Collection of fish
M. ancietae is an overexploited species. Therefore, its fishing is difficult, and catches are not always successful. Fishing trips were made during 2019. Fishes were caught by free diving using a modified harpoon off the shore of Pisagua (Latitude: 19°00´/20°00´ and Longitude: 69°00´/70°30´), Chile. Morphometric measurements were obtained from each fish, 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, certificate UNAP/VRIIP Nº003/2018. This study is reported in accordance with ARRIVE guidelines [17].
Collection of reference patterns and Stomach Content Analysis
From the components commonly observed in the diet by divers in the area of individuals of this species, comparison patterns of epidermis were obtained, organisms were collected manually in the intertidal zone and by diving off the coasts of Pisagua (Latitude: 19°00´/20°00´ and Longitude: 69°00´/70°30´) and Iquique (Latitude: -20.2167, Longitude: -70.15 20°13′0″ South, 70°9′0″ West). The macroalgae species collected were Macrocystis integrifolia, Lessonia berteroana, Corallina officinalis var. chilensis., and Glosophora kunthii.
The samples were dried in the field and fixed 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 identified. Subsequently, the epidermal tissue was removed from the macroalgae, following the diafanization methodology [16] and the sodium bicarbonate method [19].
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 inflammations 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 five 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 magnification of 40× were taken to the visualization of its histological characteristics. Descriptions and illustrations of the records were made by consultation in bibliographic references [20–23]. 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 fish died at the time of capture, so it was not necessary to apply euthanasia. Stomach samples from the caught fish 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
Macroalgae and Medialuna a. were collected manually in the intertidal zone and by diving off the coasts of Pisagua (Latitude: 19°00´/20°00´ and Longitude: 69°00´/70°30´) and Iquique (Latitude: -20.2167, Longitude: -70.15 20°13′0″ South, 70°9′0″ West). The macroalgae species collected were Macrocystis integrifolia, Lessonia berteroana, Corallina officinalis var. chilensis., and Glosophora kunthii.
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 Official Analytical Chemists (AOAC): fatty acid profile [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.