Morphology and Histochemistry of Infralabial Glands of Two Species in the Slug-Eating Family Pareidae (Reptilia: Serpentes)

Abstract

Pareidae and Dipsadidae, two independently evolved taxa in the Serpentes lineage, both feed exclusively on terrestrial mollusks (snails and slugs). Dipsadid snakes developed hypertrophic infralabial glands in their lower jaw, which are thought to be associated with their specialized feeding behaviors. However, whether a similar gland exists in pareid snakes is unknown. In this study, we examined the morphological characteristics of the infralabial glands in Pareas berdmorei and Pareas chinensis based on comparative anatomical, histochemical, and histopathological analysis. Our results demonstrated that both Pareas species had similar hypertrophied infralabial glands in the lower jaw, which consisted of tubules with mucinous properties and seromucous acini. The secretory granules of the seromucous cells also showed high electron density. The cytoplasm was rich in rough endoplasmic reticulum, mitochondria, and Golgi apparatus, suggesting that these cells may secrete protein substances, and play an important role in digesting mollusks. This study provides evidence of morphological convergence between Pareidae and Dipsadidae due to specialized diet adaptation, which will be the foundation for prospective functional research.

Headnote

Abstract Pareidae and Dipsadidae, two independently evolved taxa in the Serpentes lineage, both feed exclusively on terrestrial mollusks (snails and slugs). Dipsadid snakes developed hypertrophic infralabial glands in their lower jaw, which are thought to be associated with their specialized feeding behaviors. However, whether a similar gland exists in pareid snakes is unknown. In this study, we examined the morphological characteristics of the infralabial glands in Pareas berdmorei and Pareas chinensis based on comparative anatomical, histochemical, and histopathological analysis. Our results demonstrated that both Pareas species had similar hypertrophied infralabial glands in the lower jaw, which consisted of tubules with mucinous properties and seromucous acini. The secretory granules of the seromucous cells also showed high electron density. The cytoplasm was rich in rough endoplasmic reticulum, mitochondria, and Golgi apparatus, suggesting that these cells may secrete protein substances, and play an important role in digesting mollusks. This study provides evidence of morphological convergence between Pareidae and Dipsadidae due to specialized diet adaptation, which will be the foundation for prospective functional research.

Keywords convergent evolution, histology, Pareas berdmorei, Pareas chinensis, specialized diet

1.Introduction

Snakes display exceptionally diversified feeding habits. Some groups consume a wide variety of animals including both vertebrates and invertebrates, while others exhibit strong dietary specialization (Greene, 1997; Lillywhite, 2014; Zug, 1993) such as crustacean-eating snakes, egg-eating snakes, and ant/termite-eating blind snakes (Scolecophidia) (Broadley, 1979; Coleman et al, 1993; Jayne et al., 2018; Webb et al., 2000). Many snakes within the family Dipsadidae are considered specialists, feeding solely on terrestrial mollusks, e.g., snails of the genus Drymaeus and slugs of the genus Sarasinula (Cundall and Greene, 2000; Dunn, 1951; Peters, 1960; Sazima, 1989). Some genera within Dipsadidae have developed hypertrophied and specialized infralabial glands in their lower jaw. These glands produce secretory products that are predominantly serous in nature and may be related to the specialized mollusk diet (de Oliveira et al, 2014; de Oliveira et al, 2008; Zaher et al, 2014). Notably, injection of infralabial gland extracts in the visceral mass of snails can result in paralysis and, in some cases, death (Laporta-Ferreira and Salomāo, 1991; Salomāo and Laporta-Ferreira, 1994). For example, the infralabial glands of the neotropical slug-eating snake Sibynomorphus mikani contain a high protein content, and extract-injected slugs show an increase in mucus secretion, contortions, and immobilization of the body (Salomāo and Laporta-Ferreira, 1994). Thus, infralabial gland specialization in some genera of Dipsadidae may be useful for neutralization of mollusk secretions and later digestion.

Snakes in the Southeast Asian family Pareidae are also considered dietary specialists, preying exclusively on terrestrial snails and slugs (Cundall and Greene, 2000). The family Pareidae, which is widely distributed across South and Southeast Asia, currently consists of four genera: i.e., Pareas Wagler, 1830; Aplopeltura Duméril, 1853; Asthenodipsas Peters, 1864; and Xylophis Beddome, 1878 (Deepak et al., 2018; Wang et al., 2020; Zhao, 2006; Poyarkov et al., 2022). These snakes exhibit several morphological changes associated with specialized foraging behavior, such as asymmetry in the number of mandibular teeth (Hoso et al., 2007). Almost all pareid snakes, except for non-snail-eating specialists, have more teeth in the right mandible than the left for functional specialization in feeding on the dextral majority of land snails (Hoso, 2017). Fewer teeth in the left mandible may be useful for smoothly biting the sticky tissue of snails, while more teeth in the right mandible may be useful for firmly grasping (Chang et al, 2021; Hoso, 2017; Hoso et al, 2007). Specialization of skull morphology is also found in some pareid and dipsadid snakes, including short snout and pterygoids, reduced supratemporals, long mandibles, and complete separation of the posterior end of the pterygoid from the quadrato-mandibular joint. This connection makes the mandible more flexible, which can help when preying on terrestrial snails (dos Santos et al, 2017; Kojima et al, 2020). Although specialized feeding behavior and associated morphological changes are well documented in the family Pareidae (Danaisawadi et al, 2016; Götz, 2002), it remains unknown whether well-developed infralabial glands similar to those found in dipsadid snakes also exist in this Asian slugeating group.

In the current study, we investigated infralabial glands and associated structures in pareid snakes and correlated gland structure with secretion biochemistry and the peculiar feeding habits of this group. We focused on morphology, histochemistry, and arrangement of different cell types within the secretory units of the infralabial glands of two species of Pareas. This work will provide a foundation for further research on the functional mechanisms of these specialized glands.

2.Materials and Methods

2.1. Samples collection and processing Three individuals of P. berdmorei were collected from Mengla county, Xishuangbanna, Yunnan Province, China and three individuals of P. chinensis were from Mt. Emei, Leshan city, Sichuan Province, China in this work. An adult male of P. berdmorei (total length 620 mm) was used to predation experiment. The snake was starved for three days before the predation experiment, and the snake was then placed on a dissecting tray with dead leaves (415 cm length x 312 cm width x 35 cm height), three snails of equal size were placed in front of the snake. At a distance of one meter from the snake, we used the camera equipped in the laboratory to film the whole process of preying on the snail. On the basis of existing specimens in the laboratory, we selected snakes with different feeding habits. A total of other four families, including nine genera and 16 species were used for comparison of infralabial gland (Table 1). The specimens were euthanized via an intraperitoneal injection of sodium thiopental, and then we removed the entire head from the specimens at the first cervical vertebra and skinned off the head. The infralabial glands were dissected individually for histological and histochemistry study. All specimens in this work were preserved in the Herpetological Museum, Chengdu Institute of Biology (CIB), Chinese Academy of Sciences (CAS), Chengdu, Sichuan, China.

The entire head was fixed in 10% neutral formalin fixative solution for 24 h and subsequently submitted to decalcification in 4.13% aqueous EDTA, pH 7.2, renewed every other three days, and kept in constant stirring for a month. After head decalcification was completed, the entire head was divided into left and right parts from the middle, put the head in gradient ethanol for dehydration, and embedded in paraffin, and submitted to serial sagittal or transversal sectioning. We used pathology slicer of Shanghai Leica Instruments Co., Ltd, model RM2016 to obtain sections of 4 pm. Dissected infralabial glands were fixed in 4% paraformaldehyde with PBS 0.1 M, pH 7.2 for 24 h, and in gradient ethanol for dehydration, and embedded in paraffin, and conducted to serial sagittal or longitudinal sectioning. We used same pathology slicer to obtain glandular sections of 4 pm.

2.2. Histology and histochemistry All sections of the head were stained with hematoxylin-eosin (HE) staining for general study of the tissues. A group of three sections of infralabial glands were stained with hematoxylin-eosin (HE) staining for general study of the tissues. The remaining four groups glandular sections were used to following histochemical staining steps, the alcian blue (AB) pH 25, periodic acid-Schiff (PAS) and combined alcian blue (AB) pH 2.5 and periodic acid-Schiff (PAS) for identification of acid and natural mucosubstances, and Coomassie brilliant blue R250 for identification of protein (Kiernan, 2015).

2.3. Transmission electron microscopy Fresh tissues were selected to minimize mechanical damage such as pulling, contusion and extrusion. We used a sharp blade to quickly cut and harvest fresh tissue blocks within 1-3 min. The size of tissue block should be no more than 1 mm3. The removed tissue block was immediately put into 2.5% glutaraldehyde fixative solution for 24 h (Sabatini et al, 1963), which was fixed at 4 °C for preservation and transportation. Tissues were fixed in 1% OsO4 with PBS 01 M, pH 7.4 for 2 h at room temperature and kept from light, and then dehydrated in ethanol and embedded in resin (EMBed 812). The resin block was cut to 60-80 nm ultrathin sections on the ultramicrotome, 2% uranium acetate saturated alcohol solution avoid light and 2.6% Lead citrate avoid CO2 staining. The ultrathin sections are observed and recorded under TEM (Transmission Electron Microscope).

2.4. Photomicrograph acquisition Photomicrographs were obtained with a Cnoptec B302 microscope and this microscope equipped with a digital camera (Sony ICX285AQ CCD) and with Image acquisition software the ImageView in computer.

3.Results

3.1. Feeding behavior and presence of infralabial glands In this work, we observed the predatory processes and relevant behaviors of P. berdmorei through feeding experiments, demonstrating similar results to previous studies (Danaisawadi et al, 2016; Götz, 2002), i.e., pre-capture, feeding, and post-feeding stages (Figure 1). Based on comparative anatomy, our results showed that, compared with other snake species with different feeding habits, members of the genus Pareas had hypertrophied infralabial glands in the lower jaw (Figure 2).

The infralabial gland in P. berdmorei was whitish, extended, and hypertrophied. The gland was attached to the mandible, from the anterior tip of the dentary to the anterior portion of the compound bones, and the entire gland was wrapped and dispersed in muscle (Figure 3).

3.2. General morphology of infralabial gland We investigated the glandular structure, cell types, and physiological and biochemical properties of cell contents in P. berdmorei and P. chinensis using histological and histochemical methods. Results showed that the infralabial glands were wrapped in a thin layer of connective tissue, which divided the glandular body into acinar lobules of varying size in a mesh formation (Figures 4A, 5A). The acini were typically arranged with multiple polygonal cells, predominantly mucous and seromucous cells, with large ducts formed in the middle of the mucous cells (Figures 4A, 5A). The acini contained multiple ducts formed by long columnar mucous cells surrounded by seromucous cells, which were confined to the peripheral area of the mucous cells (Figures 4A, 5A).

In P. berdmorei, the long columnar mucous cells usually formed ducts with mucinous properties in the central region of the acini, which were characterized by cytoplasmic secretory granules and flat basal nuclei pressed to the edge of the cell by cellular content (Figure 4C, 4D). The seromucous cells were irregular polygonal cells with round basal nuclei and cytoplasm full of secretory granules (Figure 4B, 4D). Two different types of secretory granules were observed in the seromucous cells (sm1 and sm2). Seromucous cell type 2 (sm2) showed stronger reaction to periodic acid-Schiff (PAS) than seromucous cell of type 1 (sm1). The former was less abundant, while the latter was more abundant (Figure 4E, 4F).

In P. chinensis, the glandular body was mainly composed of mucous and seromucous cells, with different-sized lumina. The seromucous cells were more dominant than the mucous cells in the acini (Figure 5B-E). Columnar mucous cells formed different-sized lumina, with flat basal nuclei and cytoplasm full of secretory granules (Figure 5C, 5D). We observed two different types of mucous cells (i.e., m1 and m2) based on the staining properties of their secretory granules: i.e., cells with medium granules (m1) and cells with strong-stained granules (m2), indicating different mucosubstances (Figure 5D). The seromucous cells formed different-sized vesicles (v), with round basal nuclei and cytoplasm containing secretory granules. We also observed two different types of seromucous cells, i.e., the more abundant type 1 (sm1) and less abundant type 2 (sm2), similar to P. berdmorei (Figure 5E, 5F).

3.3. Histochemistry of infralabial gland In P. berdmorei, most secretory cells of the acini were positive to both periodic acidSchiff (PAS) and Coomassie brilliant blue R250, and negative to Alcian blue pH 2.5, indicating the seromucous nature of these cells (Figure 4B-E). The seromucous cells showed stronger positive reaction to PAS than the mucous cells, and the secretory granules in the sm2 cells showed a stronger positive reaction to periodic acid-Schiff (PAS) than the sm1 cells, indicating differences in protein content (Figure 4E, 4F). Duct and columnar mucous cells around the duct were strongly positive to Alcian blue pH 25 and negative to Coomassie brilliant blue R250, indicating their mucinous properties (Figure 4B, 4D).

In P. chinensis, most secretory cells were positive to both periodic acid-Schiff (PAS) and Coomassie brilliant blue R250, and negative to Alcian blue pH 25, similar to that observed in P. berdmorei (Figure 5B-E). The m2 cells showed stronger positive reaction to Alcian blue pH 2.5 than the m1 cells, indicating differences in mucosubstance content (Figure 5D).

3.4. Ultrastructure of infralabial gland The ultrastructure can reflect the subcellular components of secretory cells. Ultrastructural criteria are mainly based on the electron density of secretory granules, with high electron density indicating rich protein content (related to serous glands) and low electron density indicating poor protein and rich mucous content (related to mucous cells) (Kardong and Luchtel, 1986; Yoshie et al, 1982). The ultrastructure of the infralabial gland in P. berdmorei showed that the acini were predominantly composed of secretory granules with different electron densities and polygonal cells of different sizes. A large duct was formed in the middle of the secretory cells, and lumen-facing microvilli protuberances were found near the bottom of the secretory cells (Figure 6A). These microvilli protuberances may participate in exuding secretions to the outside of the cell. The electron density of the seromucous cells was higher than that of the mucous cells. The seromucous cells contained unique spherical or elliptical granules, which were more electron-dense and heterogeneous when compared to mucous cells, indicating differences in cell content between the cell types (Figure 6C). Mucous cells around the duct contained uniform secretory granules with medium electron density (Figure 6B), whereas the seromucous cells contained secretory granules of varying size with higher electron density. Cytoplasm of the perinuclear region of the seromucous cells was rich in mitochondria (mi) and rough endoplasmic reticulum (er), typical characteristics of protein-secreting cells. Desmosomes were found at the junction of adjacent cells (Figure 6D).

4. Discussion

Convergent evolution usually refers to the evolution of the same or similar phenotypes from independent lineages under similar selective pressure (Christin et al, 2010; Peng et al, 2022; Stern, 2013; Storz, 2016). In Serpentes, the South American dipsadid snakes (Dipsadidae) and Asian pareid snakes (Pareidae) have evolved independently to feed exclusively on terrestrial mollusks (snails and slugs) (Götz, 2002; Sazima, 1989; Zaher et al, 2019). The Pareidae family is one of the basal groups of Colubroidea, which split with other Colubroidea snakes in ~62.5 million years ago (Ma), whereas Dipsadidae is a recently evolved family, which diverged ~32.4 Ma (Li et al, 2020). Previous studies have indicated that snakes in Dipsadidae developed hypertrophied infralabial glands as an adaptation to their specialized feeding behaviors (de Oliveira et al, 2014; de Oliveira et al, 2008; Taub, 1966). Based on comparative anatomy, our results confirmed that hypertrophic infralabial glands similar to those found in dipsadid snakes also exist in Pareas, suggesting that these two groups evolved convergent phenotypes to target their specialized diets.

In Dipsadidae snakes that feed exclusively on mollusks, e.g., Dipsas and Sibynomorphus, the infralabial glands are primarily composed of mucous and seromucous cells (Dunn, 1951; Kochva, 1978; Taub, 1966). In contrast, in the dipsadid genus Atractus, which feeds predominantly on earthworms, the infralabial glands are primarily composed of mucous cells (de Oliveira et al, 2008). These results suggest that different dietary behaviors in these predators may cause differences in the composition of the infralabial glands. In our study, the two species of Pareas showed an infralabial gland composition similar to that of dipsadid snakes (de Oliveira et al., 2008), suggesting that the seromucous cells in the infralabial glands may be specialized to snakes that feed exclusively on terrestrial mollusks. In addition, based on ultrastructural analysis, the seromucous cells were rich in cell structures correlated with protein secretion, indicating that such cells may secrete abundant proteases to aid in the digestion of food (Ichikawa and Ichikawa, 1975; Yoshie et al, 1982). This is consistent with previous research reporting high protein content in infralabial gland extracts of the neotropical slug-eating snake S. mikani (Salomäo and Laporta-Ferreira, 1994). Therefore, we speculate that seromucous cells are unique in the infralabial glands of snakes that feed on terrestrial mollusks in both Dipsadidae and Pareidae, and these cells may be responsible for digesting mollusks.

Previous studies have suggested that the secretions produced by the glands are discharged to the outside through ducts (de Oliveira et al, 2016). Ducts inside glands and ducts leading to the oral cavity have been observed in neotropical goo-eating snakes (de Oliveira et al, 2014; Laporta-Ferreira and Salomāo, 1991). The former are receptacles for secretions generated by secretory cells, with the latter transporting these secretions to mediate physiological functions of the organism (Rosenberg, 1967). In our work, the mucous cells of the two Pareas species were only found in the ducts inside the glands, thus showing a similar distribution pattern as found in some dipsadid snakes (de Oliveira et al, 2014; de Oliveira et al, 2008). This pattern facilitates the transportation and release of secretions generated by glandular cells (Taub, 1967; 1966; Yoshie et al, 1982). However, we did not observe ducts leading the oral cavity, possibly due to wrong anatomical methods and sectional angles. Therefore, how seromucous cell secretions are transported through the surrounding ducts in Pareas needs to be further researched.

Acknowledgements We thank Junfeng GUO, Chenyang TANG and Hanliu AI for the fieldwork. We thank Zhongliang PENG for his help in the sampling work. We thank Dihao WU and Junjie HUANG for their help in taking the photographs and editing photographs, respectively. We also thank Zeng WANG for her help in reviewing the manuscript. This work was supported by the National Natural Science Foundation of China (32220103004 and 32200363); the International Partnership Program of Chinese Academy of Sciences (151751KYSB20190024); the Sichuan Science and Technology Program (2020YFH0005); the Key Research Program of Frontier Sciences, CAS (QYZDB-SSWSMC058); the Youth Innovation Promotion Association of CAS (2021370); CAS President's International Fellowship Initiative (2021VBA0003) (Hussam El Dine ZAHER).

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Received: 22 December 2021 Accepted: 17 February 2022

Handling Editor: Heling Zhao

How to cite this article:

Wang D., Ren J. L., Jiang K., Wu W., Peng C. J., Zaher H., Jiang D. C., Li J. T. Morphology and Histochemistry of Infralabial Glands of Two Spe-cies in the Slug-Eating Family Pareidae (Reptilia: Serpentes). Asian Herpetol Res, 2022, 13(3): 180-189. DOI: 10.16373/j.cnki.ahr.210071

Footnote

* Corresponding authors: Dr. Jiatang LI, from Herpetological Museum, Chengdu Institute of Biology (CIB), Chinese Academy of Sciences (CAS), Chengdu, Sichuan, China, with his research focusing on taxonomy, phylogenetics, biogeography, genomics and evolution of amphibians and reptiles; Dr. Dechun JIANG, from CIB, CAS, Chengdu, Sichuan, China, with her research focusing on phylogenetics, biogeography, and evolution of amphibians and reptiles.

E-mail: [email protected] (Jiatang LI); [email protected] (Dechun JIANG)

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