Linking anatomical and histological traits of the digestive tract to resource consumption and assimilation of omnivorous tetra fishes

Description

Notes

Methods

Model species and sampling

We studied three sympatric omnivorous tetra fish from two headwater streams in the lower Iguacu River basin: Psalidodon bifasciatus (Garavello & Sampaio 2010), Psalidodon aff. gymnodontus Eigenmann 1911, and Bryconamericus ikaa 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., 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. bifasciatus UFRGS 26235; P. aff. gymnodontus UFRGS 25725; B. ikaa UFRGS 26246).

2.2 Stomach content and stable isotope analyses

To estimate stomach contents, we analysed 244 adult fish specimens (B. ikaa: 31; P. aff. gymnodontus: 96; P. bifasciatus: 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 (B. ikaa: 10; P. aff. gymnodontus: 15; P. bifasciatus: 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 (Aegla 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 (¹⁵N/¹⁴N) and carbon (¹³C/¹²C) 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 (¹³C:¹²C and ¹⁵N:¹⁴N) 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 δ¹³C, 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).

Gross anatomy

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 (B. ikaa: 10; P. aff. gymnodontus: 15; P. bifasciatus: 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.

Histology and histochemistry

To describe the microscopic anatomy and histology of the digestive tract of tetra fishes, we examined a total of 40 specimens (B. ikaa: 10; P. aff. gymnodontus: 10; P. bifasciatus: 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 vs 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.