©Copyright by Sten M



AN ABSTRACT OF THE THESIS OF

Sten M. Erickson for the degree of Honors Baccalaureate of Science in Biology presented on June 6, 2007. Title: Sexual dimorphism and seasonal changes in the Harderian gland

of the red-sided garter snake, Thamnophis sirtalis parietalis.

Abstract approved: __________________________________

Robert T. Mason

The Harderian gland (HG) of the red-sided garter snake, Thamnophis sirtalis parietalis, is a secretory structure that plays a role in the vomeronasal system by solubilizing semiochemicals such as prey chemoattractants and the female sexual attractiveness pheromone. The HG produces secretions containing pheromone-binding proteins that form a complex with pheromone molecules making them soluble, allowing for vomeronasal detection. Detection of prey chemoattractants and pheromones is essential for both feeding and reproduction. Feeding occurs only in summer and mating only in spring. Because the semiochemicals and the pheromone-binding proteins are likely different in each season, changes in the gross anatomy and histology of the HG were expected to be seen throughout the year. We found this to be true as glands in the summer had larger masses, greater cell heights, and larger lumen diameters than in other seasons. Sexual dimorphism was observed in the histology and morphology of the gland as males had significantly larger HGs, greater cell heights, and larger lumen diameters than those of females. Finally, sexual dimorphism was observed in the greater cell height of males than females in the spring, likely because males respond to and solubilize the sex pheromone in spring, while females do not.

Key Words: Harderian gland, sexual dimorphism, seasonal change, sex pheromone, vomeronasal organ

Corresponding email address: ericksos@onid.orst.edu

©Copyright by Sten M. Erickson

June 6, 2007

All Rights Reserved

Sexual dimorphism and seasonal changes in the Harderian gland

of the red-sided garter snake, Thamnophis sirtalis parietalis

by

Sten M. Erickson

A PROJECT

submitted to

Oregon State University

University Honors College

in partial fulfillment of

the requirements for the

degree of

Honors Baccalaureate of Science in Biology

Presented June 6, 2007

Commencement June 2007

Honors Baccalaureate of Science in Biology project of Sten M. Erickson presented on June 6, 2007.

APPROVED:

______________________________________________________________________________

Mentor, representing Biology

______________________________________________________________________________

Committee Member, representing Biochemistry and Biophysics

______________________________________________________________________________

Committee Member, representing Zoology

______________________________________________________________________________

Chair, Biology Program

______________________________________________________________________________

Dean, University Honors College

I understand that my project will become part of the permanent collection of Oregon State University, University Honors College. My signature below authorizes release of my project to any reader upon request.

______________________________________________________________________________

Sten M. Erickson, Author

Acknowledgment

I am very grateful to my primary investigator and mentor, Dr. Robert T. Mason for his guidance and support. He always challenged me and encouraged me throughout the thesis experience. I would also like to thank Rocky Parker for his ideas, instruction, and encouragement; Dr. Barbara Taylor for the use of her microscope and in the analysis of slides; Dr. Indira Rajagopal for advice and encouragement; and Chris Friesen for his help with the statistics involving Harderian gland mass. I would lastly like to thank Heather Waye for all of her direction, critique, and valued input. I could not have done this thesis without her.

Funding for this research was provided by a grant from the National Science Foundation to R.T. Mason.

TABLE OF CONTENTS

Page

INTRODUCTION 1

Chemical Senses and the Vomeronasal System 1

Role of the Harderian Gland in the VN System 4

Anatomy of the Harderian Gland 6

Histology of the Harderian Gland 7

Ultrastructure 8

Seasonality 9

Sexual Dimorphism 10

Garter Snakes as Models 11

MATERIALS AND METHODS 13

Collection of Snakes with Respect to Seasonality 13

Perfusion 14

Dissection 14

Morphometric Measurements 15

Embedding and Sectioning 15

Staining 16

Structural Anatomy 16

Histological Measurements 16

Statistics 17

RESULTS 18

Anatomy/Gross Morphology 18

Histology 20

Difference between Left and Right Gland 21

Sexual Dimorphism 22

Seasonal Change 24

CONCLUSION AND DISCUSSION 26

Anatomy/Gross Morphology 26

Left and Right Harderian Gland Similarity 27

Sexual Dimorphism and Behavior 28

Seasonality and Behavioral Significance 30

BIBLIOGRAPHY 33

LIST OF FIGURES

Figure Page

1. The vomeronasal system of the red-sided garter snake 6

2. The gross anatomy and morphology of the Harderian gland 18

3. Black India ink injected into the Harderian gland 19

4. The histology of the Harderian gland of garter snakes at 200X 20

5. Linear regression of Harderian gland mass against head length 21

6. Cell height of acini in male and female garter snakes 22

7. Residuals of male and female garter snake Harderian glands compared over three seasons 23

8. Lumen diameter of acini for male and female garter snakes compared over three seasons 24

Sexual dimorphism and seasonal changes in the Harderian gland

of the red-sided garter snake, Thamnophis sirtalis parietalis

INTRODUCTION

Chemical Senses and the Vomeronasal System

The detection of chemicals and odorants is essential for the survival of many terrestrial vertebrates (Halpern 1987). Many creatures gather information about their surroundings as well as find their next meal using the sense of smell, known as olfaction. Along with the olfactory system, another sensory system exists known as the vomeronasal (VN) system. The VN system has not yet been fully described and is possibly the last major vertebrate sensory system that remains largely unknown. Using their VN sense, many animals detect pheromones which are specific chemical messages that signal information to other members of the same species (Halpern & Martinez-Marcos 2003). Pheromones can be broken into the major categories of sex pheromones, aggregation pheromones, and alarm pheromones. There are other species-specific pheromones that induce behavior, but these three categories are the most prominent. In addition, other semiochemicals (chemicals with signal function) are detected by the VN system. Principal among these are prey chemoattractants that predators use to locate and identify favored food items.

One of the most studied VN systems is that of garter snakes (Rehorek 2000a). Recent research has shown that a functioning VN system is absolutely crucial for mating to occur in the red-sided garter snake, Thamnophis sirtalis parietalis (Huang et al. 2006, Kubie et al. 1978, Halpern 1983). Upon emergence from hibernation in the spring, female garter snakes produce sexual attractiveness pheromones in their skin lipids that attract males and induce species-specific courtship behaviors (Mason et al. 1989, 1990). These pheromones are so specific that males can determine a female’s size and overall body condition which combine to determine her sexual attractivity to males (Shine et al. 2003). The pheromone specifically responsible for the sexual attractiveness of female red-sided garter snakes has now been isolated and synthesized (Mason et al. 1989, 1990).

Semiochemicals are not only involved with reproduction, but also play a role in identifying and locating prey (Halpern & Frumin 1979, Halpern 1987). Earthworms, when alarmed, produce a pheromone named Earthworm Shock Secretion that excites the neurons in the garter snakes VN system (Halpern et al. 1987, Jiang et al. 1990, Wang et al. 1988). Earthworm Shock Secretion is a potent feeding chemoattractant that allows garter snakes to locate and identify their preferred earthworm prey (Wang et al. 1988). Therefore the two crucial behaviors of feeding and mating are associated with the VN system through detection of species-specific pheromones and prey chemoattractants (Halpern 1987).

Chemical stimuli are brought into the mouth by the familiar vertical flicking of the tongue seen in many different species of snakes. This is known as “tongue-flick” behavior and functions to transport chemical cues from the environment to the roof of the mouth (Gillingham & Clark 1981, Halpern & Kubie 1980, 1983). Two small ducts known as the vomeronasal ducts connect the buccal cavity to the vomeronasal organ (VNO), a bone-encapsulated structure where pheromone detection takes place (Rehorek et al. 2003, Halpern et al. 2006). The mechanism by which the pheromones are transported to the VNO is still debated, though some believe a form of suction may occur (Meredith & O’Connell 1979, Meredith et al. 1980). Whatever the mechanism, pheromones are detected in the sensory epithelial tissue of the VNO by surface receptors and a second messenger system that amplifies the signal (Huang et al. 2006). The signal then transmits through neural tracts to the brain where it alerts the organism of new chemical information and causes a change in behavior (Rehorek et al. 2000c).

For a chemical stimulus like a pheromone to be detected by the vomeronasal system, it must be dissolved, or solubilized, in aqueous solution (Huang et al. 2006). The fluid filling the VNO is aqueous (Rehorek et al. 2000b) however, the sexual attractiveness pheromone of the red-sided garter snake consists of a homologous series of long-chain aliphatic lipids identified as saturated and monounsaturated methyl ketones (Mason et al. 1989, 1990). This pheromone is very nonpolar and therefore will not mix or dissolve in the aqueous solution of the VNO (Huang et al. 2006). Thus, the pheromone must be modified in some manner for solubilization to occur. The specific mechanism by which this occurs remains unknown, but a glandular cephalic organ known as the Harderian gland (HG) seems to play a critical role in the solubilization of pheromones and other chemical cues (Huang et al. 2006).

Pheromone detection in garter snakes was studied by Huang et al. (2006). In their study, male garter snake were anesthetized and individual VN neurons were isolated. The neurons were then stimulated by the female garter snake sexual attractiveness pheromone and the voltage along the neuron was recorded (Huang et al. 2006). It was discovered that the neurons respond to the pheromone with the opening of ion channels causing membrane depolarization (Huang et al. 2006). It appears that a G protein-coupled second messenger system is involved because generation of IP3 increased by 200% when male VN neurons were exposed to female sex pheromone (Huang et al. 2006). The depolarization of the membrane initiated action potentials which allowed the signal to be transduced into an electric signal (Huang et al. 2006). Detection of pheromones leads to specific changes in sexual behavior exhibited by the male garter snake, allowing it to locate females and initiate mating behavior. Male garter snakes lacking a fully functional vomeronasal system are unable to detect or respond appropriately to the sex pheromone and no mating behavior is possible (Huang et al. 2006). The vomeronasal system, however, cannot properly function without the presence of the HG, a secretory structure located posterior to and behind the eye (Huang et al. 2006).

Role of the Harderian Gland in the VN system

Essential to the functioning of the VN system is the HG, the topic of study in this thesis. Little is currently known about the HG and the role it plays in snakes or any other vertebrates. First discovered in 1694 by Johann Jacob Harder in deer, it has since received little interest (Payne 1994). This lack of in depth study for 300 years is remarkable given that the gland is easily identified and in snakes is often larger than the eye (Minucci et al. 1992). Recently, more attention has been given to it due to its possible link to the VN system (Saint Girons 1982). The HG is directly connected to the VNO via the nasolacrimal duct as it was observed that autofluorescent dyes injected into the HG later appeared on the tongues of snakes (Heller & Halpern 1982). More recently, radioactive labeling traced the path of the HG secretions as they flowed through the nasolacrimal duct into the VNO and onto the tongue (Rehorek et al. 2000c). This anatomy is unique to snakes in that it allows HG secretions direct access to the VNO, suggesting some function in the VN system (Rehorek et al. 2000c).

Secretions of the HG facilitate the uptake of chemical stimuli, including pheromones, into the VNO (Rehorek et al. 2000c, Huang et al. 2006). For odorants to access the VN sensory epithelium they must be solubilized in the aqueous medium filling the VNO (Rehorek 1997, Rehorek et al. 2000b, 2000c). The HG is the major source of the fluid in the VNO and therefore is expected to have some role in dissolving the odorants (Rehorek 1997, Rehorek et al. 2000b, Kubie et al. 1978). The pheromones, being nonpolar, cannot be dissolved in aqueous solution until they are bound by some substance that makes them polar (Huang et al. 2006). It has recently been discovered that HG homogenate exposed to the sex pheromones allows them to be solubilized in the aqueous environment of the VNO (Huang et al. 2006). This discovery lends support to the hypothesis that HG secretions solubilize pheromones.

Secretions from the HG contain many different substances, but recent research shows that pheromone-binding proteins are present (Mason et al. unpub. data). These pheromone-binding proteins identify and bind to certain sites on the pheromone, polarizing them for solubilization in aqueous environments (Beynon & Hurst 2004). Without the HG homogenate, the pheromones are not solubilized, not detected by the VNO, and therefore do not induce responses in the VN neurons of garter snakes (Huang et al. 2006). Thus, HG secretions appear to be essential to pheromone detection, solubilization, and pheromonal transport.

Anatomy of the Harderian Gland

The HG of snakes is still relatively unexamined and many of the existing studies used varying techniques making it difficult to compare results. Snakes have very highly developed HGs made up of incomplete lobules, bound together by connective tissue (Chieffi et al. 1992). Snakes have two HGs, one on each side of the head behind and within the orbit (Rehorek et al. 2000c). The HG of garter snakes possess only one lobe, but consists of two portions: a small ocular portion and a large postorbital portion, that lies just under the scales posterior to the eye (Rehorek et al. 2003, Chieffi et al. 1992). The large postorbital portion makes up the majority of the gland’s mass (Payne 1994, Chieffi et al. 1992). Located along the wall of the ocular orbit, the orbital portion contains little glandular material (Rehorek et al. 2003). The duct of the HG opens into the lacrimal canaliculi, short tubes connecting to the surface of the eye, which join to form the nasolacrimal duct (Rehorek et al. 2000c). The duct then exits the orbit encased in bone and travels laterally along the nasal capsule towards the VNO (Rehorek et al. 2000c, 2003). It then turns medially where it opens into the vomeronasal ducts (Rehorek et al. 2000c). These ducts ultimately connect the mouth cavity to the VNO allowing HG secretions and chemical stimuli from the environment to come into contact (Rehorek et al. 2000c). The lack of a direct opening to the orbit of the eye eliminates orbital lubrication as a possible function for the gland, as was once believed (Rehorek et al. 2000c). The entire pathway is shown in Figure 1.

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Histology of the Harderian Gland

At the cellular level, there are only two different types of cells in the HG of garter snakes: the acinar serous-secreting cells and the ductal mucous-secreting cells (Rehorek et al. 2003, Saint Girons 1982). These two types of cells differ in their chemical staining properties due to their differing products (Rehorek et al. 2003). Acinar secretory cells (similar to those found in salivary glands) are arranged in a circular structure called an acinus surrounding a small, oval lumen (Rehorek et al. 2003, Minucci et al. 1992). Duct cells are adjacent to each other and secrete mucous products into a large, irregularly-shaped lumen (Rehorek et al. 2003). The serous (protein-containing) secretions from the acinar cells are transported to the vomeronasal organ (Rehorek et al. 2003, Saint Girons 1982). Since the serous secretions must pass through the ducts to exit the gland, the mucous may simply act as a carrier medium for the serous proteins (Rehorek et al. 2003).

In the Western whip snake, Coluber viridiflavus, some mucous-secreting acinar cells are present in the anterior and posterior portions of the gland, but the majority of the acinar cells produce proteins (Minucci et al. 1992). The acinar cells in this species are columnar glandular cells while the secretory duct cells are pseudostratified columnar in the central duct and stratified cuboidal in the smaller ducts. The secretory duct cells have low density mucous products, the function of which is unknown (Minucci et al. 1992). The histology of neither the acinar type cell nor the mucous duct cell type has been well-described in garter snakes and no sexual dimorphism in the gland has been observed in any snake species thus far examined (Rehorek 2003, Minucci et al. 1992).

Ultrastructure

Acinar cells of garter snakes have extensive rough endoplasmic reticulum which is associated with protein production (Lodish 1988). Protein-containing secretions, after being secreted into the central lumen of each acinus, pass through to the secretory ducts which join to form larger ducts before exiting the gland (Payne 1994). The secretions ultimately make their way into the nasolacrimal duct leading then to the VNO (Rehorek et al. 2000c).

The serous cell proteins are transported through the cytoplasm in secretory granules (Chieffi et al. 1992). These occupy most of the cytoplasm of the apical portion of the cell. In the whip snake, two different types of acinar cells were discovered with differing secretory granules (Chieffi et al. 1992). Type 1 cells secreted proteins and had a well-developed rough ER with “special secretory granules” present in the cytoplasm (Chieffi et al. 1992). Type 2 cells secreted mucous and had tightly packed homogenous secretory granules of low density in the cytoplasm (Chieffi et al. 1992). Type II cells were found only in the most posterior and anterior portions of the gland (Chieffi et al. 1992).

Seasonality

Garter snakes display distinct and easily identifiable behaviors in every season (Aleksiuk & Stewart 1971). During the warm summer months, they feed to store energy for hibernation (Reichenbach & Dalrymple 1986). In Manitoba, snakes migrate back to their dens in early fall and enter hibernation shortly thereafter. Upon emergence in the spring, snakes mate using the energy stored from the previous summer (Reichenbach & Dalrymple 1986). There is little variation from these seasonal behavioral changes, however, limited mating has been noted in the fall (Aleksiuk & Gregory 1974).

It remains unclear exactly how snakes regulate their behaviors to correspond with each season. The female sexual attractiveness pheromone is present during both the mating and non-mating season, and yet the males only respond to it in the spring (LeMaster & Mason 2001, Mason et al. 1987, Smith & Mason 1997). The reproductive behavior of males is independent of circulating sex hormone control, so another method of regulation must be present (Krohmer et al. 2004). Some component of pheromone transport, signal transduction, or the central neural pathway must be altered during the non-mating season, significantly changing the response to the pheromone stimulus. One method of behavioral regulation could be a change in pheromone-binding proteins that snakes produce in their HG during different times of the year. Pheromone-binding protein production is hormonally regulated and sex-specific (Pelosi 1996). This change in production of pheromone-binding proteins could result in significant morphological changes in the acinar cell height or in the area of the acinar lumen. Studies of the HG of the pool frog, Rana esculenta, have shown that the acinar glandular cell height varies depending on its functional state, with the greatest heights seen during the periods of highest glandular activity (Baccari et al. 1991, Di Matteo et al. 1989, Minucci et al. 1989). Therefore, we would expect greater glandular cell heights during those seasons when the HG is most active.

Sexual Dimorphism

Sexual dimorphism is the difference in form of a specific body feature between males and females of the same species. This occurs throughout the animal kingdom and even in humans. The function of sexual dimorphism becomes clear when viewed in evolutionary terms. For example, larger female snakes are able to produce more offspring than smaller females and are therefore favored by natural selection (Shine et al. 2003). Larger females then have larger offspring until females are ultimately larger than males (Shine et al. 2003, Crews et al. 1985). Males, however, lack any specific relationship between their size and their fecundity so no selection pressure is placed on them to increase in size. Therefore we see a discrepancy in the size of individual male and female garter snakes.

In snakes, sexual dimorphism is present in many forms such as females being larger than males (Crews et al. 1985), males having smaller heads (Shine & Crews 1988), males having longer tails (Shine et al. 1999), females having larger cloacal glands than males (Kissner et al. 1998), and males having larger kidneys than females (Bonnet et al. 1998). Most of these differences are likely the result of differences in essential behaviors for reproductive success in that males have enlarged systems related to sexual competition and females have enlarged systems related to the storage of energy (Bonnet et al. 1998). Sexual dimorphism has not been reported at the histological level in any snake species observed (Chieffi et al. 1992, Minucci et al. 1992, Rehorek et al. 2003). Specifically, the red-sided garter snake, Thamnophis sirtalis parietalis, and the Eastern brown snake, Pseudonaja textilis, were recently examined by Rehorek et al. (2003), yet no evidence of sexual dimorphism was noted at any level. However, Huang et al. (2006), discovered that exposing female sexual attractiveness pheromone to VN neurons of males caused the neurons to respond and depolarize. Conversely, exposing the female pheromone to female VNO neurons did not elicit any response (Huang et al. 2006). Thus, there is a sexually dimorphic response to the sexual attractiveness pheromone in this species.

Garter Snakes as Models

Garter snakes (Thamnophis spp.) make excellent subjects for the study of chemical ecology because they rely heavily on chemoreception for both feeding and mating (Burghardt 1970, Kubie et al. 1978, Halpern & Kubie 1983, Halpern & Frumin 1979). Snake reproductive behavior is also noteworthy in that it is entirely dependent on the VN system (Halpern 1987). Colubrid snakes including garter snakes, have highly developed VN sensory epithelial tissue responsible for semiochemical detection and contain the largest squamate HGs (Rehorek 2000a). The garter snake has even been called a “vomeronasal specialist” because of its well-developed neural connection between the VNO and the brain (Rehorek 2000b, Halpern 1992). Garter snakes congregate in large numbers in Manitoba, Canada allowing for easy collection. Finally, garter snakes display distinct stereotypical behaviors only seen in specific seasons (Aleksiuk & Stewart 1971). The present study will examine the HG of red-sided garter snakes including comparing the gross anatomy and histology of the left and right glands within individuals; examine the presence of sexual dimorphism; and observe seasonal changes in gland structure and mass.

MATERIALS AND METHODS

Collection of Snakes with Respect to Seasonality

Adult male (15) and female (12) red-sided garter snakes were collected in the spring and fall of 2006 near Inwood, Manitoba, Canada. “Summer” male and female snakes were collected in late spring and kept under summer conditions in a temperature-controlled environmental chamber for 20 weeks prior to sacrifice. “Winter” male and female snakes were collected in late fall, housed under a temperature regime similar to Manitoba seasonal conditions, and experienced winter conditions for 10 weeks before being sacrificed. “Spring” male and female snakes were also collected in the fall and housed under winter conditions, then sacrificed 1 week after emergence at which time they were exhibiting vigorous courtship behavior.

Fall snakes were excluded from this study for a number of reasons. To begin with, there is a very low incidence of mating in the fall and only select individuals participate. Because spring animals are already being measured for the effect of mating on the HG, fall season animals are not needed for comparison. Also, it does not seem likely to find significant changes in the morphology of the gland during the fall, because fall is a transitioning period between summer and winter. In a side study, males sacrificed in the fall showed HG masses, cell heights, and lumen diameters in transition between the seasons of summer and winter. However, lacking females from the fall, these results were not recorded. It is difficult to distinguish if a female is truly acclimated to fall because there is no specific behavior they exhibit during this time. Because of these reasons, fall was excluded and the remaining seasons of winter, spring, and summer were examined.

Perfusion

All snakes were anaesthetized with a subcutaneous injection of 0.003 ml of 0.5% Brevital sodium per gram of body mass. This method of anaesthetizing snakes follows IACUC protocol for animal care. After 15 minutes, the underbelly scales were cut along the length of the body for about 6 cm, and by meticulous dissection, the heart of the snake was revealed. A 23ga. needle connected to a Rainin peristaltic pump® was carefully inserted into the posterior end of the ventricle while the right atrium was cut using microscissors. The pump circulated 10% phosphate-buffered formalin into the bloodstream, fixing tissues instantly throughout the entire body. This method fixes the tissue very quickly allowing for enhanced histological images.

Dissection

After fixation, the post-orbital scales were removed to reveal the HG with the subcutaneous posterior portion partially covered by a ribbon of muscle. This muscle was removed and the HG very carefully dissected from the orbit. Much care was taken to remove any traces of the optic nerve, orbital membrane, and nerves/blood vessels from the gland so as not to compromise the measurements of mass.

Morphometric Measurements

Snakes were weighed and snout-vent lengths measured prior to anesthesia. After the gland had been dissected from the orbit, digital calipers were used to measure specific dimensions of the gland to a tenth of a millimeter. The measurements included the width, length, and height of the enlarged posterior lobe, the thickness of the intraorbital section as well as length, width, and height of the “thumb” of the gland. The entire body of the gland including the intraorbital portion was padded dry and weighed. Both the left and right glands from each snake were measured to a thousandth of a gram using a Mettler AT400® digital scale. Due to a high degree of individual variation in the morphology of the gland, mass was chosen as the most appropriate measurement for analysis.

Embedding and Sectioning

Harderian glands were put through an ethanol dehydration series in which they were placed in progressively higher ethanol concentrations. The water in the tissue was replaced by ethanol and ultimately completely replaced by xylene. The tissue then sat in melted paraffin wax for 2 hours. The glands were arranged in specific orientations in the paraffin and allowed to harden in plastic molds. A razor blade was used to trim the excess wax and the blocks were sectioned vertically at a thickness of 10 µm using a Spencer® mictrotome. Approximately 20 sections were placed on each slide which was labeled with the specific ID number of the snake and the orientation of the glands. Slides were heat-fixed on a Precision Scientific® slidewarmer and later observed microscopically.

Staining

Between five and ten slides from of each snake were stained using Harris’ hematoxylin and eosin. The slides with tissue ribbons were placed in a rehydration process in which they were placed in progressively lower ethanol concentrations and ultimately water. Hematoxylin was then used to stain the slides for approximately 3 minutes followed by a water rinse and finally eosin for 1 minute. The slides were then dehydrated again using the steps described above in the embedding process. After the application of the binding product Permount®, the slides were covered with cover slips and allowed to dry.

Structural Anatomy

Snakes previously fixed in 10% buffered formalin were decapitated and decalcified in Jenkin’s fluid to soften the bones of the skull. The heads were then dehydrated through the process described above, and were embedded in the same manner as before. Several snake heads were sectioned vertically while others were sectioned horizontally to observe the anatomy of the gland in multiple dimensions. Sectioning, mounting, and staining were as described in the previous section.

Histological Measurements

Adobe Photoshop 5.0® was used to capture images off the Olympus Vanox-T® microscope after which Image Pro Discovery 4.5® was used to measure the cell heights and the lumen diameters. Acini were chosen for their circular shape and clarity of structure. The first 20 acini meeting these requirements were measured in both the left and right gland.

Statistics

Paired t-tests were used to compare the left and right gland measurements of HG mass, acinar cell height, and lumen diameter to determine significant differences, if any. Left and right glands were not statistically significantly different within an individual so the measurements were combined to give a total of 40 acini measurements of both cell height and lumen diameter per individual. A linear regression was used to generate the residuals of HG mass versus head length after the regression was checked for normality. A two-way ANOVA was used to examine HG mass residuals, with sex and season as factors. Two-way ANOVAs were also performed on cell height and lumen diameter with sex and season as factors. All statistics were performed using SigmaStat 3.1®.

RESULTS

Anatomy/Gross Morphology

The HG of garter snakes is a white, slightly yellowish structure found under the eyeball and postorbital scales. The majority of the gland is beneath the postorbital scales and a thin layer of muscle. Upon removal of the scales and muscle, the HG is surrounded by a thin connective tissue capsule and resembles a “mitten” in shape from the lateral view (Fig. 2). A small portion of the gland resembling the “thumb” of the mitten was seen in nearly every specimen; however the relative size and shape of this feature along with the rest of the gland was highly variable between individuals. The postorbital portion of the gland is ridged and consists of incomplete lobules that are held together by connective tissue which concurs with the anatomical observations made by Chieffi et al. (1992). The postorbital portion is generally about 1.5 mm thick and extends approximately 3.4 mm posteriorly from the postorbital bone. Projecting anteriorly under the postorbital bone and into the orbit, the gland follows the wall of the orbit and compresses into a thin ribbon medial to the eye. This ribbon has two small protuberances that vary in shape as well as orientation. These encircle the optic nerve as it exits the orbit caudally. The gland continues along the back wall of the orbit and follows it around the eye until it is positioned anteriorly to the eye. Several nerves as well as a small blind-ended segment of the gland exit the orbit through a deep anterior and slightly more dorsal opening in the orbit, while the nasolacrimal duct exits through a lateral anterior opening of the orbit. As described by Rehorek, the duct is encased in bone and travels along the nasal capsule before finally making a medial turn and meeting up with the vomeronasal ducts (Rehorek et al. 2000c).

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India ink injected into the HGs of several anaesthetized snakes was seen flowing out of the vomeronasal ducts into the snakes’ mouths (Fig. 3). This experiment shows that the HG secretions do indeed reach the mouth and are ultimately deposited on the tongue. Stimulation of the roof of the mouth with a blunt probe caused the snake to repeatedly suck the ink in and out of the vomeronasal ducts very quickly while the mouth was still open. The mechanism by which snakes transport the pheromones into the ducts remains unknown; however, this observation lends support to the hypothesis that perhaps suction is the mechanism by which transport of odorants into the VN ducts is accomplished (Meredith & O’Connell1979, Meredith et al. 1980).

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Histology

The HG of garter snakes is a lobular, compound acinar gland with mostly uniform, slightly pyramidal acinar cells surrounding a small, centrally located lumen. These cells appear pinkish red when stained with hematoxylin and eosin and appear very dense because of numerous secretory granules present in the cytoplasm (Fig. 4). Generally 14 cells comprise an acinus. The nuclei stain dark red and are basally located. The acini are nearly spherical with lumen diameters approximately 30% the length of the cell height. Connective tissue appears between the acini and a membrane encompasses the entire gland. The secretory duct cells are columnar in shape and surround a lumen that varies in shape and area. The nuclei of these cells stain a dark purple while the cytoplasm stains extremely lightly and remains nearly transparent. The gland is made up of protein-secreting serous acinar cells with scattered mucous-secreting cells interspersed among them. The duct cells of the gland are not very numerous and are often clustered together as noted by Chieffi et al. (1992). The number of acini per unit area did not appear to change throughout the year.

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Difference between Left and Right Glands

No differences were found between the left and right HGs of the garter snakes observed. The masses of the HGs from both sides of the animal were also not significantly different (p = 0.861). Histologically, neither the cell height (p = 0.406) nor the lumen diameter (p = 0.421) were significantly different. Therefore, the left and right glands can be considered equivalent for this study, and the data for the left and right glands were collapsed into a single group for further analyses.

Sexual Dimorphism

Sexual dimorphism exists in the HGs of garter snakes when examined with respect to season. In every season, males have larger HGs than those of females with similar head lengths (p = 0.012) (Fig. 5).

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Acinar cell height of males is significantly greater than that of females (p < 0.001) (Fig. 6). Males also have larger average residuals for HG mass than females (p = 0.012) (Fig. 7). The same pattern holds true for the lumen diameter with males having a significantly larger diameter than that of females (p = 0.021) (Fig. 8). Therefore, male garter snakes have significantly larger HGs, acinar cell heights, and lumen diameters than those of females with similar head lengths. There was a striking difference between male and female acinar cell height in spring, with males having significantly larger cell heights than females (p < 0.001) (Fig. 6). These observations are the first to report sexual dimorphism in the HG of any snake species.

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Seasonal Change

Harderian gland mass in summer was significantly higher than the mass of the HG in the winter and spring (p = 0.009, p = 0.032 respectively) (Fig. 7). There was no significant difference between the masses of the HGs in the winter and spring (p = 0.836).

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Acinar cell height was different in every season (p < 0.001). The largest acinar cell height was seen in the summer, followed by a large drop in the winter, and a slight increase in the spring (Fig. 6). For males, the cell height was different in every season, being largest in the summer and smallest in the winter (p < 0.001, p < 0.005 respectively). For females, the acinar cell height did not change significantly in every season, but instead was only significantly different when comparing summer to winter and spring (p < 0.001).

The lumen diameter followed the overall trend of being largest in the summer while significantly decreasing in spring and winter (p < 0.001). The lumen diameter of males was significantly larger than that of females (Fig. 8). Interestingly, the lumen diameter seemed to decrease very slightly in spring, however the decrease was not significant until compared to summer (p < 0.001).

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CONCLUSION AND DISCUSSION

Anatomy/Gross Morphology

The anatomy of the HG of the red-sided garter snake was found to be fairly accurately described by Rehorek et al. (2000c, 2003). Anatomical observations of the incomplete lobules revealed one specific lobule on the most dorsal side of the gland referred to earlier as the “thumb” of the mitten-shaped gland. In addition, the HG was found to be highly variable in morphology between individuals, including changes in the shape of the thumb, the horn-like protrusions on the most posterior part of the gland, and protuberances on the section immediately posterior to the orbit of the snake. The cause for this high degree of variability is unknown. In general, the HG of squamates varies in shape between species (Rehorek et al. 1993). According to Rehorek et al. (2003), there seems to be a minimum size requirement for the HG. The smaller orbit size of garter snakes may have caused the development of the postorbital portion (Rehorek et al. 2003). Future research should examine the variation throughout the gland and try to elucidate the reasons for this variability. A starting point would be to compare the different lobules and note any changes in activity throughout different periods of the year and under different steroid hormone treatments, as steroids likely play a role in the regulation of the gland (Minucci et al. 1994, Di Matteo et al. 1995, Chieffi-Baccari et al. 1993, Variale et al. 1992).

The mass of the HG was greatest in the summer, but not significantly different between winter and spring. One explanation could be the change in cell height in the summer. Harderian gland cell height also increases dramatically in the summer while winter and spring are essentially the same. While the number of acini remains essentially the same, the increase in cell height results in more tissue and thus more mass. During the longer summer period, the amount of binding proteins needed for feeding is much greater than that needed for the relatively brief spring reproductive period. It seems very likely that different pheromone-binding proteins are needed to bind the sex pheromones than the earthworm chemoattractant. Therefore, the pheromone-binding proteins produced in the spring and their quantities would be expected to be different than those in the summer. The data collected in this study suggest that pheromone-binding proteins are produced by males only, and that the duration of their production is limited to the spring. Both males and females produce prey chemoattractant-binding proteins and due to the longer summer feeding period, it would be expected that the HGs of both sexes would be most active at this time. Finally, little is known about either the pheromone-binding proteins or the chemoattractant-binding proteins, nor their structure, so future research should seek to identify the proteins responsible for binding the sex pheromones and the prey chemoattractants.

Left and Right Harderian gland Similarity

It is not surprising that the left and right HGs of garter snakes are very similar in their structure, in particular the mass, cell height, and lumen diameter. Although small variations in morphology do occur in the same animal, the overall features of the glands are conserved on both sides of the body. None of the previously published studies of the HG in this species compared the left and right glands within individuals (Rehorek et al. 2000c, 2003, Huang et al. 2006). It is known however, that each gland is connected only to the VN duct on the same respective side of the body. Autoradiographic labeling of injected compounds into the HG appear only in the VNO of the respective side injected (Rehorek et al. 2000c). Therefore it is likely that each HG is responsible for providing the necessary pheromone-binding proteins only to the VN ducts on their respective side of the snake. The left and right HGs of snakes likely function identically and independently and contribute equally to the function of the vomeronasal system.

Sexual Dimorphism and Behavior

The HG of male garter snakes functions in the transport and solubilization of both prey chemoattractants and sex pheromones (Huang et al. 2006). Male VNO neurons respond to both Earthworm-Shock Secretion and female sexual attractiveness pheromone (Huang et al. 2006). Female garter snakes however, only respond to the Earthworm-Shock Secretion and therefore their HGs likely only function in feeding (Huang et al. 2006). Female snakes have never been observed to respond to the sexual attractiveness pheromone (Huang et al. 2006). If females do not need their HG for reproduction, the largest difference in glandular activity between males and females should be in the spring when female HGs are not active and those of males are very active. This trend was seen in the data collected and supports the hypothesis that males have a dual role for their HGs while the gland of females plays only a single role. While females show a significant increase in gland activity in the summer due to feeding and decreased levels in the other seasons, males show high levels of activity both in the spring and summer. The glands of males must function at both times of the year due to mating in spring and feeding in summer. Moreover, when comparing acinar cell heights in springtime, males have significantly greater cell heights than females. Interestingly, the production of pheromone-binding proteins has been shown to be hormonally-controlled and sex-specific (Pelosi 1996). Therefore, the proteins needed to solubilize the sex pheromone may only be present in males. The higher activity seen in the spring is likely a result of the males producing pheromone-binding proteins in order to respond appropriately to the sexual attractiveness pheromone, while the lowered activity in females reflects a lack of this production.

In general, males have larger HG masses, cell heights, and lumen diameters than females. One could hypothesize that the dual requirements of males would necessitate a larger gland than females. Also, a more highly developed HG could be selected for in males because it would allow males to quickly identify the best mates. Whatever the mechanism by which it occurs, the increase in all three parameters of mass, cell height, and lumen diameter is very apparent. If cell height increases and the number of acini remains relatively the same, the mass of the gland increases as seen in summer. Also, larger lumen diameters could be a result of increased flow of secretions in the summer months. The prey chemoattractant binding proteins are used extensively in exploratory behavior while searching for food. Thus, both males and females have larger lumen diameters in summer to accommodate the increased binding-protein production and the corresponding mucous carrier medium. Future research should address these issues and attempt to account for the differences seen in each season. No other studies thus far have shown lumen diameter to vary between males and females.

Sexual dimorphism has been seen in the HG of the European green toad, Bufo viridis (Minucci et al. 1989). The acinar cells of the female HG contain lipid droplets in the smooth endoplasmic reticulum while the cells of the males do not (Minucci et al. 1989). Also in hamsters, lipid droplet production, porphyrin levels, vacuole size, and protein sizes are dimorphic between males and females (Buzzell 1996, Bucana & Nadakavukaren 1972, Elofsson et al. 1997). Therefore, the HG is sexually dimorphic in other animals, but this has not been noted previously in garter snakes (Rehorek et al. 2003). The sexual dimorphism observed in this study is likely due to the dual role of the HG of males to solubilize both prey chemoattractants and sex pheromones while the gland of females likely only solubilizes prey chemoattractants.

Seasonality and Behavioral Significance

Considering that the reproductive cycle of garter snakes is seasonal, it is not surprising that the HG itself appears to be seasonally regulated. Multiple accounts of HG seasonality have been found in frogs, toads, and geckos (Di Matteo et al. 1989; Minucci et al. 1990, 1991; Baccari et al. 2000). The HG secretory activity of the pool frog, Rana esculenta, was found to be directly modulated by temperature and most active during the hottest months of the year as determined by greater cell height (Minucci et al. 1990, Di Matteo et al. 1988, 1989, Chieffi et al. 1992). In addition, the mast cell number of the HG of the pool frog is cyclic, reaching its peak in the summer months (Baccari et al 1991). The seasonal pattern of high summer activity found in frogs coincides with that in garter snakes in that summer months had the highest levels of secretory activity (as reflected by larger total masses and taller cell heights). There is, however, some variation in highest secretory periods of the HG. Using Mallory stain in the gecko, Tarentola mauritanica, the presence of “blue nuclei” in the HG secretory cells indicated a more intense secretory activity in April than in July (Baccari et al. 2000). However, April is the beginning of the mating season, which may be related to high secretory levels (Picariello et al. 1989). It is somewhat difficult to compare frog, gecko, and snake HG studies as the HG of frogs and geckos appears to function in orbital lubrication while this is not anatomically possible for garter snakes (Rehorek et al. 2003). Instead, the HG in garter snakes likely has an entirely different additional function; that of solubilizing pheromones (Huang et al. 2006). This difference in function may also explain the difference in lumen diameters at the peak secretory periods. The HG of frogs in the summer had the greatest glandular cell heights and narrower lumens (Minucci et al. 1990) while garter snake glandular cells in summer had the greatest heights and largest lumens. One interesting similarity between pool frogs and garter snakes was that the average acinar cell height was 15.36 μm for red-sided garter snakes and 15.31 μm for frogs (Minucci et al. 1990). It appears that the HG acinar cell height may be somewhat conserved across species of similar size, regardless of gland function.

Because more secretory activity is present in the summer months than other seasons, it appears that more is demanded of the HG for prey detection than for sexual behavior. This could be simply a difference in the flow rate needed to bind the prey chemoattractants. Future research should attempt to isolate the HG secretions and identify the different binding proteins. The seasonality of the entire VN system should also be studied to identify the means by which it is regulated. The pathway contains multiple areas where regulation could take place such as the pheromones themselves, the proteins that bind them, the transduction pathway in the VNO sensory epithelia, and the action potentials being sent to the brain. Hormones, especially gonadal steroids, also likely regulate the level of activity in the HG. Several endocrine studies have been conducted on the hormonal control of the HG of the pool frog, but it has not been studied to date in garter snakes (Minucci et al. 1994, Di Matteo et al. 1995, Chieffi-Baccari et al. 1993, Variale et al. 1992). Seasonal studies may also show differing levels of activity throughout the year on different areas of the gland, particularly the thumb of the gland. Future research on the HG secretions would allow us to better understand the mechanism by which pheromones and other semiochemicals interact with proteins and how this interaction orchestrates behaviors critical to life history events in vertebrates.

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Figure 7. Residuals of male and female garter snake Harderian glands compared over three seasons. Residuals were calculated from the general linear regression of Harderian gland mass against head length in males and females (n = 5 males, 4 females per season). Bars indicate standard error.

Figure 5. Linear regression of Harderian gland mass against head length. Harderian gland mass against head length for male and female red-sided garter snakes is depicted (n = 27).

Figure 6. Cell height of acini in male and female garter snakes. Mean cell heights of acini from male and female garter snakes compared over three seasons (n = 5 males, 4 females per season). Bars indicate standard error.

Figure 8. Lumen diameter of acini for male and female garter snakes compared over three seasons (n = 5 males, 4 females per season). Bars indicate standard error.

Figure 2. The gross anatomy and morphology of the Harderian gland. The large lobular portion on the right is located just underneath the postorbital scales while the ribbon-like section on the left is located behind the eye along the orbit wall. Secretions would flow from right to left in this image.

Figure 3. Black India ink injected into the Harderian gland. Ink is seen flowing out of the roof of the mouth through the vomeronasal ducts. Secretions from the gland follow the same pathway moving down the nasolacrimal duct into the vomeronasal duct and finally onto the tongue.

Figure 4. The histology of the Harderian gland of garter snakes at 200X. The red-staining acinar cells make up the bulk of the gland while the translucent duct cells are dispersed among the acinar cells. The cells on the far right of the image show acinar cells transitioning into a duct.

Females

Males

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Female

Male

Female

Male

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Female

Male

Thumb

Orbital portion

Postorbital lobe

Ink from VN ducts

Acinus

Duct

1 mm

30 μm

Figure 1. The vomeronasal system of the red-sided garter snake.

HG = Harderian gland NC = Nasal cavity

LC = Lacrimal canal VNO = Vomeronasal organ

LD = Lacrimal duct

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