DOI: 10.5061/dryad.x95x69ps2
title: Linking anatomical and histological traits of the digestive tract to resource consumption and assimilation of omnivorous tetra fishes

description: This study explores the interplay between digestive tract traits, food
 intake, and assimilation in omnivorous tetra fishes (Psalidodon
 bifasciatus, P. aff. gymnodontus, and Bryconamericus ikaa) from the Iguaçu
 River basin, an ecologically significant region known for high endemism.
 We hypothesize that variations in digestive tracts across species would
 associate with differences in diet, isotopic composition in fish tissues,
 and overall diet assimilation. To test this, we employed stereoscopic and
 light microscopy to characterize the gross anatomy, histomorphology, and
 histochemistry of fish digestive tracts. Additionally, the study used
 stomach content and stable isotope analyses to trace fish diets. While
 these tetra fishes shared histological structures, disparities were noted
 in anatomical digestive traits and diet preferences. The smallest species,
 B. ikaa, with a shorter intestine, had fewer pyloric caeca and primarily
 consumed animal-based diets. Conversely, P. bifasciatus and P. aff.
 gymnodontus, with longer intestines, displayed numerous pyloric caeca and
 consumed a balanced mix of animal and plant items. Despite anatomical and
 dietary differences, all three species predominantly assimilated
 animal-origin food. Histological variations were found among digestive
 tract segments, with the esophagus having the thickest muscular layer,
 gradually thinning towards the posterior intestine. The final portion of
 the intestine exhibited a significant expansion in lumen perimeter, while
 the esophagus had the smallest lumen area. Goblet cells were most
 concentrated in the posterior intestine for all species. The gross anatomy
 of these tetra fishes aligns with their ingestion of animal or plant
 diets, while diet assimilation is dominated by animal-origin food. These
 findings provide crucial insights into the structural and tissue
 characteristics of their digestive systems, laying the groundwork for
 deeper exploration into the physiological aspects of their digestive
 tracts and enhancing our understanding of their feeding strategies.
description: <strong>Model species and sampling</strong>
 We studied three sympatric omnivorous tetra fish from two
 headwater streams in the lower Iguacu River basin: <em>Psalidodon
 bifasciatus</em> (Garavello & Sampaio 2010),
 <em>Psalidodon</em> aff. <em>gymnodontus</em>
 Eigenmann 1911, and <em>Bryconamericus ikaa</em> Casciotta,
 Almirón & Azpelicueta 2004. We sampled the fishes in July and
 December 2017 using electrofishing with three passes of 40 minutes. First,
 we anesthetized the specimens in eugenol and measured the standard length
 and weight. We then visually inspected the presence of mature gonads of
 all sampled fishes and selected adult specimens of each species for
 further analysis. For the histological analysis, during each sampling
 period, we selected five individuals of each species from each stream. We
 dissected their digestive tracts with a longitudinal incision along the
 ventral region. Then, we fixed the digestive tract in ALFAC (alcohol 80%,
 85 mL; formaldehyde PA, 10 mL; and acetic acid PA, 5 mL; Caputo et
 al<em>.</em>, 2011) and conserved in 70% alcohol. We also
 fixed additional specimens in formaldehyde 10% and conserved them in 70%
 alcohol for stomach content analysis. Finally, for stable isotope
 analysis, in the summer (December 2017), we dissected samples of dorsal
 muscle tissue from 10 to 15 adult specimens per species and stream.
 Following the methodology from Neves et al. (2021, 2023), we manually
 sampled basal resources (terrestrial plants and sedimentary organic matter
 – SOM) and putative prey (aquatic and terrestrial invertebrates). We
 immediately stored all samples on ice for further processing in the
 laboratory. We collected the fishes under authorization
 from the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio)
 (license number 25039). The project was ethically and methodologically
 approved by the Ethics Committee on Animal Use of the Universidade Federal
 do Rio Grande do Sul (CEUA – 32734). To comply with their protocols for
 fish usage, we deposited the voucher specimens in the fish collection of
 the Departamento de Zoologia at the Universidade Federal do Rio Grande do
 Sul (P<em>. bifasciatus</em> UFRGS 26235;
 <em>P.</em> aff. <em>gymnodontus</em> UFRGS 25725;
 <em>B. ikaa</em> UFRGS 26246).
 <strong>2.2 Stomach content and stable isotope
 analyses</strong> To estimate stomach contents,
 we analysed 244 adult fish specimens (<em>B. ikaa</em>: 31;
 <em>P.</em> aff. <em>gymnodontus</em>: 96;
 <em>P. bifasciatus</em>: 117). We carefully extracted the
 stomachs of the fishes under optical and stereoscopic microscopes (Opton
 TIM-2B WF10X) to identify the stomach contents with the highest possible
 taxonomic precision. For the identification of algae we used specialized
 literature by Bicudo & Bicudo (1970), while for invertebrates, we
 used Mugnai et al. (2010). We applied the volumetric method to quantify
 the food items (Hyslop, 1980) with graduated test tubes and glass counting
 plates (Hellawell & Abel, 1971). For stable
 isotope analysis, we examined a total of 55 fish specimens (<em>B.
 ikaa</em>: 10; <em>P.</em> aff.
 <em>gymnodontus</em>: 15<em>; P. bifasciatus</em>:
 30, Table S7). Muscle samples were first washed with distilled water, then
 lyophilized and homogenized with a mortar and pestle. We then stored all
 samples in 2 mL Eppendorf tubes until they were weighed into tin capsules,
 with each capsule containing approximately 1.6 ± 0.2 mg of dried animal
 tissue. Similarly, we washed all basal resources and prey items with
 distilled water. We then lyophilized and homogenized aquatic and
 terrestrial invertebrates (separated by taxonomic groups), and basal
 resources (Table S7). The small aquatic insects, such as Ephemeroptera,
 Chironomidae, Coleoptera, and Hymnoptera, underwent complete maceration.
 Larger prey items, including shrimps and crabs (<em>Aegla</em>
 sp.) were freeze-dried and their muscle tissue was macerated. We preserved
 all specimens in 2 mL Eppendorf tubes before measuring and transferring
 them into tin capsules (1.6 ± 0.2 mg of dry animal tissue and 3.6 ± 4.2 mg
 of basal resources). We carried out the analysis of nitrogen
 (<sup>15</sup>N/<sup>14</sup>N) and carbon
 (<sup>13</sup>C/<sup>12</sup>C) stable isotope
 ratios at the Center for Nuclear Energy in Agriculture (CENA) at the
 University of São Paulo, Brazil. To determine the stable isotope ratios,
 CENA used a mass spectrometer system operating in continuous-flow
 (CF-IRMS) mode. This system was fit with a Carlo Erba elemental analyzer
 (CHN 1110) connected to a Delta Plus mass spectrometer (Thermo
 Scientific). The results of the stable isotope analysis were presented in
 the delta notation, which represents the deviation of stable isotope
 ratios (<sup>13</sup>C:<sup>12</sup>C and
 <sup>15</sup>N:<sup>14</sup>N) from universal
 standards. Specifically, the carbon ratios were compared to the PDB
 limestone standard, while the nitrogen ratios were compared to atmospheric
 nitrogen. For the analysis of fish muscle δ<sup>13</sup>C, we
 did not correct lipids due to the low C:N ratios observed (below 3.5),
 indicating negligible lipid content in the samples (Hoffman et al.,
 2015). <strong>Gross
 anatomy</strong> To investigate the gross
 morphology of the digestive tract of tetra fishes, we measured the
 intestinal length with digital calipers (accuracy of 0.01 mm) and counted
 the number of pyloric caeca of a total of 55 adult fish specimens
 (<em>B. ikaa</em>: 10; <em>P.</em> aff.
 <em>gymnodontus</em>: 15; <em>P. bifasciatus</em>:
 30). We computed the intestinal coefficient (IC) using the Hynes (1950)
 model, following the formula IC = IL/SL, where IL denotes the length of
 the intestine in mm, while SL denotes the standard length, also measured
 in mm. We photographed anatomical and macroscopic characteristics of the
 digestive tract of species using a Multipurpose Zoom Microscope Nikon
 AZ100M. <strong>Histology and
 histochemistry</strong> To describe the
 microscopic anatomy and histology of the digestive tract of tetra fishes,
 we examined a total of 40 specimens (<em>B. ikaa</em>: 10;
 <em>P.</em> aff. <em>gymnodontus</em>:
 10<em>; P. bifasciatus</em>: 20). We used the ALFAC-fixed
 material and selected tissue fragments from the esophagus, stomach, and
 three portions of the intestine (anterior, middle, and posterior; Figure
 1) of species and washed with 70% ethanol to remove the food items. These
 tissue fragments were then dehydrated in graded ethanol solutions and
 embedded in historesin (Leica®). We then made transverse and longitudinal
 histological sections (2–3 µm) with a Leica microtome (model RM 2145).
 Next, we stained the histological sections with 1% toluidine blue (TB),
 periodic acid Schiff (PAS) with alcian blue (AB -pH 2.5), and Masson’s
 trichrome staining (MT). We used PAS+AB to detect acid and neutral mucins
 (Cao & Wang, 2009). Lastly, we used Masson’s trichrome staining to
 better visualize the collagen fibers. We observed all sections using a
 BX60 Olympus microscope and recorded the images using an Olympus DP71
 digital camera and DP Controller 3.2.1.276 software.
 For a quantitative examination of the digestive tract traits, we
 took three histological photographs of each segment from five individuals
 per species. Following the methodology outlined by Bellinate et al. (2022)
 and Curvo et al. (2020), we used histological photographs and Fiji-ImageJ
 software (Schindelin et al., 2012) to determine the number of goblet
 cells, thickness of the muscle layer, and the perimeter and area of the
 lumen, as a proxy for the absorption surface. We analysed these variables
 only for the esophagus and intestine segments, as the degree of stomach
 fullness (i.e., full <em>vs</em> empty) could cause
 measurement bias. Because goblet cells completely made up the esophageal
 epithelium, we focused on accounting for this cell type in the anterior,
 middle, and final segments of the intestine. We counted the number of
 goblet cells by randomly choosing four microvilli in each segment of the
 intestine. We standardized the measurements in micrometers using the
 scales of the histological photographs.  
description: # Linking anatomical and histological traits of the digestive tract to
 resource consumption and assimilation of omnivorous tetra fishes
 [https://doi.org/10.5061/dryad.x95x69ps2](https://doi.org/10.5061/dryad.x95x69ps2) We have submitted our raw data (**Gross_anatomy_data.exe; Histological_data.exe; Stable_isotopes_data.exe; and Stomach_content_data.exe**) and R script (**Neves_statistical_analysis.R**). ## Description of the data and file structure The data is organized in the following spreadsheets: **Gross_anatomy.xlsx**: contains the standardized size (mm) of the species, intestinal length (mm), intestinal coefficient and number of pyloric caeca. * *ID*: specimen identification number * *Species*: code for species name (Bik: *Bryconamericus ikaa*;  Pbi: *Psalidodon bifasciatus*; Pgy: *Psalidodon aff.* gymnodontus) * *Stream*: stream where the individual was collected (S1 or S2) * *Standart_length*: standard length of the individual (mm) * *Intestine_length*: intestine length of the individual (mm) * *Intestinal_coeficient*: intestinal coefficient of the individual - IC = IL/SL, where IL denotes the length of the intestine in mm, while SL denotes the standard length, also measured in mm * *Number_of_pyloric_ceca*: number obtained from counting pyloric caeca **Histological_data.xlsx:** contains the thickness (one), perimeter (one), and area (one) of the muscular layer and number of goblet cells in each segment of the digestive tract of the species studied. * *ID*: specimen identification number and species  (Bik: *Bryconamericus ikaa*;  Pbi: *Psalidodon bifasciatus*; Pgy: *Psalidodon aff.* gymnodontus ) * *Species*: code for species name (Bik: *Bryconamericus ikaa*;  Pbi: *Psalidodon bifasciatus*; Pgy: *Psalidodon aff.* gymnodontus  * *Segment*: segment of the digestive tract (Esophagus; II: initial intestine; IM: middle intestine; IF: final intestine) * *Muscular_layer*: muscle layer measured in micrometers * *Permiter_lumen*: perimeter of the intestinal lumen measured in micrometers * *Area_lumen*: area of ​​the intestinal lumen measured in square micrometers **Stable_isotopes_data.xlsx:** contains the d13C and d15N isotopic compositions of each species, food resources and TDF per stream sampled. * Sheet S1_consumers: stable isotopes values for fish species in stream S1 * *Species_code*: species code (1: *Psalidodon bifasciatus*; 2: *Psalidodon aff.* gymnodontus * *Species*: code for species name (Pbi: *Psalidodon bifasciatus*; Pgy: *Psalidodon aff.* gymnodontus) * *d13C*: carbon 13 delta value * *d15N*: nitrogen 15 delta value * Sheet S1_resources: stable isotopes values for food resources in stream S1 * *Sources*: sources code (Animal; Plant, SOM: sedimentary organic matter) * *d13C*: carbon 13 delta value * *d15N*: nitrogen 15 delta value * Sheet S1_TDF: trophic discrimination factor (TDF) values for food resources in stream S1 * *Sources*: sources code (Animal; Plant, SOM: sedimentary organic matter) * *Meand13C*: average value of  carbon 13 delta value * *SDd13C*: standard deviation of carbon 13 delta value * *Meand15N*: average value of nitrogen 15 delta value * *SDd15N*: standard deviation of nitrogen 15 delta value * Sheet S2_consumers: stable isotopes values for fish species in stream S2 * *Species_code*: species code (1: *Psalidodon bifasciatus*; 2: *Bryconamericus ikaa*) * *Species*: code for species name (Bik: *Bryconamericus ikaa*;  Pbi: *Psalidodon bifasciatus*) * *d13C*: carbon 13 delta value * *d15N*: nitrogen 15 delta value * Sheet S1_resources: stable isotopes values for food resources in stream S2 * *Sources*: sources code (Animal; Plant, SOM: sedimentary organic matter) * *d13C*: carbon 13 delta value * *d15N*: nitrogen 15 delta value * Sheet S1_TDF: trophic discrimination factor (TDF) values for food resources in stream S2 * *Sources*: sources code (Animal; Plant, SOM: sedimentary organic matter) * *Meand13C*: average value of  carbon 13 delta value * *SDd13C*: standard deviation of carbon 13 delta value * *Meand15N*: average value of nitrogen 15 delta value * *SDd15N*: standard deviation of nitrogen 15 delta value **Stomach_content_data.xlsx:** contains the volumes of food resources consumed by each species in each stream sampled. * *Site*: stream where the individual was collected (S1 or S2) * *ID*: code for species name (Bik: *Bryconamericus ikaa*;  Pbi: *Psalidodon bifasciatus*; Pgy: *Psalidodon aff.* gymnodontus) * *Undertemined*: volume (mm3) of indeterminate food items observed in the individual's stomach contents * *Plant*: volume (mm3) of plant food items observed in the individual's stomach contents * *Animal*: volume (mm3) of indeterminate food items observed in the individual's stomach contents ## Code/Software The script contains the statistical analyzes carried out with the data set: ANOVA, ANCOVA, PERMANOVA, Mixing models and graphs.