Development of a plausible ‘pan-specific primer’ targeting fish CYP1A gene for a rapid streamlined assessment of aquatic ecosystem integrity

Development of a plausible ‘pan-specific primer’ targeting fish CYP1A gene for a rapid streamlined assessment of aquatic ecosystem integrity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of a plausible ‘pan-specific primer’ targeting fish CYP1A gene for a rapid streamlined assessment of aquatic ecosystem integrity Himangshu Goswami, Nilakshi Sarma, Simanta Kalita, Partha Pratim Das, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7999597/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aquatic ecosystems, one of the vital resources of earth’s biosphere, are deviously threatened by anthropogenic pollution from compounds like polycyclic aromatic hydrocarbons (PAHs), heavy metals, and persistent organic pollutants. Though their early detection at trace levels in the aquatic environment is critical, fish CYP1A gene expression, induced via the AhR pathway, stands as a dominant and reliable sensitive biomarker. However, lack of a broad-range primer has limited large-scale ecological monitoring across diverse fish taxa. Addressing this gap for maintaining the healthy status of aquatic ecosystems, the present study aims to develop and validate a pan-specific primer targeting the fish CYP1A gene for multi-species biomonitoring in Northeast India, especially Assam. Seasonal samples of five freshwater fish species that includes Indian major carps, exotic carp, and indigenous fishes, were collected from an ecologically important site, the Deepor Beel, the only Ramsar site in Assam. Fish specimens were collected in two batches for each season: one batch was immediately dissected on-site for liver tissue isolation to extract RNA, while the other batch was acclimatized in the laboratory for 15 days to serve as the control group for further analyses. Following RNA isolation and cDNA synthesis, primers were designed and optimized via gradient PCR and qRT-PCR. The designed primer efficiently amplified CYP1A across species with 295 bp product size and sensitivity up to 0.1 ng/µL cDNA (LOD = 10⁻⁴). Significant upregulation of CYP1A expression was observed in the wild species compared to the control, indicating pollution-induced stress. This optimized primer enables time and cost-effective, species-independent assessment of aquatic pollution and can streamline conservation strategies in biodiverse regions. Future applications may expand its use to establish it as a universal tool for ecological risk assessment. CYP1A biomarker broad range primer aquatic pollution fish health Assam wetlands Figures Figure 1 Figure 2 Figure 3 1. Introduction Aquatic ecosystems are one of the pivotal foster resources of earth’s biosphere for stabilizing the global climate as well as other developmental purposes of human activities. But increase in the saturation levels of toxicological compounds like polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), organochlorines, heavy metals, oil spills etc., released from growing industrial sector, urbanization and over-exploitation of natural resources lead a serious threat for this naturally abundant ecosystem and their aquatic biota (Kolawole and Iyiola 2023; Malik et al. 2020 ). It puts the entire balanced state of earth in an alarming position. Restoring the vitality of these aquatic ecosystems hence authenticates one of the most concerned issues of present-day technology to trace devious pollutants in aquatic habitats at early level. Fish, having a vast array of potential biomarkers, are suitable for this purpose including the aspect of environmental assessment (Alves et al. 2022 ). CYP1A is one of the best biomarkers to detect the pollutants in the microscopic levels because of AhR pathway activation in presence of xenobiotics in the fish body (Shankar et al. 2020 ). It works for metabolism of pollutants through oxidative reactions in fish, allowing either detoxification or bioactivation of the xenobiotic substances (Mao, Li, and Zheng 2023; Schlenk et al. 2024 ). Their elevated levels indicate the exposure to contaminants and provide early signs to the pollution that further helps in the mitigation of environmental evaluation strategy (Amutha and Subramanian 2010 ). This study lights up the effect of contamination in the natural water ecosystems with the help of seasonal variation of CYP1A expression in multiple freshwater fish species of Northeast India, particularly in Assam, collected from a single ecologically important site, the Deepor Beel, it is the only Ramsar site of Assam. Previous study works showed that a particular primer was designed for each particular species. It also causes a lack in CYP1A expression analysis in a large number of species. Hence pointing the urgency of earlier toxicants detection due to increasing contamination levels, there is a need of designing a universal primer that is specific for more than one species. Due to the presence of large number of species in an aquatic environment, there is a need of universal primer which is specific for multiple species. It validates CYP1A expression as a robust marker for ecological integrity, binding with more than one species. This regulation of CYP1A expression provides an insight to the mechanistic view of chemical toxicity and potential risks due to environmental contaminants. Furthermore, due to inevitable role in the removal of toxicity, this gene can be considered as one of the best and potential biomarkers for two stepped biomonitoring of toxicological substances and environmental assessment (Abdallah et al. 2024 ). 2. Materials and Methods 2.1 Sampling Sites: In this study, five freshwater fish species named Cyprinus carpio , Labeo rohita , Catla catla , Clarias batrachus and Anabas testudineus , were selected as representative sample organisms. All specimens were collected from the Deepor Beel, the only Ramsar site in Assam, situated in the Kamrup (M) district (Rongpi 2024 ). The selected species are locally available in the beel, comprising two Indian major carps ( Labeo rohita and Catla catla ), an exotic carp ( Cyprinus carpio ), and two other indigenous fishes that naturally occur in this ecosystem. 2.2 Sample Collection: Fish samples of all five species were collected seasonally in post-monsoon, summer, and winter with the assistance of local fishermen. The collections were made in two batches. The first batch was used for immediate liver tissue isolation, where specimens were dissected on-site and liver tissues preserved in RNAlater for RNA extraction. The second batch was transported to the laboratory for acclimatization. This acclimatization procedure was followed for all specimens across the three seasons, each maintained for a period of about 15 days. During acclimatization, Cyprinus carpio, Labeo rohita , and Catla catla were maintained in tanks with dechlorinated, well-oxygenated water covered with protective nets in accordance with Ullah et al. 2024 (Ullah et al. 2024 ). Fishes were fed to satiation with a 35% protein small pelleted feed twice daily at 5% of their body weight. Tank hygiene was maintained by removing uneaten feed and excreta by manually using small net, and approximately 50% of the water was exchanged daily to maintain optimal water quality. Regular monitoring was performed. Water parameters such as temperature, pH, and dissolved oxygen were measured to ensure that they remained within optimal ranges. According to Ahammad et al. 2021 (Ahammad et al. 2021 ), Clarias batrachus and Anabas testudineus were maintained separately under a 12h:12h light/dark cycle at a constant temperature of 25°C to mimic natural environmental conditions. They were hand-fed commercial fish feed once daily under continuous monitoring. All specimens that underwent this acclimatization period were considered as the experimental control group for subsequent laboratory procedures. Following acclimatization, the fishes were dissected and liver tissues were immediately collected and preserved in RNAlater for RNA extraction. 2.3 Primer Designing and Optimization: Broad range primer targeting conserved regions of the gene were designed by aligning coding sequences from multiple teleost fish species using ClustalW in BioEdit v7.2.5. Conserved regions were selected, and primers were generated using NCBI Primer-BLAST. Along with CYP1A gene specific primer for quantitative expression analysis, β-actin was selected as the internal control gene. The primer sequences for β-actin were adopted from a previously published study [AF057040] (Lu et al. 2025 ), which reported stable expression of β-actin across multiple fish tissues. Both the primer sequences were validated in silico through NCBI Primer-BLAST to confirm the absence of off-target binding and were synthesized commercially. The accuracy and optimization of each primer sets were verified through PCR amplification. Gradient PCR has been performed with an annealing temperature profile ranging from 50°C to 60°C based on the melting temperature (Tm) of each of the CYP1A and β-Actin specific target primer set. After that specificity of the broad range primer has been analysed via observing the amplicon size against a GeneRuler 50 bp ladder (Thermo Scientific). Then we proceed to the primer sensitivity with the help of limit of detection (LOD). For analysing the sensitivity of the designed primer, a ten-fold serial dilution of cDNA template ranging from 10 − 1 to10 − 5 was used with our optimized PCR mixture. 2.4 RNA Extraction and Expression Analysis: Total RNA was isolated using TRIzol methods. cDNA synthesis was performed using the iScript™ cDNA Synthesis Kit (Bio-Rad, USA). Quantitative real-time PCR (qRT-PCR) was conducted using SYBR® Premix Ex Taq™ II (2x) (Takara Bio, Japan) following the manufacturer’s protocol. Relative expression levels of CYP1A were normalized to β-actin expression and quantified using the ΔΔCt method (Livak and Schmittgen 2001 ). 2.5 Statistical Analysis: For statistical analysis, IBM SPSS Statistics version 21.0 was used. ANOVA was conducted to compare mean expression differences between the groups. A p-value < 0.05 was considered statistically significant for all tests conducted in this study. 3. Results 3.1. Pan species primer for Fish CYP1A expression analysis: The primer pair obtained after through in silico analysis has a forward sequence 5′-GGRATCGTCAAYGACCTCTTCGGAGC-3′ and a reverse sequence 5′-ACAGGTGTCTTTGGGAATGAAATATCC-3′ with codon degeneracy in the forward sequence at position 3 ( R , A or G) and 12 ( Y , C or T). Following through standardization procedure, the optimum annealing temperature for the pair has been found to be 55°C with amplicon size of 295 bp for all the tested fish species. 3.2 Specificity and Sensitivity of the primer pairs: Upon performing the PCR with the optimised annealing temperature using cDNA template prepared from aforesaid five different species, it has been observed that all the visible band were of amplicon size 295bp, there by indicating the absence of any nonspecific amplicons and that the primer binds specifically to the target gene only. Further, the binding affinity of the primer was observed to be of higher magnitude as evident from the fluorescence intensity of the band on agarose gel for most of the species except Anabus testudineus and Clarias batrachus. Non reactivity with human and mice template nullifies the possibilities of appearing false positive reaction. In silico BLAST analysis also exhibit supportive evidence for fish specific amplification of CYP1A gene by the wide range primer designed. Further, analysis using Sanger sequencing of the amplicons and subsequent In silico global alignment analysis also exhibits 100% similarity with fish CYP1A gene. Table 1 Detection Limit and Amplification Efficiency of the Designed Broad-Range Primer Across Serial Dilutions Dilution Replicates detected Average Ct value Standard deviation SE P value 10 − 1 5/5(100%) 23.68 1.29 0.57 P < 0.05 10 − 2 5/5(100%) 28.90 3.55 1.56 10 − 3 5/5(100%) 32.49 2.50 1.11 10 − 4 2/5(40%) 33.73 2.47 1.10 10 − 5 0/5(0%) - - - * p Value < 0.05 was considered statistically significant. For analyzing the sensitivity of the designed primer, a ten-fold serial dilution of cDNA template ranging from 10 − 1 to10 − 5 was used with our optimized PCR mixture and it has been observed that the primer can successfully amplify the target gene in all (100%) replicate up to 10 − 3 dilution (1ng/ul) with increasing Ct value from 23.68 to 32.49, (Table 1 ). At 10 − 4 dilution (0.1ng/ul) of cDNA, 40% amplification has been observed with Ct value of 33.73 and no amplification was observed at 10 − 5 dilution. As such it can be stated that the primer pair can efficiently amplify up to a template concentration as low as 0.1ng/ul (L.O.D is 10 − 4 ) without any nonspecific amplification, which is indicative of the enhanced sensitivity of the designed broad range primer pairs. (Fig. 1 ) 3.3 Broad range amplification of the primer: Analysis of the amplification ability of the optimised primer pair using cDNA template from five different fish species exhibit efficient amplification with 295 bp amplicon, (Fig. 2 ). Among all the tested species Cyprinus carpio , Labeo rohita , Catla catla shows intense band on agarose gel that indicates probable efficient binding of the primer pair with the targeted CYP1A gene. Apart from that, cDNA prepared from Anabas testudineus and Clarias batrachus also showed amplification but with lower intensity band on agarose gel, probably due to lower template concentration or may be due to some degree of sequency divergence arises out of lower sequence information availability in global databases reinforced by geographical variation resulting in picking primer sequence with some degree of sequence variability. 3.4 CYP1A expression profiling and fold change analysis of all collected species: The CYP1A expression shows a consistent upregulation in terms of mean fold-change of wild groups compared to control groups across all collected five species ( Cyprinus carpio, Labeo rohita, Catla catla, Anabas testudineus and Clarias batrachus ) and seasons (post-monsoon, winter and summer) (Fig. 3 ). A significant fold-change differences (p < 0.05) in Cyprinus carpio are observed among both control and wild groups. The mean fold-change in Cyprinus carpio is higher in summer (1.15 ± 0.35) and post-monsoon (1.03 ± 0.14). Similar pattern has also been observed in other species with significant mean fold-change differences (p < 0.05) (Table 2 ). During the winter season, the mean CYP1A expression of wild groups are also higher than that of the control groups of all collected species. But these elevated levels were comparatively lower than that of the other two seasons. In the indigenous species Anabas testudineus and Clarias batrachus , a consistently reduced pattern in mean CYP1A expression in wild groups were observed throughout all seasons as compared to the carp family (Table 2 ). Table 2 Mean Fold Change Analysis and Statistical Correlation Species Season Post-Monsoon Winter Summer Control (N = 7) Wild (N = 7) P Value* Control (N = 10) Wild (N = 10) P Value* Control (N = 7) Wild (N = 7) P Value* Cyprinus carpio 0.69 ± 0.28 1.03 ± 0.14 0.014 0.70 ± 0.28 0.98 ± 0.10 0.008 0.66 ± 0.34 1.15 ± 0.35 0.021 Labeo rohita 0.59 ± 0.40 1.06 ± 0.30 0.032 0.66 ± 0.27 0.97 ± 0.16 0.006 0.50 ± 0.38 1.07 ± 0.21 0.009 Anabas testudineus 0.74 ± 0.07 0.99 ± 0.14 0.002 0.71 ± 0.16 0.90 ± 0.12 0.008 0.70 ± 0.15 0.94 ± 0.13 0.008 Catla catla 0.57 ± 0.37 1.02 ± 0.18 0.014 0.63 ± 0.38 0.94 ± 0.17 0.033 0.56 ± 0.25 1.18 ± 0.23 0.000 Clarias batrachus 0.52 ± 0.22 1.00 ± 0.16 0.001 0.51 ± 0.20 0.98 ± 0.13 0.000 0.60 ± 0.11 0.99 ± 0.19 0.001 *Post-Monsoon (October-November), Winter (November to March), Summer (March to April) * p Value < 0.05 was considered statistically significant. It was observed that the overall expression of CYP1A was comparatively higher in wild samples than that of the controls irrespective of species and seasons which indicates persistent environmental exposure to pollutants. These results highlighted pollution-induced induction of CYP1A expression with seasonal and species-specific intensity patterns for these collected species. . 4. Discussion North East India being a famous biodiversity hotspot with four major Ramsar site (Deepor Beel in Assam, Loktak Lake in Manipur, Rudrasagar in Tripura and Pala wetland located in Mizoram) harbors a rich diversity of indigenous fish species with reported 422 different species, belonging to 133 genera and 38 families (Goswami et al. 2012 ). Apart from that, it also harbors 216 SIFS (small indigenous fish species) which accounts for 48% of total SIFs reported in India (Mandal, F. B., & Nandi 2015). However, due to modern anthropogenic activities, this diverse population is gradually become endangered over the last few decades. Prime factors are overfishing, habitat destruction, pollution and climate change, added to it are paddy field wash off, industrial effluent with heavy metal contaminants and pesticides. These xenobiotic components induce oxidative stress, affects normal reproduction and leads to immune dysfunction (Ninawe, A.S., Chippu Shakir, Aseer Manilal, Indulkar, S. T., Sana K. M Patel, Shabna 2025). In an approach towards mitigating the problem of xenobiotic contaminants in aquatic environment tissue CYP1A expression analysis may plays a major role. Dar.et.al,2020 reported CYP1A as an important biomarker for emamectin benzoate detoxification in Labeo rohita (Dar et al. 2020 ). CYP1A expression level in fish liver has also been extensively used for identifying potential exposure to PAH and PCBs in different environmental integrity and ecotoxicological studies (Chivittz et al. 2016 ). Thus, analysing the CYP1A expression level in fish liver tissue exhibits reliable and caused effective option for analysing aquatic environmental integrity. However, scarce number of studies in this regard has been reported from this region, probably due to unavailability of sufficient amount of sequence information for this diverse number of species in global database, making it difficult to design a specific primer or may be due to unavailability of a broad range primer or pan species primer which will significantly ease the process. As a consequence, the region is slowly losing its fish species diversity (Marques et al. 2021 ). In this study, a broad range pan species primer pair has been reported and evaluated for its specificity and sensitivity, that can successfully amplify five locally available species ( Cyprinus carpio , Labeo rohita , Catla catla , Clarias batrachus , and Anabas testudineus ), from all the possible target species as predicted by in-silico similarity analysis, in a wet lab setting. With further research, this broad-range primer could potentially be used to include additional species data, including indigenous small fish from the region. Additionally, the primer pair can successfully amplify the target region of all the five tested fish species at a single annealing temperature (55 0 C) with uniform product size of 295 bp. Thus, the reported primer pair enables the analysis of different species in a single batch reaction which is both cost and time effective. Notably, the primer pair efficiently amplified the CYP1A gene in Anabas testudineus and Clarias batrachus , despite the limited availability of only one or two partial sequences in the NCBI database. This indicates its potential to facilitate the submission of additional sequences. Apart from this, the applicability of the reported broad range primer will made the screening of CYP1A expression across diverse fish species easy and affordable by eliminating the need of species-specific primer. Overall CYP1A expression was higher in wild fish than controls irrespective of species and seasons, reflecting persistent environmental pollutant exposure. However, the relatively lower expression in indigenous species may result from their metabolism and adaptive resistance, which can modulate CYP1A induction to reduce toxic effects (Carrothers et al. 2025 ; Williams et al. 2022 ). This adaptive downregulation has been observed in fishes chronically exposed to pollutants which indicates species-specific detoxification strategies. Seasonal and species-specific patterns, such as higher expression in Cyprinus carpio, Labeo rohita and Catla catla during post-monsoon and summer, further emphasize the complexity of pollutant responses in these ecosystems. However, the study includes only five different species out of all the similarity hit showed by NCBI BLAST due to resource limitation. As such future endeavours, considering more and more no. species, which make this broad range primer as a universal primer, with few modifications if needed, thereby easing the screening process for the environmental integrity, which is the need of these hours. Under such an alarming environmental circumstance this finding will help the agencies and conservationist with a strong scientific base for taking bold step towards safeguarding the natural biodiversity within the region. 5. Conclusion and Future Perspectives This study authenticates a broader range, pan-species primer which is specially designed for CYP1A gene amplification of indigenous fish species of Northeast India, specifically in Assam. It demonstrates a higher specificity yielding alongside uniform product size as well as efficient performance at a single annealing temperature, which plays a pivotal role to establish it as an universal tool for early-stage pollution detection along with environmental monitoring. Future studies can be worked on to validate this primer across a multiple range of species from a diverse ecological region. Such advancements are crucial as it is both time-effective as well as cost-effective and help to mitigate large-scale screening of aquatic pollution that ultimately leads safeguarding of Northeast India's fragile ichthyofaunal diversity. Declarations Acknowledgement: The authors thoroughly acknowledge the Department of Zoology Pub Kamrup College, Kamrup for providing all the necessary support needed during the research work. Declarations competing interests : The authors have no competing interests to declare that are relevant to the content of this article. Funding source: The study did not receive any financial support from any other external or internal source Author Contributions: All authors contributed to the conception and design of the study. Material preparation, data collection, and analysis were performed by Himangshu Goswami and Nilakshi Sharma . The study design, methodology development, and supervision were provided by Dr. Manash Jyoti Kalita (Corresponding Author) . Laboratory support was provided by Dr . Subash Medhi , while Dr. Simanta Kalita contributed to validation, and Dr. Partha Partim Das assisted in visualization. The first draft of the manuscript was written by Himangshu Goswami , and all authors commented on previous versions of the manuscript. All authors read and approved the final version of the manuscript. Ethical Approval: This is not applicable Consent to Participate: This is not applicable Consent to Publish: All authors declare that they have given their consent for publication of this manuscript. The authors confirm that the work described is original, has not been published previously, and is not under consideration for publication elsewhere. All authors have read and approved the final version of the manuscript and agree to its submission to Environmental Science and Pollution Research . Data Availability Statement: Data are available from the corresponding author on reasonable request References Abdallah, Salwa M., Reham E. Muhammed, Reda El Mohamed, Hala El Daous, Dina M. Saleh, Mohamed A. Ghorab, Shaohua Chen, and Gharieb S. 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University","correspondingAuthor":false,"prefix":"","firstName":"Subhash","middleName":"","lastName":"Medhi","suffix":""},{"id":551120251,"identity":"a77eb720-bb9b-4e97-8e21-4d1962a0b9cc","order_by":5,"name":"Manash Jyoti Kalita","email":"data:image/png;base64,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","orcid":"","institution":"Pub Kamrup College Department of Zoology","correspondingAuthor":true,"prefix":"","firstName":"Manash","middleName":"Jyoti","lastName":"Kalita","suffix":""}],"badges":[],"createdAt":"2025-10-31 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13:14:40","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89120,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7999597/v1/07c7b683c7cc9e7d39d4a91f.html"},{"id":97169901,"identity":"e189b10e-3a12-431b-829e-f12184febb66","added_by":"auto","created_at":"2025-12-01 14:30:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":60735,"visible":true,"origin":"","legend":"\u003cp\u003eDetection sensitivity of the pan-specific CYP1A primer across decreasing cDNA concentrations, showing reliable amplification down to 0.1 ng/µL.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7999597/v1/5ca86d3253fd0e3de89423b9.png"},{"id":97250036,"identity":"43f3f3f0-01d2-4bd9-82a0-3c955ed8222d","added_by":"auto","created_at":"2025-12-02 13:13:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80791,"visible":true,"origin":"","legend":"\u003cp\u003eBroad range amplification of the primer with different tested species\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7999597/v1/cddc449f51b383a637b2b1d2.png"},{"id":97169902,"identity":"6a72d130-d454-4d08-9ea2-02a69d800416","added_by":"auto","created_at":"2025-12-01 14:30:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43721,"visible":true,"origin":"","legend":"\u003cp\u003eCYP1A expression profiling and fold change analysis of five collected species during \u003cem\u003ea.\u003c/em\u003e Post Monsoon \u003cem\u003eb. \u003c/em\u003eWinter and \u003cem\u003ec.\u003c/em\u003e Summer\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7999597/v1/00cbffb9e30a7599a9cf2679.png"},{"id":103505924,"identity":"3eb39d6f-cfd1-40dd-b5dc-b04149da9b52","added_by":"auto","created_at":"2026-02-26 13:33:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1033150,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7999597/v1/a54bbba8-a54e-4253-9e0d-1e2cd129fedd.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eDevelopment of a plausible ‘pan-specific primer’ targeting fish CYP1A gene for a rapid streamlined assessment of aquatic ecosystem integrity\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAquatic ecosystems are one of the pivotal foster resources of earth\u0026rsquo;s biosphere for stabilizing the global climate as well as other developmental purposes of human activities. But increase in the saturation levels of toxicological compounds like polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), organochlorines, heavy metals, oil spills etc., released from growing industrial sector, urbanization and over-exploitation of natural resources lead a serious threat for this naturally abundant ecosystem and their aquatic biota (Kolawole and Iyiola 2023; Malik et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It puts the entire balanced state of earth in an alarming position.\u003c/p\u003e\u003cp\u003eRestoring the vitality of these aquatic ecosystems hence authenticates one of the most concerned issues of present-day technology to trace devious pollutants in aquatic habitats at early level. Fish, having a vast array of potential biomarkers, are suitable for this purpose including the aspect of environmental assessment (Alves et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). CYP1A is one of the best biomarkers to detect the pollutants in the microscopic levels because of AhR pathway activation in presence of xenobiotics in the fish body (Shankar et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It works for metabolism of pollutants through oxidative reactions in fish, allowing either detoxification or bioactivation of the xenobiotic substances (Mao, Li, and Zheng 2023; Schlenk et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Their elevated levels indicate the exposure to contaminants and provide early signs to the pollution that further helps in the mitigation of environmental evaluation strategy (Amutha and Subramanian \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study lights up the effect of contamination in the natural water ecosystems with the help of seasonal variation of CYP1A expression in multiple freshwater fish species of Northeast India, particularly in Assam, collected from a single ecologically important site, the Deepor Beel, it is the only Ramsar site of Assam. Previous study works showed that a particular primer was designed for each particular species. It also causes a lack in CYP1A expression analysis in a large number of species. Hence pointing the urgency of earlier toxicants detection due to increasing contamination levels, there is a need of designing a universal primer that is specific for more than one species. Due to the presence of large number of species in an aquatic environment, there is a need of universal primer which is specific for multiple species. It validates CYP1A expression as a robust marker for ecological integrity, binding with more than one species. This regulation of CYP1A expression provides an insight to the mechanistic view of chemical toxicity and potential risks due to environmental contaminants. Furthermore, due to inevitable role in the removal of toxicity, this gene can be considered as one of the best and potential biomarkers for two stepped biomonitoring of toxicological substances and environmental assessment (Abdallah et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Sampling Sites:\u003c/h2\u003e\u003cp\u003eIn this study, five freshwater fish species named \u003cem\u003eCyprinus carpio\u003c/em\u003e, \u003cem\u003eLabeo rohita\u003c/em\u003e, \u003cem\u003eCatla catla\u003c/em\u003e, \u003cem\u003eClarias batrachus\u003c/em\u003e and \u003cem\u003eAnabas testudineus\u003c/em\u003e, were selected as representative sample organisms. All specimens were collected from the Deepor Beel, the only Ramsar site in Assam, situated in the Kamrup (M) district (Rongpi \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The selected species are locally available in the beel, comprising two Indian major carps (\u003cem\u003eLabeo rohita\u003c/em\u003e and \u003cem\u003eCatla catla\u003c/em\u003e), an exotic carp (\u003cem\u003eCyprinus carpio\u003c/em\u003e), and two other indigenous fishes that naturally occur in this ecosystem.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Sample Collection:\u003c/h2\u003e\u003cp\u003eFish samples of all five species were collected seasonally in post-monsoon, summer, and winter with the assistance of local fishermen. The collections were made in two batches. The first batch was used for immediate liver tissue isolation, where specimens were dissected on-site and liver tissues preserved in RNAlater for RNA extraction. The second batch was transported to the laboratory for acclimatization. This acclimatization procedure was followed for all specimens across the three seasons, each maintained for a period of about 15 days.\u003c/p\u003e\u003cp\u003eDuring acclimatization, \u003cem\u003eCyprinus carpio, Labeo rohita\u003c/em\u003e, and \u003cem\u003eCatla catla\u003c/em\u003e were maintained in tanks with dechlorinated, well-oxygenated water covered with protective nets in accordance with Ullah et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e (Ullah et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Fishes were fed to satiation with a 35% protein small pelleted feed twice daily at 5% of their body weight. Tank hygiene was maintained by removing uneaten feed and excreta by manually using small net, and approximately 50% of the water was exchanged daily to maintain optimal water quality. Regular monitoring was performed. Water parameters such as temperature, pH, and dissolved oxygen were measured to ensure that they remained within optimal ranges.\u003c/p\u003e\u003cp\u003eAccording to Ahammad et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e (Ahammad et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), \u003cem\u003eClarias batrachus\u003c/em\u003e and \u003cem\u003eAnabas testudineus\u003c/em\u003e were maintained separately under a 12h:12h light/dark cycle at a constant temperature of 25\u0026deg;C to mimic natural environmental conditions. They were hand-fed commercial fish feed once daily under continuous monitoring. All specimens that underwent this acclimatization period were considered as the experimental control group for subsequent laboratory procedures. Following acclimatization, the fishes were dissected and liver tissues were immediately collected and preserved in RNAlater for RNA extraction.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Primer Designing and Optimization:\u003c/h2\u003e\u003cp\u003eBroad range primer targeting conserved regions of the gene were designed by aligning coding sequences from multiple teleost fish species using ClustalW in BioEdit v7.2.5. Conserved regions were selected, and primers were generated using NCBI Primer-BLAST. Along with CYP1A gene specific primer for quantitative expression analysis, β-actin was selected as the internal control gene. The primer sequences for β-actin were adopted from a previously published study [AF057040] (Lu et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), which reported stable expression of β-actin across multiple fish tissues. Both the primer sequences were validated in silico through NCBI Primer-BLAST to confirm the absence of off-target binding and were synthesized commercially. The accuracy and optimization of each primer sets were verified through PCR amplification. Gradient PCR has been performed with an annealing temperature profile ranging from 50\u0026deg;C to 60\u0026deg;C based on the melting temperature (Tm) of each of the CYP1A and β-Actin specific target primer set. After that specificity of the broad range primer has been analysed via observing the amplicon size against a GeneRuler 50 bp ladder (Thermo Scientific). Then we proceed to the primer sensitivity with the help of limit of detection (LOD). For analysing the sensitivity of the designed primer, a ten-fold serial dilution of cDNA template ranging from 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e was used with our optimized PCR mixture.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 RNA Extraction and Expression Analysis:\u003c/h2\u003e\u003cp\u003eTotal RNA was isolated using TRIzol methods. cDNA synthesis was performed using the iScript\u0026trade; cDNA Synthesis Kit (Bio-Rad, USA). Quantitative real-time PCR (qRT-PCR) was conducted using SYBR\u0026reg; Premix Ex Taq\u0026trade; II (2x) (Takara Bio, Japan) following the manufacturer\u0026rsquo;s protocol. Relative expression levels of CYP1A were normalized to β-actin expression and quantified using the ΔΔCt method (Livak and Schmittgen \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Statistical Analysis:\u003c/h2\u003e\u003cp\u003eFor statistical analysis, IBM SPSS Statistics version 21.0 was used. ANOVA was conducted to compare mean expression differences between the groups. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant for all tests conducted in this study.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Pan species primer for Fish CYP1A expression analysis:\u003c/h2\u003e\u003cp\u003eThe primer pair obtained after through in silico analysis has a forward sequence 5\u0026prime;-GGRATCGTCAAYGACCTCTTCGGAGC-3\u0026prime; and a reverse sequence 5\u0026prime;-ACAGGTGTCTTTGGGAATGAAATATCC-3\u0026prime; with codon degeneracy in the forward sequence at position 3 (\u003cem\u003eR\u003c/em\u003e, A or G) and 12 (\u003cem\u003eY\u003c/em\u003e, C or T). Following through standardization procedure, the optimum annealing temperature for the pair has been found to be 55\u0026deg;C with amplicon size of 295 bp for all the tested fish species.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Specificity and Sensitivity of the primer pairs:\u003c/h2\u003e\u003cp\u003eUpon performing the PCR with the optimised annealing temperature using cDNA template prepared from aforesaid five different species, it has been observed that all the visible band were of amplicon size 295bp, there by indicating the absence of any nonspecific amplicons and that the primer binds specifically to the target gene only. Further, the binding affinity of the primer was observed to be of higher magnitude as evident from the fluorescence intensity of the band on agarose gel for most of the species except \u003cem\u003eAnabus testudineus\u003c/em\u003e and \u003cem\u003eClarias batrachus.\u003c/em\u003e Non reactivity with human and mice template nullifies the possibilities of appearing false positive reaction. In silico BLAST analysis also exhibit supportive evidence for fish specific amplification of CYP1A gene by the wide range primer designed. Further, analysis using Sanger sequencing of the amplicons and subsequent In silico global alignment analysis also exhibits 100% similarity with fish CYP1A gene.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDetection Limit and Amplification Efficiency of the Designed Broad-Range Primer Across Serial Dilutions\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDilution\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReplicates detected\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAverage Ct value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStandard deviation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5/5(100%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5/5(100%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5/5(100%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e32.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2/5(40%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e33.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0/5(0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e* \u003cem\u003ep Value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor analyzing the sensitivity of the designed primer, a ten-fold serial dilution of cDNA template ranging from 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e was used with our optimized PCR mixture and it has been observed that the primer can successfully amplify the target gene in all (100%) replicate up to 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e dilution (1ng/ul) with increasing Ct value from 23.68 to 32.49, (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). At 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e dilution (0.1ng/ul) of cDNA, 40% amplification has been observed with Ct value of 33.73 and no amplification was observed at 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e dilution. As such it can be stated that the primer pair can efficiently amplify up to a template concentration as low as 0.1ng/ul (L.O.D is 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e) without any nonspecific amplification, which is indicative of the enhanced sensitivity of the designed broad range primer pairs. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Broad range amplification of the primer:\u003c/h2\u003e\u003cp\u003eAnalysis of the amplification ability of the optimised primer pair using cDNA template from five different fish species exhibit efficient amplification with 295 bp amplicon, (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Among all the tested species \u003cem\u003eCyprinus carpio\u003c/em\u003e, \u003cem\u003eLabeo rohita\u003c/em\u003e, \u003cem\u003eCatla catla\u003c/em\u003e shows intense band on agarose gel that indicates probable efficient binding of the primer pair with the targeted CYP1A gene. Apart from that, cDNA prepared from \u003cem\u003eAnabas testudineus\u003c/em\u003e and \u003cem\u003eClarias batrachus\u003c/em\u003e also showed amplification but with lower intensity band on agarose gel, probably due to lower template concentration or may be due to some degree of sequency divergence arises out of lower sequence information availability in global databases reinforced by geographical variation resulting in picking primer sequence with some degree of sequence variability.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.4 CYP1A expression profiling and fold change analysis of all collected species:\u003c/h2\u003e\u003cp\u003eThe CYP1A expression shows a consistent upregulation in terms of mean fold-change of wild groups compared to control groups across all collected five species (\u003cem\u003eCyprinus carpio, Labeo rohita, Catla catla, Anabas testudineus\u003c/em\u003e and \u003cem\u003eClarias batrachus\u003c/em\u003e) and seasons (post-monsoon, winter and summer) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A significant fold-change differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in \u003cem\u003eCyprinus carpio\u003c/em\u003e are observed among both control and wild groups. The mean fold-change in \u003cem\u003eCyprinus carpio\u003c/em\u003e is higher in summer (1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35) and post-monsoon (1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14). Similar pattern has also been observed in other species with significant mean fold-change differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). During the winter season, the mean CYP1A expression of wild groups are also higher than that of the control groups of all collected species. But these elevated levels were comparatively lower than that of the other two seasons. In the indigenous species \u003cem\u003eAnabas testudineus\u003c/em\u003e and \u003cem\u003eClarias batrachus\u003c/em\u003e, a consistently reduced pattern in mean CYP1A expression in wild groups were observed throughout all seasons as compared to the carp family (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMean Fold Change Analysis and Statistical Correlation\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"9\" nameend=\"c10\" namest=\"c2\"\u003e\u003cp\u003eSeason\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePost-Monsoon\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003e\u003cb\u003eWinter\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003eSummer\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl (N\u0026thinsp;=\u0026thinsp;7)\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eWild\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e(N\u0026thinsp;=\u0026thinsp;7)\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eP Value*\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eControl (N\u0026thinsp;=\u0026thinsp;10)\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eWild\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e(N\u0026thinsp;=\u0026thinsp;10)\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eP Value*\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eControl (N\u0026thinsp;=\u0026thinsp;7)\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eWild\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e(N\u0026thinsp;=\u0026thinsp;7)\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003eP Value*\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCyprinus carpio\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.014\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e0.021\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLabeo rohita\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.032\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.006\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e0.009\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAnabas testudineus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCatla catla\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.014\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.033\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eClarias batrachus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003e\u003cem\u003e*Post-Monsoon (October-November), Winter (November to March), Summer (March to April)\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e* p Value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eIt was observed that the overall expression of CYP1A was comparatively higher in wild samples than that of the controls irrespective of species and seasons which indicates persistent environmental exposure to pollutants. These results highlighted pollution-induced induction of CYP1A expression with seasonal and species-specific intensity patterns for these collected species.\u003c/p\u003e\u003cp\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eNorth East India being a famous biodiversity hotspot with four major Ramsar site (Deepor Beel in Assam, Loktak Lake in Manipur, Rudrasagar in Tripura and Pala wetland located in Mizoram) harbors a rich diversity of indigenous fish species with reported 422 different species, belonging to 133 genera and 38 families (Goswami et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Apart from that, it also harbors 216 SIFS (small indigenous fish species) which accounts for 48% of total SIFs reported in India (Mandal, F. B., \u0026amp; Nandi 2015). However, due to modern anthropogenic activities, this diverse population is gradually become endangered over the last few decades. Prime factors are overfishing, habitat destruction, pollution and climate change, added to it are paddy field wash off, industrial effluent with heavy metal contaminants and pesticides. These xenobiotic components induce oxidative stress, affects normal reproduction and leads to immune dysfunction (Ninawe, A.S., Chippu Shakir, Aseer Manilal, Indulkar, S. T., Sana K. M Patel, Shabna 2025).\u003c/p\u003e\u003cp\u003eIn an approach towards mitigating the problem of xenobiotic contaminants in aquatic environment tissue CYP1A expression analysis may plays a major role. Dar.et.al,2020 reported CYP1A as an important biomarker for emamectin benzoate detoxification in \u003cem\u003eLabeo rohita\u003c/em\u003e (Dar et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). CYP1A expression level in fish liver has also been extensively used for identifying potential exposure to PAH and PCBs in different environmental integrity and ecotoxicological studies (Chivittz et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Thus, analysing the CYP1A expression level in fish liver tissue exhibits reliable and caused effective option for analysing aquatic environmental integrity.\u003c/p\u003e\u003cp\u003eHowever, scarce number of studies in this regard has been reported from this region, probably due to unavailability of sufficient amount of sequence information for this diverse number of species in global database, making it difficult to design a specific primer or may be due to unavailability of a broad range primer or pan species primer which will significantly ease the process. As a consequence, the region is slowly losing its fish species diversity (Marques et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, a broad range pan species primer pair has been reported and evaluated for its specificity and sensitivity, that can successfully amplify five locally available species (\u003cem\u003eCyprinus carpio\u003c/em\u003e, \u003cem\u003eLabeo rohita\u003c/em\u003e, \u003cem\u003eCatla catla\u003c/em\u003e, \u003cem\u003eClarias batrachus\u003c/em\u003e, and \u003cem\u003eAnabas testudineus\u003c/em\u003e), from all the possible target species as predicted by in-silico similarity analysis, in a wet lab setting. With further research, this broad-range primer could potentially be used to include additional species data, including indigenous small fish from the region. Additionally, the primer pair can successfully amplify the target region of all the five tested fish species at a single annealing temperature (55\u003csup\u003e0\u003c/sup\u003eC) with uniform product size of 295 bp. Thus, the reported primer pair enables the analysis of different species in a single batch reaction which is both cost and time effective.\u003c/p\u003e\u003cp\u003eNotably, the primer pair efficiently amplified the CYP1A gene in \u003cem\u003eAnabas testudineus\u003c/em\u003e and \u003cem\u003eClarias batrachus\u003c/em\u003e, despite the limited availability of only one or two partial sequences in the NCBI database. This indicates its potential to facilitate the submission of additional sequences. Apart from this, the applicability of the reported broad range primer will made the screening of CYP1A expression across diverse fish species easy and affordable by eliminating the need of species-specific primer.\u003c/p\u003e\u003cp\u003eOverall CYP1A expression was higher in wild fish than controls irrespective of species and seasons, reflecting persistent environmental pollutant exposure. However, the relatively lower expression in indigenous species may result from their metabolism and adaptive resistance, which can modulate CYP1A induction to reduce toxic effects (Carrothers et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Williams et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This adaptive downregulation has been observed in fishes chronically exposed to pollutants which indicates species-specific detoxification strategies. Seasonal and species-specific patterns, such as higher expression in \u003cem\u003eCyprinus carpio, Labeo rohita and Catla catla\u003c/em\u003e during post-monsoon and summer, further emphasize the complexity of pollutant responses in these ecosystems.\u003c/p\u003e\u003cp\u003eHowever, the study includes only five different species out of all the similarity hit showed by NCBI BLAST due to resource limitation. As such future endeavours, considering more and more no. species, which make this broad range primer as a universal primer, with few modifications if needed, thereby easing the screening process for the environmental integrity, which is the need of these hours. Under such an alarming environmental circumstance this finding will help the agencies and conservationist with a strong scientific base for taking bold step towards safeguarding the natural biodiversity within the region.\u003c/p\u003e"},{"header":"5. Conclusion and Future Perspectives","content":"\u003cp\u003eThis study authenticates a broader range, pan-species primer which is specially designed for CYP1A gene amplification of indigenous fish species of Northeast India, specifically in Assam. It demonstrates a higher specificity yielding alongside uniform product size as well as efficient performance at a single annealing temperature, which plays a pivotal role to establish it as an universal tool for early-stage pollution detection along with environmental monitoring. Future studies can be worked on to validate this primer across a multiple range of species from a diverse ecological region. Such advancements are crucial as it is both time-effective as well as cost-effective and help to mitigate large-scale screening of aquatic pollution that ultimately leads safeguarding of Northeast India's fragile ichthyofaunal diversity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u0026nbsp;\u003c/strong\u003eThe authors thoroughly acknowledge the Department of Zoology Pub Kamrup College, Kamrup for providing all the necessary support needed during the research work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;competing interests\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003e\u003cem\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding source:\u0026nbsp;\u003c/strong\u003eThe study did not receive any financial support from any other external or internal source \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eAll authors contributed to the conception and design of the study. Material preparation, data collection, and analysis were performed by \u003cstrong\u003eHimangshu Goswami\u003c/strong\u003e and \u003cstrong\u003eNilakshi Sharma\u003c/strong\u003e. The study design, methodology development, and supervision were provided by \u003cstrong\u003eDr. Manash Jyoti Kalita (Corresponding Author)\u003c/strong\u003e. Laboratory support was provided by \u003cstrong\u003eDr\u003c/strong\u003e. \u003cstrong\u003eSubash\u003c/strong\u003e \u003cstrong\u003eMedhi\u003c/strong\u003e, while \u003cstrong\u003eDr. Simanta Kalita\u003c/strong\u003e contributed to validation, and \u003cstrong\u003eDr. Partha Partim Das\u003c/strong\u003e assisted in visualization. The first draft of the manuscript was written by \u003cstrong\u003eHimangshu Goswami\u003c/strong\u003e, and all authors commented on previous versions of the manuscript. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval:\u0026nbsp;\u003c/strong\u003eThis is not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate:\u0026nbsp;\u003c/strong\u003eThis is not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish:\u0026nbsp;\u003c/strong\u003eAll authors declare that they have given their consent for publication of this manuscript. The authors confirm that the work described is original, has not been published previously, and is not under consideration for publication elsewhere. All authors have read and approved the final version of the manuscript and agree to its submission to \u003cem\u003eEnvironmental Science and Pollution Research\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eData are available from the corresponding author on reasonable request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdallah, Salwa M., Reham E. Muhammed, Reda El Mohamed, Hala El Daous, Dina M. Saleh, Mohamed A. Ghorab, Shaohua Chen, and Gharieb S. El-Sayyad. 2024. \u0026ldquo;Assessment of Biochemical Biomarkers and Environmental Stress Indicators in Some Freshwater Fish.\u0026rdquo; \u003cem\u003eEnvironmental Geochemistry and Health\u003c/em\u003e 46(11):1\u0026ndash;19. doi:10.1007/S10653-024-02226-6/TABLES/5.\u003c/li\u003e\n\u003cli\u003eAhammad, A. K. Shakur, Md Asaduzzaman, Md Borhan Uddin Ahmed, Salma Akter, Md Sadiqul Islam, M. 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Schmittgen. 2001. \u0026ldquo;Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2\u0026minus;\u0026Delta;\u0026Delta;CT Method.\u0026rdquo; \u003cem\u003eMethods\u003c/em\u003e 25(4):402\u0026ndash;8. doi:10.1006/METH.2001.1262.\u003c/li\u003e\n\u003cli\u003eLu, Ping Hsun, Sheng Fen Tung, Chi Chung Wen, Ching Yuan Huang, Jhih Wei Huang, Chih Hsin Chen, and Yau Hung Chen. 2025. \u0026ldquo;Pro-Angiogenic Effects of Guo Min Decoction in a Zebrafish Model.\u0026rdquo; \u003cem\u003eTzu Chi Medical Journal\u003c/em\u003e 37(1):58\u0026ndash;64. doi:10.4103/TCMJ.TCMJ_59_24,.\u003c/li\u003e\n\u003cli\u003eMalik, D. S., Amit Kumar Sharma, Arvind Kumar Sharma, Rishi Thakur, and Munish Sharma. 2020. \u0026ldquo;A Review on Impact of Water Pollution on Freshwater Fish Species and Their Aquatic Environment.\u0026rdquo; \u003cem\u003eAdvances in Environmental Pollution Management: Wastewater Impacts and Treatment Technologies\u003c/em\u003e 10\u0026ndash;28. doi:10.26832/aesa-2020-aepm-02.\u003c/li\u003e\n\u003cli\u003eMandal, F. B., Nandi, N. C. 2015. \u0026ldquo;Loss of Small Indigenous Fish Species in India: A Case of Concern.\u0026rdquo; \u003cem\u003eJ. Environ. \u0026amp; Sociobiol .\u003c/em\u003e 12(1):35\u0026ndash;45.\u003c/li\u003e\n\u003cli\u003eMao, Xu, Hui Li, and Jiang Zheng. 2023. \u0026ldquo;Effects of Xenobiotics on CYP1 Enzyme-Mediated Biotransformation and Bioactivation of Estradiol.\u0026rdquo; \u003cem\u003eDrug Metabolism Reviews\u003c/em\u003e 55(1\u0026ndash;2):1\u0026ndash;49. doi:10.1080/03602532.2023.2177671,.\u003c/li\u003e\n\u003cli\u003eMarques, Virginie, Tristan Milhau, Camille Albouy, Tony Dejean, St\u0026eacute;phanie Manel, David Mouillot, and Jean Baptiste Juhel. 2021. \u0026ldquo;GAPeDNA: Assessing and Mapping Global Species Gaps in Genetic Databases for EDNA Metabarcoding.\u0026rdquo; \u003cem\u003eDiversity and Distributions\u003c/em\u003e 27(10):1880\u0026ndash;92. doi:10.1111/DDI.13142.\u003c/li\u003e\n\u003cli\u003eNinawe, A.S., Chippu Shakir, Aseer Manilal, Indulkar, S. T., Sana K. 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Azzam. 2024. \u0026ldquo;Ameliorative Role of Vitamin C against Cypermethrin Induced Oxidative Stress and DNA Damage in Labeo Rohita (Hamilton, 1822) Using Single Cell Gel Electrophoresis.\u0026rdquo; \u003cem\u003eToxics\u003c/em\u003e 12(9):664. doi:10.3390/TOXICS12090664/S1.\u003c/li\u003e\n\u003cli\u003eWilliams, Rebecca, Theresa Taggart, Kayla Ganger, Teri Koetsier, Seema Johnson, and Amber Dinchman. 2022. \u0026ldquo;CYP1A Expression in Freshwater Fish of Western New York as an Indicator of Pollution Levels.\u0026rdquo; \u003cem\u003eEcology and Evolution\u003c/em\u003e 12(1):e8526. doi:10.1002/ECE3.8526.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CYP1A biomarker, broad range primer, aquatic pollution, fish health, Assam wetlands","lastPublishedDoi":"10.21203/rs.3.rs-7999597/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7999597/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAquatic ecosystems, one of the vital resources of earth\u0026rsquo;s biosphere, are deviously threatened by anthropogenic pollution from compounds like polycyclic aromatic hydrocarbons (PAHs), heavy metals, and persistent organic pollutants. Though their early detection at trace levels in the aquatic environment is critical, fish CYP1A gene expression, induced via the AhR pathway, stands as a dominant and reliable sensitive biomarker. However, lack of a broad-range primer has limited large-scale ecological monitoring across diverse fish taxa. Addressing this gap for maintaining the healthy status of aquatic ecosystems, the present study aims to develop and validate a pan-specific primer targeting the fish CYP1A gene for multi-species biomonitoring in Northeast India, especially Assam. Seasonal samples of five freshwater fish species that includes Indian major carps, exotic carp, and indigenous fishes, were collected from an ecologically important site, the Deepor Beel, the only Ramsar site in Assam. Fish specimens were collected in two batches for each season: one batch was immediately dissected on-site for liver tissue isolation to extract RNA, while the other batch was acclimatized in the laboratory for 15 days to serve as the control group for further analyses. Following RNA isolation and cDNA synthesis, primers were designed and optimized via gradient PCR and qRT-PCR. The designed primer efficiently amplified CYP1A across species with 295 bp product size and sensitivity up to 0.1 ng/\u0026micro;L cDNA (LOD\u0026thinsp;=\u0026thinsp;10⁻⁴). Significant upregulation of CYP1A expression was observed in the wild species compared to the control, indicating pollution-induced stress. This optimized primer enables time and cost-effective, species-independent assessment of aquatic pollution and can streamline conservation strategies in biodiverse regions. Future applications may expand its use to establish it as a universal tool for ecological risk assessment.\u003c/p\u003e","manuscriptTitle":"Development of a plausible ‘pan-specific primer’ targeting fish CYP1A gene for a rapid streamlined assessment of aquatic ecosystem integrity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 14:30:30","doi":"10.21203/rs.3.rs-7999597/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ac19d763-5cce-43fe-9da1-5239e57cb678","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-24T14:58:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-01 14:30:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7999597","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7999597","identity":"rs-7999597","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}