Summary of study ST001721

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001103. The data can be accessed directly via it's Project DOI: 10.21228/M84988 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST001721
Study TitleDetecting sex-related changes to the metabolome of a critically endangered freshwater crayfish during the mating season
Study TypeLC-MS analysis of crustacean haemolymph
Study SummaryCaptive breeding is a vital tool in the conservation of highly endangered species, as it is for the Margaret River hairy marron, Cherax tenuimanus, from the south west of Australia. A close relative, Cherax cainii, has almost completely displaced C. tenuimanus in the wild and is a successful aquaculture species, whereas C. tenuimanus has performed poorly in captivity. We used untargeted liquid chromatography-mass spectrometry to obtain metabolomic profiles of female and male C. tenuimanus held in controlled aquarium conditions during their reproductive period. Using repeated haemolymph sampling we tracked the metabolomic profiles of animals just prior to and for a period of up to 34 days after pairing with a similar sized potential mate. We identified 54 reproducible annotated metabolites including amino acids, fatty acids, biogenic amines, purine and pyrimidine metabolites and excretion metabolites. Hierarchical clustering analysis distinguished five metabolite clusters. Principal component-canonical variate analysis clearly distinguished females from males, both unpaired and paired; similar trends in profile changes in both sexes after pairing; and a striking shift in males upon pairing. We discuss three main patterns of metabolomic responses: differentiation between sexes; reactive responses to the disturbance of pairing; and convergent response to the disturbance of pairing for males. Females generally had higher concentrations of metabolites involved in metabolic rate, mobilisation of energy stores and stress. Responses to the disturbance of pairing were also related to elevated stress. Females were mobilising lipid stores to deposit yolk, whereas males had a rapid and strong response to pairing, with shifts in metabolites associated with gonad development and communication, indicating males could complete reproductive readiness only once paired with a female. The metabolomic profiles support a previously proposed potential mechanism for displacement of C. tenuimanus by C. cainii in the wild and identify several biomarkers for testing hypotheses regarding reproductive success using targeted metabolomics.
Institute
Edith Cowan University
DepartmentSchool of Science
Last NameLette
First NameEmily
Address270 Joondalup Drive, Joondalup, WA, 6027, Australia
Emaile.lette@ecu.edu.au
Phone+61 8 6304 5513
Submit Date2021-02-25
Total Subjects10
Num Males5
Num Females5
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2021-03-17
Release Version1
Emily Lette Emily Lette
https://dx.doi.org/10.21228/M84988
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:


Project:

Project ID:PR001103
Project DOI:doi: 10.21228/M84988
Project Title:Detecting sex-related changes to the metabolome of a critically endangered freshwater crayfish during the mating season
Project Type:MS analysis of crustacean haemolymph
Project Summary:Captive breeding is a vital tool in the conservation of highly endangered species, as it is for the Margaret River hairy marron, Cherax tenuimanus, from the south west of Australia. A close relative, Cherax cainii, has almost completely displaced C. tenuimanus in the wild and is a successful aquaculture species, whereas C. tenuimanus has performed poorly in captivity. We used untargeted liquid chromatography-mass spectrometry to obtain metabolomic profiles of female and male C. tenuimanus held in controlled aquarium conditions during their reproductive period. Using repeated haemolymph sampling we tracked the metabolomic profiles of animals just prior to and for a period of up to 34 days after pairing with a similar sized potential mate. We identified 54 reproducible annotated metabolites including amino acids, fatty acids, biogenic amines, purine and pyrimidine metabolites and excretion metabolites. Hierarchical clustering analysis distinguished five metabolite clusters. Principal component-canonical variate analysis clearly distinguished females from males, both unpaired and paired; similar trends in profile changes in both sexes after pairing; and a striking shift in males upon pairing. We discuss three main patterns of metabolomic responses: differentiation between sexes; reactive responses to the disturbance of pairing; and convergent response to the disturbance of pairing for males. Females generally had higher concentrations of metabolites involved in metabolic rate, mobilisation of energy stores and stress. Responses to the disturbance of pairing were also related to elevated stress. Females were mobilising lipid stores to deposit yolk, whereas males had a rapid and strong response to pairing, with shifts in metabolites associated with gonad development and communication, indicating males could complete reproductive readiness only once paired with a female. The metabolomic profiles support a previously proposed potential mechanism for displacement of C. tenuimanus by C. cainii in the wild and identify several biomarkers for testing hypotheses regarding reproductive success using targeted metabolomics.
Institute:Edith Cowan University
Department:School of Science
Last Name:Lette
First Name:Emily
Address:270 Joondalup Drive, Joondalup, WA, 6027, Australia
Email:e.lette@ecu.edu.au
Phone:+61 8 6304 5513

Subject:

Subject ID:SU001798
Subject Type:Invertebrate
Subject Species:Cherax tenuimanus
Taxonomy ID:99755
Weight Or Weight Range:116g-198g
Gender:Male and female

Factors:

Subject type: Invertebrate; Subject species: Cherax tenuimanus (Factor headings shown in green)

mb_sample_id local_sample_id Sex Timepoint
SA162029FH4_1Female 1
SA162030FH5_1Female 1
SA162031FH3_1Female 1
SA162032FH1_1Female 1
SA162033FH2_1Female 1
SA162034FH2_2Female 2
SA162035FH4_2Female 2
SA162036FH1_2Female 2
SA162037FH3_2Female 2
SA162038FH5_2Female 2
SA162039FH5_3Female 3
SA162040FH3_3Female 3
SA162041FH1_3Female 3
SA162042FH2_3Female 3
SA162043FH4_3Female 3
SA162044FH5_4Female 4
SA162045FH3_4Female 4
SA162046FH2_4Female 4
SA162047FH4_4Female 4
SA162048FH1_4Female 4
SA162049MH4_1Male 1
SA162050MH2_1Male 1
SA162051MH3_1Male 1
SA162052MH1_1Male 1
SA162053MH5_1Male 1
SA162054MH5_2Male 2
SA162055MH4_2Male 2
SA162056MH3_2Male 2
SA162057MH2_2Male 2
SA162058MH1_2Male 2
SA162059MH1_3Male 3
SA162060MH2_3Male 3
SA162061MH5_3Male 3
SA162062MH4_3Male 3
SA162063MH3_3Male 3
SA162064MH3_4Male 4
SA162065MH2_4Male 4
SA162066MH4_4Male 4
SA162067MH5_4Male 4
SA162068MH1_4Male 4
SA162022QC11- -
SA162023QC9- -
SA162024QC14- -
SA162025QC12- -
SA162026QC13- -
SA162027QC15- -
SA162028QC10- -
Showing results 1 to 47 of 47

Collection:

Collection ID:CO001791
Collection Summary:All haemolymph collections for all animals occurred at the same time of day and the animals were handled in the same order on each of the four collection dates. Haemolymph (200mL) was extracted from the ventral sinus of each crayfish using a 21G needle and 1mL syringe inserted into the soft tissue at the base of the 5th pereopod. Haemolymph was added to 2mL Eppendorf tubes preloaded with 600µL LC-MS grade acetonitrile (Optima, Thermo Fisher Scientific, AUS) containing deuterated internal standards (d8- valine, d9 –trimethylamine-N-oxide (TMAO) , d3-leucine, d6-trans-cinnamic acid, d5-tryptophan, 1g/mL) from Cambridge Isotope Laboratories (Cambridge, MA, USA) and stored on ice. Samples were mixed at 1400rpm (Thermal Mixer, Thermo Fisher Scientific, AUS) for 60 seconds at 4°C, then centrifuged (Heraeus Megafuge 8R, Thermo Fisher Scientific, AUS) for 20 minutes (1800  g) at 4°C. After centrifuging, 100µL of supernatant was aliquoted into five separate vials. The samples were then dried using a SpeedVac centrifugal vacuum concentrator (Thermo Fisher Scientific, AUS). The dried samples were stored at -80°C for subsequent metabolomics analysis.
Collection Protocol Filename:EmilyLette_20210225_014138_PR_CO_Methods__Lette_marron.pdf
Sample Type:Hemolymph
Collection Method:aspiration
Collection Location:ventral sinus accessed from soft tissue at base of 5th pereopod
Storage Conditions:-80℃
Collection Vials:2mL Eppendorf tubes
Storage Vials:2mL Eppendorf tubes

Treatment:

Treatment ID:TR001811
Treatment Summary:Cherax tenuimanus (n=10) were housed in aquaria as individuals prior to sampling at timepoint 1. After timepoint 1 they were placed in pairs with a potential mate of the opposite sex and housed in aquaria over the next four weeks which included timepoints 2-4.

Sample Preparation:

Sampleprep ID:SP001804
Sampleprep Summary:The dried haemolymph samples were reconstituted using 100µL of LC-MS water containing 0.1% Formic acid. Samples were manually swirled, then placed in a thermomixer for 2 minutes at 4oC, before being centrifuged at (1800  g) for 5 minutes at 4oC. Next, 40µL of the supernatant was transferred into LC-MS amber vials with inserts and placed in the autosampler kept at 6oC. The order in which samples were analysed was randomised to avoid any potential instrument bias. A pooled quality control (QC) sample was prepared by adding 40 µL from each reconstituted sample to a single Eppendorf tube, which was then mixed to homogenise in a thermal mixer and centrifuged as above. This pooled sample was aliquoted (40µL) into LC-MS amber vials to create 16 QC samples and placed into the autosampler tray kept at 6°C ready for analysis. Samples were analysed within 24 hours from preparation. At the start of the analytical batch, a solvent blank, matrix blank, and ten conditioning QC samples were analysed (Broadhurst et al., 2018). QC samples were then injected after every fifth marron haemolymph sample with two QCs analysed at the end of the batch, following the standard protocols for metabolomics (Broadhurst et al., 2018).
Processing Storage Conditions:Described in summary

Combined analysis:

Analysis ID AN002804 AN002805
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Dionex Ultimate 3000 RS Thermo Dionex Ultimate 3000 RS
Column Thermo Hypersil GOLD C18 (100 x 2.1mm,1.9µm) Thermo Hypersil GOLD C18 (100 x 2.1mm,1.9µm)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE
Units peak area values peak area values

Chromatography:

Chromatography ID:CH002072
Methods Filename:Methods_Lette_marron
Instrument Name:Thermo Dionex Ultimate 3000 RS
Column Name:Thermo Hypersil GOLD C18 (100 x 2.1mm,1.9µm)
Column Temperature:45
Flow Gradient:isocratic at 99% A: 1 min; 50% B:1-2 min; linear increase to 99% B: over 7min; 99% B: 2min; initial conditions returned over 2 min; 100% A: 3 min.
Flow Rate:0.3mL/min
Solvent A:0.1% formic acid in LC-MS water
Solvent B:0.1% formic acid in LC-MS acetonitrile
Chromatography Type:Reversed phase

MS:

MS ID:MS002599
Analysis ID:AN002804
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Full scans with data-dependent tandem mass spectrometry were acquired on the Orbitrap mass analyzer. Full scans were acquired at a resolution of 70,000 at mass-to-charge ratio (m/z) 200 over the m/z range 70–1000 with the ESI conditions as follows: source heater = 350°C, sheath gas = 35 (arbitrary units), auxiliary gas = 10 (arbitrary units), capillary temperature 350°C, ion spray voltage = 3.0 kV (positive ion mode) and 2.5 kV (negative ion mode), S-lens 50%, and automatic gain control = 110¬¬¬-6. Tandem mass spectrometry experiments were performed at a resolution of 17,500 at m/z 200 on each sample with the higher energy collisional dissociation energy set at 20 eV. Data acquisition was carried out using Xcalibur software (Thermo Fisher Scientific). Before analysis, the Orbitrap was externally calibrated using ready-made calibration solutions (ESI-negative ion calibration and ESI-positive ion calibration solutions) obtained from Thermo Fisher Scientific.
Ion Mode:POSITIVE
  
MS ID:MS002600
Analysis ID:AN002805
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Full scans with data-dependent tandem mass spectrometry were acquired on the Orbitrap mass analyzer. Full scans were acquired at a resolution of 70,000 at mass-to-charge ratio (m/z) 200 over the m/z range 70–1000 with the ESI conditions as follows: source heater = 350°C, sheath gas = 35 (arbitrary units), auxiliary gas = 10 (arbitrary units), capillary temperature 350°C, ion spray voltage = 3.0 kV (positive ion mode) and 2.5 kV (negative ion mode), S-lens 50%, and automatic gain control = 110¬¬¬-6. Tandem mass spectrometry experiments were performed at a resolution of 17,500 at m/z 200 on each sample with the higher energy collisional dissociation energy set at 20 eV. Data acquisition was carried out using Xcalibur software (Thermo Fisher Scientific). Before analysis, the Orbitrap was externally calibrated using ready-made calibration solutions (ESI-negative ion calibration and ESI-positive ion calibration solutions) obtained from Thermo Fisher Scientific.
Ion Mode:NEGATIVE
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