Histological and histochemical characterisation of the salivary glands of the palatine fold and the mandibular venom gland of the Komodo dragon (Varanus komodoensis)

Abstract

The Komodo dragon (Varanus komodoensis) is the largest living lizard whose hunting strategy allows it to attack large animals. It inflicts serious damage to its prey with specially developed teeth, called ziphodonts. Although the mandibular venom gland system was previously detected in the Komodo dragon, there is still a lack of its histochemical analysis. Thus, the objective of the current study was a detailed description of the mandibular venom gland of this species. In addition, a histological examination of the salivary glands of the palatine fold was performed. The research material was collected post-mortem from the captive adult female Komodo dragon. Hematoxylin and eosin and Masson-Goldner trichrome staining methods were used for the histological analysis of the glands, while periodic acid-Schiff, alcian blue pH 1.0, alcian blue pH 2.5, alcian blue pH 2.5 PAS and Hale’s dialysed iron methods were included for the histochemical study. The venom gland was composed of distinctly marked and very numerous individual lobes surrounded by dense, irregularly structured, highly developed connective tissue that forms the interlobar septa. The salivary glands of the palatine fold were surrounded by a thick connective tissue capsule made of dense, irregularly structured connective tissue. The single ducts of the mandibular venom gland open into the sheaths surrounding the consecutive teeth. The presence of numerous muscle cells in the stroma of the venom gland between its lobes may indicate their participation in the emptying of the vesicles of their secretion.

Background

Venoms are widespread in the animal kingdom. They occur in fish, insects, amphibians, and, although rarely, in mammals (Całkosiński et al. 2009; Morgenstern and King 2013; Arbuckle 2020; Ligabue-Braun et al. 2012). The use of venom can be defensive or offensive in hunting. In reptiles, venom has been found in snakes, Heloderma lizards, Monitor lizards, and Iguana (Fry et al. 2006; Koludarov et al. 2012). In the case of Heloderma, venom glands are found in Heloderma suspectum and Heloderma horridum. In the case of Monitor lizards, the presence of venom has been confirmed in Varanus komodensis and Varanus niloticus stellatus, while in iguanas it was detected in Iguana iguana (Fry et al. 2010a, b; Sanggaard et al. 2015). The extinct bird-like raptor Sinornithosaurus has been hypothesized to have venom. This conclusion is based on the presence of structures that could have served to drain venom from the surface of the maxillary teeth (Gong et al. 2009). Therefore, these teeth would resemble snake teeth specialized in venom depositing (Palci et al. (2021). Venom in modern reptiles is produced by modified salivary glands in the oral cavity. In snakes, they are located in the maxilla and in lizards, in the mandible (Fry et al. 2006, 2009); Sunilkumar et al. 2016). In Sinornithosaurus, they would appear in the jaw (Gong et al. 2009).

The Komodo dragon is the largest living lizard. Its body weight can reach about 80 kg. The Komodo monitor was recorded on the islands of Komodo, Rinca, Padar, Gili Motang, Gili, Dasarni and Flores (Ciofi and de Boer 2004; Jessop et al. 2007). The adult lizard also hunts large prey such as Rusa deer (Rusa timorensis) and wild pigs (Sus scrofa). Although its bite force is relatively weak, its hunting strategy allows it to kill large animals. It inflicts serious damage to its prey with its specially developed teeth, called ziphodonts (Moreno et al. 2008; Janeczek et al. 2023). Teeth break the continuity of the integumentum communae and cause extensive damage to the soft tissues, facilitating the penetration of the venom into the victim’s body (Fry et al. 2009). The venom of the Komodo dragon is a mixture of many bioactive proteins. In the Komodo dragon, classes of toxin identified as AVIT, cysteine-rich secretory proteins (CRISP), kallikrein, natriuretic peptide, and type III phospholipase A2 protein scaffolds have been found. They have multidirectional effects, including blood clotting, hypotension, and shock induction (Fry et al. 2009, 2012). Although the participation of microorganisms contained in the saliva of the dragon cannot be completely ruled out in weakening the victims, it seems that the action of the venom plays a key role (Fry et al. 2009, 2012). Not all researchers agree with this concept; they believe that the ambush attack and injuries inflicted cause massive bleeding, so the role of the venom in killing the victim would not be particularly important, and the primary function of the venom was to participate in digestive processes (Arbuckle 2009).

In reptiles, salivary glands of mucous and seromucous nature are present in the oral cavity. The presence of unicellular and multicellular glands has been noted. In lizards, the lower labial glands are transformed into venom glands (Baccari et al. 2002; Srichairat et al. 2022). The location and general structure of the Komodo dragon venom gland have been described by Fry et al. (2006); however, detailed histochemical characterization of this gland is lacking.

Case Presentation

Animal

The 7-year-old female Komodo dragon (Varanus komodoensis) was collected from the Wrocław Zoological Garden. The cause of death of the examined lizard was not related to pathological changes in the oral cavity (Janeczek et al. 2023). Research material was obtained after the natural death of the animal. According to Polish and European law, studies of tissues obtained after post-mortem do not require the approval of the Ethics Committee (Journal of Laws of the Republic of Poland, the Act of January 15, 2015, on the protection of animals used for scientific or educational purposes (Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes). General observation of the mucosa of the hard palate, buccal vestibule, and floor of the oral cavity, the sheath surrounding the mandibular teeth, and the venom gland was conducted with a stereoscopic Zeiss Stemi 2000-C microscope (Carl Zeiss, Jena, Germany).

Histological and histochemical examination

Samples of the hard palate mucosa, buccal vestibule and floor of the oral cavity, the sheath surrounding the mandibular teeth, and the venom gland were collected. The research material was placed in 4% buffered formaldehyde for at least 72 h and then rinsed in tap water for 24 h. The samples were then processed in a vacuum tissue processor– ETP (RVG3, Intelsint, Italy) and embedded in paraffin. The samples were cut using a Slide 2003 (Pfm A.g., Germany) sliding microtome in 4 μm sections. Hematoxylin and eosin and Masson-Goldner trichrome staining methods were applied. The slides obtained were then observed using the Zeiss Axio Scope A1 light microscope (Carl Zeiss, Jena, Germany) and assessed by a scoring system that was based on a standard protocol previously described (Burck 1975). Histochemical evaluation of the examined structure was performed according to Spicer and Henson (1967), where (–) indicated a negative reaction; (–/+) and (+) a weak reaction; (++) a mild reaction and (+++) a strong reaction. The following histochemical stains were performed: periodic acid-Schiff (PAS) visualizes glycans, glycoconjugates, and neutral glycoproteins, alcian blue pH 1.0 (AB pH 1.0) characterizes strongly sulfated mucosubstances, the alcian blue pH 2.5 (AB pH 2.5) identifies acid sialylated glycosaminoglycans, the alcian blue pH 2.5 PAS (AB pH 2.5 PAS) visualizes sulfated and carboxylated acid mucopolysaccharides, sulfated and carboxylated sialomucins (glycoproteins) (blue colour), and neutral mucins (magenta colour), and the Hale’s dialysed iron (HDI) identifies sulfated acid mucosubstances (SAM) and carboxylated acid mucosubstances (CAM) (Burck 1975; Spicer and Henson 1967).

Macroscopic analysis

Fig. 1.

Photomacrographs of the maxilla and mandible of the Komodo dragon (Varanus komodoensis). a - head with numerous osteoderms - dorsal view; b - maxilla with palate - lateral view (palatine fold - black arrow); and mandible - ventral view; c - maxilla with numerous caudally orientated teeth and palatine fold with partially removed mucosa, as well as the palatine fold glands (orange arrow); d - mandible with the teeth and well visible openings of the salivary glands; the mandibular venom gland is located beneath the mucosa (area of pink oval with arrows) (Janeczek et al. 2023); e - magnification of the mandibular region with several teeth; the venom gland is covered with mucosa; see the single ducts of the mandibular venom gland (green arrows); f - magnification of the mandibular region with two teeth surrounded by a cuffs (blue asterisks)

Salivary glands - palatine fold

The salivary glands of the palatine fold were arranged in large glandular packets composed of 3–4 to 6–7 lobes. These packets were surrounded by dense irregularly structured connective tissue. Numerous blood vessels were observed in the interlobar septa. The glandular lobes were made up of secretory tubules. The mucous cells that built the tubules were low pyramidal cells with a wide base. In the basal part of the cells, there were kidney-shaped cell nuclei. The mucous tubules were surrounded by myoepithelial cells (Fig. 2a-d).

Fig. 2.

Salivary glands of the palatine fold of the Komodo dragon (Varanus komodoensis). l – lobes, dct – dense connective tissue, bv – blood vessels, icd - intercalated duct, is – interlobar septa, mc – mucous cells, mec - myoepithelial cells (arrows). a, c, d – H&E stain; b – Masson-Goldner trichrome stain; e – PAS stain; f – Alcian blue pH 1.0 stain; g – Alcian blue pH 2.5 stain; h – Alcian blue pH 2.5/PAS stain; i – HDI stain. Scale bars: a – 200 μm; b, e-i – 100 μm; c – 20 μm, d – 5 μm

The PAS method showed a negative reaction in glandular units. AB pH 1.0 and AB pH 2.5 stains showed a strong (+++) positive reaction in the mucous tubules. AB pH 2.5/PAS staining showed a strong (+++) positive blue colouration in the mucous units. The HDI method resulted in a moderately strong (+++) positive reaction (Fig. 2e-i).

Mandibular venom gland

Venom glands are made up of distinctly marked and very numerous individual lobes surrounded by highly developed, irregularly structured, dense connective tissue that forms the interlobar septa. Numerous muscle cells were observed between the individual lobes. Numerous blood vessels were present in this connective tissue. Each lobe was composed of serous acini characterized by tall conical cells with an oval-shaped nucleus. In addition, numerous intercalated ducts lined with a single-layered cubic epithelium were observed. The intercalary duct passed into a striated duct lined with a single-layered cylindrical epithelium, which extended into an interlobular duct lined with a multi-row epithelium. A single excretory duct lined with a double-layered columnar epithelium emerged from each lobe of the venom glands, which in its terminal section passed into multilayered squamous epithelium (Figs. 3a-d).

Fig. 3.

Mandibular venom gland of the Komodo dragon (Varanus komodoensis). l – lobes, dct – dense connective tissue, ed - excretory duct, mm – muscles, is – interlobar septa, sa – serous acini. a, d – H&E stain; b, c – Masson-Goldner trichrome stain; e – PAS stain; f – Alcian blue pH 1.0 stain; g – Alcian blue pH 2.5 stain; h – Alcian blue pH 2.5/PAS stain; i – HDI stain. Scale bars: a – 200 μm; b, c, e - i – 100 μm; d – 50 μm

The PAS method showed a negative reaction in serous acini. AB pH 1.0 and AB pH2.5 stainings present a medium (++) positive reaction in glandular units. The AB pH 2.5/PAS staining identifies a strongly (+++) positive (blue colour) in the acini. The HDI method was a moderately strongly (+++) positive reaction (Figs. 3e-i).

Discussion

Until recently, it was believed that the bite of a Komodo dragon caused a fatal infection and possibly sepsis, causing the death of the victim. The bacteria living in the Komodo dragon’s mouth were said to be responsible for this (Auffenberg 1981; Goldstein et al. 2013; Montgomery et al. 2002). The discovery of venom glands changed the perception of Varana’s hunting strategy and the mechanism of death of its prey (Fry et al. 2006). Biologically active substances contained in the venom have a wide spectrum of action that contribute to the death of the prey in various ways. Biting and penetration of venom into tissues and the circulatory system causes heavy bleeding, which is intensified by the anticoagulant effect of venom (PLA2 toxins). Salivary kallikrein has also been shown to have a destructive effect on fibrinogen, which is crucial in the blood clotting process (Dobson et al. 2019). Furthermore, venom causes a drop in blood pressure in the victim, which can cause collapse and shock (CRISP, kallikrein and natriuretic toxin types). In addition, the victim is immobilized by hyperalgesic cramping AVIT toxins (Fry et al. 2006). Due to their content of natriuretic peptides, these toxins lower blood pressure by relaxing the aortic wall musculature (Dobson et al. 2019). The specific structure of the lizard’s teeth causes the damage to facilitate the penetration of the venom into the tissues and bloodstream of the victim. However, no special structures have been shown in lizard teeth that would serve to drain and deposit venom (Janeczek et al. 2023). Studies have shown a large number of aerobic and anaerobic bacteria in the oral cavity of the lizard (Glodstein et al. 2013; Montgomery et al. 2002). Some of them were opportunistic pathogens, but the risk of causing a fatal infection or sepsis does not appear to be high (Goldstein et al. 2013). Of course, it cannot be ruled out that, as with any bite, infection and related complications may occur (Arbuckle 2009). However, no specific pathogen common to Komodo dragons has been shown to be responsible for infections in their victims. The microbiological composition of the saliva of the Komodo dragon appears to be diverse and depends on many factors (Goldstein et al. 2013).

The salivary glands of the oral cavity of reptiles are characterized by a species-differentiated morphology. They are mucous or seromucous (Jarrar and Taib 2004; Baccari et al. 2002). Their structure varies within individual squamata. The salivary glandular cells of Tarentola mauritanica were observed to lack intercellular canaliculi, which, in contrast, are present between the seromucous cells in the salivary glands of Coluber viridiflavus and Podarcis sicula sicula. In the water monitor lizard (Varanus salvator), the mandibular salivary glands are mucous and no venom gland was found in this species (Srichairat et al. 2022). In Uromastyx microlepis, sublingual glands have a mixed structure (Taib 1988). In Abronia graminea, the mandibular venom gland was found to be mucous and consisted of multiple compartments (Koludarov et al. 2012). In our case, the venom gland extended along the alveolar arch of the mandible and, similar to the studies conducted by Koludarov et al. (2012) on Abronia graminea, each tooth had its own duct leading from the gland and a corresponding secretory complex. A similar morphological structure was described in Heloderma, Lanthanotus, and Varanus by Fry et al. (2010a, b).

Conclusions

Limitation of our study is the a low number of research material. Thus, our conclusions are related to the one case and are not general. Our observations indicate that the venom gland ducts open into sheaths surrounding the teeth. The presence of numerous muscle cells in the stroma of the venom gland between its lobes may indicate their important participation in emptying the vesicles of their secretions. Ziphodontic teeth, together with the venom gland, are adapted to maximise effective venom deposition in the victim’s body, optimising hunting by these animals.

Author contributions

MJ - conceptualization, visualization and writing– original draft; KGH - transportation and collection of the material, visualization and writing– original draft, writing– review & editing; AM - writing - original draft - collecting literature; LH - collection of the research samples; JKN - transportation and collection of the material, visualization and writing– original draft. All authors revised and approved the final version of the manuscript.

Funding

The current research received no external funding.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.