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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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 — Mahdotonta, mahdotonta, ovethan ovat lukitut, — nyyhkytti
Ursula.

— Vanno rukoilevasi, niin päästän sinut, ovet kyllä aukenevat! Ja

Ursula vannoi pyhän valan.

Tuskin oli Ursula vapautunut luisesta syleilystä, kun kirkon ovet

juhlallisen hitaasti aukenivat. Kirkas valo loisti alttarilta, jonka
ääressä kolme impeä hohtavan valkeissa puvuissa polvistuneina
rukoili. Heidän ohimoillaan väreili jumalallinen kirkkaus, ja kultaiset
kirjat heidän käsissänsä välkkyivät kuin aurinko. Urut soivat hiljaa,
niin utuisen vienosti, ja heleät naisäänet yhtyivät urkujen hyminään.
—

Ääniaallot paisuivat vähitellen valtavammiksi, tuntui kuin temppeli

olisi avartunut — soitto paisui paisumistaan, ja valkeiden neitojen
kiitosvirsi kaikui mahtavana. -Ursulan valtasi syvä hartaus, ja hän
lankesi maahan impien eteen.

— Vapauttakaa Pitkän-Pienan sielu —, rukoili hän. Valo

ristiinnaulitun edessä himmeni, ja päätään pudistaen herkesivät
immet laulustaan.

— Päästä, oi päästä minut, olet tuomittu, armoa ei sinulle suoda!

—

— Rukoile innolla, rukoile palavasti! –

Ursula polvistui toisen kerran ja rukoili:

— Lepoa, rauhaa suokaa hänen sielulleen rakkautensa tähden! —

 Valo ristiinnaulitun edessä kokonaan sammui, urkujen pauhu
taukosi, ja kaamea hiljaisuus täytti temppelin.

— Ikuisesti olet kadotettu, syntisi ovat liian suuret, oi päästä minut!

—

— Rukoile kauniimmin, rukoile! Armonsa he minulle suovat, onhan

jouluaamu, rauhanhuomen! —

Ursula ryömi pimeässä alttarin juureen ja rukoili:

— Kristuksen tähden armoa hänelle! - Samassa leimahti kirkas

liekki ristiinnaulitun edessä, urut soivat pauhaten, ilman täytti pyhä
suitsutus, ja hohtavat siivet puhkesivat impien hartioille, vieno tuulen
henki väreili temppelissä, ja hohtosiipiset kohosivat kohti korkeutta
häipyen katon hämärään.

Ursula palasi eteiseen, kuului kova kolahdus, kirkon ovet

sulkeutuivat, ja laatta lattiassa liikahti. Tyhjä oli Pitkän-Pienan
komero, tyhjä vihkivesimalja sen edessä. - Avoimesta eteisen ovesta
näkyi jouluaamun taivas; vaaleat olivat taivaankannen kiiluvaiset,
vaaleat olivat Ursulan posket. —
SCALA SANTA.

Laterani-kirkon edustalla Roomassa tunkeili paljon väkeä. — Scala

santa, scala santa oli joka toinen sana odottavien keskustelussa.

Aina Jerusalemista saakka olivat ne laivalla tuodut Roomaan,

Pyhän isän mahtavaan Roomaan. Lautaisissa laatikoissa ne olivat
tulleet niin, ettei odottava väkijoukko ollut nähnyt vilahdustakaan siitä
valkoisesta, hohtavasta marmorikivestä, mutta nyt olivat ne
kivilohkareet rakennetut Laterani-kirkkoon, oli niiden vihkitilaisuus, ja
kansa odotti levottomana ovien avautumista.

Vihdoinkin, kuin sulkunsa murtanut kevätkoski, tulvehti

ihmisjoukko ovesta sisään, mutta vain pieni osa sinne mahtui, ja
ulkopuolelle jääneet odottivat jännittyneinä kertomusta siitä mitä
siellä sisällä tapahtui. Ja aina vähä väliä kertoi joku ulostuleva
munkki odottavalle joukolle ihmeellisiä asioita niistä
marmoriportaista, jotka olivat olleet Pilatuksen palatsissa ja joita
myöten Kristus oli astunut, kun hän lähti ristinkuolemaan.

Ja eräs munkki kertoi:

— Tuskin olivat ovet avautuneet, kun eräs vaimo alkoi nousta niitä
valkoisia portaita. Aluksi tuntui hänen käyntinsä niin helpolta ja
kevyeltä, mutta mitä ylemmäksi hän tuli sitä raskaammilta tuntuivat
hänen jalkansa, ja kolmannella portaalla ylhäältä lukien kompastui
hän, mutta yrittäessään nousta tunsi hän itsensä voimattomaksi ja
polvillaan ryömi ne viimeiset askeleet.

Mutta ylös tultuaan kaatui hän hengetönnä valkoiselle

marmorilaatalle —, hän oli saanut armon Herran edessä! — — —

Kun tämä tapahtuma oli tullut Pyhän isän tietoon, julisti hän, että
scala santaa on noustava polvillaan kulkien ja ken sen tekee hän
saa suuren anteen.

Niin tapahtui, että ihmiset kilvan riensivät Laterani-kirkkoon ja

polvillaan nousivat näitä portaita, vaikka se oli raskasta ja
vaivaloista, ja he suutelivat kolmea ylintä porrasta, joissa Kristuksen
veren jäljet olivat valkoiseen kiveen syöpyneet.

Mutta suuri oli katuvaisten syntisten luku, ja marmoriportaat

alkoivat kulua, särmät pyöristyivät. Silloin laudoitettiin portaat, ja
kilvaten kiipesi kristikunta näitä pyhiä portaita.

*****

Pölyisenä, matkasta uupuneena astui vaaleatukkainen, sinisilmä

nuorukainen eräänä iltapäivänä Laterani-kirkon pääovesta sisään:
jaloissaan oli hänellä tuohiset kuluneet taniaiset ja kädessään pitkä,
katajainen ryhmysauva.

Heti ovensuussa lankesi hän polvillensa ja alkoi ryömiä portaita

kohti. Lähellä seisova avulias munkki tarjoutui tekemään tämän
vaivaloisen työn pienestä maksusta, mutta pyhiinvaeltaja sanoi:

— Itse on minun noustava levottoman sieluni portaita! —

 Ja hän suuteli ensimmäistä porrasta. Näin suudellen jokaista
porrasta saapui hän vihdoin ylimmälle askeleelle ja hän rukoili:

— Herra minä kiitän Sinua, että sinä annoit minulle voimaa nousta
minun sieluni sameista syvyyksistä sen kukkuloille! —

Mutta silloin puhkesi hänen sauvansa ryhmyistä ohuita oksia,

hienot vehreät neulaset eivät koskettaissa pistäneet, ja vasta
syntyneet katajanoksat kantoivat pieniä kukkia, vaan vaalea
nuorukainen itki, sillä kukkivan katajan tuoksu oli tervehdys hänen
kaukaisesta kotimaastaan.

Kun Pyhä isä kuuli sauvaihmeestä, kutsui hän sen hurskaan,

vaalean nuorukaisen luoksensa ja pyysi nähdä sitä ihmeellistä
sauvaa, ja Pyhän isän huone täyttyi kukkivan katajan tuoksulla.

— Pyhä, pyhä on tämän puun tuoksu oleva, sillä Jumalan armo on

sinun kädessäsi kukalle puhjennut, palaja kotimaahasi
puhdistuneena —, sanoi Pyhä isä ja siunasi polvistuvaa nuorukaista.
—

*****

Lunta sataa, suurina tähtinä putoilevat hennot lumihyötyvät. —

Lunta sataa, Turun punaiset tiilikatot peittyvät valkoiseen untuvaan.
Uupunut matkamies astuu verkalleen pitkin Auran siltaa, kädessään
on hänellä katajainen ryhmysauva. — Lunta sataa, kevyet kiteet
kimmeltävät hänen keltaisilla kiharoillaan. — —

Hän tulee tuomiokirkon portaiden eteen ja puhelee hiljaa:

— Scala santa, kaikki nousu elämässä kulkee sinua myöten! —

 Nöyränä ja pelokkaana lähestyy hän kuoria, jossa harmaaparta
pappi messuaa. Pappi tuntee jo kaukaa lähestyvän nuorukaisen ja
huutaa: — Pakene Herran pyhästä, sillä vielä veljesi veri hänen
hautakivellänsä kukkii! —

Mutta kalpea veljensurmaaja kulkee nöyränä kuorissa olevan

veljensä hautakiven luo ja langeten polvillensa rukoilee:

— Herra, minä nousin kerran, älä syökse minua syvyyteen! —

Mutta sauvaan puhkesivat katajaiset kukkivat oksat, ja pienet

katajankukat imivät itsensä punaisiksi hautakiven hyytyneestä
verestä, ja kalpea nuorukainen lausui:

— Scala santa, raskas on elämässä nousu, scala santa, jokaisen

on sinua kuljettava kerran, scala santa, sinä olet jokaisen sielussa!
—

Ja veljensurmaajan sieluun palasi uuden elämän kirkas toivo, ja

korkean temppelin ilma tuli pyhälle katajan tuoksulle — —
VERIKIVI.

Kopeana astui Herska suureen tupaan, jossa pitkän, yhdestä

hongasta veistetyn pöydän ääressä istui joukko meluavia miehiä
oluthaarikat edessään.

Herska oli pahamaineisen talon pahamaineisin vieras, hänellä oli

pitkä, pörröinen, vaalea tukka, joka lakin alta valui otsalle niin, että
miltei peitti hänen pienet, palavat silmänsä, Herska oli seudun
kuuluisin tappelupukari.

— Lisää olutta, emäntä, tätä poikaa janottaa —, huusi hän ovea

kiinni läimähyttäessään. Hänen lähestyessään pöytää antoivat
nuoremmat miehet hänelle sijaa, vaikka olikin jo ahdasta.

Punakka tihrusilmä eukko laskeusi lattialuukusta kellariin

hampaittensa välissä roihuava päre ja kädet täynnä tyhjiä haarikoita.

— Katosi kuin kärppä kivenkoloon —, nauroi Herska.

Keskustelu ei tahtonut enään oikein sujua, sillä Herskan tullessa

tuntui kuin tuvan ilma olisi käynyt raskaammaksi. Nuori Iikka, joka oli
tunnettu rohkeudestaan ja sukkeluudestaan, sanoa tokaisi kotvan
kuluttua:
 — On niistä pyhäpäivistä ainakin se apu, ettei tarvitse ruumistaan
kiusata! —

— Onpa niinkin, mutta sielu se pyhäisin vaivaantuu —, yritti joku

laskea leikkiä.

— Minusta sentään voisi kirkkoa rakentaa pyhänäkin, sillä sehän

on pyhää työtä —, puheli eräs vanhanpuoleinen mies, joka jo silmin
nähtävästi oli päihtynyt.

— Jaa rakentakaa te kirkkoa koska haluatte, minä en enään niihin

töihin tule —, sanoi Herska vilkuttaen pieniä silmiään.

Mutta samassa palasi juomalan emäntä unkeasta kammiostaan,

tuoksuva olut vaahtosi haarikoissa, ja lukemattomat kädet
kurottautuivat niitä tavoittelemaan.

Nopsajalka Iikka ehti ennen ylpeätä Herskaa, mutta tuostapa

Herska tulistui, hän sieppasi haarikan Iikan kädestä ja heitti sen
sisällön vasten Iikan kasvoja. Iikka sivalsi puukkonsa, syntyi kauhea
meteli. Päihtyneet miehet koittivat rauhoittaa kamppailevia, jotka
taistelivat kunniansa puolesta.

Muori oli pudottanut päreen hampaittensa välistä ja pakeni

huutaen peräkamarin ovelle, sillä silmänräpäyksessä oli tuvan lattia
muuttunut päihtyneiden miehien hurjaksi temmellystantereeksi.
Pöydällä palava korri kaatui ja sammui.

Kuului kova, sydäntä viiltävä kiljahdus, yhtäkkiä kaikki hetkeksi

vaikeni — —, kuului sitten pehmeä jysähdys niinkuin joku olisi
pudonnut maahan, ja äänettöminä pakenivat tappelijat tuvasta
taivasalle.
 Kauhuissaan oli muori vetäytynyt kamariinsa ja koitti
kehoituksillaan ja huudoillaan saada ylös talon nuorta renkiä Hintiä,
joka makasi sairaana ovivuoteessa, mutta turhaan, sillä kuumeen
uuvuttama Hinti ei voinut liikuttaa itseään.

Eukko painoi päänsä oveen, tuvassa oli kaikki hiljaista. Hän sytytti
päreen ja hiipi varovaisesti tupaan. Sinne tultuaan näki hän lattiassa
punaisen veriviirun, avoimesta lattialuukusta kuului syvä huokaus,
päre putosi, ja tiedotonna vaipui eukko lattialle.

*****

Varhaisesta aamusta illan hämärään asti kuului maantien sivulta

kiven kilkatusta, sillä suurta kivikirkkoa siellä rakennettiin.

Oli helteinen päivä, työväki piti paraikaa ruokalepoa.

He keskustelivat hiljaa miltei kuiskaten siitä kamalasta sunnuntai-

illasta; siitä oli jo kohta pari viikkoa kulunut.

— Sitäköhän se Herska tarkoitti, kun se sanoi, ettei se enään

kirkon töihin ryhtyisi, — puheli eräs joukosta.

— Ei se mitään, mitäs niistä juopuneen sekaisista puheista, puhui

mitä sattui —, sanoi toinen.

— Mutta ei sitä vaan sen koommin ole työhön kuulunut —, puuttui

puheeseen kolmas, — eikä se ole uskaltanut näyttäytyäkään sen
jälkeen, pelkääköhän se joutuvansa rautoihin, mutta kukas sitä
taitaisi todistaa, että juuri hän Iikkaan osui, silloinhan oli pilkkosen
pimeä!
 —Sitäpaitsi on talon renki, se kalpea Hinti jo vangittu, sillä häntä
laki epäilee! —

— Mutta olihan se silloin sairaana! —

— Jaa mitäs siitä, mutta Hinti oli ainoa mies, jonka siinä talossa
sinä iltana tiedettiin olevan, niin sanotaan! —

Miehet nauroivat hiljaa, mutta samassa kiintyi heidän huomionsa

toisaalle. Pitkin pölyistä tietä näkyi kulkevan kolme miestä aivan
lähekkäin, kun ne tulivat lähemmäksi saattoi helposti erottaa, että se
keskimmäinen kulki sidottuna niiden kahden välissä.

— Siellä sitä kalpeata Hintiä kuletetaan, sanoi yksi työmiehistä.

Kirkon kohdalle tultuaan istuttivat saattomiehet sen nuoren köysillä

sidotun eräälle kivilohkareelle kiviröykkiössä, joka oli ajettu kirkon
rakennusaineiksi.

— Hintikö se on —, sanoi eräs työmiehistä.

Puhuteltu katseli väsyneesti miestä, joka oli lausunut hänen

nimensä, ja sanoi:

— Niin, minä! —

— Mihinkä matka? —

— Kokemäelle —, vastasi Hinti.

__ Kokemäelle —, kertasivat miehet kauhun valtaamina, sillä he

tiesivät mitä se nimi merkitsee miehelle, joka sinne sidottuna
kuljetetaan!
 — Murhanko olet tehnyt —, kysyi sama mies, hän ei tiennyt koko
tapahtumasta mitään.

— En, olen viaton! —

‒ Viaton —, toisti toinen kuljettajista, — sinä tapoit Iikan siinä

huonomaineisessa talossa! —

‒ En minä —, useat miehet säpsähtivät kuullessaan nuo sanat ja

muuttuivat kasvoiltaan oudon näköisiksi.

‒ Vai et sinä, sinä olet sen talon renki, olet emännän kätyri, sinut
sieltä peräkamarista tavattiin, kun Iikan ystävät tulivat häntä
hakemaan, murhaaja olet, ja kuolema on palkkasi —, puhui toinen
kuljettajista.

— Kysykää emännältä! —

— Niin siltä parhaimmalta, sitä paitsi ei se taida puhua, Herra

rankaisi sitä halvauksella! —

— Jos olisin murhaaja kuolisin ilolla tekoni sovitukseksi —,

nyyhkytti Hinti, -mutta viattomana kuolla, kuolla nuorena se on
katkeraa! — Suuret kyyneleet vierivät pitkin kalpean nuorukaisen
poskia, ja useat ympärillä seisovista miehistä käänsivät kasvonsa
poispäin, sillä he tiesivät kaiken, mutta heistä tuntui edullisemmalta
vaieta.

— Niin totta kuin olen viatoin itkeköön tämä kivi, jolla istun,
ikuisesti verta —, sanoi Hinti ja nousi seisomaan.

Verkalleen asteli pitkin pölyistä tietä kolme miestä Kokemäkeä

kohti, mutta se keskimmäinen hoiperteli, sillä hän oli kuoleman oma
———

*****

Rauhallisena virtaa Kokemäenjoki loppujuoksussaan merta kohti,

keltaiset upukat sen liejuisilla rannoilla ovat sulkeneet upunsa ja
keinuvat hiljaa, uupuneina, sillä on kesäinen ilta.

Jylhänä seisoo korkea, musta hirsipuu Kokemäenjoen partaalla,

sen varjo makaa maassa niin pitkänä ja raskaana, sillä on kesäinen,
myöhä ilta.

Rauhallisena astuu kookas, kalpea nuorukainen rahille hirsipuun

alla, hänen kalpeat poskensa saavat auringolta punaisen hohteen,
sillä on kesäinen ilta.

Kokemäen kellot kumajavat kesäisenä iltana, kuumasta maasta

nousee viileä sumu, ja niinkuin kellojen soitto liitelee vapautunut,
viaton sielu korkeuksia kohti — — —

*****

Mutta siellä missä uutta kivistä kirkkoa rakennettiin, siellä oli ääntä
ja liikettä. Herska oli palannut kirkon työhön, sillä vaara oli ohi, eikä
kukaan enään uskaltanut puhua siitä tapahtumasta.

Eräänä iltana nosti Herska muiden miesten kanssa suurta kiveä

kirkon seinään, se oli jo paikallaan, ja yksin hän täytteli savella
muurissa olevia koloja; aurinko laski punaisena korkean harjun taa.

Yhtäkkiä huomasi hän, että hänen kätensä olivat veriset, eikä hän
kuitenkaan voinut huomata niissä haavaa, hän katsahti kiveen, jota
vasten hän nojasi toisella kädellään, ja huomasi, että kivi tihkui verta,
viattoman verta! Hän alkoi huutaa kauheasti, menetti tasapainonsa
ja putosi maahan lyöden ohimonsa terävään kiveen.

Kun toverit saapuivat paikalle löysivät he Herskan kuolleena

maasta ja näkivät, että kivi kirkon seinässä tihkui verta, ja yksi
miehistä lausui:

— Jumala on armollinen —, ja kaikki polvistuivat maahan verikiven

kohdalla, johonka laskevan auringon viimeiset säteet lankesivat. —
—

II.
SYDÄMENUSKO.

— Miksi karjuu kohiseva koski, mitä huutaa Nokianvirta, hyytävät

vedet, hyiset pyörteet, mitä itkette yössä! — Mitä ulvot, miksi uliset
Jäämimaan pyhä virta —, näin puhui suurkodan uljas Pii-sydän ja
kääntyi toiselle kyljelleen kodan pehmeällä poron taljalla.

— Emo, kuuletko sa Nokian laulun, kuuletko himohuokunan

kamalan, — — nukut akka, naisesta ei valvojaksi! —

Hiillos riutuu paaden päällä, nukkuu emo, lapset nukkuu, Pii-sydän

vaan ei nuku, silmänsä tuijottavat, korvansa kuuntelevat jokaista
säveltä kosken laulussa. Hän tarttuu poronsuoniseen rihmaan
hiilipaaden vierellä ja kiskaisee voimakkaasti siitä, talja siirtyy
savuaukolta, tanot katossa aukon suulla väräjävät, ne soivat
matalaäänisesti. —

Pii-sydän makaa selällään ja tutkien katselee korkeata taivaan

kantta, tuuli vinkuu, koski huutaa, ja kevyet tuohet soivat — — —

Pii-sydän kuuntelee henkeään pidätellen ja tähystelee; tähtiä

lentää tummalla laella.
 — Mitä, lensikö sekin, pohjantähti itää kohden —, mutisee Pii-
sydän ja nousee levotonna vuoteeltaan, hän vetää nahkamekkonsa
ylleen ja mataa kodasta taivasalle.

Koskelle hän rientää, kuumana verensä kohisee, sydän niin oudon

rajusti lyö.

— Mitä ulvot, koski pyhä, miksi riitteesi sulavat, miksi iljankos

alenee! — Pii-sydän hyppää putouksen alla vesipaadelta toiselle ja
pääsee vihdoin Hälläpyörteen kivelle, jonka juuria koski kuumimmin
jauhaa. Hän kohottaa kätensä ja huutaa yli pauhun:

— Kuole ääni hurjan rinnan, vaikene itku vesien! —

Sitten laskeutuu hän polvilleen paadelle ja silmäilee hiidenkirnua

kiven kupeella, se on tyhjä, ilman vettä. Hän sieppaa nahkaisen
lakkinsa ja alkaa lippiä sillä vettä hiidenkirnuun, mutta se ei täyty,
vesi on syönyt puhki kirnunpohjan.

— Jo tiedän, uhria Ukko ulisee —, lausuu Pii-sydän ja painaa

jäätyvän lakkinsa suortuvilleen. Ketterästi kuin ilves hyppii hän
kiveltä kivelle iljankoihin lipeämättä.

Tuuli ulvoo, pohja pölyttää lunta, Pii-sydän hiipii kotaansa, lämmin,

unkea ilma lehahtaa hänen kylmille kasvoillensa, hän sulkee
savuräppänän ja koittaa nukkua. - Otava ei vielä ole kiertoansa
tehnyt, kun Pii-sydän akkansa herättää:

— Uhria Ukko ulisee, hae valkein uuhilammas, en tiedä mistä

vihansa heitti! —

Emo palaa uuhikodasta, jäärän tuo.

 — Unohdit isäntä, eihän Ukko uuhta huoli! —

Pii-sydän sitoo jäärän jalat ja heittää sen olallensa, sitaisee vielä

uhripussinsa kupeellensa ja nousee suksilleen —

*****

Pyrynä lumi tuprusi, kun Pii-sydän hiihti. Kuun viimeinen sakara

paloi taivaan rannalla kellervänä, tähdet kelmenivät päivän puolla,
nouseva rusko ennusti aamua, mutta levotonna kulki pohjantähti.

Sukset luisuivat, lammas määki hartioilla, ja rajusti hiihti Pii-sydän.

Kuu katosi, hän hiihti yhä.

Päivä nousi, ja itsekin hän nousi uhrivaaran harjanteelle.

Pyynikin pyhät petäjät hiljaa huojuivat, ja latu nousi, se taittoi

polven ja nousi vieläkin ylemmäksi, jo kuului kosken laulu, Tammer
lähellä huurusi huurtaen uhrilehdon puut.

Kuumat hikihelmet putoilivat Pii-sydämen kulmaluilta ja muuttuivat

rakeiksi jäisellä hangella. Hän iski suksikeihäänsä suureen petäjään,
oksat satoivat huurrettaan, ja kevyet lumihiuteet kimaltelivat
uhrijäärän päällä puun juurella.

Pii-sydän katkaisi oksan puusta ja alkoi puhdistaa lumesta

uhripaatta, asetti paadelle puita ja reunalle uhrikaluja. Sitten sieppasi
hän aseen ja aukaisi teuraan rinnan, asetti sen paadelle ja kaikkosi
itse suuren kiven taa tuulen suojaan tulta iskemään.

Punainen veri valui höyryten hangelle ja piirsi kauniita kuvioita.

 Mutta äkkiä hyppäsi tupsukorva ilves pyhästä petäjästä maahan,
juoksi uhrijäärään käsiksi ja pakeni sen kanssa ylös puuhun. Pii-
sydän tyrmistyi kiukusta kun hän näki, että ilves raateli Ukon uhria,
viimeiset veripisarat valuivat puusta valkoiselle hangelle.

Jo nousi hidas jäämiveri, Pii-sydän tempasi jousensa, pingoitti sen

rivakasti, jousi joudutti nuolta, ilma suhisi, petäjänoksat huojuivat, ja
raadellun lampaan ruumis putosi maahan. — Pii-sydän katsahti ylös
petäjään, mutta silmänsä sattuivat pohjantähteen, joka levottomasti
vilkkui taivaan laella. Ilves kiepsahti lumelle ja verinen sydän
suussaan kiipesi toiseen puuhun. Piisydän tuosta enemmän
sydäntyi.

Hän sieppasi keihäänsä ja tavoitti sillä ilvestä, mutta rauhallisena

se vain pureskeli jäärän veristä sydäntä — — — mutta pohjantähti
lepatti levottomasti — —

Ilves hyppäsi hangelle ja juoksi puiden lomissa. Pii-sydän nousi

suksilleen ja ajoi pakenevaa petoa. Äkkiä pysähtyi ilves, sen silmät
hehkuivat kuin tuli, ja punainen sydän sen suussa säteili kuin tulinen
hiili. Pii-sydän kohotti keihäänsä ja iski, mutta osumatta.

Ilves vilisti, pitkin pyhän vaaran harjannetta, vihdoin alas Pyynikin

mäkeä Pyhäjärven jäälle.

Siellä pysähtyi se ja katseli uhkamielisesti Pii-sydäntä, joka piirsi

sauvallaan lumeen kolme tähteä, sitten hiihti hän sydämen
muotoisen ladun pedon ympärille ja piirsi neljä ruusua sydämen
kärkipuoleen, nyt ei peto enään voinut liikahtaa. Pii-sydän mutisi
loihtuja ja iski keihäällään ilveksen hankeen, ja punainen uhriteuraan
sydän värisi hangella.
 Pii-sydän lankesi maahan ja kiitti Ukkoa, mutta hän näki, että
pohjantähti oudosti taivaalla lepatti. Oli jo kirkas päivä, ja kuitenkin
katseli yksinäinen pohjantähti lumisia metsämaita.

Pii-sydän uhrasi jäärän Ukolle, mutta sen punaisen sydämen kätki

hän uhripussiinsa ja palasi kodalleen — —

*****

Mutta pohjantähti seurasi häntä koko matkan ja pysähtyi hänen

kotansa kohdalle.

Pii-sydän kuuli kodasta lapsen itkua ja ilokseen näki hän emon

sylissä pienen lapsen. Hän asetti uhrikalunsa kodan pyhälle
hiilipaadelle ja rukoili Ukkoa. Mutta vastasyntynyt nähdessään
sydämen paadella avasi suunsa ja puhui:

— Pii-sydän, Ukko ei enään uhria kaipaa, ilves sydämen Ukolta

riisti, riistä sinäkin sydämesi häneltä ja lahjoita se Kaikkivallalle! —

Ja tapahtui, että sydän hiilipaadella syttyi suureen liekkiin, ja

säkeneet kohosivat kodan kattoa kohti, jossa ne muodostuivat
tähtirenkaaksi, eikä ylhäinen pohjantähti enään värissyt
korkeudessaan.

Mutta uhrisydän paloi poroksi paadella, ja tähtikehä laskeusi alas

ja asettui lapsen pään ympärille, mutta Pii-sydän lankesi maahan
lapsen eteen ja lausui:

—Ukko ei sydäntä kaipaa! — Mutta lapsi kohotti pienen kätensä ja

sanoi:
 — Sinun nimesi olkoon Sydän, nouse ja ilmoita heimollesi minun
tulostani! —

Ja Jäämien apostoli nousi ja saarnasi — — sydämen uskoa! —

LINTUJEN LAULU.

Herra ja Pyhä Pietari kulkiessaan maailmaa saapuivat kerran

Suomenkin saloille. - Matka oli ollut pitkä ja vaivaloinen, ja väsyneinä
istahtivat he sammaleiselle kivelle korkealla kivikkorinteellä.

Kevät teki tuloaan, pohjolan kevät! Ilma tuoksui puhkeavien

koivunlehtien pihalle, ja täyteläiset purot solisivat iloisesti rinteitä alas
kiitäessään. —

— Noiden purojen solinassa on kylmä sävel —, sanoi Pyhä Pietari

—, yhtä kylmä kuin tämän kansan sydän! —

Silloin katsahti Herra Pietariin, tarttui hänen kuumaan käteensä ja

sanoi:

— Simon Jonaanpoika, kuumakin koski voi joskus jäähtyä, ja

kylmä puro voi kerran lämmetä! — Mutta Pietari ei ymmärtänyt
Herran puhetta.

Hän aikoi juuri pyytää Herralta selitystä, kun harmaa lintu lensi
heidän ylitsensä ja asettui istumaan nuoren kuusen latvaan aivan
lähelle heitä.