Aplicaciones de los anfípodos (Crustacea: Peracarida: Amphipoda) en acuicultura.

Laboratorio de Biología Marina Facultad de Biología Universidad de Sevilla Uso de los anfípodos (Crustacea: Peracarida: Amphipoda) en acuicultura Use of amphipods (Crustacea: Peracarida: Amphipoda) in aquaculture Pablo Jiménez Prada Sevilla, 2018 Los directores Dr. José Manuel Guerra García, catedrático del Departamento de Zoología de la Universidad de Sevilla y Dr. Ismael Hachero Cruzado, investigador del Instituto Españos de Oceanografía de Vigo. INFORMAN: Que esta Memoria de Investigación, titulada “Uso de los anfípodos (Crustacea: Peracarida: Amphipoda) en acuicultura”, fue realizada por Pablo Jiménez Prada bajo su dirección, en el Departamento de Zoología de la Universidad de Sevilla. Considerando que reúne las condiciones necesarias para constituir un trabajo de Tesis Doctoral, autorizan su defensa ante los miembros del Tribunal para optar al titulo de Doctor. Sevilla, a 09 de mayo del 2018 Director (1) Director (2) Fdo. José Manuel Guerra García Fdo. Ismael Hachero Cruzado La presente tesis ha sido financiada gracias al Proyecto de Excelencia de la Junta de Andalucía (P11-RNM-7041) con contrato predoctoral asociado. Parte de la tesis se realizó gracias a la Beca de Movilidad del V Plan Propio de la Universidad de Sevilla conseguida por el doctorando. Agradecimentos - Hola, buenas, soy alumno de la universidad de La Laguna, estuve en la planta de visita y me gustaría poder hacer prácticas de empresa con los pulpos. - Vale, ¿Cuándo terminas los exámenes? - Dijo una voz ronca al otro lado del teléfono. - El 6… - Po’vente el 7. – Sentenció con acento andalú. De esta forma, hace casi una década conocí a Eduardo. A él le doy las gracias por abrirme las puertas de la investigación, por tratarme como a un biólogo más sin haber terminado la carrera, por darme la oportunidad de ejercer mi actual profesión e ilusión, y mandar mi primer póster que sería después ¡mi primer artículo! Y, por supuesto, que me llevara al chiringuito del puerto a comer bocadillos de mejillones, pollito y atún ¡qué sería Tenerife sin ellos! ¡O sin Ricardo! “El sureño más internacional del globo”, de risa contagiosa y conversación interminable, con quien mejor me lo he pasado en el trabajo y volveré a pasarlo, estoy seguro. No puedo hablar de Tenerife sin acordarme de “my lab-Darling”, mi querida Sarita, “mi niña” de sonrisa y lágrima fácil que, junto a su mini-familia (Ana, Cova, José, Lupe y Marga), me hicieron sentir como uno más en todo momento. Si hablamos de familia, al entrar en el departamento encontré una. Cómo no mencionar a mi Pili pili, la mujer que puso su vida en mis manos saltando al vacío y de la que heredé su mesa; Maca con sus eternas anécdotas; y, como no, “La Grinch” del departamento que tantas buenas tardes me hizo pasar, quién me diría que después su tesis sería mi biblia ¿verdad, Elena? No haré ningún comentario sobre la Beca de Colaboración y cómo algún “hombre de poca fe” siempre me omitió de todos los emails al respecto, pero no dudó en darme la oportunidad de poder escribir estos agradecimientos. Al entrar en el departamento tuve la suerte de conocer a Sara que, aunque nos abandonara por “sus honguitos”, no duda en sumarse a una peli con pizza (lo de la peli es secundario) y, sin estar, siempre será mi compi de departamento preferida. Con Carlos, conseguí el título de “Rey de lo Tanaidaceos” durante las tardes interminables mientras separabamos bichos en los ratos libres que teníamos entre ver videos de Youtube y comer galletas de dinosaurios. A estos dos individuos solo les puedo decir: os quiero. La familia siguió creciendo y llegó Gemma, mi estómago gemelo y fiel seguidora de nuestro lema THINK IN FAT. Paco y Altai, “The Couple”, con sus increíbles fotos submarinas; Martita, “la reina del Power Point”; Semperepedia, “el maestro Jedi de la estadística”; Rafa, “The Ginger Slave”; y, por último, el imperturbable hombre que todo lo hace bien: Free. Ellos/as son los/as que han hecho sentirme como en casa y les debo gran parte de esta tesis. Gracias, familia. Lo bonito de la etapa predoctoral es que la tesis se vuelve de todos, de los más cercanos, no es algo propio, no se puede hacer sólo, intervienen en el proceso cada persona que conoces, incluso las que se van, pero hay alguien que siempre ha estado desde mucho antes de saber siquiera que es un doctorado, ella es Elvira, esa niña de carácter cortante que desde los 14 años ha ido perfeccionado la habilidad de no insultarme (aunque lo parezca mucho, la verdad) sino de calificarme objetivamente para saber como soy y que, desde la distancia, siempre ha sido un hombro para mí. Anita y Valeria me hicieron descubrir que tenía una hermanita en Sevilla que, aunque no es de sangre, lo es de corazón, y que las uruguayas son las más “dramaqueen” del planeta (sin exagerar, lo prometo) pero las mejores amigas también. Belén hizo que los rincones de Cádiz para mí dejaran de tener nombre y pasaran a tener fechas de cafés y charlas. Haciendo uso de un término muy biológico, gracias a la serendipia, “descubrimiento o hallazgo realizado por accidente, casualidad, inesperado y afortunado, de cosas que no se están buscando ni investigando, pero que son la solución para otro problema que se tenía”, conocí a Eva, mi cojita escenógrafa cortacuentos que, aparte de ser la diseñadora de la magnífica portada, en tan poco tiempo ha conseguido ser tan importante en mi vida. Gracias, Pequeña. Para terminar, agradecerles a mis directores por confiar en mí para este proyecto. A Ismael, por todo lo que me ha enseñado del lipídico mundo de la acuicultura, y a Jose… ¡qué no agradecerle a Jose! Si le dedico a él más líneas que a ninguna otra persona en los agradecimientos, perdonad. Pero cómo se le agradece a un director de tesis que se acuesta a las 2 de la madrugada porque te ha hecho una tarta de cumpleaños, que te llama a cualquier hora del día para continuar la conversación que dejasteis a media, que incluso enfadado transmita cercanía haciéndote mejorar día a día como investigador pero, sobre todo, como persona, y por miles de detalles que lo hacen, sin duda alguna, la mejor persona que conozco. Me siento afortunado de haber sido doctorando tuyo, pero más aun de ser tu amigo. Y, por supuesto, a la persona que me ha enseñado todo lo que sé de la vida, a la que me ha enseñado que con esfuerzo y trabajo se consigue todo, la que me enseñó a reír llorando y a llorar de risa, la mujer de mi vida: mi MADRE. Roads go ever ever on, Over rock and under tree, By caves where never sun has shone, By streams that never find the sea; Over snow by winter sown, And through the merry flowers of June, Over grass and over stone, And under mountains in the moon. The Road goes ever on and on Down from the door where it began. Now far ahead the Road has gone, And I must follow, if I can, Pursuing it with eager feet, Until it joins some larger way Where many paths and errands meet. And whither then? I cannot say. Song of Bilbo Baggins 17 Resumen general La protección costera, el ciclo de nutrientes, el control de la erosión, la purificación del agua y el secuestro de carbono son servicios ecosistémicos proporcionados por los esteros. Además, las salinas ofrecen un hábitat de cría para peces proporcionando además abundantes invertebrados, como anfípodos, potencialmente útiles como recurso en la acuicultura. La harina de pescado y el aceite de pescado son recursos alimentarios necesarios para apoyar la acuicultura de especies carnívoras debido a sus ácidos grasos poliinsaturados de cadena larga (LC-PUFA) omega-3 y omega-6. Actualmente, la acuicultura depende de pesquerías y piensos limitados con niveles elevados de LC-PUFA n-3, pero es necesario desarrollar fuentes de alimentos más sostenibles. Los anfípodos son crustáceos con una alta calidad nutricional y un gran potencial para criarlos como alimento para especies de interés acuícola, juegan un rol fundamental en el intercambio energético de la cadena trófica como recurso de los depredadores, y parecen ser un recurso alternativo potencial de alta calidad para la acuicultura. La presente tesis trata de explorar las siguientes preguntas: i) ¿Cuál es la importancia de los anfípodos en la dieta en especies de interés comercial en acuicultura; ii) ¿Qué composición nutricional tienen las especies de anfípodos de esteros?; iii) ¿Cómo se cultiva y qué análisis nutricional tienen los anfípodos alimentados con diferentes dietas en condiciones de laboratorio? y iv) ¿Es posible utilizar anfípodos como recurso alternativo de alimentos con especies de interés comercial?. Por ello, se realizó un estudio nutricional para varias especies principales de anfípodos -Microdeutopus gryllotalpa, Monocorophium acherusicum, Gammarus insensibilis, Melita palmata y Cymadusa filosa- en esteros del sur de España. Estas especies mostraron un alto contenido de proteína (hasta 40%), altos niveles de PUFA n-3 y fosfolípidos, altos niveles de fosfatidilcolina (PC), fosfatidiletanolamina (PE) y triacilgliceroles (TAG). Isoleucina, glicina y alanina fueron los aminoácidos dominantes en todas las especies. Además, los anfípodos recolectados en estanques mostraron bajos niveles de metales pesados. 18 Por lo tanto, los anfípodos de esteros son buenos candidatos para ser utilizados como alimento y se proponen como un nuevo recurso económico sostenible para ser utilizado en la acuicultura. De las especies estudiadas, G. insensibilis puede ser la mejor para el cultivo intensivo como recurso de alimentación alternativo porque muestra: 1) composición adecuada de PUFA n-3 y fosfolípidos; 2) altos niveles de glicina, alanina, tirosina, isoleucina y lisina; 3) altas densidades naturales; 4) tamaño grande (≥1 cm) y 5) alta concentración de calcio. Además, una acuicultura combinada de anfípodos y peces en los estanques de esteros parece una forma prometedora y ambientalmente sostenible de desarrollar un sistema de Acuacultura Multitrófica Integrada (IMTA) en estos ecosistemas. Se utilizaron dos modelos de anfípodo para el cultivo, los caprélidos Caprella equilibra y C. scaura y el gammarideo Gammarus insensibilis. En ambos casos, se ensayaron distintas dietas, Artemia, fitoplancton y detritus (consistente en heces y resto de pienso, deshechos de tanques de acuicultura). Además, se añadió Ulva en el experimento de G. insensibilis. Los caprélidos adultos alimentados con detritus mostraron una tasa de supervivencia significativamente más alta, y aquellos alimentados con fitoplancton y detritus fueron más ricos en ácidos grasos poliinsaturados, especialmente DHA. Curiosamente, los caprélidos alimentados con detritus también fueron una fuente rica de LA (18:2-n6), considerada como un ácido graso esencial en los vertebrados. Se encontró que los desechos basados principalmente en heces y restos de pienso no consumidos pueden considerarse un alimento adecuado para los caprélidos adultos, proporcionando una fuente de ácidos grasos omega-3 (DHA) y omega-6 (LA). En el estudio con gammarideos, G. insensibilis se cultivó durante 21 días. Tanto los alimentados con detritus y Ulva mostraron tasas de supervivencia interesantes y contribuyeron a altas concentraciones de ácido palmítico (16: 0), ácido oleico (18: 1n9), ácido araquidónico (20: 4n6) (ARA), ácido eicosapentaenoico (20: 5n3) (EPA) y ácido docosahexaenoico (22: 6n3) (DHA). Además, Gammarus insensibilis podría desempeñar un papel clave como biorremediador siendo cultivado con detritus y Ulva obteniendo una composición bioquímica adecuada para ser utilizada en acuicultura. Este anfípodo podría reemplazar parcial o totalmente la dieta formulada con la consiguiente reducción de los costes económicos. 19 Finalmente, se realizaron dos experimentos con larvas de Seriola dumerili (modelo de especie comercial acuícola) de 22 y 44 días después de la eclosión. Se alimentaron con el anfípodo Gammarus insensibilis, procedente de esteros, y con una dieta formulada. Se midieron supervivencia, biometría y perfil nutricional de las larvas. En el primer experimento, el tratamiento con G. insensiblis mostró una mejor supervivencia, aunque los otros parámetros no fueron diferentes. Por otro lado, en el segundo experimento la supervivencia fue similar, el crecimiento fue mejor en la dieta comercial, pero los alimentados con el anfípodo tuvieron un mejor perfil químico. El tratamiento con G. insensibilis mostró valores más altos de ARA (5,53 ± 0,18%) y DHA (19,07 ± 0,19%), niveles bajo de TAG (2,18 ± 0,5%) y una coloración similar a la de los juveniles silvestres de S. dumerili. Como conclusión, la presente tesis muestra el alto valor nutricional de los anfípodos tanto en el medio natural como cultivados, y su gran potencial para poder ser usado como recurso trófico en acuicultura. Es interesante explorar futuras iniciativas en el contexto de la Acuicultura Multitrófica Integrada, donde los anfípodos parecen ser un recurso muy adecuado, pues puede combinarse su uso como biofiltros y una producción a gran escala con un coste muy reducido a partir de los desechos de cultivos asociados General summary Coastal protection, nutrient cycling, erosion control, water purification, and carbon sequestration are ecosystem services provided by salt marshes. Additionally, salt ponds offer coastal breeding and a nursery habitat for fishes and they provide abundant invertebrates, such as amphipods, which are potentially useful as a resource in aquaculture. Fishmeal and fish oil are necessary food resources to support aquaculture of carnivorous species due to their omega-3 and omega-6 long-chain polyunsaturated fatty acids (LC-PUFA). Currently aquaculture depends on limited fisheries and feed with elevated n-3 LC-PUFA levels, but the development of more sustainable food sources is necessary. Amphipods are crustacean with high nutritional quality and great potential to be reared as food for species of aquaculture interest. They play a fundamental rol in energetic exchange of trophic chain as predators’ resource. They appear to be a potential high quality alternative feed resource for aquaculture. 20 The present thesis tries to explore the next questions: i) How important are amphipods in diet of species of commercial interest in aquaculture; ii) What nutritional composition have the amphipods species terrestrial ponds?; iii) How can be cultured and what nutritional value have the amphipods fed with different diets under laboratory conditions? and iv) Is it possible use the amphipod as alternative food recourse with species of commercial interest?. Hence, a nutritional study was carried out for several main amphipod species -Microdeutopus gryllotalpa, Monocorophium acherusicum, Gammarus insensibilis, Melita palmata and Cymadusa filosain terrestrial ponds in the South of Spain. These species showed high protein content (up to 40%), high n-3 PUFA and phospholipid levels, and high levels of phophatidylcholine (PC), phosphatidylethanolamine (PE) and triacylglycerols (TAG). Isoleucine, glycine and alanine were the dominant amino acids in all species. In addition, amphipods collected from ponds showed low levels of heavy metals. Therefore, pond amphipods are good candidates to be used as feed and are proposed as a new sustainable economic resource to be used in aquaculture. Of the five studied species, G. insensibilis may be the best for intensive culture as an alternative feed resource because it shows: 1) adequate n-3 PUFA and phospholipids composition; 2) high levels of glycine, alanine, tyrosine, isoleucine and lysine; 3) high natural densities; 4) large body size (≥1 cm), and 5) high concentration of calcium. Moreover, a combined culture of amphipods and fishes in these marsh ponds seems a promising and environmentally sustainable way to develop Integrate Multi-Trophic Aquaculture (IMTA) in these ecosystems. Two models of amphipod were used to be cultured, caprellids (Caprella equilibra and C. scaura) and gammarideans (Gammarus insensibilis). In both experiment Artemia, Phytoplankton, Detritus were used as food. In addition, Ulva was added in G. insensibilis experiment. Caprellids showed an adult survival rate significantly higher for caprellids fed with Detritus, and those fed with Phytoplankton and Detritus were richer in polyunsaturated fatty acids, especially DHA. Interestingly, caprellids fed with Detritus were also a rich source of LA (18:2-n6), considered to be an essential fatty acid in vertebrates. It was found that detritus based mainly on fish faeces and uneaten feed pellets can be considered an adequate food for adult caprellids, providing a source 21 of both omega-3 (DHA) and omega-6 (LA) fatty acids. In the study with gammarideans, G. insensibilis was cultured for 21 days. When fed with Detritus and Ulva gammarideans also showed interesting survival rates and contributed to high concentrations of palmitic acid (16:0), oleic acid (18:1n9), arachidonic acid (20:4n6) (ARA), eicosapentaenoic acid (20:5n3) (EPA) and docosahexaenoic acid (22:6n3) (DHA). Furthermore, Gammarus insensibilis could play a key role as biorremediator being cultured with detritus and Ulva getting an adequate biochemical composition to be used in aquaculture. This amphipod could replace partial or totally the formulated diet with two consequences, no dependence of fish oil and reduction of economic costs. Finally, two experiments were done with Seriola dumerili larvae (model of commercial aquaculture species) of 22 and 44 days after hatchery. They were feed with the amphipod Gammarus insensibilis, collected from a terrestrial pond, and one formulated diet. Survival, biometry measures and chemical profile were measured. In the first experiment, G. insensiblis treatment showed better survival, although the other parameters were no different. On the other hand, in the second experiment the survival was similar, the growth was better in formulated diet but those fed with amphipod had the best chemical profile. Juveniles fed with G. insensibilis showed higher values of ARA (5.53±0.18%) and DHA (19.07±0.19%), low level of TAG (2.18±0.5%) and a coloration more similar to wild juveniles of S. dumerili. In conclusion, this thesis shows the high nutritional value of amphipods both in the wild and cultivated, and its great potential to be used as new trophic resource in aquaculture. It is interesting to explore future initiatives in the context of Integrated Multitrophic Aquaculture, where amphipods appear to be a very suitable resource, since their use as biofilters and a large-scale production can be combined at a very low cost. Capítulo 1Introducción general Introducción general 25 1. Introducción general La relevancia de la acuicultura radica en la importancia del consumo de productos acuáticos en las dietas equilibradas al ofrecer proteína de alta calidad, contener todos los aminoácidos esenciales y ser fácilmente digeribles (Kaushik and Seiliez, 2010). Además, son especialmente indicados por su alto contenido en ácidos grasos esenciales omega3 (EPA y DHA) y omega6 (ARA) (Tocher, 2010), vitaminas (D, A y B) (NRC, 2011) y minerales (calcio, iodo, zinc, hierro y selenio) (Watanabe et al., 1988,1997). Tradicionalmente la totalidad de los productos acuícolas han procedido de la actividad extractiva, pero desde los años 80 se estabilizó en torno a los 90 millones de toneladas anuales, techo definitivo que se había vaticinado para esta actividad (FAO, 2016). Fue precisamente desde la década de los 80’s, cuando se produce el despegue definitivo de la acuicultura. Figura 1. Producción de productos acuícola en el periodo 1950-2015. Fuente: APROMAR (2017). Tal como se muestra en la figura 1 el máximo se alcanza en 2015, produciéndose 106 millones de toneladas y superando en 12,3 millones a la cantidad extraída por la actividad pesquera la cual ese mismo año la actividad extractiva alcanzó también su máximo (93,7 millones de toneladas) (FAO, 2016). Capítulo 1 32 FAO, 2016. The State of World Fisheries and Aquaculture (SOFIA). Rome: Food and Agriculture Organization of the United Nations. Gonzalez-Silvera D, Izquierdo-Gomez D, Fernandez-Gonzalez V, Martínez- López FJ, López-Jiménez JA, Sanchez-Jerez P. 2015. Mediterranean fouling communities assimilate the organic matter derived from coastal fish farms as a new trophic resource. Marine Pollution Bulletin, 91: 45–53 Grabowski M, Bacela K and Konopacka A. 2007. How to be an invasive gammarid (Amphipoda: Gammaroidea) - comparison of life history traits. Hydrobiology 590: 75-84. Guerra-García JM, Martínez-Pita I, Pita ML. 2004. Fatty acid composition of the Caprellidea (Crustacea: Amphipoda) from the Strait of Gibraltar. Scientia Marina, 68(4): 501-510 Guerra-García JM, Tierno De Figueroa JM, Navarro-Barranco C, Ros M, Sánchez-Moyano JE and Moreira J. 2014. Dietary analysis of the marine Amphipoda (Crustacea:Peracarida) from the Iberian Peninsula. Journal of Sea Research, 85: 508-517. Hamre K, Yúfera M, Rønnestad I, Boglione C, Conceição L- EC, Izquierdo MS. 2013. Fish larval nutrition and feed formulation: knowledge gaps and bottlenecks for advances in larval rearing. Review in Aquaculture 5: S26–S58. Kaushik SJ and Seiliez I. 2010. Protein and amino acid nutrition and metabolism in fish. Aquaculture Research 41:322-332 Legezynska J, Kedra M and Walkusz W. 2012. When season does not matter: summer and winter trophic ecology of Arctic amphipods. Hydrobiologia, 684: 189-214. Moren M, Suontama J, Hemre GI, Karlsen Ø, Olsen RE, Mundheim H and Julshamn K. 2006. Element concentrations in meals from krill and amphipods. Possible alternative protein source in complete diets for farmed fish. Aquaculture 261: 174-181. Nelson WG. 1979. Experimental studies of selective predation on amphipods: consequences for amphipod distribution and abundance. Journal Experimental of Marine Biology and Ecology 38: 225-245. Nelson WG. 1978. The community ecology of seagrass amphipods: predation and community structure, life histories, and biogeography. Tesis doctoral. Duke University. Introducción general 33 O’Gorman EJ, Enright RA and Emmerson MC. 2008. Predator diversity enhances secondary production and decreases the likehood of trophic cascades. Oecología, 158: 557-567. Serrano A, Velasco F, Olaso I and Sánchez F. 2003. Macrobenthic crustaceans in the diet of demersal fish in the Bay of Biscay in relation to abundance in the environment. Sarsia, 88(1): 36-48. Shpigel M, Guttman L, Shauli L, Osintsov V, Ben-Ezra D and Harpaz S. 2017. Ulva lactuca from an Integrated Multi-Trophic Aquaculture (IMTA) biofilter system as a protein supplement in gilthead seabream (Sparus aurata) diet. Aquaculture. 481:112-118. Tocher, D. R. (2010). Fatty acid requirements in ontogeny of marine and freshwater fish. Aquaculture Research 41(5):717-732. Watanabe T, Kiron V and Satoh S. 1997. Trace minerals in fish nutrition. Aquaculture 151:185-207. Watanabe T, Satoh S and Takeuchi T. 1988. Availability of minerals in fish meal to fish. Asian Fisheries Association. 1:175-1795. Woods C. 2009. Caprellid amphipods: An overlooked marine finfish aquaculture resource? Aquaculture 289: 199–211 Introducción general 35 Objetivos generales Los objetivos del presente estudio se han abordado en forma de capítulos, con los siguientes contenidos: • Capítulo 2. Revisión bibliográfica de la importancia de los anfípodos en especies de interés comercial en la acuicultura: Se ha revisado la dieta de varias especies de interés acuícola, centrándonos en la importancia de los anfípodos, con el objetivo de evaluar su uso como alimento para estas especies, bien como alimento vivo o como componente de dietas inertes. • Capítulo 3. Descripción de la composición nutricional de diferentes especies de anfípodos de esteros: Análisis bioquímico de cinco especies de anfípodos, describiéndose la composición general (proteínas, lípidos, carbohidratos, y cenizas) así como análisis de su composición especifica de aminoácidos, clases lipídicas y ácidos grasos, con el objetico de elegir la especie más adecuada para el uso en acuicultura teniendo en cuenta además su tamaño, abundancia y su accesibilidad. • Capítulo 4. Ensayo de cultivo y análisis nutricional de anfípodos alimentados con diferentes dietas en condiciones de laboratorio para explorar su potencialidad en Acuicultura Multitrófica Integrada. Se llevaron a cabo dos experiencias diferentes, una con caprélidos y otra con gammarideos: Capítulo 4.1. Cultivo de especies de los caprélidos Caprella scaura y Caprella equilibra. Capítulo 4.2. Cultivo del gammarideo Gammarus insensibilis. • Capítulo 5. Aplicación y comparación del anfípodo Gammarus insensibilis con dietas comerciales en especies de interés comercial. Seriola dumerili como modelo de estudio. Comparación de la calidad nutricional en dos etapas del desarrollo, larvas y juveniles, de S. dumerili alimentados con liofilizado de G. insensibilis precedente de los esteros y pienso comercial. Capítulo 2Importancia de los anfípodos en la dieta de especies de interés acuícola del litoral andaluz. Adaptado de: Jiménez-Prada P, Hachero-Cruza- do I y GuerraGarcía JM. 2015. Importancia de los anfípodos en la dieta de especies de interés acuícola del litoral andaluz. Zoologica baetica, 26: 3-29 Autores de las fotografias de este capítulo: Dra. Maite Vázque-Luis Dr. Pablo Arechavala Resumen Los anfípodos son crustáceos con un alto valor nutricional y gran potencial para ser cultivados como alimento en especies de interés acuícola. Juegan un papel fundamental en el intercambio energético de la cadena trófica como recurso de muchos depredadores. Para evaluar la importancia de este grupo, se han revisado los estudios sobre alimentación de 10 especies (ocho peces y dos cefalópodos) del litoral andaluz cultivadas o con interés en acuicultura. Los anfípodos están presentes en la dieta de especies ya consolidadas en acuicultura como Sparus aurata, Dicentrarchus labrax y Solea senegalensis, aunque no se usan en ninguna de las fases de su cultivo intensivo. Existen otras muchas especies en las que los anfípodos podrían jugar un papel fundamental en su cultivo, como son Sepia officinalis, Octopus vulgaris, Solea solea, Mullus surmuletus y M. barbatus, además del género Hippocampus, de gran interés en acuariofilia. Es necesario intensificar la investigación de estos crustáceos para evaluar adecuadamente su importancia como presas en el medio natural y para cuantificar su valor nutricional como fuente alternativa de alimento. Abstract Amphipods are crustacean with high nutritional quality and great potential to be reared as food for species of aquaculture interest. They play a fundamental rol in energetic exchange of trophic chain as predators’ resource. To evaluate the importance of this group, the feeding habits of 10 species (eight finfish and two cephalopods) of Andalusian coast have been reviewed. Amphipods are present in diet of consolidated species in aquaculture like Sparus aurata, Dicentrarchus labrax and Solea senegalensis, althought they are not used in any production step. There are a lot of other species where amphipods could play an important rol in their intensive cultures, like Sepia officinalis, Octopus vulgaris, Solea solea, Mullus surmuletus and M. barbatus, in addition to the genus Hippocampus, with great interest in the field of fishkeeping. It is necessary to intensify research of these crustacean to evaluate correctly their importance as preys in the wild and to quantify their nutritional values as an alternative food source. Importancia de los anfípodos 41 Importancia de los anfípodos en la dieta de especies de interés acuícola del litoral andaluz Jiménez-Prada, P.1,2*, Hachero-Cruzado, I.2 and Guerra-García, J.M.1 1Laboratorio de Biología Marina, Dpto. Zoología, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes 6, 41012 Sevilla, España. 2IFAPA — El Toruño, Camino Tiro Pichón s/n, El Puerto de Santa María, España. * Corresponding author: Dpto. Zoología. Avda. Reina Mercedes 6. 41012 Sevilla, España. Teléfono: +34-954556229. E-mail: [email protected] Este capítulo tiene como objetivo dar respuesta a la pregunta inicial: ¿pueden ser los anfípodos un buen alimento para los peces y cefalópodos? Para ello, se realizó una revisión bibliográfica sobre la dieta de diferentes especies marinas, con especial atención a las especies con interés acuícola del litoral andaluz, sur de España. 1. Especies actualmente en producción acuícola • Dicentrarchus labrax, Linneo 1758 (Fig. 1) Nombre común: Lubina, robalo. Interés comercial e importancia en la acuicultura D.labrax es una de las especies comerciales más abundantes en las costas atlánticas. De hecho, la lubina y la dorada (Sparus aurata) son las dos especies en las que se basa la acuicultura del Mediterráneo. Después del salmón, la lubina fue la primera especie en ser cultivada, debido a su comportamiento en etapas juveniles, habitando estuarios y lagunas costeras donde se podían recolectar los ejemplares y dejar crecer confinados en estructuras naturales o creadas por el hombre, como por ejemplo los esteros de Cádiz. Aunque son Francia e Italia las que en la década de los 60 se implicaron en poder producir esta especie a gran escala (Moretti et al., 1999). Su cultivo intensivo se inició en la década de los 80, pero fue a finales de la década siguiente cuando la producción piscícola sufrió una aceleración debido al impulso de la Unión Europea mediante ayudas a la investigación y creación de empresas (Büke, 2002). Actualmente la extracción pesquera supone un volumen constante del 7 Capítulo 2 48 Importancia de los anfípodos en la dieta El salmonete de roca se alimenta de decápodos, anfípodos, poliquetos, misidáceos y peces (Labroupoulou et al., 1997; Mazzola et al., 1999). En los estudios de Labroupoulou et al. (1997) en aguas del Mediterráneo Oeste los estómagos del salmonete de roca mostraban una alta preferencia por dos grupos, los decápodos y los anfípodos. Ambos explican el 90,06% del IRI (Índice relativo de importancia; Hacunda, 1981). Como grupo, los decápodos son los más consumidos (45,3% IRI), seguidos de los anfípodos (40,1%), pero a nivel de especie son dos anfípodos los más frecuentes en los estómagos del salmonete, Apherusa chiereghinii, Giordani- Soika 1949 (45.9%) y Dexamine spinosa, Montagu 1813 (44.5%). La abundancia relativa de ambos grupos en el medio según la estación del año influye en la frecuencia de aparición. Aunque en el cómputo anual los decápodos son los más importantes, los anfípodos son más consumidos en invierno (51% IRI) y primavera (73,2 % IRI), y los decápodos en verano, coincidiendo con el periodo de reclutamiento de estos (Robertson, 1984). No sólo influye la abundancia relativa en la selección de la presa, ya que en el medio natural los grupos más representativos son los poliquetos y moluscos, y no son tan consumidos, por lo que tamaño, comportamiento, densidad y la abundancia relativa de la presa influyen en la selectividad (Labropoulou et al., 1997). • Solea solea, Linneo 1758 (Fig. 6) Nombre común: Lenguado. Interés comercial e importancia en la acuicultura Especie muy consumida y valorada nutricional y económicamente. La captura mundial en el 2012 fue de 32.746 toneladas, pudiéndose considerar constante desde el año 2000 (FAO, 2014). Solea solea es una especie que actualmente se cultiva en Grecia y España, cuya producción acuícola del año 2010 alcanzó las 125 toneladas, aunque en la actualidad dicha producción ha bajado bruscamente, no llegando a las 50 toneladas en el 2012 (FAO, 2014) exclusivamente en España. Importancia de los anfípodos 49 Hábitat y distribución Es una especie bentónica de fondos arenosos y fangosos, desde aguas superficiales hasta los 300 metros de profundidad, produciéndose la puesta entre los 50 y 100 metros de profundidad entre los meses de enero y abril (Koutsikopoulos et al., 1989), por lo que las larvas habitan desde febrero a mayo en las zonas de cría, esteros y aguas costeras poco profundas (Amara et al., 2001). Los juveniles (hasta 2 años de edad) se quedan en zonas someras hasta pasar al estado adulto en el que emigran a mar abierto (Nicolas et al., 2007). Se distribuye por las costas del Atlántico Este desde los fiordos noruegos a las costas senegalesas y el Mar Mediterráneo (Teixeira y Cabral, 2010). Importancia de los anfípodos en la dieta Los estudios realizados en el estuario del Ebro por Molinero y Flos (1992) describen una alimentación que varía según las estaciones del año en S. solea. Los crústaceos son las presas principales, excepto en otoño que son sustituidos por los poliquetos. Dentro de los crustáceos, los decápodos son las presas más consumidas en invierno, y son los anfípodos los más consumidos en primavera, verano y otoño, consumidos preferentemente por lenguados de 2 y 3 años de edad. En las aguas del Canal de la Mancha, al alcanzar tallas superiores a 50 mm, los juveniles reemplazan los copépodos harpacticoides por poliquetos y anfípodos, estos últimos con una frecuencia ascendente de aparición en los estómagos del 29 al 50% (Amara et al., 2001). Un cambio similar ha sido observado en las costas atlánticas francesas por Castel y Lasserre (1982) y Marchand y Masson (1989). En aguas portuguesas el lenguado en fase adulta consume crustáceos, poliquetos y bivalvos, siendo los anfípodos los crustáceos más consumidos y con mayor importancia en la dieta en primavera y verano (Teixeira y Cabral, 2010), comportamiento alimenticio similar al descrito por Molinero and Flos (1992) en el estuario del Ebro. Capítulo 2 50 • Sepia officinalis, Linneo 1758 (Fig. 7) Nombre común: Choco, Jibia, Sepia. Interés comercial e importancia en acuicultura Debido a su gran distribución, la sepia es una de las especies más capturadas; sólo en el año 2012 se capturaron un total de casi 30.000 toneladas (FAO, 2014). La importancia de la sepia radica en ser una especie capturada tanto por pesquerías de alta mar como por flota artesanal, teniendo gran impacto en la economía local e industrial. En la década de los 90, la sepia empezó a ser investigada como especie objetivo, junto con otras especies de cefalópodos, para ser cultivada en acuicultura (Fuentes y Iglesias, 2001; Sykes et al 2006). Túnez ha sido el único país que logró producir sepia a gran escala, produciendo 23 toneladas entre los años 1990 y 1991 (FAO, 2014), Sin proseguir con la producción de sepia después de ellos. Baeza- Rojano et al. (2010) cultivaron sepia con el uso de gammarideos y caprélidos como alimentación en etapas tempranas del desarrollo de esta especie, con lo que demostraron el posible uso de los gammarideos como fuente de alimento, a falta de experimentar la viabilidad de producción a gran escala. Hábitat y distribución Sepia officinalis es una especie necto-bentónica que ocupa un amplio rango de profundidad desde pocos metros hasta 200 m, en fondos fangosos y arenosos, con mayor abundancia en los 100 metros de profundidad. Tanto la puesta como el crecimiento de los juveniles tienen lugar en aguas poco profundas, donde la hembra fija los huevos al sustrato en verano, desplazándose los juveniles a aguas más profundas en otoño e invierno. En cuanto a su distribución geográfica, ocupa todo el Mar Mediterráneo, y aguas del Atlántico Este, desde Mauritana hasta el sur de Noruega y norte de Reino Unido (Guerra, 2006). Importancia de los anfípodos en la dieta Los anfípodos son parte de la dieta de la sepia, pero exclusivamente de las etapas tempranas. Según Pinczon et al. (2000) los anfípodos son la presa mayoritaria durante los tres primeros meses de vida de S. Importancia de los anfípodos 51 officinalis (tallas comprendidas entre 1,46 ± 0,47 a 4,70 ±,04 cm), formando parte de la dieta en un 59 %, 80%, y 23% del primer al tercer mes, desapareciendo de la dieta en meses posteriores y siendo sustituidos por Carcinus maenas Linnaeus 1758. Este cambio en la dieta también ha sido descrito por otros autores como Guerra (1985) y Le Mao (1985). Blanc y Daguzan (2000), describen las especies más consumidas por S. offinalis desde junio (mes delas primeras puestas) a septiembre; éstas son principalmente una especie de isópodo (Cyathura carinata, Krøyer 1847) y 5 especies de anfípodos, 3 gammáridos (Ampelisca bevicoris, Costa 1853, Marinogammarus marinus,Leach 1815 y Dexamine spiros, Montagu 1813), un talítrido (Orchestia gammarellus) y un caprélido (Phtisica marina, Slabber 1769), siendo esta última la más consumida. En las Ría de Vigo, Castro y Guerra (1990) describen el consumo de presas según el tamaño de la sepia, apareciendo los anfípodos exclusivamente en las tallas menores de 65 mm de longitud de manto, aunque en proporciones más bajas que lo citado anteriormente (4,5%). La desaparición de los anfípodos en la dieta puede ser debido a que dejen de ser rentables desde un punto de vista energético por el aumento de tamaño de la sepia (Pinczon et al., 2000). • Octopus vulgaris, Cuvier 1797 (Fig. 8) Nombre común: pulpo común. Interés comercial e importancia en la acuicultura Especie muy importante en las pesquerías mundiales debido a su distribución (Roper et al., 1984), cuyas capturas han ido descendiendo en las dos últimas décadas (80.247 toneladas desembarcadas en 1991) hasta alcanzar 40.453 toneladas en el año 2012 (FAO, 2014). Junto al descenso de capturas mundial, la gran demanda comercial (Vaz-Pires et al., 2004), su rápido crecimiento y su alta tasa de conversión (Iglesias et al., 2000) hacen del pulpo una especie con gran interés para la acuicultura. Se han realizado multitud de ensayos respecto a la alimentación de las paralarvas (Iglesias et al., 2007) teniendo como factor común el uso de Artemia y zoeas de crustáceos. Únicamente los trabajos de Iglesias et al. (2004) y Carrasco et al. (2006) lograron completar el ciclo de vida de O. vulgaris. El Plan Nacional de Cultivo del Pulpo (Planes JACUMAR), llevado a cabo desde 2001 al 2004, reveló que las mayores dificultades que presenta el cultivo de pulpo son la alta mortalidad y bajo crecimiento de las paralarvas, atribuido a la poca estandarización del cultivo y el Capítulo 2 52 uso de presas con bajos valores de ácidos grasos insaturados, entre otros factores (Iglesias et al., 2007). Navarro and Villanueva (2000, 2003) han demostrado la importancia de estos ácidos grasos (EPA y DHA) en las paralarvas y su alimentación, lo que convierte a los anfípodos en presas potenciales interesantes. Hábitat y distribución Especie bentónica de fondos rocosos, arenosos y de fango, que habita desde la línea costera al borde de la plataforma continental. Comúnmente considerada una especie de distribución cosmopolita de aguas templadas debido a la existencia de especies crípticas, su distribución estricta se considera el Mar Mediterráneo y este del Océano Atlántico (Jereb et al., 2014). Importancia de los anfípodos en la dieta El pulpo es un gran depredador que se alimenta en su fase adulta principalmente de moluscos, crustáceos y peces (Mather, 1991). Existe un gran desconocimiento de la dieta del pulpo en su primera fase de vida, ya que su digestión es externa, por lo que los estudios clásicos de contenidos estomacales son muy difíciles de realizar (Roura et al., 2012). Para conocer qué tipo de animales consumen, Roura et al. (2012) identificaron las presas de las paralarvas utilizando primers específicos de la región 16S de ADN ribosómico de crustáceos para realizar una PCR de las presas potenciales. Se añadieron primers específicos para los copépodos, ya qeu los generales no los amplificaron; quizás la ausencia de bandas correspondientes a los anfípodos pudiera ser debida a esa misma causa. Aun así se han encontrado anfípodos en estómagos de individuos adultos (Smith, 2003). 3. Especies con interés en acuarofilia (género Hippocampus) • Hippocampus spp. (Fig. 9 ) Nombre común: Caballito de mar. Interés comercial e importancia en la acuicultura Importancia de los anfípodos 53 La extracción/producción de especies marinas tiene como objetivo la alimentación, ornamentación, uso industrial o medicinal. Los caballitos de mar, todos pertenecientes al género Hippocampus, son usados en acuariofilia y en la medicina tradicional china (MTC), la cual consume más de 45 toneladas en peso seco anuales de caballitos de mar. Esto crea una demanda cubierta en la actualidad exclusivamente por la extracción del medio, lo que provoca una sobrexplotación en muchas especies de caballitos de mar (Koldewey y Martin-Smith, 2010). La producción a gran escala de caballito de mar empezó a desarrollarse en Estados Unidos, Australia y Nueva Zelanda, con objeto de sustituir parte de la demanda de la MTC y el uso en acuarios. Pero las dificultades para ser competitivo económicamente radican en el bajo coste de los individuos extraídos del medio natural como captura accesoria. Actualmente, las tres prioridades para su cultivo a gran escala consisten en mejorar los tratamientos de salud y enfermedades, mantener poblaciones con independencia de capturas de individuos salvajes y reducir los costes de alimentación, ya que pueden llegar a ser un tercio del coste de producción (Koldewey and Martin-Smith, 2010). En este sentido, se han ensayado dietas congeladas sin resultados positivos, por lo que la búsqueda de presas vivas de bajo coste es de gran relevancia (Woods, 2003). Actualmente el valor de los caballitos de mar vivos varía entre los 100 y 750 dólares la unidad (http:// seahorse.com), por lo que el conocimiento de las dietas naturales de estos peces es fundamental para el crecimiento y producción intensiva de los mismos, no sólo para conseguir un crecimiento más acelerado, sino para evitar mortalidad, deformaciones o mala pigmentación que devalúen el producto. Es el caso de la alta mortalidad de juveniles por aparición de burbujas en los estómagos, asociada a la alimentación con Artemia enriquecida con productos comerciales (Palma et al., 2014). En España, el Instituto de Investigaciones Marinas de Vigo está llevando a cabo la cría y reproducción de varias especies del género Hippocampus a través del proyecto “Hippocampus”, que tiene como objetivo la repoblación de caballitos de mar en el medio natural. Para su alimentación están usando copépodos y Artemia (Planas, 2012); ambas presas no son consumidas en el medio natural (Koldewey and Martin-Smith 2010). Esto conlleva problemas en la alimentación y supervivencia en los primeros días de vida (Planas, 2012), probablemente por el déficit en ácidos grasos poliinsaturados omega-3. Capítulo 2 54 Hábitat y distribución El género Hippocampus tiene una distribució global asociada a aguas cálidas y templadas. En Andalucía podemos encontrar dos especies, Hippocampus guttulatus Cuvier, 1829 e Hippocampus hippocampus Linnaeus, 1758 (Fig. 9). Son especies de escasa capacidad natatoria, cuya estrategia ofensiva es el camuflaje, por lo que suelen estar asociados a un tipo de sustrato, al que imitan, adheridos por la cola, para acercarse a las presas o esperar que entren en el rango de ataque (Woods, 2002). Importancia de los anfípodos en la dieta Tienen una dieta diversa, principalmente constituida por anfípodos, decápodos, y misidáceos (Koldewey and Martin-Smith, 2010). En cuanto a las dos especies de Andalucía, H. guttulatus consume mayoritariamente decápodos (zoeas), misidáceos y anfípodos, con una frecuencia de aparición en los estómagos del 100%, 85,71% y 47,62% respectivamente (Kitsos et al., 2008; Gurkan et al., 2011). Por su parte, H. hippocampus tiene una dieta más diversa, ocupando los anfípodos el segundo puesto de importancia (frecuencia del 21,05%) detrás de los decápodos (larvas) (frecuencia del 26,32%) (Kitsos et al. 2008; Gurkan et al., 2011). Los anfípodos son de gran importancia en varias especies alóctonas de caballitos de mar que podrían ser cultivadas en circuitos cerrados, junto con las especies autóctonas de Andalucía. Por ejemplo, los estudios sobre H. abdominalis (Lesson, 1827) describen una dieta basada en crustáceos. A nivel de grupo son los peracáridos en general y anfípodos en particular los más frecuentes (52,5 y 49,2% respectivamente), y a nivel de especie Caprella equilibra (Say, 1818) ocupa el primer puesto (25.5%). Los anfípodos son más importantes en la dieta de los juveniles que en adultos, y son especialmente consumidos en primavera e invierno (Woods, 2002). También para Hippocampus japonicus (Kaup, 1856) los anfípodos son la principal fuente de alimentación, ya que el 88,4% de la masa total encontrada en su dieta son gammáridos, siendo fundamentales en todo el ciclo de vida, seguidos en orden de importancia por los caprélidos con un 5,9% (Kwak et al., 2004). La revisión de Koldewey y Martin-Smith (2010) aporta más información respecto al papel de los anfípodos en la dieta de otras especies de éste género. El uso de los anfípodos en la dieta de los caballitos de mar podría mejorar la alimentación y crecimiento de estas Importancia de los anfípodos 55 especies pues cumplen con los requisitos nutricionales necesarios y son presas altamente consumidas en el medio natural. Por ello, podría llegarse a cubrir la demanda de estos animales para repoblación, acuariofília o usos en la medicina tradicional china. 4. Conclusiones Las especies de interés acuícola en Andalucía consumen frecuentemente anfípodos en el medio natural, aunque el consumo puede variar a lo largo del desarrollo y estacionalmente. En algunas especies, la frecuencia de aparición de anfípodos en el tracto digestivo puede llegar a superar el 70%, como es el caso de D.labrax, S. senegalensis, M. barbatus, M. surmuletus y S. officinalis. La mayor parte de los trabajos de dieta suelen identificar las presas a nivel de grandes grupos, y son muy pocos los estudios que alcanzan la resolución taxonómica de género o especie. Son necesarios, por tanto, estudios más detallados de las especies de anfípodos más consumidos para orientar adecuadamente los estudios aplicados en el uso de estos crustáceos como recurso alternativo en acuicultura. El uso de los anfípodos, directamente como alimento vivo o formando parte de piensos y harinas, puede generar nuevas líneas de investigación, no sólo por ser consumidos en el medio natural sino por su alto valor nutritivo y su potencial para ser cultivados en condiciones controladas. Importancia de los anfípodos 57 1. Dicentrarchus labrax 2. Solea senegalensis 3. Sparus aurata 4. Mullus barbatus 5. Mullus Surmuletus 6. Solea solea 7. Sepia officinalis 8. Octopus vulgaris 9. Hippocampus sp 1. 2. 3. 4. 5. 6. 7. 8. 9. Figuras 1-9. 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Caprellid amphipods: An overlooked marine finfish aquaculture resource? Aquaculture, 289: 199-211. Capítulo 3 Crustacean amphipods from marsh ponds: a nutritious feed resource with potential for application in Integrated Multi-Trophic Aquaculture Adaptado de: Jiménez-Prada P, Hachero-Cruzado I, Giráldez I, Fernández-Diaz C, Vilas C, Cañavate JP, Guerra-García JM. 2018. Crustacean amphipods from marsh ponds: a nutritious feed resource with potential for application in Integrated Multi-Trophic Aquaculture. PeerJ 6:e4194. DOI 10.7717/peerj.4194 Amphipod pictures taken from Arias and Drake (1994) Resumen La protección costera, el ciclo de nutrientes, el control de la erosión, la purificación del agua y el secuestro de carbono son servicios ecosistémicos proporcionados por las marismas. Además, las salinas ofrecen un hábitat de cría para peces y proporcionan abundantes invertebrados, como anfípodos, potencialmente útiles como recurso en acuicultura. La harina de pescado y el aceite de pescado son recursos alimentarios necesarios para mantener la acuicultura de especies carnívoras debido a sus ácidos grasos poliinsaturados de cadena larga omega-3 (n-3 LC-PUFA). Actualmente, la acuicultura depende de pesquerías y piensos limitados con niveles elevados de n-3 LC-PUFA, pero es necesario desarrollar fuentes de alimentos más sostenibles. Los anfípodos parecen ser un potencial recurso alimenticio alternativo de alta calidad para la acuicultura. Por lo tanto, se realizó un estudio nutricional para varias especies principales de anfípodos -Microdeutopus gryllotalpa, Monocorophium acherusicum, Gammarus insensibilis, Melita palmata y Cymadusa filosa- en esteros del sur de España. Estas especies mostraron un alto contenido de proteína (hasta 40%), altos niveles de PUFA n-3 y fosfolípidos, y altos niveles de fosfatidilcolina (PC), fosfatidiletanolamina (PE) y triacilgliceroles (TAG), siendo este último significativamente alto para M. acherusicum. M. gryllotalpa y M. acherusicum mostraron la mayor proporción de lípidos (19,15% y 18,35%, respectivamente). Isoleucina, glicina y alanina fueron los aminoácidos dominantes en todas las especies. Además, los anfípodos recolectados en estanques mostraron bajos niveles de metales pesados. Los perfiles bioquímicos de las cinco especies de anfípodos se compararon con otras presas alternativas. Por lo tanto, los anfípodos de esteros son buenos candidatos para ser utilizados como alimento y se proponen como un nuevo recurso sostenible y económico para ser utilizado en acuicultura. G. insensibilis puede ser la mejor especie para el cultivo intensivo como recurso alimenticio alternativo por sus características: 1) una composición adecuada de PUFA n-3 y PL; 2) altos niveles de glicina, alanina, tirosina, isoleucina y lisina; 3) altas densidades naturales; 4) cuerpo grande (≥1 cm) y 5) alta concentración de calcio. Más allá, una aquicultura combinada de anfípodos y peces parece una forma prometedora y ambientalmente sostenible de desarrollar Acuicultura Multitrófica Integrada (IMTA) en estos ecosistemas. Abstract Coastal protection, nutrient cycling, erosion control, water purification, and carbon sequestration are ecosystem services provided by salt marshes. Additionally, salt ponds offer coastal breeding and a nursery habitat for fishes and they provide abundant invertebrates, such as amphipods, which are potentially useful as a resource in aquaculture. Fishmeal and fish oil are necessary food resources to support aquaculture of carnivorous species due to their omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA). Currently aquaculture depends on limited fisheries and feed with elevated n-3 LC-PUFA levels, but the development of more sustainable food sources is necessary. Amphipods appear to be a potential high quality alternative feed resource for aquaculture. Hence, a nutritional study was carried out for several main amphipod species -Microdeutopus gryllotalpa, Monocorophium acherusicum, Gammarus insensibilis, Melita palmata and Cymadusa filosain terrestrial ponds in the South of Spain. These species showed high protein content (up to 40%), high n-3 PUFA and phospholipid levels, and high levels of phophatidylcholine (PC), phosphatidylethanolamine (PE) and triacylglycerols (TAG), the latter being significantly high for M. acherusicum. M. gryllotalpa and M. acherusicum showed the highest proportion of lipids (19.15% and 18.35%, respectively). Isoleucine, glycine and alanine were the dominant amino acids in all species. In addition, amphipods collected from ponds showed low levels of heavy metals. Furthermore, the biochemical profiles of the five species of amphipods have been compared with other studied alternative prey. Therefore, pond amphipods are good candidates to be used as feed, and are proposed as a new sustainable economic resource to be used in aquaculture. G. insensibilis may be the best for intensive culture as an alternative feed resource because it shows: 1) adequate n-3 PUFA and PL composition; 2) high levels of glycine, alanine, tyrosine, isoleucine and lysine; 3) high natural densities; 4) large body size (≥1 cm), and 5) high concentration of calcium. Moreover, a combined culture of amphipods and fishes in these marsh ponds seems a promising and environmentally sustainable way to develop Integrate Multi-Trophic Aquaculture (IMTA) in these ecosystems. 71 Anfípodos de esteros Crustacean amphipods from marsh ponds: a nutritious feed resource with potential for application in Integrated Multi-Trophic Aquaculture. Pablo Jiménez-Prada1,2*, Ismael Hachero-Cruzado2, Inmaculada Giraldez3, Catalina Fernández-Díaz2, César Vilas2, José Pedro Cañavate2, José Manuel Guerra-García1. 1 Laboratorio de Biología Marina, Dpto. de Zoología, Facultad de Biología, Universidad de Sevilla, 41012 Sevilla. 2 IFAPA - El Toruño, Camino Tiro Pichón s/n, El Puerto de Santa María. 3 Dpto. de Química “Prof. J.C. Víchez Martín”, Facultad de Ciencias Experimentales, Universidad de Huelva, Campus Universitario El Carmen, Avenida de las Fuerzas Armadas, 21071- Huelva. *Corresponding author: Pablo Jiménez-Prada. Tel.: +34954556229 E-mail address: [email protected] 1.Introduction Saltmarshes are aquatic systems with many ecosystem services arising from ecosystem processes and functions such as: coastal protection, nutrient cycling, erosion control, water purification, and carbon sequestration (Barbier et al., 2011). Nowadays, approximately 50% of salt marshes suffer deterioration due to human activity (Barbier et al., 2011). Interestingly, some salt marshes, such as those located in Southern Spain, require anthropogenic activities (hydraulic control, wall conservation, sediment removal, etc.) to be economically sustainable in the context of aquaculture (Arias and Drake, 1999). These modified saltmarshes provide, therefore, services as a coastal breeding and nursery habitat, and are used by locals for the practice of a kind of extensive aquaculture generating economic activity. The saltmarsh zone located East and South of the Parque Natural Bahía de Cádiz (PNBC), in the South of Spain, is characterized by a complex system of tidal channels and creeks that supply seawater to saltmarsh fish-ponds situated along their course. Most of the saltmarsh ponds remain permanently flooded during the greater part of the year, and constitute a semi-natural lagoon ecosystem exploited for extensive and semi-intensive fish culture (Arias and Drake, 1994). These ponds are productive ecosystems thanks to their particular hydrology and morphology, allowing for optimal use of the available light and nutrients. The marshes’ elevated primary productivity is thus associated to a 72 Capítulo 3 significant nutrient load and the flooding capacity in the adjacent lands (Cañavate et al., 2015). The macroinvertebrate saltmarsh community is subjected to intensive predation from fish and shorebirds, and is a main food source for non-intensively reared fish (Arias and Drake, 1994). Gastropods, amphipods and chironomid larvae dominate the macrofauna in terms of abundance (Arias and Drake, 1994). Fish accounted for 16.7% of the global population’s intake of animal protein and 6.5% of all protein consumed in 2010 (FAO, 2014; Tocher, 2015). Moreover, what is arguably of greatest importance to consumers in the developed world, fish and seafood are unique and rich sources of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), particularly eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic (DHA; 22:6n-3) acids. An increasing proportion of fish are farmed, accounting for almost half of all fish for human food in 2012 (Tocher, 2015). Aquaculture of carnivorous species depends on n-3 LC-PUFA from fishmeal and fish oil but these come from limited, overexploited fisheries or from fish slaughter waste. Therefore, the continued growth of aquaculture will depend on the development of more sustainable feeds with alternative ingredients, generally derived from terrestrial agriculture, with important consequences for the supply of n-3 LC-PUFA (Tocher, 2015) or, as an alternative, finding new marine resources. In that sense, amphipods could serve as an adequate alternative food resource for aquaculture. They are important low trophic position organisms which play a major role in the biological processing of algae inputs and facilitate the transfer of nutrients from the ocean to the coastline (Hamed et al., 2014). They are also regarded as important food for economically relevant fish species (chapter 2). Additionally, amphipods are abundant throughout the year in marsh zones, reach high densities (ca. 60.000 ind/m2), and show adequate protein content and fatty acid profiles, with high levels of beneficial polyunsaturated fatty acids (DHA and EPA) (Kolanowski et al., 2007; Hyne et al., 2009; Baeza-Rojano et al., 2014). Previous studies have explored amphipods as alternative protein sources in experimental diets for farmed fish (Moren et al., 2006; Opstad et al., 2006; Suontama et al., 2007) and cephalopods (Baeza-Rojano et al., 2010; González et al., 2011; Baeza-Rojano et al. 2013a) obtaining promising results. 73 Anfípodos de esteros With the aim to select the more adequate amphipods from terrestrial ponds as alternative prey, we studied the composition (protein, carbohydrates, lipids and ashes) of the dominant species. Furthermore, lipid classes, fatty acids, amino acids and trace/major elements were also studied in depth. This allowed us to compare with other alternative preys used in aquaculture, in addition to evaluating their nutritional composition and potential to be used as alternative feed in aquaculture. 2. Materials and Methods 2.1 Sample collection Amphipods were collected at terrestrial marsh ponds located at IFAPA “El Toruño” (Figure 1), El Puerto de Santa María, South Spain, within the “Bahía de Cádiz” Natural Park. Six ponds were selected (Figure 1A) for setting passive traps at shore areas during 1 week in December 2014. Salinity ranged from 23.6 to 31.8 and temperature from 12.0 to 15.4ºC. Passive traps, made up of filling oyster’s cylindrical mesh structures with nylon mesh poufs, were used to collect amphipods (Figure 1B). Traps were fixed close to or inside algae patches of Ulva spp. Figure 1. Ponds of IFAPA ``El Toruño’’. (A) Map of ponds of IFAPA ``El Toruño’’ with samples stations. (B) Traps were used to collect amphipods. (C) Ponds. 80 Capítulo 3 3.3. Lipid classes and fatty acids Regarding lipid classes, all studied species showed high PC, PE and TAG levels (Table 4). Although PERMANOVA analysis showed differences in the lipid class profile among species (Pseudo-F =4.01, p=0.046), pairwise tests did not show any differences. Axis 1 of the PCA analysis (Figure 4) explained 95.2% of total variance. TAG correlated significantly with this axis (r=-0.98, p<0.01) and separated M. acherusicum, with higher values of TAG, from the other species. Axis 2 explained 3.3% of total variance. PC correlated positively with this axis (r= 0.846, p<0.01) and separated G. insensibilis, with lower values of PC, from the other species. ANOVAs confirmed differences among species in PC (F=18.7, p=0.033) and TAG (F=5.47, p=0.045). In both lipids class, the SNK test did not show differences among species. In connection with fatty acid composition, the most abundant fatty acids in all species were: the saturated 16:0, the monounsaturated 16:1n9 and 18:1n9, and the polyunsaturated 18:2n6, 20:5n3 (EPA) and 22:6n3 (DHA) (Table 5). PERMANOVA also reflected global differences in fatty acid composition among species (pseudo-F=9.97, p=0.001) despite no differences in species pair-wise comparisons being detected. These differences were also supported by the PCA analysis (Figure 5). Axis 1 explained 67.7% of total variance and axis 2 explained 22.7%. The fatty acid 22:6 n3 (DHA) correlated positively with axis 1 (r=0.657, p<0.05), and G. insensibilis M. palmata C. filosa M. grillotalpa M. acherusicum 1 6 5 2 4 2 4 3 1 5 PC 1.96 1.75 2.36 2.52 2.19 1.93 2.36 2.54 3.08 2.90 PE 1.27 1.17 1.58 1.66 1.52 1.29 1.52 1.49 1.72 1.50 PI 0.17 0.18 0.31 0.31 0.33 0.23 0.15 0.11 0.13 0.32 PS 0.26 0.36 0.40 0.46 - 0.31 0.43 - - 0.36 TAG 3.01 2.93 3.87 2.44 2.76 1.86 1.97 3.97 5.58 7.19 Cho 0.32 0.18 0.35 0.25 0.30 0.35 0.24 0.25 0.332 0.30 Table 4. Lipid classes (μg/100μg of DW) per station of the five studied species per station. PC: Phophatidylcholine, PE: Phosphatidylethanolamine, PI: Phosphatidylinositol, PS: Phosphatidylserine, TAG: Triacylglycerols, Cho: Cholesterol. The values are a mean of two replicates. Each replicate consists of a pool of specimens. Figure 3. Principal Component Analysis (PCA) plot based on the Amino acid composition of the five amphipod species. GI: Gammarus insensibilis, MP: Melita palmata, CF: Cymadusa filosa, MG: M. gryllotalpa, MA: Monocorophium acherusicum. The numbers after the species indicate the collection station. 81 Anfípodos de esteros separated M. acherusicum and M. gryllotalpa from the other species. 20:5n3 (EPA) correlated positively with axis 2 (r=0.59, p<0.05), and separated M.gryllotalpa from M. acherisicum. ANOVAs confirmed significant differences for these fatty acids among species (EPA, F=6.03 p=0.0375; DHA, F=53.26 p=0.0003). For both fatty acids the SNK test did not show differences among species. Figure 4. Principal Component Analysis (PCA) plot based on the Lipid Classes (LCs) composition of the five amphipod species. PC: Phophatidylcholine, PE: Phosphatidylethanolamine, PI: Phosphatidylinositol, PS: Phosphatidylserine, TAG: Triacylglycerols, Cho: Cholesterol. GI: Gammarus insensibilis, MP: Melita palmata, CF: Cymadusa filosa, MG: M. gryllotalpa, MA: Monocorophium acherusicum. The numbers after the species indicate the collection station. Only variables significantly correlated with axis 1 and 2 are represented. 3.3. Lipid classes and fatty acids Regarding lipid classes, all studied species showed high PC, PE and TAG levels (Table 4). Although PERMANOVA analysis showed differences in the lipid class profile among species (Pseudo-F =4.01, p=0.046), pairwise tests did not show any differences. Axis 1 of the PCA analysis (Figure 4) explained 95.2% of total variance. TAG correlated significantly with this axis (r=-0.98, p<0.01) and separated M. acherusicum, with higher values of TAG, from the other species. Axis 2 explained 3.3% of total variance. PC correlated positively with this axis (r= 0.846, p<0.01) and separated G. insensibilis, with lower values of PC, from the other species. ANOVAs confirmed differences among species in PC (F=18.7, p=0.033) and TAG (F=5.47, p=0.045). In both lipids class, the SNK test did not show differences among species. In connection with fatty acid composition, the most abundant fatty acids in all species were: the saturated 16:0, the monounsaturated 16:1n9 and 18:1n9, and the polyunsaturated 18:2n6, 20:5n3 (EPA) and 22:6n3 (DHA) (Table 5). PERMANOVA also reflected global differences in fatty acid composition among species (pseudo-F=9.97, p=0.001) despite no differences in species pair-wise comparisons being detected. These differences were also supported by the PCA analysis (Figure 5). Axis 1 explained 67.7% of total variance and axis 2 explained 22.7%. The fatty acid 22:6 n3 (DHA) correlated positively with axis 1 (r=0.657, p<0.05), and G. insensibilis M. palmata C. filosa M. grillotalpa M. acherusicum 1 6 5 2 4 2 4 3 1 5 PC 1.96 1.75 2.36 2.52 2.19 1.93 2.36 2.54 3.08 2.90 PE 1.27 1.17 1.58 1.66 1.52 1.29 1.52 1.49 1.72 1.50 PI 0.17 0.18 0.31 0.31 0.33 0.23 0.15 0.11 0.13 0.32 PS 0.26 0.36 0.40 0.46 - 0.31 0.43 - - 0.36 TAG 3.01 2.93 3.87 2.44 2.76 1.86 1.97 3.97 5.58 7.19 Cho 0.32 0.18 0.35 0.25 0.30 0.35 0.24 0.25 0.332 0.30 Table 4. Lipid classes (μg/100μg of DW) per station of the five studied species per station. PC: Phophatidylcholine, PE: Phosphatidylethanolamine, PI: Phosphatidylinositol, PS: Phosphatidylserine, TAG: Triacylglycerols, Cho: Cholesterol. The values are a mean of two replicates. Each replicate consists of a pool of specimens. 82 Capítulo 3 G. insensibilis M. palmata C. filosa M. grillotalpa M. acherusicum 1 6 5 2 4 2 4 3 1 5 Saturated (Sat) 14:0 1.21 1.18 1.17 1.08 1.15 1.32 1.36 1.75 1.61 1.65 16:0 5.86 6.00 5.14 5.55 3.88 4.26 5.99 8.55 8.86 8.97 17:0 - - 0.73 0.50 - - 0.64 0.68 0.64 0.62 18:0 1.71 1.82 2.62 2.44 1.60 1.50 2.39 2.59 2.45 2.43 Total 8.79 9.01 9.66 9.56 6.63 7.08 10.37 13.56 13.55 13.66 Monounsaturated (MUFA) 16:1n9 2.40 3.43 1.28 1.61 0.76 0.86 2.27 4.47 1.67 1.66 17:1 0.45 0.60 0.72 0.47 - - 0.58 0.65 0.64 0.62 18:1n9 4.94 5.58 5.42 4.36 4.19 5.62 3.79 4.35 5.22 6.01 20:1n9 0.43 0.45 0.56 0.50 0.67 0.80 0.52 0.85 0.60 0.61 Total 8.22 10.05 7.99 6.95 5.61 7.28 7.16 10.32 8.13 8.91 Poliunsaturated (PUFA) 18:2n6 4.75 2.16 1.73 2.23 2.71 2.61 1.72 1.65 2.27 2.23 18:3n6 0.44 0.48 - - - - 0.66 0.85 - - 18:3n3 0.57 1.17 0.81 0.65 1.25 2.01 0.69 0.63 1.82 1.74 20:2n6 0.77 0.49 0.52 0.54 0.90 0.78 - - 0.63 0.65 20:4n6 ARA 1.40 1.47 2.08 2.28 1.71 1.47 1.99 1.94 1.41 1.43 20:3n3 DPA 0.36 0.41 0.36 0.40 0.62 0.92 0.39 0.41 1.02 1.03 20:5n3 EPA 5.02 6.03 5.41 7.03 3.47 3.63 7.26 9.98 6.47 6.77 22:6n3 DHA 3.25 3.59 5.68 4.41 2.04 1.97 4.04 4.86 8.71 8.79 Total 16.57 15.80 16.59 17.54 12.70 13.38 16.76 20.31 22.33 22.63 Table 5. Fatty acids (μg/mg of DW) of the five species studied per station. ARA: Arachidonic acid; EPA: Eicosapentanoic acid; DPA: Docosapentanoic acid; DHA: Docosaheanoic acid. The values are a mean of two replicates. Each replicate consists of a pool of specimen. Figure 5. Principal Component Analysis (PCA) plot based on the Fatty acid composition of the five amphipod species. 20:5n3 (EPA), 22:6n3 (DHA). GI: Gammarus insensibilis, MP: Melita palmata, CF: Cymadusa filosa, MG: M. gryllotalpa, MA: Monocorophium acherusicum. The numbers after the species indicate the collection station. Only variables significantly correlated with axis 1 and 2 are represented. 83 Anfípodos de esteros 3.4. Trace and major elements Silver (Ag), arsenic (As), cadmium (Cd), cobalt (Co), nickel (Ni), lead (Pb) and selenium (Se) were not detected. Cr values ranged from 8.74 to 81.62 ppm, Cu from 36.42 to 162.63 ppm and Zn from 36.64 to 322.61 (Table 6). PERMANOVA results showed differences in the composition of trace and major elements among species (pseudo-F=9.36, p=0.007). In the PCA analysis (Figure 6), calcium (Ca) was significantly correlated (r=-0.98, p<0.001) with axis 1, which explained 96.7% of the variance; this axis separated M. acherusicum and M. palmata with lower Ca levels. Axis 2 explained 1.9% of the variance; Na (r=-0.60, p<0.05) and P (r=-0.56, p<0.05) were negatively correlated. ANOVA also indicated significant differences for Ca (F=20.31, p=0.0027). The SNK test separated two groups, where M. acherusicum and M. palmata were the group with a lower Ca level. Na (F=1.68, p=0.28) and P (F=0.19, p=0.93) did not present differences according to the ANOVA test. Table 6. Trace and major elements (μg/g of DW) of the five studied species per station. The values are a mean of two replicates. Each replicate consists of a pool of specimen. Detection limit of trace elements = 33.33 µg/g of DW. Detection limit of Major elements = 83.33 µg/g of DW. Nd = under detection limit. G. insensibilis M. palmata C. filose M. grillotalpa M. acherusicum 1 6 5 2 4 2 4 3 1 5 Trace elements Ag nd nd nd nd nd nd nd nd nd nd As nd nd nd nd nd nd nd nd nd nd Cd nd nd nd nd nd nd nd nd nd nd Co nd nd nd nd nd nd nd nd nd nd Cr 16.23 12.30 8.74 15.10 8.98 9.27 27.11 81.62 14.28 14.08 Cu 87.04 133.73 38.76 126.92 134.90 52.88 78.02 162.63 52.11 36.42 Ni nd nd nd nd nd nd nd nd nd nd Pb nd nd nd nd nd nd nd nd nd nd Se nd nd nd nd nd nd nd nd nd nd Zn 76.98 104.64 36.64 171.79 53.31 44.96 57.38 322.61 111.37 45.69 Major elements Al 6870 7209 11880 8103 6364 6755 6792 7019 9611 12236 Ca 247771 248136 195680 192075 223772 257195 256192 275224 158634 172823 K 17197 17327 9136 16778 10173 17700 15643 14008 9809 8474 Fe 1805 949 1909 1289 837 1316 2114 2220 1566 1814 Mg 13123 14098 15078 15811 17111 13852 18693 20532 13396 13944 Na 31793 36698 20066 27681 26060 36693 30951 37165 28885 21476 P 14240 21992 17343 19671 14073 26080 17002 16278 21949 18223 S 18251 18691 17992 20644 24265 20945 29232 26668 12616 12263 84 Capítulo 3 4. Discussion Aquaculture development requires sources of marine protein, n-3 LC-PUFA and micronutrients that are more sustainable than those currently in use, because, according to the FAO’s (2016) report, fishery activity has stabilized around 90 million tons in the last 20 years, while aquaculture activity reached 106 million tons in 2015, exceeding fishery activity by 12.3 million tons. Because of that, it is necessary to investigate new marine feed sources to be used in aquaculture. Previous works have explored the use of alternative preys, like copepods, and traditional preys, like Artemia and rotifers (Tables 7, 8 and 9). Amphipods from marsh ponds could also be an alternative source. To date, amphipods have been used in meals for finfish with different results. Themisto libellula (Amphipoda: Hyperiidae) has been used in different studies: (1) Moren et al. (2006) found improved growth (Salmon) with a total substitution of fish meal for amphipod meal; (2), Suontama et al. (2007) described that a substitution lower than 60 % did not affect post-mortem pH and rigidity in trout; however, (3) Opstad et al. (2006) obtained negative results in meals with higher than 25% substitution for feeding Atlantic cod. Another amphipod, Jassa marmorata, was used as live prey in a cephalopod species (Robsonella fontaniana) achieving 20% higher growth than with Artemia (González et al., 2011). In Baeza-Rojano et al. (2010), Sepia officinalis had Figure 6. Principal Component Analysis (PCA) plot based on the Trace/Major elements composition of the five amphipod species. GI: Gammarus insensibilis, MP: Melita palmata, CF: Cymadusa filosa, MG: M. gryllotalpa, MA: Monocorophium acherusicum. The numbers after the species indicate the collection station. Only variables significantly correlated with axis 1 and 2 are represented. 85 Anfípodos de esteros a similar growth using amphipods or mysids, and, in Baeza-Rojano et al. (2012), marine gammarideans were better live prey than Artemia and freshwater gammarids for culture of Octopus maya. Moreover, Lari et al. (2013) showed that sturgeon species, Acipenser gueldensuedtii and A. ruthenus, preferred Gammarus to polychaetes of the genus Nereis. 4.1. General composition In general terms, the studied amphipods from marsh ponds were characterised by high levels of protein and ash and low levels of carbohydrates and lipids. Protein values were similar to those found in copepods and mysids by other authors (Wang et al., 2014), but lower than those reported for other amphipods from the Bay of Algeciras (Baeza- Rojano et al., 2014). However, amphipods present a higher percentage of carbohydrates than other studied groups (e.g. Wang et al., 2014). Taking into account the National Research Council’s (NRC, 2011) indications, no dietary requirement for carbohydrates has been demonstrated in fish. However, carbohydrates are important for sparing proteins and lipids for energy provision, and for the synthesis of important compounds derived from carbohydrates (NRC, 2011). Regarding lipid content, values of 5.9 and 7.7% of DW have been measured in Gammarus pulex and Carinogammarus roeselii respectively (Geng, 1925); similar to the Strait of Gibraltar amphipods (Baeza-Rojano et al., 2014), but lower than total lipids measured in this study. However, higher lipid levels are normally found in deep-sea amphipod species (45% in populations of Monoporeia affinis (Lehtonen, 1996)) and in Arctic and Antarctic species (27% in Onisimus affinis and 53% in Orchomonella plebs (Percy, 1979; Pearse and Giese, 1966). On the negative side, the high ash levels present in all the analysed amphipods species could be considered inadequate for feeding fish larvae (Moren et al., 2006; Opstad et al., 2006; Suontama et al., 2007). 4.2. Amino acids Dietary supplementation with ingredients rich in specific amino acids is beneficial, due to the crucial roles in cell metabolism and physiology of these molecules. Amino acids have several functions in fishes, such as: (1) increasing the chemo-attractive property and nutritional value 86 Capítulo 3 of aquafeeds with low fishmeal inclusion; (2) optimizing efficiency of metabolic transformation in juvenile and subadult fishes; (3) reducing aggressive behaviours and cannibalism; (4) increasing larval performance and survival; (5) mediating timing and efficiency of spawning; (6) improving fillet taste and texture; and (7) enhancing immunity and tolerance to environmental stresses (Li et al. 2009). The three most abundant amino acids in the analysed species are the non-essential amino acids glycine, alanine and aspartic acid. Although non-essential they have a significant role in stimulating feeding response and energy mechanisms in fishes (Li et al., 2009). Glycine participates in gluconeogenesis, sulphur amino acid metabolism, one-carbon unit metabolism and fat digestion (Fang et al., 2002), stimulates feed intake (Shamushaki et al., 2007), and has a critical role in the osmoregulatory responses of fishes and shellfishes (Li et al., 2009). Alanine and Aspartic acid are two of the major glucogenic precursors and important energy substrates for fish, and can also stimulate the feeding response (Li et al. 2009). Isoleucine, leucine and valine are three of the most abundant essential amino acids in the analysed amphipods (EAA). These EAAs play important structural roles and are primarily deposited in body protein, notably in skeletal muscles (NRC, 2011). Phenylalanine and tyrosine, an essential and conditionally essential amino acid respectively, are also abundant in amphipod species (Table 3). The former can be converted to the latter, and dietary levels of both could profoundly influence pigmentation development, feed intake, growth performance, immunity, and survival of fish in natural environments (Pinto et al., 2008). Consequently, fishes’ dietary requirements for phenylalanine and tyrosine increase substantially during metamorphosis (Pinto et al., 2008). Analysed species show higher levels of the non-essential amino acids glycine, alanine and tyrosine, but lower levels of leucine, than other amphipods (Table 7). The high levels of glycine and alanine are of special interest for fish feeding, because these amino acids are feed intake stimulators (Shamushaki et al., 2007). Kasumyan and Mikhailova (2014) and Kasumyan and Marusov (2015) showed an increase in the fish’s feed intake with alanine and glycine, respectively. Regarding essential amino acids, amphipods have higher levels of isoleucine and valine, but lower of 87 Anfípodos de esteros leucine (Table 7). These three AA are of interest for fish feeding because they play important structural roles and are primarily deposited in body protein, notably in skeletal muscles. In addition, Valine is involved in the synthesis of the myelin covering of the nerves (Cowey and Walton, 1989; Brosnan and Brosnan, 2006). 4.3. Lipid classes and Fatty acids Among lipid classes, triglycerides (TAG) are the predominant form of lipid reserve, and are always mobilized before phospholipids (PL) during starvation (Sargent et al., 1989; Hachero-Cruzado et al., 2014). The larvae presumably utilize the TAG to satisfy their energy demands while PL, which play an important structural role in the cell membrane, tend to be conserved (Rainuzzo et al., 1997) and increase the efficiency of the transport of dietary fatty acids and lipids from the gut to the rest of the body (Coutteau et al., 1997; Fontagne et al., 1998; Salhi et al., 1999; Tocher et al., 2008). Studied amphipods have a similar range of phospholipids (PL) but higher TAG dispersion, with M. acherisicum and M. palmata showing higher levels of PL and TAG than other preys (Table 8). Table 7. Essential (E) and Non-Essential (NE) amino acids (% of identified amino acids) of other prey. Taxa Species Amino acids References Essencials Non essencials Isoleucine Leucine Lysine Methionine Phenylalan ine Valine Alanine Aspartic acid Glutamic acid Glycine Proline Serine Tyrosine Amphipod Gammarus insensibilis 10,45 7,41 10,55 2,41 6,44 8,28 11,04 8,16 2,43 13,72 1,60 5,06 12,47 Present study Amphipod Melita palmata 10,35 7,65 9,71 2,28 6,31 8,37 10,76 9,82 3,70 13,07 1,83 5,39 10,74 Present study Amphipod Cymadusa filosa 11,05 6,87 9,53 1,45 6,35 8,42 11,41 10,54 3,78 13,90 2,25 5,15 9,31 Present study Amphipod M. gryllotalpa 10,95 6,93 9,65 2,41 6,41 8,29 11,41 9,73 3,07 13,59 2,15 5,42 10,00 Present study Amphipod M. acherisicum 11,50 5,51 9,41 2,30 6,33 8,07 12,45 10,17 3,18 14,39 2,40 4,78 9,50 Present study Copepod Pseudodiaptomas inopinus 4,89 9,28 8,90 2,53 5,41 6,31 8,44 11,40 17,31 7,68 6,60 5,58 5,67 Yang & Hur (2014) Copepod Paracyclopina nana 5,26 9,69 8,94 0,62 5,98 6,48 7,98 12,17 17,82 6,40 6,79 5,67 6,22 Yang & Hur (2014) Branchiop Artemia spp. 6,09 10,07 10,97 1,97 5,70 6,30 7,24 10,76 16,07 6,26 7,97 6,99 3,60 Yang & Hur (2014) Rotifers Brachionus plicatilis 5,56 10,37 8,36 0,42 6,77 5,20 5,84 10,96 16,24 4,61 14,59 7,41 3,66 Yang & Hur (2014) Copepod Pool of copepods 4,40 7,60 7,40 2,30 4,10 5,30 7,10 9,60 13,60 7,50 5,30 5,30 4,60 Hamre et al. (2013) 88 Capítulo 3 Regarding fatty acids, DHA is one of the most important fatty acids for its key role in the formation and composition of nervous tissue and the retina (Mourente and Tocher, 1992; Bell et al., 1996; Coutteau and Mourente, 1997). Recent studies search for new feed sources with higher levels of n-3 and n-6 PUFA. Table 8. Comparison of Lipid Classes (µg/100µg of DW). Taxa are organized according to increasing levels of Total Phospholipids (Total PL). PC: Phophatidylcholine. PE: Phosphatidylethanolamine. PI: Phosphatidylinositol. PS: Phosphatidylserine. TAG: Triacylglycerols. Taxa Specie TAG PC PE PI PS Total PL References Amphipod G. insensibilis 2.97 1.86 1.22 0.17 0.31 3.56 Present study Amphipod C. filosa 2.31 2.06 1.41 0.28 0.15 3.90 Present study Branchiopod Artemia franciscana 17.84 1.74 1.43 0.42 0.32 3.91 Van der Mereen et al. 2008 Amphipod M. grillotalpa 2.97 2.45 1.51 0.13 0.21 4.30 Present study Rotifer (ME) Brachionus sp. - 2.50 1.90 - - 4.40 Li et al. 2015 Rotifer (emulL) Brachionus sp. - 2.20 2.10 - - 4.41 Li et al. 2015 Zooplankton - 4.21 1.90 1.52 0.56 0.48 4.46 Van der Mereen et al. 2008 Amphipod M. palmata 3.16 2.44 1.62 0.31 0.43 4.80 Present study Copepods - 2.63 2.00 1.99 0.39 0.55 4.93 Van der Mereen et al. 2008 Amphipod M. acherusicum 6.38 2.99 1.61 0.22 0.18 5.01 Present study Rotifer Brachionus plicatilis 6.06 1.85 2.10 1.06 0.53 5.54 Van der Mereen et al. 2008 Copepod Acartia tonsa 0.00 - - - - 7.00 Olsen et al. 2014 Table 9. Comparison of percentage in lipids (% of DW) and fatty acids (μg/mg of DW). Taxa are organized according to increasing levels of DHA. ARA: Arachidonic acid; EPA: Eicosapentanoic acid; DPA: Docosapentanoic acid; DHA: Docosaheanoic acid. * Eurythemora affinis, Calanus finmarchicus and Microsetella norvegica (Copepods). Taxa Species Lipids ARA EPA DHA Refrences Copepod Pool of three species* 3.20 - 0.63 0.80 Maehre et al. 2013 Rotifer Brachionus sp. 10.00 - 0.44 1.01 Maehre et al. 2013 Rotifer Brachionus plicatilis 15.40 0.29 1.09 1.91 Van der Mereen et al. 2008 Amphipod C. filosa 13.42 1.59 3.55 2.00 Present study Zooplankton - 14.30 0.23 2.35 2.47 Van der Mereen et al. 2008 Rotifers Brachionus sp. 9.80 0.72 3.20 3.00 Li et al. 2015 Copepod P. anmandeti 16.00 0.24 1.47 3.09 Rayner et al. 2015 Amphipod G. insensibilis 12.98 1.44 5.53 3.42 Present study Copepod - 11.10 0.09 1.93 3.80 Van der Mereen et al. 2008 Amphipod M. grillotalpa 19.15 1.96 8.62 4.45 Present study Branchiopod Artemia franciscana 24.90 0.80 2.29 4.98 Van der Mereen et al. 2008 Amphipod M. palmata 15.90 2.18 6.22 5.04 Present study Amphipod M. acherusicum 18.35 1.42 6.62 8.75 Present study Rotifer Brachionus sp. 12.70 0.60 4.00 10.00 Li et al. 2015 Copepod - 7.07 - 5.17 11.76 Li et al. 2015 Copepod Acartia tonsa 9.40 0.24 8.80 13.37 Olsen et al. 2014 89 Anfípodos de esteros Table 9 compares the amounts of essential fatty acids in some crustacean species and rotifers with the amounts in “El Toruño” ponds’ amphipods. For example, rotifers studied by Li et al. (2015) and Acartia tonsa (copepod) studied by Olsen et al. (2014) have the highest values of DHA and high EPA values, but they have lower amounts of ARA and total lipids (7.0 and 9.4 %, respectively). Furthermore, A. tonsa is not easy to collect since a ship with trawls is necessary (Olsen et al., 2014). We must also take into account that the rotifers had been enriched with a lipid emulsion (Marol E, prepared by SINTEF Fisheries and Aquaculture, Norway), based on the marine oil DHASCO (Martek Biosciences, Columbia, MD, USA), which is used as a TAG source rich in DHA (Li et al., 2015). Both methods increase the economic cost. However, M. palmata and M. acherusicum are two species with high values of essential fatty acids; while Cymadusa filosais is the species with the worst fatty acid profile of the five studied amphipod species. 4.4. Major and trace elements Data of major and trace elements of amphipods from the “El Toruño” ponds can be compared with recent studies conducted in nearby areas with anthropogenic influence. In Algeciras Bay, Guerra-García et al. (2009) showed that there were elevated concentrations of heavy metals in the amphipods Caprella penantis and Hyale schmidti in the inner sites, higher than those obtained for amphipods of the PNBC’s ponds. Guerra-García et al. (2010b) studied the levels of trace elements in several species of peracaridean crustaceans (amphipods, isopods and tanaids) from different sites of Southern Spain, and reported similar concentrations of Cr and Cu, higher values of As, Ni and Pb, and a considerably higher concentration of Zn than in the present study’s amphipods. In the same area, Guerra-García et al. 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Selection of copepods as live food for marine fish larvae based on their size, fecundity, and nutritional value. Ocean and Polar Research 36:199-208. Capítulo 4Testing diets for amphipods culture in Integrated Multi-Trophic Aquaculture Capítulo 4.1 New findings for Integrated Multi- Trophic Aquaculture: Lessons from caprellids (Crustacea: Amphipoda) Adaptado de: Guerra-García JM, Hachero-Cru- zado I, González-Romero P, Jiménez-Pra- da P, Casel C, Ros M. 2016. Towards Integrated Multi-Trophic Aquaculture: Lessons from Caprellids (Crustacea: Amphipoda. PlosOne, 11(4):1-16. DOI: 10.137/journal.pone.0154776. Resumen La búsqueda de organismos alimentarios vivos alternativos y la progresión de la acuicultura multi-trófica integrada (IMTA) actualmente son una prioridad en las estrategias de la UE. Los caprélidos podrían ser un importante recurso potencialmente explotable en acuicultura debido a sus altos niveles de ácidos grasos poliinsaturados beneficiosos, crecimiento rápido natural y distribución global. Además, dado que son principalmente detritívoros, podrían ser excelentes candidatos para la integración en sistemas IMTA, podrían beneficiarse de las heces y pellets de alimento no consumido por peces cultivados en plantas de acuicultura y estructuras de jaulas marinas. A pesar de esto, faltan estudios experimentales para: (i) probar dietas económicas para caprélidos, como detritus, (ii) desarrollar técnicas de cultivo de caprélidos sostenibles e (iii) incluir caprélidos en sistemas IMTA. El objetivo principal de este estudio fue determinar si los detritus (D) en forma de heces de pescado proporcionaban una dieta adecuada para los caprélidos en comparación con otras dietas tradicionales, como nauplios de Artemia (A) o fitoplancton (P). Se demostró que la tasa de supervivencia de los adultos era significativamente más alta para los caprélidos alimentados con D. Por el contrario, las crías tenían la tasa de supervivencia más alta con A, aunque la tasa de crecimiento juvenil y el número de mudas eran similares en las tres dietas. Con respecto a la composición de lípidos, los caprélidos alimentados con A tenían concentraciones más altas de TAG y PC, mientras que los alimentados con P o D eran más ricos en ácidos grasos poliinsaturados, especialmente DHA. Curiosamente, los caprélidos alimentados con D también eran una fuente rica de LA, considerada como un ácido graso esencial en los vertebrados. Se encontró que los desechos basados principalmente en heces de pescado y gránulos de alimento no consumidos pueden considerarse un alimento adecuado para los caprélidos adultos, proporcionando una fuente de ácidos grasos omega-3 (DHA) y omega-6 (LA). Sin embargo, las crías parecen requerir una aporte adicional de TAG y PC durante las etapas juveniles para crecer adecuadamente. Capítulo 4.1 112 El Rompido, Huelva (37º12’46’’N, 7º04’51’’W) where it was the dominant amphipod, associated mainly to the bryozoans Bugula neritina and Zoobotryon verticillatum, and the hydroid Ectopleura crocea. Caprella scaura, absent in Nuevo Portil Marina, was collected from colonies of B. neritina from La Línea Marina -Club Marítimo Linense, Cádiz (36º09’36’’N, 5º21’34’’W). No specific permissions were required for collections in these marinas, and the field studies did not involve endangered or protected species. In previous samplings conducted in 2011, La Línea Marina was dominated by C. equilibra and C. scaura was absent (Guerra-García et al., 2015). In the last 4 years C. scaura has invaded this marina and reached high densities, displacing C. equilibra. Colonies of bryozoans and hydroids attached to floating pontoons, buoys and ropes of the marinas were handpicked. Preliminary caprellid sorting was conducted in situ, before the individuals were transported with aeration together with some bryozoan colonies to the laboratory (see Baeza-Rojano et al., 2013a). 2.2. Experimental designs in laboratory conditions Experiment 1: Are caprellids able to feed on a variety of diets? To test the feeding preferences of C. equilibra and C. scaura and to verify the ability of caprellids to properly ingest different food types under laboratory conditions, the following experiment was conducted. 80 adult specimens of each species were isolated in small, 120 ml glass containers with a diameter of 6.5 cm and a height of 6 cm (4 specimens per container). An equal amount of males and females were considered to avoid potential differences among sexes. A 1 mm plastic mesh was used as a substratum for attachment. Specimens were maintained without food for a duration of 24 hours to empty their guts and the water was then changed to remove faecal pellets. 16 specimens (4 glasses) of each species were then used for each different diet; Artemia nauplii (A), phytoplankton (P), detritus (D), the three items together to explore preferences (All) or no additional food (NF). Trace natural detritus would have entered all containers by attachment to specimens. Artemia nauplii were hatched from cysts maintained (1-2 days) in a Brine Shrimp Hatchery Hobby® with seawater at 25ºC. The phytoplankton consisted of a mixture of microalgae, made from lyophilized colonies of Phaeodactylum, Tetraselmis and Nannochloropsis (1:1:1). Detritus was obtained by scraping the bottoms of aquaculture tanks used for culture of meagre (Argyrosomus regius) at Innovaciones en IMTA: Caprélidos 113 the IFAPA center “El Toruño” experimental aquaculture station (Cádiz, Spain) and consisted primarily of fish faeces and also of uneaten fishfeed pellets used as food for meagre. Caprellids were fed ad libitum and after 24 hours were preserved in 90% ethanol. The caprellids were maintained at 20ºC with a photoperiod of 12 h light: 12 h dark. The seawater used for the culture was treated by filtration (through a 0.45 μm Milipore filter) and UV irradiation, and had a salinity of 35.5 psu. In addition to the specimens used in the 5 treatments of the experimental design, 16 adult specimens of each species were fixed directly after collection from the marinas to analyse the diet in field populations. For the diet study, individuals were analysed following the methodology of Bello and Cabrera (1999) with slight variations, recently proposed to study the diet of amphipods (Guerra-García et al., 2014; Guerra-García and Tierno de Figueroa, 2009). Specimens of each species were placed in vials with Hertwig’s liquid (consisting on 270 g of chloral hydrate, 19 ml of chloridric acid 1N, 150 ml of distilled water and 60 ml of glycerin) and heated in an oven at 65 ºC for 2 to 10 hours depending on the size of the specimens. After this, they were mounted on slides for study under a microscope. The percentage of the absolute gut content (at 40x or 100x), described as the total area occupied by the content in the whole digestive tract, and the relative gut content (at 100x or 400x), described as the area occupied for each component within the total gut content, were estimated using a microscope equipped with an ocular micrometer. Experiment 2: Does diet influence the lipid composition and survival of adults of C. equilibra and C. scaura? Is detritus an adequate food for them? Provided that caprellids were able to feed on the three diets considered (see experiment 1), the same diets (A, P or D) were used to test the influence of food on the lipid composition of the caprellids and therefore their nutritional value for use as a resource for aquaculture. The present experiment was designed to last 12 days, as McLeod et al. (2013) reported changes in lipid composition of littoral amphipods in relation to diet after 12 days. 18 aquaria (20 cm x 13 cm x 13 cm) containing 3l of seawater (see experiment 1) were used; 3 for each treatment (A vs P vs D) of each species (C. equilibra vs C. scaura). In this experiment “All foods” and “no food” treatments were not included, mainly for two reasons: (i) in Capítulo 4.1 114 this case, the objective was not to explore feeding selection among items; to clearly evaluate the effect of each diet on the lipid composition it was necessary to use each diet separately. (ii) The use of additional treatments such as a combination of foods or starvation was prevented due to the high amount of individuals required. The high number of specimens required is a common constraint limiting the number of experimental treatments possible. Forty adult specimens were added to each aquarium equating to a total of 360 specimens of C. equilibra and 360 specimens of C. scaura. Folded meshes, as proposed by Baeza-Rojano et al. (2013b) were used as substratum. Two small stones were used to prevent buoyancy of the meshes, maintaining their position at the bottom of the aquaria. The caprellids were fed daily and the water of the aquaria was replaced every two days. Any dead specimens, uneaten food or faecal pellets were removed daily. Aquaria were maintained with a continuous air supply, a water temperature of 20ºC, a photoperiod of 12 h light: 12 h dark, and a seawater salinity of 35.5 psu. Twelve days after the introduction of the caprellids in the aquaria, the plastic meshes were removed individually. The number of remaining adult specimens was counted, and the pool of specimens for each aquarium (considered as a replicate) was immediately frozen and stored at -80ºC for chemical analysis. Three replicates of the three food types used for feeding C. equilibra and C. scaura during the experiment (A, P and D) were also frozen for chemical analysis to characterise their lipidic nutritional value. In order to compare the treatments under laboratory conditions to the lipid composition of field populations, three replicates (three pools of specimens) for each species (C. equilibra vs C. scaura) were frozen immediately after collection from the marinas and stored at -80ºC for chemical analysis. Experiment 3: Does diet influence the survival and growing rate of juveniles of C. equilibra and C. scaura? Is detritus an adequate food for them? Thirty ovigerous females (15 of C. equilibra and 15 of C. scaura) taken directly from the marinas were isolated individually in small glass containers of 120 ml with a diameter of 6.5 cm and a height of 6 cm. A 1 mm plastic mesh, replaced every 5 days, was used as a substratum. This experiment was focused on the juvenile stage. As a reference, Baeza-Rojano and Guerra-García (2013) found the duration of the juvenile period in C. equilibra after emerging from the brood pouch to Innovaciones en IMTA: Caprélidos 115 be approximately 14-16 days at 20ºC, corresponding with instars I-III (from instar IV, sexes can be differentiated). Therefore, all the hatchlings which emerged from each female were monitored during the first 16 days of their life history, corresponding with the juvenile stage. Three treatments were also considered (A vs P vs D) and 5 replicas (consisting of the hatchlings produced by 5 females of each species) were used for each treatment. Juveniles were fed ad libitum every day, and water was also replaced daily. All of the juveniles were counted daily under a binocular microscope and observations were made of any signs of moulting or parental care. After 16 days, all of the juveniles were fixed in 90% ethanol. The number of articles of the flagellum of antenna 1, which is indicative of the number of moults (see Baeza-Rojano et al., 2011), were counted in each juvenile. Photographs of each specimen were taken with a Motic K-400L stereomicroscope and measures of the total body length were taken using the software Scion Image Alpha 4.0.3.2© (2000-2001 Scion Corporation). 2.3. Chemical analysis: lipid classes (LCs) and fatty acids (FAs) Three replicates of the following samples from experiment 2 were used for chemical analysis: (i) the three food types the caprellids were subjected to (A, P and D), (ii) adult specimens of the two caprellid species collected from marinas and frozen immediately as reference values and (iii) adult specimens of the two caprellid species fed either A, P or D during 12 days. Each replicate consisted of a pool of specimens in order to reach the minimum quantity required for chemical analysis, which was 10 mg. Quantities used in other studies usually range from 10- 40 mg (Baeza- Rojano et al., 2014; McLeod et al., 2013; Guerra-García et al., 2004; Hyne et al., 2009) to 0.5-3 g (Gonzalez-Silvera et al., 2015). Unfortunately, the small weight of caprellids prevented the analysis of LCs and FAs in juveniles, since it was not possible to reach the minimum required weight for the chemical procedure, even when all the individuals of all the replicates of each treatment were pooled. LCs and FAs were therefore analysed only for adult specimens. Taking into account that Clark et al. (1985) found differences among sexes in some lipid classes, and that (Guerra-García et al., 2004) showed sex differentiation using fatty acid signatures, the same weight of male and female specimens were considered for each replicate to avoid the influence of sex in the results. Capítulo 4.1 116 Samples were freeze-dried for 24 h at -50ºC. The lipid fraction was extracted according to the Folch-Lee method (Folch et al., 1957). Total lipid was extracted with chloroform:methanol (2:1v/v) containing 0.01% of butylated hydroxytoluene (BHT) as an antioxidant (Christie, 1982). The organic solvent was evaporated under a stream of nitrogen and the lipid content was determined gravimetrically. Lipid classes were separated by one-dimensional double development high-performance thin-layer chromatography (HPTLC) using methyl acetate/isopropanol/chloroform/ methanol/0.25% (w/v) KCl (25:25:25:10:9 by volume) as the polar solvent system and hexane/diethyl ether/glacial acetic acid (80:20:2 by volume) as the neutral solvent system. Final quantification of lipid classes was made by densitometry in a CAMAG scanner at a wavelength of 325 nm, and by comparison with external standard (Sigma-Aldrich) (see Olsen and Henderson, 1989). For fatty acid analysis, total lipid extracts were subjected to acid catalysed transmethylation for 16 hours at 50 ºC, using 1mL of toluene and 2mL of 1% sulphuric acid (v/v) in methanol. The resulting fatty acid methyl esters (FAME) were separated and quantified using a Shimadzu GC 2010-Plus (Shimadzu) gas chromatograph equipped with a fame-ionisation detector (280 ºC) and a fused Tecnokroma – Suprawax- 280TM (15 m x 0.1 mm I.D.). Hydrogen was used as carrier gas and the oven initial temperature was 100ºC, followed by an increase at a rate of 20 ºC min-1 to a final temperature of 250 ºC for 8 min. Individual FAME were identified by reference to authentic standards and to a well-characterised fish oil. 2.4. Statistical analysis To determine whether the amount of ingested food varied among species and treatments (experiment 1), a two-way ANOVA was conducted with the following factors: ‘Species’ which was a fixed factor with two levels: C. equilibra (Eq) and C. scaura (Sc), and ‘Treatment’ which was a fixed factor orthogonal with ‘Species’ consisting of six levels: Marinas (caprellids taken directly from the marinas), A (caprellids fed with Artemia), P (caprellids fed with phytoplankton), D (caprellids fed with detritus), all foods (caprellids fed with the three diets simultaneously) and no food (caprellids given no food). Innovaciones en IMTA: Caprélidos 117 The differences in survival rates of adult males (experiment 2) and juveniles (experiment 3) among species and treatments were also tested using two-way ANOVA, with factor ‘Species’ (Eq vs Sc) and factor ‘Treatment’ (A vs P vs D). For testing differences in the length of juveniles and number of flagellar articles in antenna 1 (experiment 3) among treatments, one-way ANOVAs were used for each species separately, with the single factor treatment (A vs P vs D) since the total mortality of juveniles of C. equilibra fed with D prevented the use of two-way ANOVAs. Two-way ANOVAs were also used to compare the main lipid classes and fatty acids (experiment 3) among species (Eq vs Sc) and treatments (A vs P vs D). Previously one-way ANOVA was also conducted to explore differences in lipid composition among the three types of food used in the experiment for feeding caprellids. Prior to ANOVAs, the homogeneity of variances was tested with Cochran’s C-test. Where variances remained heterogeneous even after data transformation, untransformed data was still analysed, as ANOVA is a robust statistical test and is relatively unaffected by the heterogeneity of variances, particularly in balanced experiments (Underwood, 1997). In such cases, to reduce type I error, the level of significance was reduced to <0.01. Where ANOVA indicated a significant difference for a given factor, the source of difference was identified using Student-Newman-Keul (SNK) tests (Underwood, 1997). Principal Component Analyses (PCAs) were carried out to show the relationship among treatments, foods (diets) and samples from marinas (reference values) according to the lipid classes and fatty acid matrices. Differences in fatty acid and lipid classes composition among species and diets were tested by the use of a permutational multivariate analysis of variance (PERMANOVA) with two factors: ‘species’, a fixed factor with two levels (Eq vs Sc) and ‘treatment’, a fixed factor orthogonal with ‘species’ consisting of three levels (A vs P vs D). Analysis was based on Euclidean distance measures and Monte Carlo tests were included. Significant P-values were obtained by computing 9999 permutations of residuals under a reduced model as this method gives the most accurate Type I error for complex designs (Anderson, 2005). Pairwise comparisons Capítulo 4.1 118 were then used. Additionally, to test the dispersion among samples for the factors ‘species’ and ‘treatments’, a permutational analysis of multivariate dispersions (PERMDISP) was used. Univariate analyses were conducted with GMAV5 (Underwood et al., 2002) and multivariate analyses were carried out using the PRIMER v.6 plus PERMANOVA package (Clarke and Gorley, 2001). 3. Results 3.1. Caprella equilibra and C. scaura as opportunistic feeders (Exp. 1) Gut content of the specimens of C. equilibra and C. scaura which were directly fixed following collection in the marinas consisted mainly of detritus (more than 95% of the total), and included crustaceans (harpacticoid copepods) and phytoplankton (microalgae) (fig. 2). In laboratory conditions, when caprellids were offered a variety of diets, all items were observed in the gut, showing the ability of caprellids to ingest all three types of food (A, P and D). When the three food items were provided simultaneously, both caprellid species showed no feeding preferences and unselectively ingested the three of them (Fig. 2). Caprellids were able to actively prey on the live Artemia nauplii. In the control treatment ‘No food’, a small percentage of detritus (less than 5%) was registered in the gut, indicating that specimens isolated in the glass containers ingested the detritus attached to their body surface. When detritus was the single item provided, the amount of food ingested (measured as the total area occupied by the gut content in the whole digestive tract) was significantly higher than in other treatments (table 1, fig. 2). There were no significant differences in the quantity ingested among specimens fed with P, A or a combined diet of A+P+D (table 1). Table 1. Results of the two-way ANOVA for the percentage of gut contents during the feeding experiment. ***P<0.001. Ma=Marinas, A=Artemia; P=phytoplankton; D=detritus; All=All food; NF=No food Source of variation df MS F P F versus Species (Sp) 1 2352.00 3.28 0.0720 Res Treatment (Tr) 5 5802.99 8.08 0.0000*** Res Sp x Tr 5 998.02 1.39 0.2300 Res Residual (Res) 180 717.98 Cochran's test C=0.16 n.s. Transformation None SNK D>(Ma=A=P=D=All)>NF Innovaciones en IMTA: Caprélidos 119 3.2. Lipid composition of the diets used in the experiments The three diets each had a distinct LC composition (table 2) and FA profiles (table 3). In connection with LCs, Artemia nauplii was characterised by a higher content in PC (one-way ANOVA, F=322.1, p<0.001) and TAG (one-way ANOVA, F=1125.5, p<0.001), while detritus was richer in ST (one-way ANOVA, F=6.6, p<0.05) and phytoplankton had a low content of most of the lipid classes analysed. Figure 2. - Total area occupied by the gut content in the whole digestive tract of Caprella equilibra and C. scaura. ‘Marina’ includes data of the field populations directly fixed after collection. Treatments represent the different food provided in laboratory conditions: Artemia nauplii, Detritus, Phytoplankton, All food provided together (All), and no food provided (NF). The bars indicate the percentage of each item observed in the gut under the microscope (Crustacea, Phytoplankton and Detritus). The Crustacea are represented by copepods in specimens from the field populations of the marinas and Artemia in those maintained in laboratory conditions fed with Artemia. Capítulo 4.1 120 In regard to FA, the saturated 16:0, the monounsaturated 16:1, OA, 18:1(n-7) and the polyunsaturated EPA were the dominant components in the three diets. However, the percentage of FA significantly differed among diets. Detritus had a higher content of 16:0 (one-way ANOVA, F=307.1, p<0.001), 18:0 (one-way ANOVA, F=1454.4 p<0.001), LA (oneway ANOVA, F=1157.1, p<0.001) and DHA (one-way ANOVA, F=620.1, p<0.001), but a lower content of 16:1 (one-way ANOVA, F=3562.9, p<0.001). Artemia nauplii were significantly richer in 18:1(n-7) (one-way ANOVA, F=1125.5, p<0.001) and EPA (one-way ANOVA, F=4563.4 p<0.001) while the phytoplankton had a significantly higher content of LNA than Artemia or detritus (one-way ANOVA, F=3237.4, p<0.001). 3.3. Influence of the diet on lipid composition and survival rate of adult specimens (Exp. 2) Both caprellid species showed similar results in the experiment. LC and FA composition of the three treatments considered (A, F and D) are included in tables 2 and 3, together with the reference values obtained from specimens of field populations (marinas). PCA results for LCs (fig. 3) showed differences among treatments, and these differences were also reflected by the PERMANOVA (table 4). Although an interaction between the factors ‘Species’ and ‘Treatment’ was detected, the pair-wise tests of the interaction reflected an identical pattern for both species: the caprellids fed with Artemia differed from those fed with phytoplankton or detritus, as shown by the PCA analysis. Axis 1 (fig. 3) explained 72% of the total variance and correlated with TAG. It clearly Table 2. Main lipid classes (µg/100 µg DW) of the three types of food used in the experiment as diets (A: Artemia, P: Phytoplankton, D: Detritus), the caprellids (Eq: C. equilibra, Sc: C. scaura) collected from marinas (used as reference values) and the caprellids fed either Artemia, phytoplankton or detritus during 12 days in laboratory conditions (treatments). Values are mean of 3 replicates. For caprellids, each replicate consisted on a pool of adult specimens. Dispersion among replicates can be observed in PCA (Fig. 3). FOOD CAPRELLIDS Diets Marinas Treatments A P D Eq Sc EqA EqP EqD ScA ScP ScD Phophatidylcholine (PC) 1.00 0.53 0.07 3.93 2.92 2.35 1.76 1.87 2.54 2.26 1.25 Phosphatidylethanolamine (PE) 0.40 - 0.15 1.45 1.13 0.95 0.73 0.85 0.94 0.98 0.58 Phosphatidylinositol (PI) 0.15 0.11 0.28 0.18 0.21 0.19 0.15 0.15 0.17 0.20 0.11 Phosphatidylserine (PS) - - 0.43 0.21 0.18 0.18 0.20 0.25 0.16 0.26 0.15 Triacylglycerols (TAG) 4.12 - 1.60 3.18 2.40 3.01 0.44 0.41 5.43 1.07 0.57 Sterols (ST) 0.04 0.01 1.68 0.27 0.28 0.15 0.29 0.19 0.22 0.21 0.12 Innovaciones en IMTA: Caprélidos 121 Table 3. Fatty acid composition (expressed as % of the total identified fatty acids) of the three types of food used in the experiment as diets (A: Artemia, P: Phytoplankton, D: Detritus). Eq: C. equilibra, Sc: C. scaura. Values are mean of 3 replicates. For caprellids, each replicate consisted on a pool of adult specimens. Dispersion among replicates can be observed in PCA (Fig. 4). OA: Oleic acid; LA: Linoleic acid; LNA: Alpha linoleic acid; ARA: Arachidonic acid; EPA: Eicosapentanoic acid; DPA: Docosapentanoic acid; DHA: Docosaheanoic acid. FOOD CAPRELLIDS Diets Marinas Treatments A P D Eq Sc EqA EqP EqD ScA ScP ScD Saturated (SFA) 14:0 1.84 4.06 2.42 1.82 3.17 0.93 0.70 0.53 0.81 0.63 0.62 15:0 0.43 1.20 0.68 0.53 0.65 0.28 0.85 0.46 0.39 0.75 0.60 16:0 12.33 19.44 27.58 22.06 19.58 13.80 20.06 18.96 12.60 17.71 17.85 17:0 0.92 - 0.93 1.37 1.55 0.99 1.73 0.98 1.05 2.14 1.27 18:0 4.91 0.64 9.44 6.10 5.31 4.52 5.56 4.94 4.01 5.29 4.91 20:0 - - 1.10 0.12 0.53 - - 0.09 0.15 0.19 0.17 22:0 0.34 - 0.98 0.05 0.05 0.19 - 0.09 0.18 - - 23:0 - - - - - 0.19 - - 0.43 0.94 0.89 24:0 - 0.77 0.97 0.09 - - 0.21 - 0.02 - - Total SFA 20.77 26.11 44.09 32.15 30.84 20.90 29.10 26.05 19.65 27.63 26.31 Monounsaturated (MUFA) 16:1 19.15 17.75 3.04 2.79 4.87 10.32 1.75 1.03 10.71 2.73 1.87 17:1 1.56 4.70 - - - 0.52 - - 1.17 - 0.22 18:1(n-9) (OA) 17.58 3.45 20.49 9.41 10.08 21.76 14.06 18.17 20.52 11.68 18.34 18:1(n-7) 12.91 2.05 3.78 1.78 2.50 10.89 4.46 2.89 11.59 4.46 4.20 20:1(n-11) - - 0.11 0.08 - - - - - - - 20:1(n-9) 0.49 0.40 1.38 1.35 1.47 1.65 1.78 2.11 1.58 1.69 2.27 22:1(n-11) - - 0.82 - - - - - - - - 22:1(n-9) - - - 0.15 0.06 0.24 - 0.06 0.18 0.15 0.18 24:1 - - 1.04 - - 0.29 - - 0.50 - - Total MUFA 51.69 28.37 30.66 15.56 18.97 45.68 22.05 24.26 46.25 20.70 27.08 Polyunsaturated (PUFA) 16:2(n-4) 1.11 2.70 - 0.15 0.31 0.42 - - 0.45 - -0 16:3(n-4) 0.38 2.09 - 0.11 0.09 - - - - - - 16:4(n-1) 0.06 0.79 - - 0.11 - - - - - - 18:2(n-6) (LA) 2.92 4.41 16.46 1.79 2.06 3.38 2.09 7.66 2.99 2.60 9.69 18:2(n-4) 0.45 - - - 0.03 0.14 - - 0.22 - - 18:3(n-6) 0.59 0.23 - 0.12 0.39 0.40 - - 0.37 0.07 0.06 18:3(n-4) 0.52 - - - - 0.31 - - 0.43 - - 18:3(n-3) (LNA) 4.27 20.79 1.95 1.41 1.09 3.87 1.32 0.41 3.07 2.41 0.76 18:4(n-3) 1.41 1.37 - 1.92 2.05 0.78 - - 0.75 0.46 0.26 20:2(n-6) - - - 1.06 0.77 0.42 0.64 1.50 0.33 0.97 1.78 20:3(n-6) - - - 0.07 0.06 - - - 0.07 - - 20:4(n-6) (ARA) 1.15 1.32 0.28 3.24 4.05 2.02 4.37 4.35 2.39 5.77 4.22 20:3(n-3) - 0.54 0.15 0.69 0.87 0.66 1.38 1.02 0.73 1.91 0.74 20 4(n-3) 0.43 - - 0.80 1.07 0.34 - - 0.27 0.07 0.08 20:5(n-3) (EPA) 14.23 11.28 1.62 19.64 21.11 16.60 20.84 16.28 18.37 21.34 14.39 21:5(n-3) - - - 0.39 0.38 - 0.26 - 0.03 0.18 - 22:4(n-6) - - - 0.09 0.17 - - - - 0.07 - 22:5(n-6) - - - 0.55 0.51 - 0.32 0.64 0.04 1.08 0.89 22:5(n-3) (DPA) - - 0.26 0.78 0.82 0.12 0.74 0.91 0.16 0.78 0.59 22:6(n-3) (DHA) - - 4.52 19.50 14.25 3.97 16.89 16.91 3.42 13.94 13.13 Total (n-3) 20.34 33.98 8.50 45.13 41.64 26.35 41.43 35.52 26.82 41.09 29.96 Total (n-6) 4.67 5.96 16.75 6.90 8.01 6.21 7.42 14.16 6.19 10.58 16.65 Total PUFA 27.54 45.52 25.25 52.29 50.19 33.43 48.84 49.68 34.10 51.66 46.61 Capítulo 4.1 128 four common caprellids of the Strait of Gibraltar, including C. equilibra (Baeza-Rojano et al., 2014). These authors proposed that the shallow water amphipods from the Strait of Gibraltar were suitable for use as natural live feed since their protein, lipid and carbohydrate contents are adequate for normal fish, crustacean and mollusc growth. They also pointed out that, in connection with lipid content, marine amphipods, especially caprellids, turned out to be more adequate for aquaculture than freshwater gammarids, based on their higher PUFA levels and polar lipids. Our results, based on the lipid analysis of C. equilibra and C. scaura from marinas, also show an adequate nutritional level. Therefore, the suitability of caprellids as a food resource for fin- and shellfish in aquaculture, based on an adequate nutritional composition, is clear. Future research should be focused on how to obtain inexpensive diets to maintain sustainable caprellid cultures, and how these caprellids could be incorporated into IMTA systems, becoming a combined feed resource and bioremediation mechanism. 4.2. Detritus as food for caprellid culture The present study produces evidence supporting the use of detritus (composed mainly of fish faeces, and secondarily of uneaten feed pellets) for caprellid culture. In natural habitats, although amphipods form a trophic continuum from primary herbivores to carnivores Mann (1998), Guerra-García and Tierno de Figueroa (2009) and Guerra-García et al. (2014) found that detritus is the main food item in the majority of species. The importance of detritus in benthic communities has often been reported in literature, and is considered to be highly nutritious after a short period of microbial colonization (Mann, 1998). Each of the three food types used as a diet for feeding caprellids during the present study showed a distinct composition; detritus had higher levels of ST and 16:0, 18:0, OA, LA or DHA, while Artemia nauplii was richer in PC, TAG, 18:1(n-7) and EPA, and phytoplankton was a source of LNA and PC. The lower concentrations of lipid classes measured for phytoplankton could be due to the presence of other polar lipids such as glucolipids, which were not quantified during the present study. High amounts of glucolipids in phytoplankton, and an abundance of free fatty acids in fish faecal faeces have also been measured (Hachero-Cruzado, unpublished data). In general terms, when compared with other diets described in literature for feeding amphipods or fishes (e.g. Gonzalez-Silvera et al., 2015; Hyne et al., 2009), detritus Innovaciones en IMTA: Caprélidos 129 can be considered a diet of relatively adequate nutritional value. It is a source of sterols (ST) 16:0, 18:0, and interestingly OA, LA and DHA. In fact, fragmented pellets and faeces might represent a new trophic source for fouling organisms, providing them with terrestrial fatty acids such as LA, which are unusual in marine environments (Gonzalez-Silvera et al., 2015). These authors found that the sediments associated with fish farms presented higher percentages of OA and LA than control sediments, due to the faeces contribution, and suggested that faeces could be used as a trophic resource. Indeed, LA is considered a dietary essential fatty acid in the diets of vertebrates (Hyne et al., 2009), since physiologically essential FAs, ARA (20:4n-6), EPA and DHA are obtained through diet or synthesised from LA or LNA via a desaturase-elongase system (Leonard et al., 2004). Gonzalez-Silvera et al. (2015) found that Caprella equilibra collected from fish farms were characterised by a higher concentration of PUFA(n-6) (mainly LA) than specimens from a control site. This data supports our results and the potential use of fish farm detritus as a resource. The FA profiles provided by Kawashima et al. (1999) for C. mutica from aquaculture facilities (salmon sea cages, shellfish longlines stocked with mussels and mooring lines from caged finfish aquaculture) do not differ very much from those obtained during the present study for C. equilibra and C. scaura fed with detritus, with the exception of the higher concentrations of LA measured in our study. It has been well established in recent decades that long chain PUFAs, especially (n-3) PUFAs, have a vital role in human nutrition, disease prevention and health promotion (Kolanowski et al., 2007; Garofalaki et al., 2006 and references therein). They cannot be synthesised by fishes, meaning their levels depend upon dietary intake. Among these, the most important (n-3) PUFAs are EPA, DPA and DHA. These FAs cannot be produced by most crustacean species in sufficient quantities for metabolic functioning so they too must be obtained from the diet (Folch et al., 1957 and references therein). The present study has shown that detritus seems to be an adequate source of DPA and DHA for caprellids, however a poor source of EPA. Detritus proved also to contain low levels of PC and TAG while Artemia was an excellent source of these lipids (see table 2). Phospholipids, such as PC, located mainly in biological membranes, have an essential role in regulating biophysical properties, protein sorting and in cell signalling pathways (Coutteau et al., 1997; Lahdes et al., 2010). The Capítulo 4.1 130 phospholipids of the majority of marine species including fish, molluscs and crustaceans are rich in PUFA, especially (n-3) (Coutteau et al., 1997). The principal storage lipids in marine organisms including amphipods consist of TAGs, with a sufficient amount of these compounds ensuring the survival of individuals over the non-productive season (Hyne et al., 2009). The percentage of TAGs in relation to the amount of total lipids can show an increase in energy demand allocated to gamete production and egg incubation (Dutra et al., 2007). A relatively high amount of TAG can indicate periods of starvation, where storage lipids may be important for survival (Graeve et al., 2001). During the present study, adults fed with detritus had a higher survival rate than those fed with Artemia or phytoplankton, but interestingly the survival rate of hatchlings during the first 16 days of development showed the opposite pattern. The higher survival rate obtained for caprellid hatchlings fed with Artemia is probably related with higher requirements of TAG and/or PC during the first stage of life history, which cannot be provided by detritus or phytoplankton. A highest level of TAG in amphipod females, increasing with the maturation of the ovary, has been reported by Clark et al. (1985) and it is expected that hatchlings also require higher TAG concentrations as a lipid reserve to properly face the first days of the life cycle. Clark et al. (1985) also pointed out that MUFAs are used in egg development, and in the present study Artemia proves to be a good source of MUFA, especially 16:1, in comparison with detritus. Curiously, Guerra-García et al. (2004) found higher percentages of 16:1 in caprellid females than in males, so this fatty acid is probably related with egg development and it is required in higher levels by hatchlings. For all these reasons, a supply of Artemia would be optimal in completing the requirements of TAG, PC, and MUFA during the early stages of development of caprellid cultures. In field populations, these requirements are probably provided by small crustaceans such as copepods or other amphipods, such as those observed in the guts of specimens from the marinas examined during the present study. The results of Ros et al. (2014a) seem to support this hypothesis since these authors found significantly higher amounts of prey (copepods) in juveniles of C. scaura than in adults. A diet shift during the development has also been observed in other amphipods (Olabarria et al., 2009). Ontogenetic shifts in diet may occur in order to overcome physiological constraints (Rossi et al., 2004). For instance, when juveniles have physiological limitation in the maximum rate of food uptake, they Innovaciones en IMTA: Caprélidos 131 might rely on higher quality sources of food to minimise the amount of food and maximise energy uptake (Hentschel, 1998). Hence, the juveniles could satisfy their higher requirements of TAG, PC and MUFA with an increased copepod intake. 4.3. Caprellid cultures and IMTA systems The present study indicated that caprellids fed with detritus had an adequate lipid composition to be used as an aquacultural resource. Additionally, the survival rate for adult specimens was higher for caprellids fed with detritus than for those fed with Artemia or phytoplankton. In the case of hatchlings, the results are not as promising due to higher mortalities seen in specimens subject to a diet of detritus. Despite this, hatchlings which successfully completed the juvenile stage with detritus were seen to reach similar sizes as those fed with Artemia or phytoplankton. Detritus therefore seems not to negatively affect the growing rate of survivors. For caprellids cultured under laboratory conditions at small- or medium-scales (Baeza-Rojano et al., 2013b; Baeza-Rojano et al., 2011) it is therefore recommended that a mixed diet of detritus enriched with Artemia, especially during the first 15 days of culture, is used. If the culture is to be maintained offshore, the presence of a nearby supply of detritus would be recommended. Fish farms and sea-cage structures are interesting sources of detritus (Salvo et al., 2015) so a caprellid culture associated to these aquaculture facilities would be an excellent option. In this case, the availability of detritus for caprellids is guaranteed, and the additional TAG, PC and MUFA requirements of juveniles and reproductive females could be obtained by preying on copepods or other small planktonic crustaceans, which naturally inhabit fish farm ecosystems. Copepods are frequently found in the gut contents of caprellids in field populations (Guerra-García and Tierno de Figuero, 2009) and caprellids can prey directly on them, or ingest them accidentally while filtering (pers. obs.). Copepods are rich in lipids (e.g. Wang et al., 2014) and can be an important source of TAG (Cass et al., 2014). In fact, cultured copepods have been successfully used in the larviculture of various marine fish larvae, although the upscale of copepod cultures to commercial levels is still a challenge (Ajiboye et al., 2011). Capítulo 4.1 132 The availability of detritus in fish farms, together with access to planktonic crustaceans naturally inhabiting the area, probably provide an optimum diet. This would explain the reason that the highest population densities for caprellids have been recorded in substrates of aquaculture facilities (see Woods (2009) for details). On the west coast of Scotland, densities of 319 000 ind/m2 have been reported for Caprella mutica in a fish farm (Ashton et al., 2010). In Ireland and Scotland, C. mutica exhibits greater fecundity and abundance, size and population longevity at fish farm locations, with these results being attributed to organic enrichment from the fish farms (Tierney et al., 2004; Willis et al., 2004; Ashton et al., 2010). The present study confirms experimentally that detritus in the form of fish faeces is an adequate food, although also provides evidence that caprellids must have access to a source of TAG, PC and MUFA during the first stages of their life histories. From the environmental point of view the establishment of caprellid cultures associated to fish farms and sea cage structures seems to be justified, as caprellids would act as bioremediators. Encouraging the presence and abundance of bioremediating biota alongside net cage aquaculture has been proposed as a potential mechanism to ameliorate associated negative aquaculture impacts (see Woods (2009) and references therein). In IMTA systems, the culture of caprellids alongside macroalgae is also advisable, since macroalgae provide substrate for caprellids. Caprellids may also have a direct benefit to macroalgae by reducing epiphytic overgrowth (e.g. Brawley and Fei, 1987; Duffy, 1990; see Woods (2009) for details). In addition to attaching to many different types of natural substrates (Guerra-García, 2001), caprellids easily use a range of artificial ones such as buoys, nets, plastic mesh, ropes and PVC panels. Baeza-Rojano et al. (2011) tested different kinds of plastic mesh with varying complexities for maintaining a culture of C. scaura and achieved very high densities (>10 000 ind/m2) in all the substrates. Therefore, the culture of caprellids in IMTA could only require the use of additional artificial structures, easy to set and remove, located close to the fish farms. Previous studies of water currents would be desirable to find the most adequate placement locations in order to maximise efficiency in receiving the detritus from the fish faeces and uneaten food pellets. As pointed out by Woods (2009), framed mesh panels or a frame of string lines suspended within culture tanks could be used and once sufficient Innovaciones en IMTA: Caprélidos 133 biomass is reached, frames could then be transferred with their attached caprellids to fish culture tanks for fish to graze on at their leisure, closing the loop. Alternatively, caprellids from the frames could also be collected and dehydrated to be used as a component in the fabrication of fish feed, encased in microdiets, etc. The two caprellid species used in the present study, C. equilibra and C. scaura can reach very high densities when associated with fouling communities of marinas in Southern Spain (Ros et al., 2013; Guerra-García et al., 2015; Ros et al., 2015). Recently, Caprella scaura has also been found on off-coast fish farm cages in the Mediterranean Sea (Fernández-González and Sánchez-Jerez, 2014). The life history of Caprella equilibra in smallscale laboratory conditions has been recently completed Baeza-rojano et al. (2013) and Woods (2009) considered C. equilibra as ideally suited to large-scale culture. A preliminary study of a medium-scale culture of C. scaura has also been conducted, showing promising results for this species (Baeza-Rojano et al., 2013b). In fact, during the present study hatchlings of C. scaura showed a significantly higher survival rate than those of C. equilibra. Consequently, C. scaura should also be a good candidate for large scale cultivation. However, it must be taken into account that C. scaura is a strongly invasive species which is quickly spreading across the Mediterranean and East Atlantic (Ros et al., 2014b) and seems to be ecologically displacing the native C. equilibra (Ros et al., 2015) along European coasts. Therefore, as pointed out by Woods (2009) any research elucidating the aquaculture potential of caprellid species should adopt a “precautionary principle” assuming nonindigenous species may have detrimental impacts on indigenous biota (unless proven otherwise) and concentrate on indigenous and/or already established caprellid species, such as C. equilibra. Provided that detritus is an adequate diet and that fish farms are an excellent source of detritus, the next step is to experimentally develop caprellid culture at a large-scale under controlled conditions in order to optimise and evaluate the use of detritus in terms of caprellid survival rates and reproductive success. Pilot experiments with caprellid cultures associated to IMTA systems are also needed to evaluate the biomass production and the economic and ecological sustainability of these cultures. Capítulo 4.1 134 5. References Ajiboye OO, Yakubu AF, Adams TE, Olaji ED, Nwogu NA. 2011. 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Mediterranean fouling communities assimilate the organic matter derived from coastal fish farms as a new trophic resource. Marine Pollution Bulletin, 91: 45-53. Graeve M, Dauby P, Scailteur Y. 2001. Combined lipid, fatty acid and digestive tract content analyses: a penetrating approach to estimate feeding modes of Antarctic amphipods. Polar Biology, 24: 853-862. Guerra-García JM, Martínez-Pita I, Pita ML. 2004. Fatty acid composition of the Caprellidea (Crustacea: Amphipoda) from the Strait of Gibraltar. Scentia Marina, 68: 501-510. Guerra-García JM, Ros M, Baeza-Rojano E. 2015. Seasonal fluctuations and dietary análisis of fouling caprellids (Crustacea: Amphipoda) from marina of southern Spain. Marine Biology Research, 11: 703-715. Guerra-García JM, Tierno de Figueroa JM, Navarro-Barranco C, Ros M., Sánchez-Moyano JE, Moreira J. 2014. Dietary analysis of the marine Amphipoda (Crustacea: Peracarida) from the Iberian Peninsula. Journal of Sea Research, 85: 508-517. 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Fatty acid composition of the estuarine amphipod Melita plumulosa (Zeidler): Resumen Los anfípodos son consumidos por los peces en la naturaleza y se han utilizado como presa alternativa para peces y cefalópodos. De los anfípodos de los esteros del sur de España, Gammarus insensiblis (Stock, 1996) reúne las características para ser una especie interesante para los sistemas IMTA. G. insensibilis fue cultivada durante 21 días con diferentes dietas; el experimento se replicó dos veces con 5 tratamientos, 4 dietas: Artemia, Detritus, Phytoplakton, Ulva sp. y el quinto tratamiento Ausencia de Alimento. Tanto el detritus como la Ulva generalmente se tratan como residuos en acuicultura, pero en este estudio se utilizaron como alimento consiguiendo buenas tasas de supervivencia. Los anfípodos criados con Artemia mostraron una alta supervivencia con altas concentraciones de triacilglicerol (TAG) y ácidos grasos saturados. Las dietas alternativas, detritus y Ulva, también mostraron tasas de supervivencia interesantes y contribuyeron a que los gammarideos presentaran altas concentraciones de ácido palmítico (16: 0), ácido oleico (18: 1n9), ácido araquidónico (20: 4n6) (ARA), ácido eicosapentaenoico (20: 5n3) (EPA) y ácido docosahexaenoico (22: 6n3) (DHA). Además, Gammarus insensibilis podría desempeñar un papel clave como biorremediador siendo cultivado con detritus y Ulva obteniendo una composición bioquímica adecuada para ser utilizada en acuicultura. Este anfípodo podría reemplazar parcial o totalmente las dietas formuladas consiguiendo la independencia del aceite de pescado y la reducción de costes económicos. Abstract Amphipods are consumed by fish in the nature and have been used as alternative prey for fishes and cephalopods. Among amphipods of South Spain ponds, Gammarus insensiblis (Stock, 1996) meet the characteristics to be an interesting species for IMTA. G. insensibilis was cultured during 21 days with different diets; the experiment was replicated twice with 5 treatments, 4 diets: Artemia, Detritus, Phytoplakton, Ulva sp. and the fifth was No Feed treatment. Detritus and Ulva are usually treated as residues in aquaculture but in this study were used as feed with good survival rates. Amphipods reared with Artemia showed high survival and high concentrations of triacylglycerol (TAG) and saturated fatty acids. The alternative diets detritus and Ulva also showed interesting survival rates and contributed to high concentrations in the gammaridean of palmitic acid (16:0), oleic acid (18:1n9), arachidonic acid (20:4n6) (ARA), eicosapentaenoic acid (20:5n3) (EPA) and docosahexaenoic acid (22:6n3) (DHA). Furthermore, Gammarus insensibilis could play a key role as biorremediator being cultured with detritus and Ulva getting an adequate biochemical composition to be used in aquaculture. This amphipod could replace partial or totally the formulated diet with two consequences, reduce dependence of fish oil and subsequently of economic costs. Innovaciones en IMTA: G. insensibilis 147 Evaluation of detritus and Ulva sp. as diets to culture the amphipod Gammarus insensibilis, a promising species for Integrated Multi- Trophic Aquaculture (imta). Jiménez-Prada, P.1,2*, Hachero-Cruzado, I.2 and Guerra-García, J.M.1 1Laboratorio de Biología Marina, Dpto. Zoología, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes 6, 41012 Sevilla, España. 2IFAPA — El Toruño, Camino Tiro Pichón s/n, El Puerto de Santa María, España. * Corresponding author: Dpto. Zoología. Avda. Reina Mercedes 6. 41012 Sevilla, España. Teléfono: +34-954556229. E-mail: [email protected] 1. Introduction Fish are an important source of protein, 17% of the animal protein consumed in the world is provided by fish and even 50 % in Occidental Africa and East Asia, the geographical areas with the lower rent per capita (FAO, 2016). Aquaculture generate near 50 million of tonnes of finfish surpassing 160 $ millions (FAO, 2016). Aquaculture production is supported by high fish meal and fish oil consumption, so it consumes 75% of fish oil. According to FAO (2014) fish oil and fish meals are the only economical viable resource of n-3 HUFA to produce formulated diet. Consequently, there is an urgent need to explore and investigate the potential of novel aquatic organisms to replace formulated diet or as live feed in aquaculture. Amphipod crustaceans are among the most diverse group of crustaceans with respect to life styles, trophic types, habitats and sizes (De Broyer and Jazdzewski, 1996). Amphipods inhabit a variety of marine environments and consequently show a high diversity of feeding habits (Guerra-García et al., 2014). Due to their nutritional characteristics, amphipods could serve as an adequate alternative live or dead feed resource for aquaculture (Baeza-Rojano et al., 2014, Kolanowski et al., 2007, Gonzalez et al. 2011). Amphipods are consumed by fish in the nature (chapter 2) and have been used as alternative prey for fishes (Moren et al., 2006, Sountana et al., 2007) and cephalopods (Baeza-rojano et al., 2010, 2013) with positive results too. Capítulo 4.2 148 Residues generated by aquaculture, such as detritus or macroalgae can be used by others organism (e.g. amphipods) in the context of Integrated Multi-Trophy Aquaculture (IMTA) protocols. Chapter 3 showed that caprellid amphipods can breed with detritus obtained of aquaculture fishes and they could be excellent candidates for integration into IMTA systems. Detritus comes mainly from fish faeces and uneaten fishfeed pellets associated to tanks or ponds in aquaculture facilities. Moreover, it is also common that some macroalgae, such as Ulva sp, quickly grow and spread in these facilities reaching high biomass associated to the main cultures. The potential use of detritus and macroalgae as cheap diets for amphipods is therefore an interesting topic to be addressed. In spite of the interest of amphipods as potential resource in aquaculture, there is a lack of studies focused on large scale culture testing different diets. For non-caprellids amphipods, most studies have dealt with freshwater species (see e.g. Kennedy et al., 2016) and for caprellid amphipods the first attempts have been conducted during the last years (see e.g. Baeza-Rojano et al., 2013). Therefore, the main aim of this study is to explore the viability of diets considered residues (such as detritus or macroalgae) to massive culture a marine amphipod potentially interesting as a resource in aquaculture. This would be the first step to further integrate this marine amphipod in IMTA systems 2. Material and Methods The experiment lasted 21 days and was replicated twice being conducted in two different dates (09/2015 and 02/2016). Three 70 litres tanks were used for each treatment with 200 specimens of Gammarus insensibilis. The water (17-18ºC and 35 U of salinity) was renovated daily. G. insensibilis (Amphipoda: Peracarida) was collected from marine ponds in IFAPA “El Toruño” (South Spain) (see details of study area in chaoter 3) where inhabit mainly on Ulva sp. To simulate the wild conditions of this amphipod species a net with same size that Ulva seaweed was introduced in each tank. Both experiments had 5 treatments, 4 diets: Artemia (nauply of 0 or 1 day without enrichment), Detritus, Phytoplakton, Ulva sp. and the fifth was No Feed treatment, in which specimens were maintaining without any food. The phytoplankton used consisted of a mixture of freeze-dried microalgae (Easy Reefs1) containing Phaeodactylum, Innovaciones en IMTA: G. insensibilis 149 Tetraselmis and Nannochloropsis (1:1:1, percentage in dry weight). To avoid the precipitation of Phytoplankton and Detritus, they were added in an accessory water container to adjust the continuous entry of food. In all treatments 100 mg per day of dry weight diet was supplied, except in Ulva treatment where the amphipods ate “ad libitum” the seaweed body. At the end of the experiment, survival of amphipods in each tank was measured, and the remaining alive specimens were kept for chemical analysis of lipid classes and fatty acids. In addition, wild gammarids (taken directly from the field) were analysed to compare their biochemical composition with the four treatments. Detritus and Phytoplankton diet were the same than those used in chapter 3; in fact, data of biochemical composition was taken from them. For Ulva and Artemia, six replicates, three in each experiment, were taken and analysed in the present study. 2.1. Chemical analyses. Three pools (ca. 15-20 individuals per pool) of the survivor specimens of each tank (3 tanks per treatment: Artemia, Detritus, Phytoplakton and Ulva) were used for the chemical analyses (lipid classes and fatty acids). Additionally, 3 pools of amphipods collected directly from the ponds (used as a control of the normal biochemical composition in natural conditions) were also analysed. The amphipod samples of each feed treatment were freeze-dried for 24h at -50°C. The lipid fraction was extracted according to the Folch-Lee method (Floch et al., 1957). Total lipid was extracted with chloroform:methanol (2:1v/v) containing 0.01% of butylated hydroxytoluene (BHT) as an antioxidant (Christie, 1982). The organic solvent was evaporated under a stream of nitrogen and the lipid content was determined gravimetrically. Lipid classes were separated by one-dimensional double development high-performance thin-layer chromatography (HPTLC) using methyl acetate/isopropanol/chloroform/ methanol/0.25% (w/v) KCl (25:25:25:10:9 by volume) as the polar solvent system and hexane/diethyl ether/glacial acetic acid (80:20:2 by volume) as the neutral solvent system. Final quantification of lipid classes was made by densitometry in a CAMAG scanner at a wavelength of 325nm, and by comparison with external standard (Sigma-Aldrich) (see Olsen and Henderson, 1989). For fatty acid analysis, total lipid extracts were subjected to acid catalysed transmethylation for 16 hours at 50°C, using 1mL of toluene and 2mL of 1% sulphuric acid (v/v) in methanol. The resulting Capítulo 4.2 150 fatty acid methyl esters (FAME) were separated and quantified using a Shimadzu GC 2010-Plus (Shimadzu) gas chromatograph equipped with a fame-ionisation detector (280°C) and a fused Tecnokroma–Suprawax-280 (15 m x 0.1 mm I.D.). Hydrogen was used as carrier gas and the oven initial temperature was 100°C, followed by an increase at a rate of 20°C min -1 to a final temperature of 250°C for 8 min. Individual FAME were identified and quantified using external standard (Sigma-Aldrich). 2.2. Statistic analyses To explore differences in survival rates among treatments (including the No feed treatment as a control in this case) a two-way ANOVA was used. Prior to ANOVAs, the homogeneity of variances was tested with Cochran’s test. The design included two factors: “Experiment” with two replications on the time, and “Diet” with 5 levels including the 5 treatmens (Artemia, Detritus, Phytoplankton, Ulva and No food). A Canonical Analysis of Principal Component (CAP) was conducted to show the relationship among Treatments (diets) according to the lipid classes and fatty acid matrices. Differences in fatty acid and lipid classes composition among treatments were tested by the use of a permutational multivariate analysis of variance (PERMANOVA) with two factors: “Experiment” with two replications on the time, and “Diet” with 5 levels, in this case, the four treatments (Artemia, Detritus, Phytoplakton and Ulva) and amphipods directly taken from the field (wild amphipod) as a control. In this case specimens of the no feed treatment were not included since they were not chemically analysed due to the lack of enough survivor specimens. Analysis was based on Euclidean distance measures and Monte Carlo tests were included. Significant P-values were obtained by computing 9999 permutations of residuals under a reduced model as this method gives the most accurate Type I error for complex designs (Anderson, 2005). Pairwise comparisons were then used. Data were transformed with function ln(x+1). Univariate analyses were conducted with GMAV5 (Underwood et al., 2002) and multivariate analyses were carried out using the PRIMER v.6 plus PERMANOVA package (Clarke and Gorley, 2001). Innovaciones en IMTA: G. insensibilis 151 3. Results 3.1. Survival The ANOVA test for survival rate presented interaction between Treatment and Experiment (table 1). In the first experiment, no significant differences among treatments were measured, while in the second experiment survival for Artemia treatment was statistically higher and for No Feed was statistically lower (table 1, figure 1) than treatments Detritus, Phytoplankton and Ulva. 3.2. Lipid composition of the diets used in the experiments. Biochemical composition of the different diets used in the experiment is included in table 2. Regarding the lipid classes, Artemia Diet (AD) had the highest amount of total lipids classes, triacylglycerols (TAG) and Figure 1. Means of survival percentage (and standard deviation) of three tanks per treatment of two experiment. Numbers indicated experiments. The letters indicate difference among treatment (p<0,01). Table 1. Two-way ANOVAs test for survival rate (%). Treatments: A= Artemia, D= Detritus, P= Phytoplankton, U= Ulva and NF= No Feed. *p<0.05, **p<0.001 SNK df MS F p Experiment 1 Experiment 1 433.2 4.08 0.057 A=NF=D=P=U Diet 4 1405.12 13.24 0.000** Experiment 2 Exp*Diet 4 354.08 3.34 0.030* A>D=P=U>NF Residual 20 106.1 Survival Two-way ANOVA Capítulo 4.2 152 AD DD PD UD Lipid Classes PC 2.31 0.07 0.53 0.12 PE 2.77 0.15 -0.06 PI -0.28 0.11 0.02 PS -0.43 - - TAG 11.17 1.60 -0.04 Cho 1.66 1.68 0.01 0.05 Fatty Acids SAT 14:0 0.62 2.45 4.05 1.29 15:0 0.15 0.69 1.20 0.29 16:0 11.91 27.91 19.40 24.36 17:0 0.73 0.94 - 0.75 18:0 5.95 9.55 0.64 1.57 20:0 0.19 1.11 - - 22:0 0.01 0.99 - 1.72 24:0 -0.98 0.77 - MUFA 14:1 0.16 - - 1.05 15:1 - - - 1.13 16:1n7 2.62 3.08 17.71 11.41 17:1 0.05 - 4.69 - 18:1n9 20.73 20.74 3.44 7.25 18:1n7 7.69 3.83 2.05 14.04 20:1n9 0.78 1.40 0.40 - 22:1n11 0.11 0.83 - - PUFA 16:2n4 0.08 - 2.69 - 16:2n3 0.12 - - - 16:3n4 0.01 - 2.09 - 16:3n3 1.50 - - - 16:4n1 0.79 16:4n1+18:0dma 0.02 - 0.79 - 18:2n6 5.39 16.66 4.40 2.08 18:3n6 0.46 - 0.23 - 18:3n3 31.02 1.97 20.74 8.43 18:3n4 0.08 - - - 18:4n3 4.50 - 1.37 15.91 18:4n1 - - - - 20:2n6 0.23 - - - 20:3n6 0.11 - - 2.06 20:4n6 0.83 0.28 1.32 0.58 20:3n3 0.94 - - 0.96 20:4n3 0.75 - - 0.58 20:5n3 1.59 1.64 11.25 2.08 22:5n3 -0.26 - 2.46 22:6n3 0.03 4.57 - - ∑ Sat 19.56 44.63 26.05 29.97 ∑ MUFA 32.15 29.87 28.28 34.89 ∑ PUFA 47.65 25.39 45.66 35.14 Diets Table2. Values of Lipid Classes (μg/100μg of DW) and Fatty Acids (% of total fatty acids) of used diets. AD= Artemia Diet, DD= Detritus Diet, PD= Phytoplankton Diet and UD= Ulva Diet. PC: Phophatidylcholine, PE: Phosphatidylethanolamine, PI: Phosphatidylinositol, PS: Phosphatidylserine, TAG: Triacylglycerols, Cho: Cholesterol. SFA: Saturated fatty acids, MUFA: Mono-unsatured fatty acids, PUFA: Poli-unsaturated fatty acids. Innovaciones en IMTA: G. insensibilis 153 phospholipids PC and PE. In Detritus Diet (DD), TAG and sterols were the most abundant lipids, although the former was much lower than in AD. Regarding Phytoplankton Diet (PD) and Ulva Diet (UD), both had a low content of most of the lipid classes analysed Regarding to fatty acid, all diets showed a high content of 16:0 (PA), 16:1n-7, 18:1n-9 (OA), 18:1n-7 and 18:2n-6 (LA). However, the percentage of FA significantly differed among diets. DD and AD were enriched in 18:1n-9 (OA), and AD and PD showed the higher content in 18:3n-3(LNA). Moreover, DD displayed the higher percentage of fatty acids 18:0, 18:2n-6 (LA) and 22:6n-3 (DHA), and PD of fatty acids 14:0, 16:1n-7, 17:1, 16:2n-4, 16:3n-4, 20:4n6 (ARA) and 20:5n3 (EPA). Finally, UD showed the higher content of 18:1n-7, 18:4n-3 and 22:5n-3 (EPA). 3.3. Lipid composition of treatments. The five treatments and wild amphipods are represented in tables 3 and 4. Wild amphipods were included as positive control. Lipid classes and fatty acids PERMANOVAs tests (table 5) showed difference in the interaction Experiment with Diet. Despite the pair-wise test of interaction did not show clear consistent patters and reflected differences among experiments, the CAPs (Canonical Analysis of Principal Components) of lipid classes and fatty acids described clear groups, consistent for both experiments. Table 3. Values of Lipid Classes (μg/100μg of DW) of amphipods of the 4 different treatments, values for wild amphipods are also used. A= Artemia treatment, D= Detritus treatment, P= Phytoplankton treatment, U= Ulva treatment, W= Wild amphipods. PC: Phophatidylcholine, PE: Phosphatidylethanolamine, PI: Phosphatidylinositol, PS: Phosphatidylserine, TAG: Triacylglycerols, Cho: Cholesterol. Each replicate consists on a pool of specimen. A D P U W A D P U W PC 1.66 1.23 1.22 1.61 1.74 2.07 1.61 1.84 2.02 1.25 PE 1.18 0.86 0.93 1.06 1.15 1.27 0.90 1.03 1.14 0.92 PI 0.08 0.06 0.06 0.04 0.06 0.08 0.05 0.09 0.09 0.06 PS 0.23 0.26 0.14 0.19 0.16 0.42 0.33 0.45 0.50 0.23 TAG 2.25 0.44 0.17 0.25 0.44 3.73 0.20 0.25 0.09 0.79 Cho 0.16 0.15 0.16 0.12 0.11 0.15 0.14 0.15 0.17 0.08 Experiment 1 Experiment 2