FIRST REPORT OF THE AMINO ACID AND FATTY ACID COMPOSITION OF JELLYFISH ( LOBONEMOIDES ROBUSTUS STIASNY, 1920) COLLECTED DURING JELLYFISH BLOOM ALONG THE COX’S BAZAR COAST, BANGLADESH

. Jellyfish (JF) are essential to marine ecosystems. However, JF that increases rapidly can have negative effects.


INTRODUCTION
The oceans are a nearly untapped reservoir of biochemicals that cover 70% of Earth's surface.They are home to over 194,000 known species of microorganisms, flora, and fauna (Primavera et al., 2019), but between 2011 and 2017, only a tiny number of these marine creatures were utilised, yielding roughly 9,000 unique natural compounds (Romano et al., 2022).Among these marine organisms are JF, a generic term that refers to medusae of the phylum Cnidaria, specifically the class Scyphozoa.Many people value JF for their elegant appearance, but they are also feared for their severe stings.Compared with other taxa, cnidarians have been subjected to relatively little natural product exploitation (Das et al., 2023;Haider et al., 2022).
Globally, JF populations seem to have risen in the last few decades.The overall increase and its causes are unclear because JF abundance is not routinely monitored (Brotz et al., 2012).The natural rhythms of JF blooms may be disrupted by several human-driven activities, including overfishing, pollution, and high temperatures (Haider et al., 2022).This could result in a substantial rise in JF populations in specific coastal areas and major marine ecosystems.Only a small number of bioactive substances have been recovered from oceanic cnidarians; the majority of natural goods are derived from benthal cnidarians.However, there are many significant potential human uses for the natural compounds that pelagic cnidarians synthesise (Fonseca et al., 2023).Substantial scientific data supports the idea that JF are valuable bioresources for a variety of high-end applications such as human food; feed for cultivated species; and the discovery of untapped bioactive compounds for use in pharmaceutical, cosmetic, nutraceutical, and other biotechnological applications (Das and Patel, 2020;Duarte et al., 2022;Romano et al., 2022).
During 3-4 August 2022, numerous dead JF of the species L. robustus were found along the shore at Cox's Bazar, Bangladesh.They were carried onto the beach at high tide and they stuck in the sand deposit during low tide.According to Kitamura and Omori (2010), L. robustus are marketed as 'white-type' JF and are typically seen in huge quantities during certain seasons.They live along the Bay of Bengal (BoB) coast and may be harvested for export or human use.No scientists in Bangladesh have yet researched the biochemical composition of L. robustus.Hence, the purpose of the current investigation was to ascertain L. robustus's AA and FA content.This information could increase the export of L. robustus and contribute to the blue economy of Bangladesh.

Study area
The current study was conducted in the following areas: the Sabrang coast, the Patuartek coast, the Shamlapur coast, the Bangladesh Oceanographic Research Institute (BORI) beach, Inani Beach, the Daria Nagar coast, and Bangladesh Fisheries Development Corporation (BFDC) Ghat.Each site is located along the Cox's Bazar shore, which is part of the BoB coast (Figure 1).Samples were collected on 3-4 August 2022 during a massive L. robustus bloom.

Sample collection and preservation
Using hand gloves, a total of 14 L. robustus samples (average weight 30 kg) were collected from each sampling site during the peak JF occurrence.The samples were collected in plastic buckets (due to their large size, only one specimen per bucket) and cleaned onsite with seawater.The samples were transported to BORI's Biological Oceanography Laboratory after being preserved in 10% formalin (Haider et al., 2022).The specimens had minimal damage and were in generally good condition.Along with live specimens, photographs and videos were captured in the field for species identification.As soon as possible after capture, specimens were photographed to capture their natural hue (Haider et al., 2022).

Determination of amino acids (AAs) Preparation of stock solution and intermediate stock solution
A stock solution of 2500 µM of AAs was prepared in methanol and water (50:50, v/v), sonicated for 1 min, and stored at -4°C.The stock solution was diluted in methanol and water (50:50, v/v) to produce solutions containing 2.0-100 µM of AAs.These solutions were filtered with a 0.232-µm syringe filter (PTFE).

Sample preparation
A 10-100 mg sample was weighed in a 15 ml tube.Then, 2 mL of 6 N HCl was added, and the mixture was incubated at 120°C for hours.Following digestion, the solvent was removed and the sample was resuspended in methanol and water (50:50, v/v; 2 mL).

Analytical conditions
The liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis used an ultra-fast liquid chromatography system (Shimadzu Corporation, Kyoto, Japan) with binary pumps, an autosampler, an on-line degassing unit, and a column oven connected to a Shimadzu LCMS-8050 triple quadrupole system mass spectrometer, which has an electrospray ionisation (ESI) source.Twenty genetically encoded AAs were subjected to an improved gradient elution method using a novel combined mode.
Table 1 presents the MS acquisition conditions, and Table 2 presents the multiple reaction monitoring (MRM) transition events of the AAs.

Determination of fatty acids (FAs)
A Shimadzu GC 2010 Plus gas chromatographic apparatus with a flame ionisation detector was utilised to identify FAs.One hundred milligrams of C 6 H 6 O 3 and 2 mL of ethanol were added to a flask containing 100-200 mg of material and thoroughly mixed.Then, 10 mL of 8.3 M HCl was added and the contents were stirred.The flask was incubated in a water bath heated to 70-80°C for 40 min, with gentle shaking every 10 min.Then, the flask was allowed to cool to ambient temperature (20-25°C).While stirring carefully, enough ethanol was added to fill the flask's bottom reservoir.
After adding 20 mL of diethyl ether and 20 mL of petroleum ether, the flask was centrifuged at 600 rpm for 5 min (if a centrifuge is not available, then the contents should be allowed to settle for at least 1 h until the upper layer is transparent).In a steam bath, the top layer was removed and the ether was evaporated.
After dissolving the residue in 2-3 mL of CHCl 3 and 2-3 mL of (C 2 H 5 ) 2 O, the mixture was shifted to a 3-dram glass vial and dried in a water bath at 40°C.Then, 1 mL of toluene and 2 mL of 7% BF 3 methanol were added.The vial was closed with a screwcap top with a teflon/silicone septum.The vial was heated in an oven to 100˚C for 45 min, with gentle shaking every 10 min.The vial was cooled to room temperature (20-25°C).After adding 1 mL hexane, 5 mL water, and 1 g Na 2 SO 4 , the vial was shaken.Then, the upper layer was transferred to a new vial containing 1 g of Na 2 SO 4 for gas chromatography.

RESULTS AND DISCUSSION
JF represents a vital part of marine food webs.Although their function as consumers has long been recognised, they are also consumed by a diverse range of species (Schaub et al., 2023).

Amino acids (AAs) in L. robustus
In general, L. robustus had low EAA levels.The most abundant EAA is Gly (Figure 2), followed by Glu, Asp, Thr, and Pro.The results of this investigation are consistent with those of Khong et al. (2016) and Hsieh et al. (2001).According to Khong et al. (2016), JF, regardless of the body area, contains roughly 33% EAAs, 46% conditionally EAAs, and 21% NEAAs.Kogovšek et al. (2014) reported that in JF, Asp, Lys, Arg, Gly, and Glu are the most abundant AAs per unit of dry mass, accounting for over half of the entire pool of AAs.
Consistent with our findings, Gly is the most prevalent AA in scyphomedusae.
This EAA is one of the major structural units of collagen (Merquiol et al., 2019) 3).Compared with Semaeostomeae, Rhizostomeae have more EAAs (Merquiol et al., 2019).Only Cotylorhiza tuberculata and Rhizostoma pulmo contain significant levels of His; in other scyphomedusae, this EAA is either absent or very low (Table 3).L. robustus also contains detectable amounts of Thr, Arg, Ser, Glu, and Lys (Figure 2).
Compared with the AA composition of rat tail collagen, JF had a low Pro content and higher Glu and Ala contents (Derkus et al., 2016).Rhopilema hispidum gelatine has notably high Gly (18.90%),Pro (8.15%), and hydroxyproline (13.93%) contents (Table 3) (Cho et al., 2014).Chrysaora sp. has a low concentration of Pro and hydroxyproline (Barzideh et al., 2014).According to De Rinaldis et al. (2021), the most prevalent AAs in Cassiopea andromeda are Glu, Gln, and Gly.This species contains 15.68 g of these AAs per 100 g lyophilised sample, more than twice as much as R. pulmo (6.1 ± 0.09 g per 100 g lyophilised sample) and Pelagia noctiluca (8.1 ± 0.3 g per 100 g lyophilised sample) samples analysed in parallel.De Rinaldis et al. (2021) also reported high levels of Ala and taurine in C. andromeda, namely 0.96 g per 100 g dry weight.The contents of the main AAs of wild JF gonad and cultured JF gonad -Glu, Lys, Gly, Asp, and Leu -are 51.47% and 52.52% of the total AA content, respectively.Asp and Glu are often present in enzyme-active sites and are crucial for preserving the solubility and ionic nature of proteins (Yu et al., 2014).Stabili et al. (2018) found free AAs in a gonadal extract from R. pulmo.The ovaries of this species may provide an abundant supply of AAs for pharmacological and nutraceutical purposes.Additionally, the ovaries may provide proteins needed for the creation of novel nutritional supplements intended to sustain fish.

Amino acids
Fatty acids (FAs) in L. robustus FAs are components of membranes and cell structures, but they also accumulate as energy storage units in plants and animals.They can be absorbed from food or biosynthesised by the organism (Saha and Pathak, 2021).FAs do not decompose during digestion, in contrast to other complex compounds; rather, they stay mostly unaltered or barely altered.Because they often do not change as reservoirs during normal cell metabolism (Elsamadony et al., 2021), they are regarded as traditional indicators that are used in environmental research to clarify the relationships between organisms in the food chain and to ascertain the movement of organic matter from lower trophic levels to higher trophic levels (De Troch et al., 2012).In the present study, C18:3 was the most prevalent FA (0.43%) in L. robustus, followed by heptadecanoic acid (0.29%), gamma-linolenic acid (0.24%), cis-9oleic acid (0.18%), decanoic acid (0.13%), and myristic acid (0.12%) (Figure 3).
The gonads of R. pulmo have ω3 PUFAs, primarily DHA and EPA, which suggests that these molecules could be extracted from them and used in the pharmaceutical industry (Stabili et al., 2018).DHA and EPA have antiinflammatory and antioxidant properties and may be used in treatment plans for mental health issues and memory impairments brought on by neuroinflammation (Apetz et al., 2014).Additionally, considering that fish diets are typically supplemented with extra EPA and DHA, the gonads of R. pulmo may provide these necessary FAs that could be extracted and then added to the fish feed (Stabili et al., 2018).

CONCLUSIONS
The search for substitute sources of bioactive chemicals to take the place of overfished resources is a pressing need for modern society.JF are important sources of AAs and FAs.In the present study, we found that L. robustus is rich in Gly.The most common FAs are linoleic acid, myristic acid, cis-9-oleic acid, gamma-linolenic acid, and heptadecanoic acid.Our data indicate that L. robustus could be a sustainable source of AAs and FAs for use in manufacturing natural nutraceutical, cosmeceutical, and biomedical products.Moreover, in Southeast Asia, L. robustus is commonly used for food.The commercially valuable L. robustus could be exported to other countries and contribute to a blue economy.

Figure 1 -
Figure 1 -The map displays the jellyfish collection points (denoted by blue color jellyfish) .T.I.; Conceptualisation, Methodology, Statistical analysis, Writingoriginal draft: M.S.B.; Literature survey, S.A.; Analysis, review and editing, M.K., N.K.K.; Review and editing: M.S.B., M.K., S.A.All authors have read and agreed to the published version of the manuscript.. Data availability: This study's findings are supported by the data presented in the report.

Table 1 -
The mass spectrometry acquisition conditions

Table 2 -
The multiple reaction monitoring (MRM) transition events of the amino acids Figure 2 -Amino acid percentages in Lobonemoides robustus