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Amino acid mobilization and growth of juvenile Octopus maya (Mollusca: Cephalopoda) under inanition and re-feeding
Aquaculture 314 (2011) 215–220
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Amino acid mobilization and growth of juvenile Octopus maya (Mollusca: Cephalopoda) under inanition and re-feeding Arturo George-Zamora a, Maria-Teresa Viana b, Sergio Rodríguez c, Gabriela Espinoza c, Carlos Rosas d,⁎ a
Posgrado en Ciencias del Mar y Limnología (PCMyL), Universidad Nacional Autónoma de México (UNAM), Puerto de Abrigo S/N, Sisal, Yucatán, Mexico Instituto de Investigaciones Oceanológicas (IIO), Universidad Autónoma de Baja California (UABC), Ensenada, Baja California, Mexico Facultad de Química, Unidad de Química-Sisal, Universidad Nacional Autónoma de México (UNAM), Puerto de Abrigo S/N, Sisal, Yucatán, Mexico d Unidad Multidisciplinaria de Docencia e Investigación (UMDI), Facultad de Ciencias, Universidad Nacional Autónoma de México (UNAM), Puerto de Abrigo S/N, Sisal, Yucatán, Mexico b c
a r t i c l e
i n f o
Article history: Received 22 September 2010 Received in revised form 15 February 2011 Accepted 15 February 2011 Available online 21 February 2011 Keywords: Amino acids Growth Cephalopoda Octopus maya Fasting México
a b s t r a c t Octopus maya is an endemic cephalopod from the Yucatán Peninsula in Mexico, with interest to develop their commercial culture. Like all cephalopods, protein is an important nutrient both for growing and energetic metabolism. This condition results in a high demand for protein and specifically for certain amino acids (AAs). In this study it is examined the effects of inanition and re-feeding on growth and AA content in soft tissue to detect the amino acid (AA) mobilization to identify the principal metabolic reserves in juvenile O. maya. After 25 days re-feeding of all starved groups, the octopuses were unable to reach the similar weight as the control group. However, SGR of some groups were greater than that of the controls, although the differences were not significant due to variability in the data. Therefore, it is assumed that the juveniles of O. maya would need a longer period of time to reach the control group. It is therefore demonstrated that juveniles of O. maya have a wide plasticity to tolerate, at least 10 days of food deprivation without any apparently physiological damage. Moreover, during inanition the juveniles of O. maya used preferentially Thr, Phe, Ile, Ala, Glu and Ser, suggesting a strong mobilization of both essential and non essential AA to maintain the homeostasis. Prove of that is that survival of the animals during fasting and re-feeding period was not affected by treatments. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The octopus Octopus maya (Voss and Solís-Ramírez, 1966) is an endemic cephalopod from the Yucatan Peninsula in Mexico (CONAPESCA, 2008) being one of the most important resources in the fishery sector. Earlier studies have demonstrated that O. maya has a high potential for aquaculture due to their well adaptability to captivity conditions (Van Heukelem, 1977; Boletzky and Hanlon, 1983; Hanlon and Forsythe 1985; DeRusha et al., 1989) and direct development from hatching without a larval transition (Boletzky, 1974). Moreover, O. maya shows larger eggs than those observed in other cephalopods (up to 17 mm). They are able to accept dead preys or formulated diets even during the first life cycle stadiums (Aguila et al., 2007), resulting in high growth rates (up to 8% wet body weight, wBW, per day) as a result of high feeding rates (FR) and food conversion efficiencies (FC) (Van Heukelem, 1983; Hanlon and Forsythe, 1985; Domingues et al., 2007; Rosas et al., 2007). Dependence on natural diets together with a long larval period has been the bottleneck in the cephalopod aquaculture (Domingues, 1999; Lee, 1994; Iglesias et al., 2000; Iglesias et al., 2007), due to the
⁎ Corresponding author. Tel.: +52 988 9120147/49; fax: +52 988 9120020. E-mail address: [email protected] (C. Rosas). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.02.022
high cost associated to obtain the juveniles for the growth-out phase (Nabhitabhata, 1995). Therefore, several formulated diets have been tested in O. maya, efforts that have resulted in moderate growth rates, below to those achieved with natural diets (Aguila et al., 2007; Domingues et al., 2007; Rosas et al., 2007; Rosas et al., 2008). Similar results were found in cuttlefish (Castro et al., 1993; Castro and Lee, 1994) concluding that protein quality is an important factor to get high digestibility coefficients and better growth rates, being necessary to generate more information on the amino acid requirements to promote higher growth rates. Le Bihan et al., (2006) found that cuttlefish fed with surimi from the shrimp Crangon crangon soaked in fish silage resulted in better growth and conversion rates than those fed a similar diet without the fish silage. The difference between both diets was the peptides and AA present in the fish silage. However, it is not clear if the peptides and AA promoted a better assimilation or due to the solubility inherent to the soluble protein resulted in a higher ingestion rate due to a better feed attractability and palatability. Like all cephalopods, O. maya is a carnivorous species and protein is an important nutrient for tissue accretion and energy source (Segawa and Hanlon, 1988; Rosas et al., 2007), therefore, cephalopods require high amounts of protein and amino acids (AAs) for optimum growth (Lee, 1994; Domingues et al., 2005; Solorzano et al., 2009). However, at present the protein and amino acid requirements have not been determined for O. maya.
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In rearing conditions octopuses are able to survive long periods without food due to several stress factors like system failures (pumping and power energy problems) or density stress (Domingues et al., 2010). After a period of food deprivation organisms are able to catch up their weight by increasing their growth rates as a compensatory growth. However when food deprivation is for longer periods the organisms are unable to catch up their weight compared to well feed siblings, calling this physiological phenomenon as the point of no return. Therefore when culturing organisms that undergo long periods of food deprivation it is important to know those limits to be able to catch up their lost weight without fatal consequences. Taking into consideration that octopuses have a low lipid reserves it is possible to think that during fasting condition octopuses will mobilize amino acids as the principal source of energy in attempt to maintain the homeostasis (Vidal et al., 2002; Villanueva et al., 2004; Iglesias et al., 2006; Vidal et al., 2006; Grigoriou and Richardson, 2009; Solorzano et al., 2009). Recently, Villanueva et al., (2004) determined that Leu, Lys and Arg represent almost half of the essential amino acids (EAAs) required by Octopus vulgaris, Sepia officinalis and Loligo vulgaris during early life stages. According to Akagi and Ohmori (2004) Thr could be the optimum substrate for D-lactate formation, suggesting that the principal biochemical pathway to obtain energy in cephalopods could be through gluconeogenesis, and therefore Thr may be limiting in the diet from these species as indicated by D'Mello (2003). Domingues et al. (2007) found similar results for the juvenile O. maya. In the present study it was evaluated the inanition and re-feeding effects on the overall performance to detect the amino acid (AA) mobilization and overall performance for the juvenile O. maya. 2. Materials and methods 2.1. Broodstock collection and rearing of juvenile octopuses Wild females from O. maya were caught on the coastal area from the Yucatan Peninsula (21° 9′ 55″ N, 90° 1′ 50″ W) using artisan lines with blue crabs Callinectes spp. as bait. Females were transported in 120 L circular tanks containing seawater to the UMDI-UNAM laboratory, situated at 300 m inland. In the laboratory, the females were also maintained in 250 L black tanks until the eggs were layed (Moguel et al., 2010). Hatchlings were fed for 15–20 days with living Artemia adults and crab paste until they passed 0.5 g living weight (Rosas et al., 2008; Avila-Poveda et al., 2009; Moguel et al., 2010).
inanition period, the octopuses from each treatment were re-feeding for 25 days, with the same diet and under similar protocol as explained above. The octopuses were weighed at the end of re-feeding period. 2.4. Growth rate Growth rate (g day− 1) was determined as the difference between the initial and final weight for octopuses during acclimation period (days 0 and 12), at the end of each fasting periods (2, 4, 6, 8 and 10 days) and latest day of re-feeding period. Specific growth rate (SGR, %day− 1) was determined as: SGR = ½ðlnW2 −lnW1 Þ = t 100 where W2 and W1 are the final and initial wet weights of the octopus (obtained during acclimation period, fasting periods or re-feeding period), ln is the natural logarithm, and t the duration of each time period (days). 2.5. Amino acid analysis The amino acid content (g AA/100 g protein) of individual samples from juvenile O. maya arms (N = 3 octopus per treatment) from the acclimation period (control on day 12), and after the inanition period (on the 4 and 8 days food deprivation) was determined. Defatted tissue samples (20 mg) were hydrolyzed with 200 μL of 6 N HCl and 0.06% phenol in a closed vial and heated to 110 °C for 24 h. Amino acid profiles were determined following Waters AccQ-Tag™ procedure as follows: (1) Hydrolyzed samples were dried in a termic monoblock at 60 °C with nitrogen and rehydrated with 1 mL water HPLC grade. (2) Samples were then filtered (0.45 μm) and maintained at −20 °C until used. (3) Samples were derivatized using the Waters system AccQ-Tag™. (4) Samples were chromatographed through a reverse phase column (3.9 × 150 mm) 4 μm Nova Pak™ C-18, using the water-acetonitrile gradient recommended by the Waters AccQ-Tag™ system (Milford, MA, USA), in a Waters™ HPLC and a fluorescence detector (excitation and emission wavelength; 250 and 395 nm, respectively). (6) Analyses were conducted at a constant temperature of 39 °C. (7) HPLC signal calibration and standard curves were obtained by using an amino acid standard solution at three different concentrations containing from 18.75 to 150 pmol of each amino acid. Taking into account that Met and Cys were partially destroyed by acid hydrolysis, the results of both amino acids were taken with caution.
2.2. Feeding acclimation of post-hatching 2.6. Statistical analysis Juvenile O. maya of 32 days post-hatching (DPH) were weighed (wet body weight (wBW) mean value of 0.63 ± 0.10 SD g; N = 120 octopuses). Juveniles, dried with a paper towel, were individually weighed in an electronic balance (± 0.001 g), randomly individualized (1 L tanks), and distributed in six experimental groups (N = 20 animals per group). Juveniles were fed a single diet consisting in fresh crab meat mixed with 5% natural gelatin, fed at 30% of wBW (Rosas et al., 2008; Quintana et al., 2010) twice a day at 0900 and 1700 h. After 12 days all octopuses from the six groups were individually weighed again (wBW; mean value of 0.90 ± 0.21 SD g; N = 65 octopuses). 2.3. Inanition and re-feeding treatments To determine the effect of food deprivation on the overall performance of juvenile O. maya, six groups (N = 20 per group) were deprived of food for 2, 4, 6, 8 and 10 days, while the sixth group was maintained with food as a control under a similar protocol as during the acclimation period. Inanition began at the same time for all octopuses from five experimental groups. At the end of each fasting period all octopuses from the six groups were weighed. After the
A one way analysis of variance was used to known the effect of fasting period on wet weight and growth rate (SGR, %day− 1), and when applied, a multiple comparison of means (Tuckey test) was used to known differences between groups. A significance level of P b 0.05 was established. All statistical analyses were performed using the Statistica@ program (Version 6.1). 3. Result 3.1. Acclimation period No differences were detected among during the acclimation period. Mean values of 0.63 ± 0.02 g, 0.90 ± 0.04 g and 2.81 ± 0.4% day−1 were obtained for initial, final and specific growth rate (SGR) respectively, during acclimation period, (Table 1; Figs. 1 and 2). 3.2. Inanition effect on growth After the food deprivation period, the final weight showed differences between treatments with 9% of wet weight lost in animals
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Table 1 Specific growth rates (SGR, %day− 1) of the Octopus maya juvenile of 32 days post-hatching (DPH) during the acclimation, inanition and re-feeding period for 25 days. Values are given as means (± SD). wBW = wet body weight; N, number of octopuses. Different letters in row means statistical differences at p b 0.05. Fasting period, days 0
2
Mean Acclimation wBW initial wBW final SGR, %day?−1
0.65 0.90 2.56
± ± ±
SD
N
0.09 0.26 2.35
15a 15a 15a
Fasting wBW initial wBW final SGR, %day?−1 Re-feeding wBW initial wBW final SGR, %day?−1
0.90 1.27 1.02
± ± ±
0.26 0.49 0.72
15a 11a 11a
4
Mean
SD
N
6
Mean
SD
N
0.64 0.93 3.06
± ± ±
0.12 0.25 1.25
10a 10a 10a
0.63 0.89 2.89
± ± ±
0.06 0.15 1.06
10a 10a 10a
0.93 0.85 −4.63
± ± ±
0.25 0.24 0.98
10a 10a 10a
0.89 0.74 −4.81
± ± ±
0.15 0.15 1.51
0.85 1.07 0.78
± ± ±
0.24 0.43 0.95
10a 10ab 10a
0.74 1.06 1.23
± ± ±
0.15 0.27 0.62
fasted by 2 days and 38% in octopuses fasted during 10 days; a loss of 17.0, 27.0 and 35.0% were lost in animals when food was deprived during 4, 6 and 8 days, respectively (P b 0.05; Table 1; Fig. 1). Negative growth rates were observed in all groups without detecting any significant difference among them (P N 0.05; Table 1). In consequence, a negative SGR of −4.94 ± 0.29% day−1 was calculated within the animals under inanition from days 2 to 10 (Fig. 2).
8
Mean
SD
N
0.60 0.86 2.87
± ± ±
0.07 0.19 1.12
10a 10a 10a
10a 10ab 10a
0.86 0.63 −5.26
± ± ±
0.19 0.16 1.12
10ab 8ab 8a
0.63 0.90 1.07
± ± ±
0.16 0.32 0.57
10
Mean
SD
N
0.65 0.85 2.19
± ± ±
0.14 0.18 2.12
10a 10a 10a
10a 10bc 10a
0.85 0.56 −5.23
± ± ±
0.18 0.13 0.67
10bc 7c 7a
0.56 0.75 1.34
± ± ±
0.13 0.15 0.60
Mean
SD
N
0.63 0.95 3.31
± ± ±
0.11 0.24 1.19
10a 10a 10a
10a 10cd 10a
0.95 0.59 −4.78
± ± ±
0.24 0.14 1.81
10a 10d 10a
10cd 7c 7a
0.59 0.80 1.11
± ± ±
0.14 0.22 0.90
10d 9c 9a
of 1.09 ± 0.19% day−1 was calculated for O. maya juveniles during recovering period (Fig. 2). There were no statistical differences on octopus survival between treatments with high values on animals fasted 2 days (100%) and low in those fasted 6 and 8 days (70%) (P N 0.05). A mean value 0f80% survival was calculated for re-feeding period. 3.4. Effect of fasting on the amino acid profiles
3.3. Re-feeding period
4. Discussion In the present study after 6 days of food deprivation on O. maya the wet weight was significantly lower than that observed in control group. However, 25 days were not sufficient for the octupuses to catch up their weight in comparison to that shown in the control group. This cannot be necessarily due to the phenomena called the point of no return, where animals exposed to a food deprivation for longer period of times are negatively affected, being unable to show growth rates to equal the control group or even to show higher growth rates to be able to re-catch
SGR, % day-1
A reduction on final wet weight was observed according with fasting days, with high values of animals of control group and low in octopuses exposed to 8 and 10 days fasting (P b 0.05; Fig. 1). After 25 days of re-feeding period none of the exposed groups to inanition were able to catch up their weight as that shown in the control group (Table 1). Even if the SGR shown during this period was similar among all groups due to a large variation, there is the possibility that all groups will be able to catch up over time, with values between 1.34% day−1 and 0.78% day−1 for the groups of octopuses exposed to 8 and 2 days inanition, respectively (P N 0.05; Table 1). As a result, a SGR mean value
Essential amino acids were affected by fasting period (Fig. 3a). A relative reduction of Thr (81%), Phe (15%) and Ile (40%) was observed according with inanition period while an increase on His (116%), Arg (132%), and Lys (566%) was evaluated on O. maya muscle. Non essential amino acids were also affected for the inanition period with relative increments of Tau (21%) and Gly (51%) and reductions on Ala (62%), Glu (46%) and Ser (88%) (Fig. 3b). The rest of the amino acids did not change significantly during fasting experimental periods.
4 3 2 1 0 -1 -2 -3 -4 -5 -6
Acclimation
Fasting
Re feeding
Experimental periods Fig. 1. Wet weights of Octopus maya juveniles during acclimation, fasting and re-feeding periods. Values are given as means ± SD. The arrow indicates the point of no return for wet weight (PNRWW). Different letters indicate statistical differences at p b 0.05.
Fig. 2. Specific growth rate (SGR, % day−1) of O. maya juveniles during acclimation, fasting and re-feeding periods. Values are given as means ± SD.
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Fig. 3. Amino acid content (gAA/100 g protein) of individual samples of arms of the Octopus maya juvenile of 32 days post-hatching (DPH) during the acclimation period (control on day 12), and at the fasting period (on the 4 and 8 days fasting). Each amino acid was ordered from major to minor content respect to the control. Values are given as means ± SD. NEAA, nonessential amino acid; EAA, essential amino acid.
their weight. It is interesting to note that the growth rate during fasting and re-feeding periods was not affected by treatments, demonstrating that O. maya juveniles have a wide plasticity to tolerate, at least 10 days of fasting without any apparently physiological damage that could affect the mechanisms involved into the growth rate of animals. Aquatic animal are generally exposed to long periods of inanition and therefore they are easily able to support long periods of food deprivation before suffering the point of no return by several mechanisms like lowering their metabolic rate and being able to efficiently use their reserves. Although we don't know exactly what kind of mechanisms are involved on such physiological plasticity, the results of the present study shows that AA were being used as an energetic support during the inanition. Besides the tissue that was lost during the food deprivation (−4.63 to −5.26% day−1), the O. maya juveniles the AA like Thr, Phe, Ile, Ala, Glu and Ser, were relatively more used, suggesting a strong mobilization of both essential and non essential AA to maintain the homeostasis. Prove of that is that survival of animals during fasting and re-feeding period was not affected by treatments. In fact a similar survival of control group was observed on fasting groups suggesting that natural mortality provoked by isolation or manipulation could be present. Leporati et al., (2007) found 78% survival of O. pallidus hatchlings, while in the present study a mean survival value of 80% was found for O. maya early juveniles. No studies have examined the fasting consequences on octopus early juveniles mainly due that there are not many research teams that have a production of octopus juveniles to make such type of studies. Although at the date there are some studies on the effects of
fasting condition on cephalopod paralarvae, that results should be taking with precaution because the differences between both life stages (Villanueva and Norman, 2008). Taking into account that there are not many research on this topics, the results obtained in the present study were compared with other obtained both octopus and squid paralarvae; that comparison should be taken with caution because amino acid profile obtained on O. maya juveniles were from arms while in paralarvae studies it was obtained on whole animals Vidal et al., (2006) showed that Loligo opalescens 15 days age, tolerated 3 days without food at 16 °C with no recuperation after 3 days fasting. In that study they observed that the mechanisms affected by starvation involved protein synthesis (RNA/DNA ratio), and the general lost of squid homeostasis. It was also observed that paralarvae fasted for 3 days or less not only survive but they showed a compensatory growth rate that permitted to animals reach a final wet weight similar to control group. Other different characteristic of octopus paralarvae is that after hatch they have available yolk reserves that help to animal during the first days of plancktonic life. According to Villanueva and Norman (2008) octopus paralarvae can tolerate up to 15 days of starving conditions depending on size of hatchlings while hatchlings of L. opalescens can survive until 6 days thanks to yolk reserves (Vidal et al., 2002). Several studies have shown that the ability of a marine organism to resist deprivation of food depends on temperature and the developmental stage; as larvae grow their sensitivity to lack of food diminishes (His and Seaman, 1992; Strüssmann and Takashima, 1992; Vidal et al., 2002; Zheng et al., 2005; Iglesias et al., 2006; Vidal et al., 2006; Viana et al., 2007). Thus, higher fasting resistance should be expected for older organisms; in fact, O. maya as all octopuses should tolerate starving conditions during the 50 days of maternal care period after spawn (Solis, 1967), when animals should use mainly protein to support its life. There are few studies on body AA composition in juvenile cephalopods. Among the most recent, Villanueva et al. (2004) found high concentrations of Lys, Leu and Arg in O. vulgaris paralarvae, representing nearly half of total AA. Moreover, in studies made on subadult and adult animals of O. maya have revealed that Lys, Leu and Arg are also abundant (Aguila et al., 2007). However the results obtained in the present study demonstrated that the amino acidic composition of octopus tissues could be not necessarily the most important for energetic metabolism. In fasting conditions Thr, Phe, Ile, Ala, Glu and Ser were mobilized to maintain animals without physiological damage demonstrating that the amino acidic requirement for the energetic demands of cephalopods could be around those amino acids, more than AAs that are abundant in tissues. Threonine has been suggested to be the best substrate for D-lactate formation in octopus tentacle, fructose-1,6biphosphate and Gly (Akagi and Ohmori, 2004). Also, Fujisawa et al., (2005) detected increments of D-lactate dehydrogenase, together with low values of D-lactate in the mantle of fasted Octopus ocellatus, demonstrating that the gluconeogenic pathway could be a more generalized process on cephalopods. These authors suggested that Thr and Gly contribution to total protein are high enough to form methylglyoxal in octopuses. The animals appear to use methylglyoxal more actively during starvation than under normal conditions. A possible explanation for the active utilization of methylglyoxal is that it is formed from amino-acetone, which in turn is formed from Gly and Thr. Fasted octopuses must use Thr or Gly even from body protein. Thus, it appears that fasted octopuses achieve energy acquisition more actively through methylglyoxal than from pyruvate. In O. maya, this metabolic route seems to be similar to that of O. ocellatus. The results obtained in the present study, suggests active “consumption” of Thr. Arg, His, Lys, Gly, Tau over the fasting days period. Arginine is vigorously metabolized in cephalopods (Hochachka and Fields, 1982; Hochachka et al., 1983) and participates in anaerobic metabolism. During anaerobic work, Arg-phosphate is hydrolyzed, leading to increased availability of Arg for condensation with glucose-derived pyruvate to form octopine, the main anaerobic end product that accumulates in adult cephalopods
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during periods of exercise and stress (Hochachka et al., 1976; Storey et al., 1979; Gäde, 1980). When octopuses must urgently escape from a predator or live anaerobically for a time, Arg-phosphate is drawn (Fujisawa et al., 2005). In the case of O. maya, the increment in Arg concentration and utilization of other AA (mainly NEAA and Thr) during starvation may be linked to a strategy aimed at obtaining energetic reserves to survive stressing conditions. This may also be the case for His and Lys, detected at high proportion of total protein during fasting conditions. Lysine was the only ketogenic amino acid which showed significant changes during fasting condition (D'Mello, 2003; all the other AA are glucogenic). In O. maya Ala was the most abundant, followed by Tau, Asp and Glu. References on the specific function of Ala in cephalopods are lacking. Aspartic acid and Glu acid were reported also as the most abundant AA of diverse cephalopods tissues as: mantle of O. vulgaris (Rosa et al., 2002), gonad of O. vulgaris and O. defilippi (Rosa et al., 2004), gonad of Illex coindetii and Todaropsis eblanae (Rosa et al., 2005), gonad and digestive gland of Rossia macrosoma (Rosa et al., 2006). Also, on early stages of S. officinalis, L. vulgaris and O. vulgaris and hatchlings and wild juveniles of L. vulgaris and O. vulgaris (Villanueva et al., 2004), and O. maya (Aguila et al., 2007; Domingues et al., 2007), were found high proportion of Tau of total protein. Taurine has been shown to play a metabolic role in the regulation of cell osmotic pressure (Norton, 1979; Bishop et al., 1983), and in the anaerobic metabolism of bivalve mollusks (Wells and Baldwin, 1995). In general, O. maya appears to use Thr, Ser and Ala mainly as metabolic fuel to face starvation. Simultaneous increases in His, Arg and Lys concentrations suggest possible accumulation of these AAs. It should be kept in mind that previous work to meet nutritional requirements in other mollusks and fishes species has shown that high concentrations of certain AAs are not the result of de novo synthesis but of protein hydrolysis (Gadomski and Petersen, 1988; Moran and Manahan, 2004). Our results provide insights on the relative utilization of AAs in octopuses, in order to design novel diets that could include different relative concentrations of these AAs to meet specific nutritional requirements. Since octopuses have low fat reserves, the amino acids may be used for energy supply during fasting period. Accordingly, when glycogen stores are depleted as a result of fasting, energy must be obtained from increased catabolism of amino acids and even of body proteins (Storey and Storey, 1983; Fujisawa et al., 2005), just to maintain the homeostasis. That adaptation could be reflecting capability of octopus to tolerate starvation during ecological constrains of food and during maternal care period. Acknowledgements This study is part of the Ph.D. thesis of George-Zamora A. The present study was partially supported by DGAPA-UNAM project no. IN202909 and CONACYT-Básico 24743. Thanks are given to “Posgrado en Ciencias del Mar y Limnologia, PCMyL” and to the “Pulpo Program from Sisal” of UMDI-UNAM, for the possibility to participate in CIAC-09 Symposium and Workshops. Also thanks are given to “Posgrado en Ciencias del Mar y Limnologia, PCMyL” for financial support during the stay at the “Instituto de Investigaciones Oceanologicas, at the Universidad Autónoma de Baja California”. Claudia Caamal-Monsreal, Richard Mena and Luis Yan from UMDI-Sisal UNAM, the Comite Sistema Producto Pulpo “CSPP” Moluscos del Mayab, and Victoria Patiño-Suarez from CINVESTAV-Mérida helped with technical assistance during the experiment and maintenance of octopuses. Omar Hernando Avila-Poveda is also thanked by reviewing and editing the manuscript. References Aguila, J., Cuzon, G., Pascual, C., Domingues, P.M., Gaxiola, G., Sánchez, A., Maldonado, T., Rosas, C., 2007. The effects of fish hydrolysate (CPSP) level on Octopus maya (Voss and Solis) diet: digestive enzyme activity, blood metabolites, and energy balance. Aquaculture 273, 641–655.
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Amino acid mobilization and growth of juvenile Octopus maya (Mollusca: Cephalopoda) under inanition and re-feeding Arturo George-Zamora a, Maria-Teresa Viana b, Sergio Rodríguez c, Gabriela Espinoza c, Carlos Rosas d,⁎ a
Posgrado en Ciencias del Mar y Limnología (PCMyL), Universidad Nacional Autónoma de México (UNAM), Puerto de Abrigo S/N, Sisal, Yucatán, Mexico Instituto de Investigaciones Oceanológicas (IIO), Universidad Autónoma de Baja California (UABC), Ensenada, Baja California, Mexico Facultad de Química, Unidad de Química-Sisal, Universidad Nacional Autónoma de México (UNAM), Puerto de Abrigo S/N, Sisal, Yucatán, Mexico d Unidad Multidisciplinaria de Docencia e Investigación (UMDI), Facultad de Ciencias, Universidad Nacional Autónoma de México (UNAM), Puerto de Abrigo S/N, Sisal, Yucatán, Mexico b c
a r t i c l e
i n f o
Article history: Received 22 September 2010 Received in revised form 15 February 2011 Accepted 15 February 2011 Available online 21 February 2011 Keywords: Amino acids Growth Cephalopoda Octopus maya Fasting México
a b s t r a c t Octopus maya is an endemic cephalopod from the Yucatán Peninsula in Mexico, with interest to develop their commercial culture. Like all cephalopods, protein is an important nutrient both for growing and energetic metabolism. This condition results in a high demand for protein and specifically for certain amino acids (AAs). In this study it is examined the effects of inanition and re-feeding on growth and AA content in soft tissue to detect the amino acid (AA) mobilization to identify the principal metabolic reserves in juvenile O. maya. After 25 days re-feeding of all starved groups, the octopuses were unable to reach the similar weight as the control group. However, SGR of some groups were greater than that of the controls, although the differences were not significant due to variability in the data. Therefore, it is assumed that the juveniles of O. maya would need a longer period of time to reach the control group. It is therefore demonstrated that juveniles of O. maya have a wide plasticity to tolerate, at least 10 days of food deprivation without any apparently physiological damage. Moreover, during inanition the juveniles of O. maya used preferentially Thr, Phe, Ile, Ala, Glu and Ser, suggesting a strong mobilization of both essential and non essential AA to maintain the homeostasis. Prove of that is that survival of the animals during fasting and re-feeding period was not affected by treatments. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The octopus Octopus maya (Voss and Solís-Ramírez, 1966) is an endemic cephalopod from the Yucatan Peninsula in Mexico (CONAPESCA, 2008) being one of the most important resources in the fishery sector. Earlier studies have demonstrated that O. maya has a high potential for aquaculture due to their well adaptability to captivity conditions (Van Heukelem, 1977; Boletzky and Hanlon, 1983; Hanlon and Forsythe 1985; DeRusha et al., 1989) and direct development from hatching without a larval transition (Boletzky, 1974). Moreover, O. maya shows larger eggs than those observed in other cephalopods (up to 17 mm). They are able to accept dead preys or formulated diets even during the first life cycle stadiums (Aguila et al., 2007), resulting in high growth rates (up to 8% wet body weight, wBW, per day) as a result of high feeding rates (FR) and food conversion efficiencies (FC) (Van Heukelem, 1983; Hanlon and Forsythe, 1985; Domingues et al., 2007; Rosas et al., 2007). Dependence on natural diets together with a long larval period has been the bottleneck in the cephalopod aquaculture (Domingues, 1999; Lee, 1994; Iglesias et al., 2000; Iglesias et al., 2007), due to the
⁎ Corresponding author. Tel.: +52 988 9120147/49; fax: +52 988 9120020. E-mail address: [email protected] (C. Rosas). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.02.022
high cost associated to obtain the juveniles for the growth-out phase (Nabhitabhata, 1995). Therefore, several formulated diets have been tested in O. maya, efforts that have resulted in moderate growth rates, below to those achieved with natural diets (Aguila et al., 2007; Domingues et al., 2007; Rosas et al., 2007; Rosas et al., 2008). Similar results were found in cuttlefish (Castro et al., 1993; Castro and Lee, 1994) concluding that protein quality is an important factor to get high digestibility coefficients and better growth rates, being necessary to generate more information on the amino acid requirements to promote higher growth rates. Le Bihan et al., (2006) found that cuttlefish fed with surimi from the shrimp Crangon crangon soaked in fish silage resulted in better growth and conversion rates than those fed a similar diet without the fish silage. The difference between both diets was the peptides and AA present in the fish silage. However, it is not clear if the peptides and AA promoted a better assimilation or due to the solubility inherent to the soluble protein resulted in a higher ingestion rate due to a better feed attractability and palatability. Like all cephalopods, O. maya is a carnivorous species and protein is an important nutrient for tissue accretion and energy source (Segawa and Hanlon, 1988; Rosas et al., 2007), therefore, cephalopods require high amounts of protein and amino acids (AAs) for optimum growth (Lee, 1994; Domingues et al., 2005; Solorzano et al., 2009). However, at present the protein and amino acid requirements have not been determined for O. maya.
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In rearing conditions octopuses are able to survive long periods without food due to several stress factors like system failures (pumping and power energy problems) or density stress (Domingues et al., 2010). After a period of food deprivation organisms are able to catch up their weight by increasing their growth rates as a compensatory growth. However when food deprivation is for longer periods the organisms are unable to catch up their weight compared to well feed siblings, calling this physiological phenomenon as the point of no return. Therefore when culturing organisms that undergo long periods of food deprivation it is important to know those limits to be able to catch up their lost weight without fatal consequences. Taking into consideration that octopuses have a low lipid reserves it is possible to think that during fasting condition octopuses will mobilize amino acids as the principal source of energy in attempt to maintain the homeostasis (Vidal et al., 2002; Villanueva et al., 2004; Iglesias et al., 2006; Vidal et al., 2006; Grigoriou and Richardson, 2009; Solorzano et al., 2009). Recently, Villanueva et al., (2004) determined that Leu, Lys and Arg represent almost half of the essential amino acids (EAAs) required by Octopus vulgaris, Sepia officinalis and Loligo vulgaris during early life stages. According to Akagi and Ohmori (2004) Thr could be the optimum substrate for D-lactate formation, suggesting that the principal biochemical pathway to obtain energy in cephalopods could be through gluconeogenesis, and therefore Thr may be limiting in the diet from these species as indicated by D'Mello (2003). Domingues et al. (2007) found similar results for the juvenile O. maya. In the present study it was evaluated the inanition and re-feeding effects on the overall performance to detect the amino acid (AA) mobilization and overall performance for the juvenile O. maya. 2. Materials and methods 2.1. Broodstock collection and rearing of juvenile octopuses Wild females from O. maya were caught on the coastal area from the Yucatan Peninsula (21° 9′ 55″ N, 90° 1′ 50″ W) using artisan lines with blue crabs Callinectes spp. as bait. Females were transported in 120 L circular tanks containing seawater to the UMDI-UNAM laboratory, situated at 300 m inland. In the laboratory, the females were also maintained in 250 L black tanks until the eggs were layed (Moguel et al., 2010). Hatchlings were fed for 15–20 days with living Artemia adults and crab paste until they passed 0.5 g living weight (Rosas et al., 2008; Avila-Poveda et al., 2009; Moguel et al., 2010).
inanition period, the octopuses from each treatment were re-feeding for 25 days, with the same diet and under similar protocol as explained above. The octopuses were weighed at the end of re-feeding period. 2.4. Growth rate Growth rate (g day− 1) was determined as the difference between the initial and final weight for octopuses during acclimation period (days 0 and 12), at the end of each fasting periods (2, 4, 6, 8 and 10 days) and latest day of re-feeding period. Specific growth rate (SGR, %day− 1) was determined as: SGR = ½ðlnW2 −lnW1 Þ = t 100 where W2 and W1 are the final and initial wet weights of the octopus (obtained during acclimation period, fasting periods or re-feeding period), ln is the natural logarithm, and t the duration of each time period (days). 2.5. Amino acid analysis The amino acid content (g AA/100 g protein) of individual samples from juvenile O. maya arms (N = 3 octopus per treatment) from the acclimation period (control on day 12), and after the inanition period (on the 4 and 8 days food deprivation) was determined. Defatted tissue samples (20 mg) were hydrolyzed with 200 μL of 6 N HCl and 0.06% phenol in a closed vial and heated to 110 °C for 24 h. Amino acid profiles were determined following Waters AccQ-Tag™ procedure as follows: (1) Hydrolyzed samples were dried in a termic monoblock at 60 °C with nitrogen and rehydrated with 1 mL water HPLC grade. (2) Samples were then filtered (0.45 μm) and maintained at −20 °C until used. (3) Samples were derivatized using the Waters system AccQ-Tag™. (4) Samples were chromatographed through a reverse phase column (3.9 × 150 mm) 4 μm Nova Pak™ C-18, using the water-acetonitrile gradient recommended by the Waters AccQ-Tag™ system (Milford, MA, USA), in a Waters™ HPLC and a fluorescence detector (excitation and emission wavelength; 250 and 395 nm, respectively). (6) Analyses were conducted at a constant temperature of 39 °C. (7) HPLC signal calibration and standard curves were obtained by using an amino acid standard solution at three different concentrations containing from 18.75 to 150 pmol of each amino acid. Taking into account that Met and Cys were partially destroyed by acid hydrolysis, the results of both amino acids were taken with caution.
2.2. Feeding acclimation of post-hatching 2.6. Statistical analysis Juvenile O. maya of 32 days post-hatching (DPH) were weighed (wet body weight (wBW) mean value of 0.63 ± 0.10 SD g; N = 120 octopuses). Juveniles, dried with a paper towel, were individually weighed in an electronic balance (± 0.001 g), randomly individualized (1 L tanks), and distributed in six experimental groups (N = 20 animals per group). Juveniles were fed a single diet consisting in fresh crab meat mixed with 5% natural gelatin, fed at 30% of wBW (Rosas et al., 2008; Quintana et al., 2010) twice a day at 0900 and 1700 h. After 12 days all octopuses from the six groups were individually weighed again (wBW; mean value of 0.90 ± 0.21 SD g; N = 65 octopuses). 2.3. Inanition and re-feeding treatments To determine the effect of food deprivation on the overall performance of juvenile O. maya, six groups (N = 20 per group) were deprived of food for 2, 4, 6, 8 and 10 days, while the sixth group was maintained with food as a control under a similar protocol as during the acclimation period. Inanition began at the same time for all octopuses from five experimental groups. At the end of each fasting period all octopuses from the six groups were weighed. After the
A one way analysis of variance was used to known the effect of fasting period on wet weight and growth rate (SGR, %day− 1), and when applied, a multiple comparison of means (Tuckey test) was used to known differences between groups. A significance level of P b 0.05 was established. All statistical analyses were performed using the Statistica@ program (Version 6.1). 3. Result 3.1. Acclimation period No differences were detected among during the acclimation period. Mean values of 0.63 ± 0.02 g, 0.90 ± 0.04 g and 2.81 ± 0.4% day−1 were obtained for initial, final and specific growth rate (SGR) respectively, during acclimation period, (Table 1; Figs. 1 and 2). 3.2. Inanition effect on growth After the food deprivation period, the final weight showed differences between treatments with 9% of wet weight lost in animals
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Table 1 Specific growth rates (SGR, %day− 1) of the Octopus maya juvenile of 32 days post-hatching (DPH) during the acclimation, inanition and re-feeding period for 25 days. Values are given as means (± SD). wBW = wet body weight; N, number of octopuses. Different letters in row means statistical differences at p b 0.05. Fasting period, days 0
2
Mean Acclimation wBW initial wBW final SGR, %day?−1
0.65 0.90 2.56
± ± ±
SD
N
0.09 0.26 2.35
15a 15a 15a
Fasting wBW initial wBW final SGR, %day?−1 Re-feeding wBW initial wBW final SGR, %day?−1
0.90 1.27 1.02
± ± ±
0.26 0.49 0.72
15a 11a 11a
4
Mean
SD
N
6
Mean
SD
N
0.64 0.93 3.06
± ± ±
0.12 0.25 1.25
10a 10a 10a
0.63 0.89 2.89
± ± ±
0.06 0.15 1.06
10a 10a 10a
0.93 0.85 −4.63
± ± ±
0.25 0.24 0.98
10a 10a 10a
0.89 0.74 −4.81
± ± ±
0.15 0.15 1.51
0.85 1.07 0.78
± ± ±
0.24 0.43 0.95
10a 10ab 10a
0.74 1.06 1.23
± ± ±
0.15 0.27 0.62
fasted by 2 days and 38% in octopuses fasted during 10 days; a loss of 17.0, 27.0 and 35.0% were lost in animals when food was deprived during 4, 6 and 8 days, respectively (P b 0.05; Table 1; Fig. 1). Negative growth rates were observed in all groups without detecting any significant difference among them (P N 0.05; Table 1). In consequence, a negative SGR of −4.94 ± 0.29% day−1 was calculated within the animals under inanition from days 2 to 10 (Fig. 2).
8
Mean
SD
N
0.60 0.86 2.87
± ± ±
0.07 0.19 1.12
10a 10a 10a
10a 10ab 10a
0.86 0.63 −5.26
± ± ±
0.19 0.16 1.12
10ab 8ab 8a
0.63 0.90 1.07
± ± ±
0.16 0.32 0.57
10
Mean
SD
N
0.65 0.85 2.19
± ± ±
0.14 0.18 2.12
10a 10a 10a
10a 10bc 10a
0.85 0.56 −5.23
± ± ±
0.18 0.13 0.67
10bc 7c 7a
0.56 0.75 1.34
± ± ±
0.13 0.15 0.60
Mean
SD
N
0.63 0.95 3.31
± ± ±
0.11 0.24 1.19
10a 10a 10a
10a 10cd 10a
0.95 0.59 −4.78
± ± ±
0.24 0.14 1.81
10a 10d 10a
10cd 7c 7a
0.59 0.80 1.11
± ± ±
0.14 0.22 0.90
10d 9c 9a
of 1.09 ± 0.19% day−1 was calculated for O. maya juveniles during recovering period (Fig. 2). There were no statistical differences on octopus survival between treatments with high values on animals fasted 2 days (100%) and low in those fasted 6 and 8 days (70%) (P N 0.05). A mean value 0f80% survival was calculated for re-feeding period. 3.4. Effect of fasting on the amino acid profiles
3.3. Re-feeding period
4. Discussion In the present study after 6 days of food deprivation on O. maya the wet weight was significantly lower than that observed in control group. However, 25 days were not sufficient for the octupuses to catch up their weight in comparison to that shown in the control group. This cannot be necessarily due to the phenomena called the point of no return, where animals exposed to a food deprivation for longer period of times are negatively affected, being unable to show growth rates to equal the control group or even to show higher growth rates to be able to re-catch
SGR, % day-1
A reduction on final wet weight was observed according with fasting days, with high values of animals of control group and low in octopuses exposed to 8 and 10 days fasting (P b 0.05; Fig. 1). After 25 days of re-feeding period none of the exposed groups to inanition were able to catch up their weight as that shown in the control group (Table 1). Even if the SGR shown during this period was similar among all groups due to a large variation, there is the possibility that all groups will be able to catch up over time, with values between 1.34% day−1 and 0.78% day−1 for the groups of octopuses exposed to 8 and 2 days inanition, respectively (P N 0.05; Table 1). As a result, a SGR mean value
Essential amino acids were affected by fasting period (Fig. 3a). A relative reduction of Thr (81%), Phe (15%) and Ile (40%) was observed according with inanition period while an increase on His (116%), Arg (132%), and Lys (566%) was evaluated on O. maya muscle. Non essential amino acids were also affected for the inanition period with relative increments of Tau (21%) and Gly (51%) and reductions on Ala (62%), Glu (46%) and Ser (88%) (Fig. 3b). The rest of the amino acids did not change significantly during fasting experimental periods.
4 3 2 1 0 -1 -2 -3 -4 -5 -6
Acclimation
Fasting
Re feeding
Experimental periods Fig. 1. Wet weights of Octopus maya juveniles during acclimation, fasting and re-feeding periods. Values are given as means ± SD. The arrow indicates the point of no return for wet weight (PNRWW). Different letters indicate statistical differences at p b 0.05.
Fig. 2. Specific growth rate (SGR, % day−1) of O. maya juveniles during acclimation, fasting and re-feeding periods. Values are given as means ± SD.
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Fig. 3. Amino acid content (gAA/100 g protein) of individual samples of arms of the Octopus maya juvenile of 32 days post-hatching (DPH) during the acclimation period (control on day 12), and at the fasting period (on the 4 and 8 days fasting). Each amino acid was ordered from major to minor content respect to the control. Values are given as means ± SD. NEAA, nonessential amino acid; EAA, essential amino acid.
their weight. It is interesting to note that the growth rate during fasting and re-feeding periods was not affected by treatments, demonstrating that O. maya juveniles have a wide plasticity to tolerate, at least 10 days of fasting without any apparently physiological damage that could affect the mechanisms involved into the growth rate of animals. Aquatic animal are generally exposed to long periods of inanition and therefore they are easily able to support long periods of food deprivation before suffering the point of no return by several mechanisms like lowering their metabolic rate and being able to efficiently use their reserves. Although we don't know exactly what kind of mechanisms are involved on such physiological plasticity, the results of the present study shows that AA were being used as an energetic support during the inanition. Besides the tissue that was lost during the food deprivation (−4.63 to −5.26% day−1), the O. maya juveniles the AA like Thr, Phe, Ile, Ala, Glu and Ser, were relatively more used, suggesting a strong mobilization of both essential and non essential AA to maintain the homeostasis. Prove of that is that survival of animals during fasting and re-feeding period was not affected by treatments. In fact a similar survival of control group was observed on fasting groups suggesting that natural mortality provoked by isolation or manipulation could be present. Leporati et al., (2007) found 78% survival of O. pallidus hatchlings, while in the present study a mean survival value of 80% was found for O. maya early juveniles. No studies have examined the fasting consequences on octopus early juveniles mainly due that there are not many research teams that have a production of octopus juveniles to make such type of studies. Although at the date there are some studies on the effects of
fasting condition on cephalopod paralarvae, that results should be taking with precaution because the differences between both life stages (Villanueva and Norman, 2008). Taking into account that there are not many research on this topics, the results obtained in the present study were compared with other obtained both octopus and squid paralarvae; that comparison should be taken with caution because amino acid profile obtained on O. maya juveniles were from arms while in paralarvae studies it was obtained on whole animals Vidal et al., (2006) showed that Loligo opalescens 15 days age, tolerated 3 days without food at 16 °C with no recuperation after 3 days fasting. In that study they observed that the mechanisms affected by starvation involved protein synthesis (RNA/DNA ratio), and the general lost of squid homeostasis. It was also observed that paralarvae fasted for 3 days or less not only survive but they showed a compensatory growth rate that permitted to animals reach a final wet weight similar to control group. Other different characteristic of octopus paralarvae is that after hatch they have available yolk reserves that help to animal during the first days of plancktonic life. According to Villanueva and Norman (2008) octopus paralarvae can tolerate up to 15 days of starving conditions depending on size of hatchlings while hatchlings of L. opalescens can survive until 6 days thanks to yolk reserves (Vidal et al., 2002). Several studies have shown that the ability of a marine organism to resist deprivation of food depends on temperature and the developmental stage; as larvae grow their sensitivity to lack of food diminishes (His and Seaman, 1992; Strüssmann and Takashima, 1992; Vidal et al., 2002; Zheng et al., 2005; Iglesias et al., 2006; Vidal et al., 2006; Viana et al., 2007). Thus, higher fasting resistance should be expected for older organisms; in fact, O. maya as all octopuses should tolerate starving conditions during the 50 days of maternal care period after spawn (Solis, 1967), when animals should use mainly protein to support its life. There are few studies on body AA composition in juvenile cephalopods. Among the most recent, Villanueva et al. (2004) found high concentrations of Lys, Leu and Arg in O. vulgaris paralarvae, representing nearly half of total AA. Moreover, in studies made on subadult and adult animals of O. maya have revealed that Lys, Leu and Arg are also abundant (Aguila et al., 2007). However the results obtained in the present study demonstrated that the amino acidic composition of octopus tissues could be not necessarily the most important for energetic metabolism. In fasting conditions Thr, Phe, Ile, Ala, Glu and Ser were mobilized to maintain animals without physiological damage demonstrating that the amino acidic requirement for the energetic demands of cephalopods could be around those amino acids, more than AAs that are abundant in tissues. Threonine has been suggested to be the best substrate for D-lactate formation in octopus tentacle, fructose-1,6biphosphate and Gly (Akagi and Ohmori, 2004). Also, Fujisawa et al., (2005) detected increments of D-lactate dehydrogenase, together with low values of D-lactate in the mantle of fasted Octopus ocellatus, demonstrating that the gluconeogenic pathway could be a more generalized process on cephalopods. These authors suggested that Thr and Gly contribution to total protein are high enough to form methylglyoxal in octopuses. The animals appear to use methylglyoxal more actively during starvation than under normal conditions. A possible explanation for the active utilization of methylglyoxal is that it is formed from amino-acetone, which in turn is formed from Gly and Thr. Fasted octopuses must use Thr or Gly even from body protein. Thus, it appears that fasted octopuses achieve energy acquisition more actively through methylglyoxal than from pyruvate. In O. maya, this metabolic route seems to be similar to that of O. ocellatus. The results obtained in the present study, suggests active “consumption” of Thr. Arg, His, Lys, Gly, Tau over the fasting days period. Arginine is vigorously metabolized in cephalopods (Hochachka and Fields, 1982; Hochachka et al., 1983) and participates in anaerobic metabolism. During anaerobic work, Arg-phosphate is hydrolyzed, leading to increased availability of Arg for condensation with glucose-derived pyruvate to form octopine, the main anaerobic end product that accumulates in adult cephalopods
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during periods of exercise and stress (Hochachka et al., 1976; Storey et al., 1979; Gäde, 1980). When octopuses must urgently escape from a predator or live anaerobically for a time, Arg-phosphate is drawn (Fujisawa et al., 2005). In the case of O. maya, the increment in Arg concentration and utilization of other AA (mainly NEAA and Thr) during starvation may be linked to a strategy aimed at obtaining energetic reserves to survive stressing conditions. This may also be the case for His and Lys, detected at high proportion of total protein during fasting conditions. Lysine was the only ketogenic amino acid which showed significant changes during fasting condition (D'Mello, 2003; all the other AA are glucogenic). In O. maya Ala was the most abundant, followed by Tau, Asp and Glu. References on the specific function of Ala in cephalopods are lacking. Aspartic acid and Glu acid were reported also as the most abundant AA of diverse cephalopods tissues as: mantle of O. vulgaris (Rosa et al., 2002), gonad of O. vulgaris and O. defilippi (Rosa et al., 2004), gonad of Illex coindetii and Todaropsis eblanae (Rosa et al., 2005), gonad and digestive gland of Rossia macrosoma (Rosa et al., 2006). Also, on early stages of S. officinalis, L. vulgaris and O. vulgaris and hatchlings and wild juveniles of L. vulgaris and O. vulgaris (Villanueva et al., 2004), and O. maya (Aguila et al., 2007; Domingues et al., 2007), were found high proportion of Tau of total protein. Taurine has been shown to play a metabolic role in the regulation of cell osmotic pressure (Norton, 1979; Bishop et al., 1983), and in the anaerobic metabolism of bivalve mollusks (Wells and Baldwin, 1995). In general, O. maya appears to use Thr, Ser and Ala mainly as metabolic fuel to face starvation. Simultaneous increases in His, Arg and Lys concentrations suggest possible accumulation of these AAs. It should be kept in mind that previous work to meet nutritional requirements in other mollusks and fishes species has shown that high concentrations of certain AAs are not the result of de novo synthesis but of protein hydrolysis (Gadomski and Petersen, 1988; Moran and Manahan, 2004). Our results provide insights on the relative utilization of AAs in octopuses, in order to design novel diets that could include different relative concentrations of these AAs to meet specific nutritional requirements. Since octopuses have low fat reserves, the amino acids may be used for energy supply during fasting period. Accordingly, when glycogen stores are depleted as a result of fasting, energy must be obtained from increased catabolism of amino acids and even of body proteins (Storey and Storey, 1983; Fujisawa et al., 2005), just to maintain the homeostasis. That adaptation could be reflecting capability of octopus to tolerate starvation during ecological constrains of food and during maternal care period. Acknowledgements This study is part of the Ph.D. thesis of George-Zamora A. The present study was partially supported by DGAPA-UNAM project no. IN202909 and CONACYT-Básico 24743. Thanks are given to “Posgrado en Ciencias del Mar y Limnologia, PCMyL” and to the “Pulpo Program from Sisal” of UMDI-UNAM, for the possibility to participate in CIAC-09 Symposium and Workshops. Also thanks are given to “Posgrado en Ciencias del Mar y Limnologia, PCMyL” for financial support during the stay at the “Instituto de Investigaciones Oceanologicas, at the Universidad Autónoma de Baja California”. Claudia Caamal-Monsreal, Richard Mena and Luis Yan from UMDI-Sisal UNAM, the Comite Sistema Producto Pulpo “CSPP” Moluscos del Mayab, and Victoria Patiño-Suarez from CINVESTAV-Mérida helped with technical assistance during the experiment and maintenance of octopuses. Omar Hernando Avila-Poveda is also thanked by reviewing and editing the manuscript. References Aguila, J., Cuzon, G., Pascual, C., Domingues, P.M., Gaxiola, G., Sánchez, A., Maldonado, T., Rosas, C., 2007. The effects of fish hydrolysate (CPSP) level on Octopus maya (Voss and Solis) diet: digestive enzyme activity, blood metabolites, and energy balance. Aquaculture 273, 641–655.
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