Comparison of growth and survival rates of big blue octopus ( Octopus cyanea , 1849) fed on natural and formulated diets in captivity

Comparative studies on growth and survival rates of Octopus cyanea fed on natural and formulated diets in captivity have never been conducted in Tanzania. This study aimed to investigate the growth and survival rates of O. cyanea using natural and formulated diets. The three formulated diets were made up of a mixture of sardines, fish waste, and alternating ratios of crab paste content across the different diet treatments. Treatment B had 75 % crab paste content, Treatment C had 50 % crab content and Treatment D had 0 % crab content while Treatment A was based on a natural diet of frozen crabs ( Scylla serrata ) and was used as a control. After five weeks of feeding, the effect of each diet was analyzed on growth performance and survival rates for the octopus. Results showed that there was a significant difference in growth rate in all the diet treatments (H=13.243, p=0.004, DF=3). Specific growth rates (SGR) were higher in octopuses fed in feed treatment A and lowest in feed treatment D. The survival rates were 100 % for treatment A and feed treatment B and 75 % for treatments C and D respectively. More research is needed to develop optimal nutritional diets for faster growth rates of O. cyanea in captivity.


Introduction
Octopus cyanea, also known as the big blue octopus, is one of the most common exploited species in the Western Indian Ocean regions (Guard and Mgaya, 2002;Roper and Hochberg, 1988a). O. cyanea is found within phylum Mollusca, class Cephalopoda, and family Octopodidae and is distributed throughout the Indo-Pacific Ocean from Hawaii to the eastern coast of Africa. Their preferred habitat is in either the lower reaches of the intertidal reef flat or along the reef edge where they live in small holes (dens) and crevices often hidden by small stones, rubble and pieces of shell (Norman, 2000;Guard and Mgaya, 2002). Octopus play an essential ecological role in the marine ecosystem where they act as predators and potential prey to larger fishes such as sharks and some neritic tuna Guard, 2009).
In Tanzania, octopus fishing is practiced by small-scale fishers and occurs extensively along the coast providing a source of protein and improving the livelihoods of the fishermen ( Jiddawi and Ohman, 2002 (FAO, 2014). The reported declines in octopus catches have been linked to several factors, including over-exploitation for export markets, increasing number of octopus fishers and tourists, poor management practices, seasonal change in sea temperatures, habitat degradation, disease outbreaks, pollution and predation (Katsanevakis and Verriopoulos, 2006;Van Heukelem, 1973;Sparre, 1998;Rocliffe and Harris, 2016). In 2020, the global export of cephalopods amounted to USD 10.2 billion which is equivalent to 6.8 % of the total value of exports of aquatic products; this trend resulted in the share of cephalopods in global trade increasing over time and put supplies at risk due to poor management (FAO, 2022). Efforts have been focused on octopus fishery closures to assist in fishery sustainability, and little attention has been paid to exploring the viability of successfully culturing octopus species for commercial industrial production.
The first feeding and behaviour experiments on the rearing of octopus in captivity were performed by the Instituto de Ciencias Marinas de Vigo and the Instituto Español de Oceanografía in Spain (Guerra, 1978;Nixon and Mangold, 1998). Octopus juveniles were grown in tanks and floating cages obtaining promising results and since then, many Spanish research centers have shown interest in the development of production techniques for octopus, such as Ciencias Marinas del Mar of CSIC, Barcelona (Villanueva, 1995). Such studies have not been explored across the Western Indian Ocean (WIO) region although octopus species present a series of favorable characteristics for rearing in captivity and commercial farming. These include: a great tolerance to captive conditions (Iglesias et al., 2000), very fast growth rate (Mangold, 1983;Van Heukelem, 1973;Guard, 2009, Roper andHochberg, 1998b) 1988b), high feed conversion efficiencies (Mangold and Von Boletzky, 1973), high reproductive rate of up to 400,000 paralarvae (Boyle and Rodhouse, 2005) and a high market price (Vaz-Pires et al., 2004 (Lopez et al., 2015).
Nevertheless, making formulated feeds of appropriate nutritional composition is the main challenge to the development of aquaculture of octopus (Vaz-Pires et al., 2004;Cerezo and Garcia, 2016). A series of experiments have shown that formulated feed is the determinant of successful growth and intensification of octopus aquaculture production. So far, considerable effort has been made to develop artificial diets (Aguila et al., 2007;Domingues et al., 2007;Cerezo et al., 2008).
Future development of octopus aquaculture depends on the development of nutritionally sound and cost-effective feeds that can support production levels (Kirimi et al., 2016). Despite octopus being recognized as an ideal candidate for aquaculture, studies on octopus aquaculture in relation to formulated diets remain lim-

Material and methods
Description of the sampling site The octopuses were randomly distributed in sixteen tanks of 1000 L (two octopus per tank) in a flowthrough saltwater system at the experimental site.
Water quality parameters (temperature, salinity, dissolved oxygen, and pH) were measured twice a day (morning and afternoon after feeding). Growth in weight was evaluated after every seven days. Coral fragments were kept inside the tanks to allow hiding and attachment as in the natural wild habitat. The octopuses were acclimatized for two days before the onset of the experiment and during this period they were not fed to allow them to get used to the captive conditions (after Sen, 2019).

Experimental design
The experimental tanks were distributed randomly in the experimental site and labeled based on the feed treatment provided. There were three feed treatments; each with four replicates. The feeds used in this study were composed of sardines, fish waste, and varying amounts of crab paste in the different treatments.
The crab paste content in Treatment B was 75 %, 50 % in Treatment C, and 0% in Treatment D. Treatment A used a natural diet of frozen crabs (Scylla serrata).
Each tank contained a PVC tube of 10.16 cm diameter that was placed at the bottom of the tanks for the octopus to use as a refuge. Other habitat substrates such as pieces of dead hard corals and pebbles were also kept in the tanks. Seawater was exchanged regularly to maintain a conducive condition for the survival of the O. cyanea. To prevent escape, each tank was covered with a plastic mesh lid designed with two removable hatches. Seawater inlet was through a pipe that was connected to the tank reservoir containing water from the sea. The seawater outlet was located at the end of each tank and sealed with a cork. The outlet was less than 2 cm diameter to prevent the octopus from escaping during opening and closing. Temperature, salinity, dissolved oxygen (DO), and pH were checked twice daily (morning and afternoon after feeding).
The three formulated diet treatments and the control were supplied to the octopus once a day at (0900 hrs) at 5 % body weight for all regimens (after Farìas et al., 2010). Unconsumed feed was siphoned every day (at 1700hrs). Overnight faeces was removed from the tanks before the octopus were given the initial feeding. The experiment was carried out for a period of 35 days.

Preparation of the formulated feeds for the experimental diets
Three formulated diets and a natural diet consisting of a crab diet (Scylla Serrata) were used in the experiment.
The formulated diets (Fig. 3) were a mixture of sardines (Sardinella longiceps), fish waste that consisted of fish stomach contents, intestines, heads and fins that are often discarded at fish markets, and crab paste (soft parts of the crab meat after the skeleton has been removed). Cassava flour was used as a binder.
Fifty kg of each ingredient was purchased, dried for 24 hours under the sun and ground using a grinding machine, then sieved using 0.5 mm mesh to remove indigestible parts and to obtain a fine powder for all ingredients. This was followed by mixing thoroughly by hand to form a uniform single mixture; however, for the case of crab, the hard parts including the carapace and hard shells which are less nutritious and less digestible were first removed before other processes.
Formulated diets of different crab paste content (75 %, 50 % and 0 %) for the diet treatments were obtained.
Hot boiled water was added to cassava flour as a binder   (Table 2).

Data analysis
The data obtained are presented as mean ± SE (standard error). Calculations of growth performance parameters for the different feed treatments were conducted for Specific Growth Rates (SGR), Weight Gain (WG), Average Daily Gain (ADG) and Survival Rates (SR) by using the formulae below as described by Abarike et al. (2012). To determine the growth rates of O. cyanea fed on the different formulated diets, growth data was tested for normality and homogeneity by Shapiro's test of normality and Levene's test for homogeneity respectively, using R programme 4.6.
The data were found not to be normally distributed therefore the non-parametric test of Kruskal Wallis was conducted followed by a post-hoc pairwise Wilcoxon test to depict where the significant difference in growth rates lies within the different diets.

Growth rates of O. cyanea fed on different formulated diets
Growth rates for O. cyanea were calculated and are presented in (Table 3) below, together with initial mean body weight (g), final mean body weight (g), weight gain (g), SGR (%/day) and ADG (g) with mean ± standard error. The highest SGR was displayed by

A B
a natural diet based on frozen crab followed by feed treatment B and C, with the lowest SGR displayed by feed treatment C (Fig. 1).

Survival rates
The survival rate was 100 % in treatment A and B, and 75 % in feed treatment C and D.

Water quality parameters
In all treatments the temperature ranged from 24 °C to 26.4 °C DO varied from 6.8 to 9.5 mg/l, pH varied from 8.6 to 8.8, and salinity from 38.5 to 40. (Table 4).

Growth and survival rates of O. cyanea in captivity
Octopus showed a slow positive growth rate for feed treatments B and C while a negative growth rate was recorded in feed treatment D. There was no significant difference in growth rate shown in all the three formulated feeds (B, C and D). These results can be compared to a study by Martínez et al. (2014) in which best growth rates in juvenile stages of O. maya were obtained by using a moist crustacean-based diet and this was attributed to high protein assimilation and digestibility levels of this diet. Furthermore, Aguado and García (2002) reported that growth and food intake were higher with a crab diet when rearing O. maya in captivity. Other studies agree that the natural diet is still the most reliable, especially when based on crustaceans Gutiérrez et al., 2015). The success of these studies are attributed to the longer period of the experiment. Replacement with formulated diets must be carried out using crustaceans-based diets, squid or fish, using freezedried meals (Estefanell et al., 2013;Rosas et al., 2013;Rodríguez et al., 2015). However, studies that have used a fish diet for octopus have exhibited lower growth rates and survival compared to those using a crustacean-based diet (Cagnetta and Sublimi, 2000;Domain, 2000;Aguado and García, 2002 In term of water quality, the salinity range for octopus is between 35 to 39.5 psu (Mangold, 1983).
In this study, they survived well between 38 up to 40 psu. There were occurrences of temperature fluctuations recorded within the experiment, but they fell  Other studies should focus on further research on replacing the "crustacean-based diet", which has proven crucial to the growth of O.cyanea and other octopus species worldwide, with an alternative, cheaper source which is nutritionally sufficient and acceptable to the animal. This is because crabs are expensive to purchase, and identifying an alternative best feed option will make this farming possible and affordable.