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Full Research Article

Nutritional Composition, Fatty Acid Signatures and DNA Barcoding of Filopaludina bengalensis: A Multi-analytical Assessment of an Underutilized Freshwater Resource

R
Reema Das1
Y
Yashaswi Nayak1,*
0009-0002-6999-1861
J
Jnanendra Kumar Sahoo2
1Department of Zoology, Applied Sciences, Jatni, Centurion University of Technology and Management (CUTM), Bhubaneswar campus-752 050, Odisha, India.
2School of Fisheries, Centurion University of Technology and Management (CUTM), Paralakhemundi campus, Gajapati-761 211, Odisha, India.

ABSTRACT

Background: Filopaludina bengalensis is a freshwater gastropod, one of the foremost aquatic resources with nutritional and biochemical significance.

Methods: Thirty specimens were evaluated for morphometric parameters, including body weight, shell length and shell width. Proximate composition and mineral analyses were done to verify nutritional quality. Fatty acid composition was characterized using GC-MS and LC-MS techniques, while amino acid profiling identified major amino acid constituents. Molecular identification was confirmed through COI (cytochrome c oxidase subunit I) gene sequencing.

Result: The mean body weight and shell length were 6.84±0.92 g and 3.51±0.17 cm, respectively. A stable shell length to shell width ratio (~1.53) indicated coordinated allometric growth. Shell length showed strong positive correlations with body weight (r = 0.846) and shell width (r = 0.803). Proximate analysis revealed high moisture (56.8±0.42%) and protein content (23.4±0.36%), with low fat content (2.6±0.28%), indicating a lean protein-rich resource. Iron (24.40 mg/L) and calcium (20.30 mg/L) were the major minerals. GC-MS identified polyunsaturated fatty acids, including linoleic and arachidonic acid derivatives, while LC-MS acknowledged long-chain fatty acid methyl esters as the major lipid components. COI sequencing confirmed species identity with >99% similarity. The result establish F. bengalensis as a nutritionally valuable and biochemically important freshwater resource with potential functions in nutrition and food research.

INTRODUCTION

Freshwater gastropods are very important components of aquatic ecosystems and similarly serve as beneficial nutritional resources in many Asian countries. Among them, Filopaludina bengalensis is broadly circulated across India, Bangladesh and other neighbouring countries, where it is habitually consumed as food and occasionally used in folk medicinal practices (Atanasov et al., 2021; Mondol et al., 2022; Carroll et al., 2022).
       
Current interest in aquatic bioresources has improved because freshwater organisms cover several metabolites with possible health-promoting properties, containing polyunsaturated fatty acids, antioxidant compounds, amino acids and bioactive peptides (Kumar et al., 2023; Zhao et al., 2022; Zhao et al., 2022; Ahmed et al., 2022; Food and Agriculture Organization, 2020; Li et al., 2023). Freshwater gastropods have been considered as low-fat, protein-rich food sources with potential nutraceutical value (Cai et al., 2023; Singh et al., 2022; Kumar et al., 2023; Baby et al., 2010; Engmann et al., 2013; Weyh et al., 2022; Gupta and Khanal, 2024; Moses Samuel et al., 2026; Prajapat Singh et al., 2024; Gan et al., 2025).
       
Additionally, some reports have described the nutritional composition and antioxidant properties of Filopaludina bengalensis, comprehensive evidence integrating morphometric characteristics, fatty acid composition, amino acid profile, lipidomic composition, mineral content and molecular identification is still limited (Prasetyo et al., 2025; Chen et al., 2023; Prakash et al., 2024; Das et al., 2023; Das et al., 2023).
       
Therefore, the current study aimed to present a multi-analytical assessment of Filopaludina bengalensis. The study estimated morphometric relationships, proximate composition, mineral content, fatty acid and amino acid profiles, LC-MS-based lipid composition and COI gene sequencing for molecular evidence and therapeutic agents from F. bengalensis Fig 1.

Fig 1: Conceptual framework illustrating integrated morphometric, nutritional, lipidomic.

MATERIALS AND METHODS

Area of study and period of sampling
 
The study was conducted between June and September 2025 in the coastal area of Anugul, Odisha, India with sampling zone (~100 m2) in Fig 2 (Raut et al., 2024; Pattnaik and Mishra, 2025).

Fig 2: Sampling locations at Anugul river area, Odisha, India.


 
Experimental species
 
The gastropod Filopaludina bengalensis was selected for its high local abundance and socioeconomic value to Anugul local communities. Recent studies have highlighted the nutritional and bioactive value of coastal gastropods as sustainable sources for protein (Zhang et al., 2024).
 
Sample collection and transportation
 
Samples were collected using the standard ecological sampling (EcoSamp) procedure, a systematic method for collecting data on organisms (Ahmed et al., 2022). Thirty participants, each about three months old, were selected between 07:30 and 08:00. Samples were packed in clean, pre-labelled polyethylene bags and kept frozen in insulated iceboxes (Kumar et al., 2023).
 
Morphometric analysis and preparation of samples
 
All experiments were done under aseptic laboratory conditions. Specimens were rinsed in deionized water and disinfected with 70% ethanol. The following measurements were taken using a calibrated Vernier caliper (0.1 mm): shell length (SL, distance from the apex to base), shell width (SW, widest shell point), spire length (SPL, apex to start of aperture), base length (BL, base of shell), aperture length (AL, longest opening dimension) and aperture width (AW, widest point of opening). Body and shell weights were determined with an analytical balance (a precision scale accurate to 0.001 g).
       
Soft tissues were dried in an oven at 50°C for 48 hours to reach constant weight (Chen et al., 2023). The dried samples were ground into a fine powder and stored at 4°C for later analysis.
 
Mineral composition analysis
 
Mineral contents were determined using a ZEEnit 700P Atomic Absorption Spectrophotometer (Analytik Jena, Germany) in flame mode. About 1 g of dried tissue was digested with a nitric acid/perchloric acid mixture, following an AOAC-approved method (AOAC, 2024 edition).
 
LC-MS analysis
 
LC-MS analysis was executed to characterize bioactive lipid compounds in Filopaludina bengalensis. Lipid extracts were studied using a liquid chromatography mass spectrometry system under standard operating conditions. Chromatographic separation was constructed on compound polarity and retention time. Mass spectra were documented in positive ionization mode and metabolites were recognized by comparing molecular mass, fragmentation patterns and retention times with reference spectral databases.
 
Vitamin profile analysis
 
Vitamins were analyzed by High-performance liquid chromatography (HPLC). Lipid-soluble vitamins were extracted by saponification with ethanolic KOH and ascorbic acid and extracted with n-hexane, while water-soluble vitamins were evaporated; dried extracts were reconstituted in methanol. Water-soluble vitamins were extracted with 0.1 M HCl and 1% metaphosphoric acid. Reference substances were used for external calibration quantification mg/100 g. (fresh weight and dry matter).
 
Fatty acid composition analysis
 
Lipids were extracted using a conventional solvent extraction procedure. Fatty acid methyl esters (FAMEs) were generated by saponification with 0.5 M NaOH in methanol (80°C, 60 min) and methylation using methanolic BF3  (80°C, 20 min). Saturated NaCl was added, then FAMEs were extracted using iso-octane and dried with anhydrous sodium sulfate. Gas chromatographic perspectives followed current fatty acid profiling conventions for aquatic species (García-Vázquez  et al., 2024).
 
Molecular identification
 
Genomic DNA was prepared by the Quick-DNA™ Tissue Miniprep Kit (Zymo Research, USA). DNA was examined by 0.8% agarose gel electrophoresis and quantified using a NanoDrop spectrophotometer (A260/A280 ratio 1.8 2.0). The 25 μL PCR volume contained 10 pmol of 12 primers, 2.5 mM MgCl‚ , 200 μM dNTPs and 0.5 U Taq polymerase, with amplification of 50-100 ng of DNA template. The thermal cycling profile was as described for the optimized barcoding protocols for gastropod identification (Martínez  et al., 2024) and molecular identification.

RESULTS AND DISCUSSION

Morphological characters
 
The shell size distribution and proportional shell development were quantitatively analyzed for the collected F. bengalensis (n = 30). The average body weight of individuals was 6.84 g (median: 6.71±0.92 SD), indicating moderate biomass homogeneity within the sample population. The mean shell length (SL) of ponds was 3.51 cm (±0.50) and the mean shell width was 2.30 cm (±0.75). The estimated SL: SW ratio (~1.53) suggests a moderately globose, strongly compact shell form for the species. SPL = 1.68 cm (1.70±0.15), showing proportional orientation with the total length of the shell.
       
Aperture dimensions demonstrated a mean AL of 1.77 cm (1.70±0.19) and an AW of 1.51 cm (1.40±0.16), resulting in balanced aperture dilation. Body whorl length (BWL) mean 2.55 cm (2.50±0.19), penultimate whorl length (PWL) and width (PWW) 0.76 cm (0.70±0.07) and 1.70 cm (1.70±0.09) in Table 1. The relatively small standard deviations across all parameters suggest little morphological variation and constant growth patterns. Together, these results confirm coordinated ontogenetic growth and establish a strong biometric baseline for additional biochemical and molecular analyses.

Table 1: Descriptive statistics of morphometric parameters of Filopaludina bengalensis.


 
Correlation and linear regression analyses
 
Table 2 and Fig 3 present the shell length (SL) showed strong correlations with weight (r = 0.846), shell width (r = 0.803) and aperture length (r = 0.772), but weaker associations with apical width (r = 0.709), spire length (r = 0.537), BWL (r = 0.578), PWL (r = 0.304) and PWW (r = 0.225).

Table 2: Linear regression and correlation analysis between shell length (SL) and other morphometric parameters.



Fig 3: Relationships between different parameters by taking the logarithm of the parameters. SW, SL, SPL and AL.


 
Proximate composition
 
The proximate composition of F. bengalensis meat is shown in Fig 4, with mean ± standard deviation (n = 3). The moisture content was the major component (56.8±0.42%), confirming that it was high-water containing. The high moisture content indicates good taste and freshness. Crude protein was the second-largest fraction at 23.4±0.34%, indicating a very good quality as a protein source for this species. Its relatively high protein content also indicates a potential contribution to dietary protein intake in the region. The carbohydrate content of the adductor muscle was 17.2±0.51%, demonstrating moderate glycogen reserves characteristic of molluscan physiology. However, crude fat content was relatively low (2.6±0.28%), indicating that F. bengalensis can be categorized as a lean aquatic food. The ash content was low (1.2±0.14%), indicating mild mineral deposition. Collectively, this proximate profile suggests that F. bengalensis is a protein-rich freshwater animal source with a low-fat content and other nutritional benefits, making it suitable for the development of functional feed. 

​

Fig 4: Proximate composition of Filopaludina bengalensis expressed as mean ± SD (n = 3).


 
Mineral composition
 
F. bengalensis showed a diverse mineral profile, with Fe highest (24.40 mg/L; 25.7%), followed by Ca (20.30 mg/L; 21.4%), Mg (18.12 mg/L; 19.1%), Cu (16.13 mg/L; 17.0%), Zn (12.69 mg/L; 13.4%) and K lowest (3.20 mg/L; 3.4%). This profile indicates its potential nutritional value as a source of essential macro- and micronutrients in Fig 5.

​

Fig 5: Estimated mineral content of F. bengalensis snail samples.


 
Fatty acid profile (GC-MS analysis)
 
GC-MS analysis of FAMEs identified fatty acids using the NIST library, retention indices (RI), mass spectra and high REV scores. PUFAs predominated, including linoleic acid (C18:2), methyl 11,14-octadecadienoate (RI 31.933; MW 294 g/mol), methyl 11,12-octadecadienoate (MW 294), methyl 6,9,12,15-hexadecatetraenoate (MW 262), arachidonic acid (C20:4; MW 304) and methyl eicosa-5,8,11,14,17-pentaenoate (MW 316). A MUFA derivative, 2-trimethylsiloxy-6-hexadecenoic acid methyl ester (MW 356), was also detected, indicating potential nutritional value in  Fig 6.

​

Fig 6: Relative identification strength of fatty acid methyl esters.


 
Amino acid composition
 
The amino acid profile of F. bengalensis (dry-weight basis) is shown in Fig 7. Both were essential and non-essential amino acids, showing a broad spectrum of diversity in the species’ amino acid profile towards its nutritional value. Hydroxyproline was the most abundant non-essential amino acid (6.94 mg/100 g), followed by taurine (2.94 mg/100 g) and phosphoserine (2.40 mg/100 g). The high level of hydroxyproline indicates a significant amount of collagen-associated structural proteins in the tissue matrix. The biomedically important non-protein amino acid taurine was detected at appreciable levels in plasma, suggesting physiological roles in osmoregulation and antioxidant protection. Moderate concentrations of (0.87 mg/100 g) β-aminoisobutyric acid and (0.37 mg/100 g) β-alanine were also observed. In contrast, the contents of alanine (0.13 mg/100 g), tyrosine (0.11 mg/100 g), aspartic acid (0.09 mg/100 g) and hydroxylysine (0.06 mg/100 g) were reported to be lower.

​

Fig 7: (a) Essential amino acid composition of the studied freshwater species. Concentrations are expressed as mg per 100 g dry weight equivalent. (b) Non-essential amino acid composition of the studied freshwater species.


       
The highest concentration of essential amino acids was detected for phenylalanine (0.37 mg/100 g), followed by alloisoleucine (0.08 mg/100 g) and lysine and valine (both 0.04 mg/100 g). Both essential amino acids were detected at lower levels than other non-essential amino acids, yet their presence reflects the nutritional importance of the species as dietary protein sources.
 
LC-MS profile and relative abundance of bioactive compounds
 
LC-MS chromatographic analysis of the F. bengalensis extract showed a unique distribution of major metabolites compared to those summarized in Table 3. A total of five significant peaks were observed in the retention time (RT) range of 1.812-23.607 min, suggesting structurally distinct compounds with varying polarities and molecular weights.

Table 3: LC-MS detected compounds and relative abundance.


       
LC-MS analysis revealed a trace unidentified peak at RT 1.812 min (6.07%). The major compound was heptacosanoic acid, 25-methyl-, methyl ester (MW 438 Da), at RT 12.592 min (50.61%). 12-Methyltridecanoic acid methyl ester (MW 242 Da) occurred at RT 11.524 min (26.80%), while oleic acid methyl ester (MW 282 Da) appeared at RT 23.607 min (14.29%). Methyl 11-methyl-dodecanoate (MW 228 Da) was detected at RT 13.248 min (2.23%), indicating abundant long- and branched-chain lipid derivatives with nutritional potential.
 
Molecular identification by COI gene sequencing
 
The analyzed specimen was molecularly identified by sequencing of the mitochondrial cytochrome c oxidase subunit I (COI) gene. The species identity of the F. bengalensis sample was confirmed by a BLASTn query of the obtained COI sequence against NCBI GenBank. The top five matches are listed and the sequence similarity profile (with background) is shown in Table 4.

Table 4: Top BLASTn matches for COI sequence (Sample FB).


       
The BLASTn outputs of all top-ranked matches with 100% query coverage confirm full-length alignment of the amplified COI fragment. It shared the highest sequence identity with Filopaludina bengalensis [accession number PP971158 (99.5%)], followed by two other F. bengalensis sequences, PP971058 (99.1%) and MN024130 (98.7%). Both of the top three hits had very significant E-values (0.0), suggesting an insignificant probability of random similarity and validating species-level assignment.
       
Bar graph showing percentage identity between the query COI sequence of F. bengalensis (Sample FB) and the top GenBank reference sequences. All top-ranked matches exhibit ³98% sequence identity and complete query coverage (100%), confirming species-level assignment. A higher value of 94.2% between F. bengalensis (MN793731) and F. sumatrensis (MN793744) indicates interspecific divergence within the genus, as observed in this dataset. Such a widespread discrepancy from closely related congeners also confirms that reliable species boundaries are correct within the family Viviparidae.
       
The morphometric analysis of F. bengalensis indicated allometric shell growth and moderate size homogeneity, suggesting a stable growth environment at the Anugul, Odisha habitat. The paleo-shell is moderately globose (SL: SW ~ 1.53),  like that of viviparid gastropods. Similar morphometric stasis has been documented in freshwater gastropods living under good environmental conditions, where increases in shell size are strongly correlated with biomass accumulation (Ahmed et al., 2022; Strong et al., 2021). The high correlation between shell length and body weight (r = 0.846) indicated a synchronized somatic and shell growth, as in other edible gastropods, where shell measurements are powerful predictors of biomass (Kumari et al., 2022; Butboonchoo et al., 2020).
       
The weaker relationships with penultimate whorl parameters suggest that morphological variation is more local, perhaps influenced by microhabitat variables such as calcium and hydrodynamic loading. Similarly, allometric divergence has been reported for freshwater molluscs (Rao et al., 2023; Chakraborty et al., 2024; Rout et al., 2023).
       
The proximate profile showed high moisture and high-protein fraction confirms the categorization of F. bengalensis as a lean, high-protein aquatic food resource (Chakraborty et al., 2024; Rout et al., 2023).
       
The relatively low carbohydrate content (17.2%) is probably due to glycogen stores found in other gastropods. Low ash content indicates appropriate mineralization and not the duplication of inorganic material. Overall, the proximate findings suggest that freshwater snails are an important source of protein, which is highly underutilized (Li et al., 2023). Iron was the most abundant mineral (24.40 mg/L); calcium and magnesium followed with previous research on mollusc tissues, where iron accumulation was attributed to hemocyanin-based oxygen transport and environmental bioavailability (Zhang et al., 2024).
       
The presence of essential trace elements zinc and copper also confirms the potential micronutrient importance of F. bengalensis. Similar mineral profiles have been found in edible freshwater snails of Southeast Asia (Rahman et al., 2024), indicating that viviparid species contribute significantly to the dietary supply of minerals.
       
GC-MS analysis showed an abundance of polyunsaturated fatty acids (PUFAs), including derivatives of linoleic and arachidonic acids, in addition to monounsaturated fatty acids (MUFAs). Comparable profiles of PUFA-dominant freshwater molluscs are also reported, in which omega-3 and omega-6 fatty acids have anti-inflammatory and cardioprotective effects (Silva et al., 2022). The discovery of EPA derivatives adds nutritional interest to this species.
       
LC-MS profiling confirmed high lipid content with long-chain fatty acid methyl esters, higher (more than 50% peak area). These lipid signatures often reflect bioactive and nutrient compounds of interest in aquatic animals (Chen et al., 2023; Chakraborty et al., 2024 ; Rout et al., 2023).
       
Hydroxyproline and taurine were the most abundant non-essential amino acids, suggesting substantial structural and Osmo protective roles similarly compare with Kumar et al., (2016) for freshwater species (Kumar et al., 2023). Even small amounts of essential amino acids, such as phenylalanine, lysine,  and valine similar amino acid distributions have been reported in edible gastropods with functional food potential (Nguyen et al., 2023, Roy et al., 2023).
       
Molecular identification based on COI resulted in sequence identity exceeding 98% with several GenBank accessions of F. bengalensis, consistent with species-level cutoffs recommended for DNA barcoding (Hebert et al., 2021). The divergent relationship with F. sumatrensis implies a clear genetic separation within Viviparidae, consistent with the taxonomic assignment (Prasetyo et al., 2025).
       
Sustainable utilization of Filopaludina bengalensis should study both ecological conservation and ethical harvesting practices. Therefore, controlled harvesting, habitat safety and seasonal monitoring are mandatory to maintain ecological balance and uphold long-term sustainability.

CONCLUSION

The present study specifies integrated morphometric, nutritional, lipidomic and molecular data for Filopaludina bengalensis. The species exhibited stable morphometric relationships, high protein content, appreciable mineral levels with several fatty acid and amino acid components. COI gene sequencing proved species identity with high sequence similarity to available GenBank records. Overall, the results indicate that Filopaludina bengalensis is a nutritionally valuable freshwater resource with potential relevance for food and aquaculture applications. 

ACKNOWLEDGEMENT

The authors express their sincere gratitude to the Head of the Department of Zoology for valuable guidance and institutional support throughout this work. We also acknowledge Newredmars Skill and Research Foundation for providing academic support and technical assistance that significantly contributed to the successful completion of this study.
 
Authors contribution
 
All authors contributed to the study conception and design. data collection and lab experiments and study design and analysis were performed by Reema Das. Assistance in analysis and interpretation of results was obtained by Dr. Yashaswi Nayak and Prof. (Dr) Jnanendra Kumar Sahoo.
 
Data availability
 
Data are available from the corresponding author upon reasonable request.

Disclaimers
 
The authors are solely responsible for the content, accuracy, conclusions and opinions expressed in this article.
 
Informed consent
 
All animal procedures followed institutional ethical guidelines and were approved by CUTM’s University Animal Care and Ethics Committee.
 

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Nutritional Composition, Fatty Acid Signatures and DNA Barcoding of Filopaludina bengalensis: A Multi-analytical Assessment of an Underutilized Freshwater Resource

    R
    Reema Das1
    Y
    Yashaswi Nayak1,*
    0009-0002-6999-1861
    J
    Jnanendra Kumar Sahoo2
    1Department of Zoology, Applied Sciences, Jatni, Centurion University of Technology and Management (CUTM), Bhubaneswar campus-752 050, Odisha, India.
    2School of Fisheries, Centurion University of Technology and Management (CUTM), Paralakhemundi campus, Gajapati-761 211, Odisha, India.

    ABSTRACT

    Background: Filopaludina bengalensis is a freshwater gastropod, one of the foremost aquatic resources with nutritional and biochemical significance.

    Methods: Thirty specimens were evaluated for morphometric parameters, including body weight, shell length and shell width. Proximate composition and mineral analyses were done to verify nutritional quality. Fatty acid composition was characterized using GC-MS and LC-MS techniques, while amino acid profiling identified major amino acid constituents. Molecular identification was confirmed through COI (cytochrome c oxidase subunit I) gene sequencing.

    Result: The mean body weight and shell length were 6.84±0.92 g and 3.51±0.17 cm, respectively. A stable shell length to shell width ratio (~1.53) indicated coordinated allometric growth. Shell length showed strong positive correlations with body weight (r = 0.846) and shell width (r = 0.803). Proximate analysis revealed high moisture (56.8±0.42%) and protein content (23.4±0.36%), with low fat content (2.6±0.28%), indicating a lean protein-rich resource. Iron (24.40 mg/L) and calcium (20.30 mg/L) were the major minerals. GC-MS identified polyunsaturated fatty acids, including linoleic and arachidonic acid derivatives, while LC-MS acknowledged long-chain fatty acid methyl esters as the major lipid components. COI sequencing confirmed species identity with >99% similarity. The result establish F. bengalensis as a nutritionally valuable and biochemically important freshwater resource with potential functions in nutrition and food research.

    INTRODUCTION

    Freshwater gastropods are very important components of aquatic ecosystems and similarly serve as beneficial nutritional resources in many Asian countries. Among them, Filopaludina bengalensis is broadly circulated across India, Bangladesh and other neighbouring countries, where it is habitually consumed as food and occasionally used in folk medicinal practices (Atanasov et al., 2021; Mondol et al., 2022; Carroll et al., 2022).
           
    Current interest in aquatic bioresources has improved because freshwater organisms cover several metabolites with possible health-promoting properties, containing polyunsaturated fatty acids, antioxidant compounds, amino acids and bioactive peptides (Kumar et al., 2023; Zhao et al., 2022; Zhao et al., 2022; Ahmed et al., 2022; Food and Agriculture Organization, 2020; Li et al., 2023). Freshwater gastropods have been considered as low-fat, protein-rich food sources with potential nutraceutical value (Cai et al., 2023; Singh et al., 2022; Kumar et al., 2023; Baby et al., 2010; Engmann et al., 2013; Weyh et al., 2022; Gupta and Khanal, 2024; Moses Samuel et al., 2026; Prajapat Singh et al., 2024; Gan et al., 2025).
           
    Additionally, some reports have described the nutritional composition and antioxidant properties of Filopaludina bengalensis, comprehensive evidence integrating morphometric characteristics, fatty acid composition, amino acid profile, lipidomic composition, mineral content and molecular identification is still limited (Prasetyo et al., 2025; Chen et al., 2023; Prakash et al., 2024; Das et al., 2023; Das et al., 2023).
           
    Therefore, the current study aimed to present a multi-analytical assessment of Filopaludina bengalensis. The study estimated morphometric relationships, proximate composition, mineral content, fatty acid and amino acid profiles, LC-MS-based lipid composition and COI gene sequencing for molecular evidence and therapeutic agents from F. bengalensis Fig 1.

    Fig 1: Conceptual framework illustrating integrated morphometric, nutritional, lipidomic.

    MATERIALS AND METHODS

    Area of study and period of sampling
     
    The study was conducted between June and September 2025 in the coastal area of Anugul, Odisha, India with sampling zone (~100 m2) in Fig 2 (Raut et al., 2024; Pattnaik and Mishra, 2025).

    Fig 2: Sampling locations at Anugul river area, Odisha, India.


     
    Experimental species
     
    The gastropod Filopaludina bengalensis was selected for its high local abundance and socioeconomic value to Anugul local communities. Recent studies have highlighted the nutritional and bioactive value of coastal gastropods as sustainable sources for protein (Zhang et al., 2024).
     
    Sample collection and transportation
     
    Samples were collected using the standard ecological sampling (EcoSamp) procedure, a systematic method for collecting data on organisms (Ahmed et al., 2022). Thirty participants, each about three months old, were selected between 07:30 and 08:00. Samples were packed in clean, pre-labelled polyethylene bags and kept frozen in insulated iceboxes (Kumar et al., 2023).
     
    Morphometric analysis and preparation of samples
     
    All experiments were done under aseptic laboratory conditions. Specimens were rinsed in deionized water and disinfected with 70% ethanol. The following measurements were taken using a calibrated Vernier caliper (0.1 mm): shell length (SL, distance from the apex to base), shell width (SW, widest shell point), spire length (SPL, apex to start of aperture), base length (BL, base of shell), aperture length (AL, longest opening dimension) and aperture width (AW, widest point of opening). Body and shell weights were determined with an analytical balance (a precision scale accurate to 0.001 g).
           
    Soft tissues were dried in an oven at 50°C for 48 hours to reach constant weight (Chen et al., 2023). The dried samples were ground into a fine powder and stored at 4°C for later analysis.
     
    Mineral composition analysis
     
    Mineral contents were determined using a ZEEnit 700P Atomic Absorption Spectrophotometer (Analytik Jena, Germany) in flame mode. About 1 g of dried tissue was digested with a nitric acid/perchloric acid mixture, following an AOAC-approved method (AOAC, 2024 edition).
     
    LC-MS analysis
     
    LC-MS analysis was executed to characterize bioactive lipid compounds in Filopaludina bengalensis. Lipid extracts were studied using a liquid chromatography mass spectrometry system under standard operating conditions. Chromatographic separation was constructed on compound polarity and retention time. Mass spectra were documented in positive ionization mode and metabolites were recognized by comparing molecular mass, fragmentation patterns and retention times with reference spectral databases.
     
    Vitamin profile analysis
     
    Vitamins were analyzed by High-performance liquid chromatography (HPLC). Lipid-soluble vitamins were extracted by saponification with ethanolic KOH and ascorbic acid and extracted with n-hexane, while water-soluble vitamins were evaporated; dried extracts were reconstituted in methanol. Water-soluble vitamins were extracted with 0.1 M HCl and 1% metaphosphoric acid. Reference substances were used for external calibration quantification mg/100 g. (fresh weight and dry matter).
     
    Fatty acid composition analysis
     
    Lipids were extracted using a conventional solvent extraction procedure. Fatty acid methyl esters (FAMEs) were generated by saponification with 0.5 M NaOH in methanol (80°C, 60 min) and methylation using methanolic BF3  (80°C, 20 min). Saturated NaCl was added, then FAMEs were extracted using iso-octane and dried with anhydrous sodium sulfate. Gas chromatographic perspectives followed current fatty acid profiling conventions for aquatic species (García-Vázquez  et al., 2024).
     
    Molecular identification
     
    Genomic DNA was prepared by the Quick-DNA™ Tissue Miniprep Kit (Zymo Research, USA). DNA was examined by 0.8% agarose gel electrophoresis and quantified using a NanoDrop spectrophotometer (A260/A280 ratio 1.8 2.0). The 25 μL PCR volume contained 10 pmol of 12 primers, 2.5 mM MgCl‚ , 200 μM dNTPs and 0.5 U Taq polymerase, with amplification of 50-100 ng of DNA template. The thermal cycling profile was as described for the optimized barcoding protocols for gastropod identification (Martínez  et al., 2024) and molecular identification.

    RESULTS AND DISCUSSION

    Morphological characters
     
    The shell size distribution and proportional shell development were quantitatively analyzed for the collected F. bengalensis (n = 30). The average body weight of individuals was 6.84 g (median: 6.71±0.92 SD), indicating moderate biomass homogeneity within the sample population. The mean shell length (SL) of ponds was 3.51 cm (±0.50) and the mean shell width was 2.30 cm (±0.75). The estimated SL: SW ratio (~1.53) suggests a moderately globose, strongly compact shell form for the species. SPL = 1.68 cm (1.70±0.15), showing proportional orientation with the total length of the shell.
           
    Aperture dimensions demonstrated a mean AL of 1.77 cm (1.70±0.19) and an AW of 1.51 cm (1.40±0.16), resulting in balanced aperture dilation. Body whorl length (BWL) mean 2.55 cm (2.50±0.19), penultimate whorl length (PWL) and width (PWW) 0.76 cm (0.70±0.07) and 1.70 cm (1.70±0.09) in Table 1. The relatively small standard deviations across all parameters suggest little morphological variation and constant growth patterns. Together, these results confirm coordinated ontogenetic growth and establish a strong biometric baseline for additional biochemical and molecular analyses.

    Table 1: Descriptive statistics of morphometric parameters of Filopaludina bengalensis.


     
    Correlation and linear regression analyses
     
    Table 2 and Fig 3 present the shell length (SL) showed strong correlations with weight (r = 0.846), shell width (r = 0.803) and aperture length (r = 0.772), but weaker associations with apical width (r = 0.709), spire length (r = 0.537), BWL (r = 0.578), PWL (r = 0.304) and PWW (r = 0.225).

    Table 2: Linear regression and correlation analysis between shell length (SL) and other morphometric parameters.



    Fig 3: Relationships between different parameters by taking the logarithm of the parameters. SW, SL, SPL and AL.


     
    Proximate composition
     
    The proximate composition of F. bengalensis meat is shown in Fig 4, with mean ± standard deviation (n = 3). The moisture content was the major component (56.8±0.42%), confirming that it was high-water containing. The high moisture content indicates good taste and freshness. Crude protein was the second-largest fraction at 23.4±0.34%, indicating a very good quality as a protein source for this species. Its relatively high protein content also indicates a potential contribution to dietary protein intake in the region. The carbohydrate content of the adductor muscle was 17.2±0.51%, demonstrating moderate glycogen reserves characteristic of molluscan physiology. However, crude fat content was relatively low (2.6±0.28%), indicating that F. bengalensis can be categorized as a lean aquatic food. The ash content was low (1.2±0.14%), indicating mild mineral deposition. Collectively, this proximate profile suggests that F. bengalensis is a protein-rich freshwater animal source with a low-fat content and other nutritional benefits, making it suitable for the development of functional feed. 

    ​

    Fig 4: Proximate composition of Filopaludina bengalensis expressed as mean ± SD (n = 3).


     
    Mineral composition
     
    F. bengalensis showed a diverse mineral profile, with Fe highest (24.40 mg/L; 25.7%), followed by Ca (20.30 mg/L; 21.4%), Mg (18.12 mg/L; 19.1%), Cu (16.13 mg/L; 17.0%), Zn (12.69 mg/L; 13.4%) and K lowest (3.20 mg/L; 3.4%). This profile indicates its potential nutritional value as a source of essential macro- and micronutrients in Fig 5.

    ​

    Fig 5: Estimated mineral content of F. bengalensis snail samples.


     
    Fatty acid profile (GC-MS analysis)
     
    GC-MS analysis of FAMEs identified fatty acids using the NIST library, retention indices (RI), mass spectra and high REV scores. PUFAs predominated, including linoleic acid (C18:2), methyl 11,14-octadecadienoate (RI 31.933; MW 294 g/mol), methyl 11,12-octadecadienoate (MW 294), methyl 6,9,12,15-hexadecatetraenoate (MW 262), arachidonic acid (C20:4; MW 304) and methyl eicosa-5,8,11,14,17-pentaenoate (MW 316). A MUFA derivative, 2-trimethylsiloxy-6-hexadecenoic acid methyl ester (MW 356), was also detected, indicating potential nutritional value in  Fig 6.

    ​

    Fig 6: Relative identification strength of fatty acid methyl esters.


     
    Amino acid composition
     
    The amino acid profile of F. bengalensis (dry-weight basis) is shown in Fig 7. Both were essential and non-essential amino acids, showing a broad spectrum of diversity in the species’ amino acid profile towards its nutritional value. Hydroxyproline was the most abundant non-essential amino acid (6.94 mg/100 g), followed by taurine (2.94 mg/100 g) and phosphoserine (2.40 mg/100 g). The high level of hydroxyproline indicates a significant amount of collagen-associated structural proteins in the tissue matrix. The biomedically important non-protein amino acid taurine was detected at appreciable levels in plasma, suggesting physiological roles in osmoregulation and antioxidant protection. Moderate concentrations of (0.87 mg/100 g) β-aminoisobutyric acid and (0.37 mg/100 g) β-alanine were also observed. In contrast, the contents of alanine (0.13 mg/100 g), tyrosine (0.11 mg/100 g), aspartic acid (0.09 mg/100 g) and hydroxylysine (0.06 mg/100 g) were reported to be lower.

    ​

    Fig 7: (a) Essential amino acid composition of the studied freshwater species. Concentrations are expressed as mg per 100 g dry weight equivalent. (b) Non-essential amino acid composition of the studied freshwater species.


           
    The highest concentration of essential amino acids was detected for phenylalanine (0.37 mg/100 g), followed by alloisoleucine (0.08 mg/100 g) and lysine and valine (both 0.04 mg/100 g). Both essential amino acids were detected at lower levels than other non-essential amino acids, yet their presence reflects the nutritional importance of the species as dietary protein sources.
     
    LC-MS profile and relative abundance of bioactive compounds
     
    LC-MS chromatographic analysis of the F. bengalensis extract showed a unique distribution of major metabolites compared to those summarized in Table 3. A total of five significant peaks were observed in the retention time (RT) range of 1.812-23.607 min, suggesting structurally distinct compounds with varying polarities and molecular weights.

    Table 3: LC-MS detected compounds and relative abundance.


           
    LC-MS analysis revealed a trace unidentified peak at RT 1.812 min (6.07%). The major compound was heptacosanoic acid, 25-methyl-, methyl ester (MW 438 Da), at RT 12.592 min (50.61%). 12-Methyltridecanoic acid methyl ester (MW 242 Da) occurred at RT 11.524 min (26.80%), while oleic acid methyl ester (MW 282 Da) appeared at RT 23.607 min (14.29%). Methyl 11-methyl-dodecanoate (MW 228 Da) was detected at RT 13.248 min (2.23%), indicating abundant long- and branched-chain lipid derivatives with nutritional potential.
     
    Molecular identification by COI gene sequencing
     
    The analyzed specimen was molecularly identified by sequencing of the mitochondrial cytochrome c oxidase subunit I (COI) gene. The species identity of the F. bengalensis sample was confirmed by a BLASTn query of the obtained COI sequence against NCBI GenBank. The top five matches are listed and the sequence similarity profile (with background) is shown in Table 4.

    Table 4: Top BLASTn matches for COI sequence (Sample FB).


           
    The BLASTn outputs of all top-ranked matches with 100% query coverage confirm full-length alignment of the amplified COI fragment. It shared the highest sequence identity with Filopaludina bengalensis [accession number PP971158 (99.5%)], followed by two other F. bengalensis sequences, PP971058 (99.1%) and MN024130 (98.7%). Both of the top three hits had very significant E-values (0.0), suggesting an insignificant probability of random similarity and validating species-level assignment.
           
    Bar graph showing percentage identity between the query COI sequence of F. bengalensis (Sample FB) and the top GenBank reference sequences. All top-ranked matches exhibit ³98% sequence identity and complete query coverage (100%), confirming species-level assignment. A higher value of 94.2% between F. bengalensis (MN793731) and F. sumatrensis (MN793744) indicates interspecific divergence within the genus, as observed in this dataset. Such a widespread discrepancy from closely related congeners also confirms that reliable species boundaries are correct within the family Viviparidae.
           
    The morphometric analysis of F. bengalensis indicated allometric shell growth and moderate size homogeneity, suggesting a stable growth environment at the Anugul, Odisha habitat. The paleo-shell is moderately globose (SL: SW ~ 1.53),  like that of viviparid gastropods. Similar morphometric stasis has been documented in freshwater gastropods living under good environmental conditions, where increases in shell size are strongly correlated with biomass accumulation (Ahmed et al., 2022; Strong et al., 2021). The high correlation between shell length and body weight (r = 0.846) indicated a synchronized somatic and shell growth, as in other edible gastropods, where shell measurements are powerful predictors of biomass (Kumari et al., 2022; Butboonchoo et al., 2020).
           
    The weaker relationships with penultimate whorl parameters suggest that morphological variation is more local, perhaps influenced by microhabitat variables such as calcium and hydrodynamic loading. Similarly, allometric divergence has been reported for freshwater molluscs (Rao et al., 2023; Chakraborty et al., 2024; Rout et al., 2023).
           
    The proximate profile showed high moisture and high-protein fraction confirms the categorization of F. bengalensis as a lean, high-protein aquatic food resource (Chakraborty et al., 2024; Rout et al., 2023).
           
    The relatively low carbohydrate content (17.2%) is probably due to glycogen stores found in other gastropods. Low ash content indicates appropriate mineralization and not the duplication of inorganic material. Overall, the proximate findings suggest that freshwater snails are an important source of protein, which is highly underutilized (Li et al., 2023). Iron was the most abundant mineral (24.40 mg/L); calcium and magnesium followed with previous research on mollusc tissues, where iron accumulation was attributed to hemocyanin-based oxygen transport and environmental bioavailability (Zhang et al., 2024).
           
    The presence of essential trace elements zinc and copper also confirms the potential micronutrient importance of F. bengalensis. Similar mineral profiles have been found in edible freshwater snails of Southeast Asia (Rahman et al., 2024), indicating that viviparid species contribute significantly to the dietary supply of minerals.
           
    GC-MS analysis showed an abundance of polyunsaturated fatty acids (PUFAs), including derivatives of linoleic and arachidonic acids, in addition to monounsaturated fatty acids (MUFAs). Comparable profiles of PUFA-dominant freshwater molluscs are also reported, in which omega-3 and omega-6 fatty acids have anti-inflammatory and cardioprotective effects (Silva et al., 2022). The discovery of EPA derivatives adds nutritional interest to this species.
           
    LC-MS profiling confirmed high lipid content with long-chain fatty acid methyl esters, higher (more than 50% peak area). These lipid signatures often reflect bioactive and nutrient compounds of interest in aquatic animals (Chen et al., 2023; Chakraborty et al., 2024 ; Rout et al., 2023).
           
    Hydroxyproline and taurine were the most abundant non-essential amino acids, suggesting substantial structural and Osmo protective roles similarly compare with Kumar et al., (2016) for freshwater species (Kumar et al., 2023). Even small amounts of essential amino acids, such as phenylalanine, lysine,  and valine similar amino acid distributions have been reported in edible gastropods with functional food potential (Nguyen et al., 2023, Roy et al., 2023).
           
    Molecular identification based on COI resulted in sequence identity exceeding 98% with several GenBank accessions of F. bengalensis, consistent with species-level cutoffs recommended for DNA barcoding (Hebert et al., 2021). The divergent relationship with F. sumatrensis implies a clear genetic separation within Viviparidae, consistent with the taxonomic assignment (Prasetyo et al., 2025).
           
    Sustainable utilization of Filopaludina bengalensis should study both ecological conservation and ethical harvesting practices. Therefore, controlled harvesting, habitat safety and seasonal monitoring are mandatory to maintain ecological balance and uphold long-term sustainability.

    CONCLUSION

    The present study specifies integrated morphometric, nutritional, lipidomic and molecular data for Filopaludina bengalensis. The species exhibited stable morphometric relationships, high protein content, appreciable mineral levels with several fatty acid and amino acid components. COI gene sequencing proved species identity with high sequence similarity to available GenBank records. Overall, the results indicate that Filopaludina bengalensis is a nutritionally valuable freshwater resource with potential relevance for food and aquaculture applications. 

    ACKNOWLEDGEMENT

    The authors express their sincere gratitude to the Head of the Department of Zoology for valuable guidance and institutional support throughout this work. We also acknowledge Newredmars Skill and Research Foundation for providing academic support and technical assistance that significantly contributed to the successful completion of this study.
     
    Authors contribution
     
    All authors contributed to the study conception and design. data collection and lab experiments and study design and analysis were performed by Reema Das. Assistance in analysis and interpretation of results was obtained by Dr. Yashaswi Nayak and Prof. (Dr) Jnanendra Kumar Sahoo.
     
    Data availability
     
    Data are available from the corresponding author upon reasonable request.

    Disclaimers
     
    The authors are solely responsible for the content, accuracy, conclusions and opinions expressed in this article.
     
    Informed consent
     
    All animal procedures followed institutional ethical guidelines and were approved by CUTM’s University Animal Care and Ethics Committee.
     

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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