Changes on lipid peroxidation,enzymatic activities and gene expression in planarian (Dugesia japonica) following exposure to perfluorooctanoic acid.

We investigated perfluorooctanoic acid (PFOA)-induced stress response in planarians. We administered different concentrations of PFOA to planarians for up to 10 d. PFOA exposure resulted in significant concentration-dependent elevations in lipid peroxidation, glutathione S-transferase and caspase-3 protease activities, and a significant decline in glutathione peroxidase activities compared with control groups. Exposure to PFOA significantly up-regulated the heat shock proteins hsp70 and hsp90, and p53, and down-regulated hsp40 compared with controls. PFOA exposure also increased HSP70 protein levels, as demonstrated by western blot analysis. These alterations indicated that PFOA exposure induced a stress response and affected the regulation of oxidative stress, enzymatic activities and gene expression. These results suggest that these sensitive parameters, together with other biomarkers, could be used for evaluating toxicity, for ecological risk assessment of PFOA in freshwaters.

Transcriptomic and proteomic analyses of splenic immune mechanisms of rainbow trout (Oncorhynchus mykiss) infected by Aeromonas salmonicida subsp. salmonicida.

Furunculosis caused by Aeromonas salmonicida subsp. salmonicida is an epidemic disease among salmonids, including rainbow trout (Oncorhynchus mykiss). However, the immune mechanisms that are elicited in rainbow trout against the invasion of A. salmonicida are not yet fully understood. In this study, we examined the spleen to investigate the immune response of rainbow trout at 3days post-infection by A. salmonicida at the transcriptome and proteome levels by using Illumina-seq and iTRAQ methods, respectively. A total of 1036 genes and 133 proteins were found to undergo differential expression during the immune response of the spleen against A. salmonicida infection. Gene ontology and KEGG analysis were conducted among the differentially expressed genes and proteins, revealing that immune system process and response to stimulus were the top two biological processes, and immune system, signaling molecules and interaction, and immune diseases were the differential pathways activated. Correlation analysis of transcriptomic and proteomic results showed 17 proteins (11 upregulated and 6 downregulated) having consistent expression at RNA and protein levels. Moreover, protein-protein interaction analysis showed that diseases, proteasome, aminoacyl-tRNA biosynthesis, and nucleotide metabolism were the main interactions among the consistently expressed proteins. Consequently, these upregulated proteins, namely, ferritin, CD209, IL13Rα1, VDAC2, GIMAP7, PSMA1, and two ANXA11s could be considered as potential biomarkers for rainbow trout immune responses. BIOLOGICAL SIGNIFICANCE: This study provides the first identification of immune markers through an analysis of the differential expression of both genes and their corresponding protein products in the spleen of rainbow trout after infection by A. salmonicida, shedding light on the molecular mechanisms triggered in rainbow trout against A. salmonicida infection and providing new molecular targets for further immunological research in fish.

Comparative analysis of the intestinal bacterial communities in different species of carp by pyrosequencing.

Gut microbiota is increasingly regarded as an integral component of the host, due to important roles in the modulation of the immune system, the proliferation of the intestinal epithelium and the regulation of the dietary energy intake. Understanding the factors that influence the composition of these microbial communities is essential to health management, and the application to aquatic animals still requires basic investigation. In this study, we compared the bacterial communities harboured in the intestines and in the rearing water of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius cuvieri), and bighead carp (Hypophthalmichthys nobilis), by using 454-pyrosequencing with barcoded primers targeting the V4 to V5 regions of the bacterial 16S rRNA gene. The specimens of the three species were cohabiting in the same pond. Between 6,218 and 10,220 effective sequences were read from each sample, resulting in a total of 110,398 sequences for 13 samples from gut microbiota and pond water. In general, the microbial communities of the three carps were dominated by Fusobacteria, Firmicutes, Proteobacteria and Bacteroidetes, but the abundance of each phylum was significantly different between species. At the genus level, the overwhelming group was Cetobacterium (97.29 ± 0.46 %) in crucian carp, while its abundance averaged c. 40 and 60 % of the sequences read in the other two species. There was higher microbial diversity in the gut of filter-feeding bighead carp than the gut of the two other species, with grazing feeding habits. The composition of intestine microbiota of grass carp and crucian carp shared higher similarity when compared with bighead carp. The principal coordinates analysis (PCoA) with the weighted UniFrac distance and the heatmap analysis suggested that gut microbiota was not a simple reflection of the microbial community in the local habitat but resulted from species-specific selective pressures, possibly dependent on behavioural, immune and metabolic characteristics.

Ultrastructural study of Balantidium ctenopharyngodoni Chen, 1955 (Class: Litostomatea) from China with an emphasis on its vestibulum.

A detailed description of the fine structure of Balantidium ctenopharyngodoni Chen, 1955 with an emphasis on its vestibulum is given in the present paper. As to the vestibular kinetids, special attention is paid to the characters of T1, T2 microtubules and nematodesmata. Serving as the major skeleton to the vestibular cortex, the T1, T2 and Pc microtubules are described herein and their support function is also discussed. Moreover, the well-developed nematodesmata of the vestibular kinetids that form a large basket-like complex are described in detail.

Effects of aqueous extract of Capsicum frutescens (Solanaceae) against the fish ectoparasite Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is an important fish ectoparasite that often results in significant economic losses to freshwater aquaculture. The search of alternative substances to control infections of I. multifiliis became stringent after malachite green, an effective and widely used chemotherapeutant, is banned on fish farms because of its carcinogenicity and teratogenicity. In this study, the effects of the aqueous extract of Capsicum frutescens, which is readily available and affordable, were evaluated under in vitro and in vivo conditions. The results in the in vitro conditions showed that the aqueous extracts of C. frutescens with the ratios (V (SS)/V (T)-V (SS), the volume of stock solution; V (T), the volume of total solution) of 1:32 and 1:64 led to more than 70 % mortality of I. multifiliis theronts during 4 h of exposure and significantly reduced the survival of the tomonts and the total number of theronts released by the tomonts within 22 h (P < 0.05). A 96-h bioassay was carried out to determine the acute toxicity of the aqueous extract of C. frutescens to goldfish. No visible effect was observed in the treatments with the aqueous extracts of C. frutescens with the ratios (V (SS)/V (T)) of 1:32, 1:64 and 1:128, while in the other treatments, the erratic behaviour of fish was noted. In addition, in vitro tests demonstrated that the aqueous extract of C. frutescens had an adverse effect on I. multifiliis trophonts in situ. Fish treated with the aqueous extracts of C. frutescens in ratios V (SS)/V (T) of 1:32 and 1:64 carried significantly fewer parasites than the control and the other treatments (P < 0.05). These results suggest, therefore, that aqueous extracts of C. frutescens have potential for the control of ichthyophthiriasis in the aquaculture industry, though further phytochemical studies will need to be performed for isolation and identification of the active compounds.

Effect of potassium ferrate(VI) on survival and reproduction of Ichthyophthirius multifiliis tomonts.

Ichthyophthirius multifiliis is an important freshwater teleost pathogen that can infect most species of freshwater fish worldwide and often leads to significant economic losses to the aquaculture industry. Potassium ferrate(VI), as a potential therapeutic agent for external protozoan parasite infections, has been proven to kill I. multifiliis theronts effectively; however, no information is available on effects of potassium ferrate(VI) exposure to I. multifiliis tomonts. This study evaluated the effects of potassium ferrate(VI) on the survival and reproduction of I. multifiliis tomonts. The results of experiment 1 showed that potassium ferrate(VI) at concentrations of 2.4, 4.8, 9.6, and 19.2 mg/L resulted in tomont survival rates of 82.2%, 34.2%, 14.6%, and 0, respectively, and significant differences were noted in tomont reproduction between the treatments and the control (P < 0.05). Additionally, this study was designed to determine the effect of potassium ferrate(VI) toxicity on age of the tomont. The results indicated that encysted I. multifiliis was more resistant to potassium ferrate(VI) treatments. In addition, this study was designed to investigate I. multifiliis tomont survival and reproduction when collected from infested goldfish in potassium ferrate(VI) solutions at different concentrations (0, 2.4, 4.8, 9.6, and 19.2 mg/L) for 30 min. It was found that the tomont survivals in the treatments were significantly lower than those in the control (P < 0.05). This study demonstrated that a bath with potassium ferrate(VI) possibly was an effective method to treat ichthyophthiriasis.

Prevention of Ichthyophthirius multifiliis infestation in goldfish (Carassius auratus) by potassium ferrate(VI) treatment.

Ichthyophthirius multifiliis is an important freshwater teleost pathogen that often leads to significant economic losses to the aquaculture industry. The purpose of this study was to assess the acute toxicity of potassium ferrate(VI) to I. multifiliis theront and the concentration needed to prevent I. multifiliis infestation in goldfish, Carassius auratus. Five hundred theronts were exposed to concentrations of potassium ferrate(VI) in each well of a 96-well microtiter plate and observed for 4h to determine the acute toxicity. Results showed that the exposure of I. multifiliis theronts to potassium ferrate(VI) at concentrations of 4.80mg/L or more resulted in 100% mortality by 4h; the LC(50) value was estimated to be 1.71mg/L. Aqueous static renewal 96-h bioassays were carried out to determine the acute toxicity of potassium ferrate(VI) to goldfish. The LC(50) value for potassium ferrate(VI) in goldfish was 42.51mg/L. Goldfish were exposed to 4000 theronts/fish in aerated tap water (a dose previously shown to result in consistent infestation) and treated with a single dose of potassium ferrate(VI) after 30min contact with theronts. Infection level and prevalence were recorded everyday after exposure. The results revealed that potassium ferrate(VI) at the 4.80mg/L or more concentrations can significantly reduce not only the number of trophonts on the fin of goldfish on day 3 (P<0.05), but also the prevalence of ichthyophthiriasis (P<0.05). Potassium ferrate(VI) at a concentration of 4.80mg/L was considered to be the lowest effective dose to prevent infestation of I. multifiliis in goldfish.

First report of Mucor circinelloides occurring on yellow catfish (Pelteobagrus fulvidraco) from China.

Infected yellow catfish (Pelteobagrus fulvidraco) were sent from Niushan Lake Fishery, Hubei Province, China, to our laboratory for diagnosis. Macroscopic daffodil yellow mold was observed on the heads and fins of the fish and one Mucor species was isolated. Based on the morphological and molecular analysis, the species was identified as Mucor circinelloides. Its optimum growth temperature was 30 degrees C and it could not grow at 40 degrees C. The infectivity results showed wound infection could cause 100% cumulative mortalities at all experimental CFU (10(6), 10(7) and 10(8)). The cumulative mortalities of the intraperitoneal infection increased along with the sporangiospore concentrations; the highest mortality was 90% with 10(8) CFU. Histopathological studies showed M. circinelloides could cause a series of pathological changes in the host tissues and they disseminated in different viscera, perhaps by the blood. This is the first report of M. circinelloides infection in yellow catfish.

Redescription of Balantidium polyvacuolum Li 1963 (Class: Litostomatea) inhabiting the intestines of Xenocyprinae fishes in Hubei, China.

Redescription of Balantidium polyvacuolum Li 1963, collected from the hindgut of Xenocypris davidi and Xenocypris argentea, from Niushan Lake Fishery (30 degrees 19' N, 114 degrees 31' E) in Wuhan City, Hubei Province, China in April and June 2007 is presented in this paper to complete Li's description at both light and scanning electronic microscopic levels. The unique body shape of B. polyvacuolum-highly arched dorsal side and flattened ventral surface-as well as its remarkable concave platelet present in the centroventral were well described and compared with other close Balantidium species. Besides, two types of vestibulum shape are observed in our present work, which may suggest the existence of two subspecies or genotype species of these balantidia.

4-(3-Methyl-phen-yl)-3-phenyl-5-(2-pyrid-yl)-4H-1,2,4-triazole.

In the title compound, C(20)H(16)N(4), the m-tolyl and phenyl substituents form dihedral angles of 74.20 (6) and 36.94 (8)°, respectively, with the 1,2,4-triazole ring and the dihedral angle between the triazole and pyridine rings is 36.06 (9)°. In the crystal, mol-ecules are linked by C-H⋯N and C-H⋯π inter-actions.

Saprolegnia brachydanis, a new oomycete isolated from zebra fish.

Saprolegnia brachydanis is described from zebra fish (Brachydanio rerio) in Wuhan, Hubei Province, China. The species is illustrated and compared with other species of the genus. The distinctive characteristics of S. brachydanis are the production of glomerulate oogonia wrapped around by predominantly monoclinous antheridia which can be up to eight in one oogonium. The oogonial stalks are short, straight, or curved and the antheridia, twisted, can enwind one or more oogonia. The oospores cannot mature or easily abort. Morphological features of the oomycete and the ITS sequence of its rDNA as well as the comparison with related species are discussed in this article.

Study of Apiosoma piscicola (Blanchard 1885) occurring on fry of freshwater fishes in Hongze, China with consideration of the genus Apiosoma.

Apiosoma piscicola (Blanchard 1885) was reported from fry of Carassius auratus (Var. pengze) and Ctenopharyngodon idella during parasite surveys in May 2005 and June 2006 at Hongze Lake Fish Hatchery, Jiangsu province, China. Twelve morphometric parameters were used to describe this peritrich in detail in the present study and comparisons were made with other reports. Its synonyms, Glossatella cylindriformis (Chen 1955) and Apiosoma magna (Banina 1968), were clarified based on reliable data. A. piscicola was still considered somewhat host-specific by us, especially to cyprinids, although it shows a quite wide limit of host variability. The telotroch was also observed, and its structures were described herein. Besides, the difficulties of the generic taxonomy were discussed and some suggestions were given.

First report of two Balantidium species from the Chinese giant salamander, Andrias davidianus: Balantidium sinensis Nie 1935 and Balantidium andianusis n. sp.

Two ciliated protozoa, Balantidium sinensis Nie 1935 and Balantidium andianusi n. sp., were isolated from the feces of a wild Chinese giant salamander (Andrias davidianus) captured from the mountainous area of Shiyan, Hubei Province, Central China in October 2006. It is the first report of Balantidium species inhabiting Cryptobranchoidea amphibians. The occurrence of B. sinensis in A. davidianus should be a new record because the type specimens were first discovered and named by Nie in 1935 from Rana nigromaculata and Rana plancyi. For the lack of enough descriptions of taxonomic features in the previous report, it was re-described in detail and compared with Nie's type specimens and B. giganteum to complete the morphological descriptions in the present work. B. andianusi n. sp. was considered to be a new species based on its unique morphological characteristics, especially the high length/width ratio of the vestibulum (8:1). Comparisons were also made among Balantidium species that were found from urodele amphibians.

Bis[µ-3-ethyl-4-phenyl-5-(2-pyrid-yl)-4H-1,2,4-triazole]bis-[dichloridocopper(II)].

The asymmetric unit of the title compound, [Cu(2)Cl(4)(C(15)H(14)N(4))(2)], contains two halves of two centrosymmetric dinuclear mol-ecules, A and B. The conformations of the two crystallographically independent mol-ecules are slightly different: in A, the Cu⋯Cu separation is 4.174 (9) Šand the dihedral angle between the triazole and phenyl rings is 74.23 (11)°; these values are 4.137 (9) Šand 68.58 (13)°, respectively, in B. In each mol-ecule, the copper(II) ions have a distorted trigonal-bipyramidal coordination geometry with a CuCl(2)NN'N'' chromophore. The crystal packing exhibits weak inter-molecular C-H⋯Cl inter-actions.

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.

Light and scanning electron microscopic study of Balantidium ctenopharyngodoni Chen, 1955 (Class: Litostomatea) from China.

Redescription of Balantidium ctenopharyngodoni "Chen (Acta Hydrobiol Sin 1:123-164, 1955)", collected from the hindgut of grass carp (Ctenopharyngodon idella), especially the segment of 6-10 cm upstream from the anus, from Honghu Lake, Hubei Province, central China in November 2005, is presented in this paper to complete Chen's description at both light and scanning electron microscopic levels. Some revisions were done: the vestibulum is fairly symmetrical, with compactly arranged cilia rather than assembled membrane bordering on the left vestibular side; four contractile vacuoles actually exist in the latter body, three of which surround the posterior portion of the macronucleus, whereas the fourth lies antero-left to it. Somatic monokinetids were compared among the species of genus Balantidium. The cysts were described, and possible infection routes of B. ctenopharyngodoni were also discussed.

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.

Redescription of Trypanosoma ophiocephali Chen 1964 (Kinetoplastida: Trypanosomatina: Trypanosomatidae) and first record from the blood of dark sleeper (Odontobutis obscura Temminck and Schlegel) in China.

During the parasite fauna investigation within 2004 and 2005, the freshwater fish trypanosomes were isolated from the blood of dark sleeper (Odontobutis obscura Temminck and Schlegel) and snakehead fish (Ophiocephalus argus Cantor) from Niushan Lake, Hubei Province, China. Blood trypomastigotes were used for light microscopy investigations. The detailed descriptions of three morphological groups of the genus Trypanosoma: Trypanosoma sp. I and Trypanosoma sp. II found in blood of O. obscura, and Trypanosoma sp. III found in blood of O. argus were provided. Morphological features and host species show Trypanosoma sp. III belong to Trypanosoma ophiocephali Chen 1964, an incompletely described species. Infection with trypanosomes of O. obscura was recorded for the first time. According to the size and appearance, the trypanosomes in O. obscura were also tentatively identified as T. ophiocephali Chen 1964.