Aquaculture International Archimer June 2011, Volume 19, Number 3, Pages 445-457 http://archimer.ifremer.fr http://dx.doi.org/10.1007/s10499-010-9360-5 © Springer Science+Business Media B.V. 2010 The original publication is available at http://www.springerlink.com is available on the publisher Web site Web publisher the on available is Partial characterization of hepatopancreatic and extracellular digestive proteinases of wild and cultivated Octopus maya R. Martínez1, R. Sántos2, A. Álvarez3, G. Cuzón4, L. Arena5, M. Mascaró5, C. Pascual5 and C. Rosas5, * 1 División de Posgrado, FMVZ, Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico 2 authenticated version authenticated Departamento de Nutrición, FMVZ, Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico - 3 Unidad de Ciencias Biológicas, UJAT, Villahermosa, Tabasco, Mexico 4 Ifremer, Tahiti, French Polynesia 5 Unidad Multidisciplinaria de Docencia e Investigación, Facultad de Ciencias, Universidad Nacional Autónoma de México, Puerto de abrigo s/n, Sisal, Yucatan, Mexico *: Corresponding author : C. Rosas, email address : [email protected] Abstract: Abstract Proteinases from hepatopancreas (HP) and gastric juice (GJ) from wild and cultured red octopus (Octopus maya) were characterized. Hepatopancreas assays revealed optimal activity at pH 4, 9–10 and 10 for wild and pH 3, 8, and 9, for cultured octopuses, for total proteinases, trypsin and chymotrypsin, respectively. In the gastric juice, maximum activity was recorded at pH 6, 8, and 7 for total proteinases, trypsin, and chymotrypsin, respectively for both wild and cultured octopus. A reduction on enzyme activity of 70 and 20% was observed in HP and GJ extracts, respectively when protease inhibitor Pepstatin A was used. That result suggests that the main proteases in the HP were aspartic acid proteinases type (possibly Cathepsin D) and some of them were present in the GJ. Dissociating discontinuous polyacrylamide gel electrophoresis showed activity bands between 20 and 28, 30 and 34, 35 and 45, 60 and 70 kDa, and a last one between 75 and 100 kDa. We concluded that extracellular digestion of O. maya takes place in an acid environment, around pH 6. In contrast, intracellular digestion in the HP is developed at pHs between 3 and 4, where cathepsin D could be the most important enzyme for O. maya. Keywords: Cephalopods ; Digestive proteinases ; Hepatopancreas ; Gastric juice ; Octopus maya produced PDF of an article accepted for publication following peer review. The definitive publisher definitive The review. following peer publication acceptedof article for an PDF produced - Please note that this is an author is anPlease note that this 1 48 1. Introduction 49 The red octopus (Octopus maya) is an endemic species that inhabits the coasts 50 of the Peninsula of Yucatan and represents an important fishing resource (Voss and 51 Solís-Ramírez, 1966, Solís-Ramírez and Chávez, 1986; Solís-Ramírez, 1988; Cabrera 52 and Defeo, 2001; Pérez Lozada et al., 2002). O. maya has a great potential for 53 aquaculture because this species reaches the juvenile stage 30 days after hatching, 54 whereas other species go through a 60-day planktonic life before their settlement as 55 juveniles (Moguel et al., 2010). At present, there is no adequate artificial feed that can 56 be used to foster octopus culture at an industrial level. Different types of artificial feeds 57 were fed to O. maya juveniles with limited success. Rosas et al., (2007) reports that the 58 main limitation of the use of pellets to feed O. maya juveniles was related with the low 59 digestibility of the diets (lower than 20%). Aguila et al., (2007) used artificial diets 60 supplemented with different concentrations of fish hydrolysate (CPSP). Results of this 61 study showed a positive growth rate when octopuses were fed with a diet supplemented 62 with 15% of CPSP, suggesting that with that diet the food digestibility can be enhanced. 63 Other studies done on O. maya and O. vulgaris reported the effect of the type of binder 64 used to prepare the artificial diets. In O. maya juveniles, the digestibility of crab paste 65 was improved using gelatin besides sodium alginate, indicating that octopuses can 66 digest feed better when a proteinaceous binder is used (Rosas et al., 2008). 67 It is known that octopuses are carnivorous, for which proteolytic enzymes are 68 determinant in the digestion and availability of nutrients (Boucher-Rodoni and Mangold, 69 1985; Aguila et al., 2007; Domingues et al., 2007). Digestibility depends on digestive 70 enzymes that act in the gastric juice to activate chymotrypsin. Digestion in cephalopods 71 is a two-parts process: extracellular and intracellular (Boucher-Rodoni et al., 1987; 3 72 Koueta and Boucaud-Camou, 1999; Koueta et al., 2000). The extracellular enzymes 73 secreted by the hepatopancreas produce the first hydrolysis on the ingested food 74 (Perrin, 2004). The gastric juice, containing the necessary enzymes for the extracellular 75 digestion, is mixed with the ingested food in the crop and carried to the hepatopancreas 76 to be absorbed. The intracellular digestion of the chymo takes place in the 77 hepatopancreas where the molecules are hydrolyzed to be transported or stored 78 (Boucaud-Camou et al., 1976). At present, the low digestibility of the formulated diets 79 could be explained by the incapacity of the extracellular digestive enzymes to hydrolyze 80 adequately the nutrient content in the diet. The high quantity of feces observed in 81 octopuses fed elaborated diets with high protein and energy content supports that 82 hypothesis (Rosas et al., 2009, 2010). 83 Enzymes in the hepatopancreas are of proteolytic nature. (Boucaud-Camou and 84 Boucher-Rodoni, 1983). Although trypsin and chymotrypsin (alkaline proteinases) have 85 been the most studied enzymes in cephalopods (Mangold, 1989; Ezquerra et al., 2002) 86 there are other enzymes, such as cysteine proteinases, that have a high proteolytic 87 activity (Cárdenas-Lopez and Haard, 2005). In the jumbo squid Dosidiscus gigas, 88 cysteine proteinases peaked proteolysis at pH values of 3 to 4 and 5 to 6, suggesting 89 cysteine cathepsins (B, H, and L) as the main enzymes in the hepatopancreas of jumbo 90 squid (Cardenas-Lopez and Haard, 2005). Recently a cathepsin L was identified in the 91 jumbo squid that had an optimal pH of 4.5 and optimal temperature of 55 °C (Cardenas- 92 Lopez and Haard, 2009). Unfortunately there are no studies on the enzyme activity of 93 the gastric juice and, in consequence, we do not know the characteristics of the 94 digestive enzymes that modulate external digestion and chymo formation in 95 cephalopods. 4 96 The pH is one of the main factors affecting enzymatic activity, modulating the 97 secondary conformation of the molecule or modifying the electric charge of the enzyme 98 and/or of the substrates (Murray et al., 2001; Le Bihan et al., 2006;). In an attempt to 99 characterize the digestive enzymes, in the present study the effect of the pH on the 100 activity of the digestive proteinases present in the gastric juice and hepatopancreas 101 tissue of O. maya was evaluated, in both wild and cultivated octopuses. At the same 102 time, the molecular weight of the enzymes of the gastric juice was determined 103 electrophoretically. 104 105 2. Materials and Methods 106 2.1. Origin of animals 107 The study was performed in the Unidad Multidisciplinaria de Docencia e Investigación, 108 Facultad de Ciencias (UMDI, Sisal, Yucatan, Mexico) of the Universidad Nacional 109 Autónoma de México. Wild O. maya (N = 15; 536.01 ± 125.89 g) were caught using 110 artisan lines, with only fresh crabs as bait and without hook, in front of Sisal harbor 111 (Yucatán, Mexico). Octopuses were transported from the port to the laboratory situated 112 300 m inland, in a 400-L circular tank with sea water. Laboratory animals were 113 individually placed in 80-L tanks for acclimation during 7 days at 28 + 1°C, 34 salinity, 114 oxygen dissolved higher than 5.5 mg L-1 and pH > 8. A PVC tube of 4” diameter was 115 offered as a shelter. During the acclimation time, octopuses were fed dead C. sapidus 116 crabs. 117 118 Cultured O. maya (N = 15; 11.90 ± 2.08 g) were obtained from spawning of wild mature 119 females under controlled conditions. Hatchlings (100 + 18 mg living weight; 80 5 120 octopuses m-2) were obtained from O. maya system production. In that system 121 octopuses were cultivated in 8 m2 (1600-L) tanks during two months until animals 122 reached 2g of living weight and maintained at 28 + 1°C, 34 PPT salinity, oxygen 123 dissolved higher than 5.5 mg L-1 and pH > 8. A photoperiod of 10 h light and 14 h 124 darkness was provided. During culture period, animals were fed crab pieces ad libitum 125 and provided with a clean Megalongena corona bispinosa conch (spiral-shell) to serve 126 as a refuge. Production tanks were connected to a flow through sea water system and 127 coupled to skimmer and anthracite earth filter. Survival of 40% was obtained in the 128 production tanks and it was a consequence of octopus cannibalism. When octopuses 129 reached 2 g of living weight, they were re-distributed in three 7-m2 sea water outside 130 tanks (0.7 octopuses m-2) where animals were fed crab pieces. Natural sea water was 131 supplied to tanks and connected to a recycling system. Only a 10% of total seawater 132 was renewed daily. In that system sea water was maintained at 28 + 2°C, 34 PPT 133 salinity, oxygen dissolved higher than 5.5 mg L-1 and pH > 8. Experimental tanks were 134 placed below a plastic mesh as a shade (90%).