Freshwater-adapted sea bass Dicentrarchus labrax feeding frequency impact in a lettuce Lactuca sativa aquaponics system

The aim of this study is to investigate the effect of three daily fish feeding frequencies, two, four and eight times per day (FF2, FF4, and FF8, respectively) on growth performance of sea bass (Dicentrarchus labrax)and lettuce plants (Lactuca sativa) reared in aquaponics. 171 juvenile sea bass with an average body weight of 6.80 ± 0.095 g were used, together with 24 lettuce plants with an average initial height of 11.78 ± 0.074 cm over a 45-day trial period. FF2 fish group showed a significantly lower final weight, weight gain and specific growth rate than the FF4 and FF8 groups. Voluntary feed intake was similar for all the three feeding frequencies treatmens (p > 0.05). No plant mortality was observed during the 45-day study period. All three aquaponic systems resulted in a similar leaf fresh weight and fresh and dry aerial biomass. The results of the present study showed that the FF4 or FF8 feeding frequency contributes to the more efficient utilization of nutrients for better growth of sea bass adapted to fresh water while successfully supporting plant growth to a marketable biomass.

Three (3) autonomous aquaponic systems with a total volume of 500 L each 128 were constructed. Each system consisted of 3 glass fish tanks (50 cm x 50 cm x 50 cm) 129 with a 100 L water volume each and a 26 L hydroponic cultivation tank (112 cm x73 cm 130 x 20 cm) paved with clay pebble (8-16 mm) substrate (fig 1, fig 2). Each aquaponic 131 system' s nitrification process was enchased by a biological sump filter (100 cm x 50 cm 132 x 48 cm) with a total volume of 184 L. Salinity was gradually decreased 4 units per week 133 until it was stabilized to <1 ppt .

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Sump filter construction was previously reported by Vlahos et al [36]. The sump 136 filter was divided into three sections (fig 1). Suitable media, providing a specific surface 137 area (SSA) for nitrifying bacteria colonization, covered the most of the filter area. The 138 mechanical filter covered an area of 1250 cm² and consisted of three layers of 139 fibreglass material, creating in this way a 30 cm thick layer to retain the solid residues 140 from the fish tanks (uneaten food and faeces). The biological filter covered an area of 141 2150 cm² and was fixed by a mixed media of 20 L of porous cylindrical substrate K1 (10 142 mm diameter each), a 10 L ceramic ring (15 mm diameter each) and 10 L bioballs (30 143 mm diameter each). A pump (Aqua Medic OR 2500 L/h, 38 W, 2.6 m h max ) was placed 144 in the last part of the filter to supply the aquaponic system with water through the filter 145 (Q=6.27 L/min). Clay pebble substrate of the hydroponic tank also provided sufficient 146 biofiltration, increasing the efficiency of the system. In each system, a high-pressure 147 sodium 400 W lamp (Sylvania) was placed at a distance of 65 cm from the surface of 148 the hydroponic tanks, ensuring this way the appropriate light for the plants. A winder 149 photoperiod of 10 h light, 14 h dark was set up. An air-lift pump was used to recycle the 150 water through a filter bed during the experiments (adjusted flow 1.53 L/min), thus 151 creating a filtration speed (V) of 1.79 cm/min [36]. The oxygen saturation levels were 152 between 75% and 80%. Water flow from the hydroponic tank to the fish tank and to the 153 sump filter was performed by gravity ( fig 1). The setup period of the systems lasted 2 154 months to develop the biological filter. According to Hirayama [37], 40-60 days are 155 necessary for the establishment of bacteria and the satisfactory oxidation of ammonia to 156 nitrate ions. 211 2 meals for FF4 and 4 meals for FF8) was performed by hand, and the other meals 212 were performed with automatic feeders. The feeding rate was adjusted to fish weight 213 every 15 days. Fish tanks were cleaned every day by siphoning, and uneaten food was 214 removed. Daily food consumption per fish tank is calculated by the difference between 215 the amount fed and the amount of uneaten feed collected (corrected for leaching 216 losses) [36]. At the end of the experiment, fish were anaesthetized with Tricaine 217 methanesulfonate (MS 222), and their final fish body weights and lengths were 218 measured.

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Lettuce plants (L. sativa var. Musena) were grown in an unheated greenhouse 221 until the 6-true-leaf stage. Five days prior to their transfer to the aquaponic system, Fe 222 (Fe-DTPA), Ca (foliar application) and K (KOH) fertilization was performed. A total of 24 223 lettuce seedlings were chosen, showing no statistically significant differences in their 224 morphometric characteristics (height, number of leaves). Eight lettuce plants were 225 evenly placed in each hydroponic bed, 20 cm apart. Plant number and density was 226 chosen according to the dimensions of the hydroponic tank and the plant positions were 227 carefully selected to ensure the homogeneity of the light environment; thus, each plant 228 was exposed to 400-500 μmol m -2 sec -1 of photosynthetically active radiation (PAR). As 229 the aquaponics systems were indoor systems, artificial light was used for the plants. 230 The artificial light was supplied by a 400 W HPS lamp placed 65 cm above each 231 growing area and accompanied by a timer for accurate control of the photoperiod (10 h 232 light: 14 h dark). Plant height as well as the number of leaves were monitored every 15 233 days. No extra Ca, K or Fe was added to the aquaponic system. Ammonium (NH 4 + ), ammonia (NH 3 ), nitrate and phosphate ions were monitored 237 once a week before the daily first fish feeding. Water samples were taken at the water 238 inlet point (GBin) and at the exit point (GBout) of the hydroponic cultivation tank. All 239 measurements were performed using a Hach DR3900 model photometer with special 240 pre-weighted reagents.

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The filter' s hydraulic loading ratio (HLR), recycled ratio (r), the hydraulic retention 242 time of the water in the filter bed (HRT), the specific surface area of the filter (SSA) nad 243 the volume of filter media (V media ) were calculated according to the equations described 244 by Endut et al.  Euthanasia of animals followed the EU Directive 2010/63/EU and FELASA 278 guidelines and performed through an overdose of Tricaine methanesulfonate (MS 222, 279 300+ mg/L). At the end of the experiment, five fish per tank were removed for 280 histopathological examination, as previously described by Vlahos et al. [36]. Fish were 281 placed immediately on ice after euthanization. Samples of liver, midgut, kidney and gill 282 were dissected from each fish. Tissue samples were fixed in Davidson' fixative for 24 h 283 at 4ºC followed by dehydration in graded series of ethanol, immersion in xylol and 284 embedding in paraffin wax. Thin sections of 4-7 μm were mounted, deparaffinized, 285 rehydrated, stained with Hematoxylin-Eosin, mounted with Cristal/Mount and examined 286 for alterations with a microscope (Axiostar plus Carl Zeiss Light Microscopy, Carl Zeiss 287 Ltd, Gottingen, Germany) under a total magnification of 100X and 400X. A semi -288 quantitative grading system was used in order to quantify the histopathological 289 alterations of the examined tissues [36]. Severity grading used the following system: 290 Grade 0 (not remarkable), Grade 1 (minimal), Grade 2 (mild), Grade 3 (moderate), 291 Grade 4 (severe).

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For midgut microbiota analysis, three fish at day 0 and three fish per feeding 293 frequency treatment at days 0, 15 and 45 were sacrificed.. Midgut samples were 294 removed after dissection and DNA was extracted with DNA Mini kit (QIAGEN, 295 Germany). Bacterial diversity was assessed by amplification of the V3-V4 region of the 296 bacterial 16S rDNA gene on the MiSeq Illumina platform 2x300 bp (MRDNA Ltd.,

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Raw sequence reads were processed and analyzed using the MOTHUR 304 software (v. 1.39.5) [43] After trimming barcodes and primer sequences, quality control 305 was performed through the 'screen.seqs' command and sequences were removed 306 according to the following filtering parameters: length less than 250 bp, ambiguous 307 bases, average quality score less than 25, and homopolymers longer than eight 308 nucleotides. Thereafter, the remaining sequences were aligned against the SILVA 132 309 database [44]. The VSEARCH algorithm was used to detect and remove chimeric 310 reads. Sequences were clustered into operational taxonomic units (OTUs) based on the 311 average neighbor algorithm at a 97% sequence identity threshold [45]. High-quality 312 OTU sequences were classified to different taxa according to the SILVA 132 database 313 [44] with confidence value set above 80%. Values are presented as means ± standard error of the mean (S.E.M.). Data were 317 tested for normality and homogeneity with Kolmogorov-Smirnov and Levene' s tests, 318 respectively. To determine any significant differences between different feeding 319 frequencies treatments, one-way ANOVA was used, followed by Tukey's post-hoc test. 320 Independent t-tests were considered statistically significant at p < 0.05. Statistical 321 analyses were carried out using the software package IBM SPSS Statistics V22. Manuscript to be reviewed 325 Temperature was kept constant at 20 ºC for each aquarium. The mean pH value 326 was 6.75±0.07, 6.76±0.07 and 6.77±0.70 for FF2, FF4 and FF8 respectively, while the 327 dissolved oxygen levels were 8.59 ± 0.05 mg / L, 8.50 ± 0.06 mg / L and 8.52 ± 0.06 mg 328 / L, respectively (Table1). In all aquaponic systems the electrical conductivity was 1.28 ± 329 0.006mS / cm while the average salinity was 0.64 ± 0.01ppt (Table 1). There were no 330 significant differences (p > 0.05) in the means of NH 4 + , NH 3 , NO 3 -, PO 4 3and pH 331 concerning the water quality in all of the 3 systems ( Table1, Table 2).

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The NH 4 + , NH 3 , NO 3and PO 4 3fluctuation at the water inlet point (GBin) and at 333 the exit point (GBout) of the hydroponic cultivation tank is shown in Figure 2 334 respectively for all of the three systems. 335 Hydraulic loading rate (HLR), the recirculation rate (r), the retention time of the water 336 into the filter bed (HRT), the flow rate (Q), ammonia production rate (P TAN ) the specific 337 surface area of the filter bed (SSA), the volume of filter media (V filter media ) and the filter 338 volume (V filter ) summarized in Table 3 were not statistically different (p>0.05). The fish growth performance is illustrated in Table 4. At the start of the study, 342 there were no significant differences in the means of sea bass initial body weight (gr) 343 and length (cm), (t-test, p > 0.05) for all the feeding frequencies groups (Table 4). At the 344 end of the 45-days study period FF2 group showed significant lower final weight, weight 345 gain, specific growth rate and final length than FF4 and FF8 groups, (p<0.05), (Table 4). 346 Voluntary feed intake and FCR was similar for all the three feeding frequencies 347 (p>0.05), (Table 4). Survival rate for FF2, FF4 and FF8 was 77.2±25.96%, 96.5±1.75%, 348 and 96.5±1.75% respectively.

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Liver histopathology of all the feeding frequency groups revealed mild (grade 2) 350 accumulation of lipid droplets in liver cells with some of the nuclei of the liver cells to be 351 pushed by the lipid droplets to the edge of the cells (Table 5, fig.3). Midgut and kidney 352 microscopic examination showed no histopathological alterations (grade 0) in any of the 353 feeding frequency groups (Table 4, fig. 3, 4). Minimal (grade 1) gill histopathological 354 alterations were detected in FF2 and FF4 groups (Table 5), while mild (grade 2) 355 histopathological alterations were detected in FF8 group (Table 5). Epithelium 356 detachment at the secondary lamellae and hyperplasia of primary lamellae were 357 detected in some cases of all the 3 groups ( fig. 4).

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A total of 2506 bacterial operational taxonomic units (OTUs) were found in all 359 samplings. The lowest number of OTUs occurred on day 0 (106±36.0). The average 360 number of OTUs on days 15 and 45 ranged between 232±166.7 and 467±129.0. 361 Permutanional Analysis of Variance (PERMANOVA) of the OTUs relative abundance 362 indicated that there were no statistically significant differences between sampling points 363 and feeding frequency ( Table 6). The plant growth characteristics are presented in Table 7. At the end of the 45-368 days study period, plants in all systems exhibited similar leaf fresh weight, total fresh 369 weight of leaves, total fresh and dry aerial biomass (Table 7). Nevertheless, plants in 370 system 2 showed inferior root growth and significantly lower number of leaves 371 compared to system 1 and 3 (Table 7). Additionally, plants in system 3 significantly 372 outweigh all the others in stem length.  In the present study an experimental aquaponic system for Mediterranean fish 378 (sea bass) and a vegetable (lettuce) was studied for a duration of 45 days. To the 379 authors' knowledge, this is one of a few studies using sea bass in aquaponic systems 380 [46-48] and the first one to use three different feeding frequencies per day in the same 381 aquaponic system. A successful aquaponic system provides important benefits, such as 382 water quality control, high fish and plant growth performances, plant and fish disease 383 management, and eliminating environmental impacts [36]. Such systems require less 384 than 5% of freshwater to be renewed due to evaporation or losses from daily functioning 385 [49, 50]. Plant growth and production are indirectly related to feeding strategies, fish 386 metabolic condition and microbial activity. Feeding rate and frequency affects nutrient 387 availability in solution inside the system. Increased feeding frequency for fish 388 contributes to more efficient plant nutrition [27, 51] as amounts of nitrate are available to 389 the water for a longer period during the day.

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The present study showed that the adaptation of sea bass in a fresh water 391 aquaponic system together with cultivation of leafy vegetable lettuce is possible. Sea 392 bass is an euryhaline specie. Direct transfer from sea to freshwater shows increased 393 mortality [32, 52]. However, fish gradually adapted over a period of one month [38] do 394 not show any mortality.

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In an aquaponic system, the water temperature setting is dependent on the fish 396 and plant species. Aquarium heater thermostats are used often in order to adjust the 397 water temperature for the best growth of both plants and fish. For sea water-cultured 398 sea bass, temperatures 19-22 °C show the maximum food utilization and growth rate 399 [53, 54]. According to Barnabé [55] and Lanari et al. [56], higher weight gain can be 400 achieved for sea bass at temperatures between 22 -28 °C. In the present study, water 401 temperature was kept constant at 20 ºC, meeting the requirements of both sea bass and 402 lettuce plants. The management of pH is also necessary in aquaponic systems. Plants, 403 fish and bacteria require different pH ranges. Plants require a pH value between 5.5 and 404 6.5 to enhance the uptake of nutrients, and the optimal pH range for bacteria is 7.0-8.0, 405 while the recommended pH for aquaculture is 6.5-8.5 [11]. So, an optimal pH range for 406 an aquaponic system appears to be 6.5-7.0. pH > 7.0 can cause reduced solubility of 407 phosphorus and micronutrients. Plant uptake of certain nutrients is restricted in the 408 aquaponic environment [57]. In our study, pH showed a downward trend for all the three 409 feeding frequencies with mean values of 6.75-6.77. This downward trend is not 410 unexpected, as the accumulation of nitrates (effective oxidation of ammonia) tends to 411 make the aquatic environment more acidic. The mean value of pH is lower than 7.0 and 412 within the tolerance levels for aquaponics. It is obvious that both pH and temperature 413 are important parameters for the optimization of aquaponic production both for fish 414 welfare/health issues and for plant needs.  fig. 2). In the 0-14 444 day time period, the phosphate concentration was also higher at the exit point than the 445 inlet point (fig. 2). The gradual rise of nitrate levels proved the efficiency of the filter in 446 oxidizing the produced ammonia. In the present study, the daily supply of 20-25 gr of 447 fish food efficiently provides the necessary nutrients for plants. During the experiment, 448 the water supply (Q) was adjusted to 6.27 L/min and the filtering speed (V) to 1.79 449 cm/min, ensuring the successful nitration and maximum efficiency of the filter [64]. reported that better 458 growth performance of fish in a freshwater aquaponic system was observed at a higher 459 HLR (2.56 m/d) than the HLR used in the present study. Nevertheless, Vlahos et al. [36] 460 reported that better growth performance of gilthead seabream and rock samphire was 461 observed at an HLR of 1.84 m/d, which was higher than the HLR of the present study. showed higher SGR and lower FCR in night feeding during 483 the winter months (0.26 ± 0.01%/day and 2.65 ± 0.08, respectively) in an RAS system 484 compared to morning feeding (0.19 ± 0.01%/day and 3.73 ± 0.17, respectively).

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The feeding frequency did not affect fish survival, with 77.2±25.96%, 96.5±1.75%, and 487 96.5±1.75% survival being observed under the FF2, FF4 and FF8 treatments, 488 respectively. On the day 16 th an unexplained fish mortality was observed (10 fish) for 489 the FF2 group. This was probably due to anaesthesia fish handling. According to 490 Gilderhus and Marking [77], the margin between the effective and toxic concentrations 491 of MS-222 tends to be narrow. Consequently, the observed mortality had no relation 492 with the feeding procedures. In sea bass, a feeding frequency of 1-3 meals per day 493 can deliver good growth and FCR performance [35,78]. For juvenile's sea bass (5.2-6.8 494 g) a feeding frequency of two times per day seems to be the minimum with good growth 495 results and was followed to previous studies [79][80][81]. Nevertheless, in this study feeding 496 frequency of 2,4 and 8 meals per day was tested in order to examine how it affects the 497 daily nitrate fluctuation for better plant nutrition in an aquaponic system. In a study by 498  In an RAS and consequently in an aquaponic system, a properly selected diet 510 must be managed in such a way as to meet the nutritional requirements of different fish 511 and plant species. By selecting the appropriate food amount per day and appropriate 512 feeding frequency, metabolic products (excretions) are reduced, fish growth is 513 enhanced, and water quality ultimately improves [27]. The removal of fish metabolic 514 products (nutrients) from the water is directly related to the quantity of the provided diet, 515 the feeding frequency and the food quality. Nitrogen content in fish faeces ranges (10 to 516 40%), depending on the nitrogen content of the provided diet and the fish type [85]. In 517 the present study, fish were fed daily at 5% of their body weight with a commercial 518 floating pellet diet (55% protein and 15% crude fat), showing good growth for all of the 519 feeding frequency groups. These results suggest that the provided food amount was 520 appropriate, and they are in agreement with those of Eroldogan et al. [34], where sea 521 bass with an initial weight 2.6±0.3 g cultured in seawater (40 ppm) and in fresh water 522 (0.4 ppm) with six different feeding rates (2%, 2.5%, 3%, 3.5%, 4%, saturation) showed 523 greater WG and SGR in fresh water and at a feeding rate of 3.5% until saturation. 524 Türkmen et al. [86] also showed that sea bass fed at 5% of their body weight 4 and 8 525 times per day exhibited a higher SGR. In contrast, Waller et al. [46], working with sea 526 bass fed daily to satiation, showed a lower SGR and FCR (1.5% and 0.93 respectively).

528
In aquaponic systems, increased feeding frequency seems to have positive 529 effects on fish and plant growth. Liang and Chien [27], working in a tilapia-water spinach 530 aquaponic system, reported that increasing feeding frequency increased both fish and 531 plant production and lessened the accumulation of nitrogen and phosphorus nutrients in 532 water. The same results were reported by Mohamed Abdelrahman [51] while studying 533 the effect of different daily fish feeding frequencies (1, 2 and 3 times per day) in a tilapia 534 and lettuce aquaponic system. In the present study, the higher WG, SGR and were 535 achieved at FF4 and FF8 (no significant differences were detected between these two 536 feeding frequencies). FCR and voluntary feed intake did not differ among the three 537 feeding frequencies (p>0.05). Feeding four or eight times per day seems to have the 538 best effects on fish growth. This result is in accordance with Biswas et al. [25], who 539 showed that Asian sea bass (Lates calcarifer) cultured in brackish water had the best 540 SGR when it was fed 3 or 4 times per day.

542
It is not clear if salinity is an important factor for the optimal growth of euryhaline 543 species, as it is a disagreement among researchers if acclimatization to fresh water can 544 cause a loss of appetite, increased mortality and decrement of conversion efficiency 545 [31,[87][88][89] or can cause similar or even better growth parameters than sea water [47, 546 90-92]. Eroldogan and Kumlu [90] showed that sea bass juveniles cultured in fresh 547 water, 10 and 20 ppt grew better than those at 30 or 40 ppt. In a second experiment of 548 the same study [90], young sea bass grown in fresh water had higher WG than those 549 grown in sea water, with a slightly higher FCR in sea water. Vlahos et al. [36] did not 550 detect differences in the growth performance of seabreams in two different salinities (8 551 ppt and 20 ppt). Nozzi et al. [47] showed higher WG and SGR for sea bass in fresh 552 water than in sea water. Even at extreme temperatures, sea bass seems to grow better 553 in low salinity water. According to Islam et al [91], sea bass reared for 35 days followed 554 by 10 days of extreme warm temperature (33 °C) showed higher weight gain and SGR 555 at 12‰ and 6‰ salinity water than at 32‰. Weight gain and SGR were similar in 32‰ 556 and 2‰ salinity (8.45 g and 9.42 g weight gain, respectively, and 2.03 and 1.93 SGR, 557 respectively). In our study, SGR was 2.11, 2.23 and 2.36, while weight gain was 10.66 558 g, 13.14 g, and 13.85 g under FF2, FF4, and FF8, respectively. These values are higher 559 than those reported by Islam et al [91], probably because no temperature stress 560 occurred. Yilmaz et al. [92], in a 60-day trial of the growth performance of sea bass in 561 fresh water (0‰ salinity, 20ºC), reported a 1.1% SGR and 1.2 FCR, which SGR to be 562 lower than the values in our study but FCR to be similar with our value in FF8 group.

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Kidney is an important organ for the osmoregulation of euryhaline fish [93,94]. 565 Fish like sea bass, trout, herring, and juvenile seabream show good adaptation to 566 salinity changes, surviving this way in both seawater and freshwater. Nebel et al. [30], 567 reported that sea bass juveniles lived in freshwater had smaller collecting ducts than 568 those lived in seawater. Vlahos et al. [36], when adapting seabream to lower salinity (8 569 ppt), did not detect histopathological alterations of the midgut, smaller collecting ducts, 570 granulomas or dilation of Bowman space in the kidney, hyperplasia of 571 primary/secondary lamellae or epithelial detachment of the secondary lamella in gills, 572 while liver histopathology showed inflammation and steatosis. In the present study, 573 midgut and kidney microscopic examination showed no histopathological alterations, 574 while liver showed mild accumulation of lipid droplets, and the gills showed mild 575 epithelial detachment at the secondary lamellae and mild hyperplasia of the primary 576 lamellae. Similar results for gills were reported in previous studies [95,96], thus 577 indicating the high plasticity and gill remodelling of sea bass adapted from seawater to for Classical and Next-Generation Sequencing-Based Diversity Studies. Nucleic    Aquac. 31, 181-194 (1983).    196, 191-199 (1997).            On the day 16th an unexplained fish mortality was observed (10 fish) for the FF2 group. This was probably due to anaesthesia fish handling. Consequently, it had no relation with the feeding procedures.
Data are expressed as means ± S.E.M. Means in a row followed by the same superscript are not significantly different (p > 0.05).  Manuscript to be reviewed