Trade-off between larval development rate and Post-metamorphic Traits in the Frog Rana latastei

Development rate early in the ontogeny is believed to correlate positively with fitness. Geographic variation in intrinsic development rate suggests the existence of trade-offs between development rate and other fitness related traits. We investigated whether these trade-offs exist between intrinsic larval development rate and post-metamorphic traits in an organism with a complex life cycle. In laboratory, we measured if the tadpoles of the frog Rana latastei with fast intrinsic development rate have a suboptimal post-metamorphic morphology, by comparing froglets from five populations. Then, we evaluated the relationship between age at metamorphosis, hindlimb length and jumping performance for frogs grown in nature in two populations. Under laboratory conditions, froglets with fast intrinsic development had shorter absolute and shorter size-adjusted tibiofibulas. We observed a strong, positive relationship between tibiofibula length and jumping performance. In nature, froglets from the last metamorphosing population had longer absolute and size-adjusted tibiofibulas, and were able to jump further. The cost of fast development could be the shorter legs of early metamorphosing frogs, and their poor jumping performance. Thus, a fast intrinsic development rate may not always be positively related to lifetime fitness, since delayed effects of larval development persist also across life history stages. Co-ordinating editor: V. Jormalainen     

Larval and metamorphic skeletal development in the fast-developing frog Pyxicephalus adspersus (Anura, Ranidae)

Pyxicephalus adspersus , is exceptional among living frogs. Embryonic development, larval phase, and metamorphosis can be completed in 17 days at a temperature of 29°C. The metamorphosis only takes 5 days. The present study shows that, despite the unusually short larval phase in P. adspersus, the state of skeletal differentiation reached at the end of metamorphosis is similar to that of other frog species. There is no shift of cranial bone formation postmetamorphosis as could have been expected and is known from other species. The majority of compared species are particularly similar in the sequence of bone formation in the postcranial skeleton. However, there are clear differences among species in the timing of these events relative to the larval growth trajectory, absolute time, and certain developmental markers, such as external limb differentiation. For example, skeletogenesis and externally visible limb differentiation are only loosely integrated. Interspecific comparisons show that, in P. adspersus, the early onset of skeletal ossification is an unusual feature among frogs. Freshly metamorphosed froglets of P. adspersus are already distinct from comparable stages of other species in having strong jaws, fang-like teeth, and a squamosal-maxilla contact. The latter stabilizes the maxillary arcade and the suspensorium and might relate to the ability to catch and swallow very large vigorous prey, such as siblings, shortly after metamorphosis. The presence of a complete set of dermatocranial elements and postmetamorphic ossification of only the sphenethmoid and operculum are considered plesiomorphic features, whereas the much less completely ossified skulls of metamorphosed froglets, particularly in Bufo and Hamptophryne, are likely apo- morphic developmental traits within the Anura. Accepted: 11 January 1999     

Outbreeding depression in the common frog, <Emphasis Type="Italic">Rana temporaria</Emphasis>

Theory suggests that parental relatedness is a continuous variable with a fitness optimum that we heretoforth will refer to as optimal outbreeding. In the present paper, we test this proposition from a conservation (translocation) perspective. Amphibians are facing a global decline and many amphibian populations are today small and threatened by extinction. Because genetic differentiation is often high between amphibian populations, they could be particularly sensitive to outbreeding depression, e.g. due to breakdown of locally adapted gene complexes. We tested if outbreeding would reduce fitness in common frogs, Rana temporaria, crossed from a large and an isolated, small population, separated by 130km, using artificial fertilization. For females from the large population, tadpoles were significantly smaller and more malformed in crosses with males from the small population, than with males from the large population. For the small population, however, no significant paternal genetic effects could be found. The difference in response to outbreeding between populations was accompanied with significant differences in the importance of maternal effects. We conclude that care should be taken when translocating frogs between distantly related populations to avoid outbreeding depression.     

Coding of amplitude modulation in the auditory midbrain of the bullfrog (Rana catesbeiana) across metamorphosis

The functional development of the auditory system across metamorphosis was examined by recording neural activity from the torus semicircularis of larval and postmetamorphic bullfrog froglets in response to amplitude-modulated sound. Multiunit activity in the torus semicircularis during early larval stages showed significant phase-locking to the envelopes of amplitude-modulated noise bursts, up to modulation rates as high as 250 Hz. Beginning at metamorphic climax and continuing into the froglet period, phase locking was restricted to the more limited frequency range characteristic of adult frogs. The onset of operation of the tympanic pathway does not reinstate the highly synchronous neural activity characteristic of the operation of the fenestral pathway. Modulation transfer functions based on spike count did not show tuning for modulation rate in early stage tadpoles, but a greater variety of shapes of these functions emerged as development proceeded. Most of the different kinds of modulation transfer functions seen in adult frogs were also observed in froglets, but band-pass functions were not as sharply peaked. These data suggest that different neural codes for processing of the periodicity of complex signals operate in early stage tadpoles than in postmetamorphic froglets. Accepted: 7 October 1998     

Limb regenerative capacity of four species of Japanese frogs of the families hylidae and ranidae

The regenerative capacity of limbs was investigated by amputation of limbs at the zeugopodium in postmetamorphic froglets and adults of various sizes in four species of Japanese frogs, all of which showed some regeneration at these ages. In Hyla arborea japonica and Rana brevipoda porosa most young froglets regenerated their limbs well; however, the rate of regeneration decreased with the age of amputation, and the limb became nonregenerative in adults. Limbs of adults in Rana rugosa and R. japonica, on the other hand, exhibited good regeneration. All of the regenerates in the four species were heteromorphic, consisting histologically of well-developed cartilaginous rods surronded by connective tissue and skin. Limited development of muscle was appartment in regenerates of the three ranid species. The relations between body size, innervation of limbs, and regenerative capacity are discussed.     

Large odorous frogs (<Emphasis Type="Italic">Odorrana graminea</Emphasis>) produce ultrasonic calls

We present the first data on the vocalizations of large odorous frogs (Odorrana graminea, previously Odorrana livida), from southern China. The males produce diverse broadband signals most of which contain ultrasonic harmonics. Six basic call-types were identified based on the number of call notes, fundamental frequency, call/note duration, frequency modulation patterns and spectral composition. O. graminea is one of only a few non-mammalian vertebrates able to detect ultrasound, but its tympanic membranes are not recessed. These results should stimulate further studies to provide new insights into the mechanisms underlying high-frequency communication in anurans.     

Poison frogs rely on experience to find the way home in the rainforest

Among vertebrates, comparable spatial learning abilities have been found in birds, mammals, turtles and fishes, but virtually nothing is known about such abilities in amphibians. Overall, amphibians are the most sedentary vertebrates, but poison frogs (Dendrobatidae) routinely shuttle tadpoles from terrestrial territories to dispersed aquatic deposition sites. We hypothesize that dendrobatid frogs rely on learning for flexible navigation. We tested the role of experience with the local cues for poison frog way-finding by (i) experimentally displacing territorial males of Allobates femoralis over several hundred metres, (ii) using a harmonic direction finder with miniature transponders to track these small frogs, and (iii) using a natural river barrier to separate the translocated frogs from any familiar landmarks. We found that homeward orientation was disrupted by the translocation to the unfamiliar area but frogs translocated over similar distances in their local area showed significant homeward orientation and returned to their territories via a direct path. We suggest that poison frogs rely on spatial learning for way-finding in their local area.     

Prey location,biomechanical constraints,and motor program choice during prey capture in the tomato frog, <Emphasis Type="Italic">Dyscophus guineti</Emphasis>

This study investigated how visual information about prey location and biomechanical constraints of the feeding apparatus influence the feeding behavior of the tomato frog, Dyscophus guineti. When feeding on prey at small azimuths (less than ± 40°), frogs aimed their heads toward the prey but did not aim their tongues relative to their heads. Frogs projected their tongues rapidly by transferring momentum from the lower jaw to the tongue. Storage and recovery of elastic energy by the mouth opening muscles amplified the velocities of mouth opening and tongue projection. This behavior can only occur when the lower jaw and tongue are aligned (i.e., within the range of motion of the neck). When feeding on prey at large azimuths (greater than ± 40°), frogs aimed both the head and tongue toward the prey and used a muscular hydrostatic mechanism to project the tongue. Hydrostatic elongation allows for frogs to capture prey at greater azimuthal locations. Because the tongue moves independently of the lower jaw, frogs can no longer take advantage of momentum transfer to amplify the speed of tongue projection. To feed on prey at different azimuthal locations, tomato frogs switch between alternative strategies to circumvent these biomechanical constraints.     

A Novel Reproductive Mode in Frogs: A New Species of Fanged Frog with Internal Fertilization and Birth of Tadpoles

We describe a new species of fanged frog (Limnonectes larvaepartus) that is unique among anurans in having both internal fertilization and birth of tadpoles. The new species is endemic to Sulawesi Island, Indonesia. This is the fourth valid species of Limnonectes described from Sulawesi despite that the radiation includes at least 15 species and possibly many more. Fewer than a dozen of the 6455 species of frogs in the world are known to have internal fertilization, and of these, all but the new species either deposit fertilized eggs or give birth to froglets.     

Evaluation of PIT‐tagging in cyprinids

Laboratory and field experiments were used to investigate how different marking procedures, with 23 mm PIT (passive integrated transponders) – tags, affected mortality, body condition and tag expulsion in small roach Rutilus rutilus and rudd Scardinus erythrophthalmus (117 to 163 mm total length). In a laboratory experiment mortality was low (≤ 6%) and only occurred among fishes that had their incision closed with sutures. The latter also had a specific growth rate that tended to be lower than the fish tagged without sutures. The different marking procedures did not adversely affect the body condition of fishes either in the laboratory experiment (after 37 days) or in the field experiment (up to 369 days). No tag expulsions were observed among laboratory fishes PIT‐tagged without sutures. Marking without the use of sutures to close the incision, appears to be the most appropriate PIT tagging procedure for small cyprinids.     

Development of vasotocin pathways in the bullfrog brain

The brain of adult bullfrogs (Rana catesbeiana) contains six populations of cells which are immunoreactive for the neurohypophysial peptide arginine vasotocin (AVT). It is unknown when some of these cell populations first appear during development and when the sexual differences in AVT distribution first become apparent. We therefore used immunocytochemistry to examine development of AVT pathways in developing bullfrog tadpoles and in newly metamorphosed froglets of both sexes. AVT-immunoreactive (AVT-ir) cells were already present in the three diencephalic areas (magnocellular preoptic nucleus, suprachiasmatic nucleus and hypothalamus) at stage III (Taylor and Kollros stages), the earliest stage examined. Cell size in the magnocellular nucleus was not bimodally distributed in either tadpoles or froglets. AVT-ir cells in the telencephalic septal nucleus and amygdala did not appear until stage VI. There was no sexual difference in the density of AVT-ir cells or fibers in the amygdala of tadpoles or froglets. Finally, cells in the hindbrain pretrigeminal nucleus appeared much later-after stage XX. Thus, different populations of neurons begin to express AVT at unique times during development. The sexual dimorphism in AVT content observed in the amygdala of adult bullfrogs must appear during juvenile development or at adulthood.     

Genetic evidence of <Emphasis Type="Italic">Ranavirus</Emphasis> in toe clips: an alternative to lethal sampling methods

Amphibian populations have been undergoing declines on a global scale. Among the many threats to these populations are emergent infectious diseases (EIDs). The Ranavirus in particular has been found within many declining amphibian populations. Although non-lethal sampling methods exist for some amphibian groups, such as salamanders, the anurans are traditionally tested using a lethal method. By comparing traditional liver samples and a new non-lethal method of toe clipping we prove that the Ranavirus can also be determined in frogs using a non-lethal method, a much needed tool in threatened populations. This method will allow for ranaviral detection without further impacting declining populations, and can further be used for other research questions.     

Annual variation in glycerol mobilization and effect of freeze rigor on post-thaw locomotion in the freeze-tolerant frog <Emphasis Type="Italic">Hyla versicolor</Emphasis>

This study documents post-thaw recovery of jump distance and cryoprotectant mobilization in the freeze-tolerant frog Hyla versicolor over two successive years. Cold acclimated frogs had plasma glycerol levels near 1.0 mM in 2004 but it was nearly 70× higher during 2005. Freezing of frogs induced nearly identical levels of plasma glycerol (ca. 177 mM) during 2004 and 2005. Plasma glucose was only mobilized upon somatic freezing, with averages ranging between 21 and 36 mM. Control jump distance showed no difference between the two years of the study. The post-thaw jump response was identical during the first 2 years despite large differences in glycerol mobilization between these 2 years. Recovery proceeded much faster in 2005 when frogs mobilized glycerol prior to freeze exposure. Frogs were more impaired in their locomotion performance during the initial stages of recovery period when they were frozen at a lower temperature (−3 vs. −1.5°C) but they eventually recovered. Moderate lengthening of the freeze duration (3 vs. 7 days) with the 2004 collection group did not affect recovery of jump distance when frogs were frozen at −1.5°C. Hence, postfreeze impairment of locomotion is dependent of the intensity of the freeze temperature but it is a reversible process that is mitigated when glycerol is more freely distributed to body tissues.     

Competition and the distribution of spring peeper larvae

Studies of tadpole distributions have shown that despite overlapping affinities for semipermanent and permanent ponds, distributions of the spring peeper (Pseudacris crucifer) and the green frog (Rana clamitans) tend to be nonoverlapping. Because spring peepers are believed to be poor competitors, I hypothesized that competition from green frog larvae limits the distribution of spring peeper larvae. I stocked field enclosures with a constant density of spring peeper larvae, and one of four densities of green frog larvae (a target-neighbor design). Increased green frog density had a small effect on metamorphic size and no effects on survivorship, larval period or growth rates of spring peepers. In contrast to these small interspecific effects, green frogs had a large effect on their own performance. Intraspecific competition resulted in a 50% decline in growth rate and an 11% decline in metamorphic size. These results suggest that the species are segregated in resource use, or that compared with green frogs, spring peepers are better able to cope with depressed resource densities. In either case, this field experiment provides no evidence that interspecific competition from green frogs limits distributions of spring peepers. Other factors such as predation and breeding site choice by adults may contribute to the absence of spring peeper larvae from many semipermanent and permanent ponds.     

Refined glufosinate selection in<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation of soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill]

Modern genetic analysis and manipulation of soybean (Glycine max) depend heavily on an efficient and dependable transformation process, especially in public genotypes from which expressed sequence tag (EST), bacterial artificial chromosome and microarray data have been derived. Williams 82 is the subject of EST and functional genomics analyses. However, it has not previously been transformed successfully using either somatic embryogenesis-based or cotyledonary-node transformation methods, the two predominant soybean transformation systems. An advance has recently been made in using antioxidants to enhance Agrobacterium infection of soybean. Nonetheless, an undesirable effect of using these antioxidants is the compromised recovery of transgenic soybean when combined with the use of the herbicide glufosinate as a selective agent. Therefore, we optimized both Agrobacterium infection and glufosinate selection in the presence of l-cysteine for Williams 82. We have recovered transgenic lines of this genotype with an enhanced transformation efficiency using this herbicide selection system.Abbreviations DTT Dithiothreitol - EST Expressed sequence tag - GUS -Glucuronidase Communicated by P. Ozias-Akins     

Hochstetter's frog (Leiopelma hochstetteri) egg,mobile larvae and froglet development

Egg strings and larvae of Hochstetter's frog (Leiopelma hochstetteri) were located at three widely separated North Island sites: in seeps at Brynderwyns in December 2004, in an open pool at Wharerino in March 2009, and in an underground pool near the Kaipawa Track, Coromandel, in late May 2009. Ten egg strings were also laid by captive frogs in water courses at Hamilton Zoo in April 2009. All egg strings held from 11 to 13 eggs. The egg strings laid in the Brynderwyns were regularly observed until metamorphosis was completed in March 2005. Twenty-four swimming larvae emerged from 25 capsules at c. 40 days after discovery, and at least 14 froglets were produced at c. 90 days. All of them developed in darkness, in a 120 ml pool <30 mm deep. The emerged froglets ranged from 9.8 to 10.8 mm snout-vent length. The detection of eggs, larvae and <11 mm froglets indicates that the egg laying period is at least from late September to May.     

Evidence of <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> Infection in Water Frogs of the <Emphasis Type="Italic">Rana esculenta</Emphasis> Complex in Central Italy

Batrachochytrium dendrobatidis (phylum Chytridiomycota, order Chytridiales) is the causative organism of chytridiomycosis in amphibians, a disease associated with their population decline worldwide. In this work, we report a cutaneous infection in water frogs of the Rana esculenta complex in agricultural areas of Umbria, central Italy. Histological, immunohistochemical, ultrastructural, and molecular analyses demonstrated for the first time the presence of the Batrachochytrium dendrobatidis in this complex; to date, no association between the presence of chytrid fungal infection and mortality has been found, to our knowledge. However, the presence of Batrachochytrium dendrobatidis infection in the water frogs of the Rana esculenta complex is of concern because the frogs could act as a reservoir species and contribute to the decline of less resistant species.     

PIT-tagging Italian spined loach (Cobitis bilineata): Methodology,survival and behavioural effects

The Italian spined loach (Cobitis bilineata) is an elongated, small-sized (<12 cm) spined loach native to northern Italy, Slovenia and Croatia. As for loaches in general, little is known about the individual movements of this loach in nature. Passive integrated transponders (PIT-tags) are small (typically 7–32 mm), relatively cheap and allow tracking of individual fish movements and behaviour. A fundamental assumption in animal telemetry is that the performance of a tagged animal does not deviate substantially from its natural performance. Although PIT-tagged fish often display high survival and tag retention, the effect varies between species and contexts, and few studies have looked at behavioural effects of PIT-tagging. Here we demonstrate a PIT-tagging methodology for spined loaches, and compare survival, activity and provoked escape response (maximum swimming speed) between tagged and control fish. We also track tag retention in the tagged fish. Italian spined loaches tagged with 12 mm PIT-tags displayed high tag retention and no extra mortality, and no effects of tagging on activity or maximum swimming speed were observed. The tag-to-fish weight and length ratios in our study ranged from 2% to 5% and from 10% to 16%, respectively, and we conclude that PIT-tagging, within these ratios, appears suitable for Italian spined loach.     

The impact of changing irrigation practices in rice fields on frog populations of the Kanto Plain, central Japan

Rice fields are important substitute wetlands for frogs. Traditionally, rice fields in Japan were supplied with water and drained via shallow earth ditches (old-style). In the last 30 years, however, more than 80% of rice fields have been converted to a new irrigation system in which water is typically supplied through underground pipes via taps and is drained into deep, concrete-sided ditches (new-style). We compared the occurrence of frogs in paired areas of old- and new-style rice fields at six locations in Ibaraki Pref., central Japan, from May to August 1995. The Japanese tree frog (Hyla japonica) did not differ in abundance between the two types of rice fields, but the Japanese brown frog (Rana japonica) and the Tokyo daruma pond frog (Rana porosa porosa) preferred the old-style rice fields. These findings suggest that the status ofRana species has been adversely affected by the conversion of rice fields to the new irrigation system. Modification of the deep, U-shaped concrete ditches and the water management regime during the spawning season is needed to safeguard these species.     

Prey-handling Behaviour of Newly Hatched Snakes in Two Species of the Genus Elaphe with Comparison to Adult Behaviour

The prey-handling behaviour of two species of Asian ratsnakes was studied in the laboratory. In experiment 1, effects of prey size and type (mouse, lizard and frog) on capture position, direction of ingestion, condition of prey at ingestion, and prey-handling method were investigated using newly hatched Elaphe quadrivirgata. Prey size did not affect these variables except prey-handling method in frog trials: large frogs were more frequently constricted after a delay of more than 1 s than small frogs, and small frogs were usually simply seized. On the other hand, prey type affected prey-handling method: mice and lizards were more frequently constricted or pinioned than frogs. When hatchlings of E. quadrivirgata tried to coil around a prey animal immediately after striking, they frequently failed or released their coils within 10 s after striking. In experiment 2, prey-handling behaviour of adult E. climacophora was examined with mice of various sizes. Adult E. climacophora tended to constrict large mice immediately after striking, whereas they simply seized small mice. Only one out of 38 mice which were constricted immediately after striking was released from the coils within the subsequent 10 s. Large mice were killed prior to ingestion, whereas small mice were swallowed alive. Large mice tended to be swallowed head first. Experiment 3 was conducted to investigate effects of mouse size on prey-handling behaviour of newly hatched E. climacophora. Mouse size affected condition of prey at ingestion and prey-handling method but not capture position and direction of ingestion. Possible relationships between feeding ecology and the differences of prey-handling behaviour among Elaphe are discussed.