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 Freshwater Crayfish 25(1):89–101, 2020
R E S E A R C H A RT I C L E ISSN: 2076-4324 (Print), 2076-4332 (Online)
https://doi.org/10.5869/fc.2020.v25-1.089

Metabolomic Profiling of Crayfish Haemolymph

Distinguishes Sister Species and Sex: Implications for
Conservation, Aquaculture and Physiological Studies
Emily D. Lette,¹,* Nathan G. Lawler,² Quinton F. Burnham,¹ Mary C. Boyce,1,2
Rodney Duffy,3 Annette Koenders 1 and David I. Broadhurst 2
1
Centre for Ecosystem Management, School of Science, Edith Cowan University, 270 Joondalup Drive, Joondalup, WA 6027 Australia.
*Corresponding Author.— e.lette@ecu.edu.au
2
Centre for Integrative Metabolomics & Computational Biology, School of Science, Edith Cowan University, Australia.
3
Department of Primary Industries and Regional Development, State Government of Western Australia, Australia.

A B S T R A C T A R T I C L E I N F O
Hairy marron, Cherax tenuimanus (Smith) are critically endangered freshwater crayfish found only in Article History:
a single river in southwest Australia. Conservation efforts have included a captive breeding program, Submitted: 3 May 2019
which has been largely unsuccessful, despite the closely related smooth marron, Cherax cainii Austin, Accepted: 07 JAN 2020
being successfully bred for aquaculture. Using an untargeted liquid chromatography-mass spectrometry Published Online: 15 APR 2020
(LC-MS) metabolomic approach we created a profile of the metabolites in the haemolymph for males Published Print: 30 APR 2020
and females of the two species of marron. A non-lethal method was used to collect haemolymph and
84 reproducible annotated metabolites were identified. Variation in the levels of some metabolites were Keywords:
detected between species and between sexes within species. Multivariate analyses clearly differentiated Cherax cainii;
the congeneric species and univariate analyses identified differences between species, sex, and for Cherax tenuimanus;
some metabolite interactions, between species and sex. This study created a baseline metabolome LC-MS;
dataset for the two species and began to investigate the biological significance of metabolites that varied marron;
between species. We have shown metabolomics could be used for targeted studies to potentially assist metabolome;
reproductive success. This approach will be beneficial for conservation and aquaculture practices with metabolomics;
potential applications for other aquatic taxa worldwide. species conservation;

Copyright © 2020 by The Author(s). Published by the International Association of Astacology.

This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION Recovery Program requires a captive breeding program as part of

Hairy marron, Cherax tenuimanus (Smith, 1912) are large
freshwater crayfish endemic to a single river, Margaret River
(33.9610° S, 115.0767° E, WGS-84), in the southwest of Western
Australia (Bunn et al. 2008) and are currently listed as critically
endangered on the IUCN red list (Austin and Bunn 2010). With only
approximately 500 animals in the wild the species is considered to
be on the brink of extinction (Duffy and Day 2015). The decline of
C. tenuimanus is correlated with the presence of the closely related
sister species smooth marron, Cherax cainii Austin, 2002 (Bunn
et al. 2008), which are widespread throughout southwest Western
Australia (their range having been expanded by human-mediated
translocation), and they are a common aquaculture species
(Morrissy 1992). Possible mechanisms for the replacement of C.
tenuimanus by C. cainii in the Margaret River include competition
and displacement by C. cainii and hybridization with C. cainii
(Bunn et al. 2008; Kennington et al. 2014). The Hairy Marron
89
the conservation effort, however, C. tenuimanus captive breeding
rates are low and declining (Duffy and Day 2015), despite C.
cainii readily breeding in captivity. Traditionally, improvement
to the methods for the captive breeding of crayfish rely upon
investigating the impact of temperature, light, nutrition, water
quality, stocking densities, etc., on growth rate and reproductive
success (Morrissy 1996; Lawrence 2007) with little understanding
of the actual physiological state of the animals (Viant 2007; Patti et
al. 2012; Alfaro and Young 2016; Young and Alfaro 2018).
The physiological state of an animal has an impact on its
metabolome, which can consist of over 6,000 metabolites (e.g.,
amino acids, organic acids, nucleic acids, fatty acids, amines,
sugars, vitamins, co-factors, pigments, and antibiotics), as it is
affected by both natural factors (including age, diet, diseases,
hormones, and stress) and a wide variety of foreign chemical
substances (xenobiotics) (Aizen et al. 2018). Metabolomics is
90 Freshwater Crayfish Volume 25, Number 1
Table 1. Details of the two Cherax species from which haemolymph was extracted for
metabolomic analyses. Mean occipital carapace length (OCL in mm) and mean weight (Weight
in g) both +/- standard deviation (SD).
Species Sex n OCL (mm)
C. tenuimanus female 5 56.64
C. tenuimanus male 5 67.94
C. cainii female 5 60.71
C. cainii male 5 62.74
a novel approach for assessing and monitoring physiological
changes in plants and animals. It has been used for several
species of vertebrates, mainly mammals, and in particular humans
(Dunn et al. 2011) and while there has been an increase in the
application of metabolomics to invertebrates, there are few studies
on aquatic invertebrates (e.g., Alfaro and Young 2016; Schock et
al. 2013; Viant 2007). By analysing the metabolites found within
a biological system, it is possible to conduct a comparison of the
final downstream consequence of gene expression and interaction
with the environment between individuals and species. This
comparative data can be applied within many fields (Schock et
al. 2010), including conservation biology (Lin et al. 2006; Viant
2007), however, it first requires a baseline metabolic profile for
changes to then be detected.
Many different types of biological materials can be used for
metabolomics studies. In studies of humans and other vertebrates
blood, urine or faeces are commonly collected (Miller 2007),
while for small invertebrates, the whole organism can be crushed
or homogenised and used for analysis, and for studies of larger
invertebrates, such as crayfish, samples are often collected from the
hepatopancreas or muscle tissue (Izral et al. 2018). The problem
with using such tissues or whole animals is the invertebrate must
be killed, however it is possible to use haemolymph (Costantini
et al. 2018; Leland and Furse 2012), in a manner that is non-
lethal and repeatable. There are very few metabolomics studies of
freshwater crayfish haemolymph (Costantini et al. 2018; Izral et al.
2018), and none using species in the family Parastacidae, which
includes all of the Southern Hemisphere species.The objective of
the current study was to characterise the metabolomic profile of
the haemolymph of the two Cherax sister species and determine
metabolite signatures that differentiate them and/or highlight
differences between the sexes. Developing a metabolomics
approach to assess the condition of these animals will provide
significant value for both conservation and aquaculture of these
and other similar species. A strength of this approach is that many
metabolites detected in aquatic species are not species specific, so
they can be compared across species without having to develop
new models for each organism (Alfaro and Young 2016).
MATERIALS AND METHODS
Study Organisms
Twenty marron (five female and five male C. tenuimanus,
five female and five male C. cainii) that were approximately size-
matched (Table 1) were collected from the captive-bred stock
(2+ years of age) at the Department of Primary Industries and
+/- SD (mm) Weight (g) +/- SD (g)
3.67 116.13g 20.43
7.09 197.94g 48.05
2.56 154.83g 25.20
5.90 180.70 49.98
Regional Development (DPIRD) Freshwater Research Facility
in Pemberton, Western Australia and were transferred to Edith
Cowan University in Joondalup, Western Australia. The crayfish
were housed in an aquarium room (3.5 m × 7.5 m) with controlled
light and temperature. Animals were kept separately, in non-
recirculating glass aquaria with glass lids (L 350 mm × W 200 mm
× H 230 mm, total water volume ~16 L), filled with dechlorinated
tap water. An airlift biofilter in each tank provided aeration, and
mechanical and biological filtration. Each aquarium was lined with
clean gravel (3–7 mm) and shell grit as substrate, and sections of
stormwater grade polyvinylchloride (PVC) tubes (L 200 mm ×
W 90 mm) as hides. To minimise disturbance to the animals but
still allow for observations, opaque laminated sheets were placed
between aquaria ensuring that three sides were covered, thus
visually isolating each aquarium.
The temperature of the water at the time of haemolymph
collection was 19.5°C, air temperature in the room 18°C, and
photoperiod was simulated to represent spring day length (12hr
light: 12hr dark). Water temperature, pH, NO2, NO3 and NH3/
NH4 were monitored throughout the trial and maintained within
recommended guidelines (Johnston and Jungalwalla 2005), with
10–25% water changes as required. To ensure optimal nutrition,
marron were fed a combination of food including: Skrettings Nova
ME 3mm marine Fish Pellet, New Life Optimum Freshwater
Flakes + garlic and Tropical® Spirulina Super Forte Granulat with
feed rates at 3% of body weight weekly. Excess uneaten food
and other solid waste was removed with a net. Crayfish were
acclimatised in aquaria in these conditions for eight weeks prior
to sample collection.
Sample Collection and Storage
On the 27th of October 2017, haemolymph samples were
collected from C. cainii and C. tenuimanus (20 animals, five
replicates for each species and sex). Haemolymph was drawn from
the ventral sinus of each crayfish using a 21G needle and 1 mL
syringe inserted into the soft tissue at the base of the 5th pereopod
(Leland and Furse 2012). Haemolymph (200 µL) was added to
2 mL Eppendorf tubes preloaded with 600 µL 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  µg·mL-1) Cambridge Isotope Laboratories (Cambridge, MA,
USA) and stored on ice. At the time of haemolymph extraction
each Eppendorf tube was placed on an analytical balance (Sartorius
BP210S) and 0.200 grams (± 10%) of haemolymph was added.
Tubes were capped and shaken immediately to prevent clotting
2019 Lette et al. — Metabolomic Profiling of Marron Haemolymph 91
and then placed on ice. Samples were mixed at 1400 rpm (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,
100 µL 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.
Sample Preparation for Metabolomic Analysis
The dried haemolymph samples were reconstituted using
100 µL of LC-MS water containing 0.1% Formic acid. Samples
were manually swirled, then placed in a thermomixer for 2 min at
4°C, before being centrifuged at (1800 × g) for 5 min at 4°C. Next,
40 µL of the supernatant was transferred into LC-MS amber vials
with inserts and placed in the autosampler kept at 6°C. 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 (40 µL) 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).
Liquid chromatography-mass spectrometry
All samples were analysed using an Ultra High-Pressure
Liquid Chromatography pump (Dionex UltiMate 3000 RS)
coupled to an Orbitrap Q-Exactive mass spectrometer (Thermo
Fisher Scientific) fitted with a heated electrospray ionisation probe
(HESI). Metabolites were separated on a reversed phase Hypersil
GOLD column (100 × 2.1 mm, 1.9 µm particle size; Thermo Fisher
Scientific) with an in-line filter. Sample analysis in both positive
and negative ionization modes was performed using 0.1% formic
acid in LC-MS water (solvent A) and 0.1% formic acid in LC-
MS acetonitrile (solvent B). The elution gradient was as follows:
isocratic at 99% solvent A for 1 min, followed by an increase to
50% solvent B (1–2 min) then a linear increase to 99% solvent B
over 7 min, which was maintained at 99% solvent B for 2 min.
Initial conditions were returned over 2 min and then held at 100%
solvent A to equilibrate for 3 min. The flow rate was 0.3 mL·min-1
for positive and negative; injection volume was 10 µL and column
oven temperature was 45°C.
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.
Data preprocessing
Raw spectral data were preprocessed by Compound Discoverer
3.0 software (Thermo Fisher Scientific) using the standard
untargeted metabolomic workflow. Compound Discoverer was
setup to align total ion chromatograms along retention time using
an adaptive curve, with a maximum shift of 0.5 min and 5 ppm
mass tolerance. Detected features with an intensity of no less than
1,000,000 and a signal-to-noise ratio greater than 5 in each set of
data were extracted and merged into components according to ion
adducts. Compounds detected in the blank samples were deleted
from the final data matrices. Metabolite data from both positive
and negative ionization modes were combined into a single data
matrix. For each metabolite, relative standard deviations were
calculated for the pooled QC injections (RSDQC), and for the
total sample variance (RSDSample), following standard protocols
(Broadhurst et al. 2018) metabolites with RSDQC >20%, or a ratio
RSDQC /RSDSample > 30%, were considered to be below accepted
quantification precision and removed from further statistical
analyses.
Metabolite identification
Before statistical analyses, metabolites were annotated by
matching accurate mass and mass spectrum fragmentation patterns
to an in-house MS/MS spectral database and the mzCloud online
spectral library (https://www.mzcloud.org/) and scored according
to the Metabolomics Standards Initiative (MSI) reporting protocol
(Sumner et al. 2007). A definitive match (MSI level 1) indicates
that identification was confirmed to an in-house authentic standard
using retention time. A putative match (MSI level 2) indicates that
the mass spectra were matched to mzCloud online MS/MS database
but without in-house authentication. An MSI level 3 match implies
most probable physiochemical class and name, based on reported
molecular weight and retention time.
Data modelling and statistical analysis
For each identified metabolite in turn, a two-way ANOVA was
conducted to examine the effects of sex and species on metabolite
concentration. Data are reported as p-value for each main effect
and interaction. If there was a significant interaction between sex
and species, then an analysis of simple main effects was performed
(i.e. the effect of species on metabolite concentrations analysed
for the males and females separately). Correction for multiple
comparisons was performed using the method described by
Benjamini & Hochberg (1995) and corrected p-values (q-values)
also reported.
All identified metabolites were then combined into a single data
matrix and the multivariate covariance analysed using Principal
Components Analysis (PCA) (Jolliffe 2002). Here, the highly
92 Freshwater Crayfish Volume 25, Number 1
Figure 1. Principal component analysis (PCA) plot showing A. PC1 against
PC2 and B. PC1 against PC3 for identified metabolites in the haemolymph
of Cherax tenuimanus and Cherax cainii. Each point represents a single
sample: blue squares, C. cainii (smooth marron) males; blue triangles,
C. cainii (smooth marron) females; yellow squares, C. tenuimanus (hairy
marron) males; yellow triangles, C. tenuimanus (hairy marron) females;
red circles, quality control). The mean (x) and 95% confidence interval
(CI), solid line around x, are presented for each group. The dashed lines
indicate 95% CI of the population. Significant differences are determined
when 95% CI do not overlap.
multidimensional metabolomic data is projected into (usually)
a two- or three-dimensional subspace (principal components)
describing the maximal orthogonal covariance in the data. The
results are presented as a principal component scores plot, with
multivariate Chi-squared 95% confidence intervals plotted for the
mean of each sample group. Non-overlapping confidence intervals
indicate significantly different covariance in the presented
components. Following standard protocols, bootstrap resampling
of each principal component loading (linear sum contribution of
all metabolites to that component) was then used to determine
metabolite significance for the variance described by each principal
component (Hastie et al. 2009).
Hierarchical cluster analysis (HCA) (Kaufman and
Rousseeuw 1990; Hastie et al. 2009) was then performed to assess
the similarities between individual metabolite concentrations.
This algorithm used a multivariate Euclidean distance metric and
Ward’s group linkage. The results were displayed as a circular
dendrograms plot, where the lower the linkage distances in the
dendrogram the more similar the feature. Metabolites that were
most similar across all samples form the lowest linkage in the
respective dendrograms; thus, emergent coloured clusters indicate
similar characteristics. Prior to PCA and HCA each metabolite
feature was scaled to unit variance (autoscaled), which allows each
metabolite to be compared within the analysis with no bias due to
differences in absolute concentration variance (van den Berg et al.
2006; Xia et al. 2013). All statistical analysis was performed using
Matlab scripting language version R2018a (Matlab 2018).
RESULTS
The LC-MS analysis of 20 marron haemolymph samples
yielded 84 reproducible annotated metabolites (Table 2). A
two-way ANOVA corrected for multiple comparisons found 27
metabolites showed significant differences between species, 18
metabolites showed significant differences between sexes and three
metabolites showed significant interaction effect between species
and sex (Table 2). Of the three metabolites with a significant
interaction, analysis of simple main effects was performed, and
all three metabolites were found to be significantly different for
both sexes after correction for multiple comparisons (Table 2),
though the combination of species, sex, and direction of change
was different for all three.
The results of PCA (Figure 1A, B) illustrates significant
clustering of multivariate covariance in the first three principal
components (explaining 57% of the total observed variance).
Principal component 1 (PC1) differentiates between the two
species. PC2 shows significant difference between male and
female C. tenuimanus, but not so in C. cainii, and PC3 shows
significant difference between male and female C. cainii, but
not so in C. tenuimanus. The tight cluster of circles at the centre
of both scores plots describes the comparative variance of the
pooled QC samples, which gives a measure of precision for
each of the samples, indicating very high-quality data. Bootstrap
resampling of each PC loading revealed that, 27 metabolites were
significant contributors to PC1 (species profile), 28 metabolites
were significant contributors to PC2 (C. tenuimanus sex profile),
and 21 metabolites were significant contributors to PC3 (C. cainii
sex profile) (Figure 2). The two sex profiles somewhat overlap,
suggesting a multivariate interaction between species and sex that
is not apparent in the univariate statistical analysis.
The circular hierarchical cluster analysis (HCA) dendrogram
illustrates the marron metabolite profile and identified six
metabolite clusters based on phenotypic similarity (labelled
A–F) (Figure 3). Statistical significance from both the univariate
(Table 2) and multivariate analyses (Figure 2) are indicated on the
dendrogram. Cluster A (n = 8 metabolites) contains metabolites
2019 Lette et al. — Metabolomic Profiling of Marron Haemolymph 93

Figure 2. Multivariate analysis loadings plot of metabolites detected in Cherax tenuimanus and Cherax cainii haemolymph. Data are presented
as mean-loading values ± 95% CI, with those coloured red representing the metabolites that contributed significantly to PC1 (differentiating
species), PC2 (differentiating between the sexes within C. tenuimanus), and PC3 (differentiating between the sexes within C. cainii). Metabolites
in black do not contribute significantly. Clusters indicated by letter and shading are based on hierarchical cluster analysis (see Figure 3).
Table 2. Identified metabolites from Cherax tenuimanus and Cherax cainii haemolymph, with significant metabolites based on p value <0.05 and q value <0.05 (FDR-adjusted P values) from
a two-way ANOVA examining the effects of sex and species on metabolite concentration highlighted in bold text. Clusters indicated are based on hierarchical cluster analysis (see Figure 3). 94
Retention time (Rt_min); C18 column either negative or positive (Mode); metabolomics standards initiative level (ID Level); relative standard deviations calculated for the pooled quality control
injections (%RSD); false discovery rate (%D-Ratio). Symbols represent the significant metabolites from the multivariate analysis (Figure 2) where PC1 (*) highlights metabolites that separate
species, PC2 (†) highlights metabolites that separate the sexes in C. tenuimanus, and PC 3 (§) highlights metabolites that separate the sexes in C. cainii.
Molecular RT ID % % Species Sex Species x Sex
Cluster Metabolite Name Formula Mode
Weight min Level RSD D-Ratio p value q value p value q value p value q value
A Docosahexaenoic acid C22 H32 O2 328.240 8.6 C18 Pos ms3 10.2 7.0 0.02 0.06 0.47 0.74 0.36 0.58
A Eicosapentaenoic acid* C20 H30 O2 302.224 8.2 C18 Pos ms3 10.1 7.4 0.02 0.05 0.57 0.83 0.53 0.73
A Creatinine C4 H7 N3 O 113.059 1.1 C18 Pos ms2 3.6 1.1 0.39 0.48 0.11 0.29 0.14 0.33
A Creatine C4 H9 N3 O2 131.070 1.1 C18 Pos ms3 3.3 1.3 0.30 0.44 0.04 0.16 0.18 0.38
A Methylimidazoleacetic acid C6 H8 N2 O2 140.059 1.4 C18 Pos ms1 9.5 1.8 0.98 0.98 0.005 0.03 0.05 0.23
A N-Acetyl-L-leucine† C8 H15 N O3 173.105 3.6 C18 Pos ms3 2.6 1.1 0.12 0.22 0.005 0.03 0.01 0.11
A Urocanic acid§ C6 H6 N2 O2 138.043 1.5 C18 Pos ms3 1.9 0.4 0.49 0.56 <0.001 0.003 0.40 0.60
A Folic acid C19 H19 N7 O6 441.140 3.0 C18 Neg ms3 5.9 16.4 0.42 0.51 0.14 0.35 0.40 0.60
B 4-Indolecarbaldehyde*† C9 H7 N O 145.052 3.9 C18 Neg ms3 2.5 2.7 0.006 0.02 0.36 0.68 0.89 0.95
B Stachydrine* C7 H13 N O2 143.095 1.1 C18 Pos ms3 2.9 3.8 0.001 0.005 0.08 0.23 0.14 0.33
B N2-Methylguanosine* C11 H15 N5 O5 297.107 2.1 C18 Pos ms3 2.7 3.9 <0.001 <0.001 0.95 0.96 0.09 0.29
B Acetylcholine* C7 H15 N O2 145.110 1.1 C18 Pos ms3 1.7 0.8 <0.001 <0.001 0.06 0.20 0.04 0.23
B Pipecolic acid*§ C6 H11 N O2 129.079 1.1 C18 Pos ms1 14.6 4.3 <0.001 <0.001 0.36 0.68 0.002 0.05
B Phenyllactic acid* C9 H10 O3 166.062 3.7 C18 Neg ms3 0.8 0.7 <0.001 <0.001 0.03 0.12 0.09 0.29
B Homovanillic acid* C9 H10 O4 182.057 3.3 C18 Neg ms3 0.8 1.2 <0.001 <0.001 <0.001 0.004 0.001 0.04
B 4-Hydroxycinnamic acid* C9 H8 O3 164.046 2.5 C18 Neg ms2 3.1 11.0 0.001 0.004 0.10 0.27 0.007 0.07
Freshwater Crayfish

B N3,N4-Dimethyl-L-arginine§ C8 H18 N4 O2 202.143 1.1 C18 Pos ms3 1.2 1.2 0.08 0.19 0.75 0.85 0.04 0.23
B Nicotinic acid* C6 H5 N O2 123.032 1.4 C18 Pos ms1 3.1 3.3 0.006 0.02 0.21 0.46 0.30 0.52
B Arginine* C6 H14 N4 O2 174.112 1.0 C18 Pos ms1 1.8 1.7 0.001 0.004 0.66 0.85 0.38 0.59
B Lysine*† C6 H14 N2 O2 146.105 0.9 C18 Pos ms3 2.9 4.0 0.02 0.06 0.87 0.91 0.17 0.36
B Benzoic acid* C7 H6 O2 122.036 3.6 C18 Neg ms3 4.4 8.8 0.003 0.01 0.02 0.10 0.004 0.06
B 3-Methylhistidine*§ C7 H11 N3 O2 169.085 1.0 C18 Pos ms1 7.5 8.7 0.02 0.06 0.06 0.20 0.12 0.30
B Prolylleucine C11 H20 N2 O3 228.147 1.6 C18 Pos ms3 2.9 3.2 0.12 0.22 0.37 0.68 0.62 0.80
B Adenine* C5 H5 N5 135.054 1.4 C18 Pos ms3 5.5 8.6 0.003 0.01 0.75 0.85 0.71 0.86
B 7-Methylguanine C6 H7 N5 O 165.065 1.7 C18 Pos ms3 2.0 3.1 0.11 0.21 0.008 0.04 0.14 0.33
B 3-O-Methyldopa*§ C10 H13 N O4 211.084 2.8 C18 Neg ms3 1.5 1.0 <0.001 <0.001 0.75 0.85 0.07 0.26
C Oxidized glutathione§ C20 H32 N6 O12 S2 612.151 1.7 C18 Neg ms1 1.7 0.7 0.45 0.52 0.64 0.85 0.37 0.58
C N-Acetyl-L-aspartic acid§ C6 H9 N O5 175.047 1.3 C18 Neg ms2 7.8 6.1 0.45 0.52 0.37 0.68 0.50 0.71
C Citric acid§ C6 H8 O7 192.026 1.4 C18 Neg ms1 17.7 16.2 0.10 0.19 0.46 0.74 0.53 0.73
Volume 25, Number 1

C Malic acid§ C4 H6 O5 134.020 1.1 C18 Neg ms2 1.5 2.0 0.10 0.19 0.02 0.09 0.09 0.29
Table 2. Continued.
2019
Molecular RT ID % % Species Sex Species x Sex
Cluster Metabolite Name Formula Mode
Weight min Level RSD D-Ratio p value q value p value q value p value q value
C Malic acid§ C4 H6 O5 134.020 1.1 C18 Neg ms2 1.5 2.0 0.10 0.19 0.02 0.09 0.09 0.29
C Glutamic acid C5 H9 N O4 147.052 1.0 C18 Neg ms2 1.4 1.2 0.76 0.80 0.69 0.85 0.93 0.95
C Cyclic ADP-ribose C15 H21 N5 O13 P2 541.060 1.4 C18 Neg ms3 2.1 3.0 0.19 0.31 0.79 0.86 0.90 0.95
C Azelaic acid§ C9 H16 O4 188.104 3.8 C18 Neg ms1 2.7 6.6 0.18 0.29 0.90 0.92 0.62 0.80
C 4-Oxoproline C5 H7 N O3 129.041 1.5 C18 Neg ms3 1.9 11.4 0.62 0.69 0.96 0.96 0.70 0.86
D Pyroglutamic acid† C5 H7 N O3 129.043 1.5 C18 Pos ms1 2.4 8.0 0.20 0.32 0.75 0.85 0.24 0.44
D Gamma-Aminobutyric acid§ C4 H9 N O2 103.064 1.0 C18 Pos ms3 7.4 11.3 0.31 0.45 0.19 0.43 0.04 0.23
D Acetylcarnitine§ C9 H17 N O4 203.116 1.5 C18 Pos ms1 1.5 0.7 0.33 0.45 0.74 0.85 0.10 0.30
D Citrulline† C6 H13 N3 O3 175.096 1.0 C18 Pos ms3 1.7 1.3 0.34 0.46 0.26 0.54 0.19 0.39
D Adenosine* C10 H13 N5 O4 267.097 2.4 C18 Pos ms2 2.2 1.1 0.01 0.03 0.68 0.85 0.29 0.50
D Nicotinamide adenine C21 H27 N7 O14 P2 663.108 1.5 C18 Pos ms3 3.2 2.9 0.09 0.19 0.69 0.85 0.28 0.50
dinucleotide†
D Niacinamide§ C6 H6 N2 O 122.048 1.5 C18 Pos ms1 12.6 6.5 0.36 0.48 0.72 0.85 0.50 0.71
D Spermidine C7 H19 N3 145.158 0.9 C18 Pos ms3 4.7 10.0 0.90 0.92 0.85 0.90 0.92 0.95
D Beta-Alanine† C3 H7 N O2 89.048 1.0 C18 Pos ms3 4.4 5.7 0.02 0.06 0.15 0.36 0.81 0.91
D Betaine† C5 H11 N O2 117.079 1.0 C18 Pos ms1 2.0 5.3 0.39 0.48 0.39 0.69 0.35 0.58
D Homoserine lactone† C4 H7 N O2 101.048 1.0 C18 Pos ms3 5.9 6.6 0.44 0.52 0.80 0.86 0.10 0.30
D Homoserine† C4 H9 N O3 119.058 1.0 C18 Pos ms3 2.2 4.2 0.09 0.19 0.55 0.81 0.06 0.23
D Glutamine† § C5 H10 N2 O3 146.069 1.0 C18 Pos ms1 0.3 1.0 0.10 0.19 0.43 0.72 0.007 0.07
D Methionine sulfoxide† C5 H11 N O3 S 165.046 1.4 C18 Pos ms3 2.7 11.0 0.29 0.44 0.43 0.72 0.06 0.23
D Methionine§ C5 H11 N O2 S 149.051 1.4 C18 Pos ms1 1.5 4.4 0.33 0.45 0.16 0.38 0.05 0.23
D N-Alpha-acetyllysine† § C8 H16 N2 O3 188.116 1.4 C18 Pos ms3 7.6 3.9 0.02 0.06 0.68 0.85 0.15 0.34
D Trimethylamine N-oxide† C3 H9 N O 75.069 1.0 C18 Pos ms3 4.2 2.2 0.06 0.16 0.76 0.85 0.02 0.17
D Phenylpyruvic acid† C9 H8 O3 164.047 1.9 C18 Pos ms3 1.3 2.2 0.68 0.74 0.52 0.80 0.61 0.80
D Uric acid† C5 H4 N4 O3 168.027 1.4 C18 Neg ms2 2.8 1.7 0.59 0.66 0.60 0.85 0.97 0.98
D Kynurenine† C10 H12 N2 O3 208.085 3.1 C18 Pos ms3 0.1 0.2 0.01 0.03 0.74 0.85 0.07 0.26
D 4-Hydroxyproline* C5 H9 N O3 131.057 1.8 C18 Neg ms2 1.4 1.0 <0.001 <0.001 0.52 0.80 0.77 0.89
Lette et al. — Metabolomic Profiling of Marron Haemolymph

D Pantothenic acid C9 H17 N O5 219.110 3.1 C18 Neg ms1 4.1 5.7 0.05 0.12 0.54 0.81 0.91 0.95
E Sorbose§ C6 H12 O6 180.063 1.0 C18 Neg ms1 4.6 10.4 0.004 0.02 0.0003 0.004 0.001 0.04
E Maltose C12 H22 O11 364.098 1.0 C18 Pos ms3 1.9 1.2 0.71 0.76 0.001 0.009 0.01 0.11
E Dopa§ C9 H11 N O4 197.069 1.5 C18 Pos ms2 3.7 1.8 0.24 0.37 0.14 0.35 0.86 0.95
E Uracil C4 H4 N2 O2 112.027 1.4 C18 Pos ms3 4.0 5.0 0.25 0.38 0.28 0.56 0.22 0.42
E Indole C8 H7 N 117.058 3.3 C18 Pos ms2 2.5 8.4 0.33 0.45 0.26 0.54 1.00 1.00
95
Table 2. Continued.
96
Molecular RT ID % % Species Sex Species x Sex
Cluster Metabolite Name Formula Mode
Weight min Level RSD D-Ratio p value q value p value q value p value q value
E trans-3-Indoleacrylic acid C11 H9 N O2 187.063 3.3 C18 Pos ms3 2.3 18.1 0.39 0.48 0.63 0.85 0.73 0.86
E Tryptophan C11 H12 N2 O2 204.090 3.3 C18 Pos ms1 2.3 18.6 0.38 0.48 0.62 0.85 0.73 0.86
E Riboflavin*§ C17 H20 N4 O6 376.138 3.4 C18 Neg ms3 4.0 3.6 0.01 0.03 0.45 0.74 0.74 0.86
E Phenylalanine*§ C9 H11 N O2 165.078 3.0 C18 Neg ms2 2.7 5.9 0.01 0.03 0.09 0.26 0.12 0.30
E trans-Cinnamic acid* C9 H8 O2 148.052 3.1 C18 Pos ms2 3.2 7.6 0.007 0.03 0.13 0.35 0.54 0.73
E Proline C5 H9 N O2 115.063 1.1 C18 Pos ms2 5.9 9.2 0.91 0.92 0.04 0.14 0.65 0.82
E Uridine§ C9 H12 N2 O6 244.069 1.6 C18 Neg ms1 3.9 9.2 0.13 0.22 <0.001 0.004 0.11 0.30
E Guanosine† C10 H13 N5 O5 283.092 2.4 C18 Neg ms1 1.6 1.8 0.11 0.20 0.001 0.01 0.23 0.43
E Guanine† C5 H5 N5 O 151.049 2.4 C18 Pos ms3 1.6 2.6 0.38 0.48 <0.001 0.003 0.46 0.68
E Cytidine† C9 H13 N3 O5 243.085 1.4 C18 Pos ms1 9.2 13.4 0.42 0.51 <0.001 0.002 0.91 0.95
E Cytosine† C4 H5 N3 O 111.043 1.4 C18 Pos ms3 5.4 7.6 0.08 0.19 <0.001 0.004 0.31 0.52
E Inosine† C10 H12 N4 O5 268.081 2.4 C18 Pos ms2 1.4 2.4 0.68 0.74 0.003 0.02 0.19 0.39
E Hypoxanthine† C5 H4 N4 O 136.038 2.4 C18 Pos ms3 1.6 3.4 0.10 0.19 0.001 0.006 0.06 0.23
E Propionylcarnitine† C10 H19 N O4 217.131 2.6 C18 Pos ms1 1.5 1.2 0.17 0.29 0.008 0.04 0.004 0.06
E Carnitine† C7 H15 N O3 161.105 1.0 C18 Pos ms1 2.8 0.9 0.08 0.19 0.005 0.03 0.05 0.23
E Decanoylcarnitine*† C17 H33 N O4 315.240 5.2 C18 Pos ms1 0.7 0.7 0.001 0.005 <0.001 0.004 0.16 0.36
E Hexanoylcarnitine† C13 H25 N O4 259.178 3.8 C18 Pos ms3 10.6 7.5 0.84 0.87 0.03 0.12 0.12 0.30
E Kynurenic acid† C10 H7 N O3 189.042 3.4 C18 Pos ms1 3.0 3.0 <0.001 0.002 <0.001 <0.001 <0.001 0.001
F N6,N6,N6-Trimethyl-L- C9 H20 N2 O2 188.152 1.0 C18 Pos ms3 2.5 3.0 <0.001 <0.001 0.25 0.54 0.07 0.26
Freshwater Crayfish

lysine*
F cis-4-Hydroxy-D-proline* C5 H9 N O3 131.058 1.0 C18 Pos ms3 2.8 1.0 <0.001 <0.001 0.78 0.86 0.03 0.23
F Melatonin* C13 H16 N2 O2 232.121 3.5 C18 Pos ms3 6.7 6.2 <0.001 <0.001 0.39 0.69 0.70 0.86
F Itaconic acid* C5 H6 O4 130.025 1.0 C18 Neg ms2 2.4 1.8 <0.001 <0.001 0.08 0.23 0.22 0.42
F 3-Hydroxyanthranilic acid* C7 H7 N O3 153.043 3.2 C18 Pos ms1 0.8 0.7 0.000 0.002 0.84 0.89 0.85 0.95
Volume 25, Number 1
2019 Lette et al. — Metabolomic Profiling of Marron Haemolymph 97

Figure 3. Circular hierarchical cluster analysis (HCA) dendrogram grouping individual metabolites detected in haemolymph of Cherax tenuimanus
and Cherax cainii into six clusters (A–F) based on phenotypic similarity. Colours of the metabolite name are based on the univariate analysis (q-value,
Table 2), with metabolites that are significant between species (red), sex (blue), and species × sex (purple) shown, as well as non-significant metabolites
(grey). Symbols represent the significant metabolites from the multivariate analysis (Figure 2) where PC1 (*) highlights metabolites that separate
species, PC2 (†) highlights metabolites that separate the sexes in C. tenuimanus, and PC 3 (§) highlights metabolites that separate the sexes in C. cainii.

that are primarily higher in male C. tenuimanus. Cluster B (n =
18) metabolite concentrations are higher primarily in C. cainii
(particularly the males) with most of the metabolites indicating
a significant difference between species in both univariate and
multivariate analyses. Cluster C (n = 8) was characterised by
elevated levels in the C. cainii females, with most being statistically
significant for sex in C. cainii in the multivariate analysis. Cluster
D (n = 22) contains a set of metabolites with a higher concentration
for C. tenuimanus, especially the females, with many being
statistically significant for sex in C. tenuimanus in the multivariate
analysis. Cluster E (n = 23) displays a difference between sexes
in both species (most strongly in C. tenuimanus), with more than
half of the metabolites being statistically significant, primarily
between sexes in the univariate analysis and C. tenuimanus sex in
the multivariate. Cluster F (n = 5) metabolites all had a statistically
significant difference between species in both the univariate and
98 Freshwater Crayfish Volume 25, Number 1
multivariate analyses, with all five being higher in C. tenuimanus
and lower in C. cainii.
DISCUSSION
The identification of 84 metabolites in the haemolymph of the
two Cherax species sampled creates a baseline dataset of metabolites
for these species. The results distinguish between two congeneric
species and indicate differences between sexes of the same species
within the metabolomic profile. The metabolites identified formed
six clusters (Figure 3) primarily based on whether the metabolites
varied between species, sex, both species and sex, or both species
and sex with an interaction between these two factors. Many of the
identified metabolites (as listed in Table 2) were amino acids or
their derivatives, including, but not limited to, arginine, glutamine,
lysine, methionine, phenylalanine, and proline. Other compounds
identified in this study were neurotransmitters (such as gamma-
Aminobutyric acid (GABA) and acetylcholine), nucleosides
(inosine and uridine), nucleotides, organic osmolytes (TMAO and
betaine), carbohydrates, and fatty acids. These metabolites play
a role in energy metabolism or signalling pathways and other
biologically important functions of the crayfish such as immune
function and osmotic regulation (Alfaro and Young 2016).
Understanding the biological importance for crayfish of each
of the 84 metabolites identified is a challenge and requires further
study in many cases. Furthermore, additional sampling at other times
throughout the year will help clarify if the results seen represent
species specific differences in a general sense or if they are related
to processes occurring at that time. For example, the presence and
levels of metabolites that define clusters B, E and F all are potentially
explained through the link between these metabolites and changes
that occur in relation to reproduction and moulting in these crayfish,
whereas, we cannot currently propose such hypotheses for the
biological significance of Clusters A, C and D.
Clusters B, E and F
Cluster B is largely defined by metabolites, mostly amino acids
and their products of metabolism, that show a significant difference
between species, with elevated levels of metabolites in C. cainii,
and the difference being most pronounced in C. cainii males.
Cherax cainii appear to mate earlier in the year than C. tenuimanus,
as this has been suggested for field mark-recapture studies (Bunn
2004) and was also witnessed in our laboratory trials. The date of
haemolymph sampling occurred after the C. cainii had passed their
normal mating season. During the period where they were being
held in aquaria the environmental cues to encourage breeding
were created (altered water temperature and increased daylength)
(Morrissy 1992; Huner 1994) but as they were held in individual
tanks they did not have the opportunity to be with a marron of the
opposite sex, therefore no mating occurred. Some of the C. cainii
males were also preparing to moult, which they did shortly after
sampling occurred. Moulting is another high energy part of their
life cycle and the process of moulting (ecdysis) could, at least in
part, explain the results in this cluster. During the moulting phase,
it is expected that there would be an increase in amino acids in
tissues and haemolymph as the proteins and muscle tissue are
being remodelled; for example during ecdysis, the proteins in
claw muscle of moulting animals are broken down (Mykles 1999;
Koenders et al. 2002; Jimenez and Kinsey 2015). A number of
amino acids or compounds related to amino acid metabolism, such
as arginine (metabolism) and lysine (degradation), were detected
in this cluster as well as the neurotransmitter acetylcholine, which
had significantly higher levels in C. cainii.
Cluster E is the largest and most varied group with 23
metabolites, characterised by metabolites related to lipid and
energy metabolism, the presence of fatty acids, as well as many
nucleosides and nuceleotide bases for purine and pyramidine
metabolism. This cluster is defined by a difference in sexes where
the level of each metabolite was higher in females in both species
than in males and was most pronounced in C. tenuimanus females.
This could be due to the fact that C. tenuimanus mate later in the
year than C. cainii and the C. tenuimanus females should have been
reproductively ready to mate had they been in aquaria with a male.
Other C. tenuimanus not sampled for this experiment but held
in similar aquaria with a mate did breed during the same period.
Elevated levels of carnitines, markers for fatty acid metabolism
in crustaceans where energy is produced from the tricarboxylic
acid cycle (Schock et al. 2013), were detected. In other aquatic
species, an increase in fatty acids has been observed around the
time of reproduction (Reverter et al. 2018), as fatty acids are
required for cell metabolism and signalling in reproduction in
early developmental processes (Martin and Hose 1995; Reverter et
al. 2018). Cluster E contained inosine, a purine nucleoside, which
has been used as a biomarker for general stress in Litopenaeus
vannamei (Pacific white shrimp) (Schock et al. 2013). Inosine is an
intermediate in a chain of purine nucleotide reactions required for
muscle movements, however, an increase in this nucleoside can
imply that the organism is under stress, as it has been considered
to represent an anti-inflammatory response that may inhibit
DNA synthesis (Schock et al., 2013). Although care was taken to
minimise stress for the crayfish, this cannot be ruled out.
All five metabolites in Cluster F indicate a significant difference
between species in both the univariate and multivariate analyses
with higher levels present in C. tenuimanus. Trimethyllysine, cis-4-
hydroxy-D-proline, and 3-hydroxyanthranilic acid are derivatives
or intermediate metabolites of amino acids, and itaconic acid is
a fatty acid. Also included in this group is melatonin, a hormone
which likely plays a role in the regulation of seasonal changes in
the marron, as crustaceans have a similar melatonin-biosynthetic
pathway to that found in other invertebrates and melatonin is
known to regulate the circadian rhythm in many species (Sainath
et al. 2013). The statistically significant difference between the
two species may once again potentially be linked to timing of
reproductive cycles as they were elevated in C. tenuimanus.
Clusters A, C and D
Cluster A contained eight metabolites; the fatty acids
docosahexaenoic acid and eicosapentaenoic acid, and the
amino acids (or amino acid derivatives) creatinine, creatine,
methylimidazoleacetic acid, N-acetyl-L-leucine, urocanic acid,
and folic acid (a B vitamin). Three of these had a statistically
significant difference between sexes. The concentrations of the
metabolites in Cluster A are primarily higher in male marron, and
2019 Lette et al. — Metabolomic Profiling of Marron Haemolymph 99
in particular C. tenuimanus, and all are in low concentrations in
female C. tenuimanus.
Cluster C is composed of eight metabolites (oxidized
glutathione, N-acetyl-L-aspartic acid, citric acid, malic acid,
glutamic acid, cyclic ADP-ribose, azelaic acid, 4-oxoproline)
that have no significant differences between species or sex when
using univariate analysis but a significant difference in five of
the metabolites between the male and female C. cainii in the
multivariate analysis, with higher levels detected in female C.
cainii (Figure 3). The biological explanation for this cluster of
metabolites is currently unknown but glutathione can be used as
a measure of oxidative stress (Viant 2007; Bone et al. 2015) and
glutamic acid (glutamate) is important in cellular metabolism for
the synthesis of protein (Wishart et al. 2018) and is the primary
neurotransmitter in arthropods (Smarandache-Wellmann 2016).
The collection of 22 metabolites in cluster D contains a set
of metabolites (including osmolytes (TMAO and betaine), and
several amino acids and metabolic derivatives of amino acids
such as kynurenine) with a higher concentration in C. tenuimanus,
especially the females, many of which are statistically significant
for sex in C. tenuimanus in the multivariate analysis. Detected
in this cluster were biologically important amino acids such as
methionine and glutamine which play roles in multiple biological
functions as methionine is required for cell growth and repair and
glutamine is a source of cellular energy and a precursor to the
neurotransmitter glutamate (Wishart et al. 2018).
CONCLUSION
The recognition of 84 identified metabolites, forming
six clusters containing 57 metabolites that were statistically
different in species and/or sex, creates a baseline dataset for
future investigation into the metabolome of freshwater crayfish.
Although the composition of the metabolome was similar in both
species, we observed a clear pattern of interspecific variation in
the metabolites detected which confirms the suitability of using
metabolomics as a tool for discriminating differences between
closely related species. The timing of reproduction and moulting
may explain part of the detected variation between species in this
study, as the experiments occurred after the mating season for
the C. cainii and during the mating season for the C. tenuimanus,
which is a biologically important time for any species, and prior to
moulting in some of the C. cainii. The differences detected for some
metabolites between sexes within species may also be related to
reproduction or life history generally. Further sampling at different
times of year will help clarify whether the differences in species or
sexes is related to seasonal cycles. It is of value to have information
on specific metabolic and biochemical levels that occur during
the life-cycle of these species as C. tenuimanus are critically
endangered and require active conservation, and C. cainii are an
important aquaculture species. Not only is this data valuable for
freshwater crayfish but it also provides a starting point for studies
into other crustaceans specifically, and arthropods generally, as
most biological pathways are highly conserved (Viant 2007). It is
well known that arthropods are important to aquatic ecosystems,
however only a small fraction of environmental metabolomic
studies have used organisms from this phylum (Alfaro and Young
2016; Lankadurai et al. 2013). Future comparative studies have
the potential to reveal previously unidentified metabolites with
important biological and ecological roles (Kuhlisch and Pohnert
2015; Reverter et al. 2018).
ACKNOWLEDGMENTS
We thank Professor Pierre Horwitz for expert advice designing
the initial experiments, and Dr. Stacey Reinke, the anonymous
reviewers, and journal editor for valuable feedback on this
manuscript. This work was funded by research grants from the
Western Australian Department of Primary Industries and Regional
Development, and by the School of Science, Centre for Ecosystem
Management (CEM), and Centre for Integrative Metabolomic and
Computational Biology (CIMCB), at Edith Cowan University
(Joondalup, Western Australia). We also acknowledge the
Australian Government Research Training Program for its support
of this Masters by Research project.
LIST OF ABBREVIATIONS
IUCN International Union for Conservation of Nature
LC-MS liquid chromatography-mass spectrometry
DPIRD Department of Primary Industries and Regional
Development, Western Australia
PVC polyvinylchloride
NO2 nitrite
NO3 nitrate
NH3/NH4 ammonia/ammonium
21G 21 gauge needle
QC quality control
HESI heated electrospray ionisation probe
GABA gamma-Aminobutyric acid
TMAO Trimethylamine N-oxide
DNA deoxyribonucleic acid
LITERATURE CITED
Aizen R, Tao K, Rencus-Lazar S and Gazit E (2018). Functional
metabolite assemblies-a review. Journal of Nanoparticle
Research 20(5):125. doi: 10.1007/s11051-018-4217-3
Alfaro AC and Young T (2016). Showcasing metabolomic
applications in aquaculture: a review. Reviews in Aquaculture:
10(1):135–152. doi: 10.1111/raq.12152
Austin CM and Bunn J (2010). Cherax tenuimanus The IUCN
Red List of Threatened Species 2010. The IUCN Red List
of Threatened Species. http://www.iucnredlist.org/details/
summary/4618/0 [accessed5 August 2016].
Benjamini Y and Hochberg Y (1995). Controlling the False
Discovery Rate: A Practical and Powerful Approach to Multiple
Testing. Journal of the Royal Statistical Society. Series B
(Methodological) 57(1): 289–300. doi: 10.2307/2346101
Bone JWP, Renshaw GMC, Furse JM and Wild CH (2015).
Using biochemical markers to assess the effects of imposed
temperature stress on freshwater decapod crustaceans: Cherax
100 Freshwater Crayfish Volume 25, Number 1
quadricarinatus as a test case. Journal of Comparative
Physiology B: Biochemical, Systemic, and Environmental
Physiology 185(3): 291–301. doi: 10.1007/s00360-014-0883-3
Broadhurst D, Goodacre R, Reinke SN, Kuligowski J,
Wilson ID, Lewis MR and Dunn WB (2018). Guidelines
and considerations for the use of system suitability and quality
control samples in mass spectrometry assays applied in
untargeted clinical metabolomic studies. Metabolomics 14(6):
72. doi: 10.1007/s11306-018-1367-3
Bunn J (2004). Investigation of the replacement of Margaret
River Hairy Marron Cherax tenuimanus (Smith) by Smooth
Marron C. cainii Austin. MS Thesis. Edith Cowan University,
Joondalup, Western Australia, Australia.
Bunn J, Koenders A, Austin CM and Horwitz P (2008).
Identification of Hairy, Smooth and Hybrid Marron
(Decapoda: Parastacidae) in the Margaret River: Morphology
and Allozymes. Freshwater Crayfish 16: 113–121.
Costantini S, Parrillo L, Guerriero E, Melck D, Colonna G,
Volpe MG and Paolucci M (2018). 1H-NMR metabolomic
profiling of the crayfish Astacus leptodactylus subjected to
polyphenol-enriched diets. Aquaculture Nutrition 24(1): 524–
538. doi: 10.1111/anu.12585
Duffy R and Day N (2015). Margaret River Hairy Marron
Recovery Plan 2015–2020 V9 for review. Department of
Fisheries, Western Australia.
Dunn WB, Broadhurst D, Begley P, Zelena E, Francis-
Mcintyre S, Anderson N, Brown M, Knowles JD, Halsall
A, Haselden JN, Nicholls AW, Wilson ID, Kell DB and
Goodacre R (2011). Procedures for large-scale metabolic
profiling of serum and plasma using gas chromatography and
liquid chromatography coupled to mass spectrometry. Nature
Protocols 6(7):1060–1083. doi: 10.1038/nprot.2011.335
Hastie T, Tibshirani R and Friedman J (2009). The Elements of
Statistical Learning: Data Mining, Inference and Prediction.
Springer, New York. doi: 10.1007/978-0-387-84858-7
Huner J V (1994). Freshwater Crayfish Aquaculture in North
America, Europe, and Australia: Families Astacidae,
Cambaridae, and Parastacidae. Food Products Press,
Binghamton, New York, USA.
Izral NM, Brua RB, Culp JM and Yates AG (2018). Developing
metabolomics-based bioassessment: crayfish metabolome
sensitivity to food and dissolved oxygen stress. Environmental
Science and Pollution Research 25(36):36184–36193. doi:
10.1007/s11356-018-3518-5
Jimenez AG and Kinsey ST (2015). Energetics and Metabolic
Regulation. Pp. 391–419, In: The Natural History of the
Crustacea, Volume 4. Chang ES and Thiel M (eds.). Oxford
University Press, New York, NY, USA.
Johnston C and Jungalwalla P (2005). Aquatic Animal Welfare
Guidelines; Guidelines on welfare of fish and crustaceans
in aquaculture and/or in live holding systems for human
consumption. National Aquaculture Council of Australia. 348
pp.
Jolliffe IT (2002). Principal Component Analysis, 2nd Edition.
Springer-Verlag, New York, USA.
Kaufman L and Rousseeuw P (1990). Finding Groups in Data:
An Introduction to Cluster Analysis, 1st Edition. John Wiley,
New York, USA.
Kennington WJ, Guildea C, Lukehurst SS, Hitchen Y,
Gardner MG, Duffy R, Dias PJ, Ledger JM and Snow
M (2014). Isolation and characterization of 13 polymorphic
microsatellite loci for the smooth Cherax cainii and hairy
marron C. tenuimanus (Decapoda: Parastacidae). Conservation
Genetics Resources 6:337–339. doi: 10.1007/s12686-013-
0088-1
Koenders A, Yu X, Chang ES and Mykles DL (2002). Ubiquitin
and actin expression in claw muscles of land crab, Gecarcinus
lateralis, and American lobster, Homarus americanus:
Differential expression of ubiquitin in two slow muscle fiber
types during molt-induced atrophy. Journal of Experimental
Zoology 292(7): 618–632. doi: 10.1002/jez.10081
Kuhlisch C and Pohnert G (2015). Metabolomics in chemical
ecology. Natural Product Reports 32(7):937–955. doi:
10.1039/c5np00003c
Lankadurai BP, Nagato EG and Simpson MJ (2013).
Environmental metabolomics: an emerging approach to study
organism responses to environmental stressors. Environmental
Reviews (21): 180–205. doi: 10.1139/er-2013-0011
Lawrence C (2007). Improved performance of marron using
genetic and pond management strategies. Final report to
Fisheries Research and Development Corporation on Project
No. 2000/215. Fisheries Research Contract Report No. 17.
Department of Fisheries, Western Australia, Australia. 178 pp.
Leland JC and Furse JM (2012). Potential utility of haemolymph
analysis in non-lethal conservation studies on threatened
Australasian freshwater crayfish: portability and practicality.
Crustacean Research, Special Number 7:87–95. doi:
10.18353/crustacea.Special2012.7_85
Lin CY, Viant MR and Tjeerdema RS (2006). Metabolomics:
Methodologies and applications in the environmental sciences.
Journal of Pesticide Science 31(3): 245–251. doi: 10.1584/
jpestics.31.245
Martin GG and Hose JE (1995). Chapter 17 Circulation, the
Blood and Disease. Pp. 465–495, In: Biology of the lobster
Homarus americanus. Factor JR (ed.). Academic Press, Inc.,
London, UK. doi: 10.1016/B978-0-12-247570-2.X5021-X
Matlab (2018). MATLAB and Statistics Toolbox Release 2018a.
The Mathworks Inc. Natick, Massachusetts, USA.
Miller MG (2007). Environmental metabolomics: A SWOT
analysis (strengths, weaknesses, opportunities, and threats).
2019 Lette et al. — Metabolomic Profiling of Marron Haemolymph 101

Journal of Proteome Research (6): 540–545. doi: 10.1021/ content of metabolomics data. BMC Genomics 7:142 doi:
pr060623x 10.1186/1471-2164-7-142
Morrissy NM (1992). An introduction to marron and other Viant M (2007). Metabolomics of aquatic organisms: the new
freshwater crayfish farming in Western Australia. Fisheries ‘omics’ on the block. Marine Ecology Progress Series 332:
301–306. doi: 10.3354/meps332301
Department of Western Australia, Australia. 36 pp.
Wishart DS, Feunang YD, Marcu A, Guo AC, Liang K,
Morrissy NM (1996). A summary of the Fisheries Departments
Vazquez-Fresno R, Sajed T, Johnson D, Li C, Karu N,
research to develop marron farming. Fisheries Department Sayeeda Z, Lo E, Assempour N, Berjanskii M, Singhal S,
Western Australia, Australia. Arndt D, Liang Y, Badran H, Grant J, Serra-Cayuela A,
Mykles DL (1999). Proteolytic processes underlying molt-induced Liu Y, Mandal R, Neveu V, Pon A, Knox C, Wilson M,
claw muscle atrophy in decapod crustaceans. American Manach C and Scalbert A (2018). HMDB 4.0: the human
Zoologist 39(3): 541–551. doi: 10.1093/icb/39.3.541 metabolome database for 2018. Nucleic Acids Research
46(D1):D608–D617. doi: 10.1093/nar/gkx1089
Patti GJ, Yanes O and Siuzdak G (2012). Innovation:
Xia J, Broadhurst DI, Wilson M and Wishart DS (2013).
metabolomics: the apogee of the omics trilogy. Nature
Translational biomarker discovery in clinical metabolomics:
Reviews Molecular Cell Biology 13(4):263–269. doi: 10.1038/ An introductory tutorial. Metabolomics 9(2):280–299. doi:
nrm3314 10.1007/s11306-012-0482-9
Reverter M, Tribalat MA, Pérez T and Thomas OP (2018). Young T and Alfaro AC (2018). Metabolomic strategies for
Metabolome variability for two Mediterranean sponge aquaculture research: a primer. Reviews in Aquaculture 10(1):
species of the genus Haliclona: specificity, time, and space. 26–56. doi: 10.1111/raq.12146
Metabolomics 14(9): 114. doi: 10.1007/s11306-018-1401-5
Sainath SB, Swetha CH, Sreenivasula Reddy P and Reddy PS
(2013). What do we (need to) know about the melatonin in
crustaceans? Journal of Experimental Zoology 319(7):365–
377. doi: 10.1002/jez.1800
Schock TB, Duke J, Goodson A, Weldon D, Brunson J, Leffler
JW and Bearden DW (2013). Evaluation of Pacific white
shrimp (Litopenaeus vannamei) health during a superintensive
aquaculture growout using NMR-based metabolomics. PLoS
ONE 8(3). e59521. doi: 10.1371/journal.pone.0059521
Schock TB, Stancyk DA, Thibodeaux L, Burnett KG,
Burnett LE, Boroujerdi AFB and Bearden DW (2010).
Metabolomic analysis of Atlantic blue crab, Callinectes
sapidus, hemolymph following oxidative stress. Metabolomics
6(2):250–262. doi: 10.1007/s11306-009-0194-y
Smarandache-Wellmann CR (2016). Arthropod neurons and
nervous system. Current Biology 26(20):R960–R965. doi:
10.1016/j.cub.2016.07.063
Sumner LW, Amberg A, Barrett D, Beale MH, Beger R,
Daykin CA, Fan TW-M, Fiehn O, Goodacre R, Griffin
JL, Hankemeier T, Hardy N, Harnly J, Higashi R, Kopka
J, Lane AN, Lindon JC, Marriott P, Nicholls AW, Reily
MD, Thaden JJ and Viant MR (2007). Proposed minimum
reporting standards for chemical analysis Chemical Analysis
Working Group (CAWG) Metabolomics Standards Initiative
(MSI). Metabolomics 3(3):211–221. doi: 10.1007/s11306-
007-0082-2
van den Berg RA, Hoefsloot HCJ, Westerhuis JA, Smilde
AK and van der Werf MJ (2006). Centering, scaling, and
transformations: improving the biological information
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