Ammonia is toxic to fish in natural and artificial waters.We evaluated the acute(96 h) and chronic(21 d) toxicity of un-ionized ammonia to GH transgenic common carp(Cyprinus carpio L.) and non-transgenic common carp using a static-renewal bioassay.The 24,48,72 and 96 h median lethal concentrations(LC50) of un-ionized ammonia were slightly lower in transgenic carp(2.64,2.44,2.28 and 2.16 mg N/L,respectively) than in non-transgenic carp(2.70,2.64,2.52 and 2.33 mg N/L,respectively).Similarly,the median lethal time(LT50) was significantly shorter for transgenic carp(1.41,7.91 and 117.42 h) than for non-transgenic common carp(2.53,14.06 and 150.44 h) following exposure to 3.86,3.29,or 2.09 mg N/L,respectively.Moreover,the mortality of transgenic carp was significantly higher than that of non-transgenic carp at all un-ionized ammonia concentrations((0.91 ± 0.12),(0.48 ± 0.06) and(0.12 ± 0.01) mg N/L) during the 21 d chronic toxicity test.Our results suggest that GH transgenic carp are less tolerant of unionized ammonia than non-transgenic carp.Our data are useful for evaluating potential environmental risk,optimizing stocking density in intensive aquaculture and establishing water quality criteria for ammonia in aquaculture.
Genetic engineering in filamentous N2-fixing cyanobacteria usually involves Anabaena sp. PCC 7120 and several other non-aggregating species. Mass culture and harvest of such species are more energy consuming relative to aggregating species. To establish a gene transfer system for aggregating species, we tested many species of Anabaena and Nostoc, and identified Nostoc muscorum FACHB244 as a species that can be genetically manipulated using the conjugative gene transfer system. To promote biodegradation of organophosphorus pollutants in aquatic environments, we introduced a plasmid containing the organophosphorus-degradation gene （opd） into Anabaena sp. PCC 7120 and Nostoc muscorum FACHB244 by conjugation. The opd gene was driven by a strong promoter, Pp,bA. From both species, we obtained transgenic strains having organophosphorus-degradation activities. At 25~C, the whole-cell activities of the transgenic Anabaena and Nostoc strains were 0.163~0.001 and 0.289~0.042 unit/gg Chl a, respectively. However, most colonies resulting from the gene transfer showed no activity. PCR and DNA sequencing revealed deletions or rearrangements in the plasmid in some of the colonies. Expression of the green fluorescent protein gene from the same promoter in Anabaena sp. PCC 7120 showed similar results. These results suggest that there is the potential to promote the degradation of organophosphorus pollutants with transgenic cyanobacteria and that selection of high-expression transgenic colonies is important for genetic engineering of Anabaena and Nostoc species. For the first time, we established a gene transfer and expression system in an aggregating filamentous N2-fixing cyanobacterium. The genetic manipulation system of Nostoc muscorum FACHB244 could be utilized in the elimination of pollutants and large-scale production of valuable proteins or metabolites.
