SEPIAMETA – On the metabolism of the European cuttlefish, Sepia officinalis (Linnaeus, 1758)

 

ABSTRACT

 

A considerable part of cuttlefish research during the past few years has been focused on its introduction as a new species for commercial aquaculture. This is due to the need to diversify the available number of farmable marine species. Taking into consideration biological and economical aspects, the European cuttlefish is considered a potential candidate for industrial culture. Recently, Sykes et al. (2006a) reviewed the aquaculture potential, the state of the art and future trends of research for the species. In this work, they highlighted a series of bottlenecks that need to be resolved before the technology can acquire a maturity that will enable a transition to industrial scale. The two main factors that have delayed large-scale culture are the dependence on live prey during the first part of the life cycle, and more importantly, the lack of an adequate artificial diet for this species.

 

Early experimental diets for S. officinalis were either moist or dry pellets, or surimi (fish myofibrillar protein concentrate; Domingues et al., 2005). Despite being eaten, the feeding and survival rates associated were always lower than those obtained with natural food (live or frozen grass shrimp; Domingues et al. 2003a and b). These results were due to the lack of nutritional knowledge of prey and of physiological processes in the cuttlefish, among other aspects.

 

In order to successfully design artificial diets for cephalopods, their particular metabolism has to be considered. The animal must be able to balance its gains from diet against its metabolic costs, in order to allocate an optimal distribution of surplus energy to somatic growth and, later, reproduction. To do so, the animal has to feed on a diet correctly balanced to its metabolic needs at a given temperature. This is where two theories clash in terms of what the cuttlefish uses as energy substrate. The first one considers that, under normal feeding conditions, both growth and energy use the protein fraction as fuel (Lee, 1994), while the second considers that the carbohydrate fraction is used as energy source, and the protein fraction is used for growth (Storey & Storey, 1983; Hochachka, 1994). Despite cuttlefish having a similar digestion process to vertebrates (which includes both extracellular and intracellular digestion), either theory contrasts with what happens among vertebrates, where the energetic fuel is lipid based, and to what might exist at different life stages of the cuttlefish.

 

This must be clarified for all life stages because of its importance to diet design, since it might imply the necessity of carbohydrate. The best way to determine this is to understand the species process of digestion and which enzymes are involved in this process. The cuttlefish has a highly efficient digestion (Boucher-Rodoni et al. 1987). Since the available enzymes might change according to life stage, as well as the cells present in the digestive gland, there is the need to study them in the hatchling, juvenile and adult stages. This is supported by the fact that cuttlefish digestion system matures during the first 30 DAH. It is also important to determine the nutritional content of the most commonly used prey, the grass shrimp Palaemonetes varians, in terms of its protein and carbohydrate content. This species has attained the status of prey model due to the good results obtained in cuttlefish culture (Sykes et al. 2006b).

 

Despite previous studies having already addressed the enzyme content in cuttlefish digestive gland, these are rather old and were not directly related to the issue of understanding the performance of cuttlefish uptake of both protein and carbohydrate fractions, and were performed in animals from a different region. Regarding the geographic location, existent biological data point to different metabolism due to higher temperatures (Sykes et al. 2006a and 2006b). Therefore, we may find different activities and quantities or even different enzymes in cuttlefish from South Portugal, resulting also from different natural prey. This is why we plan to conduct studies using similar enzymes.

 

After determining which enzymes are present with a normal diet, then the best way to identify possible metabolic pathways will be through the use of radiolabelled tracers. A review on this type of methods was conducted recently by Conceição et al. (2007) and we think that we could adapt some of the methodologies to cuttlefish.

 

Based on the above, the objectives of the current proposal are: to determine the nutritional and energy content of grass shrimp, to identify enzyme activity during digestion and to establish possible metabolic pathways. The accomplishment of the objectives of the current proposal will allow not only a better understanding of cuttlefish digestion, but will also determine a correct design of a prepared diet to be tested in the future.

SEPIAMETA – Estudo do metabolismo do choco Europeu, Sepia officinalis (Linnaeus, 1758)

 

SUMÁRIO

 

Uma parte considerável da pesquisa sobre o choco nos últimos anos tem-se focado sobre a sua introdução como nova espécie em aquacultura. Isto deve-se à necessidade de diversificação das espécies disponíveis nesta área. Tendo em conta os aspectos biológicos e económicos, o choco é considerado um potencial candidato ao cultivo industrial. Recentemente, Sykes et al. (2006a) reviram o seu potencial em aquacultura, o estado da arte e futura investigação sobre a espécie. Nesse trabalho, enumeraram uma série de problemas que precisam ser resolvidos de modo a que a tecnologia atinja a maturidade que permita a transição para a escala industrial. Os dois grandes factores que têm atrasado o cultivo em larga-escala são a dependência de alimento vivo durante a primeira parte do ciclo de vida, e mais importante, a inexistência de uma dieta artifical adequada para esta espécie.

 

Anteriormente utilizaram-se “pellets” húmidos ou secos ou “surimi” (concentrado de proteína miofibrilar de peixe; Domingues et al., 2005) como dieta. Apesar de serem aceites, a alimentação e as taxas de sobrevivência associadas foram sempre mais baixas do que aquelas obtidas com alimento natural (camarinha viva ou congelada; Domingues et al. 2003a e b). Isto resultou da falta de conhecimento nutricional da presa e dos processos fisiológicos do choco, entre outros aspectos.

 

Para se garantir o sucesso da dieta artificial, tem de ser considerado o metabolismo particular da espécie. A dieta deve assegurar ao animal um equilíbrio entre os ganhos contra os custos metabólicos, de modo a distribuir, de forma óptima, o excedente energético pelo crescimento somático e, mais tarde, pela reprodução. Para isso, a dieta deve ser correctamente equilibrada, baseada nas necessidades metabólicas do animal a uma dada temperatura. Neste ponto, o que o choco usa como fonte energética gerou duas teorias. A primeira considera que, sob condições normais de alimentação, tanto o crescimento como a energia resultam da metabolização da fracção proteica (Lee, 1994), enquanto que a segunda considera a fracção dos carbohidratos como fonte de energia e a fracção proteica para crescimento (Storey & Storey, 1983; Hochachka, 1994). Apesar do choco partilhar um processo de digestão similar ao dos vertebrados (que inclui digestão extracelular e intracelular), qualquer uma das teorias contrasta com o que se passa nos vertebrados, nos quais a fonte energética é de base lipídica, e com o que acontece nas diferentes fases de vida do choco.

 

Este assunto deve ser clarificado, para todas as fases de vida, devido à importância para o design da dieta, uma vez que pode implicar a inclusão de carbohidratos na mesma. A solução mais adequada é entender o processo de digestão da espécie e quais as enzimas intervenientes no mesmo. O choco possui uma digestão altamente eficaz (Boucher-Rodoni et al. 1987). Uma vez que as enzimas disponíveis podem variar de acordo com a fase de vida, tal como as células presentes na glândula digestiva, existe a necessidade de as estudar nas fases de “hatchling”, juvenil e adulto. Tal facto deve-se a que o sistema digestivo do choco matura durante os primeiros 30 dias pós-eclosão. Também é vital determinar o conteúdo nutricional da presa mais consumida, a camarinha Palaemonetes varians, em termos de proteína e carbohidratos. Esta espécie atingiu o estatuto de presa modelo devido aos bons resultados obtidos no cultivo do choco (Sykes et al. 2006b).

 

Embora alguns estudos anteriores se tenham focado no conteúdo enzimático da glândula digestiva do choco, estes são bastante antigos e não estavam directamente relacionados com o conhecimento da performance de assimilação de ambas as fracções de proteína e de carbohidratos, para além de terem sido realizados com animais de outras regiões. Tendo em conta a localização geográfica, os dados biológicos existentes apontam para metabolismos diferentes devido à temperatura (Sykes et al. 2006a and 2006b). Por conseguinte, podem verificar-se actividade e quantidade de enzimas distintas ou mesmo enzimas diferentes no choco do Sul de Portugal, que resultariam de uma presa natural diferente. Por esta razão, irão ser realizados estudos usando enzimas semelhantes.

 

Após determinar quais as enzimas presentes usando uma dieta normal, então a melhor maneira de identificar os fluxos metabólicos possíveis será através do uso de marcadores radioactivos. Conceição et al. (2007) fizeram recentemente uma revisão acerca este tipo de métodos, e pensamos ser capazes de conseguir adaptar algumas metodologias ao choco.

 

Com base em tudo o que foi referido em cima, os objectivos da actual proposta são: determinar o conteúdo nutricional e energético da camarinha, identificar a actividade enzimática durante a digestão e estabelecer as potenciais vias metabólicas. A execução dos objectivos desta proposta irá permitir não só uma melhor compreensão da digestão do choco, bem como a determinação de um correcto design de uma dieta artificial a ser testada no futuro.

RESEARCH PLAN AND METHODS

 

This proposal aims to understand the metabolism of the European cuttlefish, Sepia officinalis. This is particularly important for the correct development of a prepared diet for the species, which was identified as major bottleneck and it is preventing the culture at an industrial scale (Sykes et al. 2006a). This diet must supply all the metabolic needs of the animal, promoting normal growth and survival.

 

Unfortunately, data regarding the physiology of the species is scarce and, where it does exist, is ambiguous. For instance, information regarding the species’ metabolism is contradictory on whether cuttlefish uses carbohydrates as fuel for energy or not. On the other hand, possible different physiology between animals from other geographical locations have been suggested (Sykes et al. 2009). Therefore, the challenge in the current proposal is to understand the physiology and metabolism of a species which was never studied, at least for southern Portugal populations. This is particularly important as most of the work on the development of cuttlefish culture was conducted with animals from this geographical location and it is known that metabolism might change due to higher temperature and different prey ingested. In addition, the fact that local animals display high growth rates and short life cycles (2 generations per year: 3 months during spring-summer and 9 months during autumn-winter) suggests a plasticity of cuttlefish which is temperature driven.

 

Considering all these aspects, we gathered a workgroup with CCMAR researchers that combine expertise in biology, aquaculture nutrition and animal physiology, to use a multidisciplinary approach on the subject. Firstly, we will characterize the nutritional and energetic contents of a natural prey, the grass shrimp Palaemonetes varians, which has been used in cuttlefish culture with success for consecutive generations (Sykes et al. 2006b).Secondly, we will characterize the activity of enzymes present in cuttlefish digestive gland under different feeding regimes and for the three life stages. Thirdly, we will develop a methodology that will allow us to conduct studies with radiolabelled markers, therefore enabling the identification of possible metabolic pathways. These approaches are therefore presented as tasks. To accomplish all these tasks, both biological knowledge of the studied species plus the knowledge in the field of aquaculture nutrition and physiology of fish species will be considered through collaboration between the team members. This is particularly important since cuttlefish shares similarity in some physiological aspects with marine vertebrates, such as extracellular and intracellular digestion.

 

The nutritional characterization of grass shrimp will be determined with standard methods for this type of study, such as: a) the Lowry et al. (1951) for total protein; b) the Waters PicoTag method, using the conditions described by Cohen et al. (1989), for the determination of both free and protein amino acids; c) the methods described in Le Bihan et al. (2006) for total, low and high molecular-weight carbohydrates; d) Christie (1982) for total lipid; e) Horwitz (1980) for moisture; and f) the calorimetric pump methodology of Parr©. After these determinations we will know the correct ratios of protein:carbohydrate:lipid of juvenile and adult grass shrimp. We will also have information on the available amino acids, mineral fraction and energetic content of this model prey. This information is vital for the elaboration of a prepared diet for cuttlefish and, until now, no one has ever fully described the grass shrimp nutritional profile as cuttlefish prey. On the other hand, this knowledge will be used by comparing results with those obtained by enzyme determinations. The information gathered during this task (T2) is therefore considered as a milestone in the project.

 

The enzyme activity will be conducted according to feeding regime and life stage. Some enzymes have already been determined in cuttlefish from different geographical locations and this will be taken into account on the methods used for assess enzyme activity. In this way, the generatlist acid and alkaline phosphatase, total acid and alkaline proteases, trypsin, chymotrypsin, lipase and amylase activity will be determined according to protocols of Ribeiro et al. (2002), Perrin et al. (2004) and Le Bihan et al. (2006). Data from these determinations will allow a better knowledge on the activity of cuttlefish digestive enzymes resulting from modulation by diet composition or fastening. This will contribute for the understanding of digestive process in the species at given life stages and will allow some initial inferences on the type of metabolism of the species. . The information gathered during this task (T3) is therefore considered as a milestone in the project.

 

As for the determination of possible pathways, this will be conducted by adapting the tube-feeding methodology of fin-fish for cuttlefish and/or, alternatively, to establish labelling of live food as described in Conceição et al. (2007), with modifications due to the particular digestive apparatus of the species that includes a beak.

 

Tube-feeding will use a radio-labelled nutrient (14C-labeled), followed by quantification, after some hours, of the tracer that is present in faeces, retained in tissues and catabolised. However, the use of this method is undermined by the beak of cuttlefish, thus complicating the use of a tube-feeding technique. Nonetheless, we will try different types of tube (plastic and metallic) and determine the accuracy of this modification. The labelling of food, using either live or dead P. varians individuals is an alternative method that according to Conceição et al. (2007), may be used to characterize digestion, absorption, metabolism or retention of dietary nutrients such as amino acids, as far as the approach that is used to label the live prey is considered for losses. Feed intake can then be estimated using food labelled with radioactive isotopes, in order to study factors impinging on feed intake regulation and to improve knowledge on the effect of feed intake in nutritional requirements and digestive physiology. Based on the above considerations for the development of an acceptable tracer method, the accomplishment of this part of the task T4 is considered a milestone for the project.

 

After establishing an acceptable adapted methodology, we will determine overall amino acid metabolism by using a mixture of radio-labelled amino acids (L-[U-14C]-protein hydrolysate, containing the 20 amino acids used for protein synthesis). As for the metabolism of a given amino acid, L-[U-14C]- tracers will be used to characterize those selected from the previous determination. The carbohydrate metabolism will be assessed by quantifying glucose kinetics through the use of [U-14C]glucose. This will expose the glucose catabolism used to synthesize ATP.

 

The final milestone refers to the good completion of this project.

 

At the end of this project, we expect to have achieved our main objective of clarifying the role of carbohydrates and protein in the physiology of cuttlefish from southern Portugal. This is particularly important not only for the basic knowledge in terms of physiology of the species but will also be of extreme importance for the correct design of a cuttlefish prepared diet that will allow a similar growth and survival rates in captivity as seen in the wild.

 

This will be the first study that addresses cuttlefish metabolism instead of assuming a determined physiology, thus contributing to the knowledge of this species. Data resulting from this project, regarding both prey nutritional and energetic content and cuttlefish physiology will be used as information for the elaboration of a dynamic mechanistic model for the species at different life stages. The PI has been working in this model for the species and other cephalopods with Spanish researchers and the results of the present proposal will dramatically increase the possibility of elaborating a well designed prepared diet for each life stage.

BIBLIOGRAPHY

 

Boucher-Rodoni, R., Boucaud-Camou, E. & Mangold, K. (1987). Feeding and digestion. In: Cephalopod Life Cycles (Boyle, P.R. ed.). Vol.2 – Comparative Reviews, pp. 85-108. Academic Press, London, UK.

 

Christie, W.W. (1982) Lipid Analysis, 2nd edn. Pergamon Press, Oxford, UK.

 

Cohen, S.A., Meys, M. & Tarvin, T.L. (1989) The Pico-Tag Method - A Manual of Advanced Techniques for Amino Acid Analysis. Waters, Bedford, USA.

 

Conceição, L.E.C., Morais, S. & Rønnestad, I. (2007). Tracers in fish larvae nutrition: A review of methods and applications. Aquaculture 267, 62–75.

 

Domingues, P.M., Poirier, R., Dickel, L. Almansa, E. Sykes, A. and Andrade, J., (2003a). Effects of culture density and live prey on growth and survival of juvenile cuttlefish, Sepia officinalis. Aquaculture International 11, 225-242.

 

Domingues, P.M., Sykes, A., Sommerfield, A., & Andrade, J.P. (2003b). The effects of feeding live or frozen shrimp on growth, survival and life cycle of the cuttlefish, Sepia officinalis (Linnaeus, 1758). Aquaculture International 11, 397-410.

 

Hochachka, P.W. (1994). Oxygen efficient design of cephalopod muscle metabolism. Mar. Fresh. Behav. Physiol. 25, 61-67.

 

Horwitz, W. (1980) Methods of Analysis, 13th edn. Association of Official Analytical Chemists, Washington D.C., USA.

 

Le Bihan, Perrin, A. & Koueta, N. (2006). Influence of diet peptide content on survival, growth and digestive enzymes activities of juvenile cuttlefish Sepia officinalis. Vie et Milieu 56(2), 139-145.

 

Lee, P.G. (1994). Nutrition of cephalopods: fuelling the system. Mar. Freshw. Behav. Physiol. 25, 35-51.

 

Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265– 275.

 

Perrin, A., Le Bihan, E. & Koueta, N. (2004). Experimental study of enriched frozen diet on digestive enzymes and growth of juvenile cuttlefish Sepia officinalis L. (Mollusca Cephalopoda). J. Exp. Mar. Biol. Ecol. 311, 267-285.

 

Ribeiro, L., Zambonino-Infante, J.L., Cahu, C. & Dinis, M.T.(2002). Digestive enzymes profile of Solea senegalensis post larvae fed Artemia and a compound diet. Fish Physiology and Biochemistry 27, 61–69.

 

Storey, K.B. & Storey, J.M. (1983). Carbohydrate Metabolism in Cephalopod Molluscs in The Mollusca, Vol. I – Metabolic Biochemistry and Molecular Biomechanics. Academic Press, Inc. pp: 91-136.

 

Sykes, A.V., Almansa, E., Lorenzo, A. & Andrade, J.P. (2009). Lipid characterization of both wild and cultured eggs of cuttlefish (Sepia officinalis) throughout the embryonic development. Aquaculture Nutrition 15, 38-53.

 

Sykes, A.V., Domingues, P.M. & Andrade, J.P. (2006b). Effects of using live grass shrimp (Palaemonetes varians) as the only source of food for the culture of cuttlefish, Sepia officinalis (Linnaeus, 1758). Aquaculture International 14(6), 551-568.

 

Sykes, A.V., Domingues, P.M., Correia, M., & Andrade, J.P. (2006a). Cuttlefish culture – state of the art and future trends. Vie et Milieu 56(2), 129-137.