Aquaculture 261 (2006) 817 822 www.elsevier.

com/locate/aqua-online

Short communication

First feeding of Octopus vulgaris Cuvier, 1797 paralarvae using Artemia: Effect of prey size, prey density and feeding frequency
J. Iglesias , L. Fuentes, J. Snchez, J.J. Otero, C. Moxica, M.J. Lago
Instituto Espaol de Oceanografa, Centro Oceanogrfico de Vigo, Apdo. 1552, 36200 Vigo, Spain Received 23 June 2006; received in revised form 1 August 2006; accepted 1 August 2006

Abstract Different assays related to the first feeding of Octopus vulgaris Cuvier, 1797 are compiled in this paper. They include: age at initial feeding age, prey size selection and optimal density, attack timing after feeding, and effect of dose number on the number of captures. Prey capture and ingestion processes were also analysed. Food supplied was cultured Artemia sp. Each assay lasted 15 min. Although paralarvae already start to feed on the hatching day (day 0), it is during day 2 when a greater number of attacks is recorded (81.7 14.7% paralarvae attack). They mainly prefer (significance level = 0.05) large Artemia, 1.4 0.4 mm (77.0 5.6% of the total attacks) than small Artemia, 0.8 0.1 mm (23.0 5.6%). There is also a slight predilection for the lowest Artemia concentration (33.3 12.6% paralarvae attack in a 0.1 Artemia ml 1 density, opposite 16.7 7.6 and 18.3 7.6% in densities of 0.5 and 1 Artemia ml 1 respectively). The greatest predatory activity is recorded during the first 5 min after food is supplied (72.2 25.5%). An increase in the predatory activity was also observed when food was distributed in several doses instead of a single dose (75.0 10.0% and 46.7 17.6% respectively). It was proved for the first time that paralarvae completely ingest their preys (including their exoskeletons), in this case Artemia. Time needed for their total ingestion ranges between 4 and 10 min. 2006 Elsevier B.V. All rights reserved.
Keywords: Octopus vulgaris; Paralarvae; Artemia; First feeding; Ingestion process

1. Introduction This work intends to approach the problem of Octopus vulgaris paralarval culture, and also to act as a link between aquaculture and this species biological knowledge, because different gaps still persist regarding their first developmental stages (Roper et al., 1984). Several studies have been focused on the octopus paralarvae rearing. Itami et al. (1963) and Villanueva
Corresponding author. Tel.: +34 986 49 21 11; fax: +34 986 49 23 51. E-mail address: jose.iglesias@vi.ieo.es (J. Iglesias). 0044-8486/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.08.002.

(1995) achieved benthic paralarvae for the first time in Japan and Europe respectively. Iglesias et al. (2002, 2004) and subsequently Carrasco et al. (2003, 2005) closed the octopus culture cycle and achieved adult octopus from paralarvae cultured in intensive systems. Diet utilized in all cases consisted of Artemia and larval stages of selected crustaceans (Palaemon serrifer, Maja brachydactyla, and Pagurus prideaux). Nevertheless, survival rates after the planktonic phase of paralarvae were very low in general. Furthermore, a number of diverse prey types and sizes have been used, often under different feeding regimens (concentrations, feeding times, and food treatments). As

818

J. Iglesias et al. / Aquaculture 261 (2006) 817822

a result, no standardized methods or protocols exist at the present for paralarvae rearing. It is globally accepted that nutrition and feeding behaviour are the most important factors for paralarval survival. Therefore, in this paper we deal with items related to first feeding, such as prey age, size and density, feeding timing and frequency, and capturing and ingestion behaviour. There are several previous works related to this subject, such as Hernndez-Garca et al. (2000), that concentrates on the evidence that O. vulgaris makes an external digestion of crustaceans larvae used as a diet, fact thoroughly verified both for juveniles and adults (Nixon, 1984; Nixon and Mangold, 1996). Likewise, Villanueva et al. (1996) presents a detailed description of the natatory and food searching behaviour of the planktonic phases of O. vulgaris. Besides, Villanueva et al. (2002) also deals with the effect of food concentration on the proteolytic activity and on the growth of O. vulgaris paralarvae. 2. Material and methods The aim of this work was to determine highly remarkable aspects of first feeding of octopus paralarvae: age at which feeding starts, selected prey size and density, attack timing after food supply, effect of the number of doses, and time to completely ingest preys. Prey supplied as food was Artemia sp. cultured with Isochrysis galbana Parke (Iglesias et al., 2005). Experiments were made in white plastic cylindrical tanks (26 cm high and 25 cm in diameter) with a total volume of 10 l containing 2.5 l of seawater. 20 paralarvae were introduced in each of them. Water temperature ranged between 18 and 20 C, salinity between 34 and 35, and light intensity in the tank surface was 300400 lx. In any case, paralarvae were starved in 100 l tanks, at a density of 5 paralarvae l 1 and a temperature of 1820 C, until the experiments began. In the assays carried out to determine age at initial feeding, 0-, 1-, 2- and 3 day old paralarvae were used.

Fig. 2. Average percentage of attacks of 2-day-old paralarvae on Artemia of two different sizes (small = 0.8 0.1 mm; large = 1.4 0.4 mm).

Fig. 1. Average percentage of paralarvae that attack Artemia at each age (0, 1, 2 and 3 days-old after hatching).

These were fed on one single dose of 0.5 Artemia ml 1 (1.5 0.0 mm of total length). Triplicates were established for each age group. In the other experiments, 2day-old paralarvae were used and triplicates were prepared for each treatment. The prey size selection experiment was made feeding paralarvae on a mixture of small (0.8 0.1 mm total length) and large (1.4 0.4 mm TL) Artemia with a ratio of 0.25 Artemia ml 1 for each size. Ingestion process was determined in paralarvae fed on a dose of 0.5 Artemia ml 1 (1.4 0.4 mm); continuous observations under binocular magnifier were carried out to estimate time invested in total ingestion. Prey density selection was estimated using paralarvae fed on three different densities of (1.4 0.1 mm TL) Artemia (0.1, 0.5 and 1 Artemia ml 1). The precise moment in which attacks on Artemia (1.4 0.1 mm TL) took place were calculated taking into account the right moment of the attack after the food supply. Finally, the effect of the number of doses was studied by supplying a single dose of 0.1 Artemia ml 1 (1.6 0.2 mm TL) and the same quantity of Artemia divided into three doses (minute 0, 5 and 10). Each experiment lasted for 15 min and started when food was added to the tank, in which paralarvae had been previously placed. Though still water was maintained during the assay, culture medium was gently shacked for no more than 10 s at 0, 5 and 10 min in order to homogenize preys distribution. Each replicate was individually controlled by an observer, in charge of constantly observing paralarvae behaviour and score the number of attacks, defined as the number of paralarvae that attack a prey and keep it in their arms. For this purpose, paralarvae and prey were together removed from the tank by a Pasteur pipette to be observed by binocular magnifier, and the precise capture time was recorded. Subsequently, neither paralarvae nor preys were returned to the tank. At the end of each experiment, in minute 15, every captured prey was reviewed to describe its morphometric characteristics or to assess its ingestion degree. In the

J. Iglesias et al. / Aquaculture 261 (2006) 817822 Table 1 Ingestion process of preys in O. vulgaris paralarvae Half ingestion (min) Paralarva 1 Paralarva 2 Paralarva 3 Paralarva 4 1 min 59 s No data recorded 2 min 54 s 4 min 28 s Total ingestion (min) 4 min 3 s 5 min 20 s 8 min 54 s 10 min 25 s

819

case of the analysis of prey size selection, observation was immediately carried out after the capture. A statistical comparison among the different treatments was carried out by one-way ANOVA (significance level = 0.05). 3. Results

Fig. 4. Average percentage of 2-day-old paralarvae that attack Artemia at different densities (0.1, 0.5, and-1 Artemia ml 1).

shows significant differences (p b 0.05). Consequently, future assays were carried out with 2-day-old paralarvae. 3.2. Selected prey size

3.1. Age influence on the number of attacks Average percentages of prey attacks observed during the first three days of life of octopus paralarvae are shown in Fig. 1. On day 0 hatching day paralarvae present a scarce capacity to attack their preys (only 13.3 5.8% paralarvae attack). The number of attacks increases gradually, four-fold in day 1 (50.0 25.2% paralarvae attack), and the greater number of prey captures is observed (81.7 14.7%) in day 2. Nevertheless, if paralarvae are kept starved one day more i.e. until day 3 a clear decrease in the number of attacks is detected (47.8 24.4%). An ANOVA among the number of attacks observed in the three replicates corresponding to each age, When 2-day-old paralarvae were supplied preys having two different sizes, a clear tendency toward those having a greater size was recorded (Fig. 2). Larger Artemia (1.4 0.4 mm total length) were clearly preferred (77.0 5.6%) to smaller Artemia (23.0 5.6%), showing significant differences ( p b 0.05). The continuous observation of the prey consumption immediately after the capture, has allowed us to find out for the first time that, when fed on Artemia, paralarvae completely ingest it and they do not simply suck up its internal content. The ingestion process of four paralarvae which completed it is shown in Table 1 (12 over 20 paralarvae attacked, and only the ingestion process of 4 of them could be entirely described). In general, paralarvae hold their preys by their dorsal area, and a suction, not only of their internal content but also their complete exoskeleton takes place; this process has a length of 4 to 10 min. In Fig. 3 Artemia appendices (thoracopods) can be observed inside a paralarva digestive tract.

Fig. 3. Stomach content of an O. vulgaris paralarva which has just ingested an Artemia. Artemia thoracic appendices (thoracopods) can be observed inside a paralarva digestive tract.

Fig. 5. Average percentage of attacks to Artemia distributed depending on the observation time fraction (0 to 5, 5 to 10 or 10 to 15 min after feeding).

820

J. Iglesias et al. / Aquaculture 261 (2006) 817822

Fig. 6. Average percentage of attacks depending on the fact that food is supplied in a single dose during the 15 min of the experiment, or in 3 successive doses at 0, 5 and 10 min after food supplies.

3.3. Selected prey density In Fig. 4 it can be observed that a greater number of attacks (33.3 12.6%) exists at lower densities, 0.1 Artemia ml 1, than at 0.5 (16.7 7.6%) and 1 Artemia ml 1 (18.3 7.6%), but these differences are not significant ( p N 0.05). 3.4. Attack timing after feeding supply In order to analyse the distribution in time of prey catches, 2-day-old paralarvae attacks during 15 min, divided into three consecutive intervals of 5 min starting after food is provided, are represented in Fig. 5. It is observed that most attacks (72.2 25.5%) take place during the first 5 min, which approximately represents one paralarva attack per minute, while during the subsequent time fractions values of 19.4 17.3% and 8.3 14.4% are consecutively recorded; significant differences exist ( p b 0.05) in the number of attacks among the three fractions analysed. 3.5. Effect of the supply of a single food dose vs three doses In Fig. 6 it is observed that if a single food dose is supplied, the average percentage of paralarvae that attack in the three replicates is 46.7 17.6, while this percentage considerably increases when three doses are supplied (75.0 10.0%). Despite this fact, these differences were not significant ( p N 0.05). 4. Discussion During day 0 O. vulgaris paralarvae carry out very few attacks. This may be due to the fact that they are not physiologically ready to start their exogenous feeding. The decrease in the number of captures observed at an age of 3 days in starvation could be interpreted as reaching the point of no return, after which paralarvae would have

considerably reduced their ability to catch, ingest and digest their preys. In this work it is evidenced that at an age of 2 days after hatching paralarvae show their greater predatory activity. No previous works point out this appraisal, but the authors obtained the same results using wild zooplankton captured in the Ra de Vigo as a prey (unpublished data). It also proved the convenience of using large size preys (1.4 0.4 mm total length) in the first feeding of octopus paralarvae. Iglesias et al. (2004); Moxica et al. (2002) and Hamazaki et al. (1991) also used large size Artemia specimens; Okumura et al. (2005) pointed out that using large Artemia strains improves both growth and survival in intensive rearing of octopus paralarvae. Even Villanueva et al. (2002) and Carrasco et al. (2003), who used nauplii for their experiments, indicate that paralarvae subsequently need a prey having a greater size; specifically, Villanueva et al. (2002) concludes that enriched Artemia nauplii seem to be useful only as an initial diet until a doubling in hatchling weight is achieved. In the case of 2-day-old paralarvae, it has been observed that the number of attacks is greater at a prey density of 0.1 Artemia ml 1 than at higher densities (0.5 and 1.0 Artemia ml 1), a result different to what could be expected. An explanation to this fact could be that at lower densities they can concentrate their attention better and launch their attacks more effectively. Nevertheless, very different densities have been used to date. For example, Iglesias et al. (2004) used adult Artemia (14 mm) at 0.050.1 ind ml 1 and crustacean zoeae as a complement; the concentration used by Moxica et al. (2002) was 0.1 Artemia ml 1. In other experiments higher densities have been used: Villanueva et al. (2002) worked with densities of 0.2, 2, 4 and 10 nauplii ml 1 and recorded the best growth at the highest densities; Carrasco et al. (2005) used densities of 0.20.8 Artemia ml 1 and Okumura et al. (2005) 2.0 Artemia ml 1. As there is not a general consensus on the optimum prey density for octopus larval culture (that in any case is related to paralarvae density), we suggest that this parameter, also related to growth and survival, should be specially considered in future investigations. The fact that during the first 5 min after food supply paralarvae are most active, suggests that paralarvae attacks and captures are stimulated by the first visual contact with preys. This leads us to think that in the larval culture of O. vulgaris it is advisable to distribute the food supply in many doses along the day, instead of one or two big doses. In fact, it is clearly observed that immediately after the food dose is supplied, most paralarvae catch their preys. Nevertheless, after a few

J. Iglesias et al. / Aquaculture 261 (2006) 817822

821

minutes they scarcely show preys in their arms and the number of attacks considerably decreases. It has been observed that paralarvae make an external digestion, at least of decapods larvae, extracting the zoeae edible content and leaving the exoskeletons completely empty (Hernndez-Garca et al., 2000). According to them, paralarvae administer paralysing agents on their preys (cephalotoxin), and digestive enzymes break the muscle joints to make the extraction of the edible content of exoskeletons easier. However, the present paper has proved that, when paralarvae are fed on Artemia, the prey is completely ingested in less than 10 min; therefore it could be possible that, besides the external digestion, an internal digestion of the Artemia takes place. The presence of Artemia gills and pieces of appendices in the digestive systems of octopus paralarvae has also been mentioned by other researchers (Roo, personal communication). The fact that adult Artemia shows a thinner exoskeleton (1 m) than other crustacean zoeae could increase its probability to be ingested. 5. Conclusions and recommendations O. vulgaris paralarvae already start to feed on the hatching day, although the number of attacks is very low. They reach their maximum predatory activity 2 days after hatching. Therefore, special attention should be paid to prey size and density during the second day of life. They preferably select larger Artemia (1.4 0.4 mm total length), instead of small Artemia (0.8 0.1 mm). Therefore, when considering the establishing of an intensive octopus paralarvae culture it is advisable to use alive preys having a large size (total length greater than 1 mm). Prey density producing the greater number of attacks and captures was 0.1 Artemia ml 1, in comparison to greater densities of 0.5 and 1.0 Artemia ml 1. Greater densities have not produced a greater number of attacks. Therefore, in order to reduce time and economical costs, it is advisable to fit the prey quantity to the effective consumption of paralarvae. During the first 5 min after food supply the greater activity of attacks and captures in paralarvae is observed. This agrees with the fact that distributing food into three doses causes a greater number of attacks on the preys in comparison to when a single dose is supplied. Consequently, in octopus larval culture a supply of alive food distributed in several doses throughout the day is recommended instead of one or two daily doses. Contrary to what is generally accepted, octopus paralarvae completely ingest all their preys (adult Artemia, in this case), process that takes from 4 to 10 min to complete.

Acknowledgements This study was funded by the projects PETRI: 19950765-OP and INIA: ACU02-003. We would like to thank the staff of the aquaculture facilities of the Instituto Espaol de Oceanografa in Vigo for their cooperation in the experiments carried out. References
Carrasco, J.F., Rodrguez, C., Rodrguez, M., 2003. Cultivo intensivo de paralarvas de pulpo (Octopus vulgaris, Cuvier) utilizando como base de la alimentacin zoeas vivas de crustceos. IX Congreso Nacional de Acuicultura. Libro de Resmenes, pp. 255256. Carrasco, J.F., Rodrguez, C., Rodrguez, M., 2005. Cultivo intensivo de paralarvas de pulpo (Octopus vulgaris, Cuvier 1797), en tanques parablicos de 30 l utilizando como base de la alimentacin zoeas vivas de crustceos. IX Congreso Nacional de Acuicultura. Libro de actas, pp. 191194. Hamazaki, H., Fukunaga, K., Yoshida, Y., Maruyama, K., 1991. Effects of marine microalgae Nannochloropsis sp. on survival and growth on rearing pelagic paralarvae of Octopus vulgaris, and results of mass culture in the tank of 20 metric tons. Saibai-Giken 19, 7584. Hernndez-Garca, V., Martn, A.Y., Castro, J.J., 2000. Evidence of external digestion of crustaceans in Octopus vulgaris paralarvae. J. Mar. Biol. Assoc. U.K. 80, 559560. Iglesias, J., Otero, J.J., Moxica, C., Fuentes, L., Snchez, F.J., 2002. Paralarvae culture of octopus (Octopus vulgaris Cuvier) using Artemia and crab zoeas and first data on juvenile growth up to eight months of age. Aquac. Eur., vol. 32. European Aquaculture Society, pp. 268269. Special Publication. Iglesias, J., Otero, J.J., Moxica, C., Fuentes, L., Snchez, F.J., 2004. The completed life cycle of the octopus (Octopus vulgaris, Cuvier) under culture conditions: paralarval rearing using Artemia and zoeae, and first data on juvenile growth up to 8 months of age. Aquac. Int. 12, 481487. Iglesias, J., Lima, J., Lago, M.J., Snchez, F.J., 2005. Tcnica de engorde de Artemia (Artemia salina, L.1758) hasta los 2 mm de longitud: tipo de alimento, temperatura de cultivo y densidad ptima. Poster in: X Congreso Nacional de Acuicultura, 1721 octubre, Ganda. (Valencia, Spain). Itami, K., Izawa, Y., Maeda, S., Nakai, K., 1963. Notes on the laboratory culture of octopus larvae. Bull. Jpn. Soc. Sci. Fish. 29, 514520. Moxica, C., Linares, F., Otero, J.J., Iglesias, J., Snchez, F.J., 2002. Cultivo intensivo de paralarvas de pulpo, Octopus vulgaris Cuvier, 1797, en tanques de 9 m3. Bol. Inst. Esp. Oceanogr. 18, 3136. Nixon, M., 1984. Is there external digestion by Octopus? J. Zool. 202 (3), 441447. Nixon, M., Mangold, K., 1996. The early life of Octopus vulgaris (Cephalopoda: Octopodidae) in the plankton and at settlement: a change in lifestyle. J. Zool. 239, 301327. Okumura, S., Kurihara, A., Iwamoto, A., Takeuchi, T., 2005. Improved survival and growth in Octopus vulgaris paralarvae by feeding large type Artemia and Pacific sandeel, Ammodytes personatus. Aquaculture 244 (14), 147157. Roper, C.F.E., Sweeny, M.J., Nauen, C.E., 1984. FAO species catalogue. Vol. 3 Cephalopods of the world. An annotated and illustrated catalogue of species of interest to fisheries. Fish. Synop., vol. 3(125). FAO, Rome, pp. 1277.

822

J. Iglesias et al. / Aquaculture 261 (2006) 817822 Villanueva, R., Koueta, N., Riba, J., Boucaud-Camou, E., 2002. Growth and proteolytic activity of Octopus vulgaris paralarvae with different food rations during first feeding, using Artemia nauplii and compound diets. Aquaculture 205 (34), 269286.

Villanueva, R., 1995. Experimental rearing and growth of planktonic Octopus vulgaris from hatching to settlement. Can. J. Fish. Aquat. Sci. 52, 26392650. Villanueva, R., Nozais, C., Boletzky, S.V., 1996. Swimming behaviour and food searching in planktonic Octopus vulgaris Cuvier from hatching to settlement. J. Exp. Mar. Biol. Ecol. 208, 169184.