Issue 2 (202)/2025

ALSE and ACS Style
Bhuyan, Md.S.; Islam, Md.T.; Mondal, V. Preliminary study of the amino acids of horseshoe crabs (Carcinoscorpius rotundicauda) from the Cox’s Bazar Coast, Bay of Bengal, Bangladesh. Journal of Applied Life Sciences and Environment 2025, 58 (2), 333-353.
https://doi.org/10.46909/alse-582179

AMA Style
Bhuyan MdS, Islam MdT, Mondal V. Preliminary study of the amino acids of horseshoe crabs (Carcinoscorpius rotundicauda) from the Cox’s Bazar Coast, Bay of Bengal, Bangladesh. Journal of Applied Life Sciences and Environment. 2025; 58 (2): 333-353.
https://doi.org/10.46909/alse-582179

Chicago/Turabian Style
Bhuyan, Md. Simul, Md. Tarikul Islam, and Vinmoy Mondal. 2025. “Preliminary study of the amino acids of horseshoe crabs (Carcinoscorpius rotundicauda) from the Cox’s Bazar Coast, Bay of Bengal, Bangladesh.” Journal of Applied Life Sciences and Environment 58, no. 2: 333-353.
https://doi.org/10.46909/alse-582179

Preliminary study of the amino acids of horseshoe crabs (Carcinoscorpius rotundicauda) from the Cox’s Bazar Coast, Bay of Bengal, Bangladesh

Md. Simul BHUYAN^1,2*, Md. Tarikul ISLAM¹ and Vinmoy MONDAL³

¹Bangladesh Oceanographic Research Institute, Cox’s Bazar 4730, Bangladesh; email: taru@bori.gov.bd

²Department of Aquatic Resource Management, Sylhet Agricultural University, Sylhet 3100, Bangladesh

³Department of Marine Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu, 620024, India; email: vinmoymondal23@gmail.com

*Correspondence: simulbhuyan@gmail.com 

Received: Apr. 28, 2025. Revised: Jun. 14, 2025. Accepted: Jul. 02, 2025. Published online: Aug. 08, 2025

ABSTRACT. Carcinoscorpius rotundicauda is an ancient marine arthropod with significant ecological and biomedical importance due to its unique physiological features and bioactive compounds. Despite this critical role in pharmaceutical applications, especially in endotoxin detection assays, the amino acid (AA) profile of C. rotundicauda remains underexplored, especially the population in the northern Bay of Bengal. This study investigated the AA profile of C. rotundicauda collected from the Cox’s Bazar coast of Bangladesh, with the goal of assessing its nutritional value and biomedical potential. A preliminary biochemical analysis was conducted using samples collected from a mangrove-dominated estuarine zone. A rigorous analytical protocol involving hydrolysis, filtration, and liquid chromatography–tandem mass spectrometry was employed to accurately quantify the essential and non-essential amino acids. The results revealed a total AA content of 2.2%, with leucine (0.5%), isoleucine (0.3%), and proline (0.3%) being the most abundant. Interestingly, aspartic acid was not detected, and trace levels of methionine (0.006%) and histidine (0.0006%) were observed, suggesting unique metabolic adaptations in this species compared to other marine arthropods. These findings are significant as they represent the first detailed account of the AA profile of C. rotundicauda from Bangladesh’s coastal waters. The dominance of branched-chain amino acids such as leucine and isoleucine highlights the species’ potential role in supporting protein synthesis and metabolic regulation. This biochemical insight opens new possibilities for the species’ application in the nutraceutical and pharmaceutical industries, while also underlining the need for its conservation amid increasing habitat degradation.

Keywords: amino acid profiling; biomedical applications; Carcinoscorpius rotundicauda; nutritional composition.

 

INTRODUCTION

Horseshoe crabs (HSCs) date before the dinosaurs and have been in existence for at least 480 million years without exhibiting any apparent morphological modifications (Sarmiento et al., 2022). They are aquatic arthropods belonging to the family Limulidae in the order Xiphosura (Ashrafuzzaman et al., 2022; Baek et al., 2014; Xu et al., 2020). HSCs comprise four extant species: Limulus polyphemus in the Americas and the Gulf of Mexico, and Tachypleus tridentatus, Tachypleus gigas, and Carcinoscorpius rotundicauda in Asia (Bowden et al., 2020; Sarmiento et al., 2022). HSCs are mostly harvested for application in pharmaceutical and biological applications because of their unique blue hemolymph (Maloney et al., 2018). Throughout the past 40 years, HSCs have become essential to the safe production of vaccines and injectable drugs (Maloney et al., 2018). Additionally, the health of communities has been threatened by the emergence of infections resistant to numerous antibiotics, and HSCs may be a possible source of antibacterial peptides (Ashrafuzzaman et al., 2022; Wang et al., 2022).

C. rotundicauda, known as the mangrove HSCs, occurs in southern and southeastern Asia, where reclamation of territory and development of coastal areas have resulted in significant destruction of biodiversity (Ding et al., 2005; Lee and Morton, 2005; Mishra et al., 2009; Ng et al., 2007). It is found in the northern region of the Bay of Bengal (Cartwright and Taylor, 2009, 2011; Yennawar, 2015). The innate immune system of rotundicauda uses coagulogen, an important protein found in hemolymph, as a primary defence mechanism. It’s precisely ordered amino acid (AA) sequence causes rapid blood coagulation upon pathogenic invasion (Ashrafuzzaman et al., 2022; Reeds et al., 2000). Numerous studies have been conducted on this special coagulation process for its biological uses, especially in assays for the detection of bacterial endotoxins (Armstrong et al., 2013).

AAs are the essential components of life and are required for metabolism, protein synthesis, and cellular signaling. They are divided into two categories: essential, which must be acquired through diet, and non-essential, which the body can generate (Brosnan et al., 2000; Wu et al., 2013). Almost all protein functions are supported by their structural variety, which affects immunological responses, enzymatic activity, and general physiological balance (Brosnan et al., 2000; Reeds et al., 2000). In addition, AAs are essential for hormone synthesis, neurotransmission, and energy metabolism, which makes them necessary for life support and good health (Wu et al., 2013).

Another essential protein in C. rotundicauda, hemocyanin, binds and transports oxygen throughout the organism based on its exact AA sequence. Even in hypoxic conditions, which are frequently found in intertidal habitats, this ensures effective metabolism and energy generation (Wu et al., 2013). Moreover, short chains of AAs called tachyplesins, which are antimicrobial peptides, have strong activity against a variety of diseases, supporting the crab’s strong immune system. The AA content of these peptides optimises their structure, making them essential for avoiding microbial infections (Miyata et al., 1989; Reeds et al., 2000). AA metabolism is closely related to tissue repair and stress reactions, indicating its function in preserving physiological homeostasis (Wu et al., 2013). The comprehensive characterisation of protein molecules derived from AAs has revealed the potential for biomedical applications, including the synthesis of novel antimicrobial substances and a better comprehension of blood coagulation processes, by offering important insights into the mechanisms of coagulation, oxygen transport, and innate immunity (Planeta et al., 2021).

The study of AAs in HSCs has focused on hemolymph proteomics. Investigators determined the complete AA sequence of coagulogen from C. rotundicauda to elucidate its structure, stability, and role in the clotting cascade (Srimal et al., 1985). These proteomic investigations have not only advanced our biochemical and evolutionary understanding but have also contributed to practical applications, such as the L. amebocyte lysate test for endotoxin detection (Srimal et al., 1985). Some of the research has been conducted on T. gigas and T. tridentatus, but there is still a knowledge gap for C. rotundicauda, with data on spawning behaviour and biological application (Ding et al., 2005; Ng et al., 2007).

Classical AA analysis remains methodologically relevant for ecological and biomedical investigations involving non-model marine species such as C. rotundicauda. This species, due to its limited representation in genomic and proteomic databases, requires foundational biochemical characterization to support further molecular studies. Classical AA analysis provides baseline compositional data that can uncover unique metabolic adaptations, nutritional potential, or biomedical utility-particularly in organisms with known bioactive compounds such as coagulogen and hemocyanin.

Moreover, considering the ecological importance and conservation status of C. rotundicauda, a robust understanding of its basic biochemistry is vital for future studies on population health, habitat stress response, and sustainable utilization. Therefore, classical AA analysis is not only justified but also serves as a critical starting point for future advanced omics-based research.

In Bangladesh, research has been conducted on the morphometric variation, DNA-based identification, reproductive biology, population structure, and artificial breeding of C. rotundicauda and T. gigas (Haider et al., 2022). No study has yet been conducted on the AA composition of C. rotundicauda.

Therefore, the specific purpose of this study was to analyze the AA composition of C. rotundicauda from the Cox’s Bazar coast of Bangladesh to fill the existing knowledge gap and evaluate its nutritional and biomedical potential.

 

MATERIALS AND METHODS

Study area

The HSCs were collected from Gorokghata, Adinath Mondir, Moheskhali Estuary, Cox’s Bazar (Figure 1). The area is covered with mangrove vegetation. The sediment is clay that is about 0.61 meters deep. The abundance of C. rotundicauda is high in this area due to the richness of bloodworm polychaetes and other macrobenthos organisms (Meilana et al., 2021). This is a dynamic zone with active high and low tides. There is also salt marsh vegetation in the study area. The average salinity of the study area is 23.1 PSU, and the average water temperature is 27.47 °C.

 

 

Dissolved oxygen ranges 4.15–5.05 mg/l, and total dissolved solids are 5.05–24.87 g/l. The mean levels of NO₃-N, NO₂-N, PO₄-P, and SiO₃ were recorded as 0.45, 0.09, 0.08, and 0.07 mg/l, respectively. Average transparency was 0.69 meters, and conductivity was 36.11 mS/cm in the study area.

Sample collection and preservation

Bycatch (from fishermen’s nets) and visual search techniques (Figure 2a) were used to collect the samples (Soykan and Moore, 2008). During the low tides, HSCs of different sizes were found on the mud surface (Figure 2b).

 

 

Individuals were observed either resting on the mud or moving slowly across it while the anterior portion of the carapace was partially covered by the mud. The HSCs were hand-picked from the mud and kept in a plastic bucket with water. After collection, all HSCs were transported to the laboratory for further investigation.

Importantly, no live experimenta-tion, anesthesia, or prolonged captivity was involved on the specimens. All specimens were collected post-mortem or obtained as incidental bycatch without any deliberate harm or sacrifice. As such, an animal ethics certificate was not required under the current institutional and national guidelines.

Nevertheless, we recognize the ethical responsibility associated with specimen handling and fully support the use of formal ethical approvals in future studies involving live animals or experimental procedures.

Amino acid analysis

A stock solution of 2,500 µM of AAs was prepared using a 50:50 v/v ratio of methanol with water, sonicated for 1 min, and kept at -4°C. To prepare solutions of 2.0–100.0 µM of AAs, the stock solution was diluted in a 50:50 v/v ratio of methanol with water. A 0.232-µm polytetrafluoroethylene (PTFE) syringe filter was used to filter the solutions (Wu, 2013). A sample of HSCs muscle weighing 10–100 mg was placed in a 15-ml tube. After adding 2 mL of 6N HCl, the mixture was incubated for 20 h at 120 °C (Brosnan et al., 2000). After digestion, methanol and water (50:50 v/v; 2 mL) were used to resuspend the sample after the solvent was removed.

The ultra-fast liquid chromatography system (Shimadzu Corporation, Kyoto, Japan) with binary pumps, an autosampler, an online degassing unit, and a column oven connected to a triple quadrupole system mass spectrometer (Shimadzu LCMS-8050) with an electrospray ionisation (ESI) source was used for the liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis (Ishak et al., 2023). An innovative combination mode was used to optimise the gradient elution method to produce 20 genetically encoded AAs.

To ensure accurate quantification of the AAs in C. rotundicauda, a meticulously optimised extraction and chromatographic protocol was employed. The hydrolysis step, using 6N HCl at 120°C for 20 h, was selected based on its proven efficiency in breaking down complex protein structures into free AAs, as recommended by Brosnan et al. (2000). The incubation duration and temperature were optimised to achieve complete digestion without degradation of heat-sensitive AAs. Following hydrolysis, each muscle sample was resuspended in a 50:50 methanol-water mixture, which not only enhances solubility but also stabilises the AAs for LC-MS/MS analysis. Filtration through a 0.232-µm PTFE syringe filter ensured removal of particulates, minimizing column blockage and improving injection precision. For chromatographic separation, the use of an Intrada column (100 × 3 mm, 3 μm) maintained at 35°C provided high resolution for diverse AA profiles. The mobile phase consisted of solution A (acetonitrile, tetrahydrofuran, 25 mM NH₄HCO₂, and HCO₂H in the proportions of 9:75:16:0.3 v/v, respectively) and solution B (acetonitrile and 100 mM NH₄HCO₂ at 20:80 v/v, respectively) (Naher et al., 2025). The mobile phase composition was specifically chosen to balance polarity and ion-pairing requirements of the AAs and optimise peak shape and retention times. At an average flow rate of 0.6 mL/min, the elution program consisted of 0% B (0–3.0 min), 0–17% B (3.0–9.0 min), 17–100% B (9.0–16.0 min), 100% B (16.0–22.0 min), and 0% B (22.0 min). The gradient elution program was carefully modulated to enhance separation within a 22-min window with a chromatographic injection volume of 10 µL (Wu et al., 2013), demonstrating effective handling of both polar and non-polar AAs. Table 1 outlines the mass spectrometry acquisition parameters, and Table 2 details the multiple reaction monitoring transitions for the AAs. Detection was carried out using a Shimadzu LCMS-8050 triple quadrupole mass spectrometer with electrospray ionisation employing multiple reaction monitoring for precise quantitation.

 

Table 1
Operational Conditions for Mass Spectrometry

Parameters	State
Run time	22 minutes
Ion polarity	Positive ion mode
Ion source	Electrospray ionisation atmospheric pressure
Capillary voltage (kV)	4.0
Block temperature	400°C
Desolvation line temperature	300°C
CID gas	Argon at (270 kPa)
Nebulising gas flow	N2, 1.5 L/min
Drying gas flow	N2, 15.0 L/min
Heating gas flow	10 L/min
Interface temperature	300°C

 

Table 2
The multiple reaction monitoring (MRM) transition events of the amino acids

Amino acid	Type	m/z	Retention time (min)	MRM event
Serine	Target	106.10>60.20	1.707	7:MRM(+)
Glycine	Target	76.00>30.00	1.732	16:MRM(+)
Glutamine	Target	147.00>84.10	1.723	6:MRM(+)
Lysine	Target	147.00>84.10	1.731	15:MRM(+)
Aspartic acid	Target	134.10>73.90	1.720	3:MRM(+)
Histidine	Target	156.10>110.10	1.761	11:MRM(+)
Threonine	Target	120.10>74.00	1.758	8:MRM(+)
Alanine	Target	90.10>44.10	1.785	1:MRM(+)
Arginine	Target	175.10>70.10	1.786	2:MRM(+)
Glutamic acid	Target	148.10>84.10	1.804	4:MRM(+)
Proline	Target	116.10>70.10	1.934	17:MRM(+)
Valine	Target	118.20>72.00	2.177	10:MRM(+)
Methionine	Target	150.10>56.10	2.380	13:MRM(+)
Leucine	Target	132.10>86.30	2.979	12:MRM(+)
Isoleucine	Target	132.10>86.30	3.180	12:MRM(+)
Tyrosine	Target	182.10>136.20	3.244	9:MRM(+)
Phenylalanine	Target	166.10>120.10	4.583	14:MRM(+)

 

The LC-MS/MS parameters, including flow rate (0.6 mL/min), injection volume (10 µL), and temperature settings, were optimised to ensure sensitivity and reproducibility. Together, these optimised conditions ensured a robust, reproducible, and sensitive workflow for profiling both essential and non-essential AAs in C. rotundicauda, even at trace levels.

Statistical analysis

The sample analyses were carried out in triplicate. A one-way analysis of variance was performed on the data to detect statistical differences among the percentages of the AAs in the total AA content.

 

RESULTS AND DISCUSSION

Amino acid profiles of Carcinoscorpius rotundicauda

A total AA content of 2.2% was found in C. rotundicauda. Among the essential AAs, methionine (0.01%) appeared at a considerably lower level compared to leucine (0.5%), which was the most prevalent, followed by isoleucine (0.3%), proline (0.3%), and phenylalanine (0.2%). The concentration of AAs was recorded considerably low in C. rotundicauda. No significant differences (p = 0.08, F = 2.246) among the percentages of the AAs were detected.

Essential amino acids

Phenylalanine is essential for protein synthesis and metabolism (Wu et al., 2013). Its side chain enhances protein stability (Brosnan et al., 2000). It also produces neurotransmitters such as dopamine that influence brain function (Reeds et al., 2000). In the present study, phenylalanine was 0.2% in C. rotundicauda (Table 3).

Srimal et al. (1985) reported 12.2% in C. rotundicauda. Ishak et al. (2023) documented 0% phenylalanine in the muscles of T. gigas, while Minetti et al. (1991) reported 2.94% in the amebocytes of L. polyphemus. Miyata et al. (1984a) found 12% in L. polyphemus, which is lower than what Miyata et al. (1984b) found. Morita et al. (1985) reported 6.8% in the anticoagulant of L. polyphemus, whereas Muta et al. (1987) recorded 7% in L. polyphemus. Nakamura et al. (1982) found 13.5% in T. tridentatus, and Nakamura et al. (1985) reported 20.1% in T. tridentatus. Saito et al. (1995) recorded 2% in T. tridentatus.

Tyrosine is a non-essential AA that helps make thyroid hormones and neurotransmitters such as dopamine and supports brain function, metabolism, and melanin production (Jongkees et al., 2015; Parthasarathy et al., 2018). It also boosts cognitive function under stress (Daubner et al., 2011). The body produces tyrosine from phenylalanine. In our study, tyrosine was 0.6% in C. rotundicauda (Table 3).

Srimal et al. (1985) recorded 5.9% in C. rotundicauda, lower than the 6% found by Saito et al. (1995) in T. tridentatus. Ishak et al. (2023) found 2.38% in the muscles and 5.3% in the roe of T. gigas.

Muta et al. (1987) observed 3% in L. polyphemus, and Morita et al. (1985) documented 3.8% and Minetti et al. (1991) 5.2% from L. polyphemus amebocytes. Miyata et al. (1984b) recorded 4% in T. gigas, while Miyata et al. (1984a) found 5% in L. polyphemus. Nakamura et al. (1982) recorded a lower concentration of tyrosine in T. tridentatus than Nakamura et al. (1985).

Leucine is a key branched-chain amino acid (BCAA) for muscle protein synthesis, repair, and metabolic regulation (Blomstrand et al., 2006). It activates the mechanistic target of rapamycin pathway, supports muscle growth, and aids energy production during exercise or fasting (Kimball and Jefferson, 2006; Nair and Short, 2005). However, excessive intake may cause imbalances linked to metabolic disorders (Liu et al., 2022).

 

Table 3
Amino acid composition in Carcinoscorpius rotundicauda

Sl. No.	Parameter	Method/Instrument	Unit	Result
01	Total Amino Acid	Lc-MS/MS	%	2.2
	Amino Acids			Mean ± standard deviation
1.01	Phenyl Alanine			0.185 ± 0.015
1.02	Tyrosine			0.055 ± 0.045
1.03	Leucine			0.485 ± 0.015
1.04	Methionine			0.0055 ± 0.0045
1.05	Isoleucine			0.285 ± 0.015
1.06	Valine	Lc-MS/MS	%	0.055 ± 0.045
1.07	Glutamic acid			0.135 ± 0.065
1.08	Proline			0.275 ± 0.025
1.09	Threonine			0.015 ± 0.005
1.10	Alanine			0.055 ± 0.005
1.11	Aspartic Acid			0.00
1.12	Serine			0.025 ± 0.005
1.13	Glycine			0.0575 ± 0.0025
1.14	Histidine			0.00055 ± 0.00045
1.15	Lysine			0.035 ± 0.005
1.16	Glutamine			0.015 ± 0.005
1.17	Arginine			0.07 ± 0.01

 

We detected 0.5% leucine in C. rotundicauda (Table 3). Srimal et al. (1985) observed 8.5% in C. rotundicauda. Ishak et al. (2023) recorded 7.2% in the roe and 1.9% in the muscles of T. gigas. Saito et al. (1995) reported 3% in T. tridentatus, and Minetti et al. (1991) recorded 4.84% in L. polyphemus amebocytes. Morita et al. (1985) noted 10.1% in L. polyphemus anticoagulant. Muta et al. (1987) documented 7.3% in L. polyphemus. Miyata et al. (1984a) found 6% in L. polyphemus, and Miyata et al. (1984b) recorded 7% in T. gigas. Nakamura et al. (1985) and Nakamura et al. (1982) reported 31.3% and 28.6%, respectively, in T. tridentatus.

Methionine is a sulfur-containing AA that is important for protein synthesis, methylation, and the production of biomolecules such as glutathione (Sinclair et al., 2019). It also affects DNA methylation and gene expression through its conversion to S-adenosylmethionine (Wunderle et al., 2024). However, excessive methionine may promote unhealthy aging and cardiovascular issues (Ungvari et al., 2023). In our study, methionine was 0.006% in C. rotundicauda (Table 3). Ishak et al. (2023) documented 0% methionine in T. gigas. Saito et al. (1995) in T. tridentatus, Muta et al. (1987) and Miyata et al. (1984a) in L. polyphemus, and Morita et al. (1985) in L. polyphemus anticoagulant recorded lower concentrations than what we observed. Nakamura et al. (1985) reported 6.1% and Nakamura et al. (1982) recorded 2.7% in T. tridentatus, which is higher than what we measured in C. rotundicauda. Minetti et al. (1991) documented a higher amount of methionine in L. polyphemus amebocytes than what we observed in C. rotundicauda.

Isoleucine is an essential BCAA important for muscle metabolism, immune function, and hemoglobin production (Wagenmakers et al., 1998). It supports muscle protein synthesis, energy control, and glucose metabolism, benefiting both athletic performance and metabolic health (Shimomura et al., 2006; Tajiri and Shimizu, 2013). However, excessive intake may disrupt metabolic balance (Liu et al., 2022). In our study, the isoleucine amount was 0.3% in C. rotundicauda (Table 3). Srimal et al. (1985) recorded 2.7% in C. rotundicauda. Ishak et al. (2023) found 3.2% in the roe and 2.7% in the muscles of T. gigas, whereas Saito et al. (1995) reported 3.8% in T. tridentatus, and Morita et al. (1985) reported 5.5% in L. polyphemus anticoagulant. Muta et al. (1987) documented 5.8% in L. polyphemus, while Minetti et al. (1991) found 7.75% in L. polyphemus amebocytes. Miyata et al. (1984a) recorded 7% in L. polyphemus, and Miyata et al. (1984b) reported 5% in T. gigas. Nakamura et al. (1985) and Nakamura et al. (1982) recorded 22.6% and 20.4%, respectively, in T. tridentatus.

Valine is an essential BCAA that aids energy production, protein synthesis, and muscle metabolism alongside leucine and isoleucine (Shimomura et al., 2004). It supports muscle growth, recovery, and tissue repair by promoting metabolic signaling and nitrogen balance (Brosnan and Brosnan, 2006; Holeček et al., 2018). In our investigation, valine was 0.06% in C. rotundicauda (Table 3). Srimal et al. (1985) documented 17% in C. rotundicauda. Ishak et al. (2023) recorded 2.85% in the roe and 2.11% in the muscle of T. gigas, whereas Minetti et al. (1991) reported 4.65% in L. polyphemus amebocytes. Morita et al. (1985) documented 6.7% in L. polyphemus anticoagulant, and Muta et al. (1987) reported 4.2% in L. polyphemus. Miyata et al. (1984a,b) recorded similar amounts in L. polyphemus and T. gigas. Saito et al. (1995) recorded 7.2% in T. tridentatus, and Nakamura et al. (1982) and Nakamura et al. (1985) found 27.4% and 27.8%, respectively, in T. tridentatus.

Threonine is an essential AA crucial for protein synthesis, immune function, and maintaining collagen and elastin integrity (Brosnan et al., 2000). It also supports immune, neurological, and digestive functions and is vital for metabolic pathways (Tang et al., 2021). In our study, threonine was 0.02% in C. rotundicauda (Table 3). Srimal et al. (1985) detected 10.8% in C. rotundicauda. Ishak et al. (2023) reported 2.83% in the muscles and 3.61% in the roe of T. gigas. Muta et al. (1987) found 7.7% in L. polyphemus, while Morita et al. (1985) documented 9.6% in L. polyphemus anticoagulant. Minetti et al. (1991) recorded 2.75% in L. polyphemus amebocytes. Miyata et al. (1984b) reported 10% in T. gigas. Nakamura et al. (1982) and Nakamura et al. (1985) recorded 22% and 36.1%, respectively, in T. tridentatus. Saito et al. (1995) documented 3.1% in T. tridentatus

Histidine is an essential AA important for protein structure, pH balance, and enzyme function (Holeček, 2020). It acts as a histamine antagonist, influencing neurotransmission, immunity, and gastric acid release, and it is vital for growth and tissue repair (Alazawi et al., 2022; Brosnan et al., 2000). Histidine concentration was 0.0006% in C. rotundicauda (Table 3). Saito et al. (1995) found 2.2% in T. tridentatus, whereas Ishak et al. (2023) documented 1.86% in the muscles and 2.11% in the roe of T. gigas. Morita et al. (1985) recorded 3.5% in L. polyphemus anticoagulant, while Minetti et al. (1991) reported 3.1% in L. polyphemus amebocytes. Muta et al. (1987) and Miyata et al. (1984a) observed 3% and 4%, respectively, in L. polyphemus. Miyata et al. (1984b) recorded a lower amount (5%) in T. gigas than what Srimal et al. (1985) reported in C. rotundicauda (5.3%). Nakamura et al. (1985) and Nakamura et al. (1982) recorded 10. 6% and 11.2%, respectively, in T. tridentatus.

Lysine is an essential AA needed for tissue healing, immune response, and protein synthesis (Wu et al., 2013). It supports hormone, enzyme, and antibody production, and promotes collagen synthesis, bone health, and calcium absorption (Aggarwal and Bains, 2022; Lv et al., 2022). Its side chain aids protein interactions and enzymatic processes (Cheng et al., 2007), and proper intake prevents deficiencies such as reduced growth and weakened immunity (Gunarathne et al., 2025). In our study, lysine was 0.04% in C. rotundicauda (Table 3). The detected amount in our study is lower than the amount found by Nakamura et al. (1982) in T. tridentatus (22%), Miyata et al. (1984b) in T. gigas (9%), Morita et al. (1985) in L. polyphemus anticoagulant (12.3%), Nakamura et al. (1985) in T. tridentatus (24.7%), Srimal et al. (1985) in C. rotundicauda (6.6%), Muta et al. (1987) in L. polyphemus (7.3%), and Minetti et al. (1991) in L. polyphemus amebocytes (7%).

Non-essential amino acids

Glutamic acid is a non-essential AA crucial for protein synthesis, neurotransmission, and metabolism (Bon et al., 2023). As a neurotransmitter, glutamate boosts synaptic transmission and supports memory, learning, and brain function (Brosnan and Brosnan, 2013; Danbolt et al., 2001). We recorded the glutamic acid concentration at 0.14% in C. rotundicauda (Figure 3). Srimal et al. (1985) found 21% in C. rotundicauda. Minetti et al. (1991) recorded 9.82% in L. polyphemus amebocytes, and Morita et al. (1985) reported 14.9% in L. polyphemus anticoagulant. Ishak et al. (2023) reported 8.40% in the roe and 3.52% in the muscles of T. gigas. Muta et al. (1987) observed 11.3% in L. polyphemus. Miyata et al. (1984a) recorded 22% in L. polyphemus, and Miyata et al. (1984b) documented 19% in T. gigas. Nakamura et al. (1982) observed 36.3% in T. tridentatus, and Nakamura et al. (1985) reported 45.8% in the same species. Saito et al. (1995) documented 1.5% in T. tridentatus.

Proline is a non-essential AA vital for metabolism, connective tissue integrity, and protein structure (Morgan and Rubenstein, 2013). It aids collagen formation, energy metabolism, protein stabilisation, and cellular stress responses (Phang et al., 2008). We recorded 0.3% proline in C. rotundicauda (Figure 3). Srimal et al. (1985) reported 9% in C. rotundicauda. Miyata et al. (1984a) and Muta et al. (1987) recorded 10% and 1.8%, respectively, in L. polyphemus. Morita et al. (1985) found a higher amount (3.4%) of proline in L. polyphemus anticoagulant than what Minetti et al. (1991) found in L. polyphemus amebocytes. Miyata et al. (1984b) recorded 10% in T. gigas. Nakamura et al. (1982) and Nakamura et al. (1985) recorded 22.4% and 32.7%, respectively, in T. tridentatus. Saito et al. (1995) reported 3.4% in T. tridentatus.

Alanine is a non-essential AA crucial for energy production, protein synthesis, and glucose metabolism, supporting muscle function and stable blood sugar levels during metabolic stress (Brosnan et al., 2000; Felig et al., 1973). In our study, alanine was 0.06% in C. rotundicauda (Figure 3). Srimal et al. (1985) noted 7.9% in C. rotundicauda. Ishak et al. (2023) found 1.24% in the muscles and 2.07% in the roe of T. gigas. Minetti et al. (1991) reported 4% in L. polyphemus amebocytes, whereas Morita et al. (1985) documented 8.7% in L. polyphemus anticoagulant, and Muta et al. (1987) and Miyata et al. (1984a) found 5.2% and 7%, respectively, in L. polyphemus. Miyata et al. (1984b) recorded 8% in T. gigas. Nakamura et al. (1982) reported 19.3% in T. tridentatus, whereas Nakamura et al. (1985) documented 22% in the same species. Saito et al. (1995) recorded 12.5% in T. tridentatus.

Aspartic acid is a non-essential AA vital for the urea cycle, neurotransmission, and protein synthesis, and it plays a key role in transamination reactions (Reeds et al., 2000). It also initiates the synthesis of other AAs, such as asparagine, contributing to neural signaling and metabolic homeostasis (Johnson and Koerner, 1988). We detected 0.0% aspartic acid in C. rotundicauda (Figure 3). Srimal et al. (1985) observed 11.1% in C rotundicauda. Ishak et al. (2023) documented 6.45% in the roe and 1.2% in the muscles of T. gigas. Morita et al. (1985) found 5.3% in L. polyphemus anticoagulant, while Minetti et al. (1991) reported 10.2% in L. polyphemus amebocytes. Miyata et al. (1984a) and Muta et al. (1987) documented 11% and 7.2%, respectively, in L. polyphemus, while Miyata et al. (1984b) found a higher amount (12%) in T. gigas. Nakamura et al. (1982) and Nakamura et al. (1985) documented 44% and 45.3%, respectively, in T. tridentatus. Saito et al. (1995) measured 6.4% in T. tridentatus.

Serine is a non-essential AA important for cell signaling, protein synthesis, and metabolic processes, and helps in enzymatic activities due to its polarity (Wu et al., 2013). It serves as a precursor to key biomolecules such as glycine, cysteine, and sphingolipids, which are crucial for phospholipid and nucleotide formation, especially during metabolic stress and cell development (Holeček, 2020; Kishor et al., 2020). In our study, serine was 0.06% in C. rotundicauda (Figure 3). Srimal et al. (1985) recorded 10.5% in C. rotundicauda. Ishak et al. (2023) documented 2.42% serine in the muscles and 4.50% in the roe of T. gigas. Morita et al. (1985) detected 13.1% in L. polyphemus anticoagulant, while Minetti et al. (1991) reported 14.9% in L. polyphemus amebocytes. Muta et al. (1987) and Miyata et al. (1984a) reported 12% and 9.3%, respectively, in L. polyphemus. Miyata et al. (1984b) noted 10% in T. gigas.

 

 

Nakamura et al. (1982) noted 17.4% in T. tridentatus, and Nakamura et al. (1982) recorded 23.9% in the same species. Saito et al. (1995) found 7.1% in T. tridentatus.

Glycine is a non-essential AA vital for metabolic functions, neurotransmission, and protein synthesis, acting as an inhibitory neurotransmitter in the central nervous system (Bon et al., 2023; Pérez-Torres et al., 2017). It also supports connective tissue integrity, antioxidant defence, collagen synthesis, and energy metabolism through the production of biomolecules like glutathione, creatine, and heme (Dunstan, 2024; Liu et al., 2022; Wu et al., 2013). We found 0.06% glycine in C. rotundicauda (Figure 3). Srimal et al. (1985) observed 14% in C. rotundicauda. Ishak et al. (2023) reported 0.95% in the muscles and 1.79% in the roe of T. gigas. Minetti et al. (1991) reported 6.91% in L. polyphemus amebocytes, lower than the 9.8% Morita et al. (1985) found in L. ployphemus anticoagulant, whereas Miyata et al. (1984a) recorded 13% in L. polyphemus. Muta et al. (1987) recorded 7.4% in L. polyphemus. Miyata et al. (1984b) found 15% in T. gigas. Saito et al. (1995) documented 6.3% in T. tridentatus.

Glutamine is an essential AA important for immune function, muscle protein balance, and nitrogen metabolism (Cruzat et al., 2018; Newsholme et al., 2023). It aids nucleotide biosynthesis and acid-base regulation and provides energy to rapidly dividing cells, such as immune cells (Taylor and Curthoys, 2004). Although the body can synthesise glutamine, increased demand during stress or illness may require dietary supplements to maintain adequate levels (Cruzat et al., 2018; Wu et al., 2013). In our study, glutamine was 0.02% in C. rotundicauda (Figure 3). Ishak et al. (2023) documented 0.04% in the muscles and 0.06% in the roe of T. gigas. Saito et al. (1995) reported 1.5% in T. tridentatus, and Minetti et al. (1991) recorded 9.82% in L. polyphemus amebocytes.

Arginine is an essential AA involved in protein synthesis, nitric oxide production, and the urea cycle and supports cardiovascular health and ammonia detoxification (Wu et al., 2013; Wu et al., 2021; Champeroux et al., 2005). Its demand increases during metabolic stress or illness, and supplements aid recovery and immune function in critically ill patients (Patel et al., 2016). In our investigation, the arginine amount was 0.07% in C. rotundicauda (Figure 3). Srimal et al. (1985) noted 15.9% in C. rotundicauda. Ishak et al. (2023) reported 3.52% arginine content in T. gigas. Minetti et al. (1991) recorded 5.06% in L. polyphemus amebocytes, whereas Morita et al. (1985) detected 6.8% in L. polyphemus anticoagulant. Muta et al. (1987) found 7.1% in L. polyphemus. Nakamura et al. (1982) recorded 14.7% in T. tridentatus, and Nakamura et al. (1985) documented 19.2% in the same species. Saito et al. (1995) reported 8% in T. tridentatus. Miyata et al. (1984a) reported a lower amount of arginine in L. polyphemus compared to the findings of Miyata et al. (1984b) for T. gigas.

 

FUTURE RESEARCH DIRECTIONS

Future research should aim to collect samples from multiple regions and seasons to evaluate how environmental variables, such as salinity, pollution, and food availability, influence the AA profile of C. rotundicauda.

Studies examining the biochemical response to environmental stressors could help determine whether factors such as pollution or climate change are responsible for the observed deficiencies in key AAs. There is also a strong need to isolate and characterise the bioactive peptides of this species, followed by in vitro or in vivo testing to confirm their pharmaceutical potential. Integrating genomic and proteomic tools would offer deeper insights into the metabolic pathways governing AA synthesis and regulation.

In addition, exploring the nutritional value of C. rotundicauda through feeding trials or digestibility studies could help validate its application in nutraceuticals. Synthetic biology approaches might also be explored to replicate valuable peptides or AAs in laboratory settings, thus reducing the need for extraction from wild populations.

To ensure the species’ conservation and sustainable utilisation, future studies should establish ethical harvesting protocols and consider the development of captive breeding programs.

Finally, incorporating recent scientific findings into conservation policy and public awareness campaigns could enhance both biodiversity protection and biomedical innovation.

 

CONCLUSIONS AND RECOMMENDATIONS

Our study presents the first detailed assessment of the AA profile of C. rotundicauda from the Cox’s Bazar coast, offering valuable insights into its nutritional and biomedical potential. The findings revealed relatively low overall AA concentrations, with leucine, isoleucine, and proline as the dominant AAs, while aspartic acid was absent, and methionine and histidine were present only in trace amounts. These results suggest species-specific metabolic adaptations that differentiate C. rotundicauda from other marine arthropods. The presence of BCAA, particularly leucine and isoleucine, points to the species’ potential role in supporting protein synthesis, muscle metabolism, and recovery, making it a promising candidate for applications in the nutraceutical and pharmaceutical industries.

This potential, several strategic actions are recommended to realise this potential. Further research should involve seasonal and regional sampling to determine how environmental factors such as salinity, pollution, and food availability affect AA profiles. Bioactive peptides derived from C. rotundicauda should be isolated and tested for therapeutic applications, including antimicrobial, anti-inflammatory, and immune-boosting properties.

Ethical and sustainable harvesting practices need to be developed, including non-lethal blood extraction and the establishment of captive breeding programs. These initiatives must be supported by robust environmental regulations to reduce habitat degradation and by public education efforts that raise awareness of the species’ ecological and biomedical importance.

The prospects of this study are broad and impactful. By bridging marine biology with biotechnology, the findings support the integration of C. rotundicauda into Bangladesh’s blue economy strategy. Synthetic biology could be explored to replicate valuable AAs and peptides in laboratory settings, minimizing pressure on wild populations. If combined with conservation policies and investment in marine research infrastructure, this work could lay the foundation for future innovations in drug discovery, health supplements, and sustainable resource utilisation, ultimately contributing to both biodiversity preservation and socioeconomic development.

 

Author contributions: Conceptualization: MSB, MTI; Methodology: MSB, MTI; Data analysis: MSB, VM; Investigation: MSB, MTI; Supervision: MSB, MTI; Writing – original draft: MSB, VM; Writing – review and editing: MSB, VM. All authors declare that they have read and approved the publication of the manuscript in this present form.

Acknowledgments: The authors are grateful to the Bangladesh Oceanographic Research Institute for allowing access to their research facility.

Funding: There was no form of external funding for this study.

Conflicts of interest: The authors declare that there are no conflicts of interest.

 

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