Evaluation of scale development and its regeneration potency in the desert killifish under laboratory conditions.

Scale development and its regeneration potency were evaluated in a desert killifish Aphaniops hormuzensis (family Aphaniidae) in laboratory conditions by using light and scanning electron microscopy. Scale development in A. hormuzensis took 156 days at room temperature. Four specific regions of scale formation were detected. The first scale development began 13 days post-hatching (dph) (total length [TL] = 8.5 mm) at the caudal peduncle region and is extended anteriorly 26 dph (TL = 13.6 mm) at the area below the dorsal fin. Scales began forming independently in the head region at 33 dph (TL = 21.7 mm), and in the abdominal region, began at 41 dph (TL = 25.8 mm). Additional points of scale origin were detected on the sides of the operculum or behind and below the eyes. Scale regeneration in the caudal peduncle started 6 days after removal (dar). In 16 dar, the microstructural features appeared and the growth circles, a wide and oblong focus (focus length = 0.6 ± 0.05 µm), and lepidonts were also formed. In 36 dar, the scale shape was gradually changed from circular to a polygon, and radii were distinguishable in the anterior field. The pattern of scale formation could be useful in enhancing the understanding of systematics and phylogeny, functional morphology, and habitat use. It could also be useful in helping to define the Larval/juvenile transition period.

Isolation of Aeromonas salmonicida subspecies salmonicida from aquaculture environment in India: Polyphasic identification, virulence characterization, and antibiotic susceptibility.

This study reports the polyphasic identification, characterization of virulence potential, and antibiotic susceptibility of Aeromonas salmonicida subspecies salmonicida COFCAU_AS, isolated from an aquaculture system in India. The physiological, biochemical, 16s rRNA gene sequencing and PAAS PCR test identified the strain as Aeromonas salmonicida. The MIY PCR tests established the subspecies as 'salmonicida'. The in vitro tests showed the isolated bacterium as haemolytic with casein, lipid, starch, and gelatin hydrolysis activity, indicating its pathogenic attributes. It also showed the ability to produce slime and biofilm, and additionally, it possessed an A-layer surface protein. In vivo pathogenicity test was performed to determine the LD50 dose of the bacterium in Labeo rohita fingerlings (14.42 ± 1.01 g), which was found to be 106.9 cells fish-1. The bacteria-challenged fingerlings showed skin lesions, erythema at the base of the fins, dropsy, and ulcer. Almost identical clinical signs and mortalities were observed when the same LD50 dose was injected into other Indian major carp species, L. catla and Cirrhinus mrigala. Out of the twelve virulent genes screened, the presence of nine genes viz., aerA, act, ast, alt, hlyA, vapA, exsA, fstA, and lip were detected, whereas ascV, ascC, and ela genes were absent. The A. salmonicida subsp. salmonicida COFCAU_AS was resistant to antibiotics such as penicillin G, rifampicin, ampicillin, and vancomycin while highly sensitive to amoxiclav, nalidixic acid, chloramphenicol, ciprofloxacin, and tetracycline. In summary, we have isolated a virulent A. salmonicida subsp. salmonicida from a tropical aquaculture pond which can cause significant mortality and morbidity in Indian major carp species.

Melatonin alleviates lead-induced intestinal epithelial cell pyroptosis in the common carps (Cyprinus carpio) via miR-17-5p/TXNIP axis.

Lead (Pb) has been concerned as one of the most severe hazardous contaminants, because it can cause pyroptosis in multiple tissues of mammals and birds. Melatonin (Mel) has attracted much interest for its role in governing intestinal injury via microRNAs (miRNAs). To explore the effect of Mel on Pb exposure-induced intestinal epithelial cell pyroptosis in common carps by regulating miR-17-5p/TXNIP axis, the Pb exposure and Pb-Mel treated models were constructed in vivo. The results elucidated that the suppressed expression of miR-17-5p and intensified level of TXNIP were primarily detected in Pb-exposed gut tissues, and both abolished with Mel addition, along with downregulated Pb-mediated elevated expression of NLRP3, CASP1, IL1ß and GSDMD. Additionally, the targeting relationship between miR-17-5p and TXNIP were demonstrated by dual-luciferase reporter assay, and on this basis, miR-17-5p NC, mimic and inhibitor cell models were established. Thereby, Thereby, the expression of TXNIP in the miR-17-5p mimic groups was significant lower in the Pb-exposure but still elevated than the Control group, and the expression of NLRP3 and NLRP3-dependent pyrotposis-related genes performed consistent alterations. Noticeably, the expression of TXNIP suppressed with Mel addition even in the miR-17-5p inhibitor cell model, resulting in the inactivation of NLRP3 inflammasome-dependent pyroptosis. Overall, we draw the conclusion as Mel attenuates Pb-induced intestinal epithelial cell pyroptosis via miR-17-5p/TXNIP axis. The present study provides a novel perspective for toxicological mechanism of Pb, and new insights for the detoxification mechanism of Mel.

Labeo rohita Mx1 exhibits the critical structural motifs of the family of large GTPases of mammals and is activated by rhabdovirus vaccination and bacterial RNA stimulations.

Myxovirus resistance (Mx) proteins belonging to the dynamin superfamily of high molecular weight GTPases exist in various isoforms and play crucial role in innate immunity. In addition to the isoforms, Mx1 also plays important role in exerting its anti-viral actions against a broad range of animal RNA viruses. In rohu (Labeo rohita), mx1 full-length cDNA sequence consists of 2440 nucleotides (nt) encoding 628 amino acids (aa) polypeptide of 71.289 kDa. Structurally, it belongs to the family of large GTPases with one DYNc domain (13-257aa) comprising of dynamin family motifs (LPRGSGIVTR) and the tripartite GTP-binding motifs (GDQSSGKS, DLPG and TKPD) at the N-terminal and one GED domain (537-628aa) at C-terminus. Rohu Mx1 is closely related to zebrafish Mx1 and is widely expressed in gill, liver, kidney, spleen and blood. In response to rhabdovirus vaccinations, poly I:C stimulation and bacterial infections, mx1 gene expression in rohu was significantly (p < 0.05) induced in majority of the tested organs/tissues. Stimulation of rohu gill cell line with bacterial RNA also induced mx1 gene expression. Together these data suggest the important role of Mx1 in innate immunity in rohu against wide spectrum of fish pathogens.

Comparative analysis of unwashed and single washed mince gel from Indian major carps.

The gelling properties and quality characteristics of unwashed and single washed mince of catla, rohu and mrigal have been investigated to find out suitability of Indian major carps for the preparation of mince gel. The higher moisture content and lower protein content was reported in the single washed mince. The single washing of mince did not improve the gel strength. The gel strength showed significant difference (p < 0.05) and decreased in single washed mince than its unwashed counterparts in catla and mrigal except rohu. It has been observed that gel did not set at pre-incubation temperature of 40 °C for 30 min treatment. SDS-PAGE patterns of proteins did not show any loss of myosin heavy chain (MHC) in single washed mince of Indian major carps. Texture profile analysis showed higher hardness in washed mince gel of Indian major carps while, non-significant difference (p > 0.05) was observed in cohesiveness, adhesiveness and elasticity properties. The whiteness index of washed mince showed improvement. The overall study indicated that mince gels can be made from unwashed mince of Indian major carps, alleviating the problems of waste water disposal leading to production of more value added products with better nutritional value.

Antioxidant properties of papain mediated protein hydrolysates from fresh water carps (Catla catla, Labeo rohita and Cirrhinus mrigala) and its application on inhibition of lipid oxidation in oil sardine mince during ice storage.

Fish protein hydrolysates (FPH) from fresh water carps Catla catla, Labeo rohita and Cirrhinus mrigala were prepared with 5, 10, 15 and 20% degree of hydrolysis (DH) using papain enzyme. FPH were evaluated for antioxidant properties using in vitro assays such as DPPH free radical scavenging activity (at 10 mg/ml), ferric reducing antioxidant power assay (at 20 mg/ml) and linoleic acid peroxidation inhibition activity (at 10 mg/ml). Antioxidant properties of FPH varied with species and DH. The DPPH radical scavenging activity, linoleic acid peroxidation inhibition activity and ferric reducing antioxidant power (as absorbance at 700 nm) of FPH from carps was in the range of 59-92%, 52-85% and 0.388-0.663 respectively. Based on the overall antioxidant activity, FPH from C. catla with 20% DH was added to oil sardine mince at different concentration (0.1, 0.2 and 0.4%) and found to inhibit effectively the formation of peroxides and malonaldehyde in dose dependent manner. FPH from C. catla with 20% DH was fractionated using size exclusion chromatography and had three different peptide fractions with the approximate molecular weight of 6561-2106 Da (fraction 1), 1942-994 Da (fraction 2) and 935-383 Da (fraction 3). The present study showed promising results that the fish protein hydrolysates from fresh water carps muscle proteins can be used as natural antioxidants in food system. Production of fish protein hydrolysates with nutraceutical properties could be the way forward for better utilization and value addition.

Toll-interacting protein in the freshwater fish Labeo rohita exhibits conserved structural motifs of higher eukaryotes and is distinctly expressed in pathogen-associated molecular pattern stimulations and bacterial infections.

Toll-interacting protein (Tollip) is a critical regulator of TOLL- like receptor (TLR)-signaling pathway. It is predominantly associated with TLR2 and TLR4 during acute inflammatory conditions and inhibits the TLR-mediated nuclear factor-kappa activation by suppressing the autophosphorylation of interleukin-1 receptor-associated kinase and its kinase activity. This article describes the Tollip of Labeo rohita (LrTollip), a highly valuable freshwater fish from the Indian subcontinent. The full-length LrTollip complementary DNA (1412 nucleotides) encodes a 276-amino acid (aa) protein, depicting a highly conserved target of the Myb1 (Tom1)-binding domain (TBD; 1-53 aa), conserved core domain 2 (C2; 54-151 aa), and coupling of ubiquitin to endoplasmic reticulum degradation (CUE; 231-273 aa) domains of mouse and human counterparts. The key amino acids exerting the critical functions of Tollip, such as phospholipids recognition and ubiquitination, are present in the C2 and CUE domains of LrTollip, respectively. LrTollip is widely expressed in the kidneys, gills, spleen, liver, and blood, and among these tested tissues, the highest expression is observed in blood. In response to TLR ligands and NOD-like receptor (NLR) ligands stimulations and Aeromonas hydrophila, Edwardsiella tarda, and Bacillus subtilis infections, LrTollip gene expression is induced in various organs/tissues with remarkable difference in their kinetics. These data together suggest the important role of LrTollip in TLR- and NLR-signal transduction pathways and immune-related diseases in fish.

PEG-modified subunit vaccine encoding dominant epitope to enhance immune response against spring viraemia of carp virus.

Spring viraemia of carp (SVC) caused by spring viraemia of carp virus (SVCV) can infect almost all fish of cyprinids, which bring huge economic losses to aquaculture. Glycoprotein (G), as the most important antigenic determinant protein of SVCV, is widely considered as an effective method against SVCV. In our previous study, we found that G3 (131 aa) is the potential dominant antigen epitope that induces strong immune responses similar to G protein (510 aa). Here, in order to further improve the immune effect, we reported a subunit vaccine (PEG-G3) constructed by PEG-modified dominant epitope protein (G3). The results of serum antibody production, enzyme activities and immune-related genes expression showed that PEG-G3 induces significantly stronger immune protective responses against SVCV than G3. PEG modification significantly increased the serum antibody level of the vaccine, which increased significantly after immunization and reached the peak at 21 day post-vaccination. T-AOC and AKP activities in the lowest concentration group (5 µg) of PEG-G3 were significantly higher than those in the highest concentration group (20 µg) of G3. In PEG-G3 group, the expression of almost all genes increased at least 4 times compared with the control group. After 14-day challenge, the RPS (relative percentage survival) of the highest concentration of PEG-G3 group was 53.6%, while that of G3 group is 38.9%. Therefore, this work shows that PEG modification and dominant epitope screening may be effective methods to improve the immune protective effect of vaccines and to resist the infection of aquatic animal viral diseases.

Integrated transcriptomic and proteomic analyses of grass carp intestines after vaccination with a double-targeted DNA vaccine of Vibrio mimicus.

Intestinal mucosal immunity plays a vital role against Vibrio mimicus infection because it is an enteric pathogen causing serious vibriosis in fish. In the previous studies, we developed an oral double-targeted DNA vaccine of V. mimicus and demonstrated that the vaccine could elicit significantly higher intestinal mucosal immune response than did naked DNA vaccine. But, little is known underlying regulatory molecular mechanisms of the enhanced intestinal mucosal immunity. Here the transcriptome and proteome in the intestines of the grass carps immunized or not with the double-targeted DNA vaccine were investigated by using RNA-seq and iTRAQ-coupled LC-MS/MS. Compared with the control group, a total of 5339 differentially expressed genes (DEGs) and 1173 differentially expressed proteins (DEPs) were identified in the immunized fish intestines. Subsequently, the integrated analysis between transcriptome and proteome data revealed that 250 DEPs were matched with the corresponding DEGs (named associated DEPs/DEGs) at both transcriptome and proteome levels. Fifty of all the associated DEPs/DEGs were immune-related and mainly enriched in phagosome, antigen-processing and presentation, complement and coagulation cascades, NLRs and MAPK signaling pathways via Gene Ontology and KEGG pathway analyses, which suggested the coordination of the five activated pathways was essential to the enhanced intestinal mucosal immune response in the immunized fish. The protein-protein interaction analysis showed that 60 of the 63 immune-related DEPs to form an integrated network. Additionally, randomly selected DEGs and DEPs were respectively validated by quantitative real-time RT-PCR and multiple reaction monitoring (MRM) assay, indicating that the both RNA-Seq and iTRAQ results in the study were reliable. Overall, our comprehensive transcriptome and proteome data provide some key genes and their protein products for further research on the regulatory molecular mechanisms underlying the enhanced intestinal mucosal immunity.

Current prospects and challenges in fish vaccine development in India with special reference to Aeromonas hydrophila vaccine.

Infectious diseases are adversely affecting aquaculture practices throughout world and Asian countries are no exception. Indian aquaculture practices are facing serious setback due to a variety of infectious agent's which are responsible for severe mortality and morbidity of all the cultured freshwater fish species leading to severe economic losses. The emergence of antibiotic resistant pattern, residual effect and environmental degradation due to indiscriminate use of antibiotics has necessitates the development of suitable alternate prophylaxis measures for better protection. In this regard, vaccine(s) has proved to be an effective strategy against pathogens to improve the fish production. Over the years numerous studies have been conducted to develop vaccine(s) against different pathogens. While most of the efforts are made to develop vaccine against bacterial pathogens especially against Aeromoniasis and Edwardsiellosis, few attempts have also been made against certain other bacterial, parasitic and fungal pathogens as well. Despite various successful experimental attempts, till date no vaccines against any of the pathogens are commercially available for Indian aquaculture. This review principally focuses on the current state of art in the development of vaccine against different microbial pathogens in general and Aeromonas hydrophila in particular since the bacterium is a major pathogen which is involved in a number of disease conditions in all the cultured fish species in India. Herein in this review, details of various experimental approaches made to find out a potential vaccine candidate which in turn can induce protective immune responses in host alongwith the constraints associated with it in developing a suitable vaccine against this bacterium and its market potential have been illustrated from an Indian perspective.

Next-generation sequencing reveals significant variations in bacterial compositions across the gastrointestinal tracts of the Indian major carps, rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus cirrhosis).

Bacterial communities strongly influence the digestion, health and immune status of fish. This study investigates the microbial distribution of the anterior, middle and distal gut sections of three economically important carp species in Bangladesh, rohu, catla and mrigal (commonly known as Indian major carps), using 16S rRNA-based Illumina sequencing technology. The alpha-diversity measurement with one-way ANOVA indicated high species richness, Shannon and Simpson indices in the middle and distal gut, while the anterior gut of IMCs had the lowest diversity. At the phylum level, there was high abundance of Proteobacteria in the GITs of rohu and mrigal, whereas Fusobacteria was dominant in the anterior and middle guts of catla. At the genus level, diverse microbial communities were identified across the three GIT sections, with six indicator genera found in rohu, catla and mrigal, as revealed by linear discriminant analysis (LDA) at a 0·05 level of significance. Of the 218 genera identified, only 33 were common across the anterior, middle and distal guts of all three species. Bacterial diversity was significantly higher (P < 0·05) in mrigal, followed by catla and rohu, respectively. Alongside the common bacteria Aeromonas, Enterobacter and Serratia, the overwhelming abundance of Cetobacterium, Shewanella and Plesiomonas warrants further investigation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study investigates the microbial communities of the gastrointestinal tracts (GITs) of three Indian major carp (IMC) species-rohu, catla and mrigal, obtained from a polyculture pond under the same feeding regime. Diverse microbial communities were found, with significantly different relative abundances and diversities of phyla and genera. The results provide valuable information on GIT microbial communities that may be useful for nutrition and health management in IMCs.

Effects of dechlorane plus on intestinal barrier function and intestinal microbiota of Cyprinus carpio L.

Dechlorane Plus (DP) is a typical polychlorinated flame retardant that has been emerged in chemical products. Due to its accumulation and amplification effect, the toxicity of DP has become a widespread environmental safety issue. However, whether DP can affect the intestinal tract of teleost fish remains largely unclear. To understand its effects on the intestinal barrier, morphological characteristics and intestinal microbiome of common carp, different concentrations (30, 60 and 120 µg/L) of DP were exposed to common carps for 4 weeks. The results indicated that DP evidently shortened the intestinal folds and damaged the intestinal epithelium layer. In addition, the mRNA expression levels of occludin, claudin-2 and zonula occludens-1 (ZO-1) were significantly decreased with increasing DP concentrations. Furthermore, the relative abundance of some microbiota species were also changed significantly. Our study first demonstrated that DP could cause damage to the intestinal epithelium and destroy the intestinal barrier and increase the relative abundance of pathogenic bacteria, thereby increasing the probability of contact between intestinal epithelium and pathogenic bacteria, which in turn lead to an increased susceptibility to various diseases and poor health. In summary, our findings reveal that chronic DP exposure can have a harmful effect on the intestinal flora balance and is potentially linked to human disease.

Poly-arginine-18 (R18) Confers Neuroprotection through Glutamate Receptor Modulation, Intracellular Calcium Reduction, and Preservation of Mitochondrial Function.

Recent studies have highlighted that a novel class of neuroprotective peptide, known as cationic arginine-rich peptides (CARPs), have intrinsic neuroprotective properties and are particularly effective anti-excitotoxic agents. As such, the present study investigated the mechanisms underlying the anti-excitotoxic properties of CARPs, using poly-arginine-18 (R18; 18-mer of arginine) as a representative peptide. Cortical neuronal cultures subjected to glutamic acid excitotoxicity were used to assess the effects of R18 on ionotropic glutamate receptor (iGluR)-mediated intracellular calcium influx, and its ability to reduce neuronal injury from raised intracellular calcium levels after inhibition of endoplasmic reticulum calcium uptake by thapsigargin. The results indicate that R18 significantly reduces calcium influx by suppressing iGluR overactivation, and results in preservation of mitochondrial membrane potential (ΔΨm) and ATP production, and reduced ROS generation. R18 also protected cortical neurons against thapsigargin-induced neurotoxicity, which indicates that the peptide helps maintain neuronal survival when intracellular calcium levels are elevated. Taken together, these findings provide important insight into the mechanisms of action of R18, supporting its potential application as a neuroprotective therapeutic for acute and chronic neurological disorders.

Influence of a diet containing plant ingredients at different levels on growth performance, carcass biochemical composition, and blood parameters in Indian major carps grown in polyculture earthen ponds.

The effect of feeding levels of plant ingredient-based diet on growth, body composition, and serological constituents of Indian major carps was determined in pond culture condition. Juveniles of Indian major carps (IMCs), Catla catla (catla, 65.87 ± 2.45 g), Labeo rohita (rohu, 64.67 ± 2.15 g), and Cirrhinus mrigala (mrigal, 39.58 ± 3.49 g) were fed 1%, 1.5%, 2%, and 2.5% of the body weight for a period of 150 days. At the end, the total production was significantly higher at 2.0% feeding level and did not change thereafter. The nutrient utilization parameters were significantly (P < 0.001) affected by the feeding level and decreased both linearly and quadratically with the higher level of feed. The SGR in terms of wet weight, dry weight, protein, and lipid increased up to 2% feeding level and plateaued thereafter. The whole body crude protein content of all the three species was the lowest at 1% feeding level and the whole body lipid content increased with increased feeding levels. Tissue protein gain and lipid gain of IMCs were the highest at 2% feeding level. Blood parameter did not indicate any disease or stress condition due to feeding treatments. Considering the growth and nutrition utilization and health of fish, it can be concluded that optimum feeding level of all plant ingredient-based feed of IMC could be 2% of the body weight in pond culture condition.

Interaction of a novel Bacillus velezensis (BvL03) against Aeromonas hydrophila in vitro and in vivo in grass carp.

This study evaluated the inhibition and interaction of Bacillus velezensis BvL03 as a probiotic agent against Aeromonas hydrophila. Strain BvL03 isolated from sediment samples of fish ponds had excellent antimicrobial activity against several fish pathogenic bacteria, especially Aeromonas, including A. hydrophila, A. veronii, A. caviae, and A. sobria. The successful amplification of lipopeptide antimicrobial chemical biosynthetic genes, including iturin family (ituA, ituB, and ituD), bacillomycin family (bacA, bacD, and bacAB), surfactin family (srfAB, srfC, and srfAA), and subtilosin family (albF and sunT) from the genome of BvL03 strain, confirmed its predominant antimicrobial activity. The challenge test suggested that BvL03 significantly decreased fish mortality when challenged with A. hydrophila, which had a cumulative mortality of 12.5% in the treatment group. Toxicity and hemolytic activity of A. hydrophila after co-cultured with BvL03 were relieved as confirmed by the cell experiments, when the initial inoculated concentration of BvL03 was 109 cfu/mL or higher. Moreover, the BvL03 strain labeled with GFP protein (BvL03-GFP) and AhX040 strain labeled with mCherry protein (AhX040-mCherry) were injected into grass carps. The fluorescence levels were monitored by using In Vivo Imaging System (IVIS), in which the green color was steadily increasing, whereas the red color was gradually weakening. Whole genome sequencing revealed that strain BvL03 possesses 15 gene clusters related to antibacterial compounds, including 5 NRPS gene clusters and 3 PKS gene clusters. These results suggested that B. velezensis BvL03 has the potential to be developed as a probiotic candidate against A. hydrophila infection in aquaculture.

Carbonic anhydrase related protein expression in astrocytomas and oligodendroglial tumors.

BACKGROUND: Carbonic anhydrase related proteins (CARPs) VIII, X and XI functionally differ from the other carbonic anhydrase (CA) enzymes. Structurally, they lack the zinc binding residues, which are important for enzyme activity of classical CAs. The distribution pattern of the CARPs in fetal brain implies their role in brain development. In the adult brain, CARPs are mainly expressed in the neuron bodies but only weaker reactivity has been found in the astrocytes and oligodendrocytes. Altered expression patterns of CARPs VIII and XI have been linked to cancers outside the central nervous system. There are no reports on CARPs in human astrocytomas or oligodendroglial tumors. We wanted to assess the expression of CARPs VIII and XI in these tumors and study their association to different clinicopathological features and tumor-associated CAs II, IX and XII. METHODS: The tumor material for this study was obtained from surgical patients treated at the Tampere University Hospital in 1983-2009. CARP VIII staining was analyzed in 391 grade I-IV gliomas and CARP XI in 405 gliomas. RESULTS: CARP VIII immunopositivity was observed in 13% of the astrocytomas and in 9% of the oligodendrogliomas. Positive CARP XI immunostaining was observed in 7% of the astrocytic and in 1% of the oligodendroglial tumor specimens. In our study, the most benign tumors, pilocytic astrocytomas, did not express CARPs at all. In WHO grade II-IV astrocytomas, CARPs were associated with molecular events related to more benign behavior, which was the case with CARP VIII in oligodendrogliomas and oligoastrocytomas as well. CONCLUSIONS: The study observations suggest that the CARPs play a role in tumorigenesis of diffusively infiltrating gliomas. Furthermore, the molecular mechanisms beneath the cancer promoting qualities of CARPs have not yet been discovered. Thus, more studies concerning role of CARPs in oncogenesis are needed.

Immunolocalization of skeletal matrix proteins in tissue and mineral of the coral Stylophora pistillata.

The precipitation and assembly of calcium carbonate skeletons by stony corals is a precisely controlled process regulated by the secretion of an ECM. Recently, it has been reported that the proteome of the skeletal organic matrix (SOM) contains a group of coral acid-rich proteins as well as an assemblage of adhesion and structural proteins, which together, create a framework for the precipitation of aragonite. To date, we are aware of no report that has investigated the localization of individual SOM proteins in the skeleton. In particular, no data are available on the ultrastructural mapping of these proteins in the calcification site or the skeleton. This information is crucial to assessing the role of these proteins in biomineralization. Immunological techniques represent a valuable approach to localize a single component within a calcified skeleton. By using immunogold labeling and immunohistochemical assays, here we show the spatial arrangement of key matrix proteins in tissue and skeleton of the common zooxanthellate coral, Stylophora pistillata. To our knowledge, our results reveal for the first time that, at the nanoscale, skeletal proteins are embedded within the aragonite crystals in a highly ordered arrangement consistent with a diel calcification pattern. In the tissue, these proteins are not restricted to the calcifying epithelium, suggesting that they also play other roles in the coral's metabolic pathways.

Microelectrophoretic study of environmentally induced DNA damage in fish and its use for early toxicity screening of freshwater bodies.

This study investigates the potential of the comet and micronucleus assays of fish DNA as a means of screening the toxicity of aquatic environments. Catla catla and Cirrhinus mrigala collected from the River Chenab in Pakistan were used as a case study for the application of comet and micronucleus techniques. Comet and micronucleus assays were used to compare DNA damage in C. catla and C. mrigala collected from polluted areas of the River Chenab and farmed fish. Atomic absorption spectrophotometry showed an acute level of toxicity from Cd, Cu, Mn, Zn, Pb, Cr, Sn, and Hg in river water. Comet assay showed significant (p < 0.05) DNA damage in C. catla representing 17.33 ± 2.42, 11.53 ± 2.14, and 14.17% DNA in the comet tail, averaged from three sites of the polluted area of the river. Tail moment was observed as 10.06 ± 2.71, 3.11 ± 0.74, and 14.70 ± 1.89, while olive moment was 8.85 ± 1.84, 3.83 ± 0.76, and 7.11 ± 0.73, respectively. Highly significant (p < 0.01) damage was reported in C. mrigala as 37.29 ± 2.51, 34.96 ± 2.53, and 38.80 ± 2.42% DNA in comet tail, tail moment was 23.48 ± 3.90, 19.78 ± 4.26, and 14.30 ± 1.82, and olive moment was 16.22 ± 2.04, 13.83 ± 1.96, and10.99 ± 0.90. Significant (p < 0.05) differences were observed in genotoxicity between farmed and polluted area fish. Micronucleus assay showed a similar picture of significant difference in respect to single and double micronucleus induction: i.e., 23.20 ± 4.19 and 2.80 ± 1.07‰ in C. catla and 44.80 ± 3.73 and 06.20 ± 0.97‰, respectively, in C. mrigala. Nuclear abnormalities were found as 6.00 ± 0.84 and 09.60 ± 1.72/thousand cells, respectively, in both species. The results of this study suggest that these novel fish DNA damage assays can be used as an expedient toxicity screening for aquatic environments.

Adaptive expression of uncoupling protein 1 in the carp liver and kidney in response to changes in ambient temperature.

The expression of uncoupling protein (UCP1) is up-regulated in mammalian brown adipocytes during cold exposure. However, a previous study revealed that UCP1 was highly expressed in the liver of common carps, and that the hepatic expression of UCP1 was down-regulated during cold exposure. The present study examined the effects of temperature on the recovery of UCP1 expression levels and the expression of genes involved in UCP1 transcription in the livers and kidneys of common carps. The hepatic and renal expressions of UCP1 were decreased by acclimation from 22 °C to 8 °C, and a subsequent increase in the water temperature from 8 °C to 28 °C recovered the renal, but not hepatic expression of UCP1. Changes in the expression of peroxisome proliferator-activator receptor (PPAR) γ, retinoid X receptor (RXR) α and PPARγ co-activator (PGC)-1α, genes that are involved in the expression of UCP1 in mammals, with ambient temperature indicated that the expressions of PPARγ and RXRα, but not expression of PGC-1α was decreased in response to cold exposure; the hepatic and renal expressions of PPARγ and RXRα recovered to basal levels with the cessation of cold exposure, although this was not complete for hepatic expression of PPARγ. The results of the present study indicate that a unique regulatory mechanism is responsible for the hepatic and renal expressions of carp UCP1 during cold exposure and subsequent reacclimation, and is distinct from that in murine brown adipocytes.

Institutional development of freshwater fish stocking in Mexico.

By using freshwater fish stocking information from the Mexican government, this work described the current situation of the national stocking and its associated fishery policy. There is a lack of effective freshwater stocking programmes as a result of limited fisheries management, unharmonized fisheries regulations and institutional performance. The fry production has decreased from 140 to 20 million in the past 11 years.