The koala (Phascolarctos cinereus) prostate: a comprehensive histological and immunohistochemical investigation.

The prostate of the koala (Phascolarctos cinereus), and of marsupials more generally, is the primary contributor of seminal fluid, yet comparatively little is known about its microanatomy or biochemistry. This study explored evidence of parenchymal segmentation of the koala prostate. The prostate of three sexually mature koalas were processed for histopathology, histochemistry (Masson's trichrome, Alcian Blue, periodic acid Schiff staining), and immunohistochemistry using basal (tumor protein 63, cytokeratin 14) and luminal (cytokeratin 8/18, prostate specific antigen, androgen receptor) markers. Results confirmed clear segmentation of the koala prostate into three zones, anterior, central, and posterior, characterized by differences in the proportion of glandular tissue, as well as the thickness of collagen fibers; there were also distinct differences in the secretions produced in each zone. Based on immunohistochemistry, the koala prostate showed evidence of both basal proliferative and luminal secretory cells. The ratio of cell types varied across the three segments, with the central segment housing the highest density of basal cells. Globular bodies produced in the anterior zone were shown to possess the same markers as those described for human prostasomes. This study is the first to comprehensively document the marsupial prostate in terms of microanatomy and corresponding immunohistochemistry. While further biochemical analysis, such as proteomics of each segment will better define the relative functions of each tissue, the data presented here are consistent with the hypothesis that the koala prostate potentially represents an example of an ontological stage in the evolutionary differentiation of male eutherian accessory glands.

The anatomy of a crushing bite: The specialised cranial mechanics of a giant extinct kangaroo.

The Sthenurinae were a diverse subfamily of short-faced kangaroos that arose in the Miocene and diversified during the Pliocene and Pleistocene. Many species possessed skull morphologies that were relatively structurally reinforced with bone, suggesting that they were adapted to incorporate particularly resistant foods into their diets. However, the functional roles of many unique, robust features of the sthenurine cranium are not yet clearly defined. Here, the finite element method is applied to conduct a comprehensive analysis of unilateral biting along the cheek tooth battery of a well-represented sthenurine, Simosthenurus occidentalis. The results are compared with those of an extant species considered to be of most similar ecology and cranial proportions to this species, the koala (Phascolarctos cinereus). The simulations reveal that the cranium of S. occidentalis could produce and withstand comparatively high forces during unilateral biting. Its greatly expanded zygomatic arches potentially housed enlarged zygomaticomandibularis muscles, shown here to reduce the risk of dislocation of the temporomandibular joint during biting with the rear of a broad, extensive cheek tooth row. This may also be a function of the zygomaticomandibularis in the giant panda (Ailuropoda melanoleuca), another species known to exhibit an enlarged zygomatic arch and hypertrophy of this muscle. Furthermore, the expanded frontal plates of the S. occidentalis cranium form broad arches of bone with the braincase and deepened maxillae that each extend from the anterior tooth rows to their opposing jaw joints. These arches are demonstrated here to be a key feature in resisting high torsional forces during unilateral premolar biting on large, resistant food items. This supports the notion that S. occidentalis fed thick, lignified vegetation directly to the cheek teeth in a similar manner to that described for the giant panda when crushing mature bamboo culms.

Two mammalian species in which the intercostal nerves innervate the serratus anterior or scalenus muscles together with the cervical nerves: an important clue to clarify the homology of cervico-thoracic trunk muscles in mammals.

The levator scapulae, rhomboideus, and serratus anterior muscles (as a group referred to the dorsal shoulder girdle muscles) and the scalenus muscles in mammals are usually innervated by cervical nerves. However, in koalas, the serratus anterior is additionally innervated by the lateral cutaneous branch of the first intercostal nerve. In cats, as in some other mammalian species, a part of the scalenus muscle (scalenus longus muscle) is innervated by the lateral cutaneous branches of the intercostal nerves. A precise comparison of the innervating nerves at the same segment in these two cases could clarify the homological relationship between the cervical and thoracic trunk muscles. In this context, the aim of this study was to follow the nerve fibers included in the nerves supplying the dorsal shoulder girdle and scalenus muscles up to the level of the spinal nerve roots in two koalas and two cats. The resultant observations revealed that both of the nerves to the serratus anterior in koalas and to the scalenus muscles in cats from the lateral cutaneous branch of the intercostal nerve occupy the same position in the spinal roots as the cervical nerve branches to the dorsal shoulder girdle muscles and the thoracic nerve branches to the external intercostal muscle. Based on these results, the axial trunk muscles in the cervico-thoracic region could be classified as follows: the scalenus and dorsal shoulder girdle muscles belong to the same lateral axial trunk muscle group as the external intercostal muscle, and are clearly distinguished from the medial group, such as the internal and innermost intercostal muscle.

The remarkable vocal anatomy of the koala (Phascolarctos cinereus): insights into low-frequency sound production in a marsupial species.

Koalas are characterised by a highly unusual vocal anatomy, with a descended larynx and velar vocal folds, allowing them to produce calls at disproportionately low frequencies. Here we use advanced imaging techniques, histological data, classical macroscopic dissection and behavioural observations to provide the first detailed description and interpretation of male and female koala vocal anatomy. We show that both males and females have an elongated pharynx and soft palate, resulting in a permanently descended larynx. In addition, the hyoid apparatus has a human-like configuration in which paired dorsal, resilient ligaments suspend the hyoid apparatus from the skull, while the ventral parts tightly connect to the descended larynx. We also show that koalas can retract the larynx down into the thoracic inlet, facilitated by a dramatic evolutionary transformation of the ventral neck muscles. First, the usual retractors of the larynx and the hyoid have their origins deep in the thorax. Secondly, three hyoid muscles have lost their connection to the hyoid skeleton. Thirdly, the genioglossus and geniohyoid muscles are greatly increased in length. Finally, the digastric, omohyoid and sternohyoid muscles, connected by a common tendinous intersection, form a guiding channel for the dynamic down-and-up movements of the ventral hyoid parts and the larynx. We suggest that these features evolved to accommodate the low resting position of the larynx and assist in its retraction during call production. We also confirm that the edges of the intra-pharyngeal ostium have specialised to form the novel, extra-laryngeal velar vocal folds, which are much larger than the true intra-laryngeal vocal folds in both sexes, but more developed and specialised for low frequency sound production in males than in females. Our findings illustrate that strong selection pressures on acoustic signalling not only lead to the specialisation of existing vocal organs but can also result in the evolution of novel vocal structures in both sexes.

Ultrasonographic assessment of the male koala (Phascolarctos cinereus) reproductive tract.

Studies documenting the application of ultrasonography to depict normal and pathological changes in koalas (Phascolarctos cinereus), especially in the male, are scarce. Sixty-two wild koalas were used in this study to define ultrasonographic protocols and features for the assessment of the male koala reproductive tract. Testis, epididymis and spermatic cord were examined using a hockey stick transducer. The normal koala testis showed a homogeneous echogenicity and an obvious hyper-echoic band corresponding to the tunica albuginea. The cauda epididymis was characterised by hypo- and hyper-echoic regions and was most effectively imaged in sagittal section. The koala prostate was assessed using a micro-curved transducer positioned midline, caudal to the bladder. On transverse section, it showed distinct margins and a well-defined internal structure, although the prostatic urethra was not apparent on most scans. To image the bulbourethral glands (BGs), the hockey stick transducer was placed lateral to the cloaca. BGIII was located just below the skin, while BGII was located deeper than BGIII. BGI was too small and not sufficiently echogenic to be detected. The ultrasonographic appearance of the BGs was similar to that of the testes but with more obvious hypo-echoic stippling. This comprehensive review of the ultrasonographic appearance of normal male koala reproductive tract can be used by veterinarians and others, in zoos or those working with wild koalas, during assessment of the reproductive tract of male koalas in relation to seasonal changes in accessory gland function or for the pathological investigation of reproductive lesions and infertility problems.

Review of some pharmacokinetic and pharmacodynamic properties of anti-infective medicines administered to the koala (Phascolarctos cinereus).

Although koalas are iconic Australian animals, no pharmacokinetic studies of any first-line medicines used to treat diseased or injured koalas had been published prior to 2010. Traditionally, medicine dosages suggested for this species underwent linear extrapolation from those recommended for domesticated species. The koala, a specialist folivore whose natural diet consists of almost exclusively Eucalyptus spp. foliage has anatomical and physiological adaptations for detoxifying their diet which also affect medicine pharmacokinetic profiles. This review addresses aspects of medicine absorption, clearance, and other indices (such as medicine binding to plasma proteins) of enrofloxacin/marbofloxacin and chloramphenicol used for the systemic treatment of chlamydiosis, and fluconazole ± amphotericin, and posaconazole for the treatment of cryptococcosis. Based on observations from published studies, this review includes suggestions to improve therapeutic outcomes when administering medicines to diseased koalas.

Computed tomography and magnetic resonance for the advanced imaging of the normal nasal cavity and paranasal sinuses of the koala (Phascolarctos cinereus).

The objective of this study is to describe computed tomography (CT) and magnetic resonance (MR) for the cross-sectional imaging of the normal anatomy of the nasal cavity and paranasal sinuses of the koala (Phascolarctos cinereus), to provide reference figures for gross anatomy with corresponding CT and MR images and to compare the features of the nasal cavity and paranasal sinuses of the normal koala with that reported in other domestic species. Advanced imaging can be used to aid in diagnosis, to plan surgical intervention, and to monitor therapeutic responses to diseases of the nasal passages in koalas. One clinically normal koala was anesthetized twice for the separate acquisition of dorsal CT scan images and transverse, dorsal, and sagittal MR images of its nasal cavity and paranasal sinuses. Sagittal and transverse CT planes were reformatted. Three fresh koala skulls were also transected in one of each transverse, sagittal, and dorsal planes and photographed. The CT and MR images obtained were matched with corresponding gross anatomic images and the normal bone, tissues and airway passages were identified. All anatomic structures were readily identifiable on CT, magnetic resonance imaging (MRI), and gross images. CT and MRI are both valuable diagnostic tools for imaging the nasal cavities and paranasal sinuses of koalas. Images obtained from this project can be used as baseline references for future comparison with diseased koalas to help with diagnosis, surgical intervention, and response to therapy.

Computed tomographic anatomy of the nasal cavity, paranasal sinuses and tympanic cavity of the koala.

OBJECTIVE: To use cross-sectional imaging (helical computed tomography (CT)) combined with conventional anatomical dissection to define the normal anatomy of the nasal cavity and bony cavitations of the koala skull. METHODS: Helical CT scans of the heads of nine adult animals were obtained using a multislice scanner acquiring thin slices reconstructed in the transverse, sagittal and dorsal planes. Subsequent anatomical dissection permitted confirmation of correct identification and further delineation of bony and air-filled structures visible in axial and multiplanar reformatted CT images. RESULTS: The nasal cavity was relatively simple, with little scrolling of nasal conchae, but bony cavitations were complex and extensive. A rostral maxillary recess and ventral conchal, caudal maxillary, frontal and sphenoidal paranasal sinuses were identified and characterised. Extensive temporal bone cavitation was shown to be related to a large epitympanic recess. CONCLUSIONS: The detailed anatomical data provided are applicable to future functional and comparative anatomical studies, as well as providing a preliminary atlas for clinical investigation of conditions such as cryptococcal rhinosinusitis, a condition more common in the koala than in many other species.

The seminiferous epithelial cycle and microanatomy of the koala (Phascolarctos cinereus) and southern hairy-nosed wombat (Lasiorhinus latifrons) testis.

The koala (Phascolarctos cinereus) and southern hairy-nosed wombat (Lasiorhinus latifrons) are iconic Australian fauna that share a close phylogenetic relationship but there are currently no comparative studies of the seminiferous epithelial cell or testicular microanatomy of either species. Koala and wombat spermatozoa are unusual for marsupials as they possess a curved stream-lined head and lateral neck insertion that superficially is similar to murid spermatozoa; the koala also contains Sertoli cells with crystalloid inclusions that closely resemble the Charcot-Bottcher crystalloids described in human Sertoli cells. Eighteen sexually mature koalas and four sexually mature southern hairy-nosed (SHN) wombats were examined to establish base-line data on quantitative testicular histology. Dynamics of the seminiferous epithelial cycle in the both species consisted of eight stages of cellular association similar to that described in other marsupials. Both species possessed a high proportion of the pre-meiotic (stages VIII, I - III; koala - 62.2 ± 1.7% and SHN wombat - 66.6 ± 2.4%) when compared with post-meiotic stages of the seminiferous cycle. The mean diameters of the seminiferous tubules found in the koalas and the SHN wombats were 227.8 ± 6.1 and 243.5 ± 3.9 µm, respectively. There were differences in testicular histology between the species including the koala possessing (i) a greater proportion of Leydig cells, (ii) larger Sertoli cell nuclei, (iii) crystalloids in the Sertoli cell cytoplasm, (iv) a distinctive acrosomal granule during spermiogenesis and (v) a highly eosinophilic acrosome. An understanding of the seminiferous epithelial cycle and microanatomy of testis is fundamental for documenting normal spermatogenesis and testicular architecture; recent evidence of orchitis and epididymitis associated with natural chlamydial infection in the koala suggest that this species might be useful as an experimental model for understanding Chlamydia induced testicular pathology in humans. Comparative spermatogenic data of closely related species can also potentially reflect evolutionary divergence and differences in reproductive strategies.

Cues to body size in the formant spacing of male koala (Phascolarctos cinereus) bellows: honesty in an exaggerated trait.

Determining the information content of vocal signals and understanding morphological modifications of vocal anatomy are key steps towards revealing the selection pressures acting on a given species' vocal communication system. Here, we used a combination of acoustic and anatomical data to investigate whether male koala bellows provide reliable information on the caller's body size, and to confirm whether male koalas have a permanently descended larynx. Our results indicate that the spectral prominences of male koala bellows are formants (vocal tract resonances), and show that larger males have lower formant spacing. In contrast, no relationship between body size and the fundamental frequency was found. Anatomical investigations revealed that male koalas have a permanently descended larynx: the first example of this in a marsupial. Furthermore, we found a deeply anchored sternothyroid muscle that could allow male koalas to retract their larynx into the thorax. While this would explain the low formant spacing of the exhalation and initial inhalation phases of male bellows, further research will be required to reveal the anatomical basis for the formant spacing of the later inhalation phases, which is predictive of vocal tract lengths of around 50 cm (nearly the length of an adult koala's body). Taken together, these findings show that the formant spacing of male koala bellows has the potential to provide receivers with reliable information on the caller's body size, and reveal that vocal adaptations allowing callers to exaggerate (or maximise) the acoustic impression of their size have evolved independently in marsupials and placental mammals.

Microscopy of the koala mandibular (submandibular) glands.

Koala mandibular (submandibular) glands are compound tubuloacinar glands, the secretory units of which consist only of serous cells. Intercellular canaliculi occur between the serous cells, which are continuous with a minute lumen that courses through the centre of each secretory unit. Intercalated ducts are abundant and join striated ducts, the latter being characterized by elaborate basolateral infoldings of the plasmalemma. Secretory granules within the serous cells fail to stain with either the PAS or Alcian Blue (pH 2.5) staining procedures. Ultrastructurally, the secretory granules are membrane bound, and consist of a homogeneous electron lucent material with a fine filamentous texture. The granules tend to coalesce into irregular shaped complexes of secretory material. Discharge of secretory material into the canalicular lumen is a common observation.

An odontometric study of the maxillary molars in Australian marsupials. I. The koala (Phascolarctos cinereus).

Crown dimensions of the maxillary molars were measured in the koala (Phascolarctos cinereus). There were no significant differences in crown diameters between the first and second molars, however the fourth molars were reduced in all crown diameters. The third molar was smaller than the first or second molars in buccolingual crown diameters but there were no significant differences in mesiodistal crown diameters. It is proposed that the similar shapes of the first and second molars are associated with similar types of masticatory activity involving these teeth, The shape of the third molar, which is reduced in size buccolingually, may be linked to the koala's occlusal function which is characterized by a condylar action that leads to differences in movement between opposing anterior and posterior molar teeth during the occlusal stroke. The fourth molar, the smallest of the molar teeth in crown diameter, erupts significantly later than the other molars, and its reduction may be explained by the terminal and distal reduction theories. It is proposed that the pattern of molar morphology in the koala is associated with both masticatory activity linked to its characteristic occlusal function, as well as reflecting the sequence of tooth emergence.

Lamination of the masticatory muscles in the Phascolarctos cinereus (Koala) according to their innervations.

The masticatory muscles are usually classified into four groups: masseter, temporalis, lateral pterygoid and medial pterygoid. The communicating muscle bundle between the temporalis and masseter called the zygomaticomandibular muscle exists. The laminations within these muscles are commonly separated by aponeuroses. Nerves control the action of muscles, so improved understanding about innervation patterns in the masticatory muscles is important in the consideration of muscle function. In this study, we focus on the relationships between the nerves supply and the lamination of masticatory muscles in Phascolarctos cinereus (Koala). The masseter muscle consists of superficial and deep muscle layers. The superficial muscle layer of the masseter muscle is divided into rostro-lateral and caudo-internal nerve layers. The deep muscle layer of the masseter muscle is divided into rostral, rostro-lateral, medial and caudo-internal nerve layers. The nerves that innervate the zygomaticomandibular muscle are distributed to the lateral area of the coronoid process. The temporalis muscle was divided into internal layer of the coronoid process, a lateral layer of the coronoid process and a posterior layer by the nerve distribution pattern. The medial pterygoid muscle divided into rostro-internal, medial and caudo-lateral nerve layers.

The zona pellucida of the koala (Phascolarctos cinereus): its morphogenesis and thickness.

In this study the ultrastructural organization of the koala oocyte and the thickness of the surrounding extracellular coat, the zona pellucida, has been determined to ascertain whether there is coevolution of the morphology of the female gamete with that of the highly divergent male gamete that is found in this marsupial species. Ovaries from several adult koalas were obtained and prepared for transmission electron microscopy. Oocytes in large tertiary follicles were somewhat smaller than those of most other marsupials, although their ultrastructural organization appeared similar and included many yolk vesicles. The zona pellucida surrounding the oocytes in tertiary follicles was approximately 8 microm thick and thus is of similar thickness to that of some eutherian mammals but at least twice as thick as that of most marsupial species so far studied. The results indicate that the koala oocyte is unusually small for a marsupial species whereas the zona pellucida is, by contrast, much thicker. How this relates to sperm-egg interaction at the time of fertilization has yet to be determined.