Genes induced during the early developmental stages of the Cane Toad, Bufo (Chaunus) marinus

Metamorphosis, a critical stage in the development of toads and frogs, involves rapid levels of morphological change. In the current study, we have used microarray analysis to identify shifts in gene expression between tadpole and toadlet stages of the cane toad, Bufo (Chaunus) marinus. Here, we report on nine genes that show the greatest induction during metamorphosis; the gut-associated gastrokine and trefoil factor, blood components haemoglobins alpha/beta, apolipoprotein and serum albumin, a nasal gene olfactomedin, a lens gene gamma-crystallin, and a novel gene with low homology to frog harderin. We present both temporal and spatial expression patterns of these genes identified in developing and adult cane toads. This study extends our knowledge of the molecular basis of toad metamorphosis, and not only offers insights to the genes induced during the general remodelling that occurs but also reveals possible targets for control and manipulation of amphibian pest species, for example, the cane toad in Australia.     

Corneal accommodation in chick and pigeon

Summary We have investigated the role of changes in corneal radius of curvature in effecting accommodation in the bird's eye. It was found that in natural accommodation (measured by IR photoretinoscopy) changes of corneal radius of curvature (measured by IR photokeratometry) play an important role in both the chick and the pigeon. In the adult pigeon the cornea is indeed responsible for the largest part of natural accommodation (up to approx. 9 D). In this animal the corneal diameter (as seen from the optical axis of the eye) decreases in accommodation which can be taken to explain the change of corneal radius of curvature. In the chicken, corneal accommodation is combined with other mechanisms (total accommodative range 15–17 D, corneal accommodation about 8 D). The chicken's cornea is aspherical within the pupil area leading to large measurement variation in photokeratometry if the Purkinje images are not symmetrical to the pupillary axis.Abbreviation IR infrared     

Accommodation behaviour during prey capture in the vietnamese leaf turtle (<Emphasis Type="Italic">Geoemyda spengleri</Emphasis>)

Vietnamese leaf turtles (Geoemyda spengleri) were tested for their ability to focus on prey objects at various distances. Accommodation was continuously measured by infrared photoretinoscopy. All animals investigated during this study showed a surprisingly high precision of accommodation over a range of over 30 D. Measured accommodation matched the target distance accurately for distances between 3 and 17 cm. The turtles switched between independent and coupled accommodation in the two eyes. Independent accommodation was observed when the turtles inspected their environment visually without a defined object of interest. Coupled accommodation was only observed during binocular prey fixation. When a turtle aimed at a target, the symmetrical focus of both eyes persisted even if vision was totally blocked in one eye or altered by ophthalmic lenses. This suggests that the eyes were linked by internal neuronal mechanisms. The pupil of the eye responded clearly to changes in ambient light intensity. A strong decrease in pupil size was also observed when the eye was focused on a close target. In this case, the constriction of the pupil probably aids in the deformation of the eye lens during near-accommodation.     

Competing tadpoles: Australian native frogs affect invasive cane toads (Rhinella marina) in natural waterbodies

The cane toad (Rhinella marina) is one of the most successful invasive species worldwide, and has caused significant negative impacts on Australian fauna. Experimental work in the laboratory and in mesocosms has shown that tadpoles of native frogs can affect survival, size at metamorphosis and duration of larval period of cane toad tadpoles. To test if these effects occur in nature, we conducted a field experiment using two temporary ponds where we set up enclosures with tadpoles of native green tree frogs (Litoria caerulea) and cane toads in treatments with a range of densities and combinations. The presence of green tree frog tadpoles significantly decreased the growth rate of toad tadpoles and increased the duration of their larval period in both ponds; in one pond, frog tadpoles also significantly reduced the body length and mass of metamorph toads. Toad tadpoles did not have any significant negative effects on green tree frog tadpoles, but there was strong intraspecific competition within the latter species: increased frog tadpole density resulted in increased larval period and reduced survival, growth rate and size at metamorphosis for frogs at one or both ponds. Our results are encouraging for the possibility of using native frogs as one component of an integrated approach to the biological control of cane toads.     

Whispers from vestigial nubbins: Arrested development provokes trait loss in toads

Despite the use of acoustic communication, many species of toads (family Bufonidae) have lost parts of the tympanic middle ear, representing at least 12 independent evolutionary occurrences of trait loss. The comparative development of the tympanic middle ear in toads is poorly understood. Here, we compared middle ear development among two pairs of closely related toad species in the genera Atelopus and Rhinella that have (eared) or lack (earless) middle ear structures. We bred toads in Peru and Ecuador, preserved developmental series from tadpoles to juveniles, and examined ontogenetic timing and volume of the otic capsule, oval window, operculum, opercularis muscle, columella (stapes), and extracolumella in three‐dimensional histological reconstructions. All species had similar ontogenesis of the otic capsule, oval window, operculum, and opercularis muscle. Moreover, cell clusters of primordial columella in the oval window appeared just before metamorphosis in both eared and earless lineages. However, in earless lineages, the cell clusters either remained as small nubbins or cell buds in the location of the columella footplate within the oval window or disappeared by juvenile and adult stages. Thus, columella growth began around metamorphosis in all species but was truncated and/or degenerated after metamorphosis in earless species, leaving earless adults with morphology typical of metamorphic anurans. Shifts in the timing or expression of biochemical pathways that regulate the extension or differentiation of the columella after metamorphosis may be the developmental mechanism underlying convergent trait loss among toad lineages.     

Alterations in physical parameters and proteins of lens from Rana catesbeiana during development

Lens proteins and lens gross morphology were examined during tadpole and adult development of the bullfrog, Rana catesbeiana. Significant increases in the lens physical parameters of diameter, wet weight, dry weight (94–97% protein), and percent water were observed to accompany both natural and thyroxine-induced metamorphosis. These increases in lens parameters were not accompanied by growth of tadpoles during metamorphic change. Lens proteins were isolated from whole lenses and also from specified layers within whole lenses by a new fractionation method. Electrophoretic examination of whole lenses showed that the lens proteins did not change rapidly, one for another, prior to or during metamorphosis. However, changes became apparent during post metamorphic development. These changes included an increase in the relative concentration and mobility of alpha crystallin, a decrease in the relative concentration of gamma crystallin and an increase in the relative concentration of beta crystallin. Examination of specified layers within tadpole and frog lenses demonstrated that changes in the patterns of lens protein synthesis and modification may occur during development. Rapid and reproducible methods for quantitating changes in lens gross morphology and lens proteins, and for fractionating both tadpole and frog lenses into a number of definable layers were devised in the course of this study.     

Roles of stress hormones in food intake regulation in anuran amphibians throughout the life cycle

Towards understanding the ontogeny of energy balance regulation in vertebrates we analyzed the responses of corticotropin-releasing factor (CRF) and corticosterone to food deprivation in the Western spadefoot toad (Spea hammondii) at three developmental stages: premetamorphic tadpole, prometamorphic tadpole, and juvenile. Corticosterone responses to 5 days of food deprivation varied among developmental stages. Both pre- and prometamorphic tadpoles increased whole-body corticosterone content with food deprivation, but the magnitude of the response of premetamorphic tadpoles was significantly greater. By contrast, juvenile toads decreased plasma corticosterone concentration. Similarly, brain CRF peptide content tended to increase in food-deprived tadpoles but did not change in food-deprived juveniles. Therefore, there is an ontogenetic difference in the way the hypothalamic-pituitary-interrenal (HPI) axis responds to food deprivation in amphibians. In tadpoles, the HPI axis is activated in response to fasting as is seen in birds and mammals, and may be associated with mobilization of stored fuels and increased foraging. Juvenile toads do not respond to food deprivation by activating the HPI axis, but instead pursue a strategy of energy conservation that involves a reduction in plasma corticosterone concentration.     

Assessment of virally vectored autoimmunity as a biocontrol strategy for cane toads

Background

The cane toad, Bufo (Chaunus) marinus, is one of the most notorious vertebrate pests introduced into Australia over the last 200 years and, so far, efforts to identify a naturally occurring B. marinus-specific pathogen for use as a biological control agent have been unsuccessful. We explored an alternative approach that entailed genetically modifying a pathogen with broad host specificity so that it no longer caused disease, but carried a gene to disrupt the cane toad life cycle in a species specific manner.

Methodology/Principal Findings

The adult beta globin gene was selected as the model gene for proof of concept of autoimmunity as a biocontrol method for cane toads. A previous report showed injection of bullfrog tadpoles with adult beta globin resulted in an alteration in the form of beta globin expressed in metamorphs as well as reduced survival. In B. marinus we established for the first time that the switch from tadpole to adult globin exists. The effect of injecting B. marinus tadpoles with purified recombinant adult globin protein was then assessed using behavioural (swim speed in tadpoles and jump length in metamorphs), developmental (time to metamorphosis, weight and length at various developmental stages, protein profile of adult globin) and genetic (adult globin mRNA levels) measures. However, we were unable to detect any differences between treated and control animals. Further, globin delivery using Bohle iridovirus, an Australian ranavirus isolate belonging to the Iridovirus family, did not reduce the survival of metamorphs or alter the form of beta globin expressed in metamorphs.

Conclusions/Significance

While we were able to show for the first time that the switch from tadpole to adult globin does occur in B. marinus, we were not able to induce autoimmunity and disrupt metamorphosis. The short development time of B. marinus tadpoles may preclude this approach.     

Eye ontogeny in Pleurodema bufoninum: A comparison with Pleurodema somuncurense (Anura,Leptodactylidae)

Vision is one of the main sensory systems in amphibians, and the eye structure is highly associated with habitat conditions. The ontogeny, as well as the adult structure, of the eye has been studied in only a few species. The life change after metamorphosis is accompanied by changes in the visual environment. The aim of this work is to describe the eye ontogeny of Pleurodema bufoninum and to compare it with that of Pleurodema somuncurense . Specimens of both Pleurodema species were processed for histology analysis at different stages of development, including the tadpole, postmetamorphic, and adult forms. Eyes in both Pleurodema species are composed of the 3 tunics, tunica fibrosa , tunica vasculosa , and tunica interna , and the lens. Additionally, in both, the iris presents a projection on its dorsal and ventral free ends that screens the cornea. This structure has been reported in the eye of several anuran species and is called the umbraculum, meniscus or pupillary nodule. Our results show that the structures related to light capture (retina and lens) appear early in larval life, while the components of the terrestrial‐life eye (scleral cartilage, specialized cornea, eyelids, nictitating membrane, and Harderian's gland) do not develop until the metamorphic climax, when the tadpole leaves the water. The adult eyes of P. bufoninum and P. somuncurense are very similar in structure and development.     

Activity metabolism of the toad (Bufo americanus): Ecological consequences of ontogenetic change

Summary Following metamorphosis, juvenile toads form dense, heliothermic aggregations on the margins of the ponds from which they emerge. After a period of growth and development, during which body mass increases ten-fold, the juveniles disperse from the pond and assume the solitary, nocturnal habits that characterize adult toads.The dispersal of the toads coincides with the culmination of ontogenetic changes in activity metabolism. Unlike adults, juvenile toads exhaust rapidly when exercised and exhibit a low aerobic component of total metabolic scope. Values of aerobic scope for sustained activity ranged from 0.5 ml O₂/(g·h) for the smallest juveniles to 1.0 ml O₂/(g·h) for adults. The capacity of juveniles for oxygen consumption increases rapidly with growth, reaching the level of mature animals immediately prior to dispersal.In contrast to aerobic metabolism, anaerobic scope was independent of body size. Whole body lactate content after a 1 min bout of vigorous activity was elevated above the resting level by 0.14 mg lactate/g for both juvenile and adult animals.The elevated body temperatures that are achieved by basking juvenile toads probably hasten their physiological development and increase their capacity for aerobic metabolism.     

In vitro induction and transplantation of eye during early Xenopus development

A vertebrate eye was induced via a series of coordinated inductive interactions. Here, we describe a novel in vitro system to induce eye formation at high frequency using Xenopus early gastrulae. The eye formed in vitro is morphologically similar to the normal eye. When the in vitro eye was transplanted into a stage-33 tadpole, the optic nerve was seen extending from the grafted eye to the tectum of the host brain and the in vitro eye graft was retained after metamorphosis. In addition, we transplanted the eye formed in vitro into a tadpole with both eyes removed. The resultant juvenile frogs could perceive brightness using the grafted eye and thereby control their skin color, suggesting that the eye formed in vitro could function normally.     

The spatial ecology of cane toads (Bufo marinus) in tropical Australia: Why do metamorph toads stay near the water?

Abstract Cane toads (Bufo marinus) have invaded large areas of Australia, killing many native predators as they have done so. The metamorph stage of the life cycle – the first terrestrial phase, immediately after transformation from the tadpole – is critical for ecological impact (because these animals are small enough to be prey for many native predators) and for potential control of toad populations (because small body size renders metamorphs vulnerable to desiccation). To quantify the spatial and temporal distribution of metamorph toads, and the biotic and abiotic factors that might affect their distributions, we surveyed toad breeding sites in Australia's wet‐dry tropics (Adelaide River floodplain, NT) in both the wet season and the dry season. Metamorphs were concentrated close to the water's edge during the dry season, especially at midday when desiccation rates were highest. During the wet season, metamorphs were widely dispersed through the landscape. Our surveys indicate that abiotic factors (risk of desiccation) are most favourable for metamorph toads close to the pond edge, but biotic factors (food supply, and risk of competition and cannibalism) are most favourable away from the water. Operative temperatures were spatially homogeneous and sublethal, and so are unlikely to influence metamorph distribution. Desiccation risk fluctuated on a diel cycle as well as seasonally. We predict that metamorph toads benefit from dispersing as soon as desiccation risk allows them to do so, and hence the distribution of metamorph toads will shift dynamically in response to weather‐mediated changes in rates of evaporative water loss.     

Vestibular Lesion-Induced Developmental Plasticity in Spinal Locomotor Networks during Xenopus laevis Metamorphosis

During frog metamorphosis, the vestibular sensory system remains unchanged, while spinal motor networks undergo a massive restructuring associated with the transition from the larval to adult biomechanical system. We investigated in Xenopus laevis the impact of a pre- (tadpole stage) or post-metamorphosis (juvenile stage) unilateral labyrinthectomy (UL) on young adult swimming performance and underlying spinal locomotor circuitry. The acute disruptive effects on locomotion were similar in both tadpoles and juvenile frogs. However, animals that had metamorphosed with a preceding UL expressed restored swimming behavior at the juvenile stage, whereas animals lesioned after metamorphosis never recovered. Whilst kinematic and electrophysiological analyses of the propulsive system showed no significant differences in either juvenile group, a 3D biomechanical simulation suggested that an asymmetry in the dynamic control of posture during swimming could account for the behavioral restoration observed in animals that had been labyrinthectomized before metamorphosis. This hypothesis was subsequently supported by in vivo electromyography during free swimming and in vitro recordings from isolated brainstem/spinal cord preparations. Specifically, animals lesioned prior to metamorphosis at the larval stage exhibited an asymmetrical propulsion/posture coupling as a post-metamorphic young adult. This developmental alteration was accompanied by an ipsilesional decrease in propriospinal coordination that is normally established in strict left-right symmetry during metamorphosis in order to synchronize dorsal trunk muscle contractions with bilateral hindlimb extensions in the swimming adult. Our data thus suggest that a disequilibrium in descending vestibulospinal information during Xenopus metamorphosis leads to an altered assembly of adult spinal locomotor circuitry. This in turn enables an adaptive compensation for the dynamic postural asymmetry induced by the vestibular imbalance and the restoration of functionally-effective behavior.     

The Nutritional Efficiency of Cannibalism and an Artificial Feed for the Growth of Tadpoles of Japanese Toad (Bufo vulgaris sp.)

Tadpoles of Japanese toad (Bufo vulgaris) were cultured by an artificial feed and by forced cannibalism from the egg until metamorphosis finished. The best growth was found in the artificial feed group, however, the highest feed efficiency was observed in the cannibalism group. The tadpoles were metamorphosed by the culture with amino acid mixture feed but no body weight increase was seen in this case. The nutritional disturbance hindered the metamorphosis.

The relative amino acid composition was constant throughout all the stages of the tadpole growth. The relative amino acid composition of the toad was diversed according to the species. Several unidentified ninhydrine-positive substances were detected on the column chromatograms of amino acid analysis of tadpoles and their adult forms.     

Toads in the backyard: why do invasive cane toads (Rhinella marina) prefer buildings to bushland?

Like many invasive species, cane toads (Rhinella marina) in Australia concentrate in the disturbed habitats created by human activity, rather than in pristine areas. We surveyed cane toads in the wet–dry tropics of the Northern Territory to assess the abundances, body sizes, sexes, behaviour, hydration state and feeding rates of toads around buildings compared to those in areas remote from buildings, and conducted experimental trials to assess the effects of building-related variables (lights and increased toad densities) on the foraging success of toads. Toads around buildings were smaller than bushland conspecifics, and adult sex-ratios were female-biased. Toads were more sedentary around buildings than in the bush, but their feeding rates (based on direct observations and faeces production post-capture) were similar. That similarity, despite twofold-higher densities of competing toads around building, reflected the strong enhancement of feeding rates due to artificial lights attracting insects (in our experimental trials, a threefold increase regardless of the number of competing toads). Toads collected from around buildings were apparently in better hydric condition. Thus, access to water also may attract toads to buildings. The relative scarcity of adult male toads around buildings likely reflects waterbody-centred reproductive activities, whereas the concentration of females and juveniles around buildings is driven largely by access to the insects attracted by artificial light. We conclude that buildings enhance the persistence of cane toad populations and may facilitate their spread.     

Binocular vision and accommodation in prey-catching chameleons

We have studied the role of accommodation and binocular convergence in the predatory behaviour of two chameleon species (Chamaeleocalyptratus, C. dilepis). Accommodation measurements support earlier observations that accommodation is the major distance cue. Specifically, accommodation speed (60 D s⁻¹), amplitude (45 D) and precision (no significant under-accommodation) were superior to those of other terrestrial vertebrates. Similar to other vertebrates, accommodation was accompanied by a prominent pupillary constriction (pupillary near response). Accommodation could be coupled or uncoupled in both eyes, depending on the experimental situation or the phase of the predatory behavioural sequence. Uncoupled accommodation occurred: 1. During scanning saccadic eye movements for prey detection. Only one eye accommodated appropriately, the other adopted a hyperopic resting refractive state. Attention switched from one eye to the other at approximately 1-s intervals. 2. During initial stages of distance estimation. Coupled accommodation only occurred shortly before the tongue shot. Coupling was demonstrated by either covering one eye with a lens or covering one eye with an infrared light transmitting cut-off filter which still permitted refraction to be measured. In both cases the amount of accommodation was identical in both eyes. Search-coil measurements showed that the angle of convergence of both eyes is too variable to permit triangulation or to provide the basic requirement for stereopsis (matching corresponding points). We conclude that coupling of accommodation serves to improve accommodation precision rather than to permit stereopsis. Accepted: 3 September 1997     

Physiological basis for diurnal activity in dispersing juvenile Bufo granulosus in the Caatinga, a Brazilian semi-arid environment

Diurnal activity is characteristic of many toad species, including Bufo granulosus from the Brazilian semi-arid biome called the Caatinga. Because of their patterns of activity, juvenile toads are exposed to hot and dehydrating conditions. Our investigation focuses on temperature and water relationships, and is based on the prediction that anuran diurnal activity in a semi-arid environment must be associated with morphological, physiological and behavioral traits enhancing thermal tolerances, capacity for performance at high temperatures and water balance. To test specific hypothesis related with this prediction, we investigated postmetamorphic B. granulosus and collected data on thermal tolerances and preferences, thermal safety margins, thermal dependence of locomotor behavior, thermal and kinetic properties of citrate synthase (CS), and skin morphophysiology. This information was compared with additional data from adult conspecifics and adult toads from sympatric species or from species from more moderate environments. We found that juvenile B. granulosus exhibit the highest critical maximum temperature reported for toads (44.2 degrees C) and are well suited to move at high temperatures. However, and in contrast with juveniles of other Bufo species, they do not show thermal preferences in a gradient and appear to hydroregulate more than thermoregulate. The CS of adult and juvenile toads shows typical patterns of thermal sensibility, but the thermal stability of this enzyme is much higher in juveniles than in adult Bufo of any other species studied. The inguinal skin exhibits a complex folding pattern and seems highly specialized for capillary water uptake. Diurnal activity in juvenile B. granulosus is possible given high thermal tolerances, keen ability to detect and uptake water, and avoidance behaviors.     

Life history consequences of feeding versus non-feeding in a facultatively non-feeding toad larva

Summary Bufo periglenes, a toad endemic to montane Costa Rica, produces an unusually small clutch of large, yolk-rich eggs. The toads breed in small ephemeral pools that are unpredictable in duration and may be low in food availability. Two congeners, Bufo coniferus and Bufo marinus, occur nearby, breed in more permanent bodies of water that offer more food, and exhibit the typical toad pattern of large clutches of small eggs. Tadpoles of all three species feed on detritus and suspended organic material. By raising tadpoles of the three species individually with and without food I investigated the relationship between egg size (yolk provision) and tadpole survival. All of the unfed B. coniferus and B. marinus tadpoles grew little and died soon after developing to the hindlimb bud stage. On the other hand, all of the unfed B. periglenes tadpoles metamorphosed successfully, demonstrating that the tadpoles are facultatively non-feeding; developmental time from hatching to metamorphosis was significantly shorter for unfed tadpoles than for fed tadpoles, but fed individuals were significantly larger at transformation. Faster developmental rate and larger body size at transformation are both advantageous for frogs and toads, but cannot be attained simultaneously. Large egg size may afford flexibility in unpredictable environments. In pools where food is available, tadpoles presumably eat, take longer to metamorphose, but are larger at transformation than tadpoles developing in nutrient-poor sites. Small body size at transformation (a consequence of not eating) has potential costs, but the large quantity of yolk provided by a large egg enhances the probability of metamorphosis in food-limited environments.     

Allometry and selection in a novel predator–prey system: Australian snakes and the invading cane toad

Because many organismal traits vary with body size, interactions between species can be affected by the respective body sizes of the participants. We focus on a novel predator–prey system involving an introduced, highly toxic anuran (the cane toad, Bufo marinus ) and native Australian snakes. The chance of a snake dying after ingesting a toad depends on the size of the snake and the size of the toad, and ultimately reflects the effect of four allometries: (1) physiological tolerance (the rate that physiological tolerance to toad toxin changes with snake size); (2) swallowing ability (the rate that maximal ingestible toad size (i.e. snake head size) increases with snake body size); (3) prey size (the rate that prey size taken by snakes increases with snake head size) and (4) toad toxicity (the rate that toxicity increases with toad size). We measured these allometries, and combined them to estimate the rate at which a snake's resistance changes with toad toxicity. The parotoid glands (and thus, toxicity) of toads increased disproportionately with toad size (i.e. relative to body size, larger toads were more toxic) but simultaneously, head size relative to body size (and thus, maximal ingestible prey size relative to predator size) declined with increasing body size in snakes. Thus, these two allometries tended to cancel each other out. Physiological tolerance to toxins did not vary with snake body size. The end result was that across snake species, mean adult body size did not affect vulnerability. Within species, however, smaller predators were more vulnerable, because the intraspecific rate of decrease in relative head size of snakes was steeper than the rate of increase in toxicity of toads. Thus, toad invasion may cause disproportionate mortality of juvenile snakes, and adults of the sex with smaller mean adult body sizes.     

Lens crystallin changes associated with amphibian metamorphosis: involvement of a beta-crystallin polypeptide

Lens crystallins isolated from the tadpole and frog lenses were compared with regard to the developmental changes of crystallin compositions. The major changes during the process of metamorphosis were (1) the total contents of alpha- and gamma-crystallins decrease from more than 70% to less than 60% and (2) one of the major beta-crystallin polypeptides increases from less than 1% to about 6% and (3) an amphibian-specific rho-crystallin also increases from about 6% to more than 10% of total soluble proteins of the lens. We have characterized the metamorphosis-dependent beta-crystallin polypeptide by peptide mapping and sequence determination of the protease-digested fragments. This polypeptide showed very high sequence homology to that of the major beta Bp-crystallin chain reported for the mammalian lenses. The changes of the relative abundance of various crystallins and the gradually-elevated levels of the expression of this beta Bp-like crystallin in the developing lens during metamorphosis may also have some bearing on the maintenance of lens stability in the adult frog lenses.