Anaerobic Ammonium Oxidation Bacteria in a Freshwater Recirculating Pond Aquaculture System.

Anaerobic ammonium oxidation (anammox) is a key biochemical process to reduce nitrogen pollution in aquaculture, especially in water recirculating pond aquaculture system (RPAS). We used 16S RNA and quantified PCR to study the distribution and environmental impacts of anammox bacteria in RPAS. The results show that the anammox bacterial community distributions and diversities that are apparently unit-specific and seasonal have significant (p < 0.05) difference variation in the RPAS. Most of the anaerobic ammonium oxidation bacteria sequences (77.72%) retrieved from the RPAS belong to the Brocadia cluster. The abundance of anammox bacterial in the RPAS ranged from 3.33 × 101 to 41.84 × 101 copies per ng of DNA. The environmental parameter of temperature and nitrogen composition in water could have impacted the anammox bacterial abundance. This study provides more information on our understanding of the anammox bacteria in the RPAS, and provides an important basis for RPAS improvement and regulation.

[Community Characteristics of ANAMMOX Bacteria in Subsurface Flow Constructed Wetland (SSFCW) for Processing of Aquaculture Waster Water].

Anaerobic ammonium oxidation (ANAMMOX) is one of the important functions in waste water treatment by subsurface flow constructed wetland (SSFCW), however, there are few studies on ANAMMOX in SSFCW environment at present. The community characteristics of ANAMMOX in the SSFCW of processing aquaculture waste water were explored in this study. In order to analyze the structure, diversity and abundance of ANAMMOX bacteria, several 16S rRNA clone libraries were constructed and real-time PCR targeting specific 16S rRNA genes together with diversity analysis was adopted. The obtained results showed that the SSFCW identified a total of three unknown clusters and two known clusters including Candidatus brocadia and Candidatus kuenenia. The dominant cluster was Candidatus brocadia. The highest diversity levels of ANAMMOX bacteria occurred in autumn (H', 1.21), while the lowest in spring (H', 0.64). The abundance of ANAMMOX bacteria in SSFCW environment ranged from 8.0 x 10(4) to 9.4 x 10(6) copies x g(-1) of fresh weight and the copy number of total bacterial 16S rRNA genes ranged from 7.3 x 10(9) to 9.1 x 10(10) copies x g(-1) of fresh weight during culture cycle. There were significant differences in the ANAMMOX bacteria abundances of different stratum and seasons in SSFCW environment, but the differences in total bacterial abundances were not obvious. In addition, the differences in ANAMMOX bacteria abundances in different stratum and seasons in SSFCW environment were irregular in different culture cycle. According to the distribution characteristics of ANAMMOX bacteria in the wetland, the denitrification effect of SSFCW could be improved by changing the supplying manners of aquaculture wastewater and adjusting the structure of wetland. The research results will provide reference for further optimizing the SSFCW and improving the efficiency of purification.

Single and joint action toxicity of heavy metals on early developmental stages of Chinese rare minnow (Gobiocypris rarus).

In this work, acute toxicities of heavy metals (Cu, Zn and Cd) were evaluated singly or in mixtures on Chinese rare minnow (Gobiocypris rarus) in its early stages of development (embryos, larvae). Normal embryos not later than 3 h post-fertilization and newly hatched larvae were selected for the tests. The embryos were exposed to the metal solutions studied until developing to the stages of somite formation (15 h), tail detachment (25 h 10 min), heart-beat visible (34 h 10 min), pectoral fin bud appearance (47 h 40 min) and hatching (75 h), respectively. Exposures of the larvae were continued for 24 h. Results from the single toxicity tests revealed that the mortality of embryos increases obviously with increasing exposure duration. G. rarus appears to be more sensitive to heavy metal exposures at larval stage than at embryonic stage. The toxicity order of the tested metals was Cu>Cd>Zn for the embryos and Cu>Zn>Cd for the larvae with LC50 as the toxicity criterion. These data suggest that the sensitivity of G. rarus to heavy metals (Cu, Zn and Cd) is different and it depends on the exposition duration and the stages of fish development. Among the metals tested, Cu is the one most toxic to G. rarus at its early development stages. In addition, results of the mixture experiments showed that binary combinations of Cu-Zn and Cu-Cd had strictly synergistic lethal effects on the larvae. The enhanced toxicity suggested inclusion of mixture considerations in the risk assessment of heavy metals might be recommended.

Immunomodulatory effects of secondary metabolites from thermophilic Anoxybacillus kamchatkensis XA-1 on carp, Cyprinus carpio.

A bacterial strain with putative immunomodulatory properties was isolated from Xi'an hot springs in China. Comparison of 16S rRNA gene revealed a 97% similarity between the tested strain (designated XA-1) and Anoxybacillus kamchatkensis. Two compounds isolated from the secondary metabolites of XA-1 were identified by spectral data (infrared, nuclear magnetic resonance and mass spectrometry) as: (1) cyclo (Gly-L-Pro) and (2) cyclo (L-Ala-4-hydroxyl-L-Pro). Two cyclic dipeptides showed stimulatory properties towards a range of parameters when a dose of 20mg kg(-1) body weight was intraperitoneally injected in naive common carp, Cyprinus carpio. Innate immune parameters (serum SOD, lysozyme and bactericidal activity, and phagocytic activity by peripheral blood leucocytes) along with the expression of two immune-related genes (IL-1ß and iNOS) in blood were examined after 7, 14, 21, and 28 days of injection. In the absence of infection, immunomodulators should ideally not affect normal physiology and immunity of the host; possible negative outcomes of activated immune responses in the naive state are discussed. Protection by two bacterial dipeptides was assessed in an intraperitoneal injection challenge trial with live Aeromonas hydrophila. Both compounds reduced mortality, with the highest survival rate observed in the group that received compound 2 (80%) followed by the group that received compound 1 (65%) while control group scored the worse (15%). Elucidation of the involved protective mechanisms in carp requires future studies.

In vivo anthelmintic activity of bruceine A and bruceine D from Brucea javanica against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus).

The present study was designated to ascertain the anthelmintic activity of the dried fruits of Brucea javanica and to isolate and characterise the active constituents. The methanol extract from the fruits of B. javanica showed significant anthelmintic activity against Dactylogyrus intermedius (EC(50) (median effective concentration) value=49.96 mg l(-1)). Based on this finding, the methanol extract was fractionated on silica gel column chromatography in a bioassay-guided fractionation affording two known quassinoids showing potent activity, bruceine A and bruceine D. Both bruceine A and D exhibited significant activity against D. intermedius with EC(50) values of 0.49 mg l(-1) and 0.57 mg l(-1), respectively, which were more effective than the positive control, mebendazole (EC(50) value=1.25 mg l(-1)). In addition, the 48-h median lethal concentration (LC(50)) for bruceine A and D against the host (Carassius auratus) was 10.6-fold and 9.7-fold higher than the EC(50) for D. intermedius. These results provide evidence that the isolated compounds might be potential sources of new anti-parasitic drugs for the control of Dactylogyrus. This is the first report on an in vivo anthelmintic investigation for B. javanica against D. intermedius.

In vivo evaluation of anthelmintic potential of medicinal plant extracts against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus).

In the present study, an attempt has been made to petroleum ether, chloroform, ethyl acetate, methanol, and water extracts of Prunus amygdalus Batsch seeds (Semen amygdali), Cimicifuga foetida L. rhizomes (Rhizoma Cimicifugae), Peucedanum decursivum (Miq.) Maxim roots (Radix Peucedani), Momordica cochinchinensis Spreng. seeds (Semen Momordicae), and Bupleurum chinense DC. roots (Radix Bupleuri chinensis) for their in vivo anthelmintic activity against monogenean Dactylogyrus intermedius in goldfish (Carassius auratus). The results showed that the efficacies of methanol, chloroform, and ethyl acetate extracts from R. Bupleuri chinensis were found to be, in this order, more effective than others with the 48 h-EC(50) and EC(90) values of 3.5 and 6.9, 6.0 and 8.4, 7.4 and 11.2 mg/L, respectively, followed by ethyl acetate extract of R. cimicifugae and chloroform extract of R. peucedani with EC(50) 189.2 and 240.4 mg/L. The promising methanol, chloroform, and ethyl acetate extracts from R. Bupleuri chinensis were subjected to acute toxicity tests for the evaluation of their safety to the host. After 48-h exposure, the mortalities of goldfish were recorded, and the established LC(50) values were 10.1-, 4.2-, and 8.4-fold higher than the corresponding EC(50). These results indicated that the three extracts from R. Bupleuri chinensis exhibit potential to be used as preferred natural antiparasitics for the control of the D. intermedius, especially for the methanolic one.