An insight into comparative sex based proteomic profiling of Hilsa Tenualosa ilisha towards domestication and aquaculture

Abstract

This study investigates the serum proteome dynamics of male Hilsa (Tenualosa ilisha), a species of immense ecological and economic value, to support its conservation, aquaculture, and long-term sustainability. Using advanced LC-MS/MS-based proteomic techniques, we identified proteins across six weight groups, with the highest number (817) found in 201–300g individuals. Interestingly, protein counts did not follow a linear trend with body weight; the 401–500g group had the lowest number (84), suggesting complex physiological transitions during development. The proteome data were submitted to the ProteomeXchange Consortium via PRIDE. Bioinformatic analysis revealed proteins associated with key immune-related gene ontology (GO) terms and enriched KEGG pathways, including Neuroactive ligand-receptor interaction, phagosome, FoxO signaling, Apelin signaling, and herpes simplex virus interaction. Several immune and inflammation-related proteins showed age- and sex-dependent expression patterns. Comparative analysis of male and female Hilsa at various developmental stages revealed commonly expressed proteins like Alpha Macroglobulin, Fibulin, MEF2C, and complement proteins. Male-dominant expression was observed for MADS-box, anaphylatoxin, and cytosolic non-specific dipeptidase. These findings provide critical insights into sex-specific proteomic adaptations and immune function during freshwater migration and offer a valuable foundation for future Hilsa, domestication, and aquaculture.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-30326-5.

Introduction

Migratory fish undertake movements, either individually or in schools, from their natal habitats to growth regions, optimizing reproductive success. Typically, these fish return to their place of origin to spawn. Such migrations play a critical role in regulating population density, expanding species distribution, exploiting diverse food sources, and adapting to changing environmental conditions¹. Among the significant migratory fish species in the Indo-Pacific region is Tenualosa ilisha (Hilsa), a member of the class Actinopterygii, order Clupeiformes, sub-family Alosinae, and family Clupeidae. The Hilsa is particularly abundant in the waters of Bangladesh and the Indian state of West Bengal, notably in the Hooghly River—part of the Ganges system—and its coastal zones, as well as in the Padma River, the primary distributary of the Ganges in Bangladesh. Esteemed for its taste, flavour, and culinary qualities, the Hilsa holds significant economic value in these regions².

Proteins are acknowledged as a key and comprehensive tool for understanding biological systems and are responsible for producing energy, actions, relationships, structure, and cell division³. Proteins in a particular biological system are assessed on a large scale using proteomic analysis. In addition to figuring out the structure and function of proteins, this study also involves quantifying them and identifying their intracellular location, changes, interactions, and abundance⁴. It is commonly known that the majority of biological processes are controlled by protein complexes rather than single proteins and that the way these proteins interact may determine a specific biological function⁵. The molecular mechanisms behind these changed proteins or their expressions can be easily interpreted by using pathway analysis to compact a large number of proteins onto a small list of pathways knowledge⁶. Protein-protein interactions are the subject of all of these and experimentally determined or anticipated data regarding protein interactions is placed in a number of interaction databases. Fish is a major part of the regular diet in many countries, accounting for almost two-thirds of the world’s population and providing roughly 40% of the total protein consumed. Although all fish species are relevant, only a few have had their physiology thoroughly examined. Fish serum protein molecular characterisation is now regarded as essential in understanding the function of particular proteins associate in a range of physiological and metabolic processes^7,8. A vital component of an organism is its proteome, which includes post-transcriptional and post-translational protein expression regulation and provides information on overall gene expression⁹. Thus, proteomics can map an organism’s adaptive potential and provide an image of its present state of being¹⁰.

The application of proteomic technologies in fish biology has increased dramatically in the last decade. The relevance of signaling events during development in a rapidly changing environment is highlighted by proteomic alterations that occur during distinct life stages of fish species or by studying biological systems and their behaviour under diverse conditions¹¹. Over the past several decades, sex-specific proteomic studies in fish have been widely explored, particularly through the use of two-dimensional gel electrophoresis, which was a popular technique at the time. For instance, conducted a comparative proteomic analysis of male and female gonads in Persian sturgeon (Acipenser persicus)¹². Similarly, as early as four decades ago, the immunochemical identification of the female-specific serum protein, vitellogenin, in medaka fish¹³. Over the past ten years, tremendous advancements in technology have been made in proteomic platforms based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), which provides the ability to identify and quantify thousands of proteins from any biological material, including fish species^14,15. Recent studies employing serum and plasma proteomics in teleosts have revealed sex- and stage-specific protein expression patterns linked to reproductive physiology and biomarker discovery^8,16–19. Additionally, sample pooling techniques are used to rationalize the overall number of samples in large trials or to lessen biological variance when the number of samples is limited^20,21. Nevertheless, very few published studies on the protein profiling of fishes in the Clupeidae family²². Remarkably, no extensive protein profile of this economically significant fish species has been reported. This may be related to the fact that many fish species in the Clupeidae family lack entire genome sequences. Additionally, author has already published a detailed analysis of female Hilsa serum proteome sequence¹⁹, which supports us in developing a successful Hilsa protein sequence database submitted to ProteomeXchange Consortium via PRIDE. Therefore, the primary goal of this research was to generate a proteome profile of Hilsa male serum in different weight groups and a comparative functional profile of male and female hilsa¹⁹. This study offers the first comprehensive shotgun proteomic analysis of Hilsa serum, revealing key insights into its immune system, metabolism, and physiological adaptations. It highlights significant variations in proteome functions based on weight, sex, and growth stages, contributing to a better understanding of the species’ migration mechanisms. These findings pave the way for future conservation and domestication efforts of Hilsa.

Materials and methods

Ethics statement

The Central Inland Fisheries Research Institute (ICAR) ethical committee at Barrackpore, Kolkata 700120, West Bengal, India, authorised this work. The Committee on Animal Ethics approved this experiment. (Approval date: 10/09/2019; approval code: CIFRI/EC/2019/62) The Institute’s defined protocols were followed when handling the fish used in the experiment. The experimental design, sample collection, and fish sacrifice were conducted in accordance with ethical standards and relevant national guidelines and regulations. Furthermore, all methods reported in this study comply with the ARRIVE guidelines.

Sample collection and processing

Wild male Hilsa (T ilisha) samples of different size groups (Table 1) were collected from the Ganga River at Farakka, Murshidabad, West Bengal (24°48′1.34″ N; 87°55′21.53″ E) during August to November 2021 using a specially designed lift net. Fish were anaesthetised (No euthanasia) with MS-222 (120 mg/L water) following the protocol²³. A total of 18 (n= 3 each weight group) male Hilsa samples from various weight groups were selected for the study. Males, typically smaller than females during the intersex stage, were identified by examining abdominal shape or through dissection and observation. During dissection, a thin, thread-like transparent gonad was observed in smaller individuals, that was identified as testes, confirming them as males. In contrast, females exhibited slightly broader, yellowish lobes, characteristic of developing ovaries. Blood was drawn from the caudal vasculature using a 2 mL sterile syringe, following the standard protocol outlined by²⁴. The sampling procedure and year classes were adopted as per our previously published paper^19,25. Briefly, the Hilsa blood was transferred to 1.5 ml Eppendorf tubes and coagulated for 2–3 hours at room temperature. Serum samples were then transported to laboratory in an ice box and stored at −40°C for further analysis. Three samples from the same weight group were pooled and processed for proteomic analysis using the LC-MS/MS method. For comparative purposes, protein data from female Hilsa, previously reported by¹⁹, were utilised to examine differences between male and female proteomes with the dataset identifiers PXD054275, PXD054224, PXD054225, and PXD054228, the mass spectrometry proteomics data of female Hilsa (H3, H28, H29, and H34) have been uploaded to the ProteomeXchange Consortium through the PRIDE.

Table 1.

LC-MS and UniProt summery.

Hilsa Sample	Weight range (g)	Avg ± SD	Length range (cm)	Avg ± SD	Total proteome (no.)	UniProt mapping (no.)
H4	501-above	659.2 ± 85.3	42.5–47.9	45.2 ± 2.65	64,248	691
H22	401–500	455 ± 38.6	39.8–40.8	40.2 ± 0.50	2293	84
H37	301–400	354.1 ± 41.1	33.7–34.9	34.3 ± 0.61	25,746	336
H35	201–300	242.3 ± 31.9	25.4–27.8	26.6 ± 1.20	81,333	817
H42	101–200	155.6 ± 12.0	18.9–21.1	20.1 ± 1.11	22,915	301
H80	50–100	76.3 ± 10.2	15.3–12.8	13.8 ± 1.30	20,171	326

LC-MS/MS

The ACQUITY UPLC apparatus from Waters Corporation in the US was used for liquid chromatography. Following dilution with 100 mM NH4HCO3, the sample (100 µg) was subjected to a 1-hour treatment with 250 mM DTT (dithiothreitol) at 95 °C and a 45-minute dark treatment with 250 mM IDA (iminodiacetic acid) at room temperature. Samples were digested in trypsin, and a Waters-made ACQUITY UPLC BEH C18 column (150mm X 2.1mm X 1.7m) was used to separate the trypsinised peptide solution. The chromatographic separation has been completed using a gradient elution procedure with mobile phases A and B (0.1% formic acid in water and acetonitrile, respectively). A SYNAPT G2 QTOF (Waters, USA) equipped with an ESI source was utilised for mass spectrometric detection. The starting mass is 300,000 Da, and the final mass is 1700,000 Da for the acquisition mass range. Protein Lynx Global Server (PLGS) 3.0.2, a database search engine (Fig. 1), was used to handle the raw mass spectrometry data before being sent via PRIDE to the ProteomeXchange Consortium^26–28.

Fig. 1.

Biological function of male hilsa (H4- 501g- above, H22- 401-500g, H37- 301-400g, H35-201-300g, H42-101-200g, H80-50-100g). The red color defined highest -log10 FDR score and blue defined the lowest. Size of the circles showed no of genes.

Proteomics data interpretation and functional profiling

Advanced techniques for interpreting high-throughput proteomics data were employed to map and annotate mass spectrometry-based proteomics identifiers using the UniProtKB fish proteome sequence database (www.uniprot.org). STRING (https://string-db.org/) was used to analyze protein interaction networks., allowing insights into the functional dynamics of enriched proteins. The Danio rerio STRING background database was used for comparative analysis with the in-house Hilsa proteome dataset interactions with a confidence score ≥0.7 (high confidence) were included. Network edges were derived from experimental and co-expression evidence, using Danio rerio as the reference organism. Proteins detected in at least two biological replicates per group were considered reliable nodes in the PPI network. Network topology was examined using Cytoscape (v3.10.2), enabling visualisation and exploration of protein interactions. Gene ontology (GO) annotations—covering "molecular functions," "cellular components," and “biological processes” and additionally KEGG pathway analysis was also conducted with significant terms identified at an adjusted p-value <0.05 (Benjamini–Hochberg correction) and comparative study representation through butterfly graph—were performed using R packages (version 4.5.0). To visualise pathway-level comparisons were illustrated using a butterfly graph, representing the relative enrichment of pathways between male and female Hilsa datasets.

Comparative statistical analysis between male and female

Statistical studies were conducted using the R packages (version 4.5.0) to generate violin plots, scatter plots, and Spearman’s rank correlation. All correlations and visualizations were created to evaluate the connections between distributions (the top 15 frequently occurring proteins) and variables (male and female hilsa).

Results

Across six male groups, a total of 2,555 non-redundant proteins were identified. Although the number of detected proteins varied slightly by weight class (ranging ≈ 84–691), the overall qualitative composition remained highly conserved. A total of 81333 (maximum) peptide-matched spectra were found in 201-300g male hilsa, these data were further filtered (after removing duplicates) in UniProt mapping and the maximum number of annotated proteomes quantified were 817 (maximum). The lowest nos. of annotated proteome (84) were found in 401-500g hilsa. These proteome profiles (Supplementary file. 1) were analysed based on their PLGS score, peptides, coverage (%), molecular weight, etc. The major identified proteins were Alpha Macroglobin, fibulin, MEF2C, complement proteins (C3, C4, C5), albumin, serum response factor (SRF), anaphylatoxin, CD109, MADS-box domain and cytosolic non-specific dipeptidase protein found in male Hilsa. Fibulin, C3, serum response factor and Alpha Macroglobin were primarily dominant. The core male serum proteome, shared across all groups including H22, comprised 142 proteins, dominated by immune and carrier molecules such as Alpha-2-Macroglobulin, Apolipoproteins A-I/A-IV, Complement C3–C5, Fibulin, and Serum Response Factor. These are typical of teleost serum and reflect the essential metabolic and immunological framework. A small number of proteins showed variability in observation frequency, notably in male- MADS-box, cytosolic, and anaphylatoxin which were indicating stage-dependent immune and developmental regulation, also called variable proteins.

A Venn diagram (Supplementary Fig. S1) visualises shared and stage-specific proteins. The smaller juvenile groups (H80–H42) expressed Cytosolic Non-Specific Dipeptidase and MADS-box proteins, possibly linked to early growth and cellular differentiation. Intermediate groups (H35–H37) showed a higher frequency of Fibulin and CD109, related to tissue remodelling, while mature males (H4) displayed enrichment of Complement C3–C5 and Alpha-2-Macroglobulin, consistent with immune maturation.

Functional annotation of proteomics and gene ontology

In this study, Gene Ontology (GO) served as a functional classification system, providing detailed insights into the properties of genes and gene products in Hilsa. This GO technique is categorised into three forms, viz. biological processes, molecular function and cellular components, to complete an understanding of the functionality of regulatory processes. The top-hit categories from the assembly annotation were filtered and are illustrated in Figures 3, 4, and 5. The enrichment values, nGenes (No. of genes) and -log₁₀ (FDR) were also calculated and analysed.

Fig. 3.

Cellular component (CC) of male Hilsa (H4- 501g- above, H22- 401-500g, H37- 301-400g, H35-201-300g, H42-101-200g, H80-50-100g). The green color defined highest -log₁₀ FDR score and pink defined the lowest. Size of the circles showed no of genes.

Fig. 4.

KEGG pathway analysis of male Hilsa (H4- 501g- above, H22- 401-500g, H37- 301-400g, H35-201-300g, H42-101-200g, H80-50-100g); color showed -log₁₀(FDR) score and circle size showed no. of genes. Blue color showed highest -log₁₀ (FDR) score and pink denoted lowest.

Fig. 5.

Comparative KEGG pathway analysis of male (H4- 501g- above, H22- 401-500g, H37- 301-400g, H35-201-300g, H42-101-200g, H80-50-100g) and female H3- 401-500g, H28- 301-400g, H29- 201-300g, H34- 100-200g¹⁹.

The top 20 biological functions were presented in the male Hilsa samples (Fig. 2). Complement activation showed the highest –log₁₀ FDR score in most of the samples, except for H37 and H80, and was associated with the maximum number of genes. In contrast, proteolysis, protein metabolic processes, and regulation of protein metabolic processes exhibited relatively low –log₁₀ FDR scores (False Discovery Rate = 5.0, respectively). Other significant biological processes included negative regulation of endopeptidase activity, included negative regulation of peptidase activity, negative regulation of proteolysis, Humoral immune response etc. (Fig 1)

Fig. 2.

Molecular function (MF) of male Hilsa (H4- 501g- above, H22- 401-500g, H37- 301-400g, H35-201-300g, H42-101-200g, H80-50-100g). The blue color defined highest -log₁₀ FDR score and red defined the lowest. Size of the circles showed no of genes.

In the molecular function (MF), the male with the dominant -log10 FDR score was negative regulation of endopeptidase activity, negative regulation of peptidase activity, negative regulation of proteolytic activity, negative regulation of hydrolase, regulation of peptidase activity, regulation of proteolysis, protein metabolic process, cellular macromolecule metabolic process, regulation of the primary and metabolic process, regulation of the cellular metabolic process, regulation of nitrogen compound metabolic process, complement activation etc. In overall sample, the first four mentioned above were the highest-log₁₀ FDR score, and a maximum number of nGenes was noticeable. In lower (H80) and middle weight groups (H22 and H37) of male Hilsa expressed lower log₁₀ FDR score in metabolic processes and functions (Fig. 1). In this study, 400 g Hilsa(H22) showed lower numbers of genes and lower -log₁₀ FDR score

In the case of the cellular component (CC), only five samples were found, and extracellular space and extracellular region were enriched and mostly found to have the highest -log10 FDR value and a maximum number of nGenes. Except for sample H4, which had a significantly higher weight, and H35 (Fig. 3), with a medium weight, three additional GO terms—collagen-containing extracellular matrix, external encapsulating structure, and extracellular matrix—exhibited low -log10 FDR values and a minimal number of nGenes in the other three samples.

In the KEGG pathway study, there were nine pathways were enriched. Among them, four common pathways were enriched in all the samples of Hilsa male, which were Herpes simplex virus, Phagosome, FoxO and Neuroactive ligand-receptor interaction pathway (Fig. 4). In the larger fish, all the pathways were enriched. The Herpes simplex virus had the maximum -log₁₀ FDR value and nGenes. Among all mTOR signaling pathway had lowest -log10 FDR value and nGenes.

Comparative functional profiling of male and female proteome

For the purpose of comprehending the molecular mechanisms of functions, the proteome approach was used to identify and thoroughly characterize the protein content of the serum of male and female Hilsa fish. LC-MS/MS was applied to map the protein content and compare proteins of both genders. Male datasets (n = 6) and female datasets (n = 4) were kept as separate groups during analysis to preserve biological variability. Proteins observed in at least two datasets from each group were retained for comparative evaluation. The results showed that the female (952 nos.) (using the data from Chakraborty et al., 2024), have more frequently observed serum protein than male (817 nos.) through UniProt mapping. In the case of males, two more weight groups were added (Below 100 g and above 500 g) to cover a more exhaustive study area than before in females¹⁹. In this gender comparative serum proteome analysis, maximum revealed proteins were common, but in the case of male MADS box, anaphylatoxin and Cytosolic non-specific dipeptidase, CD109, MEF2C-like proteins, which were very less quantity found in female, were dominant in male. Comparison between pooled male and female datasets identified no statistically significant qualitative differences after correction, indicating that both sexes share a highly conserved immune-dominant serum proteome. Nevertheless, several proteins differed in observation frequency: MADS-box, Anaphylatoxin, and CD109 were more frequent in males, whereas Vitellogenin-like and lipid-transport proteins were relatively more frequent in females.

In the comparative KEGG pathway study, the highest weight group of Hilsa males comprised all nine pathways. In contrast, females with the same weight group comprised only six and also, the – log₁₀ FDR score and fold enrichment value were highest in males. Like the Herpes simplex virus 1 infection, the log₁₀ FDR score and fold enrichment value of males was 4.84E-05 and 34.5, but for females, it showed 0.0082 and 25.211, respectively. In the same weight groups of males and females, the same pathways, like Histidine metabolism and beta-alanine metabolism, were absent in 300-400g in Hilsa males, which is present in females, but mTOR was absent in females. In the weight range 200-300g, females (three) comprised half of the KEGG pathway than males (six), and the common pathways were Herpes simplex virus 1 infection, Phagosome, and Neuroactive ligand-receptor interaction. Most interestingly, it was found that in the lower weight group of Hilsa there was no common pathways were found (Fig 5).

Comparative analysis of PPI networks

Protein-protein interaction (PPI) networks provide critical insights into the molecular mechanisms underlying biological processes. The comparative analysis of male and female serum protein networks in Hilsa (Tenualosa ilisha) reveals notable differences in protein-protein interaction clusters, functional annotations, and biological implications.

Male Hilsa exhibits eight clusters, including a larger complement activation cluster (24 genes), alpha-2-macroglobulin family proteins, and NGF-stimulated transcription, indicating heightened immune activity and stress response mechanisms (Fig. 6A). Additionally, the number of nodes was 76, average local clustering coefficient – 0.508. In contrast, female Hilsa¹⁹ (Fig. 6B), displays ten clusters, highlighting acute phase responses, pentraxin-related proteins, and interleukin pathways, reflecting a broader serum protein repertoire and readiness for systemic inflammation, additionally, the number of nodes was 45, average local clustering coefficient – 0.421 and both the cases PPI enrichment p-value was same (<1.0e-16). Both sexes share key proteins such as C3, C5, and alpha-2-macroglobulin but differ in interaction density and clustering. Females exhibit additional antigen-like proteins and more diverse interaction networks. These findings underscore sex-specific immune strategies, with males emphasizing complement cascade activation and females focusing on acute phase proteins and broader immune modulation. Further experimental validations and environmental impact studies are warranted to elucidate these molecular mechanisms and their adaptive significance in T. ilisha.

Fig. 6.

Protein-Protein Interaction of Hilsa serum proteome -A. Male, B. Female ¹⁹; each color showed one cluster of the network.

Comparative statistical analysis of male and female Hilsa

The violin plot (Fig. 7A) provides information on distribution and variability by combining a box plot with a density trace. The distribution is more concentrated around lower observed frequency values and was comparatively narrower for male Hilsa (blue plot). There was less variety since the density was more compressed. The distribution was wider and more dispersed for females (orange plot), indicating higher variability in observed frequencies. There were some severe values and a larger range in the density trace. In comparison to males and females had more diverse and expansive distribution of observed protein frequencies. This result implied that whereas males exhibit a more consistent pattern but females had more individual variability in the assessed characteristic. A linear regression analysis (Fig. 7B) was performed to examine the influence of the variable male on the variable female.

Fig. 7.

(A) Violin plot (B) scatter diagram- comparing the observer frequency distribution between male and female¹⁹.

The regression model showed that the variable female explained 79.83% of the variance from the variable male Hilsa. ANOVA found that the effect was significantly different from zero, F=51.44, p = <.001, R2 = 0.8. Overall, the scatter plot shows that there was a general correlation between higher observed frequencies in females and higher observed frequencies in males. This favorable correlation may indicate that both groups are influenced by a common biological or environmental source.

A Spearman correlation presents (Fig. 8) two probability histograms, one for males (left, blue) and one for females (right, green), showing the distribution of observed frequencies. A fitted density curve (red line) is superimposed on each histogram to illustrate the overall distribution trend. The result of the Spearman correlation showed that there was a significant correlation between male and Female, r(13) = 0.69, p =.004. The distributions of observed frequencies for both males and females are positively skewed, with most observations taking place at lower frequency levels. However, females have a wider and more varied distribution, indicating that the assessed attribute is more heterogeneous among females.

Fig. 8.

Distribution of Hilsa proteins observed frequencies in the male and female.

Discussion

During this current study, proteomic profile of different weight groups of males and compared male-females of T. ilisha were presented here. Due to a paucity of resources in the fish proteins functional annotation database, the functional analysis of the Hilsa proteome collection was cross-validated by two different bioinformatics platforms utilising the Zebrafish gene reference as a thoroughly studied model fish organism²⁹. This study identified a wide range of proteins with diverse functions, indicating the complexity of Hilsa serum proteome and its role in physiological, metabolic, and immune regulation. Interestingly, the maximum number of peptide-matched spectra and the proteomics study reveals that the male Hilsa had a maximum of 817 annotated proteins, indicating higher protein levels than the female (691). This is a remarkable finding that the 200 g Hilsa had a maximum of annotated proteins, whereas females with 500 g Hilsa contained a maximum¹⁹. As a result, the number of novel proteins did not follow a notable trend as weight increased. Several common key proteins, including Alpha Macroglobulin, Fibulin, Serum Response Factor (SRF), Complement components (C3, C4, C5), and CD109, were consistently dominant. The top 10 frequent proteins found in the male-female¹⁹ were the same and complement proteins were dominant¹⁹. These proteins are widely recognized for their roles in complement activation, immunological defense, and extracellular matrix organization, are essential for surviving in dynamic aquatic environments. These represent the core serum proteome, crucial for metabolic regulation and immune defense, confirming their essential role in basic physiological processes. However, in the case of males, three more proteins were predominant: MADS-box, cytosolic, and anaphylatoxin. Three different processes in the mammalian system carry out target recognition and creating a protease complex for C3 activation. The lectin pathway is similar to the classical pathway, but C1 is replaced by a lectin–protease complex containing the mannose-binding lectin (MBL) or ficolin (FCN). Alternative pathway activation depends on complex interactions of complementary regulatory molecules like factor H and properdin (P). The classical pathway, which comprises C1, C4, and C2 components reacting in this order, primarily recognises antibodies in immune complexes. According to³⁰, this pathway is also in charge of amplifying C3-activation that is brought on by the other two pathways. To activate C5 into C5a and C5b, the target-bound C3b subsequently forms a protease complex. This serves as a scaffold for C6–C9 to create a membrane-attack complex (MAC) and exhibit cytolytic activity. Even though the aforementioned effector actions are essential for effective host defense, excessive complement activation and host cell stimulation may seriously harm host tissue, resulting in anaphylaxis and cell damage³¹. The core sex-shared proteins, Complement C3–C5, Fibulin, Alpha-2-Macroglobulin, Apolipoproteins, and Serum Response Factor—represent proteins essential for cellular defence and transport. Their ubiquitous presence suggests fundamental roles in survival, while frequency variation indicates physiological fine-tuning between sexes.

KEGG and GO analyses revealed predominant enrichment of immune-related pathways, including Complement and Coagulation Cascades, Acute-Phase Response, and Lipid-Transport processes. Smaller male groups (H80, H42) showed fewer enriched pathways, reflecting immature metabolic and immune development, whereas larger groups displayed stronger complement activation and protease-inhibition networks. The absence of common pathways in smaller males corresponds with limited hepatic maturity and reduced serum protein complexity at juvenile stages showed that developmental and physiological changes; complexity increases with maturity and stress response³², additionally, smaller fish have limited exposure to pathogens and less-developed complement and metabolic systems, resulting in fewer annotated immune pathways. Insights from the advanced LC-MS/MS technique method can be used to identify proteins that are part of a particular biological pathway or alter their expression in response to disease, toxicants, or other stressors³³. Thus, there is no current information on this MAD Box protein in fish, but in male Hilsa, it was identified and reported for the first time. The consensus CTA(T/A)4TAG/A sequence, found in the regulatory areas of many muscle-specific and growth-inducible genes, is bound by this protein, which is a family of transcription factors of MEF2 genes and its encoded proteins, in both homo- and heterodimers³⁴. There is limited research on the roles of MADS-box genes in stress resistance in animals, and none have been published in fish-related studies. The term "MADS-box" refers to four of the initially recognized members of this conserved motif, which is present in the DNA-binding domains of these proteins: MCM1, AG, DEFA, and SRF³⁵. Furthermore, fish development is significantly influenced by MADS-box transcription factors, especially the Myocyte Enhancer Factor 2 (MEF2) family. MEF2 proteins are necessary for the development of skeletal muscles and the preservation of myofibril structure in zebrafish (Danio rerio)³⁶. According to these results, MADS-box genes are essential for teleost species’ growth, reproductive development, and adaption processes. This information is particularly pertinent for comprehending the physiology of migration in species such as Hilsa (Tenualosa ilisha). Notably, Alpha macroglobin (A2m) was dominant in Hilsa, which may work in disease resistance. According to³⁷, a potential marker found in A2M protein could be used as a molecular marker to diagnose fish resistance or susceptibility to EUS disease, and they might even be able to be included in the marker panel for pilot research and this marker also anticipated in T. Ilisha also. Fibulin and Alpha Macroglobulin in particular were dominant, that play a part in controlling innate immunity and preserving structural integrity under stressful condition during migration.

Proteomics study at the molecular level can reveal crucial information on the signaling proteins and transcription factors that control fish growth patterns. Nine KEGG pathways were identified via pathway enrichment analysis, with neuroactive ligand-receptor interactions, phagosomes, FoxO signaling, and Herpes simplex virus infection being prevalent in male groups. As observed in proteomic studies of other teleosts³⁸, the steady enrichment of pathways linked to viral infections (e.g., Herpes simplex virus 1) is intriguing because it was not reflected actual viral exposure but rather emphasize conserved immune and stress-related protein networks in fish. In this study, Apelin signaling pathways found in both adult male and female (stage I and IV) Hilsa, which was also found in Salmon fish kidney cell line³⁹ were important in fish many biological functions, work as energy balance regulation, intake of food, immunity regulation⁴⁰, the FoxO signaling pathway was present and plays significant roles during stress associated with migration in Hilsa^41–43claimed that although FoxO is necessary for numerous unique cellular processes, such as differentiation, apoptosis, autophagy, metabolism, inflammation, and stress tolerance, it also contributes to heat stress. This process was notably intensified in Hilsa at elevated temperatures, suggesting a potential link between modified energy metabolism and thermal adaptation during sea-to-river migration. The mTOR (mammalian target of rapamycin) signaling pathway was found in a previous study of female adult Hilsa, which helps in immunology, but in males, it showed in each weight group, that in different life stages of Hilsa, it might work in the development and growth⁴³. studied n triploid crucian carp, and it showed that the mTOR signaling pathway helps to regulate embryonic development to rapid growth. Additionally, in the realm of cellular metabolism^44,45, regulation of essential biological processes such as cell proliferation, metabolism, differentiation, and survival of fish^46,47 this signaling pathway has received a lot of attention. In KEGG pathways analysis, a few genes were revealed which was associated with specific pathways like metabolism pathways were regulated by cndp2 genes, sgk1 and sgk3 in signaling pathway (mTOR and FOXo), mef2 in Apeline signaling pathway and c3a.1, c3a.2, c3a.3, c5 was related to immunology like phagosome, Herpes simplex virus 1 infection and Neuroactive ligand-receptor interaction. These cndp2 genes are found in mainly human serum, which is related to carnosinase⁴⁸ but also identified for the first time in the tissues of Japanese eel⁴⁹. Therefore, understanding the genes involved in the immune system is crucial for developing effective disease management strategies for wild Hilsa.

This study through PPI networking showed several key proteins and their associate neighbour interactor protein linked to the immune system of males (Fig. 6A) and females (Fig. 6B). Hilsa were identified, offering significant advancements in understanding hilsa’s self-defense mechanisms. These proteins are associated with vital immune processes, including pathogen recognition, antigen presentation, and the activation of innate and adaptive immune responses. During the spawning migration phase, Hilsa experiences starvation, which significantly impacts its immune system and overall physiology⁵⁰. Sexual maturity and spawning are reported to influence humoral immunity through interactions between sex hormones and the immune system⁵¹, while starvation suppresses the expression of genes associated with immune proteins⁵². In T. ilisha, a decline in immunological indices during migration has been observed, likely due to a combination of environmental and hormonal factors⁵³. Understanding fish immune defense mechanisms in response to chemical⁵⁴ and physical^55,56, stresses, such as pathogens^57,58, is another significant use of the proteomic technique.

Our integrated proteomic and bioinformatics approach reveals key immune-related proteins in Hilsa, highlighting sex- and weight-dependent differences in response to migration, stress, and infection. These insights provide biomarkers for disease resistance, support selective breeding of robust populations, and strengthen aquaculture and conservation strategies. As seen in salmon and trout^52,59, such molecular-level understanding lays the foundation for Hilsa domestication and sustainable management. The findings were consistent with the biology of serum, which was dominated by highly abundant, conserved immune and carrier proteins that exhibited limited qualitative variability across individuals and sexes. The absence of statistically significant differences after correction indicated that sex and developmental stage did not alter the serum proteome composition at the level of protein identity in our dataset; rather, the quantitative detectability (observation frequency) of shared proteins varied modestly across groups.

Conclusion

This study represents the first comprehensive shotgun proteomic analysis of Hilsa (Tenualosa ilisha) serum, providing an in-depth overview of its functional proteome profile with the highest enrichment in immunological properties. Across six weight groups, the maximum number of annotated proteins (817) in 201–300 g individuals, and other groups also comparative study with female reflecting non-linear proteome dynamics during growth and sex. A core set of 142 proteins formed the conserved proteomic backbone across sexes, with only stage-specific shifts in immune-related proteins (MADS-box, Anaphylatoxin, CD109) and no major qualitative sex-specific differences. Functional annotation and network interactions revealed that defence-related genes and immune pathways—including neuroactive ligand–receptor interaction, phagosome, FoxO signaling, and Apelin signaling—varied across developmental stages. Several proteins such as Alpha Macroglobulin, Fibulin, MEF2C, and complement proteins were consistently expressed across groups, whereas MADS-box, anaphylatoxin, and cytosolic non-specific dipeptidase displayed male-biased expression, underscoring sex-dependent proteomic adaptations. Collectively, biological molecular and cellular functions of the hilsa proteomics and KEGG pathways and protein-protein interaction networks helped to understand the physiological plasticity of this ecologically and commercially important species. Considering its high consumer demand and market value, domestication of Hilsa is crucial; proteomic insights into hormonal regulation, ovulation, and migration offer a strong baseline for manipulation, artificial breeding, and sustainable aquaculture of this wild species.

Author contributions

The experiment was designed by BKD. Samples were collected, prepared and manuscript writing by HC. Proteomics analysis was done by HJC. Data interpretation by BKD, HC, HJC and JM. The manuscript is prepared and edited by all the authors.

Data availability

All supporting data for this study are included in the manuscript and the supplementary materials. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via PRIDE (Perez et al., 2016; 2022; Deutsch et al., 2023) partner repository of male Hilsa H4, H22, H37 H35, H42, and H80 dataset identifier [PXD063644],(http:/ebi.ac.uk/pride/archive/projects/PXD063644/private), [PXD062032](https:/www.ebi.ac.uk/pride/archive/projects/PXD062032/private), [PXD063281](https:/www.ebi.ac.uk/pride/archive/projects/PXD063281/private), [PXD062121](https:/www.ebi.ac.uk/pride/archive/projects/PXD062121/private), [PXD061789](https:/www.ebi.ac.uk/pride/archive/projects/PXD061789/private) and [PXD063282](https:/www.ebi.ac.uk/pride/archive/projects/PXD063282/private) respectively. For additional data requests, please contact the corresponding author.

Declarations

Competing interest

The authors declare no competing interests.