Epidermal tissue homeostasis. II. Cell pool size, cell birth rate and cell loss in toads deprived of the pars distalis of the pituitary gland

Following removal of the pars distalis of the pituitary gland in toads, epidermal efflux from the stratum corneum recruitment cell pool (i.e. production of corneal layers) is greatly increased. In this investigation the cell birth rate is studied by means of the metaphase arrest technique, as a function of time after pars distalis ablation. The method allows assessment of the total cell production over 14 days after the operation, to be compared with the total efflux and changes in the epidermal cell pool size. Whereas in intact toads the rate of cell production exceeds that of cell loss by moulting by a factor of 2.7, the 'surplus' of cells neither being used for formation of corneal layers nor permanently accommodated within the living epidermis, a 'balance sheet' of efflux and influx indicates that following pars distalis ablation all cells produced are also used for the (excessive) formation of corneal cell layers. The observations lend further support to the hypothesis that controlled cell deletion is a tissue homeostatic mechanism complementary to controlled cell divisions.     

Epidermal Tissue Homeostasis.

Abstract. It has previously been shown that in toad epidermis the cell birth rate (K_b) exceeds the rate of cell loss through moulting (K_b) and that the ‘surplus’ of cells seems to be removed in a controlled manner. Assuming that the epidermis is non-expanding, a K_b/K_d ratio > 1 indicates that cell deletion additional to desquamation takes place. In normal toads this ratio is 2-3. Following implantation of hydrocortisone pellets into intact toads (release rate, 18 μ/g toad/d), the K_b/K_d ratio, over a period of 14 d of hormone treatment, had increased to about 7, due mainly to an increased K_b and to a lesser extent to a decreased K_d. No change in the epidermal cell pool size had taken place. It was previously shown that, following removal of the pars distalis of the pituitary gland, the K_b/K_d ratio decreased with time, due to a decreasing K_b and an increasing K_d, eventually leading to a decreased epidermal cell pool size. In this paper it is shown that, in pars distalisectomized toads with hydrocortisone pellets implanted, the K_b/K_d ratio is restored to control levels by a restoration of the K_b as well as the K_d. the results differ from those of previous studies in which ACTH or adrenocortico-steroids were administered discontinuously (by injection). Thus, by experimental manipulation, different K_b/K_d ratios can be obtained: low (< 1, pars distalis ablation), medium (2-3, normal toads) and high (7, hydrocortisone implantation). the potentiality of this unique situation in analysing the important question of how the ‘surplus’ cells are deleted is discussed.     

Epidermal tissue homeostasis: Apoptosis and cell emigration as mechanisms of controlled cell deletion in the epidermis,of the toad,Bufo bufo

Summary In normal, non-expanding toad epidermis more cells are produced than needed to replace cells lost by moulting. By implication, cell deletion additional to moulting must take place. This paper deals with the mechanisms by which the surplus of cells is deleted, taking advantage of the fact that the ratio between cell birth rate (K _b) and the rate of desquamation (K _d), which in normal toads is 2 to 3, can be manipulated. In toads deprived of the pars distalis of the pituitary gland it is decreased to 0.2 to 0.3, and in toads with hydrocortisone pellets implanted into the subcutaneous lymph space it is increased to 7 to 10. Thus, structures candidates for the morphological manifestation of the deletion process should occur rarely in toads in which the pars distalis has been removed and frequently in toads with hydrocortisone pellets implanted. Categorization and enumeration of such structures by light microscopy in the epidermis from operated, normal, and hormone-treated toads were performed. The incidence of structures referred to as dark cells and omega-figures were found to correlate relatively well with the K _b/K_d-ratio. A subsequent ultrastructural analysis — on a cell-by-cell basis — of dark cells showed these to reflect various stages of apoptosis. The duration of the apoptotic process was calculated to be approximately 7 h. Light- and electron microscopy of omega-figures combined with histochemical observations of PSA-lectin binding were interpreted as reflecting a release of cells from the basal epidermis and their final elimination within the dermis. It is concluded (i) that apoptosis is an important mechanism of controlled cell deletion, (ii) that emigration to, and elimination in, the dermis is a possible deletion mechanism, and (iii) that necrosis is unlikely to play a role in controlled cell deletion.Supported in part by the Danish National Science Research Council (grant no. 11-6498) (PB)Part of this work was presented at the XVth Meeting of the European Study Group for Cell Proliferation, Sundvollen, Norway, 16–20 September 1987     

Time-dependent effects of removal of the pars distalis of the pituitary gland on toad epidermal cell and tissue kinetic parameters

Abstract. Amphibian moulting and its hormonal control has been extensively studied, but the possible influence of hormones on epidermal proliferation has been less investigated. In the present contribution to studies on the control of epidermal homeostasis, the proliferative pattern of the toad epidermis following ablation of the pars distalis of the pituitary gland is analysed by an investigation of the changes in the epidermal cell number, metaphase index and [³H]thymidine incorporation at various times after the operation. During the first 24 hr after pars distalis ablation, labelling index (LI) increased concurrently with an initial decrease in the metaphase index (MI), followed by an increase. During the same period of time the mean grain count (MGC) and the grain distribution pattern also changed. From 24 hr to 7 days after operation, MI, LI, and MGC were significantly lower than those of controls, but increased to control levels at 14 days after the operation. Phase durations and their possible changes were not measured directly, but data showed that the S-phase duration (Ts) and the mitotic duration (TM) must have changed in relation to each other during the experiment. Assuming that the MGC is a rough estimate of the DNA-synthesis rate, the compatibility of a postulated change in phase duration with the observed MGC was analysed. This analysis revealed that TS and TM could have decreased up to 18 hr after the operation, whereas these phase durations, after 24 hr and during the rest of the experiment, increased compared to those of controls. Even with these possible changes in phase durations, and in the absence of direct assessment of cell division rates, the observed cell kinetic parameters appeared incompatible with an increased rate of proliferation. This was surprising since the efflux of cells from the living, subcorneal epidermis to the stratum corneum was significantly increased after pars distalis ablation, without a concurrent decrease in the stratum corneum recruitment cell pool (SCRP-number of subcorneal epidermal cells per mm surface). Possible reasons for the discrepancy between the expected increase in proliferation following pars distalis ablation, and the failure to demonstrate this, are discussed.     

Epidermal tissue homeostasis. I. Cell pool size, cell birth rate and cell loss by moulting in the intact toad, Bufo bufo

Toad epidermis is a suitable model for studies on tissue homeostasis because cell pool size, influx into and efflux from the cell pool can be easily determined. The cell pool size was obtained by cell counting on photomicrographs, the influx (cell birth rate) was assessed by the metaphase-arrest technique, and the efflux (cell loss by moulting) assessed by counting the number of cells in the corneal layer and recording of intermoult periods. The importance of the methods for assessing these parameters per square unit of skin surface is emphasized. These parameters were studied in eight groups of ten adult male toads sacrificed at various hours of the day. There were minor variations in the cell birth rate, fluctuating around a mean of 26 cells/mm2/hr (obtained at the metaphase collection period from 11.00-14.00 hours). By summation of the cell productions during the eight metaphase collection periods of 3 hr, and extrapolation to an intermoult period (time between two moults), a calculated cell production of about 6340 cells/mm2 in 10.3 days was obtained, whereas the cell loss at each moult was only 2370 cells/mm2. Thus the cell production rate exceeds the rate of cell loss through moults by a factor of 2.7 Arguments are presented that the 'surplus' of cells produced cannot be permanently accommodated within the living epidermis. Consequently a cell deletion rate beyond that by moulting of about 4000 cells/mm2 in 10.3 days or 16 cells/mm2/hr can be calculated. These results are discussed in relation to current concepts of tissue homeostatic mechanism(s). The results are consistent with the hypothesis that controlled cell deletion may be a tissue homeostatic mechanism complementary to controlled cell divisions.     

Cell kinetics in the epidermis of the toad Bufo bufo bufo (L.) after partial hypophysectomy followed by corticotrophin treatment (an autoradiographic study)

1. Cell kinetic parameters in the epidermis of partially hypophysectomized, ACTH-treated toads whose moulting frequency was increased threefold were studied using labelled thymidine and the autoradiographic technique. 2. The labelling index, length of S-phase and cycle time of stratum germinativum cells changed slowly as a function of the number of ACTH injections, and so did the migration of cells from stratum germinativum to stratum corneum. 3. A cell production in stratum germinativum of normal toads in excess of cell loss, as demonstrated by Levi & Nielsen (1982) should make it possible to maintain a status quo over a limited period of time following ablation of pars distalis and ACTH treatment.     

Effects of malathion on disease susceptibility in Woodhouse's toads.

Adult male Woodhouse's toads (Bufo woodhousi) developed clinical disease, hepatomegaly, and died at a higher rate when externally exposed once to either a high or low sublethal dose (0.011 or 0.0011 mg malathion/g toad) of field grade malathion and challenged with a sublethal dose of Aeromonas hydrophila injected intraperintoneally (1.1 x 10(4) bacteria/g toad) when compared to toads not exposed to malathion but challenged with A. hydrophila (P < 0.007). Toads exposed to malathion (high or low dose) and challenged with A. hyydrophila had clinical disease, hepatomegaly, and died at a higher rate [9 (90%) of 10] than toads exposed to malathion alone (P < 0.002). Toads exposed to the high and low doses of malathion had a 22% and 17% decrease in brain cholinesterase levels, respectively, when they were compared to nonmalathion exposed toads (P < 0.025, P < 0.006). It appears that field grade malathion applied externally to adult Woodhouse's toads may cause increased disease susceptibility when challenged with a potentially pathogenic bacteria.     

Studies on the erythropoietic effect of plasma from anemic toads both with and without testis.

Injection of plasma from experimentally induced anemic toad with intact testis increases erythropoiesis in starved toads evidenced by the increase of red blood cell, hemoglobin and hematocrit, whereas the plasma of castrated and phenylhydrazine-HCl treated anemic toad failed to do so. It can be suggested that the erythropoiesis stimulating factor (ESF) is produced from the testis of toad and the production of this factor (ESF) was found to increase during anemia in an attempt to correct the anemic condition.     

Native Australian frogs avoid the scent of invasive cane toads

Invasive species can affect the ecosystems they colonize by modifying the behaviour of native taxa; for example, avoidance of chemical cues from the invader may modify habitat use (shelter site selection) by native species. In laboratory trials, we show that metamorphs of most (but not all) native frog species on a tropical Australian floodplain avoid the scent of invasive cane toads (Bufo marinus Linnaeus 1758). Cane toads also avoid conspecific scent. This response might reduce vulnerability of metamorph frogs and toads to larger predatory toads. However, similar avoidance of one type of pungency control (garlic), and the presence of this avoidance behaviour in frogs at the toad invasion front (and hence, with no prior exposure to toads), suggest that this may not be an evolved toad‐specific response. Instead, our data support the simpler hypothesis that the metamorph anurans tend to avoid shelter sites that contain strong and unfamiliar scents. Temporal and spatial differences in activity of frogs versus toads, plus the abundance of suitable retreat sites during the wet season (the primary time of frog activity), suggest that avoiding toad scent will have only a minor impact on the behaviour of native frogs. However, this behavioural impact may be important when environmental conditions bring toads and frogs into closer contact.     

Mortality of captive Canadian toads from Basidiobolus ranarum mycotic dermatitis

Twenty-six adult free-ranging Canadian toads (Bufo hemiophrys) were collected from northeastern North Dakota (USA) during the last week of August 1994 and placed in captivity. During late December and January 1995, 21 Canadian toads died. Clinical signs included increased time sitting in water bowls, darkened dorsal skin, constant arching of their backs, and hyperemia and sloughing of ventral epidermis. The condition progressively worsened until death occurred within 5 to 7 days after onset of clinical disease. Mycotic dermatitis due to Basidiobolus ranarum was diagnosed in all toads and the fungus was isolated from 11 (52%) of 21 toads. Histology of the ventral skin and digits revealed numerous fungal spherules and occasional hyphae without significant inflammatory reaction. This condition clinically resembled red leg associated with Aeromonas hydrophila and many other bacterial organisms, and the diseases could be confused without appropriate diagnostic tests. This also is the first report of B. ranarum causing clinical disease in a toad species.     

Behavioural responses of reptile predators to invasive cane toads in tropical Australia

The ecological impact of an invasive species can depend on the behavioural responses of native fauna to the invader. For example, the greatest risk posed by invasive cane toads (Rhinella marina Bufonidae) in tropical Australia is lethal poisoning of predators that attempt to eat a toad; and thus, a predator's response to a toad determines its vulnerability. We conducted standardized laboratory trials on recently captured (toad‐naïve) predatory snakes and lizards, in advance of the toad invasion front as it progressed through tropical Australia. Responses to a live edible‐sized toad differed strongly among squamate species. We recorded attacks (and hence, predator mortality) in scincid, agamid and varanid lizards, and in elapid, colubrid and pythonid snakes. Larger‐bodied predators were at greater risk, and some groups (elapid snakes and varanid lizards) were especially vulnerable. However, feeding responses differed among species within families and within genera. Some taxa (notably, many scincid and agamid lizards) do not attack toads; and many colubrid snakes either do not consume toads, or are physiologically resistant to the toad's toxins. Intraspecific variation in responses means that even in taxa that apparently are unaffected by toad invasion at the population level, some individual predators nonetheless may be fatally poisoned by invasive cane toads.     

Wildfires modify the parasite loads of invasive cane toads

The frequency and severity of wildfires are increasing due to anthropogenic modifications to habitats and to climate. Post-fire landscapes may advantage invasive species via multiple mechanisms, including changes to host–parasite interactions. We surveyed the incidence of endoparasitic lungworms (Rhabdias pseudosphaerocephala) in invasive cane toads (Rhinella marina) in near-coastal sites of eastern Australia, a year after extensive fires in this region. Both the prevalence of infection and number of worms in infected toads increased with toad body size in unburned areas. By contrast, parasite load decreased with toad body size in burned areas. By killing moisture-dependent free-living lungworm larvae, the intense fires may have liberated adult cane toads from a parasite that can substantially reduce the viability of its host. Smaller toads, which are restricted to moist environments, did not receive this benefit from fires.     

No emotional fever in toads

Four Bufo marinus toads and one Bombina bombina toad were placed individually along a temperature gradient where they could regulate their body temperature through behavioral means. When injected intraperitoneally with killed Pseudomonas aeruginosa bacteria, the toads sought heat and raised their body core temperature. Gentle handling by the experimenter, however, was not followed by heat-seeking behavior and rising core temperature. These findings confirm the existence of behavioral fever, but not emotional fever, in the species studied.     

Visual optics in toads (Bufo americanus)

Aspects of visual optics were investigated in the American toad (Bufo americanus). The development of the refractive state of the eye during metamorphosis was followed with IR photoretinoscopy. Frozen sections documented the changes in optical parameters before and after metamorphosis. There is a difference in light sensitivity between juvenile and adult toads. Binocular accommodation in adult toads was observed. 1. IR photoretinoscopic measurements showed that the refractive state of the eye changed very rapidly during metamorphosis, about 10 D/h while the animal entered the terrestrial habitat. 2. Frozen sections showed that the almost spherical lens in a tadpole eye had flattened in a just metamorphosed toad's eye while at the same time the distance of the lens to the retina had decreased. However, the morphological measurements were not sufficiently sensitive to record the relatively small changes in ocular dimensions that were responsible for the rapid changes in refractive state during metamorphosis. 3. Schematic eyes, with homogeneous and non homogeneous lenses, were constructed for tadpoles, juvenile toads, and adult toads. 4. Nonparaxial raytracing studies in schematic eyes suggested that the lenses of animals of the three developmental stages tadpole, juvenile toad, and adult are not homogeneous but have a refractive index gradient. The raytracing studies indicated that the refractive index gradient is different for the different developmental stages, being highest in the tadpole lens. 5. The observations of toads during feeding behavior at different light levels showed an increased light sensitivity in the adult nocturnal toads in contrast to the juvenile animals, which are diurnal. The increased light sensitivity could partly be explained with an increase in aperture and an increase in red rod outer segments. To fully explain the higher light sensitivity in adult toads, changes in neuronal parameters had to be assumed. 6. Retinoscopic measurements of the resting refractive state in the adult toad showed a hyperopic defocus of about +8 D. By subtracting the measurement artefact for retinoscopy, the true resting focus was found to be nearly emmetropic. 7. The amount of natural accommodation in adult toads during normal feeding behavior was investigated with IR photoretinoscopy. Binocular accommodation of about 8 D was observed.     

The ecological impact of invasive cane toads (Bufo marinus) in Australia

Although invasive species are viewed as major threats to ecosystems worldwide, few such species have been studied in enough detail to identify the pathways, magnitudes, and timescales of their impact on native fauna. One of the most intensively studied invasive taxa in this respect is the cane toad (Bufo marinus), which was introduced to Australia in 1935. A review of these studies suggests that a single pathway-lethal toxic ingestion of toads by frog-eating predators-is the major mechanism of impact, but that the magnitude of impact varies dramatically among predator taxa, as well as through space and time. Populations of large predators (e.g., varanid and scincid lizards, elapid snakes, freshwater crocodiles, and dasyurid marsupials) may be imperilled by toad invasion, but impacts vary spatially even within the same predator species. Some of the taxa severely impacted by toad invasion recover within a few decades, via aversion learning and longer-term adaptive changes. No native species have gone extinct as a result of toad invasion, and many native taxa widely imagined to be at risk are not affected, largely as a result of their physiological ability to tolerate toad toxins (e.g., as found in many birds and rodents), as well as the reluctance of many native anuran-eating predators to consume toads, either innately or as a learned response. Indirect effects of cane toads as mediated through trophic webs are likely as important as direct effects, but they are more difficult to study. Overall, some Australian native species (mostly large predators) have declined due to cane toads; others, especially species formerly consumed by those predators, have benefited. For yet others, effects have been minor or have been mediated indirectly rather than through direct interactions with the invasive toads. Factors that increase a predator's vulnerability to toad invasion include habitat overlap with toads, anurophagy, large body size, inability to develop rapid behavioral aversion to toads as prey items, and physiological vulnerability to bufotoxins as a result of a lack of coevolutionary history of exposure to other bufonid taxa.     

The spinal nerves innervate putative chemosensory cells in the ventral skin of desert toads, Bufo alvarius

Toads normally obtain water by absorption across their skin from osmotically dilute sources. When hyperosmotic salt solutions are presented as a hydration source to dehydrated desert toads, they place the ventral skin onto the source but soon afterwards escape to avoid dehydration. The escape behavior coincides with neural excitation of the spinal nerves that innervate putative chemosensory cells in the ventral skin. In the present study, fluorescent dye translocated through the spinal nerves to those receptor cells in the epidermis was photoconverted in the presence of 3, 3'-diaminobenzidine tetrahydrochloride for electron-microscopic observation of the cells and associated nerve terminals. Most of the photoconverted cells were located in the deepest layer of the epidermis, with some being in more intermediate layers. No labeled cell was seen in the outermost layer of living cells. In desert toads, flask cells and Merkel cells are occasionally seen in the epidermis. An association of nerve fibers with these epidermal cells has been reported in some species of the anurans. In the present study, however, the cytological features of the photoconverted cells are neither reminiscent of flask cells nor Merkel cells, but are similar to those of surrounding epithelial cells in each layer of the epidermis. We hypothesize a sensory function for these cells, because they have a close association with nerve fibers and participate in the transepithelial transport of salts that must pass through all cell layers of the skin.     

Invasive cane toads might initiate cascades of direct and indirect effects in a terrestrial ecosystem

Understanding the impacts that invasive vertebrates have on terrestrial ecosystems extends primarily to invaders’ impacts on species with which they interact directly through mechanisms such as predation, competition and habitat modification. In addition to direct effects, invaders can also initiate ecological cascades via indirect population level effects on species with which they do not directly interact. However, evidence that invasive vertebrates initiate ecological cascades in terrestrial ecosystems remains scarce. Here, we ask whether the invasion of the cane toad, a vertebrate invader that is toxic to many of Australia’s vertebrate predators, has induced ecological cascades in a semi-arid rangeland. We compared activity of a large predatory lizard, the sand-goanna, and abundances of smaller lizards preyed upon by goannas in areas of high toad activity near toads’ dry season refuges and areas of low toad activity distant from toads’ dry season refuges. Consistent with the hypothesis that toad invasion has led to declines of native predators susceptible to poisoning, goanna activity was lower in areas of high toad activity. Consistent with the hypothesis that toad-induced goanna decline lead to increases in abundance the prey of goannas, smaller lizards were more abundant in areas of high toad activity. Structural equation modelling showed a positive correlation between goanna activity and distance from dry season refuge habitats used by toads. The abundances of small lizards was correlated negatively with goanna activity and distance from dry season refuges of toads. Our findings provide support for the notion that invasions by terrestrial vertebrates can trigger ecological cascades.     

Interacting impacts of invasive plants and invasive toads on native lizards

The ecological impacts of an invasive species may be reduced by prior invasions if selective pressures imposed by earlier events preadapt the native biota to deal with the newer arrival. In northwestern Australia, invasion of the cane toad (Rhinella marina) kills many native predators if they ingest the highly toxic toads. Remarkably, the toads' defensive toxins (bufadienolides) are chemically similar to those of another invasive species: an ornamental plant from Madagascar, Bryophyllum spp. (Crassulaceae, mother-of-millions). Omnivorous lizards (bluetongue skinks, Tiliqua scincoides) are imperiled by the invasion of toads in northwestern Australia, but conspecifics from other areas of the continent (those where exotic plants were introduced and including areas where toads have yet to invade) are less affected because they exhibit higher physiological tolerance of toad toxins (and also of plant toxins). The willingness of captive bluetongues to consume both toads and these plants and the high correlation in the lizards' sensitivity to toad toxins versus plant toxins suggest that exotic plants may have imposed strong selection on the lizards' physiological tolerance of bufadienolides. As a result, populations of lizards from areas previously exposed to these alien plants may be preadapted to deal with the toxins of the more recent anuran invader.