Integrating Gammarus insensibilis in biofloc systems: A sustainable approach to nutrient enrichment and waste valorisation in aquaculture

In eg a ing Gamma us insensibilis in bio loc sys ems: A sus ainable app oach o nu ien en ichmen and was e alo isa ion in aquacul u e Ma a Cas illa-Ga il´ an a,b,* , Jos´ e Manuel Gue a-Ga cía c , Ismael Hache o-C uzado b,d a Ins i u des Subs ances e O ganismes de la Me , ISOme , UR 2160, Nan es Uni e si ´ e, 44000 Nan es, F ance b IFAPA Cen o El To u˜ no, Camino Ti o Pich´ on s/n, El Pue o de San a Ma ía, 11500 C´ adiz, Spain c Labo a o io de Biología Ma ina, Depa amen o de Zoología, Facul ad de Biología, Uni e sidad de Se illa, A da. Reina Me cedes 6, 41012 Se illa, Spain d “C ecimien o Azul”, Cen o IFAPA el To u˜ no, Unidad Asociada al CSIC, El Pue o de San a Ma ía, 11500 C´ adiz, Spain ARTICLE INFO Keywo ds: Sus ainable aquacul u e Nu i ion Low- ophic aquacul u e Was e alo isa ion Ci cula economy ABSTRACT This esea ch add esses he cul i a ion o Gamma us insensibilis in bio loc sys ems. The aim o he wo k was o alo ise and o bio emedia e aquacul u e e luen s while enhancing he nu i ional alue o bio locs h ough he in oduc ion o amphipods, which can be g own in close aquacul u e sys ems. Two expe imen al die s (aqua- cul u e was e de i us and comme cial ish eed) we e es ed o amphipods in iplica ed bio loc sys ems, agains h ee con ol anks wi hou bio loc. The expe imen was conduc ed o e a six-week pe iod a a empe a u e o 20 ◦C in he absence o ligh . The wa e quali y, he su i al, lipid p o ile and a y acid composi ion o he amphipods and he bio locs p oduced we e s udied. Signi ican di e ences we e obse ed in he wa e quali y be ween he ea men g oups and he con ol, since ni i ying bac e ia we e p esen in he bio locs o med in he ea men anks. Indeed, 100 % mo ali y was eached in he con ol anks om he second week o he expe imen . The ish pelle s die and he pelle -based bio loc esul ed in a signi ican ly highe o al lipid con en han he de i us die and bio loc. Howe e , no di e ences we e ound in he essen ial long-chain poly- unsa u a ed a y acids (LC-PUFAs) con en (ARA, EPA and DHA) be ween he comme cial pelle s and he de i us-based bio loc, hus jus i ying he en ichmen o he bio loc wi h amphipods. Indeed, he wild amphipods displayed highe le els o sa u a ed a y acids and omega-6 PUFAs compa ed o he expe imen al g oups, which demons a ed highe omega-3 PUFAs con en , pa icula ly DHA. The s udy highligh s he nu i ional ad an ages o pelle -based die s o amphipods, including imp o ed su i al and lipid con en , while also emphasising he enhanced nu i ional p o ile o he en iched de i us-based bio loc. We sugges ha he in eg a ion o aquacul- u e de i us wi h supplemen al aqua eed in BFT sys ems will suppo bo h he bio loccula ion p ocess and he nu i ional needs o he amphipods, he eby c ea ing a sus ainable and e icien cycle o was e alo isa ion and li e ood p oduc ion. Fu he esea ch is equi ed in o de o s udy he comple e li e cycle o amphipods and hei ep oduc i e capaci y in hese sys ems. A lowe wa e empe a u e and he inocula ion o mic oalgae should be conside ed in o de o achie e highe su i al a es and PUFAs con en . 1. In oduc ion Bio loc echnology (BFT) was o igina ed in he 1970s (Eme enciano e al., 2021) and has been widely adop ed due o i s en i onmen al and economic bene i s (Zimme mann e al., 2023). This echnology is based on ze o-wa e exchange sys ems wi h he o ma ion o bio locs in he cul u e medium whe e unea en ood, excess ino ganic nu ien s and aeces agg ega e along wi h mic oo ganisms (bac e ia, mic oalgae such as dia oms, p o ozoa…) (Khanjani e al., 2024b). This agg ega ion is enabled by a ma ix o ex acellula polyme ic subs ances sec e ed by he mic oo ganisms (Ha g ea es, 2013). By main aining a high ca bon/ ni ogen a io (Ha g ea es, 2013) h ough he addi ion o a ca bon sou ce (molasses, glyce ol, lou s; Zhao e al. (2016) o he cul u e medium, he colonies o chemoau o ophic bac e ia p esen in he bio- locs assimila e he ammonium exc e ed by he cul u ed species du ing he ni i ica ion p ocess, ans o ming i in o ni a e (Khanjani e al., 2022). He e o ophic bac e ia can also di ec ly assimila e he ammo- nium in o bac e ial biomass. Addi ionally, mic oalgae con ibu e o ni- ogen abso p ion du ing pho osyn hesis (Ebeling e al., 2006). BFT sys ems ha e been iden i ied as a p omising echnology o sus ainable * Co esponding au ho a : Ins i u des Subs ances e O ganismes de la Me , ISOme , UR 2160, Nan es Uni e si ´ e, 44000 Nan es, F ance. E-mail add ess: [email p o ec ed] (M. Cas illa-Ga il´ an). Con en s lis s a ailable a ScienceDi ec Aquacul u e jou nal homepage: www.else ie .com/loca e/aquacul u e h ps://doi.o g/10.1016/j.aquacul u e.2024.741922 Recei ed 6 Sep embe 2024; Recei ed in e ised o m 18 No embe 2024; Accep ed 18 No embe 2024 Aquacul u e 597 (2025) 741922 A ailable online 19 No embe 2024 0044-8486/© 2024 The Au ho (s). Published by Else ie B.V. This is an open access a icle unde he CC BY license ( h p://c ea i ecommons.o g/licenses/by/4.0/ ). ood p oduc ion, p omo ing low- ophic-le el species cul i a ion, poly- cul u e, and he de elopmen o a ci cula economy model (Khanjani e al., 2024c). The eeding e iciency and was e managemen a e enhanced in hese sys ems, as hey ha e he capaci y o inc ease nu ien u ilisa ion and minimize en i onmen al impac , achie ed h ough educed wa e and e ilize use (Knowle e al., 2020; Zimme mann e al., 2023). Thus, he need o wa e exchange is minimized, which in u n educes pumping cos s, p e en s luc ua ions in empe a u e and educes he ene gy equi emen o hea ing (McCuske e al., 2023). In BFT sys ems, ood equi emen s a e also educed, and an inc ease in he su i al and g ow h a es o cul i a ed species has been obse ed (Khanjani and Sha i inia, 2024). Bio locs p esen a a ou able nu i- ional p o ile (C ab e al., 2012) and hei consump ion ha e been demons a ed o enhance g ow h and o i y he immune sys em o cul i a ed species by inc easing he ac i i y o hei diges i e enzymes, esul ing in an inc ease in he eed con e sion a e (Xu and Pan, 2013). BFT is p ima ily employed in he cul i a ion o penaeids (C ab e al., 2010b) bu nume ous s udies ha e applied his echnology o he cul i a ion o il e eede s, de i i o es and e en some he bi o ous o omni o ous in ish, which a e able o eed di ec ly on pa icula e o ganic ma e (C ab e al., 2012; Das and Mandal, 2021; Dinda e al., 2020). These species a e ole an o changes in he concen a ions o dissol ed oxygen in he wa e and ni ogenous compounds and can wi hs and high cul u e densi ies and high concen a ions o suspended solids (El-Sayed, 2021; Eme enciano e al., 2021). Despi e all hese ad an ages, bio locs ha e been obse ed o p esen low p opo ions o long-chain polyunsa u a ed a y acids (LC-PUFAs) (C ab e al., 2010a), maybe linked wi h he absence o PUFAs in bac e ia (Zhuko a and Kha lamenko, 1999), and some essen ial amino acids (EAA) such as me hionine (Abbaszadeh e al., 2022). In his sense, se e al s udies ha e demons a ed ha he inco po a ion o li e ood, such as dia oms, o i e s, A emia o copepods, in o BFT can enhance he nu i ional alue o he bio locs, as well as he g ow h and su i al a es o he cul i a ed species (Abbaszadeh e al., 2022; B i o e al., 2016; de And ade e al., 2021). Bio loc en ichmen wi h amphipods has been add essed in only one p e ious s udy (P om hale e al., 2021). These au ho s epo ed a high su i al a e o amphipods in he BFT condi ions and a high po en ial o he en iched bio locs o eplace ishmeal. Am- phipods cul i a ion in e es has inc eased in he las yea s due o hei high-quali y nu i ional p o ile (Baeza-Rojano e al., 2014; Gue a- Ga cía e al., 2016; Jim´ enez-P ada e al., 2018) adequa e o eeding high ma ke alue ishes o cephalopods (Baeza-Rojano e al., 2010; Baeza-Rojano e al., 2013; Suon ama e al., 2007). Indeed, he Spanish Ins i u e o Oceanog aphy (IEO) holds a pa en o he me hodology o cul i a ion o he common oc opus pa ala ae (Tu e al., 2020). This pa en delinea es pa icula zoo echnical condi ions and he u ilisa ion o amphipods as an al e na i e li e p ey o c us acean zoeae, and is cu en ly being u ilised by he company “Nue a Pescano a” o he cul i a ion o Oc opus ulga is. Howe e , he upscaling o he sys ems equi es inc easing amoun s o li e ood, being a bo leneck o he de elopmen o hese species aquacul u e (Ga cía-Fe n´ andez, 2022). The ma ine amphipod Gamma us insensibilis S ock, 1966 has been highligh ed as a p omising species o being in ensi ely cul u ed due o i s adequa e nu i ional p o ile, la ge body size and high na u al den- si ies (Jim´ enez-P ada e al., 2020). Mo eo e , as a de i i o ous species, G. insensibilis can eed on aquacul u e sludge and was es (Cas illa-Ga - il´ an e al., 2023). Wi h his backg ound, we sugges ha G. insensibilis has he po en ial o u ilisa ion as a li e ood sou ce and could be cul i a ed in ensi ely in BFT sys ems, he eby en iching bio locs nu i ional alue. Mo eo e , hei abili y o eed on de i us would pe mi he alo isa ion o aqua- cul u e was es and he bio emedia ion o he e luen s (Cas illa-Ga il´ an e al., 2023). In he p esen s udy, G. insensibilis, ed wi h comme cial aqua eed o de i us om aquacul u e e luen s, has been cul i a ed in BFT sys ems. The wa e quali y, he su i al a e and he nu i ional p o ile o he amphipods as well as he bio loc p oduced we e analysed. 2. Ma e ials and me hods Specimens o G. insensibilis we e sampled in Ma ch 2023 om a na u al ma sh pond a he IFAPA Cen e “El To u˜ no” (El Pue o de San a Ma ia, Cadiz Bay, Spain). Amphipods on hei o iginal sub ac (Ul a sp. halli, whe e hey mainly inhabi ) we e anspo ed o IFAPA acili ies and ans e ed in o a 400 L ank o allow hei acclima ion du ing a week p io expe imen a ion. This ank was connec ed o a eci cula ion sys em (RAS) equipped wi h a cooling, mechanical il e , p o ein skimme , ul a iole ligh s and bio il e (Cas illa-Ga il´ an e al., 2023). They we e illumina ed (1.5–1.7 μ mol pho ons m −2 s −1 ) wi h luo es- cen ubes in a 8:16 h (ligh :da kness) o acclima e amphipods o he da kness, acco ding o P om hale e al. (2021). The seawa e was main ained a he same condi ions han in he na u al en i onmen , 17 ◦C and 38 g L −1 salini y, and was enewed daily in con inuous wa e low. P io o he expe imen , 2700 adul s o G. insensibilis we e sampled om he ank, de oid o algae and placed in 9 cul u e uni s (300 spec- imens pe cul u e uni ) illed wi h 25 L o ae a ed and mixed seawa e . Th ee hund ed addi ional “wild” specimens we e sampled and s o ed a −80 ◦C o lipid p o ile analysis and compa ison wi h he specimens used in he expe imen . The cul u e uni s we e main ained in da kness wi hou any wa e exchange, ollowing P om hale e al. (2021). An a i icial subs a e o amphipods was p o ided in all cul u e uni s (plas ic mesh, ollowing Cas illa-Ga il´ an e al., 2023). Amphipods we e s a ed o 24 h o s anda dize hei nu i ional condi ions. Two expe imen al ea men s we e es ed agains a con ol, a 20 ◦C (P om hale e al., 2021), in iplica e cul u e uni s. In he i s ea men , G. insensibilis specimens we e ed wi h g ounded sea b eam comme cial pelle s (0.2 g day −1 ). In he second ea men , he eed was was e de i us (1.2 g day −1 ) ob ained h ough he cleaning o he il e s o a Senegalese sole RAS uni and consis ed p ima ily o ish aeces and unea en ish eed pelle s. The quan i y o ood was de e mined by conside ing he o ganic ma e pe cen age o each ype, wi h de i us con aining six imes less o ganic ma e han pelle s. Th ee samples o each ood ype we e also s o ed a −80 ◦C o lipid p o ile analyses. Be ween h ee and ou imes a week (20 sampling days in o al), h ee wa e samples we e aken om he anks and il e ed on GF/C ibe glass il e s ha we e ea ed ollowing Cas illa-Ga il´ an e al. (2023), in o de o de e mine o al suspended solids in he wa e and he o ganic ma e con en o hese solids. Wa e was hen analysed: dis- sol ed ni a e and ni i e concen a ions we e sequen ially quan i ied by he educ ion o NO 3 − o NO 2 − wi h anadium (VCl 3 ) ollowing he me hod o Ga cía-Robledo e al. (2014). Ammonium concen a ion was de e mined by he indophenol-blue me hod (Amino e al., 1997), and ca bon concen a ion was hen adjus ed o a 16:1C:N a io h ough he addi ion o co n lou (P om hale e al., 2021). The con ol ea men was also ed wi h de i us, and wa e was also sampled daily o moni- o ing pu poses and ammonium concen a ion analyses, bu no ca bon sou ce was added in o de o a oid he bio- loccula ion p ocess. Wa e pa ame e s (mean dissol ed oxygen, empe a u e, salini y and pH) we e checked daily in all anks and we e ound o be 6.1 ±0.04 mg L −1 DO, 21.1 ±0.33 ◦C, 40.1 ±4.14 g L −1 salini y ( ypical o ma sh en i on- men s), and 8.1 ±0.09 pH. The expe imen las ed six weeks excep o he con ol anks, since 100 % mo ali y was obse ed om he second week. A he end o he expe imen , su i ing amphipods and he sludge (bio locs) p oduced in he 6 expe imen al cul u e uni s we e sampled, amphipods we e coun ed and all samples we e s o ed a −80 ◦C p io o biochemical analysis. All he eeze samples we e eeze-d ied and g ounded in o a ine powde . Ashes, mois u e, o al lipids and a y acids p o ile we e ana- lysed. To al lipids (TL) and a y acids (FA) p o ile me hods we e hose p e iously desc ibed by Hache o-C uzado e al. (2014). B ie ly, TL we e ex ac ed om powde ollowing he me hodology o Folch e al. (1957) wi h sligh modi ica ion by Ch is ie and Han (2012). To ex ac FA, o al lipid ex ac s we e subjec ed o acid-ca alysed ansme hyla ion. Fa y M. Cas illa-Ga il´ an e al. Aquacul u e 597 (2025) 741922 2 acids me hyl es e s (FAME) we e sepa a ed and quan i ied by gas ch oma og aphy and he iden i ied FA we e compa ed o he au hen ic s anda ds (FAME Mix C37) and well-cha ac e ized ish oil (Menhaden Oil, SUPELCO, USA) Ch is ie and Han (2012). Mean and s anda d de ia ion (SD) we e calcula ed o all da a. S a- is ical analyses and plo s we e pe o med using RS udio so wa e. Su i al was analysed h ough a one-way ANOVA es be ween am- phipods in bo h ea men s and he con ol. Di e ences in he ino ganic ni ogen concen a ion o he di e en ea men s we e es ed wi h wo- way ANOVAs ollowing an expe imen al design wi h o hogonal ac o s: ea men , a ixed ac o wi h h ee le els (con ol s pelle s s de i us) and ime, a ixed ac o wi h wen y le els (sampling days). Di e ences in TL be ween amphipods in he di e en ea men s and be ween p oduced bio locs and die s we e also s udied h ough one-way ANOVAs. Whe e ANOVAs indica ed signi ican di e ences (p <0.05), he sou ce o he di e ences was iden i ied using a Tukey es . P io o ANOVAs, he no mali y was es ed wi h Shapi o-Wilk es (p >0.05) and he homogenei y o a iances was es ed wi h Le ene es (p >0.05). I a iances emained he e ogeneous e en a e da a ans o ma ion, un- ans o med da a we e s ill analysed, as ANOVA is a obus s a is ical es and is ela i ely una ec ed by he he e ogenei y o a iances (Unde wood, 1997). In such cases, he le el o signi icance was educed o <0.01 o a oid ype I e o . Addi ionally, p incipal componen ana- lyses (PCA) we e conduc ed o FA ma ixes o he o dina ion o he samples. 3. Resul s 3.1. Su i al a e Su i al was signi ican ly highe in he anks ed wi h pelle s han in hose ed wi h de i us (one-way ANOVA; d =2; SS =0.28; MS =0.14; F alue =7.95; p alue =0.0206; Fig. 1). No amphipods we e obse ed in he con ol anks om he nin h day. 3.2. Wa e quali y Rega ding ino ganic ni ogen concen a ions, s a is ics a e summa- ized in he Table 1. Ammonium (N-NH 4 + ) le els we e signi ican ly highe in he con ol anks han in he o he ea men s (Fig. 2), wi h no di e ences obse ed be ween he la e (Table 1). Ni i e (N-NO 2 − ) and ni a e (N-NO 3 − ) concen a ions we e in gene al low (Fig. 3), and no di e ences we e ound be ween anks ed wi h pelle s o de i us (Table 1). 3.3. Nu i ional p o ile The Fig. 4 illus a es he o ganic ma e (OM) and he o al lipids (TL) con en o he samples. Signi ican di e ences we e obse ed in TL con en be ween he expe imen al die s and he bio locs (one-way ANOVA; d =4, SS =228.68, MS =57.17, F alue =42.14, p alue <0.001). The pelle s die exhibi ed he signi ican ly highes TL con en (Tukey es ; p alues <0.001) and he bio loc om he pelle s ea men p esen ed a signi ican ly highe con en han he bio loc om he de i us ea men (Tukey es ; p alue =0.047). Conce ning amphipods, no di e ences we e ound be ween hem (one-way ANOVA; d =2, SS = 14.38, MS =7.19, F alue =1.65, p alue =0.268). Di e ences in he OM con en we e only ound be ween he die s and he bio locs (one- way ANOVA; d =3; SS =6447; MS =2148.9; F alue =210.1; p alue <0.001). The pelle s p esen ed he highes con en (Tukey es ; p alues <0.001), ollowed by he pelle -based bio loc, which p esen ed a signi ican ly highe con en han he wo o he ea men s (Tukey es ; p alues <0.001). No di e ences we e ound be ween de i us-based die and bio loc (Tukey es ; p alue =0.6509). The a y acid p o ile (in pe cen age) o he die s and bio locs ana- lysed and he one-way ANOVAs a e summa ized in he Table 2. The isoC 16 a y acid, which is cha ac e is ic o bac e ia (Tanaka e al., 2014), was analysed among he SFA. While he pelle s p esen ed no e idence o his FA, he de i us die and he di e en bio locs did (Table 2), he eby co obo a ing he p esence o bio loc- o ming bac- e ia. Majo FA we e he SFA 16:0 (PA), he MUFAs 18:1n7 (cis-VA) and 18:1n9 (OA), he omega-6 PUFA 18:2n6 (LA) and he omega-3 PUFA 22:6n3 (DHA). The pelle -based die p esen ed he signi ican ly highes le el o OA (Tukey es ; p alue <0.001). Howe e , he pelle -based bio loc did no show he same p opo ion o OA, and displayed a signi ican ly lowe con en han he de i us-based bio loc (Tukey es ; p alue =0.008) o no di e ences wi h he de i us-based die (Tukey es ; p alue =0.392). The pelle -based bio loc had he signi ican ly highes le el o cis-VA (Tukey es ; p alue ≤0.01), while he pelle s p esen ed he lowe con en (Table 2). These indings suppo he p esence o bio loc- o ming bac e ia, as cis-VA is a p oduc o many he e o ophic bac e ial species (Gillan and Sands om, 1985). In gene al, he de i us-based die p esen ed he signi ican ly highes con en o SFA and he lowes MUFAs con en . The pelle -based die exhibi ed he signi ican ly highes con en o omega-3 and omega-6 Fig. 1. Mean amphipods su i al a es (%) in he di e en ea men s. Ba s ep esen he maximum and minimum su i al a es. Table 1 Resul s o he wo-way ANOVAs o ammonium, ni i e and ni a e concen a ions. Sou ce o a ia ion d SS MS F alue P alue Ammonium T ea men (T) 2 11.48 5.74 15.33 0.000 Time ( ) 19 18.40 0.97 2.59 0.002 T: 21 1.80 0.09 0.23 0.999 Residuals 86 32.20 0.37 Le ene Tes 42 4.10 0.343 Tukey Tes con ol >de i us 0.000 con ol >pelle s 0.000 de i us =pelle s 0.997 Ni i e T ea men 1 0.00 0.00 0.06 0.805 ime 4 0.00 0.00 5.07 0.006 T: 4 0.00 0.00 1.04 0.410 Residuals 20 0.00 0.00 Le ene Tes 9 1.58 0.189 Ni a e T ea men 1 0.01 0.01 3.12 0.093 ime 4 0.14 0.04 9.73 0.000 T: 4 0.04 0.01 2.38 0.086 Residuals 20 0.07 0.00 Le ene Tes 9 1.37 0.266 M. Cas illa-Ga il´ an e al. Aquacul u e 597 (2025) 741922 3 PUFAs (Table 2). Howe e , he bio loc gene a ed in his ea men p esen ed he lowes p opo ion o PUFAs, showing no signi ican di - e ences wi h he de i us-based die (Tukey es ; p alue =0.990) and a signi ican ly lowe con en han he de i us-based bio loc (Tukey es ; p alue =0.010). The esul s o he PCA based on die s and bio locs we e also in ag eemen wi h he p e ious esul s and iden i ied OA, cis-VA, LA and PA as majo con ibu o s o he o al a iance. The i s p incipal componen explained 57 % o he o al a iance and he second p incipal componen explained 34.5 %. PC1 co ela ed posi i ely wi h he a y acids OA ( =0.950, n =20, p <0.005) and LA ( =0.925, n =20, p < 0.005), and nega i ely wi h cis-VA ( = − 0.756, n =20, p <0.005). PC2 Fig. 2. Ammonium concen a ion (mean ±SD) in he di e en cul u e uni s du ing he expe imen . Fig. 3. Ni a e and ni i e concen a ions (mean ±SD) in he di e en cul u e uni s du ing he expe imen . M. Cas illa-Ga il´ an e al. Aquacul u e 597 (2025) 741922 4 co ela ed posi i ely wi h PA ( =0.902, n =20, p <0.005) and nega i ely wi h cis-VA ( = − 0.603, n =20, p <0.005). The axes clea ly sepa a e he die s om he bio locs, and di e en ia e he bio locs based on hei PA and cis-VA con en (Fig. 5). The a y acid composi ion o he wild amphipods was compa ed wi h ha o he amphipods used in he expe imen ( ed wi h pelle s o de i us). Table 3 shows esul s (in %) and one-way ANOVAs. The majo a y acids we e: he sa u a ed a y acids (SFA) 16:0 (palmi ic acid; PA) and 18:0 (s ea ic acid; SA); he monounsa u a ed a y acids (MUFAs) 16:1n7 (palmi oleic acid, POA), 18:1n9 (oleic acid, OA) and 18:1n7 (cis- accenic acid; cis-VA); and he polyunsa u a ed a y acids (PUFAs) 18:2n6 (linoleic acid, LA), 20:4n6 (a achidonic acid, ARA), 20:5n3 (eicosapen aenoic acid, EPA) and 22:6n3 (docosahexaenoic acid, DHA). Only o al SFA composi ion showed signi ican di e ences be ween he amphipods, wi h he wild animals displaying a highe concen a ion han hose used in he expe imen (Tukey es ; p alue <0.001). This was mainly a ibu ed o a highe indi idual PA con en (Table 2). Con- ce ning MUFAs, wild amphipods p esen ed a signi ican ly highe con- en o POA while amphipods ed wi h pelle s showed a highe con en o OA (Table 2). Wild amphipods also showed he highes con en o omega-6 PUFAs due o he highe LA and ARA le els, while he am- phipods used in he expe imen p esen ed highe con en s o omega-3 DHA. The p incipal di e en ia ing ac o s be ween he wo eeding e- gimes we e he signi ican ly highe le els o PA (Tukey es ; p alue = 0.012) and OA (Tukey es ; p alue <0.001) obse ed in he pelle s g oup. P incipal componen s analyses (PCA) we e consis en wi h he e- sul s shown abo e. The i s p incipal componen explained 57 % o he o al a iance and he second p incipal componen explained 23 %. P incipal componen 1 (PC1) co ela ed posi i ely wi h he a y acids DHA ( =0.854, n =9, p <0.005) and cis-VA ( =0.705, n =9, p < 0.025), and nega i ely wi h PA ( = − 0.958, n =9, p <0.005), EPA ( = −0.695, n =9, p <0.025), ARA ( = − 0.934, n =9, p <0.025) and LA ( = − 0.718, n =9, p <0.025). PC2 co ela ed posi i ely wi h OA ( = 0.821, n =9, p <0.005). Axes clea ly sepa a ed samples by ea men s (wild, pelle s o de i us) (Fig. 6). 4. Discussion 4.1. Su i al a e The e a e limi ed epo s on he amphipods cul u e. Ne e heless, he su i al a e obse ed in his s udy aligns wi h he indings o Jim´ enez-P ada e al. (2020), who epo ed alues o app oxima ely 40 % o e a 21-days expe imen . Simila ly, Ribes-Na a o e al. (2022) obse ed compa able su i al a es o Gamma us locus a when wa e a 20 ◦C and/o die s ich in sho -chain PUFAs and SFAs we e u ilised in hei expe imen . Howe e , hei esul s indica ed ha lowe empe a- u es (5–15 ◦C) and die s ich in LC-PUFAs we e associa ed wi h enhanced su i al a es. 4.2. Wa e quali y No s a is ically signi ican di e ences we e ound in wa e quali y pa ame e s among he ea men s in his s udy. The esul s on nu ien s concen a ion in he ea men s anks, when compa ed o he con ol, con i med he e ec o he ca bon sou ce on he colonisa ion o he - e o ophic bac e ia and he loccula ion p ocess (C ab, 2010; Ha - g ea es, 2013; Khanjani e al., 2024a): he concen a ion o ammonium was signi ican ly lowe , while ni a e cumula es in he ea men anks, hus indica ing ha ni i ying bac e ia we e p esen in he bio loc and being consis en wi h p e ious wo ks (Abbaszadeh e al., 2022; B i o e al., 2016). 4.3. Nu i ional p o ile The loccula ion success was also co obo a ed by he lipid p o ile o he bio locs, which exhibi ed majo a y acids including PA, cis-VA, OA and LA, as al eady obse ed in p e ious wo k (Anand e al., 2014). Essen ial LC-PUFAs we e no among he majo FA. Howe e , he p esen s udy e ealed clea ly highe amoun s o ARA (0.8 %), EPA (3.1 %) and DHA (5.5 %) han Anand e al. (2014), who ound only 0.4 % ARA and did no de ec DHA o EPA in hei bio loc. These alues we e simila o he FA p o ile o he comme cial aqua eed used in he p esen s udy (pelle s), which is o mula ed o mee he nu i ional needs o ish (see Table 2: no di e ences we e ound in he ARA, EPA and DHA con en be ween he pelle s and he de i us-based bio loc). This jus i ies he en ichmen o he bio loc wi h amphipods. Indeed, amphipods showed a p o ile ich in EPA, DHA, LA and ARA, as also demons a ed by Jim´ enez- P ada e al. (2018). The gamma ids om bo h ea men s showed he same amoun o TL as he wild specimens, ye demons a ed a educ ion in ARA and EPA, which was coun e balanced by a highe amoun o DHA. In e es ingly, Ribes-Na a o e al. (2021) ha e demons a ed ha gamma id amphipods a e de oid o desa u ase genes ha a e comple- men a y o he elongases needed o he biosyn hesis o LC-PUFAs. Consequen ly, hey a e unable o syn hesise hese compounds de no o. Simila ly o he s udy conduc ed by P om hale e al. (2021), ou expe imen was pe o med in da kness. Thus, i ’s unlikely ha hese compounds we e acqui ed h ough he die (phy oplank on). I has been p oposed ha he high pe cen ages o DHA de ec ed in gamma ids, e en i ed wi h die s lacking LC-PUFAs, a e he esul o an accumula ion o p e ious die s (Ribes-Na a o e al., 2022), which could explain he esul s obse ed in he p esen s udy. These selec i e e en ion mecha- nisms would enable hem o su i e du ing pe iods o limi ed o no bioa ailabili y o hese nu ien s (Ribes-Na a o e al., 2022). Fig. 4. O ganic ma e (OM) and o al lipids (TL) con en (%) in he wo die s es ed (pelle s and de i us), in he bio locs sampled om he di e en ea - men s a he end o he expe imen , and in he amphipods in bo h ea men s and hose sampled om he wild (mean ±SD). M. Cas illa-Ga il´ an e al. Aquacul u e 597 (2025) 741922 5 The de i us-based bio loc p esen ed a highe TL con en and a mo e nu i ious FA p o ile han he de i us-based die , p omo ed by he p esence o he mic oo ganism assemblage and he amphipods. Mo e- o e , his bio loc showed highe pe cen ages o PA, OA and LA han he pelle -based bio loc. This is p obably due o he highe mo ali y a e in his ea men , wi h ewe indi iduals being sampled o biochemical analyses and u he en iching he bio loc. I is impo an o no e ha hese a e ela i e esul s and ha absolu e alues o hese FA we e simila in bo h ea men s (da a no shown), since he pelle -based bio loc showed a signi ican ly highe amoun o TL. Howe e , he Table 2 Fa y acids composi ion (mean % ±SD) o he die s used in he expe imen (i.e. pelle s, de i us) and he bio locs sampled om hese wo ea men s a he end o he expe imen . Fa y acid pelle s de i us Bio loc pelle s Bio loc de i us P alue Tukey SFA 14:0 1.44 ±0.00 3.81 ±0.13 1.57 ±0.44 2.64 ±0.16 0.000** d >all; bd >bp; p =bp 15:0 0.24 ±0.01 0.78 ±0.01 0.66 ±0.19 0.60 ±0.05 0.017* d >p; p =bp =bd; d =bp =bd 16:0 16.43 ±0.03 38.21 ±2.12 22.06 ±0.92 26.78 ±1.37 0.000*** d >bd >bp >p iso-C 16 0.00 ±0.00 0.84 ±0.05 1.48 ±0.32 0.79 ±0.15 0.000** bp >bd >d >p; d =bd 17:0 0.33 ±0.00 1.09 ±0.16 1.31 ±0.26 0.48 ±0.31 0.000** d =bp >p; p =bd; bp >bd 18:0 4.87 ±0.09 8.49 ±0.06 8.22 ±1.09 10.37 ±1.03 0.000** bd >bp >p; d =bd 20:0 0.36 ±0.00 0.64 ±0.06 0.82 ±0.09 0.70 ±0.03 0.000*** bp >bd =d >p 22:0 0.29 ±0.02 0.65 ±0.07 1.47 ±0.36 1.19 ±0.22 0.000*** p <bp; p =d =bd; bp =d =bd 24:0 0.19 ±0.00 0.76 ±0.08 1.08 ±0.27 1.16 ±0.16 0.000*** bd =bp =d >p ∑SFA 24.14 ±0.15 55.28 ±2.46 38.68 ±1.01 44.71 ±2.05 0.000*** d >bd >bp >p MUFAs 16:1n5 0.03 ±0.03 0.02 ±0.02 0.36 ±0.19 0.39 ±0.23 0.044 16:1n7 3.68 ±0.04 3.21 ±0.13 8.16 ±2.14 5.83 ±1.65 0.001** bp >p =d; bp =bd 18:1n5 0.16 ±0.00 0.06 ±0.01 0.58 ±0.06 0.41 ±0.23 0.000*** bp =bd >d; d =p; bp >p; bd =p 18:1n7 3.31 ±0.09 4.73 ±0.09 18.99 ±5.55 8.01 ±4.27 0.000*** bp >p =d =bd 18:1n9 30.67 ±0.03 9.59 ±0.71 8.30 ±0.87 10.67 ±1.64 0.000*** p >all; bd >bp: d =bp; d =bd 20:1n9 2.30 ±0.01 2.38 ±0.12 0.73 ±0.31 2.42 ±0.33 0.000*** bp <p =d =bd 22:1n11 1.22 ±0.01 2.30 ±0.06 0.87 ±0.41 3.06 ±0.45 0.003** bd >bp; bd =p =d; bp =p =d 24:1 0.51 ±0.01 1.88 ±0.06 1.02 ±0.23 2.39 ±0.28 0.000*** p =bp; d =bd; d >p; bd >bp ∑MUFAs 41.89 ±0.09 24.16 ±0.84 39.01 ±2.72 33.19 ±3.98 0.000*** p =bp >bd >d n-6 PUFAs 18:2n6 14.93 ±0.01 4.15 ±0.21 3.96 ±0.33 5.04 ±0.23 0.000*** p >bd >bp >d 20:4n6 0.85 ±0.00 0.41 ±0.12 0.68 ±0.33 0.80 ±0.24 0.440 ∑n-6 PUFAs 15.78 ±0.02 4.56 ±0.34 4.64 ±0.24 5.84 ±0.17 0.000*** p >all; d =bp; bd >d; bd >bp n-3 PUFAs 18:3n3 2.36 ±0.01 0.48 ±0.02 0.32 ±0.25 0.37 ±0.12 0.000*** p >d =bp =bd 20:5n3 3.68 ±0.00 2.28 ±0.15 1.87 ±0.59 3.11 ±0.90 0.002** p >bp; p =d =bd; bp =d =bd 22:5n3 0.94 ±0.00 0.46 ±0.01 0.21 ±0.03 0.48 ±0.08 0.000** p >all; bd >bp; bd =d 22:6n3 6.73 ±0.03 3.76 ±0.18 3.92 ±1.12 5.54 ±1.66 0.010* p >d =bp; d =bp =bd; p =bd ∑n-3 PUFAs 13.72 ±0.04 6.99 ±0.37 6.31 ±1.73 9.50 ±2.63 0.000*** p >all; bd >bp; d =bd; d =bp ∑PUFAs 29.49 ±0.03 11.56 ±0.70 10.95 ±1.87 15.34 ±2.77 0.000*** p >all; bd >bp;d =bd; d =bp p =pelle -based die ; d =de i us-based die ; bp =pelle -based bio loc; bd =de i us-based bio loc; SFA =sa u a ed a y acids; MUFA =monounsa u a ed a y acids; PUFA =polyunsa u a ed a y acid. Fig. 5. P incipal componen analysis (PCA) plo based on FA composi ion (in % o FA) o expe imen al die s (i.e. pelle s o de i us) and bio loc sampled om he expe imen al anks. Only majo FA a e shown. 16:0 =PA, 18:1n7 =cis-VA, 18:1n9 =OA, 18:2n6 =LA. D =de i us; P =pelle s; BD =de i us-based bio loc; BP = pelle -based bio loc. M. Cas illa-Ga il´ an e al. Aquacul u e 597 (2025) 741922 6 pelle -based bio loc did no p esen a be e p o ile han he pelle -based die , highligh ing he nu i ional alue o he die , bu also poin ing he in e es o alo ising aquacul u e was es in BFT sys ems (Das e al., 2023; Kala e al., 2023). Fu he mo e, he pelle -based bio loc exhibi ed a signi ican ly highe pe cen age o isoC 16 and cis-VA, commonly associa ed wi h bac e ia (Ron ani e al., 2003). This is a ibu ed o he signi ican ly highe con en o OM in he pelle s and pelle -based bio loc, which p o ides essen ial nu ien s and acili a es he es ablishmen o he e o ophic bac e ia and he con e sion o he OM in o bac e ial biomass (Zhuko a and Kha lamenko, 1999). This demons a es he ad an age o he pelle s ea men in e ms o bio loccula ion capaci y (Faus , 2014), in addi ion o he imp o ed su i al a es. I has been shown ha he FA p o ile o amphipods as well as hei su i al a e a e closely linked o he die (Ribes-Na a o e al., 2022). Thus, he in e- g a ion in BFT sys ems o aquacul u e was e de i us wi h a supple- men a ion o comme cial aqua eed could po en ially enhance he o e all nu i ional p o ile while enhancing he bio loccula ion p ocess o he bio emedia ion o he aquacul u e e luen s (Cas illa-Ga il´ an e al., 2023). Mo eo e , i will be sui able o ca y ou he expe imen unde ligh condi ions and wi h he addi ion o dia oms o chlo ophy es, which ha e been highligh ed by o he au ho s o imp o e he LC-PUFAs and EAA p o iles (Kha oon e al., 2009; Ma ins e al., 2016), and wa e quali y (B i o e al., 2016; de And ade e al., 2021). The in e es in in eg a ed BFT sys ems (a combina ion o in eg a ed mul i- ophic aquacul u e, IMTA, and BFT) has inc eased in ecen yea s (Bo ges e al., 2020; Ca alho e al., 2023; Lega da e al., 2021; Lima e al., 2021). In hese sys ems, he esidues o one ed species se e as a sou ce o OM o bio locs, which can be en iched wi h a second species, which in u n is ed o a hi d species. This app oach aims o minimize he p oduc ion o was e and he use o aqua eed. The p esen s udy shows ha he e luen o a ish RAS can be alo ised o eed he medium o a BFT sys em en iched wi h amphipods. Bo h bio locs and amphipods could be u he used as ood, as hei a y acids p o ile would sa is y he equi emen s o a ious species o in e es in aquacul u e as a e salmonids (Ca e al., 2023), ma ine c us aceans (Gonz´ alez-F´ elix e al., 2002), sea b eam (Ibeas e al., 1996) o u bo (Cas ell e al., 1994). The use o bio locs o eed ish has been demons a ed o be an e ec i e app oach, as e idenced by he indings o se e al s udies (Bo ges e al., 2020; Holanda e al., 2023; Lega da e al., 2021; Pinho e al., 2021; Poe sch e al., 2021; Sil a e al., 2022). Fu he mo e, amphipods ha e also been success ully employed as a die a y sou ce o ish (Mo en e al., 2006; Suon ama e al., 2007) and cephalopods (Baeza-Rojano e al., 2010; Baeza-Rojano e al., 2013). Finally, he p esen s udy suppo s he g owing in e es in he use o amphipods in aquacul u e, wi h a pa icula ocus on BFT sys ems. I aims o p omo e u he esea ch in his opic, gi en ha o da e, BFT sys ems ha e been p edominan ly s udied in mono-speci ic o mul i-speci ic decapod c us aceans aqua- cul u e (Baj acha ya e al., 2024; Ekasa i e al., 2014; Eme enciano e al., 2021; Galasso e al., 2024; Huang e al., 2023; Lima e al., 2021; Nguyen e al., 2024; P´ e ez de Jesús e al., 2024; P´ e ez-Velasco e al., 2023; Pimen el e al., 2024; Qiu e al., 2023; Reis e al., 2023). Few s udies ha e been conduc ed on echinode ms (Chen e al., 2018a, 2018b) and only a single s udy has been ca ied ou on amphipod c us aceans (P om hale e al., 2021). 5. Conclusion This s udy demons a es he po en ial o using G. insensibilis in BFT sys ems, ocusing on i s su i al and nu i ional p o ile, when ed wi h comme cial aqua eed o de i us om aquacul u e e luen s. The ind- ings e eal ha G. insensibilis can be e ec i ely cul i a ed in BFT sys- ems, le e aging i s abili y o eed on de i us, which p omo es bio loccula ion and enhances he nu ien p o ile o he bio loc. The p esence o bio locs en iched wi h amphipods signi ican ly imp o es he o e all a y acid composi ion, pa icula ly in essen ial LC-PUFAs such as EPA, DHA, and ARA. This en ichmen add esses he inhe en nu i- ional de iciencies o bio locs, making hem mo e sui able as ood o high- alue aquacul u e species. The s udy highligh s he dual bene i s o his app oach: enhancing he nu i ional alue o bio locs while con ibu ing o he bio emedia ion o aquacul u e e luen s. Fu he - mo e, he use o close aquacul u e sys ems educes cos s and main ain Table 3 Fa y acids composi ion (mean % ±SD) o G. insensibilis om sal ma shes and hose used in he expe imen , ed wi h ish pelle s o de i us. Fa y acid wild pelle s de i us P alue Tukey SFA 14:0 1.46 ± 0.12 0.75 ± 0.15 0.63 ± 0.17 0.000*** w >p =d 15:0 0.32 ± 0.27 0.41 ± 0.02 0.47 ± 0.02 0.538 16:0 20.40 ± 0.14 16.44 ± 0.37 15.47 ± 0.26 0.000*** w >p >d 16:0i 0.18 ± 0.03 0.90 ± 0.03 0.79 ± 0.39 0.057 17:0 0.12 ± 0.01 1.03 ± 0.04 1.22 ± 0.05 0.000*** w <p <d 18:0 3.46 ± 0.12 3.83 ± 0.15 4.39 ± 0.15 0.000*** w <p <d 22:0 0.30 ± 0.26 0.37 ± 0.16 0.70 ± 0.06 0.073 ∑SFA 26.23 ± 0.51 23.74 ± 0.42 23.67 ± 0.29 0.000 w >p =d MUFAs 16:1n7 5.97 ± 0.28 3.67 ± 0.29 4.16 ± 1.42 0.036* w >p =d 18:1n9 15.89 ± 0.27 22.17 ± 0.85 16.14 ± 0.95 0.000*** p >w =d 18:1n7 4.30 ± 0.16 6.48 ± 1.25 8.35 ± 3.17 0.115 18:1n5 0.19 ± 0.01 0.65 ± 0.28 0.77 ± 0.39 0.092 20:1n9 0.80 ± 0.03 1.22 ± 0.03 0.79 ± 0.06 0.000*** p >w =d 20:1n7 0.33 ± 0.04 0.52 ± 0.11 0.57 ± 0.26 0.243 ∑MUFAs 27.47 ± 0.63 34.70 ± 2.55 30.77 ± 4.40 0.064 n-6 PUFAs 18:2n6 7.43 ± 0.09 6.77 ± 0.63 5.69 ± 0.69 0.021* w >d; w =p; p =d 20:2n6 0.94 ± 0.02 0.85 ± 0.17 0.84 ± 0.18 0.636 20:4n6 7.59 ± 0.12 4.90 ± 0.52 5.49 ± 1.23 0.013* w >p =d 22:5n6 0.55 ± 0.01 0.77 ± 0.15 0.64 ± 0.08 0.084 ∑n-6 PUFAs 15.51 ± 0.22 13.29 ± 1.44 12.66 ± 2.13 0.040* w >d; w =p; p =d n-3 PUFAs 18:3n3 0.75 ± 0.04 0.37 ± 0.32 0.19 ± 0.34 0.108 20:5n3 15.25 ± 0.28 11.37 ± 0.87 13.43 ± 3.34 0.136 22:5n3 0.97 ± 0.03 0.56 ± 0.09 0.76 ± 0.15 0.008** w >p; w =d; p =d 22:6n3 4.82 ± 0.00 9.47 ± 0.41 11.81 ± 2.17 0.002** w <p =d ∑n-3 PUFAs 21.79 ± 0.26 21.77 ± 1.21 26.19 ± 4.83 0.176 16:2n4 0.44 ± 0.48 0.05 ± 0.05 0.39 ± 0.07 0.261 16:4n1 1.08 ± 0.47 0.87 ± 0.60 1.33 ± 0.30 0.526 ∑PUFAs 39.82 ± 1.12 35.98 ± 2.66 40.57 ± 6.36 0.386 w =wild; p =pelle s; d =de i us; SFA =sa u a ed a y acids; MUFA = monounsa u a ed a y acids; PUFA =polyunsa u a ed a y acid. M. Cas illa-Ga il´ an e al. Aquacul u e 597 (2025) 741922 7 isola ion om na u al en i onmen . The in eg a ion o aquacul u e de i us wi h supplemen al aqua eed in BFT sys ems will suppo bo h he bio loccula ion p ocess and he nu i ional needs o he amphipods, he eby c ea ing a sus ainable and e icien cycle o was e alo isa ion and ood p oduc ion. The use o G. insensibilis in BFT sys ems ep esen s a p omising s a egy o imp o ing he sus ainabili y and p oduc i i y o aquacul u e ope a ions. By op imizing he use o o ganic was e and p oducing nu i ionally ich bio locs, his app oach can signi ican ly con ibu e o he de elopmen o a ci cula economy in aquacul u e, educing en i- onmen al impac s and enhancing he g ow h and heal h o cul i a ed species. Fu he esea ch is needed in o de o s udy he comple e li e cycle o amphipods and hei ep oduc i e capaci y in hese sys ems. A lowe wa e empe a u e and he inocula ion o mic oalgae should be conside ed in o de o achie e highe su i al a es and PUFAs con en . CRediT au ho ship con ibu ion s a emen Ma a Cas illa-Ga il´ an: W i ing – e iew & edi ing, W i ing – o iginal d a , Valida ion, Me hodology, In es iga ion, Fo mal analysis, Da a cu a ion, Concep ualiza ion. Jos´ e Manuel Gue a-Ga cía: Vali- da ion, Supe ision, Me hodology, Da a cu a ion. Ismael Hache o- C uzado: Valida ion, Supe ision, Me hodology, Da a cu a ion, Concep ualiza ion. Decla a ion o compe ing in e es The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape . Da a a ailabili y Da a will be made a ailable on eques . Acknowledgemen s Au ho s wish o hank he Eu opean Ma i ime and Fishe ies Fund (EMFF) o he unding du ing his esea ch included in he p ojec “Acuicul u a y ci cula idad: alo izaci´ on de esiduos de la acuicul u a median e o ganismos de bajo ni el ´ o ico (ACUICIRC)” unde ag ee- men EI.FEM.PPA202200.003. Du ing his s udy MCG was suppo ed by a “Ma ga i a Salas” pos -doc o al g an om he Spanish Minis y o Science, Inno a ion and Uni e si ies unded by he Eu opean Union – Nex Gene a ion EU p og am. Thank you o Melissa San ’ Anna da Sil a o he con ibu ion du ing he mas e ’s in e nship. Re e ences Abbaszadeh, A., Mozanzadeh, M.T., Qasemi, A., Ouji a d, A., Na isi Bahabadi, M., 2022. E ec s o he addi ion o Calanopia ellip ica, A emia anciscana, and B achionus o undi o mis in a nu se y bio loc sys em on wa e quali y, g ow h, gu mo phology, heal h indices, and ansc ip ional esponse o immune and an ioxidan - ela ed genes in Penaeus annamei. Aquac. 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