Proteome Profile of Muscle Tissue of Indian Walking Catfish, Clarias Magur Exposed to Abiotic Temperature Stress


 The study of expression of proteins in organisms on exposure to various environmental challenges gives clues for understanding on how these challenges affects and copes with the biological system. A study was undertaken to understand the proteome profile of Clarias magur, exposed to abiotic stress of water temperature, to find how fishes evolve adaptive strategies towards stress induced by unforeseen vagaries of climate change. Specimens of Clarias magur were exposed to high temperature sub-lethal water stress of 37°C for 60 days and the muscle proteome profiling was analysed through Liquid Chromatography –Mass Spectroscopy for qualitative differential profiling . The study provides an understanding of different proteins expressed as adaptative challenge to the environment. This is the first study to see proteome expression in Clarias magur through Liquid Chromatography –Mass Spectroscopy


Introduction
Climate change is a major contributory factor towards adaptation of ecosystems from where the food emerges. UN Sustainable Development Goals (SDGs), particularly the SDG1 (end poverty), SDG2 (end hunger), SDG13 (good health and well-being) and SDG4 (action for reducing impact of climate change) has direct linkage to provide an integrative framework for bringing together the policy and the system initiatives to counter the vagaries of climate change. Climate change shall eventually impact food production due to rising temperature, affecting the adaptability of the genetic resources and their productivity. By the middle of 21 st century, the global population of ~9 billion people will be ready to be fed. The Paris Agreement of the United Nations Framework Convention on Climate Change (UNFCCC), which aims to keep the global temperature rise this century well below 2°C above pre-industrial levels, recognizes the fundamental priority of safeguarding the food security to integrate the climate change measures into national policies, strategies and planning to reduce the effects of climate change on the productivity. Aquaculture heavily depends on ambient environment, hence vulnerable to climate change, potential of epigenetic adaptation can improve stress tolerance through early life exposure for which real time complex data is needed on multiple stressor levels for prediction of long term change for planning in response to climate change adaptation and mitigation [ 1 ]. Aquaculture has adapted its self at local level, by changing cultural practices and community based adaptive planning as identi ed through a study percent of total n sh production in 2018 [ 3 ]. Nadarajah and Eide, 2020 [ 4 ] evaluated relisence of shrimp, tilapia, carp and cat sh in sequence, indicating high diversity with high adaptation capacity specially in Asia that has capacity to adapt with proper modi cations in farming system in future. Climate change also affects temperate region, where mitigation strategy would be to adapt existing species, wild and domesticated for temperate temperature tolerance along with collaboration with stakeholders, researchers and government shall help in developing mitigating strategies for climate change [ 5 ]. A metaanalysis study found that aquatic animals present higher mortalities at warmer temperatures induced due to climate change, also making them susceptible to high antimicrobial resistance [ 6 ]. Providing an environment that is stress free for aquaculture shall have its bene ts in long term, considering minimum stress and maximum welfare for the aquaculture. Fishes when stressed jeopardises their health and survival, while adjusting to the aquatic conditions [ 7 ] . Chemical imbalances in water cause direct harm to sh by disrupting physiological functions as ionic regulation, gill and kidney function, by destroying the shes' mucous coating[ 8 ],which is a primary protection against pathogenic and parasitic invasion [ 9 ]. The facultative air breathing cat sh, Clariasmagur (magur), has more commercial importance as food sh that can thrive in swampy water conditions where many teleosts cannot survive due to intolerance to high environmental ammonia. An initiative was taken to analyse stress induced changes in the sh proteome by exposing them to the sub lethal high temperature. The peptides from the Liquid Chromatography Mass Spectrometry (LC-MS) were analysed to study the effect on the adaptability of C.magur to this temperature stress as preparedness for tolerance induced by the climate change. The primary aim of the study was to understand adaptive mechanism undergoing at genomic and transcriptomic level ultimately translating to proteomic level. The complexity of the system was frozen after a time span established under experimental conditions to understand basic proteome changes underlying a stressful condition. The study was taken up to understand the complexity of the physiological changes starting with activation of sympathetic response altering the energy metabolism . In the downstream process, energy is metabolised that is coped by the activation of the glycogenolysis and gluconeogenesis from muscle or liver [ 10 ]. Lactate can be generated from the muscle due to external stressful stimuli leading to anaerobic glycolysis, which is released into plasma [ 11 ]. The stress induction leads to alterations in the overall performance impacting growth, immune response and health [ 12 ]. Proteomics has emerged as a powerful tool towards the deep understanding of the sh biology, helping aquaculture to reach its main goal of high productivity with better quality products. The conduct of experiment is represented in graphical abstract Figure 1. Compared to genomics, the proteome provides not only information at a mechanistic level but can also captures the changes in protein activity measured as post-translational modi cations. It deciphers the amount of protein present in an organism at a particular time, thus, helps to analyse the responses from the expressed proteome pro le. Whereas genome remains the blue print, protein expressed helps in adapting to changing environmental and anthropometric stress.

Materials And Methods
Healthy live specimens (n=15) of Clarias magur (weighting 50-90 g) were procured from the local area in and around Lucknow, Uttar Pradesh, India. The specimens were anaesthetised in clove oil and transported to indoor hatchery in 150 litre plastic tank with proper oxygenation. The shes were then acclimatized in a re-circulatory system for 15 days at 26°C. After acclimatization, the specimens were randomly divided into control and experimental groups. Each group contained 10 shes. The control group were kept at ambient water temperature of 26°C in re-circulatory system, while experimental were exposed to 37°C sub lethal limit by increasing the water temperature 1°C per day from the initial temperature. Both the groups were exposed for long duration of 60 days, and were monitored daily. After experimentation, 3 specimens from both groups were sacri ced under anaesthetic condition with clove oil. Muscle tissues samples were dissected and immediately frozen in liquid nitrogen and also kept at -80°C till further protein extraction. The shes were taken care of ethically by anesthetising with clove oil before dissection

Protein extraction and LC MS
Protein was extracted from the stored muscle samples of 3 control and 3 experimental groups within a week of conducting the experiment. Brie y, the samples were grinded and homogenised in 50 mM Tris Buffer containing protease inhibitor cocktail and phenylmethylsulfonyl uoride (PMSF), centrifuged at 14,000 rpm to remove the debris. The supernatant was collected in Tris buffer extract and run on SDS-PAGE to check quality for downstream processing. The protein was quanti ed by BCA method. 100 µg of extracted protein was processed for running in LC-MS. The sample was treated with 100 mM dithiothreitol (DTT) for 1 h at 95 o C for reducing disulphide bonds of cysteine, followed by treatment with 250 mM IDA for 45 min at room temperature in dark to alkylate cystine formed after reduction of the cysteine. The sample was then digested with trypsin, which speci cally cleaves at lysine and arginine amino acids, and incubated overnight at 37˚C. The peptides from the sample were extracted in 0.1% formic acid and incubated for 45 min at 37˚C followed by centrifugation at 10,000 g. The supernatant was collected and dried in vacuum. The dried sample was dissolved in 20 µl of 0.1% formic acid in water. This sample was then used for injecting in LC-MS. 10 µL injection volume was used on Ethylene Bridged Hybrid C 18 nano columns (Hydrophobic stationary phase) of UPLC for separation of peptides, based on their a nity, with hydrophilic molecules eluting rst. The peptides separated on the column were directed to Waters Synapt G2 Q-TOF instrument for Data Independent Acquisition (DIA) for MS and MS/MS analyses. The raw data was processed by Mass Lynx 4.1 WATERS. The individual peptides MS/MS spectra were matched to the database sequence for protein identi cation on PLGS software (Protein Lynx Global Server), WATERS with UniProt (https://www.uniprot.org/)reviewed sequences of zebra sh. The raw data of elutant and mass/ charge value was subjected to PLGS software and data was processed with UniProt (https://www.uniprot.org/) reviewed proteins of zebra sh. Qualitative differential expression of proteins was based on 50 ppm peptide tolerance, 100 ppm fragment tolerance with minimum fragment match of 2 peptides for proteins. Peptides were modi ed (carbamidomethylation and oxidation) and the retrieved proteins were analysed, visualised and interpreted. 25 most relevant pathways, sorted by p-values, were analysed in control and experimental groups.
C. magur shes were exposed to abiotic temperature stress and Liquid chromatography was performed on a ACQUITY UPLC system (Waters

Data Analysis
Identi cation of proteins: The peptide spectra was identi ed by its PLGS Score, that uses a Monte Carlo algorithm to analyse all available mass spectra data, providing a statistical measure of accuracy of assignation with a higher score implying a greater con dence of protein identity [ 14 , 15 ]. The identi ed proteins from experimental and control groups were analysed for their signi cance and regulatory mechanism. A representative chromatogram and peptide mass/charge ratios are shown in Figure 2 showing chromatogram of elutant and mass spectra of control and experimental samples.
The differential expression of proteins retrieved from the muscle of C. magur exposed to high water temperature stress was analysed. Since the peptides analysed were not same in all three samples due to

Results And Discussion
The objective of the study was to understand changes occurring in the metabolism of sh when exposed to temperature stress. The study throws light on the differential expression of proteins under stress. Semaphorins are extracellular signaling proteins characterized by a single cysteine-rich extracellular sema domain, function as axon guidance molecules, but it is now understood that semaphorins, are key regulators of morphology and Semaphorin signaling occurs predominantly through Plexin receptors and results in changes to the cytoskeletal and adhesive machinery that regulate cellular morphology [ 20 ]. Sema3A, a prototypical semaphorin, acts as a chemorepellent or a chemoattractant for axons by activating a receptor complex comprising neuropilin-1 as the ligand-binding subunit and plexin-A1 as the signal-transducing subunit. FARP2 is a key molecule involved in the response of neuronal growth cones to class-3 semaphorins [ 21 ]. The members of the collapsin response mediator protein (CRMP) family-ve cytosolic phosphoproteins-are highly expressed throughout brain development. Moreover, the expression of CRMPs is altered in neurodegenerative diseases, and these proteins may be of key importance in the physiopathology of the adult nervous system [ 22 ] . Vascular endothelial growth factor (VEGF) and VEGF  Figure 4 shows more proteins involved in biological process. Enzymatic activity of 275 Commonly Expressed Proteins (CEP) in control and experimental samples of muscle tissue exposed to temperature stress is shown in Figure 5, depicting oxidoreductases, transferases, hydrolases and enzymes altering polypeptide conformation. Figure 6 shows Pathway analysis of 275 Commonly Expressed Proteins in control and experimental samples of muscle tissue exposed to temperature stress with major proteins involved in protein modi cation mainly ubiquitination and glycosylation. Figure 7 shows Gene Ontology of 581 proteins expressed only in Control Samples of Muscle tissue. Figure 8 shows Gene ontology of 163 proteins expressed in experimental samples of muscle tissue exposed to temperature stress. Figure 9 depicts enzymatic analysis of 163 proteins expressed in experimental muscle tissue samples exposed to temperature stress. Figure 10 depicts pathway analysis of 163 proteins expressed in experimental muscle tissue samples exposed to temperature stress The major pathway of proteins expressed in response to high temperature stress was related to protein modi cations glycosylation and ubiquitination. Ubiquitination is a multistep exzymatically catalyzed post-translational modi cation process that targets proteins for degradation and recycling . Ubiquitination is critical in almost every cellular process, has a role in modulating diverse cellular functions like cell proliferation and differentiation, autophagy, apoptosis, immune response, DNA repair, neural degeneration, myogenesis, and stress response as well as a major player in almost any disease or   Enzymatic activity of 275 Commonly Expressed Proteins in control and experimental samples of muscle tissue exposed to temperature stress Gene ontology of 163 proteins expressed in experimental samples of muscle tissue exposed to temperature stress Page 16/17

Figure 8
Enzymatic analysis of 163 proteins expressed in experimental muscle tissue samples exposed to temperature stress Figure 9 Pathway analysis of 163 proteins expressed in experimental muscle tissue samples exposed to temperature stress