Validation and calibration of a novel GEM biosensor for specific detection of Cd2+, Zn2+, and Pb2.

BACKGROUND: In this study, we designed a novel genetic circuit sensitive to Cd2+, Zn2+ and Pb2+ by mimicking the CadA/CadR operon system mediated heavy metal homeostasis mechanism of Pseudomonas aeruginosa. The regular DNA motifs on natural operon were reconfigured and coupled with the enhanced Green Fluorescent Protein (eGFP) reporter to develop a novel basic NOT type logic gate CadA/CadR-eGFP to respond metal ions mentioned above. A Genetically Engineered Microbial (GEM)-based biosensor (E.coli-BL21:pJET1.2-CadA/CadR-eGFP) was developed by cloning the chemically synthesised CadA/CadR-eGFP gene circuit into pJET1.2-plasmid and transforming into Escherichia coli (E. coli)-BL21 bacterial cells. RESULTS: The GEM-based biosensor cells indicated the reporter gene expression in the presence of Cd2+, Zn2+ and Pb2+ either singly or in combination. Further, the same biosensor cells calibrated for fluorescent intensity against heavy metal concentration generated linear graphs for Cd2+, Zn2+ and Pb2+ with the R2 values of 0.9809, 0.9761 and 0.9758, respectively as compared to non-specific metals, Fe3+ (0.0373), AsO43- (0.3825) and Ni2+ (0.8498) making our biosensor suitable for the detection of low concentration of the former metal ions in the range of 1-6 ppb. Furthermore, the GEM based biosensor cells were growing naturally within the concentration range of heavy metals, at 37 °C and optimum pH = 7.0 in the medium, resembling the characteristics of wildtype E.coli. CONCLUSION: Finally, the novel GEM based biosensor cells developed in this study can be applied for detection of targeted heavy metals in low concentration ranges (1-6 ppb) at normal bacterial physiological conditions.

Long-term epidemiological survey of Kudoa thyrsites (Myxozoa) in Atlantic salmon (Salmo salar L.) from commercial aquaculture farms.

Kudoa thyrsites (Myxozoa) encysts within myocytes of a variety of fishes. While infected fish appear unharmed, parasite-derived enzymes degrade the flesh post-mortem. In regions of British Columbia (BC), Canada, up to 4-7% of fillets can be affected, thus having economic consequences and impacting the competitiveness of BC's farms. K. thyrsites was monitored in two farms having high (HP) or low (LP) historical infection prevalence. At each farm, 30 fish were sampled monthly for blood and muscle during the first year followed by nine samplings during year two. Prevalence and intensity were measured by PCR and histology of muscle samples. In parallel, fillet tests were used to quantify myoliquefaction. Infections were detected by PCR after 355 and 509 degree days at LP and HP farms, respectively. Prevalence reached 100% at the HP farm by 2265 degree days and declined during the second year, whereas it plateaued near 50% at the LP farm. Infection intensities decreased after 1 year at both farms. Blood was PCR-positive at both farms between 778 and 1113 degree days and again after 2000 degree days. This is the first monitoring project in a production environment and compares data between farms with different prevalence.

Hemoglobin genotype has minimal influence on the physiological response of juvenile Atlantic cod (Gadus morhua) to environmental challenges.

Hemoglobin (Hb) polymorphism in cod is associated with temperature-related differences in biogeographical distribution, and several authors have suggested that functional characteristics of the various hemoglobin isoforms (HbIs) directly influence phenotypic traits such as growth rate. However, no study has directly examined whether Hb genotype translates into physiological differences at the whole animal level. Thus, we generated a family of juvenile Atlantic cod consisting of all three main Hb genotypes (HbI-1/1, HbI-2/2, and HbI-1/2) by crossing a single pair of heterozygous parents, and we compared their metabolic and cortisol responses to an acute thermal challenge (10 degrees C to their critical thermal maximum [CTM] or 22 degrees C, respectively) and tolerance of graded hypoxia. There were no differences in routine metabolism (at 10 degrees C), maximum metabolic rate, metabolic scope, CTM (overall mean 22.9 degrees +/- 0.2 degrees), or resting and poststress plasma cortisol levels among Hb genotypes. Further, although the HbI-1/1 fish grew more (by 15%-30% during the first 9 mo) when reared at 10 degrees +/- 1 degrees C and had a slightly enhanced hypoxia tolerance at 10 degrees C (e.g., the critical O(2) levels for HbI-1/1, HbI-2/2, and HbI-1/2 cod were 35.56% +/- 1.24% , 40.56% +/- 1.99%, and 40.20% +/- 1.19% air saturation, respectively), these results are contradictory to expectations based on HbI functional properties. Thus, our findings (1) do not support previous assumptions that growth rate differences among cod Hb genotypes result from a more efficient use of the oxygen supply-that is, reduced standard metabolic rates and/or increased metabolic capacity-and (2) suggest that in juvenile cod, there is no selective advantage to having a particular Hb genotype with regards to the capacity to withstand ecologically relevant environmental challenges.

The immune and stress responses of Atlantic cod to long-term increases in water temperature.

Sea-caged cod are limited in their movements in the water column, and thus can be exposed to large seasonal ( approximately 0-20 degrees C) temperature fluctuations. To investigate the physiological response of Atlantic cod to summer-like increases in temperature, we exposed 10 degrees C acclimated juvenile cod to a graded thermal challenge (1 degrees C increase every 5 days) and measured: (1) plasma cortisol and glucose levels; (2) the respiratory burst activity of blood leukocytes; and (3) the expression of specific immune-related genes [MHC Class I, Interleukin-1beta (IL-1beta), beta2-microglobulin (beta2-M), Immunoglobulin M (IgM)-light (L) and -heavy (H) chains] in the blood using quantitative reverse transcription-polymerase chain reaction (QRT-PCR). The experiment was stopped at 19.1 degrees C, with 26.7% of the fish surviving to this point. Plasma glucose levels increased slightly at 16 and 18 degrees C (by 1.39- and 1.74-fold, respectively), in contrast, cortisol levels were elevated significantly (by 2.9-fold) at 16 degrees C but returned to control levels thereafter. The effect of increasing temperature on the expression of immune related genes in blood cells (leukocytes) was variable and depended on the gene of interest. The expression of IgM-H remained stable for the duration of the experiment. In contrast, IL-1beta expression was increased significantly (by approximately 25-fold) at 19 degrees C as compared to time-matched control fish, and changes in the expression of beta2-M, MHC Class I and IgM-L followed a pattern similar to that seen for cortisol: increasing at 16 degrees C (by 4.2-, 5.3- and 17-fold, respectively), but returning to pre-stress levels by 19 degrees C. Interestingly, increasing temperatures had no effect on respiratory burst activity. This study is the first to examine the effects of a chronic regimen of increasing temperature on the stress physiology and immunology of a marine teleost, and suggests that immune function is influenced by complex interactions between thermal effects and temperature-induced stress (elevated circulating cortisol levels).

Changes in free and total plasma cortisol levels in juvenile haddock (Melanogrammus aeglefinus) exposed to long-term handling stress.

We measured changes in free and total plasma cortisol levels, plasma glucose, gill hsp70 levels, and growth in haddock (Melanogrammus aeglefinus) subjected to a long-term handling stress (15 s out of water, each day, for 4 weeks), and the effect of this long-term stress on the ability of haddock to respond to an acute stressor. The acute stressor was a single handling stress, and fish were sampled at 1, 6, and 12 h post-stress. During the long-term stress study, free and total plasma cortisol levels increased significantly (10-fold) in the stressed group after the second week. However, the percentage of free cortisol was already significantly elevated by the first week (control 17%, stressed 55%), and remained high during the second week (control 35% and stressed 65%). After 3 and 4 weeks of handling, both free and total cortisol declined in stressed fish to levels that were not significantly different from pre-stress values. Control fish grew significantly more than stressed fish (by 32% and 18%, respectively) over the 4 week study, and condition factor only increased in control fish. Although fish from the control group showed elevated total plasma cortisol levels (to 47 ng mL(-1)) 1 h after the acute stress, and the levels in stressed fish were comparable to those for the control fish, no significant increase in plasma cortisol was measured in the group subjected to the long-term stress. Free plasma cortisol levels did not increase significantly in either group following the acute stress. However, free plasma cortisol levels were significantly higher in long-term stress group, as compared with the control group, at 6 h post-stress. Plasma glucose and gill hsp70 levels were not altered by either the long-term stress or acute stressor. Our data indicate that cortisol (free and total), but not glucose or hsp70, appears to be adequate to assess short- and long-term stress in haddock.

Sex reversal in Nile tilapia (Oreochromis niloticus) using a nonsteroidal aromatase inhibitor.

Several studies have demonstrated that steroid hormones can influence sex differentiation in nonmammalian vertebrates and it has been hypothesized that male and female sex differentiation are driven by androgen and estrogen hormones, respectively. Estrogen biosynthesis is mediated by the steroidogenic enzyme cytochrome P450 aromatase, which converts androgens to estrogens. In the present study we examined the efficacy of a potent nonsteroidal aromatase inhibitor incorporated into the food, on sex reversal of Nile tilapia (Oreochromis niloticus) larvae. Nile tilapia larvae were divided in seven groups, which were fed with diets containing different amounts of the aromatase inhibitor Fadrozole (0, 50, 75 and 100 mg/kg) during 15 and 30 days, starting 9 days after hatching. Independent of the period, the proportion of males was significantly higher in the treated groups. Treatment with the highest doses (75 and 100 mg/kg) for 30 days produced 100% males. Histological examination revealed no differences in gonadal tissues between control males and treated fish. Furthermore, one intersex fish was identified in the group treated with 50 mg Fadrozole/kg for 30 days. This study reports that a 100% Nile tilapia male population can be obtained by suppressing aromatase activity and suggests that besides steroid hormones, nonsteroidal compounds, such as aromatase inhibitors, have potential for production of monosex population in tilapia. J. Exp. Zool. 290:177-181, 2001.

Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion and ovulation rate in female coho salmon, Oncorhynchus kisutch, close to final maturation.

Plasma levels of 17beta-estradiol, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-P), and testosterone were measured in adult female coho salmon in late vitellogenesis, approximately 1.5 months before spawning and just before and following intraperitoneal injection with the aromatase inhibitor (AI) Fadrozole. Injection at dosages of 0.1, 1.0, and 10.0 mg AI/kg body wt caused a significant drop in plasma 17beta-estradiol levels relative to preinjection values within 3 or 6 h. Injection of 10 mg AI/kg body wt caused a significant increase in plasma 17alpha-20beta-P levels within 3 h. Ten days after injection 67% of the fish treated with 10 mg AI/kg body wt had ovulated in contrast with 0% in the group injected with 0.1 mg AI/kg body wt. The fertilization rate of the eggs varied between 96% in the control group and 85% in the groups injected with AI. We conclude that the shift from 17beta-estradiol to 17alpha,20beta-P biosynthesis, which is characteristic of maturing Oncorhynchus sp., was advanced significantly by treatment with AI and that Fadrozole can be used as a tool to investigate periovulatory endocrine changes in salmon.

The effect of the aromatase inhibitor fadrozole and two polynuclear aromatic hydrocarbons on sex steroid secretion by ovarian follicles of coho salmon.

A variety of endogenous and exogenous factors can influence sex steroid production by salmon ovarian follicles and ultimately impact reproductive development. We examined the effect of an aromatase inhibitor, fadrozole, and common environmental contaminants (PAHs) on sex steroid secretion by ovarian follicles. Ovarian follicles of coho salmon were incubated in vitro with various concentrations of testosterone (0.10-0.40 microM) and fadrozole (10 and 100 microM), or with varying doses (between 0.05 and 5.0 microM) of the PAHs beta-naphthoflavone (BNF) and 20-methylcholanthrene (20-MC). 17 beta-Estradiol secretion was significantly reduced when follicles were incubated in the presence of fadrozole, BNF, or 20-MC. In contrast, 17 beta-estradiol production by ovarian follicles increased in a dose-dependent manner when incubated with increasing doses of the aromatizable androgen testosterone. Although increasing doses of PAHs significantly reduced follicular 17 beta-estradiol production no effect on testosterone secretion was observed. Hence, both fadrozole and PAHs can significantly reduce 17 beta-estradiol secretion by salmon ovarian follicles and may affect female sexual development.