Improving the efficiency of production of lambari and diet assimilation using integrated aquaculture with benthic species

A single farmed fish species assimilate about 20% of the nutrients in the supplied diet. This study evaluated if the culture of complementary ecologicalfunction species can recover nutrients dispersed into the water and transform them into high-valued biomass. A completely randomized experiment was designed with three treatments and four replications of each production system: monoculture of lambari (Astyanax lacustris); integrated aquaculture of lambari and Amazon river prawn (Macrobrachium amazonicum); and integrated aquaculture of lambari, Amazon river prawn, and curimbatá (Prochilodus lineatus). Fingerlings of lambari (0.8 ± 0.8 g) were stocked in twelve earthenponds (0.015 ha) at the density of 50 fish m. Eight ponds, were stocked with juveniles of Amazon river prawn (1.1 ± 0.2 g) at the density of 25 prawn m. Four of these eight ponds were stocked with curimbatá fingerlings (0.2 ± 0.1 g) at a density of 13 fish m2. Only lambari was fed twice a day with an extruded commercial diet. The experiment lasted 60 days when lambari attained commercial size. The inclusion of prawn increased the total species yield from 1.8 to 2.4 t hacycle and reduced the FCR from 2.5 to 1.8, whereas The inclusion of prawn and curimbatá increased the total yield to 3.2 t hacycle and reduced the FCR to 1.4. Therefore, the integrated culture of lambari, prawn, and curimbatá improves the use of space, water, feed, and benthic species can recover the large quantity of nutrients accumulated in the bottom of lambari pond production, converting them into high-nutritional and monetary-valued biomass. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2021 doi:10.20944/preprints202108.0110.v1 © 2021 by the author(s). Distributed under a Creative Commons CC BY license.


Introduction
Worldwide aquaculture production surpassed 120 billion tonnes and USD 275 billion in 2019 [1]. This activity is one of the fast-growing food-producing sectors, increasing about 6% yearly in the past three decades, employing more than 20 million people [2]. Aquaculture is essential to meet the increase in animal protein demand and provide food security [3]. Recently, animal aquaculture based on allochthonous diets (fed aquaculture) surpassed unfed aquaculture [2]. Diet is the major operating cost in fed monoculture systems [4].
This situation is because a single species are not able to assimilate most diet nutrients and energy. In monocultures, the farmed species assimilates only ~20% of the diet nutrients, while almost 80% are dispersed into the water as particulate matter or dissolved nutrients and transform into pollution [4] Integrated systems are based on farming more than one species per pond, and then it is possible to occupy the three spatial dimensions and different ecological niches [4]. These systems promote synergistic interactions between farmed species. The available resources can be more efficiently used, shared, recycled, and converted into biomass of high commercial value, based on the economic circularity concept. These systems also promote animal welfare and reduce environmental impact [8,9]. The integrated multitrophic aquaculture (IMTA) system is based on the farming of species with different trophic levels and/or with complementary functions and economic potential. The IMTA systems generally combine fed species with extractive species. These species use food waste and residues from the production of the fed species to grow; thus, it is possible to recover nutrients and increase yield without increasing inputs [10]. Therefore, choose suitable species that showed compatibility and complementarity is crucial to improving aquaculture sustainability [8,9].
Lambari is a group of small native fish from Brazil common in natural freshwater. They have gained visibility and good acceptance in very profitable market niches, such as human food and live bait for sport fishing [11,12]. The yellow tail lambari (Astyanax lacustris, former A. bimaculatus) [13] has opportunistic omnivorous feeding habits, high reproductive rate, short life cycle, and easy management, showing high qualities for aquaculture [14,15]. Despite the recent increasing production [12] and significant interest, there are no established standard raising systems and practices for farming this lambari.
Fonseca et al. [15] recommend the integrated multitrophic culture to improve the sustainability of the yellow tail lambari production in Brazil. Amazon river prawn, Macrobrachium amazonicum, is another species with great potential for aquaculture and described as an excellent alternative to composing integrated multitrophic systems. This species is detritivore and omnivorous, ingesting macrozoobenthos, algae, dead plants, and other residues deposited on lakes and river bottoms [16], and has a benthic habit, which avoids competition with pelagic species. However, studies carried out previously showed that the nutrients accumulated on the pond bottom at the end of the integrated farming of pelagic fish and M. amazonicum are still large, making it possible to include another bottom detritivorous species [17][18][19][20][21][22]. Therefore, the addition of a third detritivorous species should improve nutrient recovery in the integrated system composed of a fed pelagic fish and a benthic prawn.
Curimbatá, Prochilodus lineatus, is another indigenous species in Brazil also known as curimba or curimatã. This species is exploited by fisheries and aquaculture in different regions of South America [12,[23][24][25][26][27]. This is an iliophagus fish that feeds predominantly on fine-particle organic matter and periphyton over the bottom of rivers and lakes [28,29]. Thus, curimbatá can be an excellent option for the integrated system with lambari and prawn. Curimbatá was introduced in China and Vietnam, where it has been farmed in integrated culture [29].
Considering the above rationale, this study aimed to evaluate if the introduction of benthic species with complementary niche trophic in the culture of a pelagic fish would recover lost nutrients and increase the yield, improving the utilization of the supplied diet. Lambari, Amazon river prawn, and curimbata are excellent models to test this hypothesis because they have complementary

Feeding management
The feeding for all treatments began after the stocking of the yellow tail lambari. Before that, the Amazon river prawn and curimbatá fed on natural biota [30][31][32]. A commercial diet for onívorous fish was provided (36% crude protein, granules of 2.3 mm, recommended for fish juveniles). Feed was supplied just for yellow tail lambari twice a day at 10 a.m. and 04:30 p.m. Initially, the feeding rate was 10% of the yellow tail lambari biomass. When this fish attained mean biomass of 3.8 ± 0.3 g (day 34), it was reduced to 5% of the biomass until the the experiment's conclusion. The prawns and the curimbatá fed on the remains of fish food and wastes produced by the yellow tail lambari, in addition to the ponds' natural biota.

Water quality
The water variables temperature, dissolved oxygen, pH, and conductivity were monitored daily at 08:00 a.m., using a probe YSI Professional Plus (Yellow Springs Instruments Company, Yellow Springs, USA) and maintained within the recommendations described in Boyd [33] for general aquaculture pond systems (Table 1). Aerators (Bernauer, model B-500 Aquahobby-monophasic) were used in all ponds to maintain adequate levels of dissolved oxygen. The aerators were turned on just when the dissolved oxygen was less than three mg L -1 . The water of each pond was sampled biweekly at 07:00 h to measure the concentrations of total nitrogen and phosphorus concentrations. The total nitrogen and the total organic carbon were determined using oxidation catalytic combustion (Elementar -Vario TOC Select, USA). The total phosphorus was determined by the persulfate digestion method (4500-P B5) [34] to liberate orthophosphates associated with organic material ( Table 1). The inlet water and the water inside the ponds during the culture were classified as eutrophic water, according to the classification of Brow and Simpson [35] because total nitrogen and total phosphorus concentration were within the ranges 390-6100 μg L -1 and 16-390 μg L -1 , respectively.

Harvest and productivity data collection
The ponds were drained 60 days after the yellow tail lambari stocking when all animals were harvested, and the survivors were counted. A sample of 100 prawns and fishes before and after the growth-out period were randomly

Data Analysis
The data were analyzed for normality and homoscedasticity of variances using Shapiro-Wilk and Levene tests, respectively. Then, the data were subjected to one-way ANOVA (F-test). For significant differences, means were compared using the Fisher-LSD test. Differences were assumed at significance levels of P < 0.05.

Results
The final mean individual mass, total length, and survival of the yellow tail lambari showed no differences between treatments. In all treatments, the yellow tail lambari attained on average 7 g of mass, 7 cm of length, survival was about 50%, and the yield about 1.9 t ha -1 (Table 2). The final mean mass and length, survival, and yield of the M.
amazonicum have no statistical difference among integrated culture treatments.
The final size of prawns was about 2.6 g and 6.7 cm, the mean survival was about 81%, and the yield was 0.6 t ha -1 . The curimbatá attained the final size of 6 g and 7 cm, and the survival and yield were 70% and 0.5 t ha -1 , respectively.
The total yield (biomass of all species) increased with the addition of prawn and curimbatá, being significantly higher in the LPC than other treatments. It ranged from 1.8 t ha -1 in LM, to 3.2 t ha -1 in LPC. The yellow tail lambari feed conversion ratio (FCR) was similar between all treatments (Table 2). For the integrated culture system, the FCR decreased with the addition of prawn and curimbatá. It was significantly lower in the LPC than LM treatment, but there was no difference compared with the LP treatment. The FCR ranged from 1.4 in LPC to 2.5 in LM.

Discussion
The integrated culture of yellow tail lambari, Amazon river prawn, and curimbata showed to be technically feasible, efficient, and productive. No adverse effect on the growth, survival, and yield of the yellow tail lambari was produced by the benthic species. Similarly, curimbata did not affect the prawn´s performance. All species developed well in stagnant earthen ponds while using nutrient-rich water, corroborating previous results [31,32,36]. Total annual highvalue biomass produced increased from 9 t ha -1 in lambari monoculture to 16 t ha -1 in integrated culture, using the same space, amount of freshwater, feed, and other resources, indicating a tremendous increase in system efficiency.
Annually, 7 t ha -1 of nutrients were recovered from the environment and transformed into nutrient-rich human food and marketable product.
The yellow tail lambari is typically farmed in small earthen ponds (0.03-0.3 ha) for 3 to 4 months to attain 3 to 8 cm and are sold by US$ 3.00/kg to processing plants or US$ 50.00 per thousand individuals to bait-fish market [12]. Generally, farmers have low control and records of the cultures [15]. be caused by predation by birds and aquatic insects, susceptibility of the species to management, and the lack of a scientific-based farming protocol.
In the present study, lambari reached the commercial size in 60 days in monoculture or integrated culture. This time is relatively short when compared to what is usual in commercial farms, which is 3 to 4 months [12]. This difference may be due to the accelerated growth of lambari in warm and rainy seasons, the high-quality diet, or more controlled management, such as observed in conducting the present study. Therefore, performing 5 production cycles annually, as we simulated, seems feasible after minor improvements in the technology used in commercial farms. Amazon river prawns stoked as postlarvae in growth-out ponds generally spent about 120 days to reach the commercial size [39]. Thus, 2-months juvenile prawns should be stocked in integrated culture with lambari as a strategy to combine and coincide both species cultivation periods. The curimbatá has a slow growth rate, but it should be traded as juveniles of different sizes to grow-out farms, bait-fish for the sportive fisheries market, or the environmental mitigation market [12,31].
Juveniles of different sizes produced in hatcheries are released annually into dam-impacted hydrological basins in Brazil, which support a massive artisanal fishery [12].
The present study corroborated that the Amazon river prawn can be raised with pelagic fishes in stagnant ponds filled with eutrophic or hypereutrophic water. Kimpara et al. [40] demonstrated that the use of nitrogen and phosphorus-rich water could be suitable for the monoculture of this prawn.
Rodrigues et al. [36] demonstrated the feasibility of combining M. amazonicum with Nile tilapia, and Dantas et al. [32] demonstrated the same, combining the prawn with tambaqui (Colossoma macropomum). All these studies used nutrient-rich water in stagnant ponds. Nevertheless, the yield of the Amazon river prawn is about 0.5 t ha -1 cycle -1 , when eating only diet residues and wastes of a pelagic fish, which is half of that obtained when prawn is fed with a specific commercial diet in densities below 40 prawns m -2 [32,36,41] and the present results.
Curimbatá may compete with prawns by space and food on the pond bottom. However, no effect on prawn growth, survival, or yield was observed.
These results indicate that the competition may be low, and no agonistic  [28], and Amazon river prawn eats alike during the day or at night [42]. Amazon river prawn eats mainly benthic organisms and large organic matter particles [16], while curimbatá ingest mud containing fine-particle organic matter, most particles lower than 105 µm, and periphyton that grow over inorganic particles [28,43].
This periphyton is composed mainly of microbial biomass [29] that extract carbon, nitrogen, phosphorous, and other nutrients from water and sediment, making them available for heterotrophic food webs.
The number and proportion of the unfed components of an integrated aquaculture system depend on the pelagic species' mass density because it provides residues and wastes. A co-stocking experiment that lasted 2 months showed that the yield of the Amazon river prawn stoked at 11 prawns.m −2 together to 4 g tambaqui, (the pelagic fed species), stocked at 1.4 m -2 , decreased by 25% when 5 curimbatas.m −2 were added [31]. However, total species biomass increased 35%, and FCR decreased 30%, showing that the yield of curimbatá compensates for the decrease in the prawn yield and the recovery of lost nutrients and energy increased. The higher biomass of lambari in the present experiment than the biomass of tambaqui in the study of Franchini et al. [31] was enough to provide the necessary wastes to support the density of prawn and curimbatá with no adverse effect on the prawn growth.
We observed that the addition of Amazon river prawn to yellow tail lambari culture increased the annual yield from 9 to 12 t.ha -1 and reduced FCR from 2.5 to 1.8. The addition of curimbatá, a second benthic species, with iliophagus food habit, increased annual yield to 16 t.ha -1 e reduce FCR to 1.4.
This great increase in efficiency by adding species with complementary ecological functions represented an improvement in nutrient recovery of almost 80%. The present system is ranked as the maximum level of integration (level 5), according to the scale of Boyd et al. [4], which means that one cultivated species originates by-products, which are inputs for the others and vice versa.
Lambari produces wastes that down to the bottom, providing energy and nutrients for developing benthic communities. Prawn and curimbatá will feed on the aquatic biota or directly on the wastes contributing to the mineralization of organic matter. Their bioturbation creates an upwelling of nutrient-rich water, which will fertilize the water column, boosting the development of phytoplankton, which will be eaten by the zooplankton, which is a nutrient-rich food to the lambari. Therefore, the integrated system proposed creates a looping of nutrients, increasing recycling, assimilation, and the system's circularity. This scenario is provided by the compatibility of animals, which limits competition and agonistic behavior, and the complementarity exploited by the synergistic interaction between species leading to biomitigation and production processes [9,44].
In conclusion, the integrated system proposed in the present study showed to be efficient in recovering nutrients spread in the water. This system also increases yield using the same inputs of feed, water, space and other natural resources as used in monocultures. The system transforms pollution into high-value marketable biomass, reducing the nutrients discharged in the effluents. The system also contemplates some important sustainability principles claimed by Valenti et al. [45], such as production based on the circular economy concept, reduction in using natural resources, increasing efficiency in assimilation nutrients, and allowing the producer to conquer different markets offering different products. The three species are native to Brazil, which brings some advantages, such as avoiding risks to biodiversity and exploring consolidated local markets [27,46]. Furthermore, the experiment was