Background: The study has a bifold objectives, firstly to determine the concentration of lead (Pb) in the pond water and bioaccumulation of the lead in the muscles of Danio rerio raised with the same pond water. Secondly, the study seeks to investigate its effect on the anti-oxidant enzyme catalase in Danio rerio, within the vicinity of a historical gold mine region located in the Kolar Gold fields (KGF), Karnataka, India.&nbsp; 
 
Methods: To achieve the objectives, the study employed Thermo Scientific&rsquo;s Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) and ICP-AES using Iteva software to evaluate and statistically analyze the distribution of lead in the pond water and and ICP-OES to evaluate the concentration of lead in the muscles sample. Four sample sites (A, B, C and D) were selected based on their proximity to residential area. Pond water samples were analyzed between April and October 2022 following a southwest monsoon rain.&nbsp; 
 
Result: During the study in the pond water, sample site B and D exhibited an exceedingly high Pb concentration during October with 0.98 mg/L against 0.21 and 0.16 mg/L in April 2022 against the maximum permissible value of 0.05 mg/L by WHO 2008 and USEPA 2009, followed by site A and C. The control sample had a normal range of 0.01 mg/L. The concentration of Pb in the muscles on day 30 of October&nbsp; was exceedingly high in the sample site D with 1.9 mg/Kg against 1 mg/Kg on day 30 of April followed by the sample sites B,C and A, which is far above the threshold value as per the regulatory bodies (WHO 2008). The increase in the concentration of the lead decreases the catalase enzyme in the brain region of Danio rerio, the value goes down drastically from 112.3 (&micro;M H2O2 utilized/min/mg protein) to 105.8 in sample site D between day 30 of April and October months, to that of the control value being stable from 151.7 to 150.94 (&micro;M H2O2 utilized/min/mg protein). The outcome of this experiment will facilitate the assessment of pond water contamination levels and the likelihood of contaminants entering the aquatic environment, resulting in the bioaccumulation of heavy metals in the aquatic organisms. The above results clearly indicates the effect of south-west monsoon rain in increasing the levels of lead concentration in the pond water during the month of October to that of April, which indirectly affects the physiology and metabolism of the fishes raised in the contaminated habitat of Kolar Gold Fields.&nbsp;

Bioaccumulation of Pb (Lead) in the Muscles of Danio rerio and its Effect on the Anti-oxidant Enzyme Catalase in the Mining Vicinity of Kolar Gold Fields

<div>Background: Changes in fish behavior can help identify accidental chemical pollution. Heavy metals and pesticides are two of the most found pollutants to investigate the different behavioral responses of fish to these two types of pollutants exposure. 
 
Methods: Real-time computer imaging was utilized to record parameters of fish behaviors, including swimming speed, turning frequency, depth and distance between fish. Deltamethrin and cadmium were 0.015 ppm and 3.5 ppm, respectively. It was conducted for a total period of 180 min. Fish behaviors were recorded with dechlorinated water during the first 60 mins, then deltamethrin and cadmium was introduced to observe behavioral responses of zebrafish during the next 120 mins. 
 
Result: As a result of increased swimming activity, the first response of zebrafish is avoidance followed by a changed distribution in the test chamber. The duration of hyperactivity during deltamethrin exposure was lasted 35 minutes larger than Cd exposure and the average swimming depth showed totally different trends with increased from 140 mm to 226 mm during deltamethrin exposure but decreased from 161 to 84 mm during cadmium exposure. It is proved that these different responses do exist under in the two chemicals studied and this may contribute to the development of biological early warning system to separate accidental chemical pollution types.</div>

Behavioral Responses in Zebrafish (Danio rerio) Exposed to Heavy Metal and Insecticide Induced Stress

Background: The RNA-guided recombinase (RGR) platform, combining a hyper-activated sin recombinase with catalytically inactive Cas9 (dCas9), offers a programmable, cell-independent and potentially safer alternative to CRISPR/Cas9 for targeted genome editing. This study was conducted to design and construct a donor DNA plasmid enabling targeted knockout of the lepb gene in zebrafish (Danio rerio) using the RGR platform. 
 
Methods: The lepb sequence was retrieved from the NCBI database and sin recombinase-based RGR recognition motifs were identified and analyzed functionally and structurally using CYC_REC, SOPMA and NetSurfP-3.0. SWISS-MODEL generated 3D structures of lepb, which were validated for quality. An experiment was conducted to build a donor DNA plasmid containing a mutated lepb fragment through site-directed mutagenesis (SDM) and verified by sequencing. 
 
Result: A nonsense mutation (GAG to TAG) was introduced at 1738 bp, disrupting a critical glutamate residue, leading to loss of lepb protein function. In silico analysis revealed the loss of key structural elements (partial helix-C, complete helix-CD loop and helix-D residues) and disulfide bridge-forming cysteine residues (C112 and C157), which are crucial for lepb-lepr binding, stability and folding of the lepb protein. The homology modeling confirmed substantial alteration in the lepb conformation due to truncation. The donor DNA plasmid containing a 1808 bp-long lepb fragment with the targeted mutation (T) was constructed and validated. No off-target effects were detected. This study demonstrates the precision and feasibility of RGR-mediated lepb knockout and provides a validated donor DNA plasmid with potential applications in growth trait enhancement and functional genomics in zebrafish. 
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Designing and Construction of Donor DNA for RNA-Guided Recombinase (RGR) Platform-Mediated Knockout of Leptin B (Lepb) Gene in Zebrafish