Discussion
The eyelids serve as integumentary duplications or folds of skin around the eyes, acting as a physical barrier in terrestrial vertebrates to protect against mechanical damage and desiccation while regulating light passage through the pupil (Johnson, 1927; Walls, 1942). Studies by Yasui et al. (2006) have confirmed that eyelids protect the eyeball and maintain a moist surface\RL.
The anatomical structure of the upper eyelid of T. mutabilis resembles that of the lower eyelid. However, the lower eyelid in this agama species is more mobile and can move downward through the contraction of the depressor palpebrae inferioris muscle, attaching to its base. Additionally, the lower eyelid appears larger than the upper eyelid, a common trait observed in reptiles and birds compared to mammals, where the upper eyelid is typically more mobile and larger than the lower one (Wyneken, 2012; Klećkowska-Nawrot et al., 2019)\RL.
Eyelids are movable structures supported by a connective tissue plate called the tarsal plate or tarsus (tarsus palpebralis). In most taxa, including lizards, birds, and mammals, the tarsal plate is described as a dense fibrous connective tissue, possibly containing cartilage, found within one or both of the upper and lower eyelids (Rieppel, 2000). Wyneken (2012) noted the presence of a tarsus in the eyelid of most lizards and crocodilians, with a bony structure in the upper eyelid of crocodilians and a cartilaginous structure in the lower eyelid of most lizards. However, turtles and tortoises lack these structures. In birds, the tarsal plate is located only in the lower eyelid and consists of dense connective tissue with elastic fibers (Klećkowska-Nawrot et al., 2016). In mammals, the tarsal plate is present in the upper and lower eyelids, comprising dense fibrous connective tissue (Klećkowska-Nawrot et al., 2019).
The eyelids of reptiles are covered by epidermal scales, displaying various patterns, shapes, thicknesses, and degrees of overlap among different species (Guerra-Fuentes et al., 2014; Wegener et al., 2014; Toni et al., 2007; Calsbeek et al., 2006). In T. mutabilis , the external surface of both upper and lower eyelids features highly polygonal scales of varying sizes and forms. The scales on the superior extension are notably large and plate-like acting as an umbrella for the eyes, serving as heat exchangers and aiding in water conservation in arid environments\RL.
Integument sense organs were first described in reptiles by Leydig (1868) in the lizard genera Lacerta and Angus and the snake genus Coronella . He identified small depressions on the surface of their scales, referred to as ”organs of the 6th sense,” and compared them to the taste buds of fishes and amphibians. Scortecci (1941) examined these receptors in detail in agamids, iguanids, and other lizards, distinguishing two principal types based on their structure: the first type has the Oberhäutchen with a bristle (or seta, or hair), while the second lacks bristles. The first type is typical of the integument of pygopodids, agamids, and gekkonids (Landmann, 1975)\RL.
The sense organs with the Oberhäutchen and bristle appear on the tips of some scales of the Egyptian agama’s eyelids. These organs may serve as points of contact with the external environment. Maclean (1980) and Sherbrooke and Nagle (1996) suggested that these sense organs might work in conjunction with visual and other sensory systems to detect, locate, and apprehend prey, as noted by Barrett et al. (1970) in Crotaline and Boid snakes\RL.
The present study reveals that the structure of the two eyelids inT. mutabilis is very similar. The outer skin surface of the eyelid consists of two to four nucleated cell layers of keratinized stratified squamous epithelium, while the internal surface comprises two to five nucleated cell layers of stratified squamous epithelium. Additionally, the distribution of pigment cells shows significant variability within the dermal layer, with large black melanophores (light-absorbing pigment) and dendrites that invaginate into the stratum basal of the upper and lower eyelids. These melanophores increase in number within the superior extension of the upper eyelid. Brownish-yellow iridophores (reflecting pigments) are scattered in large numbers within the dermis of the upper eyelid, its extension, and the lower eyelid. Saenko et al. (2013) and Teyssier et al. (2015) pointed out that these types of pigmented cells (iridophores) in reptiles participate in skin coloration and thermoregulation.
The third eyelid, known as the nictitans eyelid or nictitating membrane, is a prominent semilunar fold of the conjunctiva in Elasmobranchii fish, amphibians, reptiles, birds, and mammals (except humans) (Klećkowska-Nawrot et al., 2019). The nictitating membrane is sometimes lost in various taxa due to ecological differences, such as in whales, echidnas, opossums, marsupial moles, largely burrowing squamates like pygopodids, amphisbaenians, and dibamids, as well as chameleons and snakes (Caprette et al., 2004)\RL.
According to Schramm et al. (1994), the size of the nictitating membrane is inversely proportional to an animal’s ability to remove foreign bodies from its eyes. This membrane, known as the haw, is well developed in ungulate mammals such as horses and cows but relatively small in carnivores like canines and felines. Cats can efficiently remove foreign bodies from their eyes using their paws. In aquatic animals, such as fishes and some amphibians, the anterior portion of the eye is cleaned by surrounding water, making the nictitating membrane unnecessary. The most prominent nictitating membranes are found in reptiles and birds exposed to wind, storms, dust, and sand (Schramm et al., 1994). A well-developed nictitating membrane is essential for keeping the corneal portion of the eye clean in species that cannot do so otherwise. In lizards, a well-developed nictitating membrane, associated with nictitating cartilages, completely covers the cornea, as seen inColobosaura modesta and Tretioscincus oriximinensis (Guerra-Fuentes et al., 2014). Conversely, this membrane is reduced to a small projection at the base of the cornea and eyelid inAlopoglossus angulatus , Cercosaura ocellata , andMicrablepharus maximiliani (Guerra-Fuentes et al., 2014), similar to T. mutabilis . Rehorek et al. (2000) reported that reptilian species with a spectacle have lost the nictitating membrane, as inNothobachia ablephara and Calyptommatus sinebrachiatus \RL.
The nictitating membrane in squamates is a movable fold that slides over the cornea (Walls, 1942). The exocrine secretion of the Harderian gland, attached to the medial region of the orbit (Guerra-Fuentes et al., 2014), lubricates this sliding movement (Payne, 1994). Oria et al. (2015) stated that the spreading of the tear film (orbital secretion) is accomplished by nictitating membrane movement rather than eyelid movement.
Crocodiles possess a well-developed nictitating membrane that moves obliquely backwards and slightly upwards. These animals often move the nictitating membrane across the eye without closing their eyelids. When closing the eye, the lower lid closes after the nictitating membrane has moved across the eye, unlike most other reptiles, where the membrane and eyelids move simultaneously (Johnson, 1927). In anura, the nictitating membrane moves from below upwards rather than horizontally or obliquely from the inner angle across the eye, as seen in all birds and many mammals (Johnson, 1927). Stibbe (1928) noted that birds require the fastest method for this movement, while mammals prioritize thoroughness over speed\RL.
Hiller (1995) reported that the conjunctival outer morphology of agamid species is mainly characterized by epithelial folds running parallel to the eyelid margins. These folds are present in all agamid species and contain scattered goblet cells that release secretory products onto the conjunctival surface. The goblet cell content in T. mutabilis is mucus, similar to the iguanid conjunctiva that mentioned by Hiller (1995)\RL.
Goodrich (1988) stated that extraocular muscles, responsible for eyeball movement, are consistent in number, arrangement, and innervation across vertebrates. El Hassni et al. (2000) noted that chameleons and higher vertebrates use the same musculature, but the performance of each type of movement varies according to lifestyle. Reptiles have six extrinsic eye muscles (medial rectus, lateral rectus, superior rectus, inferior rectus, inferior oblique, and superior oblique) responsible for rotating the eyeball within the orbit (Kardong, 2012). These muscles are characterized by fine movement and coordinated control of the eyes with the vestibular system (Kardong, 2012; Schwab, 2012). Eye movement is essential to prevent photoreceptor fatigue (Schwab, 2012). Turtles, crocodilians, and most lizards (except Heloderma) have mobile eyes, while snake eyes are immobile (Underwood, 1970).
The dorsalis rectus muscle and dorsalis oblique muscle in T. mutabilis are divided into two parts with different origin sites on the orbitosphenoid and parabasisphenoid bones but share the same insertion in the sclera and upper eyelid. The eye of T. mutabilis lacks levator muscles (levator palpebrae superioris), so the dorsalis rectus muscle and dorsalis oblique muscle are responsible for elevating the upper eyelid.
In reptiles, the protective reflex caused by the retraction of the eyeball in response to corneal stimulation (Oelrich, 1956; Tansley, 1965) seems to be accomplished by simultaneous contractions of two retractor muscles, the retractor bulbi and bursalis, as observed in the lizard Varanus (Barbas-Henry and Lohman, 1988; El Hassni et al., 2000). Retractor oculi and protractor oculi, known as the levator bulbi or retractor bulbi muscle in reptiles, are inserted on the sclera just adjacent to the optic nerve and are responsible for inward and outward eye movement within the socket (Wyneken, 2012). The present results showed that the retractor bulbi and bursalis muscles of T. mutabilis are inserted on the scleral cartilage, with their origin sites lying ventral to the optic nerve. These muscles pull the eyeball inward and outward within the socket.
Retractor bulbi muscles are synapomorphic for tetrapods and are subsequently lost in snakes and birds. Losing these muscles in snakes results in a condition convergent with primitive aquatic animals. This loss implies visual reduction, similar to the loss observed in large-eyed forms with great visual acuity, like birds (Caprette et al., 2004).
Bour et al. (2000) stated that in the absence of retractor bulbi muscles, retraction is produced by the simultaneous contraction of multiple extraocular muscles. In some reptiles, the retractor bulbi is assisted by one of two additional muscles: the quadratus (in lizards) or the pyramidialis (in crocodiles and turtles). In birds, the globe of the eye fits so tightly into the eye socket that little room is left for retraction. Therefore, the retractor bulbi is not present and is replaced by both quadratus and pyramidalis muscles. These muscles slide the nictitating membrane over the cornea without retraction of the eye (Butler and Hodos, 2005).
In terrestrial mammals, well-developed retractor muscles are usually associated with the presence of a nictitating membrane (Duke-Elder, 1958; Spencer and Porter, 2006). Retractors are absent in some mammals, such as the aardvark, flying fox, and primates. In mammals, the retractor bulbi can be divided into two (mouse), three (dog), or four (cat) muscle bellies (Matheus et al., 1995; Meshida et al., 2020).
In conclusion, all these characteristics that mentioned above are well-suited for the remote desert environment in which the agama lives.