Effects of dietary protein sources on growth, survival and digestive capacity of Octopus maya...

Effects of dietary protein sources on growth, survival

and digestive capacity of Octopus maya juveniles

(Mollusca: Cephalopoda)

Carlos Rosas1, Ana Valero2, Claudia Caamal-Monsreal1,5, Iker Uriarte3,4, Ana Farias3,4,

Pedro Gallardo1, Ariadna Sanchez1 & Pedro Domingues6

1Unidad Multidisciplinaria de Docencia e Investigacion (UMDI), Facultad de Ciencias, Universidad Nacional Autonoma

de Mexico (UNAM), Yucatan, Mexico2Licenciatura en Biologıa, Facultad de Ciencias, Universidad Nacional Autonoma de Mexico (UNAM), Yucatan, Mexico3Instituto de Acuicultura, Universidad Austral de Chile, Puerto Montt, Chile4CIEN Austral, Puerto Montt, Chile5Posgrado en Ciencias del Mar y Limnologıa “PCMyL”, Universidad Nacional Autonoma de Mexico “UNAM”, Yucatan,

Mexico6Instituto Espanol de Oceanografıa “IEO”, Centro Oceanografico de Vigo, Vigo, Espana

Correspondence: C Rosas, Unidad Multidisciplinaria de Docencia e Investigacion (UMDI), Facultad de Ciencias, Universidad Nac-

ional Autonoma de Mexico (UNAM), Puerto de abrigo S/N Sisal, Yucatan, Mexico. E-mail: [email protected]

Abstract

We propose two hypotheses to explain the inexis-

tence of adequate prepared diet for Octopus maya

at this date: Hypothesis 1 is related to changes in

protein structure during protein cooking, which

affects the digestibility. Hypothesis 2 is related to

changes on nutritional characteristics during

ingredient process, which affects the nutritional

composition of diet. To test hypothesis 1, experi-

ments one and two were directed to determine if

protein cooking reduces digestibility and growth of

animals when compared to fresh or lyophilized

protein sources. For hypothesis 2, three experi-

ments were conducted, testing seven different die-

tary protein sources offered in isolation or

combined in artificial diets fed to O. maya. Results

demonstrated that the diets that promoted growth

were the ones based on fresh crab paste, and both

lyophilized crab and squid tentacles paste. In con-

sequence hypothesis 1 was accepted. The cooking

process also changed nutritional characteristics of

protein sources, affecting the growth of O. maya.

Results obtained when squid and crab were mixed

suggest that nutritional requirements of octopuses

were covered with that diet in similar forms

compared to when using fresh or lyophilized crab,

also confirming hypothesis 2. Based on growth

rates obtained, we can conclude that nutritional

requirements of O. maya must be between 80%

and 86% Protein (P), 5.1–5.6% Lipids and a

protein: energy ratio between (P/E) 38.9 and

42.2 g MJ�1.

Keywords: cephalopods, digestive gland, enzy-

mes, growth, metabolism, Octopus maya, dietary

protein sources

Introduction

Octopus maya is a carnivorous species and protein

is the main energy source (Van Heukelem 1976,

1977; Segawa & Hanlon 1988; Houlihan, McMil-

lan, Agnisola, Genoino & Foti 1990; Lee 1994;

Petza, Katsanevakis & Verriopoulos 2006; Rosas,

Cuzon, Pascual, Gaxiola, Lopez, Maldonado &

Domingues 2007). Protein is the main metabolic

substrate for this species, characterized by a low O/

N ratio (Segawa & Hanlon 1988; Rosas et al. 2007).

Octopus maya easily adapts to captivity, accepts

artificial diets immediately after hatching (Domin-

gues, Lopez, Munoz, Maldonado, Gaxiola & Rosas

2007), has a high market price (Cabrera & Defeo

2001; Salas, Mexicano-Cintora & Cabrera 2006),

fast growth and high feed efficiency (Rosas et al.

2007). The direct egg development and absence of

© 2012 Blackwell Publishing Ltd 1

Aquaculture Research, 2012, 1–16 doi:10.1111/j.1365-2109.2012.03107.x

a paralarval phase in O. maya (Van Heukelem

1976, 1977, 1983; Moguel, Mascaro, Avila-Pove-

da, Caamal, Sanchez, Pascual & Rosas 2010)

delivers juveniles without the high mortality

observed in other species with paralarva phases,

such as Octopus vulgaris or Loligo opalescens (Young

& Harman 1989; Iglesias, Sanchez, Bersano, Carr-

asco, Dhont, Fuentes, Linares, Munoz, Okumura,

Roo, van der Meeren, Vidal & Villanueva 2007;

Uriarte, Hernandez, Dorner, Paschke, Farıas, Crov-

etto & Rosas 2010).

Interest in octopus maintenance, rearing and

culture has risen in the past two decades (Robaina

1983; Hanlon, Forsythe, Hixon & Yang 1984;

Hanlon & Forsythe 1985; Nabhitabhata 1995;

Solis-Ramirez 1997; Vaz-Pires, Seixas & Barbosa

2004; Domingues, Garcia & Garrido 2010). Gar-

cıa-Garcıa, Rodrıguez-Gonzalez and Garcıa-Garcıa

(2004) indicating that the actual technology for

O. vulgaris fattening is a high-risk activity with

low profits, due to the dependence on juveniles

collected from the wild and prices of natural prey

used to feed them. Therefore, an adequate nutri-

tional programme is necessary to develop a profit-

able commercial diet. Research on artificial diets

for cephalopods has been increasing in importance

for the past few years (Castro 1991; Castro, DiM-

arco, DeRusha & Lee 1993; Castro & Lee 1994).

Domingues et al.(2007) and Rosas et al. (2007)

showed that a dry pellet diet made with fish meal

did not promote O. maya growth, but animals did

not lose weight and more importantly, ate regu-

larly all the food supplied, with feeding rates

higher than those reported in the literature for

prepared diets. During that experiment, higher

growth rates and assimilated energy were obtained

when feeding O. maya with frozen crabs, compared

with the dry pellet, which had high lipid content

(21%) (Domingues et al. 2007).

Those results indicated that O. maya could be a

good research animal for the development of artifi-

cial diets for cephalopods.

Quintana, Domingues and Garcıa (2008) tested

squid (Loligo gahi) and shrimp (Palaemonetes vari-

ans) pastes bound with cold gelatin using frozen

squid as a control diet for O. vulgaris. Results

obtained showed that gelatin produced similar

growth rates of octopus than those obtained of

animals fed freeze squid, suggesting that diets

bound gelatin without pre-heating could be a base

for other prepared diets. In a more recent study

Garcıa, Domingues, Navarro, Hachero, Garrido

and Rosas (2011) demonstrated that O. vulgaris

sub adults had higher growth rates when fed diets

also bound with gelatin without previous heating,

compared with alginate, and demonstrated that

digestibility was affected by alginate, with negative

energetic consequences for animals. Similarly,

(Aguila, Cuzon, Pascual, Domingues, Gaxiola, San-

chez, Maldonado & Rosas 2007; Rosas et al.

2007) also report that the use of alginate as a

binder for the artificial diets could reduce digest-

ibility, since O. maya does not have the capacity to

hydrolyze carbohydrates.

To date, different protein sources have been

used to feed octopuses. Results obtained indicate

that, among them, fresh squid paste and crab can

cover the nutritional requirements of O. vulgaris

and O. maya (Quintana et al. 2008; Garcıa et al.

2011; Rosas, Sanchez, Pascual, Aguila y Elvira,

Maldonado & Domingues 2011). Recent results

demonstrated that protein requirement for O. maya

could be of around 60%, which is the average pro-

tein content commonly found in squid meal (Rosas

et al. 2011). This suggests that if high quality pro-

tein sources are used, with gelatin as binder

(Rosas, Tut, Baeza, Sanchez, Sosa, Pascual, Arena,

Domingues & Cuzon 2008) a complex diet could

be elaborated for O. maya.

We have several hypotheses to possibly explain

the poor growth rates obtained when feeding pre-

pared diets. One could be because of changes in

protein structure during fish meal elaboration, or

due to denaturation by heating (Domingues, Mar-

quez, Lopez & Rosas 2009), preventing the enzyme

attack during the chymo formation, which could

affect the digestibility (Hypothesis 1). There are

evidences that demonstrate that meat cooking

affected myofibrillar protein susceptibility to prote-

ases, reducing the meat digestibility (Sante-Lhou-

tellier, Astruct, Marinova, Greve & Gatellier 2008;

Domingues et al. 2009; Gatellier & Sante-Lhoutel-

lier 2009). Another possible explanation is related

to ingredient condition during food elaboration.

During ingredient process (lyophilized, cooked, or

frozen) amino acids and some lipids could be lost

(through peroxidation), reducing the nutritional

quality of the meat used to make the food and

affecting the growth rate, not via the digestibility,

but via the changes of nutritional characteristics

of ingredient used (Hypothesis 2).

To test those hypotheses, five experiments were

designed. For hypothesis 1, two experiments were

conducted. The effects of type food (fish meal alone

© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–162

Effect of dietary protein sources for Octopus maya C Rosas et al. Aquaculture Research, 2012, 1–16

or in an elaborated diet) on digestibility of dry mat-

ter and protein of O. maya late juveniles were stud-

ied in experiment 1, to determine if industrial

cooked fish meal reduced digestibility of dietary

protein when compared with high quality native

protein (lyophilized and fresh squid tentacles). Late

juveniles were used due to the necessity of obtaining

marked faeces. In experiment 2, the effect of crab

meat cooking on growth and survival was evalu-

ated. Fresh crab meat was used as a reference diet.

To test hypothesis 2, three experiments were

conducted to determine if dietary protein sources

processing could affect the nutritional composition

of the prepared diets. In the first one (Experiment

3), three complex diets (based on fish and clam

meal and lyophilized squid meat) were prepared

with different fish protein concentrates (0%, 8%

and 15% CPSP) to determine if a complex diet

improved with fish protein concentrates (based on

Aguila et al. 2007) and lyophilized squid (based on

Quintana et al. 2008), could reduce the negative

effects that cooked protein sources have when

used in elaborated diets for O. maya. Experiment 4

was designed to test the effect of each protein

source used to elaborate the complex diet on

growth and survival of O. maya early juveniles. In

addition, muscle glycogen concentration and

digestive gland enzyme activities were evaluated to

know how proteins modulate part of the metabo-

lism and digestive capacity of octopus fed each

protein source. In these experiments, fresh crab

was used as a control diet. From the results

obtained in these two experiments, a fifth experi-

ment was carried out in an attempt to evaluate

the effect of three protein sources combinations on

growth and survival of O. maya juveniles and

define general (protein, lipids and protein energy

ratio) requirements for O. maya. Also, digestive

enzyme activities in the digestive gland were eval-

uated to determine how different protein sources

modulate the digestive capacity of this species.

Materials and methods

Experiment 1. The effects of protein type on dry

matter and protein digestibility

Animals

Late O. maya juveniles (550 ± 45 g) were caught

using artisan lines, with live blue crabs Callinectes

spp as bait, in front of Sisal harbour (Yucatan,

Mexico). Octopuses were transported to the labora-

tory situated 300 m inland, in a 120 L circular

tank with sea water. A total of 80 octopuses were

used during this experiment. Octopuses were

placed in individual flow-through 80-L tanks with

aerated sea water for 5 days before the start of the

experiment. A PVC tube (50 mm in diameter) was

placed in each tank as a refuge.

Experimental design

Animals were maintained in a semi-closed recircu-

lating system consisting of plastic tanks (80 L), a

rapid-rate sand filter and a skimmer filter. Salinity,

temperature and dissolved oxygen were maintained

at (mean ± standard deviation) 34 ± 1 ppt, 28 ±2°C and 5.8 ± 0.4 mg L�1 respectively. Each of the

test diets was fed to 20 replicate tanks of octopus

(one octopus per tank) over a 10-day conditioning

period followed by a 5-day collection period. Faeces

were removed from the tanks before the next feed-

ing. To ensure that previous faeces were cleared

from the digestive system, faeces from the first feed-

ing were discarded. Octopuses were allowed to feed

for 2 h after which unconsumed food was dis-

charged by siphoning from the tank. After 2-4h,

faeces were collected by siphoning and retained in a

fast paper filter. Faecal samples were immediately

rinsed with distilled water and stored at �20°C.Faecal samples collected from the same tank/treat-

ment were pooled, resulting in 20 samples per diet.

Celite (Sigma®, Sigma-Aldrich Quimica S. A.,

Toluca, Mexico) was used as a marker and appar-

ent digestible content (ADC) for dry matter (DM)

and protein (P) was measured (Cuzon & Aquacop

1998):

ADCDMð%Þ ¼ 100� ½100� ð%marker in diet=

%marker in faecesÞ

ADCPð%Þ ¼ 100� ½100� ð%marker in diet=

%marker in faecesÞ� ð%nutrient in faeces=

%nutrient in dietÞ�:

Acid-insoluble ash in feed and faeces was mea-

sured by burning samples at 500°C for 6 h. Ashes

were placed into boiling 4N HCl for 20 min and

filtered in a ash free filter (Whatman® 40; What-

man Inc., Piscataway, NJ, USA). Filters were

burned in a muffle furnace at 500°C overnight.

© 2012 Blackwell Publishing Ltd, Aquaculture Research, 1–16 3

Aquaculture Research, 2012, 1–16 Effect of dietary protein sources for Octopus maya C Rosas et al.

Diets

Digestibility of a semi-purified basal diet and some

other dietary protein sources was tested. The semi-

purified diet was prepared mixing all dry protein

sources with fresh water until a semi-moist diet

resulted (Table 1). This diet was bound by gelatin

dissolved in hot water (70°C). Salmon meal, lyoph-

ilized squid tentacles and fresh squid tentacles

were bound by gelatin and mixed with fresh water

until a semi humid paste was also obtained. Acid-

washed Celite was used as a marker for semi-puri-

fied diets and protein sources.

All diets were prepared on a weekly basis and

stored at 6°C. Octopuses were fed once a day

(09:00 hours) between 3% and 5% animal dw d�1

for all regimens. This ration is considered to be an

adequate feeding rate for this species and cephalo-

pods in general, at these rearing temperatures

(Aguila et al. 2007).

Experiment 2. Effect of crab meat cooking on

growth and survival of O. maya early juveniles

Animals

Wild Octopus maya females were caught on the

continental shelf of the Yucatan Peninsula (21° 9′

55″ N, 90° 1′ 50″ W) using artisan lines with blue

crabs Callinectes spp as bait. Females were trans-

ported in 120 L circular tanks with seawater from

the port to the UMDI-UNAM laboratory, situated

300 m inland. In the laboratory, females were

maintained in 250 L black tanks until egg laying

(Moguel et al. 2010).

Experimental design

O. maya post-hatchings (N = 100) were individu-

ally placed in a plastic tank (500 mL) connected

to re-circulatory seawater system following the

procedures of Moguel et al. (2010). Melongena cor-

ona bispinosa shells were offered as refuges. All

post-hatching octopuses were fed twice a day at

9:00 and 16:00 hours (at a ratio of 30% wet body

weight ‘wBW’) with semi-moist crab paste (native

protein 95%) mixed with natural pre-heated (70°C)gelatin as agglutinant (5%). Paste was placed on

empty clam shells (Quintana, Rosas & Moreno-

Villegas 2010).

Thirty days post-hatching (DPH) juvenile O.

maya from a single egg-laying female were

weighed at the start of the experiment (wet body

weight, wBW; mean value of 0.964 ± 0.08 SD g;

N = 40 octopuses), and separated into two experi-

mental groups; animals were fed either fresh crab

or cooked crab, both bound with pre-heated gela-

tin. Water quality was maintained through the

use of biological and mechanical filtration, and

passed through UV light prior to entering the rear-

ing system. Seawater was maintained at 26 ± 2°C,34 ± 2 salinity, 5 ± 1 mg L�1 dissolved oxygen,

total ammonia <1 mg L�1, pH 8 and at a light

Table 1 Composition of the experimental diets used in experiment one and three

Basal CPSP 8% CPSP 15% Crab§

% %CP % %CP % %CP % %CP

Chilean prime fish meal 35 25.2 30 22 30 21.6

CPSP70* 8 6 15 11

Clam meal (Lamellibranchia) 10 7 10 7 10 7

Lyophilized squid (Dosidiscus gigas) 41 30 38 28 31 23

Gelatine 7 6 7 7 5.6

Fish oil 3 3 3

Soy bean lecithin 1 1 1

Vitamins.mix† + stay-C‡ 2 2 2

Mineral mix† 1 1 1

Total percentage 100 100 100 100

Total protein 68 62 67 86

Energy, KJ g�1 18 17 19 20

g DP/MJ DE 38 36 35 42

*Fish protein concentrate, Sopropeche@, France.

†Vitamin and mineral mixes provided by DSM@ nutritional products.

‡Stay-C, 35%, DSM@ nutritional products.

§Fresh crab muscle meal (Callinectes sapidus).



intensity of 60 lux cm�2 (i.e., photoperiod of 12 h

light:12 h dark).

Diets

Crab meat (Callinectes sapidus) was obtained from

breast muscle of fresh animals. The muscle was

separated into two groups; one was maintained

fresh and the other was cooked in boiling water

for 10 min (100°C). Fresh crab muscle (native

crab protein) and cooked crab muscle were mixed

with pre-heated gelatin (5%) dissolved in hot

water to obtain both pastes. Pastes were prepared

once a week and preserved at 4°C. A ration of

30% of body weight was maintained during the

experiment.

Growth and survival rates

Specific growth rate was calculated as (SGR, %

day�1) = [(LnW2-LnW1)/t] 9 100, where W2 and

W1 are the final and initial wet weights of the

octopus, Ln is the natural logarithm, and t the

duration of each time period (days). Survival rate

(SR) was calculated as Survival, % = (N2/N1)

9 100, where N2 and N1 are the final and initial

number of octopuses remaining alive respectively.

Experiment 3. Effect of complex diets on growth

and survival of O. maya early juveniles

Animals

One day post-hatchings O. maya (N = 120; 0.102

± 0.001 g) were used in this experiment.

Experimental design

Octopuses were individually placed in a plastic

tank (500 mL) connected to re-circulatory seawa-

ter system and following the rearing procedures as

in experiment 2. Animals were fed during the first

15 days with semi-moist fresh crab protein at a

ratio of 30% bw d�1. After the conditioning per-

iod, octopuses (N = 80, 0.136 ± 0.011 g) were

weighed and four groups of 20 animals each were

randomly assigned to experimental diets: basal

diet, basal diet with 8% CPSP, basal diet with 15%

CPSP and fresh crab meat (control diet).

Diets

Protein sources were ground and passed through

a 250 lm mesh, and then agglutinated with 5%

pre-heated gelatine (Rosas et al. 2008) (Table 1).

Semi humid paste was prepared once a week, and

placed on empty clam shells (Quintana et al.

2010) and preserved at 4°C until use. Growth and

survival rates were calculated as in experiment 2.

Experiment 4. Effect of protein sources (protein

sources) used to elaborate the complex diet on

growth, survival and digestive gland metabolism

of O. maya early juveniles

Animals

Twenty days post-hatching (DPH) O. maya hatched

from a same egg-laying female were weighed at

the start of the experiment (wet body weight,

wBW; mean value of 0.630 ± 0.04 SD g; N = 140

octopuses) and separated into seven experimental

groups (N = 20 octopus each).

Diets

Animals were randomly assigned to experimental

diets made with different protein sources used in

complex diet (Experiment 3; Table 1): prime fish

meal; CPSP70, clam meal, lyophilized squid and

compared with lyophilized clam, fresh and lyophi-

lized crab meat. All protein sources were bound by

pre-heated gelatin (5%) dissolved in hot water. A

ration of 30% bw d�1 was used. Although lyophi-

lized clam was not used as a source of protein of

complex diet it was included to determine if clam

could cover the nutritional requirements for O.

maya juvenile growth. Semi humid pastes were

prepared once a week, placed on empty clam shells

and preserved at 4°C. (Quintana et al. 2010).

Growth and survival rates were calculated as in

experiment 2. At the end of the experiment, the

digestive gland and samples of the muscle of three

individuals fed each diet were removed and imme-

diately frozen at �80°C for the determination of

digestive enzymes (total proteases, acidic phospha-

tases, trypsin and chemotrypsin) and muscle gly-

cogen content.

Before the sampling all octopuses were anesthe-

tized with cool seawater at 5°C to assess physio-

logical and digestive enzymes (Roper & Sweeney

1983), according to ethics and welfare during the

manipulations (Moltschaniwskyj, Hall, Lipinski,

Marian, Nishiguchi, Sakai, Shulman, Sinn, Stau-

dinger, Van Gelderen, Villanueva & Warnke

2007).

Enzyme activity and glycogen analyses

All the octopuses were fasted for 12 h before sam-

pling. Digestive glands were dissected and stored

at �80°C until analysis. For acidic phosphatase



determination, frozen samples were homogenized

in 1% KCl and 1 mM EDTA solution at 4°C. Fortotal protease, trypsin and chymotrypsin determi-

nations, samples were homogenized using a Tris-

base buffer (0.09 M Tris-base, 0.08 M boric acid,

3 mM EDTA, 0.5 M mercaptoethanol, 10% glyc-

erol, pH 8.3) at 4°C. Homogenates were centri-

fuged at 10 000 g for 30 min at 4°C and the

supernatant was diluted in 10 volumes of ice-cold

pyrogen-free water. Homogenates were immedi-

ately used for enzyme analyses. The soluble-pro-

tein content was measured in diluted

homogenates (Bradford 1976) using the Bio-Rad

protein determination kit (BIO-RAD, Mexico City,

Mexico, 500–0006).

Total proteases were measured using Anson’s

(1938) method. Acid protease activity was assayed

using haemoglobin (1%) as substrate. The sub-

strate was dissolved using Stauffer (1989) univer-

sal buffer (1989) at pH 3 for acid. Briefly, in a

tube, 20 lL of the enzyme extract (dilution 1:10)

was mixed with 0.5 mL of the corresponding buf-

fer and 0.5 mL of freshly prepared substrate in the

corresponding buffer, and incubated for 10 min at

38°C. The reaction was stopped by adding 0.5 mL

of 20% TCA (w/v) and cooling for 15 min at 4°C.The precipitated undigested substrate was sepa-

rated by centrifugation for 15 min at 13 370 g.

The absorbance of the supernatants was measured

spectrophotometrically at 280 nm against the

substrate without enzyme extract. One unit of

enzymatic activity was defined as the change

in absorbance per minute per milligram protein

of the enzyme used in this assay (DAbsmin�1 mg protein�1). Assays were duplicated for

each sample. Trypsin activity was measured in

diluted homogenates (1:100) using benzoil-

arginine-para-nitro-anilide (1100 mM BAPNA) as

substrate in a buffer at pH 8 (0.1 M TRIS, 0.05 M

NaCl). Samples were incubated at 25°C for 1 h.

Absorbance was read at 410 nm. Chymotrypsin

activity was measured in diluted homogenates

(1:100) using succinil-Ala-Ala-Pro-Phe-p-nitroani-

lide (1142 mM SAPNA) as a substrate in a buffer

at pH 8 (0.1 M TRIS-base with 0.01 M CaCl2).

Samples were incubated at 37°C for 30 min and

read at 410 nm. Acidic phosphatases were mea-

sured using p-nitrophenil-phosphate (2%) in a buf-

fer at pH 4 (1 M TRIS-HCl). The sample was

incubated at 25°C for 30 min, and the reaction

was stopped adding 1 mL of 1 M NaOH. Absor-

bance was read at 405 nm. All enzyme activities

were expressed as international units (IU) per milli-

gram of protein.

Glycogen from the digestive gland was extracted

in the presence of sulphuric acid and phenol

(Dubois, Lilles, Hamilton, Rebers & Smith 1965).

Tissues were first homogenized in ice-cold trichlo-

roacetic acid (TCA, 5%) at 3340 g. After centrifu-

gation (7000 g) the supernatant was quantified;

this procedure was done twice. A quantity of 1 mL

of supernatant was pipetted into a tube and mixed

with five volumes of 95% ethanol. Tubes were

placed in an oven at 37–40°C for 3 h. After pre-

cipitation, the tubes were centrifuged at 7000 g

for 15 min. The glycogen pellet was dissolved by

adding 0.5 mL of boiling water. Then, 5 mL of

concentrated sulphuric acid and phenol (5%) were

added and mixed. The content in the tubes was

transferred to micro plates and read at 490 nm

using a micro plate reader (BIO-RAD, Mexico

550).

Experiment 5. Effect of three protein sources

combinations on growth, survival and biochemical

characteristics of digestive gland of O. maya

juveniles

Animals

Twenty-five days post-hatching (DPH) Octopus

maya hatched on the same day from a spawning

female were weighed at the start of the experiment

(wet body weight, wBW; mean value of 0.75 ±0.05 SD g; N = 100 octopuses), and separated

into five experimental groups (N = 20 octopus

each).

Diets

The combined effect of the diets that had produced

the best results in experiment four (CPSP® 70%

and lyophilized squid tentacles) were determined

and combined with lyophilized crab meat. Diets

used were: A-CPSP® 70% + lyophilized squid ten-

tacles; B-lyophilized squid tentacles + lyophilized

crab; C-CPSP® 70% + lyophilized crab; D-lyophi-

lized crab and E-fresh crab. Diet preparation was

similar as for the previous experiments; also agglu-

tinated with 5% gelatine dissolved in hot water. A

ration of 30% bw d�1 was used. Fresh crab meat

bound gelatine (E) was used as a control diet. Semi

humid pastes were prepared once a week, placed

on empty clam shells and preserved at 4°C. (Quin-tana et al. 2010). Growth and survival was mea-

sured as in experiment two.



Proximal analysis of diets

Protein content

Protein content was determined using the method

of Lowry (Lowry, Rosebrough, Farr & Randall

1951) modified by Bensadoun and Weinstein

(1976) and Hess, Lees and Derr (1978). Protein

content was quantified as gram per 100 g in dry

weight (g 100 g�1 DW).

Ash content

For the determination of ash content, freeze-dried

samples of 100–200 mg of each diet were placed in

porcelain crucibles previously weighed. The cruci-

bles with samples were placed in an oven at 550°Cfor 12 h; then, allowed to cool in a desiccator and

again weighed. The ash content was quantified as

gram per 100 g in dry weight (g 100 g�1 DW).

Moisture content

Moisture content was determined using the

method of Horwitz (1980) and quantified as gram

per 100 g in dry weight (g 100 g�1).

Energy content of the diet

Food energy contents were determined with a

calorimetric pump (Parr Instrument Company,

Moline, IL, USA) calibrated with benzoic acid and

expressed as kj kg�1 diet.

Total lipids

Total lipid was extracted with chloroform: metha-

nol (2:1 v/v) containing 0.01% of butylated

hydroxytoluene (BHT) (v/v) as antioxidant (Chris-

tie 1982). The organic solvent was evaporated

under a stream of nitrogen and the lipid content

determined gravimetrically. Total lipid content

from the diets was calculated as gram per 100 g

in dry weight (g 100 g�1 DW).

Biochemical evaluations

After the end of the experiment, the digestive

gland of three individuals fed each diet were

removed and immediately frozen at �80°C for the

determination of digestive enzymes (total prote-

ases, total phosphatases, trypsin and chemotryp-

sin). Enzyme evaluations were done as in

experiment four.

Statistical analysis

One way ANOVA was applied to results of growth

rate, survival rate, digestive gland glycogen and

enzyme activity. Arc sine transformation was used

before processing percentage data (Zar 1999).

Results

Experiment 1

Apparent digestibility of dry matter (ADCdm) for

O. maya fluctuated between 29% and 95%, with

lower values in octopuses fed salmon meal and

higher in animals fed fresh and lyophilized squid

tentacles. Intermediate values were recorded in ani-

mals fed the basal diet (53%) (Fig. 1a; P < 0.05).

Similarly, apparent digestibility for crude protein

(ADCP) was lower in animals fed salmon meal and

the basal diet (48% and 61% respectively) and

higher in octopuses fed fresh squid tentacles

(97.3%). Intermediate values were recorded in ani-

mals fed lyophilized squid tentacles (87%) (Fig. 1b;

P < 0.05).

Experiment 2

Growth rates and survival of octopuses were

affected by crab meat processing; animals fed fresh

crab showed a positive and higher growth rate

than those fed cooked crab (Table 2; P < 0.05). A

lower survival (15%) was recorded for animals fed

cooked crab, while for those fed fresh crab, sur-

vival was of 75% (P < 0.05; Table 2).

Experiment 3

Growth rates of octopuses fed the four diets are

shown in Table 3. The only diet that promoted

positive growth was the one based on fresh crab

meat. The other diets promoted negative growth,

and were not different between them (P > 0.05).

Survival varied between 90% and 95%, and was

similar for octopuses fed the four diets (P < 0.05;

Table 3).

Experiment 4

Only diets based on lyophilized squid, fresh crab

meat and lyophilized crab meat promoted growth,

with values between 0.7 and 1.7% d�1 (P < 0.05;

Table 4). The remaining diets promoted negative



growth, being worse than those based on clam

meat (P < 0.05; Table 4).

Higher survival (80%) was obtained with the

CPSP, while the lowest was from octopuses fed the

fish meal diet, (25%) and lyophilized clam (30%).

A survival between 70% and 80% was obtained

for octopuses fed lyophilized and fresh crab meal

(Table 4).

Enzymatic activity and glycogen

Muscle glycogen concentration was affected by the

type of diet, with high values in animals fed lyoph-

ilized clam and low those fed fish meal, CPSP and

fresh crab meat (P < 0.05). Intermediate values

were obtained in octopuses fed clam meal and

freeze-dried squid (Fig. 2).

Total acid protease activity was higher for octo-

puses fed fish meal, followed by lyophilized clam

(P < 0.05; Fig. 3a). The remaining diets showed

low acidic protease activity (P > 0.05) (Fig. 3a).

Trypsin activity varied between 10.55 and

30.81 UI mg�1 with high activities for animals fed

fish meal, lyophilized squid and lyophilized crab

(P < 0.05). Low trypsin activity was obtained

when feeding animals the diet based on clam meal

(P < 0.05) (Fig. 3b). A higher chymotrypsin activ-

ity was recorded in octopuses fed lyophilized crab,

compared to that fed on the remaining diets

(P < 0.05; Fig. 3c). There were no differences in

chymotrypsin activities of animals fed the remain-

ing diets (P > 0.05). A high acid phosphatase

activity was recorded on animals fed clam meal

Table 2 Experiment 2. Effect of the crab process (fresh

and cooked) on growth and survival of Octopus maya

juveniles (30 days post-hatching)

Diet

Fresh crab

(native protein) Cooked crab

Initial weight, g 0.99 ± 0.05a 0.94 ± 0.11a

N 20 20

Final weight, g 1.71 ± 0.12a 0.18 ± 0.01b

SGR, % day1 3.91 ± 0.28a �11.70 ± 0.38b

Time, days 14 14

Survival,% 75 15

Values as mean ± SE. Different letters mean statistical differ-

ences between treatments.

29.4

53.09

84.5

94.9

0

10

20

30

40

50

60

70

80

90

100

Salmon meal Basal diet Liofilized squidtentacles

Fresh squidtentacles

AD

CD

M. %

AD

CP. %

Type of ingredient

a

b

c

d

47.9

61.5

88.697.3

0

10

20

30

40

50

60

70

80

90

100

Salmon meal Basal diet Liofilized squidtentacles

Fresh squidtentacles

Type of ingredient

a

a

bc

Figure 1 Octopus maya in vivo apparent digestible content of dry matter (ADCDM%) and protein (ADCP%) of different

protein sources (protein sources) and elaborate diet for experiment 1. Values as Mean ± SE. Different letters mean

differences between treatments (P < 0.05). Wild late juveniles used in this experiment (550 ± 45 g). N = 10 ani-

mals per diet.



and low in animals fed fresh crab meat, with inter-

mediate activities for animals fed the remaining

diets (P < 0.05; Fig. 3d).

Experiment 5

Diet proximal composition showed that lyophilized

squid, crab and CPSP mixed diets had between

71% and 86% crude protein (CP) and between

5.1% and 13.2% crude lipid (CL). Low protein and

high total lipids levels were recorded for CPSP +Lyophilized squid while high protein and low lipid

levels for lyophilized crab bound gelatine (Table 5).

All the diets used in this experiment were practi-

cally iso-energetic with a mean value of 20.6 ±0.8 kj kg�1 (Table 5). Differences were observed

on P/E ratio, with low values for CPSP + lyophi-

lized squid (34.5 g MJ�1) and high values in

lyophilized crab paste (42.2 g MJ�1). Intermediate

values were observed on mixes made with lyophi-

lized crab and squid (38.9 g MJ�1) and CPSP +lyophilized crab (37.8 g MJ�1) (Table 5).

Positive and maximum growth rate were

obtained in animals fed lyophilized squid and crab,

and lyophilized crab and fresh crab meat with val-

ues between 1.12 and 1.77% day�1 (Table 6).

Survival varied between 55% and 65%, being

higher in octopuses fed CPSP and lyophilized crab

(65%) and lower in animals fed lyophilized and

fresh crab (50%). Lowest survival was registered

on animals fed CPSP and lyophilized squid (48%)

(Table 6)

Enzymatic activity and glycogen

Total acid protease activity was high in all diets,

with values between 86 000 and 92 000 UI mg�1

protein (Fig. 4a). The higher protease activity was

Table 3 Experiment 3. Effect of the elaborated diets on growth and survival of Octopus maya juveniles (15 days post-

hatching)

Diet BASAL BASAL + 8% CPSP BASAL + 15CPSP Crab meat

Initial weight, g 0.13 ± 0.004a 0.14 ± 0.004a 0.14 ± 0.143a 0.13 ± 0.004a

Final weight, g 0.10 ± 0.010a 0.12 ± 0.009a 0.12 ± 0.01a 0.25 ± 0.016b

SGR, % �2.49 ± 0.920a �2.17 ± 0.72a �1.81 ± 0.63a 4.46 ± 0.67b

Time, days 12 12 12 12

Survval, % 95 90 95 90

Crab meat as a control diet. Values as mean ± SE. Different letters mean statistical differences between treatments.

Table 4 Experiment 4. Effect of different protein sources on growth and survival of Octopus maya juveniles (20 days

post-hatching)

Diet Fish meal CPSP 70 Clam meal

Lyophilized

clam

Lyophilized

squid

Fresh crab

meat

Lyophilized

crab meat

Initial weight, g 0.63 ± 0.039a 0.69 ± 0.041a 0.60 ± 0.043a 0.65 ± 0.043a 0.57 ± 0.04a 0.61 ± 0.041a 0.68 ± 0.043a

Final weight, g 0.54 ± 0.050b 0.56 ± 0.037b 0.35 ± 0.029a 0.45 ± 0.068a 0.64 ± 0.087b 0.73 ± 0.133c 0.79 ± 0.124c

SGR, % �1.88 ± 0.910b �2.16 ± 0.31b �5.2 ± 0.74a �3.63 ± 1.25a 0.70 ± 1.04c 1.43 ± 1.25c 1.71 ± 0.643c

Time, days 10 10 10 10 10 10 10

Survival, % 25 80 65 30 50 75 65

Crab meat as a control diet. Values as mean ± SE. Different letters mean statistical differences between treatments

0

0.5

1

1.5

2

2.5

3

Gly

coge

n. m

g/g

Fish meal CPSP Clam meal L clam L squid Fresh crabmeat

Type of food

a a

a

b b

c

Figure 2 Effect of type of food on digestive gland

glycogen of O. maya juveniles (20 days post-hatching;

N = 20 per diet) during experiment four. Values as

mean ± SE. Different letters mean differences between

treatments (P < 0.05). L = Lyophilized meal.



recorded in animals fed lyophilized squid and

lyophilized crab, compared with the remaining

diets (P < 0.05) (Fig. 4b). The lower protease

activity was recorded in octopuses fed CPSP and

lyophilized crab (P < 0.05; Fig. 4a). Chymotrypsin

activity was between 180 and 1100 UI mg�1 pro-

tein, with high values in animals fed CPSP and

lyophilized squid. Low activity was recorded in

animals fed lyophilized squid and lyophilized crab

(P < 0.05; Fig. 4b). Acidic phosphatase activity

was between 0.7 and 3 UI mg�1 protein, with

higher values in animals fed CPSP and lyophilized

squid, compared with the remaining diets

(P < 0.05; Fig. 4c). No differences in glycogen

concentration (P < 0.05) were found on animals

fed all mixed diets; a mean value of 4.29 ±0.23 mg glycogen g�1 was calculated.

Discussion

Results obtained herein indicate that hypothesis 1

could be accepted: octopus digestibility (both early

0

100 000

200 000

300 000

400 000

500 000

Fishmeal

CPSP Clammeal

L clam L squid L crab

UL/

mg

prot

eín

Type of diet

Acidic total proteases

aa

a

b

a

c(a)

(c) (d)

05

10152025303540

Fishmeal

CPSP Clammeal


UL/

mg

prot

ein

Type of diet

Trypsinc

ab

a

c

b

c

(b)

0

10

20

30

40

50

60

Fishmeal

CPSP Clammeal


UL/

mg

pro

tein

Type of diet

Chymotrypsin

a a

a a a

b

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Fish meal CPSP Clammeal

L squid Freshcrab meat

UL/

mg

prot

ein

Type of diet

Acidic phosphatases

b bb

a

c

Figure 3 Effect of type of food on digestive gland enzymes of O. maya juveniles (20 days post-hatching; N = 20 per

diet) during experiment four. Values as mean ± SE. Different letters mean differences between treatments

(P < 0.05). L = Lyophilized meal.

Table 5 Experiment 5. Proximal analysis (g 100 g�1) of protein sources used (singles or mixed) to fed O. maya

juveniles

CPSP70 + L squid L Crab + L squid CPSP70 + L crab

L crab Callinectes

sapidus

L squid Dosidiscus

gigas

Protein 71 ± 3 80 ± 3 78 ± 3 86 ± 3 73 ± 3

Total lipids 13.2 ± 0.264 5.6 ± 0.11 12.6 ± 0.3 5.15 ± 0.10 6.4 ± 0.13

Total CHO* 9.2 6.8 2.1 0.25 14.4

Ash 6.3 ± 0.4 7.6 ± 0.5 7.3 ± 0.5 8.6 ± 0.6 6.6 ± 0.5

Energy, Kj g�1 20.7 ± 0.8 20.6 ± 0.8 20.7 ± 0.8 20.4 ± 0.8 20.5 ± 0.8

P/E ratio, g MJ�1 34.5 38.9 37.8 42.2 35.4

Values as mean ± SE.

*Calculated by difference.



and late juveniles) is affected by the cooking pro-

cess, presumably by reducing the enzyme attack

during chyme formation, limiting nutrients

absorption and in consequence the growth rate.

Domingues et al. (2009) reported similar results

when feeding cooked or raw shrimp to S. officinalis.

Present results also confirm that lyophilized crab

with native protein alone and combined with

lyophilized squid covered the nutritional require-

ments of O. maya, producing acceptable growth

rates. This indicates that lyophilization does not

alter the nutritional characteristics of those protein

sources, suggesting this as a suitable method to

process protein sources for O. maya diets. The sec-

ond hypothesis can also be accepted. Nevertheless,

other mechanisms related to anti-nutrient aspects

(such as digestive enzymes inhibitors; Hemre, San-

den, Bakke-Mckellep, Sagstad & Krogdahl 2005)

or thermo-labile bioactive compounds related with

octopus growth could have also been implicated in

the cooking process (Drew, Borgeson & Thiessen

2007).

Recent studies demonstrated that meat cooking

affected myofibrillar protein susceptibility to

proteases, with increasing or decreasing rates,

depending on the nature of the protease and the

Table 6 Experiment 5. Effect of mixes of different protein sources on growth and survival of Octopus maya juveniles

(25 days post-hatching). Fresh and lyophilized crab meat as control diets

Diet CPSP70 + L squid L squid + L crab CPSP70 + L crab L crab meat Fresh crab meat

Initial weight, g 0.75 ± 0.050a 0.76 ± 0.06a 0.73 ± 0.05a 0.77 ± 0.05a 0.73 ± 0.05a

Final weight, g 0.79 ± 0.070b 0.95 ± 0.14b 0.68 ± 0.06a 1.05 ± 0.09c 1.04 ± 0.08c

SGR, % 0.26 ± 0.540b 1.12 ± 0.58b �0.35 ± 0.04a 1.55 ± 0.58c 1.77 ± 0.35c

Time, days 20 20 20 20 20

Survival, % 55 60 65 50 50

Values as mean ± SE. Different letters mean statistical differences between treatments.

L = lyophilized.

80 000

82 000

84 000

86 000

88 000

90 000

92 000

94 000

CPSP70+L squid

L squid +crab

L cPSP70 +L crab

L crabmeat

Fresh crabmeat

Aci

dic

tota

l pro

teas

es.U

L/m

gpro

tein

Type of diet

a

b b

cb

0

200

400

600

800

1000

1200

CPSP70 +L squid

Lsquid + Lcrab

CPSP70 +L crab

L crabmeat

Fresh crabmeat

Chy

mot

ryps

in.U

L/m

gpro

tein

Type of diet

ab b

c

d

0

0.5

1

1.5

2

2.5

3

3.5

4

CPSP70 +L squid

L squid + Lcrab

CPSP70 +L crab

L crabmeat

Aci

dic

phos

fata

ses

.UL/

mg

prot

ein

Type of diet

aa a

b

c

Fresh crabmeat

(a)

(c)

(b)

Figure 4 Effect of type of food on digestive gland enzymes of O. maya juveniles (20 days post-hatching; N = 20 per

diet) during experiment five. Values as mean ± SE. Different letters mean differences between treatments (P < 0.05).

L = Lyophilized meal.



time per temperature parameters involved during

meat cooking (Sante-Lhoutellier et al. 2008; Do-

mingues et al. 2009). According to Sante-Lhoutel-

lier et al. (2008), the effects of meat cooking were

measured on myofibrillar proteins from bovine

M. Rectus abdominis. Results showed a direct and

quantitative relationship between protein carbony-

lation and aggregation induced by cooking and

proteolytic susceptibility to pepsin, affecting at the

end the meat digestibility. In that study, a nega-

tive and highly significant correlation between

pepsin activity and carbonyl group formation

affecting recognition of proteins by proteases was

also observed. Although cephalopods do not have

pepsin, recent results revealed the presence of

cathepsin L and D respectively, (all of them as

pepsin, endopeptidases) in the digestive gland of

giant squid Dosidicus gigas (Cardenas-Lopez &

Haard 2009) and in the digestive gland and gas-

tric juice of O. maya (Martinez, Santos, Alvarez,

Cuzon, Arena, Mascaro, Pascual & Rosas 2011a,b).

If meat cooking leads to aggregation and decreases

pepsin digestion capacity by reduction in cleavage

sites, it is possible to suppose that cathepsin diges-

tion capacity can also be affected in a similar form

by fish meal cooking. Therefore, after fish meal

elaboration (usually at 300°C), myofibrillar pro-

teins from fish could also be aggregated, affecting

the protein digestibility of food via reduction on

cleavage sites. A negative effect of food thermal

treatments on growth, absorption and assimilation

of Sepia officinalis was also reported by Domingues

et al. (2009). In the experiment, food lyophiliza-

tion produced good results demonstrating that

lyophilized crustacean meal could be used as a

source of protein for cephalopods other than O.

maya (Domingues et al. 2009).

Nevertheless, a question could arise: Could

digestibility, and in consequence, the growth rate

of octopuses fed an elaborated, diet be improved if

native proteins from lyophilized protein sources

are used? Results from experiment 3 suggest that

the negative effect of industrial fish meal (cooked

protein) cannot be reverted using at least CPSP

(8% and 15% of the diet), lyophilized squid and

clam, suggesting that the enzymes in the gastric

juice could be highly sensible to interference that

exert cooked protein on the digestion of the

remaining protein sources.

When different protein sources were tested in

experiment 3, all cooked protein sources provoked

negative growth rates. Surprisingly, also lyophi-

lized clam provoked a negative growth, suggesting

that not all lyophilized protein sources per se cover

the nutritional requirements of O. maya. Therefore,

there are other nutritional characteristics of the

protein sources (i.e. lipid composition, amino acids,

vitamins, etc.) that should be considered at the

time to formulate diets for cephalopods using

lyophilized or fresh protein sources. Although

there are little studies related with the fatty acids

requirement on cephalopods, the low proportion of

polyunsaturated fatty acids on raw clam (0.91 g/

100 g; Lamellibrachia; USDA Nutrient Data

Laboratory; http://ndb.nal.usda.gov/ndb/foods/list)

in comparison with 17.34 g/100 g reported for

squid when fed O. vulgaris (Loligo gahi; Domingues

et al. 2010; Garcıa, Hachero-Cruzado, Garrido, Ro-

sas & Domingues 2010) could explain why O. maya

had negative growth rate when fed clam in experi-

ment three. Nevertheless, clam composition in other

components, such as HUFA, mainly 20:5n-3

(0.043%), 22:6n�3 (0.064%) and 20:4n�6

(0.011%) (Lamellibrachia; USDA Nutrient Data

Laboratory; http://www.usda.gov/wps/portal/usda/

usdahome) could also contribute to the poor quality

of clam as food for O. maya.

Results on experiment 4 showed that although

growth obtained from feeding CPSP® 70 and gela-

tine were the worst during this experiment; sur-

vival was always higher than that when feeding

other protein sources. Recent studies demonstrated

that CPSP® is a good attractant for O. maya, possi-

bly due to the great concentrations of di-peptides

and free amino acids (aa), which should also

improve digestion (Aguila et al. 2007). The attrac-

tant effect of free aa was also demonstrated for

O. vulgaris paralarvae (Villanueva, Riba, Ruız-Capi-

llas, Gonzalez & Baeta 2004).

During experiment 4, lyophilized squid promoted

satisfactory results; together with lyophilized and

fresh crab, it was the best diet for O. maya with

relatively high growth rates. Lyophilization

removes water at low temperatures, contrary to

drying at high temperatures, and is apparently a

good method to obtain high quality protein

sources to elaborate acceptable diets for cephalo-

pods. Although the high costs associated to lyoph-

ilization make this a non-profitable method for

commercial production of cephalopod feeds, it is

still a good base for the formulation of diets at

experimental levels to understand cephalopod

digestive physiology, requirements and to develop

artificial feeds.



Results obtained in experiment 5 showed that

diets containing CPSP, although well consumed,

did not cover nutritional requirements of O. maya

in the proportion used in the mixed diets (50–

50%), nor those with mixed crab and squid.

Although CPSP could be used as an attractant,

results in experiment 2 and 5 show that its use on

cephalopod diets should be re-considered.

When squid and crab were mixed, results sug-

gest that nutritional requirements of octopuses

were covered, with similar results when compared

to when using fresh or lyophilized crab. From the

proximal composition of the experimental diets in

experiment 5, and growth rates obtained, we can

conclude that nutritional requirements must be

between 80% and 86% CP, 5.1–5.6% CL and a

protein: energy ratio between (P/E) 38.9 and

42.2 g MJ�1. To date there is not much available

information related to nutritional requirements for

cephalopods, although some information on P/E

ratio and lipid levels has been proposed regarding

dietary formulations for O. vulgaris. Aguado-Gime-

nez and Garcıa-Garcıa (2003) attributed the

increased growth of crab fed O. vulgaris compared

to that of bogue-fed animals to the higher P/E

ratio estimated for crabs (34.03 g MJ�1), com-

pared to bogue (27.70 g MJ�1), since the protein

content of both diets was similar. In addition, Garcıa-

Garcıa and Aguado-Gimenez (2002) reported

higher growth with bogue than sardine (15.02 P/

E g MJ�1). O’Dor, Mangold, Boucher-Rodoni, Wells

and Wells (1984) proposed that the P/E optimum

for O. vulgaris could be around 35 g MJ�1, as its

natural diet is composed mainly of crabs. Also

a high P/E ratio for squid Loligo vulgaris

(34.68 g MJ�1) used to feed O. vulgaris (Miliou,

Fintikaki, Tzitzinakis, Kountouris & Verriopoulos

2006) could explain the high growth rate and feed

efficiency of squid-fed octopus found for O. maya.

It is interesting to note that in the present study,

the P/E ratio was not similar for crabs

(42.2 g MJ�1) and squids (35.4 g MJ�1) suggest-

ing that the relatively lower growth rate observed

on squid-fed O. maya on experiment 4 could be

related with protein difference, since energy was

similar between both protein sources. Also, when

squid was mixed with crab, protein level and P/E

were improved and in consequence a higher

growth rate was observed.

Another aspect of the nutritional requirement of

O. maya is that related with lipid levels on diet.

Results obtained in the fifth experiment showed

that all CPSP mixes provoked low or negative

growth rates suggesting that, besides the protein

and energy levels, the relatively high total lipids

proportion of the diet (>10%CL) could be interfer-

ing into the growth of animals. In this case

CPSP70 (70% protein and 20% lipids) was used.

The effect of total lipids on growth of O. vulgaris

was also observed by Garcıa-Garcıa and Aguado-

Gimenez (2002), showing that diets with relatively

low lipid levels (5.9% CL; Boops boops) produced

higher growth rate than that observed in animals

fed high lipid levels (19% CL; Sardina pilchardus).

Previous studies have demonstrated that enzyme

activities are good indicators of how a diet affects

growth and survival in several cephalopod species

(Perrin, Le Bihan & Koueta 2004; Le Bihan, Perrin

& Koueta 2006; Aguila et al. 2007; Moguel et al.

2010). Low trypsin and chymotrypsin activity has

been reported for O. vulgaris paralarvae, when fed

inadequate diets (Villanueva et al. 2004). O. maya

have the capacity of adjusting their metabolism,

altering enzyme secretion to more efficiently assim-

ilate the nutrients in each diet (Aguila et al. 2007;

Rosas et al. 2008), but that response could depend

on age of animals and type of diet combination.

When juveniles were fed the worst diets (fish meal,

clam meal, or CPSP and squid), acidic proteases,

trypsin and acidic phosphatases were inducted

showing that in an attempt to obtain more nutri-

ents, both acidic and alkaline proteases were more

active in the digestive gland. In contrast, chymo-

trypsin and acidic proteases were inducted when

animals were fed the best diets (fresh crab or squid

and crab freeze-dried meat), suggesting that in

such nutritional circumstances both enzymes

could have a key role in digestion of protein.

Recent results obtained on O. maya showed that

cathepsins (in particular cathepsin D) could be

involved as a key enzyme in the extra and intra-

cellular digestion (Martinez et al. (2011). In fact,

an acidic environment was registered in the diges-

tive tract during digestion process, suggesting that

a relationship between digestive gland enzymes

and digestion process could co-occur to maintain

an acidic environment during digestion (Martinez

et al. 2011). Some questions emerge of those

results: Could acidic enzymes and chymotrypsin be

part of extracellular enzymes that will be used dur-

ing feed digestion? Acidic phosphatases were

described as linked to lipid metabolism of S. offici-

nalis hatchlings (Perrin et al. 2004). Are these

enzymes inducted in O. maya as a response of high



dietary levels on CPSP diet? Although other stud-

ies will be necessary to determine how the diges-

tive enzymes (intracellular and extracellular) are

modulated in O. maya, similar results have been

observed in S. officinalis (Perrin et al. 2004) and O.

vulgaris paralarvae (Villanueva & Norman, 2008).

Moguel et al. (2010) reported higher chymo-

trypsin activity in O.maya early juveniles with neg-

ative growth rates, which indicates that

chymotrypsin activity is inducted when inefficient

diets are provided. Similar results were reported by

Domingues et al. (2009) for S. officinalis with

increase chemotrypsin activity when feeding

cooked shrimp, and increase trypsin activity when

feeding frozen raw shrimp. In another study on

the sub polar Patagonian octopus (Enteroctopus

megalocyathus), Farıas, Pereda, Uriarte, Dorner,

Cuzon and Rosas (2010) reported low digestive

gland chymotrypsine activity both in octopus fed

diets that promoted growth and diets that resulted

in negative growth rates. During that study, it

was also observed that the growth rate was only

related to ingestion rate and not to digestive gland

enzyme activity or dietary digestibility.

In the present study, enzymes were inducted or

not, depending on the type of diet used. When pro-

tein sources were tested alone, acidic total prote-

ases and trypsin were inducted by fish meal while

chymotrypsin was inducted by fresh crab meat.

When protein sources were mixed, total acidic pro-

teases were inhibited when animals were fed

mixed diet CPSP + lyophilized crab, while chymo-

trypsin and acidic phosphatases were inducted by

mixed diet CPSP + lyophilized squid. Although we

do not know what kind of mechanisms are operat-

ing, actual results put in evidence that O. maya

and probably other cephalopods have a wide

enzyme flexibility to digest food, inducting enzymes

to enhance digestion of sub optimal diets when

necessary.

In conclusion, from all the high quality protein

sources tested, fresh crab, both lyophilized squid

tentacles and crab promoted better growth,

although survival was enhanced with the inclu-

sion of CPSP® in the diets. These protein sources

should be considered as main protein sources in

future studies. Also, the lyophilization process

appears to be the most adequate in maintaining

the nutritional quality of the protein sources, and

should still be used in future studies on the devel-

opment of artificial feeds for this and other cepha-

lopod species.

Acknowledgments

The present study was partially financed by DGAP-

A-UNAM project IN216006-3, CONACYT – Basico

24743 and Fundacion Produce Yucatan to Rosas

C.

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