Dietary lipids as modulators of fatty

acid profile and gene expression

patterns on body compartments of
Octopus vulgaris paralarvae M. Nande
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 Aquaculture 556 (2022) 738293

Contents lists available at ScienceDirect

Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Dietary lipids as modulators of fatty acid profile and gene expression

patterns on body compartments of Octopus vulgaris paralarvae
M. Nande a, *, Ó. Monroig b, A.M. Machado a, L.F.C. Castro a, c, M. Lopes-Marques c, d, e,
A. Capitão a, J.C. Navarro b, *
a
CIIMAR-Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de Cruzeiros de Leixões. Av., General Norton de Matos s/n,
Portugal
b
Instituto de Acuicultura Torre de la Sal (IATS-CSIC), 12595 Ribera de Cabanes, Castellón, Spain
c
Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal
d
i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal
e
IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal

A R T I C L E I N F O A B S T R A C T

Keywords: The common octopus, Octopus vulgaris, is a promising mollusc species for marine aquaculture diversification due
Octopus vulgaris paralarvae to its high growth rates and commercial value. Yet, the elevated mortalities mainly related to lipid-linked
Fatty acids nutritional deficiencies during the planktonic stage (paralarvae), hinder the development of efficient protocols
Gene expression
for a complete rearing cycle. Although the effect of dietary lipids, especially fatty acids (FA), has been the subject
Glycerophospholipid metabolism
Dietary lipids
of intense research, the available information is essentially restricted to their impact on the composition of the
whole parlarval organism. In contrast, little is known about the effects of dietary signature and the specific
requirements of each anatomical structure through the paralarvae development. In addition, knowledge about
the endogenous capacity in each paralarvae body structure for adaptation to different dietary scenarios is
necessary. In the present work, a series of experiments were carried out based on newly hatched paralarvae (PH)
and paralarvae fed (30 days post-hatch (DPH)) either with marine crustacean zoeae (PZ) or with Artemia met­
anauplii (PA). At the end of the trials, the paralarvae were dissected into functional (mantle, head, and arms) and
digestive (digestive gland (DG)) body compartment and the FA profile, as well as the expression patterns of genes
involved in the long chain polyunsaturated FA (LC-PUFA) (stearoyl-CoA desaturase (scd), ωx2 desaturase (ωx2),
ωx1 desaturase (ωx1), fatty acyl desaturase (fad), elovl2/5, elovl4), and glycerophospholipid biosynthetic pathways
(agpta, lpin, chpt, and dgat) were analysed. Results showed a positive effect of the PZ diet on growth and
development of paralarvae as compared to PA, and distinct FA composition for the 3 experimental groups. The
digestive gland was associated to 18C FA (18:3n-3, 18:4n3, 18:1n9, and 18:2n6), while n-6 and n-3 LC-PUFA
(20:4n6, 20:5n3, 22:5n3, and 22:6n3) were found in a higher proportion in functional body compartments.
The expression of LC-PUFA biosynthesis-linked genes increased significantly during development, with the
functional body compartments of the PA treatment being up-regulated as compared to PZ, pointing at a putative
compensatory mechanism. In addition, a higher amount of transcripts linked to the triggering of the phospha­
tidylcholine synthesis (chpt) was found in the digestive gland of PA and PZ and arms of PZ; whereas genes related
with triacylglycerol (TAG) synthesis (lpin and dgat) were enhanced in the digestive gland of PA and PH. Dietary
treatments affected the FA profile and the gene expression patterns in both digestive (more similar FA profile and
glycerophospholipid biosynthesis) and functional (different FA profile and LC-PUFA biosynthesis) compartments
of the paralarvae. Furthermore, LC-PUFA biosynthesis-related genes in the head and glycerophospholipid target
genes in DG could be used as biomarkers of nutritional deficiencies in paralarvae.

* Corresponding authors.
E-mail addresses: mnande@ciimar.up.pt (M. Nande), jc.navarro@csic.es (J.C. Navarro).

https://doi.org/10.1016/j.aquaculture.2022.738293
Received 11 January 2022; Received in revised form 6 April 2022; Accepted 24 April 2022
Available online 27 April 2022
0044-8486/© 2022 Elsevier B.V. All rights reserved.
M. Nande et al.
1. Introduction
The expansion of the so-called “fed aquaculture” in the last decades
has occurred through a remarkable optimisation of farming technology
for a relatively small number of high value species, mostly fish and
shrimp (FAO, 2020). Diversification of aquaculture with new species has
been pointed out as a key aspect to increase the environmental sus­
tainability and profitability of the sector (FAO, 2020). In this context,
the high growth rates and commercial value of O. vulgaris has prompted
enormous interest in developing appropriate culture protocols for
octopus species worldwide (Iglesias et al., 2014; Dan et al., 2018, 2019;
de Ortiz et al., 2021). However, high mortalities during the planktonic
stage (paralarvae) still represent a bottleneck for an efficient protocol of
integral rearing (Iglesias et al., 2007). Nutritional deficiencies of larval
food have been suggested as one of the main causes accounting for such
paralarval mortalities (Navarro and Villanueva, 2003; Navarro et al.,
2014).
Certain lipids such as long-chain (≥C20) polyunsaturated fatty acids
(LC-PUFA), cholesterol and phospholipids (PL) are regarded as essential
nutrients for cephalopods (Almansa et al., 2006). Compared to reared
individuals, wild paralarvae present fatty acid (FA) profiles that are
relatively rich in LC-PUFA, especially docosahexaenoic acid (DHA,
22:6n-3), that mostly derives from diet (Navarro and Villanueva, 2003;
Garrido et al., 2016)., but at present it is unknown if paralarvae can
trigger specific metabolic pathways to compensate for dietary lipid de­
ficiencies, as occurs in other aquatic animals like fish (Monroig and
Kabeya, 2018; Xie et al., 2021).
Dietary lipids have been shown to regulate the expression of genes
involved in the biosynthesis of LC-PUFA in aquatic animals (Monroig
and Kabeya, 2018; Xie et al., 2021). Specific genes encoding key fatty
acyl elongases and desaturases of the O. vulgaris LC-PUFA biosynthesis
have been studied in recent years. The PUFA elongases Elovl4 and
Elovl2/5, and the Δ5 front-end desaturase (Fad) and Δ9 stearoyl-CoA
desaturase (Scd), were molecularly and functionally characterised in
O. vulgaris (Monroig et al., 2012a, 2012b, 2016a, 2017). These studies
confirmed that O. vulgaris lacks key enzymatic activities within its
elongation and desaturation complement, thus making necessary the
dietary supply of the physiologically important LC-PUFA, namely
arachidonic (ARA, 20:4n-6), eicosapentaenoic (EPA, 20:5n-3) and do­
cosahexaenoic (DHA, 22:6n-3) acids. More recently, two methyl-end (or
ωx) desaturase genes encoding enzymes with Δ12 (ωx2) and Δ15 (ωx1)
desaturase regioselectivities enabling the de novo biosynthesis of the 18C
PUFA linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3),
have been reported in O. vulgaris (Garrido et al., 2019). Importantly,
the functional characterisation of the O. vulgaris ωx desaturases revealed
that EPA can be biosynthesised through several routes and therefore
should not be regarded as dietary essential, although both ARA and DHA
should still be considered essential nutrients for O. vulgaris (Garrido
et al., 2019).
To the best of our knowledge, and unlike LC-PUFA, the specific genes
involved in PL biosynthesis (glycerophospholipid metabolism) and their
regulation through diet have not been studied in O. vulgaris. A simplified
view of the biosynthetic pathway of PC, one of the most abundant PL in
O. vulgaris (Reis et al., 2019), includes a first step by which the acyl-sn-
glycero-3-phosphate acyltransferase (Agpta) mediates the reaction that
transforms 1-acyl-sn-glycero-3-phosphate into 1,2-diacyl-sn-glycero-3-
phosphate through the incorporation of a FA in the sn-2 position
(Beppu et al., 2017). Next, the enzyme phosphatidate phosphatase
(Lpin) catalyses the conversion of 1,2-diacyl-sn-glycero-3-phosphate
into 1,2-diacyl-sn-glycerol (DAG). The final reaction, catalysed by
CDP-choline and 1,2-diacylglycerol cholinephosphotransferase (Chpt),
is the condensation of CDP-choline with DAG to form PC. Alternatively,
DAG can be converted into TAG by the action of acyl-CoA diacylglycerol
acyltransferase (Dgat). Thus, Chpt and Dgat compete for DAG for the
synthesis of PC or TAG, respectively (Xu et al., 2019).
An integrative study of FA profile and lipid metabolism gene
2
Aquaculture 556 (2022) 738293
expression may be even more revealing of the paralarvae requirements
when linked to different anatomical areas of the organism. The tradi­
tional experimental approach to ascertain dietary requirements of
essential nutrients such as lipids including essential FA, has been the
scrutiny of the impact of food on the whole-body composition of
paralarvae (Navarro and Villanueva, 2000, 2003; Seixas et al., 2008;
Uriarte et al., 2011; Iglesias et al., 2014; Garrido et al., 2016). However,
a design often used in adults and juveniles consists of the analysis of the
main body compartments (García-Garrido et al., 2010; García et al.,
2011). This strategy provides critical physiological insight on the spe­
cific needs in developmental periods, and it has not yet been attempted
with paralarvae as far as we know. This is particularly interesting
because different anatomical tissues, organs, or compartments may have
specific requirements. Thus, the specific nutritional requirements may
be different in those final accepting body compartments and with
particular functions (functional) such as swimming or breathing
(mantle), vision and perception (head), or development (arms)
compared to those found in the digestive body compartment (digestive
gland) related to the initial metabolism and storage of food (Villanueva
and Norman, 2008; O’dor et al., 1984).
Thus, due to their lipid and FA composition, and for the reasons
already mentioned above, the use of two live preys like Artemia and crab
zoeae, offers an optimal scenario to scrutinize the effects of dietary lipids
(Garrido et al., 2018; Varó et al., 2017), specifically LC-PUFA, but most
importantly PC and TAG. The present study aimed to investigate the
growth and general performance of paralarvae fed these two live preys
and further explore the dietary effects at two levels. First, analysing the
dietary lipid fingerprint in different body compartment of the paral­
arvae. Second, associating this information to potential mechanisms of
nutritional regulation through the study of the expression patterns of
genes involved in LC-PUFA, PC and TAG biosynthetic pathways.
2. Materials and methods
2.1. Broodstock and spawning
During the winter of 2014 and 2015, 14 common octopuses (average
weight: 1.6 ± 0.4 kg) were captured in the Ria of Vigo (NW Spain), by
local fishermen. Animals were transported to the facilities of the Insti­
tuto Español de Oceanografía (IEO) in Vigo, Spain, in a 100 L opaque
tank with seawater at 14 ◦ C and oxygen saturation. The animals were
acclimatised in a 10 m3 tank (4 m L x 2 m W x 1.25 m H), with a seawater
flow-through system in semidarkness conditions (<100 lx (lux)) in a 3:1
females:males ratio. The males were subsequently removed from the
broodstock tank after a month, anaesthetised and humanely killed as
described below. Throughout the period, the temperature was 16 ± 2 ◦ C
and the salinity 34 ± 1 psu (Iglesias et al., 2016). Levels of dissolved
oxygen, nitrites and ammonium were monitored daily. Several sections
of PVC pipes (20 cm in diameter and 50 cm long) were placed into the
broodstock tank as a shelter and spawning dens. The animals were fed ad
libitum three times per week with frozen mussels (Mytilus gallopro­
vincialis), fish (Merluccius merluccius) and crustaceans (Polybius spp.).
Presence of egg batches inside the PVC dens was checked weekly. The
first layings were registered in March in both years, and each female plus
the spawn and den was relocated separately in 0.5 m3 tanks (1 m L x 1 m
W x 1.5 m H, with 0.5 m seawater depht), with low light intensity (<100
lx), and a seawater flow-through system. The incubation period varies
with temperature (Nande et al., 2017a, 2018), and was adjusted to
match the highest frequency of hatched zoeae (see below).
2.2. Preys
2.2.1. Crab zoeae
From April to August (2014 and 2015) 56 ovigerous females of spider
crab (Maja brachydactyla) were maintained at different stages of em­
bryonic development according to González-Gurriarán et al. (1995).
M. Nande et al.
Females were kept at low light intensity (< 100 lx) and ambient tem­
perature (14–18 ◦ C), in six 0.3 m3 flow-through seawater tanks (1 m L x
1 m W, 0.5 m H, with 0.3 m seawater depth). A 500 μm net in the outlet
tube was fitted to collect the hatched zoeae. Crab females were fed three
times a week with frozen mussels (M. galloprovincialis) in a proportion of
10% of their body weight. The hatched zoeae were manually harvested
using a 500 μm collector and transferred to the experimental tanks to
feed the O. vulgaris paralarvae.
2.2.2. Artemia metanauplii
Artemia cysts (AF 480, INVE Aquaculture, Dendermonde, Belgium)
were incubated for 24 h at 28 ◦ C until hatching. The newly hatched
nauplii were transferred to a 150 L tronco-conical tank at a final density
of 50 ± 10 ind/mL, and kept in a closed seawater system at 25 ◦ C, 36 psu
of salinity, surface light intensity from 600 to 900 lx, and constant
photoperiod (24L: 0D). Artemia was grown for 8–10 days to a total
length > 2 mm, and fed with a multi-specific diet of microalgae based on
Isochrysis galbana and Nannochloropsis sp., at a final concentration of
250,000 and 500,000 cell/mL, respectively.
2.3. O. vulgaris feeding experiments
Hatchling paralarvae (PH) of two different spawns (June 2014 and
July 2015) were used in two indentical feeding trials, consisting of
feeding paralarvae with preys, namely spider crab zoeae (PZ) and
Artemia metanauplii (PA). Both dietary treatments were performed in
100 L triplicate tanks (5 paralarvae/L) with black background, soft
central aeration, controlled temperature (21 ± 1 ◦ C), 14:10 (L:D)
photoperiod, and a light intensity range of 300–500 lx on the seawater
surface. Two microalgae, I. galbana and N. sp., were added at a final
concentration of 150,000 and 250,000 cell/mL, respectively, in order to
keep the paralarvae and prey in a green water environment (Iglesias and
Fuentes, 2014). According to a previous estimate of food ingestion
(Nande et al., 2017b), paralarvae were fed at a final prey density of 0.05
preys/mL for PZ and 0.1 preys/mL for PA, with a frequency of three
shots per day (Iglesias et al., 2006), adjusting the final concentration to
the preys still present in the rearing tank. Occasionally, older PZ
paralarvae (20 DPH) required higher food supply, and Artemia meta­
nauplii were used as a supplement every 3 d at a final concentration of
0.05 preys/mL.
At 0, 10, 15, 20, 25 and 30 DPH, 15 paralarvae were collected, and
the number of suckers per arm was counted under a binocular micro­
scope Leica MZ8®. Then, Paralarvae were washed with distilled water,
and kept for 24 h at 80 ◦ C, until weighed with an ultra-precision scale
(0.000001 g) UM3 Mettler (Mettler-Toledo International Inc., Colum­
bus, USA). The standard growth rate (SGR%) for dry weight was
calculated using the formula:
( )
LNDW f − LNDW i
SGR% = x 100
tf − ti
where DWf is the final dry weight (mg), DWi is the initial dry weight
(mg), tf is the final time (d), and ti is the initial time (d).
At 0 and 30 DPH, 60 paralarvae per treatment and sampling were
collected (3 h after the first-morning feed) and anaesthetised with
magnesium chloride (1.5% for 10 min and 3.5% for 15 min), and then
dissected using entomological needles (Ento Sphinx inox 0.2 mm,
Entomopraxis SCP, Barcelona, Spain) in a Petri dish placed over ice.
Mantle (M), head (H) and arms (A), as representative of functional body
compartments, and digestive gland (DG), representing a digestive body
compartment, were either placed individually in 1.5 mL tubes filled with
RNA stabilisation buffer (RNA later™, Invitrogen Life Technologies)) for
RNA extraction, or freeze-dried for lipid extraction and FA analyses. All
samples were stored at − 80 ◦ C.
3
Aquaculture 556 (2022) 738293
2.4. Fatty acid analysis
Total lipids from prey samples were extracted using a modification of
the method of Folch et al. (1957). Subsequently, three total lipids ali­
quots (250 μg) were pooled and fractionated into polar and neutral
lipids by thin layer chromatography (20 × 20 plates silica-gel G60,
VWR) using hexane:ethyl ether:acetic acid (75:15:1.5, in volume) as
solvent system. Spots corresponding to polar and neutral lipids were
scrapped off the plate and acid-catalysed transmethylated overnight
(Christie, 1982), previous to analysis by gas chromatography as
described in Viciano et al. (2011). The FA analyses of the body com­
partments of paralarvae were carried out from freeze-dried samples
consisting of pools (N = 20) of samples of each body compartment (M,
H, A, and DG) collected from hatchlings (PH) and 30 DPH paralarvae for
both treatments, PZ and PA, and a direct transmethylation micro-
method was used as described in Garrido et al. (2016).
2.5. Predicted target genes sequences
To obtain coding sequences of the O. vulgaris genes agpta, lpin, chpt,
and dgat, we first identified orthologous predicted from the Octopus
bimaculoides genome (Albertin et al., 2015) available at NCBI with
accession numbers XM_014914065.1, XM_014912142.1,
XM_014912849.1, XM_014914829.1, respectively. Next, the
O. bimaculoides gene sequences were used as queries in BLASTn searches
within Sequence Read Archive (SRA) databases available at NCBI for
O. vulgaris (SRR2857272, SRR2857274, and SRX006887). All reads
were collected and uploaded to Geneious (Geneious V7.1.9) and aligned
against each predictive sequence of O. bimaculoides. Reads poorly
aligned or with identity scores below 95% were removed and the
consensus sequences were obtained. Each putative gene was translated
to amino acid (aa) sequences using ExPASy free software (http://www.
expasy.org) (Artimo et al., 2012).
2.6. Phylogenetic analysis
The aa deduced sequences for each of the target genes (agpta, lpin,
chpt, and dgat) were compiled in the NCBI and Ensembl databases. The
aa sequences were aligned using MAFFT v7.402 free software (Katoh
et al., 2019) with the L-INS-i (Katoh and Standley, 2013). Then, gaps
were deleted from the alignment using GapStrip/Squeeze v2.1.0 in any
columns containing more than 95% gaps. Maximum likelihood trees
were reconstructed using the PhyML 3.0 server (Guindon et al., 2010)
using LG + G + I + F as an evolution model for Agpta, Chpt and Dgat,
and JTT + G + I + F for Lpin. The evolutionary models were calculated
automatically using built in PhyML tool Smart Model Selection (SMS)
(Lefort et al., 2017). The phylogenetic trees were visualised using
Dendroscope (Huson et al., 2007) and rooted with sequences from
Cnidaria and Porifera species. The orthologue protein sequences of
O. vulgaris, published by Zarrella et al. (2019) after the completion of our
experimental and gene expression analyses, were incorporated into the
phylogenetic study (XP_029655226.1, XP_029657873.1,
XP_029636395.1, and XP_029647479.1) to validate ortology of our aa
deduced sequences.
2.7. RNA extraction and cDNA synthesis
For the extraction of total RNA, replicate (N = 6) pools (N = 3) of
body compartments (M, H, A and DG) from PH and 30 DPH PZ and PA
paralarvae were homogenised in 0.5 mL of TRIzol® Reagent and using a
Precellys® 24 (Bertin Technologies, France). Total RNA was extracted
using total Direct-zol RNA Isolation kit (Zymo Research, Irvine, CA,
USA) following the manufacturer’s instructions. The RNA integrity was
checked by running an aliquot of total RNA (~500 ng) on a 1% (w/v)
agarose gel stained with GelRed™ nucleic acid stain (Biotium, Hayward,
CA, USA). The RNA quality was evaluated by sample absorbance
M. Nande et al.
according to the A260/280 and A260/230 ratios using a BiotTek®
microplate reader. Reverse transcription was performed from 500 ng of
total RNA for each group of samples, using the first-strand cDNA Syn­
thesis Kit (NZYTech, Lisbon, Portugal) in T100 thermal cycler (Bio-Rad,
Laboratories, CA, USA) according to the manufacturer’s
recommendations.
2.8. Quantitative RT-PCR design
Gene expression was examined by quantitative RT-PCR (Q-PCR) in
all body compartments including mantle, head, arms and, digestive
gland, from PH and paralarvae fed both dietary treatments (PZ and PA).
Primers of genes related to LC-PUFA biosynthesis, stearoyl-CoA desa­
turase (scd), ωx2 desaturase (ωx2), ωx1 desaturase (ωx1), fatty acyl
desaturase (fad), elovl2/5, and elovl4, and reference genes (Ubiquitin,
18S, Ef1-1α, β-actin, β-tubulin) were compiled from the literature
(Monroig et al., 2012a, 2012b, 2016a, 2017; Garrido et al., 2019; Gar­
cía-Fernández et al., 2016) (Supplementary Table S1). Moreover,
primers targeting the newly identified genes involved in glycer­
ophospholipid biosynthesis (agpta, lpin, chpt, and dgat) were designed on
the exon-exon junctions using Primer 3 software (Untergasser et al.,
2012).
To quantify the relative expression of each target gene in the
different samples (Q-PCR) a Mastercycler ep realplex system (Eppen­
dorf) was used. Each 96-well plate was designed to analyse each body
compartment in six replicates for each treatment (PH, PA and PZ). Each
well contained 5 μL of NZYSpeedy Q-PCR Green MasterMix (2×)
(NZYTech), 0.4 μL of each primer (forward and reversed), and 2 μL of
diluted cDNA (250 nmol) in a final volume of 10 μL. On each plate, a
four non-template control was included. The reaction was carried out
with an initial denaturation at 95 ◦ C (2 min), followed by 40 cycles of
amplification with denaturation at 95 ◦ C for (15 s) and combined
annealing and extension to 58–62 ◦ C, depending on the set of primers
(25 s) (Supplementary Table S1). A melting curve (from 55 ◦ C to 95 ◦ C)
was generated at each run to confirm the specificity of the reactions. The
efficiency of the PCR for both the target and the reference genes was
determined by a standard curve, using six serial dilutions from 1:10 of
cDNA sets of all samples. All reference gene expressions were analysed
in ReFinder online platform to obtain the best comprehensive gene
stability (Xie et al., 2012). Finally, Ef1-1α and 18S were selected to
perform the normalisation of the relative gene expression according to
the Livak method (Livak and Schmittgen, 2001).
2.9. Statistical analysis
Normality (Kolmogorov-Smirnov) and homoscedasticity (Levene)
assumptions were confirmed prior to statistical analyses. The means of
growth indicators (dry weight and number of suckers) for each treat­
ment, and FA data were compared independently by means of one-way
ANOVA and Tukey’s post-hoc test (P < 0.05). The FA data were
compared using univariate analysis of variance (one-way ANOVA) for
each body compartment as fixed factors, exposed to different treat­
ments. Also, the FA profiles from the body compartments of the paral­
arvae of the different treatments were chemometrically analysed using
Multi Dimensional Scaling (PROXSCAL) to establish patterns of simi­
larities (SPSS version 15.0). Normalised relative gene expression for
each target gene was analysed first for differences through the devel­
opment for each body compartment and dietary treatment using a one-
way analysis of variance (ANOVA, P < 0.05). Next, the dietary effect for
each target gene was analysed for each body compartment between PA
and PZ using a one-way analysis of variance (ANOVA, P < 0.05). Except
otherwise stated, the statistical analyses above were conducted using
STATISTICA 10.0© (Zar, 1999). The expression files were normalised
and analysed in Partek® Genomics Suite® in order to perform the hi­
erarchical clustering and heat map analyses. The datasets of relative
gene expression for each of the treatments and body compartments were
4
Aquaculture 556 (2022) 738293
included, and normalisation and log2 transformations were performed.
2.10. Ethics
All experiments were carried out under the Spanish legislation
(RD53/2013) and the European Directive 2010/63/EU (European
Parliament, Council of the European Union, 2010) for the protection of
animals used for experimentation and other scientific purposes. Adults
and paralarvae were anaesthetised with an initial concentration of 1.5%
magnesium chloride (magnesium chloride hexahydrate, Barcelonesa©,
Global Chemical Solutions, Barcelona, Spain) for 10 min, which was
subsequently increased to 3.5% for 15 min to minimize pain, suffering
and distress according to Fiorito et al. (2015). A humane killing method
by brain destruction using a bistoury was performed at the end of the
study according to the guidelines of experimentation with cephalopods
(Andrews et al., 2013; Fiorito et al., 2015).
3. Results
3.1. Growth rate and development
No differences in dry weight were observed in paralarvae of the same
age from the two replicated experiments (P < 0.05). Thus, growth and
development data were grouped by age. Average initial dry weight (PH)
was 0.27 ± 0.04 mg. After 30 d of dietary treatment, average dry weight
reached 1.86 ± 0.20 mg for PA and 4.46 ± 0.25 mg for PZ (P < 0.05,
Fig. 1). Considering growth related to arm length, at 30 DPH it was 17 ±
2 suckers/arm for PZ, also significantly higher than that of PA (8 ± 2
suckers/arm) (P < 0.05, Fig. 1). Such difference between dietary
treatments was already significant at 20 DPH, with 8 ± 1 suckers/arm
for PZ, and 5 ± 1 suckers/arm for PA (P < 0.05). The SGR at 30 DPH was
9.37% for PZ, whereas PA paralarvae reached 6.46% of body increment
per day.
3.2. Fatty acid composition
The FA profiles from total lipids, as well as those from the polar and
neutral lipid fractions of the preys (spider crab zoeae and Artemia), are
shown in Supplementary Tables S2 and S3, respectively. Also, the FA
composition of the total lipids from each body compartment of the
paralarvae at hatching (PH) and at 30 DPH are shown in Supplementary
Table S4.
3.2.1. Preys
The preys FA composition was significantly different (Supplemen­
tary Table S2). While Artemia metanauplii were particularly rich in 18C
FA (18:1n-9, 18:1n-7, 18:2n-6, 18:3n-3, and 18:4n − 3), spider crab
zoeae presented a high content of ARA (20:4n-6), EPA (20:5n-3), and
DHA (22:6n-3) (P < 0.05). Also, the proportion of FA in neutral and
polar lipids of preys was remarkably different for most of the FA ana­
lysed (Supplementary Table S3). Besides, 18C FA like oleic acid (18:1n-
9), vaccenic acid (18:1n-7), and LA (18:2n-6) were present in higher
proportion in the polar lipids of Artemia compared to crab zoeae, while
they were more abundant in the neutral lipids from crab zoeae. How­
ever, n-6 and n-3 LC-PUFA like ARA, EPA, and DHA were found in a
higher percentage in the polar lipid fraction of both preys, these being
significantly more abundant in the spider crab zoeae.
3.2.2. Paralarvae
Differences were found in the FA composition of PH and fed paral­
arvae (Supplementary Table S4). The impact of diet on the FA compo­
sition was higher on functional (M, H, and A) than on digestive body
compartment (DG). A higher proportion of palmitic acid (16:0) was
found in all body compartments of PH, and it decreased significantly in
both dietary treatments at 30 DPH (P < 0.05), showing the lowest
amount in the DG (Table 1; Supplementary Table S4). A significantly
M. Nande et al.
higher amount of 16:0 was found in the mantle and arms of PA as
compared to PZ (P < 0.05).
Regarding C18-FA, the amount of stearic acid (18:0) increased dur­
ing development in the mantle and digestive gland in PA and PZ
compared to PH, but decreased in the arms (Table 1; Supplementary
Table S4). Yet, ALA and stearidonic acid (18:4n-3) were not detected in
PH and thus accumulated during development, with a higher amount
found in all body compartments of PA paralarvae compared to PZ (P <
0.05). Besides, LA increased in all functiona body compartments of PA
while decreasing in PZ as compared to PH (P < 0.05), and the propor­
tion of 18C oleic (18:1n-9) and vaccenic (18:1n-7) acids, also increased
in the DG of fed paralarvae (PA and PZ) as compared to that of the
hatchlings (Table 1; P < 0.05). The most remarkable differences in the
n-3 LC-PUFA composition between PA and PZ were found in the func­
tional body compartments (Supplementary Table S2). Levels of eicosa­
trienoic acid (ETE, 20:3n-3) increased significantly in the heads of
paralarvae fed both experimental diets (P < 0.05; Supplementary
Table S4). The amount of ARA and EPA increased in paralarvae from
both dietary treatments in all functional body compartments (Table 1; P
< 0.05). Thus, the head and mantle of PZ paralarvae showed a signifi­
cantly higher amount of ARA compared to PA (P < 0.05). For EPA,
5
Aquaculture 556 (2022) 738293
Fig. 1. Image of paralarvae at 30 DPH fed with zoeae
of spider crab (M. brachydactyla; A) and with Artemia
metanauplii (Artemia franciscana; B). The graph rep­
resents growth in dry weight of paralarvae fed zoeae
(PZ, closed circle) and Artemia (PA, open square)
throughout 30 DPH. Bars indicate arms growth
measured in number of suckers of paralarvae fed
zoeae (SPZ), and Artemia (SPA). Within each sampling
period, the asterisk (*) and symbol (✚) represent
significant differences (P < 0.05) in dry weight and
number of suckers respectively.
significant differences were found in the functional body compartments
of the PA treatment compared to PZ (P < 0.05). Also, docosapentaenoic
acid (DPA, 22:5n3) increased during development in all body com­
partments except in the digestive gland of PA. DHA decreased in all body
compartments except in the arms and mantle of PZ during development,
with the digestive gland having the lowest amounts of DHA in paral­
arvae from both dietary treatments (Table 1). Compared to PZ, PA
showed a significantly lower proportion of DHA in all body compart­
ments (P < 0.05).
The integration of the FA profiles in a Multidimensional Scaling
Analysis (MDS) showed distinct patterns (Fig. 2). Hatchlings (PH) and
on-grown paralarvae (PA and PZ) were clearly distinguishable, the latter
being further segregated. Importantly, the digestive gland of both PA
and PZ were separated from the rest (Fig. 2). Fatty acids of the diets also
formed distinct clouds, with spider crab zoeae showing higher similarity
(proximity) to the hatchlings and most functional compartments of the
dietary groups, and those of Artemia being closer to the digestive gland
patterns. Along the x-axis, the FA patterns were distributed following a
left (more) to the right (low) gradient of FA unsaturation (i.e. LC-PUFA).
M. Nande et al. Aquaculture 556 (2022) 738293

Table 1
Selected fatty acids (% of total fatty acids) in body compartments (mantle, head, arm and digestive gland) of hatchlings (PH) and 30 DPH paralarvae fed with zoeae of
spider crab (Maja brachydactyla) (PZ) or Artemia metanauplii (Artemia franciscana) (PA). Results are the mean and standard deviation. For every body compartment and
fatty acid, different letters denote significant differences (one-way ANOVA P < 0.05). ND: not detected; Sat.: saturated fatty acids; Mono.: monounsaturated fatty acids;
n-3 LC-PUFA: n-3 long-chain polyunsaturated fatty acids (C ≥ 20); n-6 LC-PUFA: n-6 long-chain polyunsaturated fatty acids (C ≥ 20). Different letters within body
compartments denote significant differences. Whole fatty acid results can be found in supplementary material.
Mantle Head Arm Digestive gland

PH PA PZ PH PA PZ PH PA PZ PH PA PZ

16:0 29.48 ± 21.23 ± 18.79 ± 27.34 ± 20.23 ± 18.32 ± 33.01 ± 20.93 ± 18.74 ± 28.12 ± 16.44 ± 14.31 ±
1.36a 0.73b 0.20c 1.42a 0.11b 0.19b 3.42a 0.22b 0.38c 3.45a 1.70b 1.44b
18:0 10.77 ± 13.84 ± 12.17 ± 12.65 ± 12.54 ± 0.04 11.33 ± 19.34 ± 14.65 ± 12.58 ± 12.78 ± 14.76 ± 15.76 ±
0.82b 0.14a 0.67a 0.81 0.13 3.12a 0.05b 0.14c 1.67b 1.43b 1.56a
18:1n-9 2.15 ± 3.97 ± 2.74 ± 3.20 ± 3.83 ± 0.04a 3.17 ± 2.88 ± 4.31 ± 3.50 ± 4.44 ± 12.33 ± 10.07 ±
0.22b 0.06a 0.13b 0.13b 0.06b 0.62c 0.02a 0.04b 0.02b 1.36a 1.72a
18:2n-6 1.06 ± 1.46 ± 0.67 ± 0.72 ± 1.38 ± 0.01a 0.67 ± 1.10 ± 1.39 ± 0.76 ± 0.72 ± 4.77 ± 3.08 ±
0.04b 0.01a 0.28c 0.11b 0.20b 0.22a 0.06a 0.35b 0.15b 0.56a 2.19a
18:3n-3 ND 1.44 ± 0.30 ± ND 0.93 ± 0.02a 0.26 ± ND 1.19 ± 0.34 ± ND 3.28 ± 1.63 ±
0.03a 0.20b 0.15b 0.06a 0.23b 0.10 1.22
20:4n-6 2.70 ± 6.47 ± 6.99 ± 2.11 ± 3.42 ± 0.06b 4.12 ± 1.50 ± 5.77 ± 5.73 ± 4.28 ± 3.67 ± 4.71 ±
0.23c 0.04b 0.10a 0.20c 0.13a 0.18b 0.32a 0.26a 0.51 0.47 0.53
20:5n-3 14.29 ± 22.21 ± 17.81 ± 14.39 ± 22.08 ± 17.35 ± 8.76 ± 22.20 ± 17.75 ± 9.82 ± 11.00 ± 10.78 ±
0.74c 0.16a 0.03b 0.70c 0.10a 0.24b 1.29c 0.69a 0.12b 1.45 1.40 1.26
22:6n-3 21.01 ± 8.30 ± 20.41 ± 19.68 ± 9.61 ± 0.16b 19.41 ± 11.53 ± 6.54 ± 17.66 ± 13.84 ± 2.01 ± 8.58 ±
1.13a 0.01b 0.32a 1.01a 0.28a 1.62b 0.11c 0.21a 2.34a 0.74c 1.72b
Sat. 45.17 ± 38.38 ± 34.13 ± 44.21 ± 35.66 ± 32.48 ± 58.82 ± 39.05 ± 34.44 ± 45.68 ± 38.18 ± 34.54 ±
2.41a 0.58b 0.48b 2.56a 0.08b 0.13b 5.82a 0.39b 0.41b 6.14a 7.17ab 3.23b
Mono. 8.14 ± 11.79 ± 10.60 ± 11.11 ± 13.387 ± 13.19 ± 7.74 ± 13.44 ± 13.08 ± 11.95 ± 25.08 ± 24.05 ±
0.45b 0.37a 0.15ª 0.41b 0.08a 0.13a 1.87b 0.07a 0.29a 0.98b 5.97a 2.18a
n-3 36.86 ± 34.86 ± 40.25 ± 36.07 ± 39.24 ± 41.69 ± 21.03 ± 32.94 ± 37.84 ± 24.39 ± 19.74 ± 22.55 ±
1.94ab 0.01b 0.8ª 1.80b 0.17ab 0.15a 2.79a 0.54a 0.23a 3.98a 2.00b 2.38ab
n-6 4.81 ± 10.09 ± 9.55 ± 3.34 ± 6.58 ± 0.19a 6.36 ± 2.43 ± 9.03 ± 8.20 ± 5.48 + 9.61 ± 10.02 ±
0.28b 0.16a 0.20ª 0.30b 0.17a 0.57b 0.11a 0.09a 0.82b 1.32a 1.91a
n-3 LC- 36.86 ± 32.66 ± 39.83 ± 36.07 ± 37.880.19ab 41.32 ± 20.99 ± 31.11 ± 37.37 ± 24.39 ± 14.36 ± 20.55 ±
PUFA 1.94ab 0.01b 0.33ª 1.80b 0.33a 2.76b 0.51a 0.03a 3.98a 1.63b 2.86a
n-6 LC- 3.75 ± 8.52 ± 8.88 ± 2.62 ± 5.14 ± 0.19a 5.69 ± 1.55 ± 7.55 ± 7.45 ± 4.76 ± 5.11 ± 6.94 ±
PUFA 0.32b 0.18a 0.09a 0.30b 0.04a 0.25b 0.16a 0.26a 0.85b 0.46b 0.63a

3.3. Predicted sequences and phylogenetics analysis
Through the analysis of SRA from O. vulgaris, we were able to deduce
complete/partial open reading frames of previously unreported genes:
agpta, lpin, chpt and dgat. To determine the orthology of each sequence,
independent phylogenetic trees were constructed (Supplementary
Fig. S1). All genes were strongly clustered within others from the
phylum Mollusca with support posterior probabilties 0.99 for Agpta
(Supplementary Fig. S1.A), 0.98 for Lpin (Supplementary Fig. S1.B), 1
for Chpt and Dgat (Supplementary Fig. S1.C and D). In addition, the
sequences showed high homology with the sequences of O. bimaculoides
(<0.99).
3.4. Gene expression
Gene expression response in each body compartment (functional and
digestive) was analysed throughout development, PH to paralarvae of
30 DPH (Figs. 3 and 4). A significantly higher expression of genes
involved in LC-PUFA biosynthesis was found on the functional body
compartments compared to the digestive one. At hatching, paralarvae
showed largely lower gene expression as compared to the 30 DPH, for
scd and both for the elongases (elovl4 and elovl2/5), in all body com­
partments. Also, in PH, transcripts of ωx2 were only detectable in the
head, and in 30 DPH paralarvae, ωx2 transcripts increased, at 30 DPH
paralarvae in head. Paralleling that, expression of the desaturase fad and
ωx1 was low in all body compartments of PH, and throughout devel­
opment, although the gene expression increased in heads and arms for
30 DPH paralarvae.
The expression of glycerophospholipid-related genes showed an in­
crease of transcripts in the digestive gland both at hatchling (PH) and
throughout development (30 DPH) except for Agpta, which was up-
regulated for head and arms at 30 DPH compared with PH (P < 0.05).
The lpin expression increased during development (from PH to 30 DPH)
for all functional compartments of the body; however, for DG, it
decreased significantly for PZ. Also, chpt showed a low number of
transcripts in all body compartments of PH, increasing during devel­
opment in the digestive gland and arms of PA and PZ respectively (P <
0.05). Therefore, the expression dgat in PH was higher in the digestive
gland than in the functinal body compartments, and at 30 DPH it was up-
regulated for PA and down-regulated for PZ compared with PH (P <
0.05).
The effect of the dietary treatments (PA and PZ) on gene expression
was also analysed for each body compartment (Figs. 3 and 4). Paralarvae
fed Artemia (PA) showed a general pattern characterised by an up-
regulation of functional body compartments for LC-PUFA genes as
compared to PZ, with a significant increase (P < 0.05) of ωx2 and fad
transcripts in the head, and elovl4 being up-regulated in mantle and head
(Fig. 3). Furthermore, PA showed an expression pattern characterised by
up-regulation of genes involved in the glycerophospholipid pathway
(lpin, chpt, and dgat) in the digestive gland, as well as of agpta and lpin in
the head (Fig. 4, P < 0.05). However, chpt showed a higher increase of
transcripts in arms of PZ as compared to PA (P < 0.05).
In order to provide a global view of these results, a hierarchical
clustering and a heat map analysis of target genes were conducted. Hi­
erarchical clustering segregated the genes into three major clusters such
as elovl4, elovl2/5, and scd for the first group, agpta, fad, ωx2, ωx1 for the
second, and lpin, chpt, and dgat for the third (Fig. 5). Clustering in terms
of body compartments/treatments was determined by the similarity in
the expression between PZ and PH in all the body compartments. In fact,
the expression of PA was far from that of the other dietary treatments,
and showed a tendency towards an up-regulation of most of the genes
analysed. In particular, the pattern of gene expression in the digestive
gland of PZ was far from that of PA and very close to that of PH (Fig. 5).

6
M. Nande et al. Aquaculture 556 (2022) 738293

Fig. 2. Multidimensional Scaling of the fatty acid profiles of the body compartments of O. vulgaris paralarvae and preys. Green ovals group the scores of paralarvae
fed zoeae (PZ), orange ovals of those fed Artemia (PA), and the blue oval hatchings (PH). Zoea (green oval) and Artemia (orange oval) identify the scores of the
profiles of both preys. M, mantle; H, head; A, arms; DG, digestive gland. (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)

Fig. 3. Relative expression of genes related to LC-PUFA biosynthesis (stearoyl-CoA desaturase (scd), ωx2 desaturase (ωx2), ωx1 desaturase (ωx1), fatty acyl desaturase
(fad), elovl2/5, and elovl4) analysed in diffrerent body compartments (mantle, head, arms, and digestive gland) from hatchlings (PH) to 30 days post-hatch (30 DPH)
paralarvae, and among dietary treatments (PZ vs PA). The results are presented as the means ± sd (N = 6) of a pool of body compartments (N = 3). Asterisks (*)
indicate significant differences (P < 0.05) during development, and letters between dietary treatments (P < 0.05).

4. Discussion effect of a diet rich in LC-PUFA (zoeae, mainly ARA and DHA) and a diet
with marked content of C18-FA (Artemia) in functional body compart­
This study is the first to offer comprehensible information on the ments compared to digestive ones, and on the compensatory

7
M. Nande et al. Aquaculture 556 (2022) 738293

Fig. 4. Relative expression of genes related to glycerophospholipid biosynthetic pathway (agpta, lpin, chpt, and dgat) analysed in different body compartments
(mantle, head, arms, and digestive gland) from hatchlings (PH) to 30 days post-hatch (30 DPH) paralarvae, and among dietary treatments (PZ vs PA). The results are
presented as the means ± sd (N = 6) of a pool of body compartments (N = 3). Asterisks (*) indicate significant differences (P < 0.05) during development, and letters
between dietary treatments (P < 0.05).

Fig. 5. Hierarchical clustering of genes detected as differentially expressed in different body compartments for the hatchlings (PH) and different dietary treatments
(PA and PZ). The colour bar denotes z-score adjusted expression values, green represents lower gene expression, and red, higher. Euclidean distance and average
linkage methods were used. PH: hatched paralarvae. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)

8
M. Nande et al.
mechanisms involved in the FA, glycerophospholipids, and glycerolipids
biosynthesis during the paralarval development.
Our results show that paralarvae fed zoeae display significantly
higher growth and development at 30 DPH than those fed Artemia. This
agrees with the results reported by several authors (Villanueva, 1995;
Iglesias et al., 2004; Carrasco et al., 2006; Roo et al., 2017; Garrido et al.,
2018), indicating that the decapod zoeae favor growth over Artemia.
Likewise, in paralarvae in co-feeding experiments (Artemia + zoeae) the
SGR% varies between 7% and 8% of increment of dry weight per day
(Villanueva, 1995; Iglesias et al., 2004; Carrasco et al., 2006) and rea­
ches only values from 2% to 6% for paralarvae fed with Artemia (Nav­
arro and Villanueva, 2000; Okumura et al., 2005; Seixas et al., 2010;
Fuentes et al., 2011). It has also been reported that the number of
suckers per arm reflects its growth during the transition from the
planktonic stage to the benthic phase (Villanueva and Norman, 2008),
and holds a good correlation with weight and development (Okumura
et al., 2005), which is also agreement with the results shown in Fig. 1.
The differences in growth and development of the paralarvae fed
Artemia is related, among other factors, to a dietary essential FA depri­
vation (Navarro et al., 2014). Thus, the effect of diet on the lipid profile
of paralarvae could be linked to the appearance of LA and 18:4n-3 which
were retained in the metabolic organ (DG), mainly in the PA treatment,
and was absent in PH. The impact of the diet in the FA composition of
paralarvae has been clearly observed in co-feeding Artemia and zoea
trials, or when inert diets were used as food, with the amount of LA
decreasing as compared to those paralarvae fed only Artemia (Seixas
et al., 2010; Roo et al., 2017). Also, our results indicate a greater amount
of total n-6 LC-PUFA in energy-demanding structures, such as the
mantle, involved in active swimming and breathing (Shadwick, 1995;
Villanueva and Norman, 2008), and sensorial structures such as head
(Villanueva et al., 2017). It has been pointed out that ARA may play
pivotal roles in developmental processes (Monroig et al., 2012b). On the
other hand, the head is a functional body compartment with organs such
as eyes and brain that require large amounts of n-3 LC-PUFA for proper
development and, interestingly, not only EPA and DHA, but for example
ETE, present in high proportions in the adult eye (Monroig et al., 2012b)
is also found here in the head of the paralarvae of both treatments. It is
interesting that the digestive gland of fed paralarvae contained the
lowest levels of n-3 LC-PUFA, as well as the arms and the digestive gland
of PH (Table 1). It is tempting to hypothesize that in case of need, EPA
could be quickly mobilized (Sargent et al., 1999) from the digestive
gland to functional body compartments as an essential component of cell
membranes (Bell and Sargent, 2003). Thus, the amount of EPA in the PA
dietary treatment being higher than in the other treatments (Table 1),
could account for the lower availability of DHA in Artemia and its
replacement by EPA. These results agree with those obtained by several
authors when feeding paralarvae with enriched Artemia (Navarro and
Villanueva, 2000; Seixas et al., 2010; Fuentes et al., 2011). Although
zoeae had a higher percentage of EPA than Artemia, PA retained this FA
in the functional compartments pointing at the compensatory mecha­
nism proposed before. Fish larvae fed DHA deficient diets show an in­
crease of DPA in their FA, pointing to a similar compensatory
mechanism (Furuita et al., 1996). Interestingly, functional body com­
partments of the paralarvae from the PA treatment showed DPA
amounts similar to those of PZ (Supplementary Table S2).
Docosahexaenoic acid content was lower in the paralarvae of the PA
group as compared with the others. Besides, its concentration in func­
tional structures was notoriously higher with respect to those found in
the digestive gland. The DHA dietary contribution of Artemia is very low,
especially in the neutral lipids, so it can be hypothesized that the DHA
present in the hatchlings was mainly functional, and was not mobilized
during development because of it having a relative resistance to
β-oxidation (Bell et al., 2001) in a scenario of essential FA paucity. The
small percentage of DHA provided by Artemia can be counterbalanced
by the incorporation of other LC-PUFA like ARA and EPA esterified into
TAG (Reis et al., 2016). Compensatory mechanisms of this kind have
9
Aquaculture 556 (2022) 738293
been reported and may be dependent on the intensity of this essential
nutrient deprivation. In O. vulgaris juveniles exposed to prolonged pe­
riods of fasting it was observed that DHA was catabolized (García-Gar­
rido et al., 2010). On the contrary, when an organ needs to increase or
maintain a greater proportion of DHA, this FA can be mobilized from
other body parts as occurs in wild O. vulgaris adults (Sieiro et al., 2020)
and squid (Lin et al., 2019), or selectively retained as described in the
cuttlefish (Castro et al., 1992). The presence of LC-PUFA, and specif­
ically DHA, are particularly important in the head, because these FA are
related to neuronal development and vision, and a deficiency could lead
to serious physiological and behavioral consequences as occurs in fish
(Sargent et al., 1999; Sargent et al., 2003). Also, higher DHA in the arms
of the PZ paralarvae could be associated with their better performance in
terms of growth and development, not to mentional axonal development
and, in this sense, the fact that the arms of newly hatched paralarvae
showed a lower amount of DHA can be linked to the suckers only
starting to develop at 10 DPH.
The MDS analysis clearly shows the scores of the digestive gland of
the dietary treatments (PA and PZ) close to each other and segregated
from the rest. This points to a unique FA pattern, perhaps due to the
gland being involved in nutrient storage and mobilization (Blanchier
and Boucaud-Camou, 1984). Saturated FA (18:0), MUFA (18:1n-9,
18:1n-7), n6-PUFA (18:2n-6), and n3-PUFA (18:3n-3, 18:4n-3) were the
main FA in the digestive gland. This points again at those found in higher
proportion in functional body compartments such as 16:0, ARA, EPA,
and DHA being essential (Navarro and Villanueva, 2000; Monroig et al.,
2012a; Iglesias et al., 2014; Reis et al., 2014; Lourenço et al., 2017) since
they would be transported and selectively retained into these structures
whenever present in the diet. The MDA shows the scores of the mantle,
head, and arms of the PZ group distinguished from those of PA, closer to
PH, and also segregated from the composition of their prey (zoeae),
reflecting the dietary effect differently expressed at the level of func­
tional vs digestive (digestive gland) organs. The scores of the PA group
distribute between the PH cluster and digestive gland, reflecting that the
dietary input of a diet not fulfilling the nutritional requirements of the
paralarvae, but still produces distinct functional versus digestive FA
profiles. In the PH treatment, although sub-groups of organ-related
scores are distinguishable, the composition of all body compartments
can be grouped in the same cluster possibly reflecting the most limited
“dietary” influence of yolk (Nande et al., 2017a). Finally, the scores
produced by the FA patterns of Artemia are clearly separated from the
rest, farther from the PH and PZ (mantle, head and arms) clusters, and
next to the digestive gland ones, showing up their distinct nature.
The diet directly affects metabolic structures such as the digestive
gland and has a more conservative effect on those functional body
compartments that tend to keep their lipid composition constant. This
effect, on the one hand, is produced by the mobilization of nutrients
from the diet and, failing that, by the biosynthesis of these FA from
enzymatic pathways. The ability of marine invertebrates to de novo
biosynthesize saturated and monosaturated FA and their further trans­
formation into PUFA from elongation reactions (fatty acyl elongases)
and desaturations through methyl and front-end desaturases (Monroig
and Kabeya, 2018) has been documented. Recently, the capacity of FA
biosynthesis of cephalopods has been studied by several authors (Mon­
roig et al., 2012a, 2012b, 2016a, 2017; Reis et al., 2014, 2016; Garrido
et al., 2019; Monroig and Kabeya, 2018), as well as that of other mol­
lusks (Pirini et al., 2007).
Our results indicate a low regulation of gene expression in PH
paralarvae compared to PA and PZ (Fig. 3). This fact may be related to
the ability of the yolk to meet all the nutritional needs of the paralarvae
at the earliest life stages, so that the biosynthetic machinery is still on
standby. Indeed, hatchlings combine endogenous and exogenous
feeding and have low needs related to growth and development (Nande
et al., 2017a). In any case, the digestive gland of the paralarvae from the
PH group was the body compartment with the highest expression of lpin
and dgat indicative of activation of glycerophospholipids synthesis
M. Nande et al.
towards TAG. The increase in the production of TAG in this group may
be related to a low concentration of this lipid class in the hatchlings
(Lourenço et al., 2017) and its need in the highly demanding early stages
of development as an energy source.
The degree of regulation, being inversely proportional to the suit­
ability of the diets in providing the necessary nutrients, increased in the
PA group in which most of the genes analysed were over-expressed, with
the PZ treatment producing intermediate results. This is in agreement
with the results of García-Fernández et al. (2017) who found that the
lipid biosynthesis pathways were up-regulated in paralarvae fed Artemia
as compared to those fed zoeae. A higher relative gene expression of scd
in functional body compartments, and mostly in the mantle of the PA
treatment, was observed. The enzymatic activity of Scd, which in­
troduces the first double bond into a saturated FA, is universally present
in all organisms (Castro et al., 2011) and can be associated with the
triggering of the unsaturation pathway. The presence of enzymes
involved in the production of ωx2 (Δ12 activity) and ω3ωx1 (Δ15, Δ17
and Δ19 activities) methyl-end desaturations, has recently been iden­
tified in marine invertebrates (Kabeya et al., 2018). In the present re­
sults, we found a higher expression of the ωx2 methyl-end desaturations
in the head and of the ωx1 in head and arms of the paralarvae of the
dietary treatments, peaking in the PA and PZ dietary group, respectively.
The activity of these enzymes can thus be anatomically linked in these
first stages under development to the formation of structures like eyes
and brain, as it has been reported in adults (Garrido et al., 2019). The
activity of the Fad, supported by fad expression, was evident in the head
of the PA paralarvae, perhaps due to an increase in the need for PUFA
not provided by the diet. The gene that encodes this front-end desaturase
has also been described in Sepia officinalis (Monroig et al., 2016b). A
high expression of elongases genes was found in functional body com­
partments such as the mantle and head for elovl4 and head for elovl2/5.
The energy demand of the mantle and the neuronal and visual matu­
ration linked to development in head, increases the demand for LC-
PUFA (Monroig et al., 2017). The slower development of the arms in
the PA treatment was also reflected in a lower number of transcripts, and
therefore was coherent with a metabolic impairment scenario.
Hierarchical clustering helps to elucidate groupings and patterns that
provide an integral vision of the results of the expression tendencies of
the different genes (Bergkvist et al., 2010). In our datasets, the clustering
showed first a group including elovl4, elovl2/5, and scd, that had a more
widespread expression in all functional body compartments of PA and
PZ, with special significance in the mantle and head. Also, these en­
zymes have a preference for the use of 18C and 16C saturated FA pre­
cursors. The second clustering grouped agpta, fad, ωx2, and ωx1,
showing a more specific expression tendency putatively related to the
development of head (Fig. 4), with visual and neuronal tissue. The
clustering of different enzymes can be related to the body compartments
where they are more active, as well as to the preferred FA precursor.
From this point of view, ARA and EPA can be synthesized from 20:3n-6
and 20:4n-3 by the mediation of Fad (Monroig et al., 2012a), whereas
Δ12ωx2 and ω3ωx1 are related to the synthesis of LA, and to the con­
version of n-6 LC-PUFA to n-3 LC-PUFA, respectively (Garrido et al.,
2019).
Long-chain PUFA like EPA, DHA, and ARA are an essential compo­
nents of glycerophospholipid in cephalopods (Shen et al., 2020), how­
ever, the regulation of their biosynthetic involvement in these structural
lipids is poorly understood in O. vulgaris. Previous studies using radio­
tracer techniques have demonstrated de novo biosynthesis by esterifi­
cation of phosphatidylethanolamine (PE) and phosphatidylcholine (PC)
with a preference for ARA and EPA (Reis et al., 2014). Since the glyc­
erophospholipid biosynthetic pathway is considered key in the pro­
duction of these phospholipids and triglycerides, and PC is an abundant
phospholipid in the common octopus (Reis et al., 2019), we have ana­
lysed here genes such as agpta, lpin, chpt, critical in each of the phases for
the synthesis of PC, as well as dgat, involved in the synthesis of TAG.
Metabolites of the glycerophospholipid pathway linked to the action of
10
Aquaculture 556 (2022) 738293
Agpta, like phosphatidic acid, are related to neuronal development, with
functions such as transmission and regulation of intracellular signaling
and proliferation (Ammar et al., 2014). In our study, we found a greater
amount of transcripts of this gene in the head of PA compared to the
other treatments. This fact is also consistent with the hypothesis that
paralarvae fed suboptimal diets tend to increase biosynthetic pathways
that compensate for the effect of nutrient deficiency such as PL. Thus,
Lpin plays multiple roles in the regulation of lipid metabolism and cell
signaling and as a regulator in the production of PL (Reue and Brindley,
2008). Additionally, García-Fernández et al. (2019) also reported up-
regulated gene expression of phosphatidate phosphatase (pap or lpin)
in O. vulgaris paralarvae subjected to sub-optimal feeding conditions. In
our study, the highest activity of Chpt was found in fed paralarvae (PA
and PZ) compared to hatchlings (PH), and this fact could be due to the
high content in PL of the latter, or the limited ability of de novo PL
synthesis of the former, as has been documented in the liver of fish
larvae (Tocher et al., 2008). Indeed, like the liver in fish, the digestive
gland of cephalopods plays an important role in the digestion and
metabolism of lipids, with various functions such as secretion, digestion,
and absorption (O’dor et al., 1984). Furthermore, for chpt, a higher
number of transcripts was found in the arms of PZ, coinciding with a
significant differentiation during development, as compared to PA. The
arms growth needs axons production for the formation of the nerve cord,
which involves a high content of neuronal membranes rich in PC
(Imperadore et al., 2019), and Chpt activity is associated with PC
biosynthesis and also to the presence of synaptic membranes (Har­
greaves and Clandinin, 1987).
Our results only showed overexpression of dgat in the digestive gland
of PA and PH treatments. The biosynthesis of either PL or TAG in the
digestive gland (involving lpin, chpt, and dgat expression) may be the
result of catabolism and the re-assembly of larger molecules that facil­
itate their absorption. This is coherent with the absence of dgat
expression in the PZ paralarvae and its overexpression in the digestive
gland of the PA as a mechanism for TAG synthesis as a source of energy.
The differential anatomical gene regulation unveiled here may be of
paramount importance in organisms like the paralarvae, in which a
preponderant part of the body is dominated by a single organ, in this
case the DG. In such cases, the analysis of the whole organism would be
biased by the response of such body compartment.
In summary, the present study provides evidence of the pivotal role
that diet plays in the biosynthesis of FA, glycerophospholipids, and
glycerolipids in paralarvae. It shows how dietary treatments affect the
expression of the related pathways, both in digestive (digestive gland)
and functional (mantle, head and arms) compartments of the paralarvae,
inducing unique compensatory responses. Genes related to FA biosyn­
thesis in the head, and glycerophospholipid and glycerolipid meta­
bolism in DG, could be used as more sensitive biomarkers of dietary
effects. This anatomical approach may pave the way for further studies
on the nutritional requirements of O. vulgaris and other species of
cephalopods.
Author statement
MN, ÓM, and JCN conceived, designed, and supervised the study;
MN performed the rearing experiments; MN, AMM, LFCC, MLM, AC
performed the Q-PCR, gene target isolation, phylogenetic analysis, and
conducted data analysis. JCN performed the fatty acid profile analysis,
and JCN and MN carried out the data analysis. All authors contributed to
the writing and revision of the manuscript and approved the final
version for submission.
Declaration of Competing Interest
We declare that all authors have no conflicts of interest in this work.
M. Nande et al. Aquaculture 556 (2022) 738293

Acknowledgments European Parliament, Council of the European Union, 2010. Directive 2010/63/EU
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Tion of Animals Used for Scientific Purposes. Council of Europe, Strasbourg.
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13
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The Project Gutenberg eBook of The house of bondage
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Title: The house of bondage

Author: Reginald Wright Kauffman

Release date: March 6, 2024 [eBook #73115]

Language: English

Original publication: New York: Grosset & Dunlap, 1910

Credits: Al Haines

*** START OF THE PROJECT GUTENBERG EBOOK THE HOUSE

OF BONDAGE ***
 THE
HOUSE OF BONDAGE

By

REGINALD WRIGHT KAUFFMAN

Author of "What is Socialism?" etc.

NEW YORK
GROSSET & DUNLAP
PUBLISHERS

COPYRIGHT, 1910, BY
MOFFAT, YARD AND COMPANY
NEW YORK

All Rights Reserved

 Published August, 1910
Second Printing, November, 1910
Third Printing, December, 1910
Fourth Printing, January, 1911
Fifth Printing, February, 1911
Sixth Printing, February, 1911
Seventh Printing, March, 1911
Eighth Printing, March, 1911
Ninth Printing, April, 1911
Tenth Printing, April, 1911
Eleventh Printing, June, 1911
Twelfth Printing, July, 1911
Thirteenth Printing, August, 1911
Fourteenth Printing, October, 1911
Fifteenth Printing, February, 1912
Sixteenth Printing, April, 1912

THE QUINN & BODEN CO. PRESS

RAHWAY, N. J.

TO
ANDREW JOHN KAUFFMAN
(1840-1899)

"O strong soul, by what shore

Tarriest thou now? For that force,
Surely, has not been left vain!
Somewhere, surely, afar,
In the sounding labor-house vast
Of being, is practiced that strength,
Zealous, beneficent, firm!"
 CAVEAT EMPTOR

This story is intended for three classes of readers, and no more. It is

intended for those who have to bring up children, for those who have to
bring up themselves, and for those who, in order that they may think of
bettering the weaker, are, on their own part, strong enough to begin that task
by bearing a knowledge of the truth.

For it is the truth only that I have told. Throughout this narrative there is
no incident that is not a daily commonplace in the life of the underworld of
every large city. If proof were needed, the newspapers have, during the last
twelvemonth, proved as much. I have written only what I have myself seen
and myself heard, and I set it down for none but those who may profit by it.

REGINALD WRIGHT KAUFFMAN.

NEW YORK CITY,

16th June, 1910

THE HOUSE OF BONDAGE

"AS IF THE SPRING WERE ALL YOUR OWN"

The local weather-prophets—the cape-coated Mennonites and the

bearded Amishmen, who came into the town to market—had said, with
choral unanimity, that the spring would be brief and sudden, and the
summer parching and intense.
 Already, though April had but dawned, the pink arbutus had bloomed
and withered, and the pale first violets were peeping, purple and fragrant,
among the lush grass of the front yards on Second Street. The annual oriole
was a full fortnight ahead of his time in opening his summer-house in the
hickory-tree on the Southwarks' lawn; and up in the droning study-room of
the high-school, where all the windows were wide to the lazy sunlight, Miss
England had begun, this week, to direct the thoughts of her dwindling
senior-class toward the subjects of their graduation essays.

Swaying with the easy, languid grace of an unstudied young animal,

Mary Denbigh, the morning-session ended, turned from the graveled walk
before the school-grounds into the little town's chief thoroughfare.

Nobody had ever called her pretty, but her light serge skirt had that day
been lengthened to her ankles, and Mary was wholly conscious of the new
tokens of her growth. Lithe, strong-limbed and firm-bodied, of peasant
stock and peasant vigor, youth and health and the open country air were not
factors sufficiently unfamiliar to combine in a charm that would attract
admiration in her own community. Only a jaded city-gaze—and a well-
trained city-gaze at that—would have seen in the blue eyes, the red mouth,
the straight nose, pink cheeks, and abundant russet hair, any promise worthy
of fulfillment,—could have detected the flower in the bud; and that such a
gaze should, on this day of all days, have been leveled in the girl's direction
was, perhaps, only one of those grim jests of a Fate that loves to play upon
the harmony between man and nature, and that here observed the coming of
a human spring that must be brief and sudden, a human summer parching
and intense.

The usual group of idle residents and idling commercial drummers were
sitting at the plate-glass window of the hotel as she went by, but the girl did
not see them. Passing among objects of long familiarity, she saw, in fact,
nothing until, in a side-street, she heard a rapid step behind her, was
covered by an approaching shadow and, half-turning, found someone, a
stranger, at her side.

"How d'y'do, liddle girl?"

 Mary looked up; but she was quite too startled to observe anything save
that the speaker—she could not have told whether he were man or boy—
was at once dark and rosy, smiling and serious, hat in hand, and, beyond all
speculation, no citizen of her own borough.

"I don't know you," she said.

She flushed quickly, and strode forward. It was, she knew, no

uncommon thing for the girls of her acquaintance to be "picked up," as they
called the process, by some fellow-townsman that had never been formally
presented to them; but the process was, as she also knew, one that lost its
propriety when extended to aliens.

The present alien was, nevertheless, not easily to be dismissed. He fell

into her gait, and walked facilely beside her.

"I beg your pardon," he said in the humblest and most unobjectionable
tones. "I don't mean to be rude to you, honest, I don't. I'm a traveling-man,
you see——"

Mary was striding rapidly ahead, her full mouth now drawn firm, her
blue eyes fixed on the vanishing-point.

"I don't care what you are," she answered.

"All righd," he pleaded. "All I vant now is a chanc't to exblain. I've

chust started out traveling for my fader, who's a big distiller in N'York. I've
got to stay in this hole for a vhile, un' I'm not used to the beesness, un' I'm
lonesome, un' I only vondered if you vouldn't go vith me to a moving-
picture show, or something, this evening."

The best way to deal with such a situation is a way that is easiest for the
inexperienced and the unpolished. Mary was both. For the first time since
he had begun to walk beside her, she now, coming to a defiant stop, faced
her annoyer.

"I don't know you," she repeated. "I've told you that onc't, and you'd
better not make me tell you any more still. I live the second door round the
coming corner, and my pop is a puddler an' weighs two hundred and ten
pounds!"

Again she wheeled and again resumed her homeward march; and this
time she walked alone. If she heard, dimly, behind her a confused murmur
of response, she did not hesitate to learn whether the words were
expressions of further apology or new-born dismay, and when she ran,
flushed and panting, up the three wooden steps to the two-story brick house
that was her home, though she could not then deny herself one backward
glance, that glance revealed to her only an empty corner. The pursuit had
ended.

She flung open the light door that was never locked by day, walked
down the short, darkened hall, past the curtain of the equally darkened
parlor, through the dining-room with its pine table covered by a red cotton
cloth, and so into the small, crowded kitchen, where her mother fretted and
clattered above the highly polished range.

Mrs. Denbigh was a little Pennsylvania-German woman, whom a stern

religion and a long life of hard work had not intellectually enlarged. In spite
of the fact that she had borne eight children, of whom Mary was the
seventh, her sympathies had failed to broaden, and her equally religious and
equally hard-working Welsh husband used often to remark to her, during his
one-monthly evening of intoxication, that he was glad indeed she was to
have no more progeny, since, somehow or other, she "seemed to git wuss
tempered with every innocent youngling as koom to 'un." Whether this
criticism was or was not precise, it is at least true that much drudgery had
not improved the weary woman's temper; that the long years before her
husband rose to his present wages—years during which his wife had not
only kept a house and reared a family, but had also added to the communal
income by night-work as a dress-maker—had left her gray and stooped and
hatchet-faced; and that, though of a race in which the maternal instinct runs
almost to a passion, her patience with her remaining pair of home-biding
children was frequently fragile and short.

Just now she looked up, a spoon in one hand and a pan in the other, her
forehead damp, as always, with sweat, and her harassed eyes momentarily
bright with anger.
 "Where on earth have you been, anyways?" she shrilly inquired of
Mary.

The girl's face instantly hardened from the excitement of her recent
adventure to the sullenness behind which she always took refuge in these
more usual domestic crises. What she might have confessed had she come
home to a less overworked mother, it is, obviously, vain to conjecture; what
she actually did was to lock within her breast the story that had been
trembling on her red lips, and what she replied to Mrs. Denbigh's question
was an ungracious:

"Been at school. Where d'you think?"

The mother straightened up as far as her long-stooped shoulders would

permit.

"Think?" she echoed. "I guess I can guess still where you was. 'Less you
was kep' in, you had ought t' been home five minutes ago, an' nobody's kep'
in only five minutes. You've been flirtin' with some idiot of a boy on the
street-corner yet—that's about what you've been doin'!"

It was a random shot, and one fired from no previous knowledge, but
the girl at once realized that, had any neighbor chanced to see what had
actually occurred, this parental construction would appear to have some
foundation in fact. The thought was enough to seal the locked gate in her
breast.

"That ain't so!" she said, with childish fury. "I come straight home, like I
always do. If you want me to help more with the work than I do help, why
don't you let me quit school? I don't want to go any more, anyhow."

There are some families in which the passing of the lie is no such
uncommon or serious offense, and the Denbigh ménage was one of them. It
was, therefore, upon the latter portion of Mary's speech that her mother, at
this time, seized.

"You'll go to school as long as your pop and me say you must!" she
retorted.
 "You let our Etta quit when she was in the grammar school,"
expostulated Mary, with an appeal to the precedent of the successfully
married sister, who was now a next-door neighbor. "You let her quit then,
and now I'm in the high."

Had Mrs. Denbigh's rejoinder been in accordance with the facts, she
would have said that all she wanted to do was to give her daughter as much
of an education as was compatible with the proper conduct of the Denbigh
domestic economy. But tired women are no more apt to indulge in
analytical exposition than are tired men, and so it chanced that her next
speech, accompanied by a gesture that raised the cooking-spoon aloft, was a
torrent of words unexpectedly interrupted.

"In the high?" she repeated. "Well, I know where you'll be in one
minute, still, if you don't right away——"

She brought the spoon forward with a mighty swoop, but its parabola, in
crossing the stove, sent it into violent contact with the pot that held the stew
destined for the noon dinner. The pot was balanced on the edge of an
aperture in the stove whence the lid had been removed. The vessel fell, and
its contents belched upon the burning coals.

Mrs. Denbigh gave one look at the steaming ruin, and then seized the
already retreating Mary. The girl's struggles, her cries, the dignity of the
newly lengthened skirt, avail nothing. A dozen times the mother's arm
descended in stinging castigation, and then she hurried her daughter into the
hall.

"You git right back to school!" she ordered. "I don't care if you're a half-
hour early—you're mostly late enough. You've spoiled your own dinner and
mine and little Sallie's, so you don't git nothin' to eat still till evening. You'll
go to school, and you'll keep on goin' till your pop an' me tells you to quit!"

Mary looked at the woman without a word, and then, still without a
word, passed through the front door and banged it behind her.

But she did not walk in the direction of the school; she was not going to
school. The rebel-spirit of youth choked her, and turned her feet, almost
without will of her own, toward the river.

She crossed the railroad tracks, came to the disused towpath and
followed it for a mile beyond the town. Far westward she went, "walking,"
as she would have said, "her madness down," and, hungry though she now
was, she did not rest until at last, as late as three o'clock in the afternoon,
she sat on a rock at the point where the Susquehanna curves between the
sheer precipice of Chicques on the Lancaster County side and the hooded
nose of the high hill they call the Point, upon the other.

The flood of rebellion had ceased, but a steady and enduring stream of
resolution remained.

Across the sweep of eddies she saw the nearer hills already shedding the
browns and blacks of winter's bared limbs and pine branches for the
tenderer green of a gentler season. The cultivated portions of the summits
were already rich with coming life. Behind her rolled the Donegal Valley,
where the crops were even then germinating. Birds were mating in the sap-
wet trees beside the water, and from the flowering seeds there came the
subtle, poignant scent of a warm April.

Something—something new and nameless and wonderful—rose in her

throat and left her heart hammering an answer to the new world around her.
She was glad—glad in spite of all her anger and her hunger; glad that she
had not told her mother of the boy—for he must have been a boy—whom
she had, after all, so needlessly reprimanded; but glad, above everything
else, for some reason, for some intoxication that she might neither then nor
ever after completely understand.

Her cheeks glowed a deeper pink; her blue eyes glistened; she opened
her red mouth to the seductive sun and, with a sweep of her firm hands,
flung loose her russet hair to the breeze. Looking out at the distant fields,
she sprang to her feet again and walked, swaying with the easy, languid
grace of an unstudied young animal.

The fields reminded her of the rural prophets. It was evident, she
thought, that they were right: this year's was to be a spring brief and sudden,
a summer parching and intense.
 II

A DEED OF TRUST

Mary Denbigh could not remember the day when the holy estate of
matrimony had not been held up to her by others as the whole destiny of
woman and had not presented itself as the natural, the easy, the sole path of
escape from filial servitude.

She belonged, as has been intimated, to a race in which motherhood is

an instinctive passion and an economic necessity, and she was born into a
class in which not to marry is socially shameful and materially precarious.
When she was very small, her own dolls were her own children and her
playmates' dolls her children-in-law, and, when she grew older, she had
always before her the sedulously maintained illusion of emancipation worn
by those girls, but a few years her seniors, who had given up the drudgery
of childhood, which she hated, for the drudgery of wifehood, which they
loftily concealed. A young wife was a superior being, whose condition was
not at all to be judged by the known condition of one's mother, and all the
other and more intimate relations of marriage remained, to the uninitiate, a
charmed mystery. If it seems strange to us that this mystery and this
innocence remained to Mary at sixteen, the reflection rests not upon her
from whom the secret kept its secrecy, but upon us to whom the innocence
appears remarkable.

From a house that exacted everything and forgave nothing, a narrow

house, which she could not see as simply an inevitable result of conditions
as wide as the world, the girl looked out to that wonderful house next door
where her sister had, only three years before, been taken as a bride. This
sister was now an elegant person, who said "fore-head," "of-ten," and "a-
gain," but Mary could remember Etta, in gingham frock and apron,
performing the tasks that were now enforced upon Mary herself. And she
could now observe—as, indeed, her sister's wholly conscious pride well
intended that she should observe—Etta in clothes that were beyond the
reach of an unmarried daughter of Owen Denbigh; Etta going to dances
forbidden to a Denbigh maid. When she climbed reluctantly to bed at ten
o'clock, Etta's lights blazed always wide awake, and when she rose in the
gray of the morning, Etta's shutters were luxuriously closed.

Every dawn Mary must pack her father's dinner-bucket, as Etta used to
pack it, before Owen started for the mill. That done, and the hurried
breakfast eaten, she must make her own bed and wash the dishes before she
set out for school. At noon there were more dishes, and only every other
evening, before sitting down to detested study by the kerosene lamp in the
dining-room, was she relieved of still more dish-washing by the growing,
and apparently too favored, younger sister, Sallie.

The evening that followed Mary's truant walk along the river was one of
those when she should have been granted this modicum of relief, but now,
after the brief five o'clock supper, tow-headed Sallie set up a wail as the
table was cleared.

"What's the matter with you now?" demanded Mrs. Denbigh, her
harassed eyes blinking in the lamplight, and her hatchet-face more than
commonly sharp.

"I ain't feelin' good," said Sallie. "I'm tired; I'm sick; I don't want to
wash no dishes."

Mrs. Denbigh shot a glance through the double-doorway to the littered

parlor; but the face of her unattentive husband was hidden behind the
crinkling sheets of the Daily Spy, gripped by one great, grimy fist, while the
stubby forefinger of the other hand spelled out the short syllables of the
personal-column, facetiously headed "Our Card-Basket." His huge bulk
bulged over all the edges of the uncomfortable patent armchair in which he
was sitting: a picture of gorged contentment, there was as yet no help to be
expected from him.

It was Mary, experienced in such attacks, who made ready to defend the
law.
 "You ain't sick," she declared.

"I am, too!" sniffed Sallie. "I'm awful sick!"

"Get out: you et more'n I did. You just want to make me do the work,
an' I won't, 'cause it's your turn. So there!"

Mary's homecoming had, as it happened, not been the signal for a

renewal of hostilities between her mother and herself. The former had just
then been too hard at work to have either energy or thought in that
direction, and throughout the evening meal the girl had deemed it wise to
maintain a reticence calculated to keep her in the domestic background.
Now, however, she had impulsively come forward, and the step at once
brought her to Mrs. Denbigh's attention.

"After what you done this noon," she said to Mary, "you'd better keep
your mouth shut. Go and wash them dishes!"

But Mary knew that she had now gone too far to retreat.

"It wasn't my fault the stew was spilled," she protested; "and anyhow,
you did lick me onc't for that. Sallie just wants to shove her work off on
me."

"I don't," blubbered Sallie. "I'll do 'em some evenin' when it's your
turn."

"Yes," Mary sneered, "I know how you will."

"I will—I will—I will so!"

Sallie's voice rose to a shrill shriek, and then suddenly broke off in the
middle of a note: there was a sound of elephantine stirring from the parlor,
and the feared master of the house, moved at last from his lethargy, rolled
into the double doorway and seemed nearly to block it.

One of the young reporters of The Spy had once remarked—not in print
—that Owen Denbigh resembled nothing so much as the stern of an
armored cruiser seen from a catboat. How much of the covering of his
powerful frame was fat and how much muscle is matter for conjecture; his
life in the iron mills had certainly given him a strength at least approaching
the appearance, and had blackened his large hands, reddened his big face,
and grayed his bristling hair and his fiercely flaring mustache.

"Whad's ahl this devil's racket?" he shouted, in the voice he used in

triumphing over the turmoil of the puddling-furnace.

Both children quailed before him, each prepared regardless of its merits,
for acquittal or condemnation, as he might decide the issue. Even Mrs.
Denbigh drew back and set her lips to silence.

The giant raised a threatening hand.

"Be ye ahl gone deef?" he demanded. "Whad's ahl this devil's racket
fur?"

In a panic of self-preservation, the two girls began at once to clamor

forth their woes.

"Sallie won't wash the dishes!" cried Mary.

"I'm sick," sobbed Sarah, "an' mom says Mary must wash 'em because
she upset the stew this noon-time!"

In the merits of any case brought before him, the household Solomon
was as little interested as if he had been the judge of a law-court. His years
of overwork had limited his sense of a just division of toil among others,
and his long oppression by task-masters had made himself a merciless task-
master. Like the men that had driven him, he delighted most in driving
those who were the hardest to drive. Sallie was too young to furnish
appreciable resistance, but in the awakening Mary he now saw something
that approached worthy opposition. He turned first to his wife.

"Did you tell 'er," he inquired, his stubby forefinger leveled at Mary
—"did you tell 'er to wash 'un?"

Mrs. Denbigh bowed her sweating forehead in timid assent.

 Then the father looked again at the offender.

"Wash 'un!" he ordered, and marched back to his parlor, his armchair,
and his evening paper.

Mary knew her father too well not to know also the price of
disobedience. Sullenly, but without hesitation, she retreated to the little
kitchen and took up her uncongenial task.

Girlhood, then, must be denied much of its claim to recreation; the

social machine was pitiless. Young life was a period of menial service from
which the sole escape was marriage, whether to stranger or to friend. That a
stranger should harm her was, to Mary—as it is to most girls of her age and
environment—an idea unentertained: strangers were too few, and the world
of moral fact too closely shut and guarded. Boys she had always been
cautioned against in vague generalities; but she understood that they were
prohibited because their company was a delectable luxury reserved for
older and marriageable girls whose younger sisters were needed only to
help in the household tasks.

Rebellion once more reddened her heart—rebellion, as she thought,

against her own particular condition, but the old rebellion, actually, that
burns, at one time or another, in every heart: the revolt of the individual,
more or less conscious of its individuality, against the conditions that are
combined to crush it. She poured the water from the heavy iron tea-kettle
into the tin dishpan with a quick anger that was not eased when two or three
of the scalding drops leaped back against her bared, round arms. She flung
the home-boiled soap after the water, and she clattered the dishes as loudly
as she dared. Through the window—her soul hot with the sense of the
injustice done her—she could see the happy lights in Etta's house, and, her
hands deep in the greasy fluid, it came to her suddenly that she had been a
fool to neglect—to repudiate—to-day what might have been the golden
chance to such an estate as her sister's.

She had heard the protesting Sarah sent to bed; had heard her mother
return to the parlor with the sewing-basket, and, finally, as she was putting
away the last of the dishes in the china-closet in the dining-room, she
caught the voices of both of her parents.
 Dimly glimpsed from the small apartment beyond, she knew the scene
well enough to reconstruct it perfectly. The crowded little parlor was like a
hundred others in the immediate neighborhood, a mathematical result of the
community of which it was a part. There were the two front windows with
the horse-hair chairs before each and, between them, the marble-top table
bearing the family Bible. There was the gilt mirror over the gorgeously
lambrequined mantelpiece, which was littered with a brass clock, dried-
grass-bearing yellow vases, stiff photographs of dead or married younger
Denbighs, and "memorial cards" with illegible gilt lettering upon a ground
of black. Close by the cabinet-organ on one side and the green sofa on the
other—the sofa adorned with a lace "tidy" that would never remain neatly
in its place—her father and mother sat, separated by the purple-covered
center-table, their gaze interrupted by the tall glass case that contained the
bunch of white immortelles from the grave of their eldest son.

Mrs. Denbigh was finishing, it seemed, the narrative of the town's latest
scandal.

"I never knowed Mrs. Drumbaugh was that soft-hearted," the mother
was saying. "Nobody in town was fooled over the reason for why her Jennie
went away, an' yet here the girl comes back a'ready, and Mrs. Drumbaugh,
church-member though she is, takes her into the house ag'in—her an' her
baby along with her."

What was it in the words that brought Mary to a sudden pause? Her
mother had always been, like most drudges, a gossip, and had sought, in
repeating scandal about her acquaintances, that relief from drudgery which
she knew how to obtain only by this second-hand thrill of evil. The girl had
heard and disregarded the telling of many such a tale, and yet, to-night, she
stood there first listening in uncomprehending horror to the narrative and
then awaiting the inevitable paternal comment upon it.

"Tuke 'er bahk, hey?" rumbled Owen Denbigh. "Well, ef she bay sooch
a fule, she deserves the scandal ov't. Thank God no youngling o' ourn ever
went the devil's way. I hahve ahlways bin sure what I'd do to 'un ef she did,
though."
 He paused a moment, as if to have his wife inquire as to the terrible
punishment that he had reserved for such an error, and then, as no inquiry
was forthcoming, he gave his statement at any rate, with all the cold
ferocity of a Judge Jeffries pronouncing sentence.

"Bay 'un thirty year old an' noot another sin ag'in 'un," he declared, "I
would beat 'un within a bare inch o' 'er deeth, an' turn 'un oot to live the life
'un had picked fur herself!"

The whole intent of that speech Mary was incapable of comprehending,

but she understood enough to tremble and then to fan to destructive fury the
fire of her rebellion. Of a sudden, the atmosphere of the house had become
unendurable. She was gasping like a sparrow under a bell-glass.

Stealthily she crept into the hall. Carefully she took her coat and faded
hat from the rack. Very gently she opened the front door and stole into the
street. She felt dumbly that the world was wrong, that youth should not
have to work, and that to seize the fruit of pleasure should not be matter far
punishment, but for congratulation.

I do not think that she meant to pass by the hotel that evening. I do not
believe that most of us, in such moments, are actuated any more by motive
than we are directed by discretion. Nevertheless, when the clutch of her
emotions had enough loosened from her throat to permit her to take account
of her whereabouts, the time, and the place, it was a quarter after six by the
town-clock; Mary was just before the plate-glass window where the
drummers sat, and, only a minute later, the stranger of the morning was
again at her side.

"Von't you chust say that you're not mad vith me?" he was asking.

She was so frightened that she was conscious of no other definite

sensation, much less of any ordered thought or opinion; but she looked
fairly at him, and of what she saw she was immediately fully aware.

He was a young man, but the sort of young man that might be anywhere
from nineteen to thirty-two, because he had the figure and the face of the
former age and the eyes and the expression of the latter. The hair on his