Direct and indirect effects of the introduced toad Bufo marinus (Anura: Bufonidae) on populations of native anuran larvae in Australia

Introduced species are frequently believed to have adverse effects on native biota and ecosystems. However, much of our knowledge of the ecological impacts of introduced species is anecdotal, and the mechanisms controlling these effects are often poorly understood. I used replicated artificial pond experiments to investigate the impact of eggs and hatchlings of the introduced toad Bufo marinus on populations of native anuran larvae (Limnodynastes ornatus and Litoria rubella) in Australia. Bufo marinus eggs and hatchlings are highly toxic to predatory native tadpoles. Under naturalistic conditions, populations of predatory L. ornatus tadpoles experienced significantly reduced survival when exposed to Bufo eggs and hatchlings. The impact of Bufo on L. ornatus survival was positively correlated with Bufo density. However, the toxic effects of Bufo on L. ornatus indirectly facilitated the survival of later-breeding L. rubella by altering predator-prey interactions between L. ornatus and L. rubella. Limnodynastes ornatus tadpoles are voracious predators of L. rubella eggs and hatchlings. Consequently, the negative impact of Bufo on populations of L. ornatus tadpoles reduced the intensity of predation by L. ornatus tadpoles on L. rubella eggs and hatchlings, thereby increasing L. rubella survival. The results demonstrate that B. marinus plays an important role in structuring native larval anuran communities via direct and indirect mechanisms, and that Bufo may have both negative and positive effects on populations of native anuran larvae. As far as I am aware, these are the first quantitative data to demonstrate that introduced fauna may affect populations of native biota via toxic effects.     

Ability of predatory native Australian fishes to learn to avoid toxic larvae of the introduced toad Bufo marinus

Of two native Australian fishes naïve to the introduced toad Bufo marinus most barramundi Lates calcarifer rapidly learned to avoid B. marinus tadpoles, while sooty grunter Hephaestus fuliginosus exhibited considerable intraspecific variation in their learning ability. Some sooty grunter learned to avoid tadpoles after only a few attacks, while other individuals continued to attack and reject tadpoles throughout the entire laboratory trials. Individuals of both species recognized and avoided tadpoles 1 day after their previous encounter. None of the fishes died during the trials. The observed variation in behavioural responses of fishes to B. marinus may be due to differences in (1) learning ability, (2) fish hunger levels, and or (3) tadpole palatability and toxicity. The results demonstrate that most barramundi and sooty grunter learn to avoid B. marinus tadpoles with minimal trauma. Consequently, it is anticipated that the toads are unlikely to have a significant negative impact on wild populations of these fishes through direct toxic effects.     

Cysteine-scanning mutagenesis study of the sixth transmembrane segment of the Na,K-ATPase alpha subunit

The accessibility of the residues of the sixth transmembrane segment (TM) of the Bufo marinus Na,K-ATPase alpha subunit was explored by cysteine scanning mutagenesis. Methanethiosulfonate reagents reached only the two most extracellular positions (T803, D804) in the native conformation of the Na,K-pump. Palytoxin induced a conductance in all mutants, including D811C, T814C and D815C which showed no active electrogenic transport. After palytoxin treatment, four additional positions (V805, L808, D811 and M816) became accessible to the sulfhydryl reagent. We conclude that one side of the sixth TM helix forms a wall of the palytoxin-induced channel pore and, probably, of the cation pathway from the extracellular side to one of their binding sites.     

High-pass filtering of small signals by retinal rods. Ionic studies

The high-pass filtering of small signals by the rod photoreceptor network was studied by intracellular recording in the isolated, perfused retina of the toad, Bufo marinus. Data were analyzed and interpreted in terms of the network analysis described in the preceding paper. External concentrations of Cs+ as high as 10 mM, which blocked the relaxation from peak to plateau of the rod's response to bright light, did not affect the filtering of small signals. The effects of reducing [Na+]o were not consistent with a direct action upon the mechanism underlying this filtering property. By contrast, raising external [K+] from 2.6 to 10 mM, which caused a fourfold reduction in EK, abolished the high-pass filtering of small signals. Analysis of the effects of external [K+] changes indicates that the underlying mechanism involves a K+ conductance that decreases with a delay when the rod is hyperpolarized. This conductance is not blocked by externally applied tetraethylammonium. Other experiments did not rule out the possibility that it might be activated by Ca++.     

Bufo marinus oocytes as a model for ion channel protein expression and functional characterization for electrophysiological studies.

Oocytes from Xenopus laevis are commonly used as an expression system for ion channel proteins. The aim of this study was to determine whether oocytes from the Colombian native toad, Bufo marinus, could be used as an alternative expression system for ion channel protein expression and functional characterization using the two-microelectrode voltage clamp method. B. marinus oocytes and X. laevis were isolated and cultured in similar conditions. The mean resting membrane potential of B. marinus oocytes was similar to that of X. laevis oocytes as well as the whole-cell basal currents. The potassium ion channel Kv1.1 was successfully expressed in B. marinus oocytes and showed a typical outward rectifying current. Potassium channel blockers reduced these currents. The similarities on electrical properties and expression of ion channel proteins show that B. marinus oocytes can be used effectively to express these proteins, making these cells a viable heterologous system for the expression of ion channel proteins and their electrophysiological characterization.     

Kinematics of prey capture in the tailed frog Ascaphus truei (Anura: Ascaphidae)

The kinematics of prey capture by Ascaphus truei was investigated. High-speed films (100 fps) of 13 successful and one unsuccessful prey capture sequences from six adult frogs were analysed. Ascaphus , the sister group of all living frogs, shares several aspects of feeding kinematics, including rotation of the tongue pad about the mandibular symphysis and mandibular bending during mouth opening and closing, with more derived frogs such as Bufo marinus. The times required for tongue retraction, mouth opening and closing are similar in Ascaphus and Bufo. However, because Bufo is much larger and protracts its tongue much farther than Ascaphus , the velocities of tongue retraction, mouth opening and mouth closing are relatively lower in Ascaphus than in Bufo. Differences in prey capture between Ascaphus and Bufo marinus are (1) the distance of tongue protraction is less in Ascaphus (±0.5 cm) than in Bufo (c. 2 cm); and (2) lunging of the whole body is more pronounced in Ascaphus. Prey capture is highly variable in Ascaphus. An intraoral transport sequence is sometimes (7 of 14 observations) inserted into the prey capture cycle before the completion of mouth closing. The gape cycles range from 80–150 ms for sequences with no oral transport and from 130–280 ms for sequences with oral transport. Also, the time required for tongue retraction is significantly longer in the unsuccessful capture attempt. This variability is generally greater than that observed during prey capture in salamanders, and suggests that frogs and salamanders may differ in the importance of sensory feedback in coordinating prey capture.     

Metabolic origins of 5-hydroxytryptamine in enteric neurons in a teleostean fish (Platycephalus bassensis), a toad (Bufo marinus) and the guinea-pig

1. 5-Hydroxytryptamine (5-HT) content and synthesis in mucosa-free intestine of guinea-pig, the teleost Platycephalus bassensis and the amphibian Bufo marinus was studied by HPLC with electrochemical detection or by TLC. 2. The 5-HT content of small intestine was: guinea-pig 0.58; Bufo: 1.23; Platycephalus: 26.88 nmol/g. 3. Intestine from each species synthesized 5-HT from exogenous 5-HTP. 4. Platycephalus preparations synthesized labelled 5-HT from 14C-tryptophan, but no labelled 5-HT was detected after similar incubation of guinea-pig or Bufo preparations. 5. Incubation of guinea-pig preparations with tryptophan did not increase tissue 5-HT or 5-HIAA content. 6. 5-HT in Platycephalus enteric neurons may be synthesized from tryptophan in situ; 5-HT in Bufo and guinea-pig neurons may be synthesized elsewhere, perhaps in enterochromaffin cells.     

Pyruvate transport in isolated cardiac mitochondria from two species of amphibian exhibiting dissimilar aerobic scope: Bufo marinus and Rana catesbeiana

Cardiac mitochondria were isolated from Bufo marinus and Rana catesbeiana, two species of amphibian whose cardiovascular systems are adapted to either predominantly aerobic or glycolytic modes of locomotion. Mitochondrial oxidative capacity was compared using VO2 max and respiratory control ratios in the presence of a variety of substrates including pyruvate, lactate, oxaloacetate, beta-hydroxybutyrate, and octanoyl-carnitine. B. marinus cardiac mitochondria exhibited VO2 max values twice that of R. catesbeiana cardiac mitochondria when oxidizing carbohydrate substrates. Pyruvate transport was measured via a radiolabeled-tracer assay in isolated B. marinus and R. catesbeiana cardiac mitochondria. Time-course experiments described both alpha-cyano-4-hydroxycinnamate-sensitive (MCT-like) and phenylsuccinate-sensitive pyruvate uptake mechanisms in both species. Pyruvate uptake by the MCT-like transporter was enhanced in the presence of a pH gradient, whereas the phenylsuccinate-sensitive transporter was inhibited. Notably, anuran cardiac mitochondria exhibited activities of lactate dehydrogenase and pyruvate carboxylase. The presence of both transporters on the inner mitochondrial membrane affords the net uptake of monocarboxylates including pyruvate, beta-hydroxybutyrate, and lactate; the latter potentially indicating the presence of a lactate/pyruvate shuttle allowing oxidation of extramitochondrial NADH. Intramitochondrial lactate dehydrogenase and pyruvate carboxylase enables lactate to be oxidized to pyruvate or converted to anaplerotic oxaloacetate. Kinetics of the MCT-like transporter differed significantly between the two species, suggesting differences in aerobic scope may be in part attributable to differences in mitochondrial carbohydrate utilization.     

Natural and experimental infection of the lizard Ameiva ameiva with Hemolivia stellata (Adeleina: Haemogregarinidae) of the toad Bufo marinus

Developmental stages of a haemogregarine in erythrocytes of the lizard Ameiva ameiva (Teiidae), from Pará State, north Brazil, were shown to be those of Hemolivia by the nature of the parasite's sporogonic cycle in the tick Amblyomma rotondatum. The type species, Hemolivia stellata Petit et al., 1990 was described in the giant toad Bufo marinus and the tick Amblyomma rotondatum, also from Pará State, and in view of the fact that A. ameiva and Bufo marinus share the same habitat and are both commonly infested by A. rotondatum, the possibility that the parasite of A. ameiva is H. stellata had to be considered. Uninfected lizards fed with material from infected ticks taken from B. marinus, and others fed with liver of toads containing tissue-cysts of H. stellata, were shown to subsequently develop typical Hemolivia infections, with all stages of the development similar to those seen in the naturally infected lizards. Conversely, a juvenile, uninfected toad became infected when fed with sporocysts of Hemolivia in a macerated tick that had fed on an infected A. ameiva and pieces of liver containing tissuecysts from the same lizard. The remarkable lack of host specificity shown by H. stellata, in hosts so widely separated as an amphibian and a reptile, is discussed.     

Effects of meal size,meal type,body temperature,and body size on the specific dynamic action of the marine toad,Bufo marinus

Specific dynamic action (SDA), the accumulated energy expended on all physiological processes associated with meal digestion, is strongly influenced by features of both the meal and the organism. We assessed the effects of meal size, meal type, body temperature, and body size on the postprandial metabolic response and calculated SDA of the marine toad, Bufo marinus. Peak postprandial rates of O(2) consumption (.V(O2)) and CO(2) production (.V(CO2)) and SDA increased with meal size (5%-20% of body mass). Postprandial metabolism was impacted by meal type; the digestion of hard-bodied superworms (Zophobas larva) and crickets was more costly than the digestion of soft-bodied earthworms and juvenile rats. An increase in body temperature (from 20 degrees to 35 degrees C) altered the postprandial metabolic profile, decreasing its duration and increasing its magnitude, but did not effect SDA, with the cost of meal digestion remaining constant across body temperatures. Allometric mass exponents were 0.69 for standard metabolic rate, 0.85 for peak postprandial .V(O2), and 1.02 for SDA; therefore, the factorial scope of peak postprandial .V(O2) increased with body mass. The mass of nutritive organs (stomach, liver, intestines, and kidneys) accounted for 38% and 20% of the variation in peak postprandial .V(O2) and SDA, respectively. Toads forced to exercise experienced 25-fold increases in .V(O2) much greater than the 5.5-fold increase experience during digestion. Controlling for meal size, meal type, and body temperature, the specific dynamic responses of B. marinus are similar to those of the congeneric Bufo alvarius, Bufo boreas, Bufo terrestris, and Bufo woodhouseii.     

Clinostomum attenuatum (Digenea) from the eye of Bufo marinus

Two specimens of the cane toad, Bufo marinus, were found with single worm infestations of the eye. One worm was excysted in the eye chamber, the other still encysted in the choroid layer; both worms were identified as Clinostomum attenuatum. This finding is both a new host record and a new site of development for this amphibian parasite.     

Serosal and mucosal facilitated transport of urea in urinary bladder of of Bufo bufo: evidence for an alleged water uptake

In Bufo bufo urinary bladder an urea facilitated transport has been localised on the luminal membrane. The transport fulfils the criteria for such a mechanism, i.e. is saturable and is inhibited by phloretin, a specific inhibitor for urea transport. Similarly to that of Bufo marinus and Rana esculenta the luminal membrane of Bufo bufo urinary bladder shows an ADH stimulated facilitated transport. Experiments wtih Amphotericin B, serosal phloretin (with and without ADH), have demonstrated the presence of a facilitated urea transport localised on basolateral membrane. Urea uptake on the isolated epithelial cells of Bufo bufo urinary bladder shows a characteristic feature, different from molecules passively transported such as glycerol yet inhibited by phloretin. Allegedly with urea, water flows in to the cells by a dragging or osmotic effect.     

Mechanism of inhibition by lithium of sodium transport in the toad bladder

Lithium transport across the urinary bladder of Bufo marinus has been studied by means of the short-circuit current technique, as well as unidirectional ion flux measurements. Exposure to lithium of the epithelial (mucosal) surface of this preparation led to a slow, progressive decrease of ion transport, with increasing discrepancy between short-circuit current and lithium influx; in fact there was still an appreciable lithium influx across bladder exposed to amiloride even though short-circuit current was suppressed. Ohmic conductance and sodium efflux barely increased under these circumstances. Upon replacement of lithium by sodium on the epithelial side, the preparations recovered slowly indeed, and residual lithium could be detected in bladder tissue for more than 2 hr while the rate of sodium extrusion at the basal-lateral cell border was slowed down. Recovery from exposure to lithium was accelerated by vasopressin and amphotericin, both of which facilitate sodium entry at the apical border of the epithelium. Thus the lasting deleterious influence of lithium on sodium transport might result from the fact that this ion, once trapped in the cytoplasm, closes the sodium channels.     

Leptospires in the marine toad (Bufo marinus) on Barbados

Leptospires were isolated from the kidneys of four of 211 toads (Bufo marinus) caught on Barbados. Two of the isolates were identified as Leptospira interrogans serovar bim in the Autumnalis serogroup (the most common cause of leptospiral illness on Barbados), and two as possibly new serovars in the Australis serogroup. Sera from 198 of the toads were examined by the leptospire microscopic agglutination test. Forty-two (21%) were positive at titers of greater than or equal to 1:100, and 54 (27%) at greater than or equal to 1:50. The predominating serogroups were Australis (50%), Autumnalis (23%) and Panama (13%). The agglutination tests on the culture-positive toads showed that serologic studies alone may be of limited value in these animals. Bufo marinus can harbor pathogenic leptospires, and it may be a significant source of the Autumnalis serogroup infections in the Caribbean.     

Transport-related modulation of the membrane properties of toad urinary bladder epithelium.

Impedance analysis and transepithelial electrical measurements were used to assess the effects of the apical membrane Na+ channel blocker amiloride and anion replacement on the apical and basolateral membrane conductances and areas of the toad urinary bladder (Bufo marinus). Mucosal amiloride addition decreased both apical and basolateral membrane conductances (Ga and Gbl, respectively) with no change in membrane capacitances (Ca and Cbl). Consequently, the specific conductances of these membranes decreased without significant changes in membrane area. Following amiloride removal, an increase was obtained in the steady-state rate of sodium transport compared to values before amiloride addition. This increase was independent of the initial transport rate, suggesting activation of a quiescent pool of apical sodium channels. Chloride replacement by acetate or gluconate had no significant effects on apical or basolateral membrane capacitances. The effects of these replacements on membrane conductances depended on the anion species. Gluconate (which induces cell shrinkage) decreased both membrane conductances. In contrast, acetate (which induces cell swelling) increased Ga and had no effect on Gbl. The increase in the apical membrane conductance was due to an increase in the amiloride-sensitive Na+ conductance of this membrane. In summary, mucosal amiloride addition or chloride replacements led to changes in membrane conductances without significant effects on net membrane areas.     

Phylogeography of Bufo marinus from its natural and introduced ranges.

The marine toad, Bufo marinus, has a broad natural distribution extending from the south-west of the USA to southern Peru and the central Amazon. It was introduced to several localities in the Caribbean and Pacific Oceans to control sugar cane pests. We sequenced 468 bp of mitochondrial DNA (mtDNA) containing the ND3 gene, and flanking tRNA genes from toads spanning the broad natural and introduced ranges. Consistent with the known history of introductions and expected effects of serial bottlenecks, mtDNA within introduced populations in Hawaii and Australia was uniform and most closely related to samples from eastern Venezuela and French Guiana. However, mtDNA nucleotide diversity in the geographic region spanning the source areas is also relative low (0.18-0.46%) and the absence of variation in the introduced populations precludes quantitative assessment of the reduction in genetic diversity. Unexpectedly, there was a large phylogeographic break (5.4% sequence divergence) within the natural range separating populations east and west of the Venezuelan Andes. We hypothesize that the two major lineages of B. marinus were isolated by the uplift of the eastern Andean cordillera which was completed approximately 2.7 Ma. Another species of the marinus group, B. paracnemis, had mtDNA paraphyletic, with marinus, being nested within the eastern lineage. Thus, at least one speciation event within the marinus group postdates the split within marinus. These findings suggest that the taxonomy of B. marinus should be re-evaluated and that the search for pathogens to control Australian populations should be conducted in populations from both lineages in the natural range.     

Pepsin in the toad Bufo marinus

The macroscopic localization of pepsinogen in the upper gastrointestinal tract of the anuran Bufo marinus was studied by means of biochemical assay. The pH optimum of the anuran pepsin was determined to be 1.6. The effect of lowered ambient temperature on stored pepsinogen and proteolytic activity was studied. Both parameters were reduced. The results are extrapolated to speculate on the mechanisms of gastric brooding in gastric brooding frogs of the genus Rheobatrachus.     

Impact of the invasive cane toad (Bufo marinus) on an Australian frog (Opisthodon ornatus) depends on minor variation in reproductive timing

Invasive species are widely viewed as unmitigated ecological catastrophes, but the reality is more complex. Theoretically, invasive species could have negligible or even positive effects if they sufficiently reduce the intensity of processes regulating native populations. Understanding such mechanisms is crucial to predicting ultimate ecological impacts. We used a mesocosm experiment to quantify the impact of eggs and larvae of the introduced cane toad (Bufo marinus) on fitness-related traits (number, size and time of emergence of metamorphs) of a native Australian frog species (Opisthodon ornatus). The results depended upon the timing of oviposition of the two taxa, and hence the life-history stages that came into contact. Growth and survival of O. ornatus tadpoles were enhanced when they preceded B. marinus tadpoles into ponds, and reduced when they followed B. marinus tadpoles into ponds, relative to when tadpoles of both species were added to ponds simultaneously. The dominant tadpole-tadpole interaction is competition, and the results are consistent with competitive priority effects. However, these priority effects were reduced or reversed when O. ornatus tadpoles encountered B. marinus eggs. Predation on toxic toad eggs reduced the survival of O. ornatus and B. marinus. The consequent reduction in tadpole densities allowed the remaining O. ornatus tadpoles to grow more rapidly and to metamorphose at larger body sizes (>60% disparity in mean mass). Thus, exposure to B. marinus eggs reduced the number of O. ornatus metamorphs, but increased their body sizes. If the increased size at metamorphosis more than compensates for the reduced survival, the effective reproductive output of native anurans may be increased rather than decreased by the invasive toad. Minor interspecific differences in the seasonal timing of oviposition thus have the potential to massively alter the impact of invasive cane toads on native anurans.     

Salmonella in wildlife from Trinidad and Grenada, W.I.

Forty-four of 219 animals from Trinidad and Grenada, W.I., yielded 20 serotypes of Salmonella, 16 of which are known to have been associated with human infection in the United States in recent years. Toads (Bufo marinus) provided the greatest number of isolates. Other carriers were mammals, vultures, lizards, a tree-frog and a cave cockroach.     

The weak link: do muscle properties determine locomotor performance in frogs?

Muscles power movement, yet the conceptual link between muscle performance and locomotor performance is poorly developed. Frog jumping provides an ideal system to probe the relationship between muscle capacity and locomotor performance, because a jump is a single discrete event and mechanical power output is a critical determinant of jump distance. We tested the hypothesis that interspecific variation in jump performance could be explained by variability in available muscle power. We used force plate ergometry to measure power produced during jumping in Cuban tree frogs (Osteopilus septentrionalis), leopard frogs (Rana pipiens) and cane toads (Bufo marinus). We also measured peak isotonic power output in isolated plantaris muscles for each species. As expected, jump performance varied widely. Osteopilus septentrionalis developed peak power outputs of 1047.0 ± 119.7 W kg(-1) hindlimb muscle mass, about five times that of B. marinus (198.5 ± 54.5 W kg(-1)). Values for R. pipiens were intermediate (543.9 ± 96.2 W kg(-1)). These differences in jump power were not matched by differences in available muscle power, which were 312.7 ± 28.9, 321.8 ± 48.5 and 262.8 ± 23.2 W kg(-1) muscle mass for O. septentrionalis, R. pipiens and B. marinus, respectively. The lack of correlation between available muscle power and jump power suggests that non-muscular mechanisms (e.g. elastic energy storage) can obscure the link between muscle mechanical performance and locomotor performance.