A Review of Nontraditional Biomanipulation

The aim of this review is to identify problems, find general patterns, and extract recommendations for successful management using nontraditional biomanipulation to improve water quality. There are many obstacles that prevent traditional biomanipulation from achieving expectations: expending largely to remove planktivorous fish, reduction of external and internal phosphorus, and macrophyte re-establishment. Grazing pressure from large zooplankton is decoupled in hypereutrophic waters where cyanobacterial blooms flourish. The original idea of biomanipulation (increased zooplankton grazing rate as a tool for controlling nuisance algae) is not the only means of controlling nuisance algae via biotic manipulations. Stocking phytoplanktivorous fish may be considered to be a nontraditional method; however, it can be an effective management tool to control nuisance algal blooms in tropical lakes that are highly productive and unmanageable to reduce nutrient concentrations to low levels. Although small enclosures increase spatial overlap between predators and prey, leading to overestimates of the impact of predation, microcosm and whole-lake experiments have revealed similar community responses to major factors that regulate lake communities, such as nutrients and planktivorous fish. Both enclosure experiments and large-scale observations revealed that the initial phytoplankton community composition greatly impacted the success of biomanipulation. Long-term observations in Lake Donghu and Lake Qiandaohu have documented that silver carp (Hypophthalmichthys molitrix) and bighead carp (H. nobilis) (two filter-feeding planktivorous species commonly used in management) can suppress Microcystis blooms efficiently. The introduction of silver and bighead carp could be an effective management technique in eutrophic systems that lack macrozooplankton. We confirmed that nontraditional biomanipulation is only appropriate if the primary aim is to reduce nuisance blooms of large algal species, which cannot be controlled effectively by large herbivorous zooplankton. Alternatively, this type of biomanipulation did not work efficiently in less eutrophic systems where nanophytoplankton dominated.

Expressional induction of Paralichthys olivaceus cathepsin B gene in response to virus, poly I:C and lipopolysaccharide

Cathepsin B is a lysosomal cysteine protease of the papain-like enzyme family with multiple biological functions. In this study, Paralichthys olivaceus cathepsin B (PoCatB) cDNA was isolated from flounder embryonic cells (FEC) treated with UV-inactivated grass carp hemorrhage virus (GCHV) and subsequently identified as a vitally induced gene. The full length cDNA of PoCatB is 1801 bp encoding 330-amino acids. The deduced protein has high homology to all known cathepsin B proteins, containing an N-terminal signal peptide, cysteine protease active sites, the occluding loop segment and a glycosylation site, all of which are conserved in the cathepsin B family. PoCatB transcription of FEC cells could be induced by turbot (Scophthalmus maximus) rhabdovirus (SMRV), UV-inactivated SMRV, UV-inactivated GCHV, poly I:C or lipopolysaccharide (LPS), and SMRV or poly I:C was revealed to be most effective among the five inducers. In normal flounder, PoCatB mRNA was detectable in all examined tissues. Moreover, SMRV infection could result in significant upregulation of PoCatB mRNA, predominantly in spleen, head kidney, posterior kidney, intestine, gill and muscle with 18.2,10.9, 24.7,12, 31.5 and 18 fold increases at 72 h post-infection respectively. These results provided the first evidence for the transcriptional induction of cathepsin B in fish by virus and LPS, indicating existence of a novel function in viral defense. (C) 2008 Elsevier Ltd. All rights reserved.

Microsatellite marker isolation and cultured strain identification in Carassius auratus gibelio

Microsatellites have become the preferred molecular markers for strain selection and genetic breeding in fish. In this study a total of 105 microsatellites were isolated and identified in gibel carp (Carassius auratus gibelio) by microsatellite sequence searches in GenBank and other databases and by screening and sequencing of positive clones from the genomic library enriched for AG and GATA repeats. Moreover, nineteen microsatellites were randomly selected to design locus-specific primer pairs, and these were successfully used to identify and discriminate different cultured strains of gibel carp including strains A, D, L, and F. Three different types of microsatellite pattern were distinguished by the number and length of fragments amplified from the 19 primer pairs, and some microsatellite primer pairs were found to produce different microsatellite patterns among strains and strain-specific fragments. In addition, some duplicated alleles were also detected in two microsatellite patterns. Therefore, the current study provides direct molecular markers to discriminate among different cultured strains for selective breeding and aquaculture practice of gibel carp.

Accumulation of hepatotoxic microcystins in freshwater mussels, aquatic insect larvae and oligochaetes in a large, shallow eutrophic lake (Lake Chaohu) of subtropical China

This paper studied the seasonal changes of two common microcystins (MCs), MC-RR and -LR, in the commercially important mussel Corbicula fluminea in Lake Chaohu, where there occurred dense cyanobacteria. Occasional measurements were also made for MC in the mussel Arconaia lanceolat, the oligochaete Limnodilus hoffineisteri and the insect larva Chironomus sp. Mean MC of C. fluminea was much higher in hepatopancreas than in intestine and foot. Our study is the first to report accumulation of MCs in oligochaetes and aquatic insect larvae. The hi-h contents of MCs in the insect larvae suggest a great possibility for the transfer of MCs to benthos-feeding omnivores like common carp. According to the provisional standard by the WHO, 28.6% of the collected C. fluminea were harmful for human consumption, assuming a daily consumption of 300 by a person. It is recommended that edible mussels should not be collected for human consumption during toxic cyanobacterial blooms in Lake Chaohu.

Molecular cloning, characterization and expression analysis of the PKZ gene in rare minnow Gobiocypris rarus

Double-stranded RNA-activated protein kinase (PKR) plays an important rote in interferon-induced antiviral responses, and is also involved in intracellular signaling pathways, including the apoptosis, proliferation, and transcription pathways. In the present study, a PKR-like gene was cloned and characterized from rare minnow Gobiocypris rarus. The full length of the rare minnow PKR-like (GrPKZ) cDNA is 1946 bp in Length and encodes a polypeptide of 503 amino acids with an estimated molecular mass of 57,355 Da and a predicted isoelectric point of 5.83. Analysis of the deduced amino acid sequence indicated that the mature peptide contains two Zalpha domains and one S_TKc domain, and is most similar to the crucian carp (Carassius auratus) PKR-like amino acid sequence with an identity of 77%. Quantitative RT-PCR analysis showed that GrPKZ mRNA expression is at low levels in gill, heart, intestine, kidney, liver, muscle and spleen tissues in healthy animals and up-regulated by viruses and bacteria. After being infected by grass carp reovirus, GrPKZ expression was up-regulated from 24 h post-injection and lasted until the fish became moribund (P < 0.05). Following infection with Aeromonas hydrophila, GrPKZ transcripts were induced at 24 h post-injection (P < 0.05) and returned to control levels at 120 h post-injection. These data imply that GrPKZ is involved in antiviral defense and Toll-like receptor 4 signaling pathway in bacterial infection. (C) 2008 Elsevier Ltd. All rights reserved.

Microcystin-induced variations in transcription of GSTs in an omnivorous freshwater fish, goldfish

The glutathione S-transferases are important enzymes in the microcystin-induced detoxication processes. In this experiment, we cloned the full-length cDNA of alpha, pi and theta-class-like glutathione S-transferase genes from goldfish (Carassius auratus Q. Their derived amino acid sequences were clustered with other vertebrate alpha, pi and theta-class GSTs in a phylogenetic tree and the goldfish GST sequences have the highest similarity with those from common carp and zebrafish. Goldfish were i.p. injected with microcystins extract at two doses (50 and 200 mu g kg(-1) BW MC-LReq) and the relative changes of the mRNA abundance in liver, kidney and intestine were analyzed by real-time PCR. The transcription of GST alpha was suppressed in both liver and intestine, but induced in the kidney. Decreased transcription of GST theta was detected in liver, kidney and intestine in the low-dose group. The transcription of GST pi was suppressed in liver and intestine post-injection in both dose groups. These results suggested that the transcription of GST isoforms varied in different ways within an organ and among organs of goldfish exposed to MCs. (C) 2008 Elsevier B.V. All rights reserved.

In situ study on effect of food competition on diet shifts and growth of silver and bighead carps in large biomanipulation fish pens in Meiliang Bay, Lake Taihu

Silver and bighead carps were cultured in large fish pens to reduce the risks of cyanobacterial bloom outbreaks in Meiliang Bay, Lake Tauhu in 2004 and 2005. Diet compositions and growth rates of the carps were studied from April to November each year. Both carp species fed mainly on zooplankton (> 50% in diet) in 2004 when competition was low, but selected more phytoplankton in 2005 when competition was high. Silver carp had a broader diet breadth than did bighead carp. Higher densities and fewer food resources increased diet breadths but decreased the diet overlap in both types of carps. It can be predicted that silver and bighead carps would be released from diet competition and shift to feed mainly on zooplankton at low densities, decreasing the efficiency of controlling cyanobacterial blooms. Conclusively, when silver and bighead carps are used to control cyanobacterial blooms, a sufficiently high stocking density is very important for a successful practice.

Identical sequences but different expression patterns of Hira gene in gynogenetic and gonochoristic crucian carps

Hir/Hira (histone regulation) genes were first identified in yeast as negative regulators of histone gene expression. It has been confirmed that HIRA is a conserved family of proteins present in various animals and plants. In this paper, the cDNAs of the Hira homolog named CagHira and CaHira were isolated from gynogenetic gibel carp (gyno-carp) and gonochoristic color crucian carp (gono-carp) respectively. The full-length CagHira is 3,860 bp in length with an open reading frame (ORF) of 3,033 bp that encodes 1,011 amino acids, while the full-length CaHira is 3,748 bp in length and also has an ORF of 3,033 bp. The deduced amino acid sequences of both Hira homologs contain seven WD domains and show high identity with other HIRA family members. RT-PCR analyses revealed strong expression of Hira in the ovaries, whereas no expression was detected in the testes of either of the fishes. Hira transcription was not detected in the liver of gyno-carp, but a high level of Hira mRNA was observed in gono-carp. The temporal expression pattern showed that the Hira mRNA is consistently expressed during all embryonic development stages in gyno-carp. However, the abundance of CaHira mRNA significantly decreased (P < 0.05) shortly after fertilization and then increased again and remained stable from gastrula till hatching. The varying spatiotemporal expression patterns of Hira genes in gyno-carp and gono-carp may be associated with the differing reproductive modes used by these two closely related fishes. Our results suggest that Hira may play a role not only in the decondensation of sperm nucleus and the formation of pronucleus during fertilization, but also in gastrulation and the subsequent development of embryos.

Generation and characterization of monoclonal antibodies against the flounder Paralichthys olivaceus rhabdovirus

Two MAbs (3C7 and 3C9) against flounder Paralichthys olivaceus rhabdovirus (PORV) were generated with hybridoma cell fusion technology and characterized by an indirect enzyme-linked immunosorbent assay, isotype test, Western blot and immunodot analysis and immunofluorescence assay. Isotyping tests demonstrated that both of the two MAbs belonged to IgM subclass. Western blot analysis showed the MAbs reacted with 42, 30, and 22 kDa viral proteins, which were localized within the cytoplasm of PORV-infected grass carp ovary (GCO) cells analyzed by indirect immunofluorescences tests. The MAb 3C7 was also selected at random for detecting virus antigens in the inoculated grass carp tissues by immunohistochemistry assay. Flow cytometry tests showed that at the 36 h postinfection (0.25 PFU/cell), the 23% PORV-infected GCO cells could be distinguished from the uninfected cells with the MAb 3C7. Such MAbs could be useful for diagnosis and potential treatment of viral infection. (C) 2007 Elsevier B.V. All rights reserved.

TN : TP ratio and planktivorous fish do not affect nutrient-chlorophyll relationships in shallow lakes

1. In previous work, phytoplankton regulation in freshwater lakes has been associated with many factors. Among these, the ratio of total nitrogen to total phosphorus (TN : TP) has been widely proposed as an index to identify whether phytoplankton are N- or P-limited. From another point of view, it has been suggested that planktivorous fish can be used to control phytoplankton. 2. Large-scale investigations of phytoplankton biomass [measured as chlorophyll a, (chl-a)] were carried out in 45 mid-lower Yangtze shallow lakes to test hypotheses concerning nutrient limitation (assessed with TN : TP ratios) and phytoplankton control by planktivorous fish. 3. Regression analyses indicated that TP was the primary regulating factor and TN the second regulating factor for both annual and summer phytoplankton chl-a. In separate nutrient-chl-a regression analyses for lakes of different TN : TP ratios, TP was also superior to TN in predicting chl-a at all particular TN : TP ranges and over the entire TN : TP spectrum. Further analyses found that chl-a : TP was not influenced by TN : TP, while chl-a : TN was positively and highly correlated to TP : TN. 4. Based on these results, and others in the literature, we argue that the TN : TP ratio is inappropriate as an index to identify limiting nutrients. It is almost impossible to specify a 'cut-off' TN : TP ratio to identify a limiting nutrient for a multi-species community because optimal N : P ratios vary greatly among phytoplankton species. 5. Lakes with yields of planktivorous fish (silver and bighead carp, the species native to China) > 100 kg ha(-1) had significantly higher chl-a and lower Secchi depth than those with yields < 100 kg ha(-1). TP-chl-a and TP-Secchi depth relationships are not significantly different between lakes with yields > 100 kg ha(-1) or < 100 kg ha(-1). These results indicate that the fish failed to decrease chl-a yield or enhance Z(SD). Therefore, silver carp and bighead carp are not recommended as a biotic agent for phytoplankton control in lake management if the goal is to control the entire phytoplankton and to enhance water quality.

Genomic sequence of mandarin fish rhabdovirus with an unusual small non-transcriptional ORF

The complete genome of mandarin fish Siniperca chuatsi rhabdovirus (SCRV) was cloned and sequenced. It comprises 11,545 nucleotides and contains five genes encoding the nucleoprotein N, the phosphoprotein P, the matrix protein M, the glycoprotein G, and the RNA-dependent RNA polymerase protein L. At the 3' and 5' termini of SCRV genome, leader and trailer sequences show inverse complementarity. The N, P, M and G proteins share the highest sequence identities (ranging from 14.8 to 41.5%) with the respective proteins of rhabdovirus 903/87, the L protein has the highest identity with those of vesiculoviruses, especially with Chandipura virus (44.7%). Phylogenetic analysis of L proteins showed that SCRV clustered with spring vireamia of carp virus (SVCV) and was most closely related to viruses in the genus Vesiculovirus. In addition, an overlapping open reading frame (ORF) predicted to encode a protein similar to vesicular stomatitis virus C protein is present within the P gene of SCRV. Furthermore, an unoverlapping small ORF downstream of M ORF within M gene is predicted (tentatively called orf4). Therefore, the genomic organization of SCRV can be proposed as 3' leader-N-P/C-M-(orf4)-G-L-trailer 5'. Orf4 transcription or translation products could not be detected by northern or Western blot, respectively, though one similar mRNA band to M mRNA was found. This is the first report on one small unoverlapping ORF in M gene of a fish rhabdovirus. (c) 2007 Elsevier B.V. All rights reserved.

Molecular characterization and subcellular localization of Carassius auratus interferon regulatory factor-1

Interferon (IFN)-regulatory transcription factor-1 (IRF-1) has been studied in mammals and fish but little is known about the relationship between its gene structure and nuclear 'ion of IRF-1 protein. In this study, a cDNA encoding Carassius auratus IRF-1 (CaIRF-1) was isolated from an interferon-producing cell line, C. ouratus blastulae embryonic (CAB) cells, exposed to UV-inactivated grass carp hemorrhagic virus (GCHV). The CaIRF-1 genomic locus exhibits exon-intron arrangements similar to those of other vertebrate IRF-1 loci, with nine exons and eight introns, although together with pufferfish IRF-1, CaIRF-1 distinguishes itself from other vertebrate IRF-1 genes by a relatively compact genomic size. Similar to the known IRF-1 genes, CaIRF-1 is ubiquitously expressed, and is upregulated in vitro and in vivo in response to virus, Poty I:C, or CAB INF-containing supernatant (ICS). Subcellular localization analysis confirms the nuclear distribution of CaIRF-1 protein, and reveals two nuclear localization signals (NILS), any one of which is sufficient for nuclear translocation of CaIRF-1. One NLS Locates to amino acids 117-146, and appears to be the structural and functional equivalent of the NLS in mammalian IRF-1. The second NLS (amino acids 73-115) is found within the DNA-binding domain (DBD) of CaIRF-1, and contains two regions rich in basic amino acids (''(KDKSINK101)-K-95" and ''(75)KTWKANFR(82)"). In comparison with mammalian IRF-1, in which the corresponding amino acid stretch does not seem to drive nuclear translocation, five conserved basic amino acids (K-75, K-78, R-82, K-95, and K-101) and one non-conserved basic amino acid (K-97) are present in this NLS from CaIRF-1. This observation suggests that K97 Of CaIRF-1 might be essential for the function of its second NLS, wherein the six basic aminoacids might cooperate to drive CaIRF-1 to the nucleus. Therefore, the current study has revealed a new nuclear localization motif in the DBD of a vertebrate IRF-1. (C) 2007 Elsevier Ltd. All rights reserved.

Improvement and observation of immunoelectron microscopic method for the localization of frog Rana grylio virus (RGV) in infected fish cells

In this paper, to understand the roles of amorphous structures which were observed within the viromatrix of Rana grylio virus (RGV), an improved immunoelectron microscopy (IEM) method was developed to detect the localization of RGV in carp Epithelipma papulosum cyprinid (EPC) cells. Infected EPC cells were fixed with 4% paraformaldehyde-0.25% glutaraldehyde mixture, dehydrated completely, and embedded in LR White resin. This method allowed good ultrastructural preservation and specific labeling with anti-RGV antibodies. The results of IEM showed that colloidal gold mainly bound to the capsids of viral particles at the stage of viral assembly, while during the viral maturation colloidal gold bound to the envelop of virions. In addition, within the viromatrix, the amorphous structures, including dense floccules, membranous materials and tubules, also had strong colloidal gold signals, revealing that those amorphous structures were participated in RGV assembly. In contrast, no significant gold labeling signals were obtained in negative controls. The present study not only provided further evidence that amorphous structures within the viromatrix were involved in the process of RGV assembly, but also developed an improved IEM method for studying the interaction between iridovirus and host cells. (C) 2006 Elsevier Ltd. All rights reserved.

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.

In situ study on the control of toxic Microcystis blooms using phytoplanktivorous fish in the subtropical Lake Taihu of China: A large fish pen experiment

Three large fish pens (0.36 km(2) of each) stocked with silver and bighead carp were set up in Meiliang Bay for controlling toxic Microcystis blooms. The responses of plankton communities and food consumption of silver and bighead carp were studied. Crustacean zooplankton were significantly suppressed in the fish pens. Total phytoplankton biomass, Microcystis biomass and microcystin concentration were lower in the fish pens than in the surrounding lake water, but the difference was not statistically significant. The present stocking density of silver plus bighead carp (about 40 g/m(3) in July) was likely too low to achieve an adequate control of Microcystis. Silver carp fed mainly on phytoplankton but bighead carp mainly on zooplankton: mean zooplankton contribution in the gut was 31.5% for silver carp and 64.7% for bighead carp. Compared with previous studies, both carp species preyed upon more zooplankton because of the abundant food resource. Daily rations of silver and bighead carp were estimated by Egger's model in the main growing season. Filtration rate was calculated from the daily ration and the density of plankton in the lake. During May-October, filtration rates of silver and bighead carp for phytoplankton were 0.22-1.53 L g(-1) h(-1) and 0.02-0.68 L g(-1) h(-1), respectively, and filtration rates for zooplankton were 0.24-0.44 L g(-1) h(-1) and 0.08-1.41 L g(-1) h(-1), respectively. Silver carp had a stronger ability of eliminating phytoplankton than bighead carp. To achieve a successful bioniampulation with a minimum effect of ichthyoeutrophication, the stocking proportion of bighead carp should be controlled in the future practice. (c) 2007 Elsevier B.V All rights reserved.