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Histology of the Digestive System of Scyliorhinus retifer,

the Chain Catshark

By Lara Slough

Advisor: Dr. John Morrissey

Abstract

I prepared a histological description of Scyliorhinus retifer, the chain catshark, to

show that the digestive tissues resemble those of other vertebrates. After taking ten tissue

samples, I prepared them for observation. I observed my slides using light microscopy

and took photomicrographs. My results show similarities between the digestive tissues of

Scyliorhinus retifer and those of other vertebrates. These results indicate similar function

of the digestive systems as well as a common ancestor.

Introduction

During the summer of 2008, I conducted a research project about the digestive

system of Scyliorhinus retifer, the chain catshark. This small, deep-dwelling

elasmobranch inhabits the waters of the outer continental shelf and slope from Nova

Scotia to Nicaragua. Elasmobranchs, which include sharks and rays, are in a sub-class of

the class Chondrichthyes that have yet to be studied extensively. The chain catshark

reaches approximately 45cm in length as an adult. Seeking an optimal temperature of 11-

12°C, it is found at average depths of 70-550m. Little is known about its biology and

habits. (Compagno, 1984) I wondered if the scarcity of food in the deep sea might have

led to adaptations in the digestive system of this shark. This was certainly a possibility.

However, because all vertebrates with rare exception have similarly functioning digestive

systems, I expected it to function in basically the same way as in other vertebrates. I

studied histology to confirm or deny similarities. There have only been a couple of

publications regarding the basic range and distribution of this species, and there is some

information regarding its reproduction, but no studies about its digestive system and

histology have been published to date. (J. Morrissey, pers.com) Histology is the study of

tissues, groups of cells with similar structure that function together as a unit. If the

tissues appear similar, the function is also similar. (Gartner, 2001) My hypothesis was

that the digestive tissues of Scyliorhinus retifer would appear similar to the digestive

tissues of other vertebrates.

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I compared my results to other vertebrates to prove my hypothesis. Because

human beings have already been compared histologically to numerous vertebrates, I used

the human digestive system as a standard for comparison. (Derrickson, 2006) I also

compared my results to primitive fishes, such as sturgeons and elasmobranchs. Humans

and sharks have similar digestive systems, the basic function of which is to break down

food for absorption. The systems appear somewhat similar upon macroscopic

observation of the organs. The major organs involved include the mouth, esophagus,

stomach, intestine, and rectum. There are some differences although the function is the

same. The stomach and intestine differ slightly in humans and sharks. The stomach

serves to break down larger pieces of food into smaller pieces of food in both humans and

sharks. The intestine of the shark is adapted to the stream-lined body shape of the shark.

The spiral valve results in a compact design as well as providing maximum surface area

for nutrient absorption. (Holmgren, 1999) In the human, the intestine is long and coiled;

the small and large intestine have a combined length of nearly 9 meters. This coiled

design also provides maximum surface area for nutrient absorption, but is more

appropriately shaped for the human organism (Derrikson, 2006).

Food follows a specific path through the digestive tract. In the human, the food

enters the oral cavity, travels through the esophagus, and into the cardiac stomach. This

is the first section of the human stomach, and is heavily muscled to break larger pieces of

food into smaller pieces of food. The muscles of the stomach create a churning motion

that breaks up the food. Then the food travels through the second section of the stomach,

the fundus. Here, the smaller pieces of food are broken down into even smaller pieces of

food. The third section of the stomach is called the pyloric stomach, in which the food is

broken down into the smallest particles possible in the stomach. The pyloric stomach

opens into the intestine via the pyloric sphincter, the muscular gateway between the

pyloric stomach and the intestine. The food travels through the long coils of the

intestines, where the nutrients are absorbed, and the remaining waste is excreted through

the rectum and out the anal opening. (Derrickson, 2006)

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The path food follows in the shark is similar to the path food follows in the

human. In the shark, the food enters the oral cavity, travels through the esophagus and

into the cardiac stomach. Sharks have a two-section stomach (Figure 1a), whereas

humans have a three-section stomach. In the cardiac stomach, as in humans, larger

pieces of food are broken down into smaller pieces of food. The food enters the second

part of the stomach, the pyloric stomach, where smaller pieces of food are broken down

into even smaller pieces of food. After the pyloric stomach has sufficiently broken down

the food, it passes through the pyloric sphincter. The sphincter contracts to keep

insufficiently processed food from passing through and relaxes to allow sufficiently

processed food to pass through into the intestine. As the food travels through the

intestine, nutrients are absorbed. Because sharks have a spiral intestine, nutrients are

absorbed while traveling through an encased spiral valve (Figure 1b) rather than long

coils as in the human. The leftover waste travels through the rectum and out the anal

opening. (Holmgren, 1999)

Figure 1a. The Shark Digestive System. Figure 1b. Inside the Spiral Intestine.

(Holmgren, 1999) (Holmgren, 1999)

I studied histology because histology accounts for apparent differences such as

the human intestine and the shark intestine, which could be misleading. Histology, which

is the study of tissues, provides a description of an organism on a fundamental level.

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Similarities at this level are most important because they prove similarities in function.

There is a hierarchy to the structure of an organism. Individual cells make up tissues,

tissues make up organs, organs make up organ systems, and organ systems make up the

organism. The type of tissue indicates the function of the tissue, the function of the

organ, the function of the organ system, and the function of the organism. There are four

types of tissue: epithelial, connective, muscle, and nervous. Epithelial tissue protects,

secretes, absorbs, lines hollow organs, and also forms glands. Connective tissue provides

a supportive framework for the body. Muscle tissue allows the body to move. Nervous

tissue controls all functions of the body. The tissues of the human intestine and the shark

intestine are histologically similar. Therefore, they are functionally similar as well. I set

out to prove that the chain catshark was histologically similar to other vertebrates.

The focal tissues of my project, epithelial and muscular, are most prevalent within

the digestive system, although all four tissue types are present. There are several

different types of epithelial tissue, each descriptively named based on the number of cell

layers and cell shape. There are three types of muscle. Smooth muscle is involuntarily

controlled and is found in the digestive tract. Skeletal muscle is voluntarily controlled

and is found, for example, in the biceps. Cardiac muscle is involuntarily controlled and is

found in the heart. (Carneiro, 2005)

Methods

To compare the digestive system tissues of the chain catshark to those of other

vertebrates, I completed several preparatory steps. After the digestive system had been

removed from the dissection specimen, I took tissue samples from certain areas along the

digestive tract, collecting ten samples in all (Figure 2).

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Figure 2. Tissue Sample Locations 1-10 (in numerical order following food path).

After collecting the tissue samples, I prepared them for observation. I dehydrated

the tissue samples by soaking them in ethanol, which is water soluble, and Safe-clear, a

tissue clearing agent. This process removes water from the sample, resulting in the space

normally taken up by water, about 80% of the tissue (Derrickson, 2006), being filled with

something more conducive to sectioning. I then embedded the tissue samples in paraffin

wax, which creates a hardened sample that is ideal for sectioning. I left the samples in

the refrigerator overnight to make sure they were thoroughly hardened.

To ensure the samples were hard enough to section, yet soft enough to avoid

splintering, I removed the samples from the refrigerator 30 minutes before sectioning.

Using a microtome set to 10 micrometers, I sectioned each sample. I placed the ribbon of

wax with embedded tissue into a water bath and let it float to flatten out. The water bath

was 45°C, to soften yet not melt the wax. After the wax had softened, I submerged a

microscope slide in the water and caught the ribbon on the microscope slide as I pulled it

1. Oral Cavity

Dorsal

2. Oral Cavity

Ventral

3. Esophagus

7. Pyloric

Sphincter

8. Spiral

Intestine A

9. Spiral

Intestine B

10. Rectum

6. Pyloric

Stomach

4. Cardiac

Stomach A 5. Cardiac

Stomach B

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up and out of the water. I placed the slides on a slide warmer, also 45°C, to dry. I made

at least three slides from each tissue sample.

After the slides were dry, I began the staining process. Staining requires soaking

the slides in a series of chemicals that ultimately darken the tissue sample and stain it

purple, making the tissues visible. I loaded the slides into a tray and submerged them in

the appropriate sequence of chemicals for the appropriate amounts of time. I had to break

down the paraffin and re-hydrate the tissues so that the dye could sufficiently permeate

the tissues to make them visible. This required reversing the procedure I had followed

earlier in order to dehydrate the tissue samples. I soaked the slides in Safe-clear and

ethanol, the dyes hematoxylin and eosin, and associated chemicals. After the staining

process was complete, I let the slides dry, applied cover-slips, and let them dry a final

time. They were finally ready to observe under the microscope.

I observed my slides using light microscopy, noting muscle and epithelial layers.

I took numerous photomicrographs, taking care to capture a good image of each of the

tissue samples at different magnifications, finding that 4x, 10x, and 40x magnifications

gave the best results. These photomicrographs provided me with a histological

description of the chain catshark which I compared to humans as well as primitive fishes.

I compared tissues and layers present, noting similarities.

Results

The photomicrographs I took of the digestive tissues of Scyliorhinus retifer

appear similar to photomicrographs taken of the digestive tissues of other vertebrates.

White spaces inside the organs, epithelial linings, muscle layers, and white spaces outside

the organs are all present. Figure 3a-f shows my results from the cardiac stomach, Figure

4a-f shows my results from the pyloric stomach and the pyloric sphincter, and Figure 5a-f

shows my results from the spiral intestine. My results indicate that the basic histology of

the gut wall of the chain catshark is similar to that of other vertebrates.

Following the path of food, I examined my slides and took photomicrographs,

focusing on the stomach and the intestine. I took photomicrographs from two areas of the

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cardiac stomach to observe any structural changes traveling father down the digestive

tract. My photomicrographs of the cardiac stomach proximal to the esophagus (Figure

3a, 3c, 3e, 3f) show the layered structure of the stomach wall. There is white space

indicating the inside of the organ, mucosa (a lining composed of simple columnar

epithelium), sub-mucosa (loose connective tissue that supports the mucosa), dual muscle

layers (interior: circular muscle, CM, exterior: longitudinal muscle, LM), and white

space indicating the outside of the organ (Figure 3a). Dense surface cells for secretion

are visible upon closer examination (Figure 3c). There are dense dual muscle layers.

The layers formed are thick and distinct (Figure 3e, 3f). My photomicrographs of the

cardiac stomach distal to the esophagus (Figure 3b, 3d) show the slight differences in

structure in this area of the stomach. There is white space indicating the inside of the

organ, mucosa, submucosa, circular muscle, longitudinal muscle, and white space

indicating the outside of the organ (Figure 3b). Notice that while the same layers are

present as in the proximal stomach, yet they are neither as dense nor clearly defined

(Figure 3d).

3a. Cardiac Stomach A. 3b. Cardiac Stomach B. 3c. Cardiac Stomach A.

4x magnification 4x magnification 10x magnification

Mucosa

Sub-

mucosa

White

Space:

Inside

Organ

Mucosa

White

Space:

Inside

Organ

CM

&

LM

White

Space:

Inside

Organ

Mucosa

White Space:

Outside

Organ

Surface

Cells for

Secretion

Mucosa

CM

&

LM

Sub-

mucosa

Sub-

mucosa

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3d. Cardiac Stomach B. 3e. Cardiac Stomach A. 3f. Cardiac Stomach A.

10x magnification 10x magnification 40x magnification

Figure 3a-f. Photomicrographs of the Cardiac Stomach of Scyliorhinus retifer.

The pyloric stomach has white space indicating the inside of the organ, mucosa,

submucosa, circular muscle, longitudinal muscle, and white space indicating the outside

of the organ (Figure 4a). The mucosa is simple columnar epithelium, and the submucosa

is loose connective tissue, as in the cardiac stomach. My photomicrographs clearly show

these layers at higher magnifications (Figure 4b, 4c). The layers appear different than the

layers in the cardiac stomach (Figure 3a, 3b). There is a clear distinction between the

basal membrane of the epithelial layer and the inner layer of submucosa (Figure 4c). The

white space indicating the inside of the organ, the circular shape of the pyloric sphincter,

and the white space indicating the outside of the organ are visible in the cross-section

(Figure 4d). The smooth muscle that makes up this sphincter is shown at higher

magnifications as well, showing the density of the fibers (Figure 4e, 4f).

Close-up of

Longitudinal

Muscle

Close-up of

Circular

Muscle Sub-

mucosa

Mucosa

White Space:

Outside Organ

CM

&

LM

Dual

Muscle

Layers

Close-up of

Basal Membrane Submucosa

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4a. Pyloric Stomach. 4b. Pyloric Stomach. 4c. Pyloric Stomach.

4x magnification 10x magnification 40x magnification

4d. Pyloric Sphincter. 4e. Pyloric Sphincter. 4f. Pyloric Sphincter.

4x magnification 10x magnification 40x magnification

Figure 4a-f. Photomicrographs of the Pyloric Stomach and the Pyloric Sphincter of

Scyliorhinus retifer.

I compared the structure of the intestine at different places along the digestive

tract to see if the structure changed as I had done with the cardiac stomach. I took

photomicrographs of two samples from the spiral intestine, one proximal to the pyloric

White Space:

Inside Organ

White Space:

Outside Organ

CM

&

LM

CM & LM Mucosa

Mucosa

Close-up of

Submucosa

White Space:

Inside Organ

Muscular

Ring

White

Space:

Outside

Organ

Muscular

Ring

Close-up of

Muscular

Ring

Smooth

Muscle

Tissue

Submucosa

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sphincter (Figure 5a, 5b) and one distal to the pyloric sphincter (Figure 5c, 5d, 5e, 5f).

There is white space indicating the inside of the organ, an epithelial layer lining the

interior of the organ with pockets for absorption, a layer of muscles lining the exterior of

the organ, and white space indicating the outside of the organ (Figure 5a, 5c). The

structure of the intestinal wall is similar to the stomach wall, mucosa, submucosa, circular

muscle, and longitudinal muscle. The spiral valve is visible in cross-section (Figure 5c).

The photomicrographs clearly show the contrast between the larger pockets for

absorption proximal to the pyloric sphincter (Figure 5a), and the smaller pockets for

absorption distal to the pyloric sphincter (Figure 5b). The epithelial lining and outer

muscle layers are not easily distinguishable in the proximal sample (Figure 5a). The

epithelial lining is more easily distinguished from the outer muscle layer in the distal

sample (Figure 5d, 5e). The distal sample also shows surface cells for absorption (Figure

5d, 5f).

White

Space:

Outside

Organ

Close-

Up of

White

Space:

Inside

Organ

Close-

up of

Inner

Lining

CM &

LM

Smaller

Pockets for

Absorption

CM

&

LM

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5a. Spiral Intestine A. 5b. Spiral Intestine A. 5c. Spiral Intestine B.

4x magnification 40x magnification 4x magnification

5d. Spiral Intestine B. 5e. Spiral Intestine B. 5f. Spiral Intestine B.

10x magnification 40x magnification 40x magnification

Figure 5a-f. Photomicrographs of the Spiral Intestine of Scyliorhinus retifer.

Discussion

The photomicrographs I took showed muscle layers and epithelial layers matching

the general structure found in other vertebrates in all major digestive organs, though I

focused on the stomach and intestine. These two organs are responsible for the majority

of the digestive process, and so similarities in these organs are most important.

Close-up of

Surface

Absorptive

Cell

Surface

Absorptive

Cell

White

Space:

Inside

Organ

White

Space:

Inside

Organ

White

Space:

Outside

Organ

Larger

pockets for

absorption

White

Space:

Inside

Organ

Spiral

Valve

White

Space:

Inside

Organ

White

Space:

Inside

Organ

White

Space:

Outside

Organ

Close-

up of

CM &

LM

CM

&

LM

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Mechanical digestion occurs in the stomach, and absorption occurs in the intestine. The

gut wall of Scyliorhinus retifer has the same basic structure as human: epithelial layers

lining the inside and muscle layers lining the outside. The epithelial layers in the

stomach contain cells that secrete acids, enzymes, and mucus. The acids and enzymes

aid in digestion and the mucus protects the underlying tissue from damage. The mucosa

of the gut wall is simple columnar epithelium, which is a single layer of column-shaped

epithelial cells. It is supported by the submucosa, which is loose connective tissue, and

dual muscle layers beneath. The dual muscle layers, the circular muscle and the

longitudinal muscle, contract perpendicular to each other to create the churning motion

that moves food along the digestive tract and breaks up larger pieces into smaller pieces.

Gut wall layers are consistent throughout the digestive tract, differing slightly depending

on location and function. The mucosa of the stomach is specialized for secretion, while

the mucosa of the intestine is specialized for absorption. The layers also vary in density

and thickness. (Cormack, 1993) In my photomicrographs, the differences in layers are

visible along the digestive tract as the organ function changes, depending on the stage of

digestion. I observed the organs in order along the digestive tract: the cardiac stomach A

(Figure 3a) and B (Figure 3b), the pyloric stomach (Figure 4a), the pyloric sphincter

(Figure 4d), and the spiral intestine A (Figure 5a) and B (Figure 5b). The distinction

between the layers is most apparent in the cardiac stomach (Figure 3e, 3f).

The histological changes that occur along the digestive tract to compensate for the

smaller particles of food are apparent in my results. The first part of the intestine that the

food passes through, just after the pyloric sphincter, has larger areas for absorption which

is consistent with the larger pieces of food that would be found there. By the time the

food reaches the latter part of the intestine, it has been broken down into much smaller

particles, and the structure reflects this change in size. (Derrickson, 2006) In the case of

the spiral intestine, there are much smaller and more numerous pockets for absorption

farther down the intestine. (Holmgren, 1999) This progression is clearly visible in my

photomicrographs. The contrast between the proximal (Figure 5a) and the distal (Figure

5c) intestine is striking.

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Conclusion

I observed macroscopic and microscopic similarities between the digestive organs

of the chain catshark and the digestive organs of other vertebrates. To prove my

hypothesis, I compared my results to photomicrographs taken of tissue samples taken

from humans and primitive fishes. Of course, I took many more photomicrographs than I

was able to include in my figures for this paper. I considered all of these when making a

histological comparison between Scyliorhinus retifer and another vertebrate. When

comparing my results to the human, I found gut wall structure was similar. (Cormack,

1993) When comparing my results to the shovelnose sturgeon, Scaphirhynchus

platorynchus, I found similarities in the dorsal and ventral oral cavity, including taste

buds and cartilage. I also found similarities in the spiral intestine and other organs.

(Weisel, 1979) The similar structures of the esophagus and spiral intestine were

especially striking in a shark, the narrownose smooth-hound, Mustelus schmitti. (Codon,

1988) I noticed similarities to a ray as well, the thornback ray, Raja clavata, particularly

in the structure of the gut wall and the rectum. (Holmgren, 1999) Similarities in the oral

cavity, intestine, and other organs were apparent in freshwater fishes as well, the

longnose sucker, Catostomus catostomus, and the squawfish, Ptychocheilus oregonense.

(Weisel, 1962) All of these comparisons led me to conclude that these results indicate

similar tissues, similar tissue function, and therefore similar digestive organs and

digestive systems. Similarities at this basic structural level between seemingly vastly

different organisms also indicate a common ancestor and evolution. Basic structural

components retained from an ancestor and then built upon by evolution may not be

obvious, but can be observed. The shark intestine seems entirely different from the

human intestine, but histological comparison shows the same types of cells present in

both organs, showing that the shark intestine and the human intestine are the same type of

organ made up of the same types of cells, evolved into different shapes more suited to the

particular organism. The results support my hypothesis: the digestive tissues of

Scyliorhinus retifer appear similar to the digestive tissues of other vertebrates.

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References

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Carneiro, Jose. Junqueira, Luiz Carlos. Basic Histology Text and Atlas: 11th

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