Light and Scanning Electron Microscope Examination of the Digestive Tract in Peppered Moray Eel, Gymnothorax pictus (Elopomorpha)

Authors


Correspondence to: Hideo Akiyoshi, Department of Biological Science, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan. Fax: +81-852-32-6440. E-mail: akiyoshi@life.shimane-u.ac.jp

Abstract

The morphology of the digestive tract of the peppered moray eel, Gymnothorax pictus (G. pictus) (Elopomorpha: Anguilliformes) was examined using both light and scanning electron microscopy. The digestive tract is composed of the esophagus, the stomach, and the intestines; pyloric caeca were absent. The stomach was divided into a cardiac region that was continuous with the esophagus, a body which terminated in a long blind sac, and a pyloric region that was continuous with the intestine. The short intestine possessed several partitions that were created by the mucosal folds within the posterior region. The terminal region of the stomach was characterized by the thick longitudinal muscularis and subserosa, and the gastric glands and microvilli were absent. Ciliary tufts of ciliated cells were observed on the surface of the partition-like mucosal folds within the intestinal wall. Acidic mucus was secreted throughout the digestive tract. It was suggested that the terminal region of the stomach is specialized for storage of large food items. In addition, it is possible that the partition-like mucosal folds within the intestine perform a function similar to that of the spiral valve and, and along with ciliated cells, facilitated digestion and absorption. The acidic mucus likely maintained surface epithelium pH and protease activity. Within a phylogenetic context, the absence of a pyloric caeca in G. pictus while possessing an intestine implies that this species is affiliated to groups that had branched off earlier than basal teleosts. Anat Rec, 296:443–451, 2013. © 2013 Wiley Periodicals, Inc.

INTRODUCTION

Teleosts are a phylogenetically unique group in that they form a bridge between invertebrates and vertebrates, as well as between animals living in aquatic and terrestrial habitats. Extant teleost species have undergone considerable adaptive radiation and the group's phylogeny is relatively diverse, making teleosts well suited to evolutionary studies. Teleosts are also the most diverse group of vertebrates and inhabit a wide range of habitats, from equatorial to polar regions, mountain streams to the ocean floor, and seawater to freshwater environments. In addition, the feeding habits and behavior of teleosts are similarly diverse.

The digestive system performs the essential role of ingesting, digesting, and absorbing nutrients from food. Being composed of a digestive tract and accessory glands, such as the liver, the digestive systems of teleosts are generally similar to those of other vertebrates (Akiyoshi and Inoue, 2004; Kimura, 2010; Wilson and Castro, 2010). The digestive tract of the majority teleosts is composed of an esophagus, a stomach, pyloric caeca, and an intestine (Hossain and Dutta, 1996; Kent and Carr, 2000; Romer and Parsons, 2007).

Current knowledge of the digestive system of species belonging to Elopomorpha has primarily been derived from studies on the Anguilliformes. The digestive tract of Anguilliformes such as Anguilla anguilla (Clarke and Witcomb, 1980; Domeneghini et al., 2005) and Anguilla japonica (Kimura, 2010) lacks pyloric caeca and is composed of an esophagus, a stomach, and an intestine. The Y-shaped stomach forms a blind sac (Clarke and Witcomb, 1980; Domeneghini et al., 2005; Kimura, 2010) and is divided into three regions; the cardia, fundus, and pylorus (Clarke and Witcomb, 1980; Domeneghini et al., 2005). The simple columnar mucosal epithelium in all three regions secretes both acidic and neutral pH mucus (Clarke and Witcomb, 1980; Domeneghini et al., 2005), and the muscularis is composed of an inner circular and an outer longitudinal layer (Clarke and Witcomb, 1980). The fundic region possesses gastric glands (Clarke and Witcomb, 1980; Domeneghini et al., 2005) and characteristic pentagonal mosaic pattern present on the luminal surface of the mucosal epithelium (Clarke and Witcomb, 1980). The pyloric region lacks any gastric glands (Clarke and Witcomb, 1980; Domeneghini et al., 2005) and has the thickest circular muscle layer of all gut regions (Clarke and Witcomb, 1980).

The intestine is almost straight and is separated from the rectum by the ileo-rectal valve (Clarke and Witcomb, 1980; Kimura, 2010). Mucosa within the intestine are composed of simple columnar epithelium cells and goblet cells that produce both acidic and neutral mucus (Clarke and Witcomb, 1980; Domeneghini et al., 2005). Ducts and microvilli can be found on the luminal surfaces of the epithelium, and the muscularis is composed of an inner circular and an outer longitudinal layer (Clarke and Witcomb, 1980).

Teleosts in the Osteoglossomorpha and Protacanthopterygii are referred to as the basal teleosts. Regarding the phylogenetic relationships between these superorders, some authorities consider that the Osteoglossomorpha branched first, followed by the Elopomorpha and Clupeomorpha (Patterson and Rosen, 1977; Inoue, et al., 2001). Other scholars consider that Elopomorpha branched first followed by Osteoglossomorpha (Arratia, 1997), or that Osteoglossomorpha and Elopomorpha branched at the same time to form sister groups (Lê et al., 1993). Interestingly, while the Anguilliformes lack pyloric caeca, they are present in both Osteoglossomorpha (Al-mahrouki and Youson, 1998) and Clupeomorpha, (Hossain and Dutta, 1996; Kimura, 2010) which are considered to have branched before and after Elopomorpha, which includes Anguilliformes, respectively. However, the underlying reason for this difference in the presence or absence of pyloric caeca between these phylogenetic groups is still unclear.

In this study, we examined the digestive tract of thepeppered moray eel, Gymnothorax pictus (G. pictus) (Ahl, 1789) (Muraenidae: Muraenoidei) by light andscanning electron microscopy (SEM) to reveal features of the stomach and intestine and to infer their function. We also examined the features within a phylogenetic context, with particular reference to the relationship between stomach and pyloric caeca. The findings of the study are considered to provide a foundation for future investigations on the digestive tract of Anguilliformes.

MATERIALS AND METHODS

The present study was approved by the animal ethics committee of Shimane University, and conducted in strict accordance with the guidelines for the care and use of research animals set out by the committee.

Sample Collection

The digestive tracts of twelve peppered moray eels, G.pictus, were collected for morphological examination. All of the eels were collected from the waters surrounding Iriomote Island, Okinawa Prefecture, Japan. To control for the influence of seasonal changes and growth, only adult-stage specimens were caught [total body length (TL) was 62.8±7.64 cm]. All specimens were caught using hand nets between the months of May and November from 2007 to 2012.

Tissue Preparation

Digestive tracts of each eel were perfusion-fixed via the heart with either 4% paraformaldehyde buffered at pH 7.4 with 0.1 M phosphate or 1.5% glutaraldehyde buffered at pH 7.4 with 0.1 M phosphate over a period of 15 min. The lumen of each specimen was subsequently washed and perfused with the same solution prior to the entire treatment being immersed in the solution for 3 days at 4°C.

Light Microscopy

Small pieces of stomach and intestine were excised from the digestive tract samples fixed with 4% paraformaldehyde (Fig. 1a). Excised samples were subsequently rinsed, dehydrated and embedded in paraffin. Serial 4 µm sections were prepared and stained with either hematoxylin and eosin, Alcian blue pH 2.5 (AB, acidic mucus is stained blue), or periodic acid-Schiff (PAS; neutral mucus is stained red).

Figure 1.

The digestive tract of G. pictus. Eso, esophagus; Int.A, anterior intestine; Int.P, posterior intestine; Pha, pharynx; Re, rectum; Sto.Bo, body region of stomach; Sto.Ca, cardiac region of stomach; Sto.Py, pyloric region of stomach; Sto.Te, terminal region of stomach. (a) Gross structures (removed accessory organs). The digestive tract was composed of an esophagus, a long saccular stomach, and a short straight intestine. Bar=10 mm. (b) Sagittal section of the body-terminal region of the stomach. The terminal region of the stomach was whitish. Bar=10 mm. (c) Sagittal section in the posterior intestine. The several partitions created by mucosal folds (arrow) were observed. Bar=10 mm.

SEM

Small pieces of digestive tract samples fixed with 1.5% glutaraldehyde were excised from the same regions as the specimens fixed with 4% paraformaldehyde. Excised samples were rinsed, dehydrated, conductive-stained using 2.0% tannic acid and 1.0% osmium tetroxide, and freeze-dried with t-butyl alcohol. Samples were subsequently coated with platinum and observed using under a digital scanning electron microscope (S-4800; Hitachi High-Technologies Corp., Japan).

RESULTS

Anatomy

Digestive tracts were composed of an esophagus, a stomach, and an intestine (Fig. 1a). No caeca were observed in the pyloric region of the stomach. The esophagus length was 8.30±0.33 cm, while the TL was 62.8±7.64 cm. The stomach was divided into a cardiac region, which was continuous with the esophagus, a long saccular body with a terminal region, and a pyloric region that was continuous with the intestine. The terminal region of the stomach was whitish in color which differed from the color of the body region (Fig. 1b). The intestine was short and almost straight, with a length of 15.58±1.22 cm and a ratio to TL of 0.26±0.03. Mucosal folds forming several partitions were observed in the posterior intestine (Fig. 1c). The rectum was situated posterior to the partition-like mucosal folds.

Body Region of Stomach

The body region of the stomach was composed of four layers; the mucosa, submucosa, muscularis, and serosa. The mucosal epithelium was composed of simple columnar cells and tubular gastric glands were observed within the lamina propria (Fig. 2a). The mucus on the surface and the apical cell cytoplasm were both positive for AB (Fig. 2b) and PAS (Fig. 2c). The muscularis was composed of two layers; a thick inner circular layer and a thin outer longitudinal layer (Fig. 2d). The subserosa was thin. Wave-like folds and partially opened pits between epithelial cells were observed in SEM images (Fig. 3a). Epithelial cells were polygonal with microvilli on the apical surface of epithelial cells and in the spaces between cells (Fig. 3b).

Figure 2.

LM images in the stomach. (a–d) The body region. (a) Cross section of mucosa. Gastric glands (GG) were observed in lamina propria. HE stain. Bar=50 μm. (b) AB stain. The mucus on surface and the apical cytoplasm were positive. (c) PAS stain. The mucus on surface and the apical cytoplasm were positive. (d) Cross section of muscularis. Thick inner circular layer (CM) and thin outer longitudinal layer (LM) were observed. Subserosa (Se) was thin. HE stain. Bar=50 μm. (e–h) The body-terminal region and the terminal region. (e) Sagittal section of mucosa in the body-terminal region. Gastric glands (GG) in lamina propria were disappeared from boundary. HE stain. Bar=50 μm. (f) AB stain of mucosa in the terminal region. The mucus on surface and the apical cytoplasm were positive. (g) PAS stain of mucosa in the terminal region. The mucus on surface and the apical cytoplasm were weakly positive. (h) Cross section of muscularis in the terminal region. Thin inner circular layer (CM) and thick outer longitudinal layer (LM) were observed. Subserosa (Se) was thick. HE stain. Bar=50 μm. (i–l) The pyloric region. (i) Sagittal section of mucosa. No gastric glands were observed in lamina propria. HE stain. Bar=50 μm. (j) AB stain. The mucus on surface and the apical cytoplasm were positive. (k) PAS stain. The mucus on surface and the apical cytoplasm were positive. (l) Cross section of muscularis. Thick inner circular layer (CM) and thin outer longitudinal layer (LM) were observed. Subserosa (Se) was thin. HE stain. Bar=50 μm.

Figure 3.

SEM images in the stomach. (a and b) The body region. (a) Wave-like folds and partially opened pits (arrowheads) were observed. Bar=50 μm. (b) Microvilli were observed in the polygonal apical surface (A) and the spaces between epithelial cells (arrow). Bar=5 μm. (c and d) The terminal region. (c) Straight folds were observed. Bar=50 μm. (d) No microvilli were observed in the polygonal apical surface (A) and the spaces between epithelial cells (arrow). Bar=5 μm. (e and f) The pyloric region. (e) Wave-like folds were observed. Bar=50 μm. (f) Microvilli were observed in the polygonal apical surface (A) and the spaces between epithelial cells (arrow). Bar=5 μm.

Terminal Region of Stomach

The terminal region of the stomach was composed of four layers; the mucosa, submucosa, muscularis, and serosa. Simple columnar epithelium of the mucosa was continuous within the terminal region of the stomach body; however, gastric glands within the lamina propria disappeared in the terminal region (Fig. 2e). The mucus on the surface and the apical cell cytoplasm were both positive for AB (Fig. 2f) and PAS (Fig. 2g). The muscularis was composed of two layers; a thin inner circular layer and a thick outer longitudinal layer (Fig.2h). The subserosa was thick. Straight folds were observed in SEM images (Fig. 3c). Epithelial cells were polygonal and microvilli were not observed on the apical surface of epithelial cells or in the spaces between cells (Fig. 3d).

Pyloric Region of Stomach

The pyloric region of the stomach was composed of four layers; the mucosa, submucosa, muscularis, and serosa. The mucosal epithelium was composed of simple columnar cells and gastric glands were not observed within the lamina propria (Fig. 2i). The mucus on the surface and the apical cell cytoplasm were both positive for AB (Fig. 2j) and PAS (Fig. 2k). The muscularis was composed of two layers; a thick inner circular layer and a thin outer longitudinal layer (Fig. 2l), with the inner circular layer thicker than in the stomach body region. The subserosa was thin. Wave-like folds were observed in SEM images (Fig. 3e). Epithelial cells were polygonal and microvilli were scattered on both the apical surface of epithelial cells and in the spaces between cells (Fig. 3f).

Anterior Intestine

The anterior intestine was composed of four layers; the mucosa, submucosa, muscularis, and serosa. Long secondary mucosal folds that appeared similar to trees were observed (Fig. 4a). The mucosal epithelium was composed of simple columnar cells and a few goblet cells (Fig. 4b). Mucus within all goblet cells and the apical cell cytoplasm were positive for AB (Fig. 4c) and negative for PAS (Fig. 4d). SEM images revealed that microvilli were densely distributed as a brush border on the apical surface of enterocytes, with the ciliary tufts of ciliated cells present between enterocytes (Fig. 5a,b).

Figure 4.

LM images in the intestine. (a–d) the anterior region. (a) Whole cross section. Mucosa (Mu), submucosa (Sb), muscularis (ML), and serosa were observed. Mucosal folds were secondary. HE stain. Bar=200 μm. (b) Cross section of mucosa. Simple columnar epithelium cells and few goblet cells were observed. HE stain. Bar=50 μm. (c) AB stain. The mucus within all goblet cells and the apical cytoplasm were positive. (d) PAS stain. The mucus within all goblet cells was negative. The apical cytoplasm was positive. (e–h) The posterior region. (e) Whole sagittal section. Mucosa (Mu), submucosa (Sb), muscularis (ML), and serosa were observed. Mucosal folds were primary. HE stain. Bar=200 μm. (f) Cross section of mucosa. Simple columnar epithelium cells and many goblet cells were observed. HE stain. Bar=50 μm. (g) AB stain. The mucus within all goblet cells and the apical cytoplasm were positive. (h) PAS stain. The mucus within all goblet cells and the apical cytoplasm were weakly positive.

Figure 5.

SEM images in the intestine. (a and b) The anterior region. (a) Wave-like folds and dense microvilli on surface were observed. Bar=50 μm. (b) Ciliary tufts from ciliated cells were observed between microvilli. Bar=5 μm. (c and d) The posterior region. (c) Wave-like folds and dense microvilli on surface were observed. Bar=50 μm. (d) Ciliary tufts from ciliated cells between microvilli increased than the anterior intestine. Bar=5 μm.

Posterior Intestine

The posterior intestine was composed of four layers; the mucosa, submucosa, muscularis, and serosa. Short primary mucosal folds were observed (Fig. 4e). The mucosal epithelium was composed of simple columnar cells and numerous goblet cells (Fig. 4f). Mucus within all goblet cells and the apical cell cytoplasm were positive for AB (Fig. 4g) and weakly positive for PAS (Fig. 4h). SEM images showed that microvilli were densely distributed as brush border on the apical surface of enterocytes, with the ciliary tufts of ciliated cells present between enterocytes (Fig. 5c,d). The density of the ciliary tufts on the ciliated cells was markedly higher within the posterior intestine than within the anterior intestine.

DISCUSSION

G. pictus lacked pyloric caeca, which are typically found in the majority of gastric teleosts (Hossain and Dutta, 1996; Kimura, 2010; Wilson and Castro, 2010) and absent in all stomachless fish (Hossain and Dutta, 1996). In addition, since the differentiation of gastric glands and pyloric caeca often occurs during the final stage of digestive tract development (García et al., 2001; Chen et al., 2006), it has been proposed that there is a relationship between these two organs (Wilson and Castro, 2010); however, the details of any such relationship have yet to be clarified. In addition to G. pictus, other gastric species lacking pyloric caeca have been reported, including Anguilla anguilla (Clarke and Witcomb, 1980; Domeneghini et al., 2005) and Anguilla japonica (Anguilliformes) (Kimura, 2010), Ictalurus punctatus (Sis et al., 1979) and Silurus asotus (Siluriformes) (Kimura, 2010), Gasterosteus aculeatus (Gasterosteiformes) (Hale, 1965), and Anarhichas lupus (Perciformes) (Hellberg and Bjerkås, 2000). It has been proposed that the stomachs of these species may have features that differ from the stomachs of other fish taxa that have pyloric caeca.

Histologically, the stomach of G. pictus could be divided into the cardiac, body, terminal, and pyloric regions. Histological features of the cardiac, body, and pyloric region were consistent with those reported in the majority of teleosts (Hale, 1965; Sis et al., 1979; Ezeasor and Stokoe, 1980; Anderson, 1986; Grau et al., 1992; Caceci et al., 1997; Hellberg and Bjerkås, 2000; Albrecht et al., 2001; Akiyoshi et al., 2005). The terminal region of the stomach possessed unique histological features that differed from those of the other three regions. Specifically, the terminal region lacked gastric glands and microvilli, and had thick longitudinal muscle layer and thick subserosa. Based on these features, it seemed likely that the terminal region of the stomach in G. pictus performs a specialized function. Normally, the stomach muscularis possesses a circular layer that is thicker than the longitudinal layer (Fänge and Grove, 1979; Anderson, 1986; Caceci et al., 1997). Contractions of the circular muscle cells results in a reduction of the lumen of the digestive tract (Dean and Padykula, 1966; Romer and Parsons, 2007), which assists with the grinding and mixing of food (Anderson, 1986). The circular layer is thickest in the pyloric region, where it is also referred to as the pyloric sphincter and is considered to indicate the boundary between the stomach and the intestine (Sis et al., 1979; Caceci et al., 1997; Albrecht et al., 2001). The contractions of longitudinal muscle cells shorten the length of the digestive tract (Dean and Padykula, 1966; Romer and Parsons, 2007). The thick longitudinal muscle layer observed in the terminal region of the stomach in G. pictus indicated that this region could potentially undergo significant relaxation and contraction due to the action of the longitudinal muscle cells. Thus, when large food items, such as whole fish, enter the body region of the stomach, the longitudinal muscle cells in the terminal region of the stomach might relax in order to accommodate the food item. Conversely, after the food has been sufficiently digested in the stomach, the longitudinal muscle cells in the terminal region would then contract to transport the digesta to the pyloric region. The lack of gastric glands in the terminal region may therefore be related to the need for significant relaxation and contraction of longitudinal muscle cells in this stomach region.

G. pictus also possessed partition-like mucosal folds in the posterior intestine. The spiral valve in the intestines of members of the Elasmobranchii (Chatchavalvanich et al., 2006), Dipnoi (Icardo et al., 2010), Cladistia (Abdel, 1975; Burkhardt-Holm and Holmgren, 1992), and Chondrostei (Domeneghini et al., 1999) consists of mucosal folds composed of mucous epithelium and connective tissue. The structure extends from the one end of the intestine to the other, and is arranged in a spiral along the intestinal walls (Romer and Parsons, 2007) and is characterized as having numerous diverticula. Although the anatomical and histological characteristics of the partition-like mucosal folds in G. pictus differed from those of the spiral valve, the partition-like mucosal folds also had numerous diverticula. The spiral valve increases digestion efficiency by increasing the absorptive area of the lumen and prolonging the retention of food (Chatchavalvanich et al., 2006; Romer and Parsons, 2007; Icardo et al., 2010). Similarly, the partition-like mucosal folds observed in G. pictus may perform a similar function. Numerous ciliary tufts on ciliated cells were observed in the epithelium of the partition-like mucosal folds in G. pictus. Generally, ciliated cells are distributed in the pharynx or oviduct where they are responsible for transporting material (Romer and Parsons, 2007). Ciliated cells have been observed to line the intestinal wall of certain Osteichthyes, such as Dipnoi (Purkerson et al., 1975) and Cladistia (Abdel, 1975; Burkhardt-Holm and Holmgren, 1992). It is thus possible that the ciliated cells observed on the intestinal wall of G. pictus may facilitate the movement of digesta and mucus. With the exception of the partition-like mucosal folds and ciliated cells, the histological characteristics of the G. pictus intestine are consistent with those that have been observed in the majority of teleosts (Hale, 1965; Sis et al., 1979; Ezeasor and Stokoe, 1980; Anderson, 1986; Grau et al., 1992; Morrison and Wright, 1999; Hellberg and Bjerkås, 2000; Albrecht et al., 2001; Akiyoshi et al., 2005).

Mucus that is secreted into the lumen of the digestive tract can generally is divided into three groups (i.e., acidic, basic, and neutral) depending on the glycoproteins it contains (Bakke et al., 2010). Acidic mucus was secreted throughout the digestive tract of G. pictus. The secretion of acidic mucus by the stomach mucosal epithelium has only been reported in Anguilla anguilla (Elopomorpha) (Clarke and Witcomb, 1980; Domeneghini et al., 2005). In addition to protecting the mucosal epithelium from mechanical or chemical injuries arising from interactions with digestive tract contents or enzymes (Domeneghini et al., 2005; Manjakasy et al., 2009), mucus also plays a role in the mediation of biophylaxis and it contains immunoglobulins for fighting the bacteria in ingested substances (Swan et al., 2008). The acidic glycoprotein in acidic mucus suppresses the degradation of the mucus by proteases or bacteria (Neuhaus et al., 2007) and aids in the absorption of digesta smaller than water-soluble molecules by increasing mucus viscosity and adhesion (Tibbets, 1997; Domeneghini et al., 2005). Acidic mucus may also contribute to the formation of an acidic environment on the luminal surface. The activity of the acidic protease pepsin is highest in strongly acidic environments (Corrêa et al., 2007; Pérez-Jiménez et al., 2009; Xiong et al., 2011). Furthermore, Pérez-Jiménez et al. (2009) suggested that lysosomal protease groups, such as cathepsin, might be prevalent within low pH regions of the pyloric caeca and intestine in Dentex dentex. Thus, the acidic mucus secreted throughout the digestive tract might promote the activity of acidic protease in G. pictus.

The structure of the musculature of the terminal region of the stomach combined with the secretion of acidic mucus in the stomach of G. pictus had not previously been reported in gastric species with pyloric caeca. Indeed, these observations could be characteristic of gastric fish without pyloric caeca. As in the spiral valve, the partition-like mucosal folds in the intestine of G. pictus possessed numerous diverticula, and the intestinal wall was lined by ciliated cells. Both of these features, that is, the spiral valve and ciliated cells, have only observed in fish species that had branched earlier than the basal teleosts. Observations of the G. pictus intestine revealed that this species may be closely related to fish taxa that branched off earlier than the basal teleosts. In a study on the comparative anatomy of the coccyx and caudal vertebra in both fossils and extant fish taxa, Arratia (1997) proposed that, among the basal teleosts, the Elopomorpha branched off earlier than the Osteoglossomorpha. As Osteoglossomorpha (Al-mahrouki and Youson, 1998) and Clupeomorpha (Hossain and Dutta, 1996; Kimura, 2010) both possess pyloric caeca, the absence of a pyloric caeca and the presence of an intestine resembling that of a group that branched earlier than the basal teleosts in G. pictus, appears to support Arratia's hypothesis that, among the basal teleosts, Elopomorpha branched off before Osteoglossomorpha.

Ancillary