Journal of Thermal Biology 55 (2016) 3038

Contents lists available at ScienceDirect

Journal of Thermal Biology

journal homepage: www.elsevier.com/locate/jtherbio

Amino and fatty acid dynamics of octopus (Octopus vulgaris) early life
stages under ocean warming
Vanessa M. Lopes a, Filipa Faleiro a, Miguel Baptista a,n, Marta S. Pimentel a, Jos R. Paula a,
Ana Couto a, Narcisa Bandarra b, Patrcia Anacleto b, Antnio Marques b, Rui Rosa a
a
MARE-Marine and Environmental Sciences Centre, Faculdade de Cincias, Universidade de Lisboa, Laboratrio Martimo da Guia, Av. Nossa Senhora do
Cabo 939, 2750-374 Cascais, Portugal
b
Diviso de Aquacultura e Valorizao (DivAV), Instituto Portugus do Mar e da Atmosfera (IPMA, I.P.), Av. Braslia, 1449-006 Lisboa, Portugal

art ic l e i nf o

a b s t r a c t

Article history:
Received 10 February 2015
Received in revised form
8 November 2015
Accepted 26 November 2015
Available online 28 November 2015

The oceans are becoming warmer, and the higher temperatures are expected to have a major impact on
marine life at different levels of biological organization, especially at the most vulnerable early life stages.
Thus, we hypothesize that the future warmer scenarios (here 3 C) will affect the biochemical composition (amino acid AA, and fatty acid-FA) of octopod (Octopus vulgaris) embryos and recently-hatched
pelagic paralarvae. The main essential amino acids found in octopus embryos were arginine, leucine and
lysine; while aspartic and glutamic acids, and taurine were the main non-essential amino acids. Palmitic,
eicosapentaenoic and docosahexaenoic acids were the main FAs found in octopus tissues. Relevant ontogenetic changes were observed, namely a steep decrease in the content of many AAs, and a selective
retention of FAs, thus evidencing the protein-based metabolism of these cephalopods. Temperature per si
did not elicit signicant changes in the overall FA composition, but was responsible for a signicant
decrease in the content of several AAs, indicating increased embryonic consumption.
& 2015 Elsevier Ltd. All rights reserved.

Keywords:
Octopus vulgaris
Ocean warming
Amino acids
Fatty acids
Embryogenesis

1. Introduction
Our planet's climate is changing at alarming and unprecedented rates. A global warming of 0.6 C has occurred over
the last one hundred years. Moreover, sea surface temperature is
expected to increase up to 3 C by the end of this century (Meehl
et al., 2007). Since temperature plays a fundamental role in most
biochemical reactions occurring within the body (Gillooly et al.,
2001), it is expected that ocean warming will adversely impact
marine organisms and communities (Harley et al., 2006; Helmuth
et al., 2006; Rosa et al., 2013).
Most research on ocean and climate change has been focused
on the adult stages of marine organisms, while the effects of ocean
warming on the early, most vulnerable and sensitive stages (Byrne,
2011) remain poorly known. During early development, the body
biochemistry should be optimally balanced. For that, embryos
have to be equipped with the right amount of fatty acids (FAs), key
constituents of the cell membrane and known energy sources
(Sargent, 1995), and amino acids (AAs), the building blocks of cell
structures, vital for cellular functioning (Wilson, 2003). Some FAs
n

Corresponding author.
E-mail address: msbaptista@fc.ul.pt (M. Baptista).

http://dx.doi.org/10.1016/j.jtherbio.2015.11.006
0306-4565/& 2015 Elsevier Ltd. All rights reserved.

and AAs are referred to as being essential because the organism is

not able to synthesize them on its own, and have, therefore, to be
obtained from external sources. Larvae, juveniles and adults can
easily obtain them through food intake, but in the case of embryos,
however, such essential FAs and AAs must be made available
through parental input during gametogenesis (Leal et al., 2012).
Cephalopods play a very important role in marine food webs,
both as predator and prey (Boyle and Rodhouse, 2005). Their nutrient requirements have received a fair share of attention during
these last decades, especially due to the great efforts to develop
cephalopod aquaculture (Navarro and Villanueva, 2000, 2003;
Villanueva et al., 2004). Cephalopod tissues are especially low in
lipid content, accounting for 0.33.4% of the organism's wet body
weight (Lee, 1994), which may be explained by the need of these
voracious predators to be negatively buoyant. Comparatively, they
present much higher levels of protein content, reaching 14.5
20.9% of the wet body weight. These organisms have a proteinbased metabolism and, thus, an elevated protein requirement that
can only be achieved by an exclusively carnivorous diet (Lee, 1994).
Cephalopods are considered to be highly versatile when it
comes to adaptation to environmental conditions (Boyle and
Rodhouse, 2005; Rosa et al., 2013). Probably the most conspicuous
example of such adaptability is the common octopus, Octopus

V.M. Lopes et al. / Journal of Thermal Biology 55 (2016) 3038

vulgaris, which inhabits mostly shallow coastal habitats of the

temperate, subtropical and tropical regions of the Atlantic, Indian
and Pacic Oceans, also occurring in the Mediterranean Sea
(Guerra et al., 2010; Mangold et al., 1998). Given its wide range of
distribution, the thermal tolerance of this species seems to depend
on the natural geographic location of a given population. For instance, European populations seem to prefer temperatures between 10 and 20 C, with death ensuing above 23 C, while populations in the Caribbean tropics are reported to thrive at sustained temperatures as high as 28 C (see Vidal et al., 2014). In
Portuguese coastal waters, this species displays a year-round
spawning period with two peaks in the spring and summer
(Moreno et al., 2009). Consequently, their young undergo major
environmental shifts throughout early development, the stage
when organisms are expected to be more vulnerable to environmental variables (Pierce et al., 2008).
Embryos of O. vulgaris have already shown to be quite sensitive
to increased temperatures, presenting lower survival, smaller size
at hatching, greater metabolic costs at the transition to paralarvae,
stronger heat shock and oxidative stress responses, and greater
levels of lipid peroxidation under a warming scenario. The key to
understand how much these organisms are affected by environmental shifts is taking a closer look at their biochemistry and
physiology, where subtle differences can be explored and quantied (Repolho et al., 2014). We hypothesize that a future warmer
scenario of 3 C will affect the biochemical composition (amino
acid AA, and fatty acid-FA) of octopod (O. vulgaris) embryos and
recently-hatched pelagic paralarvae.

2. Materials and methods

2.1. Egg collection
Clutches of O. vulgaris embryos in the earliest developmental
stage [stage I; see Naef (1928)] were obtained from traps employed by local shermen, between October 2010 and November
2011 in Cascais, Portugal (384107N, 92806W). The mean sea
surface temperature (SST) in the shing ground varied between
15.2 C in the winter and 18.3 C in the summer. Egg clutches were
carefully removed from the traps and transported (for 5 min) in
thermal cases with proper aeration to Laboratrio Martimo da
Guia, Cascais, Portugal.
2.2. Egg incubation
Egg clutches were placed in recirculating aquaculture systems
comprising 24 separate glass tanks (each with an approximate
volume of 16 L) connected to a 270-L sump equipped with a wetdry lter with bioballs, a protein skimmer and a 36-W ultraviolet
sterilizer. Natural seawater was 1-mm ltered. A small pump was
placed inside each tank to generate high water currents and keep
the eggs swinging gently, in order to preserve the surface of the
eggs clean, free of fungus, and well aerated.
Embryos were incubated until hatching at two different temperatures, namely: (i) 18 C the mean summer SST, and (ii) 21 C
the future summer SST warming scenario in 2100 (3 C, Meehl
et al., 2007). Temperature was kept stable through the use of
heating or cooling systems. Salinity was maintained at 34 71 and
pH at 8.1 70.1. Ammonia and nitrite were monitored every other
day and maintained within recommended levels (see Iglesias and
Fuentes, 2014). The tanks were illuminated from above with a
photoperiod of 14 L:10 D.

31

2.3. Development stages

Three development stages were established for O. vulgaris
embryos based on Naef's (1928) main descriptions, namely:
(i) early stage (Naef's stages #110); (ii) late stage (Naef's stage
#29); and (iii) paralarvae (Naef's stage #30).
Embryos were observed every 48 h and their development
stage was identied using a dissecting microscope. After observation, embryos were returned to the aquariums. This type of
handling does not affect embryogenesis, as documented in previous studies (Oosthuizen et al., 2002; Pimentel et al., 2012; Rosa
et al., 2012). At each development stage, embryo samples from
different clutches were collected (N between 3 and 6 per stage and
temperature) for posterior biochemical analyses.
2.4. Biochemical analyses
2.4.1. Amino acids
Samples for AA analysis were homogenized with a grinder
(Retsch Grindomix GM200), vacuum-packed and frozen at  80 C.
Later, frozen samples were freeze-dried for 48 h at 50 C under
low pressure (approximately 10  1 atm), powdered and stored at
 80 C. To extract total AAs, 15 mg of sample was placed in 10-mL
ampoules with 3 mL of 6 M HCl containing 0.1% phenol (Merck),
according to the method described by AOAC (2005). Following the
establishment of inert and anaerobic conditions to prevent the
oxidative degradation of AAs, ampoules were sealed and samples
were hydrolyzed at 110 C for 24 h. Hydrolysates were ltered
(0.45 mm pore size) and dissolved with Milli-Q distilled water
making up to 20 mL. Samples were then frozen again at  80 C
and freeze-dried for 48 h at  50 C under low pressure (approximately 10  1 atm), after which they were dissolved in 5 mL of
0.1 M HCl (Merck) and stored at  80 C until AA separation. Finally, thawed samples were ltered (0.2 mm pore size), and separation was performed with high-performance liquid chromatography (Agilent 1100 HPLC). Here we used precolumn o-phthalaldehyde and 3-mercaptopropionic acid in borate buffer (OPA,
Agilent Technologies), and 9-uorenylmethylchloroformate in
acetonitrile (FMOC; Agilent Technologies) derivatization, in a
Phenomenex Gemini ODS C18 guard column (4 mm  3 mm), and
a Phenomenex Gemini ODS C18 110A column (4.6 mm15  0 mm,
5 mm). The solvents and gradient conditions were those described
by Henderson et al. (2000). Detection wavelengths were set at UV
338 and 262 nm and uorescence at 340/450 and 266/305 nm.
The identity and quantity of the AAs were assessed by comparison
with the retention times and peak areas of internal standards,
namely norvaline and sarcosine (Sigma-Aldrich). Trypthophan was
quantied in O. vulgaris but, since it is at least partially lost
throughout the acidic hydrolysis, it was not considered for
analysis.
2.4.2. Fatty acids
The determination of the FA prole was based on the experimental procedure already described in (Rosa et al., 2004a,2005).
Each sample (100 mg of dry mass) was dissolved in 5 mL of acetyl
chloride/methanol (1:19 v/v; Merck), shaken, and heated (at 80 C
for 1 h). After cooling, 1 mL of Milli-Q distilled water and 2 mL of
n-heptane pro analysis (Merck) were added, and samples were
shaken and centrifuged (at 2300g for 5 min) until phase separation. The moisture content of the upper phase was removed using
anhydrous sodium sulfate (Panreac). Afterwards, an aliquot (2 L)
of the upper phase was injected onto a gas chromatograph (Varian
Star 3800Cp, Walnut Creek) equipped with an autosampler and
tted with a ame ionization detector at 250 C for FAME (fatty
acid methyl ester) analysis. The separation was carried out with
helium as carrier gas at a ow rate of 1 mL min  1, in a capillary

32

V.M. Lopes et al. / Journal of Thermal Biology 55 (2016) 3038

column DB-WAX (30 m length  0.32 mm internal diameter;

0.25 m lm thickness; Hewlett-Packard) programmed at 180 C
for 5 min, raised to 220 C at 4 C min  1, and maintained at 220 C
for 25 min, with the injector at 250 C. FAME identication was
accomplished through comparison of retention times with those
of Sigma standards. Quantitative data (peak areas) were obtained
with Varian software using C21:0 FA (Sigma) as internal standard.

21 C

A
a

a*
B
b

4.0
DW)

1.2

18 C

-1

-1

HIS (g 100g

DW)

1.6
1.4

Ontogenetic and temperature-derived differences in the FA and

AA contents were tested with analysis of variance (two-way ANOVA) followed by post-hoc tests (Unequal N test). All data were
tested at a 0.05 level of probability using the software STATISTICA
12 (Statsoft, Inc.).

3.5

THR (g 100g

18 C

2.5. Statistical analysis

3.0

1.0

b
B
c

C
b

DW)

1.2

2.0
1.8

ab*
B

0.8
0.4

3.0

2.6

b
B

2.2

1.8

4.5

-1

5.0
DW)

4.0

Early
Late
Paralarval
Development stage

LYS (g 100g

6.4

0.0

-1

2.2

1.6

LEU (g 100g -1 DW)

3.4

ILE (g 100g

DW)

-1

PHE (g 100g

MET (g 100g

2.4

3.2

-1

3.0

4.8

1.6

DW)

DW)
-1

ARG (g 100g

3.5

5.6

2.5

4.5
4.0

21 C

4.0
3.5
3.0

Early

Late

Paralarval

Development stage

Fig. 1. Main essential amino acid contents (A Histidine, B Threonine, C Arginine, D Methionine, E Phenylalanine, F Isoleucine, G Leucine, and H Lysine) of
Octopus vulgaris embryos incubated at two different temperatures: 18 C (summer sea surface temperature) and 21 C (warming scenario). Values are mean 7SD (N between
3 and 6 per development stage and temperature). Different letters (lowercase and uppercase for 18 and 21 C, respectively) represent signicant differences between
development stages, and asterisks represent signicant differences between temperatures (Po 0.05).

V.M. Lopes et al. / Journal of Thermal Biology 55 (2016) 3038

18 C

21 C

3.0

2.5

c C

2.2

1.8

DW)
-1

a
B

2.0

8.0

7.2
6.4
5.6

2.8

2.4
2.0

3.4

A
b

2.9

2.4

a
A

5.5

a
B

5.0

b
7.0

DW)

GLU ( g 1 0 0 g -1 DW)

6.0

G
a

6.0

-1

PRO ( g 1 0 0 g -1 DW)

3.0

8.8

-1

AB

1.6

3.2

3.9

E
DW)

CYS ( g 1 0 0 g -1 DW)

2.0

2.5

TYR (g 100g

-1

1.8

b
a

DW)

2.4

b*

2.0
1.5

c*

2.5

3.6

VAL (g 100g

ALA ( g 1 0 0 g -1 DW)

2.6

2.0

GLY (g 100g -1 DW)

21 C

3.0

ASP (g 100g

3.5

18 C

Early
Late
Paralarval
Development stage

TAU (g 100g

SER ( g 1 0 0 g -1 DW)

4.0

33

5.0
4.0
3.0

A
a
Early

b
B

Late
Paralarval
Development stage

Fig. 2. Main non-essential amino acid contents (A Serine, B Glycine, CAlanine, D Tyrosine, E Cysteine, F Valine, G Proline, H Aspartic acid, I Glutamic acid, and
J Taurine) of Octopus vulgaris embryos incubated at two different temperatures: 18 C (summer sea surface temperature) and 21 C (warming scenario). Values are
mean 7 SD (N between 3 and 6 per development stage and temperature). Different letters (lowercase and uppercase for 18 and 21 C, respectively) represent signicant
differences between development stages, and asterisks represent signicant differences between temperatures (Po 0.05).

34

V.M. Lopes et al. / Journal of Thermal Biology 55 (2016) 3038

3. Results
3.1. Amino acids
The impact of environmental warming on the AA contents
during the early ontogeny of O. vulgaris is shown in Figs. 13.
Further details in AA contents can be found in Supplementary
Table 1.
The major essential amino acids (EAAs) found were leucine
(LEU), arginine (ARG), lysine (LYS), threonine (THR) and isoleucine
(ILE) (Fig. 1). EAA contents generally followed a steep decreasing
trend between the early, late and paralarval stages. Exceptions to
this were phenylalanine (PHE) and LYS. No signicant differences
(P 40.05) were found in the contents of these two AAs between all
the three stages at both temperatures. On the other hand, histidine
(HIS) and methionine (MET) contents only decreased signicantly
(P o0.05) from late to paralarval stages at both temperatures.

EAA (g 100g -1 DW)

18 C
28

21 C

3.2. Fatty acids

a
A

24

b
B

20

AA (g 100g

-1

DW)

NEAA (g 100g -1 DW)

16
40
36

B
a
A

32

ab
B

28
64
60
56
52
48

a
A

b
B
b

Moreover, temperature did not affect most of the EAAs analyzed,

with the exception of HIS, MET and PHE. The late stages of development presented signicantly lower contents of these AAs
under warming (P o0.05).
The main non-essential amino acids (NEAAs) found were glutamic acid (GLU), taurine (TAU) and aspartic acid (ASP) (Fig. 2). The
majority of NEAAs, including serine (SER), tyrosine (TYR), valine
(VAL), proline (PRO), ASP and GLU, followed a decreasing trend
between stages of development. Contrarily, glycine (GLY), alanine
(ALA), cysteine (CYS) and TAU followed an increasing trend during
early ontogeny. The development stage had a signicant effect
(P o0.05) in most NEAA contents. In contrast, temperature did not
elicit signicant changes in NEAA, except in GLY contents in late
and paralarval stages.
In general, the fractions of total AAs, EAAs and NEAAs decreased signicantly (P o0.05) during octopus development
(Fig. 3). In contrast, temperature did not play a signicant role in
total AA, EAA and NEAA contents (P 40.05).

The effect of ocean warming on the FA contents during the

early ontogeny of O. vulgaris is shown in Figs. 47. Further details
in FA contents can be found in Supplementary Table 2.
The main saturated fatty acids (SFAs) found were palmitic
(16:0), stearic (18:0) and myristic (14:0) acids (Fig. 4). The palmitic
acid content was only signicantly affected (Po 0.05) by the development stage at 18 C. The stearic acid content increased signicantly (P o0.05) throughout the embryogenesis, but was not
affected by temperature (P4 0.05). Myristic acid contents were not
affected by neither factor (P 40.05). A lack of effect (P 40.05) was
also observed in total SFAs (Fig. 7A).
The main monounsaturated fatty acids (MUFAs) were vaccenic
acid (18:1n  7), 18:1n  5 and 20:1, depending on the development stage and temperature (Fig. 5). MUFA contents were not
affected either by development stage or temperature (P 40.05).
The same was observed for the total fraction of MUFA (Fig. 7B).
The main polyunsaturated fatty acids (PUFAs) found were the
essential fatty acids (EFAs) docosahexaenoic (DHA, 22:6n  3), eicosapentaenoic (EPA, 20:5n 3) and arachidonic (ARA, 20:4n  6)
acids (Fig. 6). The development stage had a signicant effect
(P o0.05) only in linoleic acid (18:2n 6) and eicosatrienoic acid
(ETA, 20:3n  3) contents at both temperatures, with both increasing throughout development. The content of the EFAs DHA,
EPA and ARA, however, did not vary signicantly (P 40.05)
throughout development. Overall, temperature did not elicit signicant changes (P 40.05) in PUFA levels. Regarding the total
fraction of PUFA (Fig. 7C), the total content was not signicantly
affected (P 40.05) by stage or temperature. The n  3/n  6 ratio
(Fig. 7D) was signicantly affected by development stage
(P o0.05), but not by temperature (P4 0.05). In contrast, none of
the factors had a signicant effect (P40.05) on the total FA content (Fig. 7E).

Early
Late
Paralarval
Development stage

Fig. 3. Total contents of essential (A EAA), non-essential (B NEAA) and total

amino acids (C AA) of Octopus vulgaris embryos incubated at two different
temperatures: 18 C (summer sea surface temperature) and 21 C (warming scenario). Values are mean7 SD (N between 3 and 6 per development stage and
temperature). Different letters (lowercase and uppercase for 18 and 21 C, respectively) represent signicant differences between development stages (Po 0.05),
and asterisks represent signicant differences between temperatures (P o0.05).

4. Discussion
Parental input plays a key role in embryo's development. In
most animals, egg reserves are the only source of nutrients and
should satisfy the high and specic demands of the embryogenesis. The main AAs (THR, ARG, ILE, LEU, LYS, GLU, TAU and ASP) and
FAs (14:0, 16:0, 18:0, 18:1n  7, 20:1, 20:4n  6, 20:5n  3 and
22:6 n  3) found in the early stages of the embryonic development of O. vulgaris reected the AAs and FAs present in mature
gonads of female octopuses (Navarro and Villanueva, 2003; Rosa
et al., 2004b; Villanueva et al., 2004). Otherwise, the embryos

V.M. Lopes et al. / Journal of Thermal Biology 55 (2016) 3038

18C

21C

18:1 n-5 (mg g

-1

2.0

14:0 (mg g

-1

DW)

2.6

DW)

18C

1.4
0.8

18:1 n-7 (mg g

-1

12

10

C
B
A

4.0

2.5
1.0

Early
Late
Paralarval
Development stage

Fig. 4. Main saturated fatty acid contents (A 14:0, B 16:0, and C 18:0) of
Octopus vulgaris embryos incubated at two different temperatures: 18 C (summer
sea surface temperature) and 21 C (warming scenario). Values are mean 7 SD (N
between 3 and 6 per development stage and temperature). Different letters (lowercase and uppercase for 18 C and 21 C, respectively) represent signicant differences between development stages, and asterisks represent signicant differences between temperatures (P o0.05).

would greatly lack the presence of essential nutrients that cannot

be synthesized by them and that are vital for development.
As expected, there was a decreasing trend of many AAs, including many essential ones, throughout the embryonic period.
Many NEAAs also presented a decreasing trend during embryogenesis, with the most notorious exception being TAU. Studies
conducted in gastropod and bivalve mollusks (Welborn and
Manahan, 1995), along with studies performed in mammals
(Huxtable, 1992; Jacobsen and Smith, 1968), report that this free
AA can be synthesized de novo through conversion of MET into
TAU. Indeed, MET levels in octopus paralarvae decreased greatly,
while TAU levels nearly doubled, suggesting a great efciency in
converting MET into TAU as the embryo hatches. TAU has been
reported to have a key role in osmoregulation in other marine
invertebrates, most commonly in marine mollusks (Allen and
Garrett, 1972; Gilles, 1972; Simpson et al., 1959), which may explain the increase in TAU levels when the embryo no longer lives
inside the egg capsule. Regarding the AA contents found in

0.8
0.4
3.5

2.5
1.5
0.5

3.0
DW)

5.5

1.2

-1

18:0 (mg g

-1

DW)

7.0

21C

1.6

2.0

20:1 (mg g

16:0 (mg g

-1

DW)

16

DW)

0.2

14

35

1.0
0.0

Early
Late
Paralarval
Development stage

Fig. 5. Main monounsaturated fatty acid contents (A 18:1n 5, B 18:1n 7, and

C 20:1) of Octopus vulgaris embryos incubated at two different temperatures:
18 C (summer sea surface temperature) and 21 C (warming scenario). Values are
mean7 SD (N between 3 and 6 per development stage and temperature). Different
letters (lowercase and uppercase for 18 and 21 C, respectively) represent signicant differences between development stages, and asterisks represent signicant differences between temperatures (P o0.05).

octopus hatchlings, we obtained similar results to those found in

Villanueva et al. (2004) for octopus paralarvae. However, when
comparing to AA contents present in squid (Loligo vulgaris), we
veried that octopus, overall, has considerably lower AA than
squid (Villanueva et al., 2004), most likely owing to the squid's
higher energy expenditure rates required to occupy the pelagic
realm and the highly costly means of locomotion (O'dor and
Webber, 1986). Moreover, cephalopods are known for having a
very powerful protein and AA metabolism, essential to support the
rapid growth rates that characterize their life cycle. These animals
have great AA requirements in order to satisfy the high demands
of protein synthesis needed to maintain optimum growth, but AAs
are also an important source of metabolic energy in cephalopods
(Lee, 1994).
In contrast, this decreasing trend throughout the embryogenesis was not observed in the fatty acid prole of the embryos.

36

V.M. Lopes et al. / Journal of Thermal Biology 55 (2016) 3038

21 C

0.2

ab

0.0

1.2

-1

0.8

0.4

a A

a
A
16

20:5 n-3 (mg g

0.1

-1

20:4 n-6 (mg g

0.3

DW)

DW)

18 C

0.4

22:6 n-3 (mg g -1 DW)

20:3 n-3 ( m g g -1 DW)

18:2 n-6 ( m g g -1 DW)

18 C

4.5

21 C

3.5
2.5
1.5

11.0

9.5
8.0
6.5

14
12
10
8

Early
Late
Paralarval
Development stage

Fig. 6. Main polyunsaturated fatty acid contents (A 18:2n 6, B 20:4n 6, C 20:3n 3, D 20:5n  5, and E 22:6n  3) of Octopus vulgaris embryos incubated at two
different temperatures: 18 C (summer sea surface temperature) and 21 C (warming scenario). Values are mean 7 SD (N between 3 and 6 per development stage and
temperature). Different letters (lowercase and uppercase for 18 and 21 C, respectively) represent signicant differences between development stages, and asterisks represent signicant differences between temperatures (P o 0.05).

Indeed, many FAs, including the essential ones (ARA, EPA and
DHA), remained quite stable throughout development, indicating
that these EFAs were selectively retained, instead of being consumed as energy sources. This is not surprising given the important role that these essential elements have in membrane
structure and functions (Sargent et al., 2002). However, SFA fraction presented a decreasing trend throughout the embryogenesis,
while the n 3/n  6 ratio an increasing trend, in accordance with
the results obtained in Navarro and Villanueva (2003). Upon
hatching, octopus paralarvae presented much lower levels of SFA,
MUFA, PUFA, n 3/n  6 ratio and FA fractions than observed in
Navarro and Villanueva (2000), and considerably lower than the
cuttlesh (Sepia ofcinalis) and squid. These differences between
both octopus samples and the other species of cephalopods can be
explained by the different rearing conditions, namely temperature.
Out of all environmental variables, temperature is widely
known to be the key driver of biochemical, ecological, behavioral
and physiological shifts throughout an organism's lifetime, with
special emphasis on the early ontogeny (Roberts and Sauer, 1994;

Rosa et al., 2012, 2013). Thermal changes have also shown to have
a signicant effect on the fatty acid dynamics of juvenile octopus
(Miliou et al., 2006; Noyola et al., 2013). Both studies revealed that
higher temperatures (within the optimal thermal range of the
species) led to increased PUFA levels in the mantle tissue. These
trends suggest that the metabolic demands for those compounds
under such temperatures were lower. Moreover, Miliou et al.
(2005) argued that higher PUFA demands under low temperatures
were associated with the maintenance of membrane permeability
and plasticity.
Information on the effect of warming on the AA and FA proles
of cephalopod early stages is, to our knowledge, inexistent. In this
study, a 3 C warming scenario elicited signicant changes in the
content of some AAs, including the EAA GLY, and the NEAAs HIS,
MET and PHE. Temperature decreased the content of the aforementioned AAs, suggesting an increased consumption by the
embryos and recently-hatched paralarvae. In contrast, the effect of
warming in the FA prole of the embryos was almost inexistent.
Temperature is known to be inversely correlated with embryonic

V.M. Lopes et al. / Journal of Thermal Biology 55 (2016) 3038

37

Fig. 7. Total saturated (A SFA), monounsaturated (B MUFA) and polyunsaturated fatty acid (C PUFA) fractions, ratio n  3/n  6 (D) and total fatty acid contents (E FA) of
Octopus vulgaris embryos incubated at two different temperatures: 18 C (summer sea surface temperature) and 21 C (warming scenario). Values are mean 7 SD (N between
3 and 6 per development stage and temperature). Different letters (lowercase and uppercase for 18 and 21 C, respectively) represent signicant differences between
development stages, and asterisks represent signicant differences between temperatures (Po 0.05).

development time (Mangold and Boletzky, 1973; Naef, 1928; Villanueva et al., 1995). Previous experiments performed by Repolho
et al. (2014) and Rosa et al. (2012) in embryos and hatchlings of
the common octopus and squid, respectively, subjected to ocean
warming have presented shortened embryonic periods. Octopus
normal embryonic development time (38 days at 18 C) was
shortened by 13 days, when exposed to a 3 C warming scenario.
More, with increasing temperature it was also shown that yolk
consumption was faster due to higher metabolic rates, resulting in
premature hatching (Repolho et al., 2014). The greater (although
not signicant) FA contents of the paralarvae under warming can
thus be explained by premature hatching, since the main characteristic of premature hatchlings is to have the remaining yolk
still attached to the paralarvae.
Ocean warming has already proven to have a negative impact
on several physiological parameters of octopus embryos and
hatchlings, including lower survival, smaller size at hatching,
greater metabolic costs of the transition to paralarvae, stronger

heat shock and oxidative stress responses, and greater levels of

lipid peroxidation (one of the most frequent cellular injury mechanisms) despite the presence of effective antioxidant defense
mechanisms (Repolho et al., 2014). Nevertheless, besides a greater
embryonic consumption of some AAs, no other disturbances in the
AA and FA dynamics were detected during the embryogenesis of O.
vulgaris that could explain the aforementioned negative impacts of
warming.
The present study constituted the rst attempt to understand
how the predicted future ocean conditions may impact the biochemistry of the rst stages of an octopus' life, and it opens space
for further research. Given the time frame in which ocean warming is expected to occur, organisms will likely have the opportunity for acclimatization and adaptation. Recent studies have already shown that transgenerational acclimation can modify the
response of marine organisms to climate change conditions
(Murray et al., 2014; Salinas and Munch, 2012; Schade et al., 2014;
Welch et al., 2014). Adaptation over multiple generations is

38

V.M. Lopes et al. / Journal of Thermal Biology 55 (2016) 3038

therefore expected to moderate the negative impacts of future

ocean conditions, especially in marine organisms with very short
life spans, such as cephalopods. Moreover, additional studies are
sorely needed, as the future oceans will not only be warmer, but
they'll also be more acidic and hypoxic.

Acknowledgments
The Portuguese Foundation for Science and Technology (FCT)
supported this study through the project grants PTDC/BIA-BEC/
103266/2008 and PTDC/MAR/0908066/2008 to R. Rosa and Ph.D.
Scholarship to V. Lopes (SFRH/BD/97633/2013).

Appendix A. Supplementary material

Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.jtherbio.2015.11.
006.

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