Diet and histomorphological study of the gastrointestinal system of Melanophryniscus klappenbachi (Anura: bufonidae)

Arias, A.M.; Larrea, D.D.; Céspedez, J.A.; F.Quintana, C.; Olea, G.B.: Diet and histo­ morphological study of the gastrointestinal system of Melanophryniscus klappenbachi (Anura: bufonidae). Rev. Vet. 32: 2, 131-137, 2021. The diet and histomorphology of the digestive tract of Melanophryniscus klappenbachi were determined by the analysis of adult and juvenile specimens collected in a private field in Bermejo department, Chaco, Argentina. The sampling was carried out monthly from March to June 2015. 29 specimens were col­ lected, measured, sexed, and dissected for the obtainment of the digestive content and histological samples. The results showed a high tendency towards myrmecophagy; more than 95% of the recovered prey items were identified as ants. The histomorphology of the gastro­ intestinal system consisted of the main four layers of tissue observed in vertebrates: mucosa, submucosa, muscular, and serosa. This study constitutes a contribution to the knowledge of biology and the natural history of anurans of the Bufonidae family, especially the genus Melanophryniscus, which currently receives a great amount of attention regarding its conservation status.


INTRODUCTION
Amphibians have a significant role as a study model in physiology, and this group is a standard model for the study of many biological processes. However, relatively few studies have been carried out on the amphibian gastrointestinal tract 16 . Some reports are investigating the gastrointestinal epithelial transformation from the larval to adult type and apoptosis 21 22 . Additionally, histological and histochemical changes in the digestive tract of Ceratophrys ornata and Xenopus laevis during metamorphic climax were reported 18 . In general, studies related to the digestive system were carried out on reptiles, birds, and mammals. But the cell renewal system of the adult epithelium in the amphibian stomach is similar to the mammalian stomach, where cell proliferation is localized in the neck region of gastric glands. Therefore, the amphibian diges tive tract serves as a model system for studying mammalian organ regeneration. Nonetheless, there are few studies about the structure of the gastrointestinal tract in amphibians 1A 8 16 20 25 27 31 34. The genus Melanophryniscus, is distributed from the center and southeast of Brazil, south of Bolivia, Par aguay, Uruguay, and the north and center of Argentina (Bol.  to the savannas of Bue nos Aires 23. There are brief descriptions of the diets of Melanophryniscus moreirae and M. stelzneri. There is also a description of the diet of M. montevidensis 32 , a Uruguayan species of the genus, that relates the ant content in the diet to the production of skin alkaloids 9 .
Some authors described the diet of three populations of M. rubriventris 4 in Argentina. Regionally, described and analyzed the diet ofM. cupreuscapularis in comparison to other species 15 16 . For M. klappenbachi, there is knowledge about the main alkaloids present in the species skin and a general description of its diet 11 . Due to the lack of studies on the digestive system of anurans, particularly those that relate its structure with dietary habits, this work aimed to study the diet and the histomorphological structure of the gastrointestinal tract of M. klappenbachi with the hope of contributing to the natural history of anurans in general and specially bufonids of this genus.

Collection
We collected 29 adult and juvenile specimens of M. klappenbachi of March a June 2015 in a private field located 20 km from the entrance of the "Las Palmas" locality, in Bermejo department, Chaco province, Argen tina. We added the specimens to the Herpetological Collection of the National University of the Northeast (UN-NEC), numbered as follows: UNNEC 13081 a 13109.
The site is characterized by remnants of Spinello and algarrobillo woodlands associated with palm trees and grasslands with flood zones, typical of Oriental Semihumid Chaco. The field is near the Paraná River, surrounded by a degraded forest.
The methodology consisted of "Surveys at breeding sites" 32 , with monthly surveys carried out between March and June 2015. We took the samples manually, without distinction of sex or size. We transferred the specimens to the laboratory in plastic bags and dampened containers.
We euthanized the specimens using benzocaine hydrochloride 250 mg/l, standard IACUC protocol, and CICUAL Fmed-UNNE 004/18. Then, we fixated the specimens with Bouin solution and preserved them in 10% formaldehyde. We measured each specimen with a digital caliper (0.01 mm accuracy); the measures taken were: snout-vent length (SVL) and mouth width (MW).

Anatomical analysis
We performed an incision on the ventral region and extracted the complete digestive tract. We measured the tract using a digital caliper (0.01 mm accuracy). The measures taken were the width and length of the stomach and intestines and the total length of the digestive tract (TLD).
Later, we fixated the tracts with Bouin solution and preserved them in 10% buffered formaldehyde. Then, we dehydrated the samples, included them in paraffin, cut them in 6 ^m sections and stained them with hematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Gómóri trichrome stains. We observed and photographed the samples with a Leica trinocular microscope (DME model). We determined the sex of each specimen by direct observation of the gonads. Then, we analyzed the digestive content under a stereomicroscope.
We dissected the specimens and cut their digestive tracts for observation under a Scanning Electron Microscope (SEM). We dehydrated, dried, and metalized the sections with gold.
We carried out the observations with a JEOL JSM-5800 LV microscope belonging to the Scanning Electron Microscopy Service of the General Secretariat of Science and Technology (SGCyT) of the National University of the Northeast (UNNE).

Data analysis
We identified each prey item to the lowest possible taxonomic level. We calculated the trophic diversity of the species using the Shannon-Weaver diversity index 33 . We compared the size of the specimens in terms of SVL, MW, and TLD between males and females using the Mann-Whitney U Test. We determined the mean number of prey per stomach for the complete sample and each sex.
We determined the Spearman (r) correlation coefficient for SVL-MW, SVL-TLD, and SVL-number of prey per stomach. We carried out every analysis using the statistical analysis programs Past 19 and Infostat 12 and the spreadsheet Microsoft Excel.

Diet
We captured a total of 29 specimens of March a June 2015. We only considered adult specimens (19 females, 8 males) with identifiable stomach or intestine content. In the case of juvenile specimens, only their histomorphology was taken into account since they did not present gastro-intestinal content at the time of cap ture.
The diet consisted of 737 items classified into 6 prey categories (Table 1) and was numerically dominated by ants. Additionally, ants were the most frequent prey category, found in all analyzed specimens (100 %).
The trophic diversity was 0.43, according to the Shannon-Weaver index (0.48 for females, 0.24 for males). We found no significant differences between the diets of males and females of this species (Mann-Whitney U Test = 97.00; n = 27; P = 0.47).

Histomorphology
The histological structure of the digestive tract in both juveniles (Fig. 2, 4 and 5) and adults (Figs. 3 and 6) is composed of the typical layers observed in vertebrates: mucosa, submucosa, external muscular and serous (Figs. 2 A and 3 A).

Stomach
In the stomach, juveniles, and adults, the mucosa had gastric folds and a simple layer of epithelium lining composed of mucous cells in the spine. It has gastric glands located at the base of the barrel-shaped grooves called gastric fossae ( Fig. 2 A and 3 B). The apical domain of epithelial cells was found to be acidophilous and exhibited a strong positive reaction for PAS ( Fig.  2 D, 3C).
The mucosa is separated from the submucosa by a muscular layer of the mucosa. The gastric glands were structured as simple tubular glands, often branched to the lower portion of the gland and composed of mucous cells of the neck, located mainly in the neck region ( Fig. 2 C, 3 B and D). Through SEM, it was possible to observe the gastric ostioles. Superficial mucous cells, such as neck cells, showed bright purple staining after treatment with PAS, indicating the presence of glycoproteins.
The submucosa was formed by loose connective tissue rich in blood vessels, as evidenced by strong staining after treatment with Gómóri trichrome stain ing; however, we did not find glands in this region. The external muscle was also made of smooth muscle tissue. The serosa was observed as a thin layer of loose connective tissue with some blood vessels, covered with mesothelium.

Intestine
The mucosa of the intestine (thin and thick) of both juveniles and adults showed several high longitudinal folds called intestinal villi (Fig. 5 E). These villi were composed of columnar enterocytes with microvilli on their apical surface (Fig. 4 B, 5 E). We also found a large number of goblet cells in this epithelium.
These cells showed strong staining after treatment with PAS ( Fig. 4 and 6 D), indicating the presence of glycoproteins, a product of the secretion of these unicellular glands. The submucosa was composed of loose connective tissue with some vascularization. The muscle showed moderate development in the sections. The outermost layer, the serosa, was composed of a thin layer of connective tissue.
The difference between juveniles and adults in the intestine was given at the level of abundance of goblet cells, the number in the region of the large intestine of the juveniles being greater, while in adults, that difference was not evident (Figure 4 and 6).

DISCUSSION
The analysis carried out in this study revealed that in M. klappenbachi, the preponderance of hymenopterans of the family Formicidae is significant, constituting more than 95% of the prey sample. These ants belonged to three species of the subfamily Myrmicinae. These taxa were followed in abundance by mites.
Analyzed feces from M. montevidensis, revealing the presence of ants and mites 32 . Observed the pre ponderance of ants in the diets of M. cupreuscapularis  15 . Finally, in 2013 carried out a study about the diet of M. devincenzii, also showing the predominance of mites followed by ants 5 . Taking into account numerical frequencies, we consider M. klappenbachi an ant specialist. Matching the results from numerous previous studies that maintain that specialist foragers use active search for the capture of their prey and prefer small and hard prey like ants and beetles 24 , we consider M. klappenbachi an active predator since these toads pursue their prey and demonstrate a clear tendency towards myrmecophagy.
In addition to the analyzed prey, there was also plant material found in the alimentary tracts, but this was not included in the study since its ingestion was deemed accidental. Observed that a certain amount of the stomach contents of Bombina variegate (29.82%) consisted of plant fragments and suggested, too, that they were eaten by accident 19. The same accidental ingestion was postulated for M. devincenzii 5 . Some authors considered that the ingestion of plant fragments is helpful for anurans in terms of gut parasites elimination and nutritional and even hydric contribution 3 . Studies regarding the intersexual differences in the diets of anurans are scarce due to the difficulty of capturing females of many species 27 . This variation between sexes could reflect the different requirements of each sex 13 29 or different foraging strategies 6 since males need more energy for vocalization and territorial defense, and females need energy for egg production during the breeding season 30 . In the present work a significant difference between both sexes could not be found.
The gastrointestinal tract showed the typical structure observed in vertebrates; in the same way, the typical cell and mucosa arrangements were observed. Some authors have presented similar results in Rhinella icterica 28 and Hyla orientalis 1 . These results provide a basis for the analysis of the gastrointestinal histomorphology and leave an open path to a more specific study regarding each region of the digestive system. The PAS staining was positive for the gastric glands and apical portions of the mucous stomach cells, and the goblet cells present in the intestines, due to the pres ence of glycoproteins. These results concur with previous observations in other amphibians, Triturus carnifex 27, Bufo viridis 26, Hyla orientalis ', Rana aurora 17 and in some fish 12 reptiles 7 and mammals 35 . These glycoconjugates are of great importance since they possess a significant role that includes maintaining the moisture of the tissues, lubrication, and reduction of mechanical friction, including the friction produced by feces when leaving the alimentary canal 1 , and the protection against chemicals and pathogens.
We consider that the data provided by this work is of importance as a basis for future studies that include both the trophic analysis of M. klappenbachi and its role in the obtainment and presentation of skin alkaloids 10 , not only for this species but for other anurans of the genus Melanophryniscus as well.
This work also characterizes the general histological structure of the gastrointestinal tract of the species and aims to serve as a foundation for future studies, both comparative and specific.