Is it or isn't it? A reexamination of the anterior orbital glands of the fat-tailed Dunnart Sminthopsis Crassicaudata (Dasyuridae: Marsupiala) and a reevaluation of definitions for the Harderian gland.

The anterior orbital glands of tetrapods, which include the Harderian and nictitans glands, can usually be differentiated either anatomically (nictitans gland is more anterior) or histochemically (Harderian gland secretes lipids). However, conflicting statements exist in the literature about the presence and identity of these glands. Two previous studies on Sminthopsis crassicaudata (Dasyuridae: Marsupiala) either failed to note any anterior ocular glands or used no histochemical analyses. This study reexamined the structure of the anterior orbital glands of S. crassicaudata. Histological, histochemical, and ultrastructural examination revealed three glandular units: two of which are located superficially in the nictitating membrane, the third lying deeper in the connective tissue. The ducts of these three glandular units all open onto the corneal aspect of the nictitating membrane. These cells contain mainly serous granules with sparse intracellular lipid droplets. The nomenclature of these structures depends upon the definition used. According to the anatomical definition, S. crassicaudata has two glands: anteriorly the nictitans and posteriorly the Harderian gland. In contrast, if the histochemical definition is used, there is only one gland, but its precise identity cannot be confirmed until the role of the lipid droplets is established. Moreover, the histochemical definition poses additional problems with respect to the mechanism of secretion, multiple secretions, and glandular plasticity. Finally, the unitary definition identifies one deeply subdivided gland with an anterior and a posterior lobe in S. crassicaudata. This last definition is broad enough to accommodate a wide level of anatomical variation in the anterior ocular glands of tetrapods.

Marsupial uncoupling protein 1 sheds light on the evolution of mammalian nonshivering thermogenesis.

Brown adipose tissue expressing uncoupling protein 1 (UCP1) is responsible for adaptive nonshivering thermogenesis giving eutherian mammals crucial advantage to survive the cold. The emergence of this thermogenic organ during mammalian evolution remained unknown as the identification of UCP1 in marsupials failed so far. Here, we unequivocally identify the marsupial UCP1 ortholog in a genomic library of Monodelphis domestica. In South American and Australian marsupials, UCP1 is exclusively expressed in distinct adipose tissue sites and appears to be recruited by cold exposure in the smallest species under investigation (Sminthopsis crassicaudata). Our data suggest that an archetypal brown adipose tissue was present at least 150 million yr ago allowing early mammals to produce endogenous heat in the cold, without dependence on shivering and locomotor activity.

The structure of the nasal chemosensory system in squamate reptiles. 1. The olfactory organ, with special reference to olfaction in geckos.

The luminal surface of the chemosensory epithelia of the main olfactory organ of terrestrial vertebrates is covered by a layer of fluid. The source of this fluid layer varies among vertebrates. Little is known regarding the relative development of the sources of fluid (sustentacular cells and Bowman's glands) in reptiles, especially in gekkotan lizards (despite recent assertions of olfactory speciality). This study examined the extent and morphology of the main olfactory organ in several Australian squamate reptiles, including three species of gekkotans, two species of skinks and one snake species. The olfactory mucosa of two gekkotan species (Christinus marmoratus and Strophurus intermedius) is spread over a large area of the nasal cavity. Additionally, the sustentacular cells of all three gekkotan species contained a comparatively reduced number of secretory granules, in relation to the skinks or snake examined. These observations imply that the gekkotan olfactory system may function differently from that of either skinks or snakes. Similar variation in secretory granule abundance was previously noted between mammalian and non-mammalian olfactory sustentacular cells. The observations in gekkotans suggests that the secretory capacity of the non-mammalian olfactory sustentacular cells show far more variation than initially thought.

The structure of the nasal chemosensory system in squamate reptiles. 2. Lubricatory capacity of the vomeronasal organ.

The vomeronasal organ is a poorly understood accessory olfactory organ, present in many tetrapods. In mammals, amphibians and lepidosaurian reptiles, it is an encapsulated structure with a central, fluid-filled lumen. The morphology of the lubricatory system of the vomeronasal organ (the source of this fluid) varies among classes, being either intrinsic (mammalian and caecilian amphibian vomeronasal glands) or extrinsic (anuran and urodele nasal glands). In the few squamate reptiles thus far examined, there are no submucosal vomeronasal glands. In this study, we examined the vomeronasal organs of several species of Australian squamates using histological, histochemical and ultrastructural techniques, with the goal of determining the morphology of the lubricatory system in the vomeronasal organ. Histochemically, the fluid within the vomeronasal organ of all squamates is mucoserous, though it is uncertain whether mucous and serous constituents constitute separate components. The vomeronasal organ produces few secretory granules intrinsically, implying an extrinsic source for the luminal fluid. Of three possible candidates, the Harderian gland is the most likely extrinsic source of this secretion.

Thermocyclic entrainment of lizard blood plasma melatonin rhythms in constant and cyclic photic environments.

We assessed how chronic exposure to 6-h cryophase temperatures of 15 degrees C in an otherwise 33 degrees C environment entrains the rhythm of blood plasma melatonin rhythms in lizards (Tiliqua rugosa) subjected to constant dark (DD), constant light (LL), and to 12:12-h light-dark cycles (12L:12D). The peak of the melatonin rhythm was entrained by the cryophase temperature of the thermocycle in DD and LL, irrespective of the time at which the cryophase temperature was applied. Comparable thermocycles of 6 h at 15 degrees C imposed on a 12L:12D photocycle, however, affected the amplitude and phase of the melatonin rhythm, depending on the phase relationship between light and temperature. Cold pulses in the early light period and at midday resulted, respectively, either in low amplitude or nonexistent melatonin rhythms, whereas those centered in or around the dark phase elicited rhythms of high amplitude. Supplementary experiments in 12L:12D using two intermittent 6-h 15 degrees C cryophases, one delivered in the midscotophase and another in the midphotophase, elicited melatonin rhythms comparable to those in lizards subjected to constant 33 degrees C and 12L:12D. In contrast, lizards subjected to 12L:12D and a 33 degrees C:15 degrees C thermocycle, whose thermophase was aligned with the photophase, produced a threefold increase in the amplitude of the melatonin rhythm. Taken together, these results support the notion that there is an interaction between the external light and temperature cycle and a circadian clock in determining melatonin rhythms in Tiliqua rugosa.

Daily and seasonal rhythms in selected body temperatures in the Australian lizard Tiliqua rugosa (Scincidae): field and laboratory observations.

This study examined daily and seasonal activity and thermoregulatory behaviour of the sleepy lizard, Tiliqua rugosa, a large, diurnally active temperate-dwelling Australian lizard, in the field and laboratory. Activity temperatures in the field were compared with those selected by lizards in laboratory thermal gradients in order to assess the extent to which endogenous versus exogenous factors contribute to seasonal variations in thermoregulatory behaviour. In the field, lizards are most active in late winter-spring (August-November), during which their activity varies from mostly unimodal on days of mild temperature to bimodal on hot days. In late spring-summer (November-January), activity is largely restricted to early morning, and at all other seasons sleepy lizards are rarely active. The winter-spring activity of sleepy lizards is constrained by low environmental temperatures, as lizards at these seasons have low body temperatures in the field but higher temperatures in laboratory thermal gradients. The lower temperatures selected in the laboratory in the summer-autumn months suggest the avoidance of high ambient temperatures and general inactivity in the field at these times. Thermal selection in the laboratory at the eight times of year tested showed that the phase of the minimum and maximum temperature selected and the amplitude of the rhythm of temperature selected varied continuously with the time of year. These daily and seasonal shifts in thermoregulatory behaviour may be regulated by endogenous physiological mechanisms coupled with seasonal ecological constraints such as food availability.

Effect of variable temperatures, darkness and light on the secretion of melatonin by pineal explants in the gecko, Christinus marmoratus.

This study examined the combined effect of thermocycles with either variable or constant photic conditions on melatonin production by pineal organs in vitro in the gecko, Christinus marmoratus. A 30 degrees C:15 degrees C thermocycle elicited a rhythm of melatonin production under conditions of 12L:12D, constant light or constant darkness when the cryophase coincided with the dark phase of the photocycle or with the subjective night. A 6 h advance of the thermocycle with respect to the photocycle produced an advance in the onset and offset of melatonin production in subsequent nights. When the thermocycle was 180 degrees out of phase with the photoperiod, the rhythm of melatonin production was disrupted, suggesting a differential pattern of sensitivity to photothermal stimuli. It was concluded that both light and temperature are important modulators of pineal function although their combined effects on pineal melatonin production is complex and unclear.

Effect of constant temperatures, darkness and light on the secretion of melatonin by pineal explants and retinas in the gecko Christinus marmoratus.

The effects of temperature and lighting conditions on the secretion of melatonin by the pineal organ of the nocturnal gecko Christinus marmoratus was studied using in vitro perifusion. In a 12L:12D lighting regime, a high-amplitude melatonin rhythm was detectable at a constant temperature of 20 and 30 degrees C but not at 10 or 37 degrees C. There were sustained high levels of melatonin in constant darkness and sustained low levels in constant light. No retinal melatonin was detected using static and perifusion culture techniques. These results show that the pineal organ of C. marmoratus maintains light sensitivity in vitro but does not contain an oscillator coupled to the melatonin synthetic pathway.

Vascularization of the pineal complex in the lizard Tiliqua rugosa.

The vascularization of the pineal complex in the lizard Tiliqua rugosa was investigated by vascular corrosion and latex casting techniques. The fine structure of the pineal capillaries was also studied by transmission electron microscopy. The pineal complex in T. rugosa consists of an elongated pineal gland proper and a separate, distinct parietal eye. The pineal complex derives an abundant blood supply from branches of the middle and posterior cerebral arteries. Scanning electron microscopy of vascular corrosion casts revealed a dense and extensive pineal capillary bed which drains ultimately into a wide longitudinal sinus suggesting an efficient pathway for the rapid removal of substances secreted by the gland. The parietal eye, which receives a unilateral left-sided blood supply from the unpaired anterior pineal artery, is shown to be a highly vascularized structure. The close morphological relationship between the pineal gland and dorsal sac, where the two structures apparently share the same blood vessels, suggests a functional relationship between them. The pineal capillaries are fenestrated with tight junctions between adjoining endothelial cells. Podia-like abluminal extensions of the endothelial cells were observed in close relation to unmyelinated nerve bundles. The basal margin of the pineal parenchyma is highly invaginated with thin finger-like cytoplasmic protrusions into the pericapillary space. Distinct bands of microfibrils form "struts" anchoring the pineal parenchyma to the endothelial wall. These features may have a role in the transfer of materials between the pineal gland and the blood stream.

Polyunsaturated dietary lipids lower the selected body temperature of a lizard.

Cold acclimation lowers the selected body temperature (Tb) in many ectothermic vertebrates. This change in behavioural thermoregulation is accompanied by an increase in the proportion of polyunsaturated fatty acids in tissues and cellular membranes. We investigated how diets containing different fatty acids, known to significantly alter the fatty acid composition of animal tissues and membranes, affect the selected Tb of the lizard Tiliqua rugosa. Lizards on a diet containing many polyunsaturated fatty acids (10% sunflower oil) showed a 3-5 degrees C decrease in Tb, whereas Tb in animals on a diet containing mainly saturated fatty acids (10% sheep fat) did not change. Our study suggests that the composition of dietary lipids influences thermoregulation in ectothermic vertebrates and may thus play a role in the seasonal adjustment of their physiology.

Thermoperiodic influences on plasma melatonin rhythms in the lizard Tiliqua rugosa: effect of thermophase duration.

Rhythms of plasma melatonin levels were determined in lizards (Tiliqua rugosa) subjected to a 12 h photocycle (12 h light: 12 h dark) at constant 33 degrees C, and at 7 different thermoperiods (33 degrees C thermophase and 15 degrees C cryophase) whose thermophase duration ranged from 1.5 to 21 h. The melatonin secretion rate, as measured by the amplitude and duration of elevated melatonin levels and the area under the curve, was maximal at thermoperiods whose thermophase was between 9 and 18 h in duration. The results indicate that in ectothermic vertebrates the prevailing thermoperiod as well as the photoperiod may influence melatonin rhythms and hence the timing of annual physiological cycles.

Thermoperiod and photoperiod interact to affect the phase of the plasma melatonin rhythm in the lizard, Tiliqua rugosa.

Rhythms of plasma melatonin levels were determined in lizards (Tiliqua rugosa) subjected to 6 h thermocycles (6 h, 33 degrees C thermophase; 18 h, 15 degrees C cryophase) placed at 4 different phases of a 12 h photocycle (12 h light: 12 h dark). The peak of the melatonin rhythm was either shifted at different rates, or inhibited by the light phase of the photocycle, depending upon the phase relationship between the thermocycle and the photocycle. The results indicate that the pineal organ of ectotherms is part of a circadian pacemaker system, transducing photothermal environmental information into a neurochemical signal.

Thermal sensitivity of reptilian melatonin rhythms: "cold" tuatara vs. "warm" skink.

Daily rhythms in plasma melatonin levels were compared in two ecologically diverse reptilian species under natural environmental conditions in autumn. The nocturnal, cold temperature-adapted tuatara (Sphenodon punctatus) had a melatonin rhythm of much lower amplitude than did the diurnal desert-adapted sleepy lizard (Tiliqua rugosa). Experiments in controlled laboratory environments showed that, although both species are capable of attaining a comparable melatonin peak (approximately 750 pmol/l), the threshold temperature at which a significant daily rhythm occurs is approximately 15 degrees C in S. punctatus compared with approximately 25 degrees C in T. rugosa. This difference probably reflects the disparate thermoregulatory adaptations of the two species, S. punctatus favoring mean activity temperatures of 11.5 degrees C and T. rugosa, 32.5 degrees C. In ectotherms such as reptiles, therefore, species-typical thermoregulatory behavior may provide thermal cues that interact with photoperiod to provide the appropriate melatonin signal for the regulation of annual physiological cycles.

Melatonin content of the pineal, parietal eye and blood plasma of the lizard, Trachydosaurus rugosus: effect of constant and fluctuating temperature.

Lizards acclimated to a light cycle accompanied by a thermocycle of 30 degrees C/15 degrees C had a more robust rhythm in pineal and plasma melatonin levels than those acclimated to constant 30 degrees C. At constant 15 degrees C, the melatonin rhythm was abolished entirely. A similar thermosensitivity in melatonin content was found in the parietal eye, indicating that this photoreceptive structure may also be capable of synthesising methoxyindoles.

Independent effects of the pineal and a bacterial pyrogen in behavioural thermoregulation in lizards.

The pineal complex of lizards is comprised of an extracranial photoreceptive structure known as the parietal eye, and an intracranial pineal organ which is homologous to the pineal gland of birds and mammals. Studies have shown that removing the parietal eye or severing the parietal nerve causes lizards to select higher temperatures when allowed to thermoregulate behaviourally in thermal or photothermal laboratory gradients. Although comparable studies involving removal of the lizard pineal organ have not previously been attempted, field data indicate that pinealectomy may have an antagonistic effect to parietalectomy. We present evidence here which shows that (1) following pinealectomy, collared lizards (Crotaphytus collaris) behaviourally select or prefer lower temperatures than their controls in thermal laboratory gradients, and (2) the effect of surgical treatment is independent of the effects of a behavioural fever-inducing substance which elevates by a fixed amount the environmental temperatures selected.