Cold-Resistant Heterotrophic Ammonium and Nitrite-Removing Bacteria Improve Aquaculture Conditions of Rainbow Trout (Oncorhynchus mykiss).

The aim of this study was isolation and characterization of heterotrophic bacteria capable of ammonium and nitrite removal at 15 °C (optimal temperature for growing rainbow trout Oncorhynchus mykiss). Environmental isolates were grown in liquid media containing ammonium or nitrite, and best strains in terms of growth and ammonium or nitrite removal were identified via 16S rRNA sequencing. Dyadobacter sp. (no. 68) and Janthinobacterium sp. (no. 100) were selected for optimal adaptation to growth at 15 °C and best ammonium and nitrite removal (P < 0.05), respectively. A heterotrophic ammonium and nitrite removal (HAN) microbial complex, containing selected strains, was prepared and applied in a trout culture system. After 10 days, the effect of microbial HAN complex was investigated in terms of ammonium and nitrite removal, as well as stress and immune indices present in the plasma of cultivated trout. Compared to a standard cultivation setup, addition of the HAN complex had a clear beneficial effect on keeping the un-ionized ammonia and nitrite level below prescribed standards (P < 0.05). This resulted in reduction of stress and immune reactions of cultivated fish (P < 0.05), leading to an augmentation of final weight and survival. Application of the selected microbial complex resulted in a significant improvement of the aquaculture ecosystem.

DNA damage, acetylcholinesterase activity, and hematological responses in rainbow trout exposed to the organophosphate malathion.

Effects of sub-lethal concentrations (0 (control), 0.009, 0.014, and 0.023 ppm) of the organophosphate insecticide "malathion" to rainbow trout (Oncorhynchus mykiss) after the determination of LC50-96 h value (0.093 ppm) were evaluated. Changes in biomarkers of neurotoxicity (acetylcholinesterase (AChE) activity), genotoxicity (DNA damage), and hematological parameters (red (RBC) and white (WBC) blood cell count, hemoglobin (Hb), hematocrit (Hct), mean cell hemoglobin (MCH), mean cell volume (MCV), and mean cell hemoglobin concentration (MCHC)) were assessed for a 15-day exposure. A significant time- and dose-dependent reduction in AChE activities of gill, muscle, brain, and liver tissues was found. However, the AChE activity was less affected by malathion concentration than by exposure time. DNA damage of erythrocytes at different malathion concentrations increased by increasing the experimental time up to the fourth day. A decrease in the count of WBC, RBC, and Hct and an increase in the number of MCH and MCV were observed by increasing malathion exposure dose and time (p < 0.05). An increase in the malathion concentration and exposure time significantly resulted in a decrease in Hb and an increase in MCHC. A significant improvement in AChE activity; DNA damage; and RBC, Hb, Hct, MCV, and MCH indices was detected during a 30-day recovery period, but the WBC count changed insignificantly. The recovery pattern based on 100% water exchange with clean water could be a successful strategy to improve the biomarker responses of rainbow trout habituating in contaminated aquatic environments.

1H NMR metabolic profiling of the cryopreserved spermatozoa of the wild endangered Persian sturgeon (Acipenser persicus) with the use of beta-cyclodextrin as an external cryoprotectant.

Persian sturgeon (Acipenser persicus) is an endangered species and genetic resource banking such as gametes and embryo preservation could be one of the most pursued conservation approaches. In this study, deleterious effects of the traditional cryopreservation technique and the effect of different doses of 2-hydroxypropyl-beta-cyclodextrin (HßCD) on thawed spermatozoa quality (motility duration and percentage) of Persian sturgeon (Acipenser persicus) were investigated from metabolic aspects of view. For cryopreserving, semen was diluted with Tris-HCl (100 mM) extenders containing 0, 5, 10, and 15 mM of HßCD in a ratio of 1:1 (semen/extenders). Semen-extenders were filled into 0.5-mL straws and were frozen with the vapor of liquid nitrogen, and then immersed into liquid nitrogen. Cryopreserved spermatozoa were thawed in water baths in 15 s. Two treatments with the highest and the lowest motility percentages (0 and 10 mM of HßCD) were chosen to reveal the extremes of the metabolites change range and were objected to 1H NMR spectroscopy. Univariate (ANOVA) and multivariate (PCA) analysis of the obtained metabolic profiles showed significant changes (P < 0.05) in metabolites. The use of 10 mM of HßCD was completely successful in the preservation of creatinine, glucose, guanidoacetate, O-phosphocholine, and N, N-dimethylglycine and probably their corresponding biochemical pathways, but it failed to preserve lactate, carnitine, betain, ß-alanin, and trimethylamine N-oxide. It was also partially successful in preserving acetate, creatine, creatine phosphate, and glycine, all suggesting how HßCD can be effective as a cryoprotectant.

Efficient osmolyte-based procedure to increase expression level and solubility of infectious hematopoietic necrosis virus (IHNV) nucleoprotein in E. coli.

The nucleoprotein of infectious hematopoietic necrosis virus (IHNV) is considered as the main target antigen for detection of IHNV infection in salmonid fish. This study aimed at improving the expression and solubility of IHNV nucleoprotein (IHNV-NP) in E. coli expression system. The effects of several expression strategies including host strain type, protein expression temperature, heat-shock treatment prior to protein induction, and additives in the growth medium and in the cell lysis buffer were examined. Results showed that bacterial strain type had a great impact on protein expression level, whereas it was not effective in preventing protein aggregation. Production of soluble IHNV-NP was proportionally increased with decreased incubation temperature. Heat-shock treatment prior to protein induction did not change the percent of solubility. For cells grown at low temperature, the presence of additives in the lysis buffer enhanced the solubility of IHNV-NP up to 24%. The highest yield of soluble protein was obtained via incorporation of osmolytes in the growth medium of cells exposed to a mild salt stress, in the following order: sucrose > sorbitol > glycerol > glycine. Soluble protein obtained by the optimized condition was efficiently purified in high yield and successfully detected by two monoclonal antibodies in a sandwich ELISA. Taken together, a combination of proper host strain, low-temperature expression, and timely application of osmolytes in the growth medium provided sufficient quantities of soluble recombinant IHNV-NP that has the potential to be used for diagnostic purposes.

A method to quantify genotoxicity of malathion in rainbow trout using the weighted averaging.

DNA breakage has been frequently used as a biomarker of the pesticide toxicity. The present study introduced a method to quantify the DNA breakage in Oncorhynchus mykiss exposed to the pesticide malathion. Specimens were exposed to different concentrations of malathion for 1-9 days and their gill and liver were sampled. DNA was extracted and electrophoresed using agarose gel. The pixel density curves were obtained from the gel smears. The area under the curves was arbitrarily divided from three up to seven segments using a Java macro in the software ImageJ. Some weighted averaging methods were used to calculate DNA breakage in each gel lane. Akaike information criterion (AIC) was used to find the best analysis of variance. The liver was more sensitive than the gill showing a larger number of significant differences among the specimens exposed to various concentrations of malathion. The geometric weighted averaging on the data extracted from the seven-segment pixel density curve resulted to the lowest AIC. The double-strand DNA breakage of O. mykiss was able to detect malathion in freshwater in concentrations over 0.05 mg L-1.

Mechanisms of peripheral phylogeographic divergence in the indo-Pacific: lessons from the spiny lobster Panulirus homarus.

BACKGROUND: There is increasing recognition of the concordance between marine biogeographic and phylogeographic boundaries. However, it is still unclear how population-level divergence translates into species-level divergence, and what are the principal factors that first initiate that divergence, and then maintain reproductive isolation. This study examines the likely forces driving population and lineage divergences in the broadly-distributed Indo-Pacific spiny lobster Panulirus homarus, which has peripheral divergent lineages in the west and east. The study focuses particularly on the West Indian Ocean, which is emerging as a region of unexpected diversity. Mitochondrial control region (mtCR) and COI sequences as well as genotypes of 9 microsatellite loci were examined in 410 individuals from 17 locations grouped into 7 regions from South Africa in the west, and eastward across to Taiwan and the Marquesas Islands. Phylogenetic and population-level analyses were used to test the significance and timing of divergences and describe the genetic relationships among populations. RESULTS: Analyses of the mtCR revealed high levels of divergence among the seven regions (ФST = 0.594, P < 0.001). Microsatellite analyses also revealed significant divergence among regions, but at a much lower level (FST = 0.066, P < 0.001). The results reveal different patterns of mtCR v. nDNA divergence between the two distinct peripheral lineages: a subspecies in South Africa and Madagascar, and a phylogeographically diverged population in the Marquesas. The results also expose a number of other more fine-scale population divergences, particularly in the Indian Ocean. CONCLUSIONS: The divergence of peripheral lineages in the west and east of the species' range appear to have been initiated and maintained by very different processes. The pattern of mitochondrial and nuclear divergence of the western lineage, implicates processes of parapatric isolation, secondary contact and introgression, and suggests possible maintenance through adaptation and behavioural reproductive isolation. In contrast, the eastern lineage appears to have diverged through a rare colonisation event, maintained through long-term isolation, and matches expectations of the core-periphery hypothesis. The process of active peripheral speciation may be a common force in the Indo-Pacific that helps drive some of the regions' recognized biogeographic boundaries.

Transcriptome and metabolome analysis of Ferula gummosa Boiss. to reveal major biosynthetic pathways of galbanum compounds.

Ferula gummosa Boiss. is an industrial and pharmaceutical plant that has been highly recognized for its valuable oleo-gum-resin, namely galbanum. Despite the fabulous value of galbanum, very little information on the genetic and biochemical mechanisms of its production existed. In the present study, the oleo-gum-resin and four organs (root, flower, stem, and leaf) of F. gummosa were assessed in terms of metabolic compositions and the expression of genes involved in their biosynthetic pathways. Results showed that the most accumulation of resin and essential oils were occurred in the roots (13.99 mg/g) and flowers (6.01 mg/g), respectively. While the most dominant compound of the resin was ß-amyrin from triterpenes, the most abundant compounds of the essential oils were α-pinene and ß-pinene from monoterpenes and α-eudesmol and germacrene-D from sesquiterpenes. Transcriptome analysis was performed by RNA sequencing (RNA-seq) for the plant roots and flowers. Differential gene expression analysis showed that 1172 unigenes were differential between two organs that 934 (79.6%) of them were up-regulated in the flowers and 238 (20.4%) unigenes were up-regulated in the roots (FDR ≤0.001). The most important up-regulated unigenes in the roots were involved in the biosynthesis of the major components of galbanum, including myrcene, germacrene-D, α-terpineol, and ß-amyrin. The results obtained by RNA-Seq were confirmed by qPCR. These analyses showed that different organs of F. gummosa are involved in the production of oleo-gum-resin, but the roots are more active than other organs in terms of the biosynthesis of triterpenes and some mono- and sesquiterpenes. This study provides rich molecular and biochemical resources for further studies on molecular genetics and functional genomics of oleo-gum-resin production in F. gummosa.

Metabolic changes in droplet vitrified semen of wild endangered Persian sturgeon Acipenser persicus (Borodin, 1997).

Comparative quantitative metabolite profiling can be used for better understanding of cell functions and dysfunctions in particular circumstances such as sperm banking which is an important approach for cryopreservation of endangered species. Cryopreservation techniques have some deleterious effects on spermatozoa which put the obtained results in controversy. Therefore, in the present study, quantitative 1H NMR (Nuclear Magnetic Resonance) based metabolite profiling was conducted to evaluate metabolite changes related to energetics and some other detected metabolites in vitrified semen of critically endangered wild Acipenser persicus. The semen was diluted with extenders containing 0, 5, 10, and 15 µM of fish antifreeze protein (AFP) type III as a cryoprotectant. Semen-extenders were vitrified and stored for two days. Based on post-thaw motility duration and motility percentage assessments, two treatments with 10 µM and 0 µM of AFP had the highest and the lowest motility percentages respectively and they were objected to 1H NMR spectroscopy investigations in order to reveal the extremes of the metabolites dynamic range. Univariate (ANOVA) and multivariate (PCA) analysis of the resulting metabolic profiles indicated significant changes (P > 0.05) in metabolites. The level of some metabolites including acetate, adenine, creatine, creatine phosphate, lactate, betaine, sarcosine, ß-alanine and trimethylamine N-oxide significantly decreased in vitrified semen while some others such as creatinine, guanidinoacetate, N, N-dimethylglycine, and glycine significantly increased. There were also significant differences between vitrified treatments in levels of creatine, creatine phosphate, creatinine, glucose, guanidinoacetate, lactate, N, N-dimethylglycine, and glycine, suggesting how fish AFP type III can be effective as a cryoprotectant.

Boule gene expression underpins the meiotic arrest in spermatogenesis in male rainbow trout (Oncorhynchus mykiss) exposed to DEHP and butachlor.

Boule, the ancestor of the DAZ (Deleted in AZoospermia) gene family, in most organisms is mainly involved in male meiosis. The present study investigates the effects of the plasticizer DEHP (50mg/kg body weight) and herbicide butachlor (0.39mg/L) on male rainbow trout (Oncorhynchus mykiss) for a 10-day period in two independent experiments. The results showed that plasma testosterone (T) concentrations were significantly lower in fish exposed to either DEHP or butachlor compared to the control fish (P<0.05). Fish showed a significantly elevated hepatosomatic index (HSI) in the butachlor treatment (P<0.05). However, no significant difference was observed in HSI values in the DEHP treatment (P>0.05). In addition, no significant differences were found in the gonadosomatic index (GSI) in both DEHP and butachlor treatments (P>0.05). Histologically, testes of male trout in the control groups were well differentiated and filled with large numbers of cystic structures containing spermatozoa. In contrast, the testes of male trout contained mostly spermatocytes with few spermatozoa in both treated group, suggesting that DEHP and butachlor may inhibit the progression of meiosis. Also, boule gene expression was significantly lower in the testes of male trout affected by DEHP and butachlor in comparison with their control groups (P<0.05), which confirmed the meiotic arrest in affected trout. Based on the results, the present study demonstrated that DEHP and butachlor can inhibit the progression of spermatogenesis in male trout, potentially by causing an arrest of meiosis, maybe due to down-regulation of boule gene expression through T and/or IGF1 via ERK1/2 signaling in T-independent pathways. In addition, these results confirmed that boule can be considered as a predictive marker to assess meiotic efficiency.

Effects of diazinon on adaptation to sea-water by the endangered Persian sturgeon, Acipenser persicus, fingerlings.

To replenish the depleting populations of sturgeon fishes especially Persian sturgeon, Acipenser persicus in the Caspian Sea, millions of Persian sturgeon fingerlings are farmed through artificial propagation and released into the Iranian river estuaries annually. Fish osmoregulation is a vital physiological process that can be affected during the release. Many Iranian river estuaries are under the influence of pesticides originating from farming activities that may affect osmoregulation. In this study, Persian sturgeon fingerlings were exposed to sublethal concentrations (0, 0.18, 0.54, 0.9mgL(-)(1)) of diazinon for 96h (short-term trial) and 12 days (long-term trial) in fresh water (FW) and then fish were exposed in brackish water (BW) for 24h. After 96h and 12 days of exposure in FW, the lower levels of plasma triidothyronine (T3), thyroxine (T4), Na(+), Cl(-), K(+), gill Na(+)/K(+)- ATPase activity and number of chloride cells were observed in exposed fish (0.54 and 0.9mgL(-)(1) diazinon) compared to control group and 0.18mgL(-)(1) diazinon treatment. Also, higher levels of plasma cortisol (except 0.18mgL(-)(1) diazinon treatment in long-term trial) were observed in diazinon exposed fish compared to control group. However, gill Na(+)/K(+)-ATPase activity and the number of chloride cells were higher in fingerlings exposed to diazinon compared than control. When fish were exposed in BW for 24h, the following changes occurred: (a) in short-term trial: increases in cortisol and Cl(-) levels (0.54mgL(-)(1) diazinon ), Na(+) (0.9mgL(-)(1) diazinon ) and gill Na(+)/K(+)-ATPase activity (0.18mgL(-)(1) diazinon ). In control group, cortisol, T4, Na(+), gill Na(+)/K(+)-ATPase activity and the number of chloride cells increased significantly. (b) In long-term trial: increases in K(+) levels in fish exposed to 0.9mgL(-)(1) diazinon, Na+ in all diazinon concentrations and decreases in chloride cells number in fish exposed to 0.18mgL(-)(1) diazinon. In control group, significant increases were observed in cortisol, T3, Na(+) and chloride cells number. Finally, gill showed many histopathological damages during exposure in FW and BW. Our results suggest that the contamination of river estuaries with diazinon may alter the osmoregulation ability of released Persian sturgeon fingerlings, which could lead to a failure in their restocking program in the Caspian Sea.

Estimating the DNA strand breakage using a fuzzy inference system and agarose gel electrophoresis, a case study with toothed carp Aphanius sophiae exposed to cypermethrin.

The DNA breakage has been widely used in ecotoxicological studies to investigate effects of pesticides in fishes. The present study used a fuzzy inference system to quantify the breakage of DNA double strand in Aphanius sophiae exposed to the cypermethrin. The specimens were adapted to different temperatures and salinity for 14 days and then exposed to cypermethrin. DNA of each specimens were extracted, electrophoresed and photographed. A fuzzy system with three input variables and 27 rules were defined. The pixel value curve of DNA on each gel lane was obtained using ImageJ. The DNA breakage was quantified using the pixel value curve and fuzzy system. The defuzzified values were analyzed using a three-way analysis of variance. Cypermethrin had significant effects on DNA breakage. Fuzzy inference systems can be used as a tool to quantify the breakage of double strand DNA. DNA double strand of the gill of A. sophiae is sensitive enough to be used to detect cypermethrin in surface waters in concentrations much lower than those reported in previous studies.

Effects of (Anti) Androgenic Endocrine Disruptors (DEHP and Butachlor) on Immunoglobulin M (IgM) and Leukocytes Counts of Male Rainbow Trout (Oncorhynchus mykiss).

The effect of two anti-androgenic endocrine disrupting compounds, i.e. the plasticizer di (2-ethylhexyl) phthalate (DEHP) and herbicide butachlor, were evaluated for their effects on immunoglobulin M (IgM) and leukocytes in male rainbow trout. Also, plasma testosterone (T) concentration was measured to confirm their anti-androgenic effects. In the first experiment, trout were treated with 50 mg/kg (body weight) DEHP intraperitoneally, and in the second one, fish were exposed to 0.39 mg/L butachlor for 10 days. The results showed that T concentrations and white blood cells were significantly lower in fish exposed to either DEHP or butachlor compared to control fish (p < 0.05). Fish showed significantly elevated neutrophil levels and decreased lymphocyte levels in the butachlor (p < 0.05); however, no significant difference was observed in lymphocyte and neutrophils values in the DEHP treatment (p > 0.05). In addition, no significant differences were found in IgM, eosinophil and monocyte parameters in either DEHP or butachlor treatments (p > 0.05). These results confirmed that leukocytes counts can be considered as a novel marker of immunotoxicity triggered by (anti) androgenic endocrine disruptors.

Dietary fermentable fiber upregulated immune related genes expression, increased innate immune response and resistance of rainbow trout (Oncorhynchus mykiss) against Aeromonas hydrophila.

This trial was carried out to investigate the effects of dietary administration of Vitacel(®), a commercial fermentable fiber, on immune related genes (Lysozyme, TNFα and HSP70) expression, innate immune response and resistance of rainbow trout against Aeromonas hydrophila. 120 healthy rainbow trout (81.65 ± 1.49 g) were distributed in six fiberglass tanks assigned to two treatments. The treatments were feeding rainbow trout with diets supplemented with 0 (control) or 10 g kg(-1) Vitacel(®) for 45 days. The results revealed that administration of fermentable fiber significantly (P < 0.05) upregulated lysozyme and TNFα gene expression. HSP70 gene expression was significantly lower in Vitacel(®) fed fish at the end of trial (P < 0.05). Furthermore dietary administrations of Vitacel(®) remarkably elevated rainbow trout innate immune parameters include serum lysozyme, ACH50, bactericidal activity and agglutination antibody titer (P < 0.05). Administration of 10 g kg(-1) Vitacel(®) significantly increased rainbow trout resistance against A. hydrophila (P < 0.05). The results of present study revealed that dietary Vitacel(®) can upregulates immune related genes expression and elevates innate immune response and disease resistance of rainbow trout.

The effects of dietary Immunogen on innate immune response, immune related genes expression and disease resistance of rainbow trout (Oncorhynchus mykiss).

This study investigates the effects of prebiotic Immunogen on lysozyme, TNFα and HSP70 gene expression in head kidney, humoral innate immune parameters and resistant to Aeromonas hydrophila of rainbow trout. 120 healthy rainbow trout (81.65 ± 1.49 g) were distributed in six fiberglass tanks assigned to two groups fed control or diet supplemented with 2 g kg(-1) Immunogen for 45 days. The results revealed that administration of Immunogen significantly (P < 0.05) up regulated lysozyme and TNFα gene expression. HSP70 gene expression was significantly (P < 0.05) lower in Immunogen fed fish at the end of trial. Humoral innate immune parameters (lysozyme activity, ACH50 and bactericidal activity) were significantly (P < 0.05) increased whether 15 or 45 days after feeding on Immunogen supplemented diet. However, significant (P < 0.05) increase in agglutination antibody titer observed just after 45 days feeding on Immunogen. Rainbow trout fed with 2 g kg(-1) Immunogen showed remarkably higher resistance against A. hydrophila (64.44% survival) compared to the control group (24.44% survival). These results confirm that Immunogen can up regulates immune related genes expression, stimulates immune response that per se enhances disease resistance in rainbow trout.

Developmental transcription of genes putatively associated with growth in two sturgeon species of different growth rate.

In the present study, we surveyed developmental changes in the transcription of growth hormone (gh), insulin-like growth factor-I (igf-I), ghrelin (ghrl) and vascular endothelial growth factor (vegf) genes in the largest freshwater fish, European sturgeon (Beluga, Huso huso) and compared the same parameters to that of its phylogenically close moderate-sized species, Persian sturgeon (Acipenser persicus). The transcripts of gh, igf-I, ghrl and vegf were detected at all developmental time-points of Persian sturgeon and Beluga from embryos to juvenile fish. Changes in normalized gh, igf-I, ghrl and vegf transcription by using the geometric average of genes encoding ribosomal protein L6 (RPL6) and elongation factor (EF1A) over the time of development of Persian sturgeon and Beluga were statistically significant (P<0.05). Our results showed that the mRNA expression levels of both igf-I and ghrl were low during early larval development and then increased significantly to the late larval time-points when larvae started exogenous feeding. In both Beluga and Persian sturgeon, after a low mRNA expression during the embryonic stage, the transcript levels of vegf displayed an increasing trend during yolk-sac fry, consistent with organogenesis. The vegf level remained constantly high in the time of exogenous feeding. The highest detection of gh transcripts coincided with the end of the embryonic stage (hatching time) in Persian sturgeon and 3 days-post-hatching (dph) in Beluga. In Persian sturgeon, the gh transcript started to decrease to the rest of the developmental time-points, whereas in Beluga gh transcript had a marked second increase from the time of exogenous feeding (20-dph). This Beluga specific increase in gh transcription may be associated with the marked growth rate and extraordinary size of this fish species.

Effect of a glyphosate-based herbicide in Cyprinus carpio: assessment of acetylcholinesterase activity, hematological responses and serum biochemical parameters.

The objective of this study was to investigate the toxicity effects of acute and sublethal of Roundup® as a glyphosate-based herbicide on acetylcholinesterase (AChE) activity and several hematological and biochemical parameters of Cyprinus carpio. The LC50-96 h of Roundup® to C. carpio was found to be 22.19 ppm. Common carp was subjected to Roundup® at 0 (control), 3.5, 7 and 14 ppm for 16 days, and the AChE activity is verified in tissues of gill, muscle, brain and liver. After 5 days, a significant decrease was observed in the AChE activity of muscle, brain and liver tissues. Besides, a time- and dose-dependent increase in mean cell hemoglobin (MCH) and mean cell volume (MCV) was observed. In contrast, a significant decrease was found in the quantities of hemoglobin (Hb), hematocrit (HCT) and, red (RBC) and white (WBC) blood cell count. Also, the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in Roundup® treated groups were significantly higher than the controlled group at experimental periods. However, the level of alkaline phosphatase (ALP) had a significant reduction behavior during the sampling days. It seems that the changes in hematological and biochemical parameters as well as AChE activity could be used as efficient biomarkers in order to determine Roundup® toxicity in aquatic environment.

Optimization of recovery patterns in common carp exposed to roundup using response surface methodology: evaluation of neurotoxicity and genotoxicity effects and biochemical parameters.

The present study is the first report on optimization of recovery conditions of fishes exposed to pesticides using response surface methodology-central composite rotatable design (RSM-CCRD). The sub-lethal toxicity bioassay of Roundup® (2 ppm ~10 percent LC50, 96 h) in common carp (1, 4, 9, 16, 25, 35 and 40 day) was investigated. After exposure for 16 days to Roundup®, some the fishes were introduced to herbicide-free water. The effects of four recovery parameters including time (5-25 d), temperature (18-26 °C), water exchange rate (WER, 10-30), and salinity (0-8 ppt) on the levels of biomarkers of genotoxicity (DNA damage), neurotoxicity (acetylcholinesterase activity (AChE)), and the serum alanine (ALT) and aspartate (AST) aminotransferase in plasma were studied. The polynomial equations were significantly fitted for all response variables with high R² values (>0.95), which revealed no indication of lack of fit. The optimum conditions for the maximum AChE activity (37.14 nmol/min/mg protein) and the minimum levels of DNA damage (8.00 percent tail DNA), ALT (27.0 IU/L) and AST (91.0 IU/L) were time of 20 d, temperature of 20 °C, WER of 25 and water salinity of 6 ppt. Thus, a promising improvement for the recovery trend of fishes exposed to Roundup® stress was obtained under the optimized conditions using RSM-CCRD.

Successful Microsurgical Replantation of Amputated Penis: A Case Report and Review of Literature.

This study aims to explain microscopic replantation in a rare case of a wholly amputated penis after prolonged ischemia. A 36-year-old patient underwent microscopic replantation of the penis after 9 hours. The penis was completely amputated due to self-mutilation. Microvascular replantation was performed after pre-operative preparation. On the second day after surgery, congestion was observed in the penis, and three sessions of leech therapy were conducted each time the leeches were placed for 30 minutes and then detached by themselves. The patient was referred to a psychiatrist to continue treatment after discharge from the hospital. Penile amputation is a rare situation and has different causes. There are various treatments to repair the amputated penis, which are both microvascular and microvascular. The microsurgery methods have shown the best results. In the present case, due to microsurgical artery repair and the early start of leech therapy, there was limited and predictable necrosis in the area of the penoscrotal junction flap, which underwent debridement and skin graft. Complete amputation of the penis is a rare phenomenon. Efforts should be made to perform the replantation surgery as soon as possible. The venous outflow is an essential factor in the success of penile re-implantation, and completely restored vascular and sensory function in this case. Early initiation of psychological care to control underlying disease leads to further cooperation of the patient to handle complications and avoid the recurrence of self-injury.

Effect of dietary Ergosan on growth performance, digestive enzymes, intestinal histology, hematological parameters and body composition of rainbow trout (Oncorhynchus mykiss).

Present study examined the effects of Ergosan on growth performance, digestive enzyme activities, hematological parameters and gastrointestinal structure of rainbow trout. Rainbow trout (mean weight 100-110 g) were fed basal diet (control) and diet treated with Aquavac Ergosan (5 g kg⁻¹ of diet) for 50 days. Results of this study showed that Ergosan supplementation significantly increased weight gain (94.27 g vs. 65.04 g), specific growth rate (4.09 vs. 3.10) and feed intake (136.85 g vs. 111.22 g) and decreased feed conversion ratio (1.43 vs. 2.03) compared to control (P<0.05). Lipase activity and leukocyte and erythrocyte count also increased in juvenile fish fed Ergosan-treated diet compared to control (P<0.05). Light microscopy demonstrated that both groups of fish displayed normal morphology of proximal intestine and pyloric caeca. In Ergosan-treated group, higher percentage of goblet cell was shown in proximal intestine and pyloric caeca. Present study suggests that Ergosan effectively promotes growth performance, lipase activity and gastrointestinal structure in rainbow trout.

Enriched microbial communities for ammonium and nitrite removal from recirculating aquaculture systems.

The aim of this study was the enrichment of high-performance microbial communities in biofilters for removal of ammonium and nitrite from aquaculture water. Ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) were enriched from different environmental water samples. The microbial communities with higher ammonium and nitrite removal activity were selected and adapted to different temperatures [9 °C, 15 °C, room temperature (25 °C), and 30 °C]. The expression of genes involved in nitrification including ammonia monooxygenase (AMO) and nitrite oxidoreductase (NXR) were measured in temperature-adapted AOB and NOB microbiomes. The microbial species present in the selected microbiomes were identified via 16s rRNA sequencing. The microbial communities containing Nitrosomonas oligotropha and Nitrobacter winogradskyi showed the highest ammonium and nitrite removal activity at all temperatures used for adaptation. Furthermore, the microbial communities do not contain any pathogenic bacteria. They also exhibited the highest expression of AMO and NXR genes. Using the enriched microbial communities, we achieved a 288% and 181% improvement in ammonium and nitrite removal over the commonly used communities in biofilters at 9 °C, respectively. These results suggest that the selected microbiomes allowed for a significant improvement of water quality in a recirculating aquaculture system (RAS).