A peer-reviewed version of this preprint was published in PeerJ on 12 January 2018. View the peer-reviewed version (peerj.com/articles/4194), which is the preferred citable publication unless you specifically need to cite this preprint. 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 https://doi.org/10.7717/peerj.4194 Crustacean amphipods from marsh ponds: a nutritious feed resource with potential application in Integrated Multi-Trophic Aquaculture. Pablo Jiménez-Prada Corresp., 1, 2 , Ismael Hachero-Cruzado 2 , Inmaculada Giráldez 3 , Catalina Fernández-Diaz 2 , César Vilas 3 , José Pedro Cañavate 2 , José Manuel Guerra-garcía 1 1 Laboratorio de Biologia Marina. Departamento de Zoología., Universidad de Sevilla, Sevilla, Spain 2 Instituto de investigacion y Pformacion Agraria y Pesquera. El Toruño, Puerto de Santa María., Spain 3 Departament de Química "Prof. J.C. Víchez Martín", Facultad de Ciencias Experimentales, Universidad de Huelva, Huelva, Spain Corresponding Author: Pablo Jiménez-Prada Email address: [email protected] Coastal protection, nutrient cycling, erosion control, water purification, and carbon sequestration are ecosystem services provided by salt marshes. Additionally, the salt ponds offers coastal breeding and nursery habitat to fishes and invertebrates providing abundant amphipods 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). Their dependence on limited fisheries constrains growth of aquaculture and the need for n-3 LC-PUFA recommends the development of more sustainable food sources. Due to their extensive feeding on detritus and primary producers, amphipods appear as potential and high quality alternative feed resource for aquaculture. Hence, a nutritional study was carried out for main amphipods species -Microdeutopus gryllotalpa, Monocorophium acherusicum, Gammarus insensibilis, Melita palmata and Cymadusa filosa- at South Spain aquaculture terrestrial ponds . These species showed a 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 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 profile of the five amphipods have been compared with other studied alternatives preys. Therefore, amphipods of ponds are good candidates to be used as feed, and are proposed as a new sustainable economic resource to use in aquaculture. G. insensibilis may be the best to be intensively cultured as an alternative feed resource because shows: 1) adequate PUFAS n-3 and PL composition; 2) high levels of glycine, alanine, tyrosine, isoleucine and lysine; 3) high natural densities; PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3220v1 | CC BY 4.0 Open Access | rec: 2 Sep 2017, publ: 2 Sep 2017 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. 1 Crustacean amphipods from marsh ponds: a nutritious feed resource with potential 2 application in Integrated Multi-Trophic Aquaculture. 3 Pablo Jiménez-Prada1,2*, Ismael Hachero-Cruzado2, Inmaculada Giraldez3, Catalina Fernández- 4 Díaz2, César Vilas2, José P. Cañavate2, José M. Guerra-García1. 5 1Laboratorio de Biología Marina, Dpto. de Zoología, Facultad de Biología, Universidad de 6 Sevilla, Avda. Reina Mercedes 6, 41012 Sevilla. 7 2 IFAPA - El Toruño, Camino Tiro Pichón s/n, El Puerto de Santa María. 8 3Dpto. de Química "Prof. J.C. Víchez Martín", Facultad de Ciencias Experimentales, 9 Universidad de Huelva, Campus Universitario El Carmen, Avenida de las Fuerzas Armadas, 10 21071- Huelva. 11 *Corresponding author: Pablo Jiménez-Prada. Tel.: +34954556229 12 E-mail addres: [email protected] 13 14 Abstract 15 Coastal protection, nutrient cycling, erosion control, water purification, and carbon sequestration 16 are ecosystem services provided by salt marshes. Additionally, the salt ponds offers coastal 17 breeding and nursery habitat to fishes and invertebrates providing abundant amphipods 18 potentially useful as a resource in aquaculture. Fishmeal and fish oil are necessary food resources 19 to support aquaculture of carnivorous species due to their omega-3 long-chain polyunsaturated 20 fatty acids (n-3 LC-PUFA). Their dependence on limited fisheries constrains growth of 21 aquaculture and the need for n-3 LC-PUFA recommends the development of more sustainable 22 food sources. Due to their extensive feeding on detritus and primary producers, amphipods 23 appear as potential and high quality alternative feed resource for aquaculture. Hence, a 24 nutritional study was carried out for main amphipods species -Microdeutopus gryllotalpa, 25 Monocorophium acherusicum, Gammarus insensibilis, Melita palmata and Cymadusa filosa- at 26 South Spain aquaculture terrestrial ponds. These species showed a high protein content (up to 27 40%), high n-3 PUFA and phospholipid levels, and high levels of phophatidylcholine (PC), 28 phosphatidylethanolamine (PE) and triacylglycerols (TAG), the latter significantly high for M. 29 acherusicum. M. gryllotalpa and M. acherusicum showed the highest proportion of lipids 30 (19.15% and 18.35%, respectively). Isoleucine, glycine and alanine were the dominant amino 31 acids in all species. In addition, amphipods collected from ponds showed low levels of heavy 32 metals. Furthermore, the biochemical profile of the five amphipods have been compared with 33 other studied alternatives preys. Therefore, amphipods of ponds are good candidates to be used 34 as feed, and are proposed as a new sustainable economic resource to use in aquaculture. G. 35 insensibilis may be the best to be intensively cultured as an alternative feed resource because 36 shows: 1) adequate PUFAS n-3 and PL composition; 2) high levels of glycine, alanine, tyrosine, 37 isoleucine and lysine; 3) high natural densities; 4) large body size (≥1 cm) and 5) high 38 concentration of Calcium. Moreover, a combined culture of amphipods and fishes in these marsh 39 ponds seems a promising and environmentally sustainable way to develop Integrate Multi- 40 Trophic Aquaculture (IMTA) in these ecosystems. 41 42 43 Keywords: Alternative prey, amino acid, amphipods, aquaculture, fatty acid, lipid classes, major 44 metal, marsh, nutrition, ponds, trace metals. 45 46 1. Introduction 47 Saltmarshes are aquatic systems with many ecosystem services arising from ecosystem 48 processes and functions such as coastal protection, nutrient cycling, erosion control, water 49 purification, and carbon sequestration (Barbier et al. 2011). Nowadays, approximate the 50% 50 (and increasing) of salt marshes suffer deterioration due to human activities (Barbier et al. 2011). 51 Interestingly, some salt marshes, as those located in Sothern Spain, require anthropogenic 52 activities (hydraulic control, walls conservation, sediment removing, etc.) to be economically 53 sustainable in the context of aquaculture (Arias and Drake, 1999). These modified saltmarshes 54 provide, therefore services as coastal breeding and nursery habitat, which are used by local 55 people to practice a kind of extensive aquaculture generating economic activity. 56 The saltmarsh zone located East and South to Parque Natural Bahía de Cádiz (PNBC), 57 South Spain, is characterized by a complex system of tidal channels and creeks that supply 58 seawater to saltmarsh fish-ponds situated along their courses. Most of the saltmarsh ponds 59 remain permanently flooded during the major part of the year, and constitute a semi-natural 60 lagoon ecosystem exploited for extensive and semi-intensive fish culture (Arias and Drake, 61 1994). These ponds are productive ecosystems thanks to their particular hydrology and 62 morphology allowing for optimum use of the available high light and nutrients. The elevated 63 marsh primary productivity is thus associated to significant nutrient load and the flooding 64 capacity in the adjacent lands (Cañavate et al., 2015). The macroinvertebrate saltmarsh 65 community is subjected to intensive predation from fish and shorebirds, and a main food source 66 for non-intensively reared fish (Arias and Drake, 1994). Gastropods, amphipods and chironomid 67 larvae dominate the macrofauna in terms of abundance (Arias and Drake, 1994). 68 Fish accounted for 16.7% of the global population's intake of animal protein and 6.5% of 69 all protein consumed in 2010 (FAO, 2014; Tocher, 2015). Arguably, of greatest importance for 70 consumers in the developed world, fish and seafood are unique and rich sources of omega-3 (n-3) 71 long-chain polyunsaturated fatty acids (LC-PUFA), particularly eicosapentaenoic (EPA; 20:5n- 72 3) and docosahexaenoic (DHA; 22:6n-3) acids. An increasing proportion of fish are farmed, 73 accounting for almost half of