Herbivore Fish as Sustainable Alternative for Nutrition Security: Food Habits and Nutritional Composition of the Acha Fish (Medialuna Ancietae) in Northern Chile.


 Sustainability in aquaculture is a necessity of the future, not only as the most promising means of supplying the protein that the world will require to feed its growing population but to offer needed conservation of the world’s ocean resources. The use of wild fish inputs in farm-raised fish outputs has been a primary concern of sustainability in aquaculture production. Herbivorous fish are more efficient convertors of protein into fish flesh. Species of the genus Medialuna fish have been reported as a fast-growing, short-lived species. The endemic Acha fish (Medialuna ancietae) in the Northern part of Chile is an over-exploited fish that has been associated with aquatic vegetation as a food source. We studied the eating habits and nutritional composition of M. ancietae. For this, we developed a reference collection of marine macroalga (epidermis and nutritional composition) observed in the diet of individuals of this species for the study of digestive material. More than 90% of the components found were marine macroalgae, indicating that M. ancietae is an herbivorous fish. Compared to non-herbivorous fish our results showed that most of the nutrients present in the Medialuna diet are found at much lower levels including n-3 long-chain polyunsaturated fatty acids (49.7%) and protein (13–60%). M. ancietae meat provides essential components of human nutrition with a significant protein content (18.99 ± 0.26%) and the presence of n-3 long-chain polyunsaturated fatty acids. Most fed aquaculture non-herbivorous species rely on wild-captured fish for these essential nutrients, while M. ancietae can obtain and has the ability to concentrate them from potentially cultivable macroalgae. M. ancietae has potential for a sustainable aquaculture production as a contribution to nutrition security and re-stocking of wild populations.


Introduction
Aquaculture is currently the fastest growing global animal food production sector and is a key future contributor to food security; most fed aquaculture species rely on wild captured sh for essential fatty acids and micronutrients, as the fastest-growing food sector fed aquaculture demand exceeds ecological limits of these population of wild sh [1,2]. Aquaculture is the biggest global consumer of shmeal (70%) and sh oil (73%); within the next decade, sh oil production is unlikely to meet the required quantities for aquaculture [3].
As a result, the use of other alternative inputs with nutritionally equivalent pro les, such as algae, will be necessary to stabilize access to sh long-chain omega-e fatty acids. The use of non-carnivorous aquaculture is also an alternative to this problem and a good strategy for sustainable aquaculture [4]. Non-carnivorous or herbivorous shes also allow for a more productive algal community by decreasing the self-shading of algae, as well as increasing the local nutrient input via herbivore excretions; herbivorous shes can be abundant in temperate rocky-reef habitats [5] and have been reported as short-lived and fast-growing species [6]. Herbivorous shes have a specialized morphology of the digestive tract with the ability to assimilate seaweed compounds and to grow on a seaweed diet (stomach content with more than 50% plant material). The stomachs of several species of herbivorous shes are muscular and function as gizzards to grind lamentous algae and diatoms into ne particles for chemical breakdown and subsequent absorption [7].
In Chile, the cultivation of native marine sh is an incipient issue. Research into the cultivation of native marine sh began with capturing wild specimens, including Merluccius australis, Cilus gilberti, Seriola lalandi, Paralichthys adpersus, and Medialuna ancietae [8]. M. ancietae (Acha sh) is a native species with potential for aquaculture and repopulation. M. ancietae lives mainly in the waters of the rocky coasts of Northern Chile and Peru, is found in wide and deep caletones, breaking from rocky beaches [9]. The catches of this species have decreased from 9000 tons per year in the 1980s to less than 100 tons per year by the middle of the 2000s. Currently, M. ancietae is virtually never encountered during spear shing [10]. The local demand for the quality of its meat, the no size or catch regulations, and its decrease in catches make this species an interesting alternative to develop its aquaculture. M. ancietae is an important resource for artisanal and sport shing in the north of Chile, but unfortunately, it shows signs of a decline in its abundance and the sizes of the sh caught [9], which is why initiatives have been developed to cultivate it for commercial purposes and of possible repopulation actions.
M. ancietae can be found with di culty in some parts of northern Chile, especially on the coasts of the small town called Pisagua (19°36'22,57 "S, 70°12' 09,96 "W). It is characterized by the presence of large cliffs and mountain ranges, typical of the coastline coastal region I (Tarapaca region). The intertidal zone of this locality is conditioned by the existence of cliffs with a narrow Lessonia berteroana (formerly Lessonia nigrescens) belt that varies between 0.5 and 1.0 m. Among the inter-disk spaces, nine taxa have been reported (two algae and seven invertebrates). The algae present correspond to calcareous with 66% and Corallina sp with 1% [11]. The fauna associated with this environment mainly comprises Fissurella sp and Chiton sp, followed by Actinia and H. helianthus. There is a speci c dominance of Fissurella spp (27%) [12]. The black snail Tegula atra (Lesson 1830) is a gastropod of the Trochidae family, which is distributed throughout the Southeast Paci c from Pacasmayo, Peru (7°24'S) to the Strait of Magellan, Chile (53°28'S). T. atra has been observed associated with different species of macroalgae of the order Laminariales. In northern Chile, T. atra is found in intertidal environments next to Lessonia berteroana and, in subtidal areas, next to Lessonia trabeculata [11].
For e cient culture and management of sh, knowledge on food and feeding habits is of immense importance, and this is intimately associated with the ecological niche they occupy in the natural environment [13]. Good reproductive performance for the successful production of juveniles is unpredictable and limiting. It has been shown that adequate nutrition and feeding of the breeders is necessary for a good quality of the egg and sperm and the production of seeds, gonadal development, and fecundity, which are affected by certain essential nutrients of the diet, especially HUFA n-3 fatty acids. The lipid and fatty acid composition of the breeders' diet has been identi ed as the main factor determining the successful reproduction and survival of the offspring. Some sh species will incorporate highly unsaturated fatty acids in the eggs, even during spawning; these highly unsaturated HUFA fatty acids with 20 or more carbon atoms directly affect the maturation of sh and steroidogenesis through their metabolites [14,15].
The knowledge of the feeding behaviors and the diet components are fundamental aspects to be considered when developing feeding plans that contain the pro les of these fatty acids necessary for the species to be tamed for commercial aquaculture and repopulation purposes. Part of these studies can be carried out by analyzing the digestive content with different microhistological techniques that constitute reliable procedures for identifying essential elements in herbivore feeding [16].
This research aimed to study the food habits and nutritional composition of Medialuna ancietae, this work is expected to provide a basis for M. ancietae environmentally sustainable aquaculture production as a contribution to nutrition security and re-stocking purposes in Northern Chile.

Materials And Methods
Collection of sh M. ancietae is an overexploited species. Therefore, its shing is di cult, and catches are not always successful. Fishing trips were made during 2019. Fishes were caught by free diving using a modi ed harpoon off the shore of Pisagua (Latitude: 19°00´/20°00´ and Longitude: 69°00´/70°30´), Chile. Morphometric measurements were obtained from each sh, and the stomach was extracted. All samples were transported to our laboratory under ice-storage conditions. All experiments were approved and performed according to guidelines provided by Comité de Bioseguridad y Biocustodia Universidad Arturo Prat, certi cate UNAP/VRIIP Nº003/2018. This study is reported in accordance with ARRIVE guidelines [17]. The samples were dried in the eld and xed with 4% formalin in seawater [18] after collection or diving. For the transfer to the laboratory in the city of Santiago, the different species of macroalgae belonging to the marine ecosystem of the area were identi ed. Subsequently, the epidermal tissue was removed from the macroalgae, following the diafanization methodology [16] and the sodium bicarbonate method [19].

Collection of reference patterns and Stomach Content Analysis
The diafanization method: the material was boiled in 96% alcohol for 10 min at 150°C, then boiled again in an aqueous solution (1:1) of 96% alcohol and 5% sodium hydroxide for another 10 min in a hot iron at 350°C under the hood, to avoid possible in ammations of alcohol and the inhalation of gases. Then, the treated material was deposited on a Petri dish and washed with distilled water until it was cleaned of reagents. Next, a solution of 5% sodium hypochlorite diluted with 50% distilled water was applied; they were allowed to stand long enough for them to become transparent (30 min), permanently monitoring this process. Once the material was rinsed, it was passed through distilled water ve times (5 min each change) and kept in a 5% chloral hydrate solution to remove opacity. The material was kept in this solution for 10 min.
The sodium bicarbonate method The material was deposited on a Petri dish, then a solution of 17.5% of sodium bicarbonate was applied, the macroalgae were soaked for 48 h, then cleared by soaking in a solution of 50% sodium hypochlorite for 20 min, and then washed with abundant distilled water. Subsequently, histological cuts (transversal) were made in the samples treated. The cuts were made manually with a scalpel, and the sample was deposited on a slide, taking care that the face to be observed is facing up, placing a drop of distilled water and covering the sample with a 24 × 24 mm object cover. Epidermis preparations were observed under a microscope (LEICA, DM500 model equipped with ICC50W digital camera) connected to a computer to observe the epidermis on the screen of the same, photographs of all the patterns obtained using a magni cation of 40× were taken to the visualization of its histological characteristics. Descriptions and illustrations of the records were made by consultation in bibliographic references [20][21][22][23]. The photographs were used for subsequent recognition of stomach contents preparations for botanical determination of their diet.
Stomach content samples were obtained immediately from eight individuals captured. The sh died at the time of capture, so it was not necessary to apply euthanasia. Stomach samples from the caught sh were weighed. Subsequently, the stomach contents were separated macroscopically by taxonomic group, and their weight was recorded. Each taxonomic group was preserved in 10% formalin.
For the microhistological analysis, the material was washed with distilled water, and the diafanization [16] and the sodium bicarbonate [19] method were used for the subsequent observation of the samples in an optical microscope (LEICA, DM500 model equipped with ICC50W digital camera). Images were captured for analysis. In the quantitative analysis of the food components, the gravimetric method (G) and frequency of occurrence (FO) were used. For FO, the number of stomachs containing one or more components of each food category was recorded; this number was then expressed as a percentage of all stomachs [24]. The total weight of each food category is expressed as a percentage of the overall weight of the stomach contents.
Nutritional composition of collected macroalgae, stomach content, and Medialuna a. meat Macroaalge and Medialuna samples were immediately frozen in liquid nitrogen for transport to the laboratory and subsequent lyophilization. The lyophilized material was used for proximate and fatty acid composition following the recommended methods of the Association of O cial Analytical Chemists (AOAC): fatty acid pro le [25], crude protein combustion analysis [26] utilizing the calculation 6.25 × nitrogen value, crude fat [27], moisture content [28], ash [29], crude fat [27], sodium and potassium [30], and zinc and calcium [31]. Total carbohydrates were calculated by difference", 100% − %(crude protein + ash + crude fat + moisture).
The nutritional and fatty acid composition of the total lipid in the stomach contents were determined based on the three main components and the results of their nutritional composition.

Results
Biological data of shes The biological data of collected sh are given in Table 1. Among the collected female sh, high gonadosomatic indices were present during January and September. The average weight of the sh caught was 8.964 ± 1.89 kg, with a length of 75.93 ± 6.29 cm.
The stomach weight of the collected sh was 156.65 ± 39.60 g, with a stomach content of 76.86 ± 19.48 g. On average, the stomach was at 49% of capacity in these sh collected from January to September of 2019. Collection of reference patterns and stomach content analysis Remarkable differences were found when comparing reference slides prepared using the sodium bicarbonate solution method compared with the Castellaro method. The sodium bicarbonate solution maceration technique provided a much more recognizable part of the macroalgae consumed by Acha sh, including greater visibility of the different pigments present in macroalgae (Fig. 1). The main dietary component identi ed in all individuals was macroalgae, accounting for 96.08 ± 6.90%. Four different macroalgae were identi ed in the diet, in addition to incidental components ( Table 2). The most abundant and consumed components by all individuals were Lessonia berteroana (formerly known as Lessonia nigrescens), followed by Glossopohora kunthii and Corallina o cianalis var. chilensis (Fig. 2). Corallina o. was present only in the rst half of the year. Only two components were present in the diet in the second half of the year: macroalgae Lessonia b. and Glossophora k. were identi ed.  Among the three main components identi ed in the sh's diet, a high ash content was observed, with a greater presence in the red macroalgae Corallina o. (80.9%), the brown algae Lessonia b., and Glossophora k. presented a similar ash content (37% and 41%, respectively), and high carbohydrate content in both brown algae was also present (Table 3).   (Table 4). Based on the three main ingredients identi ed in the diet and its nutritional composition, we found that the diet of the Acha sh is rich in carbohydrates and minerals and has a low protein content.
Among the main fatty acids present in the sh's diet, oleic fatty acid (9c-18:1) was the most abundant. Within the polyunsaturated fatty acids, the results showed a higher presence of arachidonic fatty acid [20:4n6], followed by EPA (20:5n3) and, to a lesser extent, DHA (22:6n3). The sh meat had a low-fat content and was 18.9% protein ( Table 5).

Discussion
The different epidermis patterns of the macroalgae associated with the habitat of the M. ancietae obtained by the sodium bicarbonate technique allowed us to identify the different plant components of the M. ancietae diet. Within the components of the diet of the Acha sh, there is a greater presence of the algae Lessonia b., followed by Glossophora k. However, the calcareous red alga Corallina showed an important presence in the rst semester of the year in females, with a low gonadosomatic index. Of the total components found in the sh's diet, 96% correspond to macroalgae, so our results show that M. ancietae is an herbivore sh [7]. Our results are consistent with studies carried out in species of the same genus, the halfmoon (Medialuna californiensis: Kyphosidae), a herbivorous sh in the temperate waters of California [6, 36]. The macroalgae identi ed in the diet of the M. ancietae sh are low in fat; this fat showed a higher presence of polyunsaturated fatty acids (41.31%), followed by saturated fatty acids (32.38%) with a higher presence of palmitic and myristic fatty acids and to a lesser extent, monounsaturated fatty acids (21.92%), with a higher presence of oleic fatty acid. These proportions are similar to those found for sh oil with a greater presence of polyunsaturated fatty acids followed by saturated fatty acids (with a high presence of palmitic fatty acid) and in relation to the monounsaturated ones with a greater presence of oleic fatty acid [37]. The polyunsaturated fatty acid with a higher presence in the components of the diet was arachidonic, follow by eicosapentaenoic and docosahexaeoic, except Corallina of., which showed a high percentage of EPA. This unexpected high EPA content may be related to the presence of diatoms observed in the Corallina of. samples or by the presence of marine cryptophytes [38]. Our studies showed an ARA/EPA ratio of 2.64 for the Medialuna sh diet; this ratio is much higher when compared to sh oil (0.15) [39].
Carbohydrates (34.65%) and minerals (40.68%) were the most abundant nutrients in the diet of M. ancietae, with important contributions of calcium from Corallina of. and zinc from Lessonia b.
The M. ancietae diet was low in protein (10.23%). The contribution of minerals was relatively higher in the summer months due to the presence of Corallina, which presented a high ash content (80.9%). Our data shows that the diet of this herbivorous species is 13-60% lower in protein than the protein requirements reported for other species [33]. Nutritional and fatty acid composition of the total lipid in diet of adult specimens of Acha sh differ from those of commercially produced feeds or nutritional requirements reported for other marine and freshwater non-herbivorous sh species. Most of the nutrients present in the Medialuna diet are found at lower levels, including EPA (20:5n3) and DHA (22:6n3) [34]. Most fed aquaculture species rely on wild-captured sh for these essential fatty acids [4], while M. ancietae can obtain and has the ability to concentrate these essential fatty acids from potentially cultivable macroalgae [40,41]. M. ancietae meat can provide 268mg/100g of the essential n-3 long-chain polyunsaturated fatty acids (EPA + DPA + DHA).
The protein content present in the meat of M. ancietae (18.99 ± 0.26%) is high considering the reported protein range of 8.2-23.9 g/100 g for some freshwater and marine sh. In terms of fat (1.89 ± 1.68%), M. ancietae is considered a lean sh [42]. Our results showed a high protein content in M. ancietae meat and the presence of less protein (13-60%) in their diet when compared with the protein requirements of other non-herbivorous sh species demonstrating that M. ancietae is an e cient converter of feed into protein with no sh feed biomass needed.

Conclusions
Mycohistology of the stomach contents of M. ancietae allowed us to identify the components of the diet; this method also allowed us to obtain a reference pattern based on the epidermis of collected aquatic plants. The results of our study show that the M. ancietae sh of Northern Chile is an herbivore species. Adult sh of the M. ancietae diet consists of more than 90% of macroalgae, mostly of Lessonia bertorana, Glossosphora kuntii, and Corallina o cinalis var. chilensis. Medialuna eating habits are rich in carbohydrates and low in protein content. Minerals are of great importance in the sh's diet, especially in the rst months of the year.
Compared to non-herbivorous sh our results showed that most of the nutrients present in the Medialuna diet are found at much lower levels, including EPA and DHA (49.7%) and protein (13-60%). Most fed aquaculture non-herbivorous species rely on wild-captured sh for these essential nutrients, while M. ancietae can obtain and has the ability to concentrate them from potentially cultivable macroalgae.
The requirements for protein, fat and long-chain polyunsaturated fatty acids for M. ancietae are low and completely sh-free compared with non-herbivorous sh species that have been studied. Analyses of the eating habits of M. ancietae and the nutritional quality of its meat allow us to conclude that this species has potential for use in the development of sustainable aquaculture by easing pressure on existing wild sh stocks and contribute to nutrition security in North of Chile. Figure 1