#METABOLOMICS WORKBENCH kuohsun_20241215_074751 DATATRACK_ID:5453 STUDY_ID:ST003633 ANALYSIS_ID:AN005966 PROJECT_ID:PR002246
VERSION             	1
CREATED_ON             	December 19, 2024, 8:02 pm
#PROJECT
PR:PROJECT_TITLE                 	1H-NMR-based Metabolomic Analysis of Hypersalinity-Induced Oviparity in Brine
PR:PROJECT_TITLE                 	Shrimp
PR:PROJECT_SUMMARY               	This study investigates the metabolic adjustments in Artemia under hypersaline
PR:PROJECT_SUMMARY               	stress using 1H-NMR-based metabolomics. Hypersalinity is known to trigger
PR:PROJECT_SUMMARY               	diapause in brine shrimp, a reproductive strategy for survival in extreme
PR:PROJECT_SUMMARY               	environments. To elucidate the metabolic responses associated with
PR:PROJECT_SUMMARY               	hypersalinity-induced oviparity, we analyzed brine shrimp samples exposed to
PR:PROJECT_SUMMARY               	normal salinity (30 parts per thousand) and high salinity (80 parts per
PR:PROJECT_SUMMARY               	thousand) conditions. The extracted metabolites were analyzed using a 400 MHz
PR:PROJECT_SUMMARY               	NMR spectrometer, and metabolites were identified and quantified using Chenomx
PR:PROJECT_SUMMARY               	Profiler and the Human Metabolome Database (HMDB).
PR:INSTITUTE                     	National Kaohsiung University of Science and Technology
PR:DEPARTMENT                    	Department and Graduate Institute of Aquaculture
PR:LAST_NAME                     	Chiu
PR:FIRST_NAME                    	Kuohsun
PR:ADDRESS                       	142 Hai-tuang Rd., Kaohsiung, Not USCanada, 811, Taiwan
PR:EMAIL                         	kuohsun@nkust.edu.tw
PR:PHONE                         	+886-7-3667141#23708
#STUDY
ST:STUDY_TITLE                   	1H-NMR-based Metabolomic Analysis of Hypersalinity-Induced Oviparity in Brine
ST:STUDY_TITLE                   	Shrimp
ST:STUDY_SUMMARY                 	This study investigated the mechanisms by which high salinity conditions
ST:STUDY_SUMMARY                 	stimulate adult Artemia females to produce diapaused cysts. We used a
ST:STUDY_SUMMARY                 	1H-NMR-based metabolomic approach to elucidate the metabolic regulation between
ST:STUDY_SUMMARY                 	ovoviviparity and oviparity in Artemia exposed to different salinities. At a
ST:STUDY_SUMMARY                 	salinity of 80 parts per thousand (ppt), 100% of females produced diapaused
ST:STUDY_SUMMARY                 	cysts, compared to 20% at 50 ppt. Metabolic profiling revealed significant
ST:STUDY_SUMMARY                 	alterations in a range of metabolites, including 5,6-dihydrouracil, betaine, and
ST:STUDY_SUMMARY                 	malate, in females undergoing oviparity at 80 ppt compared to ovoviviparity at
ST:STUDY_SUMMARY                 	30 ppt. Multivariate statistical analyses indicated clear separation between the
ST:STUDY_SUMMARY                 	two reproductive strategies. The up-regulated metabolites in oviparity were
ST:STUDY_SUMMARY                 	involved in significant metabolic pathways, such as β-alanine metabolism and
ST:STUDY_SUMMARY                 	the citrate cycle, highlighting substantial metabolic differences between the
ST:STUDY_SUMMARY                 	two reproductive strategies. These identified metabolic pathways might play
ST:STUDY_SUMMARY                 	crucial roles in the maternal response to high salinity, facilitating embryo
ST:STUDY_SUMMARY                 	protection and enhancing the survival and reproductive success of brine shrimp.
ST:STUDY_SUMMARY                 	These findings provide a basis for further research into the molecular
ST:STUDY_SUMMARY                 	mechanisms underlying Artemia adaptation to high salinity environments.
ST:INSTITUTE                     	National Kaohsiung University of Science and Technology
ST:DEPARTMENT                    	Department and Graduate Institute of Aquaculture
ST:LABORATORY                    	Lab. of Live Feeds Aquaculture
ST:LAST_NAME                     	Chiu
ST:FIRST_NAME                    	Kuohsun
ST:ADDRESS                       	142 Hai-tuang Rd., Kaohsiung, Not USCanada, 811, Taiwan
ST:EMAIL                         	kuohsun@nkust.edu.tw
ST:PHONE                         	+886-7-3667141#23708
#SUBJECT
SU:SUBJECT_TYPE                  	Invertebrate
SU:SUBJECT_SPECIES               	Artemia salina
SU:TAXONOMY_ID                   	85549
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	L1	Salinity:30 ppt | Sample source:Brine shrimp	RAW_FILE_NAME(Raw file name)=L1_Proton.jdf
SUBJECT_SAMPLE_FACTORS           	-	L2	Salinity:30 ppt | Sample source:Brine shrimp	RAW_FILE_NAME(Raw file name)=L2_Proton.jdf
SUBJECT_SAMPLE_FACTORS           	-	L3	Salinity:30 ppt | Sample source:Brine shrimp	RAW_FILE_NAME(Raw file name)=L3_Proton.jdf
SUBJECT_SAMPLE_FACTORS           	-	L4	Salinity:30 ppt | Sample source:Brine shrimp	RAW_FILE_NAME(Raw file name)=L4_Proton.jdf
SUBJECT_SAMPLE_FACTORS           	-	L5	Salinity:30 ppt | Sample source:Brine shrimp	RAW_FILE_NAME(Raw file name)=L5_Proton.jdf
SUBJECT_SAMPLE_FACTORS           	-	L6	Salinity:30 ppt | Sample source:Brine shrimp	RAW_FILE_NAME(Raw file name)=L6_Proton.jdf
SUBJECT_SAMPLE_FACTORS           	-	H1	Salinity:80 ppt | Sample source:Brine shrimp	RAW_FILE_NAME(Raw file name)=H1_Proton.jdf
SUBJECT_SAMPLE_FACTORS           	-	H2	Salinity:80 ppt | Sample source:Brine shrimp	RAW_FILE_NAME(Raw file name)=H2_Proton.jdf
SUBJECT_SAMPLE_FACTORS           	-	H3	Salinity:80 ppt | Sample source:Brine shrimp	RAW_FILE_NAME(Raw file name)=H3_Proton.jdf
SUBJECT_SAMPLE_FACTORS           	-	H4	Salinity:80 ppt | Sample source:Brine shrimp	RAW_FILE_NAME(Raw file name)=H4_Proton.jdf
SUBJECT_SAMPLE_FACTORS           	-	H5	Salinity:80 ppt | Sample source:Brine shrimp	RAW_FILE_NAME(Raw file name)=H5_Proton.jdf
SUBJECT_SAMPLE_FACTORS           	-	H6	Salinity:80 ppt | Sample source:Brine shrimp	RAW_FILE_NAME(Raw file name)=H6_Proton.jdf
#COLLECTION
CO:COLLECTION_SUMMARY            	Brine shrimp samples were collected to investigate the metabolic response under
CO:COLLECTION_SUMMARY            	different salinity conditions. Two experimental groups were established: Control
CO:COLLECTION_SUMMARY            	group (30 ppt): Samples (L1–L6) were maintained under normal salinity
CO:COLLECTION_SUMMARY            	conditions (30 parts per thousand, ppt). High salinity group (80 ppt): Samples
CO:COLLECTION_SUMMARY            	(H1–H6) were exposed to hypersaline conditions (80 ppt) to induce
CO:COLLECTION_SUMMARY            	diapause-related metabolic changes. For each group, six biological replicates
CO:COLLECTION_SUMMARY            	were collected. The brine shrimp were maintained in controlled laboratory
CO:COLLECTION_SUMMARY            	conditions, and after exposure, samples were quickly harvested and processed as
CO:COLLECTION_SUMMARY            	follows: Sample collection: Whole brine shrimp were homogenized in liquid
CO:COLLECTION_SUMMARY            	nitrogen. Preservation: Homogenized samples were stored at -80°C until NMR
CO:COLLECTION_SUMMARY            	analysis. The collected samples were subjected to 1H-NMR analysis to identify
CO:COLLECTION_SUMMARY            	and quantify the metabolic changes under hypersalinity stress. The raw NMR data
CO:COLLECTION_SUMMARY            	files are named as L1_Proton.jdf–L6_Proton.jdf (30 ppt) and
CO:COLLECTION_SUMMARY            	H1_Proton.jdf–H6_Proton.jdf (80 ppt).
CO:SAMPLE_TYPE                   	Whole animals
CO:STORAGE_CONDITIONS            	Described in summary
#TREATMENT
TR:TREATMENT_SUMMARY             	Brine shrimp samples were divided into two treatment groups to investigate the
TR:TREATMENT_SUMMARY             	metabolic responses under different salinity conditions: Control group (30 ppt):
TR:TREATMENT_SUMMARY             	Brine shrimp were maintained under normal salinity conditions of 30 parts per
TR:TREATMENT_SUMMARY             	thousand (ppt) as the control group. Hypersalinity group (80 ppt): Brine shrimp
TR:TREATMENT_SUMMARY             	were exposed to hypersaline conditions of 80 parts per thousand (ppt) to induce
TR:TREATMENT_SUMMARY             	a physiological and metabolic response related to diapause.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Brine shrimp samples were prepared for 1H-NMR-based metabolomics using the
SP:SAMPLEPREP_SUMMARY            	following procedures: Sample Collection and Treatment: Brine shrimp were exposed
SP:SAMPLEPREP_SUMMARY            	to two salinity conditions: 30 ppt (control group) for ovoviviparity. 80 ppt
SP:SAMPLEPREP_SUMMARY            	(hypersalinity group) for oviparity. After 5 days of treatment, adult female
SP:SAMPLEPREP_SUMMARY            	brine shrimp were collected, ensuring the presence of white granular eggs (30
SP:SAMPLEPREP_SUMMARY            	ppt) and green stringy cysts (80 ppt). Sample Homogenization and Extraction: Ten
SP:SAMPLEPREP_SUMMARY            	females per group were homogenized in 1 mL of pre-chilled 50% methanol at 4°C
SP:SAMPLEPREP_SUMMARY            	to quench metabolic activity. The homogenates were centrifuged at 13,000 × g
SP:SAMPLEPREP_SUMMARY            	for 20 minutes at 4°C. The resulting supernatants were collected for further
SP:SAMPLEPREP_SUMMARY            	processing. Lyophilization and Reconstitution: The supernatants were lyophilized
SP:SAMPLEPREP_SUMMARY            	(freeze-dried) to remove water. Lyophilized samples were reconstituted in 600
SP:SAMPLEPREP_SUMMARY            	µL of 100 mM sodium phosphate buffer prepared in D₂O (pH 7.0), containing 1
SP:SAMPLEPREP_SUMMARY            	mM DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) as the internal reference
SP:SAMPLEPREP_SUMMARY            	for NMR analysis. Sample Preparation for NMR Analysis: The reconstituted samples
SP:SAMPLEPREP_SUMMARY            	were transferred to 5-mm NMR tubes for analysis. Samples were stored at -80°C
SP:SAMPLEPREP_SUMMARY            	until NMR data acquisition.
SP:EXTRACT_STORAGE               	Described in summary
#ANALYSIS
AN:DATA_FORMAT                   	jdf
#NMR
NM:INSTRUMENT_NAME               	JEOL-ECZS400
NM:INSTRUMENT_TYPE               	FT-NMR
NM:NMR_EXPERIMENT_TYPE           	1D-1H
NM:STANDARD_CONCENTRATION        	100 mM
NM:SPECTROMETER_FREQUENCY        	400 MHz
NM:NMR_SOLVENT                   	sodium phosphate buffer in D₂O
NM:NMR_TUBE_SIZE                 	5 mm
#NMR_METABOLITE_DATA
NMR_METABOLITE_DATA:UNITS	uM
NMR_METABOLITE_DATA_START
Samples	L1	L2	L3	L4	L5	L6	H1	H2	H3	H4	H5	H6
Factors	Salinity:30 ppt | Sample source:Brine shrimp	Salinity:30 ppt | Sample source:Brine shrimp	Salinity:30 ppt | Sample source:Brine shrimp	Salinity:30 ppt | Sample source:Brine shrimp	Salinity:30 ppt | Sample source:Brine shrimp	Salinity:30 ppt | Sample source:Brine shrimp	Salinity:80 ppt | Sample source:Brine shrimp	Salinity:80 ppt | Sample source:Brine shrimp	Salinity:80 ppt | Sample source:Brine shrimp	Salinity:80 ppt | Sample source:Brine shrimp	Salinity:80 ppt | Sample source:Brine shrimp	Salinity:80 ppt | Sample source:Brine shrimp
5,6-Dihydrouracil	10.546	7.583	13.294	9.572	14.361	9.583	35.736	27.715	37.792	26.734	31.782	28.748
Betaine	8.436	18.953	3.629	23.741	5.318	13.267	60.245	50.248	70.248	40.247	65.245	45.243
Malate	10.539	7.571	13.417	9.684	14.935	9.528	14.139	13.647	14.676	12.854	14.368	13.592
Methylamine	16.843	12.965	6.329	18.491	8.157	14.324	35.489	20.483	40.426	22.472	30.499	18.491
Methylguanidine	10.231	10.185	9.716	10.398	9.851	10.541	10.874	10.514	10.924	10.763	10.648	11.382
N-Acetylaspartate	14.729	10.592	8.315	11.837	8.594	12.743	20.674	16.672	21.639	15.641	17.678	18.691
Succinate	13.729	11.486	9.427	14.518	8.193	12.659	19.948	16.935	18.951	15.969	17.995	14.943
Trimethylamine	25.783	5.394	20.148	8.273	13.469	7.615	32.593	17.561	37.571	20.573	25.519	19.523
β-Alanine	10.582	8.239	5.917	15.389	6.789	11.591	26.493	38.435	25.342	18.624	29.578	17.769
Trehalose	12.395	9.681	11.293	9.582	11.539	9.673	14.769	11.746	14.297	12.273	12.786	13.769
Leucine	10.500	8.200	5.000	15.300	6.700	11.500	4.543	5.234	3.912	4.123	4.678	4.321
Valine	10.214	9.954	11.543	8.932	13.654	7.921	6.214	7.234	5.923	6.543	7.012	6.985
Glycine	10.500	7.500	13.000	9.000	14.000	9.500	5.432	6.214	4.945	6.543	5.734	6.123
Glucose	10.987	9.654	11.432	8.213	13.214	7.934	4.543	3.923	4.112	3.723	4.432	4.218
Glycogen	9.543	10.432	10.987	8.345	12.654	7.954	7.123	6.823	7.912	7.412	7.012	7.523
Procollagen trans-4-hydroxy-L-proline	18.000	5.000	22.000	3.000	16.000	6.000	10.500	1.500	9.500	6.500	6.500	5.000
Trigonelloside C	20.145	18.923	22.156	19.523	21.034	19.876	8.934	7.134	9.423	8.534	8.034	9.245
alpha-Cyperone	15.312	14.245	16.843	17.534	13.923	15.934	5.723	6.034	5.214	5.923	6.312	6.843
2,4-Dichloro-2,5-dihydro-5-oxofuran-2-acetate	10.156	9.812	11.045	10.523	10.934	9.723	14.934	15.423	14.023	16.213	15.823	16.523
O-Methyl-scyllo-inositol	10.213	10.823	9.923	11.213	10.523	9.723	17.523	16.823	18.213	17.123	16.523	18.923
(R)-DNPA	10.500	8.200	5.000	15.300	6.700	11.500	26.400	38.400	25.300	18.600	29.500	17.700
Primary diamine	10.023	10.523	9.723	10.213	11.123	10.923	14.523	15.023	15.234	16.034	14.923	15.823
8-Methylthiooctyl-desulfoglucosinolate	10.034	9.543	10.523	11.213	10.834	10.143	13.534	14.234	13.034	14.523	12.834	14.123
UDP-apiose	15.345	14.823	16.134	15.923	14.234	16.034	7.534	8.534	7.923	7.034	7.234	8.123
Fisetin	12.034	11.523	13.245	10.934	14.034	11.834	4.134	5.034	4.823	5.534	4.734	5.234
Isodomoic acid A	35.245	33.134	40.523	32.012	30.934	36.034	9.234	8.034	10.134	8.734	9.534	7.834
Monardaein	10.543	10.923	9.823	11.134	10.234	10.734	6.523	7.034	6.213	7.134	6.834	6.323
Taraxasterol	10.034	9.723	10.823	10.213	9.923	10.143	5.423	5.834	5.023	5.323	5.623	6.234
Anhydrochlortetracycline	10.134	10.423	9.823	11.034	10.234	9.923	11.234	12.534	10.934	11.423	11.534	12.034
Alcophosphamide	10.023	10.523	9.723	10.213	11.123	10.923	14.523	15.023	15.234	16.034	14.923	15.823
1,4-Naphthoquinone	12.543	11.834	13.023	12.934	14.123	12.034	4.034	5.134	4.523	4.834	4.234	5.034
1-Deoxypentalenate	5.012	4.523	5.234	6.123	5.523	4.823	24.034	21.523	22.834	23.923	20.123	25.323
Virol B	10.234	10.834	9.923	10.423	10.123	9.723	9.534	9.923	10.234	10.834	9.323	10.123
Cefotiam	12.034	11.523	13.245	11.034	11.834	12.123	4.134	5.034	4.923	5.534	4.734	5.234
Protein L-isoaspartate	10.012	9.923	10.834	9.523	11.234	10.523	12.534	13.034	11.923	12.123	12.834	13.534
Hydroxysteroid	10.123	10.723	9.623	11.023	10.523	9.923	17.934	18.534	16.823	19.213	17.523	18.834
N-Acetyl-O-acetylneuraminate	10.523	9.923	10.123	10.834	10.234	10.012	4.213	3.923	4.523	4.034	4.123	3.723
Cycloheximide	10.234	10.923	9.823	9.523	11.034	10.423	4.934	5.534	5.023	4.734	5.234	4.423
NMR_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	PubChem ID	KEGG ID
5,6-Dihydrouracil	649	C00429
Betaine	247	C00318
Malate	525	C00149
Methylamine	6329	C00218
Methylguanidine	10111	C02294
N-Acetylaspartate	65065	C01042
Succinate	160419	C00042
Trimethylamine	1146	C00565
β-Alanine	239	C00099
Trehalose	7427	C00689
Leucine	6106	C00123
Valine	6287	C00141
Glycine	750	C00037
Glucose	5793	C00029
Glycogen	439177	C00182
Procollagen trans-4-hydroxy-L-proline	7037	C04398
Trigonelloside C	441899	C08917
alpha-Cyperone	6452086	C17090
2,4-Dichloro-2,5-dihydro-5-oxofuran-2-acetate	440410	C04625
O-Methyl-scyllo-inositol	439990	C05163
(R)-DNPA	136120795	C18835
Primary diamine	75408	C02311
8-Methylthiooctyl-desulfoglucosinolate	49859709	C21746
UDP-apiose	439536	C01623
Fisetin	5281614	C01378
Isodomoic acid A	6442601	C20027
Monardaein	11979367	C08723
Taraxasterol	115250	C08636
Anhydrochlortetracycline	54693278	C06626
Alcophosphamide	98612	C16551
1,4-Naphthoquinone	8530	C21527
1-Deoxypentalenate	70678894	C20404
Virol B	10753985	C19994
Cefotiam	43708	C12861
Protein L-isoaspartate	6161	C03306
Hydroxysteroid	235496	C01058
N-Acetyl-O-acetylneuraminate	11953823	C03708
Cycloheximide	6197	C06685
METABOLITES_END
#END