatocytes of freshwater tilapia (Oreochromis niloticus)

Perfluorinated organic compounds (PFOCs) are emerging persistent organic pollutants (POPs) widely present in the environment, wildlife and human. We studied the cellular toxicology of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) on oxidative stress and induction of apoptosis in primary cultured hepatocytes of freshwater tilapia (Oreochromis niloticus). Cultured hepatocytes were exposed to PFOS or PFOA (0, 1, 5, 15 and 30 mg L-1) for 24 h, and a dose-dependent decrease in cell viability was determined using trypan blue exclusion method. Significant induction of reactive oxygen species (ROS) accompanied by increases in activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were found, while activities of glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were decreased. Glutathione (GSH) content was reduced following treatment of PFOA and PFOS. A dose-dependent increase in the lipid peroxidation (LPO) level (measured as maleic dialdehyde, MDA) was observed only in the PFOA exposure groups, whereas LPO remained unchanged in the PFOS exposure groups. Furthermore, a significant activation of caspase-3, -8, -9 activities was evident in both PFOS and PFOA exposure groups. Typical DNA fragmentation (DNA laddering) was further characterized by agarose gel electrophoresis. The overall results demonstrated that PFOS and PFOA are able to produce oxidative stress and induce apoptosis with involvement of caspases in primary cultured tilapia hepatocytes. (c) 2007 Elsevier B.V. All rights reserved.

Microcystin accumulation in freshwater bivalves from Lake Taihu, China, and the potential risk to human consumption

The potential risk through ingestion of microcystins (MC) in contaminated mollusks has not been well studied. The present paper studied seasonal changes of MC content (determined by liquid chromatography-mass spectrometry) in various organs of three species of bivalves (Cristaria plicata, Hyriopsis cumingii, and Lamprotula leai) in Lake Taihu, China, where toxic cyanobacterial blooms occurred. Coinciding with peaks of seston MC (maximum, 5.7 mu g/L) and MC in cyanobacterial blooms (maximum, 0.534 mg/g), most organs showed sharp MC peaks during the summer, indicating both fast uptake and fast depuration by bivalves. Because hepatopancreas and intestine had considerably higher MC content than other organs, they are the most dangerous for human consumption. Both the present and previous studies show that the hepatopancreatic MC and total tissue MC often are correlated in various aquatic invertebrates. During the peak of the cyanobacterial blooms, C. plicata had higher hepatopancreatic MC content than the other bivalves, whereas H. cumingii had higher intestinal MC content than the other bivalves. Estimated daily intakes for humans from the consumption of whole tissues of the three bivalves were 0.48 to 0.94 mu g MC-LR equivalent/kg body weight (12- to 23.5-fold the tolerable daily intake value proposed by the World Health Organization), which indicates a high risk for humans consuming these bivalves.

A possible evolutionary lag in the relationship between freshwater mussels and European bitterling

The coevolutionary dynamics between European bitterling Rhodeus amarus and freshwater unionid mussels, which the former parasitize by laying eggs on their gills, were tested. In a series of experiments fish preferences and mussel responses were compared in parasites and hosts of recent (Europe) and ancient (Asia) sympatry. Rhodeus amarus readily oviposited on the gills of all mussel species tested. Fish that laid their eggs on the gills of Asian Anodonta woodiana, however, suffered a dramatic reduction in reproductive success compared to fish that oviposited on the gills of European mussels: Unio pictorum, Unio tumidus, Anodonta anatina and Anodonta cygnea. This difference was the result of egg ejection behaviour by mussels rather than the unsuitability of the internal gill environment for European bitterling embryo development. The ejection response of mussels with a long sympatry with European bitterling was considerably more pronounced than that of mussels with a substantially shorter sympatry. The data support a coevolutionary arms race between bitterling and mussels and point to an evolutionary lag in the relationship between R. amarus and its European mussel hosts. (c) 2007 The Authors. Journal compilation (c) 2007 The Fisheries Society of the British Isles.

The co-evolutionary relationship between bitterling fishes and freshwater mussels: insights from interspecific comparisons

Hypothesis: In parasites that use hosts for offspring development, adults may base oviposition decisions on a range of host traits related either to host quality or the co-evolutionary relationship between parasite and host. We examined whether host quality or co-evolutionary dynamics drive the use of hosts in the bitterling-mussel relationship. Organisms: Six species of bitterling fish (Acheilognathinae) and eight species of freshwater mussels (Unionidae, Corbiculidae) that are used by bitterling for oviposition. Site of experiments: Experimental tanks in Wuhan, China, at the site of the natural distribution of the studied species. Methods: Three experiments that controlled for host accessibility and interspecific interactions were conducted to identify host preferences among bitterling fishes and their mussel hosts. We started with a broad interspecific comparison. We then tested bitterling behavioural choices, their temporal stability, and mussel host ejection behaviour of the eggs of generalist and specialist bitterling species. Finally, we measured host mussel quality based on respiration rate and used published studies on mussel gill structure to infer mussel suitability as hosts for bitterling eggs. Results: We found significant interspecific differences among bitterling species in their use of mussel hosts. Bitterling species varied in their level of host specificity and identity of preferred hosts. Host preferences were flexible even among apparently specialized species and fishes switched their preferences adaptively when the quality of individuals of preferred host species declined. Mussels varied considerably in their response to oviposition through egg ejections. Host preference by a generalist bitterling species correlated positively with host quality measured as the efficiency of the mussel gills to extract oxygen from inhaled water. Host ability to eject bitterling eggs correlated positively with their relative respiration rate, probably due to a higher velocity of water circulating in the mussel gill chamber.

Serum concentrations of gonadotropins and steroid hormones of Neophocaena phocaenoides asiaeorientalis in middle and lower regions of the Yangtze River

As a relatively isolated and unique freshwater population, the Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis) is the most endangered subpopulation of this species. The objective of this study was to improve our understanding of their reproductive endocrinology by measuring (radioimmunoassay) serum concentrations of FSH, LH, estradiol (E-2), progesterone (P-4) and testosterone (T-2) in free-ranging animals. Blood samples were collected from 66 Yangtze finless porpoises (41 males and 25 females) captured in the middle and lower reaches of the Yangtze River. Based on significant variation of serum T-2 concentrations in males with body length (BL) > 138 cm, we inferred that they were mature; in these animals, there were significant seasonal variations in serum T-2 concentrations, with the highest concentrations in March and April (502.0 +/- 319.8 ng/dL, mean +/- S.D.) and the lowest in December (79.4 +/- 83.2 ng/dL). Serum T-2 concentrations positively correlated with serum concentrations of LH and weakly correlated with serum concentrations of E-2, whereas there was a significant negative correlation between serum LH and FSH concentrations in males > 138 cm. The smallest apparently pregnant female porpoise had a BL of 130 cm. Serum P-4 concentrations ranged from 13.2 to 112.4 ng/mL (43.9 +/- 28.3 ng/mL) in pregnant females, and fluctuated under 1.0 ng/mL in non-pregnant females with BL > 130 cm. Serum LH concentrations were significantly higher in non-pregnant females > 130 cm versus those females < 130 cm. To our knowledge, this is the first study of endocrine-related maturity and seasonal breeding characteristics of the Yangtze finless porpoise. (c) 2006 Elsevier Inc. All rights reserved.

Bioaccumulation of the hepatotoxic microcystins in various organs of a freshwater snail from a subtropical Chinese lake, Taihu lake, with dense toxic microcystis blooms

In this paper, we describe the seasonal dynamics of three common microcystins (MCs MC-RR, MC-YR, and MC-LR) in the whole body, hepatopancreas, intestine, gonad, foot, remaining tissue, and offspring of a freshwater snail, Bellamya aeruginosa, from Gonghu Bay of Lake Taihu, China, where dense toxic Microcystis blooms occur in the warm seasons. Microcystins were determined by liquid chromatography electrospray ionization mass spectrum. Microcystin (MC-RR + MC-YR + MC-LR) content of the offspring and gonad showed high positive correlation, indicating that microcystins could transfer from adult females to their young with physiological connection. This study is the first to report the presence of microcystins in the offspring of the adult snail. The majority of the toxins were present in the intestine (53.6%) and hepatopancreas (29.9%), whereas other tissues contained only 16.5%. If intestines are excluded, up to 64.3% of the toxin burden was allocated in the hepatopancreas. The microcystin content in the intestine, hepatopancreas, and gonad were correlated with the biomass of Microcystis and intracellular and extracellular toxins. Of the analyzed foot samples, 18.2% were above the tolerable daily microcystin intake recommended by the World Health Organization (WHO) for human consumption. This result indicates that public health warnings regarding human ingestion of snails from Taihu Lake are warranted. In addition, further studies are needed to evaluate the occurrence by Microcystis in relation to spatial and temporal changes in water quality.

Redescription of Trypanosoma siniperca Chang 1964 from freshwater fish of China based on morphological and molecular data

During the parasite fauna investigation within 2005, the freshwater fish trypanosome Trypanosoma siniperca Chang 1964 was isolated from the blood of Mandarin carp (Siniperca chuatsi) from Niushan Lake, Hubei Province, central China. Blood trypomastigotes were observed only, and the density of infection was low. Light microscopy examinations of this material made it possible to study in detail the morphology of this parasite and redescribe it according to current standards. T. siniperca is characterized also on the molecular level using the sequences of SSU rRNA gene. Phylogenetic analyses based on these sequences allowed clearer phylogenetic relationships to be established with other fish trypanosomes sequenced to date.

In situ studies on the bioaccumulation of microcystins in the phytoplanktivorous silver carp (Hypophthalmichthys molitrix) stocked in Lake Taihu with dense toxic Microcystis blooms

The phytoplanktivorous silver carp is an important biomanipulation fish to control cyanobacterial blooms and is also a food fish with the greatest production in China. The accumulation of the hepatotoxic microcystins (MCs) determined by LC-MS in various organs of silver carp was studied monthly in Lake Taihu dominated by toxic Microcystis aeruginosa. Average recoveries of spiked fish samples were 78% for MC-RR and 81% for MC-LR. The highest content of MCs was found in the intestine (97.48 mu g g(-1) DW), followed by liver (6.84 mu g g(-1) DW), kidney (4.8 8 mu g g(-1) DW) and blood (1.54 mu g g(-1) DW), and the annual mean MC content was in the order of intestine > liver > kidney > blood > muscle > spleen > gallbladder > gill. Silver carp could effectively ingest toxic Microcystis cells (up to 84.4% of total phytoplankton in gut contents), but showed fast growth (from 141 g to 1759 g in I year in mean weight). Silver carp accumulated less microcystins in liver than other animals in the same site or other fish from different water bodies at similar level of toxin ingestion. There was possible inhibition of the transportation of the most toxic MC-LR across the gutwall. Muscle of silver carp in Lake Taihu should not be consumed during period of dense Microcystis blooms while viscera were risky for consumption in more months. (c) 2006 Elsevier B.V. All rights reserved.

Methylmercury, total mercury and total selenium in four common freshwater fish species from Ya-Er Lake, China

Snakehead fish (Ophiocephalus argus cantor), silver carp (Hypophthalmichthys molitrtix), crucian carp (Carassius carassius), and common carp (Cyprinus carpio) are four common freshwater fish species in China. In this study, the level of methylmercury (MeHg), total mercury (T-Hg), and total selenium (T-Se) in muscle samples of these four fish species from Ya-Er Lake, China, were analyzed using atomic fluorescence spectrometry coupled with high-performance liquid chromatography, and inductively coupled plasma mass spectrometry. The concentrations of MeHg in all the fish species were significantly correlated with those of T-Hg. Higher T-Hg and MeHg concentrations had accumulated in the snakehead fish, which is a strongly predatory fish, than in the other three species. The concentration ratios of MeHg and T-Hg in the muscles of these four fish species were almost equal. Conversely, there was negative correlation between the concentrations of T-Hg and T-Se, which implies that there is a competition between these two elements with respect to bioaccumulation. It is noteworthy that of all the muscle samples tested, the level of T-Hg exceeded the maximum allowable limit in fish [0.4 mg kg(-1) (w/w) recommended by the World Health Organization] in 38.46% of those of the silver carp, 18.18% of those of the crucian carp, and 100% of those of snakehead fish. These results show that the consumption of contaminated fish is a potential threat to human health and that necessary preventive measures to safeguard public health should be emphasized.