Received: 22 July 2016
|
Revised: 10 April 2017
|
Accepted: 14 April 2017
DOI: 10.1111/are.13387
ORIGINAL ARTICLE
Effect of starvation on the performance of baby octopus (Robsonella fontaniana) paralarvae Viviana Espinoza1,*
| Maria T. Viana2,* | Carlos Rosas3,* | Iker Uriarte1,* | Ana Farıas1,*
1
Hatchery de Invertebrados Marinos, Instituto de Acuicultura, Sede Puerto Montt, Universidad Austral de Chile, Puerto Montt, Chile 2 Instituto de Investigaciones Oceanologicas, noma de Baja California, Universidad Auto xico Ensenada, Baja California, Me 3
Unidad Multidisciplinaria de Docencia e n, Facultad de Ciencias, Investigacio noma de Me xico, Universidad Nacional Auto xico Sisal, Yucatan, Me Correspondence Ana Farıas, Hatchery de Invertebrados Marinos, Instituto de Acuicultura, Sede Puerto Montt, Universidad Austral de Chile, Puerto Montt, Chile. Email:
[email protected]
Abstract The aim of this study was to evaluate the effect of short- and long-term starvation on paralarvae from hatching and compare to fed paralarvae. In the continuous starvation treatments, paralarvae at hatching were left without food as independent treatments for 5, 8, 9, 10, 11, 12, 13 and 15 days. In the fed treatments, the newly hatched paralarvae were fed for five and eight days; then each group was left in starvation as independent treatments for 3, 5 and 7 days. After any experimental starvation period, the paralarvae were fed for five more days to evaluate their recovery. Paralarvae exposed to continuous starvation from hatching endured up to 8 days after hatching (8 SDAH) showing significant recovery. Its survival decreased proportionally to the days of starvation, without any recovery after 12 DAH. Fed paralarvae (5 and 8 FDAH), resulted in significant differences accordingly to the length of the starvation period. Paralarvae left under permanent starvation showed
Funding information n Nacional de Investigacio n Comisio gica, Grant/Award Cientıfica y Tecnolo Number: FONDEF D09I1401
a noticeable decrease in their arm/mantle length ratio and an atrophy of the digestive gland was observed. Amino acids were used primarily as energy source by paralarvae, particularly in the second week of starvation. It is discussed the critical age to avoid deleterious effects of starvation on paralarvae. KEYWORDS
amino acids, digestive gland, ontogeny, point of no return, yolk reserves
1 | INTRODUCTION
Although during embryonic development of cephalopods, most of the yolk is used for tissues and organ synthesis, the environment
The culture of planktonic octopuses paralarval stages is characterized
determines the amount of yolk in hatchlings that will be spared to
by a high mortality rate, which has so far hampered the production
be used until the beginning of planktonic stages. Vidal, DiMarco,
of merobenthic juvenile’s species under controlled culture conditions
Wormuth and Lee (2002) found in embryos of the squid, Loligo opa-
(Uriarte, Iglesias, et al., 2011). The main cause of cephalopod paralar-
lescens, that the temperature is the key factor affecting the amount
vae mortality seems to be the lack of appropriate food, that is food
of yolk used for the tissues and organs synthesis. Also, it was sug-
containing the required nutrients, prey’s size, taste and movement
gested that the nutritional female status of Enteroctopus megalocy-
behaviour to stimulate the predation activity (Iglesias et al., 2007;
athus (Farıas, Navarro, Cerna, Pino & Uriarte, 2011) and Octopus
Uriarte, Farıas, Paschke, Navarro & Rosas, 2011; Uriarte, Iglesias,
maya (Caamal-Monsreal et al., 2015; Tercero-Iglesias et al., 2015)
et al., 2011; Villanueva, Nozais & Boletzky, 1996). Octopuses are
during the reproduction conditioning influences the amount of yolk
~ez strict carnivores that become cannibals as strategy to survive (Iban
present, during the embryo development as well as during the post-
& Keyl, 2010) when there is a density imbalance or food is not avail-
hatchlings phases.
€ rner, Farıas & Uriarte, 2011). ability (Miranda, Espinoza, Do
Vidal et al. (2002) and Uriarte et al. (2010) observed in the paralarvae of squid (Loligo opalescens) and baby octopus (Robsonella
*Interdisciplinary Network of Advanced Research for Marine Larviculture of Species with Complex Lifecycles (INLARVI)
5650
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© 2017 John Wiley & Sons Ltd
fontaniana), respectively, the so-called no net growth phase during the first 22 days after hatch, which means that under this period
wileyonlinelibrary.com/journal/are
Aquaculture Research. 2017;48:5650–5658.
ESPINOZA
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ET AL.
5651
there is no growth in terms of total weight increase according to
starvation from the first day after hatching was then distributed
Uriarte et al. (2010), when the remaining yolk is used as a main
into 24 flasks for the eight treatments with three replicates each.
source of energy. In benthic hatchlings of O. maya, a similar period
The treatments were left under starvation at eight different periods
of “no net growth phase” was identified during the first 7 days,
(days) after hatching (SDAH): 8, 9, 10, 11, 12, 13 and 15 SDAH,
when the digestive gland reaches its maturity and hatchling starts
whereas treatment B only had two different times of feeding from
the digestive processes linked to the digestion of exogenous food
hatching (5 and 8 days) followed by three periods of starvation
(Moguel et al., 2010). Pereda, Uriarte and Cabrera (2009) have
each. To evaluate the capability of paralarvae to recover after star-
demonstrated that R. fontaniana paralarvae under controlled culture
vation, the unsampled paralarvae left in the flasks were fed during
conditions is able to ingest prey from the first day after hatching (1
five additional days with king crab (Lithodes santolla) zoea at a ratio
DAH). Only a proper prey will allow the development of digestive
of two prey paralarvae
enzymes during the first 2 weeks after hatching, as previously
Survival was estimated as percentage from the total paralarvae at
reported in O. maya juveniles (Moguel et al., 2010).
the end of each starvation period (index of resistance to starva-
1
day
1
according to Uriarte et al. (2010).
Earlier studies evaluating the starvation effects in fish larvae
tion). The post-starvation survival was measured as percentage
have indicated that when food is not available, the larvae can experi-
from the individuals that remained alive after the refeeding period
ence a visible external deterioration besides changes in the digestive
was used as an index of recovery from starvation. Samples for
organs (Uriarte & Balbontın, 1987). Also, Navarrete (2006) proposed
analysis were taken each time, consisting in three active paralarvae
that the maximum survival time for well-fed fish larvae with different
per treatment, one per each replicate, at the end of each period.
preys before being exposed to food deprivation could be used as an
Sampled organisms were immediately anesthetized by submersion
indicator of prey quality. Similarly, Vidal, DiMarco and Lee (2006)
in 1% ethanol dissolved in sea water, and thereafter, they were
proposed the RNA/DNA ratio as a valid indicator of nutritional con-
taken for morphological measurements, a duplicate was chosen and
dition in squid paralarvae that had undergone starvation (Vidal et al.,
fixed for histology and one for amino acid analysis was frozen at 80°C.
2006). This study was aimed to estimate how R. fontaniana paralarvae
In the group B, the paralarvae were fed with L. santolla zoeas at 1
day
1
and distributed into nine 1-L
withstand starvation after hatching, either using its own reserves or
a ratio of 2 prey paralarvae
throughout previous different exogenous feeding. Thus, the lack of a
flask to be fed at different periods of time (days) after hatching
proper development will result in a physiological impairment, which can go from morphometric changes up to mortality. With this purpose it was evaluated: (i) the maximum survival time under starva-
PARALARVAE AT HATCHING
tion, using only the endogenous reserves; and (ii) the tolerance to starvation under different previous nutritional conditions. A.STARVED (S)
B.FED (F)
2 | MATERIALS AND METHODS 2.1 | Experimental design Female octopuses of R. fontaniana and their spawns were collected
Starved days after hatching (SDAH) 5, 8, 9, 10, 11, 12, 13 and 15 SDAH
Fed days after hatching (FDAH) 5 and 8 FDAH
from their natural environment (Hueihue, 42°S, Southern Chile) and transported to the Marine Invertebrate Hatchery Laboratory of the
Starved days after feeding (SDAF) (independent treatments for each above group) 3, 5 and 7 SDAF
Universidad Austral de Chile (HIM-UACh). The females and their corresponding eggs attached to rocks were incubated in a semi-dark laboratory with photoperiod 12:12 (dark:light) and distributed into three individual 70-L tanks with aerated sea water (ie, 12°C and 30psu salinity) and connected to a sea water recirculation system. In total, 576 newly hatched paralarvae (after 24 hr) were selected from the three different spawns and randomly distributed into 42 flasks (1L) at a density of 12 paralarvae L
1
After each starvation period 3 surviving paralarvae were sampled for analyses and the rest were fed for 5 days to estimate percentage of recovery
After each starvation period, 3 surviving paralarvae were sampled for analyses and the rest were fed for 5 days to estimate percentage of recovery
for all the different treatments.
Filtered (5 lm) and sterilized (UV) sea water was used throughout the experiment with a 100% daily water replacement. For all experimental treatments, aerated sea water was used to reach a 90%100% saturation. Two feeding regimes of paralarvae were selected: (i) continuous starvation, and (ii) under feeding regime from hatching followed by a period of starvation (Figure 1). The group A of continuous
F I G U R E 1 Scheme of the experimental design. Two groups of paralarvae: group A always starved and group B fed after hatching for some days followed by periods of food deprivation (3, 5 and 7). For group A, the starvation time treatments after hatching (SDAH) were 5, 8, 9, 10, 11, 12, 13 and 15 SDAH. For group B, the feeding times after hatching (FDAH) treatments were 5 and 8 FDAH, all of them followed by starvation period after feeding (SDAF) treatments: 3, 5 and 7 SDAF. All treatments had three replicates each
5652
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ESPINOZA
ET AL.
(FDAH): 5 and 8 FDAH. When paralarvae reached 5 or 8 FDAH,
phase column (3.9 9 150 mm; from Waters). Acetonitrile, water and
respectively, they were separated into three new treatments for
AccQ*Tag solution A were used as eluents as recommended.
starvation days after feeding (SDAF), each treatment with three
Quantification was performed using a fluorescence detector 474 Mil-
replicates each. The treatments were as follows: 3, 5 and 7 SDAF
lenniumâ chromatography manager. A sole mixture of 18 AA at three
(Figure 1). After the starvation period, one paralarvae from each
different concentrations were to obtain the standard curve for each
replicate (three per treatment) was sampled for analyses. The rest of
AA. Each paralarvae was analysed in duplicate, and the mean concen-
surviving paralarvae were refed again for 5 days to assess the post-
tration of each AA was expressed as lg AA per mg of dry matter (rel-
starvation survival. The indexes for resistance and recovery to star-
ative value) or lg AA per paralarvae (absolute value).
vation were calculated as indicated above for group A.
2.5 | Statistical analysis 2.2 | Morphometric characteristics
The effects of feeding regime and time on total length and dry
The sampled paralarvae for morphological evaluation were evaluated
weight of paralarvae were evaluated using a two-way ANOVA,
for lengths of total organism, mantle and arm (TL, ML and AL
whereas the effect of starvation period on survival of starved par-
respectively) using an AxioCam camera (ICC3 Carl Zeiss) coupled to
alarvae was analysed by one-way ANOVA. For the starvation
a stereomicroscope (Stemi 2000c) with the Carl Zeiss AxioVision 4.6
effect at different periods of time (3, 5 and 7 SDAF) after the
software. TL was registered from the posterior end of the mantle to
paralarvae was fed for 5 and 8 days (FDAH), a two-way ANOVA
the tip of the longest arm, ML was estimated dorsally from the mid-
was used.
point of the imaginary line that links both eyes to the posterior end
Morphometric characteristics and digestive gland area were com-
of the mantle and AL was measured from the base of the arms to
pared between groups A and B by factorial ANOVA. The factors
the tip of the longest arm. After these measurements were carried
analysed were as follows: feeding regime (treatments: groups A and
out, the same samples were used for the rest of the analyses, ran-
B) and paralarvae age (treatments: 5, 8, 10, 11, 13 days after
domly separating two of them for histological studies and one for
hatching).
amino acid profile.
For the effect of starvation on the AA profile, a one-way ANOVA was performed and if significant differences were found, a posteriori analysis was performed using Tukey estimation. Differ-
2.3 | Histological analysis
ences were considered statistically significant if p < .05. The survival
Two paralarvae per each treatment were selected and fixed in
data given as percentage were arcsine-transformed before analysis
Bouin’s solution until histological processing. For this purpose, sam-
(Sokal & Rohlf, 1995).
ples were washed, dehydrated, cleared and embedded into paraffin
For the different morphological evaluations (length and area),
according to the procedure reported by Avila-Poveda, Colin-Flores
regression models were adjusted to data from both A and B groups
and Rosas (2009). Briefly, sagittal serial cross sections (5 lm) were
(5 and 8 FDAH): lineal: Y = bX +a, exponential: Y = aebX or logarith-
sliced manually with a rotary microtome (model RM 2125, Leica),
mic: Y = a + b ln X; where a and b are constants, and X is the time
processed and stained with haematoxylin and erythrosin (Bonet &
(days) from paralarvae hatching (DAH).
Huguet, 1985). In each sample, the area of the digestive gland (DG)
Statistical analyses were performed using Statistica 7.
was used as an indicator of the nutritional condition.
3 | RESULTS
2.4 | Amino acid (AA) analysis One paralarvae per treatment was randomly selected for AA analysis.
At hatching, paralarvae showed an average total length (TL) and dry
Samples were individually freeze-dried and weighted in a microbal-
weight (DWt) of 4.8 0.06 mm and 2.9 0.3 mg. At 5 and 8 DAH
ance (0.001 mg; METTLER-TOLEDO XP2U). Then, they were indi-
under starved (S) or fed (F) regimen, an interaction between both
vidually grinded and stored at
20°C until analysis. Each sample
factors was observed in the TL (Ftime*feed
regime
= 5.55; df=1; 115;
HCl containing
p = .02; Figure 2). At 5 DAH, no significant differences in TL were
0.01% phenol to avoid oxidation and left at 112°C for 24 hr. Samples
observed among fed and unfed paralarvae, whereas at 8 DAH, the
were then dried under nitrogen atmosphere and further rehydrated
fed paralarvae resulted in a significant higher TL (4.92 0.07 mm)
with 200 lL ultra-pure water and then filtered through a single-use
than those from the starved group (4.36 0.07 mm). The fed par-
0.45-lm Teflon filter. Filtered samples were then derivatized using
alarvae maintained a similar TL than hatchlings, while the starved
the AccQTagTM derivatization kit (Waters TM) following the proce-
ones decreased their TL (Figure 2). The paralarvae weight was only
(approx 3 lg) was hydrolysed with 200 ll of 6
M
regime=17.53;
df=1; 100;
dure described by the manufacturer and according to Cohen and de
affected by the feed regime (Ffeed
Antonis (1994) and Liu, Chang, Yan, Yu, and Liu (1995), heating the
p = .00006). The fed paralarvae kept the same weight from hatching
mixture at 55°C for 10 min. The AA separation was carried out by an
(2.9 0.1 mg) while paralarvae under starvation resulted in a reduc-
HPLC (Waters LC 626, Milford, MA, USA) using a C-18 reversed-
tion of 24% in weight (Fig. 2).
|
ET AL.
5
c
b
ab
a
4 3
b
a
a
b
2 1 0 8 SDAH
5 SDAH
5 FDAH
100
c
c
c
80
bc
bc
60 40
ab
b a
20
a
a
10
11
0
8 FDAH
8
9
Starved Fed Paralarval condition and age TL (mm)
5653
Group A; starved
(a)
Fed
Survival or recovery (%)
Starved
6
a
a
12
13
a a 14
15
14
15
Paralarvae age (DAH) Survival
Recovery
DWt (mg)
F I G U R E 2 Robsonella fontaniana. Total length (TL) and dry weight (DWt) of paralarvae under starvation for 5 and 8 days after hatching (5 SDAH and 8 SDAH respectively) and fed paralarvae of 5 and 8 days after hatching (5 FDAH and FDAH 8 respectively). Each value is the average and standard error of 20 to 30 paralarvae. Different letters on average bars indicate significant differences (p < .05).
Group B; 5 FDAH
(b) Survival or recovery (%)
Total length (mm) and dry weight (mg)
ESPINOZA
100
b
b
80 60 40
b
a
20
a
a
0 8
The survival of group A (always starved) resulted significantly
9
10
11
12
13
affected (Fig. 3a) through time (F = 11.52; df=6, 32, p = .000001) up
Paralarvae age (DAH)
to day 15. No differences were observed between days 8 and 12
Survival
Recovery
and no differences were observed between days 10 and 12 either,
accordingly to the increase in number of food deprivation days (F = 3,68; df=6, 32; p = .007), with the highest value being recorded in treatment 8 SDAH (38% 17) and the minimum value in the rest of treatments without recovery up to day 12. The survival of group B (fed paralarvae, F) after a period of food deprivation resulted in significant differences (Figure 3b,c) according to the length of the starvation period (F = 2.95; df=2, 18; p = .01). Paralarvae from both feeding times, 5 and 8 FDAH, left under starvation for 3 or 5 days (3 SDAF or 5 SDAF respectively) had a similar survival of 89%, ranging between 76 and 100% for all combinations. Paralarvae left for 7 days under starvation (7 SDAF) showed the lower survival for 5 FDAH (25.9%, Figure 3b) and 8 FDAH (62.9%, Figure 3c). No interaction was observed among feeding time and the length of starvation period in group B (Ffeeding
time*starvation length=1.70;
df=2, 18; p = .21). The recovery of paralarvae after refeeding (Fig-
Survival or recovery (%)
15. The recovery after experimental starvation periods decreased
Group B; 8 FDAH
(c)
whereas a significant decrease (40% reduced) was observed at day
b
b
100
ab
80 60 40
ab
ab
20
a
0 8
9
10
11
12
13
14
15
Paralarvae age (DAH) Survival
Recovery
F I G U R E 3 Robsonella fontaniana. Survival under starvation and post-starvation recovery. (a) Paralarvae of group A starved from hatching (SDAH), (b) paralarvae of group B fed for 5 days after hatching (FDAH) followed by 3, 5 and 7 days of feed deprivation and (c) paralarvae of group B fed for 8 days after hatching (FDAH) followed by 3,5 and 7 of feed deprivation. DAH is the age in days after hatching. Different letters on average bars indicate significant differences (p < .05)
ure 3b) failed to show statistical differences among 5 and 8 FDAH (Ffeeding
time=0.06;
days (Fstarvation
df=1, 24, p = .76), or along the number of starving
length=
2.95; df=2, 18; p = .078).
that age affected AL/ML ratio depending on feeding condition was observed, although in the first days was noted a decreasing ratio in
The morphometric parameters of paralarvae such as total length
all the treatments (Figure 4). AL/ML ratio in fed paralarvae (group
(TL), mantle length (ML) and eye diameter (EY) were not affected by
B), both 5 FDAH and 8 FDAH, increased after 10 days. However,
the age (5, 8, 9, 10, 11 DAH) or by the feeding regime (A and B)
when paralarvae was constantly starved (group A) the ratio
along the experiment. Arm length (AL) was affected only by age
decreased up to 0.65 0.02 and remained at the lowest ratio.
(Fage= 8.92; df=4, 37; p = .00004) observing the highest arm length
The unfed paralarvae did not show a development of the diges-
in paralarvae of 5 DAH (1.59) versus the lowest value in the other
tive gland; on the contrary, they suffered a sustained logarithmic
ages (1.15). Similarly, the AL/ML ratio was affected by age (Fage=
reduction (Figure 5a). The DG in paralarvae fed from hatching during
11.90; df=4, 36; p = .000003), and the highest value observed at 5
5 DAH did not show any change in the following days of starving
DAH (0.96) was followed by an intermediate value at 11 DAH (0.72)
(Figure 5b). Paralarvae that was fed during the first 8 DAH (8 FDAH)
while the other ages showed a mean lowest value of 0.36. A trend
and then left to starve for 3, 5 and 7 days (3, 5, 7 SDAF) fail to
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AL/ML
1.0 0.8 0.6 AL/ML5 FDAH = 0.017×age2 – 0.32×age + 2.06 R2 = .85
0.4 0.2
AL/ML8 FDAH = 0.029×age2 – 0.46×age + 2.47 R2 = .84
0.0 0
2
4
Group A; starved
6 8 10 Parlarval age (DAH) Group B; 5 FDAH
12
14
Group B; 8 FDAH
F I G U R E 4 Robsonella fontaniana. Variation of morphometry in the arm length/mantle length ratio (AL/ML) depending on the age of the paralarva from hatching (DAH), in paralarvae starved (group A), paralarvae fed 5 days from hatching (group B) and then subjected to feed deprivation (5FDAH), and paralarvae fed 8 days from hatching (group B) and then subjected to food deprivation (8FDAH). DAH is the age in days after hatching show differences among starvation days (F = 1.75; df=3,7; p = .24). However, a trend of increasing their DG along with time resulted in a significant regression slope of 0.65 (Figure 5c). Regarding the essential amino acids (EAA) in relative amounts (lg mg
1
0.5 0.4 DGAstarved = 0.31×DAH–0.42 R2 = .15; p = .08
0.3 0.2 0.1 0 0 1
2
3
4
FDAH; Table 1). When TAA were analysed per paralarvae (lg paralarvae
1
), most
of the AA were changed by time and feeding, with the exception of the EAAs arginine, methionine, leucine and phenylalanine (Table 1). However, the variation in the proportion of EAA and NEAA was affected during the first week for starved and fed paralarvae. A decrease in most AAs was observed due to starvation, with the exception of methionine, which declined in the fed paralarva (Table 2). The net change proportion of EAAE and NEAA from organisms starved during 2 weeks in comparison with those that
9 10 11 12 13 14 15 16
0.3 0.2 0.1 0 0 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
Paralarval age (DAH)
Group B; 8 FDAH
(c) Digestive gland area (µm2)
centration in those paralarvae fed for 5 or 8 days (column 5-8
8
DGA5 FDAH = 0.25×DAH–0.08 R2 = .008; ns
0.4
arginine (~19% of EAA). Non-essential amino acids (NEAA) such as
followed by starvation, except taurine that resulted in higher con-
7
0.5
EAA) and histidine (~4.6% of EAA), whereas the most abundant was
tion among the unfed paralarvae and paralarvae fed until 5 or 8 days
6
Group B; 5 FDAH
(b)
dw), they constituted the 45% from the total amino acids
relative amount of EAA and NEAA were maintained without varia-
5
Paralarval age (DAH)
(TAA). The least abundant among the EAA was methionine (0) or reduced (